DOI: 10.6084/m9.figshare.27273783.v1
title: 多杀性巴氏杆菌流行过程中水平基因转移对其毒力与耐药的影响-附录.xlsx

description: <i>Pasteurella multocida</i> (PM) is prevalent worldwide in the breeding industry and causes continuous economic losses by infecting a variety of domestic and wild animals as well as humans. Some strains have multidrug resistance and hypervirulent phenotypes (i.e., individual bacterial cells causing 100% mortality in hosts). Horizontal gene transfer (HGT) is ubiquitous in prokaryotes, contributing to phenotypic innovation and enhancing environmental adaptability during niche changes. Some studies on PM have suggested that its strong adaptability and the emergence of various novel phenotypes might have a possible link to HGT. Currently, the available research results that could offer insights into the comprehensive impact of HGT on PM populations are significantly limited, and there exists a notable scarcity of reference data pertaining to its transmission patterns and dynamic changes within this bacterial group. This study explored the primary factors contributing to the widespread prevalence of PM using 435 strains from 13 host-sourced 21 countries across four continents. Among them, 393 strains were publicly available from NCBI, and 42 were laboratory-isolated. The laboratory strains consist of 14 newly clinical duck-sourced isolates, 21 previously clinical duck-sourced isolates, six poultry-sourced strains from the American Type Culture Collection, and one poultry-sourced strain from the China Veterinary Culture Collection Center. Challenge tests of PM in the laboratory identified hypervirulent (causing 100% mortality with &lt;10² CFU), avirulent (100% survival with &gt;10⁶ CFU), and intermediate virulence strains with host mortality rates between these two extremes. High-throughput sequencing was used to obtain whole-genome data for these strains with varying virulence phenotypes. Serotypes of all strains were determined using offline multiplex PCR and online electronic PCR programs, combined with data from 42 PM in the laboratory and 393 PM in the NCBI database. Bioinformatics analysis was then performed to identify HGT datasets including genomic islands (GIs), phages, plasmids. By correlating these phenotypic traits with genetic data, factors associated with clinically high virulence were determined. The study found that the dominant serotype of hypervirulent poultry-sourced PM is ST129:A:L1; however, this serotype does not perfectly correlate with the hypervirulent, suggesting that its prevalence is related to PM's adaptability. HGT introduced numerous mobile elements into PM, including many virulence and resistance genes. The HGT datasets primarily impacts host metabolism-related biological processes and overall metabolic network structures. By summarizing the basic information, a correlation was observed between the number of HGT elements and virulence phenotype: the laboratory hypervirulent poultry-sourced strains typically had fewer HGT elements, while avirulent strains had more, and intermediate virulence strains fell between these two extremes. Among the various HGT elements, GIs (present in 99.7% of strains, with 1,794 suspected regions) are ubiquitous in PM and abundant, contributing significantly to the introduction of virulence and resistance genes; Phages (present in 75.7% of strains, with 862 suspected regions) are mainly associated with the introduction of some virulence genes; and plasmids (present in 4.6% of strains, with 19 plasmids) are primarily related to the introduction of resistance genes but are relatively scarce. These data highlight the significant impact of HGT on PM populations, although the specific mechanisms and patterns of this impact remain unknown. Genome-wide association studies (GWAS) enable the exploration of phenotypic genetic mechanisms by correlating large-scale genomic data with specific phenotypes. Utilizing the whole-genome data of poultry-sourced PM with varying virulence phenotypes from the laboratory, along with the HGT datasets, this study employed the GWAS approach to identify 26 exogenous genes unique to hypervirulent poultry-sourced strains. Among these, only one hypothetical protein with an unknown function (A0R64_02365) belonged to the HGT gene dataset. Examination of the genetic context of A0R64_02365 in bacteria confirmed that this gene is specific to the PM species and universally present within the SUKH superfamily. The laboratory strain DY120818 was used as the test subject to construct a knockout mutant ΔA0R64_02365. The virulence of the knockout mutant (LD50 of 8.92×10<sup>9</sup>) was significantly reduced compared to the wild strain DY120818 (LD<sub>50</sub> &lt; 