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Food safety is a new area for novel applications of metagenomics analysis, which not only can detect and subtype foodborne pathogens in a single workflow but may also produce additional information with in-depth analysis capabilities. In this study, we applied a quasimetagenomic approach by combining short-term enrichment, immunomagnetic separation (IMS), multiple-displacement amplification (MDA), and nanopore sequencing real-time analysis for simultaneous detection of Salmonella and Escherichia coli in wheat flour. Tryptic soy broth was selected for the 12-h enrichment of samples at 42°C. Enrichments were subjected to IMS using beads capable of capturing both Salmonella and E. coli MDA was performed on harvested beads, and amplified DNA fragments were subjected to DNA library preparation for sequencing. Sequencing was performed on a portable device with real-time basecalling adaptability, and resulting sequences were subjected to two parallel pipelines for further analysis. After 1 h of sequencing, the quasimetagenomic approach could detect all targets inoculated at approximately 1 CFU/g flour to the species level. Discriminatory power was determined by simultaneous detection of dual inoculums of Salmonella and E. coli , absence of detection in control samples, and consistency in microbial flora composition of the same flour samples over several rounds of experiments. The total turnaround time for detection was approximately 20 h. Longer sequencing for up to 15 h enabled serotyping for many of the samples with more than 99% genome coverage, which could be subjected to other appropriate genetic analysis pipelines in less than a total of 36 h. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella are of serious concern in low-moisture foods, including wheat flour and its related products, causing illnesses, outbreaks, and recalls. The development of advanced detection methods based on molecular principles of analysis is essential to incorporate into interventions intended to reduce the risk from these pathogens. In this work, a quasimetagenomic method based on real-time sequencing analysis and assisted by magnetic capture and DNA amplification was developed. This protocol is capable of detecting multiple Salmonella and/or E. coli organisms in the sample within less than a day, and it can also generate sufficient whole-genome sequences of the target organisms suitable for subsequent bioinformatics analysis. Multiplex detection and identification were accomplished in less than 20 h and additional whole-genome analyses of different nature were attained within 36 h, in contrast to the several days required in previous sequencing pipelines., (Copyright © 2020 American Society for Microbiology.)

A silica-protein hybrid material has been prepared by simultaneous covalent deposition of apoferritin and lactoferrin on functionalized silica. This material exhibits strong antibacterial activity against E. coli due to its high iron-uptake capacity.

Microbiological analyses of fruits and vegetables produced by farms in Minnesota and Wisconsin were conducted to determine the prevalence of Escherichia coli in pre-harvest fruits and vegetables. During the 2003 and 2004 harvest seasons, 14 organic (certified by accredited organic agencies), 30 semi-organic (used organic practices but not certified) and 19 conventional farms were sampled to analyze 2029 pre-harvest produce samples (473 organic, 911 semi-organic, 645 conventional). Before each harvest season, a farmer survey was conducted to collect relevant information on farm management practices that might affect the risk of E. coli contamination in fresh produce. The use of animal wastes for fertilization of produce plants increased the risk of E. coli contamination in organic (OR=13.2, 95% CI=2.2-61.2, P-value<0.0001) and semi-organic (OR=12.9, 95% CI=2.9-56.3, P-value<0.0001) produce significantly. Improper ageing of untreated animal manure significantly increased this risk in organic produce (OR=4.2 95% CI=1.7-12.3, P-value=0.005) grown using such manure as a fertilizer. Organic growers who used cattle manure for fertilization of their crops showed significantly greater risk of contamination with the E. coli (OR=7.4, 95% CI=1.6-36.8, P-value=0.003), compared to those who used other types of manure-based fertilizer. In Minnesota, organic and semi-organic produce collected from the southeastern (SE) part of the state were at a significantly greater risk of E. coli contamination (OR=3.45, 95% CI=1.8-35.2, P=0.008), compared to those collected from farms located in the southern (S) regions of the state. In Wisconsin, organic and semi-organic produce collected from the southern (S) cluster of farms were at approximately 3-times greater risk of E. coli contamination (OR=2.67, 95% CI=1.3-9.4, P=0.004), compared to those grown in the northern (N) cluster of farms.

