Genetic diversity and molecular analysis of metallo beta lactamases among imipenem resistant clinical isolates of Pseudomonas aeruginosa from Peshawar, Pakistan

Objectives: Pseudomonas aeruginosa is an opportunistic pathogen with remarkable adaptation ability to thrive in diverse environmental conditions. This study aimed at phenotypic and molecular analysis of metallo beta lactamases (blaIMP, blaVIM, blaNDM-1 and blaSPM-1) and genetic diversity analysis among imipenem resistant clinical isolates of Pseudomonas aeruginosa. Methods: This study was conducted from May 2017 to June 2018. The study included 187 Pseudomonas aeruginosa isolates collected from different clinical specimens from Peshawar, Pakistan. The isolates were analyzed for resistance to imipenem. Combined disc test (CDT) was then performed for phenotypic detection of metallo beta lactamases among imipenem resistant isolates of Pseudomonas aeruginosa. Molecular detection of metallo beta lactamases genes i.e. blaIMP, blaVIM, blaNDM-1 and blaSPM-1 was analyzed through polymerase chain reaction. Genetic diversity was determined through RAPD-PCR. Results: MBL production was observed in 76% (n=19) isolates. The occurrence of MBL genes blaIMP, blaNDM-1 and blaVIM was 68% (n=17), 48% (n=12), and 4% (n=1) respectively. The blaSPM-1 gene was not detected. High genetic diversity was observed in current study. Out of 182 isolates 171 isolates showed different RAPD profiles (93.95% polymorphism); 160 were unique RAPD strains and based on similarity coefficient ≥ 80%, 22 isolates were clustered into 11 distinct clones. Conclusion: A high prevalence of blaIMP and blaNDM-1 among imipenem resistant isolates of Pseudomonas aeruginosa is alarming that calls for proper control and prevention strategies. RAPD technique was found to be a good genotyping technique when limited resources are available.


INTRODUCTION
Extensive drug resistant strains of Pseudomonas aeruginosa have been reported from hospitals around the world. 1 Resistance of Pseudomonas aeruginosa to different classes of antibiotics such as penicillin, cephalosporin, quinolone, aminoglycoside and carbapenem has been demonstrated. 2 Metallo beta lactamases (MBL) are enzymes that catalyze the hydrolysis of broad-spectrum beta lactam antibiotics including carbapenems. 3 Metallo beta lactamases (MBL) production has been reported from Brazil, Iran and India among carbapenem resistant clinical isolates of Pseudomonas aeruginosa. [4][5][6] Among beta lactam antibiotics, carbapenems are the most effective against Gram negative and Gram-positive bacteria demonstrating broad range antibacterial activity. 7 Resistant to carbapenems especially in Gram negative pathogens is global public health issue because of the spread of transferable carbapenemase encoding genes. 7 Highest resistance was reported by Farooq et al., against imipenem in multi drug resistant clinical isolates of Pseudomonas aeruginosa from tertiary care hospital Karachi, Pakistan. 8 Imipenem resistant pathogenic bacterial strains have been reported from Lahore and Quetta Pakistan. 9,10 Imipenemase (IMP), Verona integron encoded metallo beta lactamase (VIM), New Delhi metallo beta lactamase (NDM), Sao Paulo metallo beta lactamase (SPM), Florence imipenemase (FIM) and Germany imipenemase (GIM) are various types of metallo beta lactamases that have been reported in carbapenem resistant isolates of Pseudomonas aeruginosa. 11 Genes for MBLs are located on integrons, plasmids, transposons or on chromosomes. 11 These specific genetic elements carry carbapenem and other antibiotics resistant determinants and thus confer multi drug resistance to Pseudomonas aeruginosa. These antibiotic resistant elements could be transferred to other Gram-negative bacterial strains and contribute towards spread of antimicrobial resistance rate making the treatment of infected patients complex. 11 It is therefore, important to know about the epidemiology and resistance mechanisms of Pseudomonas aeruginosa in order to control and prevent multi drug resistant pathogenic strains to overcome possible health risks. 11 Microbial typing is vital in order to determine the relationship between microbes. 12 Knowledge about clonal relatedness between microbial strains is important to determine source, route of infection, detect cross transmission and confirm epidemic of pathogen. 12 Microbial typing is also used in the recognition of virulent strains and to assess the effectiveness of control measures. Randomly amplified polymorphic DNA (RAPD) RAPD is a polymerase chains reaction (PCR) based genotyping method that requires short single arbitrary primers that could randomly amplify DNA and generate numerous discrete segments of DNA. 13 RAPD demonstrates quick and efficient DNA polymorphism at large number of loci in genome of an organism. RAPD is simple, efficient, low-cost technique that needs no prior knowledge of target DNA sequence. 13 No information is available on the occurrence of metallo beta lactamases and genetic diversity of Pseudomonas aeruginosa from Pakistan. Hence, this study aimed at analysis of MBL genes and genetic diversity of Pseudomonas aeruginosa isolates from the region.

