Background and aim: Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.6**, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011–13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health.

Whole genome sequencing (WGS) of foodborne pathogens has become an effective method for investigating the information contained in the genome sequence of bacterial pathogens. In addition, its highly discriminative power enables the comparison of genetic relatedness between bacteria even on a sub-species level. For this reason, WGS is being implemented worldwide and across sectors (human, veterinary, food, and environment) for the investigation of disease outbreaks, source attribution, and improved risk characterization models. In order to extract relevant information from the large quantity and complex data produced by WGS, a host of bioinformatics tools has been developed, allowing users to analyze and interpret sequencing data, starting from simple gene-searches to complex phylogenetic studies. Depending on the research question, the complexity of the dataset and their bioinformatics skill set, users can choose between a great variety of tools for the analysis of WGS data. In this review, we describe the relevant approaches for phylogenomic studies for outbreak studies and give an overview of selected tools for the characterization of foodborne pathogens based on WGS data. Despite the efforts of the last years, harmonization and standardization of typing tools are still urgently needed to allow for an easy comparison of data between laboratories, moving towards a one health worldwide surveillance system for foodborne pathogens.

Two slow-growing, Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic approach. In a recent study both isolates were assigned to the genus Brucella but could not be attributed to one of the existing species. Hence we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other Brucella species. The genome sizes of F60T and F965 were 3,236,779 bp and 3,237,765 bp, respectively. Each genome consisted of two chromosomes, with a GC content of 57.2%. A genome to genome distance (GGD) of >80%, an average nucleotide identity (ANI) of 97%, and an average amino acid identity (AAI) of 98% compared with the type species B. melitensis confirmed affiliation to the genus. Remarkably, 5% of the entire genetic information of both strains was of non-Brucella origin, including not yet characterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed B. vulpis sp. nov. well separated from all hitherto described Brucella species, forming a long-branched sister clade to the classical Brucella species. In summary, based on phenotypic and molecular data we conclude that strains F60T and F965 are members of a novel Brucella species for which the name Brucella vulpis sp. nov. with the type strain F60T (= BCCN 09-2T; = DSM 101715T) is proposed.

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant ( mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet (M) alone or together with tet (K) and/or tet (L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA , ermB , and ermC , alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG ), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac ( 6 ′ )-Ie-aph ( 2 ″ )-Ia and/or ant(4 ′ )-Ia but also to aph(3 ′ )-IIIa ], chloramphenicol (9%, fexA or cfr ), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

The polymyxin antibiotic colistin has been used in decades for treatment and prevention of infectious diseases in livestock. Nowadays, it is even considered as last-line treatment option for severe human infections caused by multidrug- and carbapenem-resistant Gram-negative bacteria. Therefore, the discovery of plasmid-mediated mobile colistin resistance (mcr) genes raised major public health concern. The aim of our study was to analyze colistin-resistant Salmonella enterica strains from animals, food, feed and the environment collected at the National Reference Laboratory for Salmonella in Germany on the presence of mcr-1 to mcr-9 genes. Altogether 407 colistin-resistant (MIC >2 mg/L) Salmonella isolates received between 2011 and 2018 were selected and screened by PCR using a published mcr-1 to mcr-5 as well as a newly developed mcr-6 to mcr-9 multiplex PCR protocol. 254 of 407 (62.4%) isolates harbored either mcr-1 (n = 175), mcr-4 (n = 53), mcr-5 (n = 18) or mcr-1 and mcr-9 (n = 8). The number of mcr-positive isolates ranged from 19 (2017) to 64 (2012) per year. WGS revealed that none of our isolates harbored the mcr-9.1 gene. Instead, two novel mcr-9 variants were observed, which both were affected by frameshift mutations and are probably non-functional. The mcr-harboring isolates were mainly derived from animals (77.2%) or food (20.1%) and could be assigned to ten different Salmonella serovars. Many of the isolates were multidrug-resistant. Co-occurrence of mcr-1 and AmpC or ESBL genes was observed in eight isolates. Our findings suggest that mcr genes are widely spread among colistin-resistant Salmonella isolates from livestock and food in Germany. Potential transfer of mcr-harboring isolates along the food chain has to be considered critically.