Detection of metallo-beta-lactamase producing Pseudomonas aeruginosa using a modified IMP-lysate assay

Since the increasing prevalence of carbapenem-resistant Pseudomonas aeruginosa spp., accurate detection of metallo-β-lactamase (MBL) such as bla VIM type enzyme producing isolates became very important. However, phenotypic MBL detection methods previously reported are not highly sensitive or highly specific. This study aimed to evaluate the performance of a modified IMP-lysate test, the double-disk-synergy-test (DDST) and the combined-disk-test (CDT) for detecting MBL bla VIM gene in P. aeruginosa. The reference technique was PCR molecular test. The study used 12 bla VIM2 producer isolates, 13 MBL-negative controls which included 4 imipenem-susceptible strains and 9 imipenem-resistant strains harbouring bla SHV-2a genes collected from two Tunisian hospitals and P. aeruginosa ATCC27853 and P. aeruginosa COL-1 as negative and positive controls respectively. CDT showed 100% of sensitivity. The highest level of specificity was shown by IMP-lysate test (76.92%). To evaluate efficiencies of methods, the study noted that the highest Youden Index (YI) was shown by IMP-lysate method (0.7), followed by DDST (0.6) than CDT (0.2). Since MBL-Etest and PCR were expensive and not adaptable for extension use in clinical microbiology laboratories, a modified IMP-lysate MBL hydrolytic activity can be chosen by laboratories with limited resources as an inexpensive, simple, and accurate test to detect . P. aeruginosa producing bla VIM enzyme.


INTRODUCTION
The infections caused by multidrug-resistant Pseudomonas aeruginosa strains are becoming increasingly prevalent and now constitute a serious threat to public health worldwide (Oliver et al., 2015;Kaye and Pogue, 2015). Due to their stability against most βlactamases, carbapenems such as imipenem have been *Corresponding author. E-mail: olfasioud@yahoo.fr. Tel: +216 21 557 394. Fax: +216 73 460 678.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License the drug of choice for treatment of infections caused by multidrug resistant P. aeruginosa. Nevertheless, the first isolate of P. aeruginosa with transferable imipenem resistance due to metallo-β-lactamase (MBL) production was reported in Japan in 1991 (Watanabe et al., 1991). Since then, it has been described from various parts of the world (Aubron et al., 2005;Mazzariol et al., 2011;Viedma et al., 2012;Sardelic et al., 2012) including Africa Jacobson et al., 2012;Touati et al., 2013) and Tunisia (Mansour et al., 2009;Hammami et al., 2010;Hammami et al., 2011;Ktari et al., 2011).
Treatment of infectious diseases caused by the carbapenem-resistant P. aeruginosa is becoming more challenging with each passing year. It have been associated with increased mortality and costs due to prolonged hospitalization and prolonged treatment with antibiotics (Liu et al., 2015). MBL producing isolates have an ability to spread and to hydrolyse virtually all β-lactam agents except monobactam (Maltezou, 2009). Detection of this resistance-phenotype by routine laboratories is essential to initiate adequate therapy and to implement proper infection control practices. However, no standardized phenotypic method is available, and previously reported phenotypic combined disk tests, are not highly sensitive or highly specific for the detection of MBL in P. aeruginosa (Peter et al., 2014). The most used phenotypic screening methods in clinical microbiology laboratories are double-disk-synergy-test (DDST), combined-disk-test (CDT) (Lee et al., 2001;Yong et al., 2002;Lee et al., 2003) and the current most widely accepted technique; the MBL E-test ( AB Biodisk, Solna, Sweeden) (Tan et al., 2008). All of these phenotypic tests are based upon the ability of chelating agents, EDTA and thiol-based compounds, to inhibit the MBL activity (Andrade et al., 2007).
A big concern with using these methods is the direct bactericidal effect of EDTA on the test strain (Conejo et al., 2003;Tan et al., 2008) . To circumvent this problem, Tan et al. (2008) proposed an IMP-lysate assay that provides a simple, inexpensive and reproducible functional screen for MBL-producing P. aeruginosa (Tan et al., 2008). The IMP-lysate assay allows EDTA to interact with the carbapenamase in vitro, effectively reducing the concentration of EDTA that interacts with the growth of the indicator strain. However, the IMPlysate test requires specific equipment, such as microcon concentration device (Millipore Bedford, MA) to concentrate the lysate which is not available in most laboratories. So, the study propose a modification in the technique (the study removed the lysate-concentration step with the microcon device, and the study incubated lysate solution with imipenem and EDTA before depositing it on the plate.) without reducing confidence of the assay. The aim of this study was to compare the performance of this modified IMP-lysate test, the DDST and the CDT for detecting MBL bla VIM gene in P. aeruginosa by referring to the molecular test; the PCR.

