Diarrheagenic Escherichia coli with Multidrug Resistance in Cattle from Mexico.

Mexico is an important producer/exporter of cattle and cattle products. In the last decade, an increase in antibiotic resistance in E. coli pathotype strains from livestock environments has been reported. This study aimed to determine the prevalence and antibiotic resistance profiles of E. coli pathotype strains from the feces of beef or dairy cattle reared in the states of Aguascalientes (AG, central) and Nuevo Leon (NL, northeastern) in Mexico. One hundred and ten fecal samples were collected (beef cattle-AG = 30; dairy cattle-AG = 20; beef cattle-NL = 30; dairy cattle-NL = 30). From these, E. coli was isolated using selective/differential media and confirmed on chromogenic media. Multiplex PCR was used to identify diarrheagenic E. coli, and the Kirby-Bauer technique was used to determine the antimicrobial susceptibilities. All the animals harbored E. coli, and pathotypes were found in 34 animals from both, beef and dairy cattle, mainly from Aguascalientes. Of the positive samples, 31 harbored a single E. coli pathotype, whereas three samples harbored two different pathotypes; EHEC was the most prevalent, followed by EPEC, ETEC, and EIEC or the combination of two of them in some samples. Most pathotype strains (19/37) were isolated from beef cattle. Neither the animals' productive purpose (beef or dairy cattle) (r = 0.155) nor the geographic regions (Aguascalientes or Nuevo Leon) (r = -0.066) had a strong positive correlation with the number of E. coli pathotype strains. However, animals reared in Aguascalientes had up to 8.5-fold higher risk of harboring E. coli pathotype strains than those reared in Nuevo Leon. All pathotype strains were resistant to erythromycin, tetracycline, and trimethoprim/sulfamethoxazole, and all dairy cattle pathotype strains were further resistant to five ß-lactams (χ2, P = 0.017). The existence of these pathotypes and multidrug-resistant pathogens in the food chain is a risk to public health.

Foodborne Viruses and Somatic Coliphages Occurrence in Fresh Produce at Retail from Northern Mexico.

Foodborne disease outbreaks linked to consumption of vegetables have been often attributed to human enteric viruses, such as Norovirus (NoV), Hepatitis A virus (HAV), and Rotavirus (RoV). Information about the occurrence of these viruses is scarce in many fresh-producing countries. Viral contamination detection of indicators, such as somatic coliphages, could indirectly reflect the presence of viral pathogens, being a valuable tool for better viral risk assessment in food industry. This study aimed to establish the occurrence and correlation of foodborne viruses and somatic coliphages in leafy greens in northern Mexico. A total of 320 vegetable samples were collected, resulting in 80 composite rinses, 40 of lettuce and 40 of parsley. Somatic coliphages were determined using the EPA 1602 method, while foodborne viruses (HAV, RoV, NoV GI, and GII) were determined by qPCR. The occurrence of RoV was 22.5% (9/40, mean 2.11 log gc/g) in lettuce and 20% (8/40, mean 1.91 log gc/g) in parsley. NoV and HAV were not detected in any samples. Somatic coliphages were present in all lettuce and parsley samples, with mean levels of 1.85 log PFU/100 ml and 2.28 log PFU/100 ml, respectively. Spearman analysis established the correlation of somatic coliphages and genomic copies of RoV, resulting in an r2 value of - 0.026 in lettuce and 0.349 in parsley. Although NoV or HAV were undetected in the samples, the presence of RoV is a matter of concern as leafy greens are usually eaten raw, which poses a potential risk of infection.

Boundaries That Prevent or May Lead Animals to be Reservoirs of Escherichia coli O104:H4.

Escherichia coli O104:H4, a hybrid serotype carrying virulence factors from enteroaggregative (EAEC) and Shiga toxin-producing (STEC) pathotypes, is the reported cause of a multicountry outbreak in 2011. Evaluation of potential routes of human contamination revealed that this strain is a foodborne pathogen. In contrast to STEC strains, whose main reservoir is cattle, serotype O104:H4 has not been commonly isolated from animals or related environments, suggesting an inability to naturally colonize the gut in hosts other than humans. However, contrary to this view, this strain has been shown to colonize the intestines of experimental animals in infectious studies. In this minireview, we provide a systematic summary of reports highlighting potential evolutionary changes that could facilitate the colonization of new reservoirs by these bacteria.

