Pathological profiles of systemic infections by Panton-Valentine leukocidin-positive, methicillin-resistant Staphylococcus aureus strains in a murine model.

AIMS: Staphylococcus aureus is one of the most common pathogens in hospital environment and community. Panton-Valentine leukocidin (PVL) production is clinically associated with skin abscesses, soft tissues infections, bacteraemia and sepsis. This study aimed to investigate the effects of the presence of genes lukF/S-PV coding for PVL, in histological and haematological features during systemic infection, using a Swiss mice experimental model. METHODS AND RESULTS: Experiments were performed using 25 mice distributed into five experimental groups, intravenously inoculated with 50 µl suspensions at density 1·0 × 107  CFU per ml of strains: methicillin-susceptible (MSSA) and pvl-negative strains isolated from nasal colonization; MSSA pvl-positive strains isolated from nasal colonization; methicillin-resistant (MRSA) and pvl-positive strains isolated from peripheral blood of a patient with severe pulmonary infection; and a MRSA pvl-positive strains isolated from a peripheral blood culture of a patient with bacteraemia. Haematological analysis was performed at 24, 48, 72 and 96 h post-infection. Morphoanatomy and histopathological analyses were performed at 96 h post-infection. For all S. aureus strains tested, the capability of intravenous dissemination and survival into mice tissues was demonstrated. Inflammatory processes at different levels were related to the presence of pvl genes, and included alterations in the format, size and colour of the organs. Staphylococcus aureus pvl-positive strains were detected in greater numbers in the organs of the infected animals. CONCLUSIONS: The pvl-positive strains isolated from blood cultures were capable to induce the greatest modifications in both haematological and histopathological profiles, and seemed to aggravate the systemic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings are valuable in characterizing infections caused by S. aureus in humans and murine.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Diphtheria toxin IgG levels in military and civilian blood donors in Rio de Janeiro, Brazil

Serologic data on diseases that are preventable by vaccines are necessary to evaluate the success of immunization programs and to identify susceptible subgroups. In the present study, we determined serum IgG levels against diphtheria toxin of military and civilian blood donors (N = 75; 69.3 percent males and 30.7 percent females) aged 18-64 years, from the Brazilian Army Biology Institute, Rio de Janeiro, using a commercial diphtheria kit (Diphtheria IgG ELISA; IBL, Germany). Most (63 percent) unprotected military donors were from the older age group of 41 to 64 years. In contrast, the majority (71 percent) of young military donors (18 to 30 years) were fully protected. About half of the military donors aged 31 to 40 years were protected against diphtheria. Among the civilians, about 50 percent of persons aged 18 to 30 years and 31 to 40 years had protective antibody levels against diphtheria as also did 64 percent of individuals aged 41 to 64 years. All civilians had a similar antibody response (geometric mean = 0.55 IU/mL) independent of age group. Military donors aged 18-30 years had higher IgG levels (geometric mean = 0.82 IU/mL) than military donors of 41-64 years (geometric mean = 0.51 IU/mL; P > 0.05). In conclusion, the existence of a considerable proportion of susceptible adults supports the position that reliable data on the immune status of the population should be maintained routinely and emphasizes the importance of adequate immunization during adulthood.

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. METHODS AND RESULTS: The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea. CONCLUSIONS: Low iron availability may modulate biofilm formation and adhesive properties of EAEC strains to HEp-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains.

Microbiological and host features associated with corynebacteriosis in cancer patients: a five-year study

During a five-year period, 932 clinical isolates from cancer patients treated in a Brazilian reference centre were identified as corynebacteria; 86 percent of the cultures came from patients who had been clinically and microbiologically classified as infected and 77.1 percent of these patients had been hospitalised (71.1 percent from surgical wards). The adult solid tumour was the most common underlying malignant disease (66.7 percent). The univariate and multivariate analyses showed that hospitalised patients had a six-fold greater risk (OR = 5.5, 95 percent CI = 1.15-26.30 p = 0.033) related to 30-day mortality. The predominant species were Corynebacterium amycolatum (44.7 percent), Corynebacterium minutissimum (18.3 percent) and Corynebacterium pseudodiphtheriticum (8.5 percent). The upper urinary tracts, surgical wounds, lower respiratory tracts, ulcerated tumours and indwelling venous catheters were the most frequent sources of C. amycolatum strains. Corynebacterium jeikeium infection occurred primarily in neutropenic patients who have used venous catheters, while infection caused by C. amycolatum and other species emerged mainly in patients with solid tumours.

