Hanane Zerrouki

Sid-Ahmed Rebiahi

Linda Hadjadj

Fatmi Ahlem

Yamina Elhabiri

Tahar Sedrati

Jean-Marc Rolain

Seydina M Diene
email: seydina.diene@univ-amu.fr

High frequency and diversity of Vancomycin-Resistant Enterococci (VRE) in Algerian healthcare settings

Keywords: Algeria, healthcare settings, vanA, vanB, Vancomycin-Resistant Enterococci

The spread of vancomycin-resistant Enterococci (VRE) in Algerian hospital settings is poorly reported. Since the first report in 2006, few data have been available on the molecular mechanism of this resistance across the country. In this study, we investigate the frequency and antibiotic resistance mechanisms of Enterococci strains isolated from hospitalised patients in the Tlemcen university hospital. 191 Enterococcus spp. strains were collected from various clinical samples and were identified using MALDI-TOF-MS. The presence of van genes was investigated by standard PCR and sequencing. Results revealed that E. faecium and E. faecalis strains are the main pathogens identified in the study.

Antibiotic susceptibility testing revealed that the resistance rate was high for the majority of antibiotic classes, including glycopeptides, and only linezolid was effective on all strains. Molecular analysis revealed that 52.2% of strains from intensive care unit (ICU) were positive for the vanA gene, including 44.44% E. faecium, 5.55% E. faecalis and 2.22% E. avium.

25.5% of these isolates co-harboured both the vanA and vanC genes, including E. gallinarum (n=16) and E. faecium (n=6). In surgical wards (SW) 29.70% of strains harboured the van genes, including 4.90% of E. faecalis harbouring the vanB gene, and of the rest of strains, (24.80%) harboured the vanC genes. Indeed, 9.90% E. gallinarum and 4.90% E. faecalis were positive for vanC1 and 9.90% of E. casseliflavus were positive for the vanC2/C3 gene. The glycopeptide resistance rate was higher among strains from the ICU and was mainly composed by E. faecium strains compared with surgical wards where resistant E. faecalis strains were predominant.

Introduction

Mostly E. faecium, and to some extent E. faecalis, are involved in healthcareassociated infections and nosocomial outbreaks. These can cause serious infections ranging from urinary tract infections to endocarditis, meningitis or bacteraemia, which are sources of significant morbidity and mortality [START_REF] O'driscoll | Vancomycin-resistant enterococcal infections: Epidemiology, clinical manifestations, and optimal management[END_REF]. Enterococci are naturally resistant to many routinely used antibiotics such as cephalosporins, low concentrations of aminoglycosides, clindamycin, fluoroquinolones, trimethoprim-sulfamethoxazole [START_REF] Cetinkaya | Vancomycin-resistant enterococci[END_REF]. They are characterised by particular genome plasticity allowing them to resist other antibiotics [START_REF] Bender | Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature[END_REF]. Vancomycin, considered as drug of last resort, has long been used against Gram-positive bacteria which are resistant to β-lactams responsible for serious infections including sepsis, meningitis, endocarditis, pneumonia, enterocolitis and osteomyelitis [START_REF] Rivera | Current concepts in antimicrobial therapy against select grampositive organisms: Methicillin-resistant Staphylococcus aureus, penicillin-resistant pneumococci, and vancomycin-resistant enterococci[END_REF]. Vancomycin-resistant Enterococci (VREs) are easily transmitted through asymptomatic patients, caregivers, and contaminated environments or instruments [START_REF] Escaut | Eradication of an outbreak of vancomycin-resistant Enterococcus (VRE): The cost of a failure in the systematic screening[END_REF]. VREs can persist on inanimate surfaces for a long time and show a remarkable ability to colonise gastrointestinal (GI) tracts of hospitalised patients [START_REF] Vehreschild | Vancomycin-resistant enterococci (VRE): a reason to isolate? Infection[END_REF]. An imbalance in hospital hygiene coupled with the excessive use of broad-spectrum antibiotics frequently results in VRE nosocomial outbreaks in high-risk departments such as transplant departments [START_REF] Kreidl | Outbreak report: A nosocomial outbreak of vancomycin resistant enterococci in a solid organ transplant unit[END_REF], oncology [START_REF] Oh | Outbreak of vancomycin resistant Enterococcus in a hematology/oncology unit in a Korean university hospital, and risk factors related to patients, staff, hospital care and facilities[END_REF] and intensive care units (ICU) [START_REF] Hughes | An outbreak of vanA vancomycin-resistant Enterococcus faecium in a hospital with endemic vanB VRE[END_REF]. As reported, the resistance mechanism to vancomycin is mediated by the modification of vancomycin-binding targets leading to alterations in the peptidoglycan synthesis. Indeed, the terminal d-Ala-d-Ala peptide of Nacetylmuramic acid is substituted with d-Lac or d-Ser, causing a significant decrease in the affinity between cell wall precursors and the antibiotic [START_REF] Hollenbeck | Intrinsic and acquired resistance mechanisms in enterococcus[END_REF]. Eight gene variants (vanA, vanB, vanD, vanE, vanG, vanL, vanM, and vanN) have been reported as acquired genes conferring resistance to vancomycin and only the vanC gene appears to be an intrinsic gene of E. gallinarum and E. casseliflavus, and E. flavescens [START_REF] Faron | Resistance mechanisms, epidemiology, and approaches to screening for vancomycin-resistant Enterococcus in the health care setting[END_REF].

