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.

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella , and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.

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.

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.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:− is a monophasic variant of S . enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes bla _TEM1-like , strA - str B , tet (B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,[5],12:i:− and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,[5],12:i:− strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.

Currently, 2,610 different Salmonella serovars have been described according to the White-Kauffmann-Le Minor scheme. They are routinely differentiated by serotyping, which is based on the antigenic variability at lipopolysaccharide moieties (O antigens), flagellar proteins (H1 and H2 antigens), and capsular polysaccharides (Vi antigens). The aim of this study was to evaluate the potential of matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for rapid screening and identification of epidemiologically important Salmonella enterica subsp. enterica serovars based on specific sets of serovar-identifying biomarker ions. By analyzing 913 Salmonella enterica subsp. enterica strains representing 89 different serovars using MALDI-TOF mass spectrometry, several potentially serovar-identifying biomarker ions were selected. Based on a combination of genus-, species-, subspecies-, and serovar-identifying biomarker ions, a decision tree classification algorithm was derived for the rapid identification of the five most frequently isolated Salmonella enterica serovars, Enteritidis, Typhimurium/4,[5],12:i:-, Virchow, Infantis, and Hadar. Additionally, sets of potentially serovar-identifying biomarker ions were detected for other epidemiologically interesting serovars, such as Choleraesuis, Heidelberg, and Gallinarum. Furthermore, by using a bioinformatic approach, sequence variations corresponding to single or multiple amino acid exchanges in several biomarker proteins were tentatively assigned. The inclusivity and exclusivity of the specific sets of serovar-identifying biomarker ions for the top 5 serovars were almost 100%. This study shows that whole-cell MALDI-TOF mass spectrometry can be a rapid method for prescreening S. enterica subsp. enterica isolates to identify epidemiologically important serovars and to reduce sample numbers that have to be subsequently analyzed using conventional serotyping by slide agglutination techniques.