Investigation of multidrug-resistant ST2 Acinetobacter baumannii isolated from Saint George hospital in Lebanon

Acinetobacter baumannii is an opportunistic pathogen causing various nosocomial infections. The spread of multidrug-resistant A. baumannii is a major public health problem. The aim of this study was to investigate the molecular epidemiology and the genetic support of multidrug-resistant A. baumannii isolates collected from Saint-Georges Hospital in Lebanon. Between January and August 2016, 31 A. baumannii isolates were collected from sputum samples of patients infected with ventilator-associated pneumonia (VAP) and treated with colistin-carbapenem combination therapy. Antibiotic susceptibility testing was performed using the disk diffusion method. Carbapenemases, extended spectrum β-lactamases encoding genes and mcr-1/2 genes were investigated by RT-PCR and standard PCR. The epidemiological relatedness of the strains was studied using MLST analysis. Most of the isolates exhibited multidrug-resistant phenotypes. All the isolates were carbapenem-resistant and among them, 30 carried the class D carbapenemase blaoxa-23 gene while one isolate carried blaoxa-72 gene. MLST results revealed three sequence types, namely ST2, ST699, and ST627. Isolates having ST2 were the most prevalent clone (29/31, 93.5%). This study shows a nosocomial spread of multidrug-resistant A. baumannii ST2 having blaOXA-23 gene in Saint-George in Lebanon. Monitoring and control measures need to be adopted to avoid the spread of A. baumannii to patients.


Background
Acinetobacter baumannii is a glucose non-fermentative, gram-negative, opportunistic pathogen and is one of the leading causes of nosocomial and community infections [1,2]. These features make A. baumannii capable of causing a wide variety of clinical complications such as pneumonia, particularly ventilator-associated pneumonia (VAP), bloodstream and urinary tract infections, meningitis, surgical site and wound infections especially in intensive care units [2,3]. Carbapenems are the first choice in the treatment of severe A. baumannii infections [4]. Carbapenem resistant A. baumannii nosocomial outbreaks have become a major concern worldwide since they lead to limited treatment options [5]. Several mechanisms contribute to carbapenem resistance in A. baumannii such as the expression of β-lactamases, alteration of cell membrane permeability, increased expression of efflux pumps, DNA gyrases, and topoisomerases [1]. In addition, the presence of three different types of β-lactamases in A. baumannii leads to β-lactame resistance such as: (1) bla GES-14, bla TEM , bla SHV, bla CTX-M and bla KPC belonging to Ambler class A β-lactamases; (2) bla IMP-like , bla VIM-like , bla SIM-1 , and bla NDM-1 belonging to metallo-β-lactamases and (3) bla OXA-23-like , bla OXA-24-like , bla OXA-58-like, bla OXA-143 , bla OXA-235-like, and the intrinsic bla OXA-51-like , which are found by nature on the chromosome of A. baumannii and belonging to oxacillinases [6][7][8]. Oxacilinases usually do not hydrolyse carbapenems, although some of them produce resistance to carbapenem and are known as "Carbapenem hydrolyzing class D beta-lactamases" or CHDLs. The most common of these enzymes were identified in Acinetobacter spp., notably in A. baumannii, and are responsible for carbapenem resistance [9].
The global dissemination of A. baumannii have been described in several countries in the world such as those of Europe, America, Asia, the Middle East, Australia, and South Africa [10]. A study done by Flamm et al demonstrated that the Mediterranean regions and Europe have the highest frequency of MDR A. baumannii isolates [11]. For example, in South and Southeast Asia, the carbapenem-resistant A. baumannii is a major challenge in public health, where these strains are predominant in nosocomial infections [12]. Regarding Europe, a lower rate of carbapenem resistance was shown in France, Germany and Sweden (10-20, 8 and 4%, respectively) whereas the rates increase up to 50-80% in Turkey, 85% in Greece, 60% in Italy and 45% in Spain [13]. It has been noticed that in many cases, one or two epidemic strains were perceived in a certain epidemiological setting. This is due to the transfer of colonized patients who transmit these strains between hospitals [14]. To date, in Lebanon, there has been a higher level of carbapenem-resistant A. baumannii (CRAB) strains [15]. While the bla OXA-24 [16] and bla OXA-58 [17] have been identified in our country, the bla OXA-23-like remains the most common among the CRAB isolates. Furthermore, the international clone ST2 was found to be broadly spread among this country [18]. At the same institution where this present study was done, the outbreak of A. baumannii began in 2010 as an epidemic with a high level of resistance due to the high usage of carbapanem [19]. Several studies showed the dissemination of CRAB isolates harboring mainly the bla OXA-23-like and belonging to the international clone ST2 [20,21] at Saint-George Hospital. The aim of the current study was to investigate the molecular epidemiology and the genetic support of multidrug-resistant (MDR) A. baumannii isolates collected from patients treated with colistin-carbapenem combination therapy in Saint-George Hospital, Beirut, to investigate if there is a change in the pattern of isolates recovered from patients at the same institution.