The dominant VRE clone harbouring vanA has been responsible for numerous hospital outbreaks worldwide [START_REF] Rangberg | Molecular analysis and epidemiological typing of Vancomycin-resistant Enterococcus outbreak strains[END_REF] [START_REF] Douglas | Utilizing genomic analyses to investigate the first outbreak of vanA vancomycin-resistant enterococcus in Australia with emergence of daptomycin non-susceptibility[END_REF] but, an emerging clone harbouring vanB has also been reported [START_REF] Hughes | An outbreak of vanA vancomycin-resistant Enterococcus faecium in a hospital with endemic vanB VRE[END_REF] [START_REF] Coombs | Molecular epidemiology of enterococcal bacteremia in Australia[END_REF].The management of VREs infections poses a real challenge for clinicians, as the therapeutic arsenal is often limited to linezolid [START_REF] Dotis | Use of linezolid in pediatrics: A critical review[END_REF]. Active surveillance based on screening and isolation of colonised or infected patients with rigorous disinfection remains the cornerstone of measures to control and limit transmission and the occurrence of outbreaks [START_REF] Kreidl | Outbreak report: A nosocomial outbreak of vancomycin resistant enterococci in a solid organ transplant unit[END_REF]. In Algerian hospitals, only a few studies have reported VRE strains, harbouring only the vanA gene [START_REF] Benammar | First molecular characterization of related cases of healthcareassociated infections involving multidrug-resistant enterococcus faecium vana in Algeria[END_REF][START_REF] Djahmi | Molecular epidemiology of Enterococcus sp. isolated in a university hospital in Algeria[END_REF].

In this study, we investigate the rate and molecular mechanisms of glycopeptide and linezolid resistance among clinical Enterococcus strains isolated from hospitalised patients at the Tlemcen university hospital in Algeria.

Materials and methods

Bacterial strains:

Between October 2016 and June 2017, a total of 191 clinical Enterococcus strains were isolated from various specimens (bronchial aspirates, gastric aspirates, ascitic fluids, cerebrospinal fluids, vaginal discharges, urine and wounds) collected from hospitalised patients in the intensive care unit (ICU) and surgical wards (SW), including Urology, Emergency Surgery, Orthopaedic-Trauma Surgery, General Surgery, and Neurosurgery at the Tlemcen university hospital, comprising a total of 646 beds.

Bacterial identification

Specimens were plated onto colistin nalidixic acid (CNA) agar with 5% Sheep Blood (bioMérieux, Marcy-l'Étoile, France) and incubated at 37°C for 48 hours. All Enterococcus spp. strains were identified using MALDI-TOF/MS (MicroflexTM; Bruker Daltonik GmbH, Bremen, Germany) as previously described [START_REF] Seng | Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry[END_REF].

