Mingsong Kang

Mammalian Campylobacter fetus (CF) is divided into two subspecies, C. fetus fetus (CFF) and C. fetus venerealis (CFV), the latter being bovine-adapted and responsible for the notifiable disease bovine genital campylobacteriosis (BGC). Differentiation between CF subspecies has traditionally been undertaken by a few biochemical tests, but these are complicated by the existence of a biotype, C. fetus venerealis intermedius (CFVi), which shares attributes of both CFF and CFV. Molecular methods targeting specific genes have gained acceptance for more accurate subtype identification and align well with whole-genome analysis. However, limited genomic diversity between subtypes has confounded efforts to understand the genetic basis for differential host tropism and pathogenesis of these organisms. A previous study of a small cohort of C. fetus isolates suggested that dam gene coding variations might correlate with CF subtype. Accordingly, this study examines a cohort of 331 C. fetus genomes, representative of all seven phylogenetic groups for their complement of adenine methylases and the genomic motifs they target in representative isolates. All CF isolates retained a cfeM1 gene, the presence of which correlates with RAATTY methylation, while seven other adenine methylase genes exhibited distinct cladal distributions. Notably, a cjeM1 gene appears to target the CCAN7TAG/CTAN7TGG motif in CFV and CFVi isolates only. Given the increasing recognition of the impact of adenine methylation on bacterial-host interactions, further exploration of the role of adenine methylation in C. fetus pathogenesis could reveal mechanisms contributing to BGC and thus aid in its eradication. IMPORTANCE: Campylobacter fetus remains an important zoonotic pathogen, for which a better understanding of its host tropism and pathogenesis is sought. However, the limited genomic variation observed between subtypes has to date confounded efforts in this regard. This study suggests that an alternative approach that examines epigenetic differences between subtypes, specifically adenine methylation patterns, may reveal mechanisms critical to the pathologies of these organisms.

Brucellosis, a reportable zoonosis with significant global economic and public health challenges, is primarily caused by Brucella melitensis , Brucella abortus , and Brucella suis in small ruminants, cattle, and swine, respectively. In addition to traditional serological tests and bacterial culture, the emergence of molecular tests, particularly quantitative PCR (qPCR), has gained attention for their rapid turnaround time, as well as their high sensitivity and specificity. However, validating existing assays using a sufficiently large and diverse data set remains challenging, and there is a pressing need for a reliable multiplex assay capable of simultaneously identifying B. abortus , B. melitensis , and B. suis . This study reports the development and evaluation of a highly sensitive and specific multiplex qPCR assay for detecting three Brucella species, alongside a novel genus-specific Brucella qPCR assay, both designed through large-scale comparative analysis of whole-genome sequencing (WGS) data. In silico validation of these primer-probe sets demonstrated sensitivities and specificities ranging from 99.8% to 100% using a large WGS data set, indicating comparable or improved in silico performance relative to currently published qPCR assays. Subsequently, the qPCR assays were evaluated using a comprehensive genomic DNA panel comprising 235 classical Brucella strains and 192 non-classical Brucella or non- Brucella samples, achieving 100% sensitivity and specificity with excellent low-level detection capability. Furthermore, these assays successfully detected Brucella DNA in tissue samples, demonstrating exceptional sensitivity and diagnostic accuracy through an advanced bacterial DNA enrichment process. These qPCR assays represent a valuable tool for rapid, accurate diagnosis of Brucella species in clinical specimens and bacterial cultures. IMPORTANCE: Brucellosis is a disease affecting animals and humans, causing major health and economic problems worldwide. It is mainly caused by Brucella abortus , Brucella melitensis , and Brucella suis . Quick and accurate identification of these bacteria is critical to control the spread of infection and protect public health. Current laboratory tests have limited ability to detect the previously mentioned Brucella species simultaneously and often lack validation across large and diverse sample sets, indicating a need for enhanced diagnostic tools. This study presents the development and evaluation of two rapid, accurate molecular tests that can detect the Brucella genus or precisely identify these three critical species simultaneously, thereby saving time and resources. With their excellent performance on large data sets across both computer-based and laboratory environments, these tests offer hope for improved diagnosis in both clinical and veterinary applications, thereby supporting public health and animal disease control efforts.

