Prevalence of antibiotic resistance and blaIMP-1 gene among Pseudomonas aeruginosa strains isolated from burn and urinary tract infections in Isfahan , central Iran

Pseudomonas aeruginosa is one of the most important opportunistic pathogens responsible for various types of hospital infections. High prevalence of antibiotic resistance in P. aeruginosa strains of human clinical samples cause more severe diseases for a longer period of time. The current research was done in order to study the distribution of blaIMP-1 gene among the imipenem-resistant P. aeruginosa strains isolated from burn and urinary tract infections of hospitalized patients. Two-hundred and forty-three P. aeruginosa isolates recovered from the cases of burn and urinary tract infections of inpatients and outpatients were analysis for antibiotic resistance pattern using the disk diffusion method. Then, imipenem-resistant isolates were further analyzed for distribution of blaIMP-1 gene using the PCR. Of 243 P. aeruginosa isolates, 146 strains (60.08%) were taken from outpatients and 97 strains (39.91%) were taken from inpatients. P. aeruginosa isolates harbored the highest levels of resistance against streptomycin (100%), nalidixic acid (100%), aztreonam (100%), cotrimoxazole (95.47%), ciprofloxacin (88.47%), cefotaxime (84.36%) and gentamycin (83.95%). Inpatients had a relatively higher levels of antibiotic resistance. One-hundred and twenty-one out of 126 (96.03%) imipenem-resistant P. aeruginosa isolates harbored the blaIMP-1 gene. Inpatients also had a relatively higher prevalence of blaIMP-1 gene. High prevalence of blaIMP-1 gene and also imipenemresistant P. aeruginosa are important public health issue. Clinical laboratories should consider the detection of the blaIMP-1 gene among the P. aeruginosa isolates of clinical samples.


Introduction
Superficial infections and especially burn infections (BIs) are important cause of emergency health care associated problems al-around the world.Superficial infections caused longer hospital stays, more expensive hospitalizations, and increased mortality. 13][4] UTIs is an important cause of mortality and morbidity alaround the world. 5,6seudomonas aeruginosa (P.aeruginosa) is a non-fermentative, aerobic, Gramnegative rod shape bacterium and is responsible for severe human clinical infections such as BIs, UTIs, pneumonia, wound, reproductive tract, respiratory tract and superficial and gastrointestinal infections, cystic fibrosis, ecthyma gangrenosum and black necrotic lesions. 7It is also related to sever cases of hospital-acquired and healthcare associated infections globally. 7esistant P. aeruginosa strains cause more severe clinical diseases which are mainly difficult to treatment with routine antibiotics. 8,9Treatment of UTIs and BIs caused by this bacterium is often started empirically and therapy is based on information determined from the antimicrobial resistance pattern. 8,9However, a large proportion of uncontrolled antibiotic usage has subsidized to the development of resistance in P. aeruginosa strains. 8,9P. aeruginosa strains exhibits the highest levels of resistance against fluoroquinolones, beta-lactams, penicilins, tetracyclines, carbapenems, aminoglycosides, macrolides and other types of antimicrobial agents.High levels of antibiotic resistance in the P. aeruginosa isolates of UTIs and BIs have been reported previously. 8,9Recently, clinical isolates of the P. aeruginosa exhibited the high levels of resistance against imipenem antibiotic agent. 10Documented data revealed that blaIMP gene is the most prevalent antibiotic resistance marker in the clinical isolates of imipenem-resistant P. aeruginosa. 10 According to the high clinical standing of P. aeruginosa in hospital infections and also lack of microbiological and epidemiological investigations about the antibiotic resistance and also distribution of blaIMP gene, the current research was done to study the antibiotic resistance pattern of P. aeruginosa isolates of BI and UTIs and study the distribution of blaIMP-1 gene among the imipenem-resistant strains.

Ethical considerations
Ethical committees of the Alzahra and Kashani Hospitals were approved the general principles and framework of the current investigation.Written informed consent was obtained from all of the study patients or their parents.Personal information of all patients were remained secret.

DNA extraction
Total genomic DNA was extracted from the bacterial colonies.A single colony was inoculated on 5 mL of brain heart infusion broth and incubated over night at 37ºC.Then 1.5 mL of a saturated culture was harvested with centrifugation for 5 min.at 14,000 rpm.The cell pellet was resuspended and lysed in 200µl of lysis buffer (40 mM Tris-acetate pH 7.8, 20 mM sodiumacetate, 1 mM EDTA, 1% SDS) by vigorous pipetting.To remove most proteins and cell debris, 66 µL of 5M NaCl solution was added and mixed well, and then the viscous mixture was centrifuged at 12,000 rpm for 10 min.at 4ºC.After transferring the clear supernatant into a new Eppendorf tube, an equal volume of chloroform was added, and the tube was gently inverted at least 50 times when a milky solution was completely formed.Following centrifugation at 14,000 rpm for 5 min, the supernatant is then removed to another eppendorf tube and double volume of 100% ethanol was added.The tubes were inverted 5 to 6 times gently, then centrifuged at 10,000rpm for 5minutes.The supernatant was discarded and 1 mL of ethanol (70%) was added to the pellet, and tubes centrifuged at 10,000 rpm for 5 minutes.Finally, the supernatant discarded and the pellet was dried for 10 min at room temperature, the pellet was resuspended by 100 µL H2O.The stock was kept at −20ºC until use.

