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

Molecular epidemiology of Acinetobacter baumannii in different hospitals in Tripoli, Lebanon... Background: A. baumannii has emerged as an important nosocomial pathogen with an outstanding ability to acquire multidrug resistant mechanisms. In this study, we investigate the molecular epidemiology and carbapenem resistance mechanisms of A. baumannii in Tripoli, Northern Lebanon. Methods: One hundred sixteen non-duplicate isolates isolated between 2011 and 2013 in different hospitals in Tripoli, Lebanon from Lebanese patients and wounded Syrian patients during Syrian war were studied. Antibiotic susceptibility testing was determined by agar disc diffusion and Etest. Carbapenemase-encoding genes were investigated by PCR. All isolates were typed by bla sequence based typing (SBT) and 57 isolates were also OXA-51-like analysed by MLST using Pasteur’s scheme followed by eBURST analysis. Results: Of the 116 isolates, 70 (60 %) showed a carbapenem resistance phenotype. The bla with an upstream OXA-23 insertion of ISAba1 was the major carbapenem resistance mechanism and detected in 65 isolates. Five isolates, including four from wounded Syrian patients and one from a Lebanese patient, were positive for bla . bla SBT revealed NDM-1 OXA-51-like thepresenceof14variants, where bla was the most common and present in 73 isolates, followed by bla in 20 OXA-66 OXA-69 isolates. MLST analysis identified 17 sequence types (ST) and showed a concordance with bla SBT. Each clonal OXA-51-like complex (CC) had a specific bla sequence such as CC2, which harboured bla variant, and CC1 harbouring OXA-51-like OXA-66 bla variant.NDM-1 producingisolatesbelongedtoST85(4Syrianisolates) andST25(1Lebaneseisolate). OXA-69 Conclusions: Our results showed a successful predominance of international clone 2 with a widespread occurrence of OXA-23 carbapenemase in Lebanese hospitals. These findings emphasise the urgent need of effective measures to control the spread of A. baumannii in this country. Keywords: Acinetobacter baumannii, Lebanon, Carbapenem resistance, bla sequence based typing, MLST, OXA-23, OXA-51 NDM-1 * Correspondence: [email protected] ATOMycA, Inserm Atip-Avenir Team, CRCNA, Inserm U892, 6299 CNRS, University and Centre Hospitalier Universitaire d’Angers, Angers, France Laboratoire de Bactériologie, Institut de Biologie en Santé - PBH, CHU, 4 rue Larrey, 49933 Angers Cedex, France Full list of author information is available at the end of the article © 2015 Rafei et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rafei et al. BMC Microbiology (2015) 15:103 Page 2 of 7 Background using blaOXA-51-like and Pasteur’s MLST typing and A. baumannii is an opportunistic pathogen mainly in- to determine the carbapenem resistance mechanisms. volved in healthcare-associated infections, with increased mortality and morbidity [1]. The serious concern associ- Results ated with this bacterium is the increasing prevalence of Bacterial isolates and identification multidrug resistant isolates, especially carbapenem re- A total of 116 non-duplicate isolates were obtained be- sistant ones [2]. Therefore, management of infections tween 2011 and 2013 from seven medical institutions in due to A. baumannii has become a real public health Tripoli: 73 isolates from Tripoli Governmental hospital, issue in many countries [3]. In a recent SENTRY Anti- 34 from Nini hospital, four from Dar Al Chiffaa hospital, microbial Surveillance program, the percentage of A. two from Monla, one isolate from each Hanane and baumannii isolates susceptible to meropenem decreased Haykal hospitals and one from the private laboratory. noticeably from 52.9 % in 2009 to 37.7 % in 2011 in Among those isolates, 59 had been obtained from Syrian European intensive care units [4]. The main mechanism refugees and 57 from Lebanese patients. They were all of resistance to carbapenems is the production of oxacil- confirmed as A. baumannii by bla real time PCR OXA-51 linases encoded mostly by blaOXA-23-like, blaOXA-24- and rpoB gene sequencing. These isolates were recov- like, blaOXA-58-like and recently blaOXA-143-like and ered from respiratory specimens (25/116; 21.6 %); blaOXA-235-like [2, 5–7]. To a lesser degree, carbape- wound (68/116; 58.6 %); urine (10/116; 8.6 %); catheter nemases from classes A and B have also been involved tips (8/116; 6.9 %), blood (1/116; 0.9 %) and other loca- [2, 5]. New Delhi metallo-ß-lactamase 1 (NDM-1), a re- tions (3/116; 2.6 %). For one isolate, no information was cent described metallo-ß-lactamase, has emerged in available. Ninety patients were male and 26 female. Ages Enterobacteriaceae as well as in Acinetobacter [8]. Gen- were between 1 month and 89 years with a mean of erally, a limited number of clones are responsible for 40 years. worldwide outbreaks, and international clones I and II are the most common ones [9]. In order to identify and Antibiotic susceptibility and carbapenem resistance track these clones during hospital outbreaks, numerous mechanisms molecular typing methods have been proposed such as Most of the isolates exhibited multidrug-resistant phe- pulsed-field gel electrophoresis (PFGE), amplified frag- notypes. Overall, 70 isolates were carbapenem resistant ment length polymorphism (AFLP), repetitive-sequence- and among them, 65 carried the bla gene while 5 OXA-23 based PCR (rep-PCR), and multilocus sequence typing isolates carried the bla gene. ISAba1 was found in NDM-1 (MLST) [10–14] . Currently, MLST is the gold standard 101 isolates. The presence of ISAba1/bla associ- OXA-23 typing tool for population structure and macro- ation was confirmed in all OXA-23 producing isolates. epidemiological investigations based on sequencing of The ISAba1/bla association was not detected. OXA-51 internal fragments of 7 genes [14]. Two MLST schemes Among carbapenem-resistant A. baumannii, 43 had have been