Hydrophobic and adhesive patterns of lactic acid bacteria and their antagonism against foodborne pathogens on tomato surface (Solanum lycopersicum L.).

AIMS: To evaluate tomato epiphyte lactic acid bacteria (LAB) hydrophobicity and auto-aggregation as an indicator of bacteria adhesion to tomato. Likewise, use LAB adhesion and co-aggregation as mechanisms to antagonize pathogen attachment. METHODS AND RESULTS: Fifty-four LAB were screened to evaluate their hydrophobic, auto- and co-aggregative properties against Salmonella Typhimurium, Saintpaul, Montevideo and Escherichia coli O157:H7. Subsequently, tomato adhesion of Enterococcus faecium Col1-1C, Weisella cibaria 11-E-2 and W. confusa Col 1-13 with high, medium and low hydrophobicity and high co-aggregation was investigated as well as their pathogen antagonism. Results indicate that bacteria hydrophobicity and auto-aggregation correspond to LAB adhesion to tomato. Enterococcus faecium Col1-1C interfered in most of the pathogen adhesion and micrographs revealed that such effect could be related to the inhibition of structures-type biofilm on E. coli O157:H7 and the aggregate formation on Salmonella. CONCLUSIONS: Lactic acid bacteria hydrophobicity and auto-aggregation can estimate bacteria adhesion to tomato and adhesive and co-aggregative properties could serve as a tool to antagonize foodborne pathogens under specific conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evidence the interference of Ent. faecium Col1-1C in E. coli O157:H7 biofilm formation and Salmonella colonization.

Evaluation of the resistance to bacterial growth of star-shaped poly(ε-caprolactone)-co-poly(ethylene glycol) grafted onto functionalized carbon nanotubes nanocomposites.

Nanocomposites of functionalized carbon nanotubes (CNTsf) as nanofillers, and a copolymer of star-shaped poly(ε-caprolactone) (stPCL) and poly(ethylene glycol) (PEG) as a polymeric matrix were synthesized, characterized, and their resistance to the growth of Staphylococcus aureus and Pseudomonas aeruginosa was evaluated. CNTsf contain hydroxyl, carboxyl and acyl chloride groups attached to their surface. Nanocomposites were prepared by mixing CNTsf to a solution of stPCL-PEG copolymer. Raman and FT-IR spectroscopies confirm the functionalization of carbon nanotubes (CNTs). Star-shaped PCL-PEG copolymer was characterized by Gel permeation chromatography (GPC), and 1H-NMR and 13C-NMR spectroscopies. X-ray photoelectron spectroscopy (XPS) shows that CNTsf are grafted to the stPCL-PEG copolymer. Crystallization behavior of the nanocomposites depends on the amount of CNTsf used in their preparation, detecting nucleation (nanocomposites prepared with 0.5 wt.% of CNTsf) or anti-nucleation (nanocomposites prepared with 1.0 wt.% of CNTsf) effects. Young's Moduli and thermal stability of nanocomposites were higher, but their resistence to the proliferation of Staphylococcus aureus and Pseudomonas aeruginosa was lower than the observed for their pure polymer matrix.

Behavior and Inactivation of Enterotoxin-Positive Clostridium perfringens in Pork Picadillo and Tamales Filled with Pork Picadillo under Different Cooking, Storage, and Reheating Conditions.

