First Description of Mergibacter septicus Isolated from a Common Tern (Sterna hirundo) in Germany.

Mergibacter septicus (M. septicus), previously known as Bisgaard Taxon 40, is a recently described species within the Pasteurellaceae family. In this study, we present a M. septicus strain isolated from a common tern (Sterna hirundo) chick that died just after fledging from the Banter See in Wilhelmshaven, Germany. The recovered M. septicus strain underwent microbiological phenotypic characterization, followed by whole genome sequencing on Illumina and Nanopore platforms. Phenotypically, M. septicus 19Y0039 demonstrated resistance to colistin, cephalexin, clindamycin, oxacillin, and penicillin G. The genome analysis revealed a circular 1.8 Mbp chromosome without any extrachromosomal elements, containing 1690 coding DNA sequences. The majority of these coding genes were associated with translation, ribosomal structure and biogenesis, followed by RNA processing and modification, and transcription. Genetic analyses revealed that the German M. septicus strain 19Y0039 is related to the American strain M. septicus A25201T. Through BLAST alignment, twelve putative virulence genes previously identified in the M. septicus type strain A25201T were also found in the German strain. Additionally, 84 putative virulence genes distributed across nine categories, including immune modulation, effector delivery system, nutrition/metabolic factors, regulation, stress survival, adherence, biofilm, exotoxin, and motility, were also identified.

Whole-genome sequencing (WGS) analysis of Brucella suis biovar 2 isolated from domestic pigs in Egypt for epidemiological and genetic diversity tracing.

In the current study, 14 Brucella suis biovar 2 (B. suis bv 2) strains isolated from slaughter pigs in Cairo were sequenced using Illumina technology to investigate genetic diversity, antimicrobial resistance (AMR) genes, and virulence-associated determinants. These strains were the first B. suis bv 2 isolates from Egypt. To place them in a global context, 92 genomes of B. suis were retrieved from the NCBI database and used for comparison. The in-silico analysis of MLST showed that all isolates have ST16. No resistome but 43 virulomes have been found without differences in distribution. The cgMLST classified the Egyptian B. suis strains into a complex type (CT) encompassing four distinct cgMLST sequence types. The closest relatives were strain B. suis 94/11 of an unknown origin and a Danish strain. Whole-genome sequencing analysis proved low diversity of Egyptian B. suis isolates; thus, a single introduction event is assumed. Investigation of a large number of B. suis isolates from different governorates is required to tailor control measures to avoid further spread.

Tracking the distribution, genetic diversity and lineage of Brucella melitensis recovered from humans and animals in Egypt based on core-genome SNP analysis and in silico MLVA-16.

Brucellosis is one of the most common neglected zoonotic diseases globally, with a public health significance and a high economic loss in the livestock industry caused by the bacteria of the genus Brucella. In this study, 136 Egyptian Brucella melitensis strains isolated from animals and humans between 2001 and 2020 were analysed by examining the whole-core-genome single-nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA-16). Almost all Egyptian isolates were belonging to the West Mediterranean clade, except two isolates from buffalo and camel were belonging to the American and East Mediterranean clades, respectively. A significant correlation between the human case of brucellosis and the possible source of infection from animals was found. It seems that several outbreak strains already existing for many years have been spread over long distances and between many governorates. The cgSNP analysis, in combination with epidemiological metadata, allows a better differentiation than the MLVA-16 genotyping method and, hence, the source definition and tracking of outbreak strains. The MLVA based on the currently used 16 markers is not suitable for this task. Our results revealed 99 different cgSNP genotypes with many different outbreak strains, both older and widely distributed ones and rather newly introduced ones as well. This indicates several different incidents and sources of infections, probably by imported animals from other countries to Egypt. Comparing our panel of isolates to public databases by cgSNP analysis, the results revealed near relatives from Italy. Moreover, near relatives from the United States, France, Austria and India were found by in silico MLVA.

Core Genome Multilocus Sequence Typing Scheme for Improved Characterization and Epidemiological Surveillance of Pathogenic Brucella.

