| Fosfomycin,an important antibiotic for the clinical treatment of multidrug-resistant Escherichia coli infections,has been banned for use in animal medicine.However,animal-sourced fosfomycin-resistant E.coli strains continue to appear frequently.The plasmid-mediated fos A3 gene is the main determinant of fosfomycin resistance in E.coli,often co-existing with genes conferring resistance to extended-spectrumβ-lactamases（ESBLs）,resulting in multidrug-resistant E.coli that can be transmitted through the environment or food chain to humans or animals,posing a serious threat to human and animal health.In this study,a total of 1059 samples were collected from water,carcass,cloaca,and cecum of ducks slaughtered in Mianyang,Chongzhou,Pengshan and other places.A total of150 strains of E.coli were isolated and identified as carrying both fos A3 and ESBLs genes,with a total isolation rate of 14.16%（150/1059）.The main ESBLs gene types carried were bla TEM（72%,108/150）,followed by bla OXA（64%,96/150）and bla CTX-M-55（52.67%,79/150）,bla CTX-M-14（18.67%,28/150）,bla CTX-M-65（9.33%,14/150）,bla CTX-M-8（8%,12/150）and bla CTX-M-64（2.67%,4/150）.The antimicrobial susceptibility testing results showed that the tested strains were highly resistant to fosfomycin（94%）,ampicillin（100%）,cefotaxime（94.67%）,ceftriaxone（94.67%）,gentamicin（82%）,streptomycin（93.33%）,tetracycline（95.33%）,chloramphenicol（94%）,florfenicol（92%）,nalidixic acid（92%）,ciprofloxacin（100%）,sulfamethoxazole（92.67%）,and polymyxin B（82%）.The strains were less resistant to amikacin（50%）and cefoxitin（20.67%）.124 strains produced ESBLs（82.67%,124/150）.The situation of multidrug resistance was severe,and strains were generally resistant to four to nine classes of antimicrobial agents.86 strains（57.33%）were resistant to eight classes of drugs simultaneously.The vast majority of isolates carried tet（A）（94.67%）,flo（88%）,and sul2（74%）,while the detection rates of rmt B（12%）,oqx A（18.67%）,aac（6’）-Ib-cr（4%）,and mcr-1（0.67%）were relatively low.fos A,fos C2,arm A,and NDM1 resistance genes were not detected.The tested strains generally carried class I integron genes（73.33%,110/150）,of which 44 strains simultaneously contained integron genes and gene cassettes.The gene cassettes mainly included the aminoglycoside-lincosamide gene cassette（aad A,aad A2,aad A5,aad A22）,the lincosamide/streptogramin gene cassette（ant1,ant3’9）,the trimethoprim gene cassette（dfr A12,dfr A17,dfr A27）,the rifampicin gene cassette（arr3）,the lincosamide gene cassette（lin F,lnu（F））,and the gene cassette encoding proteins containing the DUF1010 domain of unknown function（gcu F）.The gene cassette fragments composed 9 different combinations,with"aad A2,ant1,dfr A12"being the most common（38.64%）,and 2 strains simultaneously containing 5 gene cassette fragments"aad A2,aad A22,ant3’9,lin F,lnu（F）".Strains carrying gene cassettes showed significant multidrug resistance,all of which could tolerate six or more classes of antibiotics.Forty-one strains carried aad and/or ant gene cassettes and were resistant to aminoglycoside drugs.Most of the tested strains（130/150,86.67%）carried two or more virulence genes,up to a maximum of 6.The highest gene carriage rate was for the transport protein hemolysin hly E（96.67%）,followed by iss（60.67%）,iut A（62.67%）,iuc D（58.67%）,and iro N（55.33%）,with a low detection rate of pap C（4.67%）.Phylogenetic grouping results showed that about half of the isolates belonged to the animal infection group B1（70/150,46.67%）,a few belonged to the commensal group A（27/150,18%）,and 34 strains in the virulence clone groups D（17.33%）and B2（5.33%）carried one or more virulence genes.Pulsed-field gel electrophoresis（PFGE）typing results showed that the homology of the tested strains ranged from 78.8%to 95%,and they could be divided into 40 patterns.Different batches and sampling sites of strains were found within the same pattern,and most patterns contained 2to 6 strains.One pattern contained 17 strains,and 5 patterns had strain homology ranging from 90%to 95%,indicating that the ESBLs-carrying E.coli strains in this study were closely related and there might be a small-scale clone outbreak.The bacterial conjugation experiment showed that the resistant genes,fos A3 and bla CTX-M-55,carried by the isolated strains can be transferred horizontally into Escherichia coli J53,and the transconjugants had a 2-fold increase in the MIC value of phosphomycin,a 4 fold increase in the MIC value of cefotaxime and a 4-8 fold increase in the MIC value of cefuroxime.Analysis of the upstream and downstream gene environment of fos A3 showed that the presence of IS26 was detected around fos A3 in all 9 tested strains,and two strains had the aph（3’’）-Ib gene upstream of fos A3,indicating that aph（3’’）-Ib and fos A3 can coexist in duck-derived E.coli strains.This study investigated the prevalence and resistance characteristics of co-carrying fos A3 and ESBLs genes in duck-derived E.coli strains in Chengdu.The results showed that the isolated strains had a high and widespread resistance rate,showing multidrug resistance.They also had a variety of virulence factors and integrative gene cassette types,with a high degree of homology,suggesting the possibility of small-scale clone outbreaks.These results provide reference data for monitoring and controlling the spread of fos A3 and ESBLs resistance in duck-derived E.coli,and provide technical support for guiding clinical medication and controlling bacterial resistance spread in Chengdu’s duck farms. |
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