Distribution of carbapenemase-producing and colistin resistant Acinetobacter baumannii isolates in Batna hospitals, Algeria

Abstract

Objective

The aim of this study was to investigate the distribution and genetic determinants of carbapenemase production and colistin resistance among Acinetobacter baumannii isolates recovered from three health care facilities in the city of Batna, Algeria.

Methods

A prospective study was conducted between 2021 and 2022 on 46 Acinetobacter baumannii clinical isolates, which were collected and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility testing was performed using the disk diffusion method and colistin minimum inhibitory concentrations (MICs) were determined by broth microdilution method. Carbapenemase and colistin resistance determinants were detected by qPCR.

Results

The 46 clinical isolates were mainly from the intensive care unit (52.17%) and the burns unit (17.39%). The strains were collected primarily from pus samples (34.78%) and blood samples (17.39%). Eleven strains were classified as colistin-resistant, with MICs ranging from 4 to 128 μg/mL. The bla_OXA-24 gene was detected in 63.04% of the isolates, followed by the bla_OXA-23 gene (43.47%). Nine strains were positive for both bla_OXA-23-like and bla_OXA-24-like genes. The bla_NDM gene was detected in eight isolates (17.39%), including two which co-expressed a bla_OXA-24 gene. In contrast, all strains were negative for the plasmid-mediated colistin resistance mcr-1 to mcr-5 and mcr-8.

Conclusion

Here, we report a high prevalence of carbapenemases-producing A. baumannii isolates in Batna hospitals. Notably, this study is the first to identify A. baumannii isolates co-producing OXA-24 and NDM carbapenemases and to report the first detection of colistin-resistant A. baumannii co-producing OXA-24 and OXA-23 carbapenemases from a patient in Algeria.

Introduction

Acinetobacter baumannii is nonmotile, coccobacilli, aerobic, oxidase-negative, Gram-negative bacterium [1]. These coccobacilli are among bacteria that have evolved from an occasional respiratory pathogen to a major opportunistic pathogen primarily responsible for lung diseases related to mechanical ventilation, sepsis, urinary tract infection, skin and wound infections, and meningitis [2]. Its ability to disseminate in the hospital environment and to quickly acquire resistance mechanisms has led to therapeutic impasses [2]. The World Health Organization has classified A. baumannii among the most important bacteria of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and species of the genus Enterobacter), presenting a high capacity to escape from the action of antimicrobials [3].

A. baumannii naturally harbours two types of β-lactamases, the Ambler class C β-lactamase and the class D β-lactamase type Oxacillinase (OXA-51). Alongside the non-enzymatic mechanisms [4], carbapenem resistance in A. baumannii is most often enzymatically linked to the acquisition of oxacillinase-type carbapenemases hydrolyzing carbapenems (CHDLs) such as OXA-23-like, OXA-24/40, OXA-58 and OXA-143, or metallo-β-lactamases (MBLs) including Imipenemase (IMP), VIM (Verona Imepenemase), SIM (Seoul Imepenemase), GIM (German imipenemase) and NDM (New Delhi métallo-β-lactamase), as well as Ambler class A carbapenemases including Klebsiella pneumoniae carbapenemases (KPCs) and some Guiana extended-spectrum (GES) variants [5].

Treatment of A. baumannii infections is often problematic and is further exacerbated by the dissemination of strains with increasing rates of carbapenem resistance, reaching up to 100% in some reports worldwide [6, 7].

In Algeria, carbapenem-resistant Gram negative bacteria isolates from hospitalized patients were dominated by A. baumannii, followed by Pseudomonas aeruginosa isolates [8]. Furthermore, the situation regarding strains of A. baumannii resistant to carbapenems appears to be of concern, with OXA-23, OXA-24 and NDM-1 being the most reported resistance mechanisms and appearing to be endemic in northern Algeria [9]. Despite these national and global reports of increased morbidity and mortality associated with A. baumannii, to the best of our knowledge, few published studies have focused on this bacterium in Algeria, particularly in the city of Batna. More data are needed to objectively assess the status and epidemiology of carbapenem resistance among clinical A. baumannii isolates.

In this context, this study was conducted to understand the distribution and molecular characterization of carbapenemase production and colistin resistance in A. baumannii isolates from three healthcare facilities in Batna, Algeria.

