Salmonella enterica subsp. enterica serovar Paratyphi B from mute swan (Cygnus olor): complete genome sequence features point towards invasive variant potential.

A subgroup of Salmonella (S.) enterica subsp. enterica serovar Paratyphi B is significantly associated with invasive infections in humans. We report the complete genome sequence of a potentially invasive. S. Paratyphi B isolated from a mute swan (Cygnus olor) found dead at an urban recreation park in Berlin, Germany.

Draft genome sequence of a Shiga toxin-producing Escherichia coli strain from deer meat showing an IS-element integration in the B-subunit of the Shiga toxin Stx2b gene.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens. Here we report sequence data of the STEC strain BfR-EC-18960, which has integrated IS elements in the B-subunit of the Shiga toxin Stx2b gene. The strain was isolated from deer meat at a local butchery in Germany in 2021.

German coasts harbor non-O1/non-O139 Vibrio cholerae with clinical virulence gene profiles.

Non-O1/non-O139 Vibrio cholerae (NOVC) are ubiquitous in aquatic ecosystems. In rare cases, they can cause intestinal and extra-intestinal infections in human. This ability is associated with various virulence factors. The presence of NOVC in German North Sea and Baltic Sea was observed in previous studies. However, data on virulence characteristics are still scarce. Therefore, this work aimed to investigating the virulence potential of NOVC isolated in these two regions. In total, 31 NOVC strains were collected and subjected to whole genome sequencing. In silico analysis of the pathogenic potential was performed based on the detection of genes involved in colonization and virulence. Phenotypic assays, including biofilm formation, mobility and human serum resistance assays were applied for validation. Associated toxin genes (hlyA, rtxA, chxA and stn), pathogenicity islands (Vibrio pathogenicity island 2 (VPI-II) and Vibrio seventh pathogenicity island 2 (VSP-II)) and secretion systems (Type II, III and VI secretion system) were observed. A maximum likelihood analysis from shared core genes revealed a close relationship between clinical NOVCs published in NCBI and environmental strains from this study. NOVC strains are more mobile at 37 °C than at 25 °C, and 68% of the NOVC strains could form strong biofilms at both temperatures. All tested strains were able to lyse erythrocytes from both human and sheep blood. Additionally, one strain could survive up to 60% and seven strains up to 40% human serum at 37 °C. Overall, the genetic virulence profile as well as the phenotypic virulence characteristics of the investigated NOVC from the German North Sea and Baltic Sea suggest potential human pathogenicity.

Arcanobacterium canis strain DSM 25104 isolated from an English bulldog suffering from otitis externa: complete genome sequence.

Many species of the genus Arcanobacterium are known as opportunistic pathogens and have been isolated in association with infectious diseases in humans and animals. Here, we present the complete genome sequence of another opportunistic pathogenic representative, namely Arcanobacterium canis, isolated from the otitis externa of an English bulldog.

Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens.

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics.

Genetic characterization of a multidrug-resistant Salmonella enterica serovar Agona isolated from a dietary supplement in Germany.

