Biological activities and determination of the mode of action of Tunisian Globularia alypum and Cistus monspeliensis ethanolic extracts.

This study was designed to evaluate the antioxidant and antimicrobial activity of ethanolic extracts (EEs) of Cistus monspeliensis and Globularia alypum. C. monspeliensis showed the highest values of phenolic compounds. Further, it was shown that EE of C. monspeliensis displayed the highest DPPH (IC50 = 8.3 ± 1.08 mg/mL). The chemical profiles demonstrated a total of 12 and 13 phenolic compounds for C. monspeliensis and G. alypum, respectively. EEs of both plants possessed broad-spectrum antimicrobial activity when tested against Escherichia coli and Staphylococcus aureus. Treatment of studied strains with these extracts at their MICs reduced considerably the bacterial viability. The extracts did not induce total bacterial lysis, as determined by the measurement of optical density at 620 nm. Strains treated with EEs at Minimum Inhibitory Concentrations showed significant loss of tolerance to NaCl. Our results contribute to understanding the antibacterial mechanism of ethanolic extracts of the studied medicinal plants.

Epidemiology and Whole-Genome Analysis of NDM-1-Producing Klebsiella pneumoniae KP3771 from Tunisia.

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 blaNDM-1 and blaCTX-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.

Extended-spectrum ß-lactamases and plasmid-mediated quinolone resistance in enterobacterial clinical isolates from neonates in Tunisia.

This study was conducted to investigate extended-spectrum-ß-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 blaCTXM-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.

Emergence of OXA-204 ß-lactamase in Tunisia.

A retrospective epidemiological survey was carried out to determine the prevalence of carbapenemase producers among enterobacterial clinical isolates recovered in the center of maternity and neonatology of Monastir (Tunisia). PCR screening identified 1 OXA-48 and 2 OXA-204 producers, which coexpressed the CTX-M-15 or the CMY-4 ß-lactamases. PCR mapping showed that the bla(OXA-48) gene was carried by a Tn1999.2 transposon, whereas the bla(OXA-204) gene was part of the Tn2016 transposon-like structure. The OXA-48- or OXA-204-producing Klebsiella pneumoniae clinical isolates and the OXA-204-expressing Escherichia coli clinical isolate belonged to the widespread sequence types ST11, ST101, and ST617, respectively. The OXA-204 enzyme, which is a point derivative of the OXA-48 carbapenemase, had hitherto been reported in 2013 from K. pneumoniae isolate. Our study shows for the first time the dissemination of this resistance marker in E. coli strain. The coproduction of OXA-204 with CTX-M-15 and CMY-4 enzymes may potentiate the risk of multiresistance and may enhance the risk of dissemination.

Imipenem resistance in Klebsiella pneumoniae is associated to the combination of plasmid-mediated CMY-4 AmpC ß-lactamase and loss of an outer membrane protein.

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 ß-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 ß-lactams, including third generation cephalosporins and carbapenems. The screening of ß-lactamases showed the presence of three ß-lactamases: TEM-1, SHV-61, and CMY-4. The CMY-4 ß-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 ß-lactamase: AmpC CMY-4, together with alteration in permeability led to resistance to all available ß-lactams and carbapenems.

Emergence of SHV-2a extended-spectrum beta-lactamases in clinical isolates of Pseudomonas aeruginosa in a university hospital in Tunisia.

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.