Bacterial isolates and identification:
A total of 187 clinical isolates of Pseudomonas aeruginosa were collected from tertiary care hospitals (Khyber Teaching Hospital (KTH) and Hayatabad Medical Complex (HMC)) of Peshawar, Pakistan in 2017-2018. These included 74 isolates from pus, 34 isolates from urine, 24 isolates from sputum, 21 isolates from wound, 12 isolates from bronchial wash, 8 isolates from cerebrospinal fluid, six isolates from blood, 5 isolates from high vaginal swab and 3 isolates from diabetic foot. Patient's informed consents and permission were obtained from the participating hospitals and study was ethically approved by Post Graduate Medical Institute (PGMI) Peshawar, KP Pakistan (Ref: 1046/PGMI, Dated: 03-05-2017). Cultures were inoculated on MacConkey agar (Oxoid, UK) and were incubated for 24 hours at 37 ˚C. Using standard procedures pure isolates were identified as Pseudomonas aeruginosa through morphological (Gram staining) and biochemical tests (catalase, oxidase, citrate utilization test, triple sugar iron test, indole test and nitrate reduction test). 14

Phenotypic detection of Metallo Beta Lactamases (MBL):
Twenty-five imipenem resistant isolates among the 187 isolates were subjected to phenotypic and genotypic detection of metalo lactamases. Combine disc test (CDT) was performed for phenotypic detection of MBL genes. Two imipenem discs (10 µg; Oxoid, UK) were placed 25 mm to 30 mm apart on surface of Muller Hinton agar (Oxoid, UK) inoculated with Pseudomonas aeruginosa. One imipenem disc was impregnated with 5 µl EDTA (0.5M, pH 8.0). Culture plates were incubated for 18 to 24 hours at 37 °C. Zones of inhibition were compared. The bacterial strain was considered as MBL positive if zone of inhibition of imipenem-EDTA disc was ≥ 7 mm than the zone of imipenem disc alone. 5 DNA extraction: GeneJET Genomic DNA purification kit (Thermo Scientific, Lithuania Cat. No. K0721) was used for DNA extraction. Recommended protocol was followed, and the quality of DNA was confirmed through gel electrophoresis using 0.8% agarose gel. The extracted DNA was preserved at −20˚C for further use. Molecular detection of MBLs: Four metalo beta lactamases genes i.e., blaIMP, blaVIM, blaNDM-1 and blaSPM-1 were analyzed among 25 imipenem resistant isolates of Pseudomonas aeruginosa using previously reported primers and amplification profiles. [15][16][17] The amplified PCR products were analyzed through gel electrophoresis and 100 base pair DNA ladder (Bioron, Cat. No. 304105) was used as size marker. RAPD-PCR: Genetic diversity was determined through RAPD-PCR using previously reported primer (RAPD 272) and procedures. 18 Amplified products were analyzed through gel electrophoresis. The sizes of amplified DNA fragments were compared with 100 base pair (bp) DNA ladder (Bioron, Cat. No. 304105). RAPD PCR was repeated multiple times to ensure the reproducibility of the results and overcome the limitations of RAPD technique in a low-income setting.
Statistical Analysis: Amplified bands through RAPD-PCR were scored as absent or present to generate bivariate (0-1) data for statistical analysis. Genetic diversity among the clinical isolates of Pseudomonas aeruginosa was determined using this bivariate data. Genetic distances among isolates were calculated using Nei and Li method by the formula D xy = 1-(N xy ) / (N x + N y -N xy ) as reported. 19 The term D xy represents dissimilarity distance between two genotypes X and Y. Term N xy represent the number of common bands present between two genotypes. The term N x represent total number of bands present in genotype X and N y shows total number of bands present in genotype Y. Using MEGA 7 (Molecular Evolutionary Genetics Analysis, version 7) software based
Limitations of the study: Current study involved sampling for hospitals of Peshawar. A large sampling area could help in better understanding of the epidemiological aspects of resistant strains of Pseudomonas aeruginosa at molecular level on regional level.

CONCLUSION
The high prevalence of metallo beta lactamases genes blaIMP, blaNDM-1 and blaVIM among imipenem resistant strains of Pseudomonas aeruginosa is a serious health concern. Strict hygienic conditions, good infection control policies and appropriate use of antibiotics are essential to prevent further dissemination of MBL producing strains of Pseudomonas aeruginosa. It is also concluded that genotypic surveillance study must be performed on regular basis to identify possible sources of dissem-