Fate of Escherichia coli O157:H7 and bacterial diversity in corn silage contaminated with the pathogen and treated with chemical or microbial additives

Inhibiting the growth of Escherichia coli O157:H7 (EC) in feeds may prevent the transmission or cycling of the pathogen on farms. The first objective of this study was to examine if addition of propionic acid or microbial inoculants would inhibit the growth of EC during ensiling, at silo opening, or after aerobic exposure. The second objective was to examine how additives affected the bacterial community composition in corn silage. Corn forage was harvested at approximately 35% dry matter, chopped to a theoretical length of cut of 10 mm, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1 × 10⁵ cfu/g of EC (ECCH); (3) EC and 1 × 10⁶ cfu/g of Lactobacillus plantarum (ECLP); (4) EC and 1 × 10⁶ cfu/g of Lactobacillus buchneri (ECLB); and (5) EC and 2.2 g/kg (fresh weight basis) of propionic acid, containing 99.5% of the acid (ECA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, and 120 d and analyzed for EC, pH, and organic acids. Samples from d 0 and 120 were also analyzed for chemical composition. Furthermore, samples from d 120 were analyzed for ammonia N, yeasts and molds, lactic acid bacteria, bacterial community composition, and aerobic stability. The pH of silages from all treatments decreased below 4 within 3 d of ensiling. Escherichia coli O157:H7 counts were below the detection limit in all silages after 7 d of ensiling. Treatment with L. buchneri and propionic acid resulted in fewer yeasts and greater aerobic stability compared with control, ECCH, and ECLP silages. Compared with the control, the diversity analysis revealed a less diverse bacterial community in the ECLP silage and greater abundance of Lactobacillus in the ECLP and ECA silages. The ECLB silage also contained greater abundance of Acinetobacter and Weissella than other silages. Subsamples of silages were reinoculated with 5 × 10⁵ cfu/g of EC either immediately after silo opening or after 168 h of aerobic exposure, and EC were enumerated after 6 or 24 h, respectively. All silages reinoculated with EC immediately after silo opening (120 h) had similar low pH values (<4.0) and EC counts were below the detection limit. The ECCH and ECLP silages reinoculated with EC after 168 h of aerobic exposure had relatively high pH values (>5.0) and EC counts (5.39 and 5.30 log cfu/g, respectively) 24 h later. However, those treated with L. buchneri or propionic acid had lower pH values (4.24 or 3.96, respectively) and lower EC counts (1.32 log cfu/g or none, respectively). During ensiling, EC was eliminated from all silages at pH below 4.0. During aerobic exposure, the growth of EC was reduced or prevented in silages that had been treated with L. buchneri or propionic acid at ensiling, respectively.     

Effect of applying inoculants with heterolactic or homolactic and heterolactic bacteria on the fermentation and quality of corn silage

This study examined the effect of applying different bacterial inoculants on the fermentation and quality of corn silage. Corn plants were harvested at 35% DM, chopped, and ensiled in 20-L mini silos after application of (1) deionized water (CON) or inoculants containing (2) 1 × 10⁵ cfu/g of Pediococcus pentosaceus 12455 and Propionibacteria freudenreichii (B2); (3) 4 × 10⁵ cfu/g of Lactobacillus buchneri 40788 (BUC); or (4) 1 × 10⁵ cfu/g of Pediococcus pentosaceus 12455 and 4 × 10⁵ cfu/g of L. buchneri 40788 (B500). Four replicates of each treatment were weighed into polyethylene bags within 20-L mini silos. Silos were stored for 575 d at ambient temperature (25°C) in a covered barn. After silos were opened, aerobic stability, chemical composition, and yeast and mold counts were determined. The DNA in treated and untreated silages was extracted using lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform and used as a template for a conventional PCR with primers designed on the 16S rRNA gene to detect the presence of L. buchneri in all silage samples. Acetic acid concentration was greater in B2 silages versus others (6.46 vs. 4.23% DM). Silages treated with BUC and B500 had lower pH and propionic acid concentration and greater lactic acid concentration than others. The B500 silage had the greatest lactic:acetic acid ratio (1.54 vs. 0.41), and only treatment with BUC reduced DM losses (5.0 vs. 14.3%). Yeast and mold counts were less than the threshold (10⁵) typically associated with silage spoilage and did not differ among treatments. Consequently, all silages were very stable (>250 h). Aerobic stability was not improved by any inoculant but was lower in B500 silages versus others (276 vs. 386 h). The conventional PCR confirmed the presence of similar populations of L. buchneri in all silages. This may have contributed to the prolonged aerobic stability of all silages.     

