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
ii
In the name of ALLAH, Most Gracious, Most Merciful.
iii
DEDICATED TO
My Parents
&
My beloved brother Muhammad Zubair
For their support, prayers, and encouragement
iv
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: --------------------------
v
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.
vi
ABBREVIATIONS
ADH Arginine dihydrolase
AMR Antimicrobial resistance
API Analytical profile index
ATCC American type culture collection
BaCl2.2H2O Barium chloride dihydrate
CIT Citrate utilization test
CLSI Clinical laboratory standards institute
CoNS Coagulase Negative Staphylococcus
DNA Deoxyribonucleic Acid
DNase Deoxyribonuclease
E. cloacae Enterobacter cloacae
E. coli Escherichia coli
EONS Early-onset neonatal septicemia
ESBL Extended spectrum beta lactamase
FDA Food and drug Administration
GBS Group B streptococci
GEL Gelatin liquefaction test
GLU Glucose fermentation test
GNR Gram-negative rods
H2S Hydrogen sulfide
H2SO4 Sulfuric acid
ICU Intensive care unit
IND Indole test
vii
K. pneumoniae Klebsiella pneumoniae
LBs Live births
LDC Lysine decarboxylase
LONS Late-onset neonatal septicemia
MBL Metallo-β-lactamase
MDR Multidrug resistant
MDR Multi-drug resistance
MEM Meropenem
MHA Mueller-Hinton agar
MRCoNS Methicillin Resistant Coagulase Negative Staphylococci
MRSA Methicillin-Resistant Staphylococcus aureus
n Number of isolates
NaCl Sodium chloride
NICU Neonatal Intensive Care Unit
NS Neonatal septicemia
ODC Ornithine decarboxylase
P. aeruginosa Pseudomonas aeruginosa
P. penneri Proteus penneri
PBP Penicillin binding protein
PMS Poly-microbial septicemia
S. aureus Staphylococci aureus
S. epidemidis Staphylococcus epidemidis
S. paucimobilis Sphingomonas paucimobilis
Spp Species
viii
SPS Sodium Polyanetholsulfonate
SPSS Statistical package for social sciences
SXT Co-trimoxazole
URE Urease test
USA United State of America
v/v volume / volume
VP Vogues-Proskauer test
WHO World Health Organization
ix
TABLE OF CONTENTS
Certificate iv
Acknowledgements v
List of abbreviations vi
Table of Contents ix
List of Tables and figures x
List of Appendices xi
Summary xii
Introduction & Literature Review 1
Materials & Methods 12
Results 24
Discussion 39
Conclusion 45
Recommendations 46
Appendices 47
References 60
x
LIST OF TABLES AND FIGURES
Table I: Frequency of blood isolates (n=93) from neonatal septicemia. 26
Table II: Poly-microbial organisms in neonatal septicemia: combination
of two isolates. 27
Table III: Percent resistant pattern of Gram-positive micro-organisms. 28
Table IV: Percent resistant pattern of Gram-negative micro-organisms. 30
Table V: Total no of ESBL, carbapenem and MBL producing micro-organisms. 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. 32
Figure 4: Demonstration of ESBL phenomenon. 33
Figure 5: Percent of carbapenem susceptibility. 34
Figure 6: Percent of MBL and Non-MBL producers. 35
Figure 7: Demonstration of MHT. 36
Figure 8: Disc Potentiation Test for MBL. 37
xi
LIST OF APPENDICES
Appendix A: Blood Collection Technique 47
Appendix B: Diagrammatic representation of principle of BACTEC 9120 48
Appendix C: Antibiotic disks and their contents 49
Appendix D: Gram stain procedure 50
Appendix E: Catalase test procedure 51
Appendix F: Slide Coagulase test procedure 52
Appendix G: Tube Coagulase test procedure 53
Appendix H: DNase test procedure 54
Appendix I: Cytochrome oxidase test procedure 55
Appendix J: API 20E prcedure 56
Appendix K: API 20-NE procedure 58
Appendix L: 0.5 Turbidity standard preparation 59
xii
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 amongst carbapenemase and MBL producers. Alarmingly high
antimicrobial resistance particularly pan-resistance was observed.