Antibiotic susceptibility testing

Antibiotic susceptibility testing was performed using the disk diffusion method according to the recommendations of CA-SFM (Antibiogram Committee of the French Microbiology Society, 2019). Sixteen antibiotics were tested, including penicillin G, oxacillin, amoxicillin, ceftriaxone, clindamycin, erythromycin, pristinamycin, gentamicin, vancomycin, teicoplanin, doxycycline, minocycline, nitrofurantoin, linezolid, rifampicin, fosfomycin, (all from i2a, Montpellier, France). Minimum inhibitory concentrations (MICs) of vancomycin, teicoplanin, and linezolid were determined by the E-test method (bioMérieux, Marcy-l'Étoile, France) and interpreted in accordance with CA-SFM guidelines.

Molecular analysis

Standard PCR and sequencing was performed to confirm and screen for the presence of vanA, vanB, vanC1, vanC2/C3 genes, as previously described [START_REF] Domingo | High prevalence of glycopeptide resistance genes vanB, vanD, and vanG not associated with enterococci in human fecal flora[END_REF] [START_REF] Said | Emergence of multidrug resistance and extensive drug resistance among enterococcal clinical isolates in Egypt[END_REF]. All the primers used are presented in (Suppl. Table S1).

Results

Clinical Data: The average age of patients in the ICU was 54.1 years (22 to 82 years), and 51.3 years (20 to 86 years) in the SW. All patients had received antibiotic treatment for at least five days and had indwelling devices. In the ICU, all patients were receiving mechanical ventilation and the average length of their hospital stay was more 15 days [7-150], compared to only seven days [5][6][7][8][9][10][11][12][13][14][15][16][17][18] in SW patients. Demographic and clinical data of patients are shown in Table 1.

Bacterial strain identification: A total of 191 Enterococcus spp. strains were isolated during the study period. 90 out of the 191 strains were recovered from ICU samples and 101 were obtained from SW samples. Bronchial aspirates and wounds were the most common samples from the ICU (35.55%) and SW (41.58%) strains, followed by urine samples from both departments (Figure 1).

As shown in Figure 1A, the ICU strains consisted of five different species of which E. faecium (n= 48; 53.33%) was the most frequently isolated followed by E. faecalis (n=18), E. gallinarum (n= 19), E. avium (n=4), E. casseflavus (n=1). In contrast, the SW strains consisted of six different species and E. faecalis (n=47; 46.53%) was the predominant species, while the rest was composed of E. faecium (n=25), E. casseliflavus (n=11), E. gallinarum (n=9), E. hirae (n=8), and E. otavius (n=1) (Figure 1B). Interestingly, bronchial aspirates and surgical wounds from both departments were the two clinical specimens from which 38.74% of the studied strains (74/191) were collected.

Antibacterial susceptibility testing (AST): Surprisingly, the AST results revealed an interesting distribution of antibiotic resistance in this hospital. Indeed, as shown in Figure 2, in the ICU, with the exception of linezolid, E. faecium clearly appeared to be the predominant multidrug-resistant Enterococci in this ward. E. faecium exhibited pronounced resistance to the majority of antibiotics such as β-lactams, vancomycin, teicoplanin, clindamycin, erythromycin, gentamicin, and fosfomycin. 96% of these E. faecium strains were resistant to glycopeptides, 56% and 46% to rifampicin and nitrofurantoin respectively and, inversely, doxycycline, minocycline, and pristinamycin were effective on the majority of E. faecium strains. None of the E. faecium strains were resistant to linezolid (Fig. 2A). Moreover, five E. faecalis strains were found to be resistant to glycopeptides (vancomycin and teicoplanin) and 15 out of the 18 E. faecalis from this ICU were resistance to oxacillin, ceftriaxone, clindamycin, erythromycin, gentamycin, and fosfomycin (Fig. 2A). Other Enterococcus species such as E. gallinarum also exhibited a multidrug resistance phenotype in this ICU.

In contrast to the ICU, in the surgical wards, E. faecalis was the predominant MDR pathogen identified. Indeed, 45 out of the 47 E. faecalis (95.74%) strains were fully resistant to penicillin G, oxacillin, ceftriaxone, and clindamycin (Fig. 2B). Moreover, high resistance rates were observed for erythromycin (n=23; 48.93%), pristinamycin (n=33; 70.21%), and fosfomycin (n=26; 55.32%). The resistance rate to doxycycline was 34% (n=16), while none were resistant to minocycline. Interestingly, none of the identified E. faecium strains in the SWs were resistant to glycopeptides (vancomycin and teicoplanin) (Fig. 2B). However, all of the 25 (100%) E. faecium strains were resistant to β-lactams (i.e. penicillin G, oxacillin, amoxicillin, and ceftriaxone), 17 (28%) were resistant to clindamycin, 14 (56%) were resistant to fosfomycin, and 10 (40%) were resistant to rifampicin (Fig. 2B). As observed for strains from the ICU, none of the strains from the SWs were resistant to linezolid (MIC < 2 µg/mL) (Fig. 2 A andB).

