Characterization of the virulence, agr typing and antimicrobial resistance profile of Staphylococcus aureus strains isolated from food handlers in Brazil.

Staphylococcus aureus is one of the main pathogens associated with foodborne outbreaks in Brazil and food handlers can carry toxigenic and resistant S. aureus strains. The aims of this study were to verify the frequency of virulence genes, to identify the agr groups and to determine the antimicrobial resistance profile of S. aureus strains isolated from food handlers of pilot kitchens located in São Paulo, Brazil. A total of 74 strains of the Staphylococcus genus were detected and 50% were identified as of the species S. aureus. The enterotoxin genes detection, tst and luk-PV detection, agr typing, mecA detection, ccr complex detection and SCCmec typing were performed using PCR. The antimicrobial resistance testing was performed by the disk diffusion method. The enterotoxin genes were identified in 36 S. aureus, including sea (83.8%). The tst gene was detected in 18.92% of the strains and the luk-PV was detected in only one isolate. Agr typing classified 58.3% of the strains as type I. Seven (18.92%) strains were classified as MRSA and the ccr2 complex was detected in six of these isolates. The SCCmec typing characterized strains as type II, III, IV and V. Moreover, there were also a greater number of resistant strains to penicillin (83.78%) and clarithromycin (67.57%). In conclusion, the study revealed a significant prevalence of S. aureus, and the presence of different virulence genes and a worrying resistance profile in S. aureus strains isolated from food handlers in this country.

Characterization of the virulence, agr typing and antimicrobial resistance profile of Staphylococcus aureus strains isolated from food handlers in Brazil

Abstract Staphylococcus aureus is one of the main pathogens associated with foodborne outbreaks in Brazil and food handlers can carry toxigenic and resistant S. aureus strains. The aims of this study were to verify the frequency of virulence genes, to identify the agr groups and to determine the antimicrobial resistance profile of S. aureus strains isolated from food handlers of pilot kitchens located in São Paulo, Brazil. A total of 74 strains of the Staphylococcus genus were detected and 50% were identified as of the species S. aureus. The enterotoxin genes detection, tst and luk-PV detection, agr typing, mecA detection, ccr complex detection and SCCmec typing were performed using PCR. The antimicrobial resistance testing was performed by the disk diffusion method. The enterotoxin genes were identified in 36 S. aureus, including sea (83.8%). The tst gene was detected in 18.92% of the strains and the luk-PV was detected in only one isolate. Agr typing classified 58.3% of the strains as type I. Seven (18.92%) strains were classified as MRSA and the ccr2 complex was detected in six of these isolates. The SCCmec typing characterized strains as type II, III, IV and V. Moreover, there were also a greater number of resistant strains to penicillin (83.78%) and clarithromycin (67.57%). In conclusion, the study revealed a significant prevalence of S. aureus, and the presence of different virulence genes and a worrying resistance profile in S. aureus strains isolated from food handlers in this country.

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis.

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 µg mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from >170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.

Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis.

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

Definition of a diagnostic routine in individuals with inconclusive serology for Chagas disease.

Despite the existence of highly sensitive tests, inconclusive serological results are frequent in chronic chagasic infection. This study aimed to define a diagnostic conduct for 30 individuals with inconclusive serology (G3) for chagasic infection assisted at the Outpatient Unit for Infectious and Parasitic Diseases of the Botucatu School of Medicine. Twenty-one individuals with negative serology (G1) and 33 with positive serology (G2) were also studied. Serological methods ELISA, HAI, IFI and immunoblotting TESA-cruzi were used for G1, G2 and G3, and parasitological methods xenodiagnosis, hemoculture and PCR-LIT were used for G2 and G3 individuals. ELISA, HAI and IFI were performed in 5 different blood samples in G2 and G3. TESA-cruzi was carried out only once in G1, G2 and G3 and, since it is the most sensitive, it was utilized as standard. In G3, positivity for ELISA reached 86% in the fifth blood sample; the ELISA+HAI+IFI combination showed a maximum of 44.8% in the second sample; and TESA-cruzi, 76% in one single sample. Xenodiagnosis positivity was 9.4%; hemoculture showed 15.2%; and PCR-LIT exhibited 22% positivity in G2. Nevertheless, in G3, positivity percentage was 3.4% for xenodiagnosis, 6.7% for PCR-LIT, and no positive result was found for hemoculture. In G3, PCR-LIT resolved one case which was still inconclusive according to serology tests. In order to define inconclusive diagnoses, the results suggest the combined use of ELISA+HAI+IFI in 2 blood samples, decreasing the occurrence of false positive/negative results. If results remain inconclusive, the performance of TESA-cruzi and PCR-LIT, if necessary, is recommended.

Occurrence of enteroparasites in day care centers in Botucatu (São Paulo State, Brazil) with emphasis on Cryptosporidium sp., Giardia duodenalis and Enterobius vermicularis.

The occurrence of the enteroparasites was verified in 279 children (0 to 6 years) of four municipal day cares of Botucatu/SP. Three samples of each child's feces were collected and processed by the methods of Hoffman-Pons-Janner, Faust and Ritchie and subsequent coloration of the fecal smear by the methods of Auramina-O and Ziehl-Neelsen modified for diagnosis of Cryptosporidium sp. and Graham method for diagnosis of Enterobius vermicularis. Of the analyzed children we verified a prevalence of intestinal parasitism in 53.40%, and the most frequent parasite was Giardia duodenalis (26.88%). Significant association was verified among enteroparasitosis, family income, maternal education and age; the lowest enteroparasite frequency occurred in children of families with larger income and higher education. It was observed that G. duodenalis is more prevalent in children from 0 to 4 years and E. vermicularis is more frequent in children between three and four years old. The high enteroparasite prevalence in day cares suggests complex structure in its epidemiology, where factors beyond sanitation should be considered.