Effect of applying molasses or inoculants containing homofermentative or heterofermentative bacteria at two rates on the fermentation and aerobic stability of corn silage

This study determined how the fermentation and aerobic stability of corn silage are affected by treatment with molasses or 2 dual-purpose inoculants applied at or above the recommended rate. Corn forage (DeKalb 69-70) was harvested at 39% dry matter (DM) and ensiled after treatment with no additives (control, CON), molasses (MOL), Buchneri 500 inoculant, or Pioneer 11C33 inoculant. Molasses was applied at 3% of forage DM. Buchneri 500 was applied at the recommended rate of 8 mg/kg fresh forage to supply 1 × 10⁵ cfu/g of Pediococcus pentosaceus 12455 and 4 × 10⁵ cfu/g of Lactobacillus buchneri 40788 (BB) or at twice the recommended rate (DBB). Pioneer 11C33 inoculant was applied at the recommended rate of 1.1 mg/kg fresh forage to supply 1 × 10⁵ cfu/g of a mixture of Lactobacillus plantarum, L. buchneri, and Enteroccocus faecium (PN) or at twice the recommended rate (DPN). Each treatment was applied in quadruplicate and the treated forages were ensiled within 20-L mini silos for 135 d at 18 to 35°C. Molasses-treated silages had greater ash and starch concentrations than CON silages and greater lactate and ethanol concentrations than other silages. Like CON silages, MOL silages had high yeast counts (>10⁵ cfu/g); consequently, they deteriorated within 30 h as shown by temperature increase. Inoculant-treated silages had lower lactate to acetate ratios than CON or MOL silages largely because they had greater acetate concentrations. Consequently, all inoculant-treated silages had fewer yeasts (<10⁵ cfu/g) and were more stable (>30 h) than CON and MOL silages. When applied at recommended rates, PN and BB had similar effects on silage chemical composition, fermentation, fungal counts, and aerobic stability, except for a lower lactate concentration in PN silages. Concentrations of VFA, and NH₃-N, pH, and extent of aerobic stability were similar for PN, DPN, BB, and DBB silages. However, lactate concentration was greater in DPN than in PN. In conclusion, MOL application increased ethanol and lactate concentration and did not improve aerobic stability. Both dual-purpose inoculants made the fermentation more heterolactic and thereby improved the aerobic stability of corn silage. Doubling the rate of application of either inoculant did not further improve fermentation or aerobic stability.     

Effect of Lactobacillus buchneri LN4637 and Lactobacillus buchneri LN40177 on the aerobic stability, fermentation products, and microbial populations of corn silage under farm conditions

