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Journal of College of Medical Sciences-Nepal,2010,Vol-6,No-3, 19-23 Study 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 Microbiol 20:92-5.
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 Antimicrob Chemother 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 Med Chem2001; 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.
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