ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF BLOOD CULTURE ISOLATES FROM A NEONATAL UNIT

A THESIS SUBMITTED TO UNIVERSITY OF HEALTH SCIENCES IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF

M.Sc MEDICAL TECHNOLOGY IN MICROBIOLOGY

By Muhammad Usman Qamar

November 2009

UNIVERSITY OF HEALTH SCIENCES LAHORE, PAKISTAN

In the name of ALLAH, Most Gracious, Most Merciful.

ii

DEDICATED TO
My Parents & My beloved brother Muhammad Zubair For their support, prayers, and encouragement

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CERTIFICATE

It is hereby certified that this thesis is based on the results of experiments carried out by Mr. Muhammad Usman Qamar and it has not been previously presented for M.Sc. MT degree. Mr. Muhammad Usman Qamar has done this research work under my supervision. He has fulfilled all the requirements and is qualified to submit the accompanying thesis for the degree of Master of Medical Technology.

Professor Major General (Retd) DR. ABDUL HANNAN

M.B.B.S (Pb), D.C.P (Pb) Dp Bact (Manchester), M.R.C. Path (London), F.R.C. Path (London) Head of Microbiology Department University of Health Sciences Lahore, Pakistan

Dated: --------------------------

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ACKNOWLEDGEMENTS
All praise to Almighty Allah Who is the only source of knowledge and without Whose mercy coming this far would only have remained a dream. Prof. Malik Hussain Mubbashar, Vice Chancellor, University of Health Sciences, Lahore, Pakistan, for his strong motivation and sympathetic attitude. I would like to express my profound and sincere appreciation to my supervisor Professor Maj. Gen. (Retd.) Abdul Hannan for his patronage, unflinching support, valuable advice & able guidance, constant encouragement and most of all his kindness. I would have been lost without him. It is difficult to overstate my gratitude to Dr. Khawja Ahmad Irfan Waheed (Associate Professor of Neonatology Children Hospital & Institute of Child Health, Lahore). Throughout my research work; he provided encouragement and sound advices. Col. (Retd.) Javaid Iqbal, Director Administration, University of Health Sciences, Lahore, for his moral support and strong motivation to address my problems encountered during research work. I would like to thanks Neonatology Department of Children Hospital Lahore for providing me blood culture samples. My special thanks to Dr. Asim Mumtaz for his kindness and encouragement throughout my study period. Worth mentioning is the help, cooperation and encouragement received from my fellows; Miss Kanwal rauf, Miss Sara Karim, Miss Aneela Bukhari, Miss Samra Asghar, Ms Amna Tariq. Mr. M. Arshad, Mr. Usman Arshad, Mr. M. Barkat, Mr. Absar for their help in bench work and thesis writing. I special thanks to Mr. Abdul Quddus Tariq and Wasim Akhtar for assisting me in the laboratory and sampling collection. Lastly my gratitude goes to my family for teaching me the value of education, hard work, patience, sacrifice, and honesty.

ABBREVIATIONS
ADH AMR API ATCC BaCl2.2H2O CIT CLSI CoNS DNA DNase E. cloacae E. coli EONS ESBL FDA GBS GEL GLU GNR H2S H2SO4 ICU IND Arginine dihydrolase Antimicrobial resistance Analytical profile index American type culture collection Barium chloride dihydrate Citrate utilization test Clinical laboratory standards institute Coagulase Negative Staphylococcus Deoxyribonucleic Acid Deoxyribonuclease Enterobacter cloacae Escherichia coli Early-onset neonatal septicemia Extended spectrum beta lactamase Food and drug Administration Group B streptococci Gelatin liquefaction test Glucose fermentation test Gram-negative rods Hydrogen sulfide Sulfuric acid Intensive care unit Indole test

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K. pneumoniae LBs LDC LONS MBL MDR MDR MEM MHA MRCoNS MRSA n NaCl NICU NS ODC P. aeruginosa P. penneri PBP PMS S. aureus S. epidemidis S. paucimobilis Spp

Klebsiella pneumoniae Live births Lysine decarboxylase Late-onset neonatal septicemia Metallo--lactamase Multidrug resistant Multi-drug resistance Meropenem Mueller-Hinton agar Methicillin Resistant Coagulase Negative Staphylococci Methicillin-Resistant Staphylococcus aureus Number of isolates Sodium chloride Neonatal Intensive Care Unit Neonatal septicemia Ornithine decarboxylase Pseudomonas aeruginosa Proteus penneri Penicillin binding protein Poly-microbial septicemia Staphylococci aureus Staphylococcus epidemidis Sphingomonas paucimobilis Species

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SPS SPSS SXT URE USA v/v VP WHO

Sodium Polyanetholsulfonate Statistical package for social sciences Co-trimoxazole Urease test United State of America volume / volume Vogues-Proskauer test World Health Organization

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TABLE OF CONTENTS
Certificate Acknowledgements List of abbreviations Table of Contents List of Tables and figures List of Appendices Summary Introduction & Literature Review Materials & Methods Results Discussion Conclusion Recommendations Appendices References iv v vi ix x xi xii 1 12 24 39 45 46 47 60

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LIST OF TABLES AND FIGURES

Table I: Frequency of blood isolates (n=93) from neonatal septicemia. Table II: Poly-microbial organisms in neonatal septicemia: combination of two isolates. Table III: Percent resistant pattern of Gram-positive micro-organisms. Table IV: Percent resistant pattern of Gram-negative micro-organisms. Table V: Total no of ESBL, carbapenem and MBL producing micro-organisms.

26

27 28 30 38

Figure 1: Overall percent susceptibility pattern of Gram-positive micro-organisms.29 Figure 2: Overall percent susceptibility pattern of Gram-negative micro-organisms.31 Figure 3: Percent of ESBL and Non-ESBL. Figure 4: Demonstration of ESBL phenomenon. Figure 5: Percent of carbapenem susceptibility. Figure 6: Percent of MBL and Non-MBL producers. Figure 7: Demonstration of MHT. Figure 8: Disc Potentiation Test for MBL. 32 33 34 35 36 37

LIST OF APPENDICES

Appendix A: Blood Collection Technique Appendix B: Diagrammatic representation of principle of BACTEC 9120 Appendix C: Antibiotic disks and their contents Appendix D: Gram stain procedure Appendix E: Catalase test procedure Appendix F: Slide Coagulase test procedure Appendix G: Tube Coagulase test procedure Appendix H: DNase test procedure Appendix I: Cytochrome oxidase test procedure Appendix J: API 20E prcedure Appendix K: API 20-NE procedure Appendix L: 0.5 Turbidity standard preparation

47 48 49 50 51 52 53 54 55 56 58 59

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SUMMARY
Neonatal septicemia defined as positive blood culture in first month of life. NS is considered to be an important cause of neonatal mortality. WHO estimates over 4 million neonatal deaths occur globally; 98% of these in developing countries. Antimicrobial resistance among neonates is growing world wide problem, such as MRSA, MRCoNS, ESBL and MBL producers are highly pathogenic bugs particularly among neonates. The current study was designed to determine the antimicrobial susceptibility pattern of blood isolates from a neonatal unit. The study was carried out at the Microbiology department, University of Health Sciences, Lahore. One hundred and three blood cultures were collected from a neonatology unit of Children Hospital Lahore. Sample collection was performed aseptically. Blood cultures were processed on BACTEC 9120 (BD Dickenson). Positive samples were subcultured on blood and MacConkey agar. Biochemical identification of isolated colonies was done by API 20E and API 20NE. Antimicrobial susceptibility testing was performed according to CLSI 2009. The isolates, which showed resistance to any of the third generation cephalosporins and carbapenems, were further tested for the production of ESBL, carbapenemase and MBL by double disk diffusion phenotypic method, MHT and disc potentiation test respectively. The results were alarmingly high. Among staphylococci, out of 6 S. aureus, 4 (66.6%) were MRSA and out of 11 CoNS, 6 (54.5%) isolates were MRCoNS. Out of total 91 isolates, 20.8% were ESBL producers and 19.8 % were resistant to carbapenems. Among these carbapenems resistant 60.1% were carbapenemase and MBL producers. The highest ESBL frequency was noted among K. pneumoniae whereas E. cloacae were predominant antimicrobial amongst carbapenemase and MBL producers. Alarmingly was high

resistance

particularly

pan-resistance

observed.

