SCREENING AND VERIFICATION FOR ESBLs Page 1 of 8

LOYOLA UNIVERSITY MEDICAL CENTER

Procedure

DOUBLE DISK DIFFUSION CONFIR ATION OF ESBL

Pre!ared "# Paul Schreckenberger Violet Rekasius Re%&e' Da$e

Da$e Ado!$ed 08/10/07

Su!ersedes Procedure

Re%&s&o( Da$e

S&g(a$ure

SCREENING AND VERIFICATION FOR ESBLs Page ) of 8 PURPOSE* Beta-lactamases are bacterial enzymes that inactivate beta-lactam antibiotics Betalactamases that inactivate all the !enicillins an" ce!halos!orins inclu"ing the e#ten"e" s!ectrum ce!halos!orins are terme" $#ten"e" S!ectrum Beta-%actamases& abbreviate" as $SB%s 'here are over 1(0 "i))erent $SB%s "escribe" all o) *hich are mutations o) the classical broa"-s!ectrum beta lactamase enzymes that *ere initially name" '$+ an" S,V -'$+-1& '$+-.& S,V-1/ $SB%0s are name" '$+-1& -2 etc & S,V-.& -1 etc & 3'4+-1& -. etc & 546-1& -. etc $SB%s hy"rolyze !enicillins& ce!halos!orins an" the monobactam& con)erring resistance to all o) these "rug classes 'hey "o not hy"rolyze the ce!hamycin antibiotics -ie7 ce)o#itin/& *hich are close relatives to the ce!halos!orins $SB%0s are also inhibite" by beta-lactamase inhibitors such as clavulanate& sulbactam an" tazobactam $SB%s are generally inactive against the carba!enem antibiotics -8mi!enem& +ero!enem& $rta!enem/ 6n $SB% con)irmatory test involves testing ce)ota#ime an" ce)tazi"ime alone an" in combination *ith clavulanate 3lavulanate inhibits the activity o) the $SB% enzyme an" makes the organisms a!!ear more sensitive to "rug 9 clavulante combinations 'his :greater sensitivity; *ith clavulante can be "emonstrate" *hen the "isk containing clavulanate has a zone "iameter that is ( mm larger that the zone "iameter o) the "rug teste" alone& or *hen a zone o) enhance" sensitivity is observe" *hen a ce!halos!orin antibiotic is !lace" in close !ro#imity to a clavulanic containing "isk 'his is commonly re)erre" to as the :keyhole !henomenon ; -see )igure/ 6m!3 beta-lactamases "i))er )rom $SB%0s in that they are ce!halos!orinases an" are resistant to beta-lactamase inhibitors 'hey hy"rolyze the ce!hamycins -eg ce)o#itin/& but not the 2th generation ce!halos!orins -eg ce)e!ime/ 6m!3 is normally !ro"uce" in lo* levels by many organisms an" is not associate" *ith resistance& but it can be !ro"uce" at high levels an" cause resistance ,igh-level !ro"uction o) 6m!3 usually causes resistance to all beta-lactams& e#ce!t carba!enems an" 2th generation ce!halos!orins 'he 6m!3 gene is )oun" on the chromosome in 100< o) the )ollo*ing organisms= Enterobacter sp, Hafnia alvei, Morganella morganii, Citrobacter freundii, Serratia marcescens, Providencia sp, Aeromonas sp, Pseudomonas aeruginosa. 3hromosomal 6m!3 beta-lactamases can be !ro"uce" in"ucibly or constitutively 8n"ucible e#!ression o) the 6m!3 gene occurs *hen the enzyme is !ro"uce" at a high level *hen the organism is e#!ose" to in"ucing agents& such as ce!hamycins -ie7 ce)o#itin/& am!icillin an" carba!enems -ie7 imi!enem& mero!enem& erta!enem/ 8n"uction is tem!orary an" may be reverse" *hen the antibiotic in"ucer is remove" 8n some organisms& mutations occur that cause the am!3 gene to become !ermanently e#!resse" at high levels 'hese organisms are terme" !ermanently "ere!resse" mutants Plasmi"-me"iate" 6m!3 beta-lactamases can be )oun" in organisms that "o not carry the chromosomal 6m!3 Plasmi"-me"iate" 6m!30s have been "etecte" in organisms such as E coli, Klebsiella sp, Proteus sp and Salmonella sp 3urrently& there are no 3%S8 a!!rove" metho"s to "etect 6m!3 gene chromosomal or !lasmi"-me"iate" resistance Since "ata are urgently nee"e" to evaluate the clinical im!lications o) this resistance& an in"irect& but

