|Year : 2013 | Volume
| Issue : 1 | Page : 7-10
Phenotypic detection of β-lactamases in enterobacteriaceae using a 12-disk procedure
Chithra Valsan, Jily P Chinnan, KA Sathiavathy
Department of Microbiology, Jubilee Mission Medical College and Research Institute, Thrissur, Kerala, India
|Date of Web Publication||3-Aug-2013|
Department of Microbiology, Jubilee Mission Medical College and Research Institute, Thrissur, Kerala
Source of Support: Jubilee Mission Medical College and Research Institute, Thrissur, Conflict of Interest: None
Background: Laboratory detection of various β-lactamases like extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, carbapenemases, etc., is often complicating and challenging to the clinical laboratories. The prevalence of these resistance mechanisms varies from place to place and from hospital to hospital. Aim: This study was aimed at detecting the prevalence of these resistant phenotypes among Enterobacteriaceae isolates, along with assessing the use of a 12-disk procedure for the same. Results: We found that the prevalence of ESBLs was 60%, AmpC was 10%, and carbapenemases was 12.6% in these isolates. All the strains which were pure ESBL, AmpC, or carbapenemase producers, and were picked up by the Vitek, were also detected by the 12 disc method. But in some of the strains where ESBLs coexisted with AmpC, the presence of AmpC masked the effect of clavulanate enhancement which is used for the detection of ESBLs. Conclusion: Resistant bacteria are prevalent in this hospital and this 12 disc method helps in identifying a majority of them.
Keywords: 12-disk procedure, β-lactamases, AmpC, carbapenemases, Enterobacteriaceae
|How to cite this article:|
Valsan C, Chinnan JP, Sathiavathy K A. Phenotypic detection of β-lactamases in enterobacteriaceae using a 12-disk procedure. J Acad Clin Microbiol 2013;15:7-10
|How to cite this URL:|
Valsan C, Chinnan JP, Sathiavathy K A. Phenotypic detection of β-lactamases in enterobacteriaceae using a 12-disk procedure. J Acad Clin Microbiol [serial online] 2013 [cited 2017 Apr 29];15:7-10. Available from: http://www.jacmjournal.org/text.asp?2013/15/1/7/116090
| Introduction|| |
Multidrug resistance in Enterobacteriaceae is an alarming condition in the treatment of life-threatening infections with these organisms. They result in therapeutic failure with associated increased mortality and morbidity. Production of β-lactamases such as extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, carbapenemases, etc., is the major mechanism responsible for resistance to β-lactam agents among Enterobacteriaceae. The existing data show a wide variation in the prevalence of these mechanisms from region to region or even from hospital to hospital in the same region. ,
Accurate and timely detection of these mechanisms is very important in deciding the appropriate treatment schedule. Detection of the resistant mechanisms is always a serious challenge to the clinical laboratories. Many of such resistant mechanisms are not always detectable in routine susceptibility tests. Often, many isolates carry more than a single resistance mechanism which makes their detection and reporting even more complicating. 
The present study was carried out to find out the prevalence of resistant phenotypes and the prevalence of coexistence of these resistance mechanisms in Enterobacteriaceae isolates from clinical samples in a 1500-bedded teaching hospital. In this context, we also assessed the use of a novel 12-disk method for the simultaneous detection of these different mechanisms. 
| Materials and Methods|| |
The study was conducted in the Department of Microbiology, Jubilee Mission Medical College, Thrissur, Kerala, India. All the isolates of Enterobacteriaceae obtained from clinical samples during a 3-month period (from September 2012 to November 2012) were included in the study. The isolates were identified and antibiotic sensitivity testing (AST) was done using standard microbiological methods which included manual methods as well as automated method (Vitek 2 systems).
Along with the routine susceptibility testing, we used a 12-disk method also for the phenotypic detection of different β-lactamases such as ESBLs, AmpCs, K1 β-lactamase, and carbapenemases simultaneously using a combination of antibiotic disks in a single 150-mm Mueller Hinton agar plate  as shown in [Figure 1]. The results of the AST were interpreted as per the Clinical and Laboratory Standards Institute (CLSI) guidelines.  An isolate showing an increase in zone size of ≥5 mm with clavulanate compared with plain ceftazidime/cefotaxime disks was interpreted as ESBL positive. AmpC was detected when the isolate showed sensitivity to cefepime disk and resistance to cefoxitin/cefotetan disks. Carbapenemase production was indicated by resistance to the carbapenem agents. 
| Results|| |
During the 3-month period, a total of 398 isolates of Enterobacteriaceae were isolated from clinical samples. Among the 398 isolates, there were 231 isolates of Escherichia More Details coli, 132 Klebsiella spp, 28 Enterobacter spp, 4 Citrobacter spp, and 3 Proteus spp [Table 1]. On subjecting them to the 12-disk antimicrobial susceptibility testing, it was found that out of the 398 isolates, 242 (60%) were ESBL producers, 40 (10%) were AmpC producers, and 50 (12.6%) were carbapenemase producers [Table 2].
