| Salmonella is an important zoonotic pathogen.To date,more than 2600 serotypes have been found.Salmonella Typhimurium is one of the most advantageous serotypes,which is widely prevalent throughout the world.S.Typhimurium has a wide range of sources and can be cross-infected between humans and animals through the food chain.It has strong drug resistance and pathogenicity,which poses great challenges to animal and human health and has important public health significance.At present,China still lacks data support for the research on different ST types of Salmonella,drug resistance and biofilm,and the research on drug resistance mechanism is not deep enough.Therefore,this study aimed to discover the biological characteristics of different ST-type S.Typhimurium drug resistance laws and new drug resistance mechanisms,and provide a data basis for the dynamic monitoring of S.Typhimurium,public health risk prediction and control,and clinical medication.In this study,a total of 309 strains of Salmonella typhimurium of human origin and food animal origin were selected for MLST typing,and it was found that ST34(253 strains,81.9%)and ST19(51 strains,16.5%)were the main ST types in China.Drug susceptibility test results showed that ST34 and ST19 Salmonella typhimurium have significant drug resistance between ampicillin,nalidixic acid,ciprofloxacin,ofloxacin,florfenicol and tetracycline Difference(P <0.05).The corresponding antibiotic resistance rates of ST34 and ST19 are(ST34,ST19): Ampicillin(75.89%,29.41%),Nalidixic acid(74.31%,37.25%),Ciprofloxacin(24.11%,0.00%),Oxygen Ofloxacin(28.06%,1.96%),florfenicol(75.49%,39.22%),tetracycline(86.56%,41.18%).Among them,the multi-drug resistance rate of ST34 was found to be 81.8%,while ST19 was only 49.0%.It is worth noting that we found that there are ST34 type antibiotics resistant to important clinical infection antibiotics ciprofloxacin,cephalosporin and colistin.The quinolones genes were tested on 61 strains of ciprofloxacin-resistant bacteria.The results showed that they all had gyrA mutations and no parC mutations.Among the PMQR genes,acc6-(lb ’)-cr and oqxAB resistance gene carrying rates are high,reaching 80.3% and 88.9%,respectively.In addition,36 strains were resistant to cephalosporin and 11 strains were resistant to polymyxin B,with a total resistance rate of11.7% and 3.6%,respectively.The biofilm formation ability test found that 99.3% of the strains were able to produce biofilms,and the strains with strong biofilm formation ability were mainly ST34 type.Among them,61.3% and 20.9% of ST34 strains had strong and moderate coating formation ability,respectively,while 96.1% of ST19 strains had weak biofilm formation ability.Combined with the discovery of drug resistance,the strains with strong and medium biofilm formation ability are more resistant to ciprofloxacin,cephalosporin,and colistin than other strains.It is suggested that the resistance of Salmonella Typhimurium is very serious,and the resistance of S.Typhimurium should be monitored as soon as possible.A ST34 representative strain R16 was selected.Using the method of λ-red homologous recombination system,the transcription factor BolA in the c-di-GMP pathway was selected for knockout to obtain the R16ΔBolA deletion strain.Growth curve measurement showed that the growth of R16ΔBolA-deficient strain was not affected.The results of the microdilution method showed that the drug resistance of the missing strain was reduced,the resistance to nalidixic acid and tetracycline was reduced by 128 times,and the resistance to chloramphenicol and ofloxacin was reduced by 8 times and 4 times,respectively.The laser confocal images intuitively showed the biofilm morphology of the parental and missing strains.The parental strains can form more aggregates,aggregate into clusters and have a dense structure,while the missing strains are loose in structure and cannot aggregate into clusters.Furthermore,transcriptome analysis was performed on the deletion strain R16ΔBolA and the parent strain R16,and 967 differentially expressed genes were screened,482 were up-regulated,and 485 were down-regulated.The results of GO enrichment analysis showed that the biological processes mainly focused on the functions of cobalt metabolism;KEGG database showed that the differential genes were mainly enriched in adaptability,twocomponent system,ABC transport system and metabolic pathways.After screening transcriptomics data,it was found by fluorescent quantitative PCR that after BolA was deleted,csgD and csgB and other regulatory factors were significantly decreased,suggesting that BolA may regulate biological characteristics and drug resistance by regulating related genes in bacterial pathways.In summary,this study conducted a systematic drug-resistant molecular epidemiology study on S.Typhimurium from different sources in China and found that the ST-type distribution of S.Typhimurium is complex but mainly ST34 and ST19.The ST34 strain exhibits severe multi-drug resistance,especially resistance to ciprofloxacin.After knocking out BolA,the drug resistance and biofilm formation ability of S.Typhimurium changed significantly.This study provides a new perspective for bacterial resistance and pathogenicity,and also provides a new entry point for the next step to find the target of antibacterial drugs and develop new antibacterial drugs. |
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