The Scientific World Journal

Volume 2012 (2012), Article ID 654939, 7 pages

doi:10.1100/2012/654939

Research Article

Phenotypic Detection of Metallo--Lactamase in Imipenem-Resistant Pseudomonas aeruginosa

Yalda Khosravi,¹ Mun Fai Loke,² Eng Guan Chua,¹ Sun Tee Tay,¹ and Jamuna Vadivelu¹

¹Department of Medical Microbiology, University of Malaya, 50603 Kuala Lumpur, Malaysia

²Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Received 19 March 2012; Accepted 12 April 2012

Academic Editors: I. F. N. Hung, Z. Markiewicz, and J. Sanchez

Copyright © 2012 Yalda Khosravi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Carbapenems are the primary choice of treatment for severe Pseudomonas aeruginosa infection. However, the emergence of carbapenem resistance due to the production of metallo-β-lactamases (MBLs) is of global concern. In this study, 90 imipenem- (IPM- or IP-) resistant P. aeruginosa (IRPA) isolates, including 32 previously tested positive and genotyped for MBL genes by PCR, were subjected to double-disk synergy test (DDST), combined disk test (CDT), and imipenem/imipenem-inhibitor (IP/IPI) E-test to evaluate their MBLs detection capability. All three methods were shown to have a sensitivity of 100%. However, DDST was the most specific of the three (96.6%), followed by IP/IPI E-test interpreted based on the single criteria of IP/IPI as positive (62.1%), and CDT was the least specific (43.1%). Based on the data from this evaluation, we propose that only IRPA with IP MIC g/mL and IP/IPI by IP/IPI E-test should be taken as positive for MBL activity. With the new dual interpretation criteria, the MBL IP/IPI E-test was shown to achieve 100% sensitivity as well as specificity for the IRPA in this study. Therefore, the IP/IPI E-test is a viable alternative phenotypic assay to detect MBL production in IRPA in our population in circumstances where PCR detection is not a feasible option.

1. Introduction

Carbapenems, including imipenem (IPM or IP) and meropenem, are the most potent antibacterial agents used for the treatment of infections initiated by multidrug-resistant gram-negative bacilli [1]. However, acquired resistance to carbapenems has been increasingly reported globally, which can be attributed to the evolution of divergent β-lactamases in numerous gram-negative bacteria (including Pseudomonas aeruginosa). P. aeruginosa is an important nosocomial pathogen that is intrinsically resistant to multiple antibiotics. Amongst the various β-lactamases that have been identified to date, the genetically mobile metallo-β-lactamases (MBLs) are the most versatile ones as they are able to hydrolyse all β-lactams except monobactams [2]. Genes encoding for MBL were shown to be carried on large transferable plasmids or were associated with transposons, allowing horizontal transfer of these MBL genes among different bacterial genera and species [3]. To date, five types of acquired MBL genes (IMP, VIM, SPM, GIM, and SIM) have been identified based on their divergent protein molecular structures [4–6]. While IMP and VIM variants have been reported worldwide, members of SPM, GIM, and SIM are restricted to certain geographical regions [7, 8].

Although PCR-based genotyping remains as the golden standard for MBL detection and classification, its use is mainly restricted to research purposes. As genotyping information is necessary, diagnostic centers and laboratories still rely mostly on culture-based phenotypic test as a means for rapid detection of MBL activity. So far, many variations of phenotypic assays for MBLs detection have been reported, and these assays are not standardized. Early detection of MBL-producing organisms is critical as it allows for the prompt use of appropriate antibiotics to effectively control infection. It has been well documented that the activity of MBLs is dependent on zinc or cadmium [4, 9–13]. Several screening methods incorporating the use of metal chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and thiol-based compounds like 2-mercaptopropionic acid (2-MPA), which are capable of blocking MBL activity, have been developed to detect MBL-producing organisms [14–18]. A double-disk synergy test (DDST) using a IPM or ceftazidime (CAZ) disk and a 2-MPA disk, designed by Arakawa et al. [14], was able to indicate the presence of MBLs through the display of an enhanced zone of inhibition around the CAZ disk toward the 2-MPA disk. In addition, a microdilution test [16] and a combined IPM-EDTA disk diffusion method [18] that both utilize EDTA were also developed. The combined IPM-EDTA disk test (CDT) works by comparing the zones of inhibition obtained with IPM disks with and without EDTA [18]. In contrast, the microdilution method compares the minimal inhibitory concentrations (MIC) of IPM with and without EDTA [16]. Both methods were reported to be reliable for the detection of MBLs in carbapenem-resistant Pseudomonas and Acinetobacter strains [16, 18]. Recently a commercial E-test strip (AB BioDisk, Solna, Sweden), which offers antimicrobial resistance testing based on the reduction of MICs of IPM in the presence of chelating agents (EDTA), was also developed. The MBL imipenem/imipenem-inhibitor (IP/IPI) E-test has been reported to be sensitive for the detection of MBLs in Acinetobacter species, P. aeruginosa, Serratia species, Stenotrophomonas maltophilia, and Bacteroides fragilis [17].