Clinical isolates
The P. aeruginosa strains used in this study were isolated from clinical specimens collected from separate patients hospitalized in different medical and surgical wards at Fattouma Bourguiba Hospital in Monastir, Tunisia and Tahar Sfar Hospital in Mahdia, Tunisia on variable periods of times (Table 1). Identification of the isolates to the species level was performed by Api 20 NE (Biomerieux, France) and by using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) (AutoflexTM; Bruker Daltonics, Bremen, Germany) with the Flex control software (Bruker Daltonics) (Seng et al., 2009). A total of 12 blaVIM2 producer isolates were screened. MBL-negative controls included 4 imipenem-susceptible strains and 9 imipenemresistant strains harbouring blaSHV-2a genes. BlaVIM2 and blaSHV-2a genes were detected by PCR. Susceptibilities of stains of antimicrobials were performed by the disk diffusion method in Mueller Hinton agar (Bio-Rad, Marnes-la-Coquette, France) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2014). Two control strains were used; P. aeruginosa ATCC27853 as negative control (NC) and P. aeruginosa COL-1 carrying the bla VIM2 gene as positive control.

Combined disk tests (CDT) (method A)
Test organisms were inoculated onto plates of Mueller Hinton according to the guidelines of the CLSI. Two IMP (10 µg) disks (Bio-Rad, Marnes-la-Coquette, France) were placed in an agar plate, and 10 µl of a 0.5 M EDTA solution (pH8) was added to one of the IMP disks. After incubation overnight at 37°C, the inhibition zones of the IMP disks with and without EDTA were compared. A ≥ 7 mm increase in the zone diameter for IMP in the presence of EDTA was interpreted as a positive test result.

Double disk synergy tests (DDST) (method B)
Tests organisms were inoculated onto plates of Mueller-Hinton agar as described for the standard disk diffusion test (CLSI). An IMP (10 µg) disk was placed 20 mm (center to center) from a blank disk containing 5 µl of 0.5 M EDTA (pH 8). Enhancement of the zone of inhibition in the area between the two disks was considered positive for a MBL.

PCR amplification
DNA template preparation was performed as followed. The organisms were cultured on blood agar plate (90 mm diameter) and incubated for 24 h at 37°C. Half of the overnight culture was inoculated into 600 µl of distilled water and vortexed. The cells were lysed by heating them at 100°C for 10 min and cellular debris was removed by centrifugation at 13000 r/min for 10 min. The superntent was used as a source of template for amplification. Standard PCR analysis was used for detection of VIM MBL and SHV Extended-spectrum β-lactamases (ESBL) genes. The specific primers used were: VIM-F: 5'-TGGTCTACATGACCGCGTCT3-3', VIM-R: 5'CGACTGAGCGATTTGTGTG-3', SHV-F: 5'-TTTATGGCGTTACCTTTGACC-3'and SHV-R: 5'-ATTTGTCGCTTCTTTACTCGC-3' (Yagi et al., 2000). Total bacterial DNA was purified with QUIAGEN Kit. Cycling parameters of standard PCR were as follows: an initial denaturation step for 15 min at 95°C, 35 cycles of 1 min at 95°C, 50s at 55°C and 1min at 72°C and a final incubation step for 7 min at 72°C. Positive PCR products were sequenced using BigDye® terminator chemistry on

