Characterization Of Heavy Metal Resistance And Mechanism Of Copper Resistance In Salmonella

Salmonella is an important zoonotic and serious food-borne pathogen that can infect humans through the food chain.Contaminated animal-derived food is an important source of zoonotic Salmonella which can contaminate the soil through feces and enter the food chain through fruits and vegetables.The food-borne surveillance system in China suggested that about 17%of food poisoning incidents were caused by Salmonella,and the number of foodborne infections caused by Salmonella is as high as 9.035 million each year.Heavy metals such as copper,zinc,and manganese are widely used as additives to prevent animal diseases and as growth promoters.The unrestrained use of copper and zinc in animal-production environments increases the resistance of pathogenic bacteria such as Salmonella.Moreover,antibiotics are widely used for the prevention and treatment of human and animal diseases caused by pathogenic bacteria.The screening pressure caused by the extensive and irrational use of antibiotics facilitates Salmonella and other pathogenic bacteria to develop drug resistance.In order to control the development and spread of antibiotic-resistant bacteria,environmental disinfection control has become the main measure.However,increased use of disinfectants can also lead to bacterial resistance to disinfectants.Generally,heavy metal,antibiotic,and disinfectant resistance genes often co-exist on mobile genetic elements(Mobile Genetic Elements,MGEs)leading to cause horizontal transmission.Heavy metal contamination in meat and the prevalence of these pollutants in the environment is a risk for both food safety and human health.Notably,the use of metals may potentially promote the proliferation of antibiotic resistance through coselection as well as co-resistance increases the difficulty of disease control in livestock and poultry by making the prevention and control of drug resistance more challenging.The present study focused to isolate and identify Salmonella in the strains preserved in the laboratory for the determination of the resistance phenotype of Salmonella to heavy metals,antibiotics,and disinfectants;the characteristics of resistance;the phenotypic correlation of heavy metal with antibiotic and disinfectant in Salmonella;the epidemiological characteristics of Salmonella heavy metal resistance genes,and their correlation with antibiotics and disinfectants;selecting copper-resistant Salmonella,whole-genome sequencing technology analysis of high copper-resistant Salmonella;resistance gene species and genetic environment characteristics in copper-resistant Salmonella strains;conjugation experiments to study the spread of copper resistance genes,in vitro competition experiments to study the fitness of copper-resistant strains,and qPCR to determine copper resistance under different concentrations of copper stress as well as the expression of antibiotic resistance genes.The characteristics of Salmonella resistance to heavy metals and the correlation with antibiotic and disinfectant resistance were identified,further the mechanism of Salmonella resistance to copper was also explored.The main research and findings are as follows:(1)The phenotypic of Salmonella resistance to heavy metals and its correlation with resistance to antibiotics and disinfectantsA total of 530 Salmonella strains from livestock and poultry manures,meat,farms,and human stool in Sichuan Province were used as subjects to determine Salmonella serotypes and their resistance phenotypes to heavy metals,antibiotics,and disinfectants.Salmonella was dominated by three serotypes:Typhimurium(61.9%,n=328),Derby(8.7%,n=46),Enteritidis(8.5%,n=45).In our study,81%of the 530 Salmonella isolates were considered resistant to Cu,almost 55%to Zn and Cr,over 30%to Mn,20%Cd,and almost 5%to Co.Among them,Typhimurium(n=328)resistant to Cr,Cu,and Mn reached 97.3%(n=319),96.6%(n=317)and 47%(n=156),in Derby 95.7%(n=44),89.1%(n=41),and 80.4%(n=37)of the strains showed resistance to Cd,Cu,and Zn,whereas in Enteritidis,100%(n=45),91.1%(n=41),and 77.8%(n=35)of isolates were resistant to Cd,Cu,and Cr.Salmonella isolates