This study compared the antimicrobial susceptibility of Shiga toxin-producing Escherichia coli (STEC) isolates from organic dairy farms, conventional dairy farms, and Minnesota county fairs. A total of 83 STEC isolates (43 O157 and 40 non-O157 STEC) were tested for antimicrobial susceptibility as determined by the automated broth microdilution method. Resistance to tetracycline was identified in 19 (23%) isolates and to sulphadimethoxine in 40 (48%) isolates. Half of the STEC isolates were resistant to at least one antimicrobial agent. Resistance to at least one antimicrobial agent was observed in 18 (62%) isolates from conventional farms and in 11 (48%) isolates from organic farms. Resistance to at least one antimicrobial agent was more frequent in isolates from calves (77%) than from cows (39%). Multidrug resistant patterns were more common in non-O157 STEC than O157 STEC. This study provides data to document the degree of STEC antimicrobial resistance from dairy cattle sources in Minnesota. The use of antimicrobial agents on farms, and other environmental influences, may affect resistance patterns in isolates from cattle sources. Systematic surveillance of STEC from cattle could potentially detect emergence of antimicrobial resistance that may be spread to humans through the food chain.

Shiga toxin-encoding bacteria (STB) and shiga toxin-producing Escherichia coli (STEC) were detected and isolated from dairy cattle and their farm environment and from manure piles at Minnesota (MN) county fairs from 2001 to 2002. A total of 2,540 samples were collected from 28 dairy cattle farms (8 organic and 20 conventional), 17 calf pens (5 organic and 12 conventional), and 12 county fairs. STB were detected from 71 (3.2%) of 2208 fecal samples with 20 (71.4%) of 28 dairy farms having at least one positive animal sample. In samples collected from conventional farms, 41 (2.3%) of 1750 fecal samples were STB-positive and 13 (65%) of 20 farms had at least one positive animal. Thirty (6.6%) of 458 fecal samples from organic farms were STB-positive and 7 (87.5%) of 8 farms had at least one positive animal. STB was detected from 31 (17.4%) of 178 samples and 7 (58.3%) out of 12 manure piles at county fairs. A total of 43 STEC isolates were recovered and belonged to 26 different serotypes (19 O and 18 H types). Among STEC, 60.5% possessed only stx1, 30.2% stx2, and 9.3% both stx1 and stx2. The genes eae and hlyA were detected in more than 50% of the STEC isolates. STB can be found on most dairy cattle farms including organic and conventional herds and county fairs. The presence of these potentially pathogenic bacteria in county fairs may pose a risk to the public who have contact with cattle or their environment.

Microbiological analyses of fruits and vegetables produced by farms in Minnesota and Wisconsin were conducted to determine coliform and Escherichia coli counts and the prevalence of E. coli, Salmonella, and E. coli O157:H7. During the 2003 and 2004 harvest seasons, 14 organic farms (certified by accredited organic agencies), 30 semiorganic farms (used organic practices but not certified), and 19 conventional farms were sampled to analyze 2,029 preharvest produce samples (473 organic, 911 semiorganic, and 645 conventional). Produce varieties included mainly lettuces, leafy greens, cabbages, broccoli, peppers, tomatoes, zucchini, summer squash, cucumber, and berries. Semiorganic and organic farms provided the majority of leafy greens and lettuces. Produce samples from the three farm types had average coliform counts of 1.5 to 2.4 log most probable number per g. Conventional produce had either significantly lower or similar coliform populations compared with the semiorganic and organic produce. None of the produce samples collected during the 2 years of this study were contaminated with Salmonella or E. coli O157:H7. E. coli contamination was detected in 8% of the samples, and leafy greens, lettuces, and cabbages had significantly higher E. coli prevalence than did all the other produce types in both years for the three farm types. The prevalence of E. coli contamination by produce type was not significantly different between the three farm types during these 2 years, with the exception of organic leafy greens, in which E. coli prevalence was one-third that of semiorganic leafy greens in 2003. These results indicate that the preharvest microbiological quality of produce from the three types of farms was very similar during these two seasons and that produce type appears to be more likely than farm type to influence E. coli contamination.

