| The crested ibis（Nipponia nippon）is one of the most endangered birds in the world and is listed as threatened by the International Union for Conservation of Nature Red List.Although the population and distribution of crested ibis has being steadily increased,the species still faces many survival challenges,and bacterial infections are one of them.Escherichia coli（E.coli）and Proteus mirabilis（P.mirabilis）are the most common gram negative bacteria,which mainly live in the intestines of human and animals,and also widely exists in the environments.Some types of E.coli and P.mirabilis are also the most common bacteria in clinical infections.Due to the use of antibiotics widely,antimicrobial resistant E.coli and P.mirabilis have emerged rapidly,and the resistance has being became increasingly serious,which affected the treatment effect of antibiotics.The generation and spread of antimicrobial resistance have been considered that it is not only related to the pressure of the use of antimicrobial induction and selection,but also connection with the horizontal transmission of drug resistance genes by various mobile elements such as plasmids.In this study,we collected different samples,including dead embryos,feces,and captive living environment（water samples,ground,loach,and breeders）from the Research Base of Crested Ibis Domestication and Breeding in Sichuan Province（Shawan and Muchuan bases）during the October 2020 to July 2021.221 strains of E.coli and 310strains of P.mirabilis were isolated and identified.The susceptibility of all isolates to 10kinds of antibiotics was determined by the micro dilution method.The results showed that the isolates showed different levels of resistance to antibiotics,the resistance rate ranged from 8.39%to 96.77%,and 61.99%（137/221）E.coli and 89.68%（278/310）P.mirabilis isolates showed multiple drug resistance;E.coli and P.mirabilis isolates from different sources（bases and sample sources）exhibited similar drug resistance characteristics,especially for amoxicillin,cefazolin,oxytetracycline and trimethoprim/sulfamethoxazole（drug resistance rate:47.06%～96.77%）.Moreover,the cross resistance to different antibiotics of the same kind and different antibiotics have been described in this study.Animal pathogenicity tests were carried out on 13 E.coli and 12 P.mirabilis isolated from dead embryo samples.The results showed that all E.coli and P.mirabilis isolates could cause infection and death in mice,and the clinical symptoms in mice were observed in different strains,such as eye inflammation,dyspnea and convulsions;The pathological changes mainly include lung congestion or bleeding,liver congestion,spleen and kidney enlargement,etc.The results showed that the E.coli and P.mirabilis isolates from the dead embryos of crested ibis were pathogenic,and the main cause of the death of artificially hatched crested ibis embryos was the single or mixed infection of these strains.The production of Extended spectrum beta-lactamases（ESBLs）enzyme was detected by double-disc test.The ESBLs genes(bla TEM,bla CTX-M,bla SHV,bla OXA),tet genes（tet A,tet B and tet C）,sul genes（sul1,sul2 and sul3）and virulence genes,including E.coli virulence genes（fim C,iss,iuc D,irp2,hly E,tsh,pap C）and P.mirabilis virulence genes（uca A,pmf A,pt A,rsb A,ure C,mrp A,atf A and zap A）in all isolated strains were detected by PCR-sequencing method.The results showed that ESBLs genes were detected in 18 E.coli and 14 P.mirabilis isolates,including bla CTX-M-55,bla CTX-M-14,bla CTX-M-65and bla TEM-1,of which,bla CTX-M-55（E.coli:83.33%;P.mirabilis:42.86%）and bla TEM-1（E.coli:94.44%;P.mirabilis:100%）were most prevalent gene-subtypes.tet A,sul1 and sul2 were the main resistance genes in E.coli and P.mirabilis isolates,and the rate of tet A is the highes（E.coli:92.76%;P.mirabilis:90.97%）.The relationship betweenβ-lactam drug resistance phenotypes and drug resistance genotypes is complex,with both positive and negative correlations;E.coli isolates showed resistance to AMX and carried bla CTX-M-14gene,P.mirabilis isolates showed resistance to CRO and bla CTX-M-55gene has positive correlation;E.coli isolates showed resistance to AMX and bla CTX-M-65gene,P.mirabilis isolates showed resistance to AMX and carried bla CTX-M-14gene has negative correlation.Different types of strains are resistant to oxytetracycline with tet gene（tet A,tet B and tet C）,and trimethoprim/sulfamethoxazole with sul have positive correlation.Seven virulence genes including fim C,iss,iuc D,irp2,hly E,tsh and pap C were widely distributed among E.coli isolates,fim C was the main virulence genes（80.54%,178/221）.There was a negative correlation between resistance genes and virulence genes,such as sul1 and fim C has most strong positive correlation.Five virulence genes including uca A,pmf A,pt A,rsb A and zap A were detected in P.mirabilis,and the detection rate of uca A（39.68%,123/310）was the highest.There was a positive correlation between resistance genes and virulence genes,such as tet B and pt A has most strong positive correlation.The transfer ability of ESBLs,tet and sul drug resistance genes and virulence genes was performed by plasmid conjugation transfer.The results showed that 64 transconjugants were obtained from 110 isolates（58 E.coli isolates and 52 P.mirabilis isolates）,and plasmid electrophoresis maps displayed that only the plasmids with a size>～15 kb were able to transfer to the transconjugants.bla CTX-M-55,bla CTX-M-14and bla TEM-1,tet A,tet B,sul1,and sul2 resistance genes could be transmitted horizontally by plasmids,which made transconjugants obtained resistance to the corresponding antimicrobial.Additionally,there were co-transfer of E.coli virulence gene iss,P.mirabilis virulence gene uca A and resistance genes including bla CTX-M-55,bla CTX-M-14and bla TEM-1,tet A,tet B,sul1,and sul2.PFGE was used to detect the relationship different isolates,which from sources had similar resistance phenotypes and resistance genotypes.The results showed that patterns of PFGE were complex,the PFGE profiling generated from the E.coli isolates and P.mirabilis isolates identified 27 and 24 patterns,respectively,which indicated that there were complex genetic backgrounds and distant genetic relationship among these isolates.These results of the study suggested that drug-resistant E.coli and P.mirabilis isolates had been widespread in the gut of crested ibis,breeders and captive environment.The ESBLs,tet and sul resistant genes showed different levels of prevalence;Plasmid conjugation and molecular typing showed that the transmission of resistance genes mainly spread horizontally,indicating that crested ibis may acquire a symbiotic microbiota carrying multiple resistance genes and become a repository of resistance genes when exposed to artificial breeding environment.Furthermore,virulence genes were detected in different isolates,and drug resistance phenotypes were positively and negatively correlated with antimicrobials.There was a negative correlation between drug resistance genes and virulence genes in E.coli,and positive in P.mirabilis.The resistance and virulence genes of the tested strains can be horizontally transmitted through plasmids.In order to maintain a healthy ecological environment and the breeding of crested ibis populations,it is necessary to strengthen the detection of symbiotic bacteria in the body and environment of crested ibis,and develop and implement relevant measures to reduce the prevalence of drug-resistant strains in captive crested ibis. |
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