Isolation And Biological Characteristic Analysis Of Sheep/Goat-Derived Clostridium Perfringens In Northwestern China

Sheep/goat farming plays a crucial role in animal product consumption and economic income of local people in the northwestern regions,which is the main commercial sheep breeding area in China.An important zoonic pathogen,C.perfringens poses serious threatens to livestock breeding,foods production,and human medication.To make an insight into the prevalence and biological characters of C.perfringens strains from sheep/goats in northwestern China,and provide references for risk assessment and prevention of C.perfringens diseases.In this study,we collected rectal samples of sheep/goats and environmental samples in Shaanxi province,Gansu province,Ningxia province,and Qinghai province.Then,C.perfringens isolation,toxin typing,antibiotic resistance test,disinfectant sensitivity test,antibiotic resistance genes(ARGs)detections,biofilm formation,and wholegenome sequencing were carried out in our study.Here are the obtained results:1.Isolation,identification,and toxin typing of C.perfringensA total of 1127 samples were collected in this study,225 C.perfringens type A strains,64 C.perfringens type D strains,and 6 C.perfringens type F strains were eventually recovered from the samples,with a general isolation rate of 26.2%.Highest isolation rate of C.perfringens was observed among the goats with diarrhea(75%),followed by free-ranged healthy sheep(45%),captive healthy goats(28.3%),and captive healthy sheep(20.6%).The isolation rate of C.perfringens was significantly higher in free-ranged healthy sheep than captive healthy sheep(P<0.01),indicating the feeding modes might affect the exist of C.perfringens in the intestinal track of sheep.Additionally,C.perfringens strains were also obtained from various farm environments including ground,railing,and milking hall,demonstrating the widespread of this bacterium the intestinal track of sheep and the farm environments.2.Antibiotic resistance and biofilm formation testsAntibiotic resistance test showed the C.perfringens isolates had strong resistance to sulfonamides,mild resistance to tetracycline and clindamycin,and low-level resistance to penicillin,ampicillin,chloramphenicol,and linezolid.However,no isolate showed resistance to vancomycin.ARGs detections indicated that the tetracycline-resistant genes tet A(P)and tet B(P)had higher detection rates,followed by lincomycin-resistant gene lnu(P),macrolidesresistant genea erm(A),erm(B),and erm(Q),aminoglycoside-resistant gene aac(6’)-aph(2’’),and etc.The high isolation rates of tet A(P),tet B(P),lnu(P),and erm(Q)partly explained the stronger resistance to tetracycline and clindamycin.However,no sulfonamides-resistant genes were detected among the C perfringens isolates,which indicated the possible natural resistance of C.perfringens to sulfonamides.After biofilm formation and quantification,we found that most C.perfringens isolates had a biofilm forming ability,and 183 of them had a strong or moderate ability of biofilm forming.The biofilms of C.perfringens were absolutely removed after treated with 0.27% sodium hypochlorite,while the biofilms of some C.perfringens strains increased significantly after treated with 10% hydrogen peroxide and 2% glutaraldehyde.Disinfectants should be reasonably selected in livestock farming to prevent the spread and infections of C.perfringens.3.Whole-genome sequencing and analysisWhole-genome sequencing indicated that C.perfringens isolates harbored various toxin genes including plc,pfo A,nag K,etx,and cpe,and eleven antibiotic resistance genes,including tet A(P),tet B(P),aac(6`)-aph(2``),fex A,and optr A.Phylogenetic analysis based on single nucleotide polymorphisms(SNPs)revealed the C.perfringens strains from same source shared closer genetic relationship,and a certain degree of genetic diversity.Additionally,the C.perfringens isolates from same animal species appeared to be distinct from the other isolates in the cluster heatmap based on toxin gene profiles,demonstrating that the host animal might make a difference on the toxin gene profiles of C.perfringens strains.The etx genes among the C.perfringens type D strains shared high sequence identities and similar gene environments,IS1151-etx-tnp-ISCp1.Genes etx and cpe were found to locate on one p CW3-like plasmid,indicating the possible horizontal transfer of them.Moreover,Tn6218-like transposons,which shared high sequence identities with the Tn6218 transposons in Clostridium difficile and encoded aac(6`)-aph(2``)and erm(B),were detected on plasmids p21-D-5b and p QHY-2.Circler comparison indicated the optr Apositive plasmids had similar structure,however,both conjugation and electrotransformation experiments using QHY-2 and 21-D-5 as donor and C.perfringens ATCC 13124,E.coli C600 or E.coli DH5α as recipients,showed unsuccessful transfer of the plasmids.Molecular and phylogenetic analysis indicated the optr A-positive plasmids belonged to a novel plasmid type in C.perfringens,that differed from the p CW3-like plasmids,p CP13-like plasmids,and p IP404-like plasmids.In summary,we collected 1127 samples and recovered 295 C.perfringens strains in this study.Strong resistance against sulfonamides and mild resistance against tetracycline and clindamycin were observed among the C.perfringens isolates,and various ARGs,including optr A,were detected in them.Most of the isolates could form biofilms,and the biofilms showed strong resistance to some disinfectants.The optr A-positive plasmids in C.perfringens belonged to a novel plasmid type that can`t be conjugated.However,the ARGs on them had a possibility of co-transmission through insertion elements or transposons.The rational use of antibiotics and disinfectants should be strengthened in production to prevent the spread and increase of antimicrobial resistance(AMR)of C.perfringens.