title: Genetic analysis of enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid reveals a new deletion within the EAF probe sequence among O119 typical EPEC strains.
abstract: BACKGROUND
  Enteropathogenic Escherichia coli (EPEC) are classified into typical and atypical strains based on the presence of the E. coli adherence factor (EAF) plasmid. The EAF plasmid contains the bfp (bundle-forming pilus) operon and the perABC (plasmid encoded regulator) gene cluster. A 1-kb cryptic region of EAF plasmid has been widely used as a genetic probe for EPEC detection. However, some EPEC strains may harbor an EAF plasmid lacking the EAF probe sequence, which makes the differentiation between typical and atypical a complex task. In this study, we report the genetic analysis of the EAF plasmid-encoded genes in a collection of EPEC clinical isolates.
  
  
  METHODS
  A total of 222 EPEC clinical isolates, which were previously classified as typical (n=70) or atypical (n=152) by EAF probe reactivity, were screened for the presence of different EAF sequences by PCR and DNA hybridization.
  
  
  RESULTS
  All typical strains possessed intact bfpA and perA genes, and most of them were positive in the PCR for EAF probe sequence. However, a subset of 30 typical strains, 22 of which belonged to O119 serogroup, presented a 1652 pb deletion in the region between 1093-bp downstream perC and 616-bp of the EAF fragment. The bfpA, bfpG, and per genes were found in all typical strains. In addition, 32 (21%) atypical strains presented the perA gene, and 20 (13.2%) also presented the bfpA gene. Among the 32 strains, 16 belonged to the O119:H2, O119:HND, and ONT:HND serotypes. All 32 atypical strains contained perA mutation frameshifts and possessed an IS1294 element upstream of the per operon as detected by PCR followed by restriction fragment length polymorphism (RFLP) typing and multiplex PCR. Among the 20 bfpA probe-positive strains, eight O119 strains possessed deletion in the bfp operon at the 3'end of bfpA due to an IS66 element.
  
  
  CONCLUSIONS
  Our data show that typical O119 strains may contain a deletion within the EAF probe sequence not previously reported. This new finding suggests that care should be taken when using the previously described EAF PCR assay in epidemiological studies for the detection of typical O119 strains. In addition, we were able to confirm that some atypical strains carry vestiges of the EAF plasmid.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 3 andar, São Paulo, 04023-062, São Paulo, Brazil. na_tx0402@yahoo.com.br.
authors.forename: Nathalia B
authors.initials: NB
authors.lastname: Teixeira
authors.affiliation: Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil. thaiscjrojas@gmail.com.
authors.forename: Thais C G
authors.initials: TC
authors.lastname: Rojas
authors.affiliation: Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil. wds@unicamp.br.
authors.forename: Wanderley D
authors.initials: WD
authors.lastname: da Silveira
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 3 andar, São Paulo, 04023-062, São Paulo, Brazil. cmatheus@globo.com.
authors.forename: Cecília
authors.initials: C
authors.lastname: Matheus-Guimarães
authors.affiliation: Disciplina de Reumatologia, Universidade Federal de São Paulo, São Paulo, Brazil. npsilva@unifesp.br.
authors.forename: Neusa P
authors.initials: NP
authors.lastname: Silva
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 3 andar, São Paulo, 04023-062, São Paulo, Brazil. scaletskyunifesp@gmail.com.
authors.forename: Isabel C A
authors.initials: IC
authors.lastname: Scaletsky
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics*; D054308:Enteropathogenic Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000175:diagnosis; D005188:False Negative Reactions; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D018829:Adhesins, Bacterial; D015345:Oligonucleotide Probes