Computational assessment of phytochemicals of medicinal plants from Mexico as potential inhibitors of Salmonella enterica efflux pump AcrB protein.

The AcrAB-TolC efflux pump (EP) confers multidrug resistance to Salmonella enterica, a major etiological agent of foodborne infections. Phytochemicals that inhibit the functions of AcrAB-TolC EP present ideal candidates for reversal of antibiotic resistance. Progressive technological advancements, have facilitated the development of computational methods that offer a rapid low-cost approach to screen and identify phytochemicals with inhibitory potential against EP. In this study, 71 phytochemicals derived from plants used for medicinal purposes in Mexico were screened for their potential as inhibitors of Salmonella AcrB protein using in silico approaches including molecular docking and molecular dynamics (MD) simulation. Consequently, naringenin, 5-methoxypsoralen, and licarin A were identified as candidate inhibitors of AcrB protein. The three phytochemicals bound distal/deep pocket (DP) and hydrophobic trap (HPT) residues of AcrB protein critical for interactions with inhibitors, with estimated binding free energies of -95.5 kJ/mol, -97.4 kJ/mol, and -143.8 kJ/mol for naringenin, 5-methoxypsoralen, and licarin A, respectively. Data from the 50 ns MD simulation study revealed stability of the protein-ligand complex and alterations in the AcrB protein DP conformation upon binding of phytochemicals to the DP and HPT regions. Based on the estimated binding free energy and interactions with three out of five residues lining the hydrophobic trap, licarin A demonstrated the highest inhibitory potential, supporting its further application as a candidate for overcoming drug resistance in pathogens. Communicated by Ramaswamy H. Sarma.

Epigallocatechin gallate and Lactobacillus plantarum culture supernatants exert bactericidal activity and reduce biofilm formation in Clostridium perfringens.

Clostridium perfringens forms biofilms and spores that are a source of food contamination. In this study, the antibacterial activities of Lactobacillus plantarum culture supernatants (LP-S), LP-S fractions, and the plant-derived compound epigallocatechin gallate (EG) were evaluated. Specifically, their effects on the viability and biofilm-forming ability of C. perfringens were assessed. Moreover, the expression of quorum sensing-regulated genes associated with the pathogenesis of this microorganism and that of genes involved in biofilm formation was also investigated. The results showed that both EG and the LP-S exerted bactericidal activity against all C. perfringens strains tested. The minimal bactericidal concentration (MBC) of EG was 75 µg/mL for all strains but ranged from 61 to 121 µg of total protein per mL for LP-S. EG exerted only minor effects on biofilm formation, whereas LP-S, particularly its 10 and 30 K fractions, significantly reduced the biofilm-forming ability of all the strains. The antibiofilm activity of LP-S was lost following preincubation with proteases, suggesting that it was mediated by a proteinaceous molecule. The treatment of C. perfringens with either EG or LP-S did not change the transcript levels of two CpAL (C. perfringens quorum-sensing Agr-like system)-related genes, agrB and agrD, which are known to be involved in the regulation of biofilms, suggesting that LP-S exerted its biofilm inhibitory activity downstream of CpAL signaling. In summary, we demonstrated the bactericidal activity of EG and LP-S against C. perfringens and antibiofilm activity of LP-S at a subinhibitory dose. Our results suggested that these compounds can be further explored for food safety applications to control agents such as C. perfringens.

Natural and synthetic antimicrobials reduce adherence of enteroaggregative and enterohemorrhagic Escherichia coli to epithelial cells.

