Genetic background of aminoglycoside-modifying enzymes in various genetic lineages of clinical aminoglycosides-resistant E. coli and K. pneumoniae isolates in Tunisia.

AIMS: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n=61) and K. pneumoniae (n=44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated. MATERIALS AND METHODS: A total of 105 aminoglycoside-resistant E. coli (n=61) and K. pneumoniae (n=44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycosides compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes (aph(3´)-Ia, aph(3') IIa, aph(3´)-VIa, ant(2")-Ia, aac(3) IIa, aac(3)-IVa, aac(6')-Ib, rmtA, rmtB, rmtC, armA and npmA) and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by Multilocus Sequence Typing (MLST) for representative isolates. RESULTS: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%) and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%) followed by aac(6')-Ib (36.2%) and aph(3')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and the blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by one and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae ST11/ST13). CONCLUSION: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse ß-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.

Unveiling the emergence of SARS-CoV-2 JN.1 sub-variant: Insights from the first cases at Charles Nicolle Hospital, Tunisia.

The JN.1 sub-variant is a new variant of the SARS-CoV-2 Omicron strain, derived from the BA.2.86 sub-variant. It was first detected in late 2023 and has quickly spread to many countries, becoming the most prevalent variant in some regions. JN.1 exhibits a unique mutation (L455S) in the spike protein compared to the BA.2.86 lineage, which may affect its transmissibility and immune evasion capabilities. JN.1 has been designated as a "variant of interest" by the World Health Organization due to its rapidly increasing spread and is being closely monitored for its impact on the COVID-19 pandemic. This study describes the emergence of SARS-CoV-2 JN.1 sub-variant in Tunisia, and reports its mutation profiles.Nasopharyngeal samples collected over a four-month period (October 2023 to January 2024) were subjected to RNA extraction and real-time RT-PCR confirmation of SARS-CoV-2 infection. The whole-genome sequencing was performed by an iSeq 100 sequencer and COVIDSeq kit reagents (Illumina, USA).Mutation analysis, using the NextClade platform and GISAID database, revealed the presence of JN.1 in 15 out of 80 positive cases (18.75%) during the study period.The emergence of JN.1 highlights the ongoing evolution of SARS-CoV-2 and the need for continued surveillance and research to better understand the characteristics and impact of emerging variants.

Genomic surveillance of SARS-CoV-2 in North Africa: 4 years of GISAID data sharing.

Objectives: This study aimed to construct geographically, temporally, and epidemiologically representative data sets for SARS-CoV-2 in North Africa, focusing on Variants of Concern (VOCs), Variants of Interest (VOIs), and Variants Under Monitoring (VUMs). Methods: SARS-CoV-2 genomic sequences and metadata from the EpiCoV database via the Global Initiative on Sharing All Influenza Data platform were analyzed. Data analysis included cases, deaths, demographics, patient status, sequencing technologies, and variant analysis. Results: A comprehensive analysis of 10,783 viral genomic sequences from six North African countries revealed notable insights. SARS-CoV-2 sampling methods lack standardization, with a majority of countries lacking clear strategies. Over 59% of analyzed genomes lack essential clinical and demographic metadata, including patient age, sex, underlying health conditions, and clinical outcomes, which are essential for comprehensive genomic analysis and epidemiological studies, as submitted to the Global Initiative on Sharing All Influenza Data. Morocco reported the highest number of confirmed COVID-19 cases (1,272,490), whereas Tunisia leads in reported deaths (29,341), emphasizing regional variations in the pandemic's impact. The GRA clade emerged as predominant in North African countries. The lineage analysis showcased a diversity of 190 lineages in Egypt, 26 in Libya, 121 in Tunisia, 90 in Algeria, 146 in Morocco, and 10 in Mauritania. The temporal dynamics of SARS-CoV-2 variants revealed distinct waves driven by different variants. Conclusions: This study contributes valuable insights into the genomic landscape of SARS-CoV-2 in North Africa, highlighting the importance of genomic surveillance in understanding viral dynamics and informing public health strategies.

Evaluation of Three Carbapenemase-Phenotypic Detection Methods and Emergence of Diverse VIM and GES Variants among Pseudomonas aeruginosa Isolates in Tunisia.