developed for Acinetobacter baumannii :Pas- been isolated from Syrian refugees and 27 from teur and Bartual schemes [10, 15]. Overall, the 2 Lebanese patients. The prevalence of carbapenem resist- schemes are concordant [14]. However, because of re- ance was significantly higher among A. baumannii iso- combination bias observed on some loci in Bartual’s lated from Syrian refugees (74 % vs. 47 %; P < 0.01). MLST (gyrB and gpi) [16, 17], Pasteur’s MLST has been chosen for this study. Recently, blaOXA-51-like Epidemiological typing sequence-based typing (SBT), a simple typing method The bla SBT revealed the presence of 14 differ- OXA-51-like based on sequencing of the full length of blaOXA-51- ent nucleotide variants. Of the 116 isolates, bla OXA-66 like gene has been proposed as it has shown a similar dis- variant was the most prevalent and found in 73 isolates, criminatory power than rep-PCR, and MLST [16, 18–20]. followed by bla (20 isolates), bla (7 isolates), OXA-69 OXA-64 In Lebanon, limited numbers of epidemiological stud- bla (5 isolates) and bla (2 isolates). Other OXA-94 OXA-120 ies concerning A. baumannii have been reported, almost variants, bla , bla , bla , bla , OXA-70 OXA-71 OXA-104 OXA-121 in Beyrouth [20–23]. In a previous study, we have de- bla ,bla , KF048914, KJ584924, and OXA-132 OXA-406 scribed the dominance of international clone II within a AKAS01000012 were present sporadically in our collec- set of 42 isolates collected from Beyrouth and North tion. bla variant encoded a new protein (OXA-406) OXA-406 Lebanon between 2009–2012 [20]. In another study, we described for the first time in this study which differed reported the first detection of four NDM-1 producing from OXA-106 protein by a single amino acid at position isolates isolated in Tripoli-Northern Lebanon from Syr- 146 (lysine instead of asparagine). ian patients injured during Syrian War [24]. The aim of MLST was performed on a set of 57 randomly selected this study was to characterise A. baumannii molecular A. baumannii isolates representing all bla vari- OXA-51 epidemiology in Tripoli-Lebanon between 2011–2013 ants. A total of 17 ST(s), including 4 new ST(s), were Rafei et al. BMC Microbiology (2015) 15:103 Page 3 of 7 identified (Table 1, Fig. 1). eBURST analysis showed that situations raised questions about their origins and the 12 of the 17 ST(s) were clustered into 9 CCs which were potential involvement of an environmental reservoir CC1, CC2, CC3, CC25, CC33, CC85, CC149, CC158, [25]. The origin of infections from Syrian patients was CC462. Additionally, ST108, ST150, ST154 were single ambiguous. Although it was likely that nosocomial infec- locus variants of ST112, ST444 and ST611 respectively, tions occurred, a number of infections were present at and ST103 and ST461 were singletons. the time of admission of the patients in Lebanese hospi- Comparison between MLST and bla SBT tals [24]. These infections may have been acquired from OXA-51-like showed that isolates belonging to the same clonal com- environmental sources at the time of injury, during the plex carried all the same bla variant. Besides, each patient stay in Syrian hospitals, or during a direct evacu- OXA-51 of the remaining lineages (single locus variants or single- ation from the theatre of operations to Lebanon. Unfor- tons) that were identified had additionally a specific tunately, data concerning the date of injury, the length bla variant (Table 1). of hospitalisation, and the conditions of care and treat- OXA-51-like ment in Syrian hospitals are lacking. Discussion Approximately 60 % of our isolates showed a carba- Our study provided a global view of molecular epidemi- penem resistance phenotype. The production of OXA- ology of A. baumannii isolates from hospitals in Tripoli, 23 was the major carbapenem resistance mechanism, Lebanon. Tripoli is a city that hosts a large number of with an upstream insertion of ISAba1, thus supporting refugees and wounded Syrians, which may explain the international data about the worldwide emergence of high number of wound specimens in our collection. Al- this carbapenemase [28–30]. Alarmingly, 5 NDM-1 pro- though, A. baumannii is an opportunistic nosocomial ducing isolates have been detected from 4 Syrian pa- pathogen, it is increasingly reported from infections oc- tients [24] and one from a Lebanese patient. curring outside hospitals, particularly skin and soft- bla is an intrinsic oxacillinase gene naturally OXA-51-like tissue infections and pneumonia [25]. A. baumannii has occurring in A. baumannii, and more than 95 variants also been a well-documented pathogen associated with have been identified to date [31]. However, the occa- wound infections in USA troops from the Iraq and sional detection of bla in Acinetobacter nosoco- OXA-51 Afghanistan wars [26, 27]. The typology of these mialis and Acinetobacter genomic species “close to Table 1 Characteristics of the Acinetobacter baumannii isolates studied No. of bla ST* (No. of CC IMP R Hospitals (No. of isolates) Origin of isolation Nationality IMP R OXA-51 isolates variant tested strains) mechanism 73 bla ST2 (21) CC2 56R TGH (50), Nini (14), Monla Respiratory specimen (18), Syria (43) ISAba1/OXA-23 OXA-66 (2), private laboratory (1), catheter tip (7), wound (44), Lebanon (30) Haykal (1), Dar Al Chiffaa (4), urine (1), other (2), not Hanane (1). informed (1) 20 bla ST1 (12) CC1 9R TGH (17), Nini (3) Respiratory specimen (1), Syria (11) ISAba1/OXA-23 OXA-69 Wound (19) ST460 (2) Lebanon (9) 5 bla ST85 (4) CC85 4R TGH (5) Wound (5) Syria (4) NDM-1 OXA-94 ST6 (1) Lebanon (1) 7 bla ST25 (6) CC25 1R Nini (7) Respiratory specimens (5), Lebanon (7) NDM-1 OXA-64 blood (1), urine (1) 1 bla ST103 (1) Singleton S Nini Urine Lebanon − OXA-70 1 KF048914 ST154 (1) C S Nini Urine Lebanon − 1 bla ST3 (1) CC3 S Nini Urine Lebanon OXA-71 1 KJ584924 ST158 (1) CC158 S Nini Urine Lebanon − 1 bla ST46 (1) CC149 