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Objectives: The whole-genome sequence (WGS) of Klebsiella pneumoniae KP3771 isolate was characterized. This strain was recovered from the urine sample of an 80-year-old man hospitalized in an intensive care unit of the University Hospital Tahar Sfar in Tunisia. Materials and Methods: WGS using a MiSeq platform was used. The assembled genome was subjected to several software analyses. Results: K. pneumoniae KP3771 was resistant to all antibiotics but colistin and tigecycline. WGS analysis found 18 transmissible genes encoding resistance markers, including bla NDM-1 and bla CTX-M-15 genes, which were carried by four plasmids belonging to the Inc Ib, IIk, and R groups. Three families of genes encoding virulence factors were detected, including adhesins ( fimH , fimA , fimB , fimC , mrkD , Kpn , and ycfM ), siderophores (enterobactin, aerobactin, and yersiniabactin siderophores), and protectin/invasin ( traT ). The strain was assigned to the sequence type 147. Conclusions: This study describes the genome of a carbapenemase-producing K. pneumoniae clinical isolate recovered in Tunisia. Bacteria WGS has become the reference technology to address epidemiological issues; this high level of information is particularly well suited to enrich epidemiological workflows' output.

Background: New Delhi metallo-β-lactamase (NDM)-producing Acinetobacter baumannii have been described in several countries worldwide, and studies have suggested that Acinetobacter spp. could play the role of intermediate progenitor of the bla NDM-1 gene between environmental progenitor and Enterobacteriaceae., Materials and Methods: In total, 246 carbapenem-resistant A. baumannii isolates from a teaching hospital in Sousse, Tunisia were investigated between 1 st June 2013 and 31 st December 2015 to detect metallo-ß-lactamase (MBL) production. Polymerase chain reaction (PCR), antibiotic susceptibility testing, and genetic and whole-genome sequencing tools were used to study the underlying carbapenem resistance mechanisms., Results: PCR screening of the 246 carbapenem-resistant A. baumannii isolates revealed that 242 of 246 isolates harboured carbapenemase genes (seven of 246 positive for bla NDM-1 , four of 246 positive for bla NDM-1 and bla OXA-23 , 231 positive for bla OXA-23 ). Conjugation and electroporation experiments suggested that the bla NDM-1 gene is likely to be chromosomally located. All the NDM-1-producing A. baumannii isolates were clonally related, and belonged to ST85 according to the Pasteur Institute's multi-locus sequence typing scheme. Analysis of the immediate genetic environment of the bla NDM-1 gene revealed that the gene was located within a truncated isoform of Tn125 transposon (ΔTn125). The bla OXA-23 gene was located within transposon Tn2008., Conclusion: This study showed the dissemination of a single clone of NDM-1-producing A. baumannii in a Tunisian hospital. Countries in north Africa may constitute a significant reservoir for NDM-1-producing A. baumannii. The spread of the bla NDM-1 gene in A. baumannii was linked to clonal spread in this study., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study was conducted to investigate extended-spectrum-β-lactamase (ESBL) producing Enterobacteriaceae isolates from the Center of Maternity and Neonatology of Monastir, Tunisia. Fourty-six strains out of 283 were found to produce ESBL: Klebsiella pneumoniae (n = 37), Escherichia coli (n = 6), Enterobacter cloacae (n = 2), and Citrobacter freundi (n = 1). Genotyping analysis, using ERIC2 and RAPD, showed that strains were clonally unrelated. PCR amplification followed by sequencing revealed that all strains produced CTX-M-15. This enzyme was co-produced with TEM and SHV determinants in 34 and 36 strains respectively. The bla CTXM-15 gene was bracked by ISEcp1 and/or IS26 in 42 out of the 46 ESBL positive strains. The quinolone resistance determinants were associated to the ESBL producing isolates: we identified the qnrB1 gene in six isolates and the aac(6')-Ib-cr gene in five isolates. This epidemiological study shows the widespread of CTX-M-15 and qnr determinants among enterobacterial isolates from neonates hospitalized at the center of Maternity and Neonatology of Monastir suggesting either mother portage or horizontal transmission., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study was conducted to identify the molecular mechanisms of imipenem resistance in a Klebsiella pneumoniae (Kp16137) isolate recovered in August 2008 at the University Hospital Sahloul, Sousse, Tunisia. The strain was identified with the API 20E system; antibiotic-containing disks were used for detection of antibiotic susceptibility by a disk diffusion assay. We investigated the presence of β-lactamases by PCR, using specific primers for bla(TEM), bla(SHV), bla(CTX-M), bla(OXA), bla(CMY), bla(ACC), bla(FOX), bla(IMP), bla(KPC), bla(VIM), and by sequencing. Extraction of plasmid DNA from Kp16137 and the transconjugant was performed by the method of Kado. Southern transfer was performed on nylon. The membrane was hybridized with a specific probe for the bla(CMY-2) gene. Outer membrane proteins were isolated and were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% polyacrylamide gel. K. pneumoniae Kp16137 was resistant to all available β-lactams, including third generation cephalosporins and carbapenems. The screening of β-lactamases showed the presence of three β-lactamases: TEM-1, SHV-61, and CMY-4. The CMY-4 β-lactamase was located on an 80-kb plasmid. An analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of 42 kDa. The loss of this outer membrane protein band correlated with imipenem resistance in this strain. In K. pneumoniae 16137, synthesis of a plasmid-mediated β-lactamase: AmpC CMY-4, together with alteration in permeability led to resistance to all available β-lactams and carbapenems.

