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ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 2  |  Page : 135-138
Evaluation of phenotypic tests for the detection of AmpC beta-lactamase in clinical isolates of Escherichia coli


Department of Microbiology, Subharti Medical College, Meerut, Uttar Pradesh, India

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Date of Web Publication23-Sep-2013
 

   Abstract 

Background: AmpC beta lactamases are cephalosporinases that confer resistance to a wide range of beta lactam drugs thereby causing serious therapeautic problem. As there are no CLSI guidelines for detection of AmpC mediated resistance in Gram negative clinical isolates and it may pose a problem due to misleading results, especially so in phenotypic tests. Although cefoxitin resistance is used as a screening test, it does not reliably indicate AmpC production. Materials and Methods: We planned a study to determine the occurrence of AmpC beta lactamase in hospital and community, clinical isolates of Escherichia coli and simultaneously evaluate different phenotypic methods for detection of AmpC beta lactamases. Results: It was observed that 82.76% isolates were ESBL positive and 59% were cefoxitin screen positive. Using phenotypic confirmatory tests the occurrence of Amp C beta lactamases was found to be 40% and 39% by inhibitor based method using boronic acid (IBM) and modified three dimensional test (M3D) respectively. Conclusion: Both the test showed concordant result. Co-production was observed in 84.62% isolates Screening of ESBL and Amp C can be done in routine clinical microbiology laboratory using aztreonam and IBM respectively as it is a simple, rapid and technically less demanding procedure which can be used in all clinical laboratories.

Keywords: AmpC beta-lactamases, extended spectrum beta-lactamases, inhibitor based test, modified 3D test

How to cite this article:
Handa D, Pandey A, Asthana AK, Rawat A, Handa S, Thakuria B. Evaluation of phenotypic tests for the detection of AmpC beta-lactamase in clinical isolates of Escherichia coli. Indian J Pathol Microbiol 2013;56:135-8

How to cite this URL:
Handa D, Pandey A, Asthana AK, Rawat A, Handa S, Thakuria B. Evaluation of phenotypic tests for the detection of AmpC beta-lactamase in clinical isolates of Escherichia coli. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Feb 26];56:135-8. Available from: http://www.ijpmonline.org/text.asp?2013/56/2/135/118686



   Introduction Top


Gram-negative bacteria pose a therapeutic problem not only in the hospital settings, but also in the communfity as they have acquired resistance to multiple antibiotics. The various mechanisms of drug resistance in Gram-negative bacteria include extended spectrum beta-lactamases (ESBL) production, AmpC beta-lactamase production, efflux mechanism and porin deficiency. Amongst this, detection of ESBLs and AmpC beta-lactamases are the most common methods. [1] AmpC beta-lactamases are clinically relevant because they confer resistance to both narrow and broad spectrum cephalosporins, beta-lactam beta-lactamase inhibitor combinations and aztreonam. [1] AmpC beta-lactamases, demonstrated or presumed to be chromosomally or plasmid mediated have been described in pathogens e.g., Klebsiella pneumoniae,  Escherichia More Details coli,  Salmonella More Details spp., Proteus mirabilis, Citrobacter fruendii, Acinetobacter, Enterobacter spp. and Pseudomonas aeruginosa.[2] Although reported with increasing frequency, the true rate of occurrence of AmpC beta-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. Coudron et al. [3] used the standard disk diffusion breakpoint for cefoxitin (CX) (zone diameter <18 mm) to screen isolates and used a 3D extract test as a confirmatory test for isolates that harbor AmpC beta-lactamases. The current Clinical and Laboratory Standards Institute (CLSI) guidelines [4] do not describe any method for detection of isolates producing AmpC beta-lactamases. The aim of the present study was to detect the occurrence of ESBL and AmpC enzymes in clinical isolates of E. coli both from inpatient units and outdoor samples in our tertiary care hospital and simultaneously evaluate two different phenotypic methods that is inhibitor based method using boronic acid (BA) [5] IBM and modified 3D test (M3D) [6] for its detection.


