β-lactamase genes in carbapenem resistance Acinetobacter baumannii isolates from a Turkish university hospital

Introduction: The spread of Acinetobacter baumannii, resistant to most of the available antimicrobial agents, is a serious health problem. The high rate of carbapenem resistance among Acinetobacter baumannii isolates is considered as a threat to public health. In this study, we aimed to determine the antibiotic resistance and related genes in carbapenem-resistant Acinetobacter baumannii isolates. Methodology: Ninety six isolates of A. baumannii were included. Antimicrobial susceptibility was performed by Phoenix Automated System and disk diffusion method. Carbapenem resistane was characterized by scrneeing of resistance genes such as blaTEM, blaSHV, blaCTX-M1-2, blaPER, blaVEB, blaKPC, blaGES, blaNDM, blaVIM, blaIMP and blaOXA23-24-51-58 using multiplex polymerase chain reaction. Results: Resistance for the levofloxacin, gentamicin, amikacin, and tigecycline were determined as 96.9%, 93.7%, 72.9% and 45.8% respectively. Colistin was the only susceptible antibiotic against all clinical isolates. All isolates were defined as multidrug resistance and of these, 31.2% were extensively drug-resistant (sensitive only to colistin). BlaOXA-51 and blaOXA-23 genes were detected in 100% strains while blaTEM was found in only 2% strains. There was no amplification for the blaSHV, blaCTX-M1-2, blaPER, blaVEB, blaKPC, blaGES blaNDM, blaVIM, blaIMP and blaOXA24-58 genes. Conclusions: The high frequency of blaOXA-23 and low frequency of blaTEM gene was observed that indicate prevalence of a variety of A. baumannii strains. The rates of resistance genes vary from region to region. Studies are required for the prevention and control of A. baumannii infection and to formulate the strategies of antibiotic usage.


Introduction
Acinetobacter baumannii (A.baumannii) is the opportunistic pathogen that causes nosocomial infection such as urinary tract infection, wound infection, pneumonia and sepsis [1]. A. baumannii is resistant to stressful environmental conditions.In addition, presence of multiple resistance mechanisms and its ability to gain new resistance characteristics against available antibiotics help to cause hospitalacquired infection more easily.A. baumannii with a variety of resistance mechanisms causes difficulties in treatment by aminoglycosides, cephalosporins, carbapenems and ciprofloxacin.Its involvment in clinical infections is increased day by day [2].
It is of great concern that if multidrug resistant (MDR) A. baumannii infections are not controlled, they may cause epidemics in the hospital and may spread intercities and even cross-countries [1,2].Therefore, the investigation for the prevalence of MDR A. Baumannii is an important step in combating this infection.The aim of this study was to characterize the susceptibility profiles and genetic mechanisms of resistance of clinical strains of A.baumannii in Turkey.

Methodology
This study was approved by the Scientific and Ethical Committee of Tokat Gaziosmanpasa University Clinical Research Ethics Committee (Tokat, Turkey), (16-KAEK-013/19.01.2015).

Bacterial strains and antimicrobial susceptibility testing
Clinical isolates of A. baumannii (n = 96) were collected from several units of Duzce University Hospital in Turkey between January 2014 and July 2015.The isolates were identified by Phoenix Automated System (BD Diagnostic Systems, Sparks, MD, USA) according to the manufacturer's instructions.Antimicrobial susceptibility testing was performed by Phoenix Automated System and disc diffusion method.The results were interpreted according to the guidelines by Clinical and Laboratory Standards Institute [5].

Multiplex PCR for detection of blaOXA genes
Genomic DNA was obtained from bacterial culture grown overnight in Luria Broth [6] and used in all PCR amplification.Multiplex PCR was used for detecting blaOXA-51-like, blaOXA-23-like, blaOXA-40-like and blaOXA-58-like genes.Primers used for the detection of resistance genes are shown in Table 1.PCRs were performed in a final volume of 50 µL that included 5 μL of genomic DNA, 20 pM of each primer, 10 μL reaction buffer (Promega), 3 μL 25 mM MgCl2, 200 μM of each dNTPs and 1.5 U of Taq Polymerase (Promega, Madison,WI, USA).PCR amplification conditions were as follows: initial denaturation at 94°C for 3 minutes followed by 30 cycles of 25 seconds at 94°C, 40 seconds at 52°C and 50 seconds at 72°C with a final extension 5 minutes at 72°C.

Multiplex PCR for detection blaCTX-M1-2 genes
Multiplex PCR was used for detecting blaCTX-M1 and blaCTX-M2 group β-lactamase genes.Primers used for detection blaCTX-M genes are shown in Table 1.PCRs were performed in a final volume of 50 µL and included 5 µL of genomic DNA, 20 pM of each primer, 10 µL reaction buffer (Promega, Madison, WI, USA), 3 mL 25 mM MgCl2, 200 mM of each dNTPs and 1.5 U of Taq Polymerase (Promega, Madison, WI, USA).PCR amplification condition was as follows: initial denaturation at 95°C for 2 minutes followed by 30 cycles of 1 minute at 95°C, 1 minute at 55°C and 1 minute at 72°C, with a final extension of 10 minutes at 72°C.

PCR amplifications of the ESBLs and MBLs genes
Simplex PCR was used to amplify ESBL and MBL genes and the primers listed in Table 1 were used.PCRs were performed in a final volume of 50 µL and included 5 μL of genomic DNA, 20 pM of each primer, 10 μL reaction buffer (Promega, Madison, WI, USA), 3 μL 25 mM MgCl2, 200 of μL dNTPs and 1.5 U Go Taq Flexi Polymerase (Promega, Madison,WI, USA) in a final volume of 50 μL.PCR amplification conditions was performed according to references listed in Table 1.All PCR results were analyzed on 1% agarose containing 0.5 μg/mL ethidium bromide, and subsequently visualized under UV light.