S Nini Urine Lebanon − OXA-104 2 bla ST459 (1) CC33 S Nini Respiratory specimen (1), Lebanon − OXA-120 urine (1) ST284 (1) 1 bla ST150 (1) C S Nini Ear Lebanon − OXA-121 1 bla ST108 (1) C S Nini Urine Lebanon OXA-132 1 bla ST461 (1) Singleton S TGH Catheter tips Syria − OXA-406 1 AKAS01000012 ST462 (1) CC462 S Nini Urine Lebanon − CC : clonal complex; C : complex formed by two sequence types (no number was assigned because no more of two sequences have been identified yet); IMP : imipenem, ST : sequence type, R : resistance and S : susceptible. Bold type indicates a new ST or a new bla allele OXA-51 Rafei et al. BMC Microbiology (2015) 15:103 Page 4 of 7 Fig. 1 Population snapshot analysed by eBURST on 634 sequences present in MLST Pasteur database (last update 22.10.2014). Red circle showed identified sequence types in this study 13TU” limit its use as a single identification method. causes for its propensity and its successful predominance Therefore, it is always combined with others techniques in hospitals throughout the world [10, 36]. The bla OXA- [32, 33]. Genetic diversity of bla gene has been ex- gene variant was the second gene mostly found in OXA-5 69 plored and found to be very interesting for identifying our collection. This gene was commonly associated epidemic clones [16, 18, 19, 34, 35]. Thus, the full gene with ST1 (belonging to CC1) but interestingly, we sequencing has been proposed as a single typing method found two bla isolates belonging to the new se- OXA-69 for A. baumannii. As being simple and rapid, we have quence type ST460 (SLV of ST1). Both isolates were initially screened here the epidemiological belonging of isolated from wounded Syrian patients. The bla OXA-94 our isolates to the previous identified clonal lineages by was another bla gene variant found in our OXA-51-like bla SBT. We have then randomly selected iso- study, it was associated with clonal complex CC85 OXA-51-like lates from each of the representative bla se- (named as CC6 in the study of Pournaras et al.[19]). OXA-51-like quences for MLST analysis. In our study, bla CC85 is currently formed by ST6, ST85, ST464, and OXA-51-like gene sequencing correctly assigned all isolates to their ST528. In our study bla SBT revealed the OXA-51-like corresponding clonal complexes. Here, the bla same sequence bla variant in the two identified OXA-66 OXA-94 gene variant, which is associated with ST2 (belonging to sequences ST6 and ST85. We have recently detected CC2) [16, 19, 35, 36], was predominant and found in 63 ST464inchicken andwefound bla as a bla OXA- 94 OXA- % of the A. baumannii isolates. The isolates carrying the variant [37]. Our 4 NDM-1 producing Syrian isolates bla gene variant have been identified in samples belonged to ST85. Indeed, this ST has also been re- OXA-66 obtained from the different hospitals in Tripoli. These sponsible for an outbreak in France. It is usually findings are consistent with those observed worldwide imported from North Africa [38, 39] and seems to be since CC2 was reported in more than 34 countries in an emerging clone. Europe, Asia, Africa, Australia, USA, and South America bla is a bla variant associated with OXA-64 OXA-51-like [36]. Eighty percent of our carbapenem resistant isolates CC25 [19]. ST25 (the founder of CC25) was also an belonged to this clone and OXA-23 was the only carba- emerging clone reported in Europe, Asia, Africa and penem resistance mechanism found. The high level of USA [36, 40], and from pets in Reunion Island, a French antimicrobial resistance may represent one of the main territory located in Western Indian Ocean [41]. Rafei et al. BMC Microbiology (2015) 15:103 Page 5 of 7 Different carbapenemases have been identified in this including ticarcillin, ticarcillin/clavulanic acid, piperacillin/ clone, as OXA-23, OXA-24 and OXA-58 [36]. Otherwise, tazobactam, ceftazidime, imipenem, ciprofloxacin, amika- NDM-1 producing ST25 was also detected in Germany cin, gentamicin, tobramycin, trimethoprim/sulfamethoxa- and Kenya [41–43]. Within our four identified isolates be- zole, colistin, netilmicin, doxycycline and rifampicin. longing to ST25, one isolate was a NDM-1 producing iso- Carbapenem resistance was confirmed by determining late recovered from a urine sample of an 80-year-old minimum inhibitory concentration (MICs) against imipe- Lebanese patient suffering from amyloidosis and anaemia nem, meropenem and doripenem by Etest®strips (bioMér- at Nini hospital, Tripoli, Lebanon. Finally, compared to ieux). Carbapenem resistant isolates were investigated by carbapenem resistant isolates belonging to successful PCR assays for the presence of carbapenemase-encoding emerging clones, most of susceptible isolates were very di- genes bla [46], bla [46], bla [46], OXA-23 OXA-24 OXA-58 verse, belonging to different sequence types (Table 1). bla [6], bla [47], bla [5], bla [5], bla OXA-143 NDM-1 IMP VIM KPC [48] and the insertion sequence ISAba1 [49]. The presence Conclusions of ISAba1 upstream bla or bla genes was OXA-23 OXA-51 In conclusion, this study highlights the emergence of searched using a combination of ISAba1 primers with re- NDM-1 and OXA-23 carbapenemases in Tripoli, Lebanon verse primers of bla or bla respectively. OXA-23 OXA-51 and the urgent need of effective measures to control the spread of A. baumannii in this country. It is noteworthy Epidemiological typing that Tripoli is located near the Syrian border and the mi- Epidemiological typing of all isolates was carried out by crobial epidemiology is probably highly impacted by bla sequence based typing (SBT) as described OXA-51-like wounded Syrian refugees who can represent a reservoir of previously [16]. The sequences were compared to all var- multidrug-resistant bacteria in hospitals. The higher preva- iants present in BLAST. Each new sequence detected lence of carbapenem-resistant A. baumannii among isolates was submitted to