Identification of Contamination Sources and Assessment of Risk Factors Associated with the Occurrence of Escherichia coli O157:H7 on Small-scale Cow-calf Operations in Oklahoma and Louisiana.

Escherichia coli O157:H7 is a human pathogen that exists as part of the commensal microflora of cattle and is shed in animal feces. Little is known about the effect of management practices on its occurrence and transmission on small-scale cow-calf operations. Identification of risk factors associated with farm practices could help implement effective measures to control E. coli O157:H7. This study quantified the risk of E. coli O157:H7 occurrence associated with cow-calf farm practices using risk modeling. Management practices of small-scale cow-calf operations in OK and LA were assessed through survey-based research. Fecal, water, sediments and water-trough-swab samples were collected to determine the incidence of E. coli O157:H7, and potential on-farm contamination sources and risk factors identified. Association between the occurrence of pathogen and farm practices was determined using two risk assessment models (I and II). Model I determined the association of E. coli O157:H7 occurrence with water source, water container, feed, cattle breed, and herd density, while Model II determined its association with farm cleanliness. For both models, logistic regression was followed using a two-step approach, univariable and multivariable analysis. In OK and LA, E. coli O157:H7 was present in 5.8% and 8.8% fecal, 4.4% and 9.4% water, 10.3% and 9.6% sediments, and 1.5% and 10.6% water-trough-swab samples, respectively. In Model I, univariable analysis identified water container and feed, whereas multivariable analysis identified feed as a significant risk factor. In Model II, the univariable analysis found cleanliness of cattle-contact areas, such as, alleyways, water-trough, chute and equipment, to be a significant risk factor. In multivariable analysis, only the cleanliness of water-trough was identified to be a significant risk factor. Results from the study could aid in the development of on-farm best management practices for the reduction of E. coli O157:H7.

Effectiveness of Bacteriophages against Biofilm-Forming Shiga-Toxigenic Escherichia coli In Vitro and on Food-Contact Surfaces.

(1) Background: Formation of biofilms on food-contact surfaces by Shiga-toxigenic Escherichia coli (STEC) can pose a significant challenge to the food industry, making conventional control methods insufficient. Targeted use of bacteriophages to disrupt these biofilms could reduce this problem. Previously isolated and characterized bacteriophages (n = 52) were evaluated against STEC biofilms in vitro and on food-contact surfaces. (2) Methods: Phage treatments (9 logs PFU/mL) in phosphate-buffered saline were used individually or as cocktails. Biofilms of STEC (O157, O26, O45, O103, O111, O121, and O145) were formed in 96-well micro-titer plates (7 logs CFU/mL; 24 h) or on stainless steel (SS) and high-density polyethylene (HDPE) coupons (9 logs CFU/cm2; 7 h), followed by phage treatment. Biofilm disruption was measured in vitro at 0, 3, and 6 h as a change in optical density (A595). Coupons were treated with STEC serotype-specific phage-cocktails or a 21-phage cocktail (3 phages/serotype) for 0, 3, 6, and 16 h, and surviving STEC populations were enumerated. (3) Results: Of the 52 phages, 77% showed STEC biofilm disruption in vitro. Serotype-specific phage treatments reduced pathogen population within the biofilms by 1.9-4.1 and 2.3-5.6 logs CFU/cm2, while the 21-phage cocktail reduced it by 4.0 and 4.8 logs CFU/cm2 on SS and HDPE, respectively. (4) Conclusions: Bacteriophages can be used to reduce STEC and their biofilms.

Effectiveness of Bacteriophages Against Biofilm-Forming Shiga-Toxigenic Escherichia coli on Leafy Greens and Cucumbers.

Background: Shiga-toxigenic Escherichia coli (STEC) have caused several produce-associated outbreaks, making it challenging to control these pathogens. Bacteriophages could serve as effective biocontrol. Materials and Methods: Spinach, lettuce, and cucumbers, inoculated with STEC (O157, O26, O45, O103, O111, O121, O145), were treated with lytic bacteriophages and stored at 4°C for 3 days. Surviving STEC were enumerated and observed under scanning electron microscope (SEM), and data analyzed using one-way analysis of variance (ANOVA) (p < 0.05). Results: Bacteriophage treatments significantly reduced STEC populations, compared with the control (p < 0.05). On spinach and romaine, STEC O26, O45, and O103 were reduced to undetectable levels and STEC O157, O111, O121, and O145 by ∼2 logs CFU/cm2. Multiserotype phage cocktail reduced STEC on leafy greens by 1.4 CFU/cm2 and on cucumbers by 1.7 logs CFU/cucumber. Clusters of STEC cells, surrounded by extracellular matrix, were observed under SEM of positive control, whereas phage-treated produce surface showed fewer cells, with cellular damage. Conclusions: Bacteriophages could be utilized as biocontrol against STEC on fresh produce.