This study analyzed the behavior of Clostridium perfringens in individual ingredients and tamales containing different pathogen concentrations upon exposure to different temperatures and methods of cooking, storage, and reheating. In ground pork, C. perfringens cells were inactivated when exposed to 95°C for 30 min. Three lots of picadillo inoculated with 0, 3, and 5 log CFU/g C. perfringens cells, respectively, were exposed to different storage temperatures. At 20°C, cell counts increased 1 log in all lots, whereas at 8°C, counts decreased by 2 log. Four lots of tamales prepared with picadillo inoculated with 0, 2, 3, and 7 log CFU/g prior to the final cooking step exhibited no surviving cells (91°C for 90, 45, or 35 min). Four lots of tamales were inoculated after cooking with concentrations of 0, 0.6, 4, and 6 log CFU/g of the pathogen and then stored at different temperatures. In these preparations, after 24 h at 20°C, the count increased by 1.4, 1.7, and 1.8 log in the tamales inoculated with 0.6, 4, and 6 log inoculum, respectively. When they were stored at 8°C for 24 h, enumerations decreased to <1, 2.5, and 1.9 log in the tamales inoculated with 0.6, 4, and 6 log of C. perfringens cells, respectively. However, when the lots were exposed to 20°C and then 8°C, 0.8, 1.8, and 2.4 log changes were observed for the tamales inoculated with 0.6, 4, and 6 log, respectively. Microwaving, steaming, and frying to reheat tamales inoculated with 6 log CFU/g C. perfringens cells showed that the pathogen was inactivated after 2 min of exposure in the microwave and after 5 min of exposure to steam. In contrast, no inactivation was observed after 5 min of frying. The tamales inoculated with spores (7 log most probable number [MPN]/g) showed a decrease of 2 log after steaming or frying, and no survival was observed after microwaving. Tamales inoculated with spores (7 log MPN/g) after cooking were susceptible to microwaves, but 2.4 and 255 MPN/g remained after frying and steaming, respectively.

Microbiological safety of domestic refrigerators and the dishcloths used to clean them in Guadalajara, Jalisco, Mexico.

Household refrigerators are a potential pathogen contamination source for foods. An evaluation of the microbiological safety of 200 refrigerators in Guadalajara, Jalisco, Mexico, was made by visual inspection, ATP-bioluminescence levels, indicator microorganisms including Escherichia coli and Staphylococcus aureus, and the presence of Listeria monocytogenes and Salmonella. Additionally, interviews of the owners of the refrigerators were carried out to determine relationships between food storage practices, demographic aspects, and microbiological status. Dishcloths used to clean refrigerators were also analyzed. Operational conditions (cleanliness, fullness, organization, frequency of cleaning, and temperature) were evaluated by trained observers. Results showed deficient cleanliness in 55% of refrigerators, 22% were completely full, 43% very disorganized, 28% were usually cleaned only once in 3 to 6 months, and 53% had internal temperatures >7.1°C. ATP-bioluminescence levels were >300 relative light units on 67 and 74% of shelves and drawers, respectively, indicating that surfaces were dirty according to the luminometer manufacturer. Psychrotrophic aerobic bacteria counts on shelves, drawers, and dishcloths were 6.3, 5.2, and 6.3 log CFU/cm(2); for coliform bacteria, 5.2, 3.9, and 4.7 CFU/cm(2); for E. coli, 3.7, 3.5, and 4.8 CFU/cm(2); and for Staphylococcus aureus, 2.1, 2.5, and 2.3 CFU/cm(2), respectively. L. monocytogenes and Salmonella were isolated from 59.5, 20.5, and 17% and 32.5, 8.0 and 12.5% of shelves, drawers, and dishcloths, respectively. Four Salmonella serotypes and nine serogroups (partially serotyped isolates) were identified. The most prevalent were Salmonella Anatum (39.5%), Salmonella group E1 (19.7%), and Salmonella group E1 monophasic (12.5%). Operational conditions and microbiological status were clearly deficient in sampled refrigerators, highlighting the consequent risk of foodborne disease among users. Educational programs are needed to improve the domestic food safety in Mexico.

Frequency of Salmonella and Listeria monocytogenes in five commercial brands of chicken eggs using a combined method of enrichment and nested-PCR.

