Antibiotic resistance and presence of blaPER-1, blaVEB-1 and blaPSE-1 beta-lacamases among clinical isolates of Pseudomonas aeruginosa from ICU settings

Introduction: Pseudomonas aeruginosa isolates are among the most common pathogens causing nosocomial infections. They are intrinsically resistant to most of antibiotics such as novel β-lactams and therefore, can develop resistance during treatment, culminating in failure in remedy. Objectives: The aim of this study was to detect the genes encoding class A extended-spectrum betalactamases (ESBLs) such as PER-1, VEB-1 and PSE-1 among P. aeruginosa isolates from intensive care unit (ICU) patients. Materials and Methods: A total of 65 isolates were collected from ICU in three hospitals of Tehran in 2016. The antibiotic susceptibility test was conducted according to Clinical and Laboratory Standards Institute (CLSI) guideline. MIC of ceftazidime was done with agar dilution method. The combine disk test was performed for detection of isolates producing ESBLs. Polymerase chain reaction (PCR) was performed to detect the PER-1, VEB-1 and PSE-1 genes using specific primers. Results: Fifty-four percent (n=38) of patients were male and 46% (n=27) were female. The majority of ICU isolates were resistant to augmentin (93.8%, n=61) and cefpodoxime (84.8%, n=56). Fifty (77%) isolates were ESBL positive, among which 94% (n=47) harbored PER-1 gene followed by 52% (n=26) VEB-1 and 16% (n=8) PSE-1 genes. Conclusion: Concomitance presence of blaPER1 and blaVEB1 was observed among 10 isolates, and 7 amplified all these three genes. A high number of ICU P. aeruginosa isolates were ESBL producers. The frequency of blaVEB1 and blaPER1 were relatively high, while blaPSE1 was detected among a low number of isolates. Moreover, resistance to carbapenems was low. It is necessary to follow up ICU centers because of drugresistant P. aeruginosa isolates.


Introduction
Pseudomonas aeruginosa isolates are among the most common pathogens causing nosocomial infections.These strains are intrinsically resistant to most of drugs such as novel β-lactam antibiotics and therefore, cause failure during treatment, culminating in higher mortality (1)(2)(3).Antimicrobial resistance of P. aeruginosa strains and the rate of multidrug resistance are increasing and outbreaks caused by carbapenem-and multidrug-resistant isolates have been described by several surveys (4,5).The most common resistance mechanisms have been developed through down-regulation of the porin OprD, and via increased activity of multi-drug efflux pumps, primarily MexAB-OprM (6,7).The novel beta-lactamases, including AmpC beta-lactamases, extended-

Key point
Concomitance presence of blaPER1 and blaVEB1 was observed among P. aeruginosa isolates.A high number of ICU P. aeruginosa isolates were ESBL producers.The frequency of bla VEB1 and bla PER1 were relatively high, while bla PSE1 was detected among a low number of isolates.The frequency of carbapenem resistance was low.spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs), have emerged worldwide as a source of antimicrobial resistance in gram-negative isolates (8).The only effective β-lactam antibiotic against co-AmpC and ESBL producers are carbapenems; however, resistance to carbapenems has recently occurred increasingly with a profound rate.However, the increasing use of these compounds has culminated in the emergence of carbapenem-resistant P. aeruginosa clinical isolates, limiting treatment choices.Detection of isolates producing ESBLs in the clinical laboratory is an important issue for guidance of proper therapeutic schemes and the implementation of the infection control measures.The combined or synergistic methods are phenotypic routes of ESBLs detection (9,10).In P. aeruginosa several classes of enzymes such as class A ESBLs have been identified including bla PER (mostly from Turkey), bla VEB (from South-East Asia, France and Bulgaria), bla GES/IBC (France, Greece and South Africa) and other less common types.These six carbapenemase types have low similarity at the genetic level, and yet they have identical hydrolysis profiles.The bla PER occurs less frequently, but has clinical importance by conferring resistance to oxy-imino-beta-lactams (11,12).Unfortunately, several antibiotic resistance mechanisms often have been occurred simultaneously, thereby conferring multidrug-resistant phenotypes.

Objectives
The aim of this study was to detect the genes encoding class A ESBLs of PER-1, VEB-1 and PSE-1 among clinical isolates of P. aeruginosa in ICU patients.

Bacterial isolates
A total of 65 isolates of P. aeruginosa were collected from intensive care unit (ICU) patients in several hospitals of Tehran between the years 2015 and 2016.Tests and culture media for identification of these isolates included: catalase and oxidase tests, hydrogen sulfide (H2S), indol, motility (SIM medium), triple sugar iron agar (TSI), and methyl red (MR), Voges-Proskauer (VP), Simon citrate, urease, oxidative/fermentative (OF), Macconkey agar and cetrimide agar.