   Materials and Methods Top


A total of 300 non-repeat clinical isolates of E. coli obtained from different clinical samples (urine, blood, pus, urinary catheter tube, endotracheal tube, discharge, drain site etc.) received in Clinical Microbiology Laboratory over a period of 1 year from various inpatient units and out-patient departments were processed. The isolates were identified as per standard bacteriological technique. [7] Antimicrobial susceptibility testing was carried out using Kirby-Bauer disc diffusion method as per CLSI recommendations, [4] using commercially available antibiotic discs (Hi-media). The antimicrobial profile against ciprofloxacin, ampicillin, gentamicin amikacin, cotrimoxazole, cephalosporins (cephalexin, ceftazadime, cefotaxime, CX and cefepime), chloramphenicol, amoxicillin/clavulanic acid, norfloxacin, nitrofurantoin and imipenem were studied. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used for quality control.

Screening of AmpC Beta-lactamases and ESBL producers

Screening of clinical isolates of E. coli was performed with CX disc. [3],[8] The isolates that yielded a CX zone diameter <18 mm was CX screen positive. Both screen positive and negative isolates were subjected for confirmation of AmpC enzyme by two different phenotypic tests IBM and M3D. Screening for ESBL production was carried out by disc diffusion method using indicator drug as per CLSI screening criteria and confirmation was performed using CLSI phenotypic confirmatory test (PCT). [4]

Detection of AmpC Beta-lactamases

IBM using BA

AmpC beta-lactamase production was detected by IBM method on disk containing BA as per the method used by Hemalatha et al. [5] Briefly, a disk containing 30 μg of CX and another disk containing 30 μg of CX with 400 μg of BA were placed on the agar at a distance of 30 mm. Inoculated plates were incubated overnight at 37°C. An organism demonstrating a zone diameter around the disk containing CX and BA ≥5 mm than the zone diameter around the disk containing CX alone was considered as an AmpC producer [Figure 1].
Figure 1: Inhibitor based method for AmpC production: Isolates showing zone diameter around the disk containing (cefoxitin [CX] + boronic acid) ≥5 mm than the zone diameter around the disk containing CX alone

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M3D

AmpC enzyme production was detected by M3D test as per the method used by Manchanda and Singh.[6] The isolates showing clear distortion of zone of inhibition of CX was taken as AmpC producers [Figure 2] and the isolates with no distortion of zone of inhibition of CX was taken as AmpC non-producers [Figure 3].
Figure 2: Modified 3D method: Isolate showing clear distortion (arrow) in the zone of inhibition of cefoxitin indicating AmpC producer

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Figure 3: Modified 3D method: Isolate showing no distorti on in the zone of inhibition of cefoxitin indicationg AmpC non-producer

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   Results Top


Out of the 300 clinical isolates of E. coli tested ESBL production was confirmed in 82.76% [Table 1] and 177/300 (59%) were CX screen positive [Table 2].
Table 1: Distributi on of ESBL screening and CLSI, PCT (n=300)

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Table 2: Detecti on of AmpC beta-lactamase by IBM and M3D method in cefoxitin screen positive and negative isolates

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Detection of AmpC Beta-lactamases

AmpC enzyme was detected in 40% and 39% of clinical isolates of E. coli using IBM and M3D methods respectively [Table 2] (P = 0.8002). The McMenar's Chi-square test of significance clearly showed a significant association or difference between IBM positive and M3D positive with respect to CX screen positive and screen negative isolates (P < 0.05).

Co-production of AmpC Beta-lactamases and ESBL

Out of 39% (M3D positive) isolates almost four-fifth (84.6%) occurred in combination with ESBL showing co-production of both ESBL and AmpC enzymes. However, in 15.38% isolates, only AmpC enzyme was detected [Table 3].
Table 3: Co-production of AmpC beta-lactamases with ESBL producers

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Antibiotic susceptibility pattern and site of localization

The clinical isolates of E. coli showed resistance to multiple antimicrobial agents. The clinical isolates showed complete resistance to 3GC (third generation cephalosporin [GC]) and beta-lactam beta-lactamase inhibitor combination, which was found to coexist with resistance to two or more antibiotics that is ciprofloxacin (95.83%), co-trimoxazole (90.28%), gentamicin (73.61%), norfloxacin (66.67%) chloramphenicol (31.94%), amikacin (20.83%). All our isolates of E. coli also showed complete resistance to cefepime, a 4 th GC. Least resistance was observed with imipenem (5.56%) and nitrofurantoin (6.48%) in cases of urinary isolates. Both ESBL and AmpC producing isolates were detected more frequently from samples received from in-patient units 81.9% and 76.9% than from samples received from outdoor patients 16.9% and 23.07%.