Adherence of bacteria to the human intestinal mucosa can facilitate their internalization and the development of pathological processes. Escherichia coli O104:H4 is considered a hybrid strain (enteroaggregative hemorrhagic E. coli [EAHEC]), sharing virulence factors found in enterohemorrhagic (EHEC), and enteroaggregative (EAEC) E. coli pathotypes. The objective of this study was to analyze the effects of natural and synthetic antimicrobials (carvacrol, oregano extract, brazilin, palo de Brasil extract, and rifaximin) on the adherence of EHEC O157:H7, EAEC 042, and EAHEC O104:H4 to HEp-2 cells and to assess the expression of various genes involved in this process. Two concentrations of each antimicrobial that did not affect (p≤0.05) bacterial viability or damage the bacterial membrane integrity were used. Assays were conducted to determine whether the antimicrobials alter adhesion by affecting bacteria and/or alter adhesion by affecting the HEp-2 cells, whether the antimicrobials could detach bacteria previously adhered to HEp-2 cells, and whether the antimicrobials could modify the adherence ability exhibited by the bacteria for several cycles of adhesion assays. Giemsa stain and qPCR were used to assess the adhesion pattern and gene expression, respectively. The results showed that the antimicrobials affected the adherence abilities of the bacteria, with carvacrol, oregano extract, and rifaximin reducing up to 65% (p≤0.05) of E. coli adhered to HEp-2 cells. Carvacrol (10 mg/ml) was the most active compound against EHAEC O104:H4, even altering its aggregative adhesion pattern. There were changes in the expression of adhesion-related genes (aggR, pic, aap, aggA, and eae) in the bacteria and oxidative stress-related genes (SOD1, SOD2, CAT, and GPx) in the HEp-2 cells. In general, we demonstrated that carvacrol, oregano extract, and rifaximin at sub-minimal bactericidal concentrations interfere with target sites in E. coli, reducing the adhesion efficiency.

Eugenol, citral, and hexanal, alone or in combination with heat, affect viability, biofilm formation, and swarming on Shiga-toxin-producing Escherichia coli.

Shiga-toxin-producing Escherichia coli strains are pathogenic for humans and cause mild to severe illnesses. In this study, the antimicrobial effect of citral, eugenol, and hexanal in combination with heat shock (HS) was evaluated in terms of the growth, biofilm formation, swarming, and expression of virulence genes of STEC serotypes (O157:H7, O103, O111, and O26). Eugenol was the most effective compound against the growth of E. coli strains (MBC = 0.58 to 0.73 mg/mL), followed by citral (MBC = 0.86 to 1.26 mg/mL) and hexanal (MBC = 2.24 to 2.52 mg/mL). Biofilm formation and swarming motility have great variability between STEC strains. Natural compounds-alone or combined with HS-inhibited biofilm formation; however, swarming motility was induced by most treatments. The expression of the studied genes during biofilm formation and swarming under natural antimicrobials was affected but not in a uniform pattern. These treatments could be used to control contamination of STEC and inhibit biofilm formation.

The Cantaloupe Farm Environment Has a Diverse Genetic Pool of Antibiotic-Resistance and Virulence Genes.

Cantaloupes contaminated with pathogens have led to many high-profile outbreaks and illnesses. Since bacterial virulence genes (VGs) can act in tandem with antibiotic-resistance and mobile genetic elements, there is a need to evaluate these gene reservoirs in fresh produce, such as cantaloupes. The goal of this study was to assess the distribution of antibiotic-resistance, virulence, and mobile genetic elements genes (MGEGs) in cantaloupe farm environments. A total of 200 samples from cantaloupe melons (n = 99), farm workers' hands (n = 66), and production water (n = 35) were collected in México. Each sample was assayed for the presence of 14 antibiotic-resistance genes, 15 VGs, and 5 MGEGs by polymerase chain reaction. Our results indicated that tetracycline (tetA and tetB) (18% of cantaloupe, 45% of hand samples) and sulfonamide (sul1) (30% of cantaloupe, 71% of hand samples) resistance genes were frequently detected. The colistin resistance gene (mcr1) was detected in 10% of cantaloupe and 23% of farm workers' hands. Among VGs, Salmonella genes invA and spiA were the most abundant. There was a significantly higher likelihood of detecting antibiotic-resistance, virulence, and MGEGs on hands compared with water samples. These results demonstrate a diverse pool of antibiotic-resistance and VGs in cantaloupe production.

