Bernadette Dora Gombossy de Melo Franco

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.

Minimally processed vegetable salads: microbial quality evaluation.

The increasing demand for fresh fruits and vegetables and for convenience foods is causing an expansion of the market share for minimally processed vegetables. Among the more common pathogenic microorganisms that can be transmitted to humans by these products are Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella. The aim of this study was to evaluate the microbial quality of a selection of minimally processed vegetables. A total of 181 samples of minimally processed leafy salads were collected from retailers in the city of Sao Paulo, Brazil. Counts of total coliforms, fecal coliforms, Enterobacteriaceae, psychrotrophic microorganisms, and Salmonella were conducted for 133 samples. L. monocytogenes was assessed in 181 samples using the BAX System and by plating the enrichment broth onto Palcam and Oxford agars. Suspected Listeria colonies were submitted to classical biochemical tests. Populations of psychrotrophic microorganisms >10(6) CFU/g were found in 51% of the 133 samples, and Enterobacteriaceae populations between 10(5) and 106 CFU/g were found in 42% of the samples. Fecal coliform concentrations higher than 10(2) CFU/g (Brazilian standard) were found in 97 (73%) of the samples, and Salmonella was detected in 4 (3%) of the samples. Two of the Salmonella-positive samples had <10(2) CFU/g concentrations of fecal coliforms. L. monocytogenes was detected in only 1 (0.6%) of the 181 samples examined. This positive sample was simultaneously detected by both methods. The other Listeria species identified by plating were L. welshimeri (one sample of curly lettuce) and L. innocua (2 samples of watercress). The results indicate that minimally processed vegetables had poor microbiological quality, and these products could be a vehicle for pathogens such as Salmonella and L. monocytogenes.

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 degrees C and 37 degrees C, reaching a maximum production of 1.0 x 10(9) AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.

Microencapsulation of Bifidobacterium animalis subsp. lactis in Modified Alginate-chitosan Beads and Evaluation of Survival in Simulated Gastrointestinal Conditions

Bifidobacterium animalis subsp. lactis was entrapped in alginate, alginate-chitosan, alginate-chitosan-Sureteric and alginate-chitosan-Acryl-Eze. Survival and in vitro release of bifidobacteria from the microparticles were investigated under conditions simulating gastrointestinal fluids covering the pH range from 1.5 to 7.5, with and without pepsin (3gL−1), pancreatin (1gL−1), and bile (10gL−1). All types of microcapsules protected B. animalis, but the use of chitosan and enteric polymers in the formulation of the beads, especially Acryl-Eze, enhanced the beneficial effects of the microencapsulation technique. Besides promoting the controlled release of bifidobacteria in simulated gastrointestinal juices, the microencapsulation with enteric polymers improved the survival rate of these microorganisms.

Growth potential of Salmonella spp. and Listeria monocytogenes in nine types of ready-to-eat vegetables stored at variable temperature conditions during shelf-life.

Growth potential (δ) is defined as the difference between the population of a microorganism at the end of shelf-life of specific food and its initial population. The determination of δ of Salmonella and Listeria monocytogenes in RTE vegetables can be very useful to determine likely threats to food safety. However, little is known on the behavior of these microorganisms in several RTE vegetables. Therefore, the aim of this study was to determine the δ of both pathogens in nine different types of RTE vegetables (escarole, collard green, spinach, watercress, arugula, grated carrot, green salad, and mix for yakisoba) stored at refrigeration (7°C) and abuse temperature (15°C). The population of aerobic microorganisms and lactic acid bacteria, including those showing antimicrobial activity has been also determined. Results indicated that L. monocytogenes was able to grow (δ≥0.5 log(10)) in more storage conditions and vegetables than Salmonella. Both microorganisms were inhibited in carrots, although a more pronounced effect has been observed against L. monocytogenes. The highest δ values were obtained when the RTE vegetables were stored 15°C/6days in collard greens (δ=3.3) and arugula (δ=3.2) (L. monocytogenes) and arugula (δ=4.1) and escarole (δ=2.8) (Salmonella). In most vegetables and storage conditions studied, the counts of total aerobic microorganisms raised significantly independent of the temperature of storage (p<0.05). Counts of lactic acid bacteria were higher in vegetables partially or fully stored at abuse temperature with recovery of isolates showing antimicrobial activity. In conclusion, the results of this study show that Salmonella and L. monocytogenes may grow and reach high populations in RTE vegetables depending on storage conditions and the definition of effective intervention strategies are needed to control their growth in these products.