In our recent study we reported that prevalence of IPM-resistant P. aeruginosa (IRPA) isolates in Malaysia is high; both IMP and VIM MBLs, but not SIM, GIM, and SPM, were detected in our local isolates [19]. Integron carrying and genes in IRPA were found using a PCR-based method [20]. In this study, three phenotypic methods (CDT, DDST, and IP/IPI E-test) for MBL detection in IRPA clinical isolates were evaluated in comparison to PCR detection of MBL genes as the gold standard.

2. Materials and Methods

2.1. Clinical Isolates

A total of 90 IRPA isolated from various clinical specimens of nonrepetitive patients admitted to the University of Malaya Medical Centre (UMMC) were used for this study [6]. These included (i) 5 isolates from Intensive care unit (ICU) and 25 isolates from other wards, collected from October 2005 to March 2006; (ii) 10 isolates from ICU and 50 from other wards, collected from October 2007 to March 2008. These isolates were identified as P. aeruginosa by the Medical Microbiology Diagnostic Laboratory using routine biochemical confirmatory tests. MBL gene detection and genotyping was carried out as previously described [6].

2.2. Phenotypic Detection of MBL Activity

2.2.1. Combined Disk Test

The test was performed as described by Yong et al. [18]. Briefly, an overnight culture of an IRPA clinical isolate was diluted with peptone water (Oxoid, USA) to 10⁵ CFU/mL and spread on Mueller-Hinton (MH) agar (Difco, France) plate using cotton swab. Two IPM disks (Oxoid, UK) were placed on the surface of the agar at a distance of 4-5 cm from each other. 5 μL of 750 μg/mL EDTA solution (Gibco BRL, USA) was then added to one of the IPM disks (Oxoid, UK). The inhibition zones displayed around the IPM (Oxoid, UK) and the IPM-EDTA disks were compared after 14 to 16 hrs of incubation at 37°C (Figure 1(a)). The difference of ≥7 mm between the inhibition zone diameter of the IPM-EDTA disk and that of IPM only disk was considered to be a positive for the presence of MBLs [18]. The procedure was repeated twice to ensure the reproducibility of results.

(a)

(b)

(c)

Figure 1: Phenotypic tests to detect MBL production. (a) Combined disk test (CDT) showing enhanced inhibition zone of >7 mm around IPM + EDTA disc indicating MBL positivity. (b) Double-disk synergy test (DDST) with synergistic zone of inhibition surrounding IPM and 2-mercaptopropionic acid (2-MPA) disks indicating MBL activity. (c) MBL IP/IPI E-test demonstrating enhanced MIC of imipenem in the presence of EDTA IPI with IP/IPI of ≥8 for MBL activity.

2.2.2. Double Disk Synergy Test (DDST)

DDST was performed according to Lee et al. [13]. Briefly, an overnight culture of an IRPA clinical isolate was diluted with peptone water (Oxoid, USA) to 10⁵ CFU/mL and spread on MH agar (Difco, France) plate using cotton swab. Two IPM disks (Oxoid, UK) were placed on the surface of the agar 4-5 cm (center to center) apart. A blank filter disk (Oxoid, UK) was subsequently placed near one of the IPM disks (Oxoid, UK) at a distance from 1.0 to 1.5 cm, and 3 μL of 2-MPA (Sigma, USA) was applied onto the blank disk. The plate was incubated overnight. The presence of a synergistic inhibitory zone was regarded as MBL positive (Figure 1(b)).

