Mercedes Passos Geimba

Serological Characterization and Prevalence of spvR Genes in Salmonella Isolated from Foods Involved in Outbreaks in Brazil

Salmonella strains (n = 75) isolated from foods involved in foodborne outbreaks occurred in Rio Grande do Sul State, Brazil, during 1999 and 2000 were studied. Strains were serotyped and submitted to PCR analysis to verify the prevalence of Salmonella plasmid virulence (spvR) regulatory gene. Among the 75 isolates, 73 (97%) were classified as Salmonella enterica serovar Enteritidis. All of the Salmonella strains isolated in 1999 were classified as serotype Enteritidis, whereas in 2000 two isolates were serotyped as Salmonella Derby and Salmonella Typhimurium. Regarding the prevalence of spvR gene, 62 strains (82.7%) were PCR positive, and a positive correlation (P < 0.05) between the strains of Salmonella Enteritidis and the presence of spvR gene was demonstrated, which suggests that this gene is a characteristic of the Salmonella Enteritidis analyzed.

Enzymatic Clarification of Fruit Juices by Fungal Pectin Lyase

The activity of pectic enzymes was measured in culture filtrates of four selected fungal strains. Pectin lyase (PL) activity was produced by all fungi, but pectinesterase activity was not detected in culture filtrates of the strain Penicillium expansum F16. The use of crude enzymes resulted in good clarification of apple juice as evaluated by an increase in transmittance at 660 nm. PL activities were partially purified by dye-affinity chromatography on Blue Sepharose. The enzyme was bound to the column and eluted in a single peak, free of PE activity, and not coinciding with the major amount of protein. Apple, grape, and passion fruit juices were effectively clarified by these partially purified enzymes. Methanol content was determined by gas-liquid chromatography, being undetectable in the clarified juices. These pectic enzymes demonstrate potential for use in fruit juice processing.

Characterisation of cellulose-hydrolysing enzymes from the fungus Bipolaris sorokiniana

Cellulose-hydrolysing enzymes from the phytopathogenic fungus Bipolaris sorokiniana were partially purified and characterised. The enzyme production was variable according to the carbon source. β-Glucosidase and cellobiohydrolase activities were higher by growing the fungus on cellulose than on other carbon sources. Carboxymethyl cellulase production was stimulated by other carbohydrates, mainly lactose. Partial enzyme purification was carried out by liquid chromatography on Sepharose CL4B. The purification was about 17-fold, with a yield of 41% as judged by assay with p-nitrophenyl-β-D-glucopyranoside as substrate. The optimum pH and temperature were 5.0 and 55–60 °C respectively. The enzyme was stable at 28 and 37 °C but lost about 50% of its initial activity after 120 min at 55 °C. Saccharification of cellulosic materials such as crystalline cellulose, filter paper and wheat straws was carried out using the partially purified enzyme, resulting in the production of reducing sugars. © 1999 Society of Chemical Industry

ANTIMICROBIAL RESISTANCE AND PCR-RIBOTYPING OF SHIGELLA RESPONSIBLE FOR FOODBORNE OUTBREAKS OCCURRED IN SOUTHERN BRAZIL

Little information about Shigella responsible for foodborne shigellosis is available in Brazil. The present study aimed to investigate the antimicrobial resistance and PCR-ribotyping patterns of Shigella isolates responsible for foodborne outbreaks occurred in Rio Grande do Sul State (RS), Southern Brazil in the period between 2003 and 2007. Shigella strains (n=152) were isolated from foods and fecal samples of victims of shigellosis outbreaks investigated by the Surveillance Service. Identification of the strains at specie level indicated that 71.1% of them were S. flexneri, 21.5% S. sonnei, and 0.7% S. dysenteriae. Ten strains (6.7%) were identified only as Shigella spp. An increasing occurrence of S. sonnei was observed after 2004. Most of the strains were resistant to streptomycin (88.6%), followed by ampicillin (84.6%), and sulfamethoxazole/trimethoprim (80.5 %). Resistant strains belonged to 73 patterns, and pattern A (resistance to ampicillin, sulfamethoxazole/trimethoprim, tetracycline, streptomycin, chloramphenicol, and intermediate resistance to kanamycin) grouped the largest number of isolates (n=36). PCR-ribotyping identified three banding patterns (SH1, SH2, and SH3). SH1 grouped all S. flexneri and SH2 grouped all S. sonnei. The S. dysenteriae strain belonged to group SH3. According to the results, several Shigella isolates shared the same PCR-rybotyping banding pattern and the same resistance profile, suggesting that closely related strains were responsible for the outbreaks. However, other molecular typing methods need to be applied to confirm the clonal relationship of these isolates.

