Prevalence And Molecular Transmission Mechanisms Of 16S RRNA Methyltransferases In Escherichia Coli From Food Animals

Aminoglycoside agents(AGAs)are a kind of important antibiotics both in human medicine and veterinary medicine.The extensive use of AGAs in agriculture is bound to create a selective pressure and promote the emergence and spread of antibiotic resistance genes in isolates from food animals.16S r RNA methyltransferases(16S-RMTases),mostly plasmid-mediated and conferring high-level resistance to many AGAs,is the most concerned AGAs resistance mechanism in recent years.Long term monitoring of its epidemic transmission in animals is lack.Therefore,to provide important data for the evaluation of AGAs resistance in livestock and poultry farms,a comprehensive understanding of the prevalence and transmission mechanisms of 16S-RMTases genes in isolates of different years from food animals in Guangdong Province was conducted,as well as assessing the transmission risk of important plasmids carrying 16S-RMTases genes and uncovering new AGAs resistance genes or mechanisms using the method of functional genomics.Firstly,a comprehensive investigation was carried out on 963 E.coli of food animals from 2002 to 2012 saved in our laboratory and 1,650 newly collected samples of food animals from 2013 to 2016.Among them,a total of 794 E.coli isolates were resistant to amikacin and gentamicin.779 of them were positive for 16S-RMTases genes,and rmt B was the most prevalent 16S-RMTases gene(28.7%,749/2,613).Another 39 and 2 isolates were positive for arm A and rmt E respectively,while 11 isolates carried both rmt B and arm A genes.According to the data,the prevalence rates of rmt B gene were relatively stable(about 20%)before 2013.However,from 2014 to 2016,the resistance rates to AGAs and the prevalence rates of rmt B gene in E.coli isolated from food animals have been rising steadily and rapidly,and exceeded 60%in 2016.All the 175 tested E.coli isolates carrying rmt B or arm A genes from 2002 to 2012 were multi-drug resistant(MDR),and more than 90%of them were resistant to ampicillin,tetracycline,sulfonamide and quinolone drugs,with over 70%of them were resistant to 15or more tested antibiotics.The PFGE profiles of 173 rmt B-positive isolates were diverse,116transconjugants were successfully obtained among them.rmt B showed higher co-existence and co-transfer rates with PMQR genes,bla _CTX-M,flo R and fos A3.Plasmid replicons of rmt B showed diversity,but Inc F type was the dominant,followed by Inc N type.Plasmids with sizes of 70,75,160 and 105 kb all belonged to the Inc F were prevalent in avian and swine isolates from different years,possibly mediating the prevalence of rmt B in avian and swine E.coli.Meanwhile,almost all the rmt B genes were related to IS26.It is speculated that the prevalence of rmt B genes in E.coli from food animals was closely related to the insertion sequence,IS26.rmt E in this study observed a single base mutation of T?C at nucleotide 20 of rmt E,which causes a replacement of Val by Ala in the gene product.According to the suggestions of the nomenclature rules,it was designated as rmt E2.The two rmt E2-positive E.coli shared the same PFGE patterns and belonged to ST898.Both of them were successfully conjugated and showed the same resistance phenotypes and genotypes.The rmt E2-positive plasmids were Inc I1 type,they shared identical Eco R?-RFLP patterns and rmt E2 genes were located in the fragment of same size.The full sequence of rmt E2 plasmid p S68 was 115,010-bp in size.A BLASTn comparison against the NCBI database indicated that p S68 has a typical Inc I1 plasmid backbone of about 91 kb and 24 kb of multidrug resistance region(MRR).rmt E2 transmission might be related to the insertion sequence ISCR28.At the same time,the MRR includes multiple IS26 and the insertion recognition sites of each IS26 are all different,suggesting that this MRR was probably formed through multiple insertions and integration.To assess the transmission risk of rmt E2-positive plasmid,conjugation experiments with different host bacteria,plasmid stability and fitness cost experiments were conducted.Six recipients(E.coli,K.pneumoniae,S.typhimurium,C.freundii,E.cloacae and Acinetobacter)showed different conjugation transfer frequencies for the rmt E2 plasmid.In general,higher conjugation transfer frequencies were observed on E.coli C600 and Acinetobacter baylyi ADP1.The original E.coli S68 showed the highest plasmid stability,with no or almost no plasmid loss in both broth and plate experiments.Other transconjugants for different bacteria showed plasmid loss at different days.Comparison results of the growth rates between six recipients and the corresponding transconjugants were varied.Taken together,these results suggested that higher transmission risk of rmt E2 plasmid were observed in E.coli and Acinetobacter and this plasmid was more likely to be isolated later in the two species.Functional genomics experiments were conducted on the selected 5 isolates negative for all tested 16S-RMTases genes.After analyzing the obtained sequences,except for one gene that was slightly different,the others were all proved to be reported aminoglycoside modified enzymes genes(AMEs).This gene is very similar to the reported AAC(3)-IId gene,except for the last three amino acids before the termination codon,so it was named as AAC(3)-IId-like.AAC(3)-IId and-like genes had the same resistance spectrums and similar MIC values.The substrate specificity of acetyltransferases,Michaelis-Menten equation fitting graphics,and the corresponding enzyme parameter values of NHis6-tagged AAC(3)-IId and-like enzymes were all quite close.How the-like gene was formed was not known,but it was speculated that the sequences change was likely to be caused by DNA mutation,displacement,inversion,etc.,which resulted in the translocation of the termination codon.However,the main sequences were not changed,so the function remained stable.The AAC(3)-IId/-like gene screened from all the 5 isolates could explain the high-level resistance to gentamicin,while the mechanism of high resistance to amikacin was not obvious,which required more verification.A relatively comprehensive investigation on the prevalence and molecular characteristics of 16S-RMTases genes in E.coli isolates from food animals in Guangdong Province have been conducted,and rmt E2 was identified the first time in China,which provided important data for evaluating the AGAs resistance in livestock and poultry farms in Guangdong province.At the same time,the method of functional genomes is used to uncover new resistance genes,AAC(3)-IId-like here,which can combine phenotype with genotype directly and efficiently.