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ORIGINAL ARTICLE
Year : 2002  |  Volume : 20  |  Issue : 2  |  Page : 92--95

Extended spectrum beta lactamase (ESBL) mediated resistance to third generation cephalosporins among klebsiella pneumoniae in Chennai

A Subha, S Ananthan 
 Dept. of Microbiology, Dr. ALM PGIBMS, University of Madras, Taramani, Chennai- 600 113, India

Correspondence Address:
S Ananthan
Dept. of Microbiology, Dr. ALM PGIBMS, University of Madras, Taramani, Chennai- 600 113
India

Abstract

PURPOSE: To examine the incidence of extended spectrum b lactamase (ESbL) producing strains and multidrug resistant strains of Klebsiella pneumoniae isolated from children between 0-5 years of age. METHODS: Multidrug resistance and ESbL production was studied in a total of 120 isolates of Klebsiella pneumoniae obtained from patients aged 0-5 years. RESULTS: 95% of the isolates showed resistance or decreased susceptibility to atleast one of the three third generation cephalosporins [3GC (ceftazidime, cefotaxime, ceftriaxone)] used for the study. 87% of the isolates showed resistance to all the three 3GC antibiotics and this resistance to all the three 3GC was found to coexist with resistance to other antibiotics. All the isolates were found sensitive to the antibiotic imipenem. ESbL production was detected in 8 strains of Klebsiella pneumoniae. The ESbL activity could be experimentally transferred to recipient E.coli (K12 J62-2). Resistance to b-lactam antibiotics was co-transferred with resistance to gentamicin. CONCLUSIONS: This study has shown the incidence of ESbL producing Klebsiella pneumoniae strains among children in Chennai. Tests for the detection of ESbL producing Klebsiella strains should be carried out in all diagnostic centers routinely and the therapeutic use of all the 3GC should be avoided against Klebsiella strains that appear resistant to any third generation antibiotic.

How to cite this article:
Subha A, Ananthan S. Extended spectrum beta lactamase (ESBL) mediated resistance to third generation cephalosporins among klebsiella pneumoniae in Chennai.Indian J Med Microbiol 2002;20:92-95

How to cite this URL:
Subha A, Ananthan S. Extended spectrum beta lactamase (ESBL) mediated resistance to third generation cephalosporins among klebsiella pneumoniae in Chennai. Indian J Med Microbiol [serial online] 2002 [cited 2019 Oct 22 ];20:92-95
Available from: http://www.ijmm.org/text.asp?2002/20/2/92/8355

Full Text

Klebsiella pneumoniae is a successful opportunistic pathogen and has been associated with various ailments such as urinary tract infections, septicaemia, respiratory tract infections and diarrhoea.[1] Resistance of this species to third generation cephalosporins such as oxyimino b-lactams was first described in 1980 and since then a linear increase in resistance has occurred. The resistant strains gain their resistance by producing Extended-spectrum b-lactamases (ESbLs) which are class A enzymes. ESbLs are the derivatives of common b-lactamases (TEM and SHV b-lactamases) that have undergone one or more amino acid substitutions near the active site of the enzyme, thus increasing their affinity and the hydrolytic activity against third generation cephalosporins and monobactams. Extensive use of newer generation cephalosporins has been the strong factor for the evolution of newer b-lactamases such as ESbLs. ESbLs are encoded by transferable conjugative plasmids, which often code resistance determinants to other antimicrobial agents such as aminoglycosides. These conjugative plasmids are responsible for the dissemination of resistance to other members of gram negative bacteria in hospitals and in the community.[2],[3],[4],[5]

ESbL are distinguished into more than 30 types based on their physical properties and all are inhibited by clavulanate, sulbactam and tazobactam, a property which has been used to detect them in the laboratory.[6]

ESbLs are more prevalent in Klebsiella pneumoniae than in any other enterobacterial species, and outbreaks of infections caused by ESbL producing strains have been reported widely. ESbL producing strains are probably more prevalent than currently recognized because they are often undetected by routine susceptibility testing methods. Occurrence of ESbL producing Klebsiella spp. has been also reported from south India[7] and central India.[8] Recent reports have highlighted the emergence of ESbL producing strains endowed with an extremely wide spectrum of antibiotic resistance, including resistance to trimethoprim, amikacin, streptomycin and gentamicin.[9] Due to the extensive spread of multidrug resistant ESbL producing strains, there has been renewed interest in Klebsiella infections.