BACKGROUND: Since 2012, few reports on the molecular epidemiology of Pseudomonas aeruginosa were reported in Tunisia. OBJECTIVES: This study aimed to evaluate carbapenem-resistance determinants and molecular epidemiology and to compare the carbapenemase-phenotypic detection methods of multidrug-resistant P. aeruginosa isolates. METHODS: During a period of four years (2014 to 2017), all imipenem-ceftazidime-resistant P. aeruginosa isolates were retrospectively selected at the microbial laboratory of Charles Nicolle hospital of Tunis. These isolates were examined by the modified Hodge test, modified carbapenem inactivation method (mCIM), and another mCIM, called CIMTris, and their performance was evaluated using PCR analysis as the gold standard. RESULTS: A total of 35 isolates were recovered among patients hospitalized in different units. All strains were colistin-susceptible.All carbapenem-resistant isolates showed a high-level resistance to carbapenems. CIMTris and mCIM showed 96.15% and 46.15% sensitivity and 44.44% and 100% specificity, respectively, for detecting carbapenemase production. CONCLUSIONS: CIMTris is a promising approach for detecting carbapenemase activity in P. aeruginosa and merits further testing. Moreover, this study described the first detection of GES-5- and GES-9-producing P. aeruginosa in Tunisia as well as the co-occurrence of the blaGES-5 and blaVIM-11 carbapenemase genes in one isolate. These findings are of great concern because the rapid dissemination of MDR strains represents a major therapeutic and epidemiological threat.

Extensively drug-resistant Acinetobacter baumannii co-producing VIM-2 and OXA-23 in intensive care units: Results of a one-day point prevalence in a Tunisian hospital.

OBJECTIVE: The main objective of this study was to identify carbapenem resistance mechanisms among clinical, commensal and environmental carbapenem-resistant Acinetobacter baumannii (CRAB) strains isolated in 5 Tunisian intensive care units (ICUs). MATERIALS AND METHODS: CRAB isolates were recovered from different sources: clinical specimens, rectal and environmental swabs. Bacterial identification was carried out using conventional methods and susceptibility testing according to EUCAST recommendations. Evaluation of phenotypic carbapenemase production of was performed using seven different methods, and molecular detection of carbapenemase-coding genes (blaOXA23, blaOXA24, blaOXA58, blaNDM, blaGES, blaOXA48, blaIMP, blaVIM and blaKPC) was done by PCR. The genetic relationships between CRAB isolates were analyzed by pulsed-field gel electrophoresis. RESULTS: All in all, 46 CRAB isolates were identified in clinical specimens (n = 26/26), rectal swabs (n = 17/36) and environmental swabs (n = 3/63). Most of them (n = 41/46) were clonally related and found in the different ICUs. All of the CARB isolates harbored blaOXA-51-like and blaOXA-23 genes, while blaVIM-2 gene was detected in 42 isolates (91.3 %). Phenotypic carbapenemase activity was found in all 46 strains, using the CIM-Tris test with 100 % sensitivity for OXA-23 and VIM-carbapenemase detection, thereby justifying its routine use. CONCLUSION: The emergence and diffusion of clonal CRAB strains inducing high mortality rates in ICUs is a major public health concern. Enhanced infection control practices and mandatory staff education are needed to control the spread of these multidrug-resistant bacteria.

Tunisian Multicenter Study on the Prevalence of Colistin Resistance in Clinical Isolates of Gram Negative Bacilli: Emergence of Escherichia coli Harbouring the mcr-1 Gene.

BACKGROUND: Actually, no data on the prevalence of plasmid colistin resistance in Tunisia are available among clinical bacteria. OBJECTIVES: This study aimed to investigate the current epidemiology of colistin resistance and the spread of the mcr gene in clinical Gram-negative bacteria (GNB) isolated from six Tunisian university hospitals. METHODS: A total of 836 GNB strains were inoculated on COL-R agar plates with selective screening agar for the isolation of GNB resistant to colistin. For the selected isolates, mcr genes, beta-lactamases associated-resistance genes and molecular characterisation were screened by PCRs and sequencing. RESULTS: Colistin-resistance was detected in 5.02% (42/836) of the isolates and colistin-resistant isolates harboured an ESBL (blaCTX-M-15) and/or a carbapenemase (blaOXA-48, blaVIM) encoding gene in 45.2% of the cases. The mcr-1 gene was detected in four E. coli isolates (0.59%) causing urinary tract infections and all these isolates also contained the blaTEM-1 gene. The blaCTX-M-15 gene was detected in three isolates that also carried the IncY and IncFIB replicons. The genetic environment surrounding the mcr-carrying plasmid indicated the presence of pap-2 gene upstream mcr-1 resistance marker with unusual missing of ISApl1 insertion sequence. THE CONCLUSIONS: This study reports the first description of the mcr-1 gene among clinical E. coli isolates in Tunisia and provides an incentive to conduct routine colistin susceptibility testing in GNB clinical isolates.