This study determined the efficacy of the use of 2 commercial inoculants containing Lactobacillus buchneri alone or in combination with homofermentative lactic acid bacteria in improving aerobic stability of corn silage stored in commercial farm silos in northern Italy. In the first survey, samples were collected from 10 farms that did not inoculate their silages and from 10 farms that applied a Pioneer 11A44 inoculant (L. buchneri strain LN4637; Pioneer Hi-Bred International, Des Moines, IA). In the second survey, corn silage samples were collected from 11 farms that did not inoculate their silages and from 11 farms that applied a Pioneer 11CFT inoculant (L. buchneri strain LN40177; Pioneer Hi-Bred International). Inoculants were applied directly through self-propelled forage harvesters, at the recommended rate of 1 g/t of fresh forage, to achieve a final application rate of 1.0 × 10⁵ cfu/g of L. buchneri. One corn bunker silo, which had been open for at least 10 d, was examined in detail on each farm. The silages inoculated with L. buchneri had lower concentrations of lactic acid, a lower lactic-to-acetic acid ratio, a lower yeast count, and higher aerobic stability compared with the untreated silages. Unexpectedly, concentrations of acetic acid and 1,2-propanediol, 2 hallmarks of L. buchneri activity, did not differ between treatments and were only numerically higher in the inoculated silages compared with untreated ones, in both surveys. Aerobic stability, on average, was 107 and 121 h in the inoculated silages and 64 and 74 h in the untreated silages, for surveys 1 and 2, respectively, and decreased exponentially as the yeast count in the silage at the time of sampling increased, regardless of treatment. Inoculation with L. buchneri proved to be effective in reducing the yeast count to <2 log cfu/g of silage in 16 of 21 of the studied farm silages, confirming the ability of this inoculum to enhance the aerobic stability of corn silages in farm bunker silos.     

The development of lactic acid bacteria and Lactobacillus buchneri and their effects on the fermentation of alfalfa silage

This study was conducted to document the development of populations of lactic acid bacteria (LAB) and Lactobacillus buchneri in alfalfa silage treated with various inoculants. Wilted and chopped alfalfa (45% dry matter) was treated with 1) distilled water (untreated, U), 2) Lactobacillus buchneri 40788 (4 × 10⁵ cfu/g; LB), or 3) L. buchneri 40788 (4 × 10⁵ cfu/g) and Pediococcus pentosaceus (1 × 10⁵ cfu/g; LBPP). Forages were packed into triplicate vacuum-sealed, nylon-polyethylene bags per treatment, and ensiled for 2, 5, 45, 90, and 180 d. Viable (cfu) LAB in forage and silage were quantified by traditional plating on selective agar, and numbers of L. buchneri (cfu-equivalent, cfu-E) were quantified by real-time quantitative PCR. Fresh, untreated forage had 5.52 log cfu of LAB/g and 3.79 log cfu-E of L. buchneri/g. After 2 d of ensiling, numbers of LAB increased to >8 log cfu/g in all silages. In contrast, numbers of L. buchneri in U remained below 4 log cfu-E/g but reached approximately 7 log cfu-E/g in LB and LBPP. From d 5 onward, numbers of L. buchneri in U remained below 6 log cfu-E/g but approached 9 log cfu-E/g in LB and LBPP. The pH was lower in LBPP compared with U and LB after 2 and 5 d of ensiling, but pH was lower for U compared with LB and LBPP thereafter. Treatments LB and LBPP had more acetic acid than U at 45 d of ensiling, which coincided with detectable amounts of 1,2 propanediol. Inoculation with LBPP resulted in silage with the highest concentration of 1,2 propanediol after 180 d of ensiling. From d 45 onward, LB and LBPP silages had lower concentrations of residual water-soluble carbohydrates but had higher concentrations of ammonia-N than U. In conclusion, epiphytic L. buchneri can be detected in alfalfa but this population is unable to lead the silage fermentation. In contrast, when L. buchneri was added to silage as an inoculant, the numbers of L. buchneri (cfu-E) increased markedly but did not dictate fermentation until 45 d of ensiling. These findings help to explain why the response (in increased acetic acid) from the addition of L. buchneri in silages is not immediate.     

Survival of Escherichia coli O157:H7 in channel catfish pond and holding tank water