Brucellosis poses a significant burden to human and animal health worldwide. Robust and harmonized molecular epidemiological approaches and population studies that include routine disease screening are needed to efficiently track the origin and spread of Brucella strains. Core genome multilocus sequence typing (cgMLST) is a powerful genotyping system commonly used to delineate pathogen transmission routes for disease surveillance and control. Except for Brucella melitensis, cgMLST schemes for Brucella species are currently not established. Here, we describe a novel cgMLST scheme that covers multiple Brucella species. We first determined the phylogenetic breadth of the genus using 612 Brucella genomes. We selected 1,764 genes that were particularly well conserved and typeable in at least 98% of these genomes. We tested the new scheme on 600 genomes and found high agreement with the whole-genome-based single nucleotide polymorphism (SNP) analysis. Next, we applied the scheme to reanalyze the genome of Brucella strains from epidemiologically linked outbreaks. We demonstrated the applicability of the new scheme for high-resolution typing required in outbreak investigations as previously reported with whole-genome SNP methods. We also used the novel scheme to define the global population structure of the genus using 1,322 Brucella genomes. Finally, we demonstrated the possibility of tracing distribution of Brucella strains by performing cluster analysis of cgMLST profiles and found nearly identical cgMLST profiles in different countries. Our results show that sequencing depth of more than 40-fold is optimal for allele calling with this scheme. In summary, this study describes a novel Brucella-wide cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection. The scheme is publicly accessible and should represent a valuable resource for laboratories with limited computational resources and bioinformatics expertise.

Genomic epidemiology of Campylobacter fetus subsp. venerealis from Germany.

Campylobacter fetus subsp. venerealis (Cfv) causes bovine genital campylobacteriosis (BGC), a World Organization for Animal Health (WOAH)-listed trade-relevant disease characterized by severe reproductive losses, such as infertility, early embryonic death and abortion in cattle. BGC has significant economic implications that have prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. In Germany, there has been a low incidence of BGC cases over the past 28 years. This study aimed to illustrate the genomic diversity of German Cfv strains isolated from different federal states in Germany. This study analyzed 63 Cfv strains, collected between 1985 and 2015, by whole-genome sequencing and compared them with genome data of 91 international Cfv isolates. The phylogenetic analysis showed that the Cfv population is genetically conserved and has geographic clusters. In Germany, one phylogenetic lineage comprising all strains was identified. This German lineage was part of a subclade that probably emerged in the nineteenth century and diversified over time. The results of this study point to a non-recurrent cross-border introduction of Cfv in Germany. The BGC control interventions in Germany can be considered successful as no outbreaks were reported since 2015.

First Comparative Analysis of Clostridium septicum Genomes Provides Insights Into the Taxonomy, Species Genetic Diversity, and Virulence Related to Gas Gangrene.

Clostridium septicum is a Gram-positive, toxin-producing, and spore-forming bacterium that is recognized, together with C. perfringens, as the most important etiologic agent of progressive gas gangrene. Clostridium septicum infections are almost always fatal in humans and animals. Despite its clinical and agricultural relevance, there is currently limited knowledge of the diversity and genome structure of C. septicum. This study presents the complete genome sequence of C. septicum DSM 7534T type strain as well as the first comparative analysis of five C. septicum genomes. The taxonomy of C. septicum, as revealed by 16S rRNA analysis as well as by genomic wide indices such as protein-based phylogeny, average nucleotide identity, and digital DNA-DNA hybridization indicates a stable clade. The composition and presence of prophages, CRISPR elements and accessory genetic material was variable in the investigated genomes. This is in contrast to the limited genetic variability described for the phylogenetically and phenotypically related species Clostridium chauvoei. The restriction-modification (RM) systems between two C. septicum genomes were heterogeneous for the RM types they encoded. C. septicum has an open pangenome with 2,311 genes representing the core genes and 1,429 accessory genes. The core genome SNP divergence between genome pairs varied up to 4,886 pairwise SNPs. A vast arsenal of potential virulence genes was detected in the genomes studied. Sequence analysis of these genes revealed that sialidase, hemolysin, and collagenase genes are conserved compared to the α-toxin and hyaluronidase genes. In addition, a conserved gene found in all C. septicum genomes was predicted to encode a leucocidin homolog (beta-channel forming cytolysin) similar (71.10% protein identity) to Clostridium chauvoei toxin A (CctA), which is a potent toxin. In conclusion, our results provide first, valuable insights into strain relatedness and genomic plasticity of C. septicum and contribute to our understanding of the virulence mechanisms of this important human and animal pathogen.