Phylogenetic and amino acid signature analysis of the SARS-CoV-2s lineages circulating in Tunisia.

Since the beginning of the Coronavirus disease-2019 pandemic, there has been a growing interest in exploring SARS-CoV-2 genetic variation to understand the origin and spread of the pandemic, improve diagnostic methods and develop the appropriate vaccines. The objective of this study was to identify the SARS-CoV-2s lineages circulating in Tunisia and to explore their amino acid signature in order to follow their genome dynamics. Whole genome sequencing and genetic analyses of fifty-eight SARS-CoV-2 samples collected during one-year between March 2020 and March 2021 from the National Influenza Center were performed using three sampling strategies.. Multiple lineage introductions were noted during the initial phase of the pandemic, including B.4, B.1.1, B.1.428.2, B.1.540 and B.1.1.189. Subsequently, lineages B1.160 (24.2%) and B1.177 (22.4%) were dominant throughout the year. The Alpha variant (B.1.1.7 lineage) was identified in February 2021 and firstly observed in the center of our country. In addition, A clear diversity of lineages was observed in the North of the country. A total of 335 mutations including 10 deletions were found. The SARS-CoV-2 proteins ORF1ab, Spike, ORF3a, and Nucleocapsid were observed as mutation hotspots with a mutation frequency exceeding 20%. The 2 most frequent mutations, D614G in S protein and P314L in Nsp12 appeared simultaneously and are often associated with increased viral infectivity. Interestingly, deletions in coding regions causing consequent deletions of amino acids and frame shifts were identified in NSP3, NSP6, S, E, ORF7a, ORF8 and N proteins. These findings contribute to define the COVID-19 outbreak in Tunisia. Despite the country's limited resources, surveillance of SARS-CoV-2 genomic variation should be continued to control the occurrence of new variants.

The Delta variant wave in Tunisia: Genetic diversity, spatio-temporal distribution and evidence of the spread of a divergent AY.122 sub-lineage.

Introduction: The Delta variant posed an increased risk to global public health and rapidly replaced the pre-existent variants worldwide. In this study, the genetic diversity and the spatio-temporal dynamics of 662 SARS-CoV2 genomes obtained during the Delta wave across Tunisia were investigated. Methods: Viral whole genome and partial S-segment sequencing was performed using Illumina and Sanger platforms, respectively and lineage assignemnt was assessed using Pangolin version 1.2.4 and scorpio version 3.4.X. Phylogenetic and phylogeographic analyses were achieved using IQ-Tree and Beast programs. Results: The age distribution of the infected cases showed a large peak between 25 to 50 years. Twelve Delta sub-lineages were detected nation-wide with AY.122 being the predominant variant representing 94.6% of sequences. AY.122 sequences were highly related and shared the amino-acid change ORF1a:A498V, the synonymous mutations 2746T>C, 3037C>T, 8986C>T, 11332A>G in ORF1a and 23683C>T in the S gene with respect to the Wuhan reference genome (NC_045512.2). Spatio-temporal analysis indicates that the larger cities of Nabeul, Tunis and Kairouan constituted epicenters for the AY.122 sub-lineage and subsequent dispersion to the rest of the country. Discussion: This study adds more knowledge about the Delta variant and sub-variants distribution worldwide by documenting genomic and epidemiological data from Tunisia, a North African region. Such results may be helpful to the understanding of future COVID-19 waves and variants.

First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia.

Environmental bacteria belonging to various families were isolated from polluted water collected from ten different sites in Tunisia. Sites were chosen near industrial and urban areas known for their high degree of pollution. The aim of this study was to investigate cross-resistance between heavy metals (HM), i.e., silver, mercury and copper (Ag, Hg, and Cu), and antibiotics. In an initial screening, 80 isolates were selected on ampicillin, and 39 isolates, retained for further analysis, could grow on a Tris-buffered mineral medium with gluconate as carbon source. Isolates were identified based on their 16S rRNA gene sequence. Results showed the prevalence of antibiotic resistance genes, especially all isolates harbored the bla TEM gene. Some of them (15.38%) harbored bla SHV. Moreover, several were even ESBLs and MBLs-producers, which can threaten the human health. On the other hand, 92.30%, 56.41%, and 51.28% of the isolates harbored the heavy metals resistance genes silE, cusA, and merA, respectively. These genes confer resistance to silver, copper, and mercury. A cross-resistance between antibiotics and heavy metals was detected in 97.43% of our isolates.