Survival of Escherichia coli O157:H7 in channel catfish (Ictalurus punctatus), pond and holding tank water was investigated. Water from three channel catfish ponds was inoculated with ampicillin/nalidixic acid-resistant E. coli O157:H7 transformed with a plasmid encoding for green fluorescent protein at 10⁵, 10⁶, and 10⁷ CFU/ml. Samples were taken from surface, internal organs, and skin scrape of fish and pond water for E. coli O157:H7 enumeration on brain heart infusion (BHI) agar containing ampicillin and nalidixic acid. To determine the survival of E. coli O157:H7 in catfish holding tank water from two farmers markets, the water was inoculated with 10⁷E. coli O157:H7 CFU/ml. E. coli O157:H7 were detected by direct plating for 33 and 69 d in pond and holding tank water, respectively. A rapid decrease of the pathogen was observed in the first 2 weeks to reach 2 log CFU/ml. When E. coli O157:H7 was not recovered by direct plating, the pathogen was isolated by enrichment in TSB for approximately another 30 d from pond and holding tank water. The populations of E. coli O157:H7 found in the internal organs and skin scrape were 5.5 log and 2.5 log CFU/ml, respectively. E. coli O157:H7 from internal organs and water were recovered for at least 12 d. Results suggest that E. coli O157:H7 can survive in channel catfish pond and holding tank water and channel catfish may become a potential carrier of the pathogen.     

Quantitative detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction

A method for quantitative detection of Escherichia coli O157:H7 based on the polymerase chain reaction (PCR) was developed. The method used the NIH Image 1·61 software program to quantitatively analyse the intensity of the fluorescent image of the amplified PCR product. Based on the PCR with SLT1 and SLT2 primers used separately, a log-linear relationship between the numbers of cfu of E. coli O157:H7 inoculated into ground beef and the intensity of the PCR products was achieved with and without enrichment. Without enrichment, 150 cfu of E. coli O157:H7 per gram of ground beef were detected. In contrast, the detection limit decreased to 1·2 cfu g⁻¹ of ground beef using SLT1 and SLT2 primers after 4·5 h of enrichment using modified EC broth with 20 μg ml⁻¹ of novobiocin.     

Assessment of Escherichia coli O157:H7-specific bacteriophages e11/2 and e4/1c in model broth and hide environments

The efficacy of bacteriophages e11/2 and e4/1c as potential biocontrol agents for Escherichia coli O157:H7 in food applications was assessed under conditions relevant to the food chain environment. The stability of each phage was determined following exposure to varying environmental conditions (pH, temperature, water activity, and sodium chloride) and the ability of each phage to infect and reduce E. coli O157:H7 numbers under selected conditions was also examined. Both e11/2 and e4/1c significantly (p < 0.05) reduced numbers of E. coli O157:H7 when exposed to pH values ranging from pH > 4 to pH 9, temperatures from 4 °C to 37 °C, water activity values of 0.87 or 0.91 to 1.00 and NaCl concentrations of 1% to 2.5%. Subsequently, a cocktail of both phages was used (e11/2 and e4/1c) to assess reduction of E. coli O157:H7 on cattle hide pieces. This involved inoculating pieces of hide (20 × 20 cm) with E. coli O157:H7 (approximately 10⁶ cfu/cm²) which were subsequently treated with either a suspension of a phage cocktail, consisting of e11/2 and e4/1c (multiplicity of infection of 1000 and 10,000, respectively) or water or not treated. Two different investigations were carried out; immediately or 1 h after treatment application was performed in different experiments. Swab samples taken immediately after phage treatment showed no significant (p > 0.05) reduction of E. coli O157:H7 numbers compared to the water treated or untreated samples. However, an extended exposure time of 1 h following phage application revealed a significant reduction (p < 0.05) (1.5 log₁₀ cfu/cm² reduction) in E. coli O157:H7 numbers compared to the numbers recovered on samples treated with water only. These findings demonstrate the potential use of e11/2 and e4/1c phages as a biocontrol agent for E. coli O157:H7 within various stages of the food chain, including on cattle hide.     