The present study was conducted with an objective to examine the incidence of ESbL producing strains and multidrug resistant strains of Klebsiella pneumoniae recovered from children between 0 and 5yrs of age, as this age group is more susceptible to intestinal and extraintestinal infections. Moreover, there are very few reports detailing the evidence of ESbL producing strains in this age group. Transmissibility of drug resistance and ESbL production was also studied, since Klebsiellae are considered as important source of transferable drug resistance among different species of enterobacteria.[10]

 Materials and Methods



Clinical Isolates:

120 clinical isolates of Klebsiella pneumoniae inclusive of 12 isolates from cases of septicaemia, 78 from urinary tract infections, 24 from acute diarrhoeal cases and 4 from respiratory tract infections were obtained from patients aged 0-5 years and attending Institute of Child Health and Hospital for Children, Chennai, from October 1999 to June 2000. Klebsiella isolates that were obtained as a pure and predominant growth from the clinical specimens were only considered for the present study. The organisms were identified based on colony morphology and biochemical reactions.[11]

Antimicrobial Susceptibility testing:

The sensitivity of the isolates to Third Generation Cephalosporins (3GC) viz., ceftazidime, cefotaxime, ceftriaxone, each 30 mg/disc and to the other antibiotics such as amikacin (30 mg), ampicillin (10 mg), cefuroxime (30 mg), gentamycin (10 mg), co-trimoxazole (25 mg), cephaloridine (30 mg), tetracycline (30 mg), imipenem (30 mg), (Hi-Media, India) was determined by the disc diffusion method.[12] The results were interpreted as per National Committee for Clinical Laboratory Standards (NCCLS) recommendations.[13] Escherichia coli ATCC 25922 strain was used for quality control. Isolates with resistance or with decreased susceptibility (intermediate by NCCLS criteria) to any of the 3GC were selected for further study.

ESbL detection by Double Disc Diffusion Synergy Test (DDST):

In the DDST, synergy was determined between a disc of augmentin (20 mg amoxycillin and 10 mg clavulanic acid) and a 30 mg disc of each 3GC test antibiotic placed at a distance of 30 mm apart on a lawn culture of the resistant isolate under test on Mueller-Hinton Agar (MHA, Hi-Media).[14] The test organism was considered to produce ESbL, if the zone size around the test antibiotic disc increased towards the augmentin disc. This increase occurs because the clavulanic acid present in the augmentin disc inactivates the ESbL produced by the test organism.

Since all the 120 isolates were found to be resistant to atleast one of the three 3GC test antibiotics they were tested for ESbL production by DDST.

Transfer of 3GC resistance and ESbL production:

Conjugal transfer of 3GC resistant strains and ESbL producing strains was done at 370C using E. coli K12 J62-2 (obtained from Christian Medical College and Hospital, Vellore) as recipient. Transconjugants were selected on Mueller-Hinton Agar plates containing ceftazidime (2 mg/mL) and rifampicin (2.5mg/mL).[7]

 Results



All the 120 isolates were found to be resistant to a minimum of 3 antibiotics to which they were tested. Hence all the isolates were considered to be multidrug resistant. 95% of the isolates showed resistance or decreased susceptibility to atleast one of the three 3GC used for the study. 87% of the isolates showed resistance to all the three 3GC antibiotics (ceftazidime, cefotaxime, ceftriaxone) and this resistance to all the three 3GC was found to coexist with resistance to other antibiotics. All the isolates were found sensitive to the antibiotic imipenem. [Table:1] shows the antibiotic resistance pattern of the K. pneumoniae isolates. ESbL production against the antibiotics ceftazidime, cefotaxime, ceftriaxone was detected in 8 strains of Klebsiella pneumoniae (4 from urine, 2 from blood and 2 from stool specimens). Resistance to the 3GC antibiotics and ESbL production was transferred to the recipient E. coli strain from all our eight ESbL positive isolates indicating plasmid mediated ESbL production in our isolates. Resistance to Gentamicin was found co-transferred with resistance to other b-lactam antibiotics.