Phylogenetic Relatedness and Genome Structure of Yersinia ruckeri Revealed by Whole Genome Sequencing and a Comparative Analysis.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a serious infection that affects global aquaculture with high economic impact. The present study used whole genome sequences to perform a comparative analysis on 10 Y. ruckeri strains and to explore their genetic relatedness to other members of the genus. Y. ruckeri, Yersinia entomophaga, and Yersinia nurmii formed a species complex that constitutes the most basal lineage of the genus. The results showed that the taxonomy of Y. ruckeri strains is better defined by using a core genome alignment and phylogenetic analysis. The distribution of accessory genes in all Yersinia species revealed the presence of 303 distinctive genes in Y. ruckeri. Of these, 169 genes were distributed in 17 genomic islands potentially involved in the pathogenesis of ERM via (1) encoding virulence factors such as Afp18, Yrp1, phage proteins and (2) improving the metabolic capabilities by enhancing utilization and metabolism of iron, amino acids (specifically, arginine and histidine), and carbohydrates. The genome of Y. ruckeri is highly conserved regarding gene structure, gene layout and functional categorization of genes. It contains various components of mobile genetic elements but lacks the CRISPR-Cas system and possesses a stable set of virulence genes possibly playing a critical role in pathogenicity. Distinct virulence plasmids were exclusively restricted to a specific clonal group of Y. ruckeri (CG4), possibly indicating a selective advantage. Phylogenetic analysis of Y. ruckeri genomes revealed the co-presence of multiple genetically distant lineages of Y. ruckeri strains circulating in Germany. Our results also suggest a possible dissemination of a specific group of strains in the United States, Peru, Germany, and Denmark. In conclusion, this study provides new insights into the taxonomy and evolution of Y. ruckeri and contributes to a better understanding of the pathogenicity of ERM in aquaculture. The genomic analysis presented here offers a framework for the development of more efficient control strategies for this pathogen.

Genome Sequence Analysis of Clostridium chauvoei Strains of European Origin and Evaluation of Typing Options for Outbreak Investigations.

Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.

Establishment of a Publicly Available Core Genome Multilocus Sequence Typing Scheme for Clostridium perfringens.

Clostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution genotyping aiming to identify bacteria at strain level has become increasingly important in modern microbiology to understand pathogen transmission pathways and to tackle infection sources. This study aimed at establishing a publicly available genome-wide multilocus sequence-typing (MLST) scheme for C. perfringens. A total of 1,431 highly conserved core genes (1.34 megabases; 50% of the reference genome genes) were indexed for a core genome-based MLST (cgMLST) scheme for C. perfringens. The scheme was applied to 282 ecologically and geographically diverse genomes, showing that the genotyping results of cgMLST were highly congruent with the core genome-based single-nucleotide-polymorphism typing in terms of resolution and tree topology. In addition, the cgMLST provided a greater discrimination than classical MLST methods for C. perfringens. The usability of the scheme for outbreak analysis was confirmed by reinvestigating published outbreaks of C. perfringens-associated infections in the United States and the United Kingdom. In summary, a publicly available scheme and an allele nomenclature database for genomic typing of C. perfringens have been established and can be used for broad-based and standardized epidemiological studies. IMPORTANCE Global epidemiological surveillance of bacterial pathogens is enhanced by the availability of standard tools and sharing of typing data. The use of whole-genome sequencing has opened the possibility for high-resolution characterization of bacterial strains down to the clonal and subclonal levels. Core genome multilocus sequence typing is a robust system that uses highly conserved core genes for deep genotyping. The method has been successfully and widely used to describe the epidemiology of various bacterial species. Nevertheless, a cgMLST typing scheme for Clostridium perfringens is currently not publicly available. In this study, we (i) developed a cgMLST typing scheme for C. perfringens, (ii) evaluated the performance of the scheme on different sets of C. perfringens genomes from different hosts and geographic regions as well as from different outbreak situations, and, finally, (iii) made this scheme publicly available supported by an allele nomenclature database for global and standard genomic typing.

Genomic Characterization of Multidrug-Resistant Salmonella Serovars Derby and Rissen From the Pig Value Chain in Vietnam.