The effect of Lactobacillus buchneri 40788 on the fermentation and aerobic stability of ground and whole high-moisture corn

Experiments were conducted to evaluate the effects of inoculating high-moisture corn (HMC) with Lactobacillus buchneri40788 on silage fermentation and aerobic stability. In the first experiment, HMC (73% DM) was ground and treated with nothing, L. buchneri40788 to achieve 6.6 × 10⁵ cfu/g of HMC (LB), a mixture of enzymes (ENZ), LB + ENZ, or 0.1% (wet weight basis) of a liquid mold inhibitor and was ensiled in 20-L bucket silos for 90 d. Treatments with LB and LB + ENZ increased the concentrations of acetic acid and improved the aerobic stability of ground HMC relative to other treatments. Treatment ENZ had no effect on the chemical composition or aerobic stability of ground HMC. The only effect of the liquid mold inhibitor relative to untreated HMC was that it increased the concentration of propionic acid, but this did not improve its aerobic stability. In a second experiment, HMC (75% DM) was harvested as the intact, whole grain and treated with nothing, L. buchneri40788 to achieve 4 × 10⁵ cfu/g of HMC, L. buchneri40788 to achieve 6 × 10⁵ cfu/g of HMC, or L. buchneri40788 to achieve 8 × 10⁵ cfu/g of HMC and ensiled for 120 d. Treatments with L. buchneri40788 resulted in whole HMC with lower concentrations of water-soluble carbohydrates; higher concentrations of lactic, acetic, and propionic acids; and greater numbers of lactic acid bacteria but fewer molds when compared with untreated corn. As a group, inoculated silages were more aerobically stable than untreated silage, but increasing levels of application did not further improve the response. These experiments showed that addition of L. buchneri40788, but not addition of an enzyme mixture or a liquid mold inhibitor, improved the aerobic stability of ground and whole HMC harvested between 73 and 75% DM.     

Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives

The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 × 10⁵ cfu/g of E. coli O157:H7 (EC); (3) EC and 1 × 10⁶ cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 × 10⁶ cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 ± 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 ± 3.80%). Relative to the control, adding E. coli O157:H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = ?0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = ?0.51), Pantoea (r = ?0.46), Pseudomonas (r = ?0.45), and Stenotrophomonas (r = ?0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = ?0.59), Sphingomonas (r = ?0.66), Pantoea (r = ?0.69), Pseudomonas (r = ?0.69), and Stenotrophomonas (r = ?0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.     

Fermentation and aerobic stability of rehydrated corn grain silage treated with different doses of Lactobacillus buchneri or a combination of Lactobacillus plantarum and Pediococcus acidilactici

We investigated the effects of different types and doses of inoculants for ensiling rehydrated corn grain. Shelled corn was finely ground and rehydrated to 35% moisture. Treatments were as follows: (1) control (no additives); (2) Lactobacillus plantarum and Pediococcus acidilactici (LPPA) at a theoretical application rate of 1 × 10⁵ cfu/g; (3) LPPA at 5 × 10⁵ cfu/g; (4) LPPA at 1 × 10⁶ cfu/g; (5) Lactobacillus buchneri (LB) at 1 × 10⁵ cfu/g; (6) LB at 5 × 10⁵ cfu/g; and (7) LB at 1 × 10⁶ cfu/g. We detected no effect of inoculant dose. Gas losses were greater in silages treated with LB compared with control and LPPA silages. Treating silages with LB reduced the concentrations of lactic acid and ethanol and increased silage pH and concentrations of acetic acid, propionic acid, and 1,2-propanediol. At silo opening, silages treated with LB had higher counts of lactic acid bacteria but lower yeast counts than the control silage. Aerobic stability was greater for silages treated with LB and lower for silages treated with LPPA compared with the control. The LB reduced dry matter (DM) losses during aerobic exposure, whereas LPPA increased them. Prolamin content was lower in silages treated with LB compared with the control, resulting in greater ruminal in situ DM degradability. Inoculating LB to a dose of 1 × 10⁵ cfu/g increased aerobic stability and ruminal in situ DM degradability of rehydrated corn grain silage. The addition of LPPA did not alter the fermentation process and worsened the aerobic stability of rehydrated corn grain silage. Further studies are warranted to confirm these conclusions in other corn hybrids, inoculants, and their combinations.     