Phenotypic detection of ESBL producers
The combine disk test was performed to detect the isolates producing ESBLs.The ceftazidime and cefotaxime disks with or without clavulanic acid were used in this test.A difference of more than 5 mm between these disks along with clavulanic acid was considered as positive.

DNA extraction
One colony of each isolate was suspended in 10 mL Lauria Bertani (LB) broth and incubated an overnight at 37 C.The tubes were centrifuged at 4000 rpm for 10 minutes and the precipitate was used for extraction.DNA was isolated by a boiling method and DNA Extraction kit (DIAtom DNA Prep 100).

Ethical issues
The research followed the tenets of the Declaration of Helsinki.This study was approved by AJA University of Medical Sciences, Tehran, Iran (Grant # 9504).

Data analysis
Data were analyzed using GraphPad Prism 6 and SPSS 20.The Student's t-test was applied for analysis, and P value of < 0.05 was considered as the significance of differences.

Antibiotic susceptibility test
The majority of the isolates were resistant to augmentin (93.8%, n=61) and cefpodoxime (84.8%, n=56).The frequency of resistance to other antibiotics were as follows: CAZ (60%, n=39), CPM (47.6%, n=31), PRL (44.6, n=29), negative rods, and thus the increasing resistance is a great concern.About 0.3% of the total genes in P. aeruginosa are responsible for antibiotic resistance (14).Approximately 77% of ICU isolates were ESBL producers which is comparable with previous studies (15,16).Susceptibility to clavulanic acid is a diagnostic method.Resistance to clavulanic acid indicates the probable presence of AmpC and other enzymes that are not inhibited by this inhibitor (17).On the other hand, de-repression of the chromosomal AmpC β-lactamases can culminate in the decreased susceptibility to β-lactam antibiotics.

Discussion
Pseudomonas aeruginosa is a prevalent nosocomial pathogen that causes a wide spectrum of opportunistic and nosocomial infections.Isolates of P. aeruginosa are resistant to a variety of antibiotics with mechanisms including intrinsic capabilities and acquired (chromosome or plasmid-mediated) resistance.In the present study, most of isolates were collected from urinary tract infections.
The urine is the main route for antibiotic excretion from the body and thus, most of resistant isolates were collected from this site.In this study, the majority of the isolates were resistant to augmentin/co-amoxiclav and cefpodoxime.Moreover, most of them were resistant to third generation cephalosporins (aztreonam, ceftriaxone and cefotaxime).These antibiotics are important in the treatment of gram-  In this study, among ESBL producers, 94% and 52% amplified bla PER1 and bla VEB1 respectively, suggesting the presence of other enzymes such as Amp-C and MBLs or possibility of efflux pumps interference.In the study by Shacheraghi et al in ESBL positive isolates, the prevalence of bla VEB-1 and bla PER-1 was 100% and 68.3%, respectively (18), but a low number (5.8%) of imipenem-resistant isolates contained bla PER-1 in another study.In a study by Davodian et al, among wound isolates the 90% and 88% were resistant to augmentin and cefpodoxime, 40% of them being bla VEB1 positive (15).Besides this, in our study, resistance to imipenem and meropenem was low (18.4 and 20%, respectively), suggesting that the low presence of carbapenemhydrolyzing enzymes in addition to class A enzymes.However, continuous and prolonged antibiotic periods are likely to be a critical factor in the selection of highly antibiotic-resistant P. aeruginosa strains.Therefore, combination therapy (usually with a β-lactam and an aminoglycoside) is now important to eradicate infections due to P. aeruginosa.The broad-spectrum carbapenems are efficient drugs for the eradication of infections caused by P. aeruginosa; however their efficacy is increasingly compromised due to the emergence and the worldwide dissemination of carbapenem-resistant strains.
Fortunately, in our study the resistance to carbapenems was low.A high number isolates of P. aeruginosa from ICU were ESBL producers.The frequency of bla VEB1 and bla PER1 were relatively high, while bla PSE1 was detected among a low number of isolates.ICU wards are of main sources for infections with drug-resistant strains.Continuous and prolonged antibiotic periods, hospitalization and misuse are pivotal factors in the selection of highly resistant strains.Combination therapy (usually with a β-lactam and an aminoglycoside) is important to treat Pseudomonas infections.

Table 1 .
The specific primers used in this study