mcr-1 encoding colistin resistance in CTX-M-1/CTX-M-15- producing Escherichia coli isolates of bovine and caprine origins in Tunisia. First report of CTX-M-15-ST394/D E. coli from goats.

The objective of this study was to isolate and characterize ESBL-producing Escherichia coli (ESBL-EC) from raw bovine and caprine milk samples, as well as from bovine faeces in Tunisia. Therefore, 120 bovine faecal samples and 9 caprine raw milk samples were collected from 2 extensive dairy-cow-farms and 5 ovine farms, respectively. In addition, 94 raw bovine milk samples, from containers and holding tanks from 50 small public-markets in the North of Tunisia, were processed for the isolation of cefotaxime-resistant E. coli (CTXR). Antimicrobial susceptibility testing was carried out by disc-diffusion/broth-microdilution methods. The presence of genes encoding ESBL, as well as those encoding colistin (mcr-1 to 5 genes)- sulfonamide-, tetracycline-, gentamicin-, quinolone and chloramphenicol-resistance and class 1 integrons were tested by PCR (and sequencing in some cases). ESBL-EC isolates were further characterized by phylogrouping and MLST/PFGE typing. Eight samples (3.6%) contained ESBL-EC isolates (3/2 from raw bovine/goat milk and 3 from cattle faeces) and one isolate/sample was characterized. Four ESBL-EC isolates, all of bovine origin (3 faeces/1 milk), were resistant to colistin (MIC: 8-16 μg/ml), harboured the mcr-1 gene and carried IncP- and IncFIB-type plasmids. The 8 ESBL-EC strains had the following characteristics: a) bovine faeces: mcr-1/CTX-M-1/D-ST1642 (3 strains); b) raw milk: mcr-1/CTX-M-1/A-ST10 (1 strain); CTX-M-15/B1-ST394 (3 strains), and CTX-M-15/A-ST46 (1 strain). Most of bovine ESBL-EC isolates were multidrug-resistant (4/5). Our results showed that ESBL-EC were detected in bovine and caprine samples (CTX-M-1/CTX-M-15 producers), being some of them colistin-resistant (associated with mcr-1 gene), and they belonged to international clonal lineages.

Hassen B ，Saloua B ，Abbassi MS ，Ruiz-Ripa L ，Mama OM ，Hassen A ，Hammami S ，Torres C ... -  《-》

High prevalence of mcr-1 encoding colistin resistance and first identification of bla(CTX-M-55) in ESBL/CMY-2-producing Escherichia coli isolated from chicken faeces and retail meat in Tunisia.

Avian industries have been reported as an important contributor in the worldwide spread of antibiotic resistance owing to some particular practices especially the overuse of antibiotics. Thus in this study, we aimed to characterize extended-spectrum-beta-lactamase (ESBL) and acquired-AmpC-beta-lactamase (aAmpC)-producing Escherichia coli isolates from chicken faeces and raw meat in Tunisia. During the year 2018, 286 faecal chicken swabs and 47 raw chicken meat samples were collected and processed to recover cefotaxime-resistant E. coli. Antimicrobial susceptibility was performed by disk-diffusion and/or broth-microdilution. blaTEM, blaSHV, blaCTX-M, and blaCMY genes were investigated by PCR/sequencing. Genes encoding resistance to colistin (mcr-1 to mcr-8), tetracycline (tetA/tetB), sulfonamide (sul1/sul3), and chloramphenicol (cmlA), were analysed by PCR. Class 1 integrons were investigated by PCR/sequencing. Phylogenetic groups of all isolates were determined. PFGE and MLST were performed for representative isolates. PCR-based replicon typing was performed in mcr1-harbouring isolates. Cefotaxime-resistant E. coli was detected in 22.4% (64/286) and 63.8% (30/47) of faeces and meat samples, respectively. Ninety isolates were ESBL-producers and harboured the genes: blaCTX-M-1 +/- blaTEM-1 (n = 65), blaCTX-M-55 +/- blaTEM-1 (n = 21), blaCTX-M-14 (n = 1), and blaSHV-12 (n = 3). The blaCMY-2 gene was detected in four ESBL-negative isolates. Isolates belonged to phylogroups D (50%), A (36.2%), B1 (9.6%), and B2 (4.3%). Fifty-four were colistin-resistant and 52 carried the mcr-1 gene. The tetA, sul1/sul3 and cmlA genes were detected among resistant isolates and 76 harboured class 1 integrons. MLST analysis revealed 13 sequence types (STs). The isolates were classified into 28 PFGE types. The IncP, IncFIB, and IncI1 replicons were detected among mcr-1-positive strains. We report a high frequency of ESBL-producers and colistin-resistant E. coli in chicken and derived food and the detection for the first time of blaCTX-M-55 in poultry in Tunisia.

Hassen B ，Abbassi MS ，Ruiz-Ripa L ，Mama OM ，Hassen A ，Torres C ，Hammami S ... -  《-》

Short communication: Bovine mastitis caused by a multidrug-resistant, mcr-1-positive (colistin-resistant), extended-spectrum β-lactamase-producing Escherichia coli clone on a Greek dairy farm.