Inoculant effects on alfalfa silage: fermentation products and nutritive value

The effect of 14 microbial inoculants on the fermentation and nutritive value of alfalfa silages was studied under laboratory conditions. The first cut (477 g of dry matter/kg) and second cut (393 g of dry matter/kg) of a second-year alfalfa stand were ensiled in 2 trials. In both trials alfalfa was harvested with standard field equipment. All inoculants were applied at 1.0 × 10⁶ cfu/g of crop. Uninoculated silages served as controls. After inoculants were added, the chopped forages were ensiled in 1.0- and 0.5-L anaerobic glass jars, respectively, at a density of 500 g/L. Each trial had 15 treatments (uninoculated control and 14 inoculants), with 4 silos per treatment. Silos were stored for a minimum of 30 d at room temperature (∼22°C). In first-cut silage, all inoculants but one reduced pH relative to the uninoculated control, and all but 2 of the homofermentative strains shifted fermentation toward lactic acid. In second-cut silage, the epiphytic lactic acid bacterial population was 2.7 × 10⁷ cfu/g, and only commercial inoculants produced significant shifts in fermentation. Overall, microbial inoculants generally had a positive effect on alfalfa silage characteristics in terms of lower pH and shifting fermentation toward lactic acid with homofermentative lactic acid bacteria or toward acetic acid with heterofermentative lactic acid bacteria, Lactobacillus buchneri. These effects were stronger in the commercial products tested. In spite of the positive effects on silage fermentation, 48-h in vitro true DM digestibility was not improved by inoculation with lactic acid bacteria.     

The effects of Lactobacillus buchneri 40788 and Pediococcus pentosaceus R1094 on the fermentation of corn silage

The effect of inoculating whole-plant corn at the time of harvest with Lactobacillus buchneri 40788 (4 × 10⁵ cfu/g of fresh forage) combined with Pediococcus pentosaceus R1094 (1 × 10⁵ cfu/g) on the fermentation and aerobic stability of corn silage (37% dry matter) through 361 d of ensiling was investigated. Dry matter recovery was similar between treatments throughout the study except at one early time point (14 d), when treated silage had a lower recovery than untreated silage. The concentration of lactic acid was unaffected by inoculation but inoculated silages had greater concentrations of 1,2-propanediol and acetic acid from 56 to 361 d of storage. In general, inoculation decreased the concentration of water-soluble carbohydrates but increased the concentration of ethanol. The numbers of yeasts was lower in inoculated silage at 42, 56, 70, and 282 d of ensiling. However, inoculation did not consistently improve the aerobic stability of silage, suggesting that microbes other than yeasts may have been responsible for aerobic instability in this study. Even after prolonged storage (361 d), silage treated with L. buchneri 40788 and P. pentosaceus R1094 had normal silage fermentation characteristics.     

Efficacy of various plant hydrosols as natural food sanitizers in reducing Escherichia coli O157:H7 and Salmonella Typhimurium on fresh cut carrots and apples

In the present study, inhibitory effects of the hydrosols of thyme, black cumin, sage, rosemary and bay leaf were investigated against Salmonella Typhimurium and Escherichia coli O157:H7 inoculated to apple and carrots (at the ratio of 5.81 and 5.81 log cfu/g for S. Typhimurium, and 5.90 and 5.70 log cfu/g for E. coli O157:H7 on to apple and carrot, respectively). After the inoculation of S. Typhimurium or E. coli O157:H7, shredded apple and carrot samples were washed with the hydrosols and sterile tap water (as control) for 0, 20, 40 and 60 min. While the sterile tap water was ineffective in reducing (P > 0.05) S. Typhimurium and E. coli O157:H7, 20 min hydrosol treatment caused a significant (P < 0.05) reduction compared to the control group. On the other hand, thyme and rosemary hydrosol treatments for 20 min produced a reduction of 1.42 and 1.33 log cfu/g respectively in the E. coli O157:H7 population on apples. Additional reductions were not always observed with increasing treatment time. Moreover, thyme hydrosol showed the highest antibacterial effect on both S. Typhimurium and E. coli O157:H7 counts. Inhibitory effect of thyme hydrosol on S. Typhimurium was higher than that for E. coli O157:H7. Bay leaf hydrosol treatments for 60 min reduced significantly (P < 0.05) E. coli O157:H7 population on apple and carrot samples. In conclusion, it was shown that plant hydrosols, especially thyme hydrosol, could be used as a convenient sanitizing agent during the washing of fresh-cut fruits and vegetables.     