Characterization of Bacteriophages Targeting Non-O157 Shiga Toxigenic Escherichia coli.

Non-O157 Shiga toxigenic Escherichia coli (STEC) are an important group of foodborne pathogens, implicated in several outbreaks and recalls in the past 2 decades. It is therefore crucial to devise effective control strategies against these pathogens. Bacteriophages present an attractive alternative to conventional pathogen control methods in the food industry. Bacteriophages, targeting non-O157 STEC (O26, O45, O103, O111, O121, O145), were isolated from beef cattle operations in Oklahoma. Their host range and lytic ability were determined against several ( n = 21) non-O157 STEC isolates, by using the spot-on-lawn assay. Isolated phages were purified, and their morphology was determined under a transmission electron microscope. Infection kinetics of selected phages ( n = 19), particularly adsorption rate, rise period, latent period, and burst size, were determined. Phages were also evaluated for stability at a wide pH range (1 to 11) and temperature range (-80 to 90°C). In total, 45 phages were isolated and classified into Myoviridae, Siphoviridae, or Tectiviridae. The phages had a latent period between 8 and 37 min, a rise period between 19 and 40 min, and a large burst size (12 to 794 virions per infected cell), indicating high lytic activity. Tested phages were stable at pH 5 to 9 for 24 h, whereas a decrease in phage titer was observed at pHs 1, 2, and 11. Phages were stable at 40 and 60°C, except for O103-specific phages. At 70°C, all the phages lost viability after 20 min, except three phages targeting O26 and O121 and one phage targeting O45 and O111 STEC, which remained viable for 60 min. All the phages lost activity after 10 min at 90°C, except one each of O26 and O121 STEC-infecting phages that remained viable for 60 min. Phages remained stable for 90 days under refrigerated (4°C) and frozen (-20 and -80°C) storage. Characterization of phages, targeting diverse non-O157 STEC serotypes, could help in the development of effective biocontrol strategies for this group of pathogens in the food industry.

Isolation and Physiomorphological Characterization of Escherichia coli O157:H7-Infecting Bacteriophages Recovered from Beef Cattle Operations.

Bacteriophages, recovered from beef cattle environment and specifically targeting Escherichia coli O157:H7, were examined for their physiological and morphological characteristics. Degree of bacterial lysis and host range of isolated bacteriophages was determined against 55 isolates of E. coli O157:H7. Morphology of phages was examined under transmission electron microscope. Phage growth parameters, particularly rate of adsorption, rise period, latent period, and burst size were also determined. The stability of isolated phages was tested at acidic and alkaline pH, at high temperatures, and in cold storage. A total of 7 phages were isolated which showed lytic activity against 50 out of 55 isolates of E. coli O157:H7. Based on the morphology, phages were classified into Myoviridae or Siphoviridae family. Phages had a rise period between 19 and 40 min, a short latent period between 12 and 30 min, and a large burst size (89-631 virions per infected cell), indicating high lytic activity. Phages remained stable for 24 h at a wide pH (1-11) and temperature range (40-60°C) and for 90 d in cold storage. Characterization of bacteriophages, with a diverse host range of E. coli O157:H7, could aid in the development of effective biocontrol strategies for this pathogen in the food industry.

Apple, carrot, and hibiscus edible films containing the plant antimicrobials carvacrol and cinnamaldehyde inactivate Salmonella Newport on organic leafy greens in sealed plastic bags.

The objective of this study was to investigate the antimicrobial effects of carvacrol and cinnamaldehyde incorporated into apple, carrot, and hibiscus-based edible films against Salmonella Newport in bagged organic leafy greens. The leafy greens tested included organic Romaine and Iceberg lettuce, and mature and baby spinach. Each leafy green sample was washed, dip inoculated with S. Newport (107 CFU/mL), and dried. Each sample was put into a Ziploc® bag. Edible films pieces were put into the Ziploc bag and mixed well. The bags were sealed and stored at 4 °C. Samples were taken at days 0, 3, and 7 for enumeration of survivors. On all leafy greens, 3% carvacrol films showed the best bactericidal effects against Salmonella. All 3 types of 3% carvacrol films reduced the Salmonella population by 5 log10 CFU/g at day 0 and 1.5% carvacrol films reduced Salmonella by 1 to 4 log10 CFU/g at day 7. The films with 3% cinnamaldehyde showed 0.5 to 3 log reductions on different leafy greens at day 7. The films with 0.5% and 1.5% cinnamaldehyde and 0.5% carvacrol also showed varied reductions on different types of leafy greens. Edible films were the most effective against Salmonella on Iceberg lettuce. This study demonstrates the potential of edible films incorporated with carvacrol and cinnamaldehyde to inactivate S. Newport on organic leafy greens.

