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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 51  |  Issue : 3  |  Page : 367-369
Prevalence of extended spectrum beta lactamase and AmpC beta lactamase producers among Escherichia coli isolates in a tertiary care hospital in Jaipur


1 Department of Microbiology and Immunology, Sawai Man Singh Medical College, Jaipur, Rajasthan, India
2 Department of Medicine, Sawai Man Singh Medical College, Jaipur, Rajasthan, India
3 Department of Forensic Medicine, Sawai Man Singh Medical College, Jaipur, Rajasthan, India

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   Abstract 

Resistance to broad spectrum β lactams, mediated by extended spectrum beta lactamase (ESβL) and AmpC βL enzymes is an increasing problem worldwide. Presence of these in clinical infections can result in treatment failure if one of the second or third generation cephalosporins is used. Therefore, it is recommended that any ESβL-producing organism according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria can be reported as resistant to all extended spectrum β lactam antibiotics regardless of the susceptibility test results. In this study, a total of 250 Escherichia coli (E. coli) isolates were subjected to Double disc test and AmpC disc test for the detection of ESβL- and AmpC βL-producing strains, respectively. Prevalence of ESβL- and AmpC βL-producing strains among E. coli isolates, over a 3-month-period in the hospital-based population of Jaipur, was 64.80% (162/250). AmpC βL producers were 24.00% (60/250) and co-existence of ESβL and AmpC βL was detected in 8.00% (20/250) of the isolates.

Keywords: AmpC βL, beta lactam, extended spectrum beta lactamase

How to cite this article:
Sinha P, Sharma R, Rishi S, Sharma R, Sood S, Pathak D. Prevalence of extended spectrum beta lactamase and AmpC beta lactamase producers among Escherichia coli isolates in a tertiary care hospital in Jaipur. Indian J Pathol Microbiol 2008;51:367-9

How to cite this URL:
Sinha P, Sharma R, Rishi S, Sharma R, Sood S, Pathak D. Prevalence of extended spectrum beta lactamase and AmpC beta lactamase producers among Escherichia coli isolates in a tertiary care hospital in Jaipur. Indian J Pathol Microbiol [serial online] 2008 [cited 2019 Dec 13];51:367-9. Available from: http://www.ijpmonline.org/text.asp?2008/51/3/367/42512



   Introduction Top


Beta lactamases (βLs) are enzymes that hydrolyze the β-lactam ring of β lactam antibiotics like penicillins and cephalosporins. Third-generation cephalosporins (3GCs) were thought to be resistant to hydrolysis by βL, but, in mid 1980s, it became evident that a new type of βL was being produced which could also hydrolyze the extended spectrum cephalosporins, collectively termed as "extended spectrum beta lactamases" (ESβLs). [1] These enzymes are the result of mutations of Temorina (TEM-1 and TEM-2) and Suph hydryl variable (SHV-1) enzymes, usually plasmid mediated and are commonly found in the Enterobacteriaceae family. [1] Some derivatives of TEM and SHV, which are not inhibited by clavulanic acid, are known as inhibitor resistant TEM (IRTs) and the AmpC class of enzymes, which are intrinsically resistant to clavulanic acid, are causing great concern as Carbapenems are the only antibiotic effective against such strains. [2]

With the spread of these strains in hospitals all over the world, it is necessary to know their prevalence in a hospital so as to formulate a policy of empirical therapy in high-risk units. Equally important is to procure information on an isolate from a patient to avoid misuse of extended spectrum cephalosporins. The routine susceptibility tests performed by clinical laboratories fail to detect these strains, which may lead to inappropriate and unsuccessful therapy of the patient and unnecessary use of the drug. [2]


   Materials and Methods Top


The study was conducted to detect the prevalence of ESβL- and AmpC βL-producing strains among E. coli isolates. A total of 250 consecutive, nonrepetitive, nonenteric E. coli isolates from various clinical samples, such as urine, sputum, pus, blood and other body fluids, etc., were selected on the basis of resistance against any one of the 3GCs (as described by others also), [3],[4] for the study over a period of 3 months (November 2006-January 2007). Two tests (Double disc test and AmpC disc test) were used in the study for the detection of ESβL and AmpC βL among E. coli isolates. A suspension of the organism was prepared with turbidity equivalent to 0.5 Mc Farland standards and inoculated on Mueller-Hinton Agar (MHA) plates for disc diffusion testing, in accordance with NCCLS M100-S10 (M2) guidelines.

Double disc test

Amoxicillin-clavulanic acid (moxclav 20/10 mg) disc was placed toward the center of the plate, a ceftazidime disc (30 mg) was placed 15 mm out from the edge of moxclav disc at 90 angles, so that its inner edge is 15 mm from it. The same was performed with cefotaxime (30 mg) or ceftriaxone (30 mg), aztreonam (30 mg) and cefpodoxime (10 mg) discs so that they were spaced 90 apart and 15 mm from moxclav disc. A cefoxitin (30 mg) disc was also placed as shown in [Figure 1]. [5]

Plates prepared were incubated at 35C, aerobically for 18-24 h; zone diameters and zone-enhancement toward moxclav disc were recorded for the all cephalosporins, as per NCCLS guidelines. E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as negative and positive controls, respectively.