The effects of various antifungal additives on the fermentation and aerobic stability of corn silage

In 2 consecutive years, whole plant corn was ensiled in laboratory silos to investigate the effects of various silage additives on fermentation, dry matter (DM) recovery and aerobic stability. In yr 1, chopped forage was treated with 1) no additive (untreated, U), 2) Lactobacillus buchneri40788, 4 × 10⁵ cfu/g of fresh forage (LLB4), 3) L. buchneri 11A44, 1 × 10⁵ cfu/g (PLB), 4) Biomax 5 (Lactobacillus plantarum PA-28 and K-270), 1 × 10⁵ cfu/g (B5), 5) Silo Guard II (sodium metabisulfite and amylase), 0.05% of fresh forage weight (SG), 6) a buffered propionic acid-based additive, 0.1% (Ki-112), 7), sodium benzoate, 0.1% of fresh weight (SB), or 8) potassium sorbate:EDTA (1:1), 0.1% of fresh weight (PSE). Silage treated with LLB4 had the highest concentration of acetic acid compared with other treatments, and yeasts were undetectable in LLB4 (<log₂ cfu/g). Silages treated with SB and PSE had the highest concentrations of water-soluble carbohydrates, the greatest recoveries of DM, and the lowest concentrations of ethanol. Silages treated with B5, SG, and Ki-112 had no effects on fermentation, DM recovery, or aerobic stability. The aerobic stabilities of silages treated with LLB4, SB, and PSE were greatest among all treatments. In yr 2, treatments were: 1) U, 2) LLB4, 3) PLB, 4) PLB at 4 × 10⁵ cfu/g (PLB4), and 5) B5. Silages treated with L. buchneri had greater concentrations of acetic acid but lower concentrations of ethanol than did U- and B5-treated silages. Yeasts were undetected in all silages except in silage treated with B5, which had the poorest aerobic stability of all treatments. Treatments had no effect on DM recovery. Silages treated with PLB, PLB4, and LLB4 remained stable for >210 h.     

Detection of Escherichia coli via VOC profiling using secondary electrospray ionization-mass spectrometry (SESI-MS)

Escherichia coli O157:H7 (EC O157:H7), as well as its recently emerging non-O157 relatives, are a notorious group of pathogenic bacteria associated with foodborne outbreaks. In this study, we demonstrated that secondary electrospray ionization mass spectrometry (SESI-MS) could be a rapid and accurate detection technology for foodborne pathogens. With SESI-MS volatile organic compound (VOC) profiling, we were able to detect and separate a group of eleven E. coli strains from two major foodborne bacteria, Staphylococcus aureus and Salmonella Typhimurium in three food modeling media. In addition, heatmap analysis of relative peak intensity show that there are six core peaks (m/z of 65, 91, 92, 117, 118 and 119) present and at a similar intensity in all eleven E. coli strains at the experimental conditions we tested. These peaks can be considered conserved VOC biomarkers for E. coli species (robustly produced after just 4 h of growth). Bacterial strain-level differentiation was also attempted via VOC profiling, and we found that EC O157:H7 and EC O145 were differentiable from all other EC strains under the conditions investigated.     