2.2.3. MBL IP/IPI E-Test

An overnight culture of an IRPA clinical isolate was diluted in peptone water to a turbidity of a 0.5 McFarland standard. A cotton swab was then used to transfer the inoculum onto a MH agar plate. Once dried, an E-test MBL strip (AB BioDisk, Solna, Sweden) was applied onto the plate which was then incubated at 37°C for 16 to 18 hrs to detect the presence of metalloenzymes. Interpretation of results was carried out according to the manufacturer’s instructions. A reduction in MIC in the presence of EDTA of greater than or equal to eight-fold (IP/IPI ≥ 8) is interpreted as indicating MBL activity (Figure 1(c)).

2.3. Statistical Analysis

Statistical analysis was carried out using two-tailed Student’s t-test with considered significant. Sensitivity was determined as number of true positives/(number of true positives + number of false negatives). Specificity was calculated as number of true negatives/(number of true negatives + number of false positives). Cutoff values were determined using the SPSS software (IBM, USA) graphically displayed as a receiver-operating characteristic (ROC) curve.

3. Results and Discussion

Among 90 IRPA clinical isolates used in this study, MBL genes (, ) were detected in 32 isolates by PCR. In addition, these 32 isolates were also found to be resistant to at least six antibiotics and, hence, regarded as multidrug-resistant isolates.

In the evaluation of three selected MBL phenotypic assays (CDT, DDST, and IP/IPI E-test) (Table 1), all three methods were shown to have a sensitivity of 100%. However, specificity of phenotypic assays differs. DDST was the most specific of the three (96.6%), followed by IP/IPI E-test with the single criteria of IP/IPI ≥ 8 as positive (62.1%). CDT was the least specific (43.1%). In all three phenotypic assays, false-positive MBL producers were detected. These false-positive cases might actually be producing an unknown and weaker β-lactamases, which is worth further investigation. Here, we demonstrated that DDST was more specific in detecting MBLs in comparison to the CDT as also previously described by Lee et al. [13] and Pitout et al. [12]. On the other hand, this is unlike Qu et al. [21] who demonstrated that the CDT is the best method for screening for MBL production in P. aeruginosa from China. This discrepancy in findings may be due to differences in population structure of MBL genes between different geographical areas (predominantly VIM-2 and IMP-9 in China versus predominantly VIM-2 and IMP-7 in Malaysia). Furthermore, there is no significant difference observed in zone diameter increases for VIM-2-producing P. aeruginosa compared to those for IMP-7-producing isolates.

Table 1: Detection of MBL activity and MBL genes of IRPA clinical isolates from Malaysia.

