Phenotypic screening of metallo-β-lactamase in multi- drug-resistant Pseudomonas aeruginosa using a combined disk diffusion method

1 International Branch of Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2 Infection Research Center, Mofid Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3 Shahid Beheshti University of Medical Sciences, Tehran, Iran. 4 School of Nursing and Midwifery, Tehran University of Medical Sciences, Tehran, Iran. 5 Audiology Departement Manager, Shahid Beheshti University of Medical Sciences, Tehran, Iran.


INTRODUCTION
Carbapenems are β-lactam group of drugs that are often used as antibiotics of last resort for treating infection due to multiple-resistant Gram-negative bacilli.They are also stable even in response to extended-spectrum and βlactamases.However, this scenario has changed with the emergence of metallo-β-lactamase (MBL)-producing strains (Jesudason et al., 2005).Resistance to carbape-nem is now of global concern and being observed more frequently among nonfermenting bacteria, such as Pseudomonas aeruginosa.
These enzymes require zinc for their catalytic activity and are inhibited by metal chelators, such as ethylene diamine tetra-acetic acid (EDTA) (Livermore and Woodford, 2000).The genes responsible for the production of MBLs are typically part of an integron structure and are carried on transferable plasmids or can also be part of the chromosome (Wan Nor Amilah et al., 2012).
MBL-producing Gram-negative bacilli have been increasingly reported in Asia, Europe, Latin American and the United States (Chu et al., 2001;Iyobe et al., 2000;Kurokawa et al., 1999;Miriagou et al., 2003;Toleman et al., 2004).The proportion of carbapenem resistance attributed to MBLs has increased significantly as well; the presence of MBLs accounted for resistance in 43.9% of Brazilian and 39.1% of Italian imipenem-resistant P. aeruginosa isolates (Toleman et al., 2005).These percentages represent a dramatic escalation in the fraction of resistance caused by these enzymes.
With the increase in worldwide occurrence, types, and rate of dissemination, early detection of MBL isolates is critical.The benefits of early detection include timely implementation of strict infection control practices as well as clinical guidance regarding the potential risks for therapeutic failure.Although polymerase chain reaction (PCR) is highly accurate and reliable, its accessibility is often limited to reference laboratories (Franklin et al., 2006).Several non-molecular techniques have been studied, mostly taking advantage of the enzyme's zinc dependence by using chelating agents, such as EDTA to inhibit its activity (Bush and Fisher, 2011;Bush and Jacoby, 2010;Deshmukh et al., 2011).
Since the rapid spread of bacterial resistance due to the extensive use of antibiotics remains a matter of concern for the optimal treatment of patients, the evaluation and use of a simple, reliable and inexpensive testing method for screening of MBL-producers in routine laboratory has become necessary.
In this study, our aim was to determine the incidence of metallo-β-lactamase (MBL) enzymes in the P. aeruginosa clinical isolates using a combined disk diffusion method.

Bacterial isolates collection and characterization
In the present study, 105 P. aeruginosa clinical isolates were collected over a period of 1 year (January 2012 to January 2013) from Motahari, Milad and Taleghani hospital laboratories in Tehran.The samples were immediately transported in transport culture media under standard conditions to the central laboratory of the Infection Research Center.P. aeruginosa ATTCC27853 was used as the negative control.Determination of P .aeruginosastrains was confirmed by standard biochemical tests (Wan Nor Amilah et al., 2012).

Phenotypic detection of MBL
Screening of MBL-producing isolates was performed using a combined disk diffusion method.The isolates were evaluated phenotypically for the presence of a metallo-β-lactamase (MBL), using the metal chelating agent: EDTA (Vahdani et al., 2012).Identification of MBL activity was performed by carbapenem-EDTA combined disk method.Two imipenem or meropenem [IMP (10 μg), MEM (10 μg)] discs were applied to a Muller Hinton agar plate inoculated with a bacterial suspension of 0.5 McFarland turbidity standards, and 10 μl of a sterile 0.5 M EDTA (pH 8.0) solution was applied to one disk.A sterile 6-mm filter paper disc to which 10 μl of 0.5 M EDTA was applied was used to determine if EDTA alone might inhibit the growth of the test isolates.The plates were incubated at 35°C under ambient air for 18 h.
The zones of inhibition around the IMP and IMP-EDTA discs were measured for all 105 isolates, however the zones of inhibition around the MEM and MEM-EDTA discs were measured for 55 isolates selected randomly and zone increases of ≥7 mm in the presence of EDTA were noted and interpreted as indicative of an MBL phenotype on the basis of criteria described previously (Bashir et al., 2011;John and Balagurunathan, 2011;Mochon et al., 2011).