Prevalence and concentration of Salmonella and Campylobacter in the processing environment of small-scale pastured broiler farms.

A growing niche in the locally grown food movement is the small-scale production of broiler chickens using the pasture-raised poultry production model. Limited research exists that focuses on Salmonella and Campylobacter contamination in the environment associated with on-farm processing of pasture-raised broilers. The objective of this study was to establish data relative to Salmonella and Campylobacter prevalence and concentration in soil and mortality compost resulting from prior processing waste disposal in the small-scale, on-farm broiler processing environment. Salmonella and Campylobacter concentrations were determined in soil (n = 42), compost (n = 39), and processing wastewater (PWW; n = 46) samples from 4 small broiler farms using a 3-tube most probable number (MPN) method for Salmonella and direct plating method for Campylobacter. Salmonella prevalence and concentration (mean log10 MPN per sample weight or volume) in soil [60%, 0.97 (95% CI: 0.66 to 1.27)], compost [64%, 0.95 (95% CI: 0.66 to 1.24)], and wastewater [48%, 1.29 (95% CI: 0.87 to 1.71)] were not significantly different (P > 0.05). Although Campylobacter prevalence was not significantly different by sample type (64.3, 64.3, and 45.7% in soil, compost, and PWW, respectively), the concentration (mean log10 cfu) of this pathogen was significantly lower (P < 0.05) in wastewater [2.19 (95% CI: 0.36 to 3.03)] samples compared with soil [3.08 (95% CI: 2.23 to 3.94)], and compost [3.83 (95% CI: 2.71 to 4.95)]. These data provide insight into small-scale poultry production waste disposal practices and provides a record of data that may serve as a guide for future improvement of these practices. Further research is needed regarding the small-scale broiler production environment in relation to improving disposal of processing waste for optimum control of human pathogens.

The antimicrobial effects of cinnamon leaf oil against multi-drug resistant Salmonella Newport on organic leafy greens.

There is generally no kill-step when preparing salad vegetables, so there is a greater risk for foodborne illness from contaminated vegetables. Some essential oils have antimicrobial activities and could provide a natural way to reduce pathogens on fresh produce. The objective of this study was to investigate the antimicrobial activity of cinnamon oil wash against Salmonella enterica serotype Newport on organic leafy greens. Organic romaine and iceberg lettuce, and organic baby and mature spinach were inoculated with Salmonella Newport and then dip treated in a phosphate buffered saline (PBS) control and 3 different concentrations (0.1, 0.3, and 0.5% v/v) of cinnamon oil. The treatment time varied at either 1 or 2min, and storage temperature varied at either 4 or 8°C. Samples were collected at days 0, 1, and 3. For romaine and iceberg lettuce, S. Newport was not recovered on day 3 for 2min 0.3% and 0.5% cinnamon oil treatments. For mature spinach, S. Newport was not recovered by day 3 for the 2min 0.3% and 0.5% 4°C treatments. For baby spinach, there was no recovery of S. Newport by day 1 for all 0.5% treatments. Overall, the cinnamon oil treatments were concentration and time dependent with higher concentrations and longer treatment times providing the greatest reduction in S. Newport population on leafy greens. In addition, the treatments had a residual effect with the greatest reduction generally seen on the last day of sampling. Storage temperature did not have a significant effect on the reduction of S. Newport. Based on the results of this study, cinnamon oil has the potential to be used as a treatment option for washing organic baby and mature spinach, and iceberg and romaine lettuces.

Antimicrobial activity of oregano oil against antibiotic-resistant Salmonella enterica on organic leafy greens at varying exposure times and storage temperatures.

