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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 6  |  Issue : 2  |  Page : 54-58

Phenotypic and molecular characterization of extended spectrum beta lactamase and AmpC beta lactamases in Escherichia coli from a tertiary care centre in India


1 Department of Microbiology, Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi, India
2 Department of Orthopedics, All India Institute of Medical Sciences, New Delhi, India

Date of Web Publication10-Jan-2019

Correspondence Address:
Dr. Purva Mathur
Department of Microbiology, 2nd Floor, Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpsic.jpsic_11_18

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  Abstract 


Background: Antibiotic resistance mediated by extended spectrum beta lactamase (ESBL) and AmpC enzymes in Escherichia coli continue to be a major threat in a health care setting. This study was undertaken to calculate the prevalence and to characterize ESBL and AmpC enzymes produced by E. coli by various phenotypic and molecular methods (polymerase chain reaction [PCR]).
Materials and Methods: A total of 196 clinical isolates of E. coli were screened for ESBL production using cephalosporin disk diffusion method, minimum inhibitory concentration (MIC) determination by E-test and Vitek-2 system. Phenotypic confirmation for ESBL production was done using cephalosporin/clavulanate combination disc test method and E-test for ESBLs. For the detection of AmpC enzymes, cefoxitin disk diffusion and cefoxitin MIC testing was used and further phenotypically confirmed by three dimensional extract test, AmpC disk test, boronic acid disk test method and disk approximation method. The genotypic detection of ESBL genes and AmpC genes were done by PCR.
Statistical Analysis: The data was analyzed using SPSS software (Version 21.0). Chi-square test was used for statistical analysis of the data.
Results: The prevalence of ESBL and AmpC enzymes among E. coli isolates was found 93.12% and 28.68%. Among the various phenotypic screening methods evaluated, ceftazidime (CZD) disk diffusion test had the highest sensitivity of 90.67% and positive predict value of 92.86% followed by cefotaxime and CZD in comparison with polymerase chain reaction (PCR). For AmpC β-lactamases, the cefoxitin disk used for screening of AmpC β-lactamases had sensitivity of 91.67%, specificity of 59.14%, positive predict value of 46.48%, and negative predict value of 94.83% when compared with PCR.
Conclusion: The high prevalence of ESBL and AmpC in our study emphasises on the judicious use of antibiotics in controlling antimicrobial resistance in the hospital.

Keywords: AmpC β-lactamases, Escherichia coli, extended spectrum beta lactamase


How to cite this article:
Govindaswamy A, Bajpai V, Batra P, Malhotra R, Mathur P. Phenotypic and molecular characterization of extended spectrum beta lactamase and AmpC beta lactamases in Escherichia coli from a tertiary care centre in India. J Patient Saf Infect Control 2018;6:54-8

How to cite this URL:
Govindaswamy A, Bajpai V, Batra P, Malhotra R, Mathur P. Phenotypic and molecular characterization of extended spectrum beta lactamase and AmpC beta lactamases in Escherichia coli from a tertiary care centre in India. J Patient Saf Infect Control [serial online] 2018 [cited 2019 Oct 18];6:54-8. Available from: http://www.jpsiconline.com/text.asp?2018/6/2/54/249840




  Introduction Top


Nosocomial Infections caused by  Escherichia More Details coli producing extended spectrum beta lactamases (ESBLs) and AmpC β-lactamases are a threat to clinicians due to limited antibiotic options for treatment.[1] The ESBLs are the enzymes that can hydrolyze and inactivate the β-lactam antibiotics like penicillin, 3rd generation cephalorins and aztreonam, but are sensitive to to β-lacatamase inhibitors such as sulbactam, clavulanate, and tazobactam.[2] They are majorly encoded by plasmids thereby leading to the spread of resistance to other nosomial pathogens in a hospital setting. ESBLs are produced more commonly by E. coli and Klebsiella spp. However, other Gram-negative bacteria can also produce them.[3]

The organisms producing ESBLs can also express the AmpC β-lactamases, thus making it more complex to diagnose and treat. The AmpC β-lactamases are emerging as a new threat since they confer resistance to cephamycins and are not affected by β-lactamase inhibitors. In case of AmpC β-lactamases the mechanism of drug resistance can be chromosomal or plasmid mediated. Unlike the ESBLs which are plasmid mediated, the AmpC β-lactamases are mostly chromosomally mediated.[4] They are difficult to identify by phenotypic tests and most often falsely identified as ESBLs in a clinical laboratory. This study was undertaken to characterize ESBL and AmpC producing E. coli using various phenotypic screening and confirmatory methods followed by characterization of their resistant genes by PCR.


