Introduction: Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time.Methods: In this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%.Results and Discussion: For a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.

Salmonella enterica causes severe food-borne infections through contamination of the food supply chain. Its evolution has been associated with human activities, especially animal husbandry. Advances in intensive farming and global transportation have substantially reshaped the pig industry, but their impact on the evolution of associated zoonotic pathogens such as S. enterica remains unresolved. Here we investigated the population fluctuation, accumulation of antimicrobial resistance genes and international serovar Choleraesuis transmission of nine pig-enriched S. enterica populations comprising more than 9,000 genomes. Most changes were found to be attributable to the developments of the modern pig industry. All pig-enriched salmonellae experienced host transfers in pigs and/or population expansions over the past century, with pigs and pork having become the main sources of S. enterica transmissions to other hosts. Overall, our analysis revealed strong associations between the transmission of pig-enriched salmonellae and the global pork trade.

Food workers have frequent contact with unprocessed foods, but their carriage of Salmonella and potential influence on public health have not been comprehensively assessed. We investigated Salmonella carriage among food workers compared with non-food workers based on occupational health screening of 260,315 asymptomatic workers over an 8-year surveillance period in Yulin, China. We confirmed that healthy carriers serve as natural reservoirs for Salmonella, with higher carriage rates in food workers than non-food workers. The isolates from food workers also exhibited greater serovar diversity and likely higher levels of antimicrobial resistance than those from non-food workers. Factors such as meteorological, social, and hygiene factors potentially influenced the carriage rate. Genomic analysis revealed a consistent increase in antimicrobial resistance genes among Salmonella isolates over the study period, with the majority of these antimicrobial resistance genes located on plasmids. Additionally, we identified numerous closely related bacterial clusters, which might reflect clusters of hidden local foodborne infections. This study underscores the elevated risk posed by food workers in the persistence and dissemination of Salmonella as vectors/fomites. Enhanced monitoring and targeted interventions in this group may reduce the dissemination of pathogens and antimicrobial resistance genes.

Of the hundreds of Vibrio cholerae serogroups, O1 and O139 are the main epidemic-causing ones. Although non-O1/non-O139 serogroups rarely cause epidemics, the possibility exists for strains within them to have pathogenic potential. We selected 25 representative strains within 16 V. cholerae serogroups and examined their genomic and functional characteristics. We tentatively constructed a gene pool containing 405 homologous gene clusters, which is well organized and functions in O-antigen polysaccharide (O-PS) synthesis. Our network analysis indicate that great diversity exists in O-PS among the serogroups, and several serogroup pairs share a high number of homologous genes (e.g., O115 and O37; O170 and O139; O12 and O39). The phylogenetic analysis results suggest that a close relationship exists between serogroups O170, O89 and O144, based on neighbor-joining (NJ) and gene trees, although serogroup O159 showed an inconsistent phylogenetic relationship between the NJ tree and the gene tree, indicating that it may have undergone extensive recombination and horizontal gene transfer. Different phylogenetic structures were observed between the core genes, pan genes, and O-PS genes. The virulence gene analysis indicated that the virulence genes from all the representative strains may have their sources from four particular bacteria (Pseudomonas aeruginosa, V. vulnificus, Haemophilus somnus and H. influenzae), which suggests that V. cholerae may have exchanged virulence genes with other bacterial genera or species in certain environments. The mobile genetic element analysis indicated that O159 carries nearly complete VSP-II and partial VPI-1 and VPI-2, O170 carries partial VPI-1 and VPI-2, and several non-O1/non-O139 strains contain full or partial VPI-1 and VPI-2. Several genes showing evidence of positive selection are involved in chemotaxis, Na + resistance, or cell wall synthesis, suggestive of environmental adaptation. This study reports on the newly sequenced O159 and O170 genomes and their comparisons with other V. cholerae serogroups. The complicated O-PS network of constituent genes highlights the detailed recombination mechanisms that have acted on the serogroups’ genomes. The serogroups have different virulence-related gene profiles, and there is evidence of positive selection acting on other genes, possibly during adaptation to different environments and hosts.

The genus Vibrio is ubiquitous in marine environments and uses numerous evolutionary characteristics and survival strategies in order to occupy its niche. Here, a newly identified species, Vibrio fujianensis, was deeply explored to reveal a unique environmental adaptability. V. fujianensis type strain FJ201301^T shared 817 core genes with the Vibrio species in the population genomic analysis, but possessed unique genes of its own. In addition, V. fujianensis FJ201301^T was predicated to carry 106 virulence-related factors, several of which were mostly found in other pathogenic Vibrio species. Moreover, a comparative transcriptome analysis between the low-salt (1% NaCl) and high-salt (8% NaCl) condition was conducted to identify the genes involved in salt tolerance. A total of 913 unigenes were found to be differentially expressed. In a high-salt condition, 577 genes were significantly upregulated, whereas 336 unigenes were significantly downregulated. Notably, differentially expressed genes have a significant association with ribosome structural component and ribosome metabolism, which may play a role in salt tolerance. Transcriptional changes in ribosome genes indicate that V. fujianensis may have gained a predominant advantage in order to adapt to the changing environment. In conclusion, to survive in adversity, V. fujianensis has enhanced its environmental adaptability and developed various strategies to fill its niche.

Toxigenic Vibrio cholerae serogroup O1 is the causative pathogen in the sixth and seventh cholera pandemics. Cholera toxin is the major virulent factor but other virulence and virulence-related factors play certain roles in the pathogenesis and survival in the host. Along with the evolution of the epidemic strains, the virulence-related genes also experience variation, gain and loss, and lead to genetic divergence in different strains. In this study, we analyzed the virulence-related gene profiles in the toxigenic serogroup O1 strains isolated from 1923 to 2015, the genomes of which were publicly available. The virulence-related genes of the V. cholerae O1 strains were annotated based on the Virulence Factors Database (VFDB). An average of 230.1 virulence-related genes per strain were identified; significant differences in the average numbers were found between the classical and El Tor biotypes, and increasing trends in the number of virulence-related genes along with the isolation years were observed in the El Tor biotype strains. A total of 176 homologs of virulence-related genes were found from these strains, of which 25 belonged to the core genes, suggesting their conservative and necessary roles in V. cholerae pathogenesis. We described the diversities of the homologs by defining gene sequence type, and illustrated its association with gene duplication; we found that gene duplication clearly increased the complexity of the gene sequence types in the core virulence-related genes. In addition, we provided virulence-related gene profiles whose genetic characteristic depend on the isolation years from the view of gene gain and loss, variation, gene duplication and gene sequence type number. Our study reveals the comprehensive variation dynamics of the virulence-related genes in toxigenic V. cholerae serogroup O1 during epidemics. The increasing trend for the virulence-related genes may suggest the evolutional advantage of strains by gaining virulence-related genes with diverse functional categories.