Study On The Related Mechanism Of Polymyxin B Resistance Of Aquatic Genus Aeromonas

The aquatic environment is an important medium for accumulating and disseminating antibiotic-resistant bacteria as it is often closely related to human activities.Aquatic environments such as incoming and outgoing sewage treatment plants,surface water,groundwater,and agricultural land are major sources of antimicrobial-resistant bacteria and antimicrobial-resistance genes.Following the rapid spread of Gram-negative multi-drug resistant（MDR）bacteria and the slow development of new antimicrobial drugs in recent years,polymyxins have been reappropriated for clinical use to treat infections caused by MDR bacteria.The clinical resistance rate of polymyxins is increasing every year due to the misuse and maladaptive use of antimicrobial drugs.The emergence of the mobile colistin resistance（mcr）gene and the worldwide spread of mcr-positive bacteria have put global public health at risk.Aeromonas spp.,a Gram-negative conditionally pathogenic bacterium widely distributed in aquatic environments,can cause wound infections and gastrointestinal infections in humans.In addition to the aquatic environment,the genus Aeromonas are also naturally distributed in the terrestrial environment and can be isolated from fish,crustaceans and soil,among others.Since 2017,when mcr-3 was first detected from chicken-derived Aeromonas veronii,sporadic studies have reported that other mcr types are identifiable from human and environmental source specimens.However,studies on mcr-positive aquatic genus Aeromonas and the health of the aquatic environment have remained comparatively scarce,and the number of available studies has paid little attention to the prevalence of polymyxin-resistant bacteria and their resistance mechanisms in the aquatic environment.In this study,we collected water specimens from 16 sampling sites in the Yangtze River in 2020 and a series of studies were conducted on the isolated aquatic genus Aeromonas.The mcr gene was screened by colony PCR to analyze the prevalence of mcr in the aquatic environment,the antimicrobial resistance of the aquatic genus Aeromonas was investigated and its antimicrobial profile was analyzed;different species of genus Aeromonas was compared in groups,and the whole genome sequencing and q RT-PCR were used to investigate the reasons for the differences in resistance of aquatic genus Aeromonas to polymyxin B.To explore the mechanisms of resistance related to polymyxin B at the genomic level.Our study was conducted to provide evidences of the possible mechanism for the variety of polymyxin susceptibility in different species of the genus Aeromonas and a theoretical basis for the surveillance of the aquatic environment.Method1.To investigate the prevalence and dissemination of mcr in the genus Aeromonas from the aquatic environment,16 water samples were collected in sterile 1-L bottles from a section of the Yangtze River.All samples were stored at 4℃after collection and analyzed within 12 h.Lysogeny Broth（LB）,Mueller-Hinton agar（MHA）,and Salmonella-Shigella（SS）agar culture mediums were used to perform the cultivation and isolation of bacteria.Matrix-assisted laser desorption ionization-time of flight（MALDI-TOF）MS identification and 16S r RNA sequencing were adopted for bacterial identification.2.The minimal inhibitory concentration（MIC）was determined using the agar dilution method and broth microdilution method to clarify the resistance characterization of the genus Aeromonas isolated from the aquatic environment.3.The whole isolates were screened for the presence of mcr-1 to mcr-10 by colony PCR.And the PCR products of mcr-positive bacteria were screened by agar-gel electrophoresis and verified by Sanger sequencing.4.To investigate the distribution of mcr-positive strains of the genus Aeromonas,we retrieved the isolates in sequences deposited in Gen Bank by Genomes Browser and Isolates Browser.5.Plasmid characteristics and transferability were determined by conjugation experiment.And the location of mcr gene was identified by S1-PFGE and DNA hybridization.6.Extract genomic DNA of the selected strain and Illumina Hiseq PE150 platform was used for the whole-genome sequencing;The reads were assembled using SPAdes3.10.18;Acquired resistance genes were analyzed by Res Finder.Multiple sequence alignment of nucleic acid sequences was performed by EMBL-EBI.RAST was used to annotate the whole genome sequencing results,and NCBI BLAST was used to compare the sequences.Easyfig was used to plot the linear comparison map of the environment around mcr gene.7.The EMBL-EBI tool was used for multiple sequence alignment of amino acid sequences,and the PROVEAN PROTEIN tool was used to predict and evaluate whether amino acid alteration affected biological function.The domains of protein were analyzed by SMART,and the secondary and complex structures of proteins were predicted by PSIPRED and SWISS-MODEL.8.q RT–PCR was performed to detect the gene expression of several different strains.The data were normalized to 16S r RNA levels and analyzed using the 2^-ΔΔCT method to calculate the fold-change relative to the control.Results1.In this study,61 strains of the genus Aeromonas were identified from 163 non-repeating Gram-negative bacteria collected from a section of the Yangtze River in November 2020,including 29 strains of Aeromonas hydrophila,21 strains of Aeromonas caviae and 11 strains of Aeromonas veronii.2.The total drug resistance rate of the genus Aeromonas bacteria to polymyxin B was 49.18%（30/61）,and the MIC₅₀ and MIC₉₀ values of polymyxin B of different species were significantly different（p<0.001）.The MIC₅₀ and MIC₉₀ values of PMB in Aeromonas hydrophila were both above 128 mg/L,while the MIC₅₀ and MIC₉₀ values of Aeromonas caviae and Aeromonas veronii were both 2 mg/L.3.The prevalence of mcr among Gram-negative bacteria isolated from the section of the Yangtze River was 1.84%（3/163）,among which two strains were mcr-3.42 positive Aeromonas veronii and one strain was mcr-3.16 positive Aeromonas caviae.4.The genetic background of mcr-3.42 was different,and the mcr-3-like gene was separated and truncated by insertion sequence ISAs20（1674 bp）and ISAs2（1084 bp）.5.According to the statistics of the countries and sample types in NCBI database from which mcr positive Aeromonas bacteria were uploaded,the majority of mcr positive Aeromonas bacteria came from the United States and China,accounting for 24.93%（93/373）and 22.25%（83/373）,respectively.Nearly half of the samples of mcr positive Aeromonas bacteria were from animal sources,accounting for 42.63%（159/373）,followed by environmental sources and clinical sources,accounting for 24.40%（91/373）and 23.59%（88/373）,respectively.6.Phylogenetic tree analysis showed that the evolutionary distance of A.caviae S611 was close to that of A.caviae SCAc2001（WUTZ00000000.1）from Chinese clinical origin.However,the evolutionary distance of A.veronii M683 and M694 is close to that of the animal-derived A.veronii MS-17-88（GCA＿00361198.1）in the United States.7.According to the results of genome comparison,arn BCADTEF only exists in Aeromonas hydrophila,and the AAA domain and 231 amino acid of Mla F protein are different among different species.The expression level of Mla F in Aeromonas veronii was lower than that in Aeromonas hydrophila and Aeromonas caviae.Conclusion1.This is the first report about the identification of mcr-3.42 positive A.veronii strains and mcr-3.16 positive A.caviae strain isolated from the aquatic environment in China.This study proved and showed that the genus Aeromonas and aquatic environment might be the potential container and reservoir of mcr-3.2.We speculated that the existence of arn BCADTEF and sequence differences in Mla F might contribute to the variety of polymyxin susceptibility in different species of the genus Aeromonas.This is the first report describing the variety in the phenotype of polymyxin susceptibility in different species of the genus Aeromonas.3.This study initially explored the possible mechanisms underlying the differences in polymyxin susceptibility of different aquatic Aeromonas species,providing a theoretical basis for the surveillance of the health of the aquatic environment.