Statistical analysis
The results were transferred to a Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA) for analysis.Statistical analysis was performed using SPSS/21.0 software (SPSS Inc., Chicago, IL) for significant relationship between pattern of antibiotic resistance and also distribution of the blaIMP-1 gene of P. aeruginosa isolates.The chi-square test and Fisher's exact 2-tailed test analysis were performed in this study.Statistical significance was regarded at a P<0.05.

Discussion
The present investigation focused on the antibiotic resistance pattern of the P. aeruginosa strains of BIs and UTIs.As it showed, P. aeruginosa had significant impact in the occurrence of BIs and UTIs.High impact of P. aeruginosa in hospital infections has been reported from other sites of the world including Brazil, 13 Italy, 14 Germany, 15 United States 16 and South Africa. 17We also found that P. aeruginosa strains harbored the high levels of resistance against commonly used antibiotic and especially streptomycin, nalidixic acid, aztreonam, cotrimoxazole, ciprofloxacin, cefotaxime and gentamycin.Of studies which were conducted in this field, [18][19][20][21][22][23] all have shown a high distribution of antibiotic resistance against ampicillin, gentamycin, cotrimoxazole, ciprofloxacin, cefotaxime and amikacin.Presence of considerable levels of resistance against imipenem have been reported previously from Iran, 19,20 Turkey, 24 India 25 and Indonesia. 26 Onguru et  al. (2008)  27 reported that the P. aeruginosa strains of various clinical sources were resistant to imipenem (44.1%) which was entirely high.They showed that imipenem resistant strains were also resistant to amikacin (70%), gentamycin (85%), tobramycin (87%), cefepime (81%), piperacillin (61%) and ciprofloxacin (77%).Our results revealed that 51.85% of P. aeruginosa strains were resistant to imipenem.Indiscriminate, unauthorized and illegal prescription on antibiotic agents and especially imipenem are the main factors causing high prevalence of resistance of P. aeruginosa strains.
Fazeli and Momtaz (2014) 29 reported more similar results with our findings.They showed that P. aeruginosa strains of clinical infections harbored the highest levels of resistance against penicillin (100%), tetracycline (90.19%), streptomycin (64.70%) and erythromycin (43.13%).The most actual antibiotics that can be used against P. aeruginosa infections are beta-lactam antibiotics in which imipenem as a carbapenem is considered as the most suitable antibiotic.However, carbapenem resistance occurs because of decrease in antibiotics absorption due to lack of an outer membrane porin, exclusion from the cell by efflux pump, decrease in outer membrane permeability and production of metallo-beta-lactamases (MBL).The IMP type is the most clinically significant carbapenemases which encoded by blaIMP gene and harden all treatments done using the imipenem. 31Our results also showed that about 96.03% of imipenem-resistant P. aeruginosa strains harbored the blaIMP-1 gene which was considerable.Peymani et al. (2015) 32 revealed that 107 (35.66%)P. aeruginosa isolates were non-susceptible to imipenem and/or meropenem and among them, 56 (52.3%) isolates were MBL producer.Twenty-four isolates of 56 (42.85%)MBL producer strains harbored MBLencoding genes.Prevalence of blaIMP-1 gene was 25% which also was lower than our findings.Moosavian et al. (2015)  33 reported that of 236 examined P. aeruginosa isolates, 122 isolates (51.40%) were resistant to imipenem.They showed that 67 strains (55%) of imipenem-resistant P. aeruginosa isolates harbored blaIMP-1 gene which was lower than our findings.Abiri et al. (2015)  34 reported that among the 225 P. aeruginosa isolates, 33.7% (76/225) and 18.1% (41/225) were resistant to imipenem and meropenem, respectively.They showed that of the 76 imipenem-resistant P. aeruginosa strains, 45 (59.2%) were positive for MBLs and 34 (75%) strains carried the blaIMP-1 gene which was similar to our findings.Tarashi et al. (2016) 35 indicated that among 278 imipenem-resistant P. aeruginosa strains, 178 (64.02%) were MBL producers.The blaIMP-1 gene was detected in 16.8% of P. aeruginosa isolates.As far as we know, the present study reported the highest prevalence of blaIMP-1 gene among the imipenem-resistant P. aeruginosa strains of human clinical samples.Differences in types of samples is the main factor for various prevalence rate of blaIMP-1 gene reported in different studies.

Conclusions
In conclusion, we identified a large number of P. aeruginosa strains in the BIs and UTIs samples taken from inpatients and outpatients.Resistance against streptomycin, nalidixic acid, aztreonam, cotrimoxazole, ciprofloxacin, cefotaxime and gentamycin was considerable in the P. aeruginosa strains of clinical samples.Moderate prevalence of resistance against imipenem was supported by the high prevalence of blaIMP gene.High prevalence of this gene poses an important public health threat regarding the occurrence of resistance against imipenem in the clinical isolates of P. aeruginosa.It seems that prescription of imipenem is not effective for treatment of the cases of P. aeruginosa infection.