1
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. Early-
onset 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 South-
Central 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
2
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 4–56% 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
3
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 Gram-
positive 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
4
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 27, 28. WHO estimates, that 8.7% of all hospital
patients have nosocomial infections 29. 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
5
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 micro-
organism from a single blood culture specimen at a given time 33. PMS is relatively
common in hospitalized neonates ranging 3 to 10% in NICU in USA 34. 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 β-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 β-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
6
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 ≥ 4µg/ml
and ≥ 0.5µg/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
7
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 β-lactamases, encoded by either plasmid or
chromosome 52. Chromosomally mediated β-lactamases are possessed by many genera of
GNR 53. In 1960s, among GNR first plasmid-mediated β-lactamase in TEM-1 was
reported. TEM-1 β-lactamase spread quickly throughout the world and are now found in
many different species of the family Enterobacteriaceae, P. aeruginosa, Haemophilus
8
influenzae, and Neisseria gonorrhoeae. The other common plasmid mediated β-lactamase
found in K. pneumoniae and E. coli is SHV-1. The SHV-1 β-lactamase is chromosomally
encoded in the majority of the isolates of K. pneumoniae but is usually plasmid mediated
in E. coli 53.
β-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 β-lactamases into four major classes
(A to D) on the basis of amino acids homology. In this scheme, β-lactamases of classes
A, C, and D are serine β-lactamases, whereas, the class B enzymes are Metallo-ß-
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 β-lactamases into functional groups. There are four main
groups and multiple subgroups in this system. Extended spectrum β-lactamases (ESBLs)
are placed in group 2be in this scheme 54, 55.
ESBLs are β-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 β-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 TEM-
types β-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.
9
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-β-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 β-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 β-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 β-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
10
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 and
erythromycin were 89%, 85%, 75%, 71% and 64% respectively 18. 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
NS Hubli Hospital, Karnataka India 65. Moniri et al (2005) documented that MDR P.
aeruginosa accounted for 74% of all isolates from NS in Beheshti Hospital, Iran 66. A 5
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
11
(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.
12
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-
13
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
14
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 35˚C 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 70˚C 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
15
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. Gram stain
2. Catalase test
3. Coagulase test (Slide and Tube)
4. DNase test
5. Phenotypic detection of Methicillin Resistance
6. Cytochrome oxidase test
7. 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
16
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 µg 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
17
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.
18
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 35°C 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.
19
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 µg), ceftazidime 30 µg, cefuroxime 30 µg,
ceftriaxone 30 µg, and cefepime 30 µg, 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.
20
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 co-
amoxiclav were confirmed as ESBL producers. While no increase in the zone indicates
ESBL negative isolates 87.
Quality Control
a) Positive Control K. pneumoniae (Known ESBL Positive)
b) Negative Control 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. Diluted the 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of saline.
3. The 1:10 dilution of E. coli (ATCC 25922) was streak on MHA plate and
allowed to dry 3-5 minutes.
4. Meropenem (10µg) disk was placed in the centre of the test area.
21
5. The organism was streaked in straight line from edge of organism to the edge
of plate.
6. Plate was incubated at 35°C 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 E. cloacae (Known carbapenemase producer)
b) Negative Control S. paucimobilis (Known carbapenemase negative)
METALLO- β –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. The pH of the solution was adjusted to 8.0 by adding NaOH to the solution.
3. The solution was sterilized by autoclaving.
22
4. Two of each imipenem (10 µg) and meropenem (10 µg) disks (Oxoid) were placed
on MHA.
5. To one of each of imipenem and meropenem disks, 5 µl of 0.5 M EDTA (which is
equal to 750 µg EDTA) solution was added.
6. Plate was incubated at 35°C 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 E. cloacae (Known MBL Positive)
b) Negative Control E. cloacae (Known MBL Negative)
SERINE CARBAPENEMASE DETECTION
Principle
These types of enzymes have serine at their active site. Like other serine β-
lactamases these are also inhibited by β-lactam inhibitors (Clavulanic Acid).
Procedure
1. A 1000µg/ml clavulanic acid solution was prepared by dissolving 1 g of clavulanic
acid in 1000 ml of water.
2. The solution was made under strict aseptic conditions.
3. Two 10 µg imipenem disks (Oxoid) were placed on MHA and to one of them 10 µl
of clavulanic acid solution was added.
4. Plate was incubated at 35°C for 18-24 hours
23
Interpretation of Results
Inhibition zones of Imipenem alone and IMP plus clavulanic acid disks will be
read after 18-20 hours incubation at 35°C. The increase in zone size of IMP plus
Clavulanic acid as compared to IMP alone is indicative of Serine carbapenemase
presence 89.
24
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 Gram-
negative 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
25
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.