Occurrence of plasmid-mediated quinolone resistance determinants among Escherichia coli strains isolated from animals in Tunisia: Specific pathovars acquired qnr genes.

OBJECTIVES: The aim of this study was to characterise Escherichia coli strains harbouring plasmid-mediated quinolone resistance (PMQR) genes recovered from various samples (n = 116) from healthy and diarrhoeic animals in Tunisia. METHODS: All nalidixic acid-resistant E. coli isolates were screened for the presence of PMQR genes. Isolates positive for PMQR genes were investigated by PCR for chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC, the presence of class 1 and class 2 integrons, genes encoding tetracycline and sulfonamide resistance, genes encoding virulence factors, and phylogenetic group. Genetic relationships was determined by pulsed-field gel electrophoresis (PFGE). RESULTS: Amongst 51 nalidixic acid-resistant isolates, 9 harboured PMQR genes (5 co-harbouredqnrS1 and qnrB1, 3 harboured qnrS1 and 1 harboured qnrB1). Two types of mutation in the QRDR of GyrA were observed: S83L and D87N (eight isolates) and S83L (one isolate). For the QRDR of ParC, the substitution S80I was observed in four isolates. A class 1 integron was found in six isolates. The tetA or tetB gene was observed in six isolates and both tetA and tetB were co-harboured by two isolates. The sul1, sul2 and sul3 genes were detected in six, four and one isolates, respectively. According to the presence of specific virulence genes, the nine strains were classified as UPEC (5), EAEC (3) and EPEC (1). Three isolates from turkey faeces were clonally related by PFGE. CONCLUSION: These findings highlight the plausible role of the avian industry as a reservoir of human pathogenic E. coli strains.

Assessment of sample pooling for SARS-CoV-2 molecular testing for screening of asymptomatic persons in Tunisia.

The aim of this study is to test a pooling approach for the RT-PCR test to detect low viral loads of SARS-CoV-2. We found that a single positive specimen can still be detected in pools of up to 10. Each laboratory should conduct its own evaluation and validation of pooling protocols according to its specific context.

Escherichia coli colonizing healthy children in Tunisia: High prevalence of extra-intestinal pathovar and occurrence of non-extended-spectrum-ß-lactamase-producing ST131 clone.

This study was performed to investigate the distribution of antimicrobial resistance genes and extra-intestinal virulence determinants in a collection of 98 Escherichia coli strains isolated from rectal swabs of healthy children. Forty-six isolated strains were resistant to at least one of the tested antibiotics (usually active against enterobacteria). They were mainly resistant to ampicillin and ticarcillin (42.97%), tetracyclin (26.5%), and trimethoprim/sulfamethoxazole (18.4%). No resistance to the third generation of cephalosporins, carbapenems, aminoglycosides and colistin was found. Resistance to penicillins was encoded by blaTEM-1 (n=34) and blaSHV-1 genes (n=4). Tetracyclin resistance was encoded by tetB (n=12), tetA (n= 5), and tetC (n=1) genes. Amongst resistant quinolones isolated (n=5), chromosomal mutations in gyrA and parC genes were detected in four isolates and qnrS1 gene in two strains. Nine plasmid replicon types were detected; IncFIB (n=36) and IncI1 (n=7) were the most frequent ones. Isolates frequently belonged to phylogenetic groups A (51.1%) and D (27.5%). Extra-intestinal pathovar (n=38) occurred mainly in B2 phylogroup (P=0.0002). Amongst them, two isolates (non-extended-spectrum-ß-lactamase (ESBL)-producers) belonged to the pandemic clone ST131. A significant distribution of virulence determinants and pathogenicity island marker was observed within strains belonging to B2 and D phylogroups. Interestingly, our results showed that ExPEC strains, including ST131 pandemic clone, are present within fecal isolates in healthy children. These findings highlight the importance of intestinal microbiota as a reservoir for virulent and resistant strains. Thus, reinforcing hand hygiene and antibiotic rational use is imperative to avoid the diffusion of these pathogens in the community.