Analysis of Bacterial Communities by 16S rRNA Gene Sequencing in a Melon-Producing Agro-environment.

Cantaloupe melons, which have been responsible of an increasing number of foodborne disease outbreaks, may become contaminated with microbial pathogens during production. However, little information is available on the microbial populations in the cantaloupe farm environment. The purpose of this work was to characterize the bacterial communities present on cantaloupe farms. Fruit, soil, and harvester hand rinsates were collected from two Mexican cantaloupe farms, each visited three times. Microbiome analysis was performed by sequencing 16sRNA and analyzed using qiime2 software. Correlations were determined between sample type and microbial populations. The α and ß diversity analysis identified 2777 sequences across all samples. The soil samples had the highest number and diversity of unique species (from 130 to 1329 OTUs); cantaloupe (from 112 to 205 OTUs), and hands (from 67 to 151 OTUs) had similar diversity. Collectively, Proteobacteria was the most abundant phyla (from 42 to 95%), followed by Firmicutes (1-47%), Actinobacteria (< 1 to 23%), and Bacteroidetes (< 1 to 4.8%). The most abundant genera were Acinetobacter (20-58%), Pseudomonas (14.5%), Erwinia (13%), and Exiguobacterium (6.3%). Genera with potential to be pathogenic included Bacillus (4%), Salmonella (0.85%), Escherichia-Shigella (0.38%), Staphylococcus (0.32%), Listeria (0.29%), Clostridium (0.28%), and Cronobacter (0.27%), which were found at lower frequencies. This study provides information on the cantaloupe production microbiome, which can inform future research into critical food safety issues such as antimicrobial resistance, virulence, and genomic epidemiology.

Norovirus Is the Most Frequent Cause of Diarrhea in Hospitalized Patients in Monterrey, Mexico.

Little information is available regarding the pathogens that cause diarrhea in hospitalized patients who also have various clinical problems. The purpose of this study was to determine the presence of pathogens in fecal samples of hospitalized patients all suffering diarrhea in addition to other problems in Mexico. Diarrheic stools from 240 patients were obtained in a third-level hospital in Monterrey, Mexico. PCR was used for the detection of Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., Aeromonas spp., Clostridioides difficile, and norovirus GI and GII. The presence of trophozoites, cysts of protozoa, eggs, and/or helminth larvae was determined by microscopic observation. Of the 240 patients analyzed, 40.4% presented at least one of the pathogens analyzed. Norovirus was the pathogen most frequently found (28.6%), followed by bacteria (11.7%), and parasites (8.3%). The majority of co-infections were parasites + norovirus, and bacteria + norovirus. Norovirus was detected mainly in children aged 0 to 10 years (9/15, 60%). Patients aged 0-20 years did not present co-infections. Entamoeba coli and Entamoeba histolytica were the most common parasites, (8/240), and Salmonella was the most prevalent bacteria (10/240). This information can help design specific strategies useful for hospitalized people with a compromised status.

Both Handwashing and an Alcohol-Based Hand Sanitizer Intervention Reduce Soil and Microbial Contamination on Farmworker Hands during Harvest, but Produce Type Matters.

