Objective: To analyze the bioinformatics of domestic Clostridium difficile toxin B(TcdB) and prepare it to provide data support for the development of effective vaccines. Methods: Using bioinformatics software such as Snippy, Blast, Muscle, and the dist.alignment() and hclust() functions in R, 1 355 strains of Clostridium difficile from NCBI GenBank in China were compared and analyzed, and TcdB were grouped. The maximum likelihood tree and phylogenetic tree were beautified and displayed using iTOL. An online bioinformatics analysis website was used to predict and analyze the spatial structure and antigenic epitopes of the two largest subgroups, TcdB1 and TcdB2. The antigen protein TcdB was expressed and purified by prokaryotic system. Results: According to the genotype of toxin B, the 1 355 prevalent strains of Clostridium difficile in China could be roughly divided into 12 subtypes, among which TcdB1 and TcdB2 were the main subtypes, accounting for more than 93.94% of all isolated strains, and about 17.20% of the strains were nontoxigenic or lack TcdB. The antigen epitope prediction of TcdB1 and TcdB2 showed that their antigen epitope distributions were basically the same, and many of them were distributed outside the C-terminal combined repetitive oligopeptides domains. Conclusion A specialized typing system for C. difficile TcdB in China has been established, and its main subtypes have been predicted for antigenic epitopes. The screened TcdB has been expressed for recombinant preparation.

Objective : To use linear PCR fragment containing antibiotic resistance cassette to carry out homologous recombination and replacement of target gene fragment of Acinetobacter baumannii to achieve rapid gene knockout and functional verification． Methods: Acinetobacter baumannii Ab4294 was used as the research object，and the upper (901 bp) and lower ( 1 028 bp) reaches of fim gene cluster (4 980 bp in length) were amplified by PCR , which was used as the recombinant homologous arm．Kanamycin antibiotic resistance cassette (KanR) was ampli- fied from pUC57 plasmid．The above three fragments were connected by overlapping extended PCR technique，and the connected fragments were transformed into wild Acinetobacter baumannii strains．The gene deletion mutant was screened，and the plasmid complement strain was constructed．The phenotype of the obtained strains was identi- fied，and the function of fim gene cluster was explored. Results : A mutant strain of Acinetobacter baumannii Ab4294 with deletion of fim gene cluster was successfully constructed by homologous substitution of linear PCR frag- ment containing antibiotic resistance cassette．Compared with the wild strain，the growth curve of the deletion strain had no significant difference ，and the rubbing ability significantly decreased ，and the phenotype recovered after complementing the gene cluster． Conclusion The fim family genes of Acinetobacter baumannii Ab4294 is success- fully knocked out by homologous substitution of linear PCR fragment containing antibiotic resistance cassette，which encodes the product involved in the motile movement of Acinetobacter baumannii.

Objective:To evaluate the virulence levels of carbapenem-resistant Acinetobacter baumannii ST191, ST195, and ST208, and to analyze the differences in virulence factors among these epidemic clones. Methods:The study involved the genomic sequencing of 233 Acinetobacter baumannii strains that were isolated from the Fifth Medical Center of the Chinese People′s Liberation Army General Hospital (North Hospital) between 2011 and 2019. The genomic data was cross-referenced with the Virulence Factor Database (VFDB) to examine the presence of virulence genes in the strains. Furthermore, a Galleria mellonella infection survival model was used to evaluate the virulence levels of the strains, and the association between virulence levels and virulence genes was analyzed. Results:The study included 38 strains of the ST191 clone, 104 strains of the ST195 clone, and 91 strains of the ST208 clone. In the Galleria mellonella infection survival experiment, the average mortality rate for ST191 was 23.0%, with 3 (7.9%) highly virulent strains. For ST195, the average mortality rate was 53.0%, with 34 (32.7%) highly virulent strains. For ST208, the average mortality rate was 47.0%, with 20 (21.9%) highly virulent strains. There was a significant statistical difference in mortality rates between ST191 and ST195 ( χ 2=13.9, P<0.001) as well as between ST191 and ST208 ( χ2=15.2, P<0.001). A comparison of the strains with the VFDB revealed significant differences in the virulence genes carried by the clones. Specifically, the type Ⅵ secretion system-related genes ( clpV/tssH, hcp/tssD, tagX, tssA, tssB, tssC, tssE, tssF, tssG, tssK, ssL, tssM) and the sugar transferase gene ACICU_RS00475 were found to be universally absent in ST191 strains (0%) while being prevalent in ST195 (100.0%) and ST208 (>82.0%) strains. Statistical analysis revealed an association between the mortality rate of the clones and the presence of virulence genes( clpV/tssHP<0.001, hcp/tssDP=0.001, tagXP<0.001, tssAP<0.001, tssBP=0.001, tssCP=0.001, tssE P=0.001, tssF P=0.001, tssGP<0.001, tssKP<0.001, tssLP<0.001, tssMP=0.001, ACICU_RS00475 P=0.001). Conclusion:Among the carbapenem-resistant epidemic clones of Acinetobacter baumannii, the ST191 clone shows lower mortality rates in Galleria mellonella, possibly because of the lack of type Ⅵ secretion system and sugar transferase genes.

