Antibiotic Resistance and Genotypes of Nosocomial Strains of Acinetobacter baumannii in Kazakhstan

The aim of this study was to determine the prevalence of A. baumannii antibiotic-resistant strains in Kazakhstan and to characterize genotypes related to epidemic “high-risk” clones. Two hundred and twenty four A. baumannii isolates from four cities of Kazakhstan in 2011–2019 were studied. Antibiotic susceptibility testing was performed by using broth microdilutions method according to EUCAST (v 11.0) recommendations. The presence of blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like, blaVIM, blaIMP, and blaNDM genes was determined by PCR. Genotyping was performed using high-throughput real-time PCR detection of 21 SNPs at 10 chromosomal loci used in existing MLST schemes. Resistance rates to imipenem, meropenem, amikacin, gentamicin, and ciprofloxacin were 81.3%, 78.6%, 79.9%, 65.2%, and 89.3%, respectively. No colistin resistant isolates were detected. The values of the MIC 50% and the MIC 90% of tigecycline were 0.125 mg/L, only four isolates (1.8%) had the ECOFF value >0.5 mg/L. The presence of acquired carbapenemase genes was found in 82.2% strains, including blaOXA-23-like (78.6%) or blaOXA-58-like (3.6%) genes. The spreading of carbapenem resistant A. baumannii strains in Kazakhstan was associated with epidemic “high-risk” clonal groups, predominantly, CG208(92)OXF/CG2PAS (80.8%) and less often CG231(109)OXF/CG1PAS (1.8%).


Introduction
Acinetobacter baumannii is an opportunistic pathogen that often causes diseases in immunocompromised patients [1]. A. baumannii is common in hospitals and causes a variety of nosocomial infections and iatrogenic diseases that include bloodstream infections, urinary tract infections, meningitis, wound infections, and more [1,2]. The World Health Organization categorizes included MDR A. baumannii as the "highest priority pathogen" for which antibiotic development is urgently needed [3]. However, carbapenems are considered as effective antibiotics against many drug-resistant microorganisms [4]. At once, the number of carbapenem-resistant A. baumannii isolates has been increasing recently [5].
The prevalence of carbapenem resistance of A. baumannii in etiological structure of nosocomial infections during last decade was heterogeneous. According to the EARS-Net data from 2013 to 2017, the part of carbapenem resistant A. baumannii in West Europe was 35.6%, at this time the prevalence of carbapenem resistant A. baumannii in Greece and Turkey were 79% and 95% respectively. However, according to longitude study in Austria the level of carbapenem resistant A. baumannii was more than 97% [6]. In alignment, the CHINET surveillance system in China from 2004 to 2015 the level of carbapenem resistant A. baumannii increased from 31 to 66.7% [7]. Russian multicenter surveillance study shown the comparable data when carbapenem resistant A. baumannii were detected in 77% [8]. According to multicenter study nosocomial infections caused by A. baumannii in Pakistan the rate of carbapenem resistance in 2017 was 89% [9].
Most isolates are resistant to carbapenems, and also become resistant to other classes of antimicrobial drugs such as aminoglycosides, fluoroquinolones, and to polymyxin in sporadic cases [10,11]. The rapid spread of multidrug-resistant nosocomial strains of Acinetobacter is a global concern. The present situation determines the need for regular monitoring of the sensitivity of nosocomial strains of Acinetobacter spp. and, if necessary, correction of the therapy strategy for infections caused by them [12,13]. The global spread of multidrug resistant strains, including resistant to carbapenems, among nosocomial A. baumannii strains was associated with expansion of two international "high-risk" clonal lineages, called ICL1 and ICL2 [14][15][16][17].
Microbial genotyping methods is an important tool for molecular epidemiological studies, particularly, for understanding of the population dynamics and transmission of pathogens. Even the ubiquity of whole-genome data, multilocus sequence typing (MLST) is still a "gold standard" for molecular typing of A. baumannii due to standardized landscape about population structure [18,19]. Two available MLST approaches for A. baumannii (Oxford [20] and Pasteur [14] schemes) characterized by different resolution ability but provide concordant discrimination at the level of predominant clonal groups [21,22]. In epidemiological studies, the epidemic "high-risk" clones are typically defined using by MLST nomenclature of sequence types (STs) and clonal complexes (CCs) or clonal groups (CGs) [23] and are characterized as CC231(109) OXF /CC1 PAS (for ICL1) and CC208(92) OXF /CC2 PAS (for ICL2) [16]. However, such approaches is still expensive and laborious especially for analysis of large sample collections, whereas high-throughput approaches allow to study in detail the entire diversity of A. baumannii strains. A lot of existing high-throughput approaches have been proposed [24][25][26][27][28][29][30] but only single nucleotide typing (SNP)-typing scheme provide direct compare SNP-typing and MLST data due to identify a set of SNPs of MLST loci [30].
In the present study, we aimed to investigate the rates of antibiotic resistance and production of acquired carbapenemase genes and to determine the genotypes and prevalence of "international high-risk clones" among A. baumannii isolates in Kazakhstan.