Among them, we found that 182 (46%) isolates were pure ESBL producers [Figure 2], 18 (4.5%) isolates were only AmpC producers [Figure 3], and 12 (3%) isolates were pure carbapenemase producers. Also, in 22 (5%) isolates, ESBL coexisted with AmpC β-lactamases [Figure 4] and in 38 (9%) isolates, ESBL coexisted with carbapenemases [Figure 5]. The results of 12-disk method were compared with those of Vitek and are shown in [Table 3].
|Figure 2: ESBL-positive E. coli (a difference in zone sizes of ≥5 mm between cefotaxime/ceftazidime with and without clavulanic acid containing disks indicates ESBL production)|
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|Figure 3: AmpC-positive E. coli (cefepime sensitive and cefoxitin/ cefotetan resistant; no clavulanate enhancement - ESBL negative)|
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|Figure 4: Klebsiella pneumonia producing ESBL and AmpC b-lactamases (ESBL positive - clavulanate enhancement present; AmpC positive - cefepime sensitive and cefoxitin/cefotetan resistant)|
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|Figure 5: Klebsiella pneumonia producing ESBL and carbapenemases (clavulanate enhancement present - ESBL positive; resistance to carbapenem agents indicates carbapenemase production)|
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All the 182 isolates which were detected as pure ESBL producers and the 18 isolates which were detected as pure AmpC producers by the Vitek 2 systems could also be detected by the 12-disk method. Among the 22 isolates in which ESBLs coexisted with AmpC as detected by Vitek, the 12-disk method could not detect the presence of ESBL in three isolates and were interpreted as pure AmpC producers. Similarly, 11 isolates in which Vitek showed the presence of both ESBL and carbapenemases were detected as only carbapenemase producers by the 12-disk method.
| Discussion|| |
The newer β-lactamases like ESBLs, AmpC, and carbapenemases have emerged as a cause of antibiotic resistance in Gram-negative bacteria worldwide in the recent years. The presence of ESBLs and AmpC β-lactamases in a single isolate often reduces the effectiveness of β-lactam-β-lactamase inhibitor combinations. Timely detection of resistance mechanisms is very crucial in deciding the treatment. Even though there are several tests for the laboratory detection of them, none of them can accurately detect all of them with 100% sensitivity and specificity. Also, co-expression of these mechanisms makes their detection more complicated. We conducted this study to detect the different resistant phenotypes in Enterobacteriaceae among our isolates and also to assess the use of a novel 12-disk procedure for the same.
A total of 398 isolates of Enterobacteriaceae were subjected to antimicrobial susceptibility testing by manual and automated method using Vitek 2 compact systems. We found that among our isolates, the prevalence of ESBL was 60%, AmpC was 10%, and carbapenemases was 12.6%. In general, previous studies have shown an overall prevalence of ESBL that varies from 6 to 87%. ,, Since ESBL-positive strains often show false susceptibility to extended-spectrum cephalosporins in standard disk diffusion tests, it is difficult to reliably detect ESBLs by routine disk diffusion methods. Since their presence is so rampant in Gram-negative bacteria, all laboratories should look for them by CLSI recommended methods.
In the present study, AmpC was detected in 10% of the isolates of which 55% coexisted with ESBLs. A similar study reports that Amp-CCphenotypes were found in 12.5% of the isolates, with 92% of them coexisting with ESBLs.  The prevalence of carbapenemases among the Enterobacteriaceae isolates in our study was 12.6%, which is low compared to some previous reports from India. Another Indian study has reported a prevalence of 7.87% carbapenemase producers among Enterobacteriaceae isolates, which correlates with our finding.  Among these carbapenemase-producing Enterobacteriaceae, 76% showed co-carriage of ESBLs in them. Hence, the present study indicates that there is high level of co-expression of various resistance mechanisms among Enterobacteriaceae, which has been reported in many previous studies also. 
We also assessed the usefulness of a novel 12-disk procedure where combinations of different antibiotic disks are used for the detection of various resistant phenotypes among the Gram-negative bacilli. All the strains which were pure ESBL, AmpC, or carbapenemase producers, and were picked up by the Vitek, were also detected by this method. But in some of the strains where ESBLs coexisted with AmpC, the presence of AmpC masked the effect of clavulanate enhancement which is used for the detection of ESBLs. Hence, in them, the presence of ESBLs could not be detected and were detected as only AmpC producers by the 12-disk method. But for the clinical laboratories, this is not a major issue as the treatment options in both these conditions are similar. When ESBL coexisted with carbapenemases, the presence of ESBL could not be detected in some of these strains. But in general, we found this a useful and cost-effective method that can be adopted by the routine clinical laboratories where automated systems or molecular facilities are not available. The chance of a resistant strain being reported as sensitive is very little with this method. The other resistance mechanisms like K1 β-lactamase, Serratia marcesans (SME) carbapenemases, etc., which also can be detected by this 12-disk method as claimed in the original paper, were not detected in this study. The reason may be the small sample size and short duration of our study.
The drawback of the present study is that we have not confirmed the results of our study with molecular method. But we would like to highlight the need for some simple, reliable methods like this 12-disk method that can be adopted by routine clinical laboratories where advanced automated methods or molecular facilities are not available. Regular antimicrobial susceptibility monitoring by the clinical laboratories by some standard methods has got an important role in identifying the resistant pathogens, thus facilitating efficient infection control measures to curb their spread and improve patient outcome.
| Conclusion|| |
Early detection of changing clinical patterns is very important in preventing the dissemination of resistant bacteria and modifying the treatment strategies. The prevalence of these resistant bacteria varies from one place to another or even from one hospital to another in the same region. Hence, clinical laboratories should look for the presence of these resistance mechanisms by standard methods and accurately report them so that timely action can be taken to curb their spread.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]