SCREENING AND VERIFICATION FOR ESBLs Page + of 8 stan"ar"ize" metho" has been utilize" to "etermine i) there is !resence o) an 6m!3 betalactamase SPECI EN* 'he s!ecimen consists o) a !ure isolate o) the $nterobacteriaceae& *hich has a susce!tibility result that is consistent *ith an $SB% or an 6+P3 !attern& ie 7 ce)tazi"ime is 8/R& ce)rtria#one is 8/R& aztreonam is 8/R an" re>uires con)irmation by a "isk metho" ATERIALS* 1 6ntibiotic "isks in "is!enser& labele" :$SB% / 6+P 3;& ke!t in )ri"ge -.-83/& until use= 6ztreonam -10/ 3e)tazi"ime -10/ 3e)tazi"ime 9 clavulante -10/10/ 3e)ota#ime -10/ 3e)ota#ime 9 clavulante -10/10/ 3e)o#itin -10/ 3e)tria#one -10/ 3e)e!ime -10/ $rta!enem -10/ 8mi!enem-10/ +ueller ,inton -+,/ agar !late& 1(0 mm & ke!t in )ri"ge -.-83/& until use sterile saline or try!tic soy broth -'SB/ sterile s*abs 0 ( +c?arlan" barium turbi"ity stan"ar" / !hotometer -colorimeter/ ET,OD* 1 . 1 6llo* the +, agar !late an" "isk "is!enser to come to room tem!erature be)ore use Pre!are a 0 ( +c?arlan" stan"ar" o) the organism to be teste" in sterile saline or 'SB Stan"ar"ize the inoculum using the colorimeter Streak the bacterial sus!ension evenly in 1 !lanes onto the sur)ace o) the +, agar !late& using a cotton s*ab Rim the e"ge o) the !late Re)er to !roce"ure B@1.(0-28& @irby Bauer metho" )or a "etaile" an" com!lete "escri!tion Place the :$SB% / 6+P 3; labele" "isk "is!enser over the +, agar !late an" "e!ress the knob 'his *ill allo* the antibiotic "isks to "is!ense an" automatically :tam!; the "isk into !lace Re)er to !roce"ure B@1.(0-.2 )or !ro!er usage o) the "isk "is!enser

. 1 2 (

SCREENING AND VERIFICATION FOR ESBLs Page - of 8

SCREENING AND VERIFICATION FOR ESBLs Page . of 8 ( A 7 6ll o) the "isks must be !lace" on the same +, agar !late in a s!eci)ie" or"er -See ?igure 1/ 8ncubate the +, agar !late overnight in a non-35. incubator at 1(3 'he )ollo*ing "ay& rea" an" recor" all zones o) inhibition

RESULTS* 1 8) the zone size increases ( mm or more *hen clavulanate is a""e" com!are" to the "rug alone the isolate is consi"ere" an $SB% 5nly one antibiotic must be Breverse"B by the clavulanate to be an $SB% ?or e#am!le= Combination Disk Method 22 mm 36C/3%6 D .. mm 36C D 11 mm 11mm .. D11 E F (mm E $SB%

8n a""ition& an :enhancement; bet*een the !arent antibiotic an" the clavulanate "isk may take !lace 'his is seen as inhibition or a :@$G,5%$; e))ect& an" is in"icative o) $SB% !ro"uction Double Disk Method P. mirabilis with ESBL

@eyhole ?ormation E $SB%

SCREENING AND VERIFICATION FOR ESBLs Page / of 8 1 6m!3 !ro"ucing organisms can be recognize" utilizing the same "isks 6m!3 strains are resistant to the ce!hamycins -ie7 ce)o#itin/ ,igh level 6m!3 !ro"uction causes resistance to all .n" an" 1r" generation ce!halos!orins& e#ce!t not to the 2th generation ce!halos!orins -ie7 ce)e!ime/ H'hey are also resistant to the beta lactams an" the monobactam& aztreonam I Double Disk Method - E. coli with plasmid mediated AmpC