Hand hygiene interventions are critical for reducing farmworker hand contamination and preventing the spread of produce-associated illness. Hand hygiene effectiveness may be produce-commodity specific, which could influence implementation strategies. This study's goal was to determine if produce commodity influences the ability of handwashing with soap and water or two-step alcohol-based hand sanitizer (ABHS) interventions to reduce soil and bacteria on farmworker hands. Farmworkers (n = 326) harvested produce (cantaloupe, jalapeño, and tomato) for 30 to 90 minutes before engaging in handwashing, two-step ABHS (jalapeño and cantaloupe), or no hand hygiene. Hands were rinsed to measure amounts of soil (absorbance at 600 nm) and indicator bacteria (coliforms, Enterococcus sp., generic Escherichia coli, and Bacteroidales universal [AllBac] and human-specific [BFD] 16S rRNA gene markers). Without hand hygiene, bacterial concentrations (0.88 to 5.1 log10 CFU/hand) on hands significantly differed by the produce commodity harvested. Moderate significant correlations (ρ = -0.41 to 0.56) between soil load and bacterial concentrations were observed. There were significant produce-commodity-specific differences in the ability of handwashing and two-step ABHS interventions to reduce soil (P < 0.0001), coliforms (P = 0.002), and Enterococcus sp. (P = 0.003), but not the Bacteroidales markers AllBac (P = 0.4) or BFD (P = 0.3). Contamination on hands of farmworkers who harvested cantaloupe was more difficult to remove. Overall, we found that a two-step ABHS intervention was similar to handwashing with soap and water at reducing bacteria on farmworker hands. In summary, produce commodity type should be considered when developing hand hygiene interventions on farms.IMPORTANCE This study demonstrated that the type of produce commodity handled influences the ability of handwashing with soap and water or a two-step alcohol-based hand sanitizer (ABHS) intervention to reduce soil and bacterial hand contamination. Handwashing with soap and water, as recommended by the FDA's Produce Safety Rule, when tested in three agricultural environments, does not always reduce bacterial loads. Consistent with past results, we found that the two-step ABHS method performed similarly to handwashing with soap and water but also does not always reduce bacterial loads in these contexts. Given the ease of use of the two-step ABHS method, which may increase compliance, the two-step ABHS method should be further evaluated and possibly considered for implementation in the agricultural environment. Taken together, these results provide important information on hand hygiene effectiveness in three agricultural contexts.

In-House Validation of a Rinse-Membrane Filtration Method for Processing Fresh Produce Samples for Downstream Cultural Detection of Salmonella, Escherichia coli O157:H7, and Listeria.

More efficient sampling and detection methods of pathogens on fresh produce are needed. The purpose of this study was to compare a novel rinse-membrane filtration method (RMFM) to a more traditional sponge rubbing or stomaching method in processing jalapeño peppers and cantaloupe samples for detection of Escherichia coli, Salmonella enterica, and Listeria monocytogenes. For jalapeño peppers inoculated with 106, 104, and 102 CFU of each pathogen and cantaloupes inoculated at 106 and 104 CFU, all pathogens were detected in all (100%) samples by RMFM at a 10-mL filtration volume, as well as by the stomacher and sponge rubbing methods. However, for cantaloupe inoculated at 102 CFU, detection differed by pathogen: S. enterica (20% RMFM, 60% stomacher, and 20% sponge), L. monocytogenes (40% RMFM, 60% stomacher, and 20% sponge), and E. coli O157:H7 (100% RMFM, 75% stomacher, and 75% sponge). When RMFM was compared with the other methods, in accordance with guidelines in the International Organization for Standardization 16140:2003 protocol, it produced values >95% in relative accuracy, relative specificity, and relative sensitivity. Overall, the RMFM performed similar to or better than the homogenization and sponge surface rubbing methods and is a good alternative for processing large numbers of produce samples for bacterial pathogen detection.

Phenolics with Bactericidal Activity Alter Motility and Biofilm Formation in Enterotoxigenic, Enteropathogenic, and Enterohemorrhagic Escherichia coli.

