Molecular epidemiology of Acinetobacter baumannii in different hospitals in Tripoli, Lebanon using bla _OXA-51-like sequence based typing

A. baumannii is an opportunistic pathogen mainly involved in healthcare-associated infections, with increased mortality and morbidity [1]. The serious concern associated with this bacterium is the increasing prevalence of multidrug resistant isolates, especially carbapenem resistant ones [2]. Therefore, management of infections due to A. baumannii has become a real public health issue in many countries [3]. In a recent SENTRY Antimicrobial Surveillance program, the percentage of A. baumannii isolates susceptible to meropenem decreased noticeably from 52.9 % in 2009 to 37.7 % in 2011 in European intensive care units [4]. The main mechanism of resistance to carbapenems is the production of oxacillinases encoded mostly by blaOXA-23-like, blaOXA-24-like, blaOXA-58-like and recently blaOXA-143-like and blaOXA-235-like [2, 5–7]. To a lesser degree, carbapenemases from classes A and B have also been involved [2, 5]. New Delhi metallo-ß-lactamase 1 (NDM-1), a recent described metallo-ß-lactamase, has emerged in Enterobacteriaceae as well as in Acinetobacter [8]. Generally, a limited number of clones are responsible for worldwide outbreaks, and international clones I and II are the most common ones [9]. In order to identify and track these clones during hospital outbreaks, numerous molecular typing methods have been proposed such as pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP), repetitive-sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST) [10–14] . Currently, MLST is the gold standard typing tool for population structure and macro-epidemiological investigations based on sequencing of internal fragments of 7 genes [14]. Two MLST schemes have been developed for Acinetobacter baumannii : Pasteur and Bartual schemes [10, 15]. Overall, the 2 schemes are concordant [14]. However, because of recombination bias observed on some loci in Bartual’s MLST (gyrB and gpi) [16, 17], Pasteur’s MLST has been chosen for this study. Recently, blaOXA-51-like sequence-based typing (SBT), a simple typing method based on sequencing of the full length of blaOXA-51-like gene has been proposed as it has shown a similar discriminatory power than rep-PCR, and MLST [16, 18–20].

In Lebanon, limited numbers of epidemiological studies concerning A. baumannii have been reported, almost in Beyrouth [20–23]. In a previous study, we have described the dominance of international clone II within a set of 42 isolates collected from Beyrouth and North Lebanon between 2009–2012 [20]. In another study, we reported the first detection of four NDM-1 producing isolates isolated in Tripoli-Northern Lebanon from Syrian patients injured during Syrian War [24]. The aim of this study was to characterise A. baumannii molecular epidemiology in Tripoli-Lebanon between 2011–2013 using blaOXA-51-like and Pasteur’s MLST typing and to determine the carbapenem resistance mechanisms.

Our study provided a global view of molecular epidemiology of A. baumannii isolates from hospitals in Tripoli, Lebanon. Tripoli is a city that hosts a large number of refugees and wounded Syrians, which may explain the high number of wound specimens in our collection. Although, A. baumannii is an opportunistic nosocomial pathogen, it is increasingly reported from infections occurring outside hospitals, particularly skin and soft-tissue infections and pneumonia [25]. A. baumannii has also been a well-documented pathogen associated with wound infections in USA troops from the Iraq and Afghanistan wars [26, 27]. The typology of these situations raised questions about their origins and the potential involvement of an environmental reservoir [25]. The origin of infections from Syrian patients was ambiguous. Although it was likely that nosocomial infections occurred, a number of infections were present at the time of admission of the patients in Lebanese hospitals [24]. These infections may have been acquired from environmental sources at the time of injury, during the patient stay in Syrian hospitals, or during a direct evacuation from the theatre of operations to Lebanon. Unfortunately, data concerning the date of injury, the length of hospitalisation, and the conditions of care and treatment in Syrian hospitals are lacking.