Salmonella enterica subsp. enterica serovar Agona has a history of causing food-borne outbreaks and any emergence of multidrug-resistant (MDR) isolates in novel food products is of concern. Particularly, in food products frequently consumed without sufficient heating prior to consumption. Here, we report about the MDR isolate, 18-SA00377, which had been isolated from a dietary supplement in Germany in 2018 and submitted to the German National Reference Laboratory for Salmonella. WGS-based comparative genetic analyses were conducted to find a potential reservoir of the isolate itself or mobile genetic elements associated with MDR. As a phylogenetic analysis did not yield any closely related S. Agona isolates, either globally or from Germany, a detailed analysis of the largest plasmid (295,499 bp) was performed as it is the main carrier of resistances. A combined approach of long-read and short-read sequencing enabled the assembly of the isolate's chromosome and its four plasmids. Their characterization revealed the presence of 23 different antibiotic resistance genes (ARGs), conferring resistance to 12 different antibiotic drug classes, as well as genes conferring resistance to six different heavy metals. The largest plasmid, pSE18-SA00377-1, belongs to the IncHI2 plasmid family and carries 16 ARGs, that are organized as two distinct clusters, with each ARG associated with putative composite transposons. Through a two-pronged approach, highly similar plasmids to pSE18-SA00377-1 were identified in the NCBI database and a search for Salmonella isolates with a highly similar ARG resistance profile was conducted. Mapping and structural comparisons between pSE18-SA00377-1 and these plasmids and Salmonella isolates showed that both the plasmid backbone and identical or similar ARG clusters can be found not only in Salmonella isolates, originating mostly from a wide variety of livestock, but also in a diverse range of bacterial genera of varying geographical origins and isolation sources. Thus, it can be speculated that the host range of pSE18-SA00377-1 is not restricted to Salmonella and its spread already occurred in different bacterial populations. Overall, this hints at a complex history for pSE18-SA00377-1 and highlights the importance of surveilling multidrug-resistant S. enterica isolates, especially in novel food items that are not yet heavily regulated.

Genetic and Phenotypic Virulence Potential of Non-O1/Non-O139 Vibrio cholerae Isolated from German Retail Seafood.

Non-O1 and non-O139 Vibrio cholerae (NOVC) can cause gastrointestinal infections in humans. Contaminated food, especially seafood, is an important source of human infections. In this study, the virulence potential of 63 NOVC strains isolated from retail seafood were characterized at the genotypic and phenotypic levels. Although no strain encoded the cholera toxin (CTX) and the toxin-coregulated pilus (TCP), several virulence factors, including the HlyA hemolysin, the cholix toxin ChxA, the heat-stable enterotoxin Stn, and genes coding for the type 3 and type 6 secretion systems, were detected. All strains showed hemolytic activity against human and sheep erythrocytes: 90% (n = 57) formed a strong biofilm, 52% (n = 33) were highly motile at 37 °C, and only 8% (n = 5) and 14% (n = 9) could resist ≥60% and ≥40% human serum, respectively. Biofilm formation and toxin regulation genes were also detected. cgMLST analysis demonstrated that NOVC strains from seafood cluster with clinical NOVC strains. Antimicrobial susceptibility testing (AST) results in the identification of five strains that developed non-wildtype phenotypes (medium and resistant) against the substances of the classes of beta-lactams (including penicillin, carbapenem, and cephalosporin), polymyxins, and sulphonamides. The phenotypic resistance pattern could be partially attributed to the acquired resistance determinants identified via in silico analysis. Our results showed differences in the virulence potential of the analyzed NOVC isolated from retail seafood products, which may be considered for further pathogenicity evaluation and the risk assessment of NOVC isolates in future seafood monitoring.

Arcanobacterium pinnipediorum Strain DSM 28752 Isolated from a Harbour Seal: Complete Genome Sequence.

The genus Arcanobacterium is constantly growing as novel species are identified. In particular, harbor seals have proven to be a common reservoir for bacteria of this genus. Here, we announce the complete genome sequence of another Arcanobacterium species-namely, Arcanobacterium pinnipediorum strain DSM 28752, isolated from a harbor seal.

Genomic Insights into the Mobilome and Resistome of Sentinel Microorganisms Originating from Farms of Two Different Swine Production Systems.

