Introduction

INTRODUCTION
Enterococcusspeciesaremembersofthenormal intestinalfloraandarethemostcommonaerobic grampositivecoccifoundinthelargebowelof humans.Theymaybeassociatedwithinvasive disease.Commonsitesofclinicalinfectionincludethe urinarytract,thebloodstream,andintraabdominal orpelvicwounds(Treitmanetal.,2005). Inrecentyears,theirclinicalimportancehas beenamplifiedbyanincreasedresistanceto antimicrobialsand,inparticular,anacquired resistancetoglycopeptidesfirstdocumentedin1988 (Cuzonetal.,2008).Sincethisfirstreported outbreak,vancomycinresistantEnterococci(VRE)has becomeacommoncauseofhealthcareassociated infectionacrossmanypartsoftheworld(Topetal., 2008).

Twoclinicallyrelevantspecies,Enterococcus faecalisandEnterococcusfaecium,havebeenshownto acquireresistancetoglycopeptidesandaccountfora majorityofenterococcalinfections(Treitmanetal., 2005).Severalgenesencoderesistanceto 1

Introduction glycopeptides;howeverVanAandVanBareof greatestconcern.VanAistransposonmediatedand confersinduciblehighlevelresistancetoboth vancomycinandteicoplanin,whereasVanBcanbe plasmidorchromosomallymediatedandconfers moderatetohighresistancetovancomycinonly (Courvalin,2005). Theclinicalimpactofinfectionbyvancomycin resistantEnterococci(VRE)hasbeenexaminedin severalstudies,withthemostnotableconsequences beingincreasesinmortality,lengthofhospitalstay, andcostofhospitalization(Ledeboeretal.,2007). ManagementofaVREoutbreakrequires strategiestocontaincasesanddecreaseratesof transmission,includingisolationofVREinfectedor colonizedpatients(ZirakzadehandPatel,2006). EarlydetectionofVREinfecalspecimensisimportant fornosocomialpreventionmeasures,epidemiologic infectiousdiseasefollowup,andalsopreventionof vancomycinresistantStaphylococcusaureusemergence (Delmasetal.,2007). Thereisacontinuousneedforimprovementof culturemethodsforVREdetection,especiallyfor thoseclinicalmicrobiologylaboratoriesthatdonot 2

Introduction haveaccesstonucleicacidamplificationtechnologies (Pauleetal.,2003).Recently,chromogenicmedia incorporatingchromogenicenzymaticsubstratesanda varietyofantimicrobialagents(chromIDVRE, CHROMagarGRE)havebecomeavailableforVRE detectioninonesinglestep,butonlyChromIDVRE containingvancomycininaconcentrationof8g/ml, canadditionallydirectlydifferentiatebetween vancomycinresistantE.faeciumandE.faecalis strainsfromclinicalspecimensbasedondistinct colonycolor(Kuchetal.,2009).

Aim of the work

AIM OF THE WORK

Theaimofthisstudyistoevaluatethe usefulnessofchromIDVREagarforrapidisolationof VancomycinresistantEnterococcusfromstoolsamples orrectalswabsofimmunocompromisedpatientsin comparisonwithmodifiedbileesculinagar.

Review of literature

ENTEROCOCCI
Taxonomy:
The name Enterococcus is derived from the French word Entrocoque, which was used at the turn of the century to describe the enteric origin of thisgrampositivecoccus(Murray,2000). Enterococci were originally classified as enteric grampositive cocci and later included in the genus Streptococcus.Inthe1930s,withtheestablishmentof the Lancefield serological typing system, Enterococci wereclassifiedasgroupDStreptococciandwerediffer rentiatedfromthenonenterococcalgroupDStreptococci such as Streptococcus bovis by distinctive biochemical characteristics. In the 1980s, based on genetic differ rences,EnterococciwereremovedfromthegenusStrep tococcus andplaced intheir own genus, Enterococcus. The previously used species designations such as faecalis,faecium,durans,andsoforthwereretainedbut wereprecededbythegenusnameEnterococcusinplace ofStreptococcus(Cetinkayaetal.,2000). The taxonomy of Enterococcus species has undergone considerable change since the mid1980s. 5

Review of literature Before the advent and widespread use of genetic techniques for taxonomic analysis, Enterococci were distinguished from Streptococci and related taxa by the ability to grow at 10C and 45C, growth in the presence of 6.5%NaCl, and at pH 9.6, ability to hydrolyze esculin in the presence of 40% bile, and production of pyrrolidonyl arylamidase (PYR). More than 90% of strains also contained the Lancefield groupDlipoteichoicantigenintheircellwall.Taxo nomicstudieshavesubsequentlyrevealedspeciesthat are members of the genus Enterococcus by genetic criteria,butthatlackmanyofthephenotypiccharac teristics typical of the genus. Fortunately, most of these species are not commonly found in human clinicalspecimens(Konemanetal.,2006a).

Natural habitat:
Several intrinsic characteristics of the Enterococci allowthemtogrowandsurviveinharsh environment and to persist almost everywhere (Teixeiraetal.,2007). Enterococci canbefoundin soil, on plants, in milk products, other foods and in highnumbers,aspartofthenormalmicroflorainthe gastrointestinal tract as well as the faeces of vertebrates(Domigetal.,2003b).Buttheymayalso 6

Review of literature colonize the upper respiratory tracts, biliary tracts, and vaginas of otherwise healthy persons. The isolation of clinical isolates of Enterococci generally denotescolonizationratherthaninfection(Zhanelet al.,2001). The prevalence of the different enterococcal speciesappearstovaryaccordingtothehostandis also influenced by age, diet, and other factors that may be related to changes in the physiologic conditions(Teixeiraetal.,2007).

Enterococcal species:
Enterococcus faecalis and Enterococcus faecium arethepredominantenterococcalspeciesidentifiedin clinical microbiology laboratories. Historically, these laboratoriesreportthat80to90%of Enterococci are E.faecalis,whereas E.faeciumaccountsfor5to10% ofEnterococci.TheE.durans,E.avium,E.raffinsus, E.gallinarum,andE.casseliflavusarereportedmuch less frequently than E. faecalis and E. faecium (Chaudharyetal.,2007). Overthepastfewyears,severalnewenterococcal species have been described from human clinical sources(i.e.,E.gilvus,E.pallens,CDCPNS[forprobable 7

Review of literature newspecies]E1,CDCPNSE2,CDCPNSE3),animals (i.e., E.canis, E.villorum [sameas E.porcinus], E.ratti, E.asini, E.phoeniculicola), and the environment (i.e., E.haemoperoxidus, E.moraviensis). Based on their phenotypiccharacteristics,the Enterococci aredivided into 5 phenotypic groups (Table 1). With the help of molecular taxonomic techniques, some former Enterococcus specieshavebeenreclassifiedorfoundto be the same as previously recognized species. For example, E.seriolicida was found to be identical to Lactococcusgarvieae,E.flavescenswasshowntobethe same as E.casseliflavus, and E.solitaries was to be identicalto Tetragenococcushalophilus (Konemanet al.,2006a).

Review of literature Table(1): Different enterococcal species (Koneman etal.,2006a)

GROUP/Species GROUP1 E.avium Isolatedfromavian,canine,andhumangastrointestinaltracts; strains may carry both Lancefield group D and group Q carbohydrateantigens;thisspecies(and E.malodoratus)arethe twoenterococcal speciesthatproduceH2S. E.avium hasbeen isolatedfromcasesofbacteremiaandosteomyelitis. Newspeciesdescribedin2002;originallyisolatedfromabile specimenofapatientwithcholecystitis. Isolated from Gouda cheese and unpasteurized milk products; namemeansillsmelling;alsoproducesH2S. Newspeciesdescribedin2002;isolatedfromaperitonealdialysate specimen of a patient in whom peritonitis developed from a perforatedintestine. Geneticrelatednessstudiesandcertainphenotypiccharacteristics differentiatethisspeciesfromE.avium;typestrainisolatedfrom acaseofbovinemastitis. Originallyconsideredtoberelatedto E.avium (alongwith E. solitaries and E.pseudoavium);namedforitsabilitytoproduce acidfromraffinose;recoveredfromhumaninfections,including bloodcultures,urine,abscesses,andvertebralosteomyelitis. OriginallycalledStreptococcussaccharolyticus;describedagroup of S. bovislike strains recovered from cows; genetic analysis showed that this organisms is more closely related to the Enterococci than the Streptococci; has certain phenotypic characteristics that are similar to Enterococcus species (eg., growthat10 OC and45 OC,growthin6.5%NaCl),butdoesnot reactwithgroupDantisera;proposedthatthisspeciesbemoved fromtheviridiansStreptococcusgrouptothegenusEnterococcus asE.saccharolyticus. CDCproposednewspecies,isolatedfrombraintissueobtained froman11montholdpatientinHonolulu,Hawaii,in2001. Mostfrequentisolatefromhumanspecimensandfromthehuman Comments

E.gilvus E.malodoratus E.pallens

E.pseudoavium

E.raffinosus

E.saccharolyticus

Enterococcussp. CDCPNSE3 GROUP2 E.faecalis

Review of literature
GROUP/Species Comments gastrointestinaltract;alsofoundintheintestinaltractsofpoultry, cattle,pigs,dogs,horses,sheep,andgoats. E.faecium Foundinhumanclinicalspecimens;generallymoreresistantto antimicrobial agents than E. faecalis; also found in the gastrointestinaltractsofvariousspeciesofanimals. Recoveredfromplants,soil,andrarely,fromthefecesofchickens; originally classified as a subspecies of E. faecium; produces a yellow pigment and is also motile. This organism is an opportunistic agent in human infections and has been isolated frompatientswithbacteremia. Isolatedfromchickenfeces;originallyclassifiedasS.gallinarum chickengroupD;oneofthetwomotileEnterococcusspecies;has alsobeenisolatedfromaninfectioninahemodialysispatient,asa causeofnativevalveendocarditis,andfrombloodcultures. Yellowpigmentednonmotileorganism;isolatedfromplants,soil, andthegastrointestinaltractsofcattle,pigs,andhorses;named afterJ.O.Mundt,anAmericanmicrobiologist.Thisspecieshas beenisolatedfromahumanthighabscessandfromanoperatively obtainedsinusmucosalspecimen. New environmental enterococcal species described in 2001; isolatedfromsurfacewaters,swimmingpools,anddrinkingwater intheNorthMoraviaregionoftheCzechRepublic. CDC proposed new species, isolated from blood cultures of a patientinLosAngelesin1997 SpeciesoriginallythoughttobeabiochemicalvariantofE.hirae, butanalysisof16SrRNAindicatedthatthisorganismisindeeda previouslyundescribedspecies;recoveredfromhumanspecimens, includingstoolandsynovialfluid. Thisspeciesisfoundmainlyinmilkandotherdairyproductsand isarareclinicalisolate.In2004E.duranswasisolatedasacause ofnativevalveendocarditis Causesgrowthdepressioninchickens;isolatedfromchickendrops and feces, and the gastrointestinal tracts of cattle, pigs, dogs, horses,sheep,goats,andrabbits;typestrain(E.hiraeATCC8043) has complex nutritional requirements and is used in the food industryasabioassayorganismforaminoacidsandvitamins.E.

E.casseliflavus

E.gallinarum

E.mundtii

E.haemoperoxidus

Enterococcussp. CDCPNSE2 GROUP3 E.dispar

E.durans

E.hirae

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Review of literature
GROUP/Species Comments hirae wasisolatedfromthebloodofa49yearoldpatientwith endstagerenaldiseasewhowasundergoinghemodialysis. E.ratti E.villorum Describedin2001,thisnewspecieswasoriginallyisolatedfrom theintestinesandfecesofratswithdiarrhea. Isolated from the gastrointestinal tract of dogs; phenotypically identical with E. porcinus, which is considered as a junior synonym of E. villorum and was originally isolated from the intestinesandfecesofpigswithdiarrhea. Describedin1998,thisspeciesisfoundinthececumofdonkeys; mostcloselyrelatedtoE.avium,E.pseudoavium,andE.faecium. Newlyreclassifiedstreptococcalspeciesfoundintheintestinesof chickens;lacksthegroupDantigen,isPYRnegativeandisunable togrowin6.5%NaClbroth;similarto E.columbae,itmaybe confusedwith E.avium andwith S.bovis. E.cecorum hasbeen isolated as a cause of peritoneal dialysisassociated peritonitis, recurrent bacteremic peritonitis, and spontaneous bacterial peritonitis with empyema, and has been isolated from blood culturesofapatientwithseveremalnutrition. Newlydescribedyellowpigmentedspecies;recoveredfromplants; hasnotyetbeenisolatedfromhumans. Describedin2003,thisspeciesisfoundinthepreenglandsofwild redbilledWoodhoopoes;doesnotgrowonBEagarorin6.5%NaCl broth. CDCproposednewspecies,isolatedfromthebloodofapatientin Evanston,Illinois,in1991 Newly reclassified streptococcal species isolated from the intestinaltractofpigeons;closelyrelatedto E.cecorum and E. avium;characterizedbybeingPYRnegativeandunabletogrow in6.5%NaClbroth. Originallyisolatedfromadogwithchronicotitisexterna;probably aresidentofthecaninegut;growsonBEagar,growsin6.5% NaClbroth,andisPYRpositive. New environmental enterococcal species described in 2001; isolatedfromsurfaceanddrinkingwaterintheNorthMoravia

GROUP4 E.asini E.cecorum

E.sulfureus E.phoeniculicola

Enterococcussp. CDCPNSE1 GROUP5 E.columbae

E.canis

E.moraviensis

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Review of literature
GROUP/Species Comments regionoftheCzechRepublic.

Virulence factors of Enterococci including VRE:

Itisnotasinglefactorwhichisresponsiblefor the virulence of the organism. A number of studies haveidentifieddifferentvirulencefactors,mostimpor tantamongthembeinghaemolysin,gelatinase,ente rococcalsurfaceprotein[Esp],aggregationsubstance [AS], MSCRAMM Ace (Microbial surface component recognizing adhesive matrix molecule adhesion of collagen from Enterococci), serine protease, capsule, cellwallpolysaccharideandsuperoxide(Hassetal., 2002). Somestrainsof E.faecalis produceacytolysin/ hemolysin that acts on human, rabbit, equine, and bovineerythrocytes(butnotsheeperythrocytes)and has demonstrated significant toxicity in rabbit endophthalmitisandendocarditismodels (Koneman etal.,2006a). Gelatinase is an extracellular protease capable of hydrolyzing gelatin, collagen, casein, haemoglobin and other peptides. Gelatinaseproducing strains of Enterococcihavebeenshowntobevirulentinanimal 12

Review of literature models inducing caries formation in germfree rats. Epidemiologicalstudiessuggestassociationsbetween proteaseproductionandinfection (Jankoskaetal., 2008). EnterococcalsurfaceproteinESPisacellwall associated protein significantly more common in clinicalisolatesthanamongstrainsfromnormalflora (Miskeenetal.,2002). ESPisimportantforbiofilm formation and cell adherence on enterococcal patho genesis (Hsiehetal.,2009). Thisprotein,likeother surfaceproteins,mayfacilitateadherencetothehost epithelium. Some studies have suggested that ESP plays an important role in the adherence of Enterococci totheuroepithelium (Jankoskaetal., 2008). Aggregation substance is a surfacebound, plasmidencodedproteinthatpromotesclumpingofthe organisms to facilitate the plasmid exchange. This substance also functions in facilitating enterococcal adherence to cultured intestinal and renal epithelial cellsandpromotesgrowthofcardiacvegetationsinthe rabbitendocarditismodel(Konemanetal.,2006a). MSCRAMM Ace: Ace is collagen binding MSCRAMM(Microbialsurfacecomponentrecognizing 13

Review of literature adhesive matrix molecule adhesion of collagen from Enterococci)onEnterococciandisstructurallyrelated to staphylococcal Cna adhesion. Its presence among bothcommensalandpathogenicisolatesofE.faecalis is apparently expressed during infection in humans (Upadhyayaetal.,2009). LipoteichoicacidsconstitutethegroupDantigen of Enterococci andmayalsofunctioninvirulenceby inducing the production of tumor necrosis factor (TNF) and interferon, leading to modulation of the immuneresponse(Konemanetal.,2006a). Extracellular Superoxide: E. faecalis isolates from blood stream are unique in their ability to produce superoxide. Superoxide production was observedtoenhanceinvivosurvivalof E.faecalis in mixed infection with bacteroides fragilis in a sub cutaneousinfection.Althoughnotaclassicvirulence factor, the resistance of Enterococci to multiple antimicrobial agents allows them to survive and proliferate(Upadhyayaetal.,2009).

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Review of literature

ENTEROCOCCAL INFECTION
Pathogenesis of enterococcal infection:
Enterococci, including vancomycinresistant Enterococci(VRE),areclearlylessvirulentasopposed toothercommonpathogenicbacteriasuchas Staph. aureus or Strept. pyogenes. They do not produce exotoxinsorsuperantigens(Moellering,2005). Keytounderstandingenterococcalinfectionatthe molecular and cellular level is that nosocomial enterococcaldiseaseispredominantlyatwostagepro cess.Thereisusuallyinitialasymptomaticcolonization of gastrointestinal tract by enterococcal strains possessingvarioustraits,suchasantibioticresistances, cytolytictoxingenes,orpossiblyaggregationsubstance or the protease gelatinase upon hospital admission (Sood et al., 2008). Subsequently this population is expanded, often facilitated by antibiotic elimination of competitors. For a select number of patients, there is subsequenttissueinvasion,directlyorindirectly, from theexpandedgastrointestinaltractreservoir.Giventhe two stage model, virulence factors may act by

15

Review of literature functioningateitherorbothlevels.Thisexplainshow antibioticresistancehelps(Mundyetal.,2000).

Clinical significance of Enterococci including VRE:

Themostfrequentinfectionscausedby Entero cocciareurinarytractinfections(UTIs)whichinclude cystitis, pyelonephritis, prostatitis, and perinephric abscess; most such infections are nosocomial and associated with urinary instrumentation. Patients withVREhaveahighprevalenceofskincolonization, which may result in colonization of intravascular catheters and subsequent intravascular catheter relatedsepsis(ZirakzadehandPatel,2006). The second mostfrequent enterococcal infections generally have been intraabdominal and intrapelvic abscesses or postsurgery wound infections (Moellering,2000). Inlivertransplantrecipients,the most common types of VRE infection include intra abdominal infections associated with biliary leaks, stenosis,orobstruction;hepaticorperihepaticabscesses; stenosis or thrombosis of the hepatic artery; or perforated viscera. In intraabdominal and pelvic infections, Enterococci usually are found mixed with other indigenous aerobic and anaerobic organisms 16

Review of literature (Zirakzadeh and Patel, 2006). Pure spontaneous enterococcal peritonitis and enterococcal peritonitis associated with continuous ambulatory peritoneal dialysis have also been reported (Koneman et al., 2006a). The third most frequent infection caused by these organisms is blood stream infections (BSIs). Bacteremiaiscommonandtypicallyderivesfromthe aforementioned infections. In kidney and pancreas transplant recipients, bacteremia typically derives fromwoundorurinarytractinfections (Joelsetal., 2003). Neutropenicpatients colonizedwithVREare at risk for VRE bacteremia as a result of gut translocation, central venous catheter infections, or urinarytractinfections(Shaikhetal.,2001). Mortality rates for patients with VRE bacteremiavarydependingonthepopulationatrisk. Recipients of autologous peripheral blood stem cell transplantshavebeenshowntohavemortalityrates as low as 10% (Kapur et al., 2000). Furthermore, differences in enterococcal species and susceptibility toampicillinmayalsoaffectmortalityrisk.E.faecium, frequently resistant to ampicillin, currently is most oftenassociatedwithnosocomialbacteraemiaandhas 17

Review of literature beenassociatedwithhighermortalitythan E.faecalis (MasciniandBonten,2005). Enterococci have been considered to be an importantcauseofendocarditisandaccountforabout 20%ofcasesofnativevalvebacterialendocarditisand for about 6 to 7% of prothetic valve endocarditis (Teixeiraetal.,2007). Endocarditisislesscommon thanbacteremia(Andersonetal.,2004). PatientswithendocarditiscausedbyVREhave been reported to have mortality rates higher than 30%,thosewithsolidtumorshavedeathrateshigher than50%,andsomestudiesofcriticallyillandliver transplant patients have shown more than 70% mortality(Chaversetal.,2003). Enterococcal meningitis is a rare enterococcal infectionthatmaybeseeninbothadultsandchildren. Enterococcalmeningitismaydevelopspontaneouslyor as a postoperative infection. Individuals with spon taneousenterococcalmeningitisusuallyhaveentero coccalinfectionatothersites,havesevereunderlying disease, and have concomitant enterococcal bacte remiamorefrequentlythanthosewithpostoperative infections(Pintadoetal.,2003).

