Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment.

Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Effects of wild blueberry (Vaccinium angustifolium) pomace feeding on gut microbiota and blood metabolites in free-range pastured broiler chickens.

There is a need to develop cost-effective approaches to modulate gut microbiota, promote bird health, and prevent infections in pasture-raised broiler chickens. The present study evaluated the efficacy of organic wild blueberry (Vaccinium angustifolium) also called low-bush blueberry pomace (LBBP)-supplemented feed to modulate the chicken gut microbiota, and blood metabolites in order to improve bird health and productivity. Slow-growing broiler chickens were reared on pasture up to 64 D for sampling after 2 wk of treatment during brooding with 0, 1, and 2% LBBP in feed. Intestinal samples were collected at different time-points throughout the trial for bacterial culture and microbial community analysis by 16S rRNA gene sequencing using Illumina MiSeq. Blood sera were also analyzed for metabolites at each sampling time. Of the 14 bacterial phyla, the predominant taxa across all sampling time-points were Firmicutes, Proteobacteria, Bacteroidetes, and Tenericutes, representing >97% of all sequences. Bacteroidetes seemed to be replacing Firmicutes by LBBP supplementation, with the most noticeable effect at day 64 with 1% LBBP. LBBP inclusion enriched Lactobacillus, Bacteroides, and Bifidobacterium, while Escherichia coli, Clostridium_Clostridiaceae, Helicobacter, and Enterococcus showed higher abundances in control birds at the end of trial. Principal co-ordinate analysis showed a clear clustering of the intestinal samples from control and LBBP-treated groups at day 29. Application of LBBP resulted in a decrease (P < 0.05) in cholesterol at day 21, and an increase (P < 0.05) in high-density lipoprotein cholesterol in 14-day-old broilers. Higher (P < 0.05) levels of phosphorus, magnesium, and globulin at day 21 as well as iron and albumin at day 36 were also observed in 1% LBBP-fed birds. Despite limitations consisting essentially of low sampled birds for measurements, this study indicated that dietary supplementation of LBBP could positively influence gut microbiota and blood metabolites that may contribute to the overall health of pasture-raised broiler chickens.

Optimized extraction and characterization of antimicrobial phenolic compounds from mangosteen (Garcinia mangostana L.) cultivation and processing waste.

BACKGROUND: Applications for antimicrobials derived from the mangosteen (Garcinia mangostana L.) plant are presently restricted by high production costs. Extraction from cultivation or processing waste streams using a solvent-free approach could lessen to permit commercial applications in food processing and preservation. RESULTS: Phenolics were extracted from mangosteen bark, leaf and fruit pericarp in methanol and in water using response surface methodology to optimize recovery. Initial examination of antimicrobial effects revealed a lack of antimicrobial activity against fungi and weak activity against the Gram-negative bacteria Escherichia coli and Salmonella typhimurium. In contrast, extracts prepared from bark or fruit pericarp exhibited strong pH-dependent bacteriostatic and bactericidal effects against Listeria monocytogenes and Staphylococcus aureus. Activity was slightly weaker in aqueous extracts due to lower concentrations of tartaric acid esters and flavonols than in methanolic extracts. Measurement of propidium iodide uptake and ATP leakage indicated that the extracts induced damage to the membrane of Gram-positive bacteria. CONCLUSION: Extracts of mangosteen bark and fruit pericarp contain mixtures of phenolic compounds with activity against Gram-positive bacteria, notably Listeria monocytogenes. Extraction of phenolics from mangosteen waste could yield fractions for potential applications in the formulation of low-cost processing aids or sanitizers for the food industry.

Thermal tolerance and survival of Cronobacter sakazakii in powdered infant formula supplemented with vanillin, ethyl vanillin, and vanillic acid.

The thermal tolerance Cronobacter sakazakii was examined in sterile powdered infant formula (PIF) rehydrated at 58 °C in water or apple juice supplemented with vanillin, ethyl vanillin, or vanillic acid. All three compounds decreased thermal tolerance during-rehydration and the lowest decimal reduction time (D-value, 0.19 ± 0.01 min) was measured in PIF rehydrated in apple juice supplemented with 20 mM vanillic acid. At this level of supplementation no C. sakazakii were detected in PIF stored for 48 h at 10 and 24 h at 21 °C subsequent to a sublethal heat treatment. Thermal tolerance during rehydration and survival in reconstituted PIF were influenced by compound type, concentration, and temperature. Supplementation of PIF with vanillin, ethyl vanillin, or vanillic acid could enhance the safety of PIF or other dehydrated foods contaminated with C. sakazakii.

Characterization of antibiotic-resistant and potentially pathogenic Escherichia coli from soil fertilized with litter of broiler chickens fed antimicrobial-supplemented diets.

The objective of this study was to characterize antimicrobial resistance and virulence determinants of Escherichia coli from soil amended with litter from 36-day-old broiler chickens ( Gallus gallus domesticus ) fed with diets supplemented with a variety of antimicrobial agents. Soil samples were collected from plots before and periodically after litter application in August to measure E. coli numbers. A total of 295 E. coli were isolated from fertilized soil samples between August and March. Antibiotic susceptibility was determined by Sensititre, and polymerase chain reaction was performed to detect the presence of resistance and virulence genes. The results confirmed that E. coli survived and could be quantified by direct plate count for at least 7 months in soil following litter application in August. The effects of feed supplementation were observed on E. coli numbers in November and January. Among the 295 E. coli, the highest antibiotic resistance level was observed against tetracycline and ß-lactams associated mainly with the resistance genes tetB and bla(CMY-2), respectively. Significant treatment effects were observed for phylogenetic groups, antibiotic resistance profiles, and virulence gene frequencies. Serotyping, phylogenetic grouping, and pulsed-field gel electrophoresis confirmed that multiple-antibiotic-resistant and potentially pathogenic E. coli can survive in soil fertilized with litter for several months regardless of antimicrobials used in the feed.

Antimicrobial effect of natural preservatives in a cooked and acidified chicken meat model.

The inhibitory effect of Microgard 100, Microgard 300, nisin, Alta 2002, Perlac 1902, sodium lactate and essential oil of mustard on microorganisms experimentally inoculated was screened in an acidified chicken meat model (pH = 5.0) and stored for 2 weeks at a none restrictive growth temperature of 22 degrees C. All antimicrobials tested were used at the highest concentration recommended by their manufacturer. Sausage batter made with mechanically deboned chicken was inoculated with a mixed culture of Escherichia coli ATCC 25922, Brochothrix thermosphacta CRDAV452, and a protective culture Lactobacillus alimentarius BJ33 (FloraCan L-2). A final cell concentration of 3-4 log CFU g (-1) was targeted after cooking at a core temperature of 55 degrees C for each microorganism in order to assess cell count variation effectively. Composition, water activity (a(w)), pH and redox potential of the sausage model was also evaluated. The E. coli population decreased steadily during storage and was close or below detection level (< 1 log CFU g (-1)) for all treatments, including the control, after 14 days. Sodium lactate was most effective against B. thermosphacta; population was 4 log lower than the control after 14 days of storage. When essential oil of mustard was used, aerobic mesophilic bacteria and lactic acid bacteria were significantly lower than the control after 2 days of storage (P < or = 0.05). The other antimicrobial agents tested had no significant effect on the aerobic mesophilic bacteria, E. coli, B. thermosphacta and lactic acid bacteria counts, when compared to the control.