Following classification was used. [5]

  • Class A ESβL present:

    (i) Potentiation of the inhibition zone (IZ) of any one of cefpodoxime, ceftazidime, ceftriaxone, or aztreonam when combined with clavulanic acid.

    (ii) Susceptibility (S) to cefoxitin.

    (iii) S or resistance (R) to any one of ceftazidime, ceftriaxone, or aztreonam.


  • Class A and Class C ESβL present:

    (i) Potentiation of the IZ of any one of cefpodoxime, ceftazidime, ceftriaxone, or aztreonam when combined with clavulanic acid.

    (ii) R or intermediate (I) to cefoxitin.

    (iii) S or R to any one of ceftazidime, ceftriaxone, or aztreonam.


  • Class C ESβL present:

    (i) No potentiation with clavulanic acid.

    (ii) R or I to cefoxitin.

    (iii) R to any one of ceftazidime, ceftriaxone, or aztreonam.


  • ESβL not Class A or Class C present:

    (i) No potentiation with clavulanic acid.

    (ii) S to cefoxitin.

    (iii) R to any one of ceftazidime, ceftriaxone, or aztreonam.


  • ESβL absent:

    (i) No potentiation with clavulanic acid.

    (ii) S or R to cefoxitin.

    (iii) S to all of ceftazidime, ceftriaxone, or aztreonam.


All the isolates were simultaneously subjected to AmpC disc test for AmpC βL detection.

AmpC disc test

A lawn culture of E. coli ATCC 25922 was prepared on MHA plate. Sterile discs (6 mm) were moistened with sterile saline and inoculated with several colonies of test organisms. The inoculated disc was then placed beside a cefoxitin disc (almost touching) on the inoculated plate. The plates were incubated overnight at 35C, aerobically. A positive test was indicated as flattening or indentation of cefoxitin IZ in the vicinity of the test disc. A negative test had an undistorted zone. [6]

In addition to the above, all the strains were tested with Cefepime and Imipenem.

Routine susceptibility results against other antibiotics were also noted.


   Results Top


Of the total 250, E. coli isolates, 64.80% (162/250) were found to be R to at least three 3GCs. Fourty-two percent were found to be R to Amikacin, 27.20% to Gatifloxacin, 8.8% to Cefoperazone+ Sulbactam combination and only 3.2% to Piperacillin + Tazobactem combination. Among non urinary isolates, R to Aztreonam and Netilmycin was found to be 60% and 41.60%, respectively. Among urinary isolates; R to Nitrofurantoin was found to be only 4.61%.

As per the classification mentioned, percentage of each class of ESβL is shown in [Table 1]. Out of 60 AmpC βL-producing isolates, flattening of zone was observed in 81.66% (49/60) isolates, indicating weak AmpC βL producers and indentation indicating strong AmpC βL producers in only18.33% (11/60) isolates. 27.45% (28/102) of ESβL producers were found to be R to Cefoxitin. Around 76.66% (46/60) of AmpC βL-producing strains were found to be S to Cefepime. Sensitivity among Class A isolates was 24.19%, Class A + Class C 53.45%, Class C 59.89% and among ESβL not ClassA or ClassC was 62.00%. All the 250 isolates were found to be S to Imipenem.


   Discussion Top


Failure to detect ESβL- and AmpC βL-producing strains has contributed to their uncontrolled spread and therapeutic failures. [6] Hence, their appearance in a hospital setting should be identified quickly so that appropriate antibiotic usage and containment measures can be implemented.

Prevalence of overall ESβL and AmpC βL among E. coli isolates in the present study was found to be 64.80% (162/250); Singhal et al . [6] Mathur et al. [2] and Manchanda et al . [7] have reported the prevalence ranging from 55% to 69%.

For detection of various types of ESβLs, molecular methods are the best but the facilities are not available especially in the developing countries. Therefore, various phenotypic methods are recommended for routine use to detect ESβL production in Gram-negative bacilli. [3] Supplemental disk approximation testing has proved to be a useful detection method; however, suboptimal placement of the disks leaves the method with limited sensitivity but acceptable specificity. [8] Recently, co-existence of both AmpC βL and ESβL in some Gram-negative organisms has also been reported. This could be because plasmid-mediated AmpC βL enzymes have been shown to disseminate among Enterobacteriaceae , sometimes in combination with ESβL. [6] Such strains may give false negative tests for the detection of ESβLs. Eight percent (20/250) of the isolates (ESβL not Class A or Class C) in the present study probably represent co-existence of phenotype of both ESβL and AmpC βL; as mentioned by Mathur et al . [2] Singhal et al . [6] had demonstrated 1.25% (1/80) of such co-existence among E. coli isolates. In the presence of AmpC βL along with ESβL in Gram-negative organisms, the Double disc test may not show positivity as AmpC βL inhibits the action of clavulanate and hence obscures the synergistic effect of clavulanic acid and 3GC. 4GC-Cefepime has been recommended as an alternative cephalosporin for ESβL detection in the presence of AmpC βL. [9] In our study, 76.66% (46/60) of AmpC βL-producing strains and 62.00% of co-existing phenotypes were found to be sensitive to Cefepime. Although in vitro susceptibility shows that Cefepime should also be active in vivo , there is a clear inoculum effect (large concentration of organisms can overwhelm the effect of this antibiotic). [1]

