Virulence and antimicrobial resistance profiles of Salmonella enterica isolated from foods, humans, and the environment in Mexico.

Salmonella enterica genotypic and phenotypic characteristics play an important role in its pathogenesis, which could be influenced by its origin. This study evaluated the association among the antimicrobial resistance, virulence, and origin of circulating S. enterica strains in Mexico, isolated from foods, humans, and the environment. The antimicrobial susceptibility to fourteen antibiotics by the Kirby-Bauer method (n = 117), and the presence of thirteen virulence genes by multiplex PCR (n = 153) and by sequence alignments (n = 2963) were evaluated. In addition, a set of S. enterica isolates from Mexico (n = 344) previously characterized according to their genotypic and phenotypic print was included to increase the coverage of the association analysis. Strains with the presence of sopE and strains with the absence of sspH1 were significantly associated with multidrug-resistant (MDR) phenotypes (p < 0.05). The origin of the strains had significant associations with the antimicrobial profiles and some virulence genes (hilA, orgA, sifA, ssaQ, sseL, sspH1, pefA, and spvC) (p < 0.05). Animal-origin food isolates showed the highest frequency of MDR (57.2 %), followed by human isolates (30.0 %). Also, sspH1, pefA, and spvC were found in major frequency in human (32.4 %, 31.0 %, 31.7 %) and animal-origin foods (41.6 %, 10.6 %, 10.6 %) isolates. The findings highlighted that antimicrobial profiles and specific virulence genes of S. enterica strains are related to their origin. Similar genotypic and phenotypic characteristics between human and animal-origin foods isolates were found, suggesting that animal-origin foods isolates are the most responsible for human cases. The revealed associations can be used to improve risk estimation assessments in national food safety surveillance programs.

Attachment and Survival of Salmonella enterica and Listeria monocytogenes on Tomatoes (Solanum lycopersicum) as Affected by Relative Humidity, Temperature, and Storage Time.

ABSTRACT: Tomatoes (Solanum lycopersicum) are one of the most commonly consumed fruits worldwide. The fruit can become contaminated with Salmonella and Listeria monocytogenes at various stages of the production and supply chain, and these pathogens may survive under various storage conditions. The effects of relative humidity, temperature, and duration of storage on the attachment and survival of both pathogens on the surface of tomatoes were investigated. Fresh whole Roma tomatoes were inoculated with a cocktail of Salmonella or L. monocytogenes strains and stored at 5, 12, 25, 30, or 35°C for up to 10 days. Every day during storage, relative humidity and temperature were measured and tomatoes were removed to enumerate pathogens cells that were loosely attached (LA; cells were detached from the tomato surface by rinsing) and strongly attached (SA; sonication was required to detach cells from the tomato surface). The attachment strength (SR) was calculated to express the proportion of surviving SA cells on the tomato surface. The initial levels of Salmonella and L. monocytogenes on the tomato surface after inoculation were 6.6 and 6.5 log CFU per tomato for LA cells and 5.1 and 5.6 log CFU per tomato for SA cells, respectively. For both pathogens, the LA levels were higher (P < 0.05) than the SA levels. The LA and SA levels differed significantly as a function of temperature, relative humidity, and duration of storage. The SR for Salmonella was affected by storage time but not temperature, whereas the SR for L. monocytogenes was affected by storage time and temperature and relative humidity (P < 0.05). An understanding of the attachment and survival of Salmonella and L. monocytogenes on tomatoes stored under various temperature conditions may be useful for preventing or reducing the establishment of pathogens and for designing improved decontamination methods.

Utilization of by-products of Hibiscus sabdariffa L. as alternative sources for the extraction of high-quality pectin.

The by-products of Hibiscus sabdariffa L. (HsL), obtained after soaking or decoction of the calyces of Colima and Sudan cultivars, were used for pectin extraction. After soaking, the wastes of both cultivars gave higher yields of pectin than those obtained by decoction. The pectin of the wastes by soaking presented high methoxyl groups, galacturonic acid content, viscosity and gelling capacity. Pectin of this treatment also exhibited bands in the regions of 1750 cm-1 and 1630 cm-1 that represents the C=O stretching vibrations of methyl ester and the amounts and degree of esterification of the HsL pectin. Interestingly, the pectin retained the typical red color of fresh HsL calyces. The amounts of anthocyanins and ascorbic acid of pectin did not show effects against pathogenic microorganisms. Nonetheless, pectin of the Sudan HsL wastes obtained by soaking, exhibited higher properties than those of the citric pectin, thus, demonstrating its potential for industrial applications.

