Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 8  |  Issue : 5  |  Page : 599-605  

Evaluation of phenotypic tests and screening markers for detection of metallo-β-lactamases in clinical isolates of Pseudomonas aeruginosa: A prospective study


Department of Microbiology, M. S. Ramaiah Medical College, Bengaluru, Karnataka, India

Date of Web Publication10-Sep-2015

Correspondence Address:
Shikha Ranjan
Department of Microbiology, K. G. Hospital and Postgraduate Medical Institute, Coimbatore - 641 018, Tamil Nadu
India
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Source of Support: Nil., Conflict of Interest: None declared.


DOI: 10.4103/0975-2870.164977

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  Abstract 

Purpose: This study was conducted to estimate the prevalence of metallo-β-lactamases (MBL)-producing Pseudomonas aeruginosa isolates obtained from various clinical samples and to compare the diagnostic strength of different phenotypic MBL-detection tests and also to know the performance of ethylene diamine tetraacetic acid (EDTA) disk potentiation test (PT) which is the least studied. Materials and Methods: This study included 160 nonconsecutive isolates of P. aeruginosa collected over a period of 1-year. Resistance to carbapenems and ceftazidime was used for screening of isolates. Positively screened isolates were further subjected to five different MBL detecting phenotypic tests-MBL Epsilometer test (E-test), combined disk test (CDT), double-disk synergy test (DDST), EDTA disk PT using four cephalosporins and modified Hodge test (MHT). MBL E-test was considered as gold standard for MBL detection. Results: Based on the screening criteria for MBL production, 66 isolates were screened positive. The prevalence of MBL producing isolates of P. aeruginosa was 15% (24/160) based on E-test result. MHT showed the highest sensitivity (87.5%), followed by CDT (79.2%), while specificity was highest for DDST (100%), followed by PT (95.2%). Out of 24 MBL producers, 15 isolates (62.5%) were resistant to both imipenem (IPM) and meropenem. Conclusion: The early detection of MBL-producing P. aeruginosa may help inappropriate antimicrobial therapy and avoid the development and dissemination of these strains. Hence, routine detection of MBL production in P. aeruginosa should be undertaken. We recommend that all IPM and/meropenem-resistant P. aeruginosa isolates should be routinely screened for MBL production using CDT and the positive isolates may further be confirmed by MBL E-test or PCR. EDTA disk PT had low sensitivity.

Keywords: Epsilometer-test, metallo-β-lactamases, phenotypic detection methods, prevalence, Pseudomonas aeruginosa


How to cite this article:
Ranjan S, Banashankari GS, Babu PR. Evaluation of phenotypic tests and screening markers for detection of metallo-β-lactamases in clinical isolates of Pseudomonas aeruginosa: A prospective study. Med J DY Patil Univ 2015;8:599-605

How to cite this URL:
Ranjan S, Banashankari GS, Babu PR. Evaluation of phenotypic tests and screening markers for detection of metallo-β-lactamases in clinical isolates of Pseudomonas aeruginosa: A prospective study. Med J DY Patil Univ [serial online] 2015 [cited 2020 Sep 19];8:599-605. Available from: http://www.mjdrdypu.org/text.asp?2015/8/5/599/164977


  Introduction Top


Carbapenems have been used as the last resort antimicrobials in the treatment of serious infections caused by Gram-negative bacteria. However, the clinical utility of these antimicrobials is under threat with the emergence of carbapenemases, particularly metallo-β-lactamases (MBLs).[1] MBLs belong to Ambler class B and have the ability to hydrolyze a wide variety of β-lactam agents, such as penicillins, cephalosporins, and carbapenems.[2] These enzymes require zinc for their catalytic activity and are inhibited by metal chelators, such as an ethylene diamine tetraacetic acid (EDTA) and thiol-based compounds.[2],[3]

Pseudomonas aeruginosa producing MBLs was first reported from Japan in 1991 and since then has been described from various parts of the world, including Asia, Europe, Australia, South America, and North America.[4],[5] MBL-producing P. aeruginosa isolates have been responsible for serious infections, treatment failure and several nosocomial outbreaks in different parts of the world resulting in high morbidity and mortality, increased economic burden and an urgent need to establish a strong infection control protocol.