Eggs or egg-based foods, either raw or undercooked, have been identified as vehicles of Salmonella outbreaks. The low numbers of Salmonella organisms in eggs makes it difficult to detect them in frequency studies. The nested-PCR (n-PCR) technique shows more sensitivity and specificity than bacteriological culture methods (BCMs). A preenrichment method followed by enrichment and n-PCR is a good alternative for the investigation of Salmonella and Listeria monocytogenes in eggs. A total of 2,650 chicken eggs representing five commercial brands were purchased from 10 grocery stores. Ten eggs of each brand were combined in order to obtain 265 pooled samples (53 per brand). The shells and yolks of 100 pooled samples were analyzed for Salmonella, while the shells of 65 pooled samples were analyzed for L. monocytogenes, using BCM and a combined method of enrichment and n-PCR (CM-n-PCR). Sixteen eggshell pooled samples tested positive for Salmonella by CM-n-PCR, compared with only two by BCM. Three egg yolk pooled samples tested positive for this pathogen by CM-n-PCR; none tested positive by BCM. Three eggshell pooled samples tested positive for L. monocytogenes by CM-n-PCR and none by BCM. In Mexico, as in other countries, official methods for detection of Salmonella and L. monocytogenes in foods are based on standard bacteriological culture techniques. The inclusion of more sensitive methods such as the one used in the present investigation would increase the probability of detecting positive samples, particularly in those foods in which a very low number of cells is expected.

Incidence of Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Staphylococcal enterotoxin in two types of Mexican fresh cheeses.

Handcrafted fresh cheeses are popular among consumers in Mexico. However, unsafe raw materials and inadequate food safety practices during cheese manufacture and preservation make them a potential public health risk. The incidence of Salmonella, Listeria, Escherichia coli O157:H7, and staphylococcal enterotoxin was analyzed in two types of fresh cheese (panela and adobera) commonly marketed in Mexico. A total of 200 samples, 100 panela and 100 adobera, were acquired from 100 wholesale milk product distributors who supply small retailers in the Guadalajara metropolitan area, Jalisco State, Mexico. Pathogens were identified using culture and immunoassay (miniVidas) methods. The presence of staphylococcal enterotoxin was determined by an immunoassay method. Of the 200 analyzed samples, 92 were positive for at least one of the pathogens. The incidence in the panela samples was 56%: 34% Salmonella, 16% E. coli O157:H7, and 6% L. monocytogenes. In the adobera samples, incidence was 36%: 20% Salmonella, 4% E. coli O157:H7, and 12% L. monocytogenes. Staphylococcal enterotoxin was not detected in any of the 200 samples. Choice of technique had no effect on detection of pathogen incidence, although the immunoassay method identified more Salmonella serotypes than the culture method. Handcrafted panela and adobera fresh cheeses in Mexico frequently contain pathogenic bacteria and therefore pose a public health risk.

Isolation and characterization of Shiga toxin-producing Escherichia coli O157:H7 and non-O157 from beef carcasses at a slaughter plant in Mexico.

The contamination of beef carcasses with Shiga toxin-producing O157:H7 and non-O157 Escherichia coli (STEC) obtained from a slaughter plant in Guadalajara, Mexico was investigated. A total of 258 beef carcasses were sampled during a 12-month period. All samples were assayed for STEC by selective enrichment in modified tryptone soy broth supplemented with cefixime, cefsulodin and vancomycin, followed by plating on Sorbitol MacConkey Agar supplemented with cefixime and tellurite (CT-SMAC). Simultaneously, all samples were assayed by immunomagnetic separation (IMS) and plated on CT-SMAC and CHROMagar. The presence of the stx1, stx2, eaeA and hly933 genes, recognized as major virulence factors of STEC, was tested for O157:H7 and non-O157 E. coli isolates by multiplex polymerase chain reaction (PCR). STEC was detected in two (0.8%) samples. One of these STEC isolates corresponded to the serotype O157:H7 showing stx2, eaeA and hyl933 genes. The other isolate corresponded to non-O157 STEC and only had the stx1 gene. Thirteen carcasses (5%) were positive for nonmotile E. coli O157 and 7 (2.7%) were positive for E. coli O157:H7. The presence of O157:H7 and non-O157 STEC on beef carcasses in this slaughter plant in Guadalajara, Mexico, emphasizes the importance of implementing the Hazard Analysis and Critical Control Point (HACCP) system, as well as the need for implementing, evaluating, and validating antimicrobial interventions to reduce the presence of potential pathogenic microorganisms.