Simultaneous and individual quantitative estimation of Salmonella, Shigella and Listeria monocytogenes on inoculated Roma tomatoes (Lycopersicon esculentum var. Pyriforme) and Serrano peppers (Capsicum annuum) using an MPN technique.

Simultaneous and individual enumeration of Salmonella, Shigella and Listeria monocytogenes was compared on inoculated Roma tomatoes and Serrano peppers using an Most Probable Number (MPN) technique. Samples consisting of tomatoes (4 units) or peppers (8 units) were individually inoculated with a cocktail of three strains of Salmonella, Shigella or L. monocytogenes, or by simultaneous inoculation of three strains of each pathogen, at low (1.2-1.7 log CFU/sample) and high (2.2-2.7 log CFU/sample) inocula. Samples were analyzed by an MPN technique using universal pre-enrichment (UP) broth at 35 °C for 24 ±â€¯2 h. The UP tubes from each MPN series were transferred to enrichment and plating media following adequate conventional methods for isolating each pathogen. Data were analyzed using multifactorial analysis of variance (p < 0.05) and LSD multiple rang test. There were differences (p < 0.05) in recovery of simultaneous and individual bacteria inoculated (individual > simultaneous), type of bacteria (Salmonella > Shigella and L. monocytogenes), type of sample (UP broth > pepper and tomato), and inoculum level (high > low). The MPN technique was effective for Salmonella on both commodities. Shigella counts were higher on tomatoes compared to peppers, (p < 0.05), and for L. monocytogenes on peppers (p < 0.05).

Use of a novel medium, the Polymyxin Ceftazidime Oxford Medium, for isolation of Listeria monocytogenes from raw or non-pasteurized foods.

Polymyxin Ceftazidime Oxford Medium (PCOM), a novel selective and differential plating medium for Listeria monocytogenes was compared with Modified Oxford Agar (MOX) for efficacy to isolate L. monocytogenes and other Listeria spp. naturally present in non-pasteurized Mexican-style cheese (n = 50), non-pasteurized fresh squeezed orange juice (n = 50), raw beef chunks (n = 36), and fresh cabbage (n = 125). Samples were collected from retail markets and farms in Mexico and tested following the US Department of Agriculture enrichment technique. Listeria spp. were isolated from 23.4% of analyzed samples, and from those, 75.0% corresponded to raw beef chunks, 38.0% to non-pasteurized Mexican-style cheese, and 30.0% to fresh squeezed orange juice. No Listeria spp. were isolated from fresh cabbage samples. L. monocytogenes was recovered from 15.3% of food samples analyzed. Non-pasteurized Mexican-style cheese showed the highest proportion of L. monocytogenes positive samples (36.0%), followed by orange juice (26.0%) and raw beef (25.0%). The frequency of isolation of Listeria spp. and L. monocytogenes was not different (P > 0.05) between PCOM and MOX. The advantages of using PCOM when comparing to MOX, include the easier way to identify Listeria species, the lower cost per plate and the availability of its ingredients for Latin-American countries.

Quantitative distribution of Salmonella spp. and Escherichia coli on beef carcasses and raw beef at retail establishments.

Salmonella is a foodborne pathogen that commonly inhabits the gastrointestinal tract of a healthy feedlot cattle and can be transferred to the carcass surface during hide removal and evisceration procedures. Numerous investigations on Salmonella prevalence throughout different stages of the beef chain have been conducted. In contrast, limited studies are available on quantitative determinations of Salmonella at different steps in raw meat production. Quantitative data, particularly for pathogenic bacteria such as Salmonella are important for quantitative risk assessment. Salmonella spp. and Escherichia coli populations were enumerated on beef carcass samples collected at abattoirs and also in beef chunks and ground beef samples collected from butcher's shops at retail in Jalisco State, Mexico. Sponge samples from beef carcass sides (n=142) were collected immediately after final water wash and before chilling at three non-federally inspected abattoirs following USDA-FSIS sampling protocols. Beef chunks (n=84) and ground beef (n=65) samples were obtained from 86 butcher's shops. Salmonella enumeration was conducted by the Most Probable Number method and E. coli counts were determined using Petrifilm plates. Salmonella was isolated from 18% of beef carcasses, 39% of beef chunks and 71% of ground beef samples. Salmonella mean counts were 1.3±0.9 Log MPN/300 cm(2) on beef carcasses, 1.9±0.9 and 2.3±1.1 Log MPN/25 g in beef chunks and ground beef samples, respectively. Twenty-six Salmonella serotypes and 11 serogroups were identified among 432 isolates recovered. Salmonella typhimurium (14%), Salmonella sinstorf (12%) and S. Group E1 monophasic (10%) were the most frequent. Escherichia coli was present on 97, 84 and 100% of beef carcasses, beef chunks and ground beef samples, respectively. Escherichia coli mean counts were 3.2±0.7 Log CFU/300 cm(2), 3.9±1.1 and 4.5±1.2 Log CFU/25 g on beef carcasses, beef chunks and ground beef, respectively. Salmonella prevalence and mean counts found in raw beef were higher than previously reported in studies from other countries. The data collected in this study show a trend in the prevalence of Salmonella to be higher as meat processing is extended at retail. This, together with the diversity of serotypes found, indicates that raw meat is exposed to multiple contamination sources during slaughter and retail processing and highlights the necessity to implement Sanitation Standard Operating Procedures for those establishments. Finally, this study provides quantitative information for future risk assessments associated with the risk of human salmonellosis.

