Indian Journal of Pathology and Microbiology

: 2008  |  Volume : 51  |  Issue : 1  |  Page : 139--142

Prevalence of extended-spectrum β-lactamases among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital

Parul Agrawal, AN Ghosh, Satish Kumar, B Basu, K Kapila 
 Department of Microbiology, Armed Forces Medical College, Pune, India

Correspondence Address:
Parul Agrawal
Department of Microbiology, Armed Forces Medical College, Pune - 411 040


Extended-spectrum β-lactamases (ESBLs) continue to be a major problem in clinical setups the world over, conferring resistance to the expanded-spectrum cephalosporins. Knowledge about their prevalence is essential to guide towards appropriate antibiotic treatment. The aim of the present study is to determine the prevalence of ESBL producers among Escherichia coli and Klebsiella pneumoniae isolates at a tertiary care institution. A total of 357 clinical isolates comprising E. coli (n = 181) and K. pneumoniae (n = 176) were recovered from various clinical samples over a period of six months from April to September 2006. Antibiogram profile of these isolates was determined to commonly used antibiotics, along with screening for ESBL production by the screening test as recommended by the Clinical Laboratory Standards Institute (CLSI). Isolates which showed positive results with screening test were shortlisted for confirmatory tests of ESBL production. Two tests were performed: phenotypic confirmatory test with combination disk and the minimum inhibitory concentration (MIC) reduction test. Out of 357 isolates of E. coli and K. pneumoniae screened for ESBL production, 120 were found to be potential ESBL producers. Of these, 80 isolates were confirmed to be ESBL producers. Thus the prevalence of ESBL-producing isolates of E. coli and K. pneumoniae was found to be 22% (80 out of 357). This was significantly lower than the data available from other hospitals.

How to cite this article:
Agrawal P, Ghosh A N, Kumar S, Basu B, Kapila K. Prevalence of extended-spectrum β-lactamases among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital.Indian J Pathol Microbiol 2008;51:139-142

How to cite this URL:
Agrawal P, Ghosh A N, Kumar S, Basu B, Kapila K. Prevalence of extended-spectrum β-lactamases among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital. Indian J Pathol Microbiol [serial online] 2008 [cited 2022 Sep 29 ];51:139-142
Available from:

Full Text


Resistant bacteria are emerging worldwide as a threat to favorable outcome in the treatment of common infections in community and hospital settings. [1] Among the wide array of antibiotics, β-lactams are the most widely used agents. The most common cause of resistance to β-lactam antibiotics is the production of β-lactamases. Emergence of resistance to β-lactam antibiotics began even before the first β-lactam, penicillin, was developed. The first plasmid-mediated β-lactamase TEM-1 was originally isolated from blood culture of a patient named Temoniera in Greece, in the early 1960s. [2] TEM-1 being plasmid and transposon mediated has facilitated its spread to other species of bacteria. Another common plasmid-mediated β-lactamase is SHV-1 (sulfhydryl variable), which is chromosomally encoded in the majority of isolates of K. pneumoniae but is usually plasmid-mediated in E. coli . Over the years, many new β-lactam antibiotics have been developed; however, with each new class of antibiotic, a new β-lactamase emerged that caused resistance to that class of drug. Presumably, the selective pressure imposed by the use and overuse of new antibiotics in the treatment of patients has resulted in the emergence for new variants of β-lactamase.

The introduction of third-generation cephalosporins into clinical practice in the early 1980s was heralded as a major breakthrough in the fight against β-lactamase-mediated bacterial resistance to antibiotics. Soon after the introduction, the first report of plasmid-encoded β-lactamase capable of hydrolyzing the extended-spectrum cephalosporins was published in 1983 from Germany. [3] Hence these new β-lactamases were coined as extended-spectrum β-lactamases (ESBLs). The total number of ESBLs characterized exceeds 200 today. There is no consensus on the precise definition of ESBLs. A commonly used working definition is that the ESBLs are β-lactamases capable of conferring bacterial resistance to the penicillins; first-, second- and third-generation cephalosporins; and aztreonam (but not the cephamycins and carbapenems) by hydrolysis of these antibiotics and which are inhibited by β-lactamase inhibitors such as clavulanic acid. [4]

