Lidia del Carmen Velázquez

Effectiveness of various disinfectants in the elimination of Yersinia enterocolitica on fresh lettuce

The effectiveness of various disinfectants against two potentially pathogenic Yersinia enterocolitica strains (Y. enterocolitica W1024 O:9 [strain A] and Y. enterocolitica B O:5 Lis Xz [strain B]) on shredded lettuce was examined. Dip-wash treatments using 25, 100, and 300 ppm of chlorine at 4 and 22 degrees C, 0.2% Orenco Peel 40, 0.1% Tergitol, 0.5% acetic acid, and 0.5% lactic acid at 22 degrees C were performed. Surfactants and organic acids were also tested in combination with 100 ppm of chlorine. Reductions of Y. enterocolitica counts with 100 ppm (2.68 log10 for strain A and 2.36 log10 for strain B at 22 degrees C) and 300 ppm of chlorine (3.15 log10 for strain A and 2.55 log10 for strain B at 4 degrees C) were observed after 10 min. Inhibitory effect of different chlorine solutions was not significantly (P < 0.05) influenced by temperature. Surfactants in combination with chlorine were more effective than surfactants alone. Treatment with 0.2% Orenco Peel 40 plus 100 ppm of chlorine resulted in reductions of 2.69 log10 CFU/g for strain A and 3.18 1og10 CFU/g for strain B at 10 min. Dip solutions containing 0.1% Tergitol plus 100 ppm of chlorine produced a significant reduction of 2.73 log10 CFU/g in strain A (P < 0.05). With the 0.5% lactic acid plus 100 ppm of chlorine combination, inactivation of Y. enterocolitica was >6 log10. The bactericidal effect of disinfectants was related to the concentration, exposure time, combination with chlorine (surfactants and organic acids), and susceptibility of each strain. Since the presence of pathogenic Y. enterocolitica on ready-to-use vegetables represents a health hazard, treatments as effective as 0.5% lactic acid plus 100 ppm of chlorine are recommended for washing of fresh lettuce.

Pulsed field, PCR ribotyping and multiplex PCR analysis of Yersinia enterocolitica strains isolated from meat food in San Luis Argentina

The characterization of phenotypic and genotypic virulence markers of Yersinia enterocolitica strains belonging to biotypes (B) 1A, 2 and 3, mostly isolated from food in San Luis, Argentina, and the assessment of their genotypic diversity using PFGE and PCR ribotyping, were performed in our laboratory for the first time. Thirty five Y. enterocolitica strains, two reference strains and 33 strains isolated in our laboratory were studied. The presence of virF, ail, ystA, and myfA genes was investigated by multiplex PCR. The pathogenic potential of B1A strains, the most predominant biotype of Y. enterocolitica strains isolated from meat in our region, was investigated by simple PCR. Four B1A strains were positive for ystB gene. Four Y. enterocolitica 2/O:9 (bio/serotype) and two 3/O:5 strains isolated in our laboratory showed virulence-related results in the phenotypic tests and multiplex PCR. A good correlation between the expression of virulence markers and their corresponding genotypes was observed for most strains. Sixteen genomic types (GT) and 9 different intergenic spacer region (SR) groups were generated by PFGE and PCR ribotyping, respectively. In both cases the Y. enterocolitica 2/O:9 strains were separately clustered from 1A and 3/O:5 strains. Meat foods might be vehicles of transmission of pathogenic Y. enterocolitica strains in our region.