The Polymyxin Ceftazidime Oxford Medium as an alternative selective and differential medium for isolation of Listeria monocytogenes from raw or unpasteurized food.

The Polymyxin Ceftazidime Oxford Medium (PCOM) was developed to recover Listeria monocytogenes from raw or unpasteurized foods. It contains esculin-ferric ammonium citrate as indicator system for Listeria growth, and ceftazidime and polymyxin B as selective agents, which are available in several Latin American countries. Comparison of PCOM, Modified Oxford Medium (MOX) and Tryptic Soy agar with 0.6% yeast extract (TSAYE) indicated that both selective media were equally effective at recovering four individual strains of L. monocytogenes (Scott A, V7, California and broccoli), and a mixture of these strains (LMM) (P > 0.05). The ability of PCOM, MOX, TSAYE and TSAYE supplemented with 4% NaCl to recover heat, acid and freeze-damaged LMM was similar for all media (P > 0.05). The PCOM proved to be effective at isolating colonies of LMM from inoculated raw beef chunks, unpasteurized orange juice, cabbage, and Mexican-style cheese by direct plating and by the US Department of Agriculture's Food Safety and Inspection Service enrichment method. Differentiation of L. monocytogenes colonies was easier on PCOM than on MOX for foods with high levels of background microbiota. Based on the evaluations performed on foods naturally contaminated with L. monocytogenes, PCOM was a more economical alternative than MOX for selective and differential isolation of Listeria from raw or unpasteurized foods.

Survival and germination of Clostridium perfringens spores during heating and cooling of ground pork.

The effect of heating rate on the heat resistance, germination, and outgrowth of Clostridium perfringens spores during cooking of cured ground pork was investigated. Inoculated cured ground pork portions were heated from 20 to 75°C at a rate of 4, 8, or 12°C/h and then held at 75°C for 48 h. No significant differences (P > 0.05) in the heat resistance of C. perfringens spores were observed in cured ground pork heated at 4, 8, or 12°C/h. At heating rates of 8 and 12°C/h, no significant differences in the germination and outgrowth of spores were observed (P > 0.05). However, when pork was heated at 4°C/h, growth of C. perfringens occurred when the temperature of the product was between 44 and 56°C. In another set of experiments, the behavior of C. perfringens spores under temperature abuse conditions was studied in cured and noncured ground pork heated at 4°C/h and then cooled from 54.4 to 7.2°C within 20 h. Temperature abuse during cooling of noncured ground pork resulted in a 2.8-log CFU/g increase in C. perfringens. In cured ground pork, C. perfringens decreased by 1.1 log CFU/g during cooling from 54.4 to 36.3°C and then increased by 0.9 log CFU/g until the product reached 7.2°C. Even when the initial level of C. perfringens spores in cured ground pork was 5 log CFU/g, the final counts after abusive cooling did not exceed 3.4 log CFU/g. These results suggest that there is no risk associated with C. perfringens in cured pork products under the tested conditions.

Influence of transportation stress and animal temperament on fecal shedding of Escherichia coli O157:H7 in feedlot cattle.

To test the influence of transportation stress and temperament on shedding of Escherichia coli O157:H7, cattle (n=150) were classified at various stages of production as Excitable, Intermediate or Calm based on a variety of disposition scores. Presence of E. coli O157:H7 was determined by rectal swabs from live animals and from colons collected postmortem. Percentage of cattle shedding E. coli O157:H7 at arrival at the feedlot was approximately equal among temperament groups. Before shipment to the processing facility, a higher (P=0.03) proportion of cattle from the Calm group shed E. coli O157:H7 compared to the other temperament groups. When pooled across all sampling periods, cattle from the Calm group had a greater percentage test positive for E. coli O157:H7. Neither the acute stressor of transportation nor a more excitable temperament led to increased shedding of E. coli O157:H7 in cattle.