Emergence of plasmid-mediated colistin-resistance in CMY-2-producing Escherichia coli of lineage ST2197 in a Tunisian poultry farm.

Our study aimed to investigate colistin resistance and the mechanisms involved in a collection of 35 extended-spectrum beta-lactamase (ESBL) and 13 CMY-2-producing E. coli strains which were previously recovered from chicken gut microbiota in Tunisia, as well as to determine the genetic location of mcr genes. Forty-eight ESBL and CMY-2-producing E. coli strains were obtained from 137 fecal samples of healthy chickens during 2013. These strains were tested for colistin resistance by the broth microdilution method, and screened for mcr-1 and mcr-2 genes by PCR. Two of these strains were colistin-resistant (MIC = 8 mg/L). Both harbored the mcr-1 gene, were CMY-2 producers, and were additionally resistant to tetracycline, ciprofloxacin, chloramphenicol, gentamicin, tobramycin and trimethoprim-sulfamethoxazole. They shared phylogroup A, the same pulsed-field gel electrophoresis (PFGE)-pattern, and were typed as ST2197. In both strains, ISApl1 and pap2 were detected upstream and downstream of mcr-1 gene, respectively. The analysis of the two mcr-1-positive strains and their transconjugants by PCR-based replicon typing and S1-PFGE, demonstrated that mcr-1 gene is linked to an IncP plasmid (~242 kb), and blaCMY-2 to an IncI1 plasmid (97 kb). The occurrence of E. coli harboring mcr-1 gene among intestinal microbiota in poultry and its location on a conjugative plasmid could represent a risk for public health. The evolution of this type of resistant microorganisms should be evaluated in the future.

First report of extensively-drug-resistant Proteus mirabilis isolate carrying plasmid-mediated blaNDM-1 in a Tunisian intensive care unit.

Emergence of the New Delhi metallo-ß-lactamase (NDM-1), an Ambler class B metallo-ß-lactamase able to hydrolyse all ß-lactams except monobactams, constitutes a critical and increasingly important medical issue. The acquisition of blaNDM-1 is of particular concern for Proteus mirabilis, which is intrinsically resistant to tetracycline, tigecycline and colistin, as this will make clinical treatment extremely difficult. To the authors' knowledge, this is the first report of the blaNDM-1 gene in an extensively-drug-resistant P. mirabilis clinical isolate carrying plasmid-mediated resistance to carbapenems (blaNDM-1), cephalosporins (blaCMY-4), aminoglycosides (aph3 VIa and aph3 Ia) and fluoroquinolones (qnrA6).

Role of association of OmpK35 and OmpK36 alteration and blaESBL and/or blaAmpC genes in conferring carbapenem resistance among non-carbapenemase-producing Klebsiella pneumoniae.

In Klebsiella pneumoniae, loss of the two major outer membrane porins (OMPs) OmpK35 and OmpK36 confers resistance to carbapenems in strains producing extended-spectrum ß-lactamases (ESBLs) or plasmid-mediated AmpC-type ß-lactamases. This study investigated mechanisms responsible for carbapenem resistance in non-carbapenemase-producing K. pneumoniae (NCPK). All carbapenem-resistant Enterobacteriaceae (CRE) at Charles Nicolle Hospital (Tunis, Tunisia) were collected over a 6-year period (2010-2015). Among the 334 CRE strains collected, 44 (13.2%) were NCPK. MIC ranges for ertapenem, imipenem and meropenem were 1 to >32 mg/L, 0.125-8 mg/L and 0.125-32 mg/L, respectively. All strains showed a multidrug-resistant (MDR) phenotype and were negative for carbapenemase activity. None of the carbapenemase genes searched for were found. ESBL production was confirmed in all isolates except one [CTX-M-15 (n = 39) and SHV-5 (n = 4)]. Three isolates produce DHA-1 (associated with CTX-M-15 in two strains). Molecular fingerprints grouped the 44 NCPK isolates into seven clusters. In seven representative strains of these clusters, SDS-PAGE results showed that four isolates lacked the OmpK35 porin, one isolate lacked OmpK36 and two isolates lacked both OmpK35 and OmpK36. Sequencing of the corresponding porin genes showed amino acid insertions and deletions leading to early termination of translation, point mutations in the promoter region, or insertion sequences disrupting the gene coding sequence. Loss or deficiency of OMPs, coupled with ESBL and/or AmpC production, plays an important role in conferring carbapenem resistance in K. pneumoniae. Dissemination of these MDR bacteria in our hospital may create serious therapeutic problems in the future.