Antibacterial Effects of Different Food-Related Phosphates Using Aeromonas hydrophila

Aeromonas hydrophila is considered to be an emergent food-related bacterium. Phosphates are used as additives, mainly in meat products, to improve the quality of these foods. The antibacterial properties of phosphates are also well known. In this work, two A. hydrophila strains in early exponential phase were used: (A) A. hydrophila ATCC 7965 and (B) A. hydrophila derived from food, isolated in our laboratory. MIC and MBC studies were performed to assess the antibacterial effects of four phosphates assayed in brain heart infusion broth (BHI) and modified complete defined synthetic medium (mCDS) as compared to cooked ground meat medium (CM). The MBC values of the phosphates in CM were significantly higher than MIC values in BHI broth and mCDS medium (P < 0.05). In the two latter media, the growth of both A. hydrophila strains was totally inhibited by concentrations between 0.5 and 3.0%. Although all the assayed phosphates proved to have bactericidal effects on A. hydrophila, 0.5% sodium acid pyrophosphate (SAPP) exhibited greater effects in both strains and was selected for subsequent experiments. The bacteriolytic effect of SAPP was spectrophotometrically determined (260 nm of absorbance) by means of the leakage of intracellular nucleotides and microscopically confirmed by the presence of massive gelatinous aggregates. These were identified by enzymes (RNase, DNase, and proteinase) that hydrolyzed the nucleotides and proteins released during cellular lysis in the presence of SAPP. It was concluded that 0.5% SAPP can have bactericidal and bacteriolytic effects in early exponential phase A. hydrophila cells.

Survival of Aeromonas hydrophila in fresh tomatoes (Lycopersicum esculentum Mill) stored at different temperatures and treated with chlorine.

This study examines the survival of two Aeromonas hydrophila strains (A. hydrophila ATCC 7965 [strain A] and A. hydrophila isolated from food [strain B] on the surface and core tissue of fresh tomatoes stored at different temperatures and the efficacy of chlorine treatment on their survival. Counts of A. hydrophila on the surface of tomatoes stored at 25 and 35 degrees C were significantly higher between days 1 and 4 for both strains as compared to results obtained at 6 degrees C. Core tissue counts of A. hydrophila cells on tomatoes dipped in a cellular suspension at 25 degrees C and stored at 25 degrees C were significantly higher (P < 0.05) than counts obtained with dip suspensions at 6 or 35 degrees C. In chopped tomatoes stored at 25 and 35 degrees C, populations of aerobic mesophiles showed significant increases after 96 and 70 h, respectively. The populations of both A. hydrophila strains in chopped tomatoes stored at 6 degrees C increased significantly after 96 h, while at 25 and 35 degrees C the counts increased in the first hours of incubation. Viable counts of A. hydrophila on the surface and central tissue of tomatoes significantly decreased (P < 0.05) when the samples were dipped for 2 min in chlorine at a concentration of 50 ppm (50 microgram/ml). The results suggest that tomatoes should be kept at low temperatures during storage, shipping, and retail stocking and that chlorine at a concentration of 50 ppm should be used to reduce the levels of A. hydrophila.

Humoral Immune Response in Yersinia enterocolitica O:5 Induced Arthritis in Hamsters

Yersinia enterocolitica can cause extraintestinal sequelae such as reactive arthritis. The immunopathogenic mechanisms of this disease have not been completely clarified. Autoimmunity and persistent immune responses against bacterial antigens have been related to Yersinia‐induced arthritis. The arthritogenic capacity of Y. enterocolitica O:5 and the kinetics of the development of autoantibodies and Yersinia antigen‐specific antibodies were studied in hamsters. The results indicated that Y. enterocolitica O:5 was arthritogenic in the animal model studied. The animals developed septic arthritis on day 2 post‐infection (p.i.) and reactive arthritis on day 65 p.i. An important IgG response to types I and II collagen and the persistence of antibodies against lipopolysaccharide and bacterial cellular extract were observed. By immunoblotting, it was obtained that IgG reacted against a large number of bacterial antigens, the strongest being the responses against 88, 76, 63 and 36‐33 kDa peptides. From the results obtained, it can be concluded that serovar O:5 was experimentally arthritogenic, and that both autoimmune mechanisms and Yersinia‐specific antibodies participated in the development of Yersinia‐induced reactive arthritis in the animal model studied.

