Resistance and Virulence Features in Carbapenem-resistant Acinetobacter baumannii Community Acquired and Nosocomial Isolates in Romania

IRINA GHEORGHE1,2, VIOLETA CORINA CRISTEA3,4*, LUMINITA MARUTESCU1,2, MARCELA POPA1,2,, CARMEN MURARIU5, BIANCA SIMONA TRUSCA6, ELVIRA BORCAN6, MIHAIELA CAMELIA GHITA6, VERONICA LAZAR1,2,, MARIANA CARMEN CHIFIRIUC1,2 1 University of Bucharest, Faculty of Biology, Department of Microbiology and Immunology, 36-46 Kogalniceanu Blvd., Bucharest, Romania 2 Research Institute of the University of Bucharest (ICUB), 4-12 Regina Elisabeta Blvd., 030018, Bucharest, Romania 3 Synevo Central Laboratory, Medicover, 25 Industriilor Str, Chiajna, Romania 4 University of Medicine and Pharmacy Carol Davila, 88 Mircea Vulcanescu Str., 01082, Bucharest, Romania 5 Emergency Hospital for Children Marie S. Curie, 20 Constantin Brancoveanu Blvd., 077120, Bucharest, Romania 6 Fundeni Hospital, 258 Fundeni Road, 022322, Bucharest, Romania

Acinetobacter baumannii is recognized as an opportunistic nosocomial pathogen, mainly in immunocompromised patients being frequently associated with therapeutic failures, due to its multi-drug (MDR), extended-drug (XDR) or even pan-drug resistance (PDR) phenotypes. Carbapenems were the antibiotics of choice for infections treatment caused by this organism, but resistance to carbapenems is becoming common, and very few therapeutic options remain. Mortality rates associated with Carbapenem Resistant Acinetobacter baumannii (CRAB) isolates are steadily growing at present [1,2]. In A. baumannii clinical isolates five groups of acquired, chromosomal or plasmid located CHDLs (class D β-lactamases) with variable geographic distribution have been identiûed, i.e.: OXA-23, OXA-24/-40, OXA-58, OXA-143 and OXA-235 [3]. OXA-23 is the most worldwide distributed enzyme in A. baumannii, having been implicated in outbreaks in multiple European (including Romania and other Eastern European countries), Asian and American countries and Oceania [4][5][6]. There have been revealed that overexpression of the bla OXA-51-like gene intrinsic in A. baumannii was responsible for carbapenem resistance. The overexpression is due to the acquisition of a promoter provided by an insertion sequence (IS) element, ISAba1, inserted upstream of the carbapenemase gene [7].
Numerous potential virulence factors have been revealed in A. baumannii strains, including biofilm formation [several factors contribute to biofilm formation such as the Csu pili [11] encoded by csuE gene, the autoinducer synthase AbaI, part of the quorum sensing (QS) system [12], the outer membrane protein A (encoded by OmpA gene) which facilitates the adhesion to host epithelial cells], complement resistance [13], iron acquisition characteristics, capsule, outer membrane protein phospholipases, alteration in penicillin-binding proteins [14]. Phospholipases C and D are responsible for epithelial cell invasion [15,16]; the siderophore acinetobactin [17]; the polysaccharidic capsule [18]; and a penicillin-binding protein 7/8 [19] are important for survival and dissemination in human serum. Growth in serum has been demonstrated to upregulate iron acquisition systems, genes associated with epithelial cell adherence and DNA uptake, as well as numerous putative antibiotic efflux pumps, leading to increased antibiotic tolerance [20]. In addition, lipopolysaccharide (LPS) is an important cell envelope component, which influence the pathogenic potential of A. baumannii by the Opolysaccharide chain (O-antigen) [21].
Regarding A. baumannii infections the most predisposed are patients from ICU in which this pathogen may cause serious infection and, thus, contributes substantially to the considerable mortality of this population [22]. Although the attention of A. baumannii infections has been focused on hospitalized patients, there is another patient population that may be affected by this important pathogen, namely, patients in the community setting that have some form of morbidity, especially in the tropical and sub-tropical area [23].
Originally, we are interested in identifying the relationship between virulence and antimicrobial resistance in CRAB strains from both hospital settings and the community, the ARGs, their transfer and dissemination into the community.

Experimental part Material and methods
The study included 93 recently isolated (Aug-Nov 2017) A. baumannii strains, which were isolated in majority from hospitalized patients (H) (n=75) and from CA infections (n=18). The hospital strains were identified by BD Phoenix and the CA ones by mass spectrometry using MALDI Biotyper and MicroScan Walk Away 96. Carbapenemases and virulence genes were searched by PCR.

Evaluation of the soluble enzymatic factors
The virulence phenotypes were investigated by performing enzymatic tests for the expression of the following soluble virulence factors: haemolysins, pore forming toxins (lecithinase, lipase), proteases (caseinase, gelatinase), amylase and aesculin hydrolysis.