Molecular analysis: Very surprisingly, the resistance level to glycopeptides (vancomycin and teicoplanin) was significantly different when comparing both departments.

It is well established that the vanA gene confers a high MIC to vancomycin and teicoplanin and that the vanB gene confers resistance only to vancomycin. Indeed, in strains from the ICU, a very high level of glycopeptide resistance (i.e., MICVAN > 256 µg/mL and MICTEC > 256 µg/mL) was observed for all resistant strains (Table 2); while this resistance level to glycopeptides in strains from the SWs ranged from 4 to 32 µg/mL, with the exception of five strains with MICVAN = 128 µg/mL (Table 3). Interestingly, these findings align perfectly with the molecular investigation of glycopeptide resistance genes, including vanA, vanB, vanC gene variants in all resistant Enterococci strains. In fact, molecular analysis targeting the van gene variants has revealed that the high resistance level observed in the ICU strains was clearly associated with the presence of the vanA gene in 70 out of the 90 strains in different species (Table 2). Its distribution in the ICU strains was as follows: E. faecium (n = 46), E. faecalis (n = 5), E. gallinarum (n= 16), E. avium (n=2) and E. casseliflavus (n =1). The simultaneous presence of vanA/vanC was identified in 24.4% (n=22 strains). In the SWs, vanB and vanC gene variants were mainly identified in strains and were associated with the observed resistance level in this ward (Table 3). In this ward, the vanC1 gene was detected in five E. faecalis and ten E. gallinarum strains and the simultaneous presence of vanC2/vanC3 was identified in ten E. casseliflavus strains. No vanA genes were detected in the SW strains (Table 3).

Discussion

Since their first detection in 1978, E. faecium vancomycin-resistant, and to some extent E. faecalis vancomycin-resistant strains have spread worldwide and are responsible for nosocomial outbreaks, substantial mortality, and additional costs to healthcare facilities every year [START_REF] Deshpande | Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe: a report from the SENTRY antimicrobial surveillance program[END_REF][START_REF] Simner | Epidemiology of vancomycin-resistant enterococci in Canadian hospitals (CANWARD study, 2007 to 2013[END_REF] . In 2017, the World Health Organization (WHO) classified vancomycin-resistant E. faecium-in a list of high priorities, requiring research and the development of new effective antibiotics [START_REF] Markwart | The rise in vancomycin-resistant enterococcus faecium in germany: Data from the german antimicrobial resistance surveillance (ars)[END_REF]. In Algeria, the Algerian Antimicrobial Resistance Network (ARN) reported a low rate of vancomycinresistant E. faecium-and vancomycin-resistant E. faecalis-(http://www.sante.dz/aarn/rapports.htm). Between 2017 and 2018, ARN reported low VRE percentages in Algeria, with 1.9% for E. faecalis and 24.4% for E. faecium. These national surveillance data are not exhaustive, however, since they do not report the genetic support of the glycopeptide resistance mechanism and concern only ten Algerian hospitals. In 2006, the first VRE strain identified was an E. faecalis strain from a urine sample taken in the Beni-Messous university hospital in Algiers [START_REF] Aggoune | Premier cas d'Enterococcus faecalis résistant à la vancomycine en Algérie[END_REF]. However, a limited number of molecular studies in Algeria to date have reported the resistance mechanism to glycopeptides.

One such study described five strains of vanA-producing E. faecium from the centre and east of country, more specifically from the university hospital in Algiers [START_REF] Hamidi | Emergence of glycopeptide resistant Enterococcus faecium in Algeria: a case report[END_REF] and the Batna university hospital [START_REF] Benammar | First molecular characterization of related cases of healthcareassociated infections involving multidrug-resistant enterococcus faecium vana in Algeria[END_REF].

