| Salmonella can cause acute gastroenteritis,seriously endangering human health,and causing serious economic losses to the poultry industry.In the post-antibiotic era,face with the dilemma of no antibiotics available,colistin has been used as an important weapon to treat the infections of "super bacterial",but there are still many questions and debates about its antibiotic-resistant mechanism.In 2015,the transferable colistin resistance gene mcr-1 was first discovered and reported in China,placing public health in a state of significant risk and challenge."What is the prevalence of colistin resistant Salmonella in the farm animal production,whether it will spread the antibiotic resistance to humans through the animal-origin food,whether the mechanism of colistin resistance in Salmonella is caused by plasmid mediated mcr-1 resistance genes only or others,how to prevent the colistin resistant Salmonella strains in the future,and whether finding new antibiotic alternatives or antibiotic combination can be a new way out",These issues are not yet clear and deserve further study.In the first part of the study,699 Salmonella strains(370 strains of chicken origin,were from the breeding companies;329 strains of retail meats origin,were from the supermarkets and open markets)isolated and identified by our laboratory during the years 2011-2021 in Guangxi were used in the study.The changes in the antibiotic resistance characteristics of Salmonella strains to clinically common antibiotics during the past 11 years were analyzed.The links of the epidemic characteristics of colistin resistant Salmonella between the chicken production and the retail meat were investigated.In order to provide data reference for the effective usage of antibiotics in Salmonella prevention and control process.The results showed that,Salmonella isolates from retail meat and chicken showed different degrees of resistance to the 16 antibiotics tested,sulfamethoxazole(93.42%)and nalidixic acid(78.40%)showed strong resistance degree,amoxicillin(68.67%),florfenicol(48.78%),tetracycline(53.65%),and colistin E(24.46%)showed moderate resistance degree.In different years,the resistance rates of the Salmonella to nalidixic acid and amoxicillin showed an upward trend,while that to florfenicol,tetracycline,and colistin showed a downward trend.In particular,the resistance rate to colistin E decreased from 65.61% in 2011 to 0% in 2019.Salmonella with antimicrobial multi-resistance(AMR)were found in a large number of strains and with rich resistant phenotype profiles.The coincidence rates between the phenotype and the resistant genes of the resistance were low in the antimicrobials except quinolones.The positive rate of the mcr-1 gene in the colistin-resistant Salmonella isolates was 1.00%,resulted in the coincidence rate between the mcr-1 positive and the resistance phenotype was only 4.09%.There was no significant mutation in the target genes through the analysis of the two-component system(only some strains exhibit silent mutations).All these results indicated that the relationship between colistin resistance and the mcr-1 gene was not one-to-one,and the resistance mechanism needs to be further explored.In the second part of the study,the Salmonella isolates positive for the mcr-1 gene found in the first part were used.Combined with whole genome sequencing(WGS)analysis and plasmid transfer conjugation experiments,relationship between the antibiotic resistance phenotype and the molecular biological characteristics of the strains,as well as the transmission characteristics of the mcr-1 gene,were analyzed to respond to the new changes in colistin resistance in the clinic.The results showed that six MLST subtypes ST19,ST40,ST214,ST516,ST413,and ST1541 were found from the seven mcr-1 positive strains(isolated from retail meat),and there was no significant ST type among the strains,indicating that there was a low possibility of clonal transmission of the same strain in the mcr-1 positive strains.The results also found that there were two types of plasmids Inc X4 and Inc I2 harbored the mcr-1 gene.Most strains(6/7)can transmit the plasmid through transfer the conjugation experiments,indicating that not all plasmids carrying mcr-1 can transmit horizontally,and the reason may be related to the lack of transposase.Due to the low coincidence rate between the mcr-1 gene and antibiotic resistance phenotype and the absence of significant mutations in the target genes in the two-component system of the colistin resistance Salmonella isolates,the third part of the study was further to investigate the mechanism of non-specific antimicrobial resistant mechanisms in the 171 colistin resistant Salmonella strains.The results showed that the higher the MIC of colistin,the stronger the strain’s BF forming ability.The positive rates of biofilm(BF)genes and the efflux pump genes in the colistin-resistant Salmonella isolates were high,and the positive rate of BF gene csg D was even hight up to 100%.The ability of BF formation test by both the crystal violet staining and the scanning electron microscope showed that the complete and mature BF formation of the colistin resistant Salmonella strains was found at 48 hours,the stronger the BF formation ability,the higher the minimum inhibitory concentration(MIC)of colistin,and there is a positive correlation between the two.By q PCR analysis,it was found that the efflux pump and BF genes of the colistin resistant Salmonella strains were differentially expressed compared to the sensitive strains,with a significant difference in the expression of acr A(up-regulated 1.70 times),tol C(up-regulated 1.76 times),repressor gene sox R(down-regulated 1.72 times),csg D(up-regulated 1.53 times),csg A(up-regulated 1.62 times)(P<0.05);There were very significant difference in the expressions of the efflux pump gene acr B(up-regulated 2.23 times)and sox S(up-regulated 2.34 times)(P<0.01).These results suggested that the colistin resistance mechanism of the Salmonella isolates involves in the efflux pumps and the biofilm formation in the non-specific antimicrobial resistant mechanisms.The fourth part of the study aims to provide new ideas for the development of new clinical drugs to prevent and control the colistin-resistant strains,provide references for the selection of antibiotic alternatives against SP.The result showed that when glycyrrhizin acid(GA)and colistin E were combinatory used in vitro,the minimum inhibitory concentration(MIC)of colistin decreased by 128 times,from 16 μg/m L to 0.125 μg/m L,and their growth activity was also lower than that of GA or colistin E used alone.GA as a antibacterial sensitizers against the colistin-resistant SP strains had also been verified by the challenge experiment.The group of GA combined with colistin treatment had not been recovered the challenge strain,while the recovery positive of either GA and colistin treatment groups were 30% and 10%,respectively,indicating that the synergistic antibacterial effect was found in chickens by the combination usage of GA and colistin.Selected yeast fraction performed the best result in terms of the weight gain due to the health care of the experimental birds.However,for the treatment of SP infection,the combined treatment of GA and colistin was more reliable.The results of the study demonstrated that the coincidence rate between mcr-1 and colistin-resistant phenotype was low in Salmonella isolates from the retail meat and chickens during 2011-2021.The positive rate of the BF genes and efflux pump genes in colistin-resistant Salmonella isolates was high,while there was a positive correlation between the strength of the biofilm formation and the colistin resistance,and the expression of efflux pump and BF genes m RNA in the colistin-resistant Salmonella strains was different from the colistin-sensitive Salmonella strains,and the mcr-1 gene cannot correspond to colistin resistance phenotype one by one,indicating that there are non-specific drug resistance mechanisms like efflux pump and biofilm formation in to colistin resistance.The study found that GA and colistin joint used showed good antibacterial sensitization effects both in vivo and in vitro experiments against the colistin-resistant SP strains.GA has the potential to be used as an antibacterial synergistic drug. |
PDF Full Text Request