Complete high-quality genome sequence of Clostridium limosum (Hathewaya limosa) isolate 14S0207, recovered from a cow with suspected blackleg in Germany

Clostridium limosum can be found in soil and the intestinal tract of animals. In 2014, C. limosum was isolated from a suspected blackleg outbreak in cattle in Schleswig-Holstein, Germany. We present a complete genome sequence of a C. limosum strain represented by a circular chromosome and three plasmids

Clostridioides difficile in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates

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

Presence of Clostridium difficile in poultry and poultry meat in Egypt

C. difficile has been recognized as a potential zoonotic agent encouraging investigations of C. difficile prevalence and ribotypes in animals. Here we report the prevalence and diversity of Egyptian C. difficile in I) samples from healthy poultry (n = 50), II) samples from diseased poultry (n = 54), and III) poultry meat (n = 150). Thirteen isolates were obtained from seven healthy and five diseased animals, but no C. difficile was cultured from poultry meat. The isolated C. difficile strains belonged to 3 different PCR-ribotypes (039/2, 205 and 001/FLI01). The detection of strains related to RT 001 known for its ability to cause disease in humans makes poultry a potential reservoir for pathogenic C. difficile

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

The zoonotic potential of Clostridium difficile from small companion animals and their owners

Background: Clostridium difficile infections (CDI) in humans range from asymptomatic carriage to life-threatening intestinal disease. Findings on C. difficile in various animal species and an overlap in ribotypes (RTs) suggest potential zoonotic transmission. However, the impact of animals for human CDI remains unclear. Methods: In a large-scale survey we collected 1,447 fecal samples to determine the occurrence of C. difficile in small companion animals (dogs and cats) and their owners and to assess potential epidemiological links within the community. The Germany-wide survey was conducted from July 2012-August 2013. PCR ribotyping, Multilocus VNTR Analysis (MLVA) and PCR detection of toxin genes were used to characterize isolated C. difficile strains. A database was defined and logistic regression used to identify putative factors associated with fecal shedding of C. difficile. Results: In total, 1,418 samples met the inclusion criteria. The isolation rates for small companion animals and their owners within the community were similarly low with 3.0% (25/840) and 2.9% (17/578), respectively. PCR ribotyping revealed eight and twelve different RTs in animals and humans, respectively, whereas three RTs were isolated in both, humans and animals. RT 014/0, a well-known human hospital-associated lineage, was predominantly detected in animal samples. Moreover, the potentially highly pathogenic RTs 027 and 078 were isolated from dogs. Even though, C. difficile did not occur simultaneously in animals and humans sharing the same household. The results of the epidemiological analysis of factors associated with fecal shedding of C. difficile support the hypothesis of a zoonotic potential. Conclusions: Molecular characterization and epidemiological analysis revealed that the zoonotic risk for C. difficile associated with dogs and cats within the community is low but cannot be excluded

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

A publicly accessible database for Clostridioides difficile genome sequences supports tracing of transmission chains and epidemics

Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10−4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile

The Gene 10 (UL49.5) Product of Equine Herpesvirus 1 Is Necessary and Sufficient for Functional Processing of Glycoprotein M

The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-β-N-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyAΔ49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyAΔ49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyAΔ49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-M(r) protein was coprecipitated with gM in KyA- or KyAΔ49.5R-infected cells or virions. This protein was absent in cells infected with KyΔ49.5 or KyAΔgM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication

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.Peer Reviewe