Biological Characteristics Of Escherichia Coli From Giant Panda

Escherichia coli(E.coli)is a normal bacterium in the intestine of humans and animals and is widespread in nature.When the body occurs stress response,or immunity decreases,some pathogenic E.coli are an important cause of bacterial diarrhea in humans and animals,which brings serious harm to human health,animal husbandry development and wild animal protection.The giant panda(Ailuropoda melanoleuca),as a national level protected animal,is recognized as vulnerable wildlife in the world.The intestinal disease caused by E.coli is one of the important reasons endangering the health of giant pandas.Up to now,the research on E.coli of giant panda origin here and abroad has mainly focused on serotypes and drug resistance phenotypes.At the same time,there are few reports on drug resistance genotypes and pathogenicity.In this study,100 strains of E.coli isolated and identified from 100 fecal samples of captive giant pandas in Chengdu Giant Panda Breeding Research Base from 2020 to 2021 were subjected to serotype identification,drug resistance phenotype and genotype analysis,virulence gene carriage and pathogenicity study,which clarified the basic biological characteristics of E.coli derived from captive giant panda populations in this base and provided guidance for clinical rational drug use,while it was important for the protection of wild animals such as giant pandas.Main study results are as follows:1 Serotype and pathogenic type of E.coli O antigen of giant panda originSeventeen dominant O antigen serotypes of 100 giant pandas derived E.coli strains were identified by slide agglutination test.The results showed that 32 strains of 100 strains of E.coli could be typed and belonged to 12 different serotypes.In this captive giant panda population,O20,O158,O15 and O9 were the dominant serotypes among the typeable strains accounting for 59.3% of the typeable strains.2 Phenotype and Genotype of Drug-resistant E.coli Isolated from Giant PandaThe Kirby-Bauer disc diffusion method was used to test the susceptibility of 100 strains of E.coli against 33 antimicrobial agents.Giant panda-derived E.coli has a broad spectrum of drug resistance.The results showed that the drug resistance rate to enrofloxacin was the highest(68%),followed by ampicillin(56%),imipenem(55%),amoxicillin(54%),cephalexin(52%),and the sensitivity to meropenem(100%)and cefoxitin(100%)was the highest.In addition,48% of the 100 E.coli strains were multidrug-resistant,including 38%of strains resistant to 20 or more antibiotics and 52% of strains resistant to 15 or more antibiotics.The age and sex of giant pandas had a particular effect on the drug resistance of E.coli.The drug resistance rates of E.coli derived from juvenile giant pandas to sultamicillin,tazocin,ceftazidime,amikacin and azithromycin were significantly higher than those of other age groups(P < 0.01).In comparison,the drug resistance rates of E.coli derived from female giant pandas were higher than those of male piperacillin and neomycin,with significant differences(P < 0.05).Wafergen Smartchip high-throughput PCR(HT-q PCR)detected 47 resistance genes in100 giant panda-derived E.coli strains.The results showed that all strains carried at least 6drug resistance genes and the highest 31 drug resistance genes;the detection rates of multidrug resistance genes acr A-04(99%)and acr A-05(99%)were the highest,followed by aminoglycoside aac C(98%),β-lactam bla CTX-M-04(98%),amp C-04(97%),bla SHV-01(94%)and bla TEM(90%).The resistance genes of E.coli derived from aged giant pandas to bla OXY,aac(6 ’)I1 and tet D-02 were significantly higher than those in other age groups(P < 0.01),and the resistance genes of E.coli derived from adult and juvenile giant pandas to bla CTX-M-03,bla SHV-02 and cat A1 were higher than those in aged and subadult groups(P < 0.05)The carriage of bla NDM in E.coli derived from male giant pandas was higher than that in females.The resistance genes to amp C-04 and bla SHV-02 in E.coli derived from females were higher than those in males,with a significant difference(P <0.05).3 The Virulence Gene Carriage of E.Coli from Giant Panda and the Pathogenicity of Representative Strains to MiceSeventeen virulence genes including E.coli adhesins,toxins,iron uptake and protective elements were detected by common PCR in 100 strains of E.coli derived from giant pandas.The results showed that 100 strains of E.coli from giant pandas carried 1 ~ 11 virulence genes.Pap A had the highest detection rate(99%),followed by fim C(74%),fyu A(48%),omp T(45%),ast A(37%),sit A(35%),and iss(30%);no hly A,elt,or eat A genes were detected.Five strains(GP8,GP67,GP58,GP54,GP22)were selected according to serotype,pathotype,and virulence gene carriage,and their pathogenicity in Kunming mice was studied by intraperitoneal injection.The results showed that the five strains were lethal to mice(100%)at the bacterial doses of 1.36 × 10 ^ 9 CFU(GP8),8.7 × 10 ^ 8 CFU(GP67),5.7 × 10 ^ 9 CFU(GP58),2.99 × 10 ^ 10 CFU(GP54),and 2.27 × 10 ^ 10 CFU(GP22),respectively,but not to mice at other doses;the main lesions occurred in the small intestinal mucosa,suggesting that the intestine is the most likely site of invasion of the bacterium,and although the above representative strains are pathogenic to mice,they are not virulent.This study showed that E.coli of giant panda origin had a wide range of serotypes,mainly serotype O20;a wide spectrum of drug resistance,and severe multi-drug resistance phenomenon.It carried 45 drug resistance genes,with the highest detection rates of multidrug resistance acr A-04 and acr A-05 genes,and the drug resistance phenotype and drug resistance genotype of the strains had a certain link;the virulence genes were mainly carrying pap A(99%),Some strains have some pathogenicity,but the pathogenicity is not strong.