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.

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.

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.

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.

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.

Natural plant products inhibits growth and alters the swarming motility, biofilm formation, and expression of virulence genes in enteroaggregative and enterohemorrhagic Escherichia coli.

The purpose of this study was to determine the effects of plant products on the growth, swarming motility, biofilm formation and virulence gene expression in enterohemorrhagic Escherichia coli O157:H7 and enteroaggregative E. coli strain 042 and a strain of O104:H4 serotype. Extracts of Lippia graveolens and Haematoxylon brassiletto, and carvacrol, brazilin were tested by an antimicrobial microdilution method using citral and rifaximin as controls. All products showed bactericidal activity with minimal bactericidal concentrations ranging from 0.08 to 8.1 mg/ml. Swarming motility was determined in soft LB agar. Most compounds reduced swarming motility by 7%-100%; except carvacrol which promoted motility in two strains. Biofilm formation studies were done in microtiter plates. Rifaximin inhibited growth and reduced biofilm formation, but various concentrations of other compounds actually induced biofilm formation. Real time PCR showed that most compounds decreased stx2 expression. The expression of pic and rpoS in E. coli 042 were suppressed but in E. coli O104:H4 they varied depending on compounds. In conclusion, these extracts affect E. coli growth, swarming motility and virulence gene expression. Although these compounds were bactericidal for pathogenic E. coli, sublethal concentrations had varied effects on phenotypic and genotypic traits, and some increased virulence gene expression.

S-adenosyl-L-methionine modifies antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells.

AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression. METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. RESULTS: SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the transcriptional and translational level. CONCLUSION: A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.