The objective of this study was to evaluate the effectiveness of oregano oil on four organic leafy greens (Iceberg and Romaine lettuces and mature and baby spinaches) inoculated with Salmonella Newport as a function of treatment exposure times as well as storage temperatures. Leaf samples were washed, dip inoculated with S. Newport (6-log CFU/ml) and dried. Oregano oil was prepared at 0.1, 0.3, and 0.5% concentrations in sterile phosphate buffered saline (PBS). Inoculated leaves were immersed in the treatment solution for 1 or 2 min, and individually incubated at 4 or 8 °C. Samples were taken at day 0, 1, and 3 for enumeration of survivors. The results showed that oregano oil was effective against S. Newport at all concentrations. S. Newport showed reductions from the PBS control of 0.7-4.8 log CFU/g (Romaine lettuce), 0.8-4.8 log CFU/g (Iceberg lettuce), 0.8-4.9 log CFU/g (mature spinach), and 0.5-4.7 log CFU/g (baby spinach), respectively. The antibacterial activity also increased with exposure time. Leaf samples treated for 2 min generally showed greater reductions (by 1.4-3.2 log CFU/g), than those samples treated for 1 min; however, there was minimal difference in antimicrobial activity among samples stored under refrigeration and abuse temperatures. This study demonstrates the potential of oregano oil to inactivate S. Newport on organic leafy greens.

Inactivation of Listeria monocytogenes on ham and bologna using pectin-based apple, carrot, and hibiscus edible films containing carvacrol and cinnamaldehyde.

UNLABELLED: Edible films can be used as wrapping material on food products to reduce surface contamination. The incorporation of antimicrobials into edible films could serve as an additional barrier against pathogenic and spoilage microorganisms that contaminate food surfaces. The objective of this study was to investigate the antimicrobial effects of carvacrol and cinnamaldehyde, incorporated into apple, carrot, and hibiscus-based edible films against Listeria monocytogenes on contaminated ham and bologna. Ham or bologna samples were inoculated with L. monocytogenes and dried for 30 min, then surface wrapped with edible films containing the antimicrobials at various concentrations. The inoculated, film-wrapped samples were stored at 4 °C. Samples were taken at day 0, 3, and 7 for enumeration of surviving L. monocytogenes by plating on appropriate media. Carvacrol films showed better antimicrobial activity than cinnamaldehyde films. Compared to control films without antimicrobials, films with 3% carvacrol induced 1 to 3, 2 to 3, and 2 to 3 log CFU/g reductions on ham and bologna at day 0, 3, and 7, respectively. Corresponding reductions with 1.5% carvacrol were 0.5 to 1, 1 to 1.5, and 1 to 2 logs, respectively. At day 7, films with 3% cinnamaldehyde reduced L. monocytogenes population by 0.5 to 1.5 and 0.5 to 1.0 logs on ham and bologna, respectively. Inactivation by apple films was greater than that by carrot or hibiscus films. Apple films containing 3% carvacrol reduced L. monocytogenes population on ham by 3 logs CFU/g on day 0 which was 1 to 2 logs greater than that by carrot and hibiscus films. Films were more effective on ham than on bologna. The food industry and consumers could use these films to control surface contamination by pathogenic microorganisms. PRACTICAL APPLICATION: Antimicrobial edible, food-compatible film wraps prepared from apples, carrots, and hibiscus calyces can be used by the food industry to inactivate Listeria monocytogenes on widely consumed ready to eat meat products such as bologna and ham. This study provides a scientific basis for large-scale application of edible fruit- and vegetable-based antimicrobial films on foods to improve microbial food safety.

Antimicrobial activity of apple, hibiscus, olive, and hydrogen peroxide formulations against Salmonella enterica on organic leafy greens.

Salmonella enterica is one of the most common bacterial pathogens implicated in foodborne outbreaks involving fresh produce in the last decade. In an effort to discover natural antimicrobials for use on fresh produce, the objective of the present study was to evaluate the effectiveness of different antimicrobial plant extract-concentrate formulations on four types of organic leafy greens inoculated with S. enterica serovar Newport. The leafy greens tested included organic romaine and iceberg lettuce, and organic adult and baby spinach. Each leaf sample was washed, dip inoculated with Salmonella Newport (10(6) CFU/ml), and dried. Apple and olive extract formulations were prepared at 1, 3, and 5% concentrations, and hibiscus concentrates were prepared at 10, 20, and 30%. Inoculated leaves were immersed in the treatment solution for 2 min and individually incubated at 4°C. After incubation, samples were taken on days 0, 1, and 3 for enumeration of survivors. Our results showed that the antimicrobial activity was both concentration and time dependent. Olive extract exhibited the greatest antimicrobial activity, resulting in 2- to 3-log CFU/g reductions for each concentration and type of leafy green by day 3. Apple extract showed 1- to 2-log CFU/g reductions by day 3 on various leafy greens. Hibiscus concentrate showed an overall reduction of 1 log CFU/g for all leafy greens. The maximum reduction by hydrogen peroxide (3%) was about 1 log CFU/g. The antimicrobial activity was also tested on the background microflora of organic leafy greens, and reductions ranged from 0 to 2.8 log. This study demonstrates the potential of natural plant extract formulations to inactivate Salmonella Newport on organic leafy greens.

