E.cloacae has taken on clinical significance and has emerged as a troublesome multi-drug resistance pathogen for healthcare institutions globally.However,little is known about sulfonamide-resistance properties of out-hospital E.cloacae.The objective of this study was to isolate E.cloacae from sediment sample from Huai River and characterize its sulfonamide resistance.Five sulfonamide resistance bacteria,including one E.cloacae(named as E.cloacae HR-1),which was identified by phylogenetic analysis of bacterial 16S rRNA gene sequences,were isolated from one sediment sample from Huai River.The isolate E.cloacae HR-1 presented a high level of resistance to sulfamethoxazole,sulfapyridine and sulfadiazine.Moreover,E.cloacae HR-1 was also resistant to six β-Lactams(ampicilli,penicillin G,cloxacillin,cefazolin,cephalothin,cefoxitin),three Macrolides(erythromycin,clarithromycin,midecamycin)and four other antibiotics(streptomycin,vancomycin,chloramphenicol,clindamycin).Most reports associate the sulfamethoxazole(SMX)resistance are due to sulfonamide(sul)genes(sul1,sul2,sul3 and sulA)and/or mutation in the chromosomal(folP)gene encoding dihydropteroate synthetase.Both the folP gene and the sul2 gene were detected in E.cloacae HR-1.Whole genome sequencing revealed that thefolP gene was similar with that of the susceptible strain E.coli K-12 MG1655,suggesting the resistance was not due to folP gene mutation.The sul2 gene was identical to that of E.cloacae sub cloacae 13047,a multiple-drug resistant opportunistic pathogen strain isolated from human brain fluids,indicating resistance was attributed mainly to sul2 gene.Meanwhile,we found the sul2 gene in tandem with the strA and strB gene located on the non-classic class 1 integron,which increased the potential environmental risks of HR-1.Based on the first comprehensive data concerning the sulfonamides resistance for E.cloacae HR-1,we propose that monitoring of this bacterial species should be implemented to appropriately safeguard human health.