Discovery Of A Novel Multidrug-resistant Integrative And Conjugative Element In Proteus Mirabilis Isolated From Swine And The Study On Cu²⁺ To Facilitate Its Horizontal Transfer

Proteus mirabilis,Escherichia coli and Haemophilus parasuis are the common opportunistic pathogens in swine farms,which can infect pigs and play a vital role in the spread of bacterial resistance.Integrative and conjugative elements（ICEs）are kinds of self-transmissible chromosomal mobile genetic elements.As the largest ICEs family,the variable regions and hot spots in the structure of SXT/R391 ICEs are often the high frequency insertion sites of antibiotic resistance genes（ARGs）,which make their genetic structures complex and changeable.Therefore,to explore the diversity genetic environment of SXT/R391 ICEs and analyze their genetic characteristics can provide theoretical support for the spread of bacterial resistance.Copper is a necessary trace element for physiological function of organisms.Copper salt,such as copper sulfate,is often added to feed to promote animal growth.Except for inducing antibiotic resistance by coselection,copper can also promote the conjugative transfer of drug-resistant plasmids within or across bacterial genera.However,whether Cu²⁺can facilitate the horizontal transfer of SXT/R391 ICEs across bacterial genera is still largely unknown.In this study,a novel multidrug-resistant（MDR）SXT/R391 ICE（ICEPmi Chn Ch SC1905）was found in a P.mirabilis strain isolated from swine.Furthermore,the effect and mechanism of Cu²⁺on the horizontal transfer of the novel ICEPmi Chn Ch SC1905 to E.coli were explored under artificial culture.Meanwhile,the changes of intestinal flora and metabolites were analyzed in vivo when Cu²⁺promoted the horizontal transfer of the novel ICEPmi Chn Ch SC1905 in the intestinal tract of mice.Thus,it can provide a new clue to explain the transmission mechanism of antimicrobial resistance and evaluate the synergistic effect of copper on it.The details were as follows:1.Discovery of the novel multidrug-resistant integrative and conjugative element（ICEPmi Chn Ch SC1905）and the analyses of its genetic characteristics.In order to explore whether the common opportunistic pathogens from swine carry new MDR SXT/R391 ICE and its genetic characteristics.139 P.mirabilis strains,491 E.coli strains and 15 H.parasuis strains were isolated and identified from 564 nasal swabs of swine.The results of drug-resistant phenotype showed that all the P.mirabilis strains showed multidrug resistance.The multiple resistance rate of E.coli strains was 72.7%.All the 15 H.parasuis strains carried aminoglycoside and tetracycline resistance genes.The investigation of SXT/R391 ICE showed that no SXT/R391 ICE was detected from E.coli and H.parasuis strains.Only 6positive strains were detected from P.mirabilis strains,among which 2 strains carrying SXT/R391 ICE were resistant to 11 kinds of antibiotics.Based on the consistent drug resistance spectrum of the 2 P.mirabilis strains,in order to further explore the genetic environment of MDR phenotype and whether it is mediated by SXT/R391 ICE,one（No.Ch SC1905）of them was selected for whole genome sequencing（Oxford Nanopore）.The result showed that there were 23 ARGs in the genome of P.mirabilis strain Ch SC1905,among which 21 ARGs were located on a novel SXT/R391 ICE,designated ICEPmi Chn Ch SC1905（with a full-length sequence of 143689 bp）.Blast analysis showed that the regions HS5,HS1 and HS2 of ICEPmi Chn Ch SC1905 were arranged in reverse order,and its MDR regions VRIII and HS4 were rearranged and divided into two parts,which have not been reported to date in SXT/R391 ICEs family involved in great difference in structure.In addition,the 21 ARGs(including the clinically important multidrug resistance gene cfr,fosfomycin resistance gene fos A3,fluoroquinolone resistance