Nontyphoidal Salmonella (NTS) is the most reported cause of bacterial foodborne zoonoses in Vietnam, and contaminated pork is one of the main sources of human infection. In recent years, the prevalence of NTS carrying multiple antimicrobial resistance genes (ARGs) have been increased. The genomic characterization along the pig value chain and the identification of ARGs and plasmids have the potential to improve food safety by understanding the dissemination of ARGs from the farm to the table. We report an analysis of 13 S. Derby and 10 S. Rissen isolates, collected in 2013 at different stages in Vietnamese slaughterhouses and markets. VITEK 2 Compact System was used to characterize the phenotypical antimicrobial resistance of the isolates. In addition, whole-genome sequencing (WGS) was used to detect ARGs and plasmids conferring multidrug resistance. Whole genome single nucleotide polymorphism typing was used to determine the genetic diversity of the strains and the spread of ARGs along the pig value chain. Altogether, 86.9% (20/23) of the samples were resistant to at least one antibiotic. Resistance to ampicillin was most frequently detected (73.9%), followed by piperacillin and moxifloxacin (both 69.6%). At least one ARG was found in all strains, and 69.6% (16/23) were multidrug-resistant (MDR). The observed phenotype and genotype of antimicrobial resistance were not always concordant. Plasmid replicons were found in almost all strains [95.6% (22/23)], and the phylogenetic analysis detected nine clusters (S. Derby, n = 5; S. Rissen, n = 4). ARGs and plasmid content were almost identical within clusters. We found six MDR IncHI1s with identical plasmid sequence type in strains of different genetic clusters at the slaughterhouse and the market. In conclusion, high rates of multidrug resistance were observed in Salmonella strains from Vietnam in 2013. Genomic analysis revealed many resistance genes and plasmids, which have the potential to spread along the pig value chain from the slaughterhouse to the market. This study pointed out that bioinformatics analyses of WGS data are essential to detect, trace back, and control the MDR strains along the pig value chain. Further studies are necessary to assess the more recent MDR Salmonella strains spreading in Vietnam.

A Whole-Genome-Based Gene-by-Gene Typing System for Standardized High-Resolution Strain Typing of Bacillus anthracis.

Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical single-nucleotide-polymorphism (SNP) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cutoff threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis with human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly available for application by users, including those with little bioinformatics knowledge.

Comparative in silico genome analysis of Clostridium perfringens unravels stable phylogroups with different genome characteristics and pathogenic potential.

Clostridium perfringens causes a plethora of devastating infections, with toxin production being the underlying mechanism of pathogenicity in various hosts. Genomic analyses of 206 public-available C. perfringens strains´ sequence data identified a substantial degree of genomic variability in respect to episome content, chromosome size and mobile elements. However, the position and order of the local collinear blocks on the chromosome showed a considerable degree of preservation. The strains were divided into five stable phylogroups (I-V). Phylogroup I contained human food poisoning strains with chromosomal enterotoxin (cpe) and a Darmbrand strain characterized by a high frequency of mobile elements, a relatively small genome size and a marked loss of chromosomal genes, including loss of genes encoding virulence traits. These features might correspond to the adaptation of these strains to a particular habitat, causing human foodborne illnesses. This contrasts strains that belong to phylogroup II where the genome size points to the acquisition of genetic material. Most strains of phylogroup II have been isolated from enteric lesions in horses and dogs. Phylogroups III, IV and V are heterogeneous groups containing a variety of different strains, with phylogroup III being the most abundant (65.5%). In conclusion, C. perfringens displays five stable phylogroups reflecting different disease involvements, prompting further studies on the evolution of this highly important pathogen.

Phylogenomic Analysis of Campylobacter fetus Reveals a Clonal Structure of Insertion Element ISCfe1 Positive Genomes.