Most Escherichia coli strains are innocuous to human beings; however, some strains can cause diarrhea and are grouped into pathotypes. Since current trends promote the use of natural-origin compounds to control bacteria, in this study, the effects of the phenolic compounds (PCs) tannic acid (TA), gallic acid (GA), methyl gallate (MG), and epigallocatechin gallate (EG) on the growth, swarming motility, biofilm formation, and expression of selected virulence genes of three E. coli pathotypes (enteropathogenic Escherichia coli [EPEC], enterohemorrhagic Escherichia coli [EHEC], and enterotoxigenic Escherichia coli [ETEC]) were evaluated. Minimum bactericidal concentrations (MBCs) were determined by using microtiter plates, and the effects of sublethal PC concentrations on swarming motility were evaluated on Luria-Bertani agar. Biofilm formation was assessed in microtiter plates via crystal violet staining, and the expression levels of genes involved in biofilm formation (flhC, fliA, fliC, and csgA) and swarming motility (csgD and cyaA) were evaluated via quantitative PCR. All PC were bactericidal with minimal bactericidal concentrations ranging from 0.07 to 2.1 mg/mL. At concentrations lower than the MBC, PCs decreased swarming motility (14.8-100%). GA reduced biofilm formation in all of the tested strains; however, TA, MG, and EG induced biofilm formation in some strains at specific concentrations. TA induced the overexpression of csgA, csgD, and cyaA, whereas the other PCs did not have any effects or reduced their expression levels. The PCs tested in this study showed potential to control E. coli strains belonging to the EHEC, ETEC, and EPEC pathotypes by affecting their growth, swarming motility, and virulence gene expression; however, proper concentrations must be used to avoid the induction of undesirable virulence factor genes.

Persistence of Bacteroidales and other fecal indicator bacteria on inanimated materials, melon and tomato at various storage conditions.

In order to determine the microbial safety of produce, conventional fecal indicator bacteria (CFIB) such as Escherichia coli and Enterococcus are quantified as a standard practice. Bacteroidales are also fecal indicators mostly used for water samples; however, their use and persistence in foods has been rarely studied. In this study, persistence of both CFIB and genetic markers of host-specific Bacteroidales was determined in artificially contaminated materials and vegetables with different textured surfaces under different storage conditions. Sterile feces were contaminated with E. coli, E. faecalis, Bacteroidesthetaiotaomicron (human origin), and Bacteroidales from porcine and bovine origin. Feces were applied to filters of mixed cellulose esters and tomatoes (smooth surface) and flat cork coupons and melons (rough surface) and stored at 10 °C/95% relative humidity (RH) and 25 °C/65%RH for up to 25 days. Bacteroidales markers were analyzed by real-time polymerase chain reaction (qPCR), whereas CFIB were plated onto selective agars. CFIB detection on filters and cork surfaces declined over time. E. coli decreased 2.9 log CFU and 1.2 log CFU per filter and cork, respectively, at 10 °C/95%RH and 5.8 log CFU and 1.8 log CFU per filter and cork, respectively, at 25 °C/65%RH. E. faecalis decreased 1.9 log CFU on filters and 1.3 log CFU on cork at 10 °C/95%RH and 2.6 log CFU/filter and cork under both storage conditions. Although E. coli levels in tomatoes slightly increased during storage, the levels decreased by the end of the assays. However, CFIB levels in melons stored at 10 °C/95%RH increased after 20 days; when stored at 25 °C/65%RH, these levels increased after five days. Bacteroidales levels (universal and host-specific markers) in inanimated material and produce did not show significant differences (P ≤ 0.01) over time. Stability and persistence of Bacteroidales genetic markers make them superior to CFIB as markers and are alternatives for determining the risk of exposure to feces-contaminated produce.

Phylogroups, pathotypes, biofilm formation and antimicrobial resistance of Escherichia coli isolates in farms and packing facilities of tomato, jalapeño pepper and cantaloupe from Northern Mexico.

