The rising prevalence of multidrug-resistant pathogens poses a substantial threat to global healthcare systems, demanding urgent therapeutic interventions. Microorganisms exhibit diverse resistance mechanisms against various classes of antibiotics, highlighting the urgent need to discover novel antimicrobial agents for combating bacterial infections. Anti-virulence therapy has emerged as a promising therapeutic strategy that neutralizes pathogens by targeting their virulence determinants. The strategies for screening virulence arresting drugs (VADs) in bacteria represent a multifaceted approach that involves elucidating molecular pathogenesis mechanisms of bacterial pathogenicity, identifying evolutionarily conserved virulence factors across different pathogens, and employing integrated approaches combining in silico prediction with experimental validation. Recent technological advancements have established standardized protocols for effective identification and validation of anti-virulence compounds. This review systematically examines contemporary screening methodologies, primarily focusing on quorum-sensing disruption and biofilm suppression strategies, including in silico screening, activity-based screening with bioassays, in vitro and in vivo models. Additionally, we emphasize the imperative for standardized preclinical validation through physiologically relevant animal models, while proposing framework recommendations for developing next-generation VAD screening platforms. This synthesis not only outlines current best practices but also proposes innovative avenues for future antimicrobial discovery research.

Pathogenic Salmonella strains causing gastroenteritis typically can colonize and proliferate in the intestines of multiple host species. They retain the ability to form red dry and rough (rdar) biofilms, as seen in Salmonella enterica serovar Typhimurium. Conversely, Salmonella serovar like Typhi, which can cause systemic infections and exhibit host restriction, are rdar-negative. In this study, duck-derived strains and swine-derived strains of S. Typhimurium locate on independent phylogenetic clades and display relative genomic specificity. The duck isolates appear more closely related to human blood isolates and invasive non-typhoidal Salmonella (iNTS), whereas the swine isolates were more distinct. Phenotypically, compared to duck isolates, swine isolates exhibited enhanced biofilm formation that was unaffected by the temperature. The transcriptomic analysis revealed the upregulation of csgDEFG transcription as the direct cause. This upregulation may be mainly attributed to the enhanced promoter activity caused by the G-to-T substitution at position −44 of the csgD promoter. Swine isolates have created biofilm polymorphisms by altering a conserved base present in Salmonella Typhi, iNTS, and most Salmonella Typhimurium (such as duck isolates). This provides a genomic characteristics perspective for understanding Salmonella transmission cycles and evolution.

Antibiotic resistance has become a serious challenge to global public health. The spread of antibiotic resistance genes (ARGs) among humans, animals, and the environment has become a critical issue within the “One Health” framework. Chinese wet market with live poultry trade provides an interface for close interaction between humans and chickens, and is considered as potential source for disease dissemination. However, the understanding of ARGs in this kind of market, including their shared profiles, influencing factors, and potential horizontal transfer subtypes and directions, remains limited. In this study, we explored the microbiome, resistome, and mobility of ARGs, and identified putative horizontal gene transfer (HGT) events in the Chinese wet market system by utilizing metagenomic assembly and binning. Consequently, a total of 1080 ARG subtypes were identified from 36 metagenomes, and 221 subtypes were shared among human feces, chicken feces, chicken carcasses, and the environment. The composition of ARGs was influenced by mobile genetic elements (MGEs) and bacterial communities. As for the host of ARGs, 89 ARG-carrying genomes (ACGs) were identified, with 18 of them carrying multiple ARGs and MGEs, indicating the potential mobility of ARGs. Notably, six ACGs were identified as opportunistic pathogens carrying multiple ARGs and MGEs, which were annotated as Escherichia coli, Acinetobacter johnsonii, Klebsiella variicola, Klebsiella pneumoniae, and Citrobacter freundii. In addition, 164 potential HGT events were identified based on ACGs, and ParS, vanB, ugd, and macB were annotated as potentially transferred ARG subtypes in humans and the wet market. This study offers new insights into the potential for HGT of ARGs within a Chinese wet market setting, highlighting putative transmission patterns among humans, poultry, and the environment. To our knowledge, few studies have explored ARG transfer potential in this context using metagenome-assembled genomes, making this a valuable contribution to One Health surveillance.

The fourth mobile sulfonamide resistance gene sul4 has been discovered in many metagenomic datasets. However, there is no reports of it in cultured bacteria. In this study, a sul4 positive clinical Salmonella enterica SC2020597 was obtained by conventional Salmonella isolation methods and characterized by species identification and antimicrobial susceptibility testing. Meanwhile, the genomic DNA was sequenced using both long-read and short-read methods. Following that, the complete genome was analyzed by bioinformatic methods. The sul4 gene in S. enterica SC2020597 differed from the sul4 identified in metagenomic data by one amino acid and could confer full resistance to sulfamethoxazole. Genetic location analysis showed that the sul4 in SC2020597 was carried by a complex chromosomally integrated hybrid plasmid. ISCR20-like was strongly associated with the mobilization of sul4 by core genetic context analysis. To the best of our knowledge, this is the first report of the emergence of sul4 in clinically cultured S. enterica. More important, the sul4 has the potential to spread to other bacteria with the help of mobile elements.

In 2019, transferable plasmid-borne tet(X3)/tet(X4) genes conferring high-level tigecycline resistance were identified in Enterobacteriaceae and Acinetobacter. These transferable tet(X) variants are associated with different plasmids and could further spread and disseminate through associated mobile plasmids, raising public concern. In this study, we report the identification and characterization of two novel plasmids carrying tet(X4) in Escherichia coli isolated from retail chicken meat. We believe that our study contributes to our understanding of the genetic environment of tet(X4) and their transferability and capability of recombination and integration with other resistance genes in plasmids.