Materials and methods

Study design and area

This prospective study was carried out on A. baumannii strains collected between August 2021 and November 2022 in three health care institutions in the city of Batna, Algeria including the University Hospital (Benflis Touhami, 650 beds), the Cancer Center (Hamdiken Belkacem, 240 beds) and the Public Hospital (Haouas Salah, 140 beds). These strains were isolated from different wards, and came from various clinical samples (Fig. 1 and Table 1).

Fig. 1.

Sankey diagram illustrating the demographic features of the study population. ICU, intensive care unit; PDS, protected distal sampling; CSU, Catheter Specimen of Urine; CC, cancer center; PH, public hospital; HU, university hospital

Table 1.

Specimen related data of A. baumannii clinical isolates

Strain	Source	Sex	Age	Ward	Sample
AB1	CC	Female	75 years	ICU	PDS
AB2	CC	Female	9 years	Hematology	Pus
AB3	CC	Female	49 years	Hematology	Ascites fluid
AB4	CC	Male	6 years	ICU	Catheter
AB5	CC	Female	45 years	Hematology	Blood
AB6	UH	Male	58 years	ICU	Blood
AB7	CC	Female	50 years	Surgery	Pus
AB8	CC	Female	65 years	Hematology	Blood
AB9	UH	Male	36 years	ICU	Blood
AB10	UH	Male	9 months	ICU	Pus
AB11	UH	Male	60 years	ICU	Pus
AB12	CC	Female	66 years	ICU	Pus
AB13	UH	Male	68 years	ICU	PDS
AB14	UH	Female	59 years	Burns	Pus
AB15	UH	Female	50 years	ICU	Pus
AB16	UH	Male	2 years	ICU	PDS
AB17	CC	Male	66 years	ICU	Peritoneal fluid
AB18	CC	Male	75 years	Oncology	Urine
AB19	CC	Female	55 years	Oncology	Urine
AB20	CC	Female	75 years	ICU	Catheter
AB21	CC	Female	65 years	ICU	CSU
AB22	CC	Female	8 years	Surgery	Pus
AB23	UH	Female	78 years	ICU	PDS
AB24	Public hospital	Female	86 years	Infectious diseases	Urine
AB25	UH	Male	7 years	ICU	Pus
AB26	CC	Male	60 years	ICU	Urine
AB27	CC	Male	3 years	ICU	Pleural fluid
AB28	UH	Male	80 years	ICU	PDS
AB29	CC	Female	4 years	ICU	Blood
AB30	UH	Male	49 years	ICU	PDS
AB31	UH	Female	66 years	ICU	Blood
AB32	CC	Female	9 years	Surgery	Pleural fluid
AB33	UH	Female	50 years	Burns	Pus
AB34	CC	Male	72 years	Oncology	Catheter
AB35	CC	Male	36 years	Surgery	Peritoneal fluid
AB36	CC	Male	3 years	Oncology	Catheter
AB37	UH	Female	29 years	Burns	Pus
AB38	UH	Male	80 years	Burns	Pus
AB39	UH	Male	44 years	Burns	Pus
AB40	UH	Female	8 years	Burns	Pus
AB41	UH	Female	3 years	Burns	Pus
AB42	UH	Male	30 years	Burns	Pus
AB43	UH	Male	72 years	ICU	PDS
AB44	UH	Female	3 years	ICU	Respiratory sampling
AB45	CC	Female	78 years	Oncology	Blood
AB46	UH	Female	4 years	ICU	Blood

CC Cancer Center, UH University Hospital, ICU intensive care unit, CSU Catheter Specimen of Urine, PDS Protected Distal Sampling

Bacterial species identification

Strains were identified using the API20 NE (bioMérieux, Marcy-l’Étoile, France), and confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (MicroflexTM; Bruker Daltonic, Bremen, Germany) [10].

Antimicrobial susceptibility testing and phenotypic characterization of carbapenemases

Antimicrobial drug susceptibility was determined by the standard disc diffusion method on Mueller–Hinton agar as recommended by the Antibiogram Committee of the French Society for Microbiology (CASFM 2019) [11].

The following antibiotic discs were used: ticarcillin (75 μg), ticarcillin/clavulanic acid (20/10 μg), piperacillin (75 μg), piperacillin/tazobactam (75/10 μg), ceftazidime (30 μg), imipenem (10 μg), ciprofloxacin (5 μg), gentamicin (15 μg), amikacin (30 μg) and tobramycin (10 μg). Antibiotic susceptibility testing results were interpreted according to the Antibiogram Committee of the French Society for Microbiology (CASFM 2019) [11].