NDM-1- and OXA-23-producing Acinetobacter baumannii isolated from intensive care unit patients in Tunisia.

Gastrointestinal colonisation by carbapenem-resistant Acinetobacter baumannii (CRAB) is a critical step before nosocomial infection. This study evaluated CRAB intestinal carriage in patients admitted to a Tunisian ICU and determined the antimicrobial resistance mechanisms involved. From December 2014 to February 2015, all 63 patients admitted to the ICU were screened for rectal CRAB colonisation upon admission and once weekly thereafter. ICU patients who acquired a CRAB nosocomial infection were also included. ß-Lactamases and associated resistance genes were screened by PCR sequencing, and molecular typing was performed by PFGE and MLST. The CRAB faecal carriage rate at admission was 4.8% (3/63). The CRAB acquisition rate during ICU stay was analysed in 39 of the remaining 60 patients and the rate of acquired CRAB faecal carriage was 15.4% (6/39); 4 patients also showed an ICU-acquired CRAB infection (one patient was a faecal carrier and suffered infection). Overall, 13 CRAB isolates were collected from 12 patients, of which 11 isolates showed resistance to all antibiotics tested except colistin. blaOXA-23 and blaNDM-1 were detected in 11 and 2 isolates, respectively. All OXA-23-producing strains carried armA, tetB, sul1 and catB, and some of them carried aph(3')-VIa, blaTEM-1, aph(3')-Ia and ant(2'')-Ia. The blaNDM-1-positive isolates harboured aph(3')-VIa and catB. Three PFGE patterns and two STs were identified [ST195 (n = 11), ST1089 (n = 2, NDM-1-positive)]. Whether imported or acquired during ICU stay, CRAB colonisation is a major risk factor for the occurrence of serious nosocomial infection. Systematic screening of faecal carriage is mandatory to prevent their spread.

An Outbreak of NDM-1-Producing Klebsiella pneumoniae, Associated with OmpK35 and OmpK36 Porin Loss in Tunisia.

OBJECTIVES: To describe clinical and molecular characteristics of an outbreak due to metallo-ß-lactamases (MBLs) producing Klebsiella pneumoniae collected at Charles Nicolle Hospital of Tunis and to analyze the impact of outer membrane porin (OMP) loss on carbapenem resistance levels. METHODS: Between 2010 and 2015, 178 carbapenem-resistant Enterobacteriaceae were isolated. Screening for MBL production was performed using combined disk diffusion method, with imipenem and ethylene diamine tetraacetic acid (EDTA) as inhibitors. Resistance genes and virulence factors were identified by polymerase chain reaction (PCR) and sequencing. Genotyping was performed by pulsed-field gel electrophoresis and multilocus sequence typing. Genetic environment of carbapenemase genes was determined by PCR mapping. Conjugation assays were performed, and plasmids were assigned to incompatibility groups by PCR-based replicon typing. OMPs were profiled by sodium dodecyl sulfate-polyacrilamide gel electrophoresis, and porin genes were sequenced. RESULTS: Nineteen K. pneumoniae (10.6%) showing MBL activity were isolated from patients hospitalized on four different wards. NDM-1 was the only MBL identified, in association with blaOXA-48. All strains lacked at least one OMP, and carbapenem resistance levels were remarkably elevated in strains lacking OmpK35 and OmpK36. blaNDM-1 was located in IncFIA-type conjugative plasmid, with the same genetic context in all strains. The epidemiological diffusion of blaNDM-1 was due to two clones, one major clone belonging to sequence type (ST) 147 (n = 16) and the other clone belonging to ST307 (n = 3). CONCLUSIONS: This study describes an outbreak of NDM-1-producing K. pneumoniae strains, isolated from a Tunisian hospital, caused by two clones belonging to ST147 and ST307; and highlights the role of OMPs loss, in combination with ß-lactamase expression, in conferring high carbapenem resistance.

Diverse Escherichia coli pathovars of phylogroups B2 and D isolated from animals in Tunisia.