gene aac（6′）-Ib-cr and extended-spectrumβ-lactamase gene bla _CTX-M-65)located on ICEPmi Chn Ch SC1905 were distributed in one rearranged region VRIII and two interrupted regions HS4,respectively.Furthermore,evolutionary analysis showed that ICEPmi Chn Ch SC1905 and nine SXT/R391ICEs from Proteus were clustered into one branch in ML（Maximum likelihood）tree,and the support rate was 100%.Result of the nested PCR showed that ICEPmi Chn Ch SC1905 could form a cyclization structure outside the chromosome.The conjugation test further showed that ICEPmi Chn Ch SC1905 could be successfully transferred to E.coli C600 at a spontaneous frequency of 5.2×10^-5.These results suggested that ICEPmi Chn Ch SC1905 has potential risk of horizontal transmission.In addition,genetic stability test showed that ICEPmi Chn Ch SC1905 could be stably inherited in P.mirabilis strain Ch SC1905,ICEPmi Chn Ch SC1905 in transconjugants was lost after the 25th passage.Growth curve test showed that the growth ability of transconjugants carried ICEPmi Chn Ch SC1905 were lower than that of recipient C600.Meanwhile,transconjugants carried ICEPmi Chn Ch SC1905 were at a disadvantage in the competitive test.These results indicated that ICEPmi Chn Ch SC1905would affect the growth properties of recipient C600,suggesting that it has a fitness cost.This study was the first to discover the new MDR ICEPmi Chn Ch SC1905 and analyzed its genetic characteristics.The rearrangement of core structure in SXT/R391 ICEs was revealed.Moreover,It was confirmed that this new ICEPmi Chn Ch SC1905 had the ability of conjugative transfer.Besides,ICEPmi Chn Ch SC1905 carried 21 ARGs including the clinically important resistance genes cfr,fos A3,aac（6′）-Ib-cr and bla _CTX-M-65.The results provided data support for the analysis of the genetic evolution of SXT/R391 ICEs and the spread of bacterial resistance.2.Study on the synergistic effect and mechanism of Cu²⁺on the horizontal transfer of the novel ICEPmi Chn Ch SC1905 under artificial culture.To explore whether and how Cu²⁺can facilitate the horizontal transfer of the novel MDR ICEPmi Chn Ch SC1905.In this study,P.mirabilis strain Ch SC1905 harboring ICEPmi Chn Ch SC1905 was used as a donor,and E.coli C600 was used as a recipient,the effects of different concentrations of Cu²⁺on the conjugative transfer of ICEPmi Chn Ch SC1905 were evaluated under artificial culture.The results showed that Cu²⁺（1μmol/L,5μmol/L and 10μmol/L）significantly promoted the horizontal transfer of ICEPmi Chn Ch SC1905（p<0.05 or p<0.01）.Further,5μmol/L Cu²⁺also significantly facilitated the horizontal transfer of ICEPmi Chn Ch SC1905 from the newly generated transconjugants to another recipient（E.coli J53）（p<0.01）.Subsequently,the mechanisms were explored by testing the intracellular reactive oxygen species（ROS）level,detecting cell membrane permeability,and checking changes in the expression of genes related to oxidative stress,cell membrane,cell adhesion and ATP synthesis.The results of flow cytometer showed that Cu²⁺（1μmol/L,5μmol/L and 10μmol/L）significantly increased the production of ROS in both donor and recipient strains（p<0.01）.The addition of ROS scavenger significantly decreased the conjugative frequency of ICEPmi Chn Ch SC1905（p<0.05 or p<0.01）.Besides,transcriptional analyses showed that the m RNA expression level of antioxidant-related genes,such as genes coding for alkyl hydroperoxide reductase（ahp C and ahp F）in the donor strain,as well as genes coding for superoxide dismutase（sod A and sod B）and thioredoxin reductase（trx B）in the recipient strain,was significantly upregulated in the conjugation mating system with 5μmol/L Cu²⁺（p<0.05）.Therefore,it was considered that ROS generation might be a critical factor for Cu²⁺to promote the horizontal transfer of ICEPmi Chn Ch SC1905.ROS generation could lead to DNA damage