Detection of metallo-β-lactamases-encoding genes among clinical isolates of Pseudomonas aeruginosa in a tertiary care hospital, Kathmandu, Nepal

Objectives This study was carried out to determine the prevalence of metallo-β-lactamases (MBLs) producing Pseudomonas aeruginosa in imipenem-nonsusceptible isolates and to detect MBL-encoding genes among MBLs-positive isolates. Results Metallo-β-lactamases production was detected in 68.6% isolates of P. aeruginosa with reduced susceptibility to imipenem. The bla VIM-2 gene was detected in 75% isolates and bla IMP-1 was detected in 25% isolates. All MBLs-positive isolates were multidrug resistant with a high level of resistance to imipenem (MIC 16 to ≥ 32 µg/ml), meropenem (MIC 16 to ≥ 32 µg/ml), and ceftazidime (MIC 64 to ≥ 512 µg/ml). All MBL-positive isolates were susceptible (MIC ≤ 2 µg/ml) to colistin. We found high prevalence of MBL-producing P. aeruginosa. To our knowledge this is the first report of detection of bla VIM-2 and bla IMP-1 in P. aeruginosa from Nepal. This indicates the need for awareness to prevent the spreading of these resistant isolates in hospital setting.


Introduction
Pseudomonas aeruginosa is a Gram-negative, non-fermentative organism found in diverse environmental settings [1]. It is an opportunistic pathogen, causing serious infection in patients with weakened immune systems [2]. This organism is generally intrinsically resistant to a wide variety of antimicrobial agents as well as it has the capacity to develop resistance by mutation or acquisition of foreign resistance genes against different antibiotic classes [3]. Carbapenem, including imipenem, meropenem and doripenem are often used as a last resort for treatment of infections caused by P. aeruginosa and other Gramnegative bacteria [4,5]. However, carbapenem-resistant P. aeruginosa has become prevalent globally [6,7]. Carbapenem resistance may arise in P. aeruginosa via OprD channel deficiencies, up-regulation of efflux pumps and production of various kinds of carbapenemases, including serine β-lactamases of Ambler classes A and D and metallo-β-lactamases (MBLs) of Ambler class B [8].
Among various mechanism of resistance for carbapenem in P. aeruginosa, production of MBLs is of particular concern because of their rapid spread, potent carbapenemase activity, resistance to β-lactamase inhibitors and ability to hydrolyze all β-lactam antibiotics with the exception of aztreonam [9]. Furthermore, MBLs encoding genes are usually located on integrons, the mobile genetic elements that also carry genes encoding for resistance to aminoglycoside and other antibiotics resulting in multidrug resistance (MDR) [10].
Several types of MBLs, such as IMP, VIM, SPM, GIM, AIM, FIM and NDM and their variants have been identified in P. aeruginosa [11][12][13]. Among them, variants of VIM and IMP types such as bla VIM-1 , bla VIM-2 , bla IMP-1 and bla IMP-2 are the most commonly found MBL genes in P. aeruginosa and responsible for many nosocomial outbreaks [9].
Although, production of MBLs in clinical isolates is a serious therapeutic problem, so far limited information is available on MBL production in P. aeruginosa clinical isolates from Nepal. The objectives of this study were to determine the prevalence of MBL producing P. aeruginosa and to detect MBL-encoding genes (bla VIM-1 , bla VIM-2 , bla IMP-1 and bla IMP-2 ).

Bacterial isolates
Clinical specimens submitted for routine culture and antibiotic susceptibility testing from hospitalized patients during the period of October 2015 to July 2016, at the microbiology laboratory of the Annapurna Neurological Institute and Allied Sciences were processed. P. aeruginosa were isolated and identified based on conventional biochemical test [14]. All P. aeruginosa isolates were screened for reduced susceptibility to imipenem (MIC ≥ 8 µg/ml) by E-test strips.

