The Escherichia coli efflux pump TolC promotes aggregation of enteroaggregative E. coli 042.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an E. coli efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its tolC mutant (042tolC). However, characteristic aggregation in high-glucose Dulbecco's minimal essential medium for the wild type was diminished for 042tolC. In a microtiter plate assay, there were significantly more planktonic cells for 042tolC than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042tolC than for the wild type. In a HEp-2 cell adherence test, 042tolC showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with tolC. In a transwell assay, planktonic cells of 042tolC decreased when cocultured with the wild type or the complement, while precipitated cells of 042tolC increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.

Role of the carboxy-terminal region of the outer membrane protein AatA in the export of dispersin from enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. AatA, an outer-membrane protein that is a homolog of E. coli TolC, facilitates the export of the dispersin protein Aap across the outer membrane in EAEC. To identify which amino acids are important for this export activity, site-directed mutagenesis of the carboxy terminus was performed. An insertional mutant of aatA was complemented with each of several deletion mutants, and was examined for Aap secretion. The results showed that three nonpolar amino acids at positions 381-383 (Phe-Leu-Leu) were required for the activity, and these residues were located at the base of carboxy-terminal elongation in the equatorial domain of AatA.

Biofilm formation by and accessory gene regulator typing of methicillin-resistant Staphylococcus aureus strains recovered from patients with nosocomial infections.

The association between biofilm formation and the accessory gene regulator (agr) types of methicillin-resistant Staphylococcus aureus (MRSA) strains in our hospital were investigated. The biofilm index and the incidence of MRSA strains carrying agr-2 in the infection group (n=91) were significantly higher than were those in the carrier group (n=225), suggesting that biofilm formation and agr type are associated with nosocomial MRSA infections.

Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli.

The gold standard for identification of Enteroaggregative Escherichia coli (EAEC) remains the HEp-2 cell adherence test, which is time-consuming and requires specialized facilities. We evaluated the usefulness of a quantitative biofilm assay to screen for EAEC from a total of 1,042 E. coli strains from children with diarrhea. Bacteria were incubated overnight in high-glucose Dulbecco's modified Eagle's medium using a polystyrene microtiter plate. The plate was stained with crystal violet after washing, and the biofilm was quantified using an enzyme-linked immunosorbent assay plate reader. The aggR gene was evaluated by a polymerase chain reaction. Forty-eight (77.4%) of 62 strains with an optical density at 570 nm (OD(570)) > 0.2 were identified as EAEC by the HEp-2 adherence test, while no EAEC was found in strains with an OD(570) < or = 0.2. Twenty-one aggR+ and 27 aggR - EAEC strains could be screened by an OD(570) > 0.2 using this assay. Although confirmation by a HEp-2 cell adherence test is needed, this biofilm assay is convenient and useful in screening for EAEC.

Relapsing cellulitis associated with Campylobacter coli bacteremia in an agammaglobulinemic patient.

Campylobacter coli rarely causes bacteremia or extraintestinal infection. We report herein a case of agammaglobulinemia in which cellulitis associated with C. coli bacteremia relapsed after a disease-free interval of >5 years. Pulsed field gel electrophoresis revealed that the organisms in this patient were genetically identical, suggesting a latent C. coli infection.

Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children.

Diarrhea remains one of the main sources of morbidity and mortality in the world, and a large proportion is caused by diarrheagenic Escherichia coli. In Mongolia, the epidemiology of diarrheagenic E. coli has not been well studied. A total of 238 E. coli strains from children with sporadic diarrhea and 278 E. coli strains from healthy children were examined by PCR for 10 virulence genes: enteropathogenic E. coli (EPEC) eae, tir, and bfpA; enterotoxigenic E. coli (ETEC) lt and st; enteroinvasive E. coli (EIEC) ipaH; enterohemorragic E. coli stx1 and stx2; and enteroaggregative E. coli (EAEC) aggR and astA. EAEC strains without AggR were identified by the HEp-2 cell adherence test. The detection of EAEC, ETEC, EPEC, and EIEC was significantly associated with diarrhea. The incidence of EAEC (15.1%), defined by either a molecular or a phenotypic assay, was higher in the diarrheal group than any other category (0 to 6.0%). The incidence of AggR-positive EAEC in the diarrheal group was significantly higher than in the control group (8.0 versus 1.4%; P = 0.0004), while that of AggR-negative EAEC was not (7.1 versus 4.3%). Nineteen AggR-positive EAEC strains harbored other EAEC virulence genes-aggA, 2 (5.5%); aafA, 4 (11.1%); agg-3a, 5 (13.8%); aap, 8 (22.2%); aatA, 11 (30.5%); capU, 9 (25.0%); pet, 6 (16.6%); and set, 3 (8.3%)-and showed 15 genotypes. EAEC may be an important pathogen of sporadic diarrhea in Mongolian children. Genetic analysis showed the heterogeneity of EAEC but illustrated the importance of the AggR regulon (denoting typical EAEC) as a marker for virulent EAEC strains.