High prevalence of TEM, VIM, and OXA-2 beta-lactamases and clonal diversity among Acinetobacter baumannii isolates in Turkey

Introduction: Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections with high mortality. Treatment options are limited owing to its resistance to numerous antibiotics. Here, we sought to determine the antibiotic susceptibilities of A. baumannii isolates, investigate clonal relationship among the strains, and determine the frequency of beta-lactamase resistance genes. Methodology: The identification and antibiotic susceptibilities of 69 A. baumannii strains were determined using a BD-Phoenix automated system. The presence of blaOXA-2, blaOXA-10, blaOXA-23, blaOXA-24/40, blaOXA-51, blaOXA-58, blaTEM, blaSHV, blaIMP, blaVIM, and blaGIM genes were investigated using polymerase chain reaction (PCR), and clonal relatioships among the isolates were determined using pulsed-field gel electrophoresis (PFGE). Results: All strains were resistant to ampicillin–sulbactam, gentamicin, cefepime, ciprofloxacin, and ceftriaxone. While 65 of the 69 strains (94.2%) were resistant to piperacillin-tazobactam, amikacin, imipenem, and meropenem, all strains were susceptible to tigecycline and colistin. The frequencies of blaOXA-51, blaOXA-23, blaTEM, blaOXA-2, blaVIM, and blaSHV were 100%, 94.2%, 53.6%, 21.7%, 14.5%, and 2.9%, respectively. Based on PFGE results, 56 of the 69 strains were clonally related, and the clustering rate was 81.2%. No common outbreak isolate was detected. Conclusions: The most prevalent OXA genes were blaOXA-51, blaOXA-23, and blaOXA-2. Furthermore, blaTEM, blaSHV, and blaVIM, which are common in Enterobacterales and Pseudomonas spp, were detected, suggesting horizontal gene transfer had occurred between bacteria. No single clone outbreak was detected by PFGE. However, multiclonal spread and the high clustering rate suggest cross-contamination. Therefore, in future, more effective infection control measures must be implemented.


Introduction
Acinetobacter baumannii is a gram-negative coccobacillus that is strictly aerobic, motile and nonfermentative.As it is resistant to dryness and disinfectants, it can survive in hospital equipment and fabrics for long periods [1][2][3].Therefore, in the last three decades, A. baumannii has been reported as a cause of nosocomial infections and outbreaks.It can cause healthcare-associated infections such as meningitis, sepsis, and ventilator-associated pneumonia, which have high mortality rates, particularly in patients that are hospitalized for long periods, are undergoing invasive procedures, or are attached to mechanic ventilators [1,2,4].Treatment options are limited as the bacteria are resistant to a large number of antibiotics, including carbapenems.Therefore, in 2009, the Infectious Diseases Society of America identified A. baumannii as one of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which show multidrug resistance and can escape the biocidal effects of antibiotics via diverse resistance mechanisms, including drug inactivation/alteration, modification of drug binding sites/targets, and changes in cell permeability [5].Similarly, in 2017, the World Health Organization defined A. baumannii as a critical priority pathogen requiring urgent development of novel antibiotics [6].
The increase in antibiotic resistance has considerably limited the treatment options that are available against nosocomial pathogens worldwide.Therefore, in addition to antimicrobial treatment, effective control measures must be put into place to prevent nosocomial infections.Moreover, it is crucial to understand the molecular characterization and epidemiology of the nosocomial pathogen.
In this study, we aimed to determine the antibiotic susceptibility, the resistance genes, and the clonal relationship among A. baumannii strains isolated from a Turkish hospital.

Ethical approval
The study protocol was approved by the Noninterventional Clinical Research Ethics Board of Karabuk University.

Bacterial Strains
The study included 69 non-repetitive A. baumannii strains that were isolated from clinical samples from inpatients at Karabuk University Training and Research Hospital from 2014 to 2015.The strains were stored in tryptic soy broth containing 10% (v/v) glycerol at −80°C until use.