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INTRODUCTION AND LITRATURE REVIEW

Neonatal septicemia (NS) is the term particularly used to describe any systemic bacterial infection documented by a positive blood culture in the first month (0-30 days) of life1. NS can be classified into two types according to the time of onset of the disease. Earlyonset neonatal sepsis (EONS) and late-onset neonatal sepsis (LONS); both are defined as illness appearing from birth to seven days and from eight to twenty-eight days postnatal respectively1, 2. Major causes of infections in early-onset are related to maternal risk factors and birth canal acquisition while in late-onset these infections are acquired from home or hospital environment 3. The reported incidence of NS varies from 7.1 to 38/1000 live births (LBs) in Asia, 5 to 23/1000 LBs in Africa, and 3.5 to 8.9/1000 LBs in South America. In contrast, rate of EONS reported in the United States of America (USA) and Australia ranges from 1.5 to 3.5/1000 LBs and up to 6/1000 LBs for LONS 4. NS is considered to be an important cause of neonatal mortality (death in the first 28 days of life) 5. World Health Organization (WHO) estimated that over 4 million neonatal deaths occur per year globally; 3 million of these deaths occur in early neonatal period. Ninety-eight percent of these deaths take place in the developing countries. In developing countries, the risk of deaths in neonates is six times greater than developed countries. Neonatal mortality rate in Africa is 41/1000 LBs, in sub-Saharan region of Eastern, Western and Central Africa mortality rate is 42 and 49/1000 LBs, in SouthCentral Asia neonatal death rate is 43/1000 LBs and in Latin America and Caribbean deaths rate is 15/1000 LBs 6. Over 40% global neonatal deaths take place in south-central Asia 6. Bangladesh and India estimated mortality rate was 43/1000 LBs and 25-42/1000

LBs respectively 7, 8. Whereas, in developed countries, the mortality rate is about 5/1000 LBs 4.According to UNICEF report published in 2009, more than 500 newborns die everyday in Pakistan. Neonatal mortality rate is 54/1000 LBs in Pakistan. About 40% of these deaths are due to asphyxia and infections. Amongst Asian countries, Pakistan has the eighth highest rate of newborns deaths ranking below only Afghanistan and Iraq 9 .It is generally assumed that neonatal mortality in developing countries is under-reported by at least 20% 7. According to WHO, most common causes of deaths in neonatal period are infections (32%) including septicemia, meningitis, pneumonia, diarrhea and neonatal tetanus, followed by birth asphyxia and injuries (29%) and prematurity (24%)
10

. A

review by Stoll reported that in under developed countries neonatal infections are responsible for 456% and 8-84% of neonatal deaths in 17 hospital-based studies and 24 community-based studies respectively11. Overall, Gram negative bacteria are more frequent causes of NS than Gram positive. Commonly isolated micro-organisms include Klebsiella spp., Escherichia coli, Pseudomonas spp., Salmonella spp., Staphylococcus aureus, coagulase negative staphylococci (CoNS), Streptococcus pneumoniae, Group B streptococci (GBS) and Streptococcus pyogenes1, 2, 4, 7 The bacteriological profile of NS is different in EONS and LONS and also varies in different parts of the world 7. Neonatal surveillance reports from developed countries indicate that GBS, E. coli and Listeria monocytogenes are the most frequent causative micro-organisms of EONS while CoNS are the predominant LONS pathogens followed by GBS and S. aureus. However, in developing countries, E. coli, GBS, Enterobacter spp., Enterococcus spp., CoNS and Listeria spp. are mostly associated with EONS. On

the other hand, Pseudomonas spp., Salmonella spp., CoNS and Serretia spp. are associated with LONS. Klebsiella spp., Acinetobacter spp. and S. aureus are associated with both EONS and LONS1, 4, 12, 13. The type and number of blood isolates reported are variable among different countries and institutions. A neonatal intensive care unit (NICU) study in Georgia, USA has reported that Gram negative rods (GNR) were isolated from 55% of blood cultures 14. While a study from Columbus, OHIO showed that recovered blood isolates in EONS were GBS (41%) and Enterobacteriaceae (34%) while CoNS (68%), GNR (18%) and Fungus spp. (14%) were documented in LONS
15

. Two studies from Middle East have

shown that CoNS were the predominant isolates in NS 1, 16. However, study carried out in Saudi Arabia has documented that Salmonella enteritidis (31%) was the most frequent isolate. This study also reported a case of NS and meningitis caused by Bacteroides fragilis17. A study performed in Nigeria showed that predominant blood isolate was Klebsiella pneumoniae (86%) among GNR and S. aureus (81%) was among Grampositive cocci (GPC)
18

. Chaturvedi et al reported that among 1059 neonatal blood

cultures, 60.1% were GNR predominantly with Klebsiella spp. However, among GPC most common isolates were CoNS
19

. Research performed in India reported that GNR

(58.5%) were predominant over GPC (41.5%) and among GPC the most common isolate was S. aureus (35.0%) 8. Whereas, another study conducted in Amritsar India, showed that GPC (52.7%) were more frequent neonatal blood isolates than GNR (47.3%) and S. aureus (27.4%) was the predominant organism among GPC followed by CoNS (20.1%). Amongst GNR, Enterobacter spp. (14.2%) were predominant followed by E. coli (9.3%), Pseudomonas spp. (7.6%) and Acinetobacter spp. (6.7%) 20. A similar study carried out in

Nepal reported that the predominant micro-organisms were CoNS (48%) followed by Enterobacter spp. (11.2%), Acinetobacter spp. (9.7%) and Klebsiella spp. (9.4%)
21

. In

Pakistan, little data is available on NS. A study conducted in Peshawar showed that E. coli (36.6%) was the most common isolate followed by S. aureus (29.5%), Pseudomonas spp. (22.4%), Klebsiella spp. (7.6%), and Proteus spp. (3.8%)
22

. While, another study

carried out in Karachi reported that most common micro-organisms causing EONS were Klebsiella app. (53%) and E. coli (10%) whereas pathogens causing LONS were Salmonella paratyphi (21%), Group A streptococci (21%), E. coli (14%), and Pseudomonas spp. (14%) 23. Two similar studies conducted in Children Hospital Lahore. One study reported that E. coli (47.8%) was the commonest micro-organism among GNR in both EONS and LONS while S. aureus was the most common among GPC 24. Second study showed that the predominant isolate was E. coli (31.6%) followed by S. epidermidis (24.8%), Klebsiella spp. (19.08%) and pseudomonas spp. (14.69%) 25. The main reason for high level of neonatal infections is mainly due to nosocomial infections. In neonates, nosocomial infections have been associated with prolonged morbidity, higher mortality and significantly greater hospital costs
26

. Nosocomial

infection is defined as an infection which develops 48 hours after hospital admission or with in 48 hours after being discharged patients have nosocomial infections
29 27, 28

. WHO estimates, that 8.7% of all hospital

. Neonates are more susceptible to acquired

hospital infections because their immune system is not fully developed. In USA, nosocomial infections rate is 15-20% in NICU
29

. Nosocomial infections are serious

problems of public sector hospitals of Pakistan where there are no well defined guidelines for hospital infections control and preventions
30

. In Pakistan, the frequency of

nosocomial infections in intensive care unit (ICU) was 29.1% and 45% in neonates
23,28

.The most common nosocomial pathogens in neonates are Klebsiella spp., E. coli, S.

aureus, CoNS, Pseudomonas spp., GBS, Serratia spp. and Candida spp.28,29, 31,32. Polymicrobial septicemia (PMS) is defined as isolation of more than one microorganism from a single blood culture specimen at a given time
33

. PMS is relatively
34

common in hospitalized neonates ranging 3 to 10% in NICU in USA

. In pediatric

population, incidence of PMS reported is 3.2 to 23% 35. Jarvis et al reported incidence of polymicrobial bactraemia to be 9.8 to 25% during an out break of K. pneumoniae and Enterobacter cloacae sepsis in NICU 36. The mortality rate of PMS is much higher than mono microbial infections 37. Fax and Kovarik reported incidence of PMS in neonates to be 3.9% with high mortality rate i.e. 70%38. The potential risk factors for PMS are central venous catheters (including umbilical and arterial catheter, percutaneous central venous catheters and arrow catheters) during and after surgery and mechanical ventilators 34. For the treatment of bacterial infections, a number of antibiotics have been used, most of them are -lactam agents e.g. penicillins, cephalosporins, cephamycins, clavulanic acid, monobactams and carbapenems. Other commonly used antimicrobial groups are aminoglycosides (amikacin, gentamicin, and kanamycin) macrolides (erythromycin, azithromycin, and clarithromycin), Quinolones (ciprofloxacin, ofloxacin, levofloxacin) and glycopeptides (vancomycin)
39

. New -lactam agents have been

introduced continuously since 1940s during that time many bacteria have developed resistance to the older drugs 40. Penicillin was immensely successful in curing previously fatal infections caused by common bacterial pathogens such as staphylococci, streptococci and pnumococci. The first S. aureus strain resistant to penicillin was

identified in 1944. At that time more than 94% of clinical isolates of S. aureus were sensitive to penicillinase labile penicillins but 50% had developed resistance in the late 1950s.41. At present, more than 90% of S. aureus isolates and 80 to 90% of CoNS produce beta-lactamase
41

and are thus resistant to penicillinase-labile penicillins

(penicillin G). Currently, the most common type of resistance among staphylococci is methicillin resistance
42

