title: Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.
abstract: Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we have not identified any plasmid genes specifically present in all LA/AA-like+ strains and absent in the LA+ strains, these results suggest the presence of an unknown mechanism to promote the AA-like pattern production and biofilm formation by the LA/AA-like+ strains. Because their ability to produce A/E lesions and biofilm concomitantly could exacerbate the clinical condition of the patient and lead to persistent diarrhea, the mechanism underlying the enhanced biofilm formation by the LA/AA-like+ O119:H6 strains and their spread and involvement in severe diarrheal diseases should be more intensively investigated.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. Electronic address: bruna.gil25@unifesp.br.
authors.forename: Bruna G
authors.initials: BG
authors.lastname: Garcia
authors.affiliation: Department of Microbiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. Electronic address: taohoka1@m.kufm.kagoshima-u.ac.jp.
authors.forename: Tadasuke
authors.initials: T
authors.lastname: Ooka
authors.affiliation: Department of Infectious Diseases, Division of Microbiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan. Electronic address: yasgotoh@med.miyazaki-u.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Gotoh
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. Electronic address: monica.vieira@unifesp.br.
authors.forename: Mônica A M
authors.initials: MA
authors.lastname: Vieira
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. Electronic address: dyamamoto@unifesp.br.
authors.forename: Denise
authors.initials: D
authors.lastname: Yamamoto
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan. Electronic address: y-ogura@bact.med.kyushu-u.ac.jp.
authors.forename: Yoshitoshi
authors.initials: Y
authors.lastname: Ogura
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Prof. Paulo de Góes, Centro de Ciências da Saúde-Ilha do Fundão, Rio de Janeiro, Brazil. Electronic address: giraodm@micro.ufrj.br.
authors.forename: Dennys M
authors.initials: DM
authors.lastname: Girão
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. Electronic address: scf.sampaio20@unifesp.br.
authors.forename: Suely C F
authors.initials: SC
authors.lastname: Sampaio
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. Electronic address: bonfimmeloa@gmail.com.
authors.forename: Alexis Bonfim
authors.initials: AB
authors.lastname: Melo
authors.affiliation: Seção de Bacteriologia, Instituto Adolfo Lutz, Secretaria de Estado da Saúde, São Paulo, Brazil. Electronic address: ikinue@hotmail.com.
authors.forename: Kinue
authors.initials: K
authors.lastname: Irino
authors.affiliation: Department of Infectious Diseases, Division of Microbiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan. Electronic address: thayash@bact.med.kyushu-u.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. Electronic address: tatg.amaral@unifesp.br.
authors.forename: Tânia A T
authors.initials: TA
authors.lastname: Gomes
mesh_terms: D018830:Adhesins, Escherichia coli / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D004269:DNA, Bacterial / Q000235:genetics; D054308:Enteropathogenic Escherichia coli / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D058885:Multilocus Sequence Typing; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D065288:Serogroup; D014774:Virulence
chemical_list: D018830:Adhesins, Escherichia coli; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins
keywords: Aggregative adherence; Attaching/effacing lesion; Biofilm; Diarrhea; Enteroaggregative Escherichia coli; Enteropathogenic Escherichia coli; Localized adherence; O119:H6