PCR-generated chimeric amplicons undermine the accuracy and reliability of subsequent amplicon-sequencing analyses. AmpliFuse, a Python-based tool, simulates realistic amplicon datasets through in silico PCR, chimera formation, and read simulation. By providing biologically relevant chimera-containing amplicon reads, AmpliFuse would facilitate benchmarking of chimera-detection algorithms and workflows, advancing microbiome and AMR research.

Serratia species are widespread, opportunistic Gram-negative microbes found in diverse environments, with some species posing risks to humans, potentially through food. In this announcement, we present six complete genome sequences of two strains for each of S. liquefaciens , S. fonticola , and S. nevei, respectively, isolated from food in Canada.

Ingestion of antibiotic-resistant bacteria following antibiotic treatments may lead to the transfer of antimicrobial resistance genes (ARGs) within a disturbed gut microbiota. However, it remains unclear whether and how microbes present in food matrices influence ARG transfer. Thus, a previously established mouse model, which demonstrated the conjugative transfer of a multi-drug resistance plasmid (pIncA/C) from Salmonella Heidelberg (donor) to Salmonella Typhimurium (recipient), was used to assess the effects of food-borne microbes derived from fresh carrots on pIncA/C transfer. Mice were pre-treated with ampicillin, streptomycin, sulfamethazine, or left untreated as a control to facilitate bacterial colonization. Contrary to previous findings where high-density colonization of the donor and recipient bacteria occurred in the absence of food-borne microbes, the presence of these microbes resulted in a low abundance of S. Typhimurium and no detection of S. Typhimurium transconjugants in the fecal samples from any of the mice. However, in mice pre-treated with streptomycin, a significant reduction in microbial species richness allowed for the significant enrichment of Enterobacteriaceae and pIncA/C transfer to bacteria from the genera Escherichia, Enterobacter, Citrobacter, and Proteus. These findings suggest that food-borne microbes may enhance ARG dissemination by influencing the population dynamics of bacterial hosts within a pre-disturbed gut microbiome.

The global spread of antimicrobial resistance genes (ARGs) poses a significant threat to public health. While antibiotics effectively treat bacterial infections, they can also induce gut dysbiosis, the severity of which varies depending on the specific antibiotic treatment used. However, it remains unclear how gut dysbiosis affects the mobility and dynamics of ARGs. To address this, mice were pre-treated with streptomycin, ampicillin, or sulfamethazine, and then orally inoculated with Salmonella enterica serovar Typhimurium and S. Heidelberg carrying a multi-drug resistance IncA/C plasmid. The streptomycin pre-treatment caused severe microbiome perturbation, promoting the high-density colonization of S. Heidelberg and S. Typhimurium, and enabling an IncA/C transfer from S. Heidelberg to S. Typhimurium and a commensal Escherichia coli. The ampicillin pre-treatment induced moderate microbiome perturbation, supporting only S. Heidelberg colonization and the IncA/C transfer to commensal E. coli. The sulfamethazine pre-treatment led to mild microbiome perturbation, favoring neither Salmonella spp. colonization nor a conjugative plasmid transfer. The degree of gut dysbiosis also influenced the enrichment or depletion of the ARGs associated with mobile plasmids or core commensal bacteria, respectively. These findings underscore the significance of pre-existing gut dysbiosis induced by various antibiotic treatments on ARG dissemination and may inform prudent antibiotic use practices.

