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).

Performance and Accuracy of Four Open-Source Tools for In Silico Serotyping of Salmonella spp. Based on Whole-Genome Short-Read Sequencing Data.

We compared the performance of four open-source in silico Salmonella typing tools (SeqSero, SeqSero2, Salmonella In Silico Typing Resource [SISTR], and Metric Oriented Sequence Typer [MOST]) to assess their potential for replacing laboratory serological testing with serovar predictions from whole-genome sequencing data. We conducted a retrospective analysis of 1,624 Salmonella isolates of 72 serovars submitted to the German National Salmonella Reference Laboratory between 1999 and 2019. All isolates are derived from animal and foodstuff origins. We conducted Illumina short-read sequencing and compared the in silico serovar prediction results with the results of routine laboratory serotyping. We found the best-performing in silico serovar prediction tool to be SISTR, with 94% correctly typed isolates, followed by SeqSero2 (87%), SeqSero (81%), and MOST (79%). Furthermore, we found that mapping-based tools like SeqSero and SeqSero2 (allele mode) were more reliable for the prediction of monophasic variants, while sequence type and cluster-based methods like MOST and SISTR (core-genome multilocus sequence type [cgMLST]), showed greater resilience when confronted with GC-biased sequencing data. We showed that the choice of library preparation kit could substantially affect O antigen detection, due to the low GC content of the wzx and wzy genes. Although the accuracy of computational serovar predictions is still not quite on par with traditional serotyping by Salmonella reference laboratories, the command-line tools investigated in this study perform a rapid, efficient, inexpensive, and reproducible analysis, which can be integrated into in-house characterization pipelines. Based on our results, we find SISTR most suitable for automated, routine serotyping for public health surveillance of SalmonellaIMPORTANCESalmonella spp. are important foodborne pathogens. To reduce the number of infected patients, it is essential to understand which subtypes of the bacteria cause disease outbreaks. Traditionally, characterization of Salmonella requires serological testing, a laboratory method by which Salmonella isolates can be classified into over 2,600 distinct subtypes, called serovars. Due to recent advances in whole-genome sequencing, many tools have been developed to replace traditional testing methods with computational analysis of genome sequences. It is crucial to validate that these tools, many already in use for routine surveillance, deliver accurate and reliable serovar information. In this study, we set out to compare which of the currently available open-source command-line tools is most suitable to replace serological testing. A thorough evaluation of the differing computational approaches is highly important to ensure the backward compatibility of serotyping data and to maintain comparability between laboratories.

Arcanobacterium bovis sp. nov., isolated from the milk of a cow with mastitis.

A polyphasic taxonomic study was performed on an unidentified Arcanobacterium-like Gram-stain-positive bacterium designated strain C605018/01/1T isolated from a milk sample collected from the udder of a cow at post mortem. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strain of Arcanobacterium pluranimalium (99.76 %); sequence similarities to all other Arcanobacterium species were below 97 %. The wet-lab DNA-DNA hybridization values among strain C605018/01/1T and A. pluranimalium DSM 13483ᵀ were low, 16.9 % (reciprocal, 49.8 %). Pertaining to the whole genome sequence with a total length of 2.02 Mb and 1654 protein counts, the novel strain C605018/01/01T displayed a G+C content of 51.6 % mol%. The presence of the major menaquinone MK-9(H4) supported the affiliation of this strain to the genus Arcanobacterium. The polar lipid profile consisted of the major components diphosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylinositol-mannoside and unidentified glycolipid and aminophospholipids. Based on these results it is proposed that strain C605018/01/1T should be classified as representing a novel species, Arcanbacterium bovis sp. nov. The type strain C605018/01/1T (CCUG 45425T=DSM 107286T=BCCM/LMG 30783T).

Characterization of mcr-5-Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany.

We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution.

The blaNDM-1-Carrying IncA/C2 Plasmid Underlies Structural Alterations and Cointegrate Formation In Vivo.

In 2012, a carbapenemase-producing Salmonella enterica serovar Corvallis isolate carrying a blaNDM-1 multiresistance IncA/C2 plasmid, apart from IncHI2 and ColE-like plasmids, was detected in a wild bird in Germany. In a recent broiler chicken infection study, we observed transfer of this blaNDM-1-carrying IncA/C2 plasmid to other Enterobacteriaceae Here, we focused on the stability of this plasmid and gained insight into the type and frequency of its structural alterations after an in vivo passage in a broiler chicken infection study.