Here we report, for the first time, a high rate (52.35%) of clinical VRE strains in western Algeria, including strains carrying the vanB gene. The vast majority of VREs (77.7%) were found in the ICU and only a few strains were revealed in samples taken from SWs. The ICU admits critically-ill patients for extended stays [START_REF] Cetinkaya | Vancomycin-resistant enterococci[END_REF], and as such is one of the largest wards, and largest consumers of broad-spectrum antibiotics such as glycopeptides and third-generation cephalosporins in our hospital, which explains the high rate of resistance strains associated with the presence of vanA resistance gene. Linezolid appears to be the only effective drug against all Enterococcus spp. isolated in this study and could be an exciting therapeutic option. A high rate of E. faecium was identified in the ICU compared to the SWs, where E. faecalis was the most predominant. Their distribution differs according to departments, specimen types, and geographical locations [START_REF] Kolář | Vancomycin-resistant enterococci[END_REF].

Pronounced resistance to glycopeptides is often detected in E. faecium, elucidating the high rate of VRE strains in our ICU mediated by the vanA gene.

A considerable number of E. gallinarum and E. casseliflavus strains were identified in this study, which have been sporadically found in clinical specimens and were often misidentified or underestimated in the past [START_REF] Monticelli | Clinical management of non-faecium non-faecalis vancomycin-resistant enterococci infection. Focus on Enterococcus gallinarum and Enterococcus casseliflavus/flavescens[END_REF]. Despite their low pathogenicity, these strains are increasingly incriminated in invasive infections in immunocompromised patients [START_REF] Choi | Clinical Features and Outcomes of Bacteremia Caused by Enterococcus casseliflavus and Enterococcus gallinarum: Analysis of 56 Cases[END_REF][START_REF] Reid | Clinical and Epidemiological Features of Enterococcus casseliflavus/flavescens and Enterococcus gallinarum Bacteremia: A Report of 20 Cases[END_REF], and even nosocomial outbreaks [START_REF] Contreras | Nosocomial outbreak of Enteroccocus gallinarum: untaming of rare species of enterococci[END_REF]. As reported, E. gallinarum and E. casseliflavus can acquire transferable plasmids carrying vancomycin-resistance genes such as the vanA or vanB gene [START_REF] Haenni | VanA in enterococcus faecium, enterococcus faecalis, and enterococcus casseliflavus detected in french cattle[END_REF][START_REF] Kateete | Species, antibiotic susceptibility profiles and van gene frequencies among enterococci isolated from patients at Mulago National Referral Hospital in Kampala, Uganda[END_REF]. In this study, 16 E. gallinarum and one E. casseliflavus strains harboured the vanA gene. The presence of VREs must be rapidly controlled to avoid spreading resistant strains among patients and to prevent hospital outbreaks [START_REF] Cetinkaya | Vancomycin-resistant enterococci[END_REF]. Our study reports the first E. faecalis strains carrying the vanC1 gene originating from human clinical specimens. The vanC genotype has only been revealed in small number of E. faecalis strains isolated from animal samples, including pig manure (Germany) [START_REF] Schwaiger | Presence of the resistance genes vanC1 and pbp5 in phenotypically vancomycin and ampicillin susceptible Enterococcus faecalis[END_REF], sheep milk (Spain) [START_REF] De Garnica | Presence of the vanC1 gene in a vancomycin-resistant Enterococcus faecalis strain isolated from ewe bulk tank milk[END_REF] and cloacal swabs from broilers (Brazil) [START_REF] Moura | Detection of vanC 1 gene transcription in vancomycinsusceptible Enterococcus faecalis[END_REF]. The detection of vanC1 genes in E.

faecalis and E. faecium is remarkable because this gene is intrinsic to E. gallinarum and has been used for species identification [START_REF] Schwaiger | Presence of the resistance genes vanC1 and pbp5 in phenotypically vancomycin and ampicillin susceptible Enterococcus faecalis[END_REF]. Previous reports have demonstrated that E. faecium and E. faecalis strains can acquired the vanC gene by horizontal transfer from E. gallinarum in animal guts [START_REF] Moura | Detection of vanC 1 gene transcription in vancomycinsusceptible Enterococcus faecalis[END_REF] and in aquatic environments [START_REF] Nishiyama | Identification of Enterococcus faecium and Enterococcus faecalis as vanC-type Vancomycin-Resistant Enterococci (VRE) from sewage and river water in the provincial city of Miyazaki, Japan[END_REF]. These observations underline a considerable ability that Enterococci strains have to exchange resistance genes.