Presence of enterotoxigenic Staphylococcus aureus in artisan fruit salads in the city of San Luis, Argentina

An increase in the consumption of fruit juices and minimally processed fruits salads has been observed in recent years all over the world. In this work, the microbiological quality of artisan fruit salads was analysed. Faecal coliforms, Salmonella spp, Shigella spp, Yersinia enterocolitica and Escherichia coli O157:H7 were not detected; nevertheless, eleven strains of Staphylococcus aureus were isolated. By multiplex PCR, all isolates showed positive results for S. aureus 16S rRNA gene and 63.6% of them were positive for sea gene. Furthermore, PCR sea positive strains were able to produce the corresponding enterotoxin. Finally, the inactivation of these strains in fruit salads by nisin, lysozyme and EDTA, was studied. EDTA produced a total S. aureus growth inhibition after 60 h of incubation at a concentration of 250 mg/L. The presence of S. aureus might indicate inadequate hygiene conditions during salad elaboration; however, the enterotoxigenicity of the strains isolated in this study, highlights the risk of consumers’ intoxication. EDTA could be used to inhibit the growth of S. aureus in artisan fruit salads and extend the shelf life of these products.

Biovars, Serovars, and Phagovars of Yersinia enterocolitica Isolated From 450 Samples of Cold Food in San Luis, Argentina

A search of Yersinia enterocolitica in foods of animal origin has been carried out. Isolates were obtained from 450 samples of cold foods: 100 samples of cooked ham, 150 samples of salami, 100 samples of porcine cheese (artisan cold foods), and 100 samples of mortadella. Enrichments were performed in 0.067 M phosphate buffered saline solutions, pH 7. 6, containing 1% sorbitol and 0.15% biliary salts. The samples were postenriched in 0.5% KOH. Subcultures were done in Salmonella-Shigella agar and MacConkey agar. Isolates were identified through biochemical, serological, and phagotyping methods. The following biovars (B), serovars (O), and phagovars (Lis) were isolated from cooked ham B2 0:9 Lis X3 (1%), from salami B1 0:5 Lis Xz and B2 0:9 Lis X3 (1.33%), from porcine cheese B2 0:9 Lis X3 (2%), and from mortadella (0%). Virulence tests (calcium dependent growth at 37°C and autoagglutination activity at 37°C) were always negative. Serovar B2 0:9 Lis X3 associated with human disease was isolated. It is concluded from the results of this study that Y. enterocolitica isolates from cold foods lack of pathogenic importance.

Yersinia spp. in surface water in San Luis, Argentina

Yersinia spp. was examined in three rivers and two lakes located in the Province of San Luis, Argentina, over a 1-year period. Water samples were concentrated either by Moores gauze technique or by filtering through diatomaceous earth. Five enrichment media: yeast extract-Bengal rose broth (YER) with bile-oxalate-sorbose broth (BOS); 67 mmol/L phosphatebuffered saline (pH 7.6; PBS); PBS enriched with 1% mannitol and 1% peptone (PBSMP); PBS with lyzed 0.5% sheep blood (PBSB); Wauters broth (W); and five plating media: Mac Conkey agar (MC);Salmonella—Shigella agar (SS); 5% sheep blood agar (BA); lactose-sucrose-urea agar (LSU) and irgasan-novobiocin agar (IN) were used. The following strains were isolated:Y. intermedia B1 O:4,32–4,33 Lis Xz (four strains),Y. intermedia B1 O:57 Lis Xo (one strain),Y. intermedia B2 0:57 Lis Xo (one strain),Y. enterocolitica B1 O:10–34 Lis Xz (one strain), andY. frederiksenii undetermined biovar, O:16–16,29 Lis Xz (two strains). The incidence of isolation ofYersinia spp. was 7.14%. YER-BOS proved to be the best enrichment method since it allowed the highest recovering ofYersinia spp. strains. Among plating media, the best results were obtained with MC. Apparently, the isolation ofYersinia spp. can be related to environmental variables such as temperature differences between cold and warm seasons. Negative results obtained during virulence assays suggest that isolated strains lack the pathogenic potential against man.