Association Between ARGs And VAGs Of Escherichia Coli From Captive Giant Pandas And Horizontal Gene Transfer Of ESBLs-producing Escherichia Coli

The widespread use of antibiotics has promoted the emergence of antibiotic-resistant bacteria,resulting in huge medical,social,and economic burdens.In particular,the emergence of ESBLs-producing strains with multi-drug-resistant（MDR）further limits the clinical treatment options.As a symbiotic bacterium,Escherichia coli（E.coli）is a reservoir of antibiotic resistance genes（ARGs）and virulence-associated genes（VAGs）,creating conditions for the spread of ARGs and VAGs among different strains.In this study,the antibiotic resistance phenotype,ARGs,and VAGs of E.coli from captive giant pandas were detected and analyzed the association between ARGs and VAGs.At the same time,ESBLs-producing E.coli were detected and analyzed for the horizontal gene transfer（HGT）,providing a theoretical basis for controlling the development of antimicrobial resistance of E.coli from giant pandas.In this study,117 E.coli were isolated from clinically healthy captive giant pandas at the Chengdu Research Base of Giant Panda Breeding.After the antimicrobial susceptibility test,ARGs,and VAGs screening,the associations among ARGs,VAGs,and ARGs vs VAGs were analyzed.The results showed that the MDR rate of E.coli was 20.51%（24/117）,and the resistance rate toβ-lactam AMP（47.01%,55/117）was the highest.The resistance rates of the remaining 12 antibiotics ranged from 1.71%to 27.35%.A total of 13 ARGs were detected in E.coli from captive giant pandas,among which sul1（75.21%,88/117）had the highest detection rate,and 13 VAGs were detected,among which fyuA（66.67%,78/117）had the highest detection rate.The association analysis results of ARGs and VAGs showed that28 pairs of ARGs were significant positive association（OR>1）,and 3 pairs were significant negative association（OR<1）,of which bla _TEM and sul3 showed the strongest positive association（OR=81.125）.There was a significant positive association（OR>1）between30 pairs of VAGs,and a significant negative association（OR<1）between 6 pairs,of which papA and ast A showed the strongest positive association（OR=19.270）.A total of 38 pairs showed significant positive association（OR>1）between ARGs and VAGs,and 21 pairs showed significant negative association（OR<1）,of which cmlA and iroN showed the strongest positive association（OR=23.528）.In this study,ESBLs-producing strains were screened from 117 E.coli by double-disc diffusion testing.A total of 12（10.26%）ESBLs-producing E.coli were identified,and the MDR rate was 50%（6/12）.The carrying of ARGs,VAGs,and mobile genetic elements（MGEs）of ESBLs-producing E.coli were further analyzed by PCR and sequencing,and plasmid-mediated HGT of ESBLs-producing E.coli from captive giant pandas were identified by plasmid replicon typing and conjugation experiment.The results showed that the incidence of bla _CTX-M,bla _TEM,and bla _SHV in 12 ESBLs-producing E.coli were 100.00%,66.67%,and 8.33%,respectively.The bla _CTX-M genotype analysis showed that the most prominent was bla _CTX-M-55（33.33%,4/12）.Besides theβ-lactam resistance genes,the most prevalent ARGs in the ESBLs-producing E.coli were tetA（58.33%,7/12）and sul1（58.33%,7/12）.The most prevalent VAG was papA（83.33%,10/12）.And the most prevalent MGEs were ISEcp1（66.67%,8/12）,IS26（66.67%,8/12）.Conjugation experiments showed that all bla _CTX-M genes could be horizontally transferred by conjugative plasmids of diverse replicon types(IncF_{rep B},IncHI1,IncFIB,IncHI2,IncX,and IncFIA).And the ARGs of rmt B and sul2,the VAGs of papA,fimC,and ompT,and the MGEs of ISEcp1 and IS26 carried by ESBLs-producing E.coli were all co-transferred with bla _CTX-M.In addition,phylogenetic grouping and multilocus sequence typing（MLST）were used in this study to identify the clonal lineages of ESBLs-producing E.coli.The results showed that the ESBLs-producing E.coli mainly belonged to group B2（75.00%,9/12）and ST48（25.00%,3/12）.This study showed that E.coli from captive giant pandas had the phenomenon of MDR,which was the reservoir of ARGs and VAGs,and there was a significant association between ARGs and VAGs.ESBLs-producing strains were screened from E.coli isolated from captive giant pandas.The bla _CTX-M genotypes were diverse and prevalent,and all were located within conjugative plasmids allowing the horizontal transfer.The spread of antibiotic-resistant E.coli（carried various ARGs and VAGs）from captive giant pandas poses a potential health threat to both pandas and humans.