Several phenotypic methods are available to detect MBLs. The principle of these methods is based on the ability of metal ion chelators like EDTA or thiol compounds to inhibit the activity of MBLs.[6] These tests include the MBL Epsilometer-test (E-test),[7],[8] a combined disk test (CDT) using EDTA with imipenem (IPM) or ceftazidime (CAZ),[9] double-disk synergy test (DDST) using EDTA and IPM or CAZ,[10] EDTA disk potentiation test (PT) using CAZ, ceftizoxime, cefepime, and cefotaxime,[11] the modified Hodge test (MHT)[10],[12] and 2-mercaptopropionic acid with CAZ or IPM.[11]

This study was conducted to estimate the prevalence of MBL-producing P. aeruginosa isolates obtained from various clinical samples and to compare the sensitivity of different phenotypic MBL-detection tests in a tertiary healthcare center in Southern India. Also to know the performance of EDTA disk PT which is the least studied.


  Materials and Methods Top


This prospective study was conducted over a period of 1-year from January 2011 to December 2011 at a tertiary hospital. Institutional review board approval was obtained, and the principle of the declaration of Helsinki was followed during the study. Considering the prevalence rate of Pseudomonas in hospitalized patients to be about 10%,[13] the sample size for this study was estimated with a relative precision of 15% and desired confidence limit of 95%. The required sample size was 160 after making an allowance of 4% for inadequate or contaminated samples. This study included all nonconsecutive P. aeruginosa isolates obtained from various clinical samples such as urine, pus, sputum, pleural fluid, peritoneal fluid, blood, and cerebrospinal fluid received for culture and sensitivity in the department of microbiology. However, Pseudomonas isolated from stool samples were excluded due to their presence as commensal. Furthermore, Pseudomonas isolated from the sputum samples which were inappropriate as per Gram's stain (Bartlett's grading) were excluded from the study.

All clinical specimens collected were subjected to direct microscopy, growth on culture media and a series of tests for identification of P. aeruginosa. After identification, these isolates were subjected to antibiotic susceptibility testing by Kirby-Bauer disk diffusion technique according to Clinical and Laboratory Standards Institute (CLSI) guidelines. All the disks were procured commercially (Hi-Media Laboratories Limited, Mumbai, India). The diameter of the zone of inhibition was measured and interpreted according to the CLSI guidelines.

The isolates resistant to IPM or meropenem were further tested with IPM and meropenem E-strips. Results were interpreted by the zone of inhibition in the form of an ellipse and the values at which zone intersected the strips were taken as Minimum Inhibitory Concentration (MIC) values. Those isolates showing resistance in the first technique, but susceptibility in later technique were considered as sensitive and were not included in the further study.

Resistance to IPM, meropenem or CAZ or any two or all the three antimicrobials was used as a screening marker for probable MBL production.[3] All positively screened probable MBL producing isolates were subjected to five phenotypic tests for MBL detection, methods of which have been described briefly as follows.


  Metallo-β-lactamase Epsilometer-test Top


The suspension of the test organism (density matching 0.5 McFarland Turbidity Standards) was inoculated onto Mueller-Hinton Agar (MHA) plates by performing lawn culture with a sterile cotton swab. The MBL E-strip containing a double sided seven-dilution range of IPM (4-256 µg/mL) and IPM in combination with a fixed concentration of EDTA (1-64 µg/mL) was placed on the inoculated agar surface. The plate was then incubated overnight at 35°C in ambient air for 18-20 h. MIC ratio of IPM/IPM + EDTA of >8, or reduction of IPM MIC by >3log2 dilutions in the presence of EDTA indicated MBL production.[7],[8]

Imipenem-ethylene diamine tetraacetic acid combined disk test

Two 10 µg IPM disks were placed on the MHA plate inoculated with the test organism, and 10 µL of EDTA solution (750 µg) was added to one of them. The plate was incubated for 16-18 h in ambient air at 35°C. If the increase in inhibition zone with the IPM + EDTA disk was >7 mm than the IPM disk alone, it was considered as MBL positive.[9]

