Study On The Dissemination Mechanism Of 16S RRNA Methylase Gene In Salmonella From Food

Salmonella is an important zoonotic pathogen and one of the main causes of food-borne disease outbreaks around the world.The increasing prevalence of multi-drug-resistant Salmonella restricts the clinical treatment options for Salmonella infections,and poses a threat to human health.Aminoglycosides are one of the commonly used antibacterial drugs in human medicine and veterinary clinics.The 16S rRNA methylase genes can mediate high-level resistance to a variety of aminoglycoside drugs.In this study,we aimed to investigate the prevalence of 16S rRNA methylase genes in Salmonella from food in China,and to explore its molecular transmission mechanism,in order to provide a theoretical basis for the rational use of drugs in the breeding industry and delay the development of Salmonella resistance.From 2019 to 2020,a total of 1037 pork and chicken samples were collected from 11 provinces and cities in China,and 226 Salmonella strains including 23 serotypes were isolated with Salmonella Typhimurium,Salmonella Kentucky,Salmonella Rissen,Salmonella London and Salmonella Derby being the main serotypes.We used PCR and sequencing to detect the presence of 16S rRNA methylase genes in 226 Salmonella strains,and we found 17 Salmonella Kentucky strains and 3 Salmonella Indiana strains carried 16S rRNA methylase genes,including rmtB(n=20)and armA(n=1);one Salmonella Indiana isolate contained rmtB and armA.Twenty rmtB/armA-positive Salmonella were tested for their susceptibility to antimicrobial agents by the agar dilution method.The results showed that all rmtB/armA-positive Salmonella exhibited resistance to at least seven antimicrobial agents,the multi-drug resistance was serious.The whole genomes of 20 rmtB/armA-positive Salmonella strains were sequenced by Illumina Hiseq.The results showed that 17 Salmonella Kentucky strains all belonged to ST198,and three Salmonella Indiana strains belonged to ST17.The phylogenetic tree based on the cgMLST showed that 17 Salmonella Kentucky strains were closely related,and 12 of them from different regions were divided into the same cluster,suggesting that clonal transmission of Salmonella Kentucky may occur.All rmtB/armA-positive isolates were performed conjugation experiments,but all of them failed to transfer rmtB/armA to E.coli C600 via conjugation.One representative Salmonella Kentucky isolate and three Salmonella Indiana isolates were selected for sequencing via PacBio,and complete genome sequences were obtained.The results showed that multiple resistance genes including rmtB were located on the chromosome of Salmonella Kentucky.The rmtB gene in Salmonella Kentucky LN19MCS9 was located in a 67,566-bp multiresistance region(MRR)bounded by two copies of IS26 with the same orientation.This MRR inserted into chromosomally located bcfG which was interrupted and produced 8-bp direct repeats.Further analysis of the rmtB region revealed that IS26 and an incomplete transposon Tn2 containing blaTEM-1 were located upstream of rmtB,the second copy of IS26 was downstream.No plasmids were identified in Salmonella Indiana strain WH19MCS1.The rmtB gene was located in the first MRR region on the chromosome of WH19MCS1.The genetic enviroment of rmtB was similar to that of LN19MCS9,except that the upstream IS26 was located with opposite orientation.In YZ19MCS15,the armA gene was located in the typical Tn1548 structure(tnpD-armA-tnpU-?ISCR1-sull-?qacE)within the chromosome.In LN19MCS15 and YZ19MCS15,the rmtB gene was located on the plasmids pYULNMCS15 and pYUYZMC15-2,respectively.pYULNMCS15 had the typical structure of IncHI2 plasmid,including replication region,leader region,conjugative transfer region and MRR,but it lacked some genes related to conjugative transfer.The genetic structure of rmtB was 1S26-?Tn2(?blaTEM-1b)-rmtB-orf1-IS26.Compared with those of LN19MCS9 and WH19MCS1,Tn2 was shorter and blaTEM-1b was truncated.Plasmid pYUYZMC15-2 contained IncX1 replication region(ddp3-repX-pir),but lacked most of IncX1 plasmid backbone structures including the conjugation transfer region,thus the size of plasmid was only 24,480 bp.The genetic structure of rmtB is ISCfrl-?Tn2(blaTEM-1b)-rmtB-orfl-ISCR1,tnpR of Tn2 was absent compared with other structures in this study.In summary,rmtB is the main 16S rRNA methylase gene prevalent in Salmonella from animal-derived food products.The clone spread of ST 198 Salmonella Kentucky and the horizontal transmission of IncHI2 and IncX1 plasmids mediate the dissemination of rmtB in Salmonella from food,transimission of armA is associated with Tn1548.