MBL hydrolytic activity test (method C)
Test organisms were inoculated onto blood agar plate (90 mm diameter) and incubated overnight at 37°C. Half of plate was resuspended in 1.5 ml sodium phosphate buffer (pH 7). The cells were subsequently lysed by cycling between (-20°C) and room temperature (RT) and a vortex (30 s) for a total of five times. The lysed cells were separated from the other debris by centrifugation (10 min, 3.200xg, 4°C) and filtration through a 0.22 µm syringe filter (Minisart, Sartorius Stedim Biotech, Goettingen, German). About 1 ml of filtrate was recuperated each time. Instead of concentrating the filtrate through a microcon device as described by Tan et al. (2008), it was divided equally into two tubes, one of which contained 25 µl of 0.5 M EDTA at pH 8. The two samples were incubated with 10 µg imipenem per ml for 20 min at 37°C and then 15 µl from each tube was transferred into 8 mm blank disks (spaced by 6 cm) in Mueller Hinton agar plates with indicator isolate Escherichia coli ATCC 25922 spread on the surface. On the same plate the study tested P. aeruginosa ATCC27853 (harboring no resistance mechanism) as a negative control and a blank disk with 15 µl of 10 µg imipenem per milliliter solution. The diameter of inhibition zones were measured after overnight incubation at 37°C. The IMP-lysate MBL hydrolytic activity assay was confirmed by comparing the sizes of zone inhibitions for three disks; the lysate-IMP disk impregnated with lysates from P. aeruginosa strain and IMP, the lysate-IMP-EDTA disk with the same lysates incubated with IMP and EDTA and the third IMP disk with imipenem solution. Negative and positive controls must be tested each time. The inhibition zone from the disk (IMP) provided a reference for comparison; it was about 20 mm (data not shown). The diminution of inhibition zone around lysate-IMP disk, compared to the IMP disk, proved the hydrolysis of IMP by the lysate solution. The increase of the zone around the lysate-IMP-EDTA disk compared to the lysate-IMP disk proved that the enzyme hydrolysing IMP was inhibited by EDTA was therefore a MBL.

The Youden's Index (YI)
The YI is the difference between the true positive rate and the false Figure 1. Results for IMP-lysate MBL hydrolytic activity test using disk 1 with lysates from P.aeruginosa COL-1 strain incubated with imipenem (IMP) at 10 µg per ml, disk 2 with the same lysates incubated with IMP and EDTA (0.5 M), disk 3 negative control with lysates from P. aeruginosa ATCC27853 incubated with IMP and disk 4 with EDTA (0.5) alone.
positive rate. It is a commonly used measure of overall diagnostic effectiveness. This index ranges between 0 and 1, with values close to 1 indicating that the biomarker's effectiveness is relatively large and values close to 0 indicating limited effectiveness (Schisterman et al., 2005) YI=sensibility+specificity-1.

RESULTS
The strains used in this study were unrelated; they were collected from two different hospitals, different medical and surgical wards, and at different time periods (Table  1). The positive control sample showed with method C no inhibition zone around disk (lysate-IMP) and 15 mm inhibition zone around disk (lysate-IMP-EDTA) ( Figure 1). P. aeruginosa ATCC27853 sample (harbouring no resistance mechanism) showed, under the study experimental conditions, 16.87±1.76 inhibition zone around the disk (lysate-IMP).
Several tests were carried out on the negative control strain (P. aeruginosa ATCC27853). The lysate-IMP disk showed no upper than 5 mm decrease compared to the IMP disk, which represents the upper bound of MBLnegative strain and obviously the lower bound of MBLpositive strain. In fact, a decrease in the inhibition zone of >5 mm represents a strong argument for judging a positive strain by this assay. Disks impregnated with EDTA (0.5M) alone and lysate alone showed no inhibition zone with the indicator strain (Figure1).
Method C was compared to the two phenotypic methods considered in this study (method A and method B) and to the molecular method (PCR) as a reference method. Among the three phenotypic methods considered in this study, method A showed 100% of sensitivity. Method B and method C showed 91.66% of sensitivity. The highest level of specificity was shown by method C (76.92%), followed by method B (69.23%) than method A (23.07%) ( Table 1). To evaluate efficiencies of methods, the study noted that the highest Youden Index (YI) was shown by method C (0.7), followed by method B (0.6) than method A (0.2).

