Bacteroides Fragilis induces conformational alterations in Trichomonas Vaginalis ultrastructure in an in vitro interaction

Bacteroides genus are commonly found on mucous membranes, including the female genital tract, acting as agents for several site infections. Anaerobic infections are usually polymicrobial and endogenous. Trichomonas vaginalis, the trichomoniasis etiologic agent, is a facultative anaerobic flagellated parasite spread worldwide. The purpose of this study was to explore the association between vaginal bacteria and T. vaginalis, as well as to understand factors that may favour the infection of T. vaginalis. We have, therefore, used T. vaginalis trophozoites and the species Bacteroides fragilis, which is considered the most important in its genus, once it is the most commonly isolated bacteria from endogenous infections. The parasite-bacteria interaction was performed in different proportions in periods varying from 1 to 12 hours applying viability tests. The data were analyzed to compare the parasite viability in vitro in the presence and absence of B. fragilis. The results indicate that in the 1:100 proportion postinteraction analysis, ultrastructural alterations were noticeable after 6 hours. After 8 hours, T. vaginalis viability decreased, and after 12 hours of interaction no viable trophozoites were found. These data suggest that the parasite can deal with B. fragilis in short interaction periods. However, in longer interaction periods the trophozoites collapse, indicating that B. fragilis may produce toxic metabolites against T. vaginalis activity.

Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium minutisporum and Lomentospora prolificans: a comparative study of surface molecules produced by conidial and germinated conidial cells

BACKGROUND Scedosporium/Lomentospora species are opportunistic mould pathogens, presenting notable antifungal resistance. OBJECTIVES/METHODS We analysed the conidia and germinated conidia of S. apiospermum (Sap), S. aurantiacum (Sau), S. minutisporum (Smi) and L. prolificans (Lpr) by scanning electron microscopy and exposition of surface molecules by fluorescence microscopy. FINDINGS Conidia of Sap, Smi and Sau had oval, ellipsoidal and cylindrical shape, respectively, with several irregularities surrounding all surface areas, whereas Lpr conidia were rounded with a smooth surface. The germination of Sap occurred at the conidial bottom, while Smi and Sau germination primarily occurred at the centre of the conidial cell, and Lpr germination initiated at any part of the conidial surface. The staining of N-acetylglucosamine-containing molecules by fluorescein-labelled WGA primarily occurred during the germination of all studied fungi and in the conidial scars, which is the primary location of germination. Calcofluor white, which recognises the polysaccharide chitin, strongly stained the conidial cells and, to a lesser extent, the germination. Both mannose-rich glycoconjugates (evidenced by fluoresceinated-ConA) and cell wall externally located polypeptides presented distinct surface locations and expression according to both morphotypes and fungal species. In contrast, sialic acid and galactose-containing structures were not detected at fungal surfaces. MAIN CONCLUSIONS The present study demonstrated the differential production/exposition of surface molecules on distinct morphotypes of Scedosporium/Lomentospora species.

Sphygmomanometers and thermometers as potential fomites of Staphylococcus haemolyticus: biofilm formation in the presence of antibiotics

BACKGROUND The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.

Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology.

Biofilm production by multiresistant Corynebacterium striatum associated with nosocomial outbreak

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum 1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatum bound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.

Apoptosis-like death in parasitic protozoa

Apoptosis is an essential physiological process that plays a critical role in development and tissue homeostasis in multicellular organisms, but which is also observed in several eukaryotic microorganisms such as yeast and protozoa. Here, the authors briefly review the most used techniques to detect apoptosis in mammalian cells, especially those that can be applied to parasitic protozoa after different conditions such as drugtreatment. Apoptosis-like processes have been described in protozoa which present mitochondria, such as members of the Kinetoplastida and Apicomplexa groups as well as in protozoa which do not have a mitochondrion, as Entamoeba, Trichomonas and Giardia do. These observations are of interest from an evolutive point of view, especially due to the fact that the participation of the mitochondria in apoptosis has been extensively analyzed in several biological systems. The authors also reviewed the available data showing that several drugs in use as anti-protozoa agents, as well as others which are in the development phase, kill the protozoa through an apoptotic-like process.