Strain Identification and Determination of Antibiotic Susceptibility
All clinical samples were inoculated on Columbia agar enriched with 5% sheep blood [Becton Dickinson (BD, Sparks, MD, USA)], Eosin Methylene Blue agar (BD) and chocolate agar (BD) followed by incubation for 18-24 hours at 35°C.Identification and antibiotic susceptibility of the strains were determined using the Phoenix 100 Automated Microbiology System (BD, Sparks, MD, USA).Strain identifications were also confirmed using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method using the VITEK®-MS device (Biomerieux, Marcy-l'Étoile, France).The susceptibility of isolates to imipenem, meropenem, tigecycline, and colistin was determined using the gradient minimum inhibitory concentration (MIC) method (Liofilchem, Roseto degli Abruzzi, Italy).The antibiotic susceptibility test results were evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [7].The Food and Drug Administration tigecycline breakpoints for Enterobacterales were applied to Acinetobacter spp. as breakpoint criteria are not outlined in the CLSI guidelines [8].Escherichia coli (E.coli) ATCC 29212 and P. aeruginosa ATCC 27853 were used as quality control strains.

PCR detection of antibiotic resistance genes
Genomic DNA was isolated using the QIAsymphony DSP Virus/Pathogen kit in the QIAsymphony system according to the manufacturer's instructions (Qiagen, Valencia, CA, USA).DNA samples were stored at −25°C until use.Previously defined primers were used for the detection of betalactamase genes (Table 1) [9][10][11][12].
Presence of the bla OXA-23 , bla OXA-24/40, bla OXA-51 , and bla OXA-58 genes was investigated using the multiplex PCR method, and bla OXA-2 , bla OXA-10, bla TEM, bla SHV, bla IMP , bla VIM , and bla GIM genes by the in-house PCR method [10].The Gene Amp PCR System 9700 system (Applied Biosystems, Waltham, MA, USA) was used for amplification.The amplification conditions for bla OXA-23 , bla OXA-24/40, bla OXA-51 , and bla OXA-58 were the following: initial denaturation for 3 minutes at 94°C, followed by 35 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and a final extension at 72°C for 10 minutes.The following conditions were used to amplify bla OXA-2 , bla OXA10 , bla TEM, bla SHV , bla IMP , bla VIM , and bla GIM : initial denaturation for 10 minutes at 95°C, followed by 35 cycles consisting of denaturation at 94°C for 1 minute, annealing at 57°C for 1 minute, and extension at 72°C for 1.5 minutes, and a final extension at 72°C for 3 minutes.
Amplification products were analyzed by electrophoresis for 1 hour at 100V in a 1.5% agarose gel followed by ethidium bromide staining.Imaging was performed using the Gel Logic 2200 imaging system (Kodak Co., Rochester, NY, USA) under ultraviolet light.Band sizes for each gene were measured by comparison with a 100-bp DNA ladder (New England Biolabs, Ipswich, MA, USA).

Molecular Typing by pulsed-field gel electrophoresis (PFGE)
The clonal relationship among A. baumannii isolates was determined using a method described by Durmaz et al. [13].Genomic DNA was cut with 10 u/µl of ApaI restriction endonuclease enzyme (Fermentas, Vilnius, Lithuania) after which PFGE was performed by clamped homogeneous electric field (CHEF) electrophoresis on a 1% agarose gel with a CHEF-DR II system (Bio-Rad Lab, Nazareth, Belgium).The fragments were separated over a period of 24 hours at 14°C with a gradient of 6 V/cm, a reorientation angle of 120° and an initial switch time of 5 seconds and a final switch time of 30 seconds.For band analysis, similarities were calculated using the Dice similarity coefficient (DSC) and cluster analysis was performed using the unweighted pair group using arithmetic means (UPGMA).If the DSC was < 90%, isolates were considered to be of different genotypes, and if DSC was ≥ 90%, isolates were classed as the same genotype.A dendrogram was created from the PFGE data using the UPGMA-based clustering algorithm.