18

Review of literature Themortalityrateofpatientswithenterococcal meningitisishigh,rangingfrom13to33%,becauseof the increasing rates of multidrug resistance Enterococci and difficulty in achieving therapeutic drug concentrations in the cerebrospinal fluid (CSF) (Tsaietal.,2006). Otherinfectionscausedwithlowerfrequencyare neonatal infections. Enterococci rarely cause respi ratory tract infections, osteomyelitis, or cellulitis (Soodetal.,2008). In general, enterococcal infections are distributed equally between the sexes. However, in published series of enterococcal endocarditis, men oftenoutnumberwomen(Megran,1992). Other significant predictors of VRE carriage included:age>65yrs,femalegender,historyofdiabetes mellitusandprolongedhospitalstay (Chlebickiand Kurup,2008). The patients most likely to be colonized are elderly, severely ill individuals with multiple co morbidities.Thesepatientstendtobehousedforlong periodsoftimeinareasofthehospital,suchasICUs, whereVREendemicityishighest(Mazuski,2008). 19

Review of literature In neonates, Enterococci occasionally cause bacteremia or meningitis. Outbreaks of enterococcal infectioninneonatalintensivecareunitsarereported. A few VRE outbreaks are reported in neonatal intensive care units, but the overall percentage of enterococcal isolates with vancomycin resistance is lowinpediatricpatientscomparedtoadults(Haslam etal.,2003).

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Review of literature

ANTIMICROBIAL RESISTANCE
IN ENTEROCOCCI
Enterococci exhibits intrinsic resistance to penicillinasesusceptible penicillin (low level), penici llinaseresistantpenicillin,cephalosporin,nalidixicacid aminoglycosideandclindamycin(Soodetal.,2008). The intrinsic resistance of Enterococci to many commonlyusedantimicrobialagentsmayhaveallowed themacumulativeadvantageforfurtheracquisitionof genesencodinghighlevelresistancetoaminoglycosides, penicillins, tetracycline, chloramphenicol, and now vancomycin(Cetinkayaetal.,2000). In the USA, the first resistance to antibiotics acquiredbyEnterococciappearedduring70swiththe emergence of E.faecium resistant to amoxicillin. Resistance to aminoglycosides then followed during the80s(Birgand,2009). Enterococci have become increasingly multi resistant in the late 1980s and early 1990s due to widespread cephalosporin use at the time and, to a lesser extent, to the usage of aminoglycosides, fluoroquinolonesandcoamoxiclav,allofwhichhave 21

Review of literature spectrapredominantlyfocusedagainstGramnegative pathogens(Amyes,2007). Althoughtheenterococcalresistancetopenicillin and ampicillin has been reported in recent years, many E.faecium strains have acquired high level resistance to these antibiotics. Increased ampicillin resistancein Enterococci isattributabletoeitherthe production of betalactamase or alterations in the expression or structure of penicillinbinding protein (PBP),PBP5(Rajaetal.,2006). Table(2): ListofIntrinsicandacquiredantimicrobial etal.,2000)
Intrinsicresistance Lactams(particularly cephalosporinsand penicillinaseresistant penicillins) Lowconcentrationsof aminoglycosides Clindamycin Fluoroquinolonesand nalidexicacid Trimethoprim sulfamethoxazole

drugresistanceinEnterococci(Cetinkaya
Acquiredresistance Highconcentrationsof lactams,throughalterationof PBPsorproductionof lactamase Highconcentrationsof aminoglycosides Glycopeptides(vancomycin, teicoplanin) Tetracycline Erythromycin Rifampin Chloramphenicol Fusidicacid

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Review of literature
Nitrofurantoin

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Review of literature

Phenotypic resistance in Enterococci:

1 lactamresistance: Enterococcibeginwithintrinsicresistancetomost lactam antibiotics. While most isolates of E.faecalis can be inhibited by concentration of penicillin achie vableintheplasma(MICof1to8g/ml)thisisusually not the case with E.faecium (MIC of 16 to 64g/ml). Enterococci having high level resistance to penicillin (HLPR) and ampicillin (MIC 256g/ml) have been reportedfromvariouslocations(Marothietal.,2005). Lowlevelampicillinresistancein Enterococci is attributable to the production of a low affinity peni cillinbindingprotein(PBP),PBP5.Williamsonetal. (1983) noted that penicillin resistance expressed by E.faeciumwasrelatedtotheamountandtheaffinityof PBP5. PBP5s havebeenidentifiedinseveralentero coccal species. Highlevel ampicillin resistance in Enterococci isattributabletoeithertheproductionof betalactamase or alterations in the expression or structureofPBP5.Betalactamaseproductionhasbeen described almost exclusively in E. faecalis and is attributable in most cases to the acquisition of the Staph.aureusbetalactamaseoperon(Rice,2001).

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Review of literature 2Aminoglycosideresistance: Enterococci exhibit low level resistance to all aminoglycosides(MIC8to256gm/ml)whichappears tobeduetolowuptakeoftheseagentsastheydonot possesscytochromeenzymesandthuscannotproduce theenergyrequiredtotakeupantibioticsintothecell (Klareetal.,2003). However, aminoglyciside uptake is enhanced when Enterococci are exposed to lactams. This synergy underlies the long standing practice of com biningbothclassesofantibioticstotreatseriousentero coccalinfectionsascombinationovercomestheintrinsic resistance exhibited by Enterococci and a synergistic effect is usually achieved since the intracellular penetrationofaminoglycosidesisfacilitatedbycellwall activeagent(Marothietal.,2005). Enterococci developed highlevel resistance to gentamycin (HLGR) that caused resistance to synergism between gentamycin and penicillin. This acquired resistance is highly specific and renders bacteria resistant to high levels of aminoglycosides and as a result resistance to synergism. Highlevel resistance to aminoglycosides (HLAR) is defined as occurringwhendrugconcentrationof 2000g/mlare 25

Review of literature requiredforinhibitionoforganism (Mohantyetal., 2005). Highlevelresistancetoaminoglycosides(HLAR) is being conducted by series of aminoglycoside modifyingenzymes(AME)codedbyplasmidandare transferable. The most frequently encountered enzymeincludea)dualfunction2'phosphotransferase and 6'acetyl transferase conferring HLR to all available aminoglycoside (Kanamycin, gentamycin, amikin,netilmycin,tobramycin)exceptstreptomycin; b)3'phosphotransferasecodingforHLRtokanamycin and penicillinamikacin synergy without HLR to gentamycin; c) 6'adenyl transferase which produces HLR to streptomycin but does not inactivate other useful aminoglycosides (Table 4) (Marothi et al., 2005). 3Vancomycinresistance: Six glycopeptideresistant enterococcal pheno types, VanA, VanB, VanC, VanD, VanE, and VanG, havebeendescribed.Theycanusuallybedistinguished onthebasisofthelevel,inducibility,andtransferability ofresistancetovancomycinandteicoplanin.Thefirst two types are the most clinically relevant (Table 3) (ZirakzadehandPatel,2006). 26

Review of literature VanAismorewidelydistributedandthusthe predominant type of resistance reported. It has inducible high level resistance to both vancomycin (MIC 64g/ml) and teicoplanin (MIC 16g/ml). VanBshowsvariablelevelsofinducibleresistanceto vancomycin (MIC 8 to 64g/ml) and sensitive to teicoplanin(MIC 1g/ml)(Marothietal.,2005). 4Linezolidresistantstrains: Linezolidresistant strains (six E.faecium and oneE.faecalisisolates)weredetected,butonlyamong theUnitedStatessites.Alllinezolidresistantisolates carried the G2576U mutation of 23S rRNA. These sevenisolatesoccurredassingleincidenceseachata differentmedicalcenterandwerenotconsideredtobe clonallyrelated(Deshpandeetal,2007).

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Review of literature Table(3): ResistancetoGlycopeptidesin Enterococci (ZirakzadehandPatel,2006)

Vancomycin Teicoplanin MIC (g/mL) MIC (g/mL) Expression

Pheno type

Genotype (resistance operon present)

Abilityto transfer resistance E.faecium E.faecalis Eavium E.gallinarum Species

VanA

VanA

64>1000

16512

Inducible

Yes

E.durans E.mundtii E.casseliflavus E.raffinosus E.hirae E.faecium

VanB

vanB

41000

0.252

Inducible

Yes

E.faecalis E.gallinarum E.durans

VanC

vanC1

232

0.122

Constitutive Inducible Constitutive Constitutive

No

E.gallinarum

VanC VanC

vanC2 vanC3

232 232

0.122 0.122

No No

E.casseliflavus E.flavescens E.faecium E.faecalis E.faecalis E.faecalis

VanD

vanD

16256

264

Constitutive

No

VanE VanG

vanE vanG

16 16

0.5 0.5

Inducible Inducible

No No

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Review of literature Table(4): Majorpatternandmechanismsofresistance to antimicrobial agents in Enterococci (TeixeiraandFacklam,2005)

Patternofresistance Highlevelresistance to Aminoglycosides Gentamycin Kanamycin Streptomycin
a

Mechanismofresistance Enzymatic (production of aminoglycosidemodifying enzymes, AMEs)b AAC(6`)Ie+APH(2``)Ia AAC(6`)Ii APH(2``)Ib;APH(2``)Ic;APH(2``)Id APH(3`)IIIa ANT(3`)Ia;ANT(4`);ANT(6`)Ia Alterationofthetarget (leadingtodecreasedribosomalbinding)

Resistanceto glycopeptides Vancomycin Teicoplanin

c

Alterationofthetarget

(modificationofthepeptidoglycanbiosyntheticpathway) VanA VanB VanC(VanC1,VanC2,andVanC3) VanD VanE VanG Alterationofthetarget (alteredpenicillinbindingproteins,PBPs) Enzymatic (productionoflactamase) Alterationofthetarget (changestothesubunitaofDNAgyrase) Enzymatic (productionofchloramphenicolacetyltransferase,CAT)

Resistanceto lactams
d

Penicillin Ampicillin Resistanceto quinolones Resistanceto chloramphenicol group Macrolides (erythromycin) Lincosamide(clindmycin)

ResistancetotheMLS Enzymatic(productionofmethylatingenzymes)

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Review of literature
StreptograminB

a)
b)

FoundinincreasingfrequenciesinE.faecalisandE.faecium AAC,acetyltransferases;ANT,nucleotidyltransferses;APH,phosphortransferases VanAandVanBareusuallyfoundinE.faecalisandE.faecium:VanAhasoccasionally beenfoundinseveralotherenterococcalspecies:VanC1isusuallyassociatedwithE gallinarumandVanC2andVanC3withE.casseliflavusrespectively.VanDresistance hasbeendescribedinE.faeciumstrainswhileVanEandVanGwerefoundinE.faecalis. FoundinE.faecalisandE.faecium.AlsodescribedamongE.raffinosus

c)

d)

Genetic mechanisms of resistance in Enterococci

There are at least three major reasons for the emergence of multidrug resistant Enterococci: (i) baseline intrinsic resistance to several antimicrobial agents, (ii) acquired resistant via mobility of the resistance genes on plasmids and transposons, and chromosomalexchange,and(iii)thetransferabilityof resistance (Cetinkaya, et al., 2000). In addition, these genetic transfers often occur in the gastrointestinaltractsofhumansandanimals,many ofwhichhaveotherbacteriaunderpotentialselective pressurefromtherapeuticorsubtherapeuticlevelsof ongoingantimicrobialexposure(Mundy,2000). Antibioticresistancein Enterococcus speciescan be transferred by pheromonemediated conjugative plasmidsortransposons.Theresistancegenesmaybe passedonnotonlytoantibioticsusceptibleEnterococci, butalsotootherpathogens(Giraffa,2002).Incontrast 31

Review of literature to Gramnegative conjugation systems, conjugation of Enterococcusspeciesdoesnotrequirepili,andinvolves a pheromoneinduced system. Bacteria containing conjugativeplasmidsrespondtopheromones(plasmid specific)forgeneticexchange;thesebacteriagenerally haveanarrowrecipientrangeforconjugation,including only closely related species. This lateral transfer of genetic elements leads to rapid dissemination of antibiotic resistance. The plasmids occurring in Enterococcus speciescanalsobetransmissionvehicles fortransposons(WilliamsandHergenrother,2008). The most extensively investigated pheromone inducibleplasmidsinE.faecalisarepCF10,pAD1and pPD1.InthecaseofpAD1thetransactingregulatory proteinencodedbythetraEgeneisexpressed(Folliet al., 2008). The transfer of these plasmids occurs in response to specific sex pheromone peptides secreted by plasmidfree recipient cells. Uptake of the exogenous pheromone by the donor cell causes it to express proteins involved in the conjugation process. Productionofaggregationsubstance(Agg)onthedonor cellsurfacefacilitatescontactwiththerecipientcellby binding to enterococcal binding substance (EBS) displayedonthesurface,resultinginconjugationand

32

Review of literature the ability to pass antibiotic resistance on to the recipientcell(Fig.1)(Clewelletal.,2002). Another system of conjugation, also not well understood, involves broad hostrange plasmids that can transfer among species of Enterococci and other grampositive organisms such as Streptococci and Staphylococci. The transfer frequency is generally much lowerthan with thepheromonesystem. Since Staphylococci, Streptococci, and Enterococci share a number of resistance genes, these broad hostrange plasmidsmaybeamechanismbywhichsomeofthese resistancegeneshavespreadamongdifferentgenera (Murray,1998). A third type of conjugation, which involves conjugativetransposons,mayalsoexplainthespread of resistance genes to many different species. As opposed to ordinary transposons, which can jump within a cell from one DNA location to another, conjugative transposons also encode the ability to bring about conjugation between different bacterial cells.Sinceplasmidstypicallyrequirerathercomplex machinery for replication (often depending on successful interactions with hostproteins) and must face additional problems of surface exlusion and 33

Review of literature incompatibility,conjugativetransposons(whichdonot replicate, butinstead insert intothechromosome or intoaplasmidofthenewhost)appeartobeaneven moreefficientandfarreachingwayofdisseminatinga resistancegene(Murray,1998).

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Review of literature

Fig.(1): Illustrates the pheromoneresponsive conjugative system. Enterococcus faecalis pheromoneresponsive conjugativesystem.PheromoneAreleasedfromthe potentialrecipientcell(right)interactswithplasmid Ainthepotentialdonorcell(left)toinducesynthesis ofaggregationsubstance.Attachmentofaggregation substance to binding substance causes the cells to clumpintovisibleaggregates.Oncethepheromone responsiveplasmidAhastransferredfromdonorto recipient cell,synthesisofpheromone Aisshut off (Murray,1998).

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Review of literature

VANCOMYCIN RESISTANT ENTEROCOCCI

Withthedevelopmentandtransmissionamong Enterococci of highlevel resistance to lactams and aminoglycosides, the use of the glycopeptide vancomycin to treat enterococcal and other serious Grampositive infections increased dramatically. As vancomycin use escalated, the first isolates of vancomycinresistant Enterococci were identified in rapidsuccessioninEuropeandintheUnitedStates (ShepardandGilmore,2002). Theoccurrenceofvancomycinresistant Entero cocci(VRE)hasprogressivelyincreasedintheUnited States and in some parts ofEuropeover the past5 years. An observation from the early years of the SENTRYProgram(19971999)illustratedthatUnited states(US)isolateswereconsiderablymoreresistant tovancomycin(17%in1999)thanthosefrompatients in other geographic regions. Approximately 25% of enterococcalinfectionsfrompatientsintheintensive care unit (ICU) have been found to be VRE (Deshpandeetal,2007). So,therapidincreaseofglycopeptideresistance among Enterococci causes great concern because of 36

Review of literature limitedtreatmentoptionsincaseofhumaninfections (Whiteneretal.,2004).

Mechanism of action of vancomycin:

Thesynthesisofpeptidoglycanintheproduction of bacterial cell walls requires several steps. In the cytoplasm,aracemaseconvertslalaninetoDalanine (DAla),andthentwomoleculesofDAlaarejoinedby aligase,creatingthedipeptideDAlaDAla,whichis then added to uracil diphosphateNacetylmuramyl tripeptidetoformuracildiphosphateNacetylmuramyl pentapeptide. Uracil diphosphateNacetylmuramyl pentapeptideisboundtotheundecaprenollipidcarrier, which, after the addition of NacetylDglucosamine (GlcNAc) from uracil diphosphateGlcNAc, allows translocationoftheprecursorstotheoutersurfaceof the cytoplasmic membrane. Nacetylmuramylpenta peptideisthenincorporatedintonascentpeptidoglycan bytransglycosylationandallowstheformationofcross bridgesbytranspeptidation(Fig.2)(Courvalin,2006). Glycopeptideantibioticsareproducedbyaction mycetesandinhibitthesynthesisofbacterialcellwall byblockingpeptidoglycanassembly.Theybindtothe DalanylDalanine (DAlaDAla) C terminus of the nascent peptidoglycan and prevent it from being 37

Review of literature utilized in the following crosslinking reactions catalyzed by transglycosylases and transpeptidases (Beltramettietal.,2007).Thisfailuretoformcross linksbetweenpeptidoglycanintermediateslowersthe rigidity of the cell wall and renders these bacteria susceptibletoosmoticlysis(Healyetal.,2000).

Fig.(2): Peptidoglycanbiosynthesisandmechanismofactionof vancomycin.BindingoftheantibiotictotheCterminal DAlaDAlaoflatepeptidoglycanprecursorsprevents reactions catalyzed by transglycosylases, trans peptidases, and the D,Dcarboxypeptidases. DDl, D Ala:DAla ligase; MurF, a synthetase protein; UDP, uracildiphosphate(Courvalin,2006).

Genetic mechanism resistance:

38

of

vancomycin

Review of literature Resistancetovancomycinisduetothepresence of operons that encode enzymes (1) for synthesis of lowaffinityprecursors,inwhichtheCterminalDAla residue is replaced by Dlactate (DLac) or Dserine (DSer),thusmodifyingthevancomyinbindingtarget; and(2)foreliminationofthehighaffinityprecursors thatarenormallyproducedbythehost,thusremoving thevancomycinbindingtarget(Courvalin,2006). Glycopeptide resistance has emerged in the Enterococci by acquisition of transposon Tn1546, whichmediatesproductionofprecursorsterminating inDlactate(DLac)insteadofDAla.Thesubstitution leads to a 1,000fold reduction in the affinity of vancomycin for its target. Tn1546 encodes two biosyntheticenzymesforreductionofpyruvateintoD Lac(VanH)andsynthesisofthedepsipeptideDAla DLac (VanA). In addition, eliminationofprecursors ending in DAla is required for resistance. This function is carried out by two Tn1546encoded D,D peptidases that hydrolyze DAlaDAla (VanX) and cytoplasmic precursors containing a stem pentapeptide (VanY). Dissemination of Tn1546 and related elements specifying the production of precursors ending in DAlaDLac is responsible for the high incidence of glycopeptide resistance in the 39

Review of literature Enterococcianditsemergenceinmethicillinresistant Staph. aureus. In addition, low level resistance to vancomycincanresultfromproductionofprecursors endinginDSer.Finally,substitutionofDAlabyD2 hydroxybutyrate and D2hydroxyvalerate has been obtainedinmutantsconstructedinvitro(Cremniter etal.,2006).

Vancomycinresistanceisconferredbyoneoftwo functionally similar operons, VanA or VanB. The VanA and VanB operons are highly sophisticated resistance determinants, which suggests that they evolved in other species and were acquired by Enterococci.Thedifferenceintheguaninecytosine(G C)contentofthegenesoftheVanBoperon(roughly 50%GC)incomparisontotypicalenterococcalgenes (35% to 40% GC) is compelling evidence for this acquisition(Rice,2001). Glycopeptideresistancecanbeintrinsic,present in all the strains of a given species, or acquired, presentonlyincertainisolatesbelongingtothesame species(Courvalin,2005).

Intrinsic glycopeptide resistance:

VanCtyperesistance 40

Review of literature The VanC type of resistance is specific to E.gallinarum, E.casseliflavus, and E.flavescens. Strainsbelongingtothesespeciesareresistanttolow levels of vancomycin but remain susceptible to teicoplanin.ThreecloselyrelatedvanCgenesencoding DAla:DSer ligases have been described: vanC1 in E.gallinarum, vanC2 in E.casseliflavus, and vanC3 inE.flavescens(ReynoldsandCourvalin,2005). Asexpectedforanintrinsicresistance,thevanC operonischromosomallylocatedandnottransferable. Certain species of Lactobacillus, Lactococcus, Leuco nostoc, and Pediococcus are intrinsically resistant to high levels of glycopeptides by synthesis of peptide glycan precursors ending exclusively in DLac. These are opportunistic pathogens rarely responsible for infections(Courvalin,2005). Three genes are required for VanC type resistance:vanTencodes theVanTmembranebound serineracemase,whichproducesDSer;thevanCgene productVanCsynthesizesDAlaDSer,whichreplaces DAlaD Ala in late peptidoglycan precursors; and vanXYcencodestheVanXYCprotein,whichpossesses both D,Ddipeptidase and D,Dcarboxypeptidase activitiesandallowshydrolysisofprecursorsendingin 41

Review of literature DAla.SubstitutionoftheultimatedAlabyaDSer resultsinsterichindrancethatreducesitsaffinityfor vancomycin(Courvalin,2006).

