Detection Rate of Metallo-β-Lactamase-Expressing Genes; blaVIM-1, blaVIM-2 and blaSPM-1 in Pseudomonas aeruginosa Isolates

Introduction: Imipenem-resistant Pseudomonas aeruginosa is an organism expressing metalloβ-lactamase (MBL) enzyme, and is a serious agent of hospital infection holding a serious universal therapeutic challenge. Carbapenems are potent options for the treatment of P. aeruginosa infections. The rate of MBLs expression has been variable among imipenem-resistant P. aeruginosa isolates. In the present study, we investigated the presence of MBL in the clinical isolates of P. aeruginosa. Methods: A total of 60 P. aeruginosa isolates were obtained from Kerman hospitals during 20142015. The antibiotics susceptibility was assessed using disk diffusion test. MBL positivity in P. aeruginosa was investigated using double disk synergy test (DDST) and polymerase chain reaction (PCR) with amplification of blaVIM-2, blaVIM-1 and blaSPM-1. Results: From 60 P. aeruginosa isolates, 28 (46.6%) were imipenem-resistant. Among these, 17 (60.7%) were identified as MBL-producing P. aeruginosa isolates using DDST. Results of PCR test demonstrated the existence of 8 (28.5%) P. aeruginosa, producing blaSPM-1. Conclusion: The frequency of blaSPM-1-producing P. aeruginosa isolates from Kerman Hospitals was relatively high. Therefore, it is recommended that the distribution of MBL-mediated resistances be managed.


Introduction
Pseudomonas aeruginosa is broadly recognized as an opportunistic aggressor rather than a cause of early infection in healthy persons. 1 It constitutes around 10%-20% of hospital-acquired infections in intensive care units (ICUs), cystic fibrosis, respiratory and renal infections, and body surface infections. 2 Multidrug-resistant mechanisms of P. aeruginosa include expression of efflux pumps, production of biofilms, and secretion of resistance-inducing enzymes such as β-lactamases and aminoglycosidases. 3This bacterium is a concerning issue considering antimicrobial chemotherapy. 4nzyme production is a prominent β-lactam resistance mechanism in P. aeruginosa.Penicilloyl-serine transferases separate the amide bond of the β-lactamase ring removing the antibacterial activity (5).β-lactamases have been recently classified in 2 ways: the molecular and the functional.Regarding the molecular classification, several categories of β-lactamases have been identified; these are known as class A, B, C and D. Enzymes belonging to the classes A, C and D use serine amino acid for their activity, while enzymes of class B are dependent on divalent cations for their activity. 1On the basis of molecular features, metallo-β-lactamases (MBLs) are comprised of the following 6 groups: IMP, VIM, SIM, SPM, GIM, and AIM. 6arbapenems resistances may result from decreased outer membrane permeabil ity, exclusion from the cell by efflux pumps, changes of penicillin-binding protein, and production of β-lactamase. 7arbapenems are currently used for the treatment of Pseudomonas infections.Resistance genes such as MBLs can easily be tramsitted by genomic compartments shuch as plasmids and calss I integrons, and this may be a source of dissemination of antibiotic resistance throughout the clinics. 8,9MBLs can intensely hydrolyze all betalactam antibiotics except azetreonam.These enzymes need zinc as cofactor. 10Sulbactam, tazobactam, and clavulanic acid which are often used to inhibit betalactamase enzymes are not useful against MBLs. 11,12Due to our limitation in providing efficient antibiotic alternatives in cases of resistant organisms, the presence of MBL in clinical isolates of P. aeruginosa were investigated.In addition, prolonged hospitalization and high fatality rates have been associated with MBL positive P. aeruginosa infections.The aim of this study was to investigate the antibiotic resistance profile and the prevalence of blaVIM-1, blaVIM-2 and blaSPM-1 genes encoding MLBs among clinical isolates of P. aeruginosa obtained from Afzalipour and Bahonar hospitals of Kerman city, by double disk synergy test (DDST) and multiplex polymerase chain reaction (PCR).

Methods
In the present cross-sectional study, 60 suspected isolates of Pseudomonas were collected out of 234 examined isolates obtained from hospitalized patients in Kerman, Iran, during 2014-2015.These bacteria had been recovered from several nosocomial samples such as urine, wound, blood, trachea and other clinical samples.The colonies were again cultured in MacConkey agar medium and pure colonies were recognized as P. aeruginosa according to Gram staining and bacteriological tests such as oxidase, catalase, oxidative-fermentative test, growth on media such as TSI, SIM, cetrimide agar (Merk, Germany) and growth at 42°C.Isolates were conserved in Trypticase soy broth media (TSB) including 20% glycerol and stored at -70°C until use. 13tibiotic Susceptibility Tests Disk diffusion assay (Kirby-Bauer) on Muller-Hinton agar plates (Merck, Germany) was accomplished with the antibiotic susceptibility test according to the Clinical & Laboratory Standards Institute (CLSI). 14The antimicrobial disks used (Mast Co, UK) are shown in Table 1.Pseudomonas aeruginosa ATCC27853 was applied as a control strain for the susceptibility testing.DDST was applied for phenotypic examination of MBLs.Therefore, 0.5 M EDTA solution was prepared using 186.1 g of disodium EDTA.H2O2 was dissolved in 1000 mL distilled water (pH 8.0 which was adjusted by addition of NaOH).Then, 930 μg of this solution was appllied on imipenem disk and incubated to be dried.These disks were then placed on a plate of Muller-Hinton agar with cultured P. aeruginosa.After an incubation period of 16-18 hours at 37°C, a positive result for MBL expression was considered as a diameter equal or greater than 7 mm of IMP-EDTA disk respective to the imipenem disk.1).Primer sequences were designed using the NCBI Primer BLAST database.Primers sequences used for detection of MBL genes and PCR temperature profile for amplification of MBL genes are shown in Tables 2 and 3, respectively.For the statistical analyses, SPSS software version 19.0 was applied.