Subspecies of the species Campylobacter fetus are associated with specific host niches including mammals and reptiles. Campylobacter fetus subsp. fetus is a zoonotic pathogen infecting humans. Infections can vary from an acute intestinal illness to severe systemic infections, with sheep and cattle as major reservoirs. In contrast, Campylobacter fetus subsp. venerealis causes bovine genital campylobacteriosis, which leads to abortion in cattle and a high economic burden for the farmers. Therefore, high-quality molecular subtyping is indispensable for interventional epidemiology. We used whole-genome sequencing (WGS) data of 283 Campylobacter fetus strains from 18 countries and compared several methods for Campylobacter fetus subtyping, including WGS, multilocus sequence typing, PCR assays, and the presence of the insertion element ISCfe1. We identified a highly clonal clade (designated as clade 1) that harbors the insertion sequence ISCfe1. The presence of this insertion sequence is an essential diagnostic tool for the identification of the subspecies Campylobacter fetus subsp. venerealis, serving as a target for several PCR assays. However, we have found a high sequence variability for the ISCfe1 besides the presence of ISCfe1-paralogues in certain other genomes (n = 7) which may cause incorrect diagnostic results. Clade 1 seems to be the cattle-specific clade of this species. We propose that only this clade might be designated as Campylobacter fetus subsp. venerealis as it harbors the ISCfe1 marker sequence, which is a major target for molecular methods currently used for Campylobacter fetus subspecies identification. Fostering this proposal, we defined eleven stable nucleotide markers specific for this clade. Additionally, we developed a bioinformatics toolbox for the fast identification of this clade based on WGS data. In conclusion, our results demonstrate that WGS can be used for Campylobacter fetus subtyping overcoming limitations of current PCR and MLST protocols.

Using affinity propagation clustering for identifying bacterial clades and subclades with whole-genome sequences of Francisella tularensis.

By combining a reference-independent SNP analysis and average nucleotide identity (ANI) with affinity propagation clustering (APC), we developed a significantly improved methodology allowing resolving phylogenetic relationships, based on objective criteria. These bioinformatics tools can be used as a general ruler to determine phylogenetic relationships and clustering of bacteria, exemplary done with Francisella (F.) tularensis. Molecular epidemiology of F. tularensis is currently assessed mostly based on laboratory methods and molecular analysis. The high evolutionary stability and the clonal nature makes Francisella ideal for subtyping with single nucleotide polymorphisms (SNPs). Sequencing and real-time PCR can be used to validate the SNP analysis. We investigate whole-genome sequences of 155 F. tularensis subsp. holarctica isolates. Phylogenetic testing was based on SNPs and average nucleotide identity (ANI) as reference independent, alignment-free methods taking small-scale and large-scale differences within the genomes into account. Especially the whole genome SNP analysis with kSNP3.0 allowed deciphering quite subtle signals of systematic differences in molecular variation. Affinity propagation clustering (APC) resulted in three clusters showing the known clades B.4, B.6, and B.12. These data correlated with the results of real-time PCR assays targeting canSNPs loci. Additionally, we detected two subtle sub-clusters. SplitsTree was used with standard-setting using the aligned SNPs from Parsnps. Together APC, HierBAPS, and SplitsTree enabled us to generate hypotheses about epidemiologic relationships between bacterial clusters and describing the distribution of isolates. Our data indicate that the choice of the typing technique can increase our understanding of the pathogenesis and transmission of diseases with the eventual for prevention. This is opening perspectives to be applied to other bacterial species. The data provide evidence that Germany might be the collision zone where the clade B.12, also known as the East European clade, overlaps with the clade B.6, also known as the Iberian clade. Described methods allow generating a new, more detailed perspective for F. tularensis subsp. holarctica phylogeny. These results may encourage to determine phylogenetic relationships and clustering of other bacteria the same way.

Aliarcobacter butzleri from Water Poultry: Insights into Antimicrobial Resistance, Virulence and Heavy Metal Resistance.

Aliarcobacter butzleri is the most prevalent Aliarcobacter species and has been isolated from a wide variety of sources. This species is an emerging foodborne and zoonotic pathogen because the bacteria can be transmitted by contaminated food or water and can cause acute enteritis in humans. Currently, there is no database to identify antimicrobial/heavy metal resistance and virulence-associated genes specific for A. butzleri. The aim of this study was to investigate the antimicrobial susceptibility and resistance profile of two A. butzleri isolates from Muscovy ducks (Cairina moschata) reared on a water poultry farm in Thuringia, Germany, and to create a database to fill this capability gap. The taxonomic classification revealed that the isolates belong to the Aliarcobacter gen. nov. as A. butzleri comb. nov. The antibiotic susceptibility was determined using the gradient strip method. While one of the isolates was resistant to five antibiotics, the other isolate was resistant to only two antibiotics. The presence of antimicrobial/heavy metal resistance genes and virulence determinants was determined using two custom-made databases. The custom-made databases identified a large repertoire of potential resistance and virulence-associated genes. This study provides the first resistance and virulence determinants database for A. butzleri.