.The methicillin resistance in staphylococci is mostly due to

horizontal acquisition of mec-A gene. It encodes transpeptidase, penicillin-binding protein2a (PBP) or PBP2 which is a low-affinity PBP
43

. Methicillin resistant S. aureus

(MRSA) and methicillin resistant coagulase-negative staphylococci (MRCoNS) strains are defined as having minimum inhihibitory concentration (MIC) to oxacillin of 4g/ml and 0.5g/ml respectively 44. Antimicrobial resistance (AMR) is a growing problem world wide and it is estimated that approximately 50 to 60% of more than two million nosocomial infections in the USA each year are caused by antimicrobial resistant bacteria
45

. In developing

countries AMR can result in extra financial burden, prolongation of hospital stay, and devastating or even fatal consequences
46

. According to Food and Drug Administration

(FDA), approximately US $4-5 billion is attributable to treat infections due to antimicrobial resistant pathogens 47. AMR is used to describe the phenomenon when a microbe can grow or multiply despite the presence of antimicrobial agents 48. Multi-drug resistance (MDR) is defined as resistance of a bacterial isolate against three or more antibiotics at the same time 49. There are certain bacteria that are intrinsically resistant to certain antibiotics e.g. Enterococci spp. are intrinsically resistant to cephalosporins. Micro-organisms can acquire antibiotic

resistance by four mechanisms; drug inactivation by enzymes or modification, target alteration, decreased permeability through outer membrane, and efflux pump 50 as shown in Figure: i

Figure i: Mechanism of AMR. Several factors promote AMR in neonatal units; firstly, cross contamination of antimicrobial resistant pathogens carried from patient to patient via the unwashed hands of health care workers. Secondly, colonization of antimicrobial resistant pathogens can lead to clinical infections because of immature host defense of neonates. Thirdly, irrational use of antimicrobial drugs in neonates leads to AMR 51. The most common type of resistance among GNR is the production of -lactamases, encoded by either plasmid or chromosome 52. Chromosomally mediated -lactamases are possessed by many genera of GNR
53

. In 1960s, among GNR first plasmid-mediated -lactamase in TEM-1 was

reported. TEM-1 -lactamase spread quickly throughout the world and are now found in many different species of the family Enterobacteriaceae, P. aeruginosa, Haemophilus

influenzae, and Neisseria gonorrhoeae. The other common plasmid mediated -lactamase found in K. pneumoniae and E. coli is SHV-1. The SHV-1 -lactamase is chromosomally encoded in the majority of the isolates of K. pneumoniae but is usually plasmid mediated in E. coli 53. -lactamases are commonly classified according to two general schemes: the Ambler Molecular classification scheme and the Bush-Jacoby-Medieros Functional classification system 54. The Ambler scheme divides -lactamases into four major classes (A to D) on the basis of amino acids homology. In this scheme, -lactamases of classes A, C, and D are serine -lactamases, whereas, the class B enzymes are Metallo-Lactamases (MBL). The Bush-Jacoby-Medeiros classification scheme uses the biochemical properties of the enzyme plus the molecular structure and nucleotide sequence of the genes to place -lactamases into functional groups. There are four main groups and multiple subgroups in this system. Extended spectrum -lactamases (ESBLs) are placed in group 2be in this scheme 54, 55. ESBLs are -lactamases capable of conferring bacterial resistance to the penicillins, first, second, and third generation cephalosporins, and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, and which are inhibited by -lactamase inhibitors such as clavulanic acid 56. TEM and SHV enzymes are the most prevalent types of ESBLs among Enterobacteriaceae 55, 56. There are more than 165 TEMtypes -lactamases and more than 115 SHV types enzymes 57. In both of these types of enzymes, a few point mutations at selected loci within the gene give rise to the extended spectrum phenotype.

Carbapenems have broad spectrum activity against micro-organisms 58. The broad spectrum activity of carbapenems is explained by their rapid penetration inside bacteria, stability to hydrolysis by almost all clinically important serine--lactamases and high affinity for essential PBPs of GNR including PBPs1a and 1b which have cell-lytic consequences
58

. Ambler Class A carbapenemases includes Non Metalloenzyme

Carbapenemase/ Imipenem Hydrolyzing -Lactamase (NMC/IMI), Serratia marcescens enzyme (SME), and K. Pneumoniae Carbapenemase (KPC) enzymes. Their hydrolytic mechanism requires an active site serine 59. MBL belongs to Ambler Class B and has the ability to hydrolyze a wide variety of -lactam agents, such as penicillins, cephalosporins, and carbapenems which requires zinc cations as cofactor for enzymatic activity. MBL families include the Verona Integron-encoded Metallo -Lactamase (VIM), Active on Imipenem (IMP), German Imipenemase (GIM) and Seoul Imipenemase (SIM) 54. AMR among neonates vary greatly from one geographical area to other. Neonatologists of developed countries in North America, Europe, and Australia have been reporting problems with MDR pathogens such as MRSA and ESBL producing GNR
60

. Thaver et al reported AMR of neonatal pathogens in developing countries with three

major pathogens (E. coli, S. aureus, and K. pneumoniae) in 2009. They documented that E. coli were resistant to ampicillin (72%), cotrimoxazole (SXT) (78%), gentamicin (13%) and third generation cephalosporins (19%). All Klebsiella spp. were resistant to ampicillin, while 45% and 66% were resistant to SXT and third generation cephalosporins respectively. Resistance to gentamicin was low among E. coli (13%) but much higher among Klebsiella spp. (60%). MRSA was rare (1 out of 33 S. aureus isolates) but 46% were resistant to SXT
61

. One year prospective study on neonatal

infections in Asian countries showed that over 50% of GNR were MDR 62. In Gaza city, MDR Acinetobacter baumannii was reported in NICU. These isolates were resistant to commonly used antibiotics, while 90% were susceptible to carbapenems, 75% to ciprofloxacin, 57.5% to gentamicin and 50% to ceftriaxone 63. A retrospective study of all the 390 neonatal blood samples carried out in National Hospital Abuja, Nigeria (Jan 2002-Dec 2004) reported that among GNR, K. pneumoniae isolates were MDR and ESBL producing. Among GPC, the predominant micro-organism was S. aureus and their sensitivity to co-amoxiclav, cefuroxime, ciprofloxacin, chloramphenicol
18

and

erythromycin were 89%, 85%, 75%, 71% and 64% respectively

. Whereas, an Indian

study documented that 86% of Klebsiellae spp., 73% of Enterobacter spp. and 63% of E. coli strains were ESBL producer
64

. Similarly, MDR K. pneumoniae was isolated from

65

NS Hubli Hospital, Karnataka India

. Moniri et al (2005) documented that MDR P.

66

aeruginosa accounted for 74% of all isolates from NS in Beheshti Hospital, Iran

.A5

years study on NS conducted at Patan Hospital, Nepal, reported that gentamicin resistance was observed in 65.6% Klebsiella spp., 50% in Enterobacter spp., 39.4% in Acinetobacter spp. and 25% in E. coli. cefotaxime resistance was seen in 53% Klebsiella spp., 31.6% Enterobacter spp., 21.2% Acinetobacter spp. and 16.6% E. coli isolates
21

Study conducted in Peshawar on NS found that E. coli and P. aureuginosa showed were highly resistant to commonly used antibiotics (ampicillin, augmentin and gentamicin), and moderately resistant to cephalosporins. S. aureus showed low resistance all three antibiotics 22. High drug resistance (76%) to ampicillin and gentamicin among GNR was reported in neonatal septicemia from Karachi
23

. K. pneumoniae producing ESBL has

been reported from Hungarian NICU 67. An out break of K. pneumoniae producing ESBL

10

(SHV-12, CMY-2) was found in NICU of Korean tertiary care hospital 68. Gundes et al reported an out break of K. pneumoniae producing ESBL in NICU
69

. Lebessi et al also

reported similar study 70. IMI-1 was first isolated in USA in California from two isolates of E. cloacae in 198471. A plasmid mediated IMI-2 producing isolate of E. cloacae was found in China in 200172. A study from India reported one case of Acinetobacter spp producing MBL in NS 73. Up till now there is no such data available on MBLs and ESBLs in neonatal septicemia in Pakistan. A study conducted at Armed Forces Institute of Pathology, Rawalpindi reported ESBL production in 35% of the Enterobacteriaceae isolates 74. Shah et al (2002) reported the frequency of ESBL to be 48%75. Similar studies conducted in Karachi and Rawalpindi in 2002 reported 30%, 40%, and 45% ESBL production76-78. Cases reported from Pakistan in general population indicate the presence of carbapenemases in P. aeruginosa and Acinetobacter spp but there is not any report or study for the detection of carbapenemases in Enterobacteriaceae in Pakistan79. In developing countries like Pakistan, there is lack of proper microbiological diagnostic facilities. Therefore most of the physician prescribes antibiotics to treat neonatal septicemia on empirical grounds. Besides antibiotics prescribed are broad spectrum which can leads to more resistance among micro-organisms 7. The present study was designed to know the antimicrobial susceptibility pattern of blood culture isolates from a neonatal unit.