ESBLs are encoded by transferable conjugative plasmids which often code resistant determinants to other antibiotics. The plasmid-mediated resistance against cephalosporins can spread among related and unrelated gram-negative bacteria. ESBLs are mostly the products of point mutations at the active site of TEM and SHV enzymes. [5] Nosocomial outbreaks of infections caused by ESBL-producing gram-negative bacteria have also been reported, which are mainly the result of extensive and inappropriate use of third-generation cephalosporins. Majority of ESBL-producing organisms are E. coli and K. pneumoniae. Others include Enterobacter spp. , Salmonella spp. , Morganella, Proteus mirabilis, Serratia marcescens, and Pseudomonas aeruginosa . The major risk factors implicated are long-term exposure to antibiotics, prolonged ICU stay, nursing home residency, severe illness, instrumentation, or catheterization. [6]

Since ESBL-positive isolates show false susceptibility to expanded-spectrum cephalosporins in standard disk diffusion test, it is difficult to reliably detect ESBL production by the routine disk diffusion techniques. Specific detection methods recommended by CLSI have to be adopted. ESBLs are specifically inhibited by β-lactamase inhibitors like clavulanic acid, and this property is utilized for the detection and confirmation of ESBLs. [7]

Prevalence of ESBLs varies from institute to institute. Previous studies from India have reported ESBL production varying from 6% to 87%. [8],[9],[10],[11],[12] Keeping in view the above facts, this study was undertaken to find the prevalence of ESBL producers among E. coli . and K. pneumoniae isolates at our institute. Moreover, the aim of this study was also to compare the antibiotic susceptibility pattern of ESBL producers with that of non-ESBL producers and to delineate the magnitude of the problem and to define appropriate therapeutic options.

 Materials and Methods

Clinical isolates

A total of 357 consecutive non-repeat culture isolates of Escherichia coli and Klebsiella pneumoniae were obtained from different clinical specimens such as urine, pus, blood, body fluids, etc., over a period of six months (April to September 2006). The isolates were identified on the basis of conventional microbiological procedures. [13]

Antimicrobial susceptibility test

Antimicrobial susceptibility was determined by Kirby-Bauer disk diffusion method as per CLSI recommendations. [14] Antimicrobial disks used were Ampicillin (10 g), Amoxycillin-clavulanic acid (20/10 g), Piperacillin (100 g), Piperacillin-tazobactam (100/10 g), Cephotaxime (30 g), Ceftriaxone (30 g), Ceftazidime (30 g), Gentamicin (10 g), Amikacin (30 g), Netilmicin (30 g), Tetracycline (30 g), Ciprofloxacin (5 g), Chloramphenicol (30 g), Trimethoprim-sulfamethoxazole (1.25/23.75 g), and Imipenem (10 g).

Screening test for ESBLs

According to the CLSI guidelines, isolates showing inhibition zone size of ≤ 22 mm with Ceftazidime (30 g), ≤ 25 mm with Ceftriaxone (30 g), and ≤ 27 mm with Cefotaxime (30 g) were identified as potential ESBL producers and shortlisted for confirmation of ESBL production [Figure 1]

Confirmatory tests for ESBLs

The following two procedures were carried out in the present study as per CLSI guidelines:

Phenotypic confirmatory test with combination disk: This test requires the use of a third-generation cephalosporin antibiotic disk alone and in combination with clavulanic acid. In this study, a disk of Ceftazidime (30g) alone and a disk of Ceftazidime + Clavulanic acid (30 g/10 g) were used. Both the disks were placed at least 25 mm apart, center to center, on a lawn culture of the test isolate on Mueller Hinton Agar (MHA) plate and incubated overnight at 37C. Difference in zone diameters with and without clavulanic acid was measured [Figure 2].

Interpretation: When there is an increase of ≥ 5 mm in inhibition zone diameter around combination disk of Ceftazidime + Clavulanic acid versus the inhibition zone diameter around Ceftazidime disk alone, it confirms ESBL production.

MIC reduction test: The isolates positive with combination disk test were further confirmed for ESBL production by this test. Minimum inhibitory concentration of the isolates was determined by Broth dilution method. The values of range of concentration of antibiotics tested were as follows:

Ceftazidime: 0.25 g/mL to 128 g/mL

Ceftazidime-clavulanic acid: 0.25/4 g/mL to 128/4 g/mL

Interpretation: A ≥ 3 two-fold decrease in MIC for Ceftazidime when tested in combination with clavulanic acid versus its MIC when tested alone indicates that the strain is an ESBL producer.

Control strain used to validate susceptibility tests was E. coli ATCC 25922.