Antimicrobial susceptibility testing of isolated strains
A total of 31 strains isolated from Saint-George Hospital in Beirut were identified by MALDI-TOF as A. baumannii. These isolates were collected from the sputum of the respiratory tract (Table 1). Antibiotic susceptibility testing results revealed high levels of resistance rates of all isolates to ticarcillin, ticarcillin clavulanic acid, piperacillin tazobactam, ceftazidime, cefepime, cefotaxime, imipenem, meropenem, ciprofloxacin, and levofloxacin. In addition, 3.2% of the isolates were resistant to gentamicin, tobramycin, and amikacin. E-tests showed high-level of resistance to imipenem, with MIC greater than 32 μg/ml for all the isolates. None of the isolates was resistant to colistin (MIC < 2 μg/ml).

Detection of beta lactamase genes
Results of PCR for carbapenemase-encoding genes showed that 30/31 of the isolates harbored the acquired OXA carbapenemase bla OXA-23-like and one isolate expressed the bla OXA-24-like gene.
In addition the ß-lactamases gene bla TEM was detected in all isolates. Using ARG-ANNOT, the analysis of the sequenced genes revealed that the bla OXA-23-like encoded for the OXA-23 whereas the bla OXA-24-like encoded for the OXA-72 variant. All the sequences of the bla TEM gene were identified as bla TEM-1 . None of the isolates harbored bla NDM-1 , bla OXA-58 , bla VIM gene, bla SHV , bla CTX-M and mcr-1, 2, 3, 4 and 5 genes.

MLST analysis
MLST analysis showed that 93.5% (29/31) of the A. baumannii isolates belonged to ST2 sequence type, whereas two isolates were assigned to ST699 and ST627, respectively. The most common clone (ST2), harboring the bla OXA-23 and bla TEM-1 genes, was found to be circulating in the hospital. The isolate belonging to ST627 was associated with the production of the bla OXA-72 and bla-TEM-1 genes (Table 1).

Discussion
Acinetobacter baumannii has been identified as one of the most successful pathogens responsible for nosocomial infections especially for patients admitted to intensive care units (ICUs) [22]. A. baumannii is able to acquire resistance to broad types of antibiotics including carbapenems. Carbapenem-resistant A. baumannii has been reported worldwide and has become a significant health problem due to the limited options for antibiotic treatment [23,24].
Between 1999 to 2009, carbapenem-resistant A. baumannii harboring the bla OXA-58 gene were predominant in the hospital flora of many Mediterranean countries such as Lebanon, Italy, Greece, and Turkey [25]. After 2009, a huge shift from OXA-58 A. baumannii to OXA-23 producing belonging to the international clonal I and II lineages has been observed globally [25].
An outbreak of MDR A. baumannii has been observed in Saint George Hospital in Beirut, Lebanon between November 2004 and October 2005 [17].
In our study, the bla OXA-23 gene was found in 30 carbapenem-resistant A. baumannii isolates (96.7%) recovered from Saint George hospital in Beirut. In 2015, a study done in Lebanon showed the predominance of Imipenem-resistant A. baumannii bla OXA-23 and bla GES-11 gene -among the majority of A. baumannii. This dissemination of OXA-23 carbepenemase in Lebanon is consistent with the worldwide epidemiology of OXA-23 [16]. Also in 2016, Al Atrouni et al. showed the high dissemination of carbapenem-resistant A. baumannii harboring the bla OXA-23 gene and belonging to the international clone II lineage [26] (Table 2).
All the isolates in our study were resistant to imipenem. In Lebanon in 2012, it has been shown that 88% of A. baumannii were imipenem-resistant. This number is closely related to the rates of 70% in Egypt, 24-72% in Turkey, 25-75% in Spain, and approximating 100% in Italy [16].
In addition to the bla OXA-23 gene, we identified the presence of the bla OXA-24-like gene in one isolate. A study in Lebanon showed that there was a replacement of the predominant OXA-58 producing A. baumannii by OXA-23 producers and belonged to the International Clone II, using isolates recovered from patients from the same institution as those used in the present study, as well as there was the presence of bla OXA-24-like gene in two isolates that harbored also the bla OXA-23-like gene [20]. Our study revealed that the bla OXA-24-like encoded for the OXA-72 variant, which was firstly reported in 2004 in an A. baumannii isolated from Thailand. After that the Acinetobacter spp. who carried this gene have been reported in different countries [27]. In Lebanon, the bla OXA-72 was firstly described in A. calcoaceticus isolated from purchased vegetables in Beirut [28]. It is also found in A. pittii isolated from patients admitted to Lebanese hospital in North of Lebanon, in 2015 [29]. Moreover, Rafei et al reported that among 31 carbapenem-resistant strains collected from different hospitals in Beirut and Northern Lebanon, 28 isolates carried the bla OXA-23 gene, 1 strain the bla OXA-24 gene and 2 strains the bla OXA-58 gene [30]. She also reported in 2015 the spread of the international clone II lineage with high incidence of bla OXA-23 carbapenemase, in addition to the presence of bla NDM-1 , bla OXA-51 , bla OXA-66 and bla OXA-69 in different hospitals in Tripoli, Lebanon [31]. Moreover, we found that the majority of our strains harbored the ß-lactamases bla TEM-1 gene. A study in Egypt in 2017 showed that bla TEM is the most frequent gene for ESBL [32]. In Saudi Arabia, Aly et al revealed that some of the isolates harbored the bla TEM resistance genes as well as the bla PER-1 gene [33] . Also in Turkey, a study by Beris showed that bla TEM was the most prevalent ESBL type amongst A. baumannii strains isolated from different regions [34].