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10(7) CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log(10) CFU/g was observed, with a maximum decrease of 1.8 log(10) CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10(8) CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.

Microbiological analyses of fresh fruits and vegetables produced by organic and conventional farmers in Minnesota were conducted to determine the coliform count and the prevalence of Escherichia coli, Salmonella, and E. coli O157:H7. A total of 476 and 129 produce samples were collected from 32 organic and 8 conventional farms, respectively. The samples included tomatoes, leafy greens, lettuce, green peppers, cabbage, cucumbers, broccoli, strawberries, apples, and seven other types of produce. The numbers of fruits and vegetables was influenced by their availability at participating farms and varied from 11 strawberry samples to 108 tomato samples. Among the organic farms, eight were certified by accredited agencies and the rest reported the use of organic practices. All organic farms used aged or composted animal manure as fertilizer. The average coliform counts in both organic and conventional produce were 2.9 log most probable number per g. The percentages of E. coli-positive samples in conventional and organic produce were 1.6 and 9.7%, respectively. However, the E. coli prevalence in certified organic produce was 4.3%, a level not statistically different from that in conventional samples. Organic lettuce had the largest prevalence of E. coli (22.4%) compared with other produce types. Organic samples from farms that used manure or compost aged less than 12 months had a prevalence of E. coli 19 times greater than that of farms that used older materials. Serotype O157:H7 was not detected in any produce samples, but Salmonella was isolated from one organic lettuce and one organic green pepper. These results provide the first microbiological assessment of organic fruits and vegetables at the farm level.

A previously identified set of anti-Escherichia coli O157:H7 colicinogenic E. coli were characterized to assess the suitability of these isolates as a preharvest food safety intervention in cattle. This collection of 23 E. coli strains were screened for virulence factors, antibiotic resistance, type of colicin(s) present, and their ability to inhibit other pathogenic E. coli. With the use of PCR, pathogen genes were detected in six of the 23 colicinogenic E. coli. When the nonpathogenic strains were assessed for antibiotic resistance, four strains showed resistance to at least one antibiotic. The remaining set of 14 strains were evaluated for the presence of previously identified colicins. Seven colicins (B, El, E2/E7, E7, Ia/Ib, K, and M) were detected. One half of the strains possessed multiple types of colicins. The most commonly detected colicins were B, E2/E7, and M, which were found in six strains each. DNA sequencing was also performed in order to classify the E2/E7 colicins separately from E7 colicins. The 14 colicinogenic E. coli also were evaluated for their ability to inhibit 10 different non-O157 pathogenic E. coli. Six of the colicinogenic E. coli were capable of inhibiting all 10 pathogens, and the remaining eight strains could each inhibit between six to eight of the pathogenic E. coli. This strain collection has great potential for inhibiting E. coli O157:H7 in cattle.

Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E. coli per animal species ranged from 14% for horse isolates to 64% for sheep strains. Those isolates that inhibited E. coli K12 were screened against E. coli O157:H7, and 42 strains were found to be capable of inhibiting all 22 pathogenic strains tested. None of these 42 strains produced bacteriophages, and only 24 isolates inhibited serotype O157:H7 in liquid culture. The inhibitory activity of these strains was completely eliminated by treatment with proteinase K. When mixtures of these 24 colicinogenic strains were grown in anaerobic continuous culture, the four-strain E. coli O157:H7 population was reduced at a rate of 0.25 log10 cells per ml per h, which was fivefold faster than the washout rate. Two strains originally isolated from cat feces (F16) and human feces (H30) were identified by repetitive sequences polymerase chain reaction as the predominant isolates in continuous cultures. The results of this work indicate that animal species other than cattle can be sources of anti-O157 colicinogenic strains, and these results also lead to the identification of at least two isolates that could potentially be used in preharvest control strategies.

Enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 is carried asymptomatically by cattle gastrointestinal tract and the inclusion of distillers grains solids (DGS) in feed is thought to be a factor in the prevalence and persistence of EHEC O157 in a herd. The present study surveys the faecal prevalence of E. coli O157 in cattle processed at an abattoir in the Upper Midwest and its association with environmental factors and feeding practices. Faecal samples were collected from pre-processing cows during a 1-year period. E. coli O157 prevalence was estimated isolation of putative positives and confirmation of isolates by immunoassay and multiplex virulence genes PCR analysis. Overall, E. coli O157 was confirmed in 11.2% of samples. Prevalence during winter was the highest at 14% followed by summer (11.6%) and declined to less than 8% the rest of the year. Winter was the only season that had a statistically significant effect on prevalence. As a category unto itself, DGS feeding before arrival had no significant influence on faecal prevalence. However, we found a significant interaction of DGS feeding and summer. This observation is extremely relevant because it corroborates a previous study and suggests possible feeding practices to abate EHEC O157 presence during harvest. [ABSTRACT FROM AUTHOR]

Salmonella can survive for long periods under extreme desiccation conditions. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular regulation of this adaptation mechanism. To determine the genetic components involved in Salmonella’s cellular response to desiccation, we performed a global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low water activity (aw 0.11) and cells equilibrated to high water activity (aw 1.0). The analysis revealed that 719 genes were differentially regulated between the two conditions, of which 290 genes were up-regulated at aw 0.11. Most of these genes were involved in metabolic pathways, transporter regulation, DNA replication/repair, transcription and translation, and, more importantly, virulence genes. Among these, we decided to focus on the role of sopD and sseD. Deletion mutants were created and their ability to survive desiccation and exposure to aw 0.11 was compared to the wild-type strain and to an E. coli O157:H7 strain. The sopD and sseD mutants exhibited significant cell viability reductions of 2.5 and 1.3 Log (CFU/g), respectively, compared to the wild-type after desiccation for 4 days on glass beads. Additional viability differences of the mutants were observed after exposure to aw 0.11 for 7 days. E. coli O157:H7 lost viability similarly to the mutants. Scanning electron microscopy showed that both mutants displayed a different morphology compared to the wild-type and differences in production of the extracellular matrix under the same conditions. These findings suggested that sopD and sseD are required for Salmonella’s survival during desiccation. [ABSTRACT FROM AUTHOR]

Escherichia coli O157: H7 (EHEC) is a major foodborne pathogen largely transmitted to humans through the consumption of undercooked ground beef. This study investigated the efficacy of two food-grade, plant-derived antimicrobials, namely rutin (RT), and resveratrol (RV) with or without chitosan (CH) in enhancing EHEC inactivation in undercooked hamburger patties. Further, the effect of aforementioned treatments on beef color and lipid oxidation was analyzed. Additionally, the deleterious effects of these antimicrobial treatments on EHEC was determined using scanning electron microscopy (SEM). Ground beef was inoculated with a five-strain mixture of EHEC (7.0 log CFU/g), followed by the addition of RT (0.05%, 0.1% w/w) or RV (0.1, 0.2% w/w) with or without CH (0.01% w/w). The meat was formed into patties (25 g) and stored at 4?C for 5 days. On days 1, 3, and 5, the patties were cooked (65?C, medium rare) and surviving EHEC was enumerated. The effect of these treatments on meat color and lipid oxidation during storage was also determined as per American Meat Science Association guidelines. The study was repeated three times with duplicate samples of each treatment. Both RT and RV enhanced the thermal destruction of EHEC, and reduced the pathogen load by at least 3 log CFU/g compared to control (P < 0.05). The combination of RT or RV with CH was found to be more effective, and reduced EHEC by 5 log CFU/g (P < 0.05). EHEC counts in uncooked patties did not decline during storage for 5 days (P > 0.05). Moreover, patties treated with RV plus CH were more color stable with higher a* values (P < 0.05). SEM results revealed that heat treatment with antimicrobials (CH + RV 0.2%) resulted in complete destruction of EHEC cells and extrusion of intracellular contents. Results suggest that the aforementioned antimicrobials could be used for enhancing the thermal inactivation of EHEC in undercooked patties; however, detailed sensory studies are warranted. [ABSTRACT FROM AUTHOR]