from the farm,meat,animal manure,and human stool showed high resistance to copper,which were 89.0%,84.0%,65.0%,and 58.0%,respectively.Cu-MnCr(25.1%,n=133)and Cu-Zn-Mn-Cr(23.7%,n=126)were the most frequent heavy metal resistance profiles.461 Salmonella isolates were resistant to at least one antibiotic and 226 were multidrug-resistant.The drug susceptibility test revealed that Salmonella resistance to Benzalkonium chloride was 85.6%(n=443),where the resistance in Salmonella from animal manure(90.7%),meat isolates(88.6%),and farm environment(87.7%)was significantly higher than that of Salmonella from human stool(28.9%).Salmonella resistant to tetracycline,trimethoprim,and amoxicillin/clavulanic acid reached 78.1%,64.5%,and 30.7%,among which the resistance in Salmonella isolates from meat,animal manure,and human stool were significantly higher.However,TET-TMP(17.9%,n=95),TET(14.5%,n=77)and TET-TMP-CN(12.6%,n=67)were the main antibiotic resistance profiles.Correlation analysis revealed a significant correlation between heavy metals and resistance to at least two or more antibiotics(P<0.05)in Salmonella.High resistance to heavy metals,antibiotics,and disinfectants in Salmonella as well as a significant correlation between these resistances might be related to the overuse of heavy metals,antibiotics,and disinfectants in animal husbandry.(2)Salmonella resistance genotypes to heavy metals and their correlation between antibiotic and disinfectant resistanceFurther,we investigated the prevalence of heavy metal resistance genes in Salmonella and their association with antibiotic and disinfectant resistance.As result,the prevalence of HMRGs was most common among all Salmonella isolates,particularly a high prevalence of genes resistance to copper was observed.In total,97.9%(n=518)of the isolates carried at least one HMRG and the high prevalence of Cu-resistance genes pcoR,pcoC was found in 58.8%(n=311),53.9%(n=285)and pcoA in 18%(n=95)of all isolates.Besides,genes nccA(resistant to Ni-Cr-Cd),cadD(resistant to Cd),and merA(resistant to Hg)were carried by 43.7%(n=231),40.6%(n=215)and 38.4%(n=203)of Salmonella isolates.For different serotypes,the high prevalence of czcD,pcoR and pcoC genes was found in Typhimurium(n=328)and Derby(n=46)where 82.9%(n=272),65.5%(n=215),and 59.8%(n=196),of Typhimurium,while 67.4%(n=31),50%(n=23)and 32.6%(n=15)of Derby isolates were carrying these genes.In Enteritidis(n=45),the most prevalent gene was pcoR,accounting for 71.1%(n=32),followed by pcoC and czcD,accounting for 64.4%(n=29)and 60.0%(n=27),respectively.The prevalence of pcoR,ncrA,and pbrA genes in animal manure isolates were significantly higher than those from other isolates,while merA and arsB genes were more prevalent in human stool than those from other isolates.A total of 240 heavy metal resistance gene combinations were found,among which czcD(5.09%,n=27),pcoC-czcD(2.45%,n=13),and czcD-pcoR(2.26%,n=12)were the top three combinations.Correlation analysis revealed the significant associations between 11 heavy metal resistance genes and resistance to at least two antibiotics(P<0.05).pcoR,pcoC and pcoA genes were significantly associated with resistance to tetracycline,amoxicillin/clavulanic acid,ciprofloxacin,trimethoprim,and gentamicin in meat,cultured environment,and human stool isolates(P<0.05).The pcoC,pcoA,cadD,pbrA,nccA,merA and arsB genes were commonly associated with disinfectant(benzalkonium chloride)resistance in all Salmonella isolates.Heavy metal resistance genes,particularly copper resistance genes were more common in Salmonella,and there is a significant correlation between copper resistance genes and resistance to antibiotics and disinfectants.Thus,it is necessary to develop the mechanism of copper resistance in Salmonella,as well as resistance to other antimicrobial drugs.