11

MATERIALS AND METHODS

Setting: The study was conducted at the Department of Microbiology, University of Health Sciences, Lahore. Study Design: Analytical observational cross sectional. Study based on antimicrobial susceptibility pattern of blood culture isolates from a neonatal unit. Duration of this study was six months; April September 2009. Blood Samples Collection: One hundred and three blood culture specimens were collected from a neonatology unit of Children Hospital Lahore. Using aseptic precautions 1-3ml venous blood was collected from each suspected neonate. Blood was immediately inoculated into pediatric blood culture bottles (Bactec Peds plus/F). Blood collection technique used is given in Appendix A. Inclusion criteria: Age range from 0-28 days. Neonates with suspected septicemia on clinical grounds.

BACTEC 9120: The BACTEC 9120 (Becton Dickinson Diagnostic Instrument System, Spark, Md) instrument is a continuous monitoring blood culture system that uses internal flourecent-CO2 sensors, used for rapid detection of growth of micro-organisms. It can process up to 120 bottles simultaneously and serves as incubator, agitator. Each blood culture bottle has a sensor disk bonded to the inner surface of the bottom. If micro-

12

organisms present in blood culture bottles (BACTEC Peds Plus/F), they metabolize nutrients and release CO2 in culture medium. As the CO2 is produced in each bottle, its sensor emits florescent light that passes an emission filter on the way to a photo-detector. The instrument photo- detector measures the level of fluorescence, which correspond to the amount of CO2 released by micro-organisms. Bottles are placed bottom down in to receiving wells that are monitored once every 10 minutes. The voltage of current reading of diode is compared with the previous reading. If the voltage change exceeds a present delta value, the microcomputer flags the bottles as positive. The position of positive bottle is indicated on the computer screen. Different media have been developed to enhance the recovery and detection of growth of micro-organisms. Soybean-Casein Digest broth medium with resins and Sodium Polyanetholsulfonate (SPS) is used in BACTEC Peds Plus/F vials. Resins in the medium neutralizing the effect of antibiotics, whereas, SPS is used for lysis of leukocytes, complement and subsequent release of viable pahogocytized micro-organisms 80.Diagrammatic representation of BACTEC 9120 principle is given in Appendix B. Bacterial Identification: Blood cultures samples were incubated in the BACTEC 9120 instrument (Becton Dickinson, USA) for 7 days. The specimens were declared negative if no growth were indicated in 7 days. Positive specimens were sub-cultured on blood agar and MacConkey agar and incubated at 35 C for 24 hours. The isolates were preliminary identified on the basis of morphology and cultural characteristics. Gram-positive isolates were biochemically identified by catalase, slide coagulase and DNase test. Tube coagulase method was used as confirmatory test in selected slide coagulase negative cases. The

13

phenotypic resistance to methicillin was evaluated using Cefoxitin disk (Oxoid Ltd., Basingstoke, Hampshire, England) whereas, Gram-negative isolates were biochemically identified by cytochrome oxidase and confirmed by API 20E and 20NE (BioMerieux France). Antimicrobial Susceptibility Testing: Antimicrobial susceptibility of isolates was performed by Kirby-Bauer disk diffusion method using Mueller-Hinton agar (Oxoid UK), according to Clinical Laboratory Standards Institute (CLSI) 2009 guidelines 44. Antibiotic disks used are given in appendix C. The plates were incubated at 35C for 24 hours. The interpretation of susceptibility results were done according to CLSI guidelines 2009
66

. Micro-organisms

resistant to antibiotics were further evaluated for methicillin resistance, ESBL and MBL production. Storage of the Micro-organisms: The isolates were preserved in 16% (v/v) glycerol in brain heart infusion (Oxoid Ltd, UK) and were stored at minus 70C 81. ATCC Strains: Reference strains, S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212 were included to monitor quality control. A known ESBL producing strain of K. pneumoniae was included as a positive control while E. coli ATCC 25922 was used as a negative control. A known carbapenemase producing strain of E. cloacae was included as positive control whereas; a known S. paucimobilis strain was used as a negative control. A known E. cloacae was

14

also used as a positive control and a known S. paucimobilis strain as negative control for the detection of MBL. Biochemical Tests: Following tests were performed; 1. 2. 3. 4. 5. 6. 7. Gram stain Catalase test Coagulase test (Slide and Tube) DNase test Phenotypic detection of Methicillin Resistance Cytochrome oxidase test API-20-E and NE

1. Gram Stain: A thin smear of the isolates was prepared on a clean glass slide, using a sterile loop. The slide was air dried and then heat fixed by passing it through a flame, making sure that it did not become too hot 81. The gram stain method is given in appendix D. 2. Catalase Test: This test is useful in distinguishing organisms that produce catalase from those that lack this ability. The test was performed from a blood-free non-inhibitory medium 82. The detailed method is given in appendix E. 3. Coagulase Test: Coagulase test is used to detect free coagulase and or bound coagulase (clumping factor) by slide and tube method respectively. This test differentiates coagulase producing S. aureus from other Staphylococcus spp.; collectively termed as CoNS
83

. The detail

15

method of both slid and Tube method is given in Appendix F, G. 4. DNase Test: DNase is an extra cellular enzyme that hydrolyzes DNA inside the medium into smaller nucleotides units. The DNase production was detected by flooding the surface of the spot inoculated medium with 1 N HCl and observing for clear zones in the medium surrounding growth. In the absence of DNase activity, the reagent reacts with the intact nucleic acid forming a cloudy precipitate. The test is useful for differentiating, S. aureus from CoNS 84. The detail method is given in Appendix H. 5. Phenotypic Detection of Methicillin Resistance: The Phenotypic detection of methicillin resistance was determined by disk diffusion method, using 30 g Cefoxitin disk on MHA according to CLSI guidelines 2009. The cefoxitin disk method is more reliable than oxacillin disk method in screening methicillin-resistant staphylococci 44. 6. Cytochrome Oxidase Test: The test is used to differentiate between oxidase producing and non oxidase producing bacteria82. The detailed method is given in appendix I. 7. API-20-E: The API-20-E strip consists of 20 microtubes containing dehydrated substrates. These tests were inoculated with a bacterial suspension that reconstitutes the media. During incubation, metabolic reactions produced colour changes that were either spontaneous or revealed by the addition of reagents. The reactions were read according to the reading table and the identification was obtained by calculating 7 digit numerical code referring to the Analytical Profile Index or using the API web (identification

16

software) 85. The detailed method is given in appendix J. 8. API 20 NE: The API 20 NE strip consists of 20 microtubes containing dehydrated substrates. These tests were inoculated with a bacterial suspension that reconstitutes the media. During incubation, metabolism produced colour changes that were either spontaneous or revealed by the addition of reagents. The reactions were read at 24 and 48 hours according to the Reading Table and the identification was obtained by calculating 7 digit numerical code referring to the API or using the API web (identification software)85. The detail method is given in Appendix K. ANTIMICROBIAL SUSCEPTIBILITY TESTING Preparation of the Inoculum: Direct colony suspension method of inoculum preparation is a convenient alternative to the growth method, the inoculum can be prepared by making a saline suspension of isolated colonies selected from a 18-24 hours old agar plate (a nonselective medium was used). The suspension was adjusted to match the 0.5 McFarland turbidity standard. Details of McFarland turbidity standard preparation is given in appendix L. Direct colony suspension method of preparing a standardized inoculum was followed: 1. With a sterile wire loop, three or four isolated morphologically similar colonies were transferred to a tube containing 5 ml of sterile saline. 2. The saline was well mixed to make a homogenous suspension. 3. Turbidity was compared with and adjusted to the 0.5 McFarland turbidity standard 86.

17

Inoculation of Plates: Within 15 minutes of adjusting the turbidity of the inoculum suspension, a sterile cotton swab was dipped into the standardized bacterial suspension. The swab was rotated several times and pressed firmly against the wall of the tube above the fluid level. This removed excess inoculum from the swab. The dried surface of a MHA plate was inoculated by streaking the swab over the entire agar surface. This procedure was repeated three times, rotating the plate approximately 60 each time to ensure an even distribution of the inoculum. Inoculated plate was left for 3 to 5 minutes, to allow for any excess surface moisture to be absorbed before applying the drug impregnated disks 86. Application of Disks: Antibiotic impregnated disks (Oxoid) were placed on the inoculated plates (Greiner, Germany) and pressed gently against the agar surface with a sterile forceps (alcohol-flamed) to ensure complete contact with the agar. The disks were placed not closer than 15 mm from the edge of the petri dish and not closer than 24 mm from center to center. Once placed, the disk was not removed. The plates were then inverted and placed in 35C incubator within 15 minutes after the disks had been applied 86. Reading and Interpretation of Results: After 18-24 hours of incubation, each plate was examined. The diameters of the zones of complete inhibition, as judged by the unaided eye, were measured by digital Vernier calipers (Sylvac Fowler ultra-cal II). The plate was held a few inches above a black, nonreflecting background and illuminated with reflected light. The diameters of the zones of inhibition were interpreted according to the CLSI 2009 guidelines 44.

