Identification,Antimicrobial Resistance And Genotyping Of Chicken Clostridium Perfringens Isolated From Different Sources In Taian,Shandong Province

Clostridium perfringens is an important bacterial pathogen in animals and humans.It is of particular importance in poultry as a causative agent of chicken necrotic enteritis(NE)which is resulted from various exotoxins produced by Clostridium perfringens.In order to investigate the prevalence and characteristics of Clostridium perfringens in chicken farms and poultry markets in Tai'an,samples of suspected necrotic enteritis and two batches of eliminated chicken were collected from six chicken farms in three districts and products of retail chicken were collected at the poultry market.The isolation of Clostridium perfringens was carried out by selective medium and the main toxin genes of the isolated strain were identified by PCR.The antimicrobial sensitive disk diffusion method(Kirby-Bauer's method)was used to study the antimicrobial sensitive of the isolate strains.Some isolated strains were used to typing analysis by the method of Multilocus Sequence Typing.209 strains of Clostridium perfringens were isolated from the 420 samples collected with the positive rate of 49.7% that includes 153 strains from products of retail chicken,25 strains from the samples of suspected necrotic enteritis and 31 strains from eliminated chickens.The results showed that all the isolates belonged to Clostridium perfringens type A and the ?2toxin gene was also detected by PCR with the detection rate of 11.5% which was lower than that reported in the related reports.The results of drug resistance by antimicrobial sensitive disk diffusion method showed that the rate of the strains that against gentamicin was the highest with the parcentage of 87%;The rate of the strains against bacitracin reached 73% and the resistance rate to lincomycin is more than 50%.100% of the strains isolated from eliminated chickens in the poultry market are multidrug resistance which have five-drug resistant;85% of the strains isolated from samples of suspected necrotic enteritis aremultidrug resistance which have seven-drug resistant.From the above 70% of the retail chicken isolates in the poultry market are multidrug resistant with the highest six-drug resistance.According to the literature,the multilocus sequence typing technique was used for the typing analysis of 34 isolated strains of Clostridium perfringens and 24 ST types,4 clonal clusters were presented.In order to better study the genetic evolution of isolated strains,10 strains of Clostridium perfringens were downloaded from the GenBank database for comparative analysis,which showed that 9 new ST types were produced.In order to investigate whether Clostridium perfringens can pose a threat to humans,five human feces isolated strains from the poultry market were added to the study.The results showed that the MLST classification of Clostridium perfringens chickens was diversified,but the clonal cluster CC-2 strain was an prevalent genotype in the analyzed strains accounting for 38% of the total analysis strain and the dominant ST type was ST-4,accounting for 18% of the total analysis strain.The isolated strains from different parts of the same suspected necrotic enteritis chicken showed a single ST type.Some isolates of chicken liver and heart sold at different stalls showed the same ST type.Five new ST types were formed by human strains.It has also been found that isolated strains from humans are closely related to some strains from retail chicken products which located in the same clonal cluster suggesting that Clostridium perfringens strains of a certain genotype might be prevalent among humans and chickens.