Salmonella and Shigella in freshly squeezed orange juice, fresh oranges, and wiping cloths collected from public markets and street booths in Guadalajara, Mexico: incidence and comparison of analytical routes.

A survey of the presence of Salmonella and Shigella in freshly squeezed orange juice and related samples was conducted in Guadalajara, Mexico. One hundred samples of freshly squeezed orange juice were collected from 49 street booths and 51 small food service establishments. In addition, 75 fresh orange samples, each consisting of five orange units, and 75 wiping cloths were collected from the same establishments from which juice had been collected. Salmonella was isolated from 14, 20, and 23% of samples of orange juice, orange surfaces, and wiping cloths collected from street vendors, while Shigella was isolated from 6, 17, and 5% of these samples. In general, the frequency of isolation of these pathogens in samples from juice serving establishments at public markets was significantly lower than that found among street vendors (P < 0.05). Salmonella enterica serotypes Agona, Typhimurium, and Anatum were found in orange juice, fresh oranges, and wiping cloth samples, while serotype Mexico was found on fresh oranges and in wiping cloths and serotypes Muenchen and Panama were found only in wiping cloth samples. Regarding Shigella species, Shigella sonnei was found in all three types of sample tested; Shigella dysenteriae was found in juice and orange samples, Shigella boydii in orange and wiping cloth samples, and Shigella flexneri on oranges only. Thirty-one percent and 39% of the juice samples showed aerobic plate counts of > or = 5.0 log CFU/ml and Escherichia coli counts of > 3.0 log CFU/ml, respectively. These high counts may indicate poor sanitation and potential exposure to fecal contamination either in the raw materials or during the orange-crushing and juice-serving process. These data may be useful for a further risk assessment of Salmonella or Shigella in unpasteurized, freshly squeezed juice.

Spread of bacterial pathogens during preparation of freshly squeezed orange juice.

To study the potential of three bacterial pathogens to cross-contaminate orange juice during extraction, normal operation conditions during juice preparation at food service establishments were simulated. The spread of Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes from inoculated oranges to work surfaces and to the final product was determined. The transference of these three bacterial pathogens to orange juice made from uninoculated oranges with the use of contaminated utensils was also studied. Fresh oranges were inoculated with a marker strain of rifampicin-resistant Salmonella Typhimurium, E. coli O157:H7, or L. monocytogenes. Final pathogen levels in juice were compared as a function of the use of electric or mechanical juice extractors to squeeze orange juice from inoculated oranges. Pathogen populations on different contact surfaces during orange juice extraction were determined on sulfite-phenol red-rifampicin plates for Salmonella Typhimurium and E. coli O157:H7 and on tryptic soy agar supplemented with 0.1 g of rifampicin per liter for L. monocytogenes. After inoculation, the average pathogen counts for the orange rind surface were 2.3 log10 CFU/cm2 for Salmonella Typhimurium, 3.6 log10 CFU/cm2 for E. coli O157:H7, and 4.4 log10 CFU/cm2 for L. monocytogenes. This contamination was spread over all utensils used in orange juice squeezing. Mean pathogen counts for the cutting board, the knife, and the extractor ranged from -0.3 to 2.1 log10 CFU/cm2, and the juice contained 1.0 log10 CFU of Salmonella Typhimurium per ml, 2.3 log10 CFU of E. coli O157:H7 per ml, and 2.7 log10 CFU of L. monocytogenes per ml. Contact with contaminated surfaces resulted in the presence of all pathogens in orange juice made from uninoculated oranges. These results give emphasis to the importance of fresh oranges as a source of pathogens in orange juice.

Survival of Vibrio cholerae O1 in ceviche and its reduction by heat pretreatment of raw ingredients.