Statistical analysis

Chi-square test was used with appropriate correction for the observation. Where the cell frequency was less than five, Fisher exact test was applied to see the significance of difference between the resistance levels of various drugs in ESBL producer strains and non-ESBL producer strains using EPI 6 software. p ≤ 0.05 was considered significant.


A total of 357 isolates of E. coli ( n = 181) and K. pneumoniae ( n = 176) were recovered from different clinical specimens submitted for routine microbiological analysis from both indoor and outdoor patients of a tertiary care hospital during a six-month period. The number of potential ESBL producers shortlisted by screening test was 120 out of the total 357. All of them showed inhibition zone size of ≤ 22 mm with Ceftazidime during screening test. Confirmatory tests for ESBL production were performed on these 120 isolates.

Out of 120 isolates, 52 E. coli and 28 K. pneumoniae isolates were found to be ESBL producers by phenotypic confirmatory test with combination disk. This was further confirmed by MIC reduction test, and the results of both tests were in concordance [Table 1].

Thus 30% (52/181) of E. coli and 16% (28/176) of K. pneumoniae isolates were found to be ESBL producers. Distribution of these ESBL-positive isolates was highest among urinary isolates, accounting for 70% of all isolates recovered [Table 2].

Antimicrobial susceptibility pattern

The multidrug resistance was significantly higher among ESBL producers than in non-ESBL producers. ESBL producers were almost always resistant to Ampicillin and Piperacillin. However, all the isolates were sensitive to Imipenem. [Table 3] shows the comparison of antibiotic resistance among ESBL producers and non-ESBL producers.


In the present study, the prevalence of ESBL producers was found to be 22% (80 out of 357) among E. coli and K. pneumoniae isolates collected over a period of six months. The overall prevalence of ESBL producers was found to vary greatly in different geographical areas and in different institutes. Previous studies from India have reported ESBL production varying from 6% to 87%. [8],[9],[10],[11],[12] One reason for such variability may be the very low number of samples studied. In recent years, a significant increase in ESBL producers was reported from USA, [15] Canada, [16] China, [17] and Italy. [18] A recent large survey of 1610 Escherichia coli and 785 Klebsiella pneumoniae isolates from 31 centers in 10 European countries found that the prevalence of ESBL in these organisms ranged from as low as 1.5% in Germany to as high as 39-47% in Russia, Poland, and Turkey. [19]

ESBLs have emerged due to selective pressure imposed by extensive use of antimicrobials, especially in intensive care units. Since ESBL-positive isolates show false susceptibility to expanded-spectrum cephalosporins in standard disk diffusion test, it is essential to adopt the specific detection methods recommended by CLSI. Though the phenotypic confirmatory tests are highly sensitive and specific, there are a number of instances when these tests may be falsely positive or negative. False-positives may occur if the isolate lacks ESBL but produces an excess of TEM-1 or SHV-1. On the other hand, isolates harboring both ESBLs and AmpC-type β-lactamases may result in false-negative results. The likely explanation is that AmpC-type β-lactamases resist inhibition by clavulanic acid; moreover, clavulanic acid induces high level AmpC production, thus preventing ESBL recognition. [2]

The isolates which have a positive phenotypic confirmatory test for ESBL production should be reported as resistant to all cephalosporins (except cephamycins, cefoxitin, and cefotetan) and aztreonam, regardless of the MIC of that particular cephalosporin. Penicillins are reported as resistant and β-lactam/β-lactamase inhibitor combinations are reported as susceptible if the zone diameters are within the appropriate range.

The present study shows higher antimicrobial resistance among ESBL producers than among non-ESBL producers [Table 3]. Almost all the ESBL-positive isolates were found to be resistant to Ampicillin and sensitive to Imipenem, which again advocates the usage of carbapenem antibiotics as the therapeutic alternative to β-lactam antibiotics as indicated in many previous studies.


The prevalence of ESBL producers at our institute was lower in comparison to the prevalence reported from other hospitals in India. Routine detection of ESBL-producing microorganisms is required to be done by each laboratory by the standard detection methods so as to control the spread of these infections and also to institute proper therapeutic strategies. Phenotypic confirmatory test using combination disk is simple and cost effective for the detection of ESBL production as it has shown 100% concordance with MIC reduction test. The control measures include judicious use of antibiotics, strict hand-hygiene protocols, and implementation of appropriate infection-control measures in the hospital, especially while treating high-risk patients.