Emergence of Multidrug-Resistant Salmonella enterica Subspecies enterica Serovar Infantis of Multilocus Sequence Type 2283 in German Broiler Farms.

During the last decade, Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) has become more prevalent across Europe with an increased capability to persist in broiler farms. In this study, we aimed to identify potential genetic causes for the increased emergence and longer persistence of S. Infantis in German poultry farms by high-throughput-sequencing. Broiler derived S. Infantis strains from two decades, the 1990s (n = 12) and the 2010s (n = 18), were examined phenotypically and genotypically to detect potential differences responsible for increased prevalence and persistence. S. Infantis organisms were characterized by serotyping and determining antimicrobial susceptibility using the microdilution method. Genotypic characteristics were analyzed by whole genome sequencing (WGS) to detect antimicrobial resistance and virulence genes as well as plasmids. To detect possible clonal relatedness within S. Infantis organisms, 17 accessible genomes from previous studies about emergent S. Infantis were downloaded and analyzed using complete genome sequence of SI119944 from Israel as reference. In contrast to the broiler derived antibiotic-sensitive S. Infantis strains from the 1990s, the majority of strains from the 2010s (15 out of 18) revealed a multidrug-resistance (MDR) phenotype that encodes for at least three antimicrobials families: aminoglycosides [ant(3")-Ia], sulfonamides (sul1), and tetracyclines [tet(A)]. Moreover, these MDR strains carry a virulence gene pattern missing in strains from the 1990s. It includes genes encoding for fimbriae clusters, the yersiniabactin siderophore, mercury and disinfectants resistance and toxin/antitoxin complexes. In depth genomic analysis confirmed that the 15 MDR strains from the 2010s carry a pESI-like megaplasmid with resistance and virulence gene patterns detected in the emerged S. Infantis strain SI119944 from Israel and clones inside and outside Europe. Genotyping analysis revealed two sequence types (STs) among the resistant strains from the 2010s, ST2283 (n = 13) and ST32 (n = 2). The sensitive strains from the 1990s, belong to sequence type ST32 (n = 10) and ST1032 (n = 2). Therefore, this study confirms the emergence of a MDR S. Infantis pESI-like clone of ST2283 in German broiler farms with presumably high tendency of dissemination. Further studies on the epidemiology and control of S. Infantis in broilers are needed to prevent the transfer from poultry into the human food chain.

Genomic Analysis and Antimicrobial Resistance of Aliarcobacter cryaerophilus Strains From German Water Poultry.

Aliarcobacter cryaerophilus (formerly Arcobacter cryaerophilus) is a globally emerging foodborne and zoonotic pathogen. However, little is known about the species' genomic features and diversity, antibiotic resistance and virulence. In this study, 27 A. cryaerophilus strains from water poultry in Thuringia, Germany, were investigated using whole-genome sequencing. Four of these strains were sequenced using long- and short-read sequencing methods to obtain circularized genomes. The German strains belong to the A. cryaerophilus cluster I. Cluster I genomes exhibited a high degree of genetic diversity in which variable sites comprised 9.1% of the core genome. The German strains formed three subgroups that contained 2, 6, and 9 strains, respectively. The genomic analysis of cluster I revealed variable presence of mobile elements and that 65% of the strains lack CRISPR systems. The four circularized genomes carried a ∼2 Mbp chromosome and a single megaplasmid (size 98.1-154.5 Kbp). The chromosome was densely packed with coding sequences (∼92%) and showed inversions and shifts in the gene blocks between different strains. Antimicrobial resistance was assessed using a gradient strip diffusion method and showed that all 27 strains were resistant to cefotaxime and susceptible to erythromycin, gentamicin, and ampicillin. Sixteen strains were also resistant to ciprofloxacin, whereas 23 were resistant to streptomycin. The genetic prediction of antibiotic resistance identified numerous efflux pumps similar to those found in A. butzleri. All strains harbored two beta-lactamase genes which may explain the cefotaxime resistance. A correlation between the gyrA point mutation (Thr-85-Ile) and ciprofloxacin resistance was partially discovered in 15 out of 16 strains. In silico virulence profiling showed a wide range of virulence factors including a full chemotaxis system and most of the flagellar genes. In contrast to A. butzleri, no urease cluster was found. This study provides new insights into the genomic variability of A. cryaerophilus strains of cluster I. The different genetic makeup of these strains may contribute to the virulence of strains and the severity of the infections in humans.