Acquired glycopeptide resistance:

VanAtyperesistance VanAisthemostcommontypeofglycopeptides resistance in Enterococci and the only one, so far, to have disseminated to Staphylococci. VanAtype glycopeptide resistance is characterized by acquired inducible resistance to both vancomycin and teicoplanin. It is mediated by transposon Tn1546 or closely related genetic elements. Tn1546 contains the vanAgeneclusterthatencodes8polypeptides.Tn1546 wasfirstdetectedonaconjugativeplasmidinaclinical isolateofE.faecium.Howeverthistransposonmaybe locatedonplasmidsorbacterialchromosomes (Weigel et al., 2003). This nonselftransferable element belongstotheTn3familyoftransposons.Inadditionto the seven genes involved in glycopeptide resistance (vanHAXYZ) and its regulation (vanRS), Tn1546 contains two genes, ORF1 and ORF2, responsible for replicativetransposition.Sincethistypeofmigrationis associated with local DNA duplication, movements of

42

Review of literature theseelementsresultinanexponentialincreaseofthe resistancegenestheycarry(Fig.4)(Courvalin,2005). Coordinated expression of the proteins VanA, VanH,andVanXisessentialforresistance.VanAisa ligase that catalyzes the formation of ester bonds between Dalanine and Dlactate, preferentially yielding modified peptidoglycan precursors with D alanylDlactate (DAlaDLac) termini that bind glycopeptideswithdecreasedaffinity.BecauseDlactate (DLac)isneitherintrinsicallyproducedbyEnterococci nortypicallyfoundintheenvironmentofthebacteria, DLacincorporatedintothemodifiedprecursorsmust beproducedthroughthereductionofpyruvatebytheD hydroxyaciddehydrogenaseactivityofVanH.VanXisa D,Ddipeptidase that specifically hydrolyzes DAlaD Ala, thereby limiting the competitive synthesis and availabilityofthenormalpeptidoglycanprecursors(Fig. 3)(ShepardandGilmore,2002). The transcription of the genes encoding VanA, VanH, and VanX is regulated by a twocomponent regulatorysystemformedbyVanSandVanR.VanS servesasthesensorkinaseproteinthatrespondsto vancomycin, teicoplanin, or the early effects of both glycopeptidesoncellwallsynthesis.Followingreceipt of the signal through VanS, VanR serves as the 43

Review of literature responseregulatortoactivatetranscription ofvanA, vanH,andvanX(Fig.3)(Depardieuetal.,2007). Accessory proteins VanY and VanZ appear to augment glycopeptide resistance, but neither is essential. VanY is a D,Dcarboxypeptidase that removesDAlaattheterminiofnormalpeptidoglycan precursors that escaped hydrolysis by VanX. VanZ promotesmoderateincreasesinresistancespecifically toteicoplanin(ShepardandGilmore,2002).

Fig.(3): Shows VanAtype glycopeptide resistance. Top, SynthesisofpeptidoglycanprecursorsinaVanAtype resistantstrain.DDl,DAla:DAlaligase;penta,lAla gDGlulLysDAlaDAla; Pentadepsi, lAlagD GlulLysDAlaDLac; Tetra, lAlagDGlulLysD Ala;Tri,lAlagDGlulLys.Bottom,Organizationof the vanA operon. Open arrows represent coding

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Review of literature
sequencesandindicatethedirectionoftranscription. Theregulatoryandresistancegenesarecotranscribed frompromotersPRandPH,respectively(Courvalin, 2006).

Fig.(4):

ShowsreplicativetranspositionofTn1546. Thedonor replicon(lowerleft)containsacopyofthetransposable element (close bar; the direction of replication is indicatedbyanarrow)whereastheacceptorreplicon (upper left) does not. Selective replication of the transposable element associated with replicon fusion generatesabirepliconthatcontainstwocopiesofthe element,inthesameorientation,atthebordersofthe replicons.Duetorecombinationbetweenthetwocopies oftheelement,whentranspositioniscompletedeach repliconcontainsacopy oftheelementandcanthusact asadonor(Courvalin,2005).

VanBtyperesistance 45

Review of literature AlthoughVanAisthepredominanttype,VanB strainsarefairlycommonintheUnitedStates (PerzHernndezetal.,2002). The VanB operon is related to VanA but its induction system recognises only vancomycin, to which the operon usually confers moderate levels of resistance.TheVanH,VanBandVanXgenesofthis operonarealsoabletoconferresistancetoteicoplanin butfailtodosobecauseteicoplanindoesnotactasan inducer.VanBislesscommonthanVanAandislikely to haveemerged in areas where teicoplaninhas not beenused(Amyes,2007). The VanB operon has been described in most casesaspartoflargeconjugativeelementsintegrated intothehostchromosome.Theseelementswerefound to contain transposons conferring resistance to vancomycin,suchasTn1547orTn5382.Vancomycin resistantE.faecalisE93/268andE.faecium654were isolatedin1994intheUnitedKingdom.Resistancein thesestrainswasassociatedwiththepresenceofthe VanBgene,bornebyconjugativeplasmids (Garnier etal.,2000).

InVanBtypestrains,twoproteinsareinvolvedin theproductionofpeptidoglycanprecursorsendinginD 46

Review of literature AlaDLac: VanHB, a dehydrogenase that reduces pyruvate to DLac, and the VanB ligase that synthesizes DAlaDLac. The VanXB D,Ddipeptidase cleaves the DAlaDAla dipeptide synthesized by the host Ddl, and the VanYB D,Dcarboxypeptidase contributestovancomycinresistancebyhydrolyzingthe CterminalDAlaofthepentapeptideprecursorswhen eliminationbyVanXBisnotcomplete.Thefunctionof theadditionalVanWproteinfoundintheVanBcluster isunknown(SanMillanetal.,2009)andthereisno generelatedtoVanZ(Courvalin,2006). Onthebasisofsequencedifferences,theVanB geneclustercanbedivided into3 subtypes: VanB1, VanB2,andVanB3.Thereisnocorrelationbetween the VanB subtype and the level of resistance to vancomycin. Enterococci harboring clusters of the VanB class remain susceptible to teicoplanin since thisantibioticisnotaninducer.However,mutations intheVanSBsensorgenehavebeenobtainedinvitro and in vivo in animal models following selection by teicoplanin,whichhaveresultedinthreephenotypic classes(constitutive,teicoplanininducible,orhetero geneous expression of the resistance genes) due to threetypes ofalterationsofVanSB function.Deriva tives of VanBtype strains that are resistant to 47

Review of literature teicoplanin have been isolated from two patients followingtreatmentwithvancomycinorteicoplanin, buttheisolateswerenotstudiedfurther(Depardieu etal.,2007). VanDtyperesistance VanD, has been characterized in E. faecium BM4339,whichisresistanttointermediatelevelsof vancomycin and to low levels of teicoplanin (Perichonetal.,2000). VanDhasbeenfoundinrelativelyfewstrainsof E. faecium and E. faecalis. All VanDtype strains characterizedtodatecontainamutatedDAla:DAla ligase gene (DDl), and thus expression of the vanD operon is necessary for peptidoglycan synthesis and growthoftheorganism.Constitutiveexpressionofthe VanDoperoninthesestrainsisachievedbymutations in either the VanS gene from the VanD operon (VanSD)ortheVanRDgene(Boydetal.,2006). Acquired VanDtype resistance is due to constitutive production of peptidoglycan precursors endinginDAlaDLac.TheorganizationofthevanD operon, which is located exclusively in the chromosome in strains that have been studied, is 48

Review of literature similartothatofVanAandVanB.However,nogenes homologous to VanZ or VanW from the VanA and VanB operons, respectively, are present. VanDtype strains share other characteristics that distinguish them from VanA and VanB type Enterococci. Resistance is constitutive and is not transferable by conjugation to other Enterococci. VanDtype strains havenegligibleD,Ddipeptidaseactivity,whichshould result in a susceptible phenotype, because these bacteria are unable to eliminate peptidoglycan precursorsendinginDAlaDAla,whichisthetarget forglycopeptides.However,inVanDtypestrains,the susceptible pathway does not function, because the DDlisinactiveastheresultofvariousmutationsin thechromosomalDDlgene(Courvalin,2006). VanEtyperesistance Synthesisofpeptidoglycanprecursorsendingin DAlaDSer has been detected in two other vancomycinresistant types of E. faecalis designated VanE and VanG (Depardieu etal., 2003). In both instances,resistanceisacquiredandlowlevelandthe genes conferring resistance are chromosomal. VanE type resistance, which is not transferable by conju gation,isvirtuallyidenticaltothatinVanCstrains 49

Review of literature apartfromthedifferentrelativeactivityofenzymes. Five genes are involved: the three essential genes encodingaVanEligase,VanXYEDDdipeptidase/DD carboxypeptidase, and VanTE serine racemase, with genes encoding a twocomponent regulatory system downstreamfromtheessentialgenes(Reynoldsand Courvalin,2005).

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Review of literature VanGtyperesistance IncommonwithVanCandVanEtypes,strains of E.faecalis of the VanG type synthesize peptido glycan precursors terminating in DAlaDSer. In severalrespects VanG strains differ from VanCand VanEisolates(ReynoldsandCourvalin,2005). Lowfrequency transfer of VanGtype glycol peptides resistance among Enterococci is associated withthemovement,fromchromosometochromosome, ofgeneticelementsofca.240kbcarryingalsoermB encodederythromycinresistance (Depardieuetal., 2003). Glycopeptidedependentstrains An intriguing and clinically important phenomenon that has developed in some VanA and VanBtype Enterococci is vancomycin dependence. Thesestrainsarenotonlyresistanttovancomycinor tobothvancomycinandteicoplanin,butalsorequire their presence for growth. Variants of glycopeptide resistantE.faecalisandE.faeciumthatgrowonlyin the presence of glycopeptides have been isolated in vitro, in animal models, and from patients treated withvancomycinforlongperiods.Inthepresenceof 51

Review of literature vancomycin,thevanAorvanBencodedDAla:DLac ligase is induced, which overcomes the defect in synthesisofpeptidoglycanprecursorsendinginDAla DAlabecauseofthelackofafunctionalDDlfollowing variousmutationsintheDDlgeneand,thus,permits growthofthebacteria.Becausethesestrainsrequire particular growth conditions, their prevalence is probably underestimated and not easily detected by routine laboratory testing. Reversion to vancomycin independence has been observed and occurs as a result of a mutation in either the VanSA or VanSB sensor, which leads to constitutive production of D AlaDLac and, thus, to teicoplanin resistance, or in theDDlgene,whichrestoressynthesisofDAlaDAla and leads to a VanB phenotype inducible by vancomycin(SanMillanetal.,2009).

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EPIDEMIOLOGY OF VRE
Vancomycinresistant Enterococci (VRE) have emerged as a significant nosocomial pathogen, since 1980, with implications for mortality, duration of hospital stay, need for intensive care and extensive surgery as well as increased costs of treatment (Ratheetal.,2009).

Reservoir:
Conversely, asymptomatic carriage among healthy people is relatively common in Europe compared to the USA. The differences in the epidemiology of VRE transmission between Europe andtheUSAcouldberelatedtothreemajorfactors. First,thewidespreaduseofavoparcin,avancomycin analogue, in the farm animals industry until 1997 (whenthedrugwasbanned),mightberesponsiblefor the reservoir of VRE among healthy subjects in Europe.Individualsinclosecontactwithanimalswith highVREcolonizationratessuchaspigs,calvesand turkeys,showedhighratesofcolonization.Secondly, nosocomialstrainsisolatedintheUSAandinEurope belonged to a distinct genetic lineage of E.faecium 53

Review of literature characterised by ampicillin resistance. Thirdly, the greateruseofvancomycinintheUSAcomparedtothe European countries might also have played a role (TacconelliandCataldo,2008). There is also evidence for direct exogenous acquisition of infection. Oropharyngeal colonization may provide a source for crosscolonization, particularly if hospital staff consider manipulations (suchastracheostomyorendotrachealtubecare)tobe clean and do not subsequently wash their hands (Soodetal.,2008). Additionally, skin colonization associated with diarrhoea or faecal incontinence has been reported. Environmentalsurfacesandmedicalequipmentitems inthepatientsroomfrequentlybecomecontaminated withVREandmayalsoserveasareservoirespecially intheroomsofpatientswhohavediarrhoea.Diarrhea (occurringdirectlyasaresultofantibiotictreatment, indirectlybecauseofClostridiumdifficileinfection,or as a result of other factors) may result in greater sheddingofallstoolorganisms,includingVRE(Drees etal.,2008a). Examples of items that may be contaminated includepatientgownsandlinen,beds,bedsiderails, 54

Review of literature overbed tables, floors, door knobs, wash basins, glucose meters, blood pressure cuffs, electronic thermometers,ECGmonitors,ECGwires,etc. (Sood et al., 2008). VRE may remain viable on such surfacesfordaysorweeksevenmonthsandmayact asasourcefromwhichhealthcareworkers(HCWs) maycontaminatetheirhandsorclothing(Duckroet al.,2005). Also Snyder et al. (2008) demonstrated that glovesandgownswerefrequentlycontaminatedwith MRSAandVREduringroutinepatientcare:therate of detection of MRSA and/or VRE on gowns and/or gloveswas18%.Wefoundthatpatientassociatedrisk factorsfordetectionincludepresenceoftheHCWin the room of a patient with a gastrostomy and/or jejunostomyfeedingtube. Oncecolonized,thepatientsremainsoforweeks ormonths(evenuptooneyear).Thus,ascolonized patients leave the hospital environment, the possi bilitythattransmissionmightoccurinthecommunity cannotbediscounted. Infact, twocasesofapparent communityacquired VRE urinary tract infection in New York City have been reported (Sood et al., 2008).

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Review of literature

Mode of transmission:
Most nosocomial acquisition of VRE is due to proximity to unisolated VREpositive patients, and crosstransmission which has been identified as an important risk factor for VRE acquisition (Zhou et al.,2008).

Crosstransmission of vancomycinresistant Enterococcus (VRE) between patients in hospitals occurs primarily via the contaminated hands of healthcare workers (HCWs). This process requires severalsequentialsteps,includingthetransferofVRE from a contaminated site on a patient or in the environmenttoanHCWshand.Becausethedensityof VREonpatientsskinisusuallygreaterthanthatfound onenvironmentalsurfaces,handcontaminationmight beassumedtooccurmorefrequentlyafterdirectcontact withapatientcolonizedwithVREthanaftercontact with the environment. Touching a VREcontaminated inanimate surface has been shown to result in glove contamination, however, in both experimental and clinicalsettings(Haydenetal.,2008). However that Drees et al. (2008b) reported thatVREtransmissionhasbeenattributedtofomites, such as fluidized beds and thermometers as VRE 56

Review of literature colonizesenvironmentalsurfacesandequipmentand maypersistforprolongedperiodsfordaystomonths (range,7daysto4months)despiteroutinecleaning (ChlebickiandKurup,2008). IthasbeensuggestedthatE.faeciummayenter thecommunityviathefoodchain.VandenBogaard etal.(1997) foundindistinguishablepulsedfieldgel electrophoresis (PFGE) patterns of vancomycin resistant Enterococci (VRE) strains isolated from a Dutchfarmerandoneofhisturkeys,indicatingthat humans and animals in close contact may harbor identicalstrains.Theyalsoshowedthatvancomycin resistant E.faecium isolates from pigs, poultry, and humans could be divided according to basepair variation in the VanX gene. All poultry isolates belonged to one type, whereas all but one of the porcine isolates belonged to another, indicating that horizontal exchange of vancomycinresistant E.faecium or Tn1546like elements between poultry andpigsisuncommon.Ontheotherhand,bothtypes were found in humans, indicating that human acquisitionmayarisefrombothanimalsources.This alsosuggestsdirectionaltransmissionfromlivestock tohumans.

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Review of literature Jensen et al. (1999) reported highly similar PFGE patterns in vancomycinresistant E. faecium strains containing similar Tn1546like elements isolated from pigs and a hospitalized patient in Denmark,furthersuggestingfoodbornetransmission ofVREfromanimalstohumans(Luetal.,2002). Riskfactors: Colonization of the gastrointestinal tract is a prerequisite to infection. A number of factors are known to contribute to acquiring colonization by resistantEnterococciaccordingtostudiesconductedin the West. Gastrointestinal carriage of resistant Enterococciisakeyfactordeterminingdevelopmentof infection by the same organism. Furthermore, colonizing flora provides an important clinically hidden reservoir of resistant enterococcal strains for subsequent hospital acquired infection and transmission. If factors promoting colonization are identified and controlled, it may be possible to decrease the incidence of colonization and thereby clinicalinfection(GunasekeraandPerera,2007). Therearenumerousriskfactorsfornosocomial colonization and infection with VRE. These risk factors include advanced age, renal and hepatic 58

Review of literature failure,haematologicalmalignancy,severityofillness, invasive procedures and devices, gastrointestinal surgery, transplantation, proximity to another VRE positive patient and antimicrobial therapy. Affected patientsareusuallyill,withprolongedhospitalstay andwhohadreceivedlongcoursesofbroadspectrum antibiotics(Karimietal.,2009). One of the most important factors involved in VREspreadinhospitalsisthecolonizationpressure. Colonization Pressure means the patients who are exposedtoVREdailyandgetcolonized.Ifmorethan 50%ofpatientsinICUarecolonizedwithVRE,other risk factors such as using antibiotics become less significant(Hautemanireetal.,2009). Antibiotic exposure has been consistently identified as a risk factor for VRE positivity. It facilitates VRE transmissionby2 mechanisms: a) it suppresses normal competing bowel flora providing selective advantage for VRE and b) it increases concentrationofVREinstoolofpreviouslycolonized patientsrenderingthemmorecontagious(Chlebicki andKurup,2008). Antibiotic selective pressure exerted by extensiveuseofthirdgenerationcephalosporinsand 59

Review of literature drugs with potent activity against anaerobes have beenreportedtopredisposetoVREcolonizationand infection(Tanejaetal,2004). While few clinical studies delineate the role of oralvancomycininVREacquisition,oralvancomycin useprobablyexercisedsomeinitialselectionpressure, contributing to the emergence of this type of resistance.Indeed,oneofthefirstdocumentedcases ofVREwasdescribedinapatientwhoreceivedoral vancomycin(Harbarthetal.,2002). Fulleretal.(1998) suggestedthatvancomycin exposure may exert selective pressure on the gut, raising undetectable levels of preexisting VRE to detectable levels. In such a case, vancomycin administration does not cause VRE to develop, nor does it increase the odds that the individual patient willacquireVRE.However,itresultsinanapparent increaseinthelikelihoodofVREdetection(Harbarth etal.,2002). VRE can be isolated from the stool of healthy adults and hospitalized patients during vancomycin therapy. Parenteral vancomycin treatment does not eliminateallgrampositivecocciintheoralandfecal floraandmayincreasetheintestinalVREloadinVRE 60

Review of literature carriers. This may also facilitate VRE transmission, sincethenumberofVREinagivenclinicalsampleis proportionaltotheeasewithwhichVREistransmitted tootherbodysitesortoanotherpatient.Forinstance, Beezhold et al. (1997) demonstrated that while all patientswithVREbacteremiahadrectalcolonization with VRE, 86 and 57% also had colonization of the inguinal skin and the antecubital fossa, respectively (Harbarthetal.,2002). Prevalence: Glycopeptideresistancein Enterococci wasfirst reportedfromEuropeandthenfromUnitedStatesin 1980s. Since then vancomycinresistant Enterococci (VRE)hasbeenreportedfromseveralregionsofthe world(Rajaetal.,2005). Vancomycinresistant Enterococci (VRE) were firstdetectedinEurope(UnitedKingdom&France) in 1986 and soon after, a VanB E.faecalis clinical isolatewasreportedintheUnitedStates.Thesehave nowbeenreportedfromAustralia,Belgium,Canada, Denmark, Germany, Italy, Malaysia, Netherland, SpainandSweden(Soodetal.,2008).

