Avian ESBL-producing Escherichia coli isolates have been increasingly reported worldwide. Animal to human dissemination, via food chain or direct contact, of these resistant bacteria has been reported. In Tunisia, little is known about avian ESBL- producing E. coli and further studies are needed. Seventeen ESBL-producing Escherichia coli isolates from poultry feces from two farms (Farm 1 and farm 2) in the North of Tunisia have been used in this study. Eleven of these isolates (from farm 1) have the same resistance profile to nalidixic acid, sulfonamides, streptomycin, tetracycline, and norfloxacine (intermediately resistant). Out of the six isolates recovered from farm 2, only one was co-resistant to tetracycline. All isolates, except one, harbored blaCTX-M-1 gene, and one strain co-harbored the blaTEM-1 gene. The genes tetA and tetB were carried, respectively, by 11 and 1 amongst the 12 tetracycline-resistant isolates. Sulfonamides resistance was encoded by sul1, sul2, and sul3 genes in 3, 17, and 5 isolates, respectively. The qnrB1 was detected in nine strains, one of which co-harbored qnrS1 gene. The search for the class 1 and 2 integrons by PCR showed that in farm 1, class 1 and 2 integrons were found in one and ten isolates, respectively. In farm 2, class 1 integron was found in only one isolate, class 2 was not detected. Only one gene cassette arrangement was demonstrated in the variable regions (VR) of the 10 int2-positive isolates: dfrA1- sat2-aadA1. The size of the VR of the class 1 integron was approximately 250 bp in one int1-positive isolate, whereas in the second isolate, no amplification was observed. All isolates of farm 1 belong to the phylogroup A (sub-group A0). However, different types of phylogroups in farm 2 were detected. Each of the phylogroups A1, B22, B23 was detected in one strain, while the D2 phylogroup was found in 3 isolates. The virulence genes iutA, fimH, and traT were detected in 3, 7, and 3 isolates, respectively. Two types of gene combination were detected: iutA+fimH+traT in 3 isolates and iutA+fimH in one isolate. The isolates recovered in farm 1 showed the same profile of PFGE macro-restriction, while isolates of farm 2 presented unrelated PFGE patterns. We conclude that these avian ESBL-producing E. coli isolates show homo- and heterogenic genetic background and that plasmids harboring ESBL genes could be involved in the dissemination of this resistance phenotype.

Significance and Impact of the Study: Enterococci possess interesting properties not only for the food industry, but also for animal and human health. The antimicrobial potential of these bacteria includes principally bacteriocin-like molecules. With the aim of identifying bacteriocinogenic strains, a collection of 300 enterococci isolated from different origins were screened and their spectrum of action, as well as the gene encoding the bacteriocin, was determined. Fifty-nine bacteriocin-producing Enterococcus showed high activity against Listeria monocytogenes and Paenibacillus larvae, the causative agent of American foulbrood. Enterocins A, P and L50A/B were found in various combinations. The most important finding of this study is the growth inhibition of P. larvae due to bacteriocin-producing Enterococcus, which opens up the possibility to use these strains to control the disease in honeybees. AbstractA total of 300 isolates of Enterococcus, from different sources including faeces of poultry, cow and sheep, raw milk, ricotta cheese and water, in Tunisia, were screened for their antibacterial activity. Amongst them, 59 bacteriocinproducing strains were detected and identified by molecular methods. Genes encoding for entA, entP, entB, entL50A/B, AS-48 and bac31 bacteriocins were targeted by PCR. The bacteriocin-producing strains were assigned to the species Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, Enterococcus mundtii and Enterococcus durans, respectively, 34, 19, 3, 2 and 1 isolates. Antimicrobial activity was specifically observed against different spoilage and pathogenic micro-organisms, such as Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Escherichia coli, Ent. faecalis, Staphylococcus aureus, Salmonella enterica serovar Enteritidis and Paenibacillus larvae. The inhibitory activity was totally lost after proteinase K treatment, thereby revealing the proteinaceous nature of the antimicrobial compound. Only three bacteriocin genes, namely entP, entA and entL50A/B were detected in the isolates included in this study. Enterocins A and P were the most frequent genes and they were found in 55 (93Á2%) and 39 isolates (66Á1%), respectively, followed by enterocin L50A/B present in 27 isolates (45Á7%). These newly identified bacteriocin-producing enterococci have the potential to be used in bio-preservation of food as well as biological control of foulbrood disease.