Genetic support of AR and virulence in CRAB
The genetic support of the resistance (carbapenemases and aminoglycosides table 1), Plasmid analysis included identification of replicase genes:19 PCR amplifications were devised to detect 27 replicase genes, which were grouped into 19 homology groups (GRs) on the basis of their nucleotide sequence similarities (table 2), BIOPFILM biofilm producing virulence factors (table 3) and global lineage in CRAB (table 4) was investigated by simplex and multiplex PCR, using a reaction mix of 20µL (PCR Master Mix 2X, Thermo Scientific) containing 1 µL of bacterial DNA extracted using the alkaline extraction method. In this purpose, 1-5 colonies of bacterial cultures were suspended in 1.5 ml tubes containing 20 µL solution of NaOH (sodium hydroxide) and SDS (sodium dodecyl sulphate). The following step was the addition of 180 µL of TE buffer (TRIS+EDTA) 1X and centrifugation at 13000 rpm for 3 min. All PCR reactions were performed using the Thermal Cycler machine Bio-Rad.

Results and discussions
The antibiotic resistance profiles in H and CA A. baumannii isolates revealed high percentages of carbapenem-resistance in both H and CA isolates, i.e. imipenem (82.66%/77.77%), meropenem (84%/83.33%) as well as for SXT (85.33%/77.77%) and aminoglycosides (85.33%/66.66%). The ciprofloxacin resistance was closed in both types of isolates (84%/83.33%) ( fig. 1). A. baumannii has acquired a huge genetic repertoire via horizontal gene transfer that makes it virulent and resistant to any environmental pressures [30][31][32][33]. Antibiotic susceptibility testing in this study showed that all A. baumannii isolates were resistant to the commercially available antibiotics with the exception of colistin. Previously in Romania, Vaduva et al., revealed the presence of beta-lactamase producers nosocomial A. baumannii strains from Timisoara hospital and a very closer aminoglycosides resistance profile [34].
CRAB H and CA isolates revealed the intrinsec carbapenemase OXA-51(58.6%/55.5%) and the acquired   [36]. Data revealed by our research team (2012-2013) demonstrated that A. baumannii strains that were investigated harboured the class D carbapenemase OXA-23 [37]. Previous studies in Timisoara, Arad and Resita indicated that CHLD in A. baumannii is encoded by chromosomally -located bla OXA-23 , with the insertion sequence ISAba1 detected upstream and the strains belonged to the ST2 and ST1 clones [6]. More recently one study from our department highlights a remarkable mobility for bla OXA-23 -Tn2008 and surrounding structures (identified in plasmid or chromosome of different clones) and also describes for the first time the spread of Tnaph6-carrying pACICU2-like plasmids in A. baumannii in Europe [27]. A pilot study from three Romanian hospitals -Iasi and Targu In our study bla OXA-23 gene was identified in different plasmid types (GR2-Aci1, GR6-pACICU2). rep135040 Antibiotic resistance profiles in CRAB The multidrug resistant profile of the isolates was also heightened by the presence of AME's namely the phosphotransferase AphA6 (24%/16.6% of the strains), AphA1 (16%/16.6%) and acetyltransferases aadB (9.3%/ 5.5%). Nowak et al., in 2014 reported in MDR A. baumannii isolated in Poland the following aminoglycoside resistance genes: aphA1, aphA6,and aacC1 [41]. The genes encoding AMEs can be disseminated via integrons, and expression of AMEs enable bacteria to catalyze the modification of amino and hydroxyl groups on sugar moieties, such as aminoglycosides [42].
Carbapenem and aminoglycosides resistance has been associated to nonenzymatic mechanisms revealed by changes on the outer membrane proteins [ompA biofilmproducing virulence factor (66.66% of the analysed strains)], mechanism [43] demonstrated also by different authors. The outer membrane protein A of A. baumannii represent one of the most abundant surface protein associated with the apoptosis of epithelial cells through mitochondrial targeting [44]. OmpA is also the major nonspecific channel in A. baumannii and appears to be essential for this organism's high levels of intrinsic resistance to different antibiotics [45]. Several reports have been demonstrated that A. baumannii possesses Omp's which interfere with carbapenem resistance, for e.g. in 2002, Limansky et al. demonstrated that imipenem resistance was associated with the loss of a 29-kDa Omp in clinical isolates of A. baumannii in which no carbapenemase activity had been detected [46].
OXA-51-like alleles corresponding to the two main sequence groups were identified as bla OXA66 (63.63% of the isolates) and respectively, bla OXA-69 (38.39%) and revealed the corresponding type of ompA and csuE sequence grouping ( fig. 2, 3). There have been revealed a link between the production of some of the naturally occurring OXA-51/69-like oxacillinases and carbapenem resistance in A. baumannii. Despite the relatively weak ability of these enzymes to hydrolyse carbapenems, it has been shown that these oxacillinases may sometimes be overexpressed, resulting in a decreased level of susceptibility to carbapenems and the presence of ISAba1element upstream of the bla OXA-51/69 -like gene (J. Turton, N. Woodford and T. Pitt, personal communication).

Conclusions
The significant levels of antibiotic resistance in CRAB strains highlights the need for continuous surveillance and epidemiological studies, of not only hospital, but also CA isolates. The elucidation of the genetic context of resistance in CRAB isolates with different origins could reveal further clinically important associations, and help to better understand the interaction between antimicrobial resistance and virulence in A.baumannii.