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Review of literature TheEuropeanepidemiologywithrespecttoVRE is almost completely opposite to the American situation.InEurope,onlyafewnosocomialoutbreaks have been reported (although this incidence is increasing)andincidencesofnosocomialinfectionare low. However, asymptomatic carriage amongst healthy European individuals is relatively common (Ridwanetal.,2002). The incidence of human VRE infections in Europeancountriesisrelativelylow(13%)compared with the high and rising rate in the US. The rates havesteadilyincreasedfrom0.3to7.9%(CDC).The increase is mainly due to 34fold rise (0.413.6%) of VRE infections in the intensive care unit (ICU) patients,althoughanincreasehasbeennotedinnon ICUpatientsalso(Soodetal.,2008). DatafromtheEuropeanantimicrobialresistance surveillancesystemshowarateof E.faecium isolated fromglycopeptidesresistant Enterococci (GRE)bacte raemiahigherthan20%inseveralcountries(Ireland, Portugal,Greece,UnitedKingdom.)andlessthan1% in others countries like the Scandinavians countries. Anincreaseoftheresistanceisalsobeingseeninsome

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Review of literature countries such as Germany, Ireland, Israel, and Slovenia(Birgand,2009). Infections with oxacillin (methicillin)resistant Staph. aureus (MRSA) and vancomycinresistant Enterococci (VRE) have increased dramatically in recentyears,andtogether,theycause12%ofallhealth careassociated infections. Patients infected with MRSAorVRE,aswellasasymptomaticallycolonized patients,serveasareservoirfortransmissionofthese bacteriatootherhospitalizedpatients (Huckabeeet al.,2009). Since2002threecasesofvancomycinresistant Staph.aureus(VRSA)possessingtheVanAgenehave beenreportedfromclinicalsettings.Inthefirstcase, theVanAgenefromtheVRSAwaspostulatedtohave originated from VRE which were also isolated from thepatient(Changetal.,2003).

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LABORATORY DIAGNOSIS
Laboratory diagnosis of Enterococcus species:
Staining:
The Enterococci are grampositive cocci that occursingly,inpairsandinshortchains.Thecellsare sometimes coccobacillary when gram stains are preparedfromagarplategrowth.Theyaremoreoval and are found in chains when gram stains are preparedfromthioglycolatebroth.

Isolation:
The Enterococci arefacultativelyanaerobic.All strains grow in broth containing 6.5% NaCl and hydrolyzeesculininthepresenceof40%bilesalts. Trypticasesoy5%sheepbloodagar,brainheart infusion5%sheepbloodagar,oranybloodagarbase containing 5% animal blood product supports the growthof Enterococci. Enterococci growwellat35to 370C and do not require an atmosphere containing increased levels of CO2, although some strains grow betterinthisatmosphere.Ifthesampletobecultured is likely to contain gramnegative bacteria, 64

Review of literature commerciallypreparedmediacontainingbile,esculin, andazideareexcellentoptionsforprimaryisolation (Teixeiraetal.,2007).

Identification:
Motilityisobservedwithseveralstrainsofsome enterococcal species (E.casseliflavus, E.gallinarum), whicharereportedtobemotilebyscantyflagella,butin general, Enterococcus is a nonfilamentous bacterium (Domigetal.,2003a). Theusefulnessofpyroglutamylaminopeptidase (PYRase)activityasanaidintheidentificationhas beenreported.However,thisenzymeactivitycannot be detected with E.saccharolyticus, E.cecorum and E.columbae(Domigetal.,2003a). All strains hydrolyze leucinenaphthylamide (LAP). Enterococci do not contain cytochrome enzymes,andthereforegiveanegativecatalasetest, however,apseudocatalaseissometimesproduced,and aweakeffervescenceisobservedinthecatalasetest. This occurs when strains of E.feacalis are grown on blood containing medium particularly on primary isolation; it is not known if any other Enterococcus species behave in this manner. The strength of this

65

Review of literature reactionmaydiminishafterafewserialsubcultures (Koneman,etal.,2006a). Enterococci are salt, bile, acid and alkali resistant, factors which aid their survival in the hospitalenvironment.Moststrainsproduceacellwall associated glycerol teichoic acid antigen, which is identified as the streptococcal group D antigen. Demonstratingbyextractionandserologicalreaction thatanunknownstrainhasgroupDantigenmaybe helpful in identification of some strains, but these reactionsshouldbeinterpretedcautiously.Mostofthe PediococcusstrainsandabouthalfoftheLeuconostoc strains isolated from human infections also have groupDantigens(Teixeira,etal.,2007). No phenotypic criteria are available for clearly distinguishing the genus Enterococcus unequivocally from others, since there are no particular criteria, whicharetypicalofall Enterococci.Thismeansthat identificationatthegenuslevelnecessarilyisfollowed byspeciesidentification:e.g.,whenastrainshowsthe characteristics of an enterococcal species, it can be presumedthatthestrainisan Enterococcus (Domig etal.,2003b).

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Review of literature

Typing methods of Enterococci:

A)Phenotypictechniques Classicaltypingmethodshaveincludedbiotyping, antimicrobial resistance profiles, serologic charac terization,bacteriocintyping,andbacteriophagetyping. Although these approaches have occasionally yielded usefulinformation,theygenerallyaretimeconsuming and difficult to reproduce and/or interpret (Arbeit, 1999). Classical phenotyping methods used to invest tigate the diversity among isolates of a given entero coccalspecieshavefrequentlyfailedtoadequetlydiscri minateamongstrains,andtheyhavelimitedvaluein epidemiologicstudies.However,phenotypicinformation in association with molecular data can contribute valuableinformation(Facklametal.,2002). 1) Carbohydrate fermentation and enzymatic activity(biotyping): Biotyping,meaningthedetectionofcarbohydrate fermentationandenzymepattern,canberegardedasa traditionalwayofmicrobialdifferentiation.Usually,a battery of bacteriological tubes containing different carbohydrate sources and indicator dyes is used, followed by numerical analysis for evaluating the results. Having their origin mainly in the field of 67

Review of literature routineanalysisofmedicalspecimen,miniaturizedtest combinationsandtestkitshavebecomeanimportant issueinthephenotypicdifferentiationof Enterococci. Becauseoftheirtimesavinguse,someofthesetests can be considered as rapid methods even with increased selectivity. In addition, the activity of esculinaseandpyrasecanbeassessedusingtheAPI zym(BioMerieux)testorrelatedformats.Khnetal. (1995) concluded that the PhenePlatek PhP plate system (PhPlate Microplate Techniques, Stockholm, Sweden) is useful for epidemiological studies of enterococcalisolates,yieldingresultssimilartothose obtained by pulsedfield gel electrophoresis (PFGE) (Domigetal.,2003a). 2) Enterocintyping: Bacteriocins are ribosomally synthesized, extracellular released antimicrobial peptides that showactivityagainstcloselyrelatedbacterialspecies. Enterococcusspeciesareknowntoproducearangeof enterocinsincludingenterocinsA,B,I,LandP,which areactiveagainstListeriaspecies,Clostridumspecies andStaph.aureus(FisherandPhillips,2009). Thedetection ofenterococcallyogroupsproved to be as reliable for species identification as 68

Review of literature conventional methods. The bacteriolytic pattern groupscorrelatewithspeciesgroupsphylogenetically determined(Domigetal.,2003a). 3) Serotyping: In 1933, Lancefield reported that haemolytic faecalStreptococciaretypicalforpossessingthegroup Dantigen.Asmanyas39serologicalsubgroupshave beenreportedingroupD Streptococci. Petts,(1995) evaluated a modified nitrous acid extraction latex agglutination kit for grouping betahaemolytic StreptococciandEnterococciandproposedthismethod tobereliable(Domigetal.,2003a). 4) Antimicrobialsusceptibilitytesting: Dicuonzoetal.(2001) analysedtheantibiotic resistanceofclinicalandenvironmentalisolates.The intrinsic lowlevel vancomycin resistance of E.casseliflavus, E.flavescens and E.gallinarum is an excellentcriterionfortheirspeciesidentification.The susceptibility or resistance to efrotomycin is one typical attribute next to others applied in the identificationschemeproposedby DaGlriaetal. (1998)(Table5)(Domigetal.,2003a).

69

Review of literature Table(5): ShowsthekeytestsincludingMethylD

Glucopyranoside (MGP) and Efrotomycin (EFRO) susceptibility tests, for the identification of enterococcal species (Da Glriaetal.,1998) Resultfor MOT +b +b PIG +b + MGP + + EFRO R S R S R

Species E.faecalis E.faecium E.casseliflavus E.mundtii E.gallinarum

MOT, motility; PIG, pigmentation; b, occasional variant reaction.

5) Pyrolysismassspectrometry(pyMS): nosocomial enterococcal infections. Morrison et al. (1999) demonstrated that pyMS could distinguish vancomycin resistant E.faecium strains similar to PFGEgrouping,withfewexceptions (Domigetal., 2003a). 70 Freemanetal.(1994) appliedpyMStoverify

Review of literature 6) BacteriophageTyping: Bacteriophageandbacteriocintypingasepidem iologic tools are limited to bacteria. Bacteriophage (phage)typingclassifiesbacteriabasedonthepattern of resistance or susceptibility to a certain set of phages. Bacteriophages are viruses that are able to attach to the cell walls of certain bacteria, enter, multiply,andlysethecells.Thedifferentialabilityof phages to infect certain cells is based upon the availability of corresponding receptors on the cell surfaceforthephagetobind.Oftendifferentstrains of pathogens have a different cohort of receptors, leadingtovariablelysisprofiles(Singhetal.,2006). B)Genotypictechniques Today, a growing range of techniques with varying taxonomical resolution is used to identify, characterize and to type Enterococci. Although the classicalphenotypebased(biotyping)methodsarestill of importance for daily routine analyses, genotypic methodshaveincreasinglycontributedtotheindepth characterization of microorganisms and their differentiation and have become a powerful tool in epidemiologicalresearch.Thecombinationofdifferent fundamental and advanced methods in a polyphasic 71

Review of literature approachwillprovideasuitablesolutionforreliable identification(Domigetal.,2003a). Improved discrimination among enterococcal strains involved analysis of chromosomal DNA restriction endonuclease profiles by pulsedfield gel electrophoresis (PFGE). Multilocus enzyme electrophoresis, ribotyping, and PCRbased typing methods,suchasrandomlyamplifiedpolymorphicDNA PCRassayandrepetitiveelementsequencebasedPCR, havealsobeenusedtoinvestigategeneticrelationships among enterococcal strains. Sequencing of PCR productsandrestrictionfragmentlengthpolymorphism analysesofPCRproductshavebeenusedtotraceand determinedifferencesamongantibioticresistancegenes inEnterococci(Donabedianetal.,2000). 1) Pulsedfieldgelelectrophoresis(PFGE): Pulsedfield gel electrophoresis (PFGE) is capable of separating large DNA fragments by periodically changing the direction of the electrical field in which the DNA is separated. Ideally, the PFGEcandifferentiatebetweenisolatesrepresenting an outbreak strain and those of epidemiologically unrelated strains but can not distinguish between differentisolatescausingtheoutbreak.Ifthisoccurs, 72

Review of literature the outbreak is relatively easy to identify. Alternatively, random genetic variations observed between individual isolates and the outbreak strain provideanestimateofthelikelihoodthattheisolateis partoftheoutbreak(Singhetal.,2006). Pulsedfield gel electrophoresis (PFGE) is consideredapracticalgoldstandardduetoitshigh discriminatoryabilities,butishamperedbyavariety of disadvantages: (i) the method is time consuming, (ii)comparabilitybetweenlaboratoriesisunsatisfying, andthusinternationaldatabasescanbecompiledonly by applying rigorous quality assurance and quality control,asinthecaseofPulseNet,and(iii)banding patternsareonlypartiallyinformativewithregardto phylogeneticrelationships(AbeleHornetal.,2006). 2) Multilocussequencetyping(MLST): For many different bacterial species, the most appropriate technique for global and longterm epidemiology studies is multilocus sequence typing (MLST). MLST provides an unambiguous nomenclatureforgenotypes,andclonesanddataare easily stored in databases that can be exchanged between different laboratories via the Internet. For E.faecium, the development of a MLST scheme has 73

Review of literature been critical in the understanding of global epidemiology, genetic evolution, and population structure(RuizGarbajosaetal.,2006). Multilocus sequence typing (MLST) and multiplelocus variablenumber tandem repeat analysis (MLVA) have been developed recently to recognize genetically related and potential epidemic isolatesof E.faecium.Itwasshownthatfewclones emerged carrying the vancomycin resistance determinant. MLST confirmed the unrelatedness of humanandnonhumanVRE,ashadbeenestablished byPFGE,andconfirmedsubgroupC1ofVangenesas being responsible for human cases. Several authors used MLST for outbreak investigations thereafter. MLVAhasbecomepopularinthisrespectforavariety oforganismsasthoserelatedtobioterrorism (Abele Hornetal.,2006). 3) QuantitativerealtimePCR(qPCR): Quantitative realtime PCR (qPCR) is a molecular method for detecting and quantifying specificgenetargetsinbacteria.Themethodinvolves the examination of amplification patterns, as displayed in realtime, over a number of designated PCR cycles utilizing spectrally distinct fluorogenic 74

Review of literature probes or DNAbinding dyes for detecting and quantifying a gene target of interest. During the exponential phase of repeated PCR amplification cycles,increasingfluorescentsignalabovebackground (threshold cycle, CT) can be determined and is theoretically proportional to increasing amounts of amplified DNA product. Because fluorescence is monitoredduringeachcycle,areliablequantification assessment can be achieved. Moreover, the high sensitivityofthismethodaddstoitspotentialvalue for detecting low numbers of cells in mixed culture (Sedgleyetal.,2005). 4) Fluorescenceinsituhybridization(FISH): Fluorescence in situ hybridization (FISH) with fluorescently labeled oligonucleotide probes targeting therRNAhasalreadybeenusedforthedetectionof E.faecalis and E.faecium inbloodcultures.FISHwas veryusefulduringaVREoutbreakintheuniversity hospital of Ulm in Germany, allowing the rapid differentiation ofthe E.faecium outbreakstrain from VanCharboring E.gallinarum isolatesthatwerealso growing on the VRE screening plates and showing ambiguous results with the API system. The black pigmentation of the colonies caused by esculin 75

Review of literature hydrolysisdidnotinterferewiththeFISHprocedure. In addition to the identification of Enterococci from pure cultures, FISH proved highly useful for direct applicationtopositivebloodcultures,eventhosewith polymicrobial growth. Thus the FISH assay may significantlyshortenthetimetotheretrievalofresults in the clinical microbiology laboratory and to the initiation of pathogenadapted antimicrobial therapy, especiallyforsepticpatients (Wellinghausenetal., 2007).

B) Laboratory diagnosis of vancomycinresistant Enterococci (VRE):

1)DiscDiffusion: According to CLSI guidelines a standard disk diffusion testing is peformed on MullerHinton agar by using commercially prepared 30g vancomycin disks(CLSI,2008). As defined by CLSI the breakpoints for vancomycin were: resistant 32mg/L; intermediate resistant from 8.0 to 16mg/L; susceptible 4.0mg/L (Ghidanetal.,2008). Although a good correlation was observed betweenVanAE.faeciumisolates26,29and30,VanB 76

Review of literature E.faecalisisolate31andvancomycinresistanceonall three plating media [MuellerHinton (MH), MH + bloodorbrainheartinfusionmedium(agarorbroth)], the disk diffusion assay appeared unreliable for vancomycin susceptibility testing Vancomycin resistance,asdeterminedbythediskdiffusionassay, didnot correlate with thepresenceofthe vanC2/C3 genotypes(Nayaketal.,2002). 2)DilutionMethods(MICstest): MICdeterminationisthestandardquantitative techniquefortestingofantimicrobialsusceptibilityof bacteria in many clinical laboratories. The recommended method for MIC determination is the agardilution(AD)method,whichisregardedasthe reference method against which other methods are assessed. However, this test is timeconsuming and requires careful control of the technique to produce reliable results, cumbersome, and inadequate for routine testing in many clinical laboratories (Mokaddasetal.,2007). MacrodilutionBrothSusceptibilityTest: Themacrodilutionbrothsusceptibilitytestwas amongthefirsttobedevelopedandstillservesasa 77

Review of literature reference method. Serial dilutions of antimicrobial agent are made in broth or agar, after which a standardizedbacterialsuspensionisadded.Tentest tubes that contain cationsupplemented Muller Hintonbroth,antimicrobialagentareseriallydiluted from100g/mlto0.4g/mlinthefirstninetubes;tube numbertenisfreeofantimicrobialagentandserves as a growth control. Each of the ten tubes is inoculated with a calibrated suspension of the microorganismtobetestedandincubatedat35Cfor 18hrs.Attheendoftheincubationperiod,thetubes are visually examined for turbidity. Cloudiness indicatesthatbacterialgrowthhasnotbeeninhibited bytheconcentrationofantimicrobialagentcontained inthemedium(Konemanetal.,2006b). BrothMicrodilution(BMD): The microtube dilution procedure is similar in principle to the macrotube method, except that the susceptibility of microorganisms to antimicrobial agents is determined in a series of microtube wells thataremouldedintoaplasticplate.Eachplatemay contain 80, 96, or more wells, depending on the number and concentration of antimicrobial agents thataretobeincludedinthesusceptibilitytestpanel. 78

Review of literature The advantages of the microtube method are that smallvolumesofreagentsareusedandlargenumbers of bacteria can be tested simply and inexpensively againstapanelofantimicrobialagents(Konemanet al.,2006b).

Broth Micro dilution (BMD) was performed by usingcationadjustedMuellerHintonbrothII.Micro trays containing serial twofold dilutions of each antibioticwereprepared,immediatelyfittedwithan adhesivesealandfrozenforlateruse.Onthedayof testing fresh subculture isolates were adjusted to a 0.5McFarlandturbiditystandardandafinalinoculum of 5x105 CFU/ml was obtained in each well. Trays wereincubatedat35Cinatmosphericairfor24hrs. Theantimicrobialagentsweretestedusingstandard antimicrobial powders of vancomycin, linezolid, daptomycinandtigecycline.Whentestingdaptomycin the broth was supplemented to a level of 50mg/l of Ca2+. The MICs were determined as the lowest concentrationofanantibioticthatinhibitedgrowthas judgedbytheunaidedeye(Ratheetal.,2009). Etest: The Etest (ET) susceptibility testing method, developedbasedonthediffusionoftheantibioticfrom 79

Review of literature a preformed continuous antibiotic gradient from a plastic strip, appears to be much simpler and less timeconsuming than reference methods. Since the adventofET,itsversatilityandeaseofusemakethe method an attractive alternative to conventional methods. This method has been recommended for susceptibility testing of Strept. pneumoniae and Enterococcus sp. isolates in clinical diagnostic laboratories(Mokaddasetal.,2007). 3)Automatedsystems: The VITEK 2 (bioMrieux) is an automated system for identification and antimicrobial susceptibility testing. It detects bacterial growth and metabolic reactions in the microwells of plastic test cards by measuring fluorescence. Isolates were subcultured onto sheep blood agar and incubated for 24hrsat37CbeforetestingontheVITEK2system. The inoculum system was prepared by adjusting a bacterialsuspensiontoaMcFarlandstandardof0.5in a0.45%sodiumchloridesolution.TheIDGramPositive Cocci(IDGPC)cardoftheVITEK2systemwasused for identification and the ASTP524 card for the antimicrobialsusceptibilitytesting.Thetestsemployed intheIDGPCcardhavebeendescribedpreviouslyby 80

Review of literature GarciaGarroteetal.(2000);Ligozzietal.(2002). The IDGPCdatabase allows theidentification of the following Enterococcus species: E.faecium, E.faecalis, E.durans, E.casseliflavus/ E.gallinarum, E.avium,and E.hirae(Eisneretal.,2005). TheMicroscanGramPositiveBreakpointCombo Panel(DadeBehringMicroscan,WestSacramento,CA) provide simultaneous identification and antimicrobial susceptibilityresultsinamicrotitreformat.Tritzand colleagues,(1990)evaluatedthissystemforitsability to identify Enterococci andfoundthatboth E.faecalis andE.faeciumwerereliablyidentifiedbutlesscommon enterococcal species were misidentified. Iwen and colleagues, (1999) evaluated the revised MicroScan DriedovernightGramPositiveIdentificationpanelfor its ability to identify 8 different Enterococcus species andfoundthatthesystemidentified98.5%ofisolates tested;resolutionoflowprobabiltyidentificationswith additional tests (i.e., motility and pigmentation) was only necessary for E.gallinarum/casseliflavus isolates (Konemanetal.,2006a). 4)Mediacontainingantibiotics: TheNCCLShasrecommendedthatthesimplest andmostsensitivetestforrecognitionofVREisthetest 81

Review of literature that uses vancomycin screening agar, originally described by Willey et al. (1992). This method incorporates use of 6g/mL of vancomycin in brain heartinfusion(BHI)agar,spotinoculationwith105 or 106 CFU, and incubation for a full 24hrs at 35C. Studiescomparinguseofthismethodwiththepresence ofthevanA,vanB,orvanCgeneshaveconfirmedthe sensitivity of this approach for detection of VRE. However, it is important to note that vancomycin screening agar is not entirely specific for vanA and vanBtyperesistance(JorgensenandFerraro,2000). A wide range of selective media has been investigatedbutthereisnoagreementonwhichisthe best.Someincorporatehighconcentrationsofvanco mycin (2064mg/L), which will inhibit some strains withlowlevelresistance,and6mg/Lprobablyoffers the best compromise between sensitivity and specificity. The National Committee for Clinical LaboratoryStandardsmethodforscreeningisolatesof Enterococci for glycopeptide susceptibility utilizes vancomycin at 6mg/L. When screening for GRE, a dedicated indicator medium, such as Enterococcosel agar with 6mg/L vancomycin (or a medium with equivalent performance), is recommended (Cookson etal.,2006).