Effect of xylitol on adhesion of Salmonella Typhimurium and Escherichia coli O157:H7 to beef carcass surfaces.

Effects of 10% xylitol (a five-carbon sugar alcohol) on adhesion of Escherichia coli O157:H7 and Salmonella Typhimurium to meat surfaces were examined with three approaches. First, beef outside round was inoculated with rifampin-resistant E. coli O157:H7 and Salmonella Typhimurium dispersed in xylitol or peptone solution. Samples were rinsed with water or not rinsed in a 2 x 2 factorial arrangement. No interaction existed between inoculum and rinsing treatments (P > 0.84). Incubation in xylitol had minimal impact on pathogen adhesion (P > 0.76); however, rinsing reduced pathogen cell counts (P < 0.01). Second, meat samples were treated with water, xylitol, or no rinse; inoculated with pathogens dispersed in peptone solution (8.6 log CFU/ml for each pathogen); and then treated with water, xylitol, or no rinse in a 3 x 3 factorial arrangement. No interactions were observed (P > 0.50). Postinoculation rinsing reduced pathogen loads (P < 0.01) without difference between water and xylitol (P > 0.64). Third, carcass surfaces inoculated with pathogens (5.5 log CFU/cm2) were treated with 35 degrees C water wash, 2.5% L-lactic acid spray, 10% xylitol spray, lactic acid plus xylitol, or hot water plus xylitol. Lactic acid treatments reduced Salmonella Typhimurium at 0 h (P < 0.01) and 24 h (P < 0.02). Hot water treatments tended to reduce Salmonella Typhimurium at 0 h (P < 0.07). Xylitol did not reduce pathogens (P > 0.62) or increase effectiveness of other treatments. Xylitol does not influence E. coli O157:H7 and Salmonella Typhimurium adhesion to meat surfaces.

Biomechanical and microbiological changes in natural hog casings treated with ozone.

The objective of this study was to determine the biomechanical and microbiological effects of exposing natural hog casings to ozonated water ≈7mg/l for 0, 2 or 4h at 16°C. A total of 450 casing segments representing 10 hanks were used over five testing days and arranged in a randomized block split-plot design. For each treatment, pH, temperature, actual ozone concentration, bursting strength, maximum rupture force, and L(∗), a(∗) and b(∗) color space values were determined. The bursting strength and the maximum rupture force values suggested that casings can be treated by ozone up to 2h without deterioration. After ozone treatments, changes in L(∗), a(∗) and b(∗) color space values made the casings appear lighter than the control samples. Microbiological studies showed that 1 and 2h ozonation reduced counts of Escherichia coli biotype I, which expressed green fluorescent protein, by 0.4 and 0.6log(10)CFU/25.4cm casing, respectively.

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.

Levels and enterotoxigenicity of Clostridium perfringens in pozole, tamales, and birria.

A quantitative survey of Clostridium perfringens in typical foods served at local restaurants was conducted for 18 months in Guadalajara, Mexico. A total of 151 samples, including goat's birria (50), pozole (50), and beef tamales (51), were collected from small restaurants in Guadalajara. Samples were tested for C. perfringens by the most probable number (MPN) method and for mesophilic aerobic plate counts (MAPCs) and coliform, yeast, and mold counts by plate count methods. Isolates confirmed as C. perfringens were further sporulated and tested for cytotoxic or cytotonic effect against Vero cells as an indication of enterotoxin production. C. perfringens was detected in 78 (52%) of all samples at concentrations that ranged from 2.3 to 5.4 log MPN/g. Average MAPCs were 1.3 to 2.7 log CFU/g, depending on the type of dish. Coliform counts ranged from less than 1.0 to 1.5 CFU/g, and yeast and mold counts were less than 1.0 log CFU/g in all cases. A total of 118 isolates of C. perfringens were tested for enterotoxic effect on Vero cells; 82 (70%) showed activity against Vero cells. Of them, 31 isolates induced cell lysis, indicating cytotoxic effect; 41 induced cell elongation, indicating cytotonic effect; and 10 produced both cytotoxic and cytotonic effect. Dilution of the bacterial filtrates that were still producing an effect on Vero cells ranged from 1:80 to 1:5,120. These results underscore the importance of determining enterotoxigenicity when testing for C. perfringens in foods.