Imipenem-ethylene diamine tetraacetic acid double-disc synergy test

An IPM (10 µg) disc was placed 20 mm center to center from a blank disc containing 10 µL of 0.5 M EDTA (750 µg) on a MHA plate inoculated with the test organism. After incubation at 35°C in ambient air for 16-18 h, enhancement of the zone of inhibition in the area between IPM and the EDTA disc in comparison with the zone of inhibition on the far side of the drug (IPM) was interpreted as a positive result.[10]

Ethylene diamine tetraacetic acid disc potentiation test using ceftazidime, ceftizoxime, cefepime, and cefotaxime

A filter paper blank disk was placed and the following discs (CAZ [30 µg], ceftizoxime [30 µg], cefotaxime [30 µg], cefepime [30 µg]) were placed 25 mm center to center from the blank disk on a MHA plate inoculated with the test organism. 10 µl of 0.5 M EDTA solution was added to the blank disk, and the plate was incubated overnight in ambient air at 35°C. Enhancement of the zone of inhibition in the area between the EDTA disk and any one of the four cephalosporin disks in comparison with the zone of inhibition on the far side of the drug was interpreted as a positive result.[11]

Modified Hodge test

Saline suspension of ATCC 25922  Escherichia More Details coli (matching 0.5 McFarland Turbidity Standards) was diluted 1:10 and the lawn culture of the later were done on to the MHA plate. The plate was allowed to stand for about 5 min at room temperature. A 10 µg IPM disc was placed at the center, and the test organism was streaked in a straight line from the edge of the disc to the edge of the plate. The plate was incubated overnight at 35°C in ambient air for 20-24 h. The presence of a distorted zone of inhibition or clover leaf type of indentation at the intersection of the test organism and E. coli, within the zone of inhibition of the IPM disk, was interpreted as a positive result.[10],[12]

In this study, MBL E-test was taken as a gold standard test for MBL detection and was used to estimate the prevalence of MBL producing isolates of P. aeruginosa. The strengths of other phenotypic tests for MBL detection were compared to that of MBL E-test. Though E-test is not 100% sensitive, but in a developing country like India where molecular detection is not always feasible, we considered E-test as a gold standard.


  Results Top


This prospective study included 160 nonconsecutive P. aeruginosa isolates obtained from various clinical samples. Based on the screening criteria for MBL production, 66 isolates (66/160 = 41.3%) were screened positive. The resistance pattern of these screened isolates for CAZ, IPM and meropenem, is shown in [Table 1]. The five phenotypic tests for MBL detection were performed on 66 positively screened isolates. MBL E-test was positive in 24 isolates, and being a gold standard test for MBL detection with 100% sensitivity, the prevalence of MBL producing isolates of P. aeruginosa was 15% (24 out of 160 isolates).
Table 1: Resistance pattern of screened isolates for CAZ, IPM and meropenem

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As compared to 24 MBL positive isolates, out of 66 positively screened P. aeruginosa isolates, MHT was positive in 26 isolates, CDT was positive in 23 isolates, DDST was positive in 17 isolates and PT was positive in 15 isolates. In this study, MHT showed the highest sensitivity (87.5%) while PT was the least sensitive test (54.2%). False-positive isolates were found in MHT, CDT, and PT, but DDST showed the highest specificity (100%) with no false-positive result. [Table 2] shows sensitivity and specificity all the phenotypic tests with MBL E-test being the gold standard for MBL detection. Graph 1 [Additional file 1] shows the sensitivity of these tests when compared to MBL E-test.
Table 2: Comparison of MBL detection tests with E-test

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Resistance pattern of 24 MBL positive isolates of P. aeruginosa to carbapenems and CAZ was analyzed retrospectively and has been shown in [Table 3]. This analysis showed resistance to both IPM and meropenem as the most powerful screening marker for probable MBL producing isolates accounting for 62.5% of true MBL producers.
Table 3: Sensitivity of different screening tests for 24 MBL positive isolates of Pseudomonas aeruginosa

