| Escherichia coli(E.coli),a foodborne enteropathogen and an opportunistic bacterium,can lead to asymptomatic infections that can cause life-threatening complications in humans and animals.Ruminants serve as one of the most important reservoirs for pathogenic E.coli.Yak(Bos grunniens)is an important ruminant living in the Qinghai-Tibetan Plateau and severs as an essential food and meat animal for local herdsmen.Currently,due to the widespread/misuse of antibiotics in veterinary settings,E.coli infections are getting difficult to treat due to emerging multi-drug resistant strains.As a result,many experts highlight the importance of research to assess the risk of food animal-related(such as,yaks)antimicrobial resistance(AMR)and virulence factors on public health.At present,there is no comprehensive monitoring of bacterial resistance at clinical level or the available data on animal-related drug resistance is not sufficient to form a complete provisional system.In addition,very scare information is available regarding the drug resistance of E.coli in yaks on Qinghai-Tibetan Plateau.Therefore,from a clinical and human health perspective,it is important to access the antibiotic resistance patterns,virulence potential,phylogenetic groups,serogroups,and the detailed genetic diversity in E.coli strains from healthy and yaks with diarrheic infections,in order to provide a theoretical basis for the evaluation of yak meat food safety in QinghaiTibetan Plateau area.The research contents are as follows: 1.Antibiotic resistance of Escherichia coli in free-ranging healthy and diarrheic aks(Bos grunniens)of Qinghai-Tibetan PlateauTo the best of our knowledge,distribution of antibiotic resistance,serogroups,phylogroups,and associated virulence traits are not previously observed in free ranging or diarrheic yaks in China.This necessitates additional studies in such neglected food animals.Therefore,this study aimed to characterize and compare the distribution and possible association of resistance phenotypes,genotypes,virulence factors,phylogenetic groups,and serogroups of E.coli strains isolated from yaks with diarrhea in Qinghai region,while Tibetan yaks that grazed on a semi-mountainous area and had not been exposed to antimicrobials for growth promotion or as therapeutic agents were also included for comparison.Among the E.coli isolates tested from yaks with diarrhea in Qinghai region,most of the isolates were multi-drug resistant(97%)and harbored at least one virulence gene.In contrast,Of the 488 E.coli isolates examined from Tibetan yaks,approximately 31.1% were multidrug resistant.We observed ten virulence genes,of which sfa was the most commonly found(96.9%)in yaks with diarrhea.However,Omp A(30.2%)and etr A(23.1%),and bla CTX-M(27.6%)and bla TEM(14.4%)were the mainly prevalent virulence and drug resistance genes in Tibetan free raging yaks,respectively.Significant statistical positive associations between some resistance phenotypes and virulence genes were observed(P<0.05,OR >1)in both regions.The majority of the E.coli isolates belonged to phylogroup A(almost 70-80%),and serogroups O91 and O145.Notably,we noticed footprints of positive selection in extended spectrum beta-lactamase(ESBL)-producing CTX-M gene containing SNPs genotyped inferred via various maximum likelihood models in Qinghai-Tibetan yaks.Our results suggest that yaks serve as a reservoir of pathogenic E.coli carrying various virulence genes and resistance phenotypes.Therefore,clinicians and relevant authorities must ensure the regulatory use of antimicrobial agents and prevent the spread of these organisms through manure to farm workers and food-processing plants.2.Characteristics of integrons and associated gene cassettes in antibiotic-resistant scherichia coli isolated from free-ranging food animals in ChinaFurther,to confirm the possible dissemination of resistance and virulence traits to free ranging animals,we investigated the occurrence of integrons and ST131 in antibioticresistant E.coli strains isolated from free-ranging yaks,piglets,and chickens,in China and characterized the gene cassettes harbored within the integrons.We examined 432 E.coli strains that exhibited resistance to at least one class of antibiotics.Integrase genes and associated gene cassettes were characterized by PCR analysis,restriction fragment-length polymorphism,DNA sequencing,conjugation experiments,plasmid analysis and multilocus sequence typing(MLST).Twenty-nine(6.7%)integrons were amplified from the 432 antimicrobial-resistant(AMR)isolates