f Typing of Mycobacterium avium subspecies paratuberculosis strains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of wholegenome sequencing have extended possibilities for the characterization of Mycobacterium avium subspecies paratuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133 Mycobacterium avium subspecies paratuberculosis isolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs Mycobacterium avium subspecies paratuberculosis isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit-variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the largescale global typing of Mycobacterium avium subspecies paratuberculosis isolates. Mycobacterium avium subspecies paratuberculosis causes Johne's disease, a chronic infectious enteritis principally of ruminants. The disease occurs worldwide and is responsible for significant losses to the livestock industry. M. avium subspecies paratuberculosis also has been detected in a subset of human patients with Crohn's disease (1), although the zoonotic role of the bacterium remains controversial.Strain typing is a prerequisite for tracing the sources of infection and for studying the epidemiology, population structure, and evolutionary relationships between isolates. It can also reveal the genetic diversity underlying important phenotypic characteristics, such as host specificity, pathogenicity, antibiotic resistance, and virulence. Typing of M. avium subspecies paratuberculosis strains presents a challenge, since M. avium subspecies paratuberculosis, like Mycobacterium tuberculosis, is genetically monomorphic (2). Genetic diversity among M. avium subspecies paratuberculosis strains has been investigated using molecular techniques, such as restriction fragment length polymorphism (RFLP) and IS900 analysis (IS900 RFLP) (3), pulsed-field gel electrophoresis (PFGE) (4), amplified fragment length polymorphism (AFLP) analysis (5), ran...

The emergence and dissemination of resistance to third- and fourth-generation cephalosporins among Enterobacteriaceae from different sources impose a global public health threat. Here, we characterized by whole-genome sequencing four Escherichia coli strains harboring the blaCTX–M–65 gene identified among 49 isolates from beef and pork collected at retail. The genomic content was determined using the Center for Genomic Epidemiology web tools. Additionally, the prediction and reconstruction of plasmids were conducted, the genetic platform of the blaCTX–M–65 genes was investigated, and phylogenetic analysis was carried out using 17 other genomes with the same sequence type and harboring the blaCTX–M–65 gene. All strains harbored blaCTX–M–65, blaOXA–1, and blaTEM–1B, and one also carried the blaSHV–12 gene. Other resistance genes, namely, qnrS2, aac(6′)-Ib-c, dfrA14, sul2, tetA, and mphA, were present in all the genomes; the mcr-1.1 gene was identified in the colistin-resistant strains. They belong to sequence type 2179, phylogenetic group B1, and serotype O9:H9 and carried plasmids IncI, IncFIC(FII), and IncFIB. All strains share an identical genetic environment with IS903 and ISEcp1 flanking the blaCTX–M–65 gene. It seems likely that the blaCTX–M–65 gene is located in the chromosome in all isolates based on deep in silico analysis. Our findings showed that the strains are clonally related and belong to two sub-lineages. This study reports the emergence of CTX-M-65-producing E. coli in Portugal in food products of animal origin. The chromosomal location of the blaCTX–M–65 gene may ensure a stable spread of resistance in the absence of selective pressure.

The present study aimed to characterize the extended-spectrum β-lactamases and plasmid-mediated AmpC β-lactamases (ESBL/PMAβ) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAβ [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6’)-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