Bacterial strain
CDT
DDST
IP/IPI E-test
PCR

IMP (mm)
IMP + EDTA (mm)
MBL index
MBL index
IP (μg/mL)
IPI (μg/mL)
MBL index

Ps1
10
22
12
+
+
256
1
256
+
IMP-7

Ps2
12
30
18
+
+
256
4
64
+
IMP-4

Ps3
10
24
14
+
+
256
1
256
+
IMP-4

Ps4
10
22
12
+
+
192
<1
>192
+
VIM-2

Ps5
10
20
10
+
+
192
1
192
+
VIM-2

Ps6
10
24
14
+
+
192
<1
>192
+
VIM-2

Ps7
10
25
15
+
+
192
<1
>192
+
VIM-2

Ps8
10
20
10
+
+
192
<1
>192
+
VIM-2

Ps9
11
21
10
+
+
192
<1
>192
+
VIM-2

Ps10
11
26
15
+
+
192
1
192
+
VIM-2

Ps11
10
24
14
+
+
192
<1
>192
+
VIM-2

Ps12
12
21
9
+
+
192
2
96
+
VIM-2

Ps13
12
22
10
+
+
192
2
96
+
VIM-2

Ps14
10
22
12
+
+
192
<1
>192
+
IMP-7

Ps15
12
30
18
+
+
192
4
48
+
IMP-7

Ps16
10
23
13
+
+
192
2
96
+
IMP-7

Ps17
10
23
13
+
+
192
2
96
+
IMP-7

Ps18
10
27
17
+
+
192
2
96
+
IMP-7

Ps19
10
17
7
+
+
192
2
96
+
IMP-7

Ps20
10
23
13
+
+
128
2
64
+
VIM-2

Ps21
10
25
15
+
+
128
<1
>128
+
VIM-2

Ps22
10
29
19
+
+
128
<1
>128
+
VIM-2

Ps23
10
27
17
+
+
128
1
128
+
VIM-11

Ps24
10
20
10
+
+
128
<1
>128
+
VIM-2

Ps25
10
33
23
+
+
128
2
64
+
VIM-2

Ps26
10
27
17
+
+
128
2
64
+
VIM-2

Ps27
11
21
10
+
+
128
2
64
+
VIM-2

Ps28
12
30
18
+
+
128
1
128
+
IMP-7

Ps29
12
21
9
+
+
128
1
128
+
IMP-7

Ps30
11
21
10
+
+
128
1
128
+
IMP-7

Ps31
10
23
13
+
+
128
1
128
+
IMP-7

Ps32
11
22
11
+
+
96
<1
>96
+
IMP-7

Ps33
10
22
12
+
−
32
6
5
−
−

Ps34
12
18
6
−
−
24
4
6
−
−

Ps35
10
27
17
+
−
24
4
6
−
−

Ps36
10
25
15
+
−
24
4
6
−
−

Ps37
12
23
11
+
−
24
4
6
−
−

Ps38
13
25
12
+
−
24
4
6
−
−

Ps39
10
19
9
+
−
24
4
6
−
−

Ps40
10
18
5
+
−
24
4
6
−
−

Ps41
10
25
15
+
−
24
4
6
−
−

Ps42
12
18
6
−
−
24
4
6
−
−

Ps43
10
20
10
+
−
24
4
6
−
−

Ps44
10
25
15
+
+
24
4
6
−
−

Ps45
9
30
21
+
−
24
4
6
−
−

Ps46
10
24
14
+
−
24
4
6
−
−

Ps47
10
16
6
−
−
24
4
6
−
−

Ps48
12
24
12
+
−
24
4
6
−
−

Ps49
13
20
7
+
−
24
4
6
−
−

Ps50
12
18
6
−
−
16
2
8
+
−

Ps51
10
20
10
+
+
16
2
8
+
−

Ps52
12
18
6
−
−
16
3
5
−
−

Ps53
11
21
10
+
−
16
3
5
−
−

Ps54
10
23
13
+
−
16
3
5
−
−

Ps55
13
25
12
+
−
16
4
4
−
−

Ps56
11
24
13
+
−
16
3
5
−
−

Ps57
11
21
10
+
−
16
4
4
−
−

Ps58
10
27
17
+
−
16
4
4
−
−

Ps59
12
23
11
+
−
16
3
5
−
−

Ps60
10
16
6
−
−
16
3
5
−
−

Ps61
10
22
12
+
−
16
4
4
−
−

Ps62
10
32
22
+
−
16
3
5
−
−

Ps63
10
22
12
+
−
16
4
4
−
−

Ps64
10
20
10
+
−
16
4
4
−
−

Ps65
11
21
10
+
−
12
2
6
−
−

Ps66
14
20
6
−
−
12
2
6
−
−

Ps67
13
18
5
−
−
12
2
6
−
−

Ps68
10
13
3
−
−
12
3
4
−
−

Ps69
12
14
2
−
−
12
4
3
−
−

Ps70
10
27
17
+
−
12
2
6
−
−

Ps71
10
20
10
+
−
8
<1
>8
+
−

Ps72
12
21
9
+
−
8
<1
>8
+
−

Ps73
12
21
9
+
−
8
<1
>8
+
−

Ps74
10
24
14
+
−
8
<1
>8
+
−

Ps75
10
14
4
−
−
8
<1
>8
+
−

Ps76
12
30
18
+
−
8
<1
>8
+
−

Ps77
12
18
6
−
−
8
<1
>8
+
−

Ps78
12
18
6
−
−
8
<1
>8
+
−

Ps79
10
16
6
−
−
8
1
8
+
−

Ps80
11
17
6
−
−
8
1
8
+
−

Ps81
12
18
6
−
−
8
<1
>8
+
−

Ps82
12
18
6
−
−
8
<1
>8
+
−

Ps83
14
18
4
−
−
8
<1
>8
+
−

Ps84
13
17
4
−
−
8
<1
>8
+
−

Ps85
13
19
6
−
−
8
<1
>8
+
−

Ps86
12
18
6
−
−
8
1
8
+
−

Ps87
12
18
6
−
−
8
1
8
+
−

Ps88
10
14
4
−
−
8
<1
>8
+
−

Ps89
11
15
4
−
−
8
<1
>8
+
−

Ps90
12
17
5
−
−
8
<1
>8
+
−

Unlike DDST, which is qualitative, CDT and IP/IPI E-test are both semiquantitative in nature and enabled the calculation of an MBL index. The MBL index of CDT and IP/IPI E-test is an estimate of the relative level of MBL activity and is comparable within the method. However, since there are considerable differences in methodology between CDT and IP/IPI E-test, the MBL indexes obtained by these two methods are incomparable. The main advantage of the IP/IPI E-test has been the only method among the three studied that allows the MIC to be determined. Although significant differences () exist between the MBL indexes of CDT (Figure 2(a)) and IP/IPI E-test (Figure 2(b)) as compared direct detection of MBL genes by PCR, distinct subsets were obvious only with IP/IPI E-test between IRPA with and without MBL genes (Figure 2(c)).

