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The aim of this work was the genetic characterization of cefotaxime-resistant enterobacteria from animals (53 samples), the surface water of rivers (17 samples), and wastewater treatment plants (43 samples) in Tunisia. A total of 48 (42.4%) cefotaxime-resistant isolates were recovered. An extended spectrum beta-lactamase (ESBL) phenotype with a positive double-disk synergy test (DDST) was exhibited by 34 (70.8%) and 14 (29.1%) isolates from water and animal origins, respectively. Isolates from water were identified as: Escherichia coli(n= 17), Hafniaspp.(n= 13), Citrobacterspp.(n= 1), Enterobacter cloacae(n= 1), Klebsiella pneumoniae(n= 1), and K. oxytoca(n= 1). Animal isolates were identified as: E. coli(n= 11), E. cloacae(n= 1), Hafniaspp.(n= 1), and K. pneumoniae(n= 1). PCR investigation of blaCTX-M, blaTEM, and blaSHVgenes showed that amongst the 48 isolates with a positive DDST, 41 (87.5%) carried the blaCTX-Mgene, 1 isolate harbored the blaSHVgene, and 1 isolate coharbored blaCTX-Mwith blaSHVgenes. The class 1 and 2 integrons were detected in 27 (56.2%) and 1 (2%) isolates, respectively. Our study showed a significant occurrence of ESBL-producing enterobacteria in animals and aquatic environments with a predominance of blaCTX-Mgenes.

•27 (19.1%) of 141 Staphylococcusspp. recovered from milk and nasal swabs of cows were S. aureus.•26 MSSA and 1 MRSA were detected, most of which were resistant to penicillin, fusidic acid, clindamycin and erythromycin.•25 isolates had at least one enterotoxin and 4 were positive for the tsst-1gene.•Twelve pulsotypes were identified amongst 25 MSSA isolates; 1 isolate was identified as CC398.•The MRSA isolate from milk was characterised as ST97-t267-agrI-SCCmecV and was PVL-negative.

•32 MRSA isolates recovered from wound specimens in Hospital from Tripoli, Lybia, were studied.•Isolates were clonal and belonged to CC5, harboured the SCCmecVelement, the spatype was t6065, and harboured the agrII.•Isolates were multidrug-resistant and exfoliative-, enterotoxin-, haemolysin- encoding genes and immune evasion cluster proteins were relevant.•This is the first report of a specific clonal spread of a multi-drug resistant MRSA-CC5-SCCmecV in Libyan Hospital.

Rotating biological disks and natural oxidizing lagoons are processes used to treat wastewater. These procedures were used in the present work and were situated in a pilot wastewater treatment plant situated in the residential area of Tunis City in Tunisia and received hospital wastewater from three nearby different clinics. This wastewater was very rich in various types of pathogens. Consequently, the monitoring of this treatment plant for hospital wastewater can be an appropriate approach to the study of the circulation of enteric viruses, such as human astroviruses. Wastewater samples (n = 102) were collected at the exit of the five basins from natural oxidizing lagoons and at the outlet of the rotating biodisks. Human astroviruses were identified in 55% (n = 56) of the wastewater samples by reverse transcription polymerase chain reaction (RT-PCR). An important increase was found in the frequencies of human astroviruses from the first to last lagoons of the natural oxidizing lagoon and at the exit of the rotating biological disks. Thus, these results showed the ineffectiveness of the two biological treatment methods studied for the human astroviruses removal. In addition, the data of this investigation pointed out the first detection of human astroviruses (genotype 1 and 6) in wastewater samples in Tunisia. These findings highlighted the inadequacy of the sanitary quality of treated wastewater for recycling, agricultural reuse and discharge into the receiving waters.

AbstractFifty-five Escherichia coliisolates were acquired from chicken and turkey meat obtained from two slaughterhouses in Tunis. Eighty-nine percent, 80%, 78%, 67%, 45%, 27%, 7%, 4%, and 2% of these isolates showed resistance to tetracycline, trimethoprim/sulfamethoxazole, streptomycin, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, colistine, and gentamicin, respectively. No resistance was detected to cefotaxime, ceftazidime, or amikacin. blaTEMgene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored blaOXA-1gene. Tetracycline resistance was predominately mediated by the tetAgene. The sul1, sul2, and sul3genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1and sul3were included in class 1 integrons in some cases. Sixty percent of isolates harbored integrons (class 1, 30 isolates; class 2, 5 isolates). Class 2 integrons contained in all cases the dfrA1-sat1-aadA1-orfXgene cassette arrangement. Nine gene cassette arrangements have been detected among class 1 integrons, containing different alleles of dfrA(five alleles) and aadA(2 alleles) genes, which encode trimethoprim and streptomycin resistance, respectively. An uncommon gene cassette array (sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) has been identified in three class 1 integron-positive isolates, and one additional isolate had this same structure with the insertion of IS26inside the aadA1gene (included in GenBank with accession no. FJ160769). The 55 studied isolates belong to the four phylogenic groups of E. coli, and phylogroups A and D were the most prevalent ones. At least one virulence-associated gene (fimA, papC, or aer) was detected in 44 of the 55 (80%) studied isolates. E. coliisolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.