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This Technical Research communication describes results of a study aimed at detecting the presence of Map in milk fed to calves, and identifying possible risk factors for that presence. A questionnaire was performed on 37 dairy farms and waste milk samples were collected on 3 occasions separated by a minimum of 1 week. For farms not feeding waste milk, bulk tank milk samples were collected instead. A real time PCR for the detection of the IS900 sequence was performed for the detection of Map. A majority of farms (89·2%) fed waste milk, with only one pasteurising the milk before feeding it to calves. Results of the PCR showed that 51·5% of the farms that were feeding waste milk had a positive result for Map on that milk. None of the studied risk factors were significantly associated with the presence of Map in milk samples, possibly due to the small number of farms entering the study. However, the prevalence of positive samples for Map on PCR was 3·5 times higher for farms that bought in animals from a single origin and 1·9 times higher for farms that bought from multiple farms, when compared with closed farms. Having a calving area for multiple cows also increased the risk of a positive Map result by 1·5 when compared with single pens. The risk of having a positive Map result on waste milk was 1·6 times higher for farms feeding that milk to male calves and 1·4 for farms feeding to both male and female calves, when compared with farms not feeding waste milk. This study highlights paratuberculosis as one of the potential risks of feeding waste milk to calves, and the need for mitigation strategies to be in place to avoid unnecessary disease transmission.

Enterococci are part of the commensal gut microbiota of mammals, with Enterococcus faecalis and Enterococcus faecium being the most clinically relevant species. This study assesses the prevalence and diversity of enterococcal species in cattle (n = 201) and pig (n = 249) cecal samples collected in 2017. Antimicrobial susceptibility profiles of E. faecium (n = 48) and E. faecalis (n = 84) were assessed by agar and microdilution methods. Resistance genes were screened through PCR and nine strains were analyzed by Whole Genome Sequencing. A wide range of enterococci species was found colonizing the intestines of pigs and cattle. Overall, the prevalence of resistance to critically important antibiotics was low (except for erythromycin), and no glycopeptide-resistant isolates were identified. Two daptomycin-resistant E. faecalis ST58 and ST93 were found. Linezolid-resistant strains of E. faecalis (n = 3) and E. faecium (n = 1) were detected. Moreover, oxazolidinone resistance determinants optrA (n = 8) and poxtA (n = 2) were found in E. faecalis (ST16, ST58, ST207, ST474, ST1178) and E. faecium (ST22, ST2138). Multiple variants of optrA were found in different genetic contexts, either in the chromosome or plasmids. We highlight the importance of animals as reservoirs of resistance genes to critically important antibiotics.

As the effects of global warming become increasingly complex and difficult to manage, the conservation and sustainable use of locally adapted sheep breeds are gaining ground. Portuguese native sheep breeds are important reservoirs of genetic diversity, highly adapted to harsh environments and reared in low input production systems. Genomic data that would describe the breeds in detail and accelerate the selection of more resilient animals to be able to cope with climatic challenges are still lacking. Here, we sequenced the genomes of 37 animals from four Portuguese native sheep breeds (Campaniça, Bordaleira Serra da Estrela, Merino Branco and Merino Preto) and 19 crossbred sheep to make inferences on their genomic diversity and population structure. Mean genomic diversities were very similar across these breeds (.30 ≤ Ho ≤ .34; .30 ≤ He ≤ .35; 1.7 × 10–3 ≤ π ≤ 3.1 × 10–3) and the levels of inbreeding were negligible (.005 ≤ FIS ≤ .038). The Principal Components, Bayesian clustering and Treemix analyses split the Portuguese breeds in two main groups which are consistent with historical records: one comprising Campaniça and Serra da Estrela together with other European and transboundary dairy breeds; and another of the well-differentiated multi-purpose Merino and Merino-related breeds. Runs of homozygosity analyses yielded 1,690 ROH segments covering an average of 2.27 Gb across the genome in all individuals. The overall genome covered by ROH segments varied from 27,75 Mb in Serra da Estrela to 61,29 Mb in Campaniça. The phylogenetic analysis of sheep mitogenomes grouped the Portuguese native breeds within sub-haplogroup B1a along with two animals of the Akkaraman breed from Turkey. This result provides additional support to a direct influence of Southwest Asian sheep in local breeds from the Iberian Peninsula. Our study is a first step pertaining to the genomic characterization of Portuguese sheep breeds and the results emphasize the potential of genomic data as a valid tool to guide conservation efforts in locally adapted sheep breeds. In addition, the genomic data we generated can be used to identify markers for breed assignment and traceability of certified breed-products.

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