   Discussion Top


AmpC, beta-lactamases are clinically significant, [3] since they confer resistance to cephalosporins in the oxyimino group (cefotaxime, ceftriaxone, ceftazidime), 7 alpha methoxy cephalosporins (CX) and are not affected by available beta-lactamase inhibitors (clavulanate, sulbactam). [9] Plasmid-mediated AmpC beta-lactamases differ from chromosomal AmpC in being uninducible [10] and are typically associated with broad multidrug resistance. [11] Therapeautic options for infections caused by Gram-negative organisms expressing AmpC beta-lactamases are limited except for 4 th GC such as cefepime and carbapenem. [12] This emphasizes the need for detecting AmpC beta-lactamase harboring isolates so as to avoid therapeautic failures and nosocomial outbreaks.

In the present study, the clinical isolates of E. coli showed resistance to multiple antimicrobial agents tested along with complete resistance to 3GC, beta-lactam beta-lactamase inhibitor combination including 4 th GC (cefepime). Taneja et al. [1] have also reported high-level of resistance to cefepime in ESBL producers and non-producers. The available data suggests that carbapenems are more effective than cefepime in treating serious infections that involve large numbers of AmpC producing organisms. [13] In our study, lowest resistance was observed with imipenem (5.56%), thus highlighting that imipenem should be used as treatment options in such cases. Similarly, Patel et al. [14] have also reported imipenem as the drug of choice in AmpC producers.

A total of 59% of clinical isolates was found to be CX screen positive. AmpC enzyme could be detected by IBM in 40% isolates and M3D test in 39% isolates. We observed that IBM had an advantage over M3D in being less time consuming, technically less demanding therefore less cumbersome to perform in routine clinical laboratory where work load is more. Singhal et al. [2] in their study have used CX resistance to screen isolates for detecting possible AmpC beta-lactamases and 3D extract test and AmpC disk test for confirmation and had observed that both phenotypic tests could detect equal number of samples.

Currently, CLSI documents do not indicate the screening and confirmatory tests that are optimal for detection of these beta-lactamases. However, several studies have been carried out on various test methods to name a few, the 3D test, [15] modified double disk test, [16] AmpC disk test, [2] IBMs employing inhibitors such as BAs, [5] M3D, [2],[6] etc. In spite of many phenotypic tests, a reference laboratory for beta-lactamase isoelectric focusing [17] and gene localization by genotype characterization [12] are considered as the gold standard. This will help us to know the actual prevalence of these enzymes and characterize them for epidemiological purpose. Unfortunately in the present study, we were unable to perform the genotypic tests due to limited resources. However, as the finding of the present study shows the presence of AmpC beta-lactamases in this area, future study may be carried out in more number of samples employing genotypic methods to know the actual prevalence of AmpC enzyme.

ESBL production was seen 82.76% isolates by PCT in our study [Table 1]. PCT is highly sensitive and specific when compared with genotypic confirmatory methods; however, there are a number of instances when PCT may be both falsely positive and falsely negative. K. pneumoniae and E. coli isolates which lack ESBLs enzymes, but which hyper produce SHV-1 or have a decrease in the quantity of the minor 45-kDa outer membrane can give false positive confirmatory tests. A comparatively higher (82.7%) occurrence of ESBL producing bacteria in the present study may be partly attributed to the fact that our hospital being a tertiary care center. In the remaining 45/261 ESBL non-producer (screen positive but PCT negative) isolates, we assume that there can be some other mechanism of resistance besides ESBL production. [12]

