Journal of College of Medical Sciences-Nepal,2010,Vol-6,No-3, 19-23Study of Extended spectrum beta-lactamases (ESBLs) producing Klebsiella species in various clinical specimens: A preliminary report
R.K. Shah1, Y.I. Singh2, R.K. Sanjana 3, Navin Chaudhary 1, Dominic Saldanha 4
1 Lecturer, 2 Prof & Head, 3Asst. Professor Dept. of Microbiology, COMS-TH Bharatpur , 4Associate Professor, Dept. of
Microbiology, Kasturba Medical college Mangalore. Abstract Objective
The present study has been undertaken to detect the presence of ESBLs producing Klebsiella species in
various clinical specimens and their antibiotic susceptibility pattern. Materials and methods
The study consists of 60 clinical isolates of Klebsiella species from various clinical specimens submitted to the
microbiology laboratory, Kasturba Medical College Teaching Hospital Mangalore over a period of one year,
between 1st January 2007 to 31st December, 2007. All isolates were identified morphologically and biochemically
by standard procedures and ESBLs production was detected by re-arranging routine discs in a novel predictor
disc approximation method. Antimicrobial susceptibility was performed using Kirby-Bauer disc-diffusion method
where Imipenem disc, an inducer was placed in center and on either side of it at 15mm distance were placed
ceftazidime and cefotaxime (indicator of induction). In addition, another inducer cefoxitin was placed 15mm from
cefotaxime (indicator). This was placed opposite to that of ceftazidime + clavulanic acid to avoid any affect of
inducible beta-lactamase on the zone of inhibition of the latter.
A total of 16 out of 60 Klebsiella isolates (26.66%) were found to be ESBL producers. Conclusions
Imipenem was found to be the most effective antibiotic (46.66%) followed by Cefoxitin (31.66%) and Cefotaxime
Key words: Klebsiella species, clinical specimens, ESBLs
respiratory tract infection, urinary tract infection, wound
Introduction
infection, bacteremia and diarrhoea.1 The wide spread
use of antibiotics in hospitals have led to emergence of
Enterobacteriaceae, are the successful opportunistic
multi-drugs resistant organisms causing serious
pathogens which have always been associated with
opportunistic infections.2,3 Beta-lactam antibiotics
various clinical ailments mainly in the hospitalized and
(cephalosporins) are the most varied and widely used
immunocompromised patients suffering from lower
agents accounting for 50 % of antibiotics in use.4
The linear increases in resistance to third and
fourth generation cephalosporins are the result of
Journal of College of Medical Sciences-Nepal,2010,Vol-6,No-3
plasmid mediated extended spectrum beta-lactamases
(10µg), Cefotaxime (30µg), Cefoxitin (30µg),
(ESBLs), which are class A enzymes and derivative of
Ceftazidime (30µg), Ceftazidime + Clavulanic acid (30/
beta-lactamases (TEM & SHV beta-lactamases)
10µg), Aztreonam (30µg), and Cefpodoxime (10µg).
which have undergone one or more amino acid
The test inocula were matched with 0.5 Mc Farland
substitutions near the active site of enzyme. 1,4
turbidity standard and lawn cultured onto the Mueller
The ESBLs producing organisms are reported
Hinton agar plates (from Himedia, Mumbai, India).The
worldwide in increasing numbers for which Clinical
disc placement was designed in novel fashion to detect
Laboratory Standard Institute (CLSI) recommends
ESBL production.8,9,10 The discs of ceftazidime and
screening for ESBLs producing Klebsiella species,
ceftazidime + clavulanic acid were kept 15mm apart
where the clinical microbiological tests are used to
from each other (centre to centre). Imipenem disc, an
detect ESBLs and employ a beta-lactamase inhibitor
inducer was placed in center and on either side of it at
usually clavulanate in combination with cephalosporins
15mm distance, ceftazidime and cefotaxime (indicator
(e.g. ceftazidime + clavulanate) in which clavulanate
of induction ) were placed. In addition, another
inhibits ESBLs reducing the level of resistance to
inducer cefoxitin was placed 15mm from cefotaxime
cephalosporins and thereby increasing the zone of
(indicator). This was placed opposite to that of
ceftazidime + clavulanic acid to avoid any affect of
This study was undertaken to determine the
inducible beta lactamase on the zone of inhibition of
detection and prevalence of ESBLs producing
the latter. 8 The remaining discs were placed as shown
Klebsiella species in various clinical specimens received
from Government Wenlock Hospital and Lady
Goschen Hospital Mangalore, by using the combined
disc method, according to guidelines of CLSI . Materials and methods
The study was conducted at the Department of
Microbiology, KMC Mangalore where the various
clinical specimens like urine, sputum, pus, wound swabs,
blood and other body fluids were received from the
Fig:-A. Showing scheme of disc placement to assess
indoor patients of Government Wenlock Hospital and
A total of 280 gram negative bacterial isolates
obtained over a period of one year from 1st January
5. Ceftazidime + Clavulanic acid (30/10µg)
2007 to 31st December 2007 were identified based
on standard microbiological methods.6 Among these,
The agar plates were incubated at 37ÚC for 18 to 24
60 isolates were identified as Klebsiella species and
antimicrobial susceptibility was performed by using
The following criteria have been used to decide
Kirby-Bauer disc- diffusion method, as per NCCLS
organisms to be ESBL producers. 11, 12, 13
guidelines,7 where the following antibiotics (from
Himedia, Mumbai, India) were taken: Imipenem
R.K. Shah et al, Study of Extended spectrum beta-lactamases (ESBLs).