The minimum inhibitory concentrations (MICs) colistin were determined by broth microdilution method. The eventual production of carbapenemase was searched for phenotypically using the modified Carba NP test (MCNP-test), as previously described by Bakour et al. [12].

Molecular detection of β-lactamases and mobile colistin-resistance encoding genes (mcr genes)

The most clinically common carbapenemase in A. baumannii: OXA carbapenemases (bla_OXA-23, bla_OXA-24, bla_OXA-58); metallo-β-lactamases (bla_NDM, bla_VIM); and class A β-lactamases bla_KPC [13], as well as mobile colistin-resistance encoding genes mcr-1, mcr-2, mcr-3, mcr-4, mcr-5 and mcr-8 were searched for by quantitative real-time polymerase chain reaction (qPCR) amplification using specific primers, as previously described [14].

Statistical analysis

Chi-square tests with two-sided comparisons were conducted to assess the association between strain sources (CC, UH, and Public Hospital) and antibiotic resistance. Additionally, the relationship between resistance genes (OXA-23, OXA-24, NDM) and antibiotic resistance profiles was analyzed. Statistical significance was set at P < 0.05. The statistical analyses were performed using IBM SPSS Statistics software (version 26).

Results

Demographic and clinical data

From August 2021 to November 2022, a total of 46 clinical isolates were collected and identified as A. baumannii. Half of the isolates were obtained from patients hospitalized in Batna University Hospital (23/46; 50%), followed by Cancer Center (22/46; 47,82%), and the Batna Public Hospital (1/46; 2,17%). The patients'ages ranged from 9 months to 86 years, among them, 54.34% (n = 25) were female, while 45.65% (n = 21) were male. The 46 clinical isolates were primarily recovered from the medical intensive care unit (n = 24), burns ward (n = 8), oncology ward (n = 5), surgical ward (n = 4), hematology ward (n = 4), and infectious diseases ward (n = 1). In this study, a high prevalence of A. baumannii isolates was particularly observed in two wards at Batna University Hospital: the medical intensive care unit (32.60%) and the burns ward (17.39%). Additionally, at Batna Cancer Center, notable prevalence rates were recorded in the intensive care unit (19,56%), followed by the oncology ward (10.86%), the surgical ward (8.69%) and the hematology ward (8.69%). The strains were isolated from various clinical specimens, including pus (n = 16; 34.78%), blood (n = 8; 17.39%), protected distal sampling (n = 7; 15.21%), catheter (n = 4; 8.69%), urine (n = 4; 8.69%), peritoneal fluid (n = 2; 4.34%), pleural fluid (n = 2; 4,34%), respiratory sampling (n = 1; 2,17%), ascites fluid (n = 1; 2,17%) and catheter specimen of urine (n = 1; 2,17%) (Fig. 1 and Table 1). These findings revealed significant differences in demographics, underlying conditions, and risk factors for A. baumannii infections. At the UH, the majority of the strains were originated from the intensive care unit (ICU), which treats patients with severe conditions requiring intensive monitoring, followed by the burns unit, where extensive skin damage and invasive procedures increase the likelihood of bacterial colonization and nosocomial infections. In contrast, at the CC, the strains were primarily sourced from the ICU but also from the hematology, oncology, and surgical departments. These patients, often immunocompromised due to malignancies and treatments such as chemotherapy or radiotherapy are highly predisposed to opportunistic infections. Additionally, oncological surgery, involving major interventions and prolonged hospital stays, presents an added risk factor for A. baumannii contamination and spread.

Antimicrobial susceptibility and phenotypic characterization of carbapenemases

The antibiotic susceptibility testing results showed a high level of resistance to all tested antibiotics. The antibiotic resistance rates are presented in Table 2. The modified Carba NP test was positive for all imipenem-resistant isolates. The colistin MICs obtained in our study ranged from 0.5 μg/mL to 128 μg/mL. Eleven strains were classified as colistin-resistant, with minimum inhibitory concentrations ranging from 4 to 128 μg/mL. The antibiogram results and colistin MICs of the obtained isolates are presented in Fig. 2.

Table 2.