Assessing the cross contamination and transfer rates of Salmonella enterica from chicken to lettuce under different food-handling scenarios.

Cross contamination of foodborne pathogens from raw meats to ready-to-eat foods has caused a number of foodborne outbreaks. The cross contamination and transfer rates of Salmonella enterica from chicken to lettuce under various food-handling scenarios were determined. The following scenarios were tested: in scenario 1, cutting board and knife used to cut chicken (10(6) CFU/g) were also used for cutting lettuce, without washing; in scenario 2, cutting board and knife were washed with water separately after cutting chicken, and subsequently used for cutting lettuce; and in scenario 3, cutting board and knife were thoroughly washed with soap and hot water after cutting chicken, and before cutting lettuce. In each scenario, cutting board, knife, chicken and lettuce were sampled for population of S. enterica. For scenario 1, both before and after cutting lettuce, the cutting board and knife each had about 2 logs CFU/cm(2) of S. enterica, respectively. The cut lettuce had about 3 logs CFU/g of S. enterica. In scenario 2, fewer organisms (0.5-2.4 logs CFU/g or cm(2)) were transferred. The transfer rates in both scenarios ranged from 0.02 to 75%. However, in scenario 3, <1 log CFU/g or cm(2) organisms were detected on lettuce, cutting board or knife, after washing and cutting lettuce. This shows that the FDA recommended practice for cleaning cutting boards is effective in removing S. enterica and preventing cross contamination.

Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants.

The effectiveness of current antimicrobial interventions used in reducing the prevalence or load of Escherichia coli O157 and indicator organisms on cattle hides and carcasses at two commercial beef processing plants was evaluated. Sponge sampling of beef cattle was performed at five locations from the initial entry of the animals to the slaughter floor to the exit of carcasses from the "hotbox" cooler. For each sample, E. coli O157 prevalence was determined and total aerobic bacteria, Enterobacteriaceae, and E. coli O157 were enumerated. E. coli O157 was found on 76% of animal hides coming into the plants, but no carcasses leaving the cooler were identified as contaminated with E. coli O157. A positive relationship was seen between the incidence of E. coli O157 in hide samples and that in preevisceration samples. Aerobic plate counts and Enterobacteriaceae counts averaged 7.8 and 6.2 log CFU/100 cm2, respectively, on hides, and 1.4 and 0.4 log CFU/100 cm2, respectively, on chilled carcasses. Aerobic plate counts and Enterobacteriaceae counts on preevisceration carcasses were significantly related to the respective levels on the corresponding hides; the carcasses of animals whose hides carried higher numbers of bacteria were more likely to carry higher numbers of bacteria. Implementation of the sampling protocol described here would allow processors to evaluate the efficacy of on-line antimicrobial interventions and allow industrywide benchmarking of hygienic practices.

Protocol for evaluating the efficacy of cetylpyridinium chloride as a beef hide intervention.

The objective of this study was to establish the necessary protocols and assess the efficacy of cetylpyridinium chloride (CPC) as an antimicrobial intervention on beef cattle hides. Experiments using CPC were conducted to determine (i) the methods of neutralization needed to obtain valid efficacy measurements, (ii) the effect of concentration and dwell time after treatment, (iii) the effect of CPC on hide and carcass microbial populations when cattle were treated at a feedlot and then transported to a processing facility for harvest, and (iv) the effectiveness of spray pressure and two-spray combinations of CPC and water to reduce hide microbial populations. Residual CPC in hide sponge samples prevented bacterial growth. Dey-Engley neutralization media at 7.8% and a centrifugation step were necessary to overcome this problem. All dwell times, ranging from 30 s to 4 h, after 1% CPC application to cattle hides resulted in aerobic plate counts and Enterobacteriaceae counts 1.5 log CFU/100 cm2 lower than controls. The most effective dose of CPC was 1%, which reduced aerobic plate counts and Enterobacteriaceae counts 2 and 1 log CFU/100 cm2, respectively. Low-pressure application of 1% CPC at the feedlot, transport to the processing facility, and harvest within 5 h of application resulted in no effect on Escherichia coli O157 prevalence on hides or preevisceration carcasses. Two high-pressure CPC washes lowered aerobic plate counts and Enterobacteriaceae counts by 4 log CFU/100 cm2, and two medium-pressure CPC washes were only slightly less effective. These results indicate that under the proper conditions, CPC may still be effective for reducing microbial populations on cattle hides. Further study is warranted to determine if this effect will result in reduction of hide-to-carcass contamination during processing.