High Biofilm-Forming Ability and Clonal Dissemination among Colistin-Resistant Escherichia coli Isolates Recovered from Cows with Mastitis, Diarrheic Calves, and Chickens with Colibacillosis in Tunisia.

BACKGROUND: Escherichia coli (E. coli) is one of the main etiological agents responsible for bovine mastitis (BM), neonatal calf diarrhea (NCD), and avian colibacillosis (AC). This study aimed to assess resistance and virulence genes content, biofilm-forming ability, phylogenetic groups, and genetic relatedness in E. coli isolates recovered from clinical cases of BM, NCD, and AC. MATERIALS/METHODS: A total of 120 samples including samples of milk (n = 70) and feces (n = 50) from cows with BM and calves with NCD, respectively, were collected from different farms in Northern Tunisia. Bacterial isolation and identification were performed. Then, E. coli isolates were examined by disk diffusion and broth microdilution method for their antimicrobial susceptibility and biofilm-forming ability. PCR was used to detect antimicrobial resistance genes (ARGs), virulence genes (VGs), phylogenetic groups, and Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for their clonal relationship. RESULTS: Among the 120 samples, 67 E. coli isolates (25 from BM, 22 from AC, and 20 from NCD) were collected. Overall, 83.6% of isolates were multidrug resistant. Thirty-six (53.73%) isolates were phenotypically colistin-resistant (CREC), 28.3% (19/67) were ESBL producers (ESBL-EC), and forty-nine (73.1%) formed biofilm. The blaTEM gene was found in 73.7% (14/19) of isolates from the three diseases, whilst the blaCTXM-g-1 gene was detected in 47.3% (9/19) of isolates, all from AC. The most common VG was the fimA gene (26/36, 72.2%), followed by aer (12/36, 33.3%), cnf1 (6/36, 16.6%), papC (4/36, 11.1%), and stx1 and stx2 genes (2/36; 5.5% for each). Phylogenetic analysis showed that isolates belonged to three groups: A (20/36; 55.5%), B2 (7/36; 19.4%), and D (6/36; 16.6%). Molecular typing by ERIC-PCR showed high genetic diversity of CREC and ESBL E. coli isolates from the three animal diseases and gave evidence of their clonal dissemination within farms in Tunisia. CONCLUSION: The present study sheds new light on the biofilm-forming ability and clonality within CREC and ESBL-EC isolated from three different animal diseases in Tunisian farm animals.

Frequent dissemination and carriage of an SCCmec-mecC hybrid in methicillin-resistant Mammaliicoccus sciuri in farm animals from Tunisia.

OBJECTIVES: In this study, we aimed to assess the extent of dissemination of methicillin-resistant Mammaliicoccus sciuri in animal farms in Tunisia and evaluate the distribution of virulence and methicillin resistance genes in the M. sciuri population. METHODS: Staphylococci and mammaliicocci isolated from unhealthy animals and healthy humans from adjacent farms in Tunisia were characterized for antimicrobial susceptibility, biofilm formation, agglutination, and hemolysis abilities. Mammaliicoccus sciuri relatedness and content in antibiotic resistance and virulence genes were analyzed by whole-genome sequencing (WGS). RESULTS: Mammaliicoccus sciuri was the most prevalent species (46.2%), showing the highest resistance rates to fusidic acid (94.6%), oxacillin (73%), penicillin (40.5%), clindamycin (37%), ciprofloxacin (27%), and cefoxitin (24.3%). Some isolates carried genes encoding resistance to nine different antibiotic classes. mecA was found in 35% of M. sciuri and mecC in 16.2%. All isolates carrying mecC were of S. sciuri subspecies carnaticus and carried the hybrid element SCCmec-mecC. Mammaliicoccus sciuri were able to produce strong biofilm (27%) and have clumping ability (16%). Additionally, they carried genes for capsule production (cap8, 100%), iron-regulated surface determinants (isdE, 24%; isdG, 3%), and virulence regulation (clpC and clpP, 100%). Single nucleotide polymorphisms (SNPs) analysis showed that 17 M. sciuri cross-transmission events probably occurred between different animal species and farms. Moreover, SCCmec was estimated to have been acquired five times by S. sciuri subsp. carnaticus. CONCLUSION: Multidrug resistant and pathogenic M. sciuri were frequently disseminated between different animal species within the farm environment. mecA and mecC can be disseminated by both frequent acquisition of the SCCmec element and clonal dissemination.