 Discussion



ESbL mediated resistance to 3GC was found in 6.6% of our isolates. This prevalence rate is much lower than other reports from India and abroad. Since the isolates were obtained from infections in children there might be a wide disparity in the prevalence rate of ESbL positive K. pneumoniae when compared to other reports. However, the prevalence of ESbL producing strains of K. pneumoniae and other members of enterobacteria have to be carefully monitored in children to prevent misuse and overuse of antibiotics (3GC and aminoglycosides) because antibiotic exposure could make them more vulnerable to multidrug resistant bacteria. During the past decade, ESbL producing K. pneumoniae have emerged as one of the major multidrug resistant organisms.[15] The incidence of ESbL-producing Klebsiella isolates in the United States has been reported to be 5%.[16] In France and England 14 to 16% ESbL producers among clinical Klebsiella isolates has been reported.[17] In particular regions or hospitals, the incidence can reach 25 to 40%.[18] In a previous study in central India, 76.5% of Klebsiella isolates resistant to 3GC antibiotics was found to produce ESbLs by DDST.[7] However, the percentage of 3GC resistant strains may be much higher, because the conventional disc diffusion criteria used in the routine laboratory underestimate the incidence of these isolates.

The incidence of ESbL -producing strains among clinical Klebsiella isolates has been steadily increasing over the past years and accounts for 6 to 17% of all nosocomial urinary tract infections. The detection rate of ESbL producing Klebsiella isolates in stool samples ranges from 5 to 38%, while rates in the nasopharynx range from 1 to 6%.[1] In our study 8.2% of the isolates obtained from stool, 4% of the isolates obtained from urine and 16.4% of isolates from blood were found to be ESbL producers. No ESbL positive strains were obtained from respiratory tract infections and this could be due to few number of samples taken for the study.

In addition to resistance to 3GC, 75% of the isolates showed resistance to amikacin, 70% to tetracycline, 65% to gentamicin and 65% to co-trimoxazole. In this study resistance to 3GC was found to coexist with resistance to other antibiotics. Since all the isolates showed multidrug resistance, the therapeutic strategies to control infections due to Klebsiella spp. has to be carefully formulated. The therapeutic use of all 3GC should be avoided against Klebsiella spp. that appear resistant to any such compound. Since all the isolates were sensitive to imipenem, it might serve as the drug of choice for the treatment of infections due to ESbL producing K. pneumoniae strains. Our study highlights the emergence of ESbL producing strains endowed with extremely wide spectrum of antibiotic resistance, including resistance to sulfonamides, streptomycin, gentamicin and amikacin.18 The resulting limitations on the therapeutic options demand new measures for the management of Klebsiella infections.

Klebsiella spp. have been an important source of transferable antibiotic resistance. In our study, the 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 that are prevalently located on large conjugative plasmids of 80-160 Kb in size.[1] Since these plasmids are easily transmitted among different members of the enterobacteriaceae, accumulation of resistance genes results in strains that contain multiresistant plasmids. For this reason, ESbL-producing isolates are resistant to a variety of classes of antibiotics.[19] Moreover, the emergence of these multiple resistant Klebsiella strains is unfortunately accompanied by a relatively high stability of the plasmids encoding ESbLs.[1] Conjugative dissemination of ESbL coding plasmids might facilitate the spread of antibiotic resistance among different members of enterobacteria.

Our study has shown the incidence of multidrug resistant and ESbL producing Klebsiella isolates among children in Chennai. Hence it is suggested that, routine diagnosis of ESbL producing strains should be done in hospitals in Chennai. And more importantly, avoiding misuse and overuse of antibiotics may reverse the undesired effects of multidrug resistant and ESbL producing Klebsiella.[1]

 Acknowledgements



We acknowledge the help of Dr. SK Amsavathani, Professor and Head, Department of Microbiology, Institute of Child Health & Hospital for Children, Chennai, in the collection of clinical specimen. The authors gratefully acknowledge Rameshwardas Birla Smarak Kosh, Mumbai, for their financial assistance.

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