The survival of Vibrio cholerae O1 serotypes Inaba and Ogawa was determined in ceviche prepared from inoculated ground fish. Ground mackerel purchased from a seafood distribution center was inoculated with V. cholerae and stored at 8 or 20 degrees C. Counts of V. cholerae decreased in 2.6 to 2.7 log10 CFU/g during 96 h of storage at 8 degrees C or 2.5 to 2.6 log10 CFU/g during 24 h at 20 degrees C. Survival studies indicated that serotype Inaba decreased its number following a linear or retarded trend, whereas serotype Ogawa followed an accelerated death trend. No effect of the initial level of inoculum was observed. Odor scores of ceviche indicated that this food became marginally acceptable within as little as 48 h of storage at 8 degrees C or 3 h at 20 degrees C and were related to total volatile nitrogen values but not to aerobic plate counts, pH, or coliform counts. A heat pretreatment that consisted of stirring 100 g of inoculated ground fish into 40 ml of boiling water produced an 8-log reduction of V. cholerae within 3 min without affecting the color, odor, or flavor of ceviche prepared with such pretreated fish. According to this study, V. cholerae present in contaminated ceviche will likely survive longer than the shelf life of this food. Preheating the ground raw fish used for preparing ceviche for 3 min should effectively eliminate V. cholerae O1, providing science-based conditions for implementing a critical control point if a hazard analysis critical control point plan were to be developed for preparation of ceviche.

[Contamination of fish ceviche by Salmonella in Guadalajara++, Jalisco, Mexico]. / Contaminación del ceviche de pescado por Salmonella en Guadalajara, Jalisco, México.

This study was done to find out the incidence of Salmonella contamination of fish prepared and sold in 89 fixed and mobile food-vending establishments in the city of Guadalajara, Jalisco, Mexico. The pH of the samples analyzed varied between 3.8 and 5.2 (median = 4.55), and their temperature ranged from 9 to 29 degree C. Of the 221 samples studied (20 g each), 16% were positive for Salmonella. The proportions positive in fixed and mobile establishments were, respectively, 12% and 20%. The positive percentages were higher during mild and hot periods than during cold weather. The preenrichment technique proved less efficient for isolating Salmonella than direct enrichment. Salmonella was isolated from two of eight samples with pH below 4.0. The results indicate that eating ceviche may pose a health risk, especially for persons whose resistance to food-transmitted enteropathogens is low. Therefore, it should be emphasized that lime juice does not guarantee the safety of ceviche.

[Incidence of Vibrio parahaemolyticus in raw fish, oysters, and shrimp]. / Incidencia de Vibrio parahaemolyticus en pescado, ostión y camarón crudos.

The incidence of Vibrio parahaemolyticus in fresh seafood sold in Guadalajara, was studied by two procedures. These two procedures were compared to choose a reliable technique when outbreaks of V. parahaemolyticus illness occur. For one year, 57 samples of fresh oysters, fish and shrimp were analyzed for mesophilic aerobic bacteria (MAB) content, V. parahaemolyticus and pH. Total volatile nitrogen (TVN) was also determined in samples of fish and shrimp. MAB were counted by the pour plate method, and TVN was determined by modified Conway's micro diffusion technique. V. parahaemolyticus was investigated in 20-g samples by enrichment in lauryl dextrose salt broth (LDSB), isolating on plates of thiosulfate citrate bile sucrose agar (TCBS) and bile salts No. 3 agar (BS No. 3), and by direct isolation on TCBS and BS No.3 agar plates. Vibrio was characterized by tests described in standard methods, based upon the halophilism of the organism. Global percent of Vibrio parahaemolyticus positive samples was 45.6%, being 71.4% in fish, 44.0% in oysters, and 27.6% in shrimp. The use of two techniques enhanced the ability to recover the vibrio. There was a greater number of positives during the warm months (p = 0.0038). Means of TVN and pH in both positive and negative samples were not significantly different. Means of MAB counts were similar either in positive or negative samples.