This study determined the effect of previously described phage cocktail ( BEC8) on the inactivation kinetics of a mixture of E scherichia coli ( EHEC) O157: H7 strains on food processing surface materials: stainless steel chip ( SSC), ceramic tile chip ( CTC) and high-density polyethylene chip ( HDPEC). EHEC strains were combined and spot-inoculated on surfaces in dried or liquid form and BEC8 was applied and incubated at 12, 23, 30 and 37C. EHEC survival was determined on tryptic soy agar. D values ranged from 3.9 min at 37C to 46.7 min at 12C. Z values calculated for SSC, CTC and HDEPC resulted in 26.1, 23.7 and 26.7C for dry cells, respectively, and 23.2, 23.7 and 24.5C for liquid cells, respectively. There was a significant difference in the rate of bacterial inactivation between lower (12 and 23C) and higher temperatures (30 and 37C). These results indicated that BEC8 killed EHEC on hard surfaces rapidly following first-order kinetics. Practical Applications The effect of a previously described phage cocktail ( BEC8) on the inactivation kinetics of a mixture of E scherichia coli ( EHEC) O157: H7 strains applied on hard food processing surface materials was studied. The results indicated that phages killed EHEC on hard surfaces relatively rapidly following a predictable rate. However, one must consider the long times to achieve a 5-log reduction when initial cell counts are high. It would take approximately 2-4 h for a 5-log cfu reduction of liquid cells at 23C. Both the time length and the temperature of application must fit within the industry time scales. [ABSTRACT FROM AUTHOR]

The article presents a study which identifies the herd-level risk factors associated with fecal shedding of Shiga toxinencoding bacteria (STB) on dairy cattle farms in Minnesota. It informs that stratified random sampling process was adopted for the study. It further informs that 105 cows were taken in average herd for study. Multiple factors related to cattle feeding practices associated with Shiga toxin-producing Escherichia coli (STEC) were identified as the conclusion of the study.

Samples were collected from 26 organic and conventional farms and 12 county fairs in Minnesota during 2001 and 2002 to identify the presence of Escherichia coli O 157. Immunomagnetic separation was used for isolation of E. coli O157. Isolates were further characterized by the presence of virulence marker genes (stx1, stx2, eaeA, E-hly, katP, etpD, and espP), antimicrobial susceptibility profiles, and genotypes. During 2001, E. coli O157 was isolated from 16 (5.2%) of 305 fecal samples and from 7 (36.8%) of 19 farms. During 2002, E. coli O157 was isolated from 6 (4.5%) of 132 fecal samples from weaned calves at 4 (23.5%) of 17 farms. During 2001 and 2002, cattle manure samples were collected from 12 county fairs, and E. coli O157 was isolated from 19 (11%) of 178 samples and 9 (75%) of 12 county fairs. Among 40 E. coli O157 isolates, 17 isolates (43%) had both the stx1 and stx2 genes, and 21 strains (53%) had the stx2 gene only. Thirteen percent of O157 isolates were resistant to tetracycline, and 25% were resistant to sulfadimethoxine. Heterogeneity of E. coli O157 strains was demonstrated by the presence of 22 different pulsed-field gel electrophoresis (PFGE) patterns. Four PFGE patterns matched those of isolates previously found in humans. The presence of E. coli O157 at county fairs suggests the potential for transmission to the public, who may have contact with cattle or their environment. [ABSTRACT FROM AUTHOR]

