Drug Resistance Of Pseudomonas Aeruginosa Isolates From The Wanzhou Section Of The Three Gorges Reservoir And Their Toxic Effects On Caenorhabditis Elegan

The Three Gorges Reservoir(TGR)is an important water source of our country,microbial pollution of water body is closely related to biological health.Pseudomonas aeruginosa is an important water-borne and food-borne pathogen,which is easy to spread through water and is detected in various water environments,such as rivers,lakes,swimming pools,etc.,when the body’s immunity declines,it is easy to cause infection to the body.In recent years,there have been more studies on the pathogenicity of Pseudomonas aeruginosa clinically,but less studies on drug sensitivity and toxicology of Pseudomonas aeruginosa in natural water environment.In order to investigate the drug resistance of Pseudomonas aeruginosa isolates from the Wanzhou section of TGR and its toxicological effect on Caenorhabditis elegans(C.elegans),water samples were collected from the Wanzhou section of TGR for bacterial isolation.Pseudomonas aeruginosa was screened out by morphological identification,physiological and biochemical identification,oprI specific gene identification and 16 S rDNA sequencing,and drug resistance and resistance genes of the isolates were studied.Then,using C.elegans as a model organism,the toxicity of Pseudomonas aeruginosa isolates against C.elegans was studied.Finally,transcriptome sequencing technology was used to explore the effects of Pseudomonas aeruginosa exposure on gene transcription levels in nematodes.The results show that:(1)By morphological identification,physiological and biochemical identification,oprI specific gene identification and 16 S rDNA sequencing,14 Pseudomonas aeruginosa strains were isolated and identified from the Wanzhou section of TGR.Pseudomonas aeruginosa isolates were resistant to penicillin G,ampicillin,cefazolin,kanamycin and erythromycin,and sensitive to amikacin,gentamicin,norfloxacin,ciprofloxacin,meropenem and levofloxacin.Drug resistance genes blaTEM,aac(6’)-Ⅱ,qacΔ1-sull,qnrS and mexR were positive in all isolates.Drug resistance genes aac(3)-Ⅰ,tetA,tetB,tetD,tetE,cat,cmlA,qnrA,qnrD were negative in all isolates.In addition,Pseudomonas aeruginosa isolates carried multiple virulence genes,including toxA,exoT and lasI,etc.(2)The toxicity effect of Pseudomonas aeruginosa exposure on C.elegans: PA7,PA8,PA12 isolates and Pseudomonas aeruginosa ATCC 27853 reduced the average life span of C.elegans by 15%,14%,16% and 13%,respectively(P<0.05),and the longest life span of C.elegans was shortened by about 3 days;and body bending frequency decreased by 18%,33%,23%,17%(P<0.05),and head swinging frequency decreased by 10%,13%,11%,10%(P<0.05).PA7,PA8,PA12 isolates and Pseudomonas aeruginosa ATCC 27853 significantly increased the reactive oxygen species(ROS)content in C.elegans(P<0.01),and the ROS rates of increase were 95.93%,74.10%,106.76% and 106.76%,respectively(P<0.01).PA7,PA8 and PA12 isolates caused abnormal expression of antioxidant genes in C.elegans.(3)Effect of exposure of Pseudomonas aeruginosa on regulation of gene transcription levels in C.elegans: PA12 exposure affects gene transcription levels in C.elegans.A total of 3917 differentially expressed genes were screened by transcriptome sequencing,among which 738 genes were up-regulated and 3179 genes were down-regulated.Through GO enrichment analysis,it was found that differential genes were enriched in multiple biological processes,including cellular processes,metabolic processes,development,locomotion,immune processes,etc.Through KEGG pathway analysis,it was found that differentially expressed genes were mainly concentrated in neuroactive ligand receptor interaction,calcium signaling pathway,and lysosome pathway.Cytochrome P450 pathway,peroxisome,extracellular factor signal,fatty acid degradation,steroid synthesis,fatty acid metabolism and life regulation pathway also play important roles in the mechanism of Pseudomonas aeruginosa toxicity.Pseudomonas aeruginosa in the Wanzhou section of TGR has multiple drug resistance,and some of the strains showed strong toxicity to C.elegans,indicating that Pseudomonas aeruginosa in the water body of TGR poses a threat to biological health.The results provide some reference for the prevention and control of Pseudomonas aeruginosa.