The global spread of antimicrobial resistance genes (ARGs) poses a significant threat to public health. While antibiotics effectively treat bacterial infections, they can also induce gut dysbiosis, the severity of which varies depending on the specific antibiotic treatment used. However, it remains unclear how gut dysbiosis affects the mobility and dynamics of ARGs. To address this, mice were pre-treated with streptomycin, ampicillin, or sulfamethazine, and then orally inoculated with Salmonella enterica serovar Typhimurium and S . Heidelberg carrying a multi-drug resistance IncA/C plasmid. The streptomycin pre- treatment caused severe microbiome perturbation, promoting high-density colonization of S . Heidelberg and S . Typhimurium, and enabling IncA/C transfer from S . Heidelberg to S . Typhimurium and a commensal Escherichia coli . The ampicillin pre-treatment induced moderate microbiome perturbation, supporting only S . Heidelberg colonization and IncA/C transfer to commensal E. coli . The sulfamethazine pre-treatment led to mild microbiome perturbation, favoring neither Salmonella spp . colonization nor conjugative plasmid transfer. The degree of gut dysbiosis also influenced the enrichment or depletion of ARGs associated with mobile plasmids or core commensal bacteria, respectively. These findings underscore the significance of pre-existing gut dysbiosis induced by various antibiotic treatments on ARG dissemination and may inform prudent antibiotic use practices.

Motivation: Genomic variations comprise a spectrum of alterations, ranging from single nucleotide polymorphisms (SNPs) to large-scale structural variants (SVs), which play crucial roles in bacterial evolution and species diversification. Accurately identifying SNPs and SVs is beneficial for subsequent evolutionary and epidemiological studies. This study presents VariantDetective (VD), a novel, user-friendly, and all-in-one pipeline combining SNP and SV calling to generate consensus genomic variants using multiple tools. Results: The VD pipeline accepts various file types as input to initiate SNP and/or SV calling, and benchmarking results demonstrate VD's robustness and high accuracy across multiple tested datasets when compared to existing variant calling approaches. Availability and implementation: The source code, test data, and relevant information for VD are freely accessible at https://github.com/OLF-Bioinformatics/VariantDetective under the MIT License.

Glanders is a highly contagious and life-threatening zoonotic disease caused by Burkholderia mallei (B. mallei). Without an effective vaccine or treatment, early diagnosis has been regarded as the most effective method to prevent glanders transmission. Currently, the diagnosis of glanders is heavily reliant on serological tests. However, given that markedly different host immune responses can be elicited by genetically different strains of the same bacterial species, infection by B. mallei, whose genome is unstable and plastic, may result in various immune responses. This variability can make the serodiagnosis of glanders challenging. Therefore, there is a need for a comprehensive understanding and assessment of how B. mallei genomic variations impact the appropriateness of specific target antigens for glanders serodiagnosis. In this study, we investigated how genomic variations in the B. mallei genome affect gene content (gene presence/absence) and expression, with a special focus on antigens used or potentially used in serodiagnosis. In all the genome sequences of B. mallei isolates available in NCBI’s RefSeq database (accessed in July 2023) and in-house sequenced samples, extensive small and large variations were observed when compared to the type strain ATCC 23344. Further pan-genome analysis of those assemblies revealed variations of gene content among all available genomes of B. mallei. Specifically, differences in gene content ranging from 31 to 715 genes with an average of 334 gene presence-absence variations were found in strains with complete or chromosome-level genome assemblies, using the ATCC 23344 strain as a reference. The affected genes included some encoded proteins used as serodiagnostic antigens, which were lost due mainly to structural variations. Additionally, a transcriptomic analysis was performed using the type strain ATCC 23344 and strain Zagreb which has been widely utilized to produce glanders antigens. In total, 388 significant differentially expressed genes were identified between these two strains, including genes related to bacterial pathogenesis and virulence, some of which were associated with genomic variations, particularly structural variations. To our knowledge, this is the first comprehensive study to uncover the impacts of genetic variations of B. mallei on its gene content and expression. These differences would have significant impacts on host innate and adaptive immunity, including antibody production, during infection. This study provides novel insights into B. mallei genetic variants, knowledge which will help to improve glanders serodiagnosis.

This announcement reports the complete genome sequence of a non-Shiga toxin-producing Escherichia coli strain that was isolated from municipal biosolids collected from a Canadian wastewater treatment plant. This strain contains multiple metal, antimicrobial, and heat resistance genes, as determined by genome sequencing, and could be a useful bacterial model for future studies.