Transposon mutagenesis of atypical enteroaggregative Escherichia coli reveals a hemagglutinin-associated protein that mediates cell adhesion and contributes to the Galleria mellonella virulence.

Twenty-two atypical enteroaggregative Escherichia coli isolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genes afpA2 and afpR were detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified in E. coli strain 7-233-03_S3_C2. Therefore, the ORF was named orfHA. The regions flanking orfHA were sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. An orfHA mutant (Q015BΔorfHA) was constructed from strain Q015B. Q015BΔorfHA strain did not adhere to HeLa cells, whereas Q015BΔ orfHA transformed with a pACYC184 plasmid carrying orfHA restored the AA/DA phenotype of strain Q015B. Furthermore, the Q015ΔorfHA mutant had a marked effect on the ability of strain Q015B to kill the larvae of Galleria mellonella. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in the G. mellonella model.

Complete Genomic Sequence of an Avian Pathogenic Escherichia coli Strain of Serotype O7:HNT.

Avian pathogenic Escherichia coli (APEC) is associated with colibacillosis in poultry. Here, we present the first complete sequence of an APEC strain of the O7:HNT serotype and ST73 sequence type, isolated from a broiler with cellulitis. Complete genomes of APEC with distinct genetic backgrounds may be useful for comparative analysis.

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.

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. CONCLUSION: 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.

In silico phylogenetic and virulence gene profile analyses of avian pathogenic Escherichia coli genome sequences / Análises in silico da filogenia e do perfil de genes associados à virulência, dos genomas de linhagens de Escherichia coli de origem aviária

Avian pathogenic Escherichia coli (APEC) infections are responsible for significant losses in the poultry industry worldwide. A zoonotic risk has been attributed to APEC strains because they present similarities to extraintestinal pathogenic E. coli (ExPEC) associated with illness in humans, mainly urinary tract infections and neonatal meningitis. Here, we present in silico analyses with pathogenic E. coli genome sequences, including recently available APEC genomes. The phylogenetic tree, based on multi-locus sequence typing (MLST) of seven housekeeping genes, revealed high diversity in the allelic composition. Nevertheless, despite this diversity, the phylogenetic tree was able to cluster the different pathotypes together. An in silico virulence gene profile was also determined for each of these strains, through the presence or absence of 83 well-known virulence genes/traits described in pathogenic E. coli strains. The MLST phylogeny and the virulence gene profiles demonstrated a certain genetic similarity between Brazilian APEC strains, APEC isolated in the United States, UPEC (uropathogenic E. coli) and diarrheagenic strains isolated from humans. This correlation corroborates and reinforces the zoonotic potential hypothesis proposed to APEC.

As infecções causadas por linhagens de Escherichia coli de origem aviária (APEC) são responsáveis por perdas significativas na indústria avícola em todo mundo. Risco zoonótico tem sido atribuído às linhagens APEC, devido às semelhanças existentes entre elas e linhagens de E. coli patogênicas extraintestinais (ExPEC) de origem humana, causadoras de infecções no trato urinário e meningite neonatal. Neste trabalho, apresentamos os resultados de análises in silico feitas a partir dos genomas de linhagens patogênicas de E. coli, incluindo genomas recentemente obtidos de linhagens APEC. Uma árvore filogenética foi obtida, com base na tipagem de sequência multilocus (MLST) de sete genes essenciais, revelando alta diversidade na composição de alelos, mas ainda assim possibilitando o agrupamento dos diferentes patótipos. Foi determinado também, para cada linhagem, o perfil gênico, por meio da presença ou ausência de 83 genes associados à virulência. A árvore filogenética e o perfil gênico demonstraram que existem semelhanças genéticas entre cepas APEC brasileiras, APEC isolada nos Estados Unidos, UPEC (uropathogenic E. coli) e linhagens produtoras de diarreia em humanos. Essa correlação corrobora e reforça a hipótese de que linhagens APEC apresentam potencial risco zoonótico.