The effect of a competitive microflora,pH and temperature on the growth kinetics of Escherichia coli O157:H7

Growth curves were generated for Escherichia coli O157:H7 in brain–heart infusion broth incubated at 37 or 15°C in the presence of individual and combinations of competing microflora. Broths were inoculated withE. coli O157:H7 (log₁₀3·00 cfu ml⁻¹) and competitors (log₁₀4·00 cfu ml⁻¹) and the initial pH of the broth was either neutral (7·0) or adjusted to 5·8 and then sequentially reduced to 4·8 over 10 h to simulate fermentation conditions. Growth curves were also generated for the competitors in these cultures, including Pseudomonas fragi, Hafnia alvei, Pediococcus acidilactici (pepperoni starter culture) and Brochothrix thermosphacta . Gompertz equations were fitted to the data and growth kinetics including lag phase duration, exponential growth rates and maximum population densities (MPD) calculated. In pure culture, the growth parameters for E. coli O157:H7 in neutral pH broths were significantly different from those recorded in simulated fermentation broths (P<0·05). The presence of competitors in the broth also had a significant effect on the growth kinetics of the pathogen. H. alvei significantly inhibited the growth (lag phase, growth rate and MPD) of E. coli O157:H7 at 37°C, neutral pH and outgrew the pathogen under these conditions. In neutral pH cultures, two other competitors, B. thermosphacta and P. acidilactici also inhibited the lag phase of the pathogen but had no effect on the other growth parameters. In simulated fermentation broths, the growth rate of E. coli O157:H7 was consistently slower and the MPD lower in the presence of a competitive microflora than when grown individually. At 15°C, only one competitor, P. fragi significantly inhibited the lag phase of the pathogen. The implications of these findings for food safety are discussed.     

Incidence of Escherichia coli O157:H7 and E. coli virulence factors in US bulk tank milk as determined by polymerase chain reaction

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5°C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (γ-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA⁺, γ-tir⁺, stx2⁺, rfbE⁺, fliC⁺, hlyA⁺) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.     

Adaptation of Escherichia coli O157:H7 to acid in traditional and commercial goat milk amasi

Acid resistance of Escherichia coli O157:H7 strains UT 10 and UT 15 were determined in traditional Amasi fermented for 3 days at ambient temperature (ca 30 °C) and commercial Amasi fermented at 30 °C for 24 h and stored at 7 °C for 2 days. Escherichia coli O157:H7 counts in commercial Amasi were detected at 2.7 log₁₀ cfu/ml after 3 days while those in traditional Amasi could not be detected after the same period. There was no significant difference (p ? 0.05) in the survival of acid adapted (AA) and non-adapted (NA) E. coli O157:H7 in traditional Amasi, while in commercial Amasi, the NA strain survived significantly (p ? 0.05) better than its AA counterpart. Regardless of prior adaptation to acid, E. coli O157:H7 can survive during fermentation and storage of fermented goat milk Amasi. Also, the fermentation time, pH and storage temperature affects the survival of E. coli O157:H7 in the fermented milk.     

An aptamer-exonuclease III (Exo III)–assisted amplification-based lateral flow assay for sensitive detection of Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 (E. coli O157:H7), one of the most widespread foodborne pathogens, can cause a series of diseases and even lead to death. In this study, a highly sensitive method was developed by combining aptamer-exonuclease III (Exo III)–assisted amplification with lateral flow assay (LFA) based on gold nanoparticles (AuNP). The compound of single-stranded (ss) DNA-anti-E. coli O157:H7 aptamer (ssDNA-aptamer) was formed by hybridization between designed target ssDNA and aptamer. When E. coli O157:H7 was present, target bacteria were bound with the aptamer, and the free target ssDNA was hybridized with the probes of the designed hairpin (HP) structure. Exo III digests the 3′ double-stranded blunt end of the complex and releases the enzyme product. Because the remaining sequence of the HP of the designed enzyme product was the same as the target ssDNA sequence, the target ssDNA could be amplified. Finally, the enhanced target ssDNA was combined with AuNP-LFA to achieve visual detection of E. coli O157:H7. The quantitative ability of this platform for E. coli O157:H7 was 7.6 × 10¹ cfu/mL in pure culture, and the detection limit in milk was 8.35 × 10² cfu/mL. This LFA was highly specific to E. coli O157:H7, and the time for detection of E. coli O157:H7 in milk was 4 h. Hence, this system has important application prospects in the detection of pathogenic bacteria in dairy products.