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Review of literature Detection of VRE colonization can be accomp lished by culturing the organism on a variety of selectiveanddifferentialmedia,mostcommonly,bile esculinazidewith6or8g/mLofvancomycin(BEAV) agar.Althoughthesetechniquesaresensitive,theyare also time consuming (2472hrs) and labor intensive. Ledeboer et al. (2007a) reported developmental studiesusingahighlyspecificchromogenicagarthat selects for and differentiates between vancomycin resistant E.faecalis (VREfs)andvancomycinresistant E.faecium(VREfm)directfromfeces(Ledeboeretal., 2007b). 5)Chromogenicmedia: Recently, chromogenic media incorporating chromogenic enzymatic substrates and a variety of antimicrobial agents (chromID VRE, CHROMagar GRE)havebecomeavailableforVREdetectioninone single step, but only chromID VRE containing vancomycin in a concentration of 8g/ml, can additionallydirectlydifferentiatebetweenvancomycin resistant(VR)E.faeciumandE.faecalisstrainsfrom clinical specimens based on distinct colony color (Kuch et al., 2009). So it is useful for rapid and selectiveisolationofVRE.faeciumandVRE.faecalis 83

Review of literature aswellasacquiredvancomycinresistancepresentin E.casseliflavus,E.gallinarumandE.raffinosusfrom clinicalsamples,especiallyafterinclusionofthebroth enrichmentstep(Kuchetal.,2009). Because chromID medium reliably identifies Enterococci to the species level and confirms glycol peptidesresistance,itisonlynecessarytoconfirmthe organismisGrampositivecocci,eliminatingtheneed forbiochemicalanalysisorantimicrobialsusceptibilities forconfirmation(Ledeboeretal.,2007b). ChromID incorporates chromogens targeted by enzymes specific for E.faecium and E.faecalis, and their degradation distinguishes the two species as purple and bluegreen colonies, respectively. The sensitivitiesforGREdetectionfromstoolsamplesare similar for ChromID and BEAV/Enterococcosel agar following direct inoculation or enrichment (MalhotraKumaretal.,2008). 6)Moleculartechniques Several molecular techniques are available for theidentificationofdifferententerococcalstrainsand detection of genetic resistance to miscellaneous antibioticsasdiscussedbeforeandtheyare: 84

Review of literature Pulsedfieldgelelectrophoresis(PFGE) Multilocussequencetyping(MLST) QuantitativerealtimePCR(qPCR) Fluorescenceinsituhybridization(FISH) (Ersoyetal.,2007)

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PREVENTION AND CONTROL OF VRE

The increasing incidence of infection, the limitations of effectivetherapyandthe potential for transferring vancomycinresistance genes to other bacteriahavemadethecontrolofVREapublichealth concern(Zhouetal.,2008). The first guidelines for the control of VRE in hospitals were published in 1994 by the CDC and Hospital Infection Control Practices Advisory Committee(HICPAC).Summaryofrecommendations for preventing the spread of vancomycin resistance (adapted from CDCHICPAC) (Chen and Zervos, 2009). Appropriateuseofvancomycin

Treatment of infection due to Blactamresistant Grampositiveorganisms. TreatmentofinfectionduetoGrampositiveorga nismsinpatientswithseriousbetalactamallergy. Treatmentofantibioticassociatedcolitisincasesof metronidazolefailureorpotentiallylifethreatening illness. 86

Review of literature

Endocarditis prophylaxis, as recommended by the AmericanHeartAssociation(Dajani). Prophylaxis for surgical procedures involving implantationofaprosthesisininstitutionswitha highrateofinfectionduetoMRSAormethicillin resistantS.epidermidis.

Educationprogram

Include physicians, nurses, pharmacy and laboratorypersonnel,students,andallotherdirect patientcareproviders.

Program should include information on epidemiology of VRE, and impact of VRE on cost andoutcomeofpatientcare.

Roleofthemicrobiologylaboratory

Laboratoryshouldbeabletoidentifyandspeciate Enterococci. FullyautomatedmethodsoftestingEnterococcifor susceptibilitytestingareunreliable;diskdiffusion, gradient disk diffusion, agar dilation, or manual brothdilutionareacceptable.

Vancomycin resistance should be confirmed by repeating one of the above tests, or by streaking 87

Review of literature onto brain heart infusion containing 6g/mL of vancomycin. Preliminary and confirmatory identification of VRE should be immediately reported to patient care personnel and infection control. ScreeningforVREshouldbeconductedperiodically in hospitals where VRE has not been previously detected Preventionandcontrolofnosocomial transmissionofVRE Forallhospitals,includingthosewithinfrequentorno isolationofVRE

NotifyappropriatestaffimmediatelywhenVREis detected. Educate clinical staff about hospital policies regarding VRE colonized or infected patients so that appropriate procedures can be implemented immediately.

Establish systems for monitoring process and outcomemeasures.

Isolation precautions to prevent patienttopatient transmissionofVRE

88

Review of literature InhospitalswithendemicVREorcontinuedVRE transmissiondespiteimplementationofabove measures: Focus initial control efforts on critical care units andotherareaswhereVREtransmissionratesare highest Wherefeasible,cohortstaffcaringforVREpositive andVREnegativepatients

Carriage of Enterococci by hospital staff is rarely implicated in transmission. Investigation and culturingofhospitalstaffshouldbeatthedirection ofinfectioncontrolstaff

Verify that environmental disinfection procedures are adequate and that procedures are correctly performed Consider sending representative VRE isolates to referencelaboratoriesforstraintypingasanaidin identifyingreservoirsandpatternsoftransmission. Isolationprecautionstopreventpatienttopatient transmissionofVRE(adaptedfromCDCHICPAC) PlaceVREcolonizedorinfectedpatientsinsingle rooms, or cohort with other patients with VRE. PatientswithGREanddiarrhoeaorincontinence 89

Review of literature areatahigherriskofspreadingGREandmustbe givenpriorityforsinglerooms Wear gloves when entering the room of a VRE infectedorcolonizedpatient Wear a gown when entering the room of a VRE infectedorcolonizedpatientif:
o Substantial contact with the patient or

environmental surfaces in the room is anticipated.

o Thepatientisincontinent.

o Thepatienthasanileostomy,colostomy,or wounddrainagenotcontainedbydressing

Remove gloves and gown before leaving the patientsroom,andwashhandsimmediatelywith anantisepticsoaporwaterlessantisepticagent.

Dedicate the use of noncritical items such as stethoscope,sphygmomanometer,orrectalthermo meter to a single patient or cohort of isolate patients.Devicesmustbedisinfectedbeforeuseon otherpatients.

Obtainstoolorrectalswabculturesofroommates ofpatientsnewlyfoundtobeinfectedorcolonized withVRE.Performadditionalpatientscreeningat thediscretionoftheinfectioncontrolstaff. 90

Review of literature

Adopt a policy for determining when patients infected or colonized with VRE can be removed from isolation precautions. As VRE colonization maybeprolonged,negativeculturesfrommultiple sites on three separate occasions at least 1 week apartisrecommended

The hospital should adopt a system by which infected and colonized patients can be recognized andplacedintoisolationpromptlyontransferorre admission

Developaplan,inconsultationwithpublichealth authorities,fordischargeortransferofcolonizedor infectedpatientstootherhealthfacilities,including nursinghomesandhomehealthcare.

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Review of literature

TREATMENT OF
ENTEROCOCCAL INFECTIONS
Uncomplicated enterococcal infections are usually treated with singledrug therapy such as ampicillin, penicillin or vancomycin. Serious entero coccal infections (e.g., bacteraemia and endocarditis) requiretreatmentwithabactericidalcombinationof antibioticsthatshouldincludeapenicillin(ampicillin orpenicillinG)towhichtheisolateissusceptibleand an aminoglycoside (gentamicin or streptomycin) to which it does not exhibit highlevel resistance. Vancomycincanalsobeusedincombinationwithan aminoglycoside, and is recommended as a drug of choiceinpatientswithseriouspenicillinallergyorin treatmentofampicillinandpenicillinresistantstrains ofbacteria(Soodetal.,2008).

Treatment of VRE infections:

Vancomycin has been used as the drug of choice in many resistant strains of gram positive bacterial infections, especially those caused by Enterococci.Therehasbeenanincreaseinnumberof vancomycinresistantEnterococciinrecenttimes.This 92

Review of literature hasposedaseriousproblemnotonlyinthetreatment of enterococcal infections but also because the organism can horizontally transfer this resistant determinant to other vancomycinsusceptible species (Upadhyayaetal.,2009). Teicoplanin, another glycopeptide which is activeagainstmostVREwithvanB.Teicoplaninand aminoglycosidesaregenerallyeffectiveinteicoplanin susceptibleVREwhichusuallydoesnotexhibithigh level aminoglycoside resistance. The development of teicoplanin resistance has been noted in vanB phenotypeofVRE(Rajaetal.,2006). In general, antibiotics are selected according to demonstrated invitro activity. Most vancomycin resistant E.faecalis and some E.faecium isolates are susceptible to ampicillin. Therefore, ampicillin, combinedwithgentamicininthecaseofendocarditis, remainstherecommendedtherapyforinfectionscaused bysuchorganisms(MasciniandBonten,2005). Twoclassesofantibiotichavebeenapproved specificallyforthetreatmentofVREinfections: streptograminsandoxazolidinones

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Review of literature Quinupristinanddalfopristin aretwosemi synthetic streptogramin antibiotics combined in a 30:70 (w:w) ratio. In susceptible organisms, both quinupristin and dalfopristin bind to the 50S ribosomalsubunit.Thebindingofdalfopristincauses anincreaseintheaffinityofquinupristinforthe50S ribosomal subunit. Together, quinupristin and dalfopristin act synergistically to inhibit protein synthesis; however, the activity of Q/D against E. faecium is still only bacteriostatic. A search of the literature revealed six previously reported cases of VRE CNS infections treated with intrathecal or intraventricularQ/D(Williamsonetal.,2002). While Linezolid is an oxazolidinone antibiotic andhasbeenapprovedfortreatmentofskinandsoft tissueinfectionsrecently.Itiseffectiveagainstmany grampositiveorganisms,includingVRE,methicillin resistant Staphylococcus aureus and penicillin resistant Streptococcus pneumoniae with good CSF penetration(Tsaietal.,2006). Linezolid inhibits bacterial protein synthesis by bindingtotheribosomalpeptidyltransferasecenter,in the50Ssubunitoftheribosome,andinterferingwiththe initiationoftranslation.ItisactiveagainstmostGram 94

Review of literature positive bacteria, is well absorbed when administered orally, facilitating the conversion from intravenous to oraltherapy,andisgenerallywelltolerated.Linezolid provides high rates of clinical cure and microbiologic success in complicated infections due to Enterococcus spp. However, several instances of emergence of resistanceduringlinezolidtreatmenthavebeenreported in clinical isolates of Enterococci (E.faecium and E.faecalis)andStaphylococci(Staph.aureusandStaph. epidermidis)(GmezGiletal.,2009). Quinupristin/dalfopristin(Q/D)andlinezolid aretheonlytwoantibioticscurrentlyapprovedbythe FoodandDrugAdministration(FDA)forthetreatment ofVREinfections (Williamsonetal.,2002). Patients with resistant organisms or neurosurgical devices leadingtodirectCSFaccesshavealsobeentreatedwith avarietyofintrathecallyinstilledantibioticsincluding vancomycin, quinupristin/dalfopristin or teicoplanin, particularlyiftheparenteraltherapyfailstoeradicate theinfection(Varelasetal.,2008). Daptomycin,acycliclipopeptide,isanantibiotic withpotentbactericidalactivityagainstawiderange ofmultidrugresistantorganismsforwhichthereare veryfewtherapeuticalternatives,suchasMRSAand 95

Review of literature VRE (Dhawan et al., 2009). Daptomycin was approved in the United States in 2003 for the treatmentofGrampositivecomplicatedSkinandsoft tissue infections (SSTIs), rightsided infective endocarditis (RIE) due to Staph. aureus, and Staph. aureus bacteraemia (SAB)whenassociatedwithRIE orSSTI.Thereisagrowingbodyofinvitroandclinical evidencesuggestingthatdaptomycinhasgoodactivity againstEnterococci(Cantnetal.,2010). Beneficial effects of chloramphenicol and tetracycline in the treatment of VRE bacteraemia havebeenreported,butthedevelopmentofresistance during treatment has also been documented. Nitro furantoin isactiveagainstmanystrainsofVRE,but its use is limited to urinary tract infections. Cipro floxacin is bacteriostatic for Enterococci and, in combinationwithampicillinorgentamicin,bactericidal invitro.Thenewerquinolones,suchasmoxifloxacin, clinafloxacin and sparfloxacin, possess better activity than ciprofloxacin against Enterococci and may offer additivevalueinthecaseofVRE infections (Mascini andBonten,2005). Tigecycline, a broadspectrum glycylcycline antimicrobial agent, became available in 2005. This 96

Review of literature novel tetracycline derivative has activity against grampositive and gramnegative aerobic and anaerobic bacteria, including tetracycline resistant isolates.Similartodaptomycin,thiscompoundhasin vitroactivityagainstVRE (Linden,2007). However, therearefewpublisheddataregardingtreatmentof VRE infections with this agent. Because it is bacteriostatic and achieves relatively low serum concentrations,itmaynotbeidealforpatientswith bacteremiaandendocarditis(Ariasetal.,2010). Moreover,tigecyclineisnotaffectedbymostof the known mechanisms of resistance to tetracycline encountered in bacteria. Tigecycline overcomes the two key tetracycline resistance mechanisms (efflux pumpsandribosomalprotection)andisunaffectedby other bacterial mechanisms of resistance, such as extendedspectrumblactamases,limitingthenumber of antibiotic options available. Monotherapy with tigecyclinewasobservedtobegenerallysafeandwell tolerated, anditsefficacywascomparabletothatof vancomycininpatientswithseriousinfectioncaused byMRSAandVRE(Florescuetal.,2008;Beheraet al.,2009).

97

Review of literature Fosfomycin also has good in vitro activity against Enterococci,andtherehasbeenareportofa patientwithVREprostatitissuccessfullytreatedwith fosfomycin(Shresthaetal.,2003). Someantibioticsareintheclinicalefficacytrial phase.Oritavancin,aglycopeptideactiveonGRE,is inthelatephasesofclinicaltrials.Dalbavancinisa lipoglycopeptide with better MIC ranges than vancomycin for susceptible strains and have the convenienttobeadministeredonceaweek.However, thisagenthasgenerallyasuboptimalactivityagainst vanA phenotypes. The Telavancin, ramoplanin is more bactericidal than vancomycin. In vitro, Telavancin has displayed activity against EnterococcusspeciesbutwitharelativelyhighMIC90 against GRE compared to results obtained against sensitive strains. Finally, Avilamycin evernimicin(Ziracine)areoligosaccharideantibiotics active against GRE and which could be used in the future for infections due to multiresistant gram positivecocci(Birgand,2009). Oritavancin isasemisyntheticlipoglycopeptide with activity against methicillinresistant Staphylo coccus aureus (MRSA) and vancomycinresistant 98 and

Review of literature Enterococci (VRE). Its capacity to interact with the bacterialcellmembrane,leadingtolossofmembrane potential and increased membrane permeability, confersrapidbactericidalactivity(Belleyetal.,2009). Sodiumbenzoate(SB),orbenzoateofsoda, isthesodiumsaltofbenzoicacid,anFDAapproved polyunsaturated fat used by food manufacturers to inhibitmicrobialgrowthforover80yrs.Itcanprevent growthofalmostallmicroorganisms(bacteria,yeast andmoulds),andhasbeenusedforthetreatmentof congenital metabolic diseases such as urea cycle disorders.Recently,sodiumbenzoateshowedeffective invitroactivityagainstmethicillinresistantS.aureus (MRSA)(KarabayandSahin,2005),anditmaybe effective against Enterococci in the doses between 32mg/Land128mg/L.Sodiumbenzoateseemstobea goodalternativeforthetreatmentofVREinfections (Karabayetal.,2006). Lysinsrapidlybreakdownthebacterialcellwall inordertoreleaseprogenyphage.Structurally,lysins are proteins with an aminoterminal domain that conferstheenzymaticactivityforapeptidoglycanbond and a carboxyterminal domain that confers binding specificity to a carbohydrate epitope in the bacterial 99

Review of literature cellwall.Thesehighlyevolvedenzymesarenormally very specific to the bacterial host of the phage from whichtheyarederived.Whenlysinsarepurifiedand applied extrinsically, their binding efficiency and catalyticactivitycanbeusedtoachievetargetedkilling ofselectpathogenic bacteria withminimaleffects on other commensal bacteria; this capacity is an advantageoverconventionalantibiotics.PlyV12isan amidasethathasalyticeffectonmultiple E.faecalis strainsandalsolysesvancomycinresistantstrainsof E.faecalis and strains of E.faecium. In addition to killingitshostbacterialstrain, E.faecalis strainV12, PlyV12alsohadlyticactivityagainst14clinicaland laboratory E.faecalis and E.faecium strains tested, including two vancomycinresistant E.faecalis strains and three vancomycinresistant E.faecium strains. Furthermore, the PlyV12 binding activity suggests a previously undiscovered surface structure common to several different pathogens, which could serve as a targetinthedevelopmentofnewantibiotics(Yoonget al.,2004).

TheeradicationofVREcolonizationhasnot(yet) been possible, although a combination of oral bacitracinanddoxycyclineshowedtransientefficacy. In addition, suppression of gastrointestinal coloni 100

Review of literature zation was achieved with ramoplanin, although colonizationrateshadcompletelyrecoveredbyday21 after the discontinuation of therapy (Mascini and Bonten,2005). Attempts to eradicate VRE colonization have primarilyfocusedoneliminatingintestinalcarriageof the organism, often with nonabsorbable enteral antimicrobial agents. Limited success has been demonstrated with the nonabsorbable agent ramoplanin.Inonestudy,therateofVREacquisition amongICUpatientswasreducedthroughtheuseof chlorhexidine baths to eliminate VRE on the skin (Weberetal.,2007). Attempts at decolonization of VRE using oral bacitracin with or without gentamicin have been unsuccessful. Since current data do not support eradication or prevention of invasive disease by antibacterial treatment, colonized neonates were not treated.AllthreeneonateswithinvasiveVREinfection thatreceivedeffectivetherapywithlinezolidremained colonizeduntildischarge(Ergazetal.,2009). Probiotics are living microbial food ingredients designedtohaveabeneficialeffectonhumanhealth. Almost all strains of Lactobacilli are resistant to 101

Review of literature vancomycinandprobioticshavebeenusedwithsome success in preventing colonization by enteric pathogensduringtreatmentfor Clostridiumdifficile. Ourstudydemonstratedasignificantreductioninthe detection of VRE in faecal specimens of patients receivingaprobioticyoghurtcontainingLactobacillus rhamnosus GG (LGG), whether given as initial treatmentorfollowingcontroltreatment.Ourresults suggestthattheuseofcommercialyoghurtcontaining theprobioticLGGmaybeaworthwhiletreatmentfor VREcolonization(Manleyetal.,2007).

102

Materials and methods

MATERIALS AND METHODS

Atotalof120stoolsampleswereincludedinthis study, 100 samples were collected from groups of patients from Ain Shams university hospitals (36 samplestakenfromhematologydepartment,21from the hepatology department, 21 from intensive care units (ICU), 16 from nephrology department and 6 from the rheumatology) and 20 samples from apparentlyhealthyindividualsascontrolgroup. Datawerecollectedregardingpatientsage,sex, durationofhospitalization,andantimicrobialintake. Stoolsampleswerecollectedincleancontainers and submitted immediately to the microbiology laboratoryforculture. All patients and control were subjected to the following:
1-

Stool samples were plated directly on both chromIDTM VRE agar (bioMrieux, Marcy lEtoile,France)andbileesculinagar(Himedia, India) supplemented with 6g/ml vancomycin (Merc,France)byquadranttechnique.

103

Materials and methods

2-

The suspected colonies were confirmed to be EnterococcibyGramstainandcatalasetestand identified to the species level by API 20 Strep after subculture on blood agar, some species werefurtherconfimedbydoingthemotilitytest.

Antibiotic susceptibility to the following antibiotics(vancomycin,ampicillin,doxycycline, erythromycin, tetracycline and ciprofloxacin) wasdoneusingthediscdiffusionmethod.MIC susceptibility testing for vancomycin and teicoplanin was done using Etest with 0.5Mc Farland inoculum to detect glycopeptides resistance.