GenBank and assigned by Lahey data- from Syrian refugees was consistent with this hypothesis. base for beta-lactamase classification (http://www.la- Besides, we showed that bla SBT is a reliable and hey.org/studies/webt.asp). MLST was performed on a OXA-51-like fast method able to assign our isolates to their clonal com- panel of 57 isolates according to the Pasteur scheme plexes. Other multicentre studies are required to investigate (http://www.pasteur.fr/mlst). When a new allele or a the situation in other Lebanese cities that are possibly less new sequence type (ST) was identified, it was submitted concerned by the afflux of Syrian refugees fleeing war. to Pasteur Institute MLST Database. eBURST (http:// eburst.mlst.net/) was applied to compare identified ST(s) Methods to available ST(s) present in MLST Database (last update Bacterial isolates and Identification 22.10.2014). A clonal complex (CC) was defined as a set The A. baumannii clinical isolates used in this study formed by the founder ST and its single locus variants were collected between 2011 and 2013 from microbio- (SLV) [10]. logical laboratories covering 6 hospitals in Tripoli, Lebanon: Tripoli Governmental hospital (100 beds), Nini Nucleotide sequence accession numbers and novel (120 beds), Dar Al Chiffaa (120 beds), Monla (120 beds), sequence types Hanane (45 beds), and Haykal (125 beds) hospitals and Four newly identified ST(s) have been coded by MLST one private laboratory. Isolates were sent to Azm Center Pasteur as ST459, ST460, ST461 and ST462. A new nu- for Research in Biotechnology and its application and cleotide variant of bla was submitted to GenBank OXA-51 stored at-80 °C. This study was also approved by the eth- under the submission number [GenBank: KJ584915] and ical committee of Azm center under the authorization N assigned by Lahey center as bla . OXA-406 ° 07/2012. Bacterial identification was initially performed by matrix assisted laser ionization time of flight mass Statistical analysis spectrometry (MALDI-TOF MS) using the Vitek®MS The comparison of the prevalence of carbapenem-resistant (bioMérieux, Marcyl’Étoile, France) and the identifica- A. baumannii in isolates from Syrian refugees and in tion of A. baumannii at species level was confirmed by isolates from Lebanese patients was performed with the real time PCR of the bla gene [44] and partial OXA-51 Chi-square for test. A P < 0.05 was considered significant. RNA polymerase ß-subunit (rpoB) gene sequencing [45]. Competing interests The authors declare that they have no competing interests. Susceptibility testing and detection of carbapenemases Antibiotic susceptibility testing was performed by the Authors’ contributions RR performed bacterial identifications and antibiograms, carried out disc diffusion method according to the recommenda- molecular studies. HP and MK carried out molecular studies. RR and MK tions of the French Society of Microbiology (www.sfm- drafted the manuscript. ME, MLJG and MK were involved in revision of the microbiologie.org/). A panel of 14 antibiotics was tested manuscript for important intellectual content. MH, HM and FD isolated Rafei et al. BMC Microbiology (2015) 15:103 Page 6 of 7 bacterial strains in Lebanon. All authors read and approved the final characterization of clinical isolates of Acinetobacter baumannii. J Clin manuscript. Microbiol. 2005;43:4382–90. 16. Hamouda A, Evans BA, Towner KJ, Amyes SGB. Characterization of epidemiologically unrelated Acinetobacter baumannii isolates from four Acknowledgements continents by use of multilocus sequence typing, pulsed-field gel electrophoresis, We thank the team of the curators of the Institut Pasteur MLST system (Paris, andsequence-basedtypingofblaOXA-51-like genes. J Clin Microbiol. France) for importing novel alleles, profiles and/or isolates at http://www. 2010;48:2476–83. pasteur.fr/mlst. 17. Park YK, Jung S-I, Park K-H, Cheong HS, Peck KR, Song J-H, et al. Independent We are very grateful to all laboratory directors for their collaboration in this emergence of colistin-resistant Acinetobacter spp. isolates from Korea. Diagn study: Dr. Marcel El Achkar, Dr. Fadel Moukkadem, Dr. Ibtihal Kassam, Dr. Microbiol Infect Dis. 2009;64:43–51. Rikardo Sarraf, Dr. Nathalie Sapiroua, Dr. Gilbert karayakoupoglou, Dr. Samir 18. Zander E, Nemec A, Seifert H, Higgins PG. Association between β-lactamase- Ghanem. encoding bla(OXA-51) variants and DiversiLab rep-PCR-based typing of We also thank Catherine Quinqueneau, Catherine Ramont, Cyrielle Lebreton, Acinetobacter baumannii isolates. J Clin Microbiol. 2012;50:1900–4. Mariam Yehya and Taha Abdo for excellent technical assistance. This work 19. Pournaras S, Gogou V, Giannouli M, Dimitroulia E, Dafopoulou K, Tsakris A, was supported in part by AZM and SAADE Association. et al. Single locus sequence-based typing of blaOXA-51-like gene for rapid classification of Acinetobacter baumannii clinical isolates to international Author details clones. J Clin Microbiol. 2014;52:1653–7. ATOMycA, Inserm Atip-Avenir Team, CRCNA, Inserm U892, 6299 CNRS, 20. Rafei R, Dabboussi F, Hamze M, Eveillard M, Lemarié C, Gaultier M-P, et al. University and Centre Hospitalier Universitaire d’Angers, Angers, France. Molecular analysis of Acinetobacter baumannii strains isolated in Lebanon Laboratoire Microbiologie Santé et Environnement (LMSE), Centre AZM pour using four different typing methods. PLoS One. 2014;9, e115969. la recherche en Biotechnologie et ses applications, Université Libanaise, 21. Zarrilli R, Vitale D, Di Popolo A, Bagattini M, Daoud Z, Khan AU, et al. A plasmid- Tripoli, Lebanon. 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Molecular epidemiology of Acinetobacter baumannii in different hospitals in Tripoli, Lebanon using bla OXA-51-like sequence based typing

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Copyright © 2015 by Rafei et al.; licensee BioMed Central.