Statistical Analysis
The data were analyzed using the MINITAB -17 statistical software program (Minitab Inc., State College, PA, USA).The Anderson-Darling test was performed to determine whether the data were normally distributed.Descriptive statistics are presented as numbers and percentages.The mean age of the patients is presented as mean ± standard deviation.For comparisons of beta-lactamase genes and isolated units, the chi-square test was performed, and a p-value < 0.05 was considered as statistically significant.

Distributions of antibiotic resistance genes
The PCR results indicated that the intrinsic bla OXA- 51 gene specific for A. baumannii was detected in all of the isolates (100%), bla OXA-23 in 65 isolates (94.3%) and bla OXA-2 in 15 isolates (21.7%).In addition, bla TEM was detected in 37 of the 69 isolates (53.6%).No significant relationship was determined between the source of the isolated strain and the frequency of bla TEM (p = 0.28).Although bla VIM was detected in 10 (14.5%) strains and bla SHV in two (2.9%) strains, bla GIM , bla IMP, bla OXA-10, bla OXA-24/40, and bla OXA-58 were not detected in any of the isolates.Of the 65 carbapenem-resistant strains, 16 (24.6%)possessed multiple beta-lactamase genes in addition to bla OXA-51 and bla OXA-23 .Of these strains, nine contained bla TEM and bla VIM, six contained bla TEM and bla OXA-2 , and only one had bla TEM and bla SHV .Furthermore, four carbapenem-susceptible strains contained only bla OXA-51 .

PFGE typing
A dendrogram was constructed based on the PFGE results.The distribution of the beta-lactamase genes among the strains and other demographic data are illustrated in Figure 1.The 69 A. baumannii isolates revealed 20 different profiles.We found that 56 of the 69 strains were clonally related, and these strains were gathered in seven clusters (a tolerance of 1.0 and a cutoff value of 90% similarity were applied).The clusters were named with upper case letters (A-G), and 56 of the 69 strains were found to be within a cluster, corresponding to a clustering rate of 81.2%.The numbers of strains in each of the clusters ranged from 2 to 24.The largest cluster was clone A that was formed from 24 isolates (34.8%), followed by clone D (12 isolates; 17.4%), and clone B (9 isolates; 13%) All of the clonally related strains possessed the bla OXA-23 gene, and bla OXA-23 negative strains (n = 4) were located in a sporadic group.
Of the 24 isolates present in clone A, 20 (83.3%) carried bla TEM in addition to bla OXA-51 and bla OXA-23 , and nine (37.5%) carried the bla VIM gene.In nine strains, both bla TEM and bla VIM were detected.Furthermore, in four strains, only bla OXA-51 and bla OXA-23 were detected and other beta-lactamase genes were not found.
We isolated the clone A strains over a 12-month period.Of the 24 strains present in clone A, 17 were obtained from the intensive care unit (ICU) and seven from service patients.Two of the three strains obtained from the coronary ICU were isolated at one-day intervals.One of these isolates was obtained from ETA and the other from blood.Both strains had bla OXA-23 in addition to bla TEM and bla VIM.Eight strains were isolated from blood, seven from wounds, six from ETA, and three from other samples.Four of the seven wound isolates were obtained from different ICUs and services in May 2014 over 15 days.Two of the three strains isolated from wound cultures of orthopedic surgery patients were obtained over one month.
Of the 12 isolates present in clone D, eight strains carried bla TEM , three strains carried bla OXA-2, and two strains carried the bla SHV gene.In three strains, bla OXA-2 and bla TEM were both detected, and one strain possessed both bla TEmetalloM and bla SHV .
The clone D strains were isolated over 22 months.Eleven of the strains were obtained from ICUs and one from the neurology service.Six strains were isolated from ETA, three from blood, two from urine, and one from sputum.Five of the six ETA strains were isolated from different ICUs over two months.Two of the three blood isolates were obtained from the reanimation ICU and the internal ICU on the same day (10 May 2014).
All nine strains present in clone B harbored bla OXA- 23 , while three strains possessed bla OXA-2 in addition to bla OXA-23 .Other beta-lactamases were not detected in any of these strains.The clone B strains were isolated over 14 months.Three of these were obtained from blood, three from ETA, and three from wound cultures.Three ETA strains were isolated from three different ICUs over one month.Two of the three strains were isolated from two different patients in the surgical ICU on the same day (18 October2014).One strain was obtained from ETA and one from blood.Both strains had only bla OXA-23.Two of the strains in clone G were obtained from the ETA samples of the surgical ICU over two months.Both strains had bla OXA-23 and bla TEM .All four carbapenem-susceptible strains were isolated from the surgical ICU on different dates.