AmpC βL producers were 24.00% in our study; 37.50% and 47.80% have been reported from Chennai and Kolkatta, respectively. [10],[11]

It might be highly desirable to develop an ESβL detection test that includes a substrate displaying a higher degree of resistance to AmpC βL enzymes. Cefoxitin resistance can be used to screen isolates for detecting possible AmpC βL production. But lack of permeation of porins has also been reported as one of the resistance mechanisms of Cefoxitin in AmpC nonproducers. [12] In this study, 27.45% (28/102) of AmpC-nonproducers were found to be resistant to Cefoxitin; 19.04% resistant and intermediate AmpC-non producers have also been reported from Delhi. [7] On the other hand, AmpC βL production in cefoxitin-susceptible isolates may have a mechanism similar to that of ESβL-producing organisms. Interestingly, no AmpC-harboring isolates revealed susceptibility to cefoxitin in our study or else, as we have used only cefoxitin resistance-based test, i.e., AmpC disc test for AmpC βL detection, this cannot be commented upon. Cefoxitin-sensitive AmpC βL producers (57.14%) have been reported from Delhi. [7] Thus, although studies indicate that screening methods which use cefoxitin in standardized methods to detect AmpC-harboring isolates are useful, they are not perfect. [7]

In conclusion, AmpC disc test can be used as a simple, easy and rapid screening test for the detection of isolates harboring AmpC βL in Clinical laboratory.

 
   References Top

1.Kevin RF. Extended spectrum beta lactamases. Can J Infect Dis Med Microbiol 2006;7:6-8.  Back to cited text no. 1    
2.Mathur P, Kapil A, Das B, Dhawan B. Prevalence of ESBL producing gram negative bacteria in a tertiary care hospital. India J Med Res 2002;115:153-7.  Back to cited text no. 2    
3.Kumar MS, Lakshmi V, Rajagopalan R. Occurrence of extended spectrum beta-lactamases among Enterobacteriaceae spp. isolated at a tertiary care institute. Indian J Med Microbiol 2006;24:208-11.  Back to cited text no. 3    
4.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;20:92-5.  Back to cited text no. 4    
5.Antimicrobial susceptibility testing. TML/MSH Microbiology dept. Policy and Procedure manual, 2000. p. 37-41.  Back to cited text no. 5    
6.Singhal S, Mathur T, Khan S, Upadhyay DJ, Chugh S, Gaind R, et al . Evaluation of methods for AmpC Beta -lactamase in gram negative clinical isolates from tertiary care hospitals. Indian J Med Microbiol 2005;23:120-4.  Back to cited text no. 6    
7.Manchanda V, Singh NP. Occurrence and detection of AmpC beta lactamases among gram negative clinical isolates using a modified three-dimensional test at Guru Tegh Bahadur Hospital, Delhi, India. J Clin Microbiol 2003;51:415-8.  Back to cited text no. 7    
8.Moland ES, Thompson KS. Extended-spectrum β -lactamases of Enterobacteriaceae . J Antimicrob Chemother 1994;33:666-8.  Back to cited text no. 8    
9.Tzelepi E, Gaikkoupi P, Sofianou D, Loukova V, Kemeroglou A, Tsakris A. Detection of extended spectrum beta-lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes . J Clin Microbiol 2000;38:542-6.  Back to cited text no. 9    
10.Subha A, Devi VR, Ananthan S. AmpC beta-lactamase producing multidrug resistant strains of Klebsiella spp. and Escherichia coli isolated from children under five in Chennai. Indian J Med Res 2003;117:13-8.  Back to cited text no. 10    
11.Arora S, Manjusri S. AmpC beta lactamase producing bacterial isolates from Kolkata Hospital. Indian J Med Res 2005;122:124-33.  Back to cited text no. 11    
12.Pangon B, Bizet C, Bure A, Pichon F, Philippon A, Ragnier B, et al . In vivo selection of cephamycin resistant, porin-deficient mutants of Klebsiella pneumoniae producing TEM -3 beta lactamase. J Infect Dis 1989;159:1005-6.  Back to cited text no. 12    

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Correspondence Address:
Rajni Sharma
P-3, Tilak Marg, C- Scheme, Jaipur, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.42512

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