Prevalence Survey Of ESBLs-producing Escherichia Coli In Zoos And Its Drug Resistance Elimination Study

Third-generation cephalosporin antibiotics are commonly used in the treatment of clinical bacterial infections and play a crucial role in the field of human medicine and animal husbandry,however,the emergence of ultra broad-spectrumβ-lactamases（Extended-specturmβ-lactamases,ESBLs）has to some extent limited the selection and application of cephalosporin antibacterial drugs in clinical treatment and poses a public health They pose a threat to public health and safety.The high population flow and animal density in zoos are important sites for the transmission of drug-resistant genes,yet relatively few reports on ESBLs Escherichia coli（ESBL-Ec）in zoos have been published in China,so it is particularly important to study ESBL-Ec in zoos.In this study,ESBL-Ec was sampled in an ecological zoo in Hunan Province for two consecutive years to investigate the prevalence of ESBL-Ec in the zoo,analyze the resistance phenotypes of drug-resistant strains to 11 commonly used clinical antibiotics,and analyze the carriage of drug-resistant genes and molecular typing by whole-genome sequencing to provide a theoretical basis for evaluating the risk of ESBL-Ec resistance in the zoo and provide an experimental basis for preventing and controlling the spread of ESBL-Ec resistance in the zoo.This will provide a theoretical basis for assessing the risk of ESBL-Ec resistance in zoos and an experimental basis for preventing and controlling the spread of ESBL-Ec resistance in zoos.The growth curve and micro tessellation dilution method were employed to screen the Chinese herbal ingredients that could restore the susceptibility of cephalosporin antibiotics to ESBL-Ec resistant basidiomycetes,and then the efficacy was detected by measuring the minimum bactericidal concentration（MBC）and mutation prevention concentration（MPC）,based on which the study of bacterial resistance elimination was conducted.In 2020,93 animal fecal samples were collected in the zoo,and 46 strains of cefotaxime resistant bacteria were obtained by spiked agar plate delineation screening,and 32 strains of ESBLs resistant bacteria were obtained by PCR amplification and agarose gel electrophoresis technique according to the primer sequence of CTX-M resistance gene,and23 strains were identified as E.coli by 16S r RNA technique in different animals The prevalence of ESBLs in different animals ranged from 0%to 56%,with the highest isolation rate in swans;the results of drug sensitivity tests showed that 23 strains of ESBL-Ec were highly resistant to cefotaxime（100%）,tetracycline（95.7%）,ciprofloxacin（82.6%）and cotrimoxazole（69.6%）,and some strains were highly resistant to florfenicol（26.0%）,gentamicin（13.1%）and ceftazidime（13.0%）,and all ESBL-Ec were sensitive to polymyxin E,meropenem,tigecycline,and amikacin.In 2021,427 samples were collected in the zoo,including 238 environmental samples,60 fecal samples from visitors and veterinarians,and 129 animal samples,which were screened by PCR amplification and agarose gel electrophoresis techniques according to the primer sequences of CTX-M drug resistance gene,and 41 strains of ESBLs resistant bacteria were obtained,and also 23 strains were identified by 16S r RNA technique as The results of drug sensitivity tests showed that 32 strains of ESBL-Ec were highly resistant to cefotaxime（100%）,tetracycline（84.3%）,cotrimoxazole（78.1%）and florfenicol（56.2%）,and some strains were resistant to ceftazidime（31.3%）,ciprofloxacin（21.9%）and gentamicin（15.6%）resistance;in addition,as in the 2020 results,all ESBL-Ec remained susceptible to polymyxin E,meropenem,tigecycline and amikacin.The whole genome sequencing of the 55 isolated ESBL-Ec strains showed that all strains carriedβ-lactam resistance genes,among which the dominant ESBL genes bla_CTX-M-55and bla_CTX-M-65 of animal origin were found in human samples,and the strains were mainly distributed in groups A,B1,B2,D and F by Clermont system classification.The MLST results showed that 15 phenotypes were identified in the ESBL-Ec isolated in 2020 and the same ST phenotype was found in different animals,indicating that the transmission of drug-resistant strains may take place between different animals in zoos;25 phenotypes were identified in the ESBL-Ec isolated in 2021,of which animal-derived dominant ST46 was found in human-derived samples,indicating that ESBL-Ec can also be capable of transmission between humans and animals.Twenty-nine herbal extracts were screened by micro tessellation dilution method and measurement of bacterial growth curve,among which oxidized bitter ginseng base had strong antibacterial effect on ESBL-Ec,but showed irrelevant effect in combination with cefotaxime;zincated polysaccharide also had certain antibacterial effect and showed additive effect in combination with cefotaxime,and it was also found that it could reduce the mutation prevention concentration（MPC）of cefotaxime on drug-resistant bacteria and narrow its drug-resistant mutation selection window（MSW）,thus reducing the possibility of mutation of sensitive bacteria to drug-resistant bacteria.In summary,this study reveals that ESBL-Ec is widely present in zoos and can be transmitted not only between different animals but also between humans and animals,providing experimental data and a theoretical basis for the prevalence and transmission of ESBL-Ec in zoos;at the same time,it is found that the combination of zincated polysaccharide and cefotaxime can restore the susceptibility of cefotaxime to drug-resistant bacteria,providing an effective treatment plan to deal with the production of ESBL-Ec in zoos.