Different methods to quantify Listeria monocytogenes biofilms cells showed different profile in their viability

Listeria monocytogenes is a foodborne pathogen able to adhere and to form biofilms in several materials commonly present in food processing plants. The aim of this study was to evaluate the resistance of Listeria monocytogenes attached to abiotic surface, after treatment with sanitizers, by culture method, microscopy and Quantitative Real Time Polymerase Chain Reaction (qPCR). Biofilms of L. monocytogenes were obtained in stainless steel coupons immersed in Brain Heart Infusion Broth, under agitation at 37 °C for 24 h. The methods selected for this study were based on plate count, microscopic count with the aid of viability dyes (CTC-DAPI), and qPCR. Results of culture method showed that peroxyacetic acid was efficient to kill sessile L. monocytogenes populations, while sodium hypochlorite was only partially effective to kill attached L. monocytogenes (p < 0.05). When, viability dyes (CTC/DAPI) combined with fluorescence microscopy and qPCR were used and lower counts were found after treatments (p < 0.05). Selective quantification of viable cells of L. monocytogenes by qPCR using EMA revelead that the pre-treatment with EMA was not appropriate since it also inhibited amplification of DNA from live cells by ca. 2 log. Thus, the use of CTC counts was the best method to count viable cells in biofilms.

Vaginal lactobacilli as potential probiotics against Candida spp

Urogenital infections affect millions of people every year worldwide. The treatment of these diseases usually requires the use of antimicrobial agents, and more recently, the use of probiotic lactic acid bacteria (LAB) cultures for the management of vaginal infections has been extensively studied. In this work, 11 vaginal lactobacilli isolates, previously obtained from healthy patients, were studied to screen microorganisms with probiotic properties against Candida spp. The LAB were tested for their ability of auto-aggregation, coaggregation with C. albicans, C. glabrata, C. krusei, and C. tropicalis, adhesion to Caco-2 epithelial cells and production of lactic acid and hydrogen peroxide (H2O2). All lactobacilli isolates tested were able to auto-aggregate (ranging from 25.3 percent to 75.4 percent assessed at 4 hours of incubation) and to co-aggregate with the four Candida species into different degrees; among them L. crispatus showed the highest scores of coaggregation. The highest amount of lactic acid was produced by L. salivarius (13.9 g/l), followed by L. johnsonii (6.5 g/l), L. acidophilus (5.5 g/l), and L. jensenii (5.4 g/l). All isolates produced H2O2 , but the highest levels (3 -10 mg/l) were observed for L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, and L. vaginalis. Only L. agilis, L. jensenii, L. johnsonii and L. ruminus were able to adhere to epithelial Caco-2 cells. Among the isolates evaluated, L agilis, L. jensenii, L. johnsonii, and L. ruminus exhibited simultaneously several desirable properties as potential probiotic strains justifying future studies to evaluate their technological properties in different pharmaceutical preparations for human use.

Correlation between API 20 STREP and multiplex PCR for identification of Enterococcus spp. isolated from Brazilian foods

We evaluated the suitability of API 20 STREP and multiplex PCR to speciate 52 Enterococcus spp. obtained from Brazilian foods. A high percentage of isolates (78.9 percent) presented discrepant results between evaluated tests. Similar results were obtained for six E. faecalis and five E. faecium. The PCR multiplex was more effective than API 20 STREP for complete identification of the isolates.

A identificação das espécies de 52 Enterococcus spp. isolados de amostras de alimentos foi realizada empregando-se duas metodologias: sistema API 20 STREP e PCR multiplex. Os resultados obtidos revelaram que 78,9 por cento dos isolados apresentaram resultados diferentes nos dois testes utilizados. Apenas seis E. faecalis e cinco E. faecium apresentaram resultados concordantes pelos dois métodos. PCR multiplex permitiu a identificação completa de um número significantemente maior de enterococos do que o sistema API 20 STREP.

Fate of Helicobacter pylori artificially inoculated in lettuce and carrot samples

H. pylori é o agente da maioria dos casos de gastrites e úlceras pépticas. Entretanto, sua forma de transmissão não foi completamente elucidada. Para avaliar a sobrevivência de H. pylori (uma cepa de referência e uma clínica), artificialmente inoculado em amostras de cenoura e alface, porções de 25g das amostras receberam aproximadamente 106 UFC/g de H. pylori, e foram embaladas em atmosfera normal e/ou modificada (3 per center oxigênio, 10 per center dióxido de carbono, e 87 per center nitrogênio). Em seguida, foram armazenadas a 8ºC, com enumeração diária da população de H. pylori em ágar Columbia sangue (CBA) e/ou Helicobacter pylori Special Peptone Agar (HPSPA). Quando usamos CBA com antibióticos, o isolado clínico de H. pylori (HP1) foi detectado por até 72 horas nas amostras de alface e cenoura sanitizadas. Em amostras de cenoura esterilizadas, H. pylori HP1 permaneceu viável por até 96 horas. CBA sem antibióticos permitiu a recuperação de H. pylori ATCC 43629, a partir de amostras de cenoura embaladas em ambas as atmosferas, por até 120 horas. Nossos resultados reforçam que a transmissão de H. pylori, através do consumo de água e alimento contaminados, ainda não pode ser descartado.

Antilisterial activity of lactic acid bacteria isolated from vacuum-packaged brazilian meat and meat products

Twenty samples of Brazilian meat and meat products were screened by the agar overlay method for bacteriocin-producing lactic acid bacteria, using Lactobacillus sake ATCC 15521 as indicator strain. Based on Gram staining, KOH reaction, catalase test and fermentation of 49 carbohydrates (API 50 CH), three out of seven isolates with confirmed antagonist properties were identified as Lactobacillus curvatus, one as Leuconostoc mesenteroides and one as Leuconostoc sp. Two isolates could not be properly identified using these tests. The inhibitors produced by these strains were sensitive to proteases. Inhibition due to lytic bacteriophages was ruled out, so the isolates were classified as bacteriocin-producing lactic acid bacteria. Four of them presented antilisterial activity and a potential application as biopreservatives in meat systems.

Inhibition of fooborne pathogens by bacteriocin-producing leuconostocs and lactobacillus sake isolated from "lingüiça frescal"

Two strains of lactic acid bacteria, isolated from "lingüiça" (a typical Brazilian meat product) stored under refrigeration, produced antagonistic substances active against selected foodborne pathogens. The proteinaceous nature of the inhibitors was demonstrated and so they were classified as bacteriocins. The inhibition due to acid production and phages was ruled out. The bacteriocin produced by Leuconostoc sp was active against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. The bacteriocin produced by Lactobacillus sake was active against Listeria monocytogenes and Staphylococcus aureus. Gram negative were bacteria not inhibited by the bacteriocins produced.