Antimicrobial resistance rates of the obtained A. baumannii isolates

Antibiotic	Resistance rate (%)
	Cancer Center (n = 22)	University Hospital (n = 23)	Public Hospital (n = 1)	Total (n = 46)
TC	100	100	100	100
TCC	100	100	100	100
PRL	100	100	100	100
TZP	100	100	100	100
CAZ	100	100	100	100
IMP	100	100	100	100
CIP	100	100	100	100
AK	100	100	100	100
GN	95.45	100	100	97.82
TOB	100	100	100	100
CT	18.18	26.08	100	23.91

TC Ticarcillin, TCC ticarcillin/clavulanic acid, PRL Piperacillin, TZP piperacillin/tazobactam, CAZ ceftazidime, IPM imipenem, CIP ciprofloxacin, GN gentamicin, AK amikacin, TOB tobramycin, CT colistin

Fig. 2.

Antibiogram results, colistin MIC and resistance mechanisms of A. baumannii isolates were analyzed and clustered using the MultiExperimentViewer 4_6_2. TC, ticarcillin; TCC, ticarcillin/clavulanate; PRL, piperacillin; TPZ, piperacillin/tazobactam; CAZ, ceftazidime; TOB, tobramycin; GN, gentamicin; AK, amikacin; IPM, imipenem; CIP, ciprofloxacin; CT, colistin

The statistical analysis results indicate no statistically significant association between strain source (CC, UH, Public Hospital) and antibiotic resistance or between resistance enzyme (OXA-24, OXA-23, NDM) and antibiotic resistance profiles. Since both p-values are greater than 0.05, the data does not support a meaningful relationship between these variables and antibiotic resistance in this dataset.

Molecular detection of β-lactamases and mcr genes

Screening for OXA-type carbapenemases revealed the presence of bla_OXA-24-like gene in 29/46 strains from the three studied hospitals, and the bla_OXA-23-like gene in 20 strains from the Cancer Centre and the University Hospital. Nine strains were positive for both bla_OXA-23-like and bla_OXA-24-like genes (six from the university hospital and three from the cancer hospital). However, the bla_OXA-58-like gene was not detected in any of our isolates. The metallo-β-lactamases type bla_NDM gene was detected in eight strains (17.39%) two isolates of which co-expressed the bla_OXA-24 gene. However, bla_VIM was not detected in any of our isolates. In contrast, all strains were negative for the tested plasmid-mediated colistin-resistance genes. The genotypic features of the obtained isolates are summarized in Fig. 2.

Discussion

Multidrug resistant A. baumannii represents a major threat in healthcare associated infections, mainly in ICUs, especially in light of widespread carbapenems-resistant A. baumannii [15]. These multidrug-resistant strains have been reported in many countries [16], not only in clinical setting, but also in the environment [17], and in the community [18].

In this study, the bla_OXA-23 gene was found in 20 imipenem-resistant A. baumannii strains (43.47%). In Algeria, OXA-23 was first identified by Mugnier et al. in 2010 among A. baumannii ST1 clone isolates [19]. Subsequently, Mesli et al. detected the bla_OXA-23 gene in 39 A. baumannii isolates and one A. nosocomialis isolate in western Algeria [20]. Additionally, Zenati et al. identified 29 environmental OXA-23-producing A. baumannii isolates in two hospitals in northern Algeria [21]. Recently, in Batna, the bla_OXA-23 was detected in only one A. baumannii isolate from wastewater [22]. Several studies have also reported its detection in Libya, Tunisia, and Morocco [23–25]. This carbapenemase is the most widely distributed worldwide [26], and is frequently associated with both endemic and epidemic forms. In Italy, OXA-23 has become the predominant carbapenemase type contributing to persistent nosocomial infections [27].