RESULTS
In this study, 105 clinical isolates of P. aeruginosa were collected from three hospital laboratories in Tehran during 1 year.
All samples were MDR.Antimicrobial susceptibilities were determined according to the interpretative criteria of the CLSI guidelines (Table 1).High resistance to all antimicrobial drugs was observed except for Colistin (3.81%) and Gentamicin (67.62%).
The overall prevalence of MBL-positive isolates was 88.27% using IMP and IMP-EDTA discs, meanwhile 92.73% of 55 randomly selected isolates evaluated phenotypically for the presence of a metallo-β-lactamase (MBL), using MEM and MEM-EDTA discs as well, were MBL positive.
Among 105 isolates, seven isolates showed different MBL producing pattern using IMP and IMP-EDTA or MEM  and MEM-EDTA discs (Table 2).

DISCUSSION
Carbapenems were the effective antibiotics for MDR gram-negative bacteria infections, especially in high-risk hospital settings (Qing et al., 2012).However, resistance is the result of different mechanisms, such as MBL production by micro-organisms.These enzymes belong to Ambler class B β-lactamases based on their amino acid sequence homology and to group 3 according to the Bush classification based on their substrate profiles (imipenem hydrolysis) (Ambler, 1980;Bush et al., 1995).The genes responsible for MBL production may be chromosomal or plasmid mediated (Wan Nor Amilah et al., 2012).MBL enzymes are inhibited by EDTA (Livermore and Woodford, 2000).The rapid detection of MBL positive isolates is necessary to control infection and to prevent their dissemination.The increasing use of extended spectrum antibiotics such as carbapenems would provide the selective pressure for selection of these enzymes (Irfan et al., 2008).In many studies across the world, different resistance ranges (4-60%) have been reported towards carbapenems.For instance, the incidence of MBL production in P. aeruginosa has been reported to be 10-30% from a variety of clinical specimens across India (Deshpande et al., 2010).However, in a study by Lagatolla et al. (2004) in Italy, 70% MBL was found in P. aeruginosa.Our prevalence of MBL in P. aeruginosa does not correlate with other studies across the country.In a study by Vahdani et al. (2012), 38 % of MBL production was reported in P. aeruginosa, and in another study performed in Ahwaz, 19.51% of P. aeruginosa strains isolated from burn patients were reported as MBL producers (Khosravi and Mihani, 2008), whereas in the study of Manoharan et al. (2010), in accordance with our results, 87.8% of P. aeruginosa isolates were MBLpositive, using a combined disk diffusion method.This variation reflects the different diagnostic methods and the different rates of antibiotics used in different hospitals.
Although CLSI has recommended IMP-EDTA for MBL investigation in P. aeruginosa (CLSI, 2012), as mentioned earlier, in our study, two P. aeruginosa isolates were MBL -positive using IMP and IMP-EDTA discs while these isolates were negative for MBL using MEM and MEM -EDTA discs; this contrast was observed for five other isolates, conversely.Thus, using different subclasses of carbapenems such as imipenem + EDTA and meropenem + EDTA together for investigating MBLpositive isolates by combined disk diffusion method, seems to be necessary.
The emergence of MBL-mediated resistance in our country, Iran, is a matter of concern for the treatment of patients (Vahdani et al., 2012).The phenotypic screening of resistance is an important step for epidemiological purposes and for developing policies for effective infection control measures in order to manage and prevent the spread of resistant strains.We suggest accurate surveillance, especially of MBL, in MDR P. aeruginosa isolates as an important step for optimal antibacterial treatment in the future.

Table 1 .
Antibiotic resistance among multi-drug-resistant Pseudomonas aeruginosa isolated from 3 hospital laboratories in Tehran.

Table 2 .
Different MBL producing pattern using IMP and IMP-EDTA or MEM and MEM-EDTA discs among 7 Pseudomonas aeruginosa isolates.