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BackgroundVentilator-associated pneumonia (VAP) is considered the most common nosocomial infection in the intensive care unit (ICU), but its features are not fully known in many hospitals in Brazil. We identified clinical and epidemiological aspects associated with VAP in an intensive care unit (ICU) in a general public hospital in northern Brazil and performed an analytical descriptive prospective cohort study.MethodsWe analyzed data from thirty-three patients who developed VAP while in the ICU. Clinical and epidemiological data of patients were obtained and tracheal secretions were submitted to culture. Microbial isolates were identified and evaluated for resistance against antimicrobial agents by using the automated Vitek 2 system.ResultsThe frequency of VAP was 26.2% in patients submitted to invasive mechanical ventilation for at least 48 hours, and death occurred in 78.8% of cases. Only the presence of comorbidity showed a significant association (P = 0.029) with death. The most commonly found bacteria were Pseudomonas aeruginosa, Acinetobacter spp., and Enterobacteriaceae. We also found a frequency of 54.5% of multiresistant bacteria associated with VAP, and previous antibiotic therapy was used in 97% of patients.ConclusionsVAP in our ICU presented with a high frequency and was mainly caused by multiresistant bacteria. Implementation of rational protocols for the use of antibacterial agents and rapid delivery of culture and susceptibility test results are essential. This may help decrease VAP-related mortality rates by multiresistant bacteria in the ICU.

Zanthoxylum rhoifolium Lam is a plant popularly used as antimicrobial, for malaria and inflammatory treatment. The essential oil of Z. rhoifolium was extracted and its cytotoxic effects against HeLa (human cervical carcinoma), A-549 (human lung carcinoma), HT-29 (human colon adenocarcinoma), Vero (monkey kidney) cell lines and mice macrophages were evaluated. Some of the terpenes of its essential oil (β-caryophyllene, α-humulene, α-pinene, myrcene and linalool) were also tested to verify their possible influence in the oil cytotoxic activity. The results obtained permitted to confirm that the essential oil is cytotoxic against tumoral cells (CD 50 = 82.3, 90.7 and 113.6 µg/ml for A-549, HeLa e HT-29 cell lines, respectively), while it did not show cytotoxicity against non-tumoral cells (Vero and mice macrophages). Thus, the essential oil from Z. rhoifolium leaves seems to present a possible therapeuthic role due to its selective cytotoxic activity against tumoral cell lines. KEYWORDSZanthoxylum, Essential oil, cytotoxicity, β-caryophyllene, α-humulene Avaliação citotóxica do óleo volátil extraído das folhas do

Aims:  To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. Methods and Results:  Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp‐2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp‐2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56°C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell‐free haemolytic activity similar to the ‘hot‐cold’ haemolysins. Conclusions:  S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. Significance and Impact of the Study:  This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.

Thirty-two clinical isolates of Enterococcus faecalis were screened for virulence factors. Twenty-four (75%) isolates produced hemolysin on Mueller-Hinton blood agar plates with sheep erythrocytes. However, the cell free heat-stable hemolysin was detected in all isolates (100%) of E. faecalis when grown in BHI-GA (BHI medium supplemented with 1% glucose and 0.03% L-arginine), but not in BHI broth alone. Twenty-four isolates (75%) produced caseinase and 23 (71.9%) lipase, but none of the isolates produced gelatinase. Fifteen (46.9%) culture filtrates caused rounding and membrane alterations with blebbing formation followed by death in HeLa and HEp-2 cells, but not in Vero cells. Thirteen isolates (40.6%) agglutinated rabbit erythrocytes, but did not produce hemagglutination in other bloods, containing or not 1% D-manose. Sixteen (50%) E. faecalis isolates adhered to HeLa cells and thirteen (40.6%) to HEp-2 cells, but all isolates adhered to polypropylene microtiter plates, indicating that clinical E. faecalis possess the ability to form biofilm in vitro. All the isolates were resistant to the bactericidal action of normal serum and did not produce aerobactin. These findings suggest that adherence and consequently biofilm formation on ephitelial host cells are the first steps in the E. faecalis virulence and that hemolysin, lipase, caseinase and other virulence factors act as causative of human epithelial cell damages.

Aims:  Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H−) on Caco 2 and HT‐29‐human epithelial intestinal cells. Methods and Results:  The Caco 2 and HT‐29 cells, which were treated with Enterohemolysin (EHly) within 10–15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. Conclusions:  Enterohemolysin induced apoptosis on human epithelial intestinal cells. Significance and Impact of the Study:  The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.

The value of E. coli virulence factors in patients with neurogenic bladder has not been established. The aim of this study is to correlate E. coli virulence factors with asymptomatic and symptomatic UTI in children with neurogenic bladder. Fifty E. coli strains, which were collected in sequence, underwent analysis in relation to: the association to pyuria, serotype (O:H), the presence of genes and expression of fimbriae P, type 1, S and hemagglutinin Dr, the presence of the gene and production of hemolysins and cytotoxins (CNF1). We also analyzed the cell adherence capability and pattern and presence of usp (uropathogenic-specific protein). Pyuria was present in most of the positive urine cultures, with 86% AB and 97% UTI. Low rates of uropathogenic strains were observed in the two groups, with 18% AB and 21% UTI. Type 1 fimbria predominated in 44% of the E. coli strains. Of the bacteria studied, 30% (15 strains) exhibited papG genotypes (11 class II and 4 class III). Of these, 12/15 patients presented AB. Production of hemolysins was detected in 38% of the strains (16 AB and 3 UTI) and usp in only 18% of the strains, with 8 AB and 1 UTI. Adherence tests demonstrated the adhesive capacity in all samples analyzed. Neither group (AB or symptomatic UTI) presented a statistically significant difference in relation to the virulence factors studied. E. coli clones that caused symptomatic UTI in children with neurogenic bladder expressed few virulence factors, with no statistically significant difference in comparison to the AB group.

Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl 2 , 1 mM iodoacetic acid) and the hemolysin (100 µg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 µg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.