18

ESBL DETECTION ESBL production was detected by double disk diffusion phenotypic method described by Jarlier V. et al
87

. Antimicrobial disks used were; co-amoxiclav

(clavulanate 10ug + amoxicillin 20 g), ceftazidime 30 g, cefuroxime 30 g, ceftriaxone 30 g, and cefepime 30 g, Use of more than one antimicrobial disks increases the sensitivity of the method 44. Double Disk Diffusion Phenotypic Method Principle Clavulanic acid, present in the co-amoxiclav disk, acts synergistically with the third generation cephalosporins and/or aztreonam and results in the increase of the zone size of these antimicrobials. This is considered as a positive result for ESBL production 87. Procedure 1. The suspension of the test organism was prepared in the sterile physiological saline, and the MHA plate was inoculated as described above. 2. The disks were placed with sterile forceps. Co-amoxiclav disk was placed in the center of the plate while the other antimicrobial disks were placed at 20 mm distance center to center from co-amoxiclav disk. The disks others than co-amoxiclav were placed at least 24 mm apart from each other. 3. The plates were incubated at 35 C for 18-24 hours. 4. Next day, the plates were examined.

19

Interpretation of Results Isolates which showed an enlarged zone of inhibition greater than 5 mm on the co-amoxiclav side of the disk compared to the results seen on the side without coamoxiclav were confirmed as ESBL producers. While no increase in the zone indicates ESBL negative isolates 87. Quality Control a) Positive Control b) Negative Control K. pneumoniae (Known ESBL Positive) E. coli ATCC 25922 (ESBL Negative) CARBAPENEMASE DETECTION Carbapenemase in Enterobacteriaceae are detected by Modified Hodge Test (MHT) that is screening and confirmatory tests44. There are two types of carbapenemases, Metallo-carbapenemases and Serine carbapenemases. Modified Hodge Test Principle: Carbapenemase production is detected by the MHT when the test isolate produces the enzyme and allows growth of a carbapenem susceptible strain (E.coli ATCC 25922) towards a carbapenem disk. The result is a characteristic cloverleaf-like indentation 44. Procedure: 1. A 0.5 McFarland dilution of the E. coli (ATCC 25922) was prepared in 5ml of saline. 2. 3. Diluted the 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of saline. The 1:10 dilution of E. coli (ATCC 25922) was streak on MHA plate and allowed to dry 3-5 minutes. 4. Meropenem (10g) disk was placed in the centre of the test area.

20

5.

The organism was streaked in straight line from edge of organism to the edge of plate.

6.

Plate was incubated at 35C for 18-20 hours.

Interpretation of Result: MHT Positive: A clover leaf-like indentation of the E. coli (ATCC 25922) growth along the test organism growth streak within the disk diffusion zone. MHT Negative: Test has no growth of the E. coli 25922 along the test organism growth streak within the disc diffusion 44. Quality Control a) Positive Control b) Negative Control E. cloacae (Known carbapenemase producer) S. paucimobilis (Known carbapenemase negative) METALLO- LACTAMASE DETECTION MBL detection has been done by disk potentiation test. Disk Potentiation Test Principle This type of enzyme need zinc ions at their active site for their action. If these ions are captured by some chelating agents ethylene diamine tetra-acetic acid (EDTA) then these enzymes becomes inactive 88. Procedure 1. A 0.5 M EDTA solution was prepared by dissolving 186.1 gm of disodium EDTA. 2H20 (Sigma) in 1000 ml of distilled water. 2. 3. The pH of the solution was adjusted to 8.0 by adding NaOH to the solution. The solution was sterilized by autoclaving.

21

4.

Two of each imipenem (10 g) and meropenem (10 g) disks (Oxoid) were placed on MHA.

5.

To one of each of imipenem and meropenem disks, 5 l of 0.5 M EDTA (which is equal to 750 g EDTA) solution was added.

6.

Plate was incubated at 35C for 18-20 hours.

Interpretation of Results An increase in the inhibition zone of 8-15mm by the addition of EDTA to the imipenem and meropenem disks (carbapenem plus EDTA) indicates MBL positive isolates, while 0-5 mm increase in the zone size indicates MBL-negative isolates 88. Quality Control a) Positive Control b) Negative Control E. cloacae (Known MBL Positive) E. cloacae (Known MBL Negative) SERINE CARBAPENEMASE DETECTION Principle These types of enzymes have serine at their active site. Like other serine lactamases these are also inhibited by -lactam inhibitors (Clavulanic Acid). Procedure 1. A 1000g/ml clavulanic acid solution was prepared by dissolving 1 g of clavulanic acid in 1000 ml of water. 2. 3. The solution was made under strict aseptic conditions. Two 10 g imipenem disks (Oxoid) were placed on MHA and to one of them 10 l of clavulanic acid solution was added. 4. Plate was incubated at 35C for 18-24 hours

22

Interpretation of Results Inhibition zones of Imipenem alone and IMP plus clavulanic acid disks will be read after 18-20 hours incubation at 35C. The increase in zone size of IMP plus Clavulanic acid as compared to IMP alone is indicative of Serine carbapenemase presence 89.

23

RESULTS
Out of 103 suspected cases of septicemia, 71 (68.9%) were positive for bacterial isolates. Among these, mono and poly-microbial were 49 (69%) and 22 (30.9%) cases respectively. The mortality rate in positive samples was 33 (46.4%). Neonatal mortality rate in EONS and LONS was 13 (39.3%) and 20 (66.6%) respectively. In 21 (66.6%) neonates mortality was caused by K. pneumoniae. Table 1 shows the type and number of micro-organisms and their frequency in EONS and LONS. K. pneumoniae and CoNS was predominant isolates among GNR and GPC respectively. Table 2 shows the association of different micro-organisms in poly-microbial infections. Among these, Gram positive and Gram negative micro-organisms were present in 4 (18.2%) and 17 (77.2%) cases respectively. Only one case (4.5%) of Candida albicans was found. K. pneumoniae and E. cloacae was commonest isolates in 12 (54%) cases. Table 3 and 4 shows antimicrobial resistance pattern of Gram-positive and Gramnegative micro-organisms respectively. Figure 1 and 2 shows overall percent of susceptibility pattern of Gram-positive and Gram-negative isolates respectively. More than 80% of Gram-positive micro-organisms were resistant to penicillin, ampicillin and erythromycin. However, among Gram-negative isolates, 100% GNR were resistant to ampicillin, ceftazidime, 98.5% to co-amoxiclav, 98.2% to cefuroxime, 97.1% to ceftriaxone and cefepime and 91.2% to amikacin. All Gram-positive isolates were susceptible to vancomycin and linezolid, while 76.6% of Gram-negative micro-organisms were susceptible to carbapenems (imipenem and meropenem). Among staphylococci, out

24

of 6 S. aureus, 4 (66.6%) were MRSA and out of 11 CoNS, 6 (54.5%) isolates were MRCoNS. Out of total 91 isolates, 19 (20.8%) were ESBL producers (Figure 3, 4) and 18 (19.8%) were resistant to carbapenems (Figure 5). Eleven (61.1%) out of 18 were positive for carbapenemases and MBL producers (Figure 6). Carbapenemase detection was done by using MHT on these carbapenems resistant isolates (Figure 7).All of these carbapenamases were confirmed to be MBLs by disc potentiation method (Figure 8). Micro-organisms that were ESBL producers, carbapenems resistant, carbapenamase and MBL producers are given in Table 5. Carbapenemase and MBL producing isolates were found to be pan-resistant but ESBL producing micro-organisms were susceptible to carbapenems.

25

Table 1: Frequency of blood isolates (n=93) from Neonatal Septicemia Isolates Gram-negative rods (n=68) Klebsiella pneumoniae Enterobacter cloacae Shingomonas paucimobilis Eschirichia coli Burkholderia cepacia Acinetobacter baumannii Klebsiella ornitholytica Flavomonas oryzihabitans Proteus penneri Pseudomonas aeruginosa Gram-positive cocci (n=22) Coagulase negative staphylococci ( CoNS) Staphylococcus aureus Enterococcus faecalis viridans streptococci Fungi (n=01) Candida albicans Contaminants (n=02) Sarcina Bacillus subtilis Distribution (n) EONS (n=35) LONS (n=58)

40 12 5 3 2 2 1 1 1 1

10 4 4 1 1 1 1 1 0 1

30 8 1 2 1 1 0 0 1 0

11 6 3 2

4 3 0 2

7 3 3 0

1 1

0 1

1 0

26

Table 2: Poly-microbial micro-organisms in neonatal septicemia: combination of two isolates.

