Ana Cláudia de Paula Rosa

Phenotypic and Genotypic Characteristics of Escherichia coli Strains of Non–Enteropathogenic E. coli (EPEC) Serogroups that Carry eae and Lack the EPEC Adherence Factor and Shiga Toxin DNA Probe Sequences

This study was conducted to characterize the virulence potential of 59 Escherichia coli strains carrying EAE and lacking the enteropathogenic E. coli adherence factor and Shiga toxin probe sequences. In hybridization studies, all strains carried the locus of enterocyte effacement (LEE)-associated DNA sequences. Of the other 15 virulence DNA sequences tested, HLY was the most frequent (44.1%); 17 combinations of these sequences were found, but strains carrying EAE only (EAE profile) were the most frequent (35.6%). Except for 1 cytodetaching strain, all others adhered to HeLa and Caco-2 cells, most of which (approximately 75.0%) showed variations of the localized adherence pattern. Actin accumulation was detected in 75.9% of the nondetaching strains. Most strains had LEE, probably inserted in pheU (49.2%), and presented a nontypeable intimin (83.1%). Translocated intimin receptor-derived DNA sequences correlated with enteropathogenic and enterohemorrhagic E. coli in 61.0% and 32.0% of the strains, respectively. Thirty-five different serotypes were found. Only strains with the EAE profile were associated with diarrhea (P=.039).

Enteropathogenicity markers in Escherichia coli isolated from infants with acute diarrhoea and healthy controls in Rio de Janeiro, Brazil

Faeces from urban children < 2 years old with acute diarrhoeal illness and from non-diarrhoeal infants (controls) were examined for Escherichia coli and other enteropathogens. A total of 990 E. coli isolates from 100 patients and 50 controls was tested for enteropathogenic E. coli (EPEC) serotype (O:H), adherence to HEp-2 cells after incubation for 3 and 6 h, fluorescent actin staining (FAS), DNA hybridisation with EAF, eaeA, STh, STp and EAggEC probes and production of heat-labile enterotoxin (LT) and verocytotoxin (VT) with Y1 and Vero cells. EPEC were the most prevalent enteropathogens in patients (32.7%; and 14% in controls). Enteroinvasive E. coli (EIEC) and Vero cytotoxin-producing E. coli (VTEC) were not detected. The rate of isolation of enterotoxigenic E. coli (ETEC) was identical in both groups. Among the EPEC isolates the prevalent serotypes were O111:H2, O55:NM and O119:H6. Localised adherence (LA) was found significantly more frequently in isolates from patients (19.6%) than controls (2.1%). All LA-positive EPEC isolates were FAS+ and eaeA+, but only 75.2% of them hybridised with the EAF probe. Diffusely adhering E. coli (DAEC) and enteroaggregative E. coli (EAggEC) were found with equal frequency in patients and controls. Twenty-seven E. coli isolates were negative for EAF but positive for eaeA and FAS and produced LA in 6-h adherence tests. These EAF-/eaeA+ strains were the only putative enteropathogen identified in seven patients and were not found in controls. The ability of these strains to elicit ultrastructural cell alterations and cell-signalling events was evaluated in Caco-2 cells (human colon carcinoma cell line) by the gentamicin invasion assay and by transmission electron microscopy. The numbers of intracellular bacteria in cell invasion tests varied from 0.4% to 1.6% of the cell-associated bacteria after a 6-h incubation period. Tyrosine phosphorylation of host cell proteins was assessed in HEp-2 cells by immunofluorescence microscopy and all strains gave positive results. EAF-/eaeA+ E. coli strains express most of the virulence properties found among true EPEC strains and can be a relevant cause of infant diarrhoea in developing countries.

Potential pathogenic role of aggregative-adhering Corynebacterium diphtheriae of different clonal groups in endocarditis.

Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40% of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.

Corynebacterium diphtheriae as an emerging pathogen in nephrostomy catheter-related infection: evaluation of traits associated with bacterial virulence

Corynebacterium diphtheriae still represents a global medical challenge, particularly due to the significant number of individuals susceptible to diphtheria and the emergence of non-toxigenic strains as the causative agents of invasive infections. In this study, we characterized the clinical and microbiological features of what we believe to be the first case of C. diphtheriae infection of a percutaneous nephrostomy catheter insertion site in an elderly patient with a fatal bladder cancer. Moreover, we demonstrated the potential role of adherence, biofilm formation and fibrin deposition traits in C. diphtheriae from the catheter-related infection. Non-toxigenic C. diphtheriae isolated from the purulent discharge (named strain BR-CAT5003748) was identified by the API Coryne system (code 1 010 324) and a multiplex PCR for detection of dtxR and tox genes. Strain BR-CAT5003748 showed resistance to oxacillin, ceftazidime and ciprofloxacin. In experiments performed in vitro, the catheter isolate was classified as moderately hydrophobic and as moderately adherent to polystyrene surfaces. Glass provided a more effective surface for biofilm formation than polystyrene. Micro-organisms adhered to (>1.5 x 10(6) c.f.u.) and multiplied on surfaces of polyurethane catheters. Microcolony formation (a hallmark of biofilm formation) and amorphous accretions were observed by scanning electron microscopy on both external and luminal catheter surfaces. Micro-organisms yielded simultaneous expression of localized adherence-like and aggregative-like (LAL/AAL) adherence patterns to HEp-2 cells. Interestingly, the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain. In conclusion, C. diphtheriae should be recognized as a potential cause of catheter-related infections in at-risk populations such as elderly and cancer patients. LAL/AAL strains may be associated with virulence traits that enable C. diphtheriae to effectively produce biofilms on catheter surfaces. Biofilm formation and fibrin deposition could have contributed to the persistence of C. diphtheriae at the infected insertion site and the obstruction of the nephrostomy catheter.