Results
A total of 96 clinical isolates of A. baumannii were collected from Duzce University hospital in Turkey over a period of 18 months.All patients were hospitalized into several units such as sixty one patients (63.5%) in intensive care unit, 24 patients (25%) in the internal units (cardiology, pulmonology, etc.) and 14 patients (14.6%) in surgery clinics.Most of the isolates were obtained from respiratory specimens (tracheal aspirates 54.2%, sputum 12.5%, bronchoalveolar lavage 5.2%) followed by wound (8.3%), urine (8.3%), blood (8.3%) and cerebrospinal fluid (3.1%).All strains were identified as A. baumannii by Phoenix Automated System and blaOXA-51 PCR for specify the A. baumannii species.
All of the A. baumannii strains were resistant to imipenem, meropenem, ampicillin-sulbactam, ceftazidime, cefepime, piperacillin-tazobactam and ciprofloxacin.Resistance for the levofloxacin, gentamicin, amikacin and tigecycline were 96.9%, 93.7%, 72.92% and 45.8% respectively.However colistin resistance was not observed in any strain.All strains were defined as MDR based on resistance to more than two antibiotic groups.The resistance rates of A. baumannii against antibiotics are shown in Table 2.

Discussion
A. baumannii often develops resistance against carbapenems.Since carbapenems are broad-spectrum antimicrobial and hydrolyze β-lactamases, they play a crucial role in the treatment of nosocomial infections caused by Gram-negative bacteria [7].The high genome plasticity of A. baumannii contributes to its virulence and high adaptation on inanimate surfaces particularly in hospital environment.This reduces the response to long term treatment and generates the multidrug resistant (MDR) strains that show resistance to last three groups of antibiotics [8].MDR strains are often resistant to carbapenems [9,10].In this study, all strains were defined as MDR and of these, 31.2% were extensively drug-resistant (sensitive only to colistin).
High rates of resistance against cephalosporins are seen all over the world [11][12][13][14][15].The most frequently used treatment regime for A. baumannii infections include carbapenems and aminoglycosides.Carbapenems produce synergistic bactericidal activity in combination with aminoglycosides; therefore, carbapenems are often used in combination therapy with aminoglycosides [11].Although several studies have reported different rates of resistance for aminoglycoside and quinolone, their resistance rates are still high in the world [11,13,14].According to the annual report of the European Antimicrobial Resistance Surveillance Network, MDR A. baunannii is very common in Europe and combined resistance to fluoroquinolones, aminoglycosides and carbapenems are the most frequently reported resistance phenotype and accounted for almost half of the reported isolates in 2015 [16].In this study, the resistance rate of A. baumannii strains to ciprofloxacin, levofloxacin, Colistin gentamicine, amikacin were 100%, 96.9%, 93.7% and 72.9% respectively.In our study, the rates of resistance to the indicated antibiotics were consistent with the literature.
Although, yearly tigecycline resistance rates ranged from 0 to 42%, tigecycline and colistin are used in combination or alone as the last option for the treatment of MDR A. baumannii strains [10][11][12]14,15].In our study, we found 45.8% resistance against tigecycline.Given that the history of tigecycline is not very old, rapidly increased resistance propose that MDR A. baumannii strains may not be cured by tigecycline in near future.This situation poses a serious threat to infections whose treatment options are very limited.
OXA23-24-51-58-like Class D β-lactamases produced by A. baumannii are investigated under 4 phylogenetic groups.The blaOXA-51-like genes naturally present in the genome of A. baumannii and were found as an intrinsic gene in all A. baumannii strains in this study.BlaOXA-23like is the most common source which causes plasmid or chromosomal transferable carbapenem resistance.BlaOXA-23 carraige has been reported all over the world for instance; China 46.31% [13], USA 58.3% [22], Kuwait 85% [12], Poland 27.9% [11].The BlaOXA-23 positive A. baumannii strains have been involved in nosocomial outbreaks.It was studied that a horizontal gene transfer within various isolates of the species constitutes a primary factor in the continued increase of carbapenem resistance over the years [23].In Turkey, the prevalence rate of blaOXA-23 were between 31 and 91.5% [18,20,21].In this study, all strain had the blaOXA-23 genes as blaOXA-51.
In current study, any strain that contain blaOXA58/40like are not detected.According to the centers, variation of the prevalence of blaOXA58/40-like has been drawn attention.Based on literature, strains which have this variant, are mostly reported from Asia and Middle East countries.It suggests that blaOXA58/40-like are not very common in Turkey.It was confirmed that one strain had blaOXA40-like gene in clinical A. baumannii isolate [18].

Conclusions
In conclusion, MDR A. baumannii poses a significant threat to patients and healthcare systems.A number of β-lactamase coding genes have been identified in Mediterranean, Middle East countries, Asia and Europe.Even though blaOXA-23 was present in all our isolates, it is noteworthy that frequency of blaTEM was and other resitance genes were not detected low in our study.Our results suggest that the prevalence of resistance genes vary from region to region.Therefore, studies for genotypic fingerprinting of MDR A. baumannii should be encouraged.

Table 1 .
Primers used in the amplification of selected genes.

Table 2 .
Resistance rates of A. baumannii isolates.