Objective:To evaluate the virulence levels of carbapenem-resistant Acinetobacter baumannii ST191, ST195, and ST208, and to analyze the differences in virulence factors among these epidemic clones. Methods:The study involved the genomic sequencing of 233 Acinetobacter baumannii strains that were isolated from the Fifth Medical Center of the Chinese People′s Liberation Army General Hospital (North Hospital) between 2011 and 2019. The genomic data was cross-referenced with the Virulence Factor Database (VFDB) to examine the presence of virulence genes in the strains. Furthermore, a Galleria mellonella infection survival model was used to evaluate the virulence levels of the strains, and the association between virulence levels and virulence genes was analyzed. Results:The study included 38 strains of the ST191 clone, 104 strains of the ST195 clone, and 91 strains of the ST208 clone. In the Galleria mellonella infection survival experiment, the average mortality rate for ST191 was 23.0%, with 3 (7.9%) highly virulent strains. For ST195, the average mortality rate was 53.0%, with 34 (32.7%) highly virulent strains. For ST208, the average mortality rate was 47.0%, with 20 (21.9%) highly virulent strains. There was a significant statistical difference in mortality rates between ST191 and ST195 ( χ 2=13.9, P<0.001) as well as between ST191 and ST208 ( χ2=15.2, P<0.001). A comparison of the strains with the VFDB revealed significant differences in the virulence genes carried by the clones. Specifically, the type Ⅵ secretion system-related genes ( clpV/tssH, hcp/tssD, tagX, tssA, tssB, tssC, tssE, tssF, tssG, tssK, ssL, tssM) and the sugar transferase gene ACICU_RS00475 were found to be universally absent in ST191 strains (0%) while being prevalent in ST195 (100.0%) and ST208 (>82.0%) strains. Statistical analysis revealed an association between the mortality rate of the clones and the presence of virulence genes( clpV/tssHP<0.001, hcp/tssDP=0.001, tagXP<0.001, tssAP<0.001, tssBP=0.001, tssCP=0.001, tssE P=0.001, tssF P=0.001, tssGP<0.001, tssKP<0.001, tssLP<0.001, tssMP=0.001, ACICU_RS00475 P=0.001). Conclusion:Among the carbapenem-resistant epidemic clones of Acinetobacter baumannii, the ST191 clone shows lower mortality rates in Galleria mellonella, possibly because of the lack of type Ⅵ secretion system and sugar transferase genes.

Horizontal gene transfer contributes to the spread of antibiotic-resistance cassettes, the distribution of toxin-encoding phages and the transfer of pathogenicity islands. Natural transformation, which is the process of competent cells to uptake free DNA from environment and to recombine this DNA into the chromosome, is a mode of horizontal gene transfer. Natural transformation promotes the spread of antibiotic-resistance cassettes among different bacteria, resulting in the emergence of antibiotic resistant bacteria. The emergence of antibiotic resistant pathogens poses an enormous threat to the treatment of infections. Natural transformation could occur in many bacteria, but the mechanism many be different in different bacteria. Also, the inducer and efficiency of natural transformation in different bacteria are influenced by various factors. This review focuses on the mechanism and influencing factors of natural transformation in bacteria.