Phenotypic and genotypic characterization of multidrug-resistant Acinetobacter baumannii isolated in Algerian hospitals

Introduction: the aim of this study was to investigate the drug-resistance and the molecular characterization of carbapenemases, ESBL, and aminoglycoside-modifying enzymes among Acinetobacter baumannii clinical isolates in Algerian hospitals. Methodology: a total of 92 A. baumannii isolates were collected between 2012 and 2016. Antimicrobial susceptibility testings were performed for β-lactams, aminoglycosides, fluoroquinolones, trimethoprim-sulfamethoxazole, rifampicin and colistin. The phenotypic characterization of β-lactamases was investigated. For 30 randomly targeted strains, the carriage of the carbapenemases, ESBL and aminoglycoside-modifying enzymes -encoding genes was determined by PCR. Sequencing was carried out for carbapenemases and ESBL genes. Results: most of the 92 isolates studied were recovered from hospitalized patients (93.5%) and were mainly from intensive care units (51.1%) and orthopedics (19.6%). The strains were collected primarily from low respiratory tract (33.7%), wounds (23.9%) and urine (16.3%). Multidrug-resistant A. baumannii strains were prevalent (96.7%). High rates of resistance were observed for almost all antibiotics tested (>70%) excluding rifampicin (7.6%) and colistin (5.4%). For the five colistin-resistant strains, MICs ranged between 4 and 128 μg/mL. Positive MBL (83.7%) and ESBL (23.9%) strains were identified. Regarding β-lactams, the blaNDM and both blaSHV and blaCTX-M1 genes were detected in five and two strains respectively. Sequencing of the genes revealed the presence of blaNDM-1, blaCTX-M-15, and blaSHV-33. For aminoglycosides, aac(6’)-Ib, ant(2’’)-I and aph(3’)-VI genes were detected in three, seven and six strains respectively. Conclusions: here, we report the first co-occurrence of extended-spectrum β-lactamases SHV-33 and CTX-M-15, the carbapenemase NDM-1 and the emergence of colistin-resistant A. baumannii in Algerian hospitals.


Introduction
Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen causing clinical infections and outbreaks in healthcare settings especially in intensive care units (ICUs). This species account for almost 90% of all reported Acinetobacter infections, including respiratory tract infections, bacteremia, meningitis, wound infections and urinary tract infections [1][2].
A. baumannii is able to easily acquire resistance to different groups of antimicrobials and to survive in hospital environment, leading to its persistence and transmission in healthcare settings [1,3]. A. baumannii is a member of the ESKAPE group (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) of six highly resistant pathogens that are a major cause of antibiotic-resistant infections [4,5].
Multidrug-resistant A. baumannii has become life threatening and is increasingly reported worldwide, including Europe, America, Asia and Africa [1,2]. Over the last decade, the emergence of A. baumannii resistant to carbapenems, a last-line group of β-lactams for treatment of patients infected with multidrug-resistant bacteria, was reported [6][7][8][9]. In this context, the World Health Organization recognize carbapenem-resistant A. baumannii (CRAB) as the first critical priority in its list of 12 resistant-bacteria that pose the greatest threat to human health [10].
The purpose of this study was to evaluate the antimicrobial resistance rates and molecular mechanisms of ESBLs, carbapenemases and AMEs to multidrug A. baumannii isolated in Algerian hospitals during a 5-year period starting from January 2012. Here, we report the first co-occurrence of extendedspectrum β-lactamases SHV-33 and CTX-M-15, the carbapenemase NDM-1 and the emergence of colistinresistant A. baumannii in Algerian hospitals.
All clinical isolates were identified by standard microbiological methods using Api 20NE identification system (bioMérieux, Marcy l'Etoile, France).
The minimum inhibitory concentrations (MICs) for netilmicin and colistin were determined using E-test method (bioMérieux, Marcy l'Etoile, France) and broth microdilution method respectively.
Quality control for the antimicrobial susceptibility analysis was performed with Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922.

PCR assays
Thirty A. baumannii isolates were randomly targeted, using the "alea" function of the Excel 2013 software, among the 92 strains for the detection of the carbapenemase, ESBL and AME -encoding genes according to phenotypic test results.
Bacterial DNA was extracted by boiling. Conventional PCR simplex assay was performed in a 50 μL reaction mixture containing the final concentrations of 0.5 μM primer, 2.5 U/µL Taq polymerase, 2 mM MgCl 2, 0.2 mM dNTPs, 10 μL
Regarding aminoglycosides, eleven resistance profiles including resistance to one to four aminoglycosides were defined among MDR A. baumannii isolates. The resistance to all aminoglycosides was prevalent (n = 54; 58.7%) strains, followed by the resistance to gentamicin and tobramycin (n = 13; 14.1%) strains (Table 2). MICs to netilmicin were ranged between 0.016 µg/mL to 256 µg/mL with MIC 50 and MIC90 both at 256 µg/mL.
MICs to colistin were ranged between 0.064 µg/mL and 128 µg/mL with MIC50 at 0.5 µg/mL and MIC90 at 1 µg/mL. For the five colistin-resistant strains, the MICs were 4 µg/mL for four strains and 128 µg/mL for one strain.