and induce SOS response.However,the differentially expressed genes related to SOS response were mainly enriched in DNA repair and recombination in the recipient strain in the conjugation mating system with 5μmol/L Cu²⁺.Moreover,the results of flow cytometer showed that the cell membrane permeability of the donor and recipient strains increased significantly in the presence of Cu²⁺at 5μmol/L and 10μmol/L（p<0.01）.Transmission electron microscope（TEM）images showed that apparent cell membrane damage and indistinct cell borders could be found in the conjugation mating system with 5μmol/L and 10μmol/L Cu²⁺.Transcriptional analyses showed that the m RNA expression level of relevant genes（e.g.,omp）encoding for membrane proteins was also significantly upregulated in the donor and recipient strains in the conjugation mating system with 5μmol/L Cu²⁺（p<0.05）.The outer membrane proteins played important roles in forming outer membrane pores and increasing membrane permeability.Consequently,it was presumed that increased membrane permeability might be a potential mechanism for Cu²⁺to accelerate the horizontal transfer of ICEPmi Chn Ch SC1905.Meanwhile,TEM images showed that more cell contact between these cells in the conjugation mating system was observed in the presence of Cu²⁺at 1μmol/L,5μmol/L and10μmol/L.Transcriptional analyses showed that the m RNA expression level of adhesion-relevant genes（e.g.,fim）was significantly upregulated in the recipient strain in the conjugation mating system with 5μmol/L Cu²⁺（p<0.05）.These findings indicated that the enhanced cell adhesion between bacteria might be beneficial for Cu²⁺to facilitate the horizontal transfer of ICEPmi Chn Ch SC1905.Based on the results of transcriptome sequencing,we further found that the significantly increased m RNA expression level of ATP synthesis genes（atp）and cop A gene（a component of the copper efflux system）were also observed in the recipient strain（p<0.05）,suggesting that Cu²⁺might also promote the horizontal transfer of ICEPmi Chn Ch SC1905 by increasing the energy supply and the uptake of ICEPmi Chn Ch SC1905.This study was the first to report that Cu²⁺（1μmol/L,5μmol/L and 10μmol/L）could promote the horizontal transfer of the new MDR ICEPmi Chn Ch SC1905 across bacterial genera.The mechanisms might be related to ROS production,increased membrane permeability,cell adhesion and ATP synthesis.The results provided a theoretical basis for explaining the synergistic effect of copper on the spread of bacterial resistance.3.Study on Cu²⁺to promote the transfer of the novel ICEPmi Chn Ch SC1905 in the intestinal tract of mice and the changes of intestinal flora and metabolites.In order to further explore whether Cu²⁺can accelerate the horizontal transfer of the novel MDR ICEPmi Chn Ch SC1905 in mice,and analyze the changes of intestinal flora and metabolic pathway when it promotes the transfer of the novel ICEPmi Chn Ch SC1905.30 SPF mice were randomly divided into three groups:normal control group（n=10）,positive control group（n=10）and Cu²⁺treatment group（n=10）.Mice in the Cu²⁺treatment group were given 5mg/kg Cu²⁺（body weight）by gavage in advance,and the normal control group and positive control group were given sterile double distilled water by gavage.After continuous administration of Cu²⁺for 3 days,mice in the Cu²⁺treatment group and positive control group were inoculated with P.mirabilis strain Ch SC1905 harboring the novel ICEPmi Chn Ch SC1905 by gavage respectively,and the normal control group were inoculated with sterilized LB broth.Fresh feces were collected on day 4,7,10 and 13.E.coli in the intestinal tract of mice was selected as the targeted ICEPmi Chn Ch SC1905 positive transferred strain.The numbers of the transferred strain and inoculated strain in each group were counted from the selective plates.The results showed that the