Co-production of both ESBL and AmpC enzyme was seen in 84.62% of isolates whereas in 15.38% of isolates only AmpC beta-lactamase was detected [Table 3]. This prevalence was higher when compared with reports from other parts of the world. [3] Hemalatha et al. [5] in an Indian study reported only AmpC beta-lactamase in 9.2% of their isolates. It has been seen that the AmpC beta-lactamases when present along with ESBLs can mask the phenotype of the latter. [5],[11]

The ESBL and AmpC producing E. coli were isolated more frequently from samples received from inpatient units (81.9% and 76.9%) than from patients attending outdoor clinics (16.9% and 23.07%). Detection of both ESBL and AmpC enzymes in a significant number of isolates from outdoor patients in the present study is definitely is a matter of concern as such strains may spread into the community rapidly and may cause therapeautic problem. To the contrary, some studies have shown that AmpC producers are restricted to hospitalized patients only, with nosocomial spread as their main mode of dissemination. [2],[6],[18]

To conclude, the occurrence of both ESBL and AmpC producing E. coli was relatively high in our setting. Moreover, these resistant strains have spread into the community. Effective infection control measures like hand washing and barrier precautions are required. A mixed type of drug resistance mechanism seems to operate in the isolate tested. Thus there is a need for a correct and reliable phenotypic test to identify AmpC beta-lactamases and to discriminate between only AmpC producer and/or ESBL producers. The IBM using BA appears to be a simple and cost-effective method in discriminating this type of resistant isolate.

 
   References Top

1.Taneja N, Rao P, Arora J, Dogra A. Occurrence of ESBL & Amp-C beta-lactamases & susceptibility to newer antimicrobial agents in complicated UTI. Indian J Med Res 2008;127:85-8.  Back to cited text no. 1
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2.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. 2
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3.Coudron PE, Moland ES, Thomson KS. Occurrence and detection of AmpC beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a veterans medical center. J Clin Microbiol 2000;38:1791-6.  Back to cited text no. 3
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4.Clinical Laboratory Standards Institute. Performance Standard for Antimicrobial Susceptibility Testing: 14 th Informational Supplement. Wayne, PA: USA: Clinical Laboratory Standards institute; 2004. p. M100-S17.  Back to cited text no. 4
    
5.Hemalatha V, Padma M, Sekar U, Vinodh TM, Arunkumar AS. Detection of Amp C beta lactamases production in Escherichia coli & Klebsiella by an inhibitor based method. Indian J Med Res 2007;126:220-3.  Back to cited text no. 5
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6.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 Antimicrob Chemother 2003;51:415-8.  Back to cited text no. 6
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7.Collee JG, Miles RS, Watt B. Tests for the identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A editors. Mackie and Mc Cartney Practical Medical Microbiology. 14 th ed. Edinburgh: Churchill Livingstone; 1996.p. 151-79.  Back to cited text no. 7
    
8.Ratna AK, Menon I, Kapur I, Kulkarni R. Occurrence & detection of AmpC beta-lactamases at a referral hospital in Karnataka. Indian J Med Res 2003;118:29-32.  Back to cited text no. 8
    
9.Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother 2002;46:1-11.  Back to cited text no. 9
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10.Barnaud G, Arlet G, Verdet C, Gaillot O, Lagrange PH, Philippon A. Salmonella enteritidis: AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii. Antimicrob Agents Chemother 1998;42:2352-8.  Back to cited text no. 10
    
11.Thomson KS. Controversies about extended-spectrum and AmpC beta-lactamases. Emerg Infect Dis 2001;7:333-6.  Back to cited text no. 11
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12.Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002;40:2153-62.  Back to cited text no. 12
    
13.Zanetti G, Bally F, Greub G, Garbino J, Kinge T, Lew D, et al. Cefepime versus imipenem-cilastatin for treatment of nosocomial pneumonia in intensive care unit patients: A multicenter, evaluator-blind, prospective, randomized study. Antimicrob Agents Chemother 2003;47:3442-7.  Back to cited text no. 13
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Correspondence Address:
Anita Pandey
Department of Microbiology, Subharti Medical College, Meerut - 250 005, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.118686

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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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