Antimicrobial resistance (AMR) is a threat to public health due to long-term antimicrobial use (AMU), which promotes the bacterial acquisition of antimicrobial resistance determinants (ARDs). Within food-producing animals, organic and extensive Iberian swine production is based on sustainable and eco-friendly management systems, providing an excellent opportunity to evaluate how sustained differences in AMU impact the development and spread of AMR. Here, through a whole-genome sequencing approach, we provide an in-depth characterization of the resistome and mobilome and their interaction in 466 sentinel bacteria, namely, Escherichia coli, Enterococcus spp., Campylobacter coli, and Staphylococcus spp., recovered from 37 intensive and organic-extensive pig farms. Both ARDs and mobile genetic elements (MGEs) were primarily taxon-associated, with higher similarities among bacteria which were closely phylogenetically related. E. coli exhibited the most diverse resistome and mobilome, with 85.4% mobilizable ARDs, 50.3% of which were plasmid-associated. Staphylococcus spp. exhibited a broad repertoire of ARDs and MGEs, with 52.3% of its resistome being mobilizable. Although Enterococcus spp. carried the highest number of ARDs per isolate and its plasmidome was similar in size to that of E. coli, 43.7% of its resistome was mobilizable. A narrow spectrum of ARDs constituted the C. coli resistome, with point mutations as its main AMR driver. A constrained AMU, as observed in organic-extensive herds, determined a reduction in the quantitative composition of the resistome and the complexity of the resistome-mobilome interaction. These results demonstrate taxon-associated AMR-MGE interactions and evidence that responsible AMU can contribute to reducing AMR pressure in the food chain. IMPORTANCE This study provides the first integral genomic characterization of the resistome and mobilome of sentinel microorganisms for antimicrobial resistance (AMR) surveillance from two different swine production systems. Relevant differences were observed among taxa in the resistomes and mobilomes they harbored, revealing their distinctive risk in AMR dissemination and spread. Thus, Escherichia coli and, to a lesser extent, Staphylococcus spp. constituted the main reservoirs of mobilizable antimicrobial resistance genes, which were predominantly plasmid-associated; in contrast to Campylobacter coli, whose resistome was mainly determined by point mutations. The reduced complexity of mobilome-resistome interaction in Enterococcus spp. suggested its limited role in AMR dissemination from swine farms. The significant differences in antimicrobial use among the studied farms allowed us to assess the suitability of whole-genome sequencing as a rapid and efficient technique for the assessment of mid- to long-term on-farm interventions for the reduction of antimicrobial use and the evaluation of AMR status.

Arcanobacterium buesumense sp. nov., isolated from an anal swab of a male harbour seal (Phoca vitulina).

A polyphasic taxonomic study was performed on an unidentified previously described Arcanobacterium-like Gram-positive strain 2701T isolated from an anal swab of a dead male harbour seal. Comparative 16S rRNA sequencing showed that the bacterium belonged to the genus Arcanobacterium in the family Arcanobacteriaceae. The genome sequence of the strain was obtained by Borowiak et al. [1]. The genome had a G+C content of 49 mol% and a total length of 1.94 Mb. The presence of the major menaquinone MK-9(H4) supported the affiliation of the isolate with the genus Arcanobacterium. The polar lipid profile consisted of diphosphatidylglycerol and an unidentified phospholipid as major components and two unidentified lipids, a further unidentified phospholipid, two unidentified phosphoglycolipids as well as phosphatidylglycerol. The major fatty acids were C16 : 0, C18 : 1 and C18 : 0. Biochemical and phylogenetic analyses clearly distinguished the isolate from other members of the genus Arcanobacterium and closely related other species. Based on these results, it is proposed that the unknown Arcanobacterium sp. strain 2701T should be classified as representing a novel species with the name Arcanobacterium buesumense sp. nov. The type strain is 2701T (=DSM 112952T=LMG 32446T).

Complete Genome Sequence of a Salmonella enterica subsp. enterica Serovar Tennessee Strain from Tahini.

Salmonella sp. infections are associated with contaminated low-moisture foods (with high fat content) with increasing frequency. Here, we report the complete genome sequence of Salmonella enterica subsp. enterica serovar Tennessee, which was isolated from tahini (a paste made from ground sesame seeds) purchased at a local retailer in Berlin, Germany.

Complete Genome Sequence of an Aeromonas rivuli Strain Isolated from Ready-to-Eat Food.

Aeromonads can be associated with diseases in animals and humans. Knowledge regarding Aeromonas rivuli, a species recently discovered in creek water in Germany, is still fragmentary. Here, we announce the complete genome sequence of Aeromonas rivuli strain 20-VB00005, which was recovered from ready-to-eat food.