Purification and characterization of ββ‐N‐acetylhexosaminidase from the phytopathogenic fungus Bipolaris sorokiniana

N‐acetylhexosaminidase (HEX) from the phytopathogenic fungus Bipolaris sorokiniana was isolated and characterized. The production of HEX by B. sorokiniana was not altered by growing on different carbon sources. Enzyme purification was carried out by sequential liquid chromatography on Sephacryl S‐200 HR, and p‐aminobenzyl‐2‐acetamido‐2‐deoxy‐β‐d‐thioglucopyranoside agarose. The purification was about 70‐fold, with a yield of 41%, determined with p‐nitrophenyl‐N‐acetylglucosaminide as substrate. The enzyme had pH and temperature optima of 4·5 and 55 °C, respectively. The molecular weight of non‐denatured enzyme was estimated as 120 000 Da by gel filtration chromatography, and about 55 000 Da by SDS‐PAGE. The fungal HEX had glycosylated residues as evidenced by binding to Concanavalin‐A. Bipolaris sorokiniana enzyme was also active with p‐nitrophenyl‐chitobioside and p‐nitrophenyl‐N‐acetylgalactosaminide as substrates.

Extracellular enzymatic activities of Bipolaris sorokiniana isolates

Several enzymatic activities were investigated in six isolates of the fungus Bipolaris sorokiniana, originating from different areas of Brazil. Among the glycosidases studied, β‐glucosidase, β‐N‐acetylglucosaminidase, β‐xylosidase, cellobiohydrolase, and chitobiohydrolase were the major activities. In some isolates, β‐glucuronidase, β‐galactosidase, and α‐mannosidase activities were also present. Polysaccharide‐hydrolyzing enzymes, such as pectin lyase and carboxymethyl cellulase were detected in significant amounts, and their activities were variable among the different isolates. Other enzymes, namely phosphatases, proteinases and phenol oxidase, were also examined, showing variable amounts depending on the isolate. The pH dependence of all enzymes tested was investigated. Endoproteinase, carboxymethyl cellulase, and phenoloxidase had maximum activity in the pH range of 6–8, whilst all other enzymes showed maximum activity at pH 4–6.

Bacterial contamination of human skin allografts and antimicrobial resistance: a skin bank problem

BackgroundBacterial contamination remains the major problem in skin banks, even after antimicrobial treatment, and results in high rates of tissue discarding. This study aimed to analyze bacterial contamination in 32 human skin allografts from the skin bank of Dr. Roberto Corrêa Chem from the Hospital Complex Santa Casa de Misericórdia de Porto Alegre. These samples were already discarded due to microbial contamination. The identification of the bacteria isolated from skin allografts was performed by matrix assisted laser desorption ionization–time of flight. The antimicrobial susceptibility of the isolates to six different classes of antimicrobials was determined using the disk-diffusion agar method, and the evaluation of the inhibitory potential was determined by the minimal inhibitory concentration (50/90) of antimicrobials already used in the skin bank and those that most isolates were susceptible to.ResultsA total of 21 (65.6%) skin samples were contaminated with Gram-positive bacteria: 1 (4.7%) with Paenibacillus sp., 12 (61.9%) with Bacillus sp., 6 (28.5%) with Staphylococcus sp., and 2 (9.5%) with Bacillus sp. and Staphylococcus sp. Several resistance profiles, including multiresistance, were found among the isolates. Most of the isolates were susceptible to at least one of the antimicrobials used in the skin bank. All isolates were susceptible to amikacin, gentamicin, and tetracycline, which demonstrated the best inhibitory activities against the isolates and were considered as potential candidates for new antimicrobial treatments.ConclusionsBacillus, Paenibacillus, and Staphylococcus were isolated from the skin allografts, thus demonstrating the predominance of Gram-positive bacteria contamination. Other factors not related to the resistance phenotype may also be involved in the persistence of bacterial isolates in the skin allografts after antibiotic treatment. Gentamicin, amikacin, and tetracycline can be considered as an option for a more effective treatment cocktail.