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 percent 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.

First detection of Corynebacterium ulcerans producing a diphtheria-like toxin in a case of human with pulmonary infection in the Rio de Janeiro metropolitan area, Brazil

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.

Corynebacterium diphtheriae threats in cancer patients

The aim of this study was to determine the bacteriological properties of Corynebacterium diphtheriae strains isolated from bronchiole washing and cancer lesions. Bacteriological characterization included fluorescence/double sugar urease (King/DSU) screening tests, pyrazinamidase (PYZ), CAMP-reactions and radial immunodiffusion toxigenicity assay. Microorganisms produced fluorescence under ultraviolet light and were catalase positive; urea and aesculin hydrolysis negative; fermentation of glucose, maltose and sucrose and no fermentation of mannitol and xylose; PYZ and CAMP reaction negative. The API-Coryne system was used for bacterial preliminary identification at local hospital laboratory and produced numerical profiles 1010325 and 0010325 for sucrose positive C. diphtheriae var. mitis (nitrate positive) and C. diphtheriae var. belfanti (nitrate negative), respectively. The hemagglutination, adherence to glass and polystyrene assays evaluated adhesive characteristics. Strains were toxigenic and able to adhere to glass, polystyrene and human erythrocyte surfaces (titer 4). C. diphtheriae strains isolated from cancer patients expressed adhesive characteristics similar to strains isolated from immunocompetent hosts. Circulation of toxigenic C. diphtheriae continues to present a threat for children and adults including patients with cancer in hospital environment. Laboratories should remain alert to the possibility of isolation of diphtheria bacilli from adults with neoplastic disease.(AU)

Corynebacterium diphtheriae threats in cancer patients

The aim of this study was to determine the bacteriological properties of Corynebacterium diphtheriae strains isolated from bronchiole washing and cancer lesions. Bacteriological characterization included fluorescence/double sugar urease (King/DSU) screening tests, pyrazinamidase (PYZ), CAMP-reactions and radial immunodiffusion toxigenicity assay. Microorganisms produced fluorescence under ultraviolet light and were catalase positive; urea and aesculin hydrolysis negative; fermentation of glucose, maltose and sucrose and no fermentation of mannitol and xylose; PYZ and CAMP reaction negative. The API-Coryne system was used for bacterial preliminary identification at local hospital laboratory and produced numerical profiles 1010325 and 0010325 for sucrose positive C. diphtheriae var. mitis (nitrate positive) and C. diphtheriae var. belfanti (nitrate negative), respectively. The hemagglutination, adherence to glass and polystyrene assays evaluated adhesive characteristics. Strains were toxigenic and able to adhere to glass, polystyrene and human erythrocyte surfaces (titer 4). C. diphtheriae strains isolated from cancer patients expressed adhesive characteristics similar to strains isolated from immunocompetent hosts. Circulation of toxigenic C. diphtheriae continues to present a threat for children and adults including patients with cancer in hospital environment. Laboratories should remain alert to the possibility of isolation of diphtheria bacilli from adults with neoplastic disease.

Surface vesicles: a possible function in commensal relations of Bacteroides fragilis.

Surface vesicles (SV) defined by electron microscopy as outer membrane (OM) extrusions were detected in Bacteroides fragilis strains from distinct sources. A partial identity between SV and OM electrophoretic protein profiles, in addition to the microscopic analysis, may suggest the designation of OMSV. Sialidase activity, a virulence determinant, was associated with these sub-cellular structures in all the strains, but in an inverse relation to the vesicle quantity per cell. A commensal strain, previously defined as avirulent in an animal model, presented the lowest vesicle-associated sialidase activity and the greatest SV expression as opposed to what happened with clinical and environmental strains. These results seem to suggest that these surface components have a function in commensal stages of B. fragilis.

Bacteroides fragilis: an exogenous pathogen?

Thirteen strains of Bacteroides fragilis isolated from contaminated water or from cases of intestinal and non-intestinal infections were comparatively analyzed in order to detect a possible biological relationship among them. The observation of similar profiles among the two groups, regarding the capacity of inducing abscesses and some surface properties, may support the hypothesis that the species could also act as an exogenous pathogen.