Results and Discussion
Determining the antimicrobial susceptibility of A. baumannii strains results are presented in Table 1. Resistance to carbapenems (imipenem and meropenem) were shown by 81.3% and 78.6% of A. baumannii isolates, respectively. Comparable the resistance phenotype was detected in South-Est Asia: Korea (87.0%), Singapore (95.2%), Hong Kong (50.0%) and Thailand (59.2%) [31]. Fluoroquinolones have high resistance indicators: 89.3% of A. baumannii isolates were resistant to ciprofloxacin. The frequency of resistance to aminoglycosides (amikacin and gentamicin) was 79.9% and 65.2%, respectively. Among non-beta-lactam antibiotics, colistin was highly active in vitro, none resistant isolates were detected. The values of the MIC 50% and the MIC 90% of tigecycline were 0.125 mg/l, only four isolates (1.8%) had the ECOFF value >0.5 mg/L. Combined resistance to imipenem, amikacin, and ciprofloxacin was observed in 73.2% isolates. Extremely profile of resistance make polymyxins (polymyxin B and colistin) as a last-resort treatment option [32]. The presence of genes for acquired molecular class D carbapenemases belonging to the OXA-23 (78.6%) and OXA-58 (3.6%) groups were revealed in 82.2% of A. baumannii isolates.
No MBL genes were found in A. baumannii isolates. Actually, number of isolates harboring carbapenemase genes was higher than carbapenem resistant isolates: eight isolates were susceptible to imipenem and meropenem despite the presence of carbapenemase genes. The OXA-23 carbapenemases are major mechanism of resistance A. baumannii to carbapenems in the Central Asia and early the spreading of OXA-23 produced hospitals acquired strains of A. baumannii was described in Russia [8] and South-East Asia countries [17]. The results of assessing the sensitivity of A. baumannii isolates carrying the genes of acquired OXA-carbapenemases are presented in Table 2. The majority of carbapenemase producers were characterized by associated resistance to ciprofloxacin (97.1%), amikacin (89.7%) and gentamicin (69.5%). No resistant to colistin strains were observed. All isolates were distributed into 20 genotypes (SNP-types) that grouped into 14 clonal groups (genetic clusters combining strains of related SNP-types) (Figure 1). Genotypes that differed in one or two SNPs were considered related. Despite this diversity, most of isolates (n = 181, 80.8%) from four cities were assigned to the same clonal group that combined five related genotypes (SNP-types). Through in silico analysis, nucleotide profiles of these genotypes corresponded to a set of STs that combined into a CG208 OXF (formerly known as CC92 OXF ) and CG2 PAS . Strains of clonal group CG208(92) OXF /CG2 PAS were characterized by a high frequency of bla OXA-23-like carbapenemase genes (170 of 181 isolates, 93.9%). CG208(92) OXF /CG 2PAS is related to international clonal lineage ICL2 that was associated with global dissemination of multidrug resistant A. baumannii strains, including neighboring countries [8,16,34].
Among other clonal groups, isolates harboring bla OXA-23-like carbapenemase genes have been also found in SNP-type 7 (3 of 4 isolates, 75.0%) and SNP-type 47 (3 of 3 isolates, 100.0%). Through in silico analysis, nucleotide profiles of SNP-type 7 corresponded to a set of STs that combined into a CG231 OXF (formerly known as CC109 OXF ) and CG1 PAS . CG231(109) OXF /CG1 PAS was corresponded to another international clonal lineage ICL1 [16,34], however, an incidence of CG231(109) OXF /CG1 PAS was sporadic in Kazakhstan-four isolates were collected from Nur-Sultan in 2014 and 2018. Three isolates of SNP-type 47 harboring bla OXA-23-like genes were also collected from Nur-Sultan in 2015. Interestingly enough, the epidemic "high-risk clones" CG231(109) OXF /CG1 PAS and CG208(92) OXF /CG 2PAS harboring bla OXA-23-like carbapenemases genes were concomitant with spreading of carbapenem resistant A. baumannii strains in Pakistan [9]. Genetic diversity and production of carbapenemases in A. baumannii strains in Kazakhstan. UPGMA algorithm was used for hierarchical cluster analysis by PHYLOViZ software [35]. Horizontal rectangles correspond to different genotypes (SNP-types). Length of the rectangles is proportional to the number of isolates. MLST nomenclature was used to characterize of prevalent clonal groups. Types of acquired carbapenemase genes are highlighted.
Isolates harboring bla OXA-58-like carbapenemase genes (n = 8) have been found in same genotype (SNP-type 83). All isolates of this genotype were carriers of bla OXA-58-like genes and were isolated in 2016 and 2017 from Nur-Sultan. One isolate of SNP-type 83 was investigated by MLST to clarify phylogenetic traits and was assigned to ST184 OXF and ST218 PAS . Strains of this clonal group CG184 OXF /CG218 PAS were previously found in South Korea in 2008 and in China in 2009-2010, according to the pubMLST database. Strains of CG184 OXF /CG218 PAS have not been previously detected in neighboring countries.
Despite the presence of bla OXA-58-like genes, all but one isolates of CG184 OXF /CG218 PAS were susceptible for imipenem and meropenem. For susceptible isolates, distribution of MIC ranged from 0.5 to 1 mg/L for imipenem and 0.25 to 1 mg/L for meropenem. Expression of bla OXA-58-like gene depends on the presence of insertion sequence, most often ISAba3, in association with carbapenemase gene [36,37], so, presence of bla OXA-58-like gene does not always lead to resistance to carbapenems. Furthermore, another isolate harbored bla OXA-23-like gene was found in CG208(92) OXF /CG2 PAS and had a low MIC values (0.5 mg/L) for imipenem and meropenem.

