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 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 15  |  Issue : 2  |  Page : 45-48

Comparison of various methods for detection of AmpC β-lactamase enzyme


Department of Microbiology, Bharati Vidyapeeth University Medical College, Pune, Maharashtra, India

Date of Web Publication7-Jan-2014

Correspondence Address:
Vrushali Harsh Thakar
Department of Microbiology, Bharati Vidyapeeth University Medical College, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1282.124585

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  Abstract 

Fifty multidrug-resistant bacterial isolates were tested for extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase enzyme production. Cefoxitin (30 μg) resistance was used as the screening test for AmpC enzyme detection. Phenotypic confirmation was done by conventional three-dimensional enzyme extract test (TDET), AmpC disc test and modified three-dimensional test (MDT). A total of 45 (90%) strains were AmpC positive by the screening test. Out of these 45 strains, 40 strains were positive by AmpC disc test, 39 strains by TDET and 38 strains by MDT. Cefoxitin resistance can be used as the screening test. AmpC disc test is simple to perform and gives rapid result. So, it can be used as a phenotypic method for detection of AmpC enzyme production in resource-limited settings where genotypic detection methods are not available.

Keywords: AmpC-b, disc test, lactamase, three-dimensional enzyme extract test


How to cite this article:
Thakar VH, Modak M. Comparison of various methods for detection of AmpC β-lactamase enzyme. J Acad Clin Microbiol 2013;15:45-8

How to cite this URL:
Thakar VH, Modak M. Comparison of various methods for detection of AmpC β-lactamase enzyme. J Acad Clin Microbiol [serial online] 2013 [cited 2017 Apr 26];15:45-8. Available from: http://www.jacmjournal.org/text.asp?2013/15/2/45/124585


  Introduction Top


A common mechanism of bacterial resistance to β-lactam antibiotics is the production of β-lactamase enzyme. [1] In the last two decades, extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases have gained increased attention.

AmpC β-lactamases are clinically more important because they confer resistance to a wide variety of β-lactam drugs, including Cefoxitin, narrow, expanded, and broad-spectrum Cephalosporins, β-lactam β-lactamase inhibitor combinations and Aztreonam. [2],[3] In case of inducible AmpC β-lactamases, Amoxicillin-Clavulanic acid combination can cause more harm than good. [2] So, detection of resistant isolates can help clinicians to implement appropriate antibiotic policies.

Clinical and Laboratory Standards Institute (CLSI) has not provided clear-cut guidelines for the detection of AmpC β-lactamases. There are many methods described by various authors. [1],[2],[3],[4] This study was done to compare the different methods for detection of AmpC β-lactamases. However, since there is no accepted gold standard other than detection of the specific gene by molecular methods, which is out of the scope of this study, statistical evaluation as a diagnostic test could not be done.


  Materials and Methods Top


This study was conducted during a period of 6 months from 1 October 2009 to 30 March 2010. Antibiotic sensitivity testing was done by Kirby-Bauer's method as per CLSI guidelines.  Escherichia More Details coli and Klebsiella isolates from various clinical samples showing resistance to all third-generation Cephalosporins were selected for the study.

The following antibiotics were tested: 1. Cefoxitin (30 μg); 2. Ceftazidime (30 μg); 3. Ceftazidime + Clavulanic acid (30 μg + 10 μg); 4. Piperacillin (100 μg); 5. Piperacillin + Tazobactam (100 μg + 10 μg); and 6. Imipenem (10 μg).

Screening tests for AmpC and ESBL enzyme detection were done as described by Sinha et al. [4],[5]

Screening test

  1. An organism was considered AmpC β-lactamase producer if
    1. It was resistant to Cefoxitin;
    2. There was no potentiation of zone size with Clavulanic acid and/or
    3. There was increase in zone size ≥5 mm with Piperacillin-Tazobactam combination. [4]
  2. An organism was considered ESBL producer if
    1. It was sensitive to Cefoxitin;
    2. It was resistant or intermediate sensitive to Ceftazidime and
    3. There was potentiation of inhibition zone with Clavulanic acid (≥5 mm).
  3. An organism was considered AmpC + ESBL producer if
    1. It was resistant or intermediate sensitive to Cefoxitin;
    2. It was sensitive or resistant to Ceftazidime and
    3. There was potentiation of inhibition zone with Clavulanic acid (≥5 mm).


Phenotypic detection methods for detection of AmpC enzymes

Control strains used in all methods - negative control ATCC E. coli 25922 and positive control Klebsiella pneumoniae 700603.

AmpC disc test

100× Tris ethylenediaminetetraacetic acid (EDTA) (pH 8.0) was prepared as described by Sambrook et al. [6]



Mix 61.24 ml of solution A and 38.76 ml of solution B to get 100× Tris HCl buffer pH 8.0.

Add 3.74 g of EDTA for preparing 100 × 0.1 M Tris EDTA.

AmpC discs were prepared in-house as described by Black et al. [1] by applying 20 μl of 1:1 mixture of saline and 100 × Tris EDTA to sterile filter paper discs, and then allowing the discs to dry and storing them at 2-8°C.

