Clostridium chauvoei is a spore-forming bacterium responsible for black quarter (BQ) in cattle, a disease leading to high mortality. Despite its economic significance in India, genomic characterization of C. chauvoei strains from the country remains scarce. This study reports the genome of a C. chauvoei strain, 23CCJK, associated with a black quarter outbreak in Leh, India. The genome, approximately 2.7 Mb, encodes 2557 protein-coding genes including important known virulence determinants such as CctA, NanA, hyaluronidase, and collagenase. Species pangenome analysis involving 85 global strains revealed 2357 core and 381 accessory genes, indicative of limited gene acquisition. CRISPR analysis revealed conserved repeat patterns among Indian strains, supporting regional relatedness. Phylogenetic analysis indicated three major lineages (L1, L2, and L3), with Indian strains clustering among L3. The major lineage (L3; n=80) represented mainland strains, whereas L1 (n=2) and L2 (n=3) represented strains mainly from Australia and New Zealand. Notably, several novel genes were identified in this analysis with hypermutations, possibly involved in vital metabolism, host-pathogen interaction and virulence. This included discoidin domain-containing proteins, Glycerophosphoryl diester phosphodiesterase, ABC transporters, and histidine kinases, suggesting possible roles in faster adaptation during infection. These findings enrich our understanding of C. chauvoei evolution, population structure, and virulence, while highlighting key genetic markers that may be associated with pathogen adaptation during infections.

Rapidly growing populations of South American camelids (SAC), introduced to Europe as non-indigenous species, have increased contacts at the livestock and human interfaces. This study assessed the occurrence of epizootic and zoonotic viral and bacterial pathogens of prime importance on 10 farms, selected to mirror the diversity of German SAC holdings in size and animal use. Farms were visited four times at approximately 6-months intervals, with 20 animals sampled per visit, resulting in 739 blood and 723 fecal samples from 449 animals (292 alpacas, 156 llamas, one huarizo). Wherever possible, diagnostic methods applied followed national or international recommendations. Antibodies against Schmallenberg virus were detected in 54.6% of animals. Only three animals showed reactivity against Borna disease Virus 1 and one against bovine viral diarrhea virus (BVDV) 1. All animals tested negative for BVDV-2, border disease virus and bovine herpesvirus 1. Thirty-six samples from 30 SAC yielded a non-negative, presumably false-positive results for antibodies against the Mycobacterium tuberculosis complex. Ten samples from six animals were non-negative in an iELISA for brucellae antibodies, but all tested negative by Complement Fixation Test. Coxiella burnetii-specific antibodies were detected in three individuals from two different flocks, and a single fecal sample tested PCR-positive for C. burnetii. Chlamydiaceae-specific antibodies were found in 9 of 10 flocks and in 13.6% of the animals, with chlamydial fecal shedding observed in 8 of 10 flocks and in 29.6% of the animals. The animal positivity rate for Candidatus Mycoplasma haemolamae was 31.6%. Mycobacterium avium subsp. paratuberculosis was isolated from 0.34% and C. difficile from 2.7% of the animals. While Salmonella Typhimurium was found in only 1 of 719 fecal samples, thermotolerant Campylobacter (16 C. jejuni and 7 C. coli) were isolated from 4.8% of the animals. The overall rate of stx-positive samples, indicative of Shiga toxin-producing Escherichia coli (STEC) shedding, was 32.4%. Based on these detection rates, SAC do not appear to pose a greater risk of infection than other German livestock species at present. However, SAC represent a novel reservoir host that could disturb established and monitored indigenous epizootic transmission networks including those of enteric and abortifacient zoonotic agents.

Background/Objectives: Antimicrobial agents play an important role in the pathogenesis and treatment of Clostridioides (C.) difficile infections. C. difficile isolates have shown different genotypic and phenotypic resistance patterns and could serve as antimicrobial resistance reservoirs. Methods: To gain insight into accordance and potential disagreements between genotypic and phenotypic antimicrobial resistances in C. difficile, we compared the genotypic and phenotypic resistance patterns of 108 bovine C. difficile isolates collected in Germany between 2010 and 2012. These isolates represent a collection of different ribotypes (RT) and originated from different husbandries in Germany. Whole genome sequencing of all isolates was performed with Illumina® Miseq™, and sequences were screened for antimicrobial resistance determinants. For phenotypic antimicrobial susceptibility testing, the agar dilution procedure according to the CLSI document M11 was used. Minimal inhibitory concentration values were determined for penicillin, meropenem, tetracycline, moxifloxacin, vancomycin, metronidazole, erythromycin and clindamycin. Results: Various phenotypic and genotypic antimicrobial resistances were found in the isolates examined that belonged to different ribotype/sequence type (ST) lineages, even if these originated from the same source and geographical region (bovine isolates from Germany). Agreement between phenotypic and genotypic resistance was seen for most antimicrobial agents tested. A total of 92% (83/90) of the investigated ST11 isolates showed phenotypic resistance or were classified as non-wild type to at least one of the antimicrobials tetracycline, moxifloxacin, erythromycin and clindamycin. Conclusions: The results of this comparison contribute to a better understanding of antimicrobial resistance in C. difficile by relating phenotypic susceptibility patterns to genomic resistance determinants.