Salmonellosis outbreak with novel Salmonella enterica subspecies enterica serotype (11:z41:e,n,z15) attributable to sesame products in five European countries, 2016 to 2017.

In spring 2016, Greece reported an outbreak caused by a previously undescribed Salmonella enterica subsp. enterica serotype (antigenic formula 11:z41:e,n,z15) via the Epidemic Intelligence Information System for Food- and Waterborne Diseases and Zoonoses (EPIS-FWD), with epidemiological evidence for sesame products as presumptive vehicle. Subsequently, Germany, Czech Republic, Luxembourg and the United Kingdom (UK) reported infections with this novel serotype via EPIS-FWD. Concerned countries in collaboration with the European Centre for Disease Prevention and Control (ECDC) and European Food Safety Authority (EFSA) adopted a common outbreak case definition. An outbreak case was defined as a laboratory-confirmed notification of the novel Salmonella serotype. Between March 2016 and April 2017, 47 outbreak cases were notified (Greece: n = 22; Germany: n = 13; Czech Republic: n = 5; Luxembourg: n = 4; UK: n = 3). Whole genome sequencing revealed the very close genetic relatedness of isolates from all affected countries. Interviews focusing on sesame product consumption, suspicious food item testing and trace-back analysis following Salmonella spp. detection in food products identified a company in Greece where sesame seeds from different countries were processed. Through European collaboration, it was possible to identify and recall sesame spread as one contaminated food item serving as vehicle of infection and trace it back to its origin.

In Vivo Transfer and Microevolution of Avian Native IncA/C2blaNDM-1-Carrying Plasmid pRH-1238 during a Broiler Chicken Infection Study.

The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) in wildlife and livestock animals pose an important safety concern for public health. With our in vivo broiler chicken infection study, we investigated the transfer and experimental microevolution of the blaNDM-1-carrying IncA/C2 plasmid (pRH-1238) introduced by avian native Salmonella enterica subsp. enterica serovar Corvallis without inducing antibiotic selection pressure. We evaluated the dependency of the time point of inoculation on donor (S Corvallis [12-SA01738]) and plasmid-free Salmonella recipient [d-tartrate-fermenting (d-Ta+) S Paratyphi B (13-SA01617), referred to here as S Paratyphi B (d-Ta+)] excretion by quantifying their excretion dynamics. Using plasmid profiling by S1 nuclease-restricted pulsed-field gel electrophoresis, we gained insight into the variability of the native plasmid content among S Corvallis reisolates as well as plasmid acquisition in S Paratyphi B (d-Ta+) and the enterobacterial gut microflora. Whole-genome sequencing enabled us to gain an in-depth insight into the microevolution of plasmid pRH-1238 in S Corvallis and enterobacterial recipient isolates. Our study revealed that the fecal excretion of avian native carbapenemase-producing S Corvallis is significantly higher than that of S Paratyphi (d-Ta+) and is not hampered by S Paratyphi (d-Ta+). Acquisition of pRH-1238 in other Enterobacteriaceae and several events of plasmid pRH-1238 transfer to different Escherichia coli sequence types and Klebsiella pneumoniae demonstrated an interspecies broad host range. Regardless of the microevolutionary structural deletions in pRH-1238, the single carbapenem resistance marker blaNDM-1 was maintained on pRH-1238 throughout the trial. Furthermore, we showed the importance of the gut E. coli population as a vector of pRH-1238. In a potential scenario of the introduction of NDM-1-producing S Corvallis into a broiler flock, the pRH-1238 plasmid could persist and spread to a broad host range even in the absence of antibiotic pressure.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes.

Background and aimPlasmid-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-4.3, 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.

Molecular Tracing to Find Source of Protracted Invasive Listeriosis Outbreak, Southern Germany, 2012-2016.

We investigated 543 Listeria monocytogenes isolates from food having a temporal and spatial distribution compatible with that of the invasive listeriosis outbreak occurring 2012-2016 in southern Germany. Using forensic microbiology, we identified several products from 1 manufacturer contaminated with the outbreak genotype. Continuous molecular surveillance of food isolates could prevent such outbreaks.

Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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.

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.

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.

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.

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.

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.

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.