|Year : 2004 | Volume
| Issue : 2 | Page : 87-91
Prevalence of extended spectrum -lactamase producing klebsiella pneumoniae in a tertiary care hospital
I Shukla, R Tiwari, M Agrawal
Department of Microbiology, JN Medical College, Aligarh Muslim University, Aligarh - 202 002, Uttar Pradesh, India
Department of Microbiology, JN Medical College, Aligarh Muslim University, Aligarh - 202 002, Uttar Pradesh, India
PURPOSE: The purpose of this study was to know prevalence of extended spectrum -lactamase (ESBL) in multi drug resistant (MDR) strains of Klebsiella pneumoniae isolated from different clinical samples. METHODS: A total of 120 MDR strain of K. pneumoniae were selected for the study, 106 of which were resistant to atleast one of the third generation cephalosporins (3GC). They were studied for ESBL production by phenotypic confirmatory disc diffusion test (PCDDT) and by double disc synergy test (DDST). RESULTS: 88.3% (106) of the isolates were found to be resistant to atleast one of the 3GC tested (cefotaxime, ceftazidime and ceftriaxone) and 72% of the isolates were resistant to all the 3GC tested. ESBL was detected in 30.18% (32) of the K. pneumoniae by PCDDT and in 27.3% (29) by DDST. Among the ESBL producers 6 (18.75%) were sensitive to cefotaxime, 2 (6.25%) to ceftazidime and 3 (9.37%) to ceftriaxone by disc diffusion test. The minimum inhibitory concentrations (MICs) of 3GC for these strains ranged from 2-8 µg/mL while for non ESBL producer sensitive counterparts it ranged from 0.03-1 µg/mL. Resistance to cefotaxime was transferred to recipient E. coli K12 strains J62-1. All the K. pneumoniae isolates were sensitive to imipenem. Resistance against amoxicillin, gentamicin, ciprofloxacin and amikacin was found in 93.28, 70, 10.37 and 26.14% of the isolates respectively. CONCLUSIONS: Our study shows presence of ESBL producer K. pneumoniae in clinical isolates. The routine antimicrobial sensitivity test may fail to detect ESBL mediated resistance against 3GC and detection of ESBL production should be carried out as a routine in diagnostic laboratories by PCDDT as it is a simple and cost effective test.
|How to cite this article:|
Shukla I, Tiwari R, Agrawal M. Prevalence of extended spectrum -lactamase producing klebsiella pneumoniae in a tertiary care hospital. Indian J Med Microbiol 2004;22:87-91
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Shukla I, Tiwari R, Agrawal M. Prevalence of extended spectrum -lactamase producing klebsiella pneumoniae in a tertiary care hospital. Indian J Med Microbiol [serial online] 2004 [cited 2014 Mar 11];22:87-91. Available from: http://www.ijmm.org/text.asp?2004/22/2/87/8078
The wide spread use of antibiotics in hospitals has led to emergence of multidrug resistant organisms of low virulence like Klebsiella causing serious opportunistic infections. Over the last 15 years numerous outbreaks of infection with organisms producing extended spectrum b-lactamases (ESBLs) have been observed world wide. The advent of ESBL producers has posed a great threat to the use of many classes of antibiotics particularly cephalosporins. There are indications that poor outcome occurs when patients with serious infections due to ESBL producing organisms are treated with antibiotics to which the organism is resistant.
ESBL producing Klebsiella pneumoniae were first reported in 1983 from Germany and since then a steady increase in resistance against cephalosporins has been seen. ESBLs are encoded by transferable conjugative plasmids which also quite often code resistant determinants to other antibiotics. An ESBL variant may be selected de novo in a given hospital or it may be introduced from another centre. Its further spread within the hospital can be consequence of plasmid transmission. Persistence and outbreaks of ESBL producers have been convincingly correlated with extensive use of cephalosporins. The plasmid mediated resistance against cephalosporins can be spread among related and unrelated gram negative bacteria. K. pneumoniae is an important cause of nosocomial infection and infections due to ESBL producing K. pneumoniae are of concern as third generation cephalosporins (3GC) are commonly used for treatment of infections due to gram negative organisms. These infections are difficult to control as they are usually associated with resistance to aminoglycosides. This study was undertaken to determine sensitivity of K. pneumoniae to various 3GC and to know the prevalence of ESBL producing K. pneumoniae strains in clinical samples.
| ~ Materials and Methods|| |
A total of 120 MDR clinical isolates of K. pneumoniae obtained from July 2001 to July 2002 were included in the study. These isolates include 50 cases from septicaemia, 34 from urinary tract infection and 36 from wound infection. The isolates were identified based on colony morphology on blood agar, MacConkey agar and by standard biochemical tests.
Antimicrobial susceptibility testing
The antibiotic sensitivity test was performed by disc diffusion technique with commercially available discs (Hi Media, Mumbai, India) on Mueller Hinton agar plates. The discs used were gentamicin (10 µg), ciprofloxacin (5 µg), cefazolin (30 µg), imipenem (10 µg), amoxycillin (10 µg) and amikacin (30 µg). For sensitivity to 3GC cefotaxime, ceftriaxone and ceftazidime each 30 µg disc was used. The diameter of the zone of inhibition for each antibiotic was measured and interpreted as resistant, intermediate susceptible or susceptible according to NCCLS criteria.
MIC of ESBL producing and ESBL nonproducing strains for ceftazidime, cefotaxime and ceftriaxone was determined by agar dilution test using an inoculum size of 105 cfu/mL. Mueller Hinton agar plates were prepared by incorporating serial two fold dilutions of each of the antibiotic and the range tested was 0.031-128 µg/mL of each antibiotic.
Phenotypic confirmatory disc diffusion test (PCDDT) for ESBL
This test requires use of both cefotaxime (30 µg) and ceftazidime (30 µg) disc alone and in combination with clavulanic acid (30 µg). Clavulanic acid was received from Ranbaxy Research Laboratory, New Delhi. Discs of ceftazidime and cefotaxime with clavulanic acid (30 µg/10 µg) were prepared using a stock solution of clavulanic acid at 1000 µg/mL (taken from a small aliquot kept frozen at -70°C). 10 µl of clavulanic acid solution was added to these discs within an hour before these were applied to the plates. An increase in zone diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone was observed. For ceftazidime an increase in zone diameter of > 5mm and for cefotaxime > 3 mm was considered as an ESBL producer.
Double Disc Synergy test (DDST)
DDST was done to determine synergy between a disc of agumentin (20 µg amoxycillin and 10 µg clavulanic acid) and 30 µg disc of each 3GC antibiotics. Mueller Hinton agar plates were prepared and inoculated with standardized inoculum (0.5 McFarland tube) to form a lawn culture. 30 µg disc of each 3GC antibiotics was placed on the agar at a distance of 15 mm centre to centre from augmentin disc. E. coli ATCC 25922 was used as the negative control and in house ESBL producer was used as the positive control. ESBL production was interpreted if the inhibition zone around the test antibiotic disc increased towards the agumentin disc or if neither discs were inhibitory alone but bacterial growth was inhibited where the two antibiotics diffuse together.
Transfer of 3GC resistance and ESBL production
Conjugal transfer of 3GC resistant ESBL producing strains was done at 37°C using E. coli K-12 strain J-62-1 (lac+F-nal r) as recipient. Equal volumes (1 mL) of culture of the donor and the recipient strain (109 cfu/mL) grown with agitation in tryptic soya broth were mixed and incubated statically for 18 hours at 35°C. Transconjugants were selected on MacConkey's agar containing 64-µg/mL nalidixic acid to inhibit the growth of donor and 2.5 µg/mL cefotaxime to inhibit the growth of recipient strain.
| ~ Results|| |
In the present study, 120 isolates of K. pneumoniae were included which were resistant to at least three antibiotics tested. 88.3% of the isolates were moderately sensitive or resistant to any one of the three 3GC and were studied for ESBL production by PCDDT and DDST. 72% of the isolates included in the study were resistant to all the three 3GC antibiotics. All the strains were sensitive to imipenem but among the non b lactam antibiotics ciprofloxacin and amikacin were most effective drugs and 89.63 and 73.86% of the strains were sensitive to these drugs.
ESBL production was detected in 32 (30.18%) isolates by phenotypic confirmation disc diffusion test as the zone of inhibition increased in presence of clavulanic acid. MIC of 3GC test antibiotics against ESBL producers ranged between 2 to >128 µg/mL and for non ESBL producers it was 0.03 to 64 µg/mL. The percentage of ESBL producing isolates which were susceptible (S), intermediate (I) or resistant (R) to 3GC by disc diffusion test (NCCLS criteria) are shown in table. Among the ESBL producers 18.75% isolates for cefotaxime, 6.25% for ceftazidime and 9.3% for ceftriaxone were found to be sensitive by disc diffusion test. 36.1% of the isolate from pus, 20.5% from urine and 24% from blood were ESBL producers. The DDST detected ESBLs using these 3GC in 29 (90.6) of the 32 ESBL producing isolates.
| ~ Discussion|| |
Extended spectrum b lactams are commonly included in the empirical antibiotic regimens for treatment of gram negative sepsis. The increasing use of broad spectrum cephalosporins has become one of the major factors responsible for the high rate of selection of extended spectrum beta lactamase producing microorganisms.
NCCLS established break point for expanded spectrum cephalosporin antibiotics shortly after the clinical release of these antibiotics in the early 1980s. MIC of <8 µg/mL correlated with >92% clinical success for all relevant species including K. pneumoniae and E. coli. These break points were established prior to the era of the ESBLs. In the late 1980s and early 1990s it was recognized that MICs of some cephalosporin for isolates producing ESBL may be <8 µg/mL (that is in the susceptible range). Good clinical outcomes were observed when extended spectrum cephalosporins were used to treat conditions like urinary tract infection due to ESBL producing organism but the outcome in serious infections was questionable. However, some authors are of the view that ESBL screening is not likely to affect patient outcome and hence neither clinically necessary nor cost effective for the laboratories. There are published reports that patients had good clinical outcome despite receiving cephalosporins for treatment of infections with ESBL producing organisms and has been an argument against routine screening for ESBL production.
The role of 3GC in the treatment of K. pneumoniae infection is limited as ESBL mediated resistance is on the increase in the last decade and in the present study ESBL mediated resistance against cephalosporins was seen in 30.19% strains of K. pneumoniae. In a study from south India ESBL mediated resistance was 25.8% and in another study a very low prevalence of 6% was reported. This very low prevalence in their study may be due to the fact that isolates were from infection in children and this may be the reason for disparity. However, there are various reports where incidence is reported to be 23%12 in hospital isolates and in 84% of epidemic isolates.
The real challenge is the ESBL producing organism for which MICs of 3GC is in the susceptible range and they may not be truly susceptible when serious infections are considered and these isolates may be reported susceptible. Among the ESBL producers 18.3% of isolates for cefotaxime, 9.37% for ceftazidime and 12.8% for ceftriaxone were in the susceptible range. MICs of these phenotype sensitive ESBL producing strains of 3GC were in the range of 2-8 µg/mL. Although these values were in the susceptible range they were substantially higher than the MICs for non ESBL producer sensitive counterparts (0.03 - 1 µg/mL). Imipenem was highly active against all the ESBL producer strains. Brun Bran et al described a large outbreak of ESBL producing organisms and reported that the cefotaxime and ceftriaxone MICs for some of ESBL producers were 0.5-4 µg/mL.
In view of these facts NCCLS approved the standard methods for dilution in antimicrobial susceptibility tests published in 2000 and recommended that MICs of >2 µg/mL for cefpodoxime, ceftazidime, aztreonam, cefotaxime and ceftriaxone should be regarded as possibly indicating ESBL production. It further suggested that for all ESBL producing strains the test result be reported as resistant for all penicillins, cephalosporins and aztreonam. The laboratories which do not perform tests for detection of ESBLs and do not report ESBL producers as resistant to cephalosporins risk poor outcome. Among the non-blactam antibiotics, which showed higher sensitivity against these ESBL producers, were amikacin and ciprofloxacin. Similar results were reported by Zoltan et al for their patients with serious infections with ESBL producers. But in another study from India 75% of the isolates showed resistance to amikacin. Resistance to 3GC antibiotics and ESBL mediated resistance to cefotaxime was transferable to recipient E. coli from ESBL positive isolates. In our study, resistance to 3GC was transferred to the recipient strain along with resistance to gentamicin and other b lactam antibiotics. ESBL production is coded by genes on conjugation plasmids which are easily transmitted among different members of the Enterobacteriaceae. Usually this plasmid also carries genes coding resistance for other antibiotics which result in strains that are multidrug resistant.
Of the two tests used in the study phenotypic confirmatory disc diffusion test was a more sensitive procedure for detection of ESBL than the DDST. Twenty nine (90.6%) of the 32 ESBL producing strains were detected by DDST using three drugs cefotaxime, ceftriaxone and ceftazidime MAST double disk (MDD) test which use discs containing ceftazidime and a complementary disc containing ceftazidime and clavulanate and a second pair containing cefotaxime and clavulanate was compared with the DDST and was found to be an inexpensive alternative to DDST for detection of ESBL. In another study three dimensional test was found to be more sensitive than DDST. DDST lacks sensitivity because of problem of optimal disc space and correct storage of the clavulanate containing discs. Assuming that a laboratory is currently testing sensitivity for ceftazidime and cefotaxime with disc diffusion and for the phenotypic confirmatory disc diffusion test only two discs are required to be added to the sensitivity plate and would screen all gram negative bacteria in the diagnostic laboratory for ESBL production. This method is technically simple and inexpensive.
Our study shows that ESBL producing K. pneumoniae were isolated in our hospital. The routine antimicrobial sensitivity tests may fail to detect ESBL mediated resistance against 3GC which may lead to treatment failure especially when cephalosporins are used. Since all the isolates were sensitive to imipenem and there were large number of isolates from blood it can be the drug of choice for the treatment of infections due to ESBL producing K. pneumoniae strains in seriously ill patients.
| ~ Acknowledgement|| |
Authors would like to thank Dr. Ashok Rattan, Ranbaxy Research Laboratory, New Delhi, for his help in carrying out this work.
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