Micro-organisms Gram-negative K. pneumoniae

Associated with micro-organisms (number of cases) Gram-negative/positive E. cloacae (12), E. coli (2), Proteus penneri (1), Acinetobacter baumannii (1), E. faecalis (1)

Gram-positive S. aureus viridans streptococci Fungi Candida albicans

CoNS (2), Acinetobacter baumannii (1) Burkholderia cepacia (1)

Flavimonas rehabilitans (1)

27

Table 3: Percent resistant pattern of Gram-positive micro-organisms

Antimicrobials Penicillin Ampicillin Cefoxitin Erythromycin Clindamycin Amikacin Ciprofloxacin Co-trimoxazole Vancomycin Linezolid

coagulase-negative staphylococci (n=11) 72.7 NT 54.5 90.1 60 0 27.3 63.6 0 NT

Staphylococcus aureus (n=6) 83.3 NT 66.6 66.6 50 16.6 66.6 66.6 0 0

Enterococcus faecalis (n=3) 100 66.6 NT 66.6 NT 100 100 50 0 0

viridans streptococci (n=2) 100 100 NT 100 NT 100 50 100 0 NT

NT: Not Tested

28

Figure 1: Overall percent susceptibility pattern of Gram-positive micro-organisms

100 81.8 80 58.8 80 81.8 72.7 65 60 56.2 50 50 41.2 40 27.3 20 18.2 20 18.2 43.8 35

100

100

0 0
Penicillin Ampicillin Cefoxitine Erthromycin Clindamycin Amikacin Ciprofloxacin CoVancomycin trimoxazole

0
linezolid

Sensitive

Resistant

29

Table 4: Percent resistant pattern of Gram-negative microorganisms K. pneumoniae (n=40) NT 100 100 100 100 40 100 47.5 60 0 2.6 E. cloacae (n=12) 100 100 100 100 100 100 100 100 100 83 83 S. paucimobilis ( n=5) 100 100 100 100 100 100 0 100 100 100 100 E. coli (n=3) 100 100 100 100 100 100 100 100 100 0 0 B. cepacia (n=2) 100 100 100 50 100 50 100 0 0 0 0 A. baumannii (n=2) 100 50 50 50 50 50 50 50 50 0 0 K. ornitholytica (n=1) 100 100 100 100 NT 100 100 100 100 0 0 F. oryzihabitans (n=1) 100 100 100 100 NT 100 100 0 0 100 100 P. penneri (n=1) 100 100 100 100 NT 100 100 0 100 0 0 P. aeruginosa (n=1) 100 100 100 100 100 100 100 100 100 100 100

Antimicrobials Ampicillin Co-amoxiclave Cefuroxime Ceftriaxone Ceftazidime Cefepime Amikacin Ciprofloxacin Co-trimaxazole Imipenem Meropenem

NT: Not Tested

30

Figure 2: Overall percent susceptibility pattern of Gram-negative micro-organisms

100

100

98.5

98.2

97.1

100

97.1 91.2

80 70.6 61.8 60

75

76.6

40

38.2 29.4 25 23.4

20 8.8 0 0
Ampicillin Co-amoxiclave Cefuroxime Ceftriaxone Ceftazidime Cefepime Amikacin Ciprofloxacin Co-trimoxazole Imipenem M eropenem

1.5

1.8

2.9

2.9

Sensitive

Resistant

31

Figure 3: Percentage of ESBL and non ESBL producers.

32

Figure 4: Demonstration of ESBL phenomenon.

CTX, CAZ, CRO and FEP disks placed at 20 mm distance from AMC disk. Arrows indicate the enlarged zone of inhibition of each of these.

AMC; Co-amoxiclav, CTX; Cefotaxime, CAZ; Ceftazidime, CRO; Ceftriaxone, FEP; Cefipime.

33

Figure 5: Percentage of carbapenem susceptibility

34

Figure 6: Percentage of MBL producers and Non MBL producers

35

ATCC E.coli

(25922)

Figure 7: Demonstration of MHT

T: Test sample (E. cloacae) -ve: Negative control (A known S. paucimobilis) +ve: Positive control (A known E. cloacae) MEM: Meropenem

36

IMP

MEM

IMP+EDTA

MEM+EDTA

Figure 8: Disk Potentiation Test for MBL

IMP: MEM: EDTA:

Imipenem Meropenem Ethylene diamine tetra-acetic acid

37

Table 5: Total number of ESBL, Carbapenem resistant, Carbapenamase and MBL producing micro-organisms

Micro-organisms

Percentage

Number of ESBL producing micro-organisms K. pneumoniae E. coli Proteus penneri 17 1 1 18.7 1.1 1.1

Number of Carbapenem producing micro-organisms E. cloacae S. paucimobilis K. pneumoniae Flavimonas oryzihabitans P. aeruginosa 10 5 1 1 1 10.9 5.5 1.1 1.1 1.1

Number of Carbapenamase and MBL producing micro-organisms E. cloacae K. pneumoniae 10 1 55.5 5.5

38

DISCUSSION
Neonatal septicemia is a leading cause of deaths in neonates particularly in developing countries 4. Present study showed a high rate of blood culture positivity (68.9%) in neonates. Previous studies also reported high percentage which varies from 40%-73% 19,
21, 22

. Neonatal mortality rate observed in this study was 33 (46.4%) in septicemia cases.

While a recent study (2005) conducted in Pakistan reported 44% neonatal mortality rate
90

. Similarly another study from Iran had reported 48.1% of neonatal deaths

91

. K.

pneumoniae was responsible for 63.6% of total neonatal deaths in present study. Almost similar result (66.6%) was found from an Iranian study
92

. One of study

93

documented

that P. aeruginosa and K. pneumoniae were responsible for 71% and 59% neonatal deaths respectively. Zaidi et al (2005) from Pakistan reported that 20% of 1.6 million neonatal sepsis-relate deaths in developing world are due to K. pneumoniae infections 3. This study found that Gram-negative micro-organisms were predominant pathogens causing NS. Similar results were reported in various neonatal units across the world
23-25, 61

. In contrast some studies reported that Gram-positive pathogens were the

predominant causing neonatal infections1, 12. The spectrum of neonatal blood isolates varies from one region to another. This study reported that 74.7% and 21.3% isolates were GNR and GPC respectively. Most common isolates among GNR were K. pneumoniae (43.9 %) followed by E. cloacae 13.1%, S. paucimobilis 5.49% and E. coli 3.2%. Among GPC, predominant pathogens were CoNS 12.0% and S. aureus 5.4% respectively. A similar pattern of NS has been reported in previous studies that shown predominant isolates were K. pneumoniae and CoNS 25, 92, 94, 95. Movahedian et al (2006) reported that GNR were responsible for 72.1%

39

of NS

96

. Studies from Pakistan, also reported that most common isolate was K.

pneumoniae in NS 23, 97. A review of 11471 blood cultures specimens on NS showed that GNR were isolated from 60% of positive blood cultures in all the developing region of the world and causative micro-organism was K. pneumoniae 4. Whereas, Akhter et al (2005) reported that Enterobacter spp 52% were the predominant isolates causing NS followed by Klebsiellae spp 22.3% 98. However, a Bangladeshi study revealed that GNR responsible for almost 73% of NS with E. coli (30%) followed by K. pneumoniae (23%)99 .Most common isolates among developed countries were GBS, E. coli and CoNS.4,38,100, LONS was found more frequent than EONS in this study. These findings are in contrast with previous studies reported from Iran, India and Pakistan1, 101,102. Similar data was reported by Mosayabi et al (2003) from Iran 102.This difference is most likely related to nosocomial infections. K. pneumoniae was found predominant in EONS and LONS while CoNS were more prevalent in both EONS and LONS. However, an Indian study on NS reported that S. aureus was commonest isolate in EONS and LONS 104. In contrast, Gheibi et al (2008) documented that CoNS were predominant in both EONS and LONS 1.A study from India reported that K. pneumoniae and Enterobacter aerogenes were predominant in EONS and LONS respectively while CoNS were responsible for both EONS and LONS
102

. In

contrast a Nepali study found that GBS were commonest isolates in early-onset while CoNS were prevalent in late-onset of disease
21

. A Pakistani study revealed that more

24

frequent isolates causing EONS and LONS were E. coli and S. aureus

. Bhutta et al

(1990) have documented that commonest pathogens causing EONS and LONS were Klebsiella spp and Salmonella paratyphi respectively 23.