The OXA-24-like carbapenemase is widely disseminated, however, its prevalence is less than OXA-23-like [5], which is in accordance with other studies in different regions in Algeria [9, 28] and some neighbouring countries [29]. However, in the current study we noticed a large dissemination of this enzyme among the studied A. baumannii isolates with a percentage of 63.04% (29 strains), nine of which co-expressed the bla_OXA-23 gene. This spread of the bla_OXA-24-like gene was greater than the previously reported rates in Algeria. The first description in an Algerian patient was reported by Bakour et al. 2012 [30]. Since then, several reports have described the isolation of OXA-24 producers in numerous Algerian cities including Tlemcen, Oran, Annaba, Béjaïa, and Algiers [12, 20, 29]. Several published studies conducted in some Arab countries have revealed the presence of the bla_OXA-24 gene in A. baumannii. In Egypt, a study found this gene in 90% of carbapenem-resistant isolates. In Iran, the prevalence of the bla_OXA-24 gene varied between 1.6% and 36.6% [31, 32]. Although, this enzyme has been primarily reported in Europe and North America, where it is commonly associated with endemic forms. The bla_OXA-24 gene was identified in 20.5% of isolates in Europe and 29.0% in the United States [33].

The carbapenemase gene bla_OXA-58 was not detected in our study. The first evidence of an outbreak involving OXA-58 producing A. baumannii strains in Algeria was in 2008 at the University Hospital of Tlemcen [34]. Subsequently, two studies in 2012 and 2016 detected the presence of OXA-58 in Annaba [35]. This gene has long been dominant in carbapenem-resistant A. baumannii isolates in several Mediterranean countries. Since 2009, bla_OXA-58 has reportedly been replaced by bla_OXA-23 which has become the most prevalent carbapenemase-encoding gene in the Mediterranean region [36]. The substitution of OXA-23 for OXA-58 can be explained by a selective advantage associated with the higher carbapenemase activity of OXA-23 [37].

In this study, we report eight isolates (17.39%) harbouring the metallo-β-lactamase gene bla_NDM, from different clinical specimens in two hospitals (Cancer Centre and University Hospital), including two isolates co-expressing the bla_OXA-24 gene, representing the first such detected co-occurrence in Algeria.

The New Delhi metallo-β-lactamase (NDM-1) was originally detected in Enterobacterales and later identified in Acinetobacter spp. The first description of NDM among A. baumannii isolates from an Algerian patient were reported between 2011 and 2013 [38, 39]. Subsequently, several studies reported their isolation in Algeria cities including, Setif, Bejaïa, Annaba, and Algiers [28, 40, 41]. In 2016, Chaalal et al. showed for the first time that raw milk in Algeria may be contaminated with NDM-1-producing A. baumannii ST85 [42]. Furthermore, this gene was detected for the first time in Algeria in A. nosocomialis isolates from the city of Ouargla [9]. Interestingly, a recent study has reported the detection of NDM-1 producers from wastewater sampled from a public hospital in Batna in four A. baumannii isolates belonging to the widespread international clone ST2 [18]. The presence of the bla_NDM gene in Acinetobacter baumannii has also been observed in neighboring countries, particularly in Tunisia. A study conducted in Tunisia revealed NDM-producing A. baumannii strains, highlighting the spread of carbapenem resistance in the region [43]. This metallo-β-lactamase has emerged more recently and is frequently associated with epidemic forms. In the United States, 263 cases of NDM-producing A. baumannii have been reported across 21 states, with a notable proportion of sporadic cases and clusters [44].This enzyme has now spread to many countries around the world [45, 46].

Moreover, the VIM metallo-β-lactamase type was not detected in the present study in any tested strain. In Algeria, the first description of VIM was reported by Cherak et al. in 2022, among A. baumannii ST2 isolates from hospital sewage harbouring the NDM-1 and VIM-4 MBLs [17]. Recently, in Tunisia, A. baumannii strains co-producing OXA-23 and VIM-2 carbapenemases were identified [24]. This enzyme is now geographically distributed across Europe, South America, United States [47].

In our study, eleven carbapenemases-producing A. baumannii clinical isolates were identified as resistant to colistin. This represents the second report in Algeria and the first occurrence in Batna city hospitals. Among these colistin-resistant strains, four harbored OXA-24, two carried OXA-23, and five co-expressed both OXA-23 and OXA-24. The emergence of colistin-resistant A. baumannii in Algeria is particularly concerning, as colistin is currently the last available therapeutic option.