This study focused on 77 isolates of Salmonella enterica serotype Enteritidis collected during 2009 to 2013 from healthy and sick chickens and environmental farm samples in Tunisia. Resistance to 14 antimicrobials and the encoding genes were analyzed. 66, 26, 6.5, 3.9 and 1.3% were pan-susceptible or showed resistance to nalidixic acid (Asp87 to Tyr and Asp87 to Asn substitutions in GyrA), ampicillin (bla and bla), sulfonamides (sul1and sul3) and streptomycin (strB), respectively. A single isolate with intermediate susceptibility to ciprofloxacin was positive for qnrB, whereas qnrA, qnrS or aac(6')-Ib-cr were not detected. The virulotype of the isolates was established by testing ten virulence genes. The orgA, ssaQ, mgtC, siiD, sopB genes, located on Salmonella pathogenicity islands, and spvC of the serotype-specific virulence plasmid, were common to all isolates. In contrast, the prophage-associated sopE-1, sodC1 and gipA genes and the fimbrial bcfC gene were variably represented. All isolates except one contained the virulence plasmid, which appeared either alone or together with one or more additional plasmids. One isolate carried a single plasmid of ca. 90Kb which may be derived from the virulence plasmid (60Kb). Overall, seven resistotypes, six virulotypes and six plasmid profiles were identified. XbaI-PFGE revealed four related pulsotypes (X1-X4), with 80% of the isolates sharing the X1 pattern. The latter isolates exhibited different resistance, virulence and plasmid profiles, suggesting that mobile genetic elements, particularly prophages and plasmids, are of central importance for the evolution and adaptation of S. Enteritidis circulating in chicken farms in Tunisia.

Salmonella enterica subsp. enterica serovar Eppendorf, with antigenic formula 1,4,12,[27]:d:1,5, is an infrequent serovar. However, 14% (20 of 142) of the isolates recovered during June-July 2012 in chicken farms in Tunisia belonged to S. Eppendorf. These isolates were analysed for resistance and virulence profiles. None of them were susceptible to all antimicrobials tested, while 70%, 60%, 50%, 50%, 20% and 5% were resistant to sulphonamides (sul1, sul2 and sul3), streptomycin (aadA1-like), trimethoprim (dfrA1-like), nalidixic acid (GyrA Asp87 →Asn and not identified), gentamicin (not identified) and ampicillin (blaTEM -1-like). About 30% of the isolates showed decreased susceptibility to ciprofloxacin and carried the qnrB gene; 65% of the isolates were multidrug resistant and contained class 1 integrons with sul1 or sul3 in the 3' conserved segment. The orgA, ssaQ, mgtC, siiD and sopB virulence genes located on SPI1 to SPI5 and the fimbrial bcfC gene were present in all isolates; the sopE1 and sodC1 carried by prophages were variably detected; however, the prophage gipA gene and the spvC gene of serovar-specific virulence plasmids were absent. Altogether, ten resistance and three virulence profiles were identified. Typing of the isolates with XbaI- and BlnI-PFGE supports a close relationship, although they appear to be evolving under selective pressure probably caused by antimicrobial use in chicken husbandry. As far as we know, this is the first study investigating the molecular bases of antimicrobial drug resistance, the virulence gene content and the PFGE profiles of S. Eppendorf. The epidemiological surveillance of this serovar would be necessary to evaluate its possible impact on human health, particularly in Tunisia and other African countries where it was already reported.