Co-occurrence of mcr-1 mediated colistin resistance and ß-lactamase-encoding genes in multidrug-resistant Escherichia coli from broiler chickens with colibacillosis in Tunisia.

OBJECTIVES: Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is considered a major hindrance in poultry farming worldwide. This study aimed to characterize the genetic content and the relatedness between multidrug-resistant E. coli isolates from broiler chickens died due to colibacillosis from three farms from Tunisia. METHODS: One hundred samples were collected from chickens' fresh carcasses from three poultry farms in Tunisia. E. coli isolation and identification were performed. Then, antimicrobial susceptibility regarding antibiotics, the ability to produce ß-lactamases and minimum inhibitory concentration for colistin were determined according to Clinical and Laboratory Standards Institute guidelines. ß-Lactam and non-ß-lactam antimicrobial resistance genes, integrons, virulence genes, and phylogenetic groups were investigated using polymerase chain reaction. The genetic relatedness of the E. coli isolates was analysed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: A high infection rate of E. coli (50%) in infected organs of chickens was observed. The majority of E. coli isolates were multidrug resistant (96%); among them, 24% were colistin resistant and 30% were ESBL producing. Seven of 12 colistin-resistant isolates harboured the mcr-1 gene; among them, 10 were ESBL producing and carried blaCTX-M-1, blaTEM, and blaSHV ß-lactamase-encoding genes. E. coli isolates were assigned to different phylogroups but most of them (74%) belonged to the pathogenic phylogroup B2. Molecular typing by PFGE showed that some E. coli isolates harbouring ESBL-mcr-1 genes were clonally related. MLST revealed the presence of four different ST lineages among ESBL- and mcr-1-carrying E. coli: ST4187, ST3882; ST5693, and ST8932 with clonal dissemination of E. coli ST4187 between two of the farms. CONCLUSION: This is the first report of ESBL-mcr-1-carrying E. coli isolates of a clinically relevant phylogenetic group (B2) from chickens that died due to colibacillosis in Tunisian poultry farms.

Prevalence of meticillin-resistant and -susceptible coagulase-negative staphylococci with the first detection of the mecC gene among cows, humans and manure in Tunisia.

In Europe, a novel mecA homologue - mecC (formerly mecALGA251) - has emerged recently in staphylococci from animals, humans and the environment. This paper reports the first occurrence of the mecC gene in Staphylococcus sciuri from cows and manure in Tunisia and Africa. Forty-nine coagulase-negative staphylococci (CNS) were isolated from the milk of cows with mastitis (n=20), manure (n=20) and human nares (n=9), including 16 Staphylococcus equoruim (32.6%), 12 S. xylosus (24.5%), 12 S. sciuri (24.5%), two S. saprophyticus (4.1%), two S. haemolyticus (4.1%), two S. lentus (4.1%), two S. vitulinus (4.1%) and one S. cohnii (2%). CNS from the three origins carried various resistance genes [mecA, blaZ, tet(K), erm(A), erm(B), msr(A)], suggesting an ongoing genetic exchange among CNS from the three niches. The mecA gene was detected in CNS (n=11) recovered from cows, manure and humans, whereas the mecC gene (n=3) was only detected in CNS from cows and manure. Various staphylococcal cassette chromosome mec (SCCmec) - SCCmec type I (n=1), II (n=3), IV (n=2), V/VII (n=2) and untypeable (n=3) - and diverse pulsed-field gel electrophoresis (PFGE) patterns were observed in mecA-positive CNS. Otherwise, similar SCCmec types and PFGE patterns were found in meticillin-resistant CNS within different farms and origins, showing the potential of SCCmec interspecies exchange and circulation of the same clones of meticillin-resistant CNS in the human-animal-environment interface. This study provides baseline data to support clonal dissemination of CNS between cows, humans and manure, and indicates the possible transmission of the mecC gene to humans in contact with cows and manure.