INTRODUCTION: The virulent Escherichia coli strains responsible for extraintestinal infections were mainly belonged to B2 and D phylogroups. However, no past studies have determinate via the presence of virulence genes the frequency of E. coli pathovars recovered from animals housed in farms in Tunisia. The aims of this study were to investigate 26 E. coli isolated from healthy and diarrheic animals and to determinate via the presence of virulence genes the frequency of pathovars. METHODOLOGY: Twenty-six E. coli isolates of phylogroups B2 (n = 14), B22 (n = 9), B23 (n = 5), and D2 (n = 12) were characterized. Genes encoding virulence factors (fimH,eaeA,aggC,papC, papG allele III, hlyA, east1, cnf1, exhA,stx1, stx2, iutA, fyuA, ibeA,and ipaH), and antibiotic resistance as well as class 1 and 2 integrons were searched by polymerase chain reaction (PCR). The genetic relationship of isolates was done by PFGE. RESULTS: According to the occurrence of specific genes the 26 isolates were classified as:9 EAEC, 2 EHEC, 4 UPEC, 3 EPEC/EHEC and 1 NTEC. Therefore, 2 Ex-PEC and 5 APEC were presented amongst our strains. Some isolates (12) were clonal and the remaining was unrelated. CONCLUSIONS: Higher diversity of pathovars which carried diverse combinations of virulence genes in healthy isolates. In addition, it seems that the infections were caused by different mechanisms.

Community fecal carriage of broad-spectrum cephalosporin-resistant Escherichia coli in Tunisian children.

The spread of extended spectrum ß-lactamases (ESBL) and plasmid mediated AmpC ß-lactamases (pAmpC) was evaluated in Escherichia coli strains collected from the intestinal microbiota of healthy children in Tunisia. The carriage rate of CTXRE. coli was 6.6% (7 of 105 samples) and one strain/sample was further characterized (7 isolates). These isolates harbored blaCTX-M-1 (n = 4), blaCTX-M-15 (n = 2), and blaCMY-2 gene (n = 1), which were usually located on FIB replicon type and carried class 1 integrons. The acc(6')-Ib-cr variant was identified in one isolate that harbored blaCTX-M-15. CTXRE. coli isolates were genetically unrelated and belonged to B1 (n = 3/ST155/ST398/ST58), D (n = 2/ST117/ST493), B2 (n = 1/ST127), and A (n = 1/ST746) phylogroups. Strain virulence scores varied from 3 to 12, and frequently harbored the pathogenicity island PAI IV536. The intestinal tract of healthy children constitute an important reservoir of ESBL producing E. coli. Thus, improvement of hygiene measures mainly in the school environment and rational use of antibiotics would be of great help in preventing selection and diffusion of resistant strains from intestinal microbiota.

High prevalence of extended-spectrum and plasmidic AmpC beta-lactamase-producing Escherichia coli from poultry in Tunisia.

This study was conducted to detect extended spectrum beta-lactamases (ESBLs) and plasmidic AmpC beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates in industrial poultry samples were collected from healthy chickens of the three farms. Samples were inoculated onto desoxycholate-lactose-agar plates supplemented with cefotaxime (2mg/L). E. coli was identified by biochemical and molecular methods and antibiotic susceptibility testing by the disk diffusion method. Genes encoding ESBLs and pAmpC-BL were detected by PCR and sequencing. Phylogenetic groups were determined by triplex PCR. The molecular typing of strains was done by pulsed field gel electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) in those isolates showing different PFGE patterns. Cefotaxime-resistant E. coli isolates were recovered in 48 of 137 fecal samples (35%), and one isolate/sample was further studied. The following beta-lactamase genes were detected: blaCTX-M-1 (29 isolates, isolated in all three farms), blaCTX-M-15 (5 isolates, confined in farm II), blaCTX-M-14 and blaCMY-2 (one isolate and 13 isolates, respectively, in farm III). The 48 cefotaxime-resistant isolates were distributed into phylogroups: B1 (n=21), A (n=15) and D (n=12). PFGE analysis revealed 19 unrelated patterns: 15 different profiles among ESBL-positive strains and 4 among the CMY-2-positive isolates. The following sequence types-associated phylogroups were detected: a) CTX-M-1-positive strains: lineages ST542-B1, ST212-B1, ST58-B1, ST155-B1 and ST349-D; b) CTX-M-15-positive strain: lineage ST405-D; c) CTX-M-14-positive strain: lineage ST1056-B1; d) CMY-2-positive strains: lineages ST117-D, ST2197-A, and ST155-B1. Healthy chickens constitute an important reservoir of ESBL- and pAmpC-BL-producing E. coli isolates that potentially could be transmitted to humans via the food chain or by direct contact.