isolated amount of transferred strain in positive control group were was relatively few,but the isolated amount of transferred strain increased significantly（p<0.01）in Cu²⁺treatment group.On day 4 and day 7,the amount of transferred strain reached the maximum,and then gradually stabilized.In the meantime,the inoculated strain could colonize in the intestinal tract of mice.However,the amount of strain Ch SC1905 isolated increased significantly in Cu²⁺treatment group（p<0.01）.These results suggested that treatment with 5 mg/kg Cu²⁺by gavage not only facilitated the horizontal transfer of ICEPmi Chn Ch SC1905 in the intestinal tract of mice,but also promoted the proliferation of the inoculated strain.The increased amount of inoculated strain（the donor strain）might be an important factor for Cu²⁺to promote the transfer of ICEPmi Chn Ch SC1905.Subsequently,the feces of mice on day 4 and day 13 were selected for 16S r RNA sequencing.The results showed that the differential predominant bacterium in the positive control group were f＿＿Bacteroidaceae,g＿＿Bacteroides,g＿＿Erysipelatoclostridium,g＿＿Negativibacillus on day 4.The differential predominant bacterium in the Cu²⁺treatment group were p＿＿Firmicutes,o＿＿Lactobacillales,c＿＿Bacilli,f＿＿Lactobacillaceae,g＿＿Lactobacillus,g＿＿norank＿f＿＿Muribaculaceae and g＿＿Lachnospiraceae＿NK4A136＿group on day 4.Analysis on the difference of genus level between groups showed that the abundance of g＿＿Lachnospiraceae＿NK4A136＿group,g＿＿unclassified＿f＿＿Lachnospiraceaeand g＿＿Lactobacillus were significantly increased in Cu²⁺treatment group compared with positive control group（p<0.05 or p<0.01）,while the abundance of g＿＿Bacteroides was significantly decreased（p<0.01）.On day 13,the differential predominant bacterium in Cu²⁺treatment group were o＿＿Staphylococcales,f＿＿Staphylococcaceae and g＿＿Staphylococcus.Analysis on the difference of genus level between groups showed that the abundance of g＿＿Staphylococcus was significantly increased in Cu²⁺treatment group compared with positive control group（p<0.001）,while the abundance of g＿＿Blautia and g＿＿unclassified＿f＿＿Lachnospiraceae were significantly decreased（p<0.05）.Meanwhile,based on the highest number of transferred strains isolated on day 4,the feces from each group were collected and were measured by untargeted metabolomics.The results showed that arginine biosynthesis was the most significant metabolic pathway for the enrichment of differential metabolites(Cu²⁺treatment group vs positive control group).In addition,spearman correlation analysis between the differential predominant bacterium in the Cu²⁺treatment group and differential metabolites(Cu²⁺treatment group vs positive control group)enriched in KEGG pathway on day 4 was performed.The results showed that g＿＿Lachnospiraceae＿NK4A136＿group was negatively correlated with Creatine.o＿＿Lactobacillales,c＿＿Bacilli,g＿＿Lactobacillus and f＿＿Lactobacillaceae were negatively correlated with D-Pantothenic acid.p＿＿Firmicutes was positively correlated with Pantothenic Acid,and was negatively correlated with Melibiose.g＿＿norank＿f＿＿Muribaculaceae was positively correlated with Histamine,D-Ala-D-Ala,L-Aspartic Acid,L-Arginine,Pantetheine and Citrulline.g＿＿unclassified＿f＿＿Lachnospiraceae was positively correlated with L-Aspartic Acid and Pantothenic Acid.g＿＿Bacteroides was negatively correlated with L-Aspartic Acid.This study was the first time to confirm that 5 mg/kg Cu²⁺could promote the horizontal transfer of the novel MDR ICEPmi Chn Ch SC1905 to E.coli in the intestinal tract of mice,and the intestinal flora and metabolic pathway were significantly changed.The results provided a theoretical reference for revealing that Cu²⁺promotes the transfer of MDR mobile elements among members of intestinal microbiome and its potential influencing factors.Risk assessment should be conducted on the use of copper.