(a)

(b)

(c)

Figure 2: Correlation analysis of combined disk test (CDT), MBL IP/IPI E-test, and PCR detection of MBL genes. (a) MBL index by CDT was 9.5 and 13.4 for IRPA isolates that were negative and positive for MBL genes, respectively (). (b) MBL index by IP/IPI E-test was 6.4 and 134.5 for IRPA isolates that were negative and positive for MBL genes, respectively (). Statistical analysis was carried out using two-tailed Student’s t-test with considered significant. (c) Strong correlation between IP MIC and MBL index with PCR detection of MBL genes. Together, IP > 16 μg/mL and MBL index ≥ 8 were able to distinguish IRPA with MBL genes from those without. The proposed cutoff values were determined from a receiver-operating characteristic (ROC) curve to obtain a 100% sensitivity and specificity for this data set.

The high false-positive reporting rate attributed to CDT is not surprising as the isolates tested in this study were IPM resistant, and, therefore, the inhibition zone of ≥7 mm in zone diameter in the presence of EDTA may not be considered as a definitive clear cutoff criterion to differentiate between MBL-producing and non-MBL-producing IPRA isolates. In view of that, Yong et al. [18] reported that the best separation between MBL-positive and MBL-negative isolates was obtained using a breakpoint of ≥8 mm in the presence of 750 μg of EDTA instead. It is also important to note that EDTA has membrane-permeabilising 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 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 [22].

In the present study, in accordance with our previous PCR findings, the MBL IP/IPI E-test was demonstrated to exhibit 100% accuracy in the detection of MBL production. Our results were similar to the findings of Walsh et al. [17] and also in another study on Acinetobacter baumannii by Segal and Elisha [23]. Since true MBL-producing IRPA tends to be more highly resistant to IPM (higher MIC) than non-MBL-producing IRPA, our results suggest that those isolates with IP MIC < 16 μg/mL be excluded from the determination of MBL status by the IP/IPI E-test. In other words, we suggest that only IP MIC > 16 μg/mL (proposed new criteria) together with IP/IPI ≥ 8 (criteria by manufacturer) by E-test should be taken as MBL activity-positive for IRPA isolated from Malaysian patients. With the new dual criteria, the MBL IP/IPI E-test was able to achieve 100% sensitivity and 100% specificity. A larger-scale study involving more IRPA strains with larger geographical scope will be needed to verify the validity of the new proposed criteria for interpreting the MBL IP/IPI E-test.

Despite the accuracy of MBL IP/IPI E-test detection, it is costly compared to the antibiotic disks to be used by health care institutions or clinical laboratories for routine MBL screening procedure. DDST, which exhibited up to 96.6% specificity, is perhaps a more suitable routine screening procedure to be considered for early detection of MBL-producing bacteria. However, in order to minimize false positivity, isolates positive by DDST can be further confirmed by MBL IP/IPI E-test.

In conclusion, MBL detection remains a controversial issue, and clinical laboratories are in need of a simple and direct method to recognize such resistance in gram-negative bacteria to improve disease management. Furthermore, in recognition that MBL genotypes are not homogenous in geographical distribution, a generalized criteria for interpretation of MBL phenotypic assays may not be possible. Thus, it is recommended that the phenotypic assays should be assessed and adopted based on the local situation.

Conflict of Interests

The authors declare that they have no conflict of interests.

Acknowledgments

This paper is supported by The University of Malaya PPP. Grant (Vote PS296/2007B). The authors would like to thank Professor Emeritus Yunsop Chong and Professor Kyungwon Lee from The Department of Clinical Pathology and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Republic of Korea, for kindly providing the IMP-1, SIM-1, and VIM-2 β-lactamase producing Acinetobacter spp. and P. aeruginosa strains for use as positive controls.

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