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  Discussion Top


The discovery and development of antibiotics were undoubtedly one of the greatest advances of modern medicine. Unfortunately, the emergence of antibiotic resistance bacteria is threatening the effective usefulness of many antimicrobial agents resulting in increased days of hospital stay and also an economic burden. With increasing isolation of ESBL-producing isolates in the hospital setting necessitating the use of carbapenems, the problem of MBL production is also increasing.[3] A case-controlled study from Japan showed that patients infected with MBL-producing P. aeruginosa were more likely to receive multiple antibiotics and also infection-related deaths due to MBL-producing P. aeruginosa were more frequent than the deaths caused by MBL-negative P. aeruginosa.[6]

In this study, out of 160 P. aeruginosa isolates, 66 isolates were screened out as probable MBL producers based on their resistance to carbapenems and CAZ. We found 21.3% resistance among P. aeruginosa to IPM and meropenem each while 26.9% resistance to CAZ. A study done at a Tertiary Care Hospital in Puducherry (India) in 2006 reported 10.9% resistance to carbapenems,[14] while another study in Puducherry in 2008 reported 31.1% resistance to meropenem.[15] A 5-year longitudinal study from Latin America has reported that P. aeruginosa resistance to carbapenems has risen to 40%.[16] These findings show there is a rising trend in the carbapenem resistance among the P. aeruginosa. Resistance to the carbapenems in P. aeruginosa is often due to the down-regulation of the porin channels, the up-regulation of an active efflux pump system present in these organisms or the production of MBLs.

Phenotypic tests for MBL have not been nationally or internationally standardized. A consensus methodology for this routine laboratory method remains to be defined, and questions regarding the timing and method of MBL detection remain to be answered.[17] PCR analysis is the gold standard method for the detection of MBL producers, but it is not suitable for daily testing in clinical laboratories due to the cost and inconvenience.[18] The sensitivity of MBL E-test is considered to be 100% and same was the finding by Khosravi et al.[19] and Walsh et al.[7] and also in another study on Acinetobacter baumannii by Segal and Elisha[20] Hence in this study, we used the MBL E-test to confirm the MBL production among positively screened probable MBL producing P. aeruginosa isolates. Out of 66 probable MBL producing isolates, 24 isolates were confirmed as MBL producers by MBL-E test. Thus, the rate of MBL producing isolates among various clinical samples of P. aeruginosa collected in our study was 15% (24/160). In Indian studies, the prevalence of MBL-producing P. aeruginosa has ranged from 7% to 65% with a recent study reporting 34% occurrence.[21] [Table 4] shows the rate of MBL-producing P. aeruginosa from different parts of India and worldwide, which clearly shows that it varies from place to place and also with time at the same place.
Table 4: Percentage of MBL producing  Pseudomonas aeruginosa Scientific Name Search  different studies

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However, it is not possible for all laboratories to perform the E-test due to cost constraints and availability. Though there are other economically feasible methods recommended for the detection of MBL production, no single test when used alone is optimal. The implementation of a simple and accurate laboratory method to detect MBL production in P. aeruginosa is useful, particularly in countries where MBL strains are increasingly reported.[18] We made an attempt to compare the diagnostic strength of other phenotypic tests with that of E-test.

In this study, MHT showed the highest sensitivity (87.5%) for detection of MBL producers, with good specificity (88.1%). However, specificity was lowest among four phenotypic tests. Similar findings were observed in the study conducted by Lee et al.[10] MHT, which precludes the use of EDTA, detects only carbapenemase activity.[32] Moreover, hence carbapenem-resistant P. aeruginosa isolates positive for carbapenemase by MHT may be negative for MBL due to non-MBL carbapenemase, which is not dependent on zinc ion for its action.[33] Though the MHT is a feasible test which is easy and economical to perform to check possible production of MBL, the subjective interpretation of its results and possible lack of an appreciable distorted zone of inhibition are the major drawbacks.[23]