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Life Sciences; Microbiology; Biological Microscopy; Mycology; Parasitology; Virology; Life Sciences, general
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10.1186/s12866-015-0441-5
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25976451
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Abstract

Background: A. baumannii has emerged as an important nosocomial pathogen with an outstanding ability to acquire multidrug resistant mechanisms. In this study, we investigate the molecular epidemiology and carbapenem resistance mechanisms of A. baumannii in Tripoli, Northern Lebanon. Methods: One hundred sixteen non-duplicate isolates isolated between 2011 and 2013 in different hospitals in Tripoli, Lebanon from Lebanese patients and wounded Syrian patients during Syrian war were studied. Antibiotic susceptibility testing was determined by agar disc diffusion and Etest. Carbapenemase-encoding genes were investigated by PCR. All isolates were typed by bla sequence based typing (SBT) and 57 isolates were also OXA-51-like analysed by MLST using Pasteur’s scheme followed by eBURST analysis. Results: Of the 116 isolates, 70 (60 %) showed a carbapenem resistance phenotype. The bla with an upstream OXA-23 insertion of ISAba1 was the major carbapenem resistance mechanism and detected in 65 isolates. Five isolates, including four from wounded Syrian patients and one from a Lebanese patient, were positive for bla . bla SBT revealed NDM-1 OXA-51-like thepresenceof14variants, where bla was the most common and present in 73 isolates, followed by bla in 20 OXA-66 OXA-69 isolates. MLST analysis identified 17 sequence types (ST) and showed a concordance with bla SBT. Each clonal OXA-51-like complex (CC) had a specific bla sequence such as CC2, which harboured bla variant, and CC1 harbouring OXA-51-like OXA-66 bla variant.NDM-1 producingisolatesbelongedtoST85(4Syrianisolates) andST25(1Lebaneseisolate). OXA-69 Conclusions: Our results showed a successful predominance of international clone 2 with a widespread occurrence of OXA-23 carbapenemase in Lebanese hospitals. These findings emphasise the urgent need of effective measures to control the spread of A. baumannii in this country. Keywords: Acinetobacter baumannii, Lebanon, Carbapenem resistance, bla sequence based typing, MLST, OXA-23, OXA-51 NDM-1 * Correspondence: [email protected] ATOMycA, Inserm Atip-Avenir Team, CRCNA, Inserm U892, 6299 CNRS, University and Centre Hospitalier Universitaire d’Angers, Angers, France Laboratoire de Bactériologie, Institut de Biologie en Santé - PBH, CHU, 4 rue Larrey, 49933 Angers Cedex, France Full list of author information is available at the end of the article © 2015 Rafei et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rafei et al. BMC Microbiology (2015) 15:103 Page 2 of 7 Background using blaOXA-51-like and Pasteur’s MLST typing and A. baumannii is an opportunistic pathogen mainly in- to determine the carbapenem resistance mechanisms. volved in healthcare-associated infections, with increased mortality and morbidity [1]. The serious concern associ- Results ated with this bacterium is the increasing prevalence of Bacterial isolates and identification multidrug resistant isolates, especially carbapenem re- A total of 116 non-duplicate isolates were obtained be- sistant ones [2]. Therefore, management of infections tween 2011 and 2013 from seven medical institutions in due to A. baumannii has become a real public health Tripoli: 73 isolates from Tripoli Governmental hospital, issue in many countries [3]. In a recent SENTRY Anti- 34 from Nini hospital, four from Dar Al Chiffaa hospital, microbial Surveillance program, the percentage of A. two from Monla, one isolate from each Hanane and baumannii isolates susceptible to meropenem decreased Haykal hospitals and one from the private laboratory. noticeably from 52.9 % in 2009 to 37.7 % in 2011 in Among those isolates, 59 had been obtained from Syrian European intensive care units [4]. The main mechanism refugees and 57 from Lebanese patients. They were all of resistance to carbapenems is the production of oxacil- confirmed as A. baumannii by bla real time PCR OXA-51 linases encoded mostly by blaOXA-23-like, blaOXA-24- and rpoB gene sequencing. These isolates were recov- like, blaOXA-58-like and recently blaOXA-143-like and ered from respiratory specimens (25/116; 21.6 %); blaOXA-235-like [2, 5–7]. To a lesser degree, carbape- wound (68/116; 58.6 %); urine (10/116; 8.6 %); catheter nemases from classes A and B have also been involved tips (8/116; 6.9 %), blood (1/116; 0.9 %) and other loca- [2, 5]. New Delhi metallo-ß-lactamase 1 (NDM-1), a re- tions (3/116; 2.6 %). For one isolate, no information was cent described metallo-ß-lactamase, has emerged in available. Ninety patients were male and 26 female. Ages Enterobacteriaceae as well as in Acinetobacter [8]. Gen- were between 1 month and 89 years with a mean of erally, a limited number of clones are responsible for 40 years. worldwide outbreaks, and international clones I and II are the most common ones [9]. In order to identify and Antibiotic susceptibility and carbapenem resistance track these clones during hospital outbreaks, numerous mechanisms molecular typing methods have been proposed such as Most of the isolates exhibited multidrug-resistant phe- pulsed-field gel electrophoresis (PFGE), amplified frag- notypes. Overall, 70 isolates were carbapenem resistant ment length polymorphism (AFLP), repetitive-sequence- and among them, 65 carried the bla gene while 5 OXA-23 based PCR (rep-PCR), and multilocus sequence typing isolates carried the bla gene. ISAba1 was found