Prevalence, populations and pheno- and genotypic characteristics of Listeria monocytogenes isolated from ready-to-eat vegetables marketed in Sao Paulo, Brazil

Listeria monocytogenes is a foodborne pathogen of great concern due to the high fatality rates of listeriosis. The consumption of RTE vegetables has increased in Brazil over the last two decades, but little is known about the risks associated to the consumption of these products. This study evaluated the prevalence and counts of L. monocytogenes in 512 packages of ready-to-eat vegetables marketed in São Paulo. The isolates were characterized for their serotypes, ribotypes, positivity for virulence genes inlA, inlC and inlJ, resistance to chlorine, growth rate variability and capability to form biofilm on stainless steel (AISI 304, #4) coupons. L. monocytogenes was detected in 3.1% of the samples. Only five samples presented countable levels, with counts between 1.0×10(1) and 2.6×10(2)CFU/g. Isolates belonged to serotypes 1/2b or 4b and most were positive for genes inlC and inlJ. Ribotypable isolates were grouped into four groups: 1038 (69.4%), 19175 (11.3%), 19191 (17.7%) and 18604 (one isolate). Most isolates survived to exposure to 125 ppm of a chlorine-based disinfectant for 3 min. All isolates were capable to attach to the coupons, reaching counts above 4 log(10) CFU/cm(2) and the growth rate (μ) at 25°C of the majority of the isolates varied between 0.1 and 0.2 log OD/h, but for few strains the μ was as high as 0.26 log OD/h. Results of this survey indicate that RTE vegetables may be vehicles of L. monocytogenes strains with limited variation in serotype, ribotype and virulence factors but varying significantly in resistance to chlorine disinfectants, capability of forming biofilm and growth rate. Data obtained is of foremost importance to serve as baseline for the development of scientific-based policies to control the incidence of L. monocytogenes in RTE vegetables in Brazil.

Modeling the growth rate and lag time of different strains of Salmonella enterica and Listeria monocytogenes in ready-to-eat lettuce

The growth parameters (growth rate, μ and lag time, λ) of three different strains each of Salmonella enterica and Listeria monocytogenes in minimally processed lettuce (MPL) and their changes as a function of temperature were modeled. MPL were packed under modified atmosphere (5% O₂, 15% CO₂ and 80% N₂), stored at 7-30 °C and samples collected at different time intervals were enumerated for S. enterica and L. monocytogenes. Growth curves and equations describing the relationship between μ and λ as a function of temperature were constructed using the DMFit Excel add-in and through linear regression, respectively. The predicted growth parameters for the pathogens observed in this study were compared to ComBase, Pathogen modeling program (PMP) and data from the literature. High R² values (0.97 and 0.93) were observed for average growth curves of different strains of pathogens grown on MPL. Secondary models of μ and λ for both pathogens followed a linear trend with high R² values (>0.90). Root mean square error (RMSE) showed that the models obtained are accurate and suitable for modeling the growth of S. enterica and L. monocytogenes in MP lettuce. The current study provides growth models for these foodborne pathogens that can be used in microbial risk assessment.

Viability of a Five-Strain Mixture of Listeria monocytogenes in Vacuum-Sealed Packages of Frankfurters, Commercially Prepared with and without 2.0 or 3.0% Added Potassium Lactate, during Extended Storage at 4 and 10° C†‡