(3)Whole-genome of copper-resistant Salmonella and analysis of copper-resistant gene-environmentAccording to the phenotype and genotype,9 copper-resistant strains(minimum inhibitory concentration to copper ≥800 mg/L)were selected,and the second-generation whole-genome sequencing was performed and annotated in multiple databases(VFDB,CAZY,SWISSPROT,CARD,BacMet2,KEGG,etc.).All strains were found to carry the virulence islands SPI-1 and SPI-2,and fimbriae-related gene clusters(fiwA,I,C,D,F,and H)were detected.A total of 146 antibiotic resistance genes were detected in the sequenced strain genomes,among which the efflux pump genes that mediate multidrug resistance were detected most frequently,accounting for 42.5%.Analysis of the gene structure found that four main structures mediate copper resistance:1)cus gene cluster,except for strain S140A,the cusS-pcoE gene structure was found in other strains;2)gol gene cluster,the gene structure is golBST;3)cueR-copA gene cluster;4)The pco-sil gene cluster,including pcoESRDCBAEG-silPGABFCRSE,pcoESRDCBAEG-silPAB-cusF-silCRS,and pcoCpcoD,were plasmid-mediated.Eight Salmonella strains(S251,S152,S151A,S140A,S244,S142A,S54A,and S246A)contained the pco/sil operon encoding the Cu/Ag resistance determinant,four of which were located in the integrated binding transfer element(ICE)between the genes tral and traC,in one strain,the ars operon was located in the middle of the tfc operon,and the two strains also carried the tetracycline resistance gene tet and the mercury operon mer.Strain S88A carried only the pcoC-pcoD gene cluster.Based on the gene species and gene structure environment of Salmonella,we analyzed the role of different resistance genes and mapped the working system of Salmonella to copper resistance.(4)Plasmid-mediated dissemination and expression of copper resistance genesWe conducted conjugation experiments on the selected copper-tolerant strains and determined the adaptive cost.qPCR was used to detect the expression of pco genes and antibiotic resistance genes under copper stress.In this study,horizontal transmission of copper-resistant pco genes in Salmonella was found.The conjugants of S251,and S244 strains exhibited resistance to copper,and there was no significant difference in growth rate between the zygotes and the recipient strains,and the zygotes showed no adaptive cost.Strain S251 was induced under different concentrations of copper.The results showed that:Under 0.25×MIC copper pressure,the expressions of pcoR weas activated at about two hours,and reached the highest ati four hours.The pcoC and pcoA genes were expressed a litter bit at four hours;the expressions of aph(6)-id,and sul2 genes were upregulated after 4 hours;the expression acrD and mdtH was significantly up-regulated in 30 minutes.Under 0.5×MIC copper pressure,the expression of copper resistance gene pcoR was activated at 30min,and reached the highest level after 4 hours,expression of both genes pcoC and pcoA,also increased in 2nd hour and reached the highest at 4th hour.The expression of pco genes remain significantly increased from 2nd hour to 10th under the same MIC.The tetracycline efflux resistance gene tet(D)was significantly up-regulated after 2 hours,the efflux pump genes mdtH and acrD reached the highest expression in 4 hours;aminoglycoside resistance genes aph(6)-id was significantly up-regulated in 2 hours and sulfonamide resistance genes sul2 at 6th h.These results indicated that copper resistance genes such as pcoR,pcoC and pcoA can be mediated by plasmid transmission.Under the pressure of copper sub-inhibitory concentration,Salmonella not only exhibited copper resistance but also induced both the expression of antibiotic resistance genes and efflux resistance genes,thus causing the strains to exhibit co-resistance or cross-resistance to other antibacterial drugs.In summary,heavy metal-resistant Salmonella was widely prevalent in livestock and poultry production,meat food,and farm environments.These isolates carried a variety of resistance genes,which were related to environmental selection pressure and the spread of mobile genetic elements such as plasmids.In the context of antibacterial drugs,the extensive use of heavy metals such as copper in the food-production environment could cause resistance to these metals and even co-resistance to other antibacterial drugs and could spread in animal production environments and food chains.This study explored the characteristics of Salmonella resistance to heavy metals and the mechanism of Salmonella resistance to copper.