Jo 3lavulanic $))ect 3e)e!ime -?$P/ D S 3e)o#itin -?54/ D R

2 (

Recor" all "isk "i))usion mm zone size rea"ings in the culture *ork u! 3hange/overri"e any !revious +83 result to resistant& i) the antibiotic is a !enicillin& ce!halos!orin& or monobactam ?ollo* 3%S8 inter!retive gui"elines )or $SB% Re)er to 3%S8 "ocument +100-S1A or re)er to !roce"ure BS1.(0-87-.& Suce!tibility 6ntibiotic Re!orting 8n a""ition& all $SB% !ro"ucing organisms are susce!tible to the ce!hamycins -ie7 ce)o#itin/ 6"" !ro!er $SB% organism co"e to !atient re!ort& stating that any treatment *ith any ce!halos!orin may be unreliable ?or $SB% organisms only& )a# 8n)ection 3ontrol an" log organism into Resistant 5rganism 3om!uter "ata base 5n *eeken"s or holi"ays& call the !atient0s )loor to ensure that 8n)ection 3ontrol isolation measures take !lace

A 7

0UALIT1 CONTROL*

SCREENING AND VERIFICATION FOR ESBLs Page 2 of 8 Kisk "i))usion testing is !er)orme" *eekly *ith 6'33L 700A01 @lebsiella !neumoniae . $SB% "isks& in a""ition to ce)tazi"ime an" ce)ota#ime 8) correct >uality control results are not obtaine"& the test is invali" an" !atient results cannot be re!orte" 6n alternative metho" -+83& $ test/ can be !er)orme" REFERENCE* 3%S8 Per)ormance Stan"ar"s )or 6ntimicrobial Susce!tibility 'esting& Si#teenth 8n)ormational Su!!lement +100-S1A .00A Meneral Revie* 6rticles
1 . %ivermore K+& Ninstanley 'B& Shannon @P 8nter!retative rea"ing= recognizing the unusual an" in)erring resistance mechanisms )rom resistance !henoty!es O 6ntimicrob 3hemother .001 Oul728 Su!!l 1=87-10. 'homson @S& +olan" $S Version .000= the ne* -lactamases o) Mram-negative bacteria at the "a*n o) the ne* millennium -Revie*/ +icrobes an" 8n)ection& .0007.=1..(-1.1( 3ou"ron P$& +olan" $S& San"ers 33= 5ccurrence an" "etection o) e#ten"e"-s!ectrum beta-lactamases in members o) the )amily $nterobacteriaceae at a veterans me"ical center= seek an" you may )in" O 3lin +icrobiol 1PP7 5ct71(-10/=.(P1-7 Oarlier V& Jicolas +,& ?ournier M& Phili!!on 6= $#ten"e" broa"-s!ectrum beta-lactamases con)erring trans)erable resistance to ne*er beta-lactam agents in $nterobacteriaceae= hos!ital !revalence an" susce!tibility !atterns Rev 8n)ect Kis 1P88 Oul-6ug710-2/=8A7-78 Pitout OK& Reisbig +K& Venter $3& 3hurch K%& ,anson JK +o"i)ication o) the "ouble-"isk test )or "etection o) enterobacteriaceae !ro"ucing e#ten"e"-s!ectrum an" 6m!3 beta-lactamases O 3lin +icrobiol .001 6ug721-8/=1P11-( 'zele!i $& Miakkou!i P& So)ianou K& %oukova V& @emeroglou 6& 'sakris 6= Ketection o) e#ten"e"s!ectrum beta-lactamases in clinical isolates o) $nterobacter cloacae an" $nterobacter aerogenes O 3lin +icrobiol .000 ?eb718-./=(2.-A Gan OO& ,sueh PR& %u OO& 3hang ?G& Shyr O+& Nan O,& %iu G3& 3huang G3& Gang G3& 'sao S+& Nu ,,& Nang %S& %in 'P& Nu ,+& 3hen ,+& Nu OO= $#ten"e"-s!ectrum beta-lactamases an" !lasmi"me"iate" 6m!3 enzymes among clinical isolates o) $scherichia coli an" @lebsiella !neumoniae )rom seven me"ical centers in 'ai*an 6ntimicrob 6gents 3hemother .00A +ay7(0-(/=18A1-2 Gan OO& @o N3& Nu ,+& 'sai S,& 3huang 3%& Nu OO 3om!le#ity o) @lebsiella !neumoniae isolates resistant to both ce!hamycins an" e#ten"e"-s!ectrum ce!halos!orins at a teaching hos!ital in 'ai*an O 3lin +icrobiol .002 Jov72.-11/=(117-20

Kouble-Kisk Potentiation +etho"

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SCREENING AND VERIFICATION FOR ESBLs Page 8 of 8

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