BACKGROUND:Clostridioides difficile is an anaerobic enteropathogen of clinical relevance in hospital and community settings. Its ubiquitous presence in pets, livestock, food and the environment, together with its ability to form spores, facilitates its survival and spread. Hypervirulent and multi-drug-resistant genotypes have been reported previously from Central America. AIM:To characterize 31 isolates from patients of two major hospitals in Honduras. METHODS:Second- and third-generation whole-genome sequencing and phenotypic antimicrobial susceptibility testing. FINDINGS:Two toxigenic polymerase chain reaction ribotypes RT027 (ST1) and RT002 (ST8) were detected. All RT027/ST1 isolates (N=29) were resistant to moxifloxacin, tetracycline and linezolid, whereas RT002/ST8 isolates (N=2) were susceptible. In addition, a number of mobile genetic elements associated with antimicrobial resistance were found in all RT027 isolates. Notably, core genome multi-locus sequence typing and core genome single nucleotide polymorphism analysis demonstrated the close genetic relationship among RT027/ST1 isolates, their persistence since 2016, and an interhospital transfer event with unknown sanitary and economic consequences. In addition, RT002, a genotype with known implications for community-acquired C. difficile infection and possible zoonotic implications, is a remarkable finding in the national epidemiologic context. CONCLUSIONS:Taken together, these findings highlight the presence of persistent and community-relevant C. difficile strains, and the consequent need to adopt and develop interventions to control and prevent CDI in the Honduran national health system within a One Health research approach.

Clostridioides difficile es un agente patógeno del tipo “Una sola salud”, y que es responsable de la diarrea asociada a antibióticos. Su epidemiología es dinámica, y se ha reportado que existe un cruce o solapamiento entre hospederos humanos y animales a nivel global. El objetivo de la presente investigación es caracterizar aislamientos obtenidos a partir de heces de origen animal que podrían actuar como reservorio de resistencia a los antimicrobianos (RAM). Mediante el uso de técnicas de cultivo, PCR, ribotipificación y prueba de susceptibilidad a los antimicrobianos (PSA), se caracterizaron 10 aislamientos no toxigénicos de C. difficile. Todos ellos fueron identificados como RT596, una cepa que ha sido previamente documentada en animales y humanos. Además, los aislamientos revelaron cuatro perfiles de multidrogoresistencia (MDR). Nuestros hallazgos constituyen la primera caracterización a nivel regional de un genotipo no toxigénico, con potencial zoonótico y MDR. Se plantea la posibilidad de que, debido a la falta de inclusión de la PSA en los algoritmos de diagnóstico actuales o a su circulación en ambientes hospitalarios, este genotipo pueda actuar como un vector de MDR.

Neurotoxins produced by Clostridium (C.) botulinum group III are responsible for the majority of botulism outbreaks occurring in animals and in this study we report the drug susceptibility of 71 field strains. The minimum inhibitory concentration (MIC) of 13 antimicrobials was established through the agar dilution method. The MIC50 matched or differed for one or two dilutions from MIC90 of the same antimicrobial, showing a unimodal distribution of the MIC values, irrespective of the geographical origin, the animal source and the toxinotype of the strain. Beta-lactams and rifampin showed the lowest MIC values, while gentamicin, polymyxin B and sulfamethoxazole showed the highest MICs. As for similar studies conducted in human botulism, the results could be helpful to avoid the administration of antimicrobials that could worsen the health condition of the affected animals and to develop selective media for the isolation of these fastidious anaerobes. Indeed, the isolation of the strain from affected animals and from environmental samples is important to perform epidemiological studies based on the genetic characterization and to produce tailor-made vaccines.

Clostridium difficile is ubiquitous and is found in humans, animals and in variety of environments. The substantial overlap of ribotypes between all three main reservoirs suggests the extensive transmissions. Here we give the overview of European studies investigating farm, companion and wild animals, food and environments including water, soil, sediment, waste water treatment plants, biogas plants, air and households. Studies in Europe are more numerous especially in last couple of years, but are still fragmented in terms of countries, animal species or type of environment covered. Soil seem to be the habitat of divergent unusual lineages of C. difficile. But the most important aspect of animals and environment is their role in C. difficile transmissions and their potential as a source for human infection is discussed.