Zone diameter of various third generation
Two hundred and eighty gram negative bacterial
cephalosporins like Aztreonam(30µg) d" 27mm,
isolates obtained from various clinical specimens such
Cefotaxime(30µg) d" 27mm, Cefpodoxime(10µg) d"
as urine, sputum, pus, wound swabs, blood and other
body fluids were received from Govt. Wenlock
Hospital and Lady Goshen Hospital, Mangalore. Out
Increase in zone size with addition of an inhibitor
of these sixty (21.42%) clinical isolates of Klebsiella
by e" 5mm when tested in combination with an
species were detected for the presence of ESBLs
inhibitor clavulanic acid versus zone diameter when
whereas sixteen (26.66%) isolates of Klebsiella
species were found to be ESBL producers.
Among the antibiotics tested, imipenem was found
to be the most effective (46.66%), followed by cefoxitin
(31.66%) and cefotaxime (30.00%Figure B: Table-1 Antibiotic susceptibility of Klebsiella species Sensitive Intermediate Resistant Antibiotics Table 2: Age distribution of patients in whom Table 3: Sex distribution of patients in whom ESBL isolates were detected. ESBLs detected. Age group Journal of College of Medical Sciences-Nepal,2010,Vol-6,No-3
spectrum cephalosporins be taken resistant in ESBL
producers. Thirdly, institutional outbreaks are increasing
because of selective pressure due to the heavy use of
expanded-spectrum cephalosporins and also due to
lapses in effective infection control measures.8,9,10
In our study, sixteen ESBLs producing Klebsiella
isolates (26.66%) were detected. However, studies
by Jain et al14 and Babypadmini et al15 showed 86.6%
and 40% of Klebsiella spp to be ESBL-producers
respectively. In our study Imipenem showed the highest
level of sensitivity (46.66%) against Klebsiella including
ESBL producers. Our study differs from the studies of
Subha et al and Rodrigues et al.1,8 Studies by Al-Zahrani
pneumoniae to be having the highest susceptibility to
Meropenem (94.4%).Carbapenems appear to be the
Antibiotic susceptibility of Klebsiella spp to detect
drug of choice for serious infections with ESBL
ESBL production.
producing organisms as recommended earlier.17
However, these should not be administered as empirical
Discussion
therapy for gram negative infections that are not life-
Beta lactamases continue to be the leading cause
threatening because their over-use can pose a significant
of resistance to beta-lactam antibiotics in gram negative
problem.18 Cefoxitin (31.66 %) and cefotaxime (30.00
bacilli. In recent years there has been an increased
%) were the other antibiotics which were found to be
incidence and prevalence of ESBLs that hydrolyze
sensitive against Klebsiella isolates.
and cause resistance to oxymino-cephalosporins and
This was marginally higher than that reported in
aztreonam.13 For a number of reasons, the detection
studies by Dutta et al5 but substantially lower in studies
of ESBL-producing strains is of significant importance
for all major hospitals worldwide. First, these strains
are most likely to be even more prevalent than it is
Conclusions
currently recognized. Due to the difficulty in their
Klebsiella isolates have been steadily increasing
detection by the current clinical methods, many of
over the past years and they have been important
these strains have been reported to be susceptible to
sources of transferable antibiotic resistance.
widely used and tested broad-spectrum ß-lactams.
Secondly, ESBLs constitute a serious threat to current
cephalosporins to treat gram negative bacterial
ß-lactam therapy. Treatment of ESBL infection is
infections is partly responsible for the emergence of
difficult as the CLSI recommends that all expanded-
resistance to beta-lactam antibiotics. Strict adherence
R.K. Shah Study et al, of Extended spectrum beta-lactamases (ESBLs).
to the hospital antibiotic policy and good infection
J.M.Blondeau Extended spectrum beta-lactamases.
Seminars in Respiratory Infections 2001;16(3):169-76.
control practices can play a significant role in reducing
10. Guidelines on susceptibility of antibiotic resistant
Enterobacteriaceae due to extended spectrum beta-
lactamases (ESBLs). Canadian External Quality
References
Assurance Advisory Groups on Antimicrobial
Resistance (CEQA - AGAR) and Bureau of Microbiology,
A. Subha , S. Ananthan Extended spectrum beta-
lactamase (ESBL) mediated resistance to third
generation cephalosporins among Klebsiella
11. V.A. Moritz , P.B.D.Carson Cefoxitin sensitivity as a
pneumoniae in Chennai. Ind. J Med Microbiol 2002;
marker for inducible beta-lactamases. J Med Microbiol20:92-5.