The most commonly used indicator of fecal contamination in fresh produce production and packing is Escherichia coli. In depth analysis of the prevalence and characteristics of naturally occurring E. coli strains in these environments is important because it can (1) serve as an indicator of sources of fecal contamination; and (2) provide information on strain pathogenicity, persistence, and other defining characteristics such as multidrug resistance. In this study, we analyzed 341 E. coli strains isolated from the jalapeño pepper, tomato and cantaloupe farm environments, in Northeast Mexico. Strains were isolated from produce, farmworkers' hands, soil and water. Pathotypes, genotypes, biofilm formation and antibiotic resistance were characterized. Phylogenetic subgroups and identification of diarrheagenic E. coli were determined by PCR; biofilm formation was quantified using a plate-based colorimetric method. Antibiotic resistance was analyzed by the Kirby Bauer diffusion disc method. Most isolates (N = 293, 86%) belonged to phylogenetic group A. Only four isolates (1.2%) were diarrheagenic: EPEC (N = 3) and ETEC (N = 1). Antibiotic resistance to tetracycline (23.2%) and ampicillin (19.9%) was high, and only 3.5% of the strains presented resistance to >5 antibiotics. Biofilms were produced by most strains (76%), among which 34.4% were categorized as high producers. The presence of antibiotic resistant E. coli strains that may contain gene markers for pathogenicity and which can form biofilms suggests potential health risks for consumers.

Microbiological Changes during Ripening of Chihuahua Cheese Manufactured with Raw Milk and Its Seasonal Variations.

Chihuahua cheese is a traditional cheese produced in Northwest Mexico that is consumed shortly after production. Cheeses prepared during autumn, winter and summer were collected from five dairies, and analyzed to determine seasonal influence on proximate analysis, texture profile and the microbiological dynamic during a ripening period of 270 days. Coliforms, coagulase-positive staphylococci, molds, yeast, as well as presumptive mesophilic lactobacilli, thermophilic lactobacilli, lactococci, thermophilic cocci and enterococci, were enumerated by plate count on selective agar. Manufacturing dairy had an effect on Chihuahua cheese composition and texture profile. Seasonality influence on the microbial dynamic was observed, since the highest initial counts of coliforms (5.14 log CFU/g), coagulase-positive staphylococci (4.13 log CFU/g) and mesophilic lactobacilli (7.86 log CFU/g) were detected on summer samples. Also, ripening time affected the survival of coliforms and presumptive lactococci after 270 days (1.24 and 5.89 log CFU/g respectively) while from day 90th, coagulase-positive staphylococci were absent. Microbial changes and seasonal influence provide information on the microbiota that can influence the sensorial characteristics of Chihuahua cheese.

Animals as sources of food-borne pathogens: A review.

Food-producing animals are the major reservoirs for many foodborne pathogens such as Campylobacter species, non-Typhi serotypes of Salmonella enterica, Shiga toxin-producing strains of Escherichia coli, and Listeria monocytogenes. The zoonotic potential of foodborne pathogens and their ability to produce toxins causing diseases or even death are sufficient to recognize the seriousness of the situation. This manuscript reviews the evidence that links animals as vehicles of the foodborne pathogens Salmonella, Campylobacter, Shiga toxigenic E. coli, and L. monocytogenes, their impact, and their current status. We conclude that these pathogenic bacteria will continue causing outbreaks and deaths throughout the world, because no effective interventions have eliminated them from animals and food.

Bacteroidales as Indicators and Source Trackers of Fecal Contamination in Tomatoes and Strawberries.

Most methods that investigate fecal contamination of vegetables do not address the origin of contamination. Because host-specific sequences are conserved in their genomes, bacteria of the order Bacteroidales are regarded as alternative indicators for tracking sources of contamination of produce. The objective of this study was to determine the efficacy of host-specific Bacteroidales markers to identify sources of fecal contamination and to determine whether detection of Bacteroidales markers correlated with traditional fecal indicator bacteria (FIB) in strawberries and tomatoes. Tomato and strawberry samples were artificially contaminated with human and animal feces, which contained 6 to 7 log CFU Bacteroidales per 100 mL and 3 to 6 log CFU/100 mL of the bacterial indicators Escherichia coli, total coliforms, and Enterococcus. FIB were enumerated by standard procedures. Universal and host-specific Bacteroidales markers were detected and quantified by quantitative PCR, and the detection range was 1.35 to 10.35 logarithmic gene copies, which corresponds to a limit of detection of two Bacteroidales cells. Few correlations between levels of Bacteroidales and levels of FIB were observed. For most of the contaminated tomato and strawberry samples, Bacteroidales levels were higher than FIB levels, and detection of FIB was highly variable. Detection of Bacteroidales markers was similar to total coliforms when ≥0.1 mg of feces was inoculated. These indicators were better than E. coli and Enterococcus for detection of fecal contamination in produce. The host-associated Bacteroidales markers were detected at an inoculum of 1 mg of feces per produce item (except those from bovine feces in strawberry). All of the host-associated Bacteroidales markers were detected at an inoculum of 10 mg of feces per produce item. Thus, Bacteroidales markers are promising tools to identify sources of fecal contamination; however, more research is required for their potential use to reduce the risks of contamination of produce.