Morganella, Providencia and Proteus strains were capable of surviving pH 2.0 for 1 h if glutamate was present. These strains did not have glutamic acid decarboxylase activity and the gadAB genes were not detected in any of these bacteria. When exposed to pH 2.0 acid shocks, the survival rate of these bacteria was significantly increased with glutamate concentrations as low as 0.3 mM in the acid media. Escherichia coli cells incubated at pH 3.4 consumed four times more glutamate and produced at least 7-fold more γ-amino butyric acid than Morganella, Providencia and Proteus strains. These results indicate that strains belonging to the Proteeae tribe might have novel glutamate dependent acid-resistance mechanisms. [Copyright &y& Elsevier]

Describes a procedure to eliminate Escherichia coli in dairy cattle manure. Normal inhabitant of the gastrointestinal tract of animals; Index of water quality and manure contamination; Pure cultures of bacteria; Bacterial enumeration; Analytical assays.

Investigates the ability of Escherichia coli O157:H7 to decrease its intracellular pH and resist the toxicity of acetic acid. Growth of E. coli K-12 and O157:H7 in anaerobic batch and continuous cultures and increasing amounts of sodium acetate; Detection of methylglyoxal shunt; Intracellular pH and acetate accumulation.

The presence of antibiotic-resistant bacteria in the food supply chain is a major concern. There is a great need to revalidate the effectiveness of the intervention strategies commonly used in food processing environments for multidrug-resistant strains. The purpose of this study was to determine the survival of antibiotic-resistant O26 and O103 Shiga-toxin producing Escherichia coli (STEC) strains to lactic acid (2.5, 3.5, 5%), sodium hypochlorite (0.2, 0.5, 1 ppm free chlorine), and heat (60, 61, 62.5 °C) treatments. Six strains from STEC serotypes O26 and O103 with three distinctive antibiotic-resistant profiles (susceptible, low and high resistance) were selected for the study. As expected, the rates of inactivation were faster at increasing biocide concentrations or temperature level. The Weibull model provided a better fit than the traditional log-linear model due to the appearance of nonlinear patterns. A 5% lactic acid treatment for 10 min reduced the population of all STEC strains by 5 log CFU/ml. Sodium hypochlorite treatment (1 ppm for 5 min) achieved a similar reduction. Thermal D values for O26 and O103 serotypes ranged from 0.37 to 2.09 and 0.37–1.71 min, respectively. No statistical differences (P > 0.05) in tolerance were found between the two STEC serotypes to the different treatments. The antibiotic profile was not related to the ability to tolerate any of the treatments. Only the susceptible strain showed a relative higher resistance to the lactic acid and lower resistance to sodium hypochlorite treatment. These results would contribute to assess the risk for the presence of STEC strains in food processing environments. [ABSTRACT FROM AUTHOR]