26
Table 1: Frequency of blood isolates (n=93) from Neonatal Septicemia
Isolates Distribution (n) EONS (n=35) LONS (n=58) Gram-negative rods (n=68)
Klebsiella pneumoniae
40 10 30
Enterobacter cloacae 12 4 8
Shingomonas paucimobilis 5 4 1
Eschirichia coli 3 1 2
Burkholderia cepacia 2 1 1 Acinetobacter baumannii
2 1 1 Klebsiella ornitholytica
1 1 0
Flavomonas oryzihabitans 1 1 0
Proteus penneri 1 0 1
Pseudomonas aeruginosa 1 1 0 Gram-positive cocci (n=22)
Coagulase negative staphylococci
( CoNS) 11 4 7
Staphylococcus aureus 6 3 3
Enterococcus faecalis 3 0 3
viridans streptococci 2 2 0 Fungi (n=01)
Candida albicans 1 1 0
Contaminants (n=02)
Sarcina 1 0 1
Bacillus subtilis 1 1 0
27
Table 2: Poly-microbial micro-organisms in neonatal septicemia: combination of two isolates.
Micro-organisms Associated with micro-organisms (number of cases)
Gram-negative Gram-negative/positive K. pneumoniae E. cloacae (12), E. coli (2), Proteus penneri (1),
Acinetobacter baumannii (1), E. faecalis (1)
Gram-positive S. aureus CoNS (2), Acinetobacter baumannii (1)
viridans streptococci Burkholderia cepacia (1)
Fungi Candida albicans Flavimonas rehabilitans (1)
28
Table 3: Percent resistant pattern of Gram-positive micro-organisms
Antimicrobials coagulase-negative
staphylococci (n=11)
Staphylococcus aureus (n=6)
Enterococcus faecalis (n=3)
viridans streptococci
(n=2)
Penicillin 72.7 83.3 100 100
Ampicillin NT NT 66.6 100
Cefoxitin 54.5 66.6 NT NT
Erythromycin 90.1 66.6 66.6 100
Clindamycin 60 50 NT NT
Amikacin 0 16.6 100 100
Ciprofloxacin 27.3 66.6 100 50
Co-trimoxazole 63.6 66.6 50 100
Vancomycin 0 0 0 0
Linezolid NT 0 0 NT NT: Not Tested
29
Figure 1: Overall percent susceptibility pattern of Gram-positive micro-organisms
18.2 20
41.2
18.2
43.8
72.7
50
35
100 100
58.8
81.8
56.2
27.3
50
65
0 0
81.8 80
0
20
40
60
80
100
Penicillin Ampicillin Cefoxitine Erthromycin Clindamycin Amikacin Ciprofloxacin Co-trimoxazole
Vancomycin linezolid
Sensitive Resistant
30
Table 4: Percent resistant pattern of Gram-negative microorganisms
NT: Not Tested
Antimicrobials K.
pneumoniae (n=40)
E. cloacae (n=12)
S. paucimobilis
( n=5)
E. coli
(n=3)
B. cepacia (n=2)
A. baumannii
(n=2)
K. ornitholytica
(n=1)
F. oryzihabitans
(n=1)
P. penneri (n=1)
P. aeruginosa
(n=1)
Ampicillin NT 100 100 100 100 100 100 100 100 100
Co-amoxiclave 100 100 100 100 100 50 100 100 100 100
Cefuroxime 100 100 100 100 100 50 100 100 100 100
Ceftriaxone 100 100 100 100 50 50 100 100 100 100
Ceftazidime 100 100 100 100 100 50 NT NT NT 100
Cefepime 40 100 100 100 50 50 100 100 100 100
Amikacin 100 100 0 100 100 50 100 100 100 100
Ciprofloxacin 47.5 100 100 100 0 50 100 0 0 100
Co-trimaxazole 60 100 100 100 0 50 100 0 100 100
Imipenem 0 83 100 0 0 0 0 100 0 100
Meropenem 2.6 83 100 0 0 0 0 100 0 100
31
Figure 2: Overall percent susceptibility pattern of Gram-negative micro-organisms
0 1.5 1.8 2.90
2.9
8.8
38.2
29.4
75 76.6
100 98.5 98.2 97.1 100 97.1
91.2
61.8
70.6
25 23.4
0
20
40
60
80
100
Ampicillin Co-amoxiclave Cefuroxime Ceftriaxone Ceftazidime Cefepime Amikacin Ciprofloxacin Co-trimoxazole Imipenem Meropenem
Sensitive Resistant
32
Figure 3: Percentage of ESBL and non ESBL producers.