  Materials and Methods Top


Study period and bacterial isolates

The total duration of the study was 3 years (2013–2016) conducted at the Microbiology laboratory of the JPNA Trauma Centre of AIIMS hospital, New Delhi. Bacterial isolates to be included in this study consisted of non-duplicate, consecutive nosocomial Gram-negative bacteria isolated from various clinical samples obtained at the Microbiology laboratory. All the E. coli isolates were isolated and identified by Vitek-2 Identification system (Biomeriux, France).

Screening of extended spectrum beta lactamase

A total of 196 E. coli isolates were screened for ESBL production by recording the zone diameters of ceftazidime (CZD) (30 μg), cefotaxime (CTX) (30 μg), ceftriaxone (30 μg), cefpodoxime (10 μg), aztreonam (30 μg) by disc diffusion testing on Mueller-Hinton agar (MHA) using Clinical and Laboratory Standards Institute (CLSI) recommended conditions.[5] ESBL screening also included the following criteria for Escherichia Spp., a ceftriaxone, CZD, cefepime, or aztreonam MIC of ≥2 μg/ml. The isolates showing positive results by screening were tested for further confirmatory and genotypic tests.

Phenotypic confirmatory test for extended spectrum beta lactamase

Phenotypic confirmatory test for ESBL production was performed using cephalosporin/clavulanate combination disc test method, E-test for ESBLs according to CLSI guidelines.[5] The combination disc test was performed using CTX (30 μg), CZD (30 μg) discs with and without clavulanate (10 μg). A ≥5-mm increase in zone diameter of CTX/CZD in the presence of clavulanic acid compared to when the antibiotic is tested alone is a positive test for an ESBL. In E-test minimum inhibitory concentrations (MICs) of CTX and CZD with and without clavulanic acid was tested as per the manufacturer's instructions. A ≥8 fold reduction in cephalosporin's MICs in the presence of clavulanate would be taken as confirmatory of ESBL.

Screening of AmpC beta-lactamase

A total of 173 E. coli isolates were screened for AmpC beta-lactamase production. The CLSI ESBL screen was used for detection of plasmid-mediated AmpC β-lactamases in E. coli isolates.[5] A 30-μg cefoxitin disk was placed on inoculated MHA. According to CLSI, isolates with zone diameters <14 mm were be selected for confirmation of AmpC production. Alternatively, a cefoxitin of MIC ≥32 μg/ml was also used for screening.

Phenotypic confirmatory tests for AmpC beta-lactamase

The isolates which were positive by screening test were further confirmed for AmpC beta-lactamase by three dimensional extract test (TDET), AmpC disk test, boronic acid disk test method and disk approximation method were performed.[6],[7],[8]

Detection of extended spectrum beta lactamase and AmpC β-lactamases by polymerase chain reaction

Strains suspected/phenotypically confirmed to be ESBL producing were examined for the presence of the blaTEM, blaSHV, blaCTX-M, blaPER and blaVEBβ-lactamases genes by PCR using the primers and cycling conditions as described by us previously.[9],[10],[11] In case of AmpC, since phenotypic tests do not differentiate between chromosomal and plasmid mediated AmpC β-lactamases, plasmid-mediated AmpC β-lactamases were most accurately detected with the multiplex AmpC PCR test of Pérez-Pérez and Hanson.[12]

Statistical analysis

Analysis was carried out using SPSS version 21, (IBM, Armonk, NY, United States of America). Chi-square test was used for statistical analysis of the data. A 'P < 0.05' was considered statistically significant.


  Results Top


Prevalence of extended spectrum beta lactamase among the Escherichia coli isolates

Out of the 196 E. coli isolates screened for ESBL production by CZD disc 173/196 (88.3%), CTX disc 183/196, ceftriaxone disc 162/196 (82.7%), aztreonam disc 125/196 (63.8%) and cefpodoxime disc 163/196 (83.2%) were positive for ESBL screen by disc diffusion method. In E-test used for screening, CZD E-test detected 117/196 (76.5%), CTX E-test 126/196 (63.4%), Ceftriaxone E-test 106/196 (54.1%) as depicted in [Table 1]. Among the various screening tests, the CZD disc diffusion test showed highest sensitivity of 90.67% and positive predict value of 92.86% in comparison with PCR.
Table 1: Screening test for extended spectrum beta lactamase