DISCUSSION
There is an urgent need for an early detection of MBLproducing organisms for their prevention of their inter and intra hospital dissemination. Currently, the most widely accepted standardized MBL functional screen is the MBL E-test. However, this method suffers from high cost and unavailability of E-test strips. Both CDT and DDST have been reported to be reliable in screening for MBL production in P. aeruginosa (Yan et al., 2004). It has 1 4 2 3 been reported that CDT and DDST were simple, inexpensive phenotypic resources for detection of MBL that could be easily incorporated into the routines of clinical laboratories. In Tunisian clinical laboratories, for its high cost, the MBL E-test is not frequently used. Generally this test is used for research purposes and not for routine screening of MBL. Although, MBL E-test has been evaluated to be a sensitive method for detection of MBL production in P. aeruginosa (Walsh et al., 2002). Qu et al. (2009) reported that several MBL-producing strains could not be detected by the MBL E-test. Several studies have been interested in evaluation and comparison of phenotypic tests (Yan et al., 2004;Picao et al., 2008;Qu et al., 2009). Variable results have been reported. Yan et al. (2004) found that the DDST was most sensitive for all bacterial species tested (Yan et al., 2004). However, one of the major disadvantages of the DDST is the subjective interpretation of results that depends upon the technician's expertise to discriminate true synergism from the intersection of inhibition zones (Picao et al., 2008;Qu et al., 2009). Other studies evaluated the CDT as most sensitive method (Berges et al., 2007;Picao et al., 2008). In this study, DDST was evaluated as more sensitive than CDT. Thus, it has been suggested that the selection of the best MBL screening method should be based on the isolated species, the local prevalence of MBL producers, and the ability of specialized technicians to correctly interpret MBL inhibition (Yan et al., 2004;Picao et al., 2008).
False detection of MBL in P. aeruginosa by E-test, CDT and DDST depend on EDTA susceptibility (Chu et al., 2005). It is known that EDTA may increase bacterial cell wall permeability and so it can increase the susceptibility of P. aeruginosa to various antimicrobials, including IMP (Ayres et al., 1999). Tan et al. (2008) proposed an IMPlysate MBL assay that overcomes the major disadvantage of the three phenotypic methods; the direct bactericidal EDTA effect on the test strain (Tan et al., 2008). It provides a simple, inexpensive and reproducible functional screen for MBL-producing P. aeruginosa. However, that technique requires special equipment, specifically the Microcon Device. Due to the cost and the scarcity of the device, it is typically not available in laboratories. Thus, the study modified the technique to adapt it to laboratory's conditions. After the elimination of the step of enzyme-concentration from the original protocol, screening of MBL bla VIM producing P. aeruginosa is still simple and easy. Importantly, EDTA still has little effect on the indicator strain.
In this study, method C showed 91.66% of sensitivity and 76.92% of specificity, and referring to Youden test, method C was more efficient than method A and B.

Conclusion
Since MBL-Etest and PCR were expensive and not adaptable for extension use in clinical microbiology laboratories, a modified IMP-lysate MBL hydrolytic activity can be chosen by laboratories with limited resources as an inexpensive, simple, and accurate test to detect P. aeruginosa producing bla VIM type enzyme.