Draft genome sequence of multi-resistant Salmonella enterica subsp. enterica serovar Rissen strain 19CS0416 isolated from Vietnam reveals mcr-1 plasmid mediated resistance to colistin already in 2013.

We report the first draft genome sequence of a Salmonella strain with plasmid-mediated resistance to colistin encoded by mcr-1 gene in Vietnam. Salmonella enterica subsp. enterica serovar Rissen was isolated from a Vietnamese pig slaughterhouse in 2013. We can confirm that mcr-1 gene is identical to the first reported mcr-1 gene of the Escherichia coli strain SHP45, isolated in 2015 from a Chinese pig. The plasmid containing this gene in the strain 19CS0416 was highly related (96.86% identity) to the plasmid (pHNSHP45) contained in this Chinese strain. Moreover, this plasmid was determined to be 100% identical to a plasmid (p13P477T-7) belonging to an Escherichia coli (13P477T) found in Hong Kong during the same year in pigs. Our results will aid in understanding the dissemination of mcr-1 gene in East Asia, dating back to as early as 2013.

MLVA-16 Genotyping of Brucella abortus and Brucella melitensis Isolates from Different Animal Species in Egypt: Geographical Relatedness and the Mediterranean Lineage.

Brucellosis is a common zoonotic disease in Egypt. However, there are limited data available on the genetic diversity of brucellae circulating in Egypt and other Mediterranean areas. One hundred and nine Brucella (B.) strains were isolated from different animal species in thirteen Egyptian governorates. Multi-locus variable number tandem repeats (VNTRs) analysis (MLVA-16) was employed to determine the geographical relatedness and the genetic diversity of a panel of selected Egyptian strains (n = 69), with strains originating from Italy (n = 49), Portugal (n = 52), Greece (n = 63), and Tunisia (n = 4). Egyptian B. melitensis strains clustered into two main clusters containing 21 genotypes. Egyptian B. abortus strains clustered into three main clusters containing nine genotypes. The genotypes were irregularly distributed over time and space in the study area. Egyptian strains of B. melitensis showed MLVA-16 patterns closer to that of Italian strains. Egyptian B. abortus strains isolated from cattle share the same genotype with strains from Portugal and similar to strains from Italy with low genetic diversity. Strains with similar MLVA patterns isolated from different governorates highlight the movement of the pathogen among governorates. Hence, it may also reflect the long endemicity of brucellosis in Egypt with earlier dispersal of types and great local genetic diversity. Open markets may contribute to cross-species transmission and dissemination of the new types nationwide. The presence of West Mediterranean lineages of B. melitensis and relatedness of B. abortus strains from the studied countries is a result of the socio-historical connections among the Mediterranean countries. Transnational eradication of brucellosis in the Mediterranean basin is highly demanded.

Multilocus sequence typing of Clostridium perfringens strains from neonatal calves, dairy workers and associated environment in India.

Clostridium perfringens is a globally recognized zoonotic pathogen. We report isolation and genotyping of C. perfringens from neonatal calves, dairy workers and their associated environment in India. A total of 103 fecal samples from neonatal calves, 25 stool swabs from the dairy workers and 50 samples from their associated environment were collected from two dairy farms. C. perfringens was detected in 26 out of 103 (25.2%) neonatal calf samples, 7 out of 25 (28%) human stool samples and 17 out of 50 (34%) environmental samples. C. perfringens type A strains were predominant in neonatal calves (24/26; 92.3%) and associated environment (15/17; 88.2%). In contrast, strains from dairy workers mostly belonged to type F (5/7; 71.4%), which also carried the beta2 toxin gene. Seventeen strains were analyzed by multilocus sequence typing (MLST) for studying genotypic relationship along with 188 C. perfringens strains available from public databases. A total of 112 sequence types (STs) were identified from 205 C. perfringens strains analyzed. A Clonal complex (CC) represented by three STs (ST 98, ST 41 and ST 110) representing predominantly type F (18/20 strains) were mostly associated with human illnesses. Among predominant STs, ST 54 was associated with enteritis cases in foals and dogs and ST 58 associated with necrotic enteritis in poultry. Seventeen Indian strains were assigned to 13 STs. Genetic relatedness among strains of calves, dairy worker and associated environments indicate inter-host transfers and zoonotic spreads.