Objective:To provide a comprehensive characterization of Clostridioides difficile antimicrobial resistance (AMR) data in veterinary medicine based on the minimum inhibitory concentrations (MICs) of all antimicrobial agents tested in relation to the techniques used. Methods:A systematic scoping review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for scoping reviews (PRISMA-ScR) and its associated checklist. The objective was to provide a synthesis of the evidence in a summarized and analyzed format.To this end, three scientific databases were consulted: Scopus, PubMed, and Web of Science, up until December 2021. Subsequently, all identified literature was subjected to screening and classification in accordance with the established study criteria, with the objective of subsequent evaluation. Study selection and data extraction:A comprehensive analysis was conducted on studies regarding Clostridioides difficile antimicrobial resistance (AMR) in veterinary medicine across various animal species and related sources. The analysis included studies that presented data on antimicrobial susceptibility testing using the E-test, agar dilution, or broth microdilution techniques. The extracted data included minimum inhibitory concentration (MIC) values and a comprehensive characterization analysis. Results:A total of 1582 studies were identified in scientific databases, of which only 80 were subjected to analysis. The research on Clostridioides difficile antimicrobial resistance (AMR) in veterinary medicine is most prolific in Europe and North America. The majority of isolates originate from production animals (55%) and pets (15%), with pigs, horses, and cattle being the most commonly studied species. The tested agents' minimum inhibitory concentrations (MICs) and resulting putative antimicrobial resistance profiles exhibited considerable diversity across animal species and sources of isolation. Additionally, AMR characterization has been conducted at the gene and genomic level in animal strains. The E-test was the most frequently utilized method for antimicrobial susceptibility testing (AST). Furthermore, the breakpoints for interpreting the MICs were found to be highly heterogeneous and frequently observed regardless of the geographical origin of the publication. Conclusions:Antimicrobial susceptibility testing techniques and results were found to be diverse and heterogeneous. There is no evidence of an exclusive antimicrobial resistance pattern in any animal species. Despite the phenotypic and genomic data collected over the years, further interdisciplinary studies are necessary. Our findings underscore the necessity for international collaboration to establish uniform standards for C. difficile antimicrobial susceptibility testing (AST) methods and reporting. Such collaboration would facilitate a "One Health" approach to surveillance and control, which is of paramount importance.

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage’s stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

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.

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.

South American camelids (SAC) are increasingly popular in Germany. Although frequently in close contact to livestock and humans, data on the prevalence of epizootic and zoonotic bacteria and of bacteria exhibiting antimicrobial resistance (AMR) in SAC is sparse. Therefore, 94 composite faecal samples collected in 43 private SAC holdings in the German states Saxony, Thuringia and Saxony-Anhalt were tested for the presence of Clostridium difficile, Mycobacterium avium subsp. paratuberculosis (MAP), Salmonella spp. and Chlamydia spp. Escherichia coli served as an AMR-indicator and for detecting Shiga toxin (Stx)-producers (STEC). Chlamydial presence was also probed with 136 vaginal swabs and twelve placental tissue samples. Most owners (n = 41/43) replied to a questionnaire providing information on animals and husbandry conditions. The questionnaire covered 974 animals, among them 20 llamas, mostly kept in smallholdings with less than 20 animals (75%). Frequently (n = 30/41), SAC were co-housed with other animal species. Most holdings (76%, n = 31/41) welcomed visitors regularly. Clostridium difficile was detected in 8.5% (n = 8/94) of the faecal samples, Chlamydia spp. in 7.4% (n = 7/94) of the faecal samples, 4.4% (n = 6/136) of the vaginal swabs and 8.3% (n = 1/12) of the placental samples. MAP and Salmonella spp. were not detected. Samples from 28% of the holdings were stx-positive. STEC strains isolated belonged to eae-negative, non-O157 serovars. All E. coli isolates were resistant to at least five antibiotics with 46% possible ESBL-producers. Even though shedding of bacteria of public health concern by SAC at livestock-livestock and livestock-human interfaces was found to be less common, positive results indicate the need for establishing continuous surveillance measures.

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.

Glyphosate-based herbicides are among the most used non-selective herbicides worldwide and inhibit synthesis of aromatic amino acids in plants, bacteria, and fungi. Given the broad usage, controversies concerning potential effects of glyphosate on health and especially on gut microbiomes arose. For cattle, it has been proposed based on in vitro data that glyphosate has detrimental effects on the ruminal microbiome, which manifest as a specific inhibition of bacteria involved in fiber degradation and as an enrichment of specific pathogens. In the present study, glyphosate effects on the ruminal microbiome were analyzed in vivo using glyphosate contaminated feedstuffs with strong differences in dietary fiber and dietary energy content in order to reproduce the proposed detrimental glyphosate effects on the rumen microbiome. While significant impact of dietary factors on the ruminal microbiome and its products are pointed out, no adverse glyphosate effects on ruminal microbiome composition, diversity, and microbial metabolites are observed.

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.

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.

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.

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.