| This study focused on a outbreak of highly fatal diarrhea in a large-scale snake farm causing serious economic loss.The bacterium isolated from lesions were verified to Aeromonas hydrophila by biochemical tests and sequence analysis of 16 S r RNA.MLST revealed that none of isolates' allele sequences matched those previously published and we acquired new numbers(gyr B-380,gro L-369,glt A-383,met G-374,pps A-414,rec A-406)constituting a previously unidentified sequence type:ST516.The bran-new genetype ST516 Aeromonas hydrophila was identified to the pathogenic bacteria of this outbreak via snake challenge test.Isolates were pathogenic to mice,causing diarrhoea within 4 hr post?challenge,which indicates that the bacterium can potentially infect mammals.The results suggest considerable zoonosis potential and raise concerns about public health.The parental strain WCX23(colistin MIC = 1?g/m L),a colistin-susceptible strain,were used to generate the highly resistant strain 23-C-23(colistin MIC =256?g/m L)with gradient doses of colistin in vitro.Whole-genome sequencing(WGS)and comparative genomics showed bacteria carried 570 virulence genes and 296 drug resistance genes.Moreover,non-synonymous mutation in mla F(518 A>C)was confirmed from 13 passages(23-C-13)with colistin and stably passaged until23-C-23.Transcriptome analysis indicated that significantly up-regulated genes were related to Propanoate metabolism,Cationic antimicrobial peptide(CAMP)resistance,ABC transporters,Bacterial secretion system and so on,while significantly down-regulated genes were related to Glycerolipid metabolism,Glycerophospholipid metabolism,Arginine biosynthesis,Purine metabolism and so on.To explore in A.hydrophila the role of hypothetical protein gene1038 in colistin-resistant mechanisms,we predicted the structure of gene1038 through software,removed gene1038 in 23-C-23 by gene knockout techniques,then complemented ?gene1038 in knockout strains,and analyzed transcriptome data between 23-C-23 and 23-C-23: ?gene1038.The results indicated that gene1038 including eight transmembrane regions had more than 86.81% similarity of nucleotide to Aeromonas spp,while less than 66.4% similarity of nucleotide to other bacteria.Deletion of gene1038 in the 23-C-23 notably attenuated resistance to colistin(colistin MIC declined from 256?g/m L to 32?g/m L);complementation of the deletion mutant with gene1038 restored resistance to colistin.Also to explore in A.hydrophila the role of env Z/omp R in colistin-resistant mechanisms,we removed env Z/omp R in 23-C-23 by gene knockout techniques,then complemented ?env Z/omp R in knockout strains,and analyzed transcriptome data between 23-C-23 and 23-C-23: ?env Z/omp R.The results showed that Deletion of env Z-omp R in the 23-C-23 background resulted in a32-fold decrease in the MIC of colistin(from 256?g/m L to 8?g/m L);complementation of the deletion mutant with env Z-omp R restored resistance to colistin.This is the first report of infectious disease among farm-raised snakes in domestic and overseas.We researched deeply this disease and confirmed ST516 A.hydrophila as the cause of an outbreak of diarrhea.We verified that gene1038 belonging exclusively to Aeromonas spp participated in mechanisms of resistance to colistin,speculating colistin was transported across membrane by gene1038 leading to resistance to colistin.This study shows that the mechanism of resistance to colistin by A.hydrophila is mainly associated with the modulation of env Z/omp R two-component systems,catalyzing 4-amino-4-deoxy-L-arabinose(L-Ara4N)modification of lipid A,resulting in reduction in the net negative charge of the outer membrane thus reducing the affinity of colistion to target.The addition of L-Ara4 N to the lipid A component of LPS are mediated by the arn BCADTEF operon.Our findings provide an important basis for futher studies of the complex network that regulate colistin resistance in A.hydrophila. |
PDF Full Text Request