in NDM-1 (MLST) [10–14] . Currently, MLST is the gold standard 101 isolates. The presence of ISAba1/bla associ- OXA-23 typing tool for population structure and macro- ation was confirmed in all OXA-23 producing isolates. epidemiological investigations based on sequencing of The ISAba1/bla association was not detected. OXA-51 internal fragments of 7 genes [14]. Two MLST schemes Among carbapenem-resistant A. baumannii, 43 had have been developed for Acinetobacter baumannii :Pas- been isolated from Syrian refugees and 27 from teur and Bartual schemes [10, 15]. Overall, the 2 Lebanese patients. The prevalence of carbapenem resist- schemes are concordant [14]. However, because of re- ance was significantly higher among A. baumannii iso- combination bias observed on some loci in Bartual’s lated from Syrian refugees (74 % vs. 47 %; P < 0.01). MLST (gyrB and gpi) [16, 17], Pasteur’s MLST has been chosen for this study. Recently, blaOXA-51-like Epidemiological typing sequence-based typing (SBT), a simple typing method The bla SBT revealed the presence of 14 differ- OXA-51-like based on sequencing of the full length of blaOXA-51- ent nucleotide variants. Of the 116 isolates, bla OXA-66 like gene has been proposed as it has shown a similar dis- variant was the most prevalent and found in 73 isolates, criminatory power than rep-PCR, and MLST [16, 18–20]. followed by bla (20 isolates), bla (7 isolates), OXA-69 OXA-64 In Lebanon, limited numbers of epidemiological stud- bla (5 isolates) and bla (2 isolates). Other OXA-94 OXA-120 ies concerning A. baumannii have been reported, almost variants, bla , bla , bla , bla , OXA-70 OXA-71 OXA-104 OXA-121 in Beyrouth [20–23]. In a previous study, we have de- bla ,bla , KF048914, KJ584924, and OXA-132 OXA-406 scribed the dominance of international clone II within a AKAS01000012 were present sporadically in our collec- set of 42 isolates collected from Beyrouth and North tion. bla variant encoded a new protein (OXA-406) OXA-406 Lebanon between 2009–2012 [20]. In another study, we described for the first time in this study which differed reported the first detection of four NDM-1 producing from OXA-106 protein by a single amino acid at position isolates isolated in Tripoli-Northern Lebanon from Syr- 146 (lysine instead of asparagine). ian patients injured during Syrian War [24]. The aim of MLST was performed on a set of 57 randomly selected this study was to characterise A. baumannii molecular A. baumannii isolates representing all bla vari- OXA-51 epidemiology in Tripoli-Lebanon between 2011–2013 ants. A total of 17 ST(s), including 4 new ST(s), were Rafei et al. BMC Microbiology (2015) 15:103 Page 3 of 7 identified (Table 1, Fig. 1). eBURST analysis showed that situations raised questions about their origins and the 12 of the 17 ST(s) were clustered into 9 CCs which were potential involvement of an environmental reservoir CC1, CC2, CC3, CC25, CC33, CC85, CC149, CC158, [25]. The origin of infections from Syrian patients was CC462. Additionally, ST108, ST150, ST154 were single ambiguous. Although it was likely that nosocomial infec- locus variants of ST112, ST444 and ST611 respectively, tions occurred, a number of infections were present at and ST103 and ST461 were singletons. the time of admission of the patients in Lebanese hospi- Comparison between MLST and bla SBT tals [24]. These infections may have been acquired from OXA-51-like showed that isolates belonging to the same clonal com- environmental sources at the time of injury, during the plex carried all the same bla variant. Besides, each patient stay in Syrian hospitals, or during a direct evacu- OXA-51 of the remaining lineages (single locus variants or single- ation from the theatre of operations to Lebanon. Unfor- tons) that were identified had additionally a specific tunately, data concerning the date of injury, the length bla variant (Table 1). of hospitalisation, and the conditions of care and treat- OXA-51-like ment in Syrian hospitals are lacking. Discussion Approximately 60 % of our isolates showed a carba- Our study provided a global view of molecular epidemi- penem resistance phenotype. The production of OXA- ology of A. baumannii isolates from hospitals in Tripoli, 23 was the major carbapenem resistance mechanism, Lebanon. Tripoli is a city that hosts a large number of with an upstream insertion of ISAba1, thus supporting refugees and wounded Syrians, which may explain the international data about the worldwide emergence of high number of wound specimens in our collection. Al- this carbapenemase [28–30]. Alarmingly, 5 NDM-1 pro- though, A. baumannii is an opportunistic nosocomial ducing isolates have been detected from 4 Syrian pa- pathogen, it is increasingly reported from infections oc- tients [24] and one from a Lebanese patient. curring outside hospitals, particularly skin and soft- bla is an intrinsic oxacillinase gene naturally OXA-51-like tissue infections and pneumonia [25]. A. baumannii has occurring in A. baumannii, and more than 95 variants also been a well-documented pathogen associated with have been identified to date [31]. However, the occa- wound infections in USA troops from the Iraq and sional detection of bla in Acinetobacter nosoco- OXA-51 Afghanistan wars [26, 27]. The typology of these mialis and Acinetobacter genomic species “close to Table 1 Characteristics of the Acinetobacter baumannii isolates studied No. of bla ST* (No. of CC IMP R Hospitals (No. of isolates) Origin of isolation Nationality IMP R OXA-51 isolates variant tested strains) mechanism 73 bla ST2 (21) CC2 56R TGH (50), Nini (14), Monla Respiratory specimen (18), Syria (43) ISAba1/OXA-23 OXA-66 (2), private laboratory (1), catheter tip (7), wound (44), Lebanon (30) Haykal (1), Dar Al Chiffaa (4), urine (1), other (2), not Hanane (1). informed (1) 20 bla ST1 (12) CC1 9R TGH (17), Nini (3) Respiratory specimen (1), Syria (11) ISAba1/OXA-23 OXA-69 Wound (19) ST460 (2) Lebanon (9) 5 bla ST85 (4) CC85 4R TGH (5) Wound (5) Syria (4) NDM-1 OXA-94 ST6 (1) Lebanon (1) 7 bla ST25 (6) CC25 1R Nini (7) Respiratory specimens (5), Lebanon (7) NDM-1 OXA-64 blood (1), urine (1) 1 bla ST103 (1) Singleton S Nini Urine Lebanon − OXA-70 1 KF048914 ST154 (1) C S Nini Urine Lebanon − 1 bla ST3 (1) CC3 S Nini Urine Lebanon OXA-71 1 KJ584924 ST158 (1) CC158 S Nini Urine Lebanon − 1 bla ST46 (1) CC149 S Nini Urine Lebanon − OXA-104 2 bla ST459 (1) CC33 S Nini Respiratory specimen (1), Lebanon − OXA-120 urine (1) ST284 (1) 1 bla ST150 (1) C S Nini Ear Lebanon − OXA-121 1 bla ST108 (1) C S Nini Urine Lebanon OXA-132 1 bla ST461 (1) Singleton S TGH Catheter tips Syria − OXA-406 1 AKAS01000012 ST462 (1) CC462 S Nini Urine Lebanon − CC : clonal complex; C : complex formed by two sequence types (no number was assigned because no more of two sequences have been identified yet); IMP : imipenem, ST : sequence type, R : resistance and S : susceptible. Bold type indicates a new ST or a new bla allele OXA-51 Rafei et al. BMC Microbiology (2015) 15:103 Page 4 of 7 Fig. 1 Population snapshot analysed by eBURST on 634 sequences present in MLST Pasteur database (last update 22.10.2014). Red circle showed identified sequence types in this study 13TU” limit its use as a single identification method. causes for its propensity and its successful predominance Therefore, it is always combined with others techniques in hospitals throughout the world [10, 36]. The bla OXA- [32, 33]. Genetic diversity of bla gene has been ex- gene variant was the second gene mostly found in OXA-5 69 plored and found to be very interesting for identifying our collection. This gene was commonly associated epidemic clones [16, 18, 19, 34, 35]. Thus, the full gene with ST1 (belonging to CC1) but interestingly, we sequencing has been proposed as a single typing method found two bla isolates belonging to the new se- OXA-69 for A. baumannii. As being simple and rapid, we have quence type ST460 (SLV of ST1). Both isolates were initially screened here the epidemiological belonging of isolated from wounded Syrian patients. The bla OXA-94 our isolates to the previous identified clonal lineages by was another bla gene variant found in our OXA-51-like bla SBT. We have then randomly selected iso- study, it was associated with clonal complex CC85 OXA-51-like lates from each of the representative bla se- (named as CC6 in the study of Pournaras et al.[19]). OXA-51-like quences for MLST analysis. In our study, bla CC85 is currently formed by ST6, ST85, ST464, and OXA-51-like gene sequencing correctly assigned all isolates to their ST528. In our study bla SBT revealed the OXA-51-like corresponding clonal complexes. Here, the bla same sequence bla variant in the two identified OXA-66 OXA-94 gene variant, which is associated with ST2 (belonging to sequences ST6 and ST85. We have recently detected CC2) [16, 19, 35, 36], was predominant and found in 63 ST464inchicken andwefound bla as a bla OXA- 94 OXA- % of the A. baumannii isolates. The isolates carrying the variant [37]. Our 4 NDM-1 producing Syrian isolates bla gene variant have been identified in samples belonged to ST85. Indeed, this ST has also been re- OXA-66 obtained from the different hospitals in Tripoli. These sponsible for an outbreak in France. It is usually findings are consistent with those observed worldwide imported from North Africa [38, 39] and seems to be since CC2 was reported in more than 34 countries in an emerging clone. Europe, Asia, Africa, Australia, USA, and South America bla is a bla variant associated with OXA-64 OXA-51-like [36]. Eighty percent of our carbapenem resistant isolates CC25 [19]. ST25 (the founder of CC25) was also an belonged to this clone and OXA-23 was the only carba- emerging clone reported in Europe, Asia, Africa and penem resistance mechanism found. The high level of USA [36, 40], and from pets in Reunion Island, a French antimicrobial resistance may represent one of the main territory located in Western Indian Ocean [41]. Rafei et al. BMC Microbiology (2015) 15:103 Page 5 of 7 Different carbapenemases have been identified in this including ticarcillin, ticarcillin/clavulanic acid, piperacillin/ clone, as OXA-23, OXA-24 and OXA-58 [36]. Otherwise, tazobactam, ceftazidime, imipenem, ciprofloxacin, amika- NDM-1 producing ST25 was also detected in Germany cin, gentamicin, tobramycin, trimethoprim/sulfamethoxa- and Kenya [41–43]. Within our four identified isolates be- zole, colistin, netilmicin, doxycycline and rifampicin. longing to ST25, one isolate was a NDM-1 producing iso- Carbapenem resistance was confirmed by determining late recovered from a urine sample of an 80-year-old minimum inhibitory concentration (MICs) against imipe- Lebanese patient suffering from amyloidosis and anaemia nem, meropenem and doripenem by Etest®strips (bioMér- at Nini hospital, Tripoli, Lebanon. Finally, compared to ieux). Carbapenem resistant isolates were investigated by carbapenem resistant isolates belonging to successful PCR assays for the presence of carbapenemase-encoding emerging clones, most of susceptible isolates were very di- genes bla [46], bla [46], bla [46], OXA-23 OXA-24 OXA-58 verse, belonging to different sequence types (Table 1). bla [6], bla [47], bla [5], bla [5], bla OXA-143 NDM-1 IMP VIM KPC [48] and the insertion sequence ISAba1 [49]. The presence Conclusions of ISAba1 upstream bla or bla genes was OXA-23 