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Among the 173 E. coli isolates which were positive for ESBL screen by CZD disk diffusion test, 103 (59.53%) were positive by the confirmatory CZD/CZD-clavulanic acid combination disk test. For isolates which were positive for ESBL screen by CTX disk diffusion test, 102 (55.73%) isolates were positive by CTX/CTX-clavulanic acid combination disk test. While in the E-test combination test 112/117 (95.72%) and 97/126 (76.98%) were detected by CZD/CZD-clavulanic acid and CTX/CTX-clavulanic acid E-test respectively as shown in [Table 2].
Table 2: Confirmatory test for extended spectrum beta lactamase

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Prevalence of AmpC β-lactamases among the Escherichia coli isolates

Among the 173 isolates, 73 (42.2%) were positive for AmpC β-lactamase production by cefoxitin disk diffusion test and 54/173 (31.2%) were positive by cefoxitin MIC E-test. The isolates which were positive by screening test were tested further by confirmatory test such as TDET, AmpC disk test, Boronic acid disk test and Disk approximation method and the percentage prevalence of AmpC β-lactamases detected by all the above methods are depicted in [Table 3]. Among the various phenotypic tests, the cefoxitin disc diffusion test showed sensitivity of 91.67%, specificity of 59.14%, positive predict value of 46.48%, and negative predict value of 94.83% when compared with PCR.
Table 3: Screening and confirmatory test for AmpC β- lactamases

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Genotypic characterization of extended spectrum beta lactamase and AmpC producing Escherichia coli isolates

The isolates which tested positive for ESBL production by screening and confirmatory tests were further genotyped for the presence of blaTEM, blaSHV, blaCTX-M, blaPER and blaVEBβ-lactamases genes in case of ESBL and for AmpC β-lactamases MOXM, CITM, DHAM, ACCM, EBCM, FOXM genes were detected by PCR. The genotypic prevalence of these genes are shown in [Table 4].
Table 4: Genotypes of extended spectrum beta lactamase and AmpC producing Escherichia coli isolates

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  Discussion Top


The increasing incidence of antimicrobial resistance is a major global public health issue in India. This had led to prolonged hospital stay for the patients, limited antibiotic options to treat, financial crisis, increased morbidity and mortality of patients. The current study was done to understand the prevalence of ESBL and Amp-C producing E. coli isolated from clinical specimens. The prevalence of E. coli producing ESBL and Amp-C β-lactamases varies from one geographical region to another.

In our study, the prevalence of E. coli producing ESBL was found to be 93.12% over a period of 3 years. The prevalence of ESBL in our study was high as compared to other previously published studies from India which ranged from 6% to 87%.[13],[14],[15],[16] Among the third-generation cephalosporins used for screening, ceftazdime had a better sensitivity and positive predictive value followed by CTX and CZD.

The percentage prevalence of AmpC β-lactamases in our study was low (28.6%) as compared to other studies from India.[17],[18],[19] The prevalence of ESBL + AmpC Phenotype was 19.65% which correlates well with the previously published studies.[20],[21] The cefoxitin disk used for screening of AmpC β-lactamases had a good sensitivity and negative predictive value making it a reliable marker for Amp-C production when compared with the molecular detection method (PCR). The multiplex PCR method used in our study will be an important technique for the identification of plasmid-mediated AmpC β-lactamase genes in E. coli.

Most of the typeable E. coli isolates possessed two or more ESBL genes (42.34%). Overall, the commonest genotype was bla (TEM) (67.30%) followed by bla (CTX-M) (63.26%), bla (SHV) (16.81%), bla (PER) (19.40%) and bla (VEB) (2.61%) which is similar to other studies.[19],[10],[22] In case of Amp-C β-lactamases, FOX gene was predominant (21.9%) followed by CIT AmpC (9.19%). Similar study done by Manoharan et al. have also shown to have FOXM (43.7%) gene to be commonest among E. coli isolates.[19] Thus the combination of phenotypic and molecular identification methods would serve as a better tool for the detection of ESBL and AmpC mediated resistance in diagnostic laboratories.


  Conclusion Top


The increasing prevalence of ESBL and Amp-C β-lactamases hampers the use of β-lactam antibiotics used for treatment. Early detection of the resistance mechanism mediated by these enzymes would help in controlling the antibiotic resistance in the hospital settings. Our study also indicates the need for continued surveillance of antibiotic resistance pattern of nosocomial pathogens thus aiding in control measures.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Manoharan A, Sugumar M, Kumar A, Jose H, Mathai D, Khilnani GC, et al. Phenotypic & molecular characterization of AmpC β-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. From five Indian medical centers. Indian J Med Res 2012;135:359-64.  Back to cited text no. 19
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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