Sources of Bacterial Isolates
Acinetobacter baumannii (n = 224) were collected from inpatients of four cities of Kazakhstan (Karaganda, Nur-Sultan, Almaty, and Jezkazgan) in 2011-2019. Isolation and primary identification of bacterial isolates were carried out in local clinical microbiological laboratories by using standard microbiological methods. The final identification of all bacterial isolates was made in the share resource laboratory of the Karaganda Medical University (Kazakhstan). Molecular genetic studies were performed in the Research Institute of Antimicrobial Chemotherapy (Smolensk, Russia).

Species Identification and Storage of Isolates
The isolates were identified by matrix-assisted laser desorption/ionization-timeof-flight mass spectrometry (MALDI-TOF MS) using the Microflex LT system and the MALDI Biotyper Compass v.4.1.80 software (Bruker Daltonics, Hamburg, Germany). The values known for Acinetobacter representatives were used as a criterion for reliable species identification score ≥ 2.0 according to manual. The species identification of A. baumanii isolates was confirmed by detection of species-specific bla OXA-51-like genes using real-time PCR with commercial kits "AmpliSensR MDR Ab-OXA-FL" (Central Research Institute of Epidemiology, Moscow, Russia) and the DTPrime 5X1 system (DNA-Technology, Moscow, Russia). Prior to analysis, the isolates were stored at −70 • C in trypticase-soy broth (BD, Sparks, MD, USA) supplemented with 30% glycerol. The obtained strains from local laboratories were revived on Columbia blood agar (BD, USA) aerobically at 36 ± 1 • C.