Molecular detection of resistance genes
Carbapenemase and extended-spectrum β-lactamaseencoding genes Out of the eight resistance genes investigated, five genes (blaTEM, blaCTXM-2, blaIMP, blaVIM and blaOXA-58like) were not detected in any of the tested strains.  Two strains were positive for both blaSHV and blaCTX-M-1 genes among 8/30 PCDDT positive isolates and were collected from an Algiers hospital.
Sequencing of the genes revealed the presence of blaNDM-1, blaCTX-M15, and blaSHV-33. Detailed data are shown in Table 3.

Discussion
Multidrug resistant A. baumannii represent a major threat in nosocomial infections, mostly in ICUs.
In this study, the most frequently strains were obtained from lower respiratory tract (33.7%), followed by wounds (23.9%) and were mostly from intensive care units (51.1%) and orthopedics (19.6%).

Antimicrobial susceptibility testing
In our study, most of the isolates showed high antibiotic resistance rates to β-lactams, aminoglycosides, fluoroquinolones and trimethoprimsulfamethoxazole and low resistance rates to rifampicin and colistin. Our findings are moderately higher than the Algerian Antimicrobial Resistance Network (AARN) data recorded in Acinetobacter spp. during the same period (ceftazidime 85.6%, imipenem 73.4%, amikacin 65.7%, ciprofloxacin 80.1% and trimethoprim-sulfamethoxazole 72.2%) [18], and studies carried out in Westen Algeria in 2013 and Algiers in 2015 [19,20].
In accordance with results reported around the world [11], almost all A. baumannii strains in our study were MDR and carbapenem-resistant A. baumannii (CRAB) were prevalent (83.7%). The widespread of theses strains has described worldwide [11]. The high rate of CRAB isolates is a concern, as carbapenems represent the treatment of choice for infections caused by A. baumannii, leading to limited therapeutic options.
In this study, five strains were resistant to colistin. In recent years, the emergence of colistin resistance in A.baumanni clinical isolates has been increasingly reported in several countries worldwide, becoming a public health concern as this antibiotic is used for the treatment of infections due to CRAB [21,22].
In the North African countries, a few cases of colistin resistance among A. baumannii has been described in Tunisia and Egypt [23,24]. In Algeria, Bakour et al.in 2014, described one colistin-resistant A. baumannii clinical strain that harbored a single mutation in the pmrB gene in an Algiers hospital [25]. Table 3. Detection of ESBL, MBL, aminoglycosides resistance and their related genes among MDR A. baumannii isolates. To delay the emergence of this resistance, combination therapies using tigecycline or rifampicin with colistin were found to be effective [26,27]. In addition, new drugs developed such as eravacycline and cefiderocol are promising but need to be evaluated in further studies [28]. Seven (7.6%) isolates were resistant to rifampicin. This rate is higher than that (2.8%) reported by Bakour et al.in 2013 in Setif and Tizi-Ouzou [29]. So, this increased resistance rate should prompt attention and be continuously monitored.

Molecular detection of resistance genes
Carbapenemases and extended-spectrum β-lactamasesencoding genes Five strains harbored the blaNDM gene, identified as blaNDM-1. The MBL NDM-1, detected originally in Enterobacteriaceae, was later described in Acinetobacter spp. Currently, this enzyme has spread in several countries around the world [30][31][32].
These studies support our results and confirm that NDM-producing A. baumannii isolates are nowadays endemic in Algerian hospitals.
For 16 CDT positive strains, none of blaVIM, blaIMP, blaNDM and blaOXA-58 genes were detected. Several studies reported false MBL-positive results using this test, particularly in CHDLs strains likely due to chelators as EDTA that may inhibit the activity of oxacillinases [41,42].
To the best of our knowledge, this is the first description of SHV-33 and co-occurrence of extended spectrum β-lactamase SHV-33 and CTX-M-15producing A. baumannii.
For the six PCDDT positive strains, blaTEM, blaSHV, blaCTX-M-1 and blaCTX-M-2 genes were negative. As previously described by Beceiro et al., the intrinsic susceptibility of some strains of A. baumannii to βlactamase inhibitor clavulanic acid could yield false ESBL-positive results in A. baumannii [45].
Combined AME resistance genes was observed with ant(2'')-I-aph(3')-VI (n = 2) and association of ESBL, MBL and AME resistance genes was also identified (n = 7).The association of aminoglycosides resistance, MBL and ESBL genes identified, highlight the multiple mechanisms involved leading to multidrug resistant A. baumannii strains.

Conclusions
Our results highlight that (i) the high prevalence of multidrug resistant A. baumannii in Algerian hospitals is alarming, (ii) NDM-1 isolates are actually endemic in Algerian hospitals, (iii) co-occurrence of ESBLs SHV-33 and CTX-M-15 is described for the first time in Algeria, (iv) AMEs, often combined, are mediated by aac(6')-Ib, aph(3')-VI and ant(2'')-Iand (v) colistinresistant strains have emerged in Algerian hospitals.
These findings report a critical situation in Algerian hospitals that requires rational use of antibiotics, strict compliance with hygiene rules, rapid and accurate screening of inpatients and implementation of early precautions for MDR carriers to decrease nosocomial transmission in hospitals. Further investigations are needed to better understand the molecular epidemiology of this nosocomial opportunistic pathogen.