Patients' report must state that the isolate is a suspected or proven ESBL producer and also include a note stating that ESBL production may predict therapeutic failure with penicillins, aztreonam, and all cephalosporins (except cephamycins), irrespective of their in vitro susceptibility.

Monitoring and judicious usage of extended-spectrum cephalosporins, periodic surveillance of antibiotic resistance patterns, and efforts to decrease empirical antibiotic therapy would go a long way in addressing some of the problems associated with ESBLs.


1Chaudhary U, Aggarwal R. Extended spectrum β lactamases (ESBL): An emerging threat to clinical therapeutics. Indian J Med Microbiol 2004;22:75-80.
2Bradford PA. Extended spectrum β lactamases in 21 st century: Characterization, epidemiology and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933-51.
3Shukla I, Tiwari R, Agrawal M. Prevalence of extended spectrum β lactamase producing Klebsiella pneumoniae in a tertiary care hospital. Indian J Med Microbiol 2004;22:87-91.
4Paterson DL, Bonomo RA. Extended spectrum β lactamases: A clinical update. Clin Microbiol Rev 2005;18:657-86.
5Duttaroy B, Mehta S. Extended spectrum β lactamases (ESBL) in clinical isolates of Klebsiella pneumoniae and Escherichia coli. Indian J Pathol Microbiol 2005;48:45-8.
6Kumar MS, Lakshmi V, Rajagopalan R. Occurrence of extended spectrum β lactamases among enterobacteriaceae spp. isolated at a tertiary care institute. Indian J Med Microbiol 2006;24:208-11.
7Menon T, Bindu D, Kumar CPG, Nalini S, Thirunarayan MA. Comparison of double disk and three dimentional methods to screen for ESBL producers in a tertiary care hospital. Indian J Med Microbiol 2006;24:117-20.
8Mathur P, Kapil A, Das B, Dhawan B. Prevalence of extended spectrum β lactamase producing Gram negative bacteria in a tertiary care hospital. Indian J Med Res 2002;115:153-7.
9Hansotia JB, Agarwal V, Pathak AA, Saoji AM. Extended spectrum β lactamase mediate resistance to third generation cephalosporins in Klebsiella pneumoneae in Nagpur, central India. Indian J Med Res 1997;105:160-5.
10Manchanda V, Singh NP, Goyal R, Kumar A, Thukral SS. Phenotypic characteristics of clinical isolates of Klebsiella pneumoniae and evaluation of available techniques for detection of extended spectrum beta lactamases. Indian J Med Res 2005;122:330-7.
11Tankhiwale SS, Jalgaonkar SV, Ahamad S, Hassani U. Evaluation of extended spectrum beta lactamase in urinary isolates. Indian J Med Res 2004;120:553-6.
12Jain A, Roy I, Gupta MK, Kumar M, Agarwal SK. Prevalence of extended spectrum β-lactamase producing Gram negative bacteria in septicaemic neonates in a tertiary care hospital. J Med Microbiol 2003;52:421-5.
13Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Win WC, editors. The enterobacteriaceae. In : Color atlas and textbook of diagnostic microbiology, 5 th ed. JB Lippincott Co: Philadelphia; 2006. p. 211-302.
14Clinical Laboratory Standards Institute; Performance standards for antimicrobial disc susceptibility testing. 14 th Informational Supplement: 2004.
15Saurina G, Quale GM, Manikal VM, Oydna E, Landman D. Antimicrobial resistance in enterobacteriaceaein Brooklyn NY: Epidemiology and relation to antibiotic usage patterns. J Antimicrob Chemother 2000;45:895-8.
16Cordero L, Rau R, Taylor D, Avers LW. Enteric Gram negative bacilli blood stream infections: 17 years experience in a neonatal intensive care unit. Am J Infect Control 2004;32:189-95.
17Xiong Z, Zzhu D, Zhang Y, Wang F. Extended spectrum beta-lactamase in Klebsiella pneumoniae and Escherichia coli isolates. Zhongua Yi Xue Za Zhi 2002;82:1476-9.
18Luzzaro F, Mezzatesta M, Mugnaioli C, Perilli M, Stefani S, Amicosante G, et al. Trends in production of Extended Spectrum B-Lactamases among Enterobacteria of medical interest: Report of the second Italian nationwide survey. J Clin Microbiol 2006;44:1659-64.
19Goosens H. MYSTIC programme: Summary of European data from 1997 to 2000. Diagn Microbiol Infect Dis 2001;41:183-9.