Materials:
1) ChromID
TM

MarcylEtoile,France):

VREagarmedium(bioMrieux,

The chromIDTM VRE agar (bioMrieux, Marcy lEtoile,France)isreadytouseplatesstoredat28C for shelf life. It consists of a rich nutritive base includingavariety of peptones. Italso containstwo chromogenic substrates and a mixture of antibiotics includingvancomycin(8g/ml)whichenable: ThespecificandselectivegrowthofVRE. 104

Materials and methods

The direct detection of E.faecium and E.feacalis throughthecharacteristiccolourofcolonies.

Table(6): Formula of chromIDTM

VRE agar g/liter

(bioMrieux,MarcylEtoile,France):
TM

FormulaofchromID

VRE

(bioMrieux) 18 3 1 6 15.0 0.13 28.8 1L

Caseinandmeatpeptone(bovineand porcine) Heartpeptone(bovineandporcine) Cornstarch Sodiumchloride Agar Mixtureofchromogenicsubstrates Selectivemixture Purifiedwater pH7.2

105

Materials and methods 2) Bileesculinagarmedium(Himedia,India) containing 6g/ml vancomycin (Merc, France)(BEAV):

Table(7): Formula of Bile esculin agar medium (Himedia,India): agarmedium FormulaofBileesculin g/liter

(Himedia) 5.00 3.00 40.00 2.0 0.5 15.0

Peptonedigestofanimaltissue Meatextract Esculin Bilesalts Ferriccitrate Agar pH6.6 0.2

A)Vancomycinsolutionpreparation(Merc,France):

500mgvialstoredatroomtemperature. The
antibioticsolutionwaspreparedbydissolving500mg ofvancomycinpowderin10mlofsteriledistilledwater inasterilecontainersothatthefinalconcentration 106

Materials and methods was50mg/ml.

107

Materials and methods B) Preparation of the media according to manufacturersinstruction:

-

32.25gm of the formula were suspended in 500mlofdistilledwaterandmixedthoroughly. The mixture was heated till boiling with frequent agitation to completely dissolve the powder,andsterilizedat121Cfor15min. Themediawasthenallowedtoequilibrateina constant temperature water bath to 50C before additionofantibioticsolution. 60l of vancomycin solution was added to the media, so that the final concentration of vancomycinwas6g/ml. Themediawasthenpouredaspeticallyinsterile plastic 9cm diameter petri dishes with a uniform depthof4mmandlefttosolidifythenstoredat2 8Ctobeusedwithinoneweek.

3)

API 20 Strep strips (bioMrieux, Marcy lEtoile,France):

API20Strepsrtips(bioMrieux,MarcylEtoile, France) is a standardized system combining 20 biochemicalteststhatofferwidespreadcapabilities.It enables group or species identification of most 108

Materials and methods Streptococci andEnterococci,andthosemostcommon relatedorganisms. Contentsofthekit(kitfor25tests):

25API20Strepstrips.

25incubationboxes 25ampoulesofAPIGPmedium 25resultsheets 1packageinsert reactions(Table8). Composition of the strip and types of

109

Materials and methods Table(8): ConstituentsandinterpretationoftheAPI lEtoile,France):

Tests VP(Voges Proskauer) HIP (Hippuricacid) ESC(Esculin) Activeingredients Sodiumpyruvate Hippuricacid Esculin Ferriccitrate Reactions/ enzymes Negative colorless Colorless/paleblue 4hrs glucosidase hydrolysis 24hrs Colorless Paleyellow Lightgrey Acetoin production Hydrolysis Results Positive Pinkred Darkblue/violet 4hrs Black grey 24hrs black VP1+VP2/wait10min NIN/wait10min

20 Strep strips (bioMrieux, Marcy

PYRA (Pyrrolidonyl arylamide) GAL( galactoside) GUR( giucuronide) GAL( galactoside) PAL(Alkaline phosphate) LAP(Leucine aminopeptide) ADH(Arginine dihydrolase) RIB(Ribose) ARA(Arabinose) MAN(Mannitol) SOR(Sorbitol) LAC(Lactose) TRE(Trehalose) INU(INUlin)

Pyroglutamicacid naphthylamide 6bromo2naphthyl D galactopyranoside NaphtholASBL glucuronicacid 2naphthylD galactopyranoside 2naphthyl phosphate lleucine naphthylamide Larginine Dribose Larabinose Dmannitol Dsorbitol Dlactose (bovineorigin) Dtrehalose Inulin

Pyrrolidonyl arylamidase

ZYMA+ZYMB/10min (PYRAtoLAP) Colorlessorvery paleorange colorless Colorless Colorlessorvery paleviolet Colorlessorvery paleviolet Colorless Yellow 4hrs Red Red Red Red Red Red Red 24hrs Orange/red Orange/red Orange/red Orange/red Orange/red Orange/red Orange/red 4hrs Orange/ yellow Orange/ yellow Orange/ yellow Orange/ yellow Orange/ yellow Orange/ yellow Orange/ Orange violet Blue Violet Violet Orange Red 24hrs yellow yellow yellow yellow yellow yellow yellow

Galactosidase Giucuronidase Galactosidase Alkaline phosphatase Leucineamino peptidase Arginine dihydrolase Acidification Acidification Acidification Acidification Acidification Acidification Acidification

110

Materials and methods

yellow RAF(Raffinose) AMD(Amidon) GLYG(Glycogen) Draffinose Starch glycogen Acidification Acidification Acidification Red Red Orange/red Orange/red Orange/ yellow Orange/ yellow yellow yellow

Redororange

Brightyellow

111

Materials and methods APIGPMedium: Table(9): FormulaofAPIGPMedium APIGP Medium 2ml LCystine0.5g Tryptone20g Sodiumchloride5g Sodiumsulfite0.5g Phenolred0.17g Demineralizedwatertomake1000ml pH:7.47.6 Reagents and material required but not provided:
-

APISuspensionMedium,2ml Reagents:Ninhydrin(NIN) VP1+VP2 ZYMA ZYMEB Mineraloil McFarlandStandard4 API20STREPstripAnalyticalProfileIndex

112

Materials and methods 4) Etest for vancomycin and teicoplanin (bioMrieux,MarcylEtoile,France): Etest consists of a thin, inert and nonporous plasticstrip,5mmwideand60mmlong.Onesideof the strip is calibrated with a MIC reading scale in ug/ml.Apredefinedexponentialgradientofantibiotic, dried and stabilized, is immobilized on the other surface of strip with the concentration maximum at oneendandtheconcentrationminimumattheother end.Thegradientcoversacontinuousconcentrationof 0.016 to 256g/ml for both vancomycin and teicoplanin. A twoletter code, on the handle, designatestheindentityoftheantibiotic (Rosseret al.,1999). 6) UK): Disc diffusion test (Oxoid Ltd., Basingstoke Antibiotic discs: vancomycin (30g), ampicillin (10g), ciprofloxacin (5g), erythromycin (15g), doxycycline (30g) and tetracycline (30g) (Oxoid Ltd., Basingstoke UK) were used according to (CLSI,2008). - Media:bloodagar

Methods:
113

Materials and methods 1) ChromIDTM VRE agar medium (bioMrieux, MarcylEtoile,France): Principle: ThechromIDTMVRE(bioMrieux,MarcylEtoile, France) is a chromogenic medium used for the detection of E.faecium and E.feacalis showing vancomycin resistance (VRE). The chromogenic substrates incorporated in chromIDTM VRE are targeted by enzymes specific for E.faecium or E.faecalis. Enzymatic degradation generates violet colour for galactosidaseproducing strains of E.faecium, and bluegreen colour for glucosidase producingstrainsofE.feacalis. Procedure: - Readymadeplateswereallowedtocometoroom temperatureinthedark.
-

Specimens were directly inoculated onto the chromIDTMVREplates.

Inoculated plates were then incubated at 370C in aerobicconditions,inthedark.Thecultureswere generally examined after 24 hours of incubation andreexaminedafter48hours.Ifanegativeresult 114

Materials and methods isobtained(nogrowthorcolour),themediumwas reincubatedforafurther24hrs. Interpretation:

-

Colonies with violet colour were considered characteristic of E.faecium species resistant to vancomycin, while those with a bluegreen colour wereconsideredcharacteristicofE.feacalisspecies. The presence of at least one violet or bluegreen colony was considered a positive result (i.e., presenceofVRE)(Fig.5).

LT/RT

115

Materials and methods

Fig.(5): ChromIDTMVREagarplateshowingattheRt:violet coloniesofE.faecium,andattheLt:2typesof colonies:violetcoloniesofE.faeciumandbluegreen coloniesofE.feacalis.

2) Bile esculin agar medium containing 6g/ml vancomycin(Himedia,India): Principle: Bile esculin agar is ideal for isolation and differentiationofGroupDStreptococciandEnterococci fromother Streptococci,basedonhydrolysisofEsculin inthepresenceofbile.OrganismspositiveforEsculin hydrolysishydrolyzetheglycosideesculintoesculetin and dextrose. The esculetin reacts with the Ferric citratetoformadarkbrownorblackcolony.Bilesalts doesnotinhibit Enterococci whileotherGrampositive bacteriaareinhibited.MeetextractandPeptonesupply thenutrientsessentialforgrowth.Tolerancetobileand the ability to hydrolyze esculin constitutes a reliable presumptive test for the identification of Group D Streptococci.Thebrowncolor(positivereaction)around thecoloniesappearsafter1824hrsofincubationata temperatureof35 2C. Procedure: - Plateswereallowedtocometoroomtemperature. 116

Materials and methods

-

Stoolsampleswereinoculateddirectlyontotheplates withquadranttechnique(Ledeboeretal.,2007a). Plates were incubated overnight at 37C under aerobic conditions for 24hrs and reexamined after 48hrs.

Interpretation: Positive resultsforVREwereconsideredwhen smallblackorbrowncoloniessurroundedwithablack haloappeared(Fig.6).

Fig.(6): Bile esculin agar showing black colonies of Enterococcisurroundedwithablackhalo.

3) Colonies suspected of being Enterococci growingoneithermediawerefurtherexaminedby GramstaintoensurethattheywereGrampositive cocci, and confirmed of being Enterococci by 117

Materials and methods catalase test. The colonies were subcultured on blood agar plate and incubated in ambient air at 37C overnight in order to obtain fresh and pure isolates. 4) API 20 Strep (bioMrieux, Marcy lEtoile, France):

Isolates that were grampositive cocci by gram stainandcatalasenegativewerefurtheridentifiedby theAPI20Strep(bioMrieux,MarcylEtoile,France). Principle: TheAPI20Strepstripconsistsof20microtubes containing dehydrated substrates for the demonstration of the enzymatic activity or the fermentation of sugars. The enzymatic tests are inoculated with a dense suspension of organisms of isolatedcoloniessuspendedinsteriledistilledwater, made from a pure culture, which is used to reconstitute the enzymatic substrates. During incubation,metabolismproducescolourchangesthat areeitherspontaneousorrevealedbytheadditionof reagents.Thefermentationtestsareinoculatedwith an enriched medium which rehydrates the sugar substrates.Fermentationofcarbohydratesisdetected 118

Materials and methods byashiftinthepHindicator.Thereactionsareread according to the reading table (Table 8) and the identificationisobtainedbyreferringtotheAnalytical ProfileIndex. Procedure: Preparationofthestrip:

About5mlofdistilledwaterordemineralizedwater [oranywaterwithoutadditivesorchemicalswhich may release gases (e.g., Cl2, CO2, etc.)] were distributedintothehoneycombedwellsofthetrayof theincubationboxtocreateahumidatmosphere.

The strip was removed from its packaging and placedinthetray. Preparationoftheinoculum: All the culture from the previously prepared subcultureplatewasharvestedusingaswabandput in tube containing 2ml of distilled water making a dense suspension with a turbidity greater than 4 McFarland. Preparationof4McFarlandturbiditytube:

119

Materials and methods 0.4ml of 1% BaCl2 was added to 9.6ml 1% H2 SO4andkeptinadarkbottleforsixmonths(Smibert andKreig,1994). Inoculationofthestrip:

Inthefirsthalfofthestrip(testsVPto ADH)the suspension was distributed with a sterile pipette, avoiding the formation of bubbles by tilting the strip slightly forwards and placing the tip of the pipetteagainstthesideofthecupule:
o ForthetestsVPtoLAP:approximately100l

wasdistributedintoeachcupule. o FortheADHtest:thetubeonlywasfilled.

Inthesecondhalfofthestrip(testsRIBtoGLYG): o An ampule of GP Medium (ampule with nodroppercap)wasopenedandtherestofthe suspensionwastransferredintoit(appr.0.5 ml)andmixedwell. o Thisnewsuspensionwasdistributedintothe tubesonly.

Thecupuleoftheunderlinedtests(ADHtoGLYG) was filled with mineral oil to form a convex meniscus.

120

Materials and methods

The lid was placed on the tray and incubated at 36C + 2C in aerobic conditions for 44.5hrs to obtainafirstreadingandfor24hours(+2hrs)to obtainasecondreadingifthisisrequired(Fig.7).

Readingofthestrip: After4hoursofincubation:

Thefollowingreagentswereadded:
o VPTest:1dropofeachofVP1andVP2. o HIPTest:2dropsofNINreagent. o PYRA, GAL, GUR, GAL, PAL and LAP

Tests:1dropofeachofZYMAandZYMB reagents.

After 10 minutes, the reactions were read by referringtothereadingtable(Table8).

Fig.(7): API 20 STREP strip showing a panel that was identifiedasE.faecium.

5)Etest(bioMrieux,MarcylEtoile,France):

121

Materials and methods TheisolatesthatwereidentifiedasE.faeciumor E.faecalis on either media were further subcultured onbloodagarandincubatedfor24hrsaerobicallyat 37CtoperformtheEtestforbothvancomycinand teicoplanintoconfirmtheglycopeptideresistance. Principle: WhenanEteststripisappliedtotheinoculated agarplate,thereisanimmediateandeffectiverelease of the antibiotic from the carrier surface into agar matrix. A continuous and exponentional gradient of antibioticconcentrationiscreateddirectlyunderneath the strip forming an ellipse zone of inhibition that intersectsthestrip(Ternidgeetal.,2003).

122

Materials and methods Preparationoftheinoculum: 0.5McFarlandturbiditytube:

0.05mlof1%BaCl2wasaddedto9.95ml1% H2SO4mixedwellandkeptinadarkbottleforsix months.

Fivetoeightcoloniesofeachisolatewerepicked upandsuspendeddirectlyin3mlsterilesaline tubes.

Turbiditywasadjustedto0.5McFarlandby addingeithersterilesalineincaseofheavy inoculumorbyaddingextracoloniesincaseoflight inoculumandvortexedthoroughly.

Inoculationofthemedia:
-

Suspensionoftestedisolateswaspoured onbloodagarplates,andtheexcessfromthe suspensionwasdiscardedindiscardingagarand allowedtocompletelydryfor15minbeforeapplying theEtestgradientstrips.

ThenvancomycinandteicoplaninEtest stripswereappliedin2differentdirectionsonthe sameplatewithasterileforceps(Movingofstrips wasavoidedoncetheywereapplied).

123

Materials and methods

-

Plateswereincubatedat35Candwere readafter24hrs.

124

Materials and methods Interpretation: TheMICvalueisreadfromthescaleintermsof ug/mlwheretheellipseedgeintersectsthestrip(Fig.8).

Fig.(8): Etestforbothvancomycinandteicoplaninwherethe MICforvancomycinis 4mg/LandtheMICforthe teicoplaninis 1.5mg/L.

AlltheisolatesthatwereE.faeciumorE.feacalis byAPI20STREPandresistantforvancomycinbyE testwerediagnosedasVRE.TheseVREisolateswere classifiedphenotypicallyintoVanA,VanBandVanD 125

Materials and methods accordingtotheresultsofEtestforbothvancomycin andteicoplanin.Similarly,theE.gallinarumand E.casseliflavusisolateswerefoundtobeofVanC phenotype(Table10). Table(10): Different phenotypes of Enterococci according to MICs for both vancomycin and teicoplanin (Zirakzadeh and Patel,2006)
Phenotype Vancomycin Teicoplanin Species E.faecium E.faecalis E.avium E.gallinarum E.durans E.mundtii E.casseliflavus E.raffinosus E.hirae E.faecium E.faecalis E.gallinarum E.durans E.gallinarum E.casseliflavus E.flavescens E.faecium E.faecalis

MIC(g/mL) MIC(g/mL)

VanA

64>1000

16512

VanB

41000

0.252

VanC

232 16256

0.122 264

VanD

126

Materials and methods

127

Materials and methods 6) Diskdiffusiontestusingvancomycin(30 g), ampicillin(10 g),ciprofloxacin(5 g), tetracycline(30 g)wasalsodone: Principle: Theantimicrobialcontainedinadiskwas allowedtodiffuseintothemediumandinteractina platefreshlyseededwiththetestorganisms. Inoculumpreparation: Theinoculumwaspreparedbymakingasaline suspensionofisolatedcoloniesselectedfrom18to 24hrsnonselectiveagarmedium,suchasbloodagar. Thesuspensionwasadjustedtomatchthe 0.5McFarlandturbiditystandard,usingasalineanda vortexmixer. Inoculationofthemedia: Suspensionoftestedisolateswaspouredon Bloodagarplates,andtheexcessfromthesuspension wasdiscardedindiscardingagarandallowedtodry for5min.Antibioticdiskswereplacedatadistanceof 2cmmeasuredcentertocenter(usingaruler).Then erythromycin(15 g),doxycycline(30 g)and

128

Materials and methods theplateswereincubatedat37Cfor20hrsi.e.over nightincubation. Applicationofthediscstotheinoculatedagarplates: Theantimicrobialdiscswereplacedontothe surfaceoftheinoculatedagarplates.Theywere distributedevenlysothattheyarenocloserthan 24mmfromcentertocenterandnotlessthan2mm fromedgeoftheplate.Eachdiscwaspresseddownto ensurecompletecontactwiththeagarsurface.The plateswereinvertedandplacedinanincubator adjustedat35Cwithin15minafterthediscswere applied. Readingplatesandinterpretingresults: After24hrsofincubation,eachplatewas examined.Thediametersofthezonesofcomplete inhibitionweremeasured,includingthediameterof thedisc,usingarulerwhichisheldonthebackofthe invertedplate.Theplatewasheldafewinchesabove ablack,nonreflectingbackgroundandilluminated withareflectedlight.

129

Materials and methods Table(11): Interpretive criteria (in mm) for disc diffusiontest(CLSI,2008) Antibiotic Ampicillin10g Vancomycin30g Erythromycin 15g Tetracycline30g Doxycycline30g Ciprofloxacin5g Resistant Intermediate Susceptible 16 14 13 14 12 15 1516 1422 1518 1315 1620 17 17 23 19 16 21

130

Materials and methods

Statistical analysis:
DatawereanalyzedusingtheSPSSstatistical softwarepackage(V.17,Echosoftcorp.,USA). DescriptiveStatistics: i. Themeanwasusedtodescribethelocationof quantitative(continuous)datawhereasvariation wasdescribedbythestandarddeviation(SD). ii. Qualitative(categorical)datawere representedbythenumberandpercent(%). AnalyticalStatistics: i. Comparisonbetweenpatientscolonizedand thosenoncolonizedwithVREregardingtheage, durationofhospitalization,andthediagnosis weredoneusingtheStudent'sttest. ii. TheChisquaretest(X2value)wasusedto assesstheassociationbetweencategoricaldata. iii. Thediagnosticperformanceofthestudied methodswasdoneusingthesensitivity,specificity, positivepredictivevalue(PPV),negativepredictive value(NPV)anddiagnosticaccuracy. Levelsofsignificance: P 0.05 P<0.05 =nonsignificant =significant 131

Materials and methods P<0.01 =highlysignificant

132

Results

RESULTS
ThepresentstudywascarriedoutattheCentral MicrobiologyLaboratory,ClinicalPathologyDepart ment,AinShamsUniversityovertheperiodfrom January2010toJune2010. Thestudyincluded100stoolsamplesthatwere obtainedgroupsofpatientsfromAinShams universityhospitals(36samplestakenfrom hematologydepartment,21fromthehepatology department,21fromintensivecareunits(ICU),16 fromnephrologydepartmentand6fromthe rheumatology)and20samplesfromhealthy volunteers.Thepatientswere57males(57%)and 43females(43%).Theiragesrangedfrom10to78 yearswithameanof40.2 17.3years. Thepresentstudyaimedatevaluatingthe chromogenicmedium(chromIDVREagar)forVRE identificationandcomparingitsperformanceagainst bileesculinagarsupplementedwith6g/ml vancomycin(BEAV),usingcombinedAPI20STREP forspeciesidentificationandEtestforconfirmationof glycopeptidesresistanceasreferencemethods. 133

Results OnchromIDVREagar23isolatesofVREwere isolatedfrom22/100(22%)stoolsamples,allofthem wereE.faeciumexceptonesamplewhichgavetwo typesofisolatesonewasE.faecium(violet)andthe otherwasE.feacalis(bluegreen)whichwere differentiatedonchromIDVREmediumandnotfrom BEAVmedium(bothgaveblackcolor),alsooneofthe positivesamplesforVREonchromIDVREagargave negativeresultonBEAV.WhileonBEAVagar33 VREisolateswererecoveredfrom33/100(33%)stool sampleswithoneisolaterecoveredfromchromIDVRE andnotfromBEAVand12isolatesrecoveredfrom BEAVandnotfromchromIDVRE.Totally,34/100 (34%)stoolsamplesgavepositiveresultsforVREon bothmedia.

