Genetic Background of Antimicrobial Resistance in Multiantimicrobial-Resistant Escherichia coli Isolates from Feces of Healthy Broiler Chickens in Tunisia.

Multiantimicrobial-resistant Escherichia coli isolates are a global human health problem causing increasing morbidity and mortality. Genes encoding antimicrobial resistance are mainly harbored on mobile genetic elements (MGEs) such as transposons and plasmids as well as integrons, which enhance their rapid spread. The aim of this study was to characterize 83 multiantimicrobial-resistant E. coli isolates recovered from healthy broiler chickens. Among 78 tetracycline-resistant isolates, the tetA, tetB, and tetC genes were detected in 59 (75.6%), 14 (17.9%), and one (1.2%) isolates, respectively. The sul1, sul2, and sul3 genes were detected 31 (46.2%), 16 (23.8%), and 6 (8.9%) isolates, respectively, among 67 sulfonamide-resistant isolates. The PCR-based replicon typing method showed plasmids in 29 isolates, IncFIB (19), IncI1-Iγ (17), IncF (14), IncK (14), IncFIC (10), IncP (8), IncY (3), IncHI2 (1), and IncX (1). The class 1 and 2 integrons were detected in 57 and 2 isolates, respectively; one isolate harbored both integrons. Seven and one gene cassette arrays were identified in class 1 and class 2 integrons, respectively. Our findings show that multiantimicrobial-resistant E. coli isolates from chickens serve as reservoirs of highly diverse and abundant tet and sul genes and plasmid replicons. Such isolates and MGEs pose a potential health threat to the public and animal farming.

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.

Antimicrobial drug resistance and genetic properties of Salmonella enterica serotype Enteritidis circulating in chicken farms in Tunisia.

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 (blaTEM-1-like and blaSHV), 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.

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.

Occurrence of bla CTX-M-1, qnrB1 and virulence genes in avian ESBL-producing Escherichia coli isolates from Tunisia.

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 bla CTX-M-1 gene, and one strain co-harbored the bla TEM-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.