Discussion
The increase in multidrug resistance among A. baumannii strains is a global health threat.Long-term, low-dose, or unnecessary consumption of antibiotics leads to the emergence of multidrug-resistant strains [14,15].Moreover, treatment options are limited by the ability of A. baumannii to develop novel drug resistance mechanisms and the spread of resistance via mobile genetic elements [1,4,14].In the last two decades, carbapenem resistance in A. baumannii isolates has reached high levels worldwide.In this study, the rate of carbapenem resistance among the clinical isolates was 95%.Similarly, data reported by the Central Asia and Eastern European Surveillance of Antimicrobial Resistance revealed that the carbapenem resistance rate was 93% in Turkey [16].According to the 2017 report of The European Antimicrobial Resistance Surveillance Network (EARS-Net), the rate of carbapenem resistance was 52.5% in Hungary, 78.7% in Italy, 68.2% in Spain, and 96.2% in Croatia [17].Tigecycline and colistin are often used for the treatment of carbapenem-resistant A. baumannii infections.In this study, we found that all the isolated strains were susceptible to tigecycline and colistin.However, there have been many reports of tigecycline and colistin resistance, which range from 0.6% to 45.8% [18][19][20][21] and 0 to 30.6%, respectively, around the world [22][23][24][25].Furthermore, colistin resistance was reported to be of 4% in the EARS-Net 2016 report [26].
Beta-lactamase enzymes, in particular oxacillinases (bla OXA ), which are defined as Ambler Class D, play a key role in antimicrobial resistance among Acinetobacter species [3,14].The first OXA enzymes, OXA-1, OXA-2, and OXA-3, were originally identified by Sykes and Matthew in 1976 [4].OXA-2 type betalactamases hydrolyze oxacillin and cloxacillin at a high level and are weakly inhibited by clavulanic acid, and they were first observed in P. aeruginosa from France in 2002 and in E. coli from Israel in 2005 [27][28].In this study we detected the bla OXA-2 gene in 15 of the 69 (21.7%) isolated A. baumannii strains.To the best of our knowledge, this is the first report of the presence of the bla OXA-2 gene among A. baumannii strains in Turkey.Moreover, there are few reports concerning bla OXA-2 in the literature.Rahimzadeh et al. reported that the frequency of the bla OXA-2 gene among A. baumannii isolates was 11.6% in Iran [29].In addition, Maurya et al. reported that bla OXA-2 is located within class I and class II integrons among different species including E. coli, K. pneumoniae, and P. aeruginosa that were isolated in India [30].The presence of bla OXA- 2 in different bacteria and its presence in integrons indicate horizontal gene transfer.
In this study, bla OXA-23 was detected in 65 of the 69 isolates (94.2%).This encodes the first known carbapenem-resistant OXA-enzyme that was initially reported in Scotland in 1985 [4,14].Prior to the discovery of bla OXA-23, the OXA enzymes were thought to be a small group of enzymes encoded by plasmids that affected only penicillins; the migration of these genes via transposons caused them to become the dominant mechanism of antibiotic resistance [4].The frequency of bla OXA-23 in A. baumannii strains has been reported to range from 32% to 100% in Turkey [21,31,32] and from 28% to 96% worldwide [33][34][35][36].
The metallo-beta-lactamases (MBL), which are known as Ambler Class B, are the second most commonly identified beta-lactamases in A. baumannii isolates [22].MBLs are so-called as they utilize a Zn 2+ cofactor [14,22].They are commonly isolated from Pseudomonas species [14].In this study, bla VIM was detected in 14.5% of the isolates, whereas bla GIM and bla IMP were not detected in any of the isolates.In a study of 519 isolates in Turkey, bla VIM was detected at the rate of 0.2% (n = 1), whereas bla GIM and bla IMP were not found [18].In this study, the high rate of bla VIM detection (14.5%) suggests that this resistance gene, which is most often found in Pseudomonas spp., had been transferred to Acinetobacters via plasmids over time.The frequency of bla VIM has been reported as 10.1% in Korea [37] and 34.2% in India [22].Among the Class A beta-lactamases, bla TEM and bla SHV were detected in 53.6% and 2.9% of the isolates, respectively.Similarly, Beriş et al. reported that the frequency of bla TEM and bla SHV in Turkey was 55.7% and 7.7%, respectively [18].Conversely, Coşkun et al. reported a low frequency of bla TEM (2%) [21].The frequency of bla TEM has been reported to be 2.9%-71% worldwide [19,38,39].