In our study, CDT and DDST also showed good sensitivity for MBL detection as 79.2% and 70.8% respectively. Though the specificity of DDST (100%) was better than that of CDT (90.5%) in this study, the subjective interpretation of results of DDST was its major disadvantage. In addition to the objective interpretation of results, CDT was rapid and easily performed test. Hence, we found the CDT to be satisfactory for detecting MBL despite its low specificity as it is an easy procedure and is simple to interpret in accordance with other studies.[8],[9],[34] Similar to our observation, Behera et al.[28] and Qu et al.[29] reported that CDT is better than DDST for routine MBL detection. However, some authors have found DDST as the better test for MBL detection as compared to CDT.[35],[36] DDST can be used for detection but should not be used as the sole indicator for the presence of MBLs.[18] EDTA disc PT was not a useful test for MBL detection, as in our study its sensitivity was significantly low (54.2%). Manoharan et al. observed the similar finding in their study.[3]

Except MHT, all of the MBL detecting tests, that is, CDT, DDST and PT are based on the use of EDTA. It is important to note that EDTA has membrane-permeabilizing properties and could exert a deleterious effect on P. aeruginosa; thus, the extended zone size difference between the IPM and IPM-EDTA disks in the CDT and IPM and EDTA disks in the DDST may be due to the susceptibility of the organism to EDTA rather than its metal-chelating effect that inactivates any MBL, thus resulting in false-positive detection.[37] Hence, caution must be taken in using only EDTA as the inhibitor agent when analyzing MBL production, as this method may lead to false-positive results.[17] In our study, all tests including MHT, CDT and PT detected false-positive MBL producers, except DDST, which was most specific with no false-positive result. Similarly, a number of previous studies have reported that DDST was more specific in detecting MBLs in comparison to the CDT.[5],[10],[19] Other than EDTA-related false positivity, these false-positive cases might actually be producing an unknown and weaker β-lactamases, which is worth further investigation.

CDT and DDST both are limited by factors like temperature, aeration, pH and thickness of media. However, the synergy between IPM and IPM + EDTA disk is influenced by diffusion. EDTA must diffuse close to the IPM disc and achieve a concentration with effective chelating activity to demonstrate a synergy. This may explain the difference in results of CDT and DDST.[31] Although some authors recommend using a CAZ disc instead of IPM disc for CDT/DDST,[11] MBL producing organisms may have other CAZ resistance mechanisms. With such strains, CDT/DDST using CAZ will not show MBL production and, therefore, IPM disc must be used for detection of MBL.[28] The CDT has been further validated against a PCR and has been found to outperform CAZ and meropenem EDTA combination.[3] On the basis of our result and the related discussion, we recommend CDT for routine detection of MBL as it has good sensitivity (79.2%) and specificity (90.5%), is easy to perform, has objective interpretation of result and is economical.

In addition to finding out the accurate and easy MBL detecting phenotypic test, we also analyzed the resistance pattern of MBL producing isolates in an attempt to find out the best screening marker, so that number of isolates to be tested by different phenotypic tests could be minimized. Out of the 24 MBL producers, 15 isolates (62.5%) were resistant to both IPM and meropenem, 7 isolates (29.2%) were resistant to all the three antibiotics, that is, CAZ, IPM and meropenem, while the remaining 2 (8.3%) MBL producers were resistant to meropenem. CAZ resistance alone was not seen in MBL producing isolates in our study and hence, using CAZ alone as the screening marker was not helpful for MBL detection. Based on analysis of [Table 3], we conclude that the best screening marker for MBL detection in P. aeruginosa is isolate resistant to both IPM and meropenem. However, some studies have suggested the inclusion of CAZ resistance as an additional criterion along with IPM and meropenem resistance, for MBL screening.[6],[38]

The present study underlines the unique problem with MBLs, because of their broad spectrum and unrivalled drug resistance, creating the therapeutic challenge for clinicians and microbiologists. Hence, routine detection of MBL production in P. aeruginosa should be undertaken. As there are various MBL genes identified that varies from one geographical location to another hence the MBL detection tests should be assessed and followed based on the local condition. The early detection of MBL-producing P. aeruginosa may help in appropriate antimicrobial therapy and avoid the development and dissemination of these multi-drug resistant strains. We recommend that all IPM and/meropenem-resistant P. aeruginosa isolates should be routinely screened for MBL production using CDT and the positive isolates may further be confirmed by MBL E-test or PCR.

 
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    Tables

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


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