134

Results ThesensitivityofchromIDVREwas(65.7%), specificity(97%),PPV(97%),NPV(84.2%),and efficacy(86%).WhiletheBEAVhadasensitivityof (97%),specificity(65.2%),PPV(58.9%),NPV(97.7%), andefficacy(76%).BydoingGramstainthe specificityincreasedto(100%and72.7%),thePPV increasedto(100%and64.7%),theNPVincreasedto (84.6%and97.9%)andtheefficacyincreasedto(88% and81%)forbothchromIDVREandtheBEAV respectively(Table12). Table(12): DiagnosticperformanceofchromIDVRE andBEAVmediumwithandwithout Gramstain
Method TP 22 22 33 33 FP 2 0 23 18 TN 64 66 43 48 FN 12 12 1 1 Se% 64.7% 64.7% 97% 97% Sp% 97% 100% 65.2% 72.7% PPV % 91.7% 100% 58.9% 64.7% NPV% 84.2% 84.6% 97.7% 97.9% Efficacy% 86% 88% 76% 81%

chromID VRE

BEAV

ResultsobtainedwithGramstainingareinbold. Atruepositive(TP)isdefinedasabluegreenorpurplecolonyonchromIDVREor gray to black colony on bile esculin agar that was identified as VRE by API 20 STREPandEtest. Afalsenegative (FN)isdefinedasanisolatethatwasconfirmedasaVREon1 mediumbutdidnotgrownonanothermedium.

135

Results
Atruenegative(TN)isdefinedasthelackofatypicallycoloredcolony. Afalsepositive(FP)isdefinedasanisolatethatexhibitedtypicalcolorationonthe respectivemediumbutwasnotidentifiedasVREAPI20STREPandEtest.

There was a highly significant association betweentheresultsofthechromIDVREandtheAPI 20 STREP and Etest (P<0.001) as there was agreement between both methods in 88% and disagreement in 12% of cases with no false positive rateandafalsenegativerateof12%(Table13). Table(13): AssociationbetweenthechromIDVRE andthecombineduseofAPI20STREP andEtest(referencemethod)
Referencemethod Negative forVRE Negative ChromID VRE forVRE Positive forVRE Total
HS=highlysignificant

Chi Total square Pvalue value

Positive forVRE 12

66

78 54.751 P<0.001 (HS)

0 66

22 34

22 100

136

Results Table(14)showsahighlysignificantassociation betweentheresultsofBEAVandtheAPI20STREP andEtest(P<0.001)with81%agreementbetween bothmethods,whiledisagreementwasfoundin19% ofcaseswithfalsepositiverateof18%andafalse negativerateof1%. Table(14): AssociationbetweentheBEAVmedium andthecombineduseofAPI20STREP andEtest(referencemethod)
Referencemethod Negative forVRE Negative BEA V forVRE Positive forVRE Total
HS=highlysignificant

Chi Total square value

Positive forVRE 1

P value

48

49 43.732 P<0.001 (HS)

18 66

33 34

51 100

137

Results Ahighlysignificantassociationwasalso reportedbetweentheresultsofthechromIDVREand theBEAVmedium(P<0.001)(Table15).Both methodsshowedagreementin87%ofcases. Table(15): AssociationbetweenthechromIDVRE andtheBEAVmedium

BEAV Negative forVRE Negative ChromID VRE forVRE Positive forVRE Total
HS=highlysignificant

Chi Total square value

Positive forVRE 12

P value

66

78 49.759 P<0.001 (HS)

1 67

21 33

22 100

138

Results Breakthrough growth of contaminants on each mediumwasassessedat24and48hrstodetermine the efficacy of the antimicrobial agents in chromID VRE to suppress growth of normal stool flora other thanVRE.Nofurthercontaminationwasfoundafter 48hincubationsothespecificityandthesensitivity werenotaffected.Contaminantswereclassifiedinto2 groups; i) those that were considered potential false positive because they produced colonies with blue greenorpurpleonchromIDVREorblackonBEAV, which could be misinterpreted as VRE, and ii) colorless isolates. Although BEAV grew more contaminants causing falsepositive results at 24 h versus chromID VRE, the latter was more likely to growcolorlesscontaminants. Contaminants most likely to grow on chromID VREincluded Candidaspp (whitecoloredexceptone gavevioletcolor)15/100(15%),andLactobacillusspp. (violet)1/100(1%),andGramnegativerods(colorless) 74/100(74%).Thelactobacillusisolateandoneofthe candidaisolatesrecoveredfromthechromIDgavethe typical color for VRE while the Gram negative rods could easily be differentiated by their colonial morphologyandconfirmedbyGramstain.Mostofthe contaminantswerelimitedtotheprimaryinoculumin 139

Results contrast to the BEAV medium where the colonies extendedtillthetertiaryquadrant(Fig.9). Contaminants causing potential falsepositive resultsonBEAVincluded9/100(9%)VanCEnterococci; oneisolatewasE.casseliflavusandeightisolateswere E.gallinarum,vancomycinsusceptibleE.faecium6/100 (6%), Streptococcus spp. 2/100 (2%), Gram positive catalasepositivecolonies5/100(5%)and Candidaspp. 4/100 (4%). The five Gram positive catalase positive coloniesgavethetypicalcolorofVRE.Proliferationof colorless isolates on either medium was of little consequencebecausethesecoloniesarenotlikelytobe interpretedincorrectly(Fig.9).

140

Results
Fig.(9): Distributionofthecontaminantsandfalsepositive onchromIDVREandBEAVbeforeandafterGram stainandcatalasetest.

AsregardstheEtest;isolateswithvancomycin MIC64>1000g/mLandteicoplaninMIC16 512g/mLwereconsideredVanAphenotype,while thosewithvancomycinMIC41000g/mLand teicoplaninMIC0.252g/mLwereconsideredVanB phenotype,andthosewithvancomycinMIC16256 g/mLandteicoplaninMIC264g/mLwere consideredVanDphenotype,finallythosewith vancomycinMIC 4g/mLwereconsideredas vancomycinsusceptibleEnterococci. The21VRE.faeciumisolatesthatwere recoveredfrombothchromIDVREmediumand BEAV,andthesingleVRE.feacalisisolatethatcould bedifferentiatedonchromIDVREbutcouldnotbe differentiatedonBEAV,andthesingleVRE. faeciumisolatethatwasrecoveredfromthechromID VREmediumandnotfromBEAVwereofVanA phenotypewhilethe12isolatesthatwererecovered fromtheBEAVandnotrecoveredfromthechromID VREmediumwereasfollows:8isolatesofVanB phenotype,3isolatesofVanAphenotypeandone isolateofVanDphenotype.Thenumberofisolates 141

Results recoveredfrombothmediawiththeirvancomycin MICsisshownintable(16)andthenumberof E.faeciumandE.feacalisisolatesandtheir phenotypesinrelationtothevancomycinand teicoplaninMICsisshownintable(17). Table(16): VancomycinMICsofallVREisolates recoveredfrombothchromIDVRE mediumandBEAVmedium Medium No.ofisolateswiththeir vancomycinMIC(g/mL): 4 >4<8 ChromID VRE BEAV 0 6 0 7 816 0 11(2E.faecium+ 9VanC) 64 22 24

Table(17): Numberofisolatesandtheirphenotypes inrelationtothevancomycinand teicoplaninMICs MIC(g/mL) vancomycin 4<8 Teicoplanin 0.252 142 No.of isolates 7 Probable phenotype VanB

Results 816 16<64 64 16 0.252 264 16512 0.122 1 1 24 9 VanB VanD VanA VanC

AsregardsthediscdiffusionallVanAisolates excepttheonethatrecoveredfromBEAVandnot fromchromIDVREmediumwereresistantto vancomycin(30g),whiletheeightVanBandtheone VanDisolatesweresensitivetovancomycin(30g).All theisolateswereresistanttotheantibiotics: ampicillin(10g),ciprofloxacin(5g),erythromycin (15g),doxycycline(30g)andtetracycline(30g).So thevancomycindiscdiffusionmaypredicttheVanA phenotyperesistancebutnottheotherphenotypes. Therewasahighlysignificantdifference betweenthepatientscolonizedandthosenon colonizedwithVREregardingthetotaldurationof hospitalization(14.5 13.9,7.5 5.77,respectively) (P<0.001)(Table18). Table(18): Comparisonbetweencolonizedandnon colonizedpatientsregardingthetotal durationofhospitalization 143

Results
Colonized patients (n=34) Totalduration (mean SD)
HS=highlysignificant

colonized patients (n=66)

Non

value (Sig.)

ofhospitalization

14.5 13.9

7.5 5.77

<0.001(HS)

Regardingtheuseofantibiotics,therewasa highlysignificantassociation(P<0.01)betweenthe useofantibiotics(ingeneral)andthecolonization withVRE(Table19). Table(19): Associationbetweentheuseantibiotics andthecolonizationwithVRE

group Colonize dwith VRE colonized Total Not Chi square Pvalue value

withVRE

Antibiotic s

Patients noton 10 45 55 antibiotics Patients antibiotics on 24 21 45

13.628 P<0.001 (HS)

144

Results
Total 34 66 100

145

Results Regardingtheantibioticclassthatismostly associatedwithVREcolonization,ahighlysignificant association(P<0.001)wasfoundbetweenthe administrationoflactamantibioticsandcolonizatin withVRE.Moreover,ahighlysignificantassociation (P<0.001)wasreportedbetweentheadministrationof glycopeptidesandcolonizationwithVRE,whileanon significantassociation(P>0.05)wasfoundbetween administrationofflagylandcolonizatinwithVRE (Table20).

146

Results Table(20): Associationbetweentheuseoflactam, flagyl,glycopeptidesantibioticsandthe colonizationwithVRE

Group Colonized withVRE 12 22 34 Patientsnoton lactam Antibiotics lactamantibiotics Patientson lactamantibiotics Total Chisquarevalue Pvalue Not withVRE 47 19 66 11.968 P<0.001(Highlysignificant) Group Colonized withVRE Patientsnoton Flagyl Flagylantibiotics Patientson Flagylantibiotics Total Chisquarevalue Pvalue 30 4 34 colonized withVRE 61 5 66 0.481 P>0.05(Nonsignificant) Group Colonized withVRE Glyco peptides Patientsnoton Glycopeptides antibiotics Patientson Glycopeptides 6 1 7 28 65 93 colonized withVRE Not Total 91 9 100 Not Total colonized Total

59 41 100

147

Results
antibiotics Total Chisquarevalue Pvalue 34 66 8.970 P<0.05(significant) 100

Regardingthesextherewasnosignificant associationbetweenbothsexesandVREcolonization (Table21). Table(21): Associationbetweenthesexandthe colonizationwithVRE

Group Colonized withVRE 14 20 34 Notcolonized withVRE 29 37 66 Total Chi square value 43 57 100 0.070 P>0.05 (NS)

P value

Female Sex Male Total

148

Results Regardingthediagnosistherewashighsignificant associationbetweenleukemiaandthecolonizationwith VREwhilenosignificantassociation(P>0.05)wasfound betweendiabetes,chronicliverdiseaseandchronicrenal failureandcolonizationwithVRE(Table22). Table(22): Associationbetweentheleukemia,dia betes,chronicliverdisease,chronicrenal failureandthecolonizationwithVRE
Patientsgroup Colonized withVRE Leukemia Nonleukemic Leukemic Total Chisquarevalue Pvalue 20 14 34 9.610 P<0.001(Highlysignificant) Patientsgroup Colonized withVRE Diabetes NonDiabetics Diabetics Total Chisquarevalue Pvalue 29 5 34 0.192 P>0.05(Nonsignificant) Patientsgroup Colonized withVRE Patientswithout chronicliverdisease Chronicliver disease Patientswith chronicliverdisease Total Chisquarevalue Pvalue 21 13 34 3.299 P>0.05(Nonsignificant) Patientsgroup Notcolonized Total withVRE 52 14 66 73 27 100 Notcolonized withVRE 54 12 66 Total 83 17 100 Notcolonized withVRE 57 9 66 Total 77 23 100

149

Results
Colonized withVRE Patientswithout chronicrenal failure Patientswith chronicrenal failure Total Chisquarevalue Pvalue 30 Notcolonized Total withVRE 49 79

Chronicrenal failure

4 34 2.648

17 66

21 100

P>0.05(Nonsignificant)

150

Discussion

DISCUSSION
Enterococcusspeciesaremembersofthenormal intestinalflora,butoverthelasttwodecadesthey havealsoemergedasimportantnosocomialpathogens (Cuzonetal.,2008). Inrecentyears,theirclinicalimportancehas beenamplifiedbyanincreasedresistanceto antimicrobialsand,inparticular,anacquired resistancetoglycopeptidesfirstdocumentedin1988. Sincethisfirstreportedoutbreak,VREhasbecomea commoncauseofhealthcareassociatedinfection acrossmanypartsoftheworld(Topetal.,2008). AsymptomaticintestinalcolonizationwithVRE iswidelyreportedandthiscanactasareservoirfor disseminationandsubsequentinfection.E.faecium andE.faecalisaretheenterococcalspeciesmost frequentlyisolatedfrompathologicalsamplesandare consideredtobeofthegreatestclinicalsignificance (Asiretal.,2009). Specificcontrolmeasuresneedtheproper identificationofindividualscolonizedbyVREthrough surveillancecultures.ThescreeningforVREinstool 151

Discussion orrectalsamplesusingselectiveisolationmedia,like thebileesculinazideagarwithvancomycin6g/mL needscomplementaryteststoidentifythegenusand thespeciesofEnterococcusandtoconfirmthe resistancephenotype.Biochemicaltestsfor identificationaretimeconsuming,expensiveandthey aresometimesinconclusive.Conventionalprotocolsto detectvancomycinresistanceasthediskdiffusion test,Etest,andbrothmicrodilutionaretime consumingaswell.Theautomatedmethodsneed expensiveequipmentsandmaterialsandarehighly accurateonlyforE.faecalisandE.faeciumidentifi cation(AlvesdAzevedoetal.,2009). Thereisacontinuousneedforimprovementof culturemethodsforVREdetection,especiallyfor thoseclinicalmicrobiologylaboratoriesthatdonot haveaccesstonucleicacidamplificationtechnologies. Recently,chromogenicmediaincorporatingchromo genicenzymaticsubstratesandavarietyof antimicrobialagents(chromIDVRE,CHROMagar GRE)havebecomeavailableforVREdetectioninone singlestep,butonlychromIDVREcontaining vancomycininaconcentrationof8g/ml,can additionallydirectlydifferentiatebetweenVR E.faeciumandE.faecalisstrainsfromclinical 152

Discussion specimensbasedondistinctcolonycolor(Kuchetal., 2009). Theaimofthisstudywastoevaluatethe usefulnessofchromIDVREagarforVREisolationin theclinicallaboratory.Inaddition,wecompared chromIDVREtobileesculinagarsupplementedwith 6g/mlvancomycin(BEAV)whileconsideringboth API20STREPforspeciesidentificationandEtestfor confirmationofglycopeptidesresistanceasthe referencemethod. Inthepresentstudy,VREwasisolatedfrom 34/100(34%)stoolsamplesobtainedfrompatients frombothmedia.ThechromIDVREmediumwasable todetectVREin22/100(22%)stoolsamples.Allwere identifiedasE.faecium(violet)whileonesamplegave bothE.faecium(violet)andE.faecalis(bluegreen) withasensitivityandspecificityof64.7%and100% respectively. Ourresultsarecomparablewiththoseobtained byDelmasetal.(2007)whoshowedaspecificityof (100%)after24hrsandasensitivityof(54.55%).Their sensitivityincreasedto100%afterenrichmentin brainheartinfusionbrothwith3g/mlvancomycin whilespecificitybecame99.9%.Soourlowsensitivity 153

Discussion couldbeexplainedbynotusingtheenrichmentstep whichproducesabettersensitivitypossiblydueto betterpenetrationofvancomycinintothecellsof bacteriainliquidmedia(Kuchetal.,2009). Asregardingthespecificityourresultsare similartothoseobtainedbyLedeboeretal.(2007b); Ledeboeretal.(2007b);Kuchetal.(2009)which were96.6%,98.6%,100%respectively.Whilethey havehighersensitivity(96.4%,88.2%,80% respectively)thanours.Kuchetal.(2009)foundthat afterovernightenrichmentstepwithBrainHeart Infusionbrothsupplementedwith30g/ml vancomycinthesensitivityincreasedto100%. WhileCuzonetal.(2008)obtainedasensitivity andspecificityof99.4%and96.9%respectivelyafter enrichmentfor18hrsinabilebrothsupplemented with3gofvancomycin/ml,5gofcolistin/ml,and 50gofamphotericinB/mlat37Cpriortoplatingon eithermedia. Asiretal.(2009)observedasensitivityof 77.8%forchromIDVREmediumbutitincreasedto 83.3%afterenrichmentwithBrainHeartInfusion Brothsupplementedwith3gvancomycin/ml.

154

Discussion Inthepresentwork,BEAVmediumwasableto detectVREin33/100(33%)stoolsamples.Oneisolate wasrecoveredfromchromIDVREandwasnot recoveredfromBEAV,while12isolateswere recoveredfromBEAVbutnotfromchromIDVREwith ahighersensitivity(97%)andalowerspecificity (72.7%)thanchromIDVRE.Thishighersensitivityof BEAVisattributedtothelowervancomycin concentration(6g/ml)presentinBEAVthanthat presentinchromIDVREmedium(8g/ml)which allowEnterococciwithphenotypehavinglower patternofresistancetovancomycintogrowonBEAV butnotonchromIDexample(VanB,VanCandVanD). ThelowerspecificityofBEAVisduetotherecoveryof sixisolatesofvancomycinsusceptibleE.faeciumwhich couldnotbeexplained,andnineVanCEnterococci isolates;oneisolateswasE.casseliflavusandeight isolateswereE.gallinarum.SimilarlyAsiretal. (2009)couldnotgiveanexplanationforthe bytheirstudy. 2007a;Ledeboeretal.,2007b)evaluatedtheinvitro sensitivityandspecificityoftheBEAVandreporteda Inaccordancetoourfindings,(Ledeboeretal., vancomycinsusceptibleEnterococcithatweredetected

155

Discussion sensitivityof(91.4%,92.2%)andspecificityof(77.2%, 72.4%)respectively. Similarly,Kuchetal.(2009)foundthatthe BEAVmediumhadasensitivityof80%andspecificity of60%andafterenrichmentwithBrainHeart Infusionbrothsupplementedwith30g/ml vancomycinthesensitivityincreasedto100%however thespecificityremainedthesame. Cuzonetal.(2008)obtainedanearsensitivity (93.9%)andaslightlyhigherspecificity(83.4%)than ours.Thishigherspecificitymightbeattributedtothe enrichmentstepfor18hrsinabilebroth supplementedwith3gofvancomycin/ml,5gof colistin/ml,and50gofamphotericinB/ml. Ontheotherhand,Delmasetal.(2007) reportedahigherspecificity(97.56%)butamuchlower sensitivity(40.91%)thanthatobtainedbyus.The sensitivityincreasedto100%afterenrichmentstepby brainheartinfusionbrothwith3g/mlvancomycin. Asiretal.(2009)observedasensitivityof 44.4%forabileesculinmediumsupplementedwith vancomycinwhichimprovedto77.8%after enrichment. 156

Discussion AlltheVREisolatesrecoveredfromchromID VREmediumwereofVanAphenotypewhilethose recoveredfromBEAVbutnotfromchromIDVRE wereasfollows:oneisolatewasofVanDphenotype, eightisolatesofVanBphenotypeandthreeisolatesof VanAphenotype.TheselatterVanAisolatesmightbe genotypicallydifferentthatiswhytheywerenot recoveredfromchromIDVREmedium.Also,nine VanCEnterococciisolatesoneisolateswas E.casseliflavusandeightisolateswereE.gallinarum wererecoveredfromBEAVandnotrecoveredfrom chromIDVREmedium. InourstudyBEAVhasgrownmorecontaminants causingfalsepositiveresultscomparedtochromID VREwhichhasgrownmorecolorlesscontaminants. ContaminantsmostlikelytogrowonchromIDVRE wereCandidaspp.,lactobacilliandGramnegativerods whilecontaminantscausingpotentialfalsepositive resultsonBEAVincludedVanCenterococcalisolates, vancomycinsusceptibleEnterococciandStreptococcus spp.Proliferationofcolorlessisolatesoneithermedium wasoflittleconsequencebecausethesecoloniesarenot likelytobeinterpretedincorrectly.Thegrowthofall contaminantswaslimitedtotheprimaryinoculumin chromIDVREandextendedtothetertiaryquadrantin 157

Discussion BEAVwhichdidnotchangewhenincubationtimewas extendedto48hrsforbothmedia. ThisfindingagreedwithLedeboeretal.(2007b) whostatedthatsensitivity,specificity,PPVandNPVof bothmediadidnotchangesignificantlywhen incubationwasextendedto48hrs.Thesignificant differenceinspecificityofBEAVversuschromIDVRE wasduetotheproliferationofcontaminantfloraon BEAV,whichmostcommonlyincludedGrampositive bacilli,Grampositivecocci,andEnterococcusspp.other thanVRE.faeciumandVRE.feacalis. Ontheotherhand,Kuchetal.(2009)stated thatwhenthetimeofgrowthwasextendedafter primaryplatingto48hours,contaminantsappeared inallquadrants.Whileafter24hrsincubationwith thebrothenrichmentstepbeforeplating,thegrowth ofallcontaminantswaslimitedtothefirstquadrant forchromIDVREagarandtothesecondandthird quadrantsforBEAVwhichdidnotchangewhenthe timeofincubationwasextendedto48hourspossibly duetobetterpenetrationofvancomycinintothecells ofbacteriainliquidmediasoinclusionofthebroth enrichmentstepmayresolvetheproblemof contaminants. 158

Discussion Inourstudyvancomycindiscdiffusioncould detecttheVanAphenotypicresistanceonbothmedia excepttheoneisolaterecoveredfromBEAVonly.This couldbeexplainedbybeingnonVanAgenotype.On thehand,neitherVanBnorVanDphenotypic resistancecouldbedetectedasallisolateswere sensitivetovancomycinbydiscdiffusion. (2009)whodemonstratedthatvancomycindisc ThesameresultswereobtainedbyRatheetal.

diffusioncorrectlyidentifiedallstrainscarryingthe VanAgene.Incontrast,thedetectionofvancomycin resistancebecauseofVanBwasinsufficient. Inthepresentstudy,therewasasignificant differencebetweenthepatientscolonizedandthose noncolonizedwithVREregardingthemeanduration ofhospitalization(14.5 13.9,7.5 5.77,respectively) (P<0.001).Thisfindingwasinaccordancewiththose ofHumphreysetal.(2004);Cohenetal.,(2009) whoreportedthatthedurationofhospitalizationisan independentriskfactorforVREcolonization.While, Askarianetal.(2007)statedthatdurationof hospitalizationhadnotbeenconsideredasa significantriskfactorforVREcolonization.