Detection of MBL encoding genes by multiplex PCR
Total DNAs of MBL-positive P. aeruginosa isolates were extracted using a boiling method, briefly; 3-5 colonies from an overnight culture grown on Mueller-Hinton agar (Hi-media, India) were transferred to 300 µl of nuclease-free water and boiled at 100 °C for 10 min. After centrifugation at 12,000×g for 5 min, the supernatant was used as a source of template for amplification. Multiplex PCR amplification of MBL genes including bla VIM-1 , bla VIM-2 , bla IMP-1 and bla IMP-2 were performed (5 Prime/02, Bibby Scientific, UK) [17]. Amplification was carried out with the following thermal cycling conditions: initial denaturation at 95 °C for 5 min and 36 cycles of amplification consisting of 40 s at 94 °C, 40 s at 52 °C, 50 s at 72 °C, with 5 min at 72 °C for the final extension.
Pseudomonas aeruginosa NCTC 13437 producing bla VIM and Escherichia coli NCTC 13476 producing bla IMP kindly provided by Prof. Neil Woodford from Public Health England were used as positive control and P. aeruginosa ATCC 27853 were used as negative control.

Discussion
Our study showed that 68.6% (24/35) isolates of P. aeruginosa with reduced susceptibility to imipenem were MBL producers. This finding was higher than those reported in the previous study from Nepal [19], indicating that MBL-producing P. aeruginosa is increasing. High prevalence of MBL producing P. aeruginosa was also detected in India, a neighboring country of Nepal (69.8%) [20], Taiwan (55.1%) [21], and Egypt (68.7%) [22]. The reduced imipenem susceptibilities in MBL-negative isolates might be due to other mechanisms such as deficiency in porin channel combined with production of AmpC β-lactamases [8].
In the present study, bla VIM-2 was the most commonly detectable genes among the different MBL genes analyzed; 75% of MBL-positive isolates carried bla VIM-2 . Only 25% of MBL-positive isolates carried bla IMP-1 . This finding was supported by result of previous studies from around the world demonstrating bla VIM-2 as the most common MBL genes responsible for reduced susceptibility to imipenem in P. aeruginosa and pose greatest clinical threat [23]. bla VIM-2 gene is associated with many nosocomial outbreaks due to MBL-producing P. aeruginosa [24]. Although, less than VIM-2 type, IMP-1 also have been reported from worldwide [25]. Due to the high mortality rate of infections caused by MBL-producing P. aeruginosa, their presence in hospital setting is of great concern. To evaluate the threat of MBL-producing P. aeruginosa and its associated risk factors, well-designed multicentre epidemiological studies are urgently required.
In this study, all MBL-producing isolates were MDR, leaving the limited treatment option. Based on the in vitro testing; the most effective antibiotic against MBL-producing isolates were colistin (100% susceptible, MIC ≤ 2 µg/ml), followed by aztreonam (62.5% susceptible). The existence of the MBL genes on integrons together with other resistance gene makes the MBLpositive isolates MDR [10]. Due to the limited treatment option (potentially toxic polymyxin B and colistin) for the infections caused by MBL-producing isolates and the mobile nature of MBL, early detection of MBL-producing isolates in hospital setting is necessary to initiate the appropriate infection control procedure to avoid the spread of these multidrug resistant isolates.
Aztreonam is stable against MBLs [10]; however in this study 37.5% of the MBL-producing isolates were resistant to aztreonam, suggesting a possible association with other resistance mechanisms such as AmpC type β-lactamases or ESBLs [1].
Many studies have shown that intensive care unit is the epicenter and the main source of amplification and dissemination of antimicrobial resistance [26], a finding that was also observed in this study among the 24 MBLpositive isolates; 14 (58.4%) were isolated from ICU while 10 (41.6%) were from general ward patients. Since more than 50% of MBL-producing P. aeruginosa isolates have been recovered from ICU, it is highly probable that it might be a clonal spread. Unfortunately, because of some limitations, such as lack of more equipped laboratory, we were not able to do genotypic comparisons of the isolates.

Conclusion
In conclusion, our study showed the presence of bla VIM-2 and bla IMP-1 producing P. aeruginosa for the first time in Nepal that underscores the necessity of screening all imipenem-resistant isolates for MBL production and implementation of infection control programs to prevent spread of such organisms.

Limitations
• The study was unable to determine whether these MBL genes are transferable or not. • The study was unable to investigate other MBLencoding genes such as NDM-1. • Due to the lack of laboratory equipment, we could not perform genotypic comparisons of the isolates.

Authors' contributions
MA and PRJ designed the study, collected data, analyzed the data and prepared the manuscript, KT and RA collected data, TK and SS supervised the study. All authors read and approved the final manuscript.