As reported in the literature, vanA and vanB genes are most common encountered in human clinical specimens and global nosocomial outbreaks and, thus, must be monitored as suggested [START_REF] Freitas | Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012)[END_REF][START_REF] Hughes | An outbreak of vanA vancomycin-resistant Enterococcus faecium in a hospital with endemic vanB VRE[END_REF].

In conclusion, our findings highlight a high incidence of VREs in the intensive care unit at the Tlemcen university hospital. A range of resistance mechanisms is currently emerging in our hospitals. These changes are due to epidemiological evolution in the country.

Permanent surveillance must be ensured to avoid the spread of resistance strains. Massive testing of high-risk patients, isolation of positive cases, and appropriate disinfection have always proven to be effective in preventing and managing outbreaks.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 :

 2 Figure 2: Comparison of antimicrobial resistance of clinical Enterococcus strains from the ICU and SWs. Enterococcus spp. regrouped other Enterococcus species including E. avium (n=4), E. casseflavus (n=1), E. gallinarum (n= 19) from the ICU and E. casseliflavus (n=11), E. octavius (n=1), E. gallinarum (n=9), E. hirae (n=8) from SWs.

Figure 2 .

 2 Figure 2.

Table 2 :

 2 The distribution of vancomycin, teicoplanin MIC range, and resistance genes of isolated species from the ICU VanR. vancomycin-resistance, TecR. Teicoplanin resistance, No. number of strains, MIC minimum inhibitory concentration. The vanA, vanA-vanC1 and, vanA-vanC1-vanC2/C3 genes were detected only in Enterococci spp. isolated from the ICU and the vanB, vanC1 and, vanC2/C3 genes were detected only in Enterococci spp. isolated from SWs.

	Species name	Number of strains	No. of VanR (MIC) No. of TecR (MIC)	vanA	vanA-vanC1 vanA-vanC2/C3
	E. avium	4	2 (> 256 µg/mL)	2 (> 256 µg/mL)	2	0	0
	E. casseflavus	1	1 (> 256 µg/mL)	1 (> 256 µg/mL)	0	0	1
	E. faecalis	18	5 (> 256 µg/mL)	5 (> 256 µg/mL)	5	0	0
	E. faecium	48	46 (>256 µg/mL)	46 (>256 µg/mL)	40	6	0
	E. gallinarum	19	16 (>256 µg/mL)	16 (>256 µg/mL)	0	16	0
	Total	90	70	70	47	22	1

Table 3 :

 3 The distribution of vancomycin, teicoplanin MIC range, and resistance genes of isolated species from SWs.VanR. vancomycin-resistance, TecR. Teicoplanin resistance, No. number of strains, MIC minimum inhibitory concentration. The vanA, vanA-vanC1 and, vanA-vanC1-vanC2/C3 genes were detected only in Enterococci spp. isolated from the ICU and the vanB, vanC1 and, vanC2/C3 genes were detected only in Enterococci spp. isolated from SWs.

	Species name	No of strains	No. of VanR (MIC)	No. of TecR (MIC)	vanB	vanC1	vanC2/C3
	E. casseliflavus	11	10 (4-28 µg/mL)	10 (4-28 µg/mL)	0	0	10
	E. otavius	1	0	0	0	0	0
	E. faecalis	47	10 (4-128 µg/mL)	10 (4-32 µg/mL)	5	5	0
	E. faecium	24	0	0	0	0	0
	E. gallinarum	10	10 (4-32 µg/mL)	10 (4-32 µg/mL)	0	10	0
	E. hirae	8	0	0	0	0	0
	Total	101	30	30	5	15	10

Acknowledgements

We are very grateful to the staff of Tlemcen University Hospital for helping with sample collection and for providing information. Also, TradOnline for English proof reading of the manuscript.

Funding

This work was supported by the Algerian Ministry of Higher Education and Scientific Research and by the French Government under the "Investissements d'avenir" (reference: Méditerranée Infection 10-IAHU-03).

Ethical issues

All rules of confidentiality and ethics as prescribed in the Helsinki Declaration have been respected. Prior to data collection, the respective authorisations from the Tlemcen University Hospital's medical director and the prevention and control infections team were obtained. Data were collected anonymously.