10). Transcriptome analysis revealed that the deletion of A0R64_02365 caused significant changes in the transcription levels of numerous ABC transporter-related protease genes, particularly downregulating the transcription of capsular polysaccharide transport genes. Validation confirmed a decrease in capsular content in the knockout mutant, with both transmission and scanning electron microscopy revealing damaged capsular structures in ΔA0R64_02365 strains. Additionally, genes related to biofilm systems (PST system, lsrACD) showed significant downregulation, yet unexpectedly, the biofilm content in the ΔA0R64_02365 increased, suggesting that PM might possess alternative biofilm formation mechanisms. The transcription levels of iron acquisition systems such as fecABCDE, sitABCD operons, and dppAB were also significantly reduced, but the afuAB-dependent periplasmic binding protein-mediated iron transport mechanism was significantly upregulated. Experimentally, the growth of the ΔA0R64_02365 in iron-rich and iron-limited media did not exhibit iron-related patterns. Apart from ABC transporter pathways, there were changes in the transcription levels of multiple protease genes. Upon aggregating A0R64_02365 and its significantly impacted gene set, a consistent gene-level difference emerged. Systematic clustering based on these gene sets distinctly separated them from other serotypes or host-sourced PM, clustering entirely within the Asian avian-sourced ST129:A:L1. This indicates that the highly virulent phenotype of PM is not a monogenic factor but rather a result of the collective interaction of a unique gene cluster specific to this host source. Consequently, the deletion of A0R64_02365, a unique HGT-sourced gene in the hypervirulent poultry-sourced PM DY120818, triggers significant transcriptional changes in a cluster of protease genes specific to this virulence phenotype. Notably, the reduction in capsular polysaccharide transport, hindering capsular synthesis, is the primary reason for the marked change in virulence phenotype. Furthermore, the role of HGT in shaping the antibiotic resistance of PM populations is pronounced within GIs, particularly those harboring integrative and conjugative elements (ICEs). Among 435 PM strains analyzed, 82 ICEs were identified, with 64 of them being resistance-related and primarily belonging to three subtypes of the ICEHin1056 family. Additionally, two ICEs are suspected to be metabolism-related, belonging to the SXT/R391 family; and one ICE is suspected to virulence-related belonging to the Tn916 family. ICEHin1056-like ICEs serve as the primary HGT mechanism for the dissemination of resistance genes within PM populations. These ICEs harbor numerous previously unreported insertion sequences and resistance gene-associated modules, which encompass nearly all reported clinical resistance phenotypes of PM. Notably, some ICEs have introduced carbapenem resistance gene <i>bla</i><sub><em>OXA-2</em></sub> and bleomycin resistance gene <i>bleO</i> into PM. Statistical analysis of the presence and distribution of ICEs in PM revealed that their dissemination primarily occurs during periods of high prevalence of PM, and their transmission trends correlate with global antibiotic usage policies. Two Bayesian tree constructed based on the whole genomes of PM and their ICEs confirmed that ICEs predominantly spread vertically and partially horizontally within PM populations, and the conserved structures of ICEs from different host-sourced PM exhibit distinct host specificity. Among the laboratory isolates, only one hypervirulent and multidrug-resistant PM HN141014, was found to harbor a novel multidrug-resistant ICE, designated ICE<i>Pmu</i>3. This element belongs to the ICEHin1056 subfamily. its conserved region contains an almost complete Type IV secretion system, excluding tfc1 and tfc20, and its variable region harbors multiple resistance genes such as <i>tetR</i>(<i>B</i>)-<i>tetB</i>-<i>tetC</i>, <i>aph</i>(<i>3ʹ</i>)-<i>Ia</i> and <i>sul2-strA-strB</i>. Besides the host strain, ICE<i>Pmu</i>3 was also detected in three other PM strains from Asia and America and eight avian Bacillus strains. Conjugation transfer experiments confirmed its intraspecific and interspecific transfer capabilities and frequencies, identifying key sequence sites involved in its interactions with host strains. Collectively, the study data indicate that ICEs on PM's adaptation to external environmental pressures, as evidenced by their existing resistance phenotypes. The prevalence of ICEs in PM indirectly reflects the clinical antibiotic usage, providing crucial parameters for clinical prevention and treatment strategies.