Conclusion
In conclusion, this study describes the detection of bla OXA-23 , and bla OXA-72 genes in clinical isolates of A. baumanii in Lebanon. MLST showed that there was a major circulating clone ST2. In addition, the ST699 and ST627 clones had not been previously detected in Lebanon. The resistant A. baumannii isolates found in Saint George hospital in Lebanon in the present study belonged to international clone II lineage and harbored the bla OXA-23 , which were firstly reported by Dahdouh et al and then again by Soudeiha et al in the same institution. Thus, it is clear that this clone has become endemic in the hospital and that an urgent strategy needs to be adopted to control the spread of such resistant microorganisms among patients as well as appropriate infection control measures and surveillance programs must be implemented. In addition, we believe that it is necessary to set up quality training for health professionals to promote a safe environment for staff, patients and visitors, in order that the risk of healthcare associated infections are kept to a minimum.

Bacterial isolates
Between January and August 2016, 31 A. baumannii were isolated from sputum of the respiratory tract of patients infected with ventilator-associated pneumonia (VAP) and receiving colistin-carbapenem combination therapy in Saint-George Hospital in Beirut. 29/31 samples were collected from the sputum of the upper respiratory tract and 2/31 from the sputum of the lower respiratory tract from hospitalized patients and kept at − 80°C before being transported to the laboratory in Marseille. Once arrived, the isolates were cultivated for 24 h at 37°C on Trypticase Sodium Agar medium (TSA). Colonies growing on this medium were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Microflex; Bruker Daltonics, Bremen, Germany) as previously described [40].

Antibiotic susceptibility testing
The antibiotic susceptibility testing was performed using the disk diffusion method on Mueller-Hinton agar as recommended by the European Committee of Antimicrobial Susceptibility Testing (EUCAST) 2017. Fourteen different antibiotics were tested: ticarcillin, ticarcillin clavulanic acid, piperacillin tazobactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, amikacin, tobramycin, colistin, ciprofloxacin, levofloxacin, and cefotaxime. Interpretations of the results of antibiotic sensitivity testing were made according to EUCAST recommendations. In addition, E-test method (bioMérieux) was performed to determine the minimal inhibitory concentration (MIC) of imipenem as recommended by the 2017 European Committee of Antimicrobial Susceptibility Testing (EUCAST). Moreover, the minimal inhibitory concentration (MIC) of colistin was determined using the broth microdilution method (Biocentric) according to EUCAST 2017.

DNA extraction
Bacterial DNA was extracted using the automatic robot EZ1 (Qiagen BioRobot EZ1-, Tokyo, Japan), with the extraction kit (EZ1 DNA, Qiagen, Hilden, Germany), following the manufacturer's instructions. The extracted DNA was eluted in 200 mL of elution buffer and was stored at − 20°C.

Screening of samples by real-time PCR and molecular characterization of beta lactamase genes
Real-time PCR was performed to screen for the presence of carbapenemase-encoding genes using specific primers previously described for bla NDM-1 , bla OXA-23 , bla OXA-24 , bla OXA-58 , bla VIM and bla SHV. All MDR bacteria were also screened for β-lactamases (bla CTX-M , bla TEM , bla SHV) genes and for the mcr-1, 2, 3, 4 and 5 genes as described previously [41][42][43][44]. Negative and positive controls were used in each assay. The positive PCR products for any gene tested were sequenced using BigDye1 terminator chemistry on an automated ABI 3130 sequencer (PE Applied Biosystems, Foster City, CA). The sequences of the genes obtained were analyzed using the ARG-ANNOT database [45] (http:// backup.mediterranee-infection.com/article.php?lar-ef=282&titre=arg-annot), and compared to other genes using the BlastN and BlastP of the National Center for Biotechnology Information (NCBI) database (https:// blast.ncbi.nlm.nih.gov/BlastAlign.cgi).

Multilocus sequence typing
Molecular typing of the isolates was done to determine the genetic relationship among the clinical isolates by using the seven housekeeping genes (cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB) as described on Institute Pasteur's MLST Web site (https://pubmlst.org/abaumannii). Each single locus has different allele and the allelic profile or sequence types (ST) of the seven loci were given a specific identification number.