40

Interestingly this study showed high percentage (30.9%) of poly-microbial organisms. Contrary to this, previous studies showed low percentage of poly-microbial infections which varies from 3.9%-10% in NS cases
34, 38, 95

. Neonates may be already

infected with one micro-organism at the time of admission in neonatal unit and may acquire the second pathogen from the hospital environment or both of the microorganisms could be of nosocomial in origin. Regarding susceptibility pattern, present study has found a high degree of antimicrobial resistance among pathogens isolated from NS. Among Gram positive isolates (Table 2) MRCoNS and MRSA were 54.5% and 66.6% in staphylococci respectively. These findings are in accordance with a study conducted in India (10 of 13 isolates of S. epidermidis and 3 of 4 S. aureus were methicillin resistant)
105

. Similarly,

Cordero et al (1999) also reported the increased prevalence of MRCoNS and MRSA in hospitalized neonates 15. Most of the staphylococci showed resistance against penicillin, erythromycin, and co-trimoxazole. The entire E. faecalis and viridans streptococci were resistant to penicillin and amikacin. However, 50% of E. faecalis and viridans streptococci were resistant to ciprofloxacin and co-trimoxazole (Table 3). More than 80% of Gram-positive isolates were resistant to penicillin, ampicillin, and erythromycin. However, 65% of Gram-positive isolates were resistant to co-trimoxazole, 50% to ciprofloxacin and 27.3% to amikacin. All the Gram-positive isolates were susceptible to vancomycin and linzolid (Figure 1). Increased drug resistant pattern was also observed in S. aureus and CoNS in various local studies 24, 25. Whereas, a study conducted in Peshawar revealed only 40% S.

41

aureus were resistant to ampicillin 22. In a review article, Zaidi et al (2005) reported that 56% of all Gram-positive isolates were methicillin-resistant in south Asia 3. Besides resistance to commonly used antibiotics, GNR showed resistance to broad spectrum cephalosporins and carbapenems (Table 2). All of K. pneumoniae were resistant to co-amoxiclave, cephalosporins (except 40% to cefipime) and amikacin. Forty seven percent and 60% of K. pneumoniae were resistant to ciprofloxacin and SXT respectively. Most effective drugs against these bugs were carbapenems. Second most common isolate was E. cloacae which showed pan resistance to all antibiotics. However, 83% of E. cloacae demonstrated resistance to carbapenems. All of S. paucimobilis showed similar pattern like E. cloacae while most effective drug was amikacin. E. coli also showed same resistance pattern. However, all of the E. coli was susceptible to carbapenems. All Burkholderia cepacia showed resistance against ampicillin, coamoxiclave, cefuroxime, ceftazidime and amikacin except ciprofloxacin, SXT and carbapenems. Acinetobacter baumannii exhibited resistance to ampicillin, moderate resistance against commonly used antibiotics whereas all were sensitive to carbapenems. Klebsiellae ornithiolytica and Flavomonas oryzihabitants were resistant to all drugs except carbapenems, ciprofloxacin and co-trimoxazole respectively. More than 97% of GNR showed resistance against ampicillin, co-amoxiclave, and cephalosporins. However, 91% of GNR were resistance to amikacin. Seventy and sixty one percent of GNR demonstrated resistance against SXT and ciprofloxacin respectively. GNR (75%) were susceptible to carbapenems (Figure 2). Findings of this study are accordance with previous studies that also reported high degree drug resistance
96, 106

. A review article found that more than 70% of K.

42

pneumoniae and E. coli caused resistance against ampicillin, gentamicin and cephalosporins among developing countries particularly in south Asia 3. WHO also reported the high rate incidence of AMR against common pathogens including (K. pneumoniae, E.coli, E. cloacae, and S. aureus) in NS 2.Whereas studies reported from national and other part of the world revealed that GNR showed low to moderate resistance 8, 15, 18, 23-25. ESBL in neonatal septicemia have been reported in developed countries including North America, Europe, and Australia
18

.This study has found 20.8% pathogens were

ESBL producers (Figure 3). K. pneumoniae was the predominant causative pathogen (Table 5). An Indian study also reported that K. pneumoniae (13.5%) were ESBL producers107. Number of previous studies across the world also revealed that K. pneumoniae were ESBL producer in NS
67, 69, 70

. According to our knowledge K.

pneumoniae producing ESBL in NS is not reported in Pakistan yet. High degree of carbapenems resistance was also detected in this study. Out of 91 isolates, 19.8% were carbapenem resistant (Figure 5). Predominant pathogen was E. cloacae (Table 5). Among these 61.1% were carbapenemase and MBL producers (Figure 6). E. cloacae were the commonest pathogens (Table 5). An Indian study reported four cases of Acinetobacter spp were imipenem resistance was detected. Among these one Acinetobacter spp was MBL producer
73

. According to our knowledge isolation of E.

cloacae in NS has yet not been reported by national and international studies so far. This alarming increase in the rate of multidrug resistance is mainly linked to poor infection-control practices that facilitate out breaks of infections and person to person transmission of resistant pathogens. For instance, common identified sources in our setup

43

are these resistant bugs are contaminated intravenous catheter, feeding tube and various environmental surfaces (door handles, sucker machine, incubators, mattresses, wash basins, floor, sink, emergency trolley, ventilator, ambo bag, laryngeal scopes) and colonized hands of staff. Nurseries are often seriously overcrowded and understaffed, sharing of baby beds (two to three babies in a cot). Substandard sterilization and disinfection practices are common. We also observed lack of standard practices such as preparation of medication in contaminated area and reuse of ambo bag and ventilator and laryngeal scopes to other neonates without disinfection. It is well documented that neonates have immature immune system and unable to provide defense against virulent pathogens. Premature babies are at high risk because of lack of protective maternal antibodies, underdeveloped innate immunity and fragile, easily damaged skin 3. Another major factor to acquire resistance in our setup is irrational use of empirical therapy which is not according to the WHO criteria 2.

44

CONCLUSIONS
1. Alarmingly high antimicrobial resistance pattern particularly pan-resistance was observed in present study. 2. Exentended spectrum--lactamase (ESBL) producers, carbapenemase producers, Metallo--lactamase (MBL) producers, methicillin resistant S. aureus (MRSA), and methicillin resistant coagulase negative staphylococci (MRCoNS) were major bugs which lead to high neonatal mortality rate. 3. Distressingly high rate of poly-microbial infections have been found in this study.

45

RECOMMENDATIONS
The spectrum of antibiotic resistance among bugs in neonatal units can be reduced to follow these points. 1. Develop local and national antibiotic policies to restrict the use of expensive and broad spectrum antibiotics. 2. Use of empirical therapy should be revised after every three months on the basis of antimicrobial results. 3. 4. 5. Trust on microbiology laboratory and blood culture results. Treat sepsis but not colonization Do your best to prevent nosocomial infection, by reinforcing infection control, particularly hand washing. 6. 7. Establish infections control committee in hospital. Organize monthly meeting and seminar for continuous education of nursery staff.

46

Appendix A
Blood Collection Technique 1. Before performing veni-puncture, the rubber on the blood culture bottles (BACTEC Peds Plus/F) was disinfected with 70% isopropyl alcohol swab. 2. 3. Veni-puncture site was selected. The site was disinfecting with 2% povidone iodine in circular fashion outward for a diameter of 5-6cm. 4. 5. 6. 7. 8. 9. Allowed povidone iodine to dry for 1-3 mints. Draw 1-3 ml blood by using 3 ml syringe. Immediately blood was transferred into BACTEC peds plus/F vials. Mixed blood culture bottle for 8-10 times. After sample collection, veni-puncture site was cleaned with 70% isopropyl alcohol. Label the blood culture blood with patient data.

10. Inoculated BACTEC vials were transported as quickly as possible to the laboratory.

47

Appendix B
Diagrammatic representation of principle of BACTC 9120

48

Appendix C
Antibiotic disks and their contents

Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Antimicrobial Agent Penicillin Ampicillin Cefoxitin Co-amoxiclav Cefrouxime Ceftriaxone Ceftazidime Cefepime Ciprofloxacin Co-trimoxazole Clindamycin Erythromycin Amikacin Imipenem Meropenem Vancomycin Linzolid

Abbreviation P AMP FOX AMC CXM CRO CAZ FEP CIP SXT C E AK IMP MEM V L

Disk Content 10g 10g 30g 20g /10g 30g 30g 30g 30g 5g 25g 2g 15g 30g 10g 10g 30g 30g

49

Appendix D
Gram Stain Procedure Following steps were done in the staining: 1. The smear was covered with crystal violet stain for 60 seconds. 2. The crystal violet was poured off and the smear was covered with Lugols iodine solution for 30 seconds. 3. The iodine solution was poured off and the smear was decolorized with acetoneiodine decolorizer until the colour ceased to come out of the smear. 4. The slide was thoroughly washed with water. 5. The slide was counterstained with diluted carbol fuchsin for 30 seconds, washed with water, blotted with absorbent paper and air dried. 6. Organisms that retained the crystal violet-iodine dye complexes, after decolorizing with acetone-iodine, stain purple and were termed as Gram-positive, those that lost this complex and became red due to counter stain (carbol fuchsin) were termed Gram-negative. Following organisms were stained simultaneously for monitoring the quality control. Controls: Positive Control : Negative Control: S. aureus E. coli (ATCC 25923) (ATCC 25922)

50

Appendix E
Catalase Test Procedure Catalase is an enzyme that decomposes hydrogen peroxide into water and oxygen. Hydrogen peroxide forms as one of the oxidative end products of aerobic carbohydrate metabolism. 2H2O2 2H2O + O2