evaluated.Specifically,class 1 and 2 integrons were detected in 26(6%)and 3(0.7%)strains,respectively.Meanwhile,6 different gene cassettes,dfr A1,dfr12,aad A1,aad A2,sat1,and orf F,were detected within 6 variable regions(VRs),of which the dfr A1 + aad A1 array was the most common,identified in 12 of 26 class 1 integrons(46.1%).Meanwhile,only one class 2 integron contained a cassette,andthe remaining two contained undetermined VRs.Notably,17 sequence types(ST),including 6 probably novel STs were recovered from free ranging animals.Finally,a conjugation assay confirmed the transfer of 4 different types of class 1 integrons into recipient strains,with plasmid sizes ranging from 20 kb to 30 kb.We observed integrons and ST131 were present in >6% of resistant E.coli strains,therefore,precautionary measures are required to prevent the spread of mobile genetic resistance determinants in food animals and monitor their emergence.3.Experimental mouse lethality of Escherichia coli strains isolated from free anging Tibetan YaksNext,we examined the virulence potential of E.coli isolates harboring at least one virulence gene(associated with Ex PEC or In PEC pathotype and belonging to different phylogenetic groups: A,B1,B2 or D),isolated from free ranging Tibetan yak feces,which were not exposed to antimicrobial agents.The E.coli isolates were characterized for possible linkage with different serogroups,phylogroups by using a mouse model of subcutaneous–infection to envisage the virulence within these E.coli strains.We observed that 23% of the E.coli isolates caused lethal infections in a mouse model of subcutaneous infection and were classified as killer.Moreover,the majority of the killer strains belonged to phylogroup A(65%)and serogroup O60 or O101(35%).Phylogroup B1,serogroups O60 and O101 were statistically associated with the killer status(P < 0.05).However,positive associations(OR >1)were observed between the killer status isolates and all other bacterial virulence traits.This study comprises the first report on the virulence potential of E.coli strains isolated from free-ranging Tibetan yaks feces.Our findings suggest that pathogenic E.coli of free ranging yaks is highly worrisome,as these feces are used as manures by farmers and therewith pose a health risk to humans upon exposure.4.Antibacterial effect and resistant regulation of Houttuynia cordata water extract gainst multi-drug resistant Escherichia coliTo investigate the antibacterial activity of Houttuynia cordata water extract(HCWE)against multi-drug resistant(MDR)E.coli isolates harboring the Acr A gene(multidrug efflux system of E.coli),we examined E.coli strains that exhibited resistance to at least three different classes of antibiotics.Antibacterial effect,minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),and transcriptional level of Acr A gene was assessed by using agar well diffusion technique,tube dilution method and real-time PCR analyses,respectively.The water extract of H.cordata showed antibacterial activity against MDR E.coli isolates with the highest and lowest zone diameter of inhibition(ZDI)of 29 and 13 mm at concentrations of 500 and 50 mg/m L,respectively.The MIC and MBC of HCWE against MDR E.coli isolates was 400 and 500 mg/m L,respectively.Furthermore,the expression of Acr A gene was inhibited(0.39-,0.29-and 0.16-fold)in a dose dependent approach by the HCWE when cultured with 25 mg/m L,50 mg/m L and 100 mg/m L.Our results revealed that H.cordata water extract had greatest potential value against multidrug-resistant E.coli.It can be taken as an alternative source of antibacterial agent against the MDR pathogens and further studies are highly needed for the drug development.Summarizing the whole study,we observed that yaks play an active role as reservoirs and transmitters of threatening pathogens and multidrug resistance genes in QinghaiTibetan Plateau.A number of reasons such as,positive selection in these resistance genes,co-selection,mobile genetic elements(integrons),frequent use of natural resources combined with unrestricted use of antimicrobial agents in veterinary medicine were associated with it.Moreover,the occurrence of phylogroup A and serogroup O60 or O101 in E.coli of free-ranging yaks poses a certain threat to the life and health of humans and animals.We also tried to use Chinese herbal medicine(H.cordata water extract)as an alternative of synthetic antibiotics and it showed greatest potential value against multidrug-resistant E.coli. |
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