A multiplex PCR assay for simultaneous detection of Corynebacterium diphtheriae and differentiation between non-toxigenic and toxigenic isolates

The importance of identifying Corynebacterium diphtheriae can be appreciated since diphtheria remains endemic in many countries, alongside the risk of epidemic outbreaks and the existence of a large number of non-immunized people worldwide (Galazka & Robertson, 1995; Damasco et al., 2005). The features of infections caused by C. diphtheriae have changed over the decades, and are most clearly emphasized by the emergence of non-toxigenic strains causing atypical diseases, such as endocarditis, septic arthritis or infection in unusual anatomical sites (Mattos-Guaraldi & Formiga, 1998; Efstratiou & George, 1999; Reacher et al., 2000; Mattos-Guaraldi et al., 2003). Rapid and reliable methods are needed for identifying a C. diphtheriae infection, thus aiding in appropriate and timely patient management, and improved monitoring of cases.

A PCR for dtxR gene: application to diagnosis of non-toxigenic and toxigenic Corynebacterium diphtheriae.

The significant rise in the percentage of adults susceptible to diphtheria and the emergence of non-toxigenic Corynebacterium diphtheriae strains as the causative agent of endocarditis and other systemic infections emphasize the need for alternative laboratory diagnostic procedures. In this study, for the first time, the value of a species-specific PCR assay that targets the dtxR gene is documented as a procedure for differentiating C. diphtheriae from Corynebacterium-like colonies. The results of the PCR-dtxR were all positive for 91 C. diphtheriae (54 non-toxigenic and 37 toxigenic) strains. PCR-dtxR completely correlated with the standard biochemical and commercial identification for all C. diphtheriae strains tested. Conversely, the PCR-dtxR results were negative in 100% of the 111 non-diphtherial Gram-positive rod strains obtained during identification procedures in a hospital laboratory. Thus, the PCR-dtxR assay emerged as viable, cost-effective screening method for C. diphtheriae laboratory identification.

Escherichia coli Strains of Serotype O51:H40 Comprise Typical and Atypical Enteropathogenic E. coli Strains and Are Potentially Diarrheagenic

ABSTRACT Escherichia coli strains of serotype O51:H40 were studied with regard to the presence of several virulence properties and their genetic diversity and enteropathogenicity in rabbit ileal loops. This serotype encompasses potential enteropathogenic strains mostly classified as being atypical enteropathogenic E. coli (EPEC) strains, which are genetically closer to enterohemorrhagic E. coli than to typical EPEC strains.

Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC).

Aims:  The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp‐2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases.

Group B Streptococcus serotypes III and V induce apoptosis and necrosis of human epithelial A549 cells.

Although group B Streptococcus (GBS) has been classically described as an exclusively extracellular pathogen, growing evidence suggests that it may be internalized by epithelial cells. However, the fates of intracellular GBS and of infected respiratory epithelial cells remain unclear. Little is known about the bacterial components involved in these processes. The present study investigated the bacterial internalization by A549 cells and the apoptosis/necrosis of the infected human epithelial cells. The morphological changes in A549 cells observed from 2 h post-infection with GBS included vacuolization and the formation of apoptotic bodies. Flow cytometry revealed that 81.2% of apoptotic A549 cells were infected with GBS serotype III 90356-liquor. Moreover, a double-staining assay using propidium iodide (PI)/Annexin V (AV) gave information about the numbers of viable (PI-/AV-) (18.27%) vs. early apoptotic (PI-/AV+) (73.83%) and late apoptotic cells (PI+/AV+) (7.37%) during infection of A549 cells with GBS III 90356-liquor. In addition, 37% necrotic cells were observed in A549 cells infected with GBS serotype V 90186-blood. In conclusion, GBS serotypes III and V induce apoptosis of epithelial cells in the early stages of GBS infection, resulting in tissue destruction, bacterial spreading and, in consequence, invasive disease or systemic infection.

Genotypic and Phenotypic Characterisation of Enteroaggregative Escherichia coli from Children in Rio de Janeiro, Brazil

Enteroaggregative Escherichia coli (EAEC) is a significant cause of diarrhoeal illness in both children and adults. Genetic heterogeneity and recovery of EAEC strains from both healthy and diseased individuals complicates our understanding of EAEC pathogenesis. We wished to establish if genetic or phenotypic attributes could be used to distinguish between strains asymptomatically colonising healthy individuals and those which cause disease. Genotypic screening of a collection of twenty four EAEC isolates from children with and without diarrhoea revealed no significant differences in the repertoire of putative virulence factors present in either group of strains. In contrast, EAEC strains from phylogroup A were more strongly associated with asymptomatic groups whereas strains from phylogroup D were more associated with cases of diarrhoea. Phenotypic screening revealed no differences in the ability of strains from either cohort of children to form biofilms, to adhere to and invade cells in tissue culture or to cause disease in the Caenorhabditis elegans model of infection. However, the latter assay did reveal significant reduction in nematode killing rates when specific virulence factors were deleted from human pathogenic strains. Our results suggest that current models of infection are not useful for distinguishing avirulent from pathogenic strains of EAEC but can be useful in studying the effect of specific virulence factors.