The viability of Listeria monocytogenes was monitored on frankfurters containing added potassium lactate that were obtained directly from a commercial manufacturer. Eight links (ca. 56 g each) were transferred aseptically from the original vacuum-sealed bulk packages into nylon-polyethylene bags. Each bag then received a 4-ml portion of a five-strain mixture of the pathogen. Frankfurters containing 2.0 or 3.0% potassium lactate were evaluated using 20 CFU per package, and frankfurters containing 3.0% potassium lactate were evaluated using 500 CFU per package. The packages were vacuum-sealed and stored at 4 or 10 degrees C for up to 90 or 60 days, respectively. During storage at 4 degrees C, pathogen numbers remained at about 1.6 log10 CFU per package over 90 days in packages containing frankfurters with 2.0% potassium lactate that were inoculated with about 20 CFU. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 20 CFU and stored at 4 degrees C, pathogen numbers remained at about 1.4 log10 CFU per package over 90 days. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 500 CFU and stored at 4 degrees C, pathogen numbers remained at about 2.4 log10 CFU per package over 90 days. However, in the absence of any added potassium lactate, pathogen numbers increased to 4.6 and 5.0 log10 CFU per package after 90 days of storage at 4 degrees C for starting levels of 20 and 500 CFU per package, respectively. During storage at 10 degrees C, pathogen numbers remained at about 1.4 log10 CFU per package over 60 days in packages containing frankfurters with 2.0% potassium lactate that were inoculated with about 20 CFU. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 20 CFU and stored at 10 degrees C, pathogen numbers remained at about 1.1 log10 CFU per package over 60 days of storage. In the absence of any added potassium lactate, pathogen numbers increased to 6.5 log10 CFU per package after 28 days and then declined to 5.0 log10 CFU per package after 60 days of storage at 10 degrees C. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 500 CFU per package, pathogen numbers remained at about 2.4 log10 CFU per package over 60 days of storage at 10 degrees C, whereas in the absence of any added potassium lactate, pathogen numbers increased to about 6.6 log10 CFU per package within 40 days and then declined to about 5.5 log10 CFU per package after 60 days of storage. The viability of L. monocytogenes in frankfurter packages stored at 4 and 10 degrees C was influenced by the pH and the presence or levels of lactate but not by the presence or levels of indigenous lactic acid bacteria or by the proximate composition of the product. These data establish that the addition of 2.0% (P < 0.0004) or 3.0% (P < 0.0001) potassium lactate as an ingredient in frankfurters can appreciably enhance safety by inhibiting or delaying the growth of L. monocytogenes during storage at refrigeration and abuse temperatures.

Inactivation by ionizing radiation of Salmonella Enteritidis, Salmonella Infantis, and Vibrio parahaemolyticus in oysters (Crassostrea brasiliana)

Irradiation is considered one of the most efficient technological processes for the reduction of microorganisms in food. It can be used to improve the safety of food products, and to extend their shelf lives. Oysters are considered one of the most important vehicles for pathogenic bacteria because of their feeding characteristics. The aim of this study was to evaluate the influence of a gamma radiation process on high levels of Salmonella Enteritidis, Salmonella Infantis, and Vibrio parahaemolyticus incorporated by oysters (Crassostrea brasiliana), as well as the effects of the process on the survival of the oysters and on their sensory attributes. The oysters were exposed to gamma radiation (60Co) in doses ranging from 0.5 to 3.0 kGy. A dose of 3.0 kGy was generally sufficient to reduce the level of Salmonella serotypes by 5 to 6 log10 units. A dose of 1.0 kGy was sufficient to produce a 6-log10 reduction in the level of V. parahaemolyticus. The highest irradiation dose did not kill the oysters or affect their sensory attributes. Hence, a dose of 3.0 kGy can be considered effective in inactivating Salmonella and V. parahaemolyticus in oysters without changing their odor, flavor, or appearance.

Parameters determining the quality of charqui, an intermediate moisture meat product

Charqui is a typical Brazilian meat product obtained by salting and sun-drying beef samples. The chemical, physical and microbiological characteristics of the charqui were evaluated throughout processing and storage. The results confirm charqui is an intermediate moisture meat product (A(w) = 0·70-0·75). A close relationship between moisture, pretein and ash vaiues was found, suggesting the possibility of using the resulting charqui A(w) value as a parameter to define the product instead of the official moisture and mineral residue contents. The TBA determination, which expresses the state of lipid oxidation, rapidly reached the maximum value, corroborating the previous observations on the salt pro-oxidant role, and then decreased gradually. A gradual decrease in microorganism count during processing and storage of charqui was also observed. These results indicate the feasibility of obtaining a final product with a low level of microbial count when raw materials of good quality, and adequate handling conditions, are used for charqui production.