Discussion
Carbapenems are effective β-lactam antibiotics for drastic cure of infections caused by Gram-negative bacteria. 15mong β-lactamases, imipenem is the chosen antibiotic for fighting against this bacterium, however P. aeruginosa can disintegrate this antibiotic through MBL enzyme. 16he rate of imipenem-resistant isolates (46.4%) in our study indicated a menace regarding therapy alternatives in our clinics.Imipenem and meropenem are commonly used antibiotics for the treatment of hospital-acquired infec tions; nevertheless, enhanced resistance against these agents has restricted their success. 17n our results, among 28 imipenem-resistant strains, 17 (60.7%)were positive for production of MBL as shown by DDST.Kalantar et al 18 exhibited that 22% of the imipenem-resistant P. aerugi nosa isolates were MBL-positive and 8 strains (8%) were positive for blaVIM1 as proved by PCR.Sarhangi et al 19   al 20 detected that 70 (70%) of imipenem-resistant P. aeruginosa expressed MBLs.PCR methods revealed that 70 (33%) of these strains contained the blaVIM gene. 20Based on DDST test, MBL was detected in 36 (87.8%) of the P. aeruginosa isolates as studied by Doosti et al.Among 41 imipenem-resistant isolates investigated by PCR, 23 (56%) isolates harbored blaVIM gene. 21The variation seen between the results of our study and those of the previous reports may be due to the diversity in geographical areas, diversity in sorts of the diseases, the increased consumption of antibiotics, or diversity in antibiotic treatment abstinence.Some MBL encoding genes have been mapped as mobile genes with possibility of transmission between organisms. 16The most prevalent, introduced families are IMP, VIM, GIM, SPM, and SIM. 1 In our study, 60.7% of the total 60 P. aeruginosa isolates were MBL producer, which were more than the outbreak of MBL producers in Egyptian studies (27% and 32.3%) and Indian study (28.57%). 1 VIM enzymes were also most prevalent in Korea (88%) and Greece (85%). 224][25] VIM type was also highly prevalent in Turkey. 26In previous studies in Egypt and Taiwan, VIM-2 demonstrated the highest outbreak among imipenem-resistant P. aeruginosa strains. 1,27Since most of the integrones harbored VIM-1 gene code for aminoglycosides destructing enzymes, the matter is of clinical importance. 28n the present study, SPM was the commonest (28.5%) gene detected among imipenem-resistant P. aeruginosa isolates.The other MBL genes were not identified.MBL producer imipenem resistant isolates that were negative for the MBL genes may benefit from other resistant genes like IMP, SIM, NDM GIM or other resistance mechanisms to carbapenem.Identifying the MBL non-producer isolates with DDST that expressed the MBL genes may indicate low sensitivity of our phenotyoic assay.The imi-penem resistant isolates negative for MBL genes may also have other mechanisms associated with carbapenem resistance (such as AmpC β-lactamase expression, or using membrane efflux pathways).0][31][32] SPM carbapenemase represents a major mechanism responsible for resistance to ceftazidime on ceftazidime-resistant P. aeruginosa isolates. 30Zavascki et al identified 86 MBL producing strains, 16.27% of which expressed the blaSPM gene, 33 and Graf et al also discovered the blaSPM gene in 13.51% strains that were only susceptible to polymyxin B. 34 Our data are in accordance with these studies regarding high prevalence of SPM among P. aeruginosa isolates.The results of Moosavian et al study on 122 imipenem resistant P. aeruginosa isolates showed that only 2 isolates (1.6%) contained blaVIM-2 gene, whereas none of them were positive for blaSPM-1. 13Sarhangi et al 19 discovered that 12.1% of imipenem-resistant P. aeruginosa isolates expressed blaVIM-2 gene, while no occurrence of blaSPM1 was found.In the study of Ghamgosha et al, 35 77.7% of MBL producing P. aeruginosa had VIM-1 gene, with no SPM-1 gene detected.Concurrent resistance to imipenem and meropenem may propose carbapenem resistance mechanisms other than the enzymatic pathway (such as porin loss and/or upregulation of efflux pumps). 30High resistance to carbapenems and ceftazidime has been conferred to MBL production. 36Current studies corroborate this propensity. 29,30,37,38Resistance to other antimicrobial agents has decreased the alternative treatments for managing multidrug-resistant P. aeruginosa. 38,39Widespread strains of the drug-resistant P. aeruginosa strains express either IMP or VIM-type genes that encode MBL.These are significant general health issues menacing the success of antimicrobial chemotherapy.The limitation of this study was low number of the isolated P. aeruginosa strains compared to some other studies, and the strength was the use of DDST and molecular detection of the isolates.

Conclusion
We propose applying powerful superintendence and intransigent infection controlling strategies to control infectious diseases.A controlled antibiotic usage should be considered to prevent the distribution of antibiotic resistance genes among hospital pathogens.

Ethical Approval
The informed consent was obtained from the patients before using their samples, and the data were kept confidential.

Competing Interests
Authors declare that they have no competing interests.

Table 2 .
Specific Primers Used for Detection of blaSPM-1, blaVIM-1, and blaVIM-2 and Lengths of the PCR Products

Table 3 .
PCR Programs for Amplification of MBL Genes