A lawn culture of ATCC E. coli 25922 was prepared on Mueller Hinton agar (MHA). Cefoxitin (30 μg) disc was placed. AmpC discs were rehydrated with 10 μl saline and several colonies of the test organism were applied to the disc. AmpC discs were placed almost touching the Cefoxitin disc, with the inoculated disc face in contact with the agar surface. Plates were incubated at 37°C overnight. Flattening or indentation of zone of inhibition of Cefoxitin was considered as AmpC positive and no distortion of zone as AmpC negative [Figure 1].
Figure 1: AmpC disc test showing positive control; negative control; 1, 4, 3 positive (showing indentation); and 2 borderline positive

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Conventional three-dimensional enzyme extraction method (TDET)

Enzyme preparation was done as described by Shahid et al. [3] Fifty microlitres of 0.5 McFarland's bacterial suspension prepared from an overnight blood agar (BA) plate was inoculated in 12 ml of peptone water and the culture was grown for 4 h at 37°C. The cells were concentrated by centrifugation and crude enzyme preparations were made by freeze-thawing the cell pellets 5-6 times. A lawn culture of ATCC E. coli 25922 was prepared on MHA. Cefoxitin (30 μg) disc was placed in the centre. With a sterile scalpel blade, a slit beginning on the edge of disc was cut within agar in an outward radial direction. Using a pipette, 25-30 μl of the enzyme preparation was dispensed into the slit, beginning near the disc and moving outwards. The plates were incubated overnight at 37°C. Zone distortion (flattening or indentation) was considered as AmpC positive and no zone distortion as AmpC negative [3] [Figure 2].
Figure 2: TDET showing positive control, negative control, 3 positive (showing indentation), 2 borderline and 1 negative

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Modified three-dimensional tests (MTDT)

This test was done as described by Shahid et al. [3] Instead of the enzyme extract, 5-10 colonies of the test organism were inoculated with the help of nichrome loop in the slit, beginning near the disc and moving outwards. After overnight incubation, the plates were observed for zone distortion (AmpC positive) or no zone distortion (AmpC negative) [Figure 3]. [3]
Figure 3: MTDT showing positive control; negative control; 1, 2 positive; and 3 negative

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  Results Top


A total of 50 strains which were multidrug resistant were tested for AmpC enzyme production. Of these, 30 (60%) were E. coli and 20 (40%) were Klebsiella spp. Forty-five (90%) strains were positive for AmpC enzyme production by the screening method.

Out of the 45 strains, 40 (88.8%) were positive for AmpC production by phenotypic methods. Of these 40 strains, 24 (60%) were E. coli and 36 (40%) were Klebsiella spp.

[Table 1] shows the number of strains detected by each phenotypic method. Eighteen (36%) strains were ESBL positive. All 18 isolates that were ESBL positive were also positive for AmpC production. All AmpC positive isolates were sensitive to Imipenem and Piperacillin-Tazobactam combination.
Table 1: Methods & Results

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  Discussion Top


With the spread of AmpC and ESBL producing strains all over the world, it is necessary to know the prevalence of these strains in hospitals. Conventional three-dimensional enzyme extraction test is difficult to perform. Hence, most clinical laboratories are finding it difficult to routinely report the presence of AmpC enzyme.

In our study, we found that 90% strains were AmpC positive by the screening test. 30% strains were positive for ESBL production, but they were also positive for AmpC enzyme. This shows that AmpC enzymes are outnumbering ESBL in our hospital and are becoming a cause for concern.

Of the isolates, 90% were positive by the screening test and 88% were detected by phenotypic methods. For the remaining five isolates, the cause of resistance remains unexplained, as they were ESBL negative.

There was no significant difference between the results obtained by the three phenotypic methods, but AmpC disc test was easy to perform and also gave a rapid result. Similar findings were observed by Sinha et al. [5] and Singhal et al. [7] Conventional TDET could not identify one isolate. This isolate was borderline positive (flattening of zone) by AmpC disc test. Probable reason for enhanced sensitivity of AmpC disc test is the use of Tris EDTA, which induces AmpC enzyme production.

AmpC disc test is sensitive, simple to perform and gives rapid results. So, we recommend that Cefoxitin resistance be used as a screening method for detection of AmpC β-lactamase producing strains and AmpC disc test as a phenotypic detection method in resource-limited settings where genotypic methods are not available.

 
  References Top

1.Black JA, Moland ES, Thomson KS. AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 2005;43:3110-3.  Back to cited text no. 1
[PUBMED]    
2.Arora S, Bal M. AmpC beta-lactamase producing bacterial isolates from Kolkata Hospital. Indian J Med Res 2005;122:224-33.  Back to cited text no. 2
    
3.Shahid M, Malik A, Agarwal M, Singhal S. Phenotypic detection of extended spectrum and AmpC beta-lactamases by a new spot-inoculation method and modified three-dimensional extract test: Comparison with the conventional three-dimensional extract test. J Antimicrob Chemother 2004;54:684-7.  Back to cited text no. 3
    
4.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. 4
[PUBMED]  Medknow Journal  
5.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.  Back to cited text no. 5
[PUBMED]  Medknow Journal  
6.Sambrook J, Russell D. Molecular cloning. A Laboratory Manual. Vol 3. 3 rd ed. Cold Spring Harbor Laboratory Press, New YORK; 2001. p. A1.7.   Back to cited text no. 6
    
7.Singhal S, Mathur T, Khan S, Upadhyay D, 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. 7
[PUBMED]  Medknow Journal  


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