159

Discussion Thepresentworkrevealedahighlysignificant association(P<0.001)betweentheuseofantibiotics andthecolonizationwithVRE.Thiswasinagreement withElizagaatal.(2002)whoreportedahighly significantassociationbetweenVREcolonizationand administrationofantibiotics. Thecurrentworkshowedahighlysignificant associationbetweentheadministrationoflactam antibioticsandcolonizationwithVREanda significantassociationbetweentheadministrationof glycopeptidesandVREcolonization. etal.(2007)whofoundthatprioruseofantimicrobial therapy,includingvancomycinandcephalosporin,has beenshowntobeassociatedwithacquisitionofVRE andincreasedtheriskforVREcolonizationthree folds.Similarly,Worthetal.(2007)reportedthat previousindividualexposuretovancomycinisan independentriskfactorforVREcolonization. Inourstudywefoundahighlysignificant association(P<0.001)betweenleukemiaandthe colonizationwithVRE.Thiswasinagreementwith Worthetal.,(2007). OurfindingsareinaccordancewithAskarian

160

Discussion

161

Summary and conclusion

SUMMARY AND CONCLUSION

VancomycinresistantEnterococcihasemerged worldwideasanosocomialpathogenofmajor importance,andtheincidenceofinfectionscausedby VREcontinuestoincrease. SinceVREinfectionsareusuallyprecededbya periodofcarriage(essentiallyfecalcarriage); screeningforVREcolonizationremainsacornerstone ofinfectioncontrolmeasurestolimitthespreadof thisorganism. IdentificationoftheVangenesisthemost reliablemethodofdetectingVREisolates,however, notalllaboratoriescanincludemolecularbiology techniquesintheirroutineclinicalpractice.Forthis reason,itisessentialthatphenotypictechniquesable todetectVREisolatesinarapidandaccuratemanner aremadeavailable,inordertoensurecorrect antibiotictreatmentandtoavoidthespreadofVRE isolatesinthehospitalenvironment. TheidentificationofVREfromcolonized patientscanbeaccomplishedbyscreeningculturesof stoolorrectalswabswithdifferentialand/orselective 162

Summary and conclusion media.However,theirdisadvantageisthatadditional confirmatorytestsarerequiredtoidentifyisolatesand confirmglycopeptidesresistancewhichmaybetime consumingandlaborintense.Inresponsetothis problem,anumberofmanufacturershavedeveloped novelmethodsofdetectingVREcolonizationthatdo notrequireconfirmatorytesting. Thepresentstudyaimedatevaluatingthe chromogenicmedium(chromIDVREagar)forVRE identificationandcomparingitsperformanceagainst bileesculinagarsupplementedwith6g/ml vancomycin(BEAV),whileusingtheAPI20STREP forspeciesidentificationandEtestforconfirmationof glycopeptidesresistanceasareferencemethod. OnchromIDVREagar23isolatesofVREwere isolatedfrom22/100(22%)stoolsamples,allofthem wereE.faeciumexceptonesamplewhichgavetwo typesofisolatesonewasE.faeciumandtheotherwas E.feacaliswhichweredifferentiatedonchromIDVRE mediumandnotfromBEAVmedium,alsooneofthe positivesamplesforVREonchromIDVREagargave negativeresultonBEAV.WhileonBEAVagar33 VREisolateswererecoveredfrom33/100(33%)stool sampleswithoneisolaterecoveredfromchromIDVRE 163

Summary and conclusion andnotfromBEAVand12isolatesrecoveredfrom BEAVandnotfromchromIDVRE.Totally,34/100 (34%)stoolsamplesgavepositiveresultsforVREon bothmedia. PrimaryplatingonchromIDVREmediumwas foundtobemorespecificthantheBEAVmedium (100%vs72.7%,respectively)forthepresumptive identificationofVRE.WhileBEAVwasmoresensitive thanchromIDVREmedium(97%vs64.7%, respectively).Whenincubationtimeforbothmedia wasextendedto48hours,neitherthesensitivitynor thespecificityimproved. ChromIDVREwasabletodetectanddifferentiate dualcolonizationwithVRE.faeciumandVRE.faecalis strainseasily,whileinhibitingthegrowthof vancomycinsusceptibleEnterococcusspp.Itprovedtobe morespecificthanconventionalBEAVandreducedthe needforadditionalbiochemicalanalysisorantimicrobial susceptibilitytestingintheclinicallaboratory. Ontheotherhand,BEAVagarfrequentlyallowed thegrowthofE.gallinarumandE.casseliflavuswhereas thesestrainswerenotrecoveredfromchromIDVRE. Thisisduetotheinclusionofalowerconcentrationof vancomycininBEAVagar.However,mostauthorities 164

Summary and conclusion wouldnotrecommendscreeningforE.casseliflavusand E.gallinarumastheydonotusuallyharbour transmissibleresistancetovancomycinandare arguablyoflowerpathogenicity. Inthepresentstudy,wefoundthatchromIDVRE isagoodpredictorfordetectionofVanAphenotypic resistanceofVREbutnotfordetectionofVanB,VanC andVanDphenotypicresistanceasinBEAV. Asregardingthediscdiffusion,thevancomycin discprovedtobeagoodpredictorforVanAphenotypic resistance. Prolongedhospitalizationandprioradminis trationofantimicrobialswerefoundtobeindependent riskfactorsforVREcolonization. Inconclusion,chromIDVREagarisausefultool fortherapid,easy,economical,andefficientdetection anddifferentiationofVanAharboringVRE.faecium andfeacalis.However,werecommendtheapplication ofanenrichmentstepwithabrothcontaining3g/ml vancomycintoimprovethesensitivityofchromID VREmedium.

165

References

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References of Enterococcus species.J.Clin.Microbiol.; 28:14771478. Tsai T.N., Wu C.P., Peng M.Y., Giian C.F., Lee S.Y. and Lu J.J. (2006): Short course of linezolid treatment for vancomycinresis tant E.faecium meningitis. J. Clin. Pract.; 60(6):740741. Turnidge J.D, Ferraro M.J and Jorgensen J.H (2003):Susceptibilitytestmethods:general considerations. In Manual of clinical microbiology, Murray P.R., Baron E.J., JorgensenJ.H.,PfallerM.A.,YolkenR.H., 8th edition, p :11031104. Washington. AmericanSocietyofClinicalMicrobiology. Upadhyaya P.M.G., Ravikumar K.L., and Umapathy B.L. (2009): virulence factors of Review of Enterococcs: an

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References stomyrelated meningitis caused by vanco mycinresistant Enterococcus with intra venous and intraventricular quinupristin/ dalfopristin. Clin. Neurol. Neurosurg.; 104: 5456. WilliamsonR.,CalderwoodS.B.,MoelleringR.C. and Tomasz A. (1983): Studies on the mechanism of intrinsic resistance to beta lactamantibioticsingroupD Streptococci. J.Gen.Microbiol.;129(3):813822. WorthL.J.,ThurskyK.A.,SeymourJ.F.,Slavin M. A. (2007): Vancomycinresistant Enterococcus faecium infection in patients withhematologicmalignancy:patientswith acutemyeloidleukemiaareathighrisk.J. compilation;79:226233. YoongP.,SchuchR.,NelsonD.andFischettiV.A. (2004): Identification of a broadly active against antibioticresistant E.faecalis and E.zfaecium.J.Bacteriol.;186(14):48084812. Zhanel G., Hoban D. and Karlowsky J. (2001): Nitrofurantoin is active against vanco 202 phage lytic enzyme with lethal activity

References mycinresistant Enterococci. Antimicrob. AgentsChemother.;45(1):324326. ZhouQ.,MooreC.,EdenS.,TongA.,McGeerA. InfectionControlTeam.(2008): Factors associatedwithacquisitionofvancomycin resistant Enterococci (VRE) in roommate contacts of patients colonized or infected withVREinatertiarycarehospital.Infect. ControlHosp.Epidemiol.;29:398403. Zirakzadeh A. and Patel R. (2006): Vancomycin resistant Enterococci: colonization, infection, detection, and treatment. Mayo Clin.Proc.;81(4):529536. and the Mount Sinai Hospital

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Arabic summary

. . . ) (Van . . / . . . . )chromID VRE (agar 6g/ ml , API 20 STREP E-test . 32 001/22 )22%( chromID VRE .E faecium ) .E 402

Arabic summary (faecium ) (E. faecalis chromID VRE BEAV chromID VRE .BEAV 33 001/33 BEAV chromID VRE BEAV 21 BEAV chromID .VRE 43 /001 )43%( . )ChromID (VRE agar %001( )) BEAV 7.27%( . BEAV %79( chromID VRE 7.46%(. 84 . chromID VRE VR E. faecium VR E. faecalis , BEAV . BEAV E. gallinarum E. casseliflavus chromID VRE .BEAV E. gallinarum E. casseliflavus . chromID VRE ) (VanA ) (VanB) (VanC)(VanD ).(BEAV

502

Arabic summary ).(VanA .

602

Arabic summary )agar chromID (VRE ) (E. faecium ) (E. faecalis ) .(VanA 3g/ml ).(chromID VRE

702

Selective Chromogenic Agar Medium for Detection of Vancomycin-Resistant Enterococcus species

Thesis
SubmittedforPartialFulfillmentofM.Sc.Degreein Clinical and Chemical Pathology

By
Noha Alaa El-Din Mohammed Mohammed Fahim
M.B.,B.Ch. FacultyofMedicine,AinShamsUniversity

Supervised by

Professor/ Hadia Hussein Bassim

ProfessorofClinicalandChemicalPathology, FacultyofMedicine,AinShamsUniversity

Professor/ Eman Mohamed Kamel

ProfessorofClinicalandChemicalPathology, FacultyofMedicine,AinShamsUniversity

Doctor/ Hala Badr El-Din Ali

LecturerofClinicalandChemicalPathology, FacultyofMedicine,AinShamsUniversity

,Faculty of Medicine Ain-Shams University

0102

/
-

/
-

/
-

 )311(

ThanksfirstandlasttoAllahasweowehim forhisgreatcare,supportandguidanceineverystep inourlife. toProf. Dr. Hadia Hussein Bassim ProfessorofClinicalandChemicalpathologyFaculty ofMedicine,AinShamsUniversity,forherKind supervisionandindispensablehelp. Iwouldliketoexpressmydeepthanksand gratitudetoProf. Dr. Eman Mohamed KamelProfessorofClinicalandChemical PathologyFacultyofMedicine,AinShamsUniversity, forhersupervision,helpandsupport IwouldliketoexpressmygratitudetoDr. Hala Badr El-Din AliAssistantProfessorof ClinicalandChemicalpathologyFacultyofMedicine, AinShamsUniversity,forherindispensablehelp, valuableadvicesandencouragement,offeringallthe facilitiestofinishthiswork. Iwouldliketoexpressmythanksandgratitude

Acknowledgeme nt

Noha Alaa ElDin 2010

Contents
Page Acknowledgement ................................................................................ ................................................................................ Listoftables ................................................................................ ................................................................................ Listoffigures ................................................................................ ................................................................................ Introduction ................................................................................ ................................................................................ 1 Aimofthework ................................................................................ ................................................................................ 4 Reviewofliterature ................................................................................ ................................................................................ 2 Enterococci

....................................................................... ....................................................................... 5 EnterococcalInfection ....................................................................... ....................................................................... 15 AntimicrobialResistanceInEnterococci ....................................................................... ....................................................................... 21 VancomycinResistantEnterococci ....................................................................... ....................................................................... 33 Mechanismofactionofvancomycin ............................................................. ............................................................. 34 Geneticmechanismofvancomycin resistance ............................................................. ............................................................. 35 Intrinsicglycopeptideresistance ............................................................. ............................................................. 37

Acquiredglycopeptideresistance ............................................................. ............................................................. 39 EpidemiologyOfVRE ....................................................................... ....................................................................... 49

Contents

(Cont..)

Page LaboratoryDiagnosis ....................................................................... ....................................................................... 60 Laboratorydiagnosisof Enterococcusspecies ............................................................. ............................................................. 60 Laboratorydiagnosisofvancomycin resistantEnterococci(VRE) ............................................................. ............................................................. 72 PreventionAndControlOfVRE

....................................................................... ....................................................................... 82 TreatmentOfEnterococcalInfections ....................................................................... ....................................................................... 88 Materialsandmethods ................................................................................ ................................................................................ 99 Results ................................................................................ ................................................................................ 123 Discussion ................................................................................ ................................................................................ 139 Summaryandconclusion ................................................................................ ................................................................................ 149 References

................................................................................ ................................................................................ 153 Arabicsummary ................................................................................ ................................................................................

List Of Tables
Table No.
1

Title Page

Differententerococcalspecies ................................................................. ................................................................. 9 ListofIntrinsicandacquiredantimicrobial drugresistanceinEnterococci ................................................................. ................................................................. 22 ResistancetoGlycopeptidesinEnterococci ................................................................. ................................................................. 27 Majorpatternandmechanismsofresistance toantimicrobialagentsinEnterococci ................................................................. ................................................................. 28 ThekeytestsincludingMethylD Glucopyranoside(MGP)and Efrotomycin(EFRO)susceptibilitytests, fortheidentificationofenterococcal

species ................................................................. ................................................................. 66

6

FormulaofchromIDTMVREagar (bioMrieux,MarcylEtoile,France) ................................................................. ................................................................. 101 FormulaofBileesculinagarmedium (Himedia,India) ................................................................. ................................................................. 102 ConstituentsandinterpretationoftheAPI 20Strepstrips ................................................................. ................................................................. 105

List Of Tables
Table No.
9

(Cont..)

Title Page

FormulaofAPIGPMedium ................................................................. ................................................................. 106

10 DifferentphenotypesofEnterococci

accordingtoMICsforbothvancomycin andteicoplanin ................................................................. ................................................................. 118 diffusiontest ................................................................. ................................................................. 121 andBEAVmediumwithandwithout Gramstain ................................................................. ................................................................. 125

11 Interpretivecriteria(inmm)fordisc

12 DiagnosticperformanceofchromIDVRE

13 AssociationbetweenthechromIDVREand

thecombineduseofAPI20STREPand Etest(referencemethod) ................................................................. ................................................................. 216

14 AssociationbetweentheBEAVmedium

andthecombineduseofAPI20STREP andEtest(referencemethod)

................................................................. ................................................................. 127

15 AssociationbetweenthechromID VREand

theBEAVmedium ................................................................. ................................................................. 128

16 VancomycinMICsofallVREisolates

recoveredfrombothchromIDVRE mediumandBEAVmedium ................................................................. ................................................................. 132

List Of Tables
Table No. Title Page

(Cont..)

17 VancomycinMICsofallVREisolates

recoveredfrombothchromIDVRE mediumandBEAVmedium ................................................................. ................................................................. 132

18 Comparisonbetweencolonizedandnon

colonizedpatientsregardingthetotal durationofhospitalization

................................................................. ................................................................. 133

19 Associationbetweentheuseantibiotics

andthecolonizationwithVRE ................................................................. ................................................................. 134

20 Associationbetweentheuseoflactam,

flagyl,glycopeptidesantibioticsandthe colonizationwithVRE ................................................................. ................................................................. 136 colonizationwithVRE ................................................................. ................................................................. 137 chronicliverdisease,chronicrenal failureandthecolonizationwithVRE ................................................................. ................................................................. 138

21 Associationbetweenthesexandthe

22 Associationbetweentheleukemia,diabetes,

List Of Figures
Figure No. Title Page

1 Thepheromoneresponsiveconjugative

system ................................................................. ................................................................. 32 nismofactionofvancomycin ................................................................. ................................................................. 35

2 Peptidoglycanbiosynthesisandmecha

3 VanAtypeglycopeptideresistance ................................................................. ................................................................. 41

4 ReplicativetranspositionofTn1546

................................................................. ................................................................. 42 VREagarplate ................................................................. ................................................................. 109

TM

5 ChromID

6 Bileesculinagarshowingblackcoloniesof

Enterococcisurroundedwithablack halo ................................................................. ................................................................. 111 wasidentifiedasE.faecium ................................................................. ................................................................. 115 ................................................................. ................................................................. 117 positiveonchromIDVREandBEAV beforeandafterGramstainand catalasetest ................................................................. ................................................................. 130

7 API20STREPstripshowingapanelthat

8 Etestforbothvancomycinandteicoplanin

9 Distributionofthecontaminantsandfalse

List of Abbreviations
AD Agg AME AS BEAV BMD BSIs CDC cfu CSF Dalanine DLac DSer E. E. faecalis E. faecium EBS Esp ET FISH GC GRE HICPAC Agardilution Aggregationsubstance Aminoglycosidemodifyingenzymes Aggregationsubstance Bileesculinagarwithvancomycin BrothMicrodilution Bloodstreaminfections Centerofdiseasecontrol Colonyformingunit Cerebrospinalfluid DAla Dlactate Dserine Enterococcus Enterococcusfaecalis Enterococcusfaecium Enterococcalbindingsubstance Enterococcalsurfaceprotein Etest Fluorescenceinsituhybridization Guaninecytosine GlycopeptideresistantEnterococci HospitalInfectionControlPracticesAdvisory Committee

HLAR ICUs ID IDGPC Lalanine LAP LGG MH MIC MLST MLVA MRSA MSCRAM MAce NCCLS PBP PFGE PNS pyMS PYR qPCR RNA S. SAB

Highlevelresistancetoaminoglycosides Intensivecareunits Identification IDGramPositiveCocci LAla Leucinenaphthylamide LactobacillusrhamnosusGG MuellerHinton Minimalinhibitoryconcentration Multilocussequencetyping Multiplelocusvariablenumbertandemrepeat analysis MethicillinresistantStaph.aureus Microbialsurfacecomponentrecognizing Adhesivematrixmoleculeadhesionofcollagen fromEnterococci NationalCommitteeforClinicalLaboratory Standardsmethod Penicillinbindingprotein Pulsedfieldgelelectrophoresis Probablenewspecies Pyrolysismassspectrometry Pyrrolidonylarylamidase QuantitativerealtimePCR Ribonucleicacid Streptococcus Staph.aureusbacteraemia

SSTIs Staph. Tn TNF UTIs VRE VRSA

Skinandsofttissueinfections Staphylococcus Transposon Tumornecrosisfactor Urinarytractinfections VancomycinresistantEnterococci VancomycinresistantStaph.aureus

Tarek

Introduction

Tarek

Aim of the work

Tarek

Review of literature

Tarek

Materials and methods

Tarek

Results

Tarek

Discussion

Tarek

Summary and conclusion

Tarek

References

Tarek

Arabic summary