Determination of Sensitivity to Antibiotics
Determination of sensitivity to antimicrobial drugs (amikacin, gentamicin, imipenem, meropenem, netilmicin, ciprofloxacin, tigecycline, colistin) was carried out by the microdilution method in Mueller-Hinton broth. Interpretation of susceptibility testing results was performed according to recommendation of EUCAST v 11.0 [33]. To control the quality of the sensitivity determination, we used Escherichia coli ATCC ® 25922, Escherichia coli ATCC ® 35218 strains, Pseudomonas aeruginosa ATCC ® 27853.

Identification of Carbapenemase Genes
The presence of acquired class D carbapenemases genes common for Acinetobacter spp. (groups OXA-23, OXA-24/40, and OXA-58), as well as class B carbapenemases (metallobeta-lactamases (MBL) of VIM, IMP, and NDM groups) were determined by real-time PCR using commercial kits "AmpliSensR MDR Acinetobacter-OXA-FL" and "AmpliSensR MDR MBL-FL" (Central Research Institute of Epidemiology, Moscow, Russia). For amplification, a DTPrime 5X1 real-time PCR system (DNA Technology, Moscow, Russia) was used. Strains A. baumannii, A. pittii, and P. aeruginosa, carring the known carbapenemases genes of the listed groupswere used as positive controls. DNA extraction was performed by express method using InstaGeneTM matrix (Bio-Rad, Hercules, CA, USA). Samples of extracted DNA were stored at −20 • C before testing. The results of assessing the sensitivity to antibiotics and determining the genes of various types of carbapenemases have been deposited to the AMRmap website database [49].
The selected set of 21 SNPs from MLST loci provided a comparison between obtained SNP-types with known STs and CCs according to the MLST nomenclature, including the so-called "high-risk international clones". That correspondence between SNP-typing and MLST data was provided by SQL database and software platform (called SNPTAb, http://snpt.antibiotic.ru:9002/, last accessed on 12 March 2021) [57]. Furthermore, the SNPTAb database was used to store of SNP-typing data with individual isolates data (e.g., source, geographical origin, data of isolation, resistance to carbapenems, and production of carbapenemases). Cluster analysis of obtained SNP profiles and was carried out using the PHYLOViZ 2.0 software [35].
MLST was performed for one isolate using both the University of Oxford and the Institute Pasteur schemes as described previously [14,20]. Obtained MLST sequences were uploaded to PubMLST database (https://pubmlst.org/organisms/acinetobacterbaumannii, last accessed on 12 March 2021) to identify alleles and STs.

Conclusions
Nosocomial infections by carbapenem resistant A. baumannii strains emerge sharply in Kazakhstan since 2011. The results of this study indicate a high prevalence of resistance to most antimicrobials, including all carbapenems, aminoglycosides, and fluoroquinolones used to treat infections caused by this pathogen. Colistin was highly active on all A. baumannii isolates. The main mechanism of resistance to carbapenems of A. baumannii was the production of acquired carbapenemases belonging to the OXA-23 group. The spreading of carbapenem resistant A. baumannii strains in Kazakhstan was associated with epidemic "high-risk" clonal groups, predominantly, CG208(92) OXF /CG2 PAS (80.8% isolates) and less often CG231(109) OXF /CG1 PAS (1.8% isolates). Isolates of these clonal groups were significantly more resistant to aminoglycosides and fluoroquinolones compared to other genetic lines. Furthermore, several isolates were carrying bla OXA-58-like carbapenemase genes and combined into one clonal group CG184 OXF /CG218 PAS . Given the high probability of A. baumannii strains resistance to the main antibacterial drugs for the treatment of nosocomial infections, the choice of antibiotics for empiric therapy is extremely difficult and requires regular local monitoring of sensitivity in each hospital.