Contamination of Fresh Produce by Microbial Indicators on Farms and in Packing Facilities: Elucidation of Environmental Routes.

To improve food safety on farms, it is critical to quantify the impact of environmental microbial contamination sources on fresh produce. However, studies are hampered by difficulties achieving study designs with powered sample sizes to elucidate relationships between environmental and produce contamination. Our goal was to quantify, in the agricultural production environment, the relationship between microbial contamination on hands, soil, and water and contamination on fresh produce. In 11 farms and packing facilities in northern Mexico, we applied a matched study design: composite samples (n = 636, equivalent to 11,046 units) of produce rinses were matched to water, soil, and worker hand rinses during two growing seasons. Microbial indicators (coliforms, Escherichia coli, Enterococcus spp., and somatic coliphage) were quantified from composite samples. Statistical measures of association and correlations were calculated through Spearman's correlation, linear regression, and logistic regression models. The concentrations of all microbial indicators were positively correlated between produce and hands (ρ range, 0.41 to 0.75; P < 0.01). When E. coli was present on hands, the handled produce was nine times more likely to contain E. coli (P < 0.05). Similarly, when coliphage was present on hands, the handled produce was eight times more likely to contain coliphage (P < 0.05). There were relatively low concentrations of indicators in soil and water samples, and a few sporadic significant associations were observed between contamination of soil and water and contamination of produce. This methodology provides a foundation for future field studies, and results highlight the need for interventions surrounding farmworker hygiene and sanitation to reduce microbial contamination of farmworkers' hands.IMPORTANCE This study of the relationships between microbes on produce and in the farm environment can be used to support the design of targeted interventions to prevent or reduce microbial contamination of fresh produce with associated reductions in foodborne illness.

Bacterial metabolites from intra- and inter-species influencing thermotolerance: the case of Bacillus cereus and Geobacillus stearothermophilus.

Bacterial metabolites with communicative functions could provide protection against stress conditions to members of the same species. Yet, information remains limited about protection provided by metabolites in Bacillus cereus and inter-species. This study investigated the effect of extracellular compounds derived from heat shocked (HS) and non-HS cultures of B. cereus and Geobacillus stearothermophilus on the thermotolerance of non-HS vegetative and sporulating B. cereus. Cultures of B. cereus and G. stearothermophilus were subjected to HS (42 or 65 °C respectively for 30 min) or non-HS treatments. Cells and supernatants were separated, mixed in a combined array, and then exposed to 50 °C for 60 min and viable cells determined. For spores, D values (85 and 95 °C) were evaluated after 120 h. In most cases, supernatants from HS B. cereus cultures added to non-HS B. cereus cells caused their thermotolerance to increase (D 50 12.2-51.9) when compared to supernatants from non-HS cultures (D 50 7.4-21.7). While the addition of supernatants from HS and non-HS G. stearothermophilus cultures caused the thermotolerance of non-HS cells from B. cereus to decrease initially (D 50 3.7-7.1), a subsequent increase was detected in most cases (D 50 18-97.7). In most cases, supernatants from sporulating G. stearothermophilus added to sporulating cells of B. cereus caused the thermotolerance of B. cereus 4810 spores to decline, whereas that of B. cereus 14579 increased. This study clearly shows that metabolites in supernatants from either the same or different species (such as G. stearothermophilus) influence the thermotolerance of B. cereus.