Epidemiology and Genetic Diversity of Colistin Nonsusceptible Nosocomial Acinetobacter baumannii Strains from Russia for 2013-2014

A high level of resistance to carbapenems in Acinetobacter baumannii strains severely limits therapeutic possibilities. Colistin is the last resort drug against such strains, although the cases of resistance to this drug have become more frequent. This article presents the epidemiological features and genetic diversity of colistin nonsusceptible A. baumannii strains collected as part of a national multicenter epidemiological study of the antibiotic resistance of pathogens of nosocomial infections (MARATHON), which was conducted in 2013-2014 in Russia. A total of 527 A. baumannii isolates were collected, 10 (1.9%) of which were nonsusceptible to colistin. The majority of nonsusceptible A. baumannii isolates to colistin showed resistance to carbapenems and had the genes of the acquired OXA-40-like carbapenemases (n = 6). In one case, a combination of OXA-23-like + OXA-40-like (n = 1) genes was identified. One strain had the multidrug-resistant (MDR) phenotype, 6 isolates had extensively drug-resistant (XDR) phenotype, and 3 isolates had pandrug-resistant (PDR) phenotype. Among the colistin nonsusceptible A. baumannii isolates, 6 individual genotypes were identified, most of which belonged to successful international clones (CC92OXF/CC2PAS, n = 4; CC944OXF/ST78PAS, n = 4; CC109OXF/CC1PAS, n = 1).


Introduction
Acinetobacter baumannii is one of the most troublesome pathogens of nosocomial infections [1,2]. This pathogen is characterized by intrinsic resistance to a number of drugs, as well as an outstanding ability to acquire the determinants of antibiotic resistance through horizontal gene transfer, which led to a high level of A. baumannii strains resistance to almost all available antibiotics [3,4]. A high level of in vitro sensitivity is retained only for colistin [5], which makes it possible to use it against the extensively drug-resistant A. baumannii strains [6]. At the same time, the cases of resistance to colistin become more frequent [7][8][9], which determines the necessity to monitor resistance to colistin among nosocomial A. baumannii strains. In addition, the spread of the multidrug-resistant (MDR) phenotype is mainly related to the spread of international high-risk clones and the horizontal transfer of antibiotic resistance genes [10,11], which provoke the interest in studying the molecular epidemiology of colistin nonsusceptible strains. The purpose of the research is to study the epidemiology and genetic diversity of colistin nonsusceptible A. baumannii strains, isolated from clinical samples of hospitalized patients in different regions of Russia in 2013-14.

Materials and Methods
Clinical strains of microorganisms were collected as part of a multicenter epidemiological surveillance study of the antibiotic resistance of nosocomial pathogens (MARATHON) [12] in 21 cities of Russia from January 2013 to December 2014. 527 nosocomial isolates of A. baumannii were collected. In this study, only colistin nonsusceptible strains (MIC > 2 mg/L) were analyzed. Species identification was performed by the matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) using Microflex LT mass spectrometer and MALDI Biotyper Compass software v. 4.1.70 (Bruker Daltonics, Germany). The value of "Score" ≥ 2.0 was accepted as a measure for the reliable identification.
Minimal inhibitory concentrations of antimicrobials have been determined by broth microdilution method with Mueller Hinton broth (Oxoid, United Kingdom) in accordance with ISO 20776-1:2006 [13]. Interpretation of MIC in clinical susceptibility categories of A. baumannii isolates to antimicrobial agents was performed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) Breakpoint tables v.7.1 [14] (for aminoglycosides, carbapenems, ciprofloxacin, trimethoprim/sulfamethoxazole, and colistin) and also Clinical and Laboratory Standards Institute CLSI M100-S27 [15] (for penicillins with -lactamase inhibitors and extended-spectrum cephalosporins). Isolates with tigecycline MIC of >1 mg/L were considered insusceptible to this drug [16]. Escherichia coli ATCC525922, E. coli ATCC535218, and Pseudomonas aeruginosa ATCC527853 were used as control strains.
DNA extraction was performed by express method by using InstaGene6 matrix (Bio-Rad, USA). Samples of extracted DNA were stored at −20 ∘ b before testing.
Genotyping was performed by single-nucleotide polymorphism (SNP) typing method [17]. Briefly, this method is based on analysis of 21 informative SNPs in 10 chromosomal loci (gltA, recA, cpn60, gyrB, gdhB, rpoD, fusA, pyrG, rplB, and rpoB) and is used at the University of Oxford and the Pasteur Institute multilocus sequence typing (MLST) schemes. A. baumannii strains of known sequence types from proper collection were used as positive controls. The comparison of received SNP profiles with MLST data was done using a web resource: http://snptab.antibiotic.ru [18]. Cluster analysis of SNP profiles was performed using PHYLOViZ 2.0 software (http://www.phyloviz.net/).