Approximately 60 % of our isolates showed a carbapenem resistance phenotype. The production of OXA-23 was the major carbapenem resistance mechanism, with an upstream insertion of ISAba1, thus supporting international data about the worldwide emergence of this carbapenemase [28–30]. Alarmingly, 5 NDM-1 producing isolates have been detected from 4 Syrian patients [24] and one from a Lebanese patient.

bla _OXA-51-like is an intrinsic oxacillinase gene naturally occurring in A. baumannii, and more than 95 variants have been identified to date [31]. However, the occasional detection of bla _OXA-51 in Acinetobacter nosocomialis and Acinetobacter genomic species “close to 13TU” limit its use as a single identification method. Therefore, it is always combined with others techniques [32, 33]. Genetic diversity of bla _OXA-5 gene has been explored and found to be very interesting for identifying epidemic clones [16, 18, 19, 34, 35]. Thus, the full gene sequencing has been proposed as a single typing method for A. baumannii. As being simple and rapid, we have initially screened here the epidemiological belonging of our isolates to the previous identified clonal lineages by bla _OXA-51-like SBT. We have then randomly selected isolates from each of the representative bla _OXA-51-like sequences for MLST analysis. In our study, bla _OXA-51-like gene sequencing correctly assigned all isolates to their corresponding clonal complexes. Here, the bla _OXA-66 gene variant, which is associated with ST2 (belonging to CC2) [16, 19, 35, 36], was predominant and found in 63 % of the A. baumannii isolates. The isolates carrying the bla _OXA-66 gene variant have been identified in samples obtained from the different hospitals in Tripoli. These findings are consistent with those observed worldwide since CC2 was reported in more than 34 countries in Europe, Asia, Africa, Australia, USA, and South America [36]. Eighty percent of our carbapenem resistant isolates belonged to this clone and OXA-23 was the only carbapenem resistance mechanism found. The high level of antimicrobial resistance may represent one of the main causes for its propensity and its successful predominance in hospitals throughout the world [10, 36]. The bla _OXA-69 gene variant was the second gene mostly found in our collection. This gene was commonly associated with ST1 (belonging to CC1) but interestingly, we found two bla _OXA-69 isolates belonging to the new sequence type ST460 (SLV of ST1). Both isolates were isolated from wounded Syrian patients. The bla _OXA-94 was another bla _OXA-51-like gene variant found in our study, it was associated with clonal complex CC85 (named as CC6 in the study of Pournaras et al. [19]). CC85 is currently formed by ST6, ST85, ST464, and ST528. In our study bla _OXA-51-like SBT revealed the same sequence bla _OXA-94 variant in the two identified sequences ST6 and ST85. We have recently detected ST464 in chicken and we found bla _{OXA- 94} as a bla _OXA-51 variant [37]. Our 4 NDM-1 producing Syrian isolates belonged to ST85. Indeed, this ST has also been responsible for an outbreak in France. It is usually imported from North Africa [38, 39] and seems to be an emerging clone.

bla _OXA-64 is a bla _OXA-51-like variant associated with CC25 [19]. ST25 (the founder of CC25) was also an emerging clone reported in Europe, Asia, Africa and USA [36, 40], and from pets in Reunion Island, a French territory located in Western Indian Ocean [41].

Different carbapenemases have been identified in this clone, as OXA-23, OXA-24 and OXA-58 [36]. Otherwise, NDM-1 producing ST25 was also detected in Germany and Kenya [41–43]. Within our four identified isolates belonging to ST25, one isolate was a NDM-1 producing isolate recovered from a urine sample of an 80-year-old Lebanese patient suffering from amyloidosis and anaemia at Nini hospital, Tripoli, Lebanon. Finally, compared to carbapenem resistant isolates belonging to successful emerging clones, most of susceptible isolates were very diverse, belonging to different sequence types (Table 1).

In conclusion, this study highlights the emergence of NDM-1 and OXA-23 carbapenemases in Tripoli, Lebanon and the urgent need of effective measures to control the spread of A. baumannii in this country. It is noteworthy that Tripoli is located near the Syrian border and the microbial epidemiology is probably highly impacted by wounded Syrian refugees who can represent a reservoir of multidrug-resistant bacteria in hospitals. The higher prevalence of carbapenem-resistant A. baumannii among isolates from Syrian refugees was consistent with this hypothesis. Besides, we showed that bla _OXA-51-like SBT is a reliable and fast method able to assign our isolates to their clonal complexes. Other multicentre studies are required to investigate the situation in other Lebanese cities that are possibly less concerned by the afflux of Syrian refugees fleeing war.