Case Report: Vibrio fluvialis isolated from a wound infection after a piercing trauma in the Baltic Sea.

Vibrio spp. are Gram-negative bacteria found in marine ecosystems. Non-cholera Vibrio spp. can cause gastrointestinal infections and can also lead to wound infections through exposure to contaminated seawater. Vibrio infections are increasingly documented from the Baltic Sea due to extended warm weather periods. We describe the first isolation of Vibrio fluvialis from a wound infection acquired by an impalement injury in the shallow waters of the Baltic Sea. The severe infection required amputation of the third toe. Whole genome sequencing of the isolate was performed and revealed a genome consisting of two circular chromosomes with a size of 1.57 and 3.24 Mb.

What WGS Reveals about Salmonella enterica subsp. enterica in Wildlife in Germany.

The aim of this study was to gain an overview of the genetic diversity of Salmonella found in wildlife in Germany. We were particularly interested in exploring whether wildlife acts as a reservoir of certain serovars/subtypes or antimicrobial resistance (AMR) genes. Moreover, we wanted to explore the potential of Salmonella in spreading from wildlife to livestock and humans. To answer these questions, we sequenced 260 Salmonella enterica subsp. enterica isolates sampled between 2002 and 2020 from wildlife across Germany, using short-read whole genome sequencing. We found, consistent with previous findings, that some Salmonella sequence types are associated with certain animal species, such as S. Choleraesuis ST145 with wild boar and S. Enteritidis ST183 with hedgehogs. Antibiotic resistance was detected in 14.2% of all isolates, with resistance against important WATCH group antibiotics present in a small number of isolates. We further found that wildlife isolates do not form separate phylogenetic clusters distant to isolates from domestic animals and foodstuff, thus indicating frequent transmission events between these reservoirs. Overall, our study shows that Salmonella in German wildlife are diverse, with a low AMR burden and close links to Salmonella populations of farm and food-production environments.

Complete Genome Sequence of Arcanobacterium phocisimile Strain DSM 26142, Isolated from a Harbor Seal.

Bacteria of the genus Arcanobacterium can be found in a variety of hosts. The species Arcanobacterium phocisimile was originally identified in a free-living harbor seal in the German North Sea in 2004. Here, we announce the complete genome sequence of Arcanobacterium phocisimile strain DSM 26142.

The Spatiotemporal Dynamics and Microevolution Events That Favored the Success of the Highly Clonal Multidrug-Resistant Monophasic Salmonella Typhimurium Circulating in Europe.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3' end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

Species-Specific Quality Control, Assembly and Contamination Detection in Microbial Isolate Sequences with AQUAMIS.

Sequencing of whole microbial genomes has become a standard procedure for cluster detection, source tracking, outbreak investigation and surveillance of many microorganisms. An increasing number of laboratories are currently in a transition phase from classical methods towards next generation sequencing, generating unprecedented amounts of data. Since the precision of downstream analyses depends significantly on the quality of raw data generated on the sequencing instrument, a comprehensive, meaningful primary quality control is indispensable. Here, we present AQUAMIS, a Snakemake workflow for an extensive quality control and assembly of raw Illumina sequencing data, allowing laboratories to automatize the initial analysis of their microbial whole-genome sequencing data. AQUAMIS performs all steps of primary sequence analysis, consisting of read trimming, read quality control (QC), taxonomic classification, de-novo assembly, reference identification, assembly QC and contamination detection, both on the read and assembly level. The results are visualized in an interactive HTML report including species-specific QC thresholds, allowing non-bioinformaticians to assess the quality of sequencing experiments at a glance. All results are also available as a standard-compliant JSON file, facilitating easy downstream analyses and data exchange. We have applied AQUAMIS to analyze ~13,000 microbial isolates as well as ~1000 in-silico contaminated datasets, proving the workflow's ability to perform in high throughput routine sequencing environments and reliably predict contaminations. We found that intergenus and intragenus contaminations can be detected most accurately using a combination of different QC metrics available within AQUAMIS.

Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli.

Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E.coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment.