| Aminoglycosides are important antibiotics for the treatment of clinical infection of bacteria used by veterinarians and doctors,and 16S rRNA methylase is a newly discovered mechanism of high resistance to aminoglycosides in recent years.Due to the extensive and long-term use of aminoglycoside drugs,drug resistance has become increasingly serious.Drug-resistant strains can be spread by cloning or horizontal transmission mediated by mobile elements and plasmids.Therefore,drug-resistant genes can spread within strains and even among different strains,accelerating the prevalence of drug-resistant bacteria and drug-resistant genes.Little knowledge has gained on 16S rRNA methylase genes of Salmonella at home and abroad,therefore,it is import to investigate the prevalence of 16S rRNA methylase genes in Salmonella isolated from different sources in China and further explore its genetic characteristics and transmission mechanisms.It provides reference for the rapid monitoring of drug resistant strain in production.Moreover,this study will provide a theoretical basis for rational use of aminoglycosides in animal clinic or aquaculture and for slowing down the emergence of drug-resistant strains.1 Prevalence of 16S rRNA methylase genes in Salmonella from different sourcesA total of 2,369 strains of Salmonella containing 37 serotypes isolated from animal,pets,humans,food and environmental samples collected from 24 provinces of China were used in this study.26 strains of Salmonella were screened to be resistant to amikacin isolated from Jiangsu province.16S rRNA methylase genes of the 26 Salmonella strains of was detected,and results revealed that the armA gene was the most popular(23/26,88.46%),22 of the 23 strains were from chicken,including 11 strains from market chicken samples,11 strains from clinical samples,and the rest one strain was isolated from human fecal sample.Three Salmonella strains carring rmtB gene(3/26,369 11.54%),which were collected from environmental samples of pig slaughter,feces of geeses and market meat samples of chickens,respectively.No other 16SrRNA methylase genes were detected.Moreover,16S rRNA methylase genes were only found in Salmonella Indiana(25/40,62.50%)and Salmonella London(1/106,0.94%).Agar dilution method was used to detect the drug sensitivity of 26 strains resistant to amikacin positively,and results showed that multiple drug resistance is urgent,and the most popular resistance patternsis is AMP-CFZ-CTX-ATM-GEN-AMK-STR-TET-NAL-CIP-CHL-FFC-OLA-FOS-SXT(17/26,65.38%)2 Transmission mechanism of 16S rRNA methylase genes in SalmonellaThe whole genome of 26 16S rRNA methylase positive Salmonella strains were sequenced by Illumina sequencing.The results showed that 25 strains of Salmonella Indiana belonged to ST17 and one Salmonella London strain belonged to ST155.Phylogenetic tree revealed that 25 Salmonella Indiana strains belonged to ST17 were divided into two clusters.24 strains in the main cluster and only one strain in the other cluster.Strains from different origins distributed in the same branch,indicating that their genetic relationship was very close,suggesting that there is a clonal relationship between ST17 Salmonella Indiana.The conjugation experiment of positive strains showed that armA/rmtB gene was successfully transferred to recipient C600 in 16 Salmonella strains,which suggested that armA/rmtB gene was located on the conjugable plasmid,and the conjugation frequency varied in different temperature,which was higher at 30? than 37?,which suggested that armA/rmtB gene was located on the plasimid of IncHI2.Moreover,armA/rmtB positive strains often carry extended-spectrum ?-lactam resistance genes?quinolone resistance genes and fosfomycin resistance genes.16 of 26 strains of armA/rmtB positive Salmonella contained replicators,and the results of replicon typing showed that there were 13 replicon types.The most popular is IncHI2 and IncX1(n=14),followed by IncQ(n=10).In this study,it is found that the transfer of armA gene is related to the transposon Tn1548,but it is often truncated by the insertion of IS26.23 armA positive strains had two genetic environments(? and ?),structure I,there was an ?ISCR1 upstream of armA,and the downstream of transposase gene tnpU,and IS26.Structure ?,the upstream of armA was ?ISCR1,and the downstream of transposase gene tnpU,was transposase gene tnpD,macrolide resistance genes(msrE and mphE)and ?IS26.The transfer of rmtB gene related to Tn2 transposons,and three strains of rmtB also have two genetic environments(? and ?),in structure I,upstream of rmtB is ?IS26,?IS1294 and ?Tn2,?Tn2 truncated by ?IS1294,including ?-lactam resistance gene blaTEM-1,and the downstream is ?ISCR1.The upstream of rmtB in structural II is ?IS26 and ?Tn2,?Tn2 containing blaTEM-1,and its downstream is ?IS26.Analyzing plasmid pSIN626 carrying armA gene and plasmid p910 carrying rmtB gene through the PacBio sequencing,the results showed that pSIN626 is a IncHI2-IncX1 plasmid,and highly similar with Salmonella indiana plasmid pJT01 and Salmonella plasmid pCFSA664-1,the difference is that pJT01 missing IncX1 replicator area between 50-60kbp and missing of tetracycline resistance area in 110kbp,pCFSA664-1 also lacks IncX1 replicator region between 50kbp-60kbp.P910 is a IncA/C-IncN-IncX1 plasmid,and has similar transfer region and partition region with plasmid PK202-1 from Escherichia coli and have similar replication area with plasmid pTHNK130-1 from klebsiella pneumoniae,the remaining regions have low similarity.In conclusion,the armA is mainly prevalent gene of 16S rRNA methylase in Salmonella from different sources in China,which is mainly derived from chicken-origin Salmonella Indiana,while rmtB gene is less prevalent but has a wide host source(goose,chicken and pig).The clonal transmission of ST17 and horizontal transmission of plasmid IncHI2-IncX1,Tn1548 and IS26 were the main reasons for armA gene to spread in Salmonella.The transmission of rmtB gene is related to Tn2,ISCR1 and IS26. |
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