Abstract: Electrochemically activated water (ECAW), also known as electrolyzed water, and ozonized water are typically effective in inactivating bacteria, but their generation typically uses high current and voltage. A few simpler antimicrobial technologies that are also based on the application of a mild electrical current have been recently marketed to food retail and service customers claiming to have sanitizing properties for controlling bacteria. The objective of this study was to determine the sanitizing effect of some of these commercial technologies on Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica and compare them with sterile water, generated ECAW generated with a pilot size electrolyzing unit, and salt solutions sprayed using commercial device sprays. A concentration of 100 mg/L ECAW had sanitizing effects of at least 5 log CFU/mL reductions on liquid culture and more than 4 log CFU/coupon reductions for E. coli O157:H7, L. monocytogenes and Salmonella dried on stainless steel surface, respectively. No bacterial cells were detected by direct plate counting post-ECAW treatment. In contrast, the treatment of liquid cultures with any of the commercial technologies tested resulted in non-significant bacterial cell reductions greater than 0.5 log CFU/mL. Similarly, when cells had been dried on metal surfaces and treated with any of the water generated with those technologies, no reductions were observed. When the manufacturer's instructions were followed, the reduction of cells on surface was largely due to the physical removal by cloth-wiping after water fraction application. These results indicate that treatment with any of these portable technologies had no noticeable antimicrobial activity. These results would be helpful for guiding consumers when choosing a right sanitization to ensure food safety. [Copyright &y& Elsevier]

Food safety is a new area for novel applications of metagenomics analysis, which can not only detect and subtype foodborne pathogens in a single workflow but may also produce additional information with in depth analysis capabilities. In this study we applied a quasimetagenomic approach by combining short-term enrichment, immunomagnetic separation (IMS), multiple displacement amplification (MDA) and nanopore sequencing real-time analysis for simultaneous detection of Salmonella and Escherichia coli in wheat flour. Tryptic soy broth was selected for the 12-h enrichment of samples at 42°C. Enrichments were subjected to IMS using beads capable of capturing both Salmonella and E. coli. The MDA was performed on harvested beads and amplified DNA fragments were subjected to DNA library preparation for sequencing. Sequencing was performed on a portable device with real-time basecalling adaptability and resulted sequences were subjected to two parallel pipelines for further analysis. After 1 h of sequencing the quasimetagenomic approach could detect all targets inoculated at approx. 1 CFU/g flour to the species level. Discriminatory power was determined by simultaneous detection of dual inoculums of Salmonella and E. coli, absence of detection in control samples and consistency in microbial flora composition of the same flour samples over several rounds of experiment. The total turnaround time for detection was approximately 20 h. Longer sequencing for up to 15 h enabled serotyping for many of the samples with more than 99% genome coverage which could be subjected to other appropriate genetic analysis pipelines in less than a total of 36 h. [ABSTRACT FROM AUTHOR]

An increasing number of outbreaks of gastroenteritis recently caused by Escherichia coli O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that E. coli survives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identify E. coli genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing E. coli K-12, a model system, and E. coli O157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, including tnaA (33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsA and ybiM) and curli production (csgA and csgB) were significantly upregulated in E. coli K-12 and O157:H7. Both csgA and bhsA (ycfR) mutants were impaired in the long-term colonization of the leaf surface, but only csgA mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of E. coli K-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation. [ABSTRACT FROM AUTHOR]

Abstract: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages and essential oils can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply. The objective of this study was to determine the effect of a bacteriophage cocktail, BEC8, alone and in combination with the essential oil trans-cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on whole baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were NalR mutants of EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on leaves and dried. EHEC cells were placed at low, medium, and high inoculum levels (104, 105, and 106 CFU/mL, respectively). Appropriate controls, BEC8 (approx. 106 PFU/leaf), and TC (0.5% v/v) were applied on treated leaves. The leaves were incubated at 4, 8, 23, and 37 °C in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid (50 μg/mL) Sorbitol MacConkey agar. No survivors were detected when both leaves were treated with BEC8 or TC individually at low inoculum levels after 24 h at 23 and 37 °C. When the EHEC inoculum size increased and/or incubation temperature decreased, the efficacy of BEC8 and TC decreased. However, when the two treatments were combined, no survivors were detected after 10 min at all temperatures and inoculum levels on both leafy greens. These results indicated that the BEC8/TC combination was highly effective against EHEC on both leafy greens. This combination could potentially be used as an antimicrobial to inactivate EHEC O157:H7 and reduce their incidence in the food chain. [Copyright &y& Elsevier]