Results and Discussion
Ten of 527 (1.9%) nosocomial colistin nonsusceptible A. baumannii isolates were isolated from 7 hospitals in 7 cities of Russia in 2013-2014. The results of antimicrobial susceptibility testing for these isolates are presented in Table 1. The MIC of colistin 4 mg/L was detected in 1 isolate, while the remaining isolates ( = 9) had high levels of resistance (MIC range: 32-256 mg/L).
The majority of colistin nonsusceptible A. baumannii isolates were also resistant to carbapenems and were the carriers of genes of acquired OXA type carbapenemases, mainly OXA-40-like ( = 6). One isolate was shown to possess simultaneously genes encoding two different carbapenem hydrolyzing oxacillinases: OXA-23-like and OXA-40-like. Expectedly high resistance was detected to all other -lactams. At the same time, associated resistance to drugs of other groups with high MIC values is observed (range of gentamicin MIC: 32-256 mg/L; amikacin: 16-256 mg/L; ciprofloxacin: 32-128 mg/L). Only one tobramycin susceptible isolate was found (MIC = 1 mg/L), while the MIC for the remaining isolates was in the range 16-256 mg/L. Two isolates were susceptible to combination of trimethoprimsulfamethoxazole (MIC range: 1-2 mg/L), while for the remaining isolates the MIC was in the range 4-128 mg/L. Six isolates had tigecycline MICs of 2 to 8 mg/L and thus were considered as resistant.
Most infections associated with colistin nonsusceptible A. baumannii isolates were detected as single cases in different cities of Russia. An exception is Smolensk, from which 4 isolates were isolated in one hospital. In this connection, it seemed interesting to evaluate the population structure of studied strains.
Colistin nonsusceptible A. baumannii isolates belonged to 6 genotypes ( Figure 1). Three genotypes differed from each other in no more than 2 positions and were united in a single genetic cluster as related genotypes. This cluster included 4 isolates from 4 cities and corresponded to the international clone CC92 OXF /CC2 PAS (according to the MLST schemes of the University of Oxford and the Pasteur Institute, resp.). Another genotype included 4 isolates from 3 cities and corresponded to CC944 OXF /ST78 PAS . The strains of this  Canadian Journal of Infectious Diseases and Medical Microbiology genetic lineage are widespread in the territory of Russia and Belarus [12] and, according to the literature, were found in Italy, the US, and Germany [20][21][22][23]. This allows considering this genotype as a new international clone. Two isolates belonged to individual genotypes: one of them corresponded to the international clone CC109 OXF /CC1 PAS and the other to the clonal complex CC490 OXF /CC25 PAS . All isolates with XDR and PDR phenotypes belonged to the international epidemic clones CC92 OXF /CC2 PAS , CC944 OXF /ST78 PAS , and CC109 OXF /CC1 PAS .

Conclusion
Thus, colistin nonsusceptible A. baumannii strains described in this study relate to different genetic lineages and mainly belong to distinct international high-risk clones. The accumulation of molecular typing data is an important element in understanding the roots and epidemiology of colistin resistance and in predicting its further spread.