To prevent the spread of Acinetobacter baumannii within the hospital, it is essential to determine the potential origins of the microorganism and its transmission routes.Therefore, it is important to determine whether a clonal relationship exists among the isolates.In this study, the clonal relationship among A. baumannii isolates was investigated using the PFGE, which is considered to be a gold standard method.The PFGE results indicated that there was no single clone outbreak in our hospital, and, instead, polyclonal spread had taken place.Three dominant clones carrying bla OXA-23 , clones A, B and D, were observed to circulate between units within the hospital setting.In addition, a small clonal spread was observed between some units.The main reservoir of the strains was the surgical ICU and the reanimation ICU.Some clone A and clone B isolates were obtained from the same unit over 1-2 days.For instance, two of the three clone A strains isolated from the coronary ICU were obtained with a one-day interval, and two of the three clone B strains isolated from the surgical ICU were obtained on the same day.
Nine strains in genotype B were isolated over a period of 14 months, of which five strains were obtained from different units within one month.Twelve isolates from clone D were obtained over 18 months, while eight isolates were obtained from different ICUs over a period of two months.This suggests that the dominant clone strains were able to circulate among different units, sometimes causing a small spread.Based on these findings, it is clear that crosstransmission is widespread in our hospital, and we are facing a huge infection control problem.Therefore, a comprehensive infection control program is required to be initiated.For instance, as a simple and cost-effective measure, we must ensure that all health care workers (HCWs) use hand disinfectant before and after contact with patients and monitor their compliance.Furthermore, to reduce cross-transmission and infection rates, contact precautions for colonized/infected patients should be used, and hospital equipment and surfaces must be cleaned with approved disinfectants at appropriate concentrations and times [3,40].Also, notouch disinfection methods such as ultraviolet light or vaporized hydrogen peroxide may be included in this program.

Conclusion
In this study, bla OXA-23 and bla OXA-2 were determined in 65 (94.2%) and 15 (21.7%) of 69 isolates, respectively.The frequencies of bla TEM and bla SHV , which are of plasmid origin and more common in Enterobacterales, were 53.6% and 2.9% respectively.The frequency of bla VIM , which is of plasmid origin and is often seen in Pseudomonas spp, was 14.5%.These findings indicate horizontal gene transfer between bacteria.Although we determined that no single clone outbreak had taken place in our hospital, a multiclonal spread had occurred.Three dominant clones carrying bla OXA-23 had circulated within the hospital setting.According to our findings, the surgical ICU is the main reservoir for the cross-transmission of strains.This suggests that there is a serious infection control problem in our hospital.Therefore, we should implement a comprehensive infection prevention and control program.In addition, compliance of HCWs to hand hygiene, usage of contact precautions for colonized/infected patients and cleaning of hospital equipment and surfaces with approved disinfectant should be closely monitored [3,40].

Figure 1 .
Figure 1.Dendrogram based on pulsed-field gel electrophoresis pattern analysis of the isolated A. baumannii strains.

Table 1 .
Primer sequences used for the amplification of the target genes.