33
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.
34
Figure 5: Percentage of carbapenem susceptibility
35
Figure 6: Percentage of MBL producers and Non MBL producers
36
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
37
Figure 8: Disk Potentiation Test for MBL IMP: Imipenem MEM: Meropenem EDTA: Ethylene diamine tetra-acetic acid
IMP+EDTA
IMP MEM
MEM+EDTA
38
Table 5: Total number of ESBL, Carbapenem resistant, Carbapenamase and MBL producing micro-organisms
Micro-organisms n Percentage
Number of ESBL producing micro-organisms
K. pneumoniae 17 18.7
E. coli 1 1.1
Proteus penneri 1 1.1
Number of Carbapenem producing micro-organisms
E. cloacae 10 10.9
S. paucimobilis 5 5.5
K. pneumoniae 1 1.1
Flavimonas oryzihabitans 1 1.1
P. aeruginosa 1 1.1
Number of Carbapenamase and MBL producing micro-organisms E. cloacae 10 55.5
K. pneumoniae 1 5.5
39
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%
40
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
frequent isolates causing EONS and LONS were E. coli and S. aureus 24. Bhutta et al
(1990) have documented that commonest pathogens causing EONS and LONS were
Klebsiella spp and Salmonella paratyphi respectively 23.
41
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 micro-
organisms 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.
42
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, co-
amoxiclave, 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.
43
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
44
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.
45
CONCLUSIONS
1. Alarmingly high antimicrobial resistance pattern particularly pan-resistance was
observed in present study.
2. Exentended spectrum-β-lactamase (ESBL) producers, carbapenemase producers,
Metallo-β-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.
46
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. Trust on microbiology laboratory and blood culture results.
4. Treat sepsis but not colonization
5. Do your best to prevent nosocomial infection, by reinforcing infection control,
particularly hand washing.
6. Establish infections control committee in hospital.
7. Organize monthly meeting and seminar for continuous education of nursery staff.
47
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. Veni-puncture site was selected.
3. The site was disinfecting with 2% povidone iodine in circular fashion outward for a
diameter of 5-6cm.
4. Allowed povidone iodine to dry for 1-3 mints.
5. Draw 1-3 ml blood by using 3 ml syringe.
6. Immediately blood was transferred into BACTEC peds plus/F vials.
7. Mixed blood culture bottle for 8-10 times.
8. After sample collection, veni-puncture site was cleaned with 70% isopropyl alcohol.
9. Label the blood culture blood with patient data.
10. Inoculated BACTEC vials were transported as quickly as possible to the laboratory.
48
Appendix B
Diagrammatic representation of principle of BACTC 9120
49
Appendix C
Antibiotic disks and their contents
Sr. No. Antimicrobial Agent Abbreviation Disk Content
1 Penicillin P 10µg
2 Ampicillin AMP 10µg
3 Cefoxitin FOX 30µg
4 Co-amoxiclav AMC 20µg /10µg
5 Cefrouxime CXM 30µg
6 Ceftriaxone CRO 30µg
7 Ceftazidime CAZ 30µg
8 Cefepime FEP 30µg
9 Ciprofloxacin CIP 5µg
10 Co-trimoxazole SXT 25µg
11 Clindamycin C 2µg
12 Erythromycin E 15µg
13 Amikacin AK 30µg
14 Imipenem IMP 10µg
15 Meropenem MEM 10µg
16 Vancomycin V 30µg
17 Linzolid L 30µg
50
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 Lugol’s iodine
solution for 30 seconds.
3. The iodine solution was poured off and the smear was decolorized with acetone-
iodine 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 : S. aureus (ATCC 25923)
Negative Control: E. coli (ATCC 25922)
51
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 : S. aureus (ATCC 25923)
Negative control : E. faecalis (ATCC 29212)
52
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: S. aureus (ATCC 25923)
Negative coagulase control: S. epidermidis
53
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
35°C 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: S. aureus (ATCC 25923)
Negative coagulase control: S. epidermidis
54
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 35ºC 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 DNase positive strain
No clearing around the colonies DNase negative strain
Controls
Positive DNase control: S. aureus (ATCC 25923)
Negative DNase control: S. epidermidis.
55
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: P. aeruginosa (ATCC 27853)
b) Negative Control: E. coli (ATCC 25922)
56
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 37°C 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
57
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.
58
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 µl 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 37°C 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.
59
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.
60
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 screw-
cap 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.
61
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PATIENT PERFORMA
75
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 FR OM 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: ________________________
76