All of these strains were negative for the tested plasmid-mediated colistin-resistance genes. The absence of mcr genes suggests that the resistance was not caused by the presence of this gene but may result from known chromosomal mutations or other mechanisms. In Algeria, only one study has reported colistin-resistance among A. baumannii isolate in bronchial secretions from a 46-year-old man, hospitalized in the University Hospital Centre of Beni-Messous in Algiers, where colistin resistance was due to a single mutation in the lipide A biosynthesis encoding gene pmrB [48]. In the North African countries, a few cases of colistin resistance among A. baumannii has been described in Tunisia and Egypt [46, 49]. Colistin is considered as one of the latest therapeutic options for the treatment of multidrug-resistant A. baumannii infections and is used as rescue therapy for serious infections [50]. Published data show that the rate of colistin resistance in Southeast Asian and Eastern Mediterranean countries is higher than in other regions of the world [51]. Given the alarming situation of rising antibiotic resistance, particularly with last-resort antibiotics such as colistin, must be carefully managed. Simultaneously, there is a need to explore new therapeutic alternatives and expand the range of antibiotics available in hospitals to effectively tackle these high levels of antibiotic resistance.

This study has some limitations, including the lack of a comprehensive analysis of infection control policies and a small sample size, which may affect the generalizability of the findings. Although meropenem was not part of the primary testing panel, it could be evaluated as a complementary option and may be of interest for the potential treatment of A. baumannii infections, particularly in combination with other antibiotics. While molecular analysis was performed, a more in-depth genomic study could have provided better insights into resistance transmission. Future research with larger cohorts and detailed epidemiological investigations is needed to strengthen these findings.

Conclusions

This study showed a high prevalence of multidrug-resistant carbapenemase producing A. baumannii isolates including those resistant to colistin considered as one of the last-resort antibiotics in several hospitals in the city of Batna, Algeria. The bla_OXA-24 was the most predominant carbapenemase encoding gene followed by bla_OXA-23, the association bla_OXA-23-bla_OXA-24^, bla_NDM and the association bla_OXA-24-bla_NDM, respectively. To the best of our knowledge, here we report for the first time the detection of A. baumannii clinical isolates co-producing OXA-24 and NDM carbapenemases in Algeria. Additionally, we report the first clinical isolate of colistin-resistant A. baumannii co-producing OXA-24 and OXA-23 carbapenemases from a patient in Algeria.

These findings highlight a critical health situation in Algerian hospitals that requires the implementation of effective infection prevention and control measures, along with antimicrobial stewardship programs, which should be closely monitored.

Abbreviations

CASFM

Antibiogram Committee of the French Society for Microbiology

CHDLs

Carbapenem-Hydrolyzing Class D β-Lactamases

ESKAPE group

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae A. baumannii, Pseudomonas aeruginosa, Enterobacter

GES

Guiana Extended-Spectrum

GIM

German Imipenemase

IMP

Impenemase

KPC

Klebsiella pneumoniae Carbapenemase

MBLs

Metallo-β-lactamases

MCI

Minimum Inhibitory Concentrations

MCNP-test

Modified Carba NP test

mcr gene

Mobile colistin-resistance encoding genes

NDM

New Delhi métallo-β-lactamase

OXA

Oxacillinase

qPCR

Quantitative Real-time polymerase Chain Reaction

SIM

Seoul imipenemase

ST

Sequence Type

VIM

Verona Imipenemase

Authors’ contributions

AB, A-KL, EB and LL Conceptualization, Methodology. AB and MM Sampling Procedure. AB, ZC, EB, LL performed the experiments and analysed the data (Investigation). AB Writing-Original Draft. AB, EB, ZC, LL and J-MR Writing- Reviewing and Editing.

Funding

This research was supported by the DGRSDT of the Algerian Ministry of Higher Education and Scientific Research.

Data availability

All data supporting the findings of this study are available within the paper.

Declarations

Ethics approval and consent to participate

This study is part of Asma Bouali doctoral research titled “Acinetobacter baumannii: Etude phénotypique, génotypique et mécanismes de résistance aux antibiotiques” which has been approved by the Scientific Council of the Faculty of Science (PVN°08/2012, 18/12/2012), University of Batna, Algeria. This work was performed on bacterial isolates which were routinely collected for diagnostic purposes with anonymous patients’ data, and no additional samples were taken from patients in accordance with the institutional ethical committee requirements. To safeguard patients’ privacy and confidentiality, their medical records were anonymized and de-identified. The research team maintained no direct contact or follow-up with the patients. Consent to participate was deemed unnecessary by the Ethics Committee of Batna University Hospital Center, Algeria. The procedures used in this study are in compliance with Algerian legislation and have received approval from the ethical committee of Batna 2 University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.