| Clostridium perfringens is an important zoonotic microorganism,which can cause diseases in animal and human under suitable conditions.Contamination of C.perfringens in chicken and pork meat has been reported worldwide,but it is rarely reported in duck products.The current study was undertaken to evaluate Clostridium perfringens contamination in retail duck products from major retail stores in Tai'an,Shandong province.From March to November 2018,173 samples of duck products,10 samples of environment origin,were collected from four major retail stores in the retail market,and 7 samples of fresh feces from healthy shopkeepers.After the samples were isolated and cultured,the serotypes of Clostridium perfringens were identified by multiple PCR.The antibiotic susceptibility of the isolates to 12types(8 classes)commonly used antimicrobial discs was detected by Kirby-Bauer method.The lactamine-resistant genes bla CTX-M,bla TEM,bla SHV,tetracycline resistance genes Tet(M),Tet A(P),Tet B(P)were detected according to the antibiotic resistance profiles.After preliminary identification,part of strains from different sources were analyzed by 16 Sr DNA sequence analysis,and the genetic relationship of Clostridium perfringens from different sources was further analyzed by multilocus sequence typing(MLST).The isolation and culture results showed that the positive rate of Clostridium perfringens in duck products,environment and healthy human feces samples were(48/173,27.7%),(10/10,100%)and(7/7,100%),respectively.And a total of 99 strains of Clostridium perfringens were isolated.The results of serotype identification showed that all the isolates were Clostridium perfringens type A;The 16 Sr DNA sequence analysis of some strains showed that the homology was 99% with the reference strains of Clostridium perfringens uploaded on NCBI.Toxin gene screening showed that beta2 toxin gene(cpb2)was detected in both duck and human strains,with positive rate of 64.20% and 33.33%,respectively.No Net B toxin gene was detected in all isolates.Enterotoxin cpe was only detected in human strains,with a positive rate of(1/7,14.30%).Antimicrobial susceptibility testing showed that 90.10% of the isolates from duckproducts and environment showed multiple antibiotic resistance and the multiple antibiotic profiles mainly contain sulfamethoxazole,bacitracin,lincomycin,erythromycin,and penicillin G,tetracyclinne.Antimicrobial resistance against cefotaxime and cefepime was less than 10% in duck origin isolates.The antibiotic resistance gene of tet A(P),tet(M),bla CTX-M was observed in 28(34.60%,28/81),18(22.20%,18/81)and(4.90,4/81)isolates from duck products and environment,respectively.The antibiotic resistance gene of Tet A(P)was observed in 12(66.70%,12/18)isolates from human,other resistance genes were not detected in human origin strains.After preliminary screening,58 duck origin isolates and 7 healthy adult origin isolates were used for multilocus sequence typing,and 20 groups of healthy children intestinal isolates STs(ST45-ST64)downloaded from NCBI were also used for typing.The results indicated four clonal populations,but only observed in duck origin strains,with 37.90% of all strains associated with four clonal complexes and 41 sequences types from 58 strains of Clostridium perfringens;besides,among them CC1 included duck origin isolates from four stores which contain carcass isolates and environmental isolates,with a total of 14 strains,accounting for24.13% of the total strain.It is a dominant clonal complex group,among which ST11 is the most common account for 8.60% of the total strain.Part of human isolates and duck isolates were distributed in the same sequence type.After the addition of ST45-ST64,three new clonal complex groups(CC5-CC7)were generated,and both duck and children isolates were assigned to the same clonal group at the same time.Conclusion: The retail duck products in Taian retail stores had a high Clostridium perfringens containation rate,all duck and environmental isolates were Clostridium perfringens type A,these isolates exhibit broad-spectrum antimicrobial resistance,multiple antibiotic resistance rate as high as 90.10%.Forty-five duck isolates showed rich genetic diversity,33 STs and 4 clonal complex groups(CC1-CC4)were produced,and CC1 was a dominant clonal complex group.Results of MLST show that cross contamination may occur,animal intestinal contents and retail environment is presumed to be main causes of carcass contamination;part of strains from both hosts were found to be phylogenetically close and appear in the same ST or CC,indicating that duck origin strain were at risk of spreading to human through the food chain. |
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