We performed a survey aimed at analyzing milk samples collected from cows with mastitis for the presence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. Single-quarter mastitic milk samples obtained from 400 cows in 23 Greek dairy herds with a history of E. coli mastitis were processed for the selective isolation of ESBL-producing E. coli. The antimicrobial susceptibility of the ESBL-producing isolates was analyzed using agar disk diffusion, and minimum inhibitory concentrations of colistin were determined by broth microdilution. We used PCR followed by DNA sequencing to characterize the β-lactamases and mcr-1 (colistin resistance) genes, and for phylotyping and multilocus sequence typing. We found a total of 89/400 (22.25%) E. coli isolates from 12/23 (52%) farms. Six isolates originating from 6 cows on a single farm were ESBL producers and were resistant to cefquinome, amoxicillin-clavulanic acid, aztreonam, ampicillin, and colistin. Five of these isolates were resistant to trimethoprim-sulfamethoxazole, and 5 to streptomycin. The 6 ESBL producers were mcr-1-positive and carried blaTEM-1 genes; 3 also carried blaCTX-M genes, and 3 carried blaSHV genes. All of the ESBL producers belonged to phylogroup A, multilocus sequence type ST666 (n = 5), or a single locus variant of ST666 (n = 1). To our knowledge, this is the first report of endemic bovine mastitis caused by mcr-1-positive, ESBL-producing E. coli. These results highlight the value of active surveillance of antimicrobial resistance not commonly tested by diagnostic laboratories for the early detection of novel resistant strains.

Filioussis G ，Kachrimanidou M ，Christodoulopoulos G ，Kyritsi M ，Hadjichristodoulou C ，Adamopoulou M ，Tzivara A ，Kritas SK ，Grinberg A ... -  《-》

Emergence and Comparative Genomics Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Carrying mcr-1 in Fennec Fox Imported from Sudan to China.

The aim of this study was to investigate the occurrence and genomic characteristics of extended-spectrum-β-lactamase-producing Escherichia coli (ESBL-EC) in fennec fox imported from Sudan to China. We screened 88 fecal samples from fennec fox for ESBL-EC, using cefotaxime- and meropenem-supplemented selective medium. Antimicrobial susceptibility testing was performed by the agar dilution method except for colistin and tigecycline; for colistin and tigecycline, testing was conducted by the broth microdilution method. ESBL-EC bacteria were sequenced, and their genomes were characterized. Plasmid conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE), and Southern blotting were performed for a MCR-1-producing isolate. The genetic environment of mcr-1 and ESBL genes was also investigated. A total of 29 ESBL-EC bacteria were isolated from 88 fennec fox (32.9%), while no carbapenemase producers were found. The most prevalent genotypes were the blaCTX-M-55 and blaCTX-M-14 genes, followed by blaCTX-M-15 and blaCTX-M-64 We detected nine sequence types among 29 ESBL-EC. Furthermore, the mcr-1 gene was detected in isolate EcFF273. Conjugation analysis confirmed that the mcr-1 gene was transferable. S1 PFGE, Southern blotting, and whole-genome sequencing revealed that mcr-1 and blaCTX-M-64 were both located on a 65-kb IncI2 plasmid. This study reports for the first time the occurrence of ESBL-EC in fennec fox. The high prevalence of ESBL producers and the occurrence of MCR-1 producer in fennec fox imported into China from Sudan are unexpected. In addition, it clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M and mcr-1, potentially contributing to the dissemination and transfer of such genes to pathogenic bacteria among fennec fox. Our results support the implication of fennec fox as a biological vector for ESBL-producing members of the Enterobacteriaceae family.IMPORTANCE The extended-spectrum-β-lactamase (ESBL)-producing members of the Enterobacteriaceae family are a global concern for both animal and human health. There is some information indicating a high prevalence of ESBL producers in food animals. Moreover, there have been an increasing number of reports on ESBL-producing strains resistant to the last-resort antibiotic colistin with the global dissemination of the plasmid-mediated mcr-1 gene, which is believed to have originated in animal breeding. However, little is known regarding the burden of ESBL-producing Enterobacteriaceae on wild animals. No data were available on the prevalence of antimicrobial resistance (AMR) among wild animals imported into China. This is the first study to investigate the microbiological and genomics surveillance investigation of ESBL colonization among fennec fox (Vulpes zerda) imported from Sudan to China, and we uncovered a high prevalence of ESBL-EC. Furthermore, the underlying mechanism of colistin resistance in an isolate that harbored mcr-1 was also investigated. Results of characterization and analysis of 29 ESBL-producing E. coli may have important implications on our understanding of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectoral surveillance for colistin-resistant E. coli in this region.

Feng C ，Wen P ，Xu H ，Chi X ，Li S ，Yu X ，Lin X ，Wu S ，Zheng B ... -  《mSphere》

Co-Occurrence of Plasmid-Mediated Colistin Resistance (mcr-1) and Extended-Spectrum β-Lactamase Encoding Genes in Escherichia coli from Bovine Mastitic Milk in China.

Liu G ，Ali T ，Gao J ，Ur Rahman S ，Yu D ，Barkema HW ，Huo W ，Xu S ，Shi Y ，Kastelic JP ，Han B ... -  《-》