OXA-51 In conclusion, this study highlights the emergence of searched using a combination of ISAba1 primers with re- NDM-1 and OXA-23 carbapenemases in Tripoli, Lebanon verse primers of bla or bla respectively. OXA-23 OXA-51 and the urgent need of effective measures to control the spread of A. baumannii in this country. It is noteworthy Epidemiological typing that Tripoli is located near the Syrian border and the mi- Epidemiological typing of all isolates was carried out by crobial epidemiology is probably highly impacted by bla sequence based typing (SBT) as described OXA-51-like wounded Syrian refugees who can represent a reservoir of previously [16]. The sequences were compared to all var- multidrug-resistant bacteria in hospitals. The higher preva- iants present in BLAST. Each new sequence detected lence of carbapenem-resistant A. baumannii among isolates was submitted to GenBank and assigned by Lahey data- from Syrian refugees was consistent with this hypothesis. base for beta-lactamase classification (http://www.la- Besides, we showed that bla SBT is a reliable and hey.org/studies/webt.asp). MLST was performed on a OXA-51-like fast method able to assign our isolates to their clonal com- panel of 57 isolates according to the Pasteur scheme plexes. Other multicentre studies are required to investigate (http://www.pasteur.fr/mlst). When a new allele or a the situation in other Lebanese cities that are possibly less new sequence type (ST) was identified, it was submitted concerned by the afflux of Syrian refugees fleeing war. to Pasteur Institute MLST Database. eBURST (http:// eburst.mlst.net/) was applied to compare identified ST(s) Methods to available ST(s) present in MLST Database (last update Bacterial isolates and Identification 22.10.2014). A clonal complex (CC) was defined as a set The A. baumannii clinical isolates used in this study formed by the founder ST and its single locus variants were collected between 2011 and 2013 from microbio- (SLV) [10]. logical laboratories covering 6 hospitals in Tripoli, Lebanon: Tripoli Governmental hospital (100 beds), Nini Nucleotide sequence accession numbers and novel (120 beds), Dar Al Chiffaa (120 beds), Monla (120 beds), sequence types Hanane (45 beds), and Haykal (125 beds) hospitals and Four newly identified ST(s) have been coded by MLST one private laboratory. Isolates were sent to Azm Center Pasteur as ST459, ST460, ST461 and ST462. A new nu- for Research in Biotechnology and its application and cleotide variant of bla was submitted to GenBank OXA-51 stored at-80 °C. This study was also approved by the eth- under the submission number [GenBank: KJ584915] and ical committee of Azm center under the authorization N assigned by Lahey center as bla . OXA-406 ° 07/2012. Bacterial identification was initially performed by matrix assisted laser ionization time of flight mass Statistical analysis spectrometry (MALDI-TOF MS) using the Vitek®MS The comparison of the prevalence of carbapenem-resistant (bioMérieux, Marcyl’Étoile, France) and the identifica- A. baumannii in isolates from Syrian refugees and in tion of A. baumannii at species level was confirmed by isolates from Lebanese patients was performed with the real time PCR of the bla gene [44] and partial OXA-51 Chi-square for test. A P < 0.05 was considered significant. RNA polymerase ß-subunit (rpoB) gene sequencing [45]. Competing interests The authors declare that they have no competing interests. Susceptibility testing and detection of carbapenemases Antibiotic susceptibility testing was performed by the Authors’ contributions RR performed bacterial identifications and antibiograms, carried out disc diffusion method according to the recommenda- molecular studies. HP and MK carried out molecular studies. RR and MK tions of the French Society of Microbiology (www.sfm- drafted the manuscript. ME, MLJG and MK were involved in revision of the microbiologie.org/). A panel of 14 antibiotics was tested manuscript for important intellectual content. MH, HM and FD isolated Rafei et al. BMC Microbiology (2015) 15:103 Page 6 of 7 bacterial strains in Lebanon. All authors read and approved the final characterization of clinical isolates of Acinetobacter baumannii. J Clin manuscript. Microbiol. 2005;43:4382–90. 16. Hamouda A, Evans BA, Towner KJ, Amyes SGB. Characterization of epidemiologically unrelated Acinetobacter baumannii isolates from four Acknowledgements continents by use of multilocus sequence typing, pulsed-field gel electrophoresis, We thank the team of the curators of the Institut Pasteur MLST system (Paris, andsequence-basedtypingofblaOXA-51-like genes. J Clin Microbiol. France) for importing novel alleles, profiles and/or isolates at http://www. 2010;48:2476–83. pasteur.fr/mlst. 17. Park YK, Jung S-I, Park K-H, Cheong HS, Peck KR, Song J-H, et al. Independent We are very grateful to all laboratory directors for their collaboration in this emergence of colistin-resistant Acinetobacter spp. isolates from Korea. Diagn study: Dr. Marcel El Achkar, Dr. Fadel Moukkadem, Dr. Ibtihal Kassam, Dr. Microbiol Infect Dis. 2009;64:43–51. Rikardo Sarraf, Dr. Nathalie Sapiroua, Dr. Gilbert karayakoupoglou, Dr. Samir 18. Zander E, Nemec A, Seifert H, Higgins PG. Association between β-lactamase- Ghanem. encoding bla(OXA-51) variants and DiversiLab rep-PCR-based typing of We also thank Catherine Quinqueneau, Catherine Ramont, Cyrielle Lebreton, Acinetobacter baumannii isolates. J Clin Microbiol. 2012;50:1900–4. Mariam Yehya and Taha Abdo for excellent technical assistance. This work 19. 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Published: May 16, 2015

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