One hundred extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae were recovered from the intensive care unit and the urology ward of the University Hospital of Sahloul in Tunisia between May 2005 and May 2006. The majority of strains showed a high level of resistance to cefotaxime and ceftazidime. Double-disk synergy test and E-test strips were used to confirm production of ESBLs. The molecular analysis revealed that the majority of strains (91%) carried genes encoding CTX-M-15. SHV-12 and SHV-2a were produced, respectively, by 9% and 3% of the strains. Pulsed-field gel electrophoresis of ESBL-producing Klebsiella pneumoniae isolates revealed four different clonal groups and three for Escherichia coli, showing the absence of spread of any epidemic clone. The CTX-M-15 ESBL-producing E. coli of the major clonal group belong to the B2 phylogenetic group, to the sequence type 131, and has a high virulence potential. In conclusion, CTX-M-15 ESBLs accounted for the overwhelming majority of ESBL types among Enterobacteriaceae from our hospital. This study confirms the high rate of ESBLs in Tunisia and further demonstrates the worldwide spread of genes coding for CTX-M-15 enzymes in clinical isolates.

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa are increasingly reported worldwide. In our study, a total of 70 clinical isolates of multidrug-resistant P. aeruginosa were studied. Isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to characterize the contained ESBLs. The Double Disk Synergy Test in Cloxacillin (250 microg/ml)-containing Mueller-Hinton agar plates with a 20 mm distance between disks was the most reliable ESBL-screening method. Seven out of 70 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBL and have the bla(SHV-2a) ESBL gene. The bla(SHV-2a)-positive isolates were clonally related according to Enterobacterial Repetetive Intergenic Consensus-PCR (ERIC-PCR) results. The bla(SHV-2a) gene was found to be chromosomally located, and the flanking IS26 sequence in the immediate upstream region of the bla(SHV-2a) gene was detected in all SHV-2a-producing isolates. This is the first report of SHV-2a-producing P. aeruginosa isolates from Tunisia.

This study was conducted to identify metallo-beta-lactamase (MBL) producers among a collection of 75 nonrepetitive carbapenem-resistant Pseudomonas aeruginosa isolates recovered between November 2003 and May 2007 at the University Hospital Sahloul, Sousse, Tunisia. Five isolates produced the MBL VIM-2. Those bla(VIM-2)-positive isolates were clonally related according to pulsed-field gel electrophoresis analysis. This carbapenemase was very likely chromosomally located and as a form of a gene cassette in a class 1 integron. This is the 1st report of spread of VIM-2 producers in Tunisia.

Ninety-nine carbapenem-resistant Acinetobacter baumannii isolates were obtained from patients hospitalized between October 2005 and January 2007 at the University Hospital Sahloul, Sousse, Tunisia. Thirteen of those isolates produced the carbapenem-hydrolyzing oxacillinase OXA-23. All the OXA-23-positive isolates were clonally related, and the bla(OXA-23) gene was found to be chromosomally located and associated with an upstream-located insertion sequence ISAba1. This study further highlights the worldwide emergence of OXA-23-producing A. baumannii isolates.