1986;21:203-7.
I. Shukla, R. Tiwari, M. Agrawal. Prevalence of extended
12. G. Revathi, S. Singh Detection of expanded spectrum
spectrum -lactamase producing Klebsiella pneumoniae
cephalosporin resistance due to inducible lactamases in
in a tertiary care hospital. Ind. J Med Microbiol 2004;
hospital isolates. Ind. J Med Microbiol 1997;15(3):113-5. 22(2):87-91
13. P.A.Wayne . National Committee for Clinical Laboratory
A. Palucha ,B. Mikiewiez , W. Hrynieeiez , et al.
Standards. Performance standards for antimicrobial
Concurrent outbreaks of extended spectrum beta-
susceptibility testing. Twelfth informational supplement.
lactamase producing organisms of the family
M100 -S12 NCCL. Oxford university press, USA 2002.
Enterobacteriaceae in a Warsaw Hospital. J AntimicrobChemother 1999;44:489-99.
14. A. Jain , I. Roy , M.K. Gupta et al. Prevalence of ESBL-
producing gram negative bacteria in septicaemic
J.J.Bronson , J.F. Barrett . Quinolone, Everninomycin ,
neonates in a tertiary care hospital. J Med Microbiol
Glycylcycline, Carbapenem, Lipopeptide and Cephem
2003; 52:421-5
Antibacterials in clinical development. Curr MedChem2001; 8:1697-704.
15. S. Babypadmini, B.Appalaraju. ESBLs in urinary isolates
of E.coli and Klebsiella pneumoniae – prevalence and
P.Datta , A. Thakur , B. Mishra . Prevalence of Clinical
susceptibility pattern in a tertiary care hospital. Ind J
strains resistant to various beta-lactams in a tertiary
Med Microbiol 2004; 22:172-4.
care hospital in India. Ind. J Med Microbiol 2004; 57:
16. A.J.Al-Zahrani , N. Akhtar. Susceptibility Patterns of
ESBL- producing E.coli and Klebsiella pneumoniae
M.S.Kumar, V. Laxmi, R. Rajagopalan . Occurrence of
isolated in a teaching hospital. Pakistan J Med Res 2005;
44(2):64-7.
Enterobacteriaceae spp. isolated at a tertiary care
hospital. Ind. J Med Microbiol 2006; 24:208-11.
17. B.N.Kim , J.H.Woo , Mn.Kim et al. Clinical implications
of ESBL-producing Klebsiella pneumoniae bacteraemia.
P.A. Villanova, National committee for Clinical Laboratory
J Hosp Infect 2002; 52:99-106.
Standards. Performance standards for antimicrobial disc
susceptibility test, 5th ed. ASM press, Washingoton DC
18. A. Wong-Beringer Therapeutic challenges associated
with ESBL-producing E.coli and Klebsiella pneumoniae.
Pharmacotherapy 2001; 21:583-92.
C. Rodrigues , P. Joshi , S.H. Jani, et al. Detection of
beta-lactamases in nosocomial gram negative clinical
19. S. Shivaprakasha, K. Radhakrishnan, A.R.Gireesh
isolates. Ind. J Med Microbiol 2004; 22:247- 50.
Routine screening for ESBL production, a necessity of
today. Internet Journal of Microbiol 2007; 3: 1.
Medicines in tap water not as risky as some say By GORDON DILLOW Chances are I'm taking a lot of pharmaceutical medications. I may be taking acetaminophen and ibuprofen and Valium and Xanax. I may be taking antihistamines and antibiotics and anti-convulsants and anti-cholesterols. I may be taking the three major male anti-impotence drugs, Viagra, Cialis and Levitra, which should have me r
Printed copies of this document may not be up to date, obtain the most recent version from www.cats.nhs.uk Transport Service Traumatic Brain Injury Document Control Information Author Position Document Owner Document Owner Position Document Version Replaces Version First Introduced Review Schedule Active Date Next Review CATS Document
Journal of College of Medical Sciences-Nepal,2010,Vol-6,No-3
spectrum cephalosporins be taken resistant in ESBL
producers. Thirdly, institutional outbreaks are increasing
because of selective pressure due to the heavy use of
expanded-spectrum cephalosporins and also due to
lapses in effective infection control measures.8,9,10
In our study, sixteen ESBLs producing Klebsiella
isolates (26.66%) were detected. However, studies
by Jain et al14 and Babypadmini et al15 showed 86.6%
and 40% of Klebsiella spp to be ESBL-producers
respectively. In our study Imipenem showed the highest
level of sensitivity (46.66%) against Klebsiella including
ESBL producers. Our study differs from the studies of
Subha et al and Rodrigues et al.1,8 Studies by Al-Zahrani
pneumoniae to be having the highest susceptibility to
Meropenem (94.4%).Carbapenems appear to be the
Antibiotic susceptibility of Klebsiella spp to detect