Chlamydiosis is one of the major diseases that can lead to abortion in ewes. Since 1997, in 5 regions of Tunisia, Chlamydia-related abortions have been reported in 15 sheep and goat flocks. One hundred and sixty-six sera and 50 vaginal swab samples were collected from adult ewes. Chlamydial antigens were detected in 29 (58%) of the vaginal swabs using Enzyme Linked Immunsorbent Assay (ELISA) while 9 (18%) were positive by cell culture. Five strains were recovered from 4 different sheep flocks. Monoclonal antibody profiles and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region showed that these isolates were C. abortus. Using amplified fragment length polymorphism (AFLP), these Tunisian strains were shown to exhibit the same pattern as strains isolated in France.

Pestiviruses have been isolated from live sheep pox Tunisian vaccines. Vaccination with these vaccines caused outbreaks of Border Disease in Tunisia. In order to study more precisely the pathogenicity of these isolates, three groups of eight four month old lambs from a pestivirus-free flock were infected by the intratracheal route with a French strain (AV) and two Tunisian isolates (SN3G and Lot21). Clinical, hematological, immunological and virological parameters were evaluated. The three groups developed mild fever and leucopaenia by day 3 to 6 post infection (pi). The differences in the weight curves were not significant. Viruses were isolated from the peripheral blood buffy coat cells by day 4 to 9 pi. Antibodies were present on day 16 pi following infection by the French strain and on day 21 pi with the Tunisian isolates. The results demonstrated that SN3G and Lot21 are almost similar to the French strain used as the reference strain. In field conditions, they could induce economical losses in naive flocks, alone or in association with other pathogens.

The double-stranded (ds) RNA genome segment 4 of bluetongue virus (BTV) serotype 2 was cloned and used as a serogroup-specific complementary (c) DNA probe for BTV diagnosis. A cDNA representing a 60% copy of genome segment 4 of BTV-2 prototype was produced. The specificity of the cDNA probe was determined by hybridizing this probe to a northern blot of dsRNA (separated by polyacrylamide gel electrophoresis) of plaque-purified BTV-2 prototype. This cDNA probe was then used to hybridize to the RNA samples. Because the probe hybridized to all BTV samples but not to epizootic hemorrhagic disease virus samples, it appears to be a group-specific probe that could be used in BTV diagnosis.

The dsRNA concentrated polyacrylamide gel electrophoresis (CPAGE) detected rotavirus directly from 19% of 77 stool specimens from diarrheic calves. A commercial enzyme-linked immunosorbent assay (ELISA) detected 25%, latex agglutination test, 23%, and polyacrylamide gel electrophoresis (PAGE), 19%. Establishing CPAGE as the “standard,” the commercial ELISA and the latex agglutination test both had higher sensitivity (84%) than PAGE (79%). However, PAGE produced the highest specificity (100%), followed by agglutination (88%) and ELISA (84%). The commercial ELISA had a slightly higher sensitivity than agglutination, PAGE, and CPAGE, but the ELISA specificity was generally lower. The latex agglutination test had a lower sensitivity than ELISA, but specificity was higher. Agglutination had similar negative predictive values (94%), compared with agglutination and PAGE, but had the lowest positive predictive value (a measure of accuracy) (70%). Agreement with CPAGE was highest for PAGE (94.8%), followed by agglutination (87%) and ELISA (84.4%). The calculated percentages of total disagreement with all other tests indicated that ELISA differed from the other rotavirus detection assays in 10.4% of the cases, agglutination in 7.8%, PAGE in 2.6%, and CPAGE in 1.3%. The 2 PAGE assays allowed the detection of atypical rotaviruses from feces based on the characteristic “super-short” migration pattern of the 11 genomic segments of rotaviruses and of other members of the Reoviridae.

AbstractIntegrons are important genetic elements implicated in acquisition and expression of antimicrobial resistance genes. Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. Thirty-four Escherichia coliisolates (carrying 38 class 1 integrons), recovered from poultry meat in previous studies in Tunisia and selected by their specific traits, were further characterized in this study. Integron promoter variants and the pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of isolates were determined. Three types of promoter variants were identified among the 38 class 1 integrons (PcW, PcH1, and PcS); the weak promoters were the most predominant. A high clonal diversity of the E. colistrains was demonstrated by PFGE or by MLST. Fifteen PFGE profiles were detected among 19 integron-positive isolates of phylogroup B2, and 12 different sequence types were identified by MLST among the remaining 15 isolates (ST48, ST88, ST101, ST117, ST155, ST189, ST351, ST359, ST410, ST641, ST665, and one new ST). These data reflect that the presence of integrons in these isolates is not due to the clonal dispersion but to dissemination of genetic structures carrying integrons in different clones. To the best of our knowledge, this is the first report examining the gene cassette promoter variants in class 1 integrons of E. coliisolates from poultry meat origin. The predominance of promoters implicated in weak expression/high excision activity of gene cassette arrays is of interest because they could theoretically enhance the capacity of integrons to adapt to antibiotic pressure.