A small amount of culture to be tested was picked with a glass rod and inserted into 3% hydrogen peroxide solution in a clean tube. The production of gas bubbles indicated a positive test. Following organisms were used for quality control. Controls: Positive control : Negative control : S. aureus (ATCC 25923) E. faecalis (ATCC 29212)

51

Appendix F

Coagulase test Slide test The slide test detects bound coagulase or clumping factor. A heavy suspension of the test isolate, from NA plate, was prepared in a small drop of water on a clean glass slide. Then 1 small drop of rabbit plasma was added to the suspension. The suspension was well mixed in a circular motion while observing for the formation of visible white clumps. Known positive and negative controls were set up in parallel. Negative results were confirmed by the Tube coagulase test. Controls: Positive coagulase control: Negative coagulase control: S. aureus (ATCC 25923) S. epidermidis

52

Appendix G

Tube Coagulase method Procedure 1. The tube coagulase test detects bound and free coagulase. Free (extracellular) coagulase clots plasma in the absence of calcium. 2. Dispensed 0.5 ml of rabbit plasma into a sterile tube. 3. A loopful of the test organism was inoculated into the tube and then incubated at 35C for 4 h. 4. Observed for clotting at intervals during the first 4 h because some staphylococci produce fibrolysin, which could lyse the clot. Do not shake or agitate the tube while checking for clotting. The formation of a clot is considered positive. The majority of coagulase-positive S. aureus isolates will form a clot within 4 h. 5. If no visible clot was observed after 4 h the tubes were incubated at room temperature overnight and then observed for clotting. Controls: Positive coagulase control: Negative coagulase control: S. aureus (ATCC 25923) S. epidermidis

53

Appendix H

Deoxyribonuclease Test (DNase) Procedure 1. The DNase plate was divided into the required number of strips by marking the underside of the plate. 2. While using a sterile wire loop the medium surface was spot-inoculated with the test and control strains. 3. The plates were incubated at 35C for 24 hours. 4. The plate surface was covered with 1N hydrochloric acid solution, excess solution was tipped off. 5. Any clearing around the colonies within 5 minutes was recorded. Results Clearing around the colonies No clearing around the colonies Controls Positive DNase control: Negative DNase control: S. aureus (ATCC 25923) S. epidermidis. DNase positive strain DNase negative strain

54

Appendix I
Cytochrome Oxidase Test The cytochrome oxidase test uses tetramethyl-p-phenylenediamine di-

hydrochloride, that substitutes oxygen as artificial electron acceptors. In the reduced state, the dye is colorless; however, in the presence of cytochrome oxidase and atmospheric oxygen, p-phenylenediamine is oxidized, forming indophenol blue which gives deep purple color. The test was performed by the indirect paper strip procedure, in which a few drops of 1% aqueous solution of the reagent were added to a filter paper strip. A bacterial colony to be tested was smeared into the reagent zone of the filter paper using a wooden stick. Bacterial colonies having cytochrome oxidase activity developed a deep purple colour at the inoculation site within 10 seconds. As a precautionary measure, stainless steel or Nichrome inoculating loops or wires were not used for this test because surface oxidation products formed by metals when flamed for sterilizing may result in false positive reactions. Bacterial species showing positive and negative reactions were run as controls at frequent intervals. The following organisms were used as controls: Controls: a) Positive Control: b) Negative Control: P. aeruginosa (ATCC 27853) E. coli (ATCC 25922)

55

Appendix J
API 20 E Procedure Preparation of the Strip 1) Five ml of distilled water was distributed into the honeycombed wells of the tray to create a humid atmosphere. 2) On the elongated flap of the tray, the strain reference number was recorded. 3) Strip was removed from its packaging and placed in the incubation box. Preparation of the Inoculum A single well isolated colony from overnight culture was picked up with inoculating loop and emulsified into a tube containing 4 ml of sterile distilled water. This suspension was used immediately after preparation. Inoculation of the Strip 1) Both tube and cupules of the tests CIT, VP and GEL were filled with the bacterial suspension. 2) Only the tubes of the other tests were filled (and not the cupules). 3) The tests ADH, LDC, ODC, H2S, and URE were overlaid with mineral oil to create anaerobiosis. 4) Incubation box was closed with flap and incubated at 37C for 18-24 hours. Reading the Strip 1) Reading table was consulted for reading the results of API strip after incubation. 2) All the positive tests were recorded on the result sheet and then reagents were added for following tests: i. TDA Test: One drop of TDA reagent was added. Appearance of reddish

56

brown color was considered a positive reaction and recorded on the result sheet. ii. IND Test: One drop of JAMES reagent was added and a pink colour in the whole cupule was taken as a positive reaction. The indole production test was performed last since this reaction releases gaseous products which interfere with the interpretation of other tests on the strip iii. VP Test: One drop each of VP-1 and VP-2 reagents was added and result was recorded after 10 minutes. A pink or red color indicated a positive reaction. iv. Supplementary tests were performed in case the recorded results were not conclusive. Identification was obtained with the numerical profile. Determination of the numerical profile On the result sheet, the tests were separated into groups of 3 and a value 1, 2 or 4 is indicated for a positive test. By adding together the values corresponding to positive reactions within each group, a 7-digit profile number was obtained for the 20 tests of the API 20 E strip. The oxidase reaction constitutes the 21st test and has a value of 4 if it is positive. E. coli ATCC 25922 was used for quality control.

57

Appendix K
API 20 NE Procedure Preparation of the Strip 1. Five ml of distilled water was distributed into the honeycombed wells of the tray to create a humid atmosphere. 2. On the elongated flap of the tray, the strain reference number was recorded. 3. Strip was removed from its packaging and placed in the incubation box. Preparation of the Inoculum Picked 1-4 well isolated colonies from overnight culture was picked up with inoculating loop and emulsified into a tube containing 4 ml of sterile distilled water. This suspension was used immediately after preparation. Inoculation of the Strip 1. Only tubes of the tests were filled NO3 to PNPG. (Not the cupules). 2. Added 200 l test saline in to API AUX Medium and mixed it homogenizes. 3. Both tube and cupules of the tests GLU to PAC were filled with suspension. 4. The tests ADH, GLU and URE were overlaid with mineral oil to create anaerobiosis. 5. Incubation box was closed with flap and incubated at 37C for 18-24 hours. Reading the Strip 1. Reading table was consulted for reading the results of API strip after incubation. 2. All the positive tests were recorded on the result sheet and then reagents were added for following tests: i. NO3 Test: One drop of NIT 1 and on drop of NIT 2 reagent was added.

58

Appearance of red color was considered a positive reaction and recorded on the result sheet. ii. TRP Test: One drop of JAMES reagent was added and a pink colour in the whole cupule was taken as a positive reaction. iii. Assimilation Test: Bacterial growth was observed; opaque capule indicated positive reaction. iv. Supplementary tests were performed in case the recorded results were not conclusive. v. Identification was obtained with the numerical profile.

Determination of the numerical profile On the result sheet, the tests were separated into groups of 3 and a value 1, 2 or 4 is indicated for a positive test. By adding together the values corresponding to positive reactions within each group, a 7-digit profile number was obtained for the 20 tests of the API 20 NE strip. The oxidase reaction constitutes the 21st test and has a value of 4 if it is positive. P. aeruginosa was used for quality control.

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Appendix L
0.5 McFarland standards Turbidity Standard Preparation 1. A 0.5 ml aliquot of 0.048 mol/L BaCl2 was added to 99.5 ml of 0.18 mol/L H2S04 (1 % v/v) with constant stirring to maintain a suspension. 2. The correct density of the turbidity standard was verified by using a spectrophotometer with a 1-cm light path and matched cuvette to determine the absorbance. The absorbance at 625 nm should be 0.08 to 0.10 for the 0.5 McFarland standards. 3. The barium sulfate suspension was transferred in 4 to 6 ml aliquots into screwcap tubes. 4. These tubes were sealed and stored in the dark at room temperature. 5. The barium sulfate turbidity standard was vigorously agitated on a mechanical vortex mixer before use and inspected for a uniform turbidity. 6. The barium sulfate standard was replaced if their densities were not within acceptable limits.

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PATIENT PERFORMA

74

CONSENT FORM

I _______________________son / daughter of ______________________________ willingly give my consent to the Department of Microbiology, University of Health Sciences, Lahore through the Department of Neonatology, Children Hospital, Lahore for utilizing any of the micro-organisms isolated from the blood of my son/daughter named ________________________ for the project of ANTIMICROBIAL

SUSCEPTIBILITY PATTERN OF BLOOD CULTURE ISOLATES FROM A NEONATAL UNIT.

This consent has been read to the person in the language he/she understands and he/she signed it as correct.

Name of Father / Mother / Guardian: _______________________

Signature/ thumb impression: _______________________

Dated: ________________________

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