Comparison Of Fecal Microbiota Between Diarrheal And Healthy Piglets And Study Of The Antibiotic Resistance Of Pathogens

Piglet diarrhea is a common disease in swine farms,which is also one of the most important factors affecting the benefits of the swine industry.How to prevent and cure it effectively has been the focus of attention and research for us.With the wide use of antibiotics in swine farms,the multidrug-resistant bacteria keep emerging,which can spread the resistance to humans through the food chain,being a threat to food safety and public health.In this study,fecal samples were collected from healthy?n=8?and diarrheal?n=8?piglets at the same feeding conditions from a swine farm in Guangdong province,China.The fecal samples of healthy and diarrheal piglets were analyzed using 16S rRNA gene high-throughput sequencing technology to find the pathogens.Then,these pathogenic bacteria were isolated and identified from the collected fecal samples according to sequencing results.The disk diffusion method?K-B?was used to test the susceptibility of 17 antibiotics of all isolates and the whole genome sequencing was used for one multidrug-resistant Klebsiella pneumoniae strain?KP4?.The details were as follows:1.The fecal microbiota of healthy and diarrheal piglets was compared using 16S rRNA high-throughput sequencing technology.From our results,the fecal microbiota of healthy group was more diverse than that in the diarrhea group and statistically significant difference was observed?P<0.05?.Compared with the healthy group,the relative abundance of Proteobacteria was significantly increased in the diarrhea group,while Bacteroidetes was significantly decreased?P<0.05?.At the genus level,Escherichia-Shigella,Klebsiella and Lactobacillus were the predominant genera in the diarrheal piglets,whereas the relative abundance of Bacteroides,Lachnoclostridium and Ruminococcus was lower than that in the healthy group?P<0.05?.2.A total of 33 strains of E.coli and 3 strains of K.pneumoniae were isolated from all fecal samples.All K.pneumoniae strains were multidrug-resistant and they were resistant to 10antibiotics,including trimethoprim/sulfamethoxazole,tetracycline,florfenicol,ciprofloxacin,enrofloxacin,gentamicin,amikacin,ceftazidime,cefotaxime and aztreonam.In addition,the multi-drug resistance rate of E.coli was 93.94%?31/33?.The antibiotic resistance rates for trimethoprim/sulfamethoxazole,tetracycline and florfenicol were more than 90%.The E.coli strains had severe antibiotic resistance to gentamicin,ciprofloxacin,enrofloxacin and cefotaxime and the antibiotic resistance rates were 84.85%,69.70%,69.70%and 45.45%,respectively.Additionly,the antibiotic resistance rates for amitracin and amikacin were 15.15%and 18.18%,respectively.However,all E.coli and K.pneumoniae strains were sensitive to meropenem,imipenem,cefoxitin,tigecycline and polymixin.3.By the whole genome sequencing,we found that the multidrug-resistant Klebsiella pneumoniae KP4 contained one chromosome genome and two plasmid genomes.From all genomes,83 resistance genes and 56 virulence genes were annotated through the CARD and VFDB database.Surprisingly,the genes of tet?A?,tet?R?,floR,dfrV,sul1,qacE,aadA16,aac?6'?-Ib-cr,qnrB2,arr-6 and bla _CTX-M-27 were located in the same plasmid,which mediated antibiotic resistance to tetracycline,chloramphenicol,sulfonamides,aminoglycosides,quinolones,rifampin and?-lactam antibiotics.In addition,the strain KP4 could produce ESBLs and the type was CTX-M-27.The pathogenic factors were related to type 1 fimbriae,ECP adhesin,iron carrier,heme carrier,biofilm,outer membrane protein and some others.However,the genes related to capsule synthesis were lacking,such as rmpA,magA,wabG.In this study,the fecal microbiota of healthy and diarrheal suckling piglets was analyzed and the antibiotic-resistance status of E.coli and K.pneumoniae in this swine farm was obtained by antibiotic sensitivity test,which provides scientific basis for the effective prevention and control of piglet diarrhea and the rational use of antibiotics in veterinary clinic.The study on the resistance,pathogenic and transmission mechanism of multidrug-resistant K.pneumoniae was of great significance for prevention and treatment of the diseases caused by multidrug-resistant K.pneumoniae.