Characterization of carbapenem resistant Acinetobacter baumannii isolated from intensive care units in two teaching hospitals from Algeria and Tunisia

Introduction: This study was conducted to identify the enzymatic mechanism of carbapenem resistance in A. baumannii isolated from intensive care units of 2 teaching hospitals (Charles Nicolle hospital of Tunis and University hospital of Annaba). Methods: Twenty seven non repetitive carbapenem-resistant A. baumannii were collected (7 strains in Algeria and 20 in Tunisia). Antibiotic susceptibility was performed by disk diffusion method. MICs were determined by agar dilution method. EDTA-disk synergy test was performed for metallo-β-lactamases (MBL) phenotypic detection. Detection of blaOXA-23-like, blaOXA-24-like, blaOXA-51-like and blaOXA-58-like families was performed by PCR followed by sequencing. Genetic relatedness between strains was investigated by pulsed-field gel electrophoresis (PFGE). Results: Strains were recovered especially from respiratory tract specimens (n=12) and blood (n=11). All strains were co-resistant to all β-lactams, gentamicin, amikacin and ciprofloxacin, but remainded susceptible to colistin. MBL production was negative for all isolates. blaOXA-51-like was detected in all strains and blaOXA-23-like in 23 strains. However, blaOXA-58-like and blaOXA-24-like were not found in any isolate. Six major PFGE patterns were found in the Tunisian isolates. However, the Algerian strains were clustered in one clone. Conclusion: This study shows a high distribution of blaOXA-23 in imipenem-resistant A. baumannii isolated in Tunisia and Algeria. It demonstrated the epidemic diffusion of this multidrug resistant pathogen. Thus, strengthening of prevention measures are required to control further spread of carbapenemases in the two countries. Pan African Medical Journal. 2017;28:19. doi:10.11604/pamj.2017.28.19.9713 This article is available online at: http://www.panafrican-med-journal.com/content/article/28/19/full/ © Sabrina Amiri et al. The Pan African Medical Journal ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pan African Medical Journal – ISSN: 19378688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Research Open Access


Introduction
Acinetobacter baumannii has emerged as a leading cause of nosocomial infections, particularly among critically ill patients in intensive care units [1,2] A. baumannii clinical isolates are commonly resistant to multiple antimicrobial drug classes and have the ability to survive in the environment for prolonged periods of time, which facilitates their persistence in hospitals and make them a frequent cause of hospital outbreaks and an endemic healthcare associated pathogens [3]. Carbapenems have been widely used to treat these infections [4], but a trend of increasing resistance to this antibiotic class has been reported worldwide [5]; limiting drastically the range of therapeutic alternatives. Carbapenem resistance in A.

Metallo-beta-lactamases are prevalent in East Asia and Western
Europe and confer resistance to all beta-lactams except aztreonam [1]. However, the OXA-type carbapenemases have emerged as the most widespread beta-lactamases with carbapenemase activity [8,9]. These enzymes can be sub-devised into 5 distinct groups:  [10][11][12][13]. In addition to betalactamases, carbapenem resistance in A. baumannii may also result from porin or penicillin-binding protein modifications [5]. The aim of this study was to determine the enzymatic mechanism of carbapenem resistance in A. baumannii isolates recovered from intensive care units of two teaching hospitals (Tunisia and Algeria) and to characterize nosocomial outbreaks by antibiotyping and pulsed-field gel electrophoresis (PFGE).  [3].

Methods
Screening for MBL-producing strains: Detection of MBL was done by double disk synergy test using an imipenem disk placed 10 mm from a disk saturated with 5µl of EDTA (0.5 M pH 8). An enlargement of the inhibition zone of imipenem facing the disk of EDTA was considered as a positive test [15].

Pulsed-field gel electrophoresis (PFGE): Molecular typing of
isolates was carried out, as described previously by Pulsed-Field Gel Electrophoresis (PFGE) using ApaI (Invitrogen) restriction endonuclease [14]. The ApaI restriction profiles were compared by visual inspection according to the criteria proposed by Tenover et al [17].

Discussion
A. baumannii has been stealthily gaining ground as an agent of serious nosocomial infections, including bacteremia, pneumonia, urinary tract and wound infections [18]. Historically, it has been associated with opportunistic infections; the last two decades have seen an increase in both the incidence and seriousness of A. All strains were co-resistant to all β-lactams, gentamicin, amikacin, ciprofloxacin but 6 isolates remained susceptible to netilmicin, 2 isolates to tobramycin and 3 strains to trimethoprimsulfamethoxazol. All strains were susceptible to colistin. Despite its renal toxicity, colistin has become useful antibiotic for treating infections caused by carbapenem resistant pathogens [25], but dissemination of A. baumannii resistant to colistin is worrying. In another side, many studies provide the activity of tigecycline against multidrug A. baumannii clinical isolates [26]. Antibiotic resistance in A. baumannii is frequently an interplay between several different mechanisms [5]. This bacteria produces naturally 2 intrinsic types of β-lactamases [5]. An AmpC type cephalosporinase expressed at a basal level and an oxacillinase represented by the OXA-51/69 variants [5]. When ISAba1 were inserted upstream of blaAmpC and blaOXA-51-like genes, the strains become resistant to ceftazidime and to carpenems, respectively [27,28]. The blaOXA-51-like genes are chromosomally located in all of the A. baumannii isolates studied to date and their presence has been used to confirm identification of A.
baumannii [29]. In addition to those naturally occurring βlactamases, several acquired β-lactamases have been identified as a source of carbapenem resistance in A. baumannii. They belong to either class D (oxacillinases) or class B (metallo-β-lactamases [MBLs] [5]. The first carbapenem-hydrolysing oxacillinase was OXA-23 identified in Scotland in 1985 [30]. The gene encoding this enzyme, named ARI-1, was plasmid born and was associated with the ISAbaI transposase [30]. Since then, the IS-OXA23 structure has been found among Acinetobacter isolates from various countries [31,32]. The bla OXA-23 genes have been identified as part of transposon structures, namely Tn2006 and Tn2007 [31]. Interestingly, the reservoir (natural producer) of this gene has been identified as being A. radioresistens [33]. This Acinetobacter species shares the same reservoir with A. baumannii, the skin flora in humans [31].
Our study revealed the presence of this carbapenemase in Tunisian (n=18) and Algerian (n=5) strains. However, blaOXA-24-like and bla OXA -58-like were not detected in any of ours strains. In the 4 strains, where only blaOXA-51 was detected, resistance can be explained by non enzymatic mechanisms or insertion of ISAba1 sequences [28,34]. MBLs are powerful carbapenemases [35]. They have been identified worldwide in A. baumannii [36,37]. Four groups were described in A. baumannii: IMP-like, VIM-like SIM-1 and NDM-1 enzymes [35]. MBL production was not detected in any of our

Competing interests
The authors declare no competing interests.