Rapid detection of pathogenic E. coli based on CRISPR Cas system.

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.

Antimicrobial Susceptibility Profile of Pathogenic and Commensal Bacteria Recovered from Cattle and Goat Farms.

The use of antibiotics in food animals results to antimicrobial resistant bacteria that complicates the ability to treat infections. The purpose of this study was to investigate the prevalence of pathogenic and commensal bacteria in soil, water, manure, and milk from cattle and goat farms. A total of 285 environmental and 81 milk samples were analyzed for Enterobacteriaceae by using biochemical and PCR techniques. Susceptibility to antibiotics was determined by the Kirby-Bauer disk diffusion technique. A total of 15 different Enterobacteriaceae species were identified from goat and cattle farms. Manure had significantly higher (p < 0.05) Enterobacteriaceae (52.0%) than soil (37.2%), trough water (5.4%), and runoff water (5.4%). There was a significant difference (p < 0.05) in Enterobacteriaceae in goat milk (53.9%) and cow milk (46.2%). Enterobacteriaceae from environment showed 100% resistance to novobiocin, erythromycin, and vancomycin E. coli O157:H7, Salmonella spp., Enterococcus spp., and Listeria monocytogenes displayed three, five, six, and ten. AMR patterns, respectively. NOV-TET-ERY-VAN was the most common phenotype observed in all isolates. Our study suggest that cattle and goat farms are reservoirs of multidrug-resistant bacteria. Food animal producers should be informed on the prudent use of antimicrobials, good agricultural practices, and biosecurity measures.

Incidence of Drug-Resistant Enterobacteriaceae Strains in Organic and Conventional Watermelons Grown in Tennessee.

The production and consumption of organic fresh produce have constantly increased since the 1990s. Consumers prefer organic produce because it does not contain synthetic chemical residues that are often implicated in health problems. The contamination of fresh produce by pathogenic Enterobacteriaceae strains remains a major challenge, and is responsible for frequent foodborne disease outbreaks. The use of antibiotics has proved an effective treatment, but the increase in occurrences of antibiotic resistance is becoming a health challenge. This study seeks to establish the presence of antimicrobial resistance in Enterobacteriaceae on organic and conventional watermelon fruits. Watermelons used for this study were cultivated at the Tennessee State University Certified Organic Farm, Nashville. At harvest, nine fruits were selected from among fruits lying on plastic mulch, and nine from fruits lying on the soil of both organic and conventional plots. These were placed in sterile sample bags for microbial analysis. Spread plating technique, API 20E, and apiweb software were used for microbial isolation and identification. Identified strains were tested for antimicrobial resistance against 12 common antibiotics. Seventeen Enterobacteriaceae strains were isolated and identified. Isolates were susceptible to gentamycin, ciprofloxacin, and chloramphenicol, but were resistant to cefoxitin. Citrobacter freundii showed a 14.3% resistance to Streptomycin. Pantoea spp. and Providencia rettigeri showed 50% and 100% resistance to tetracycline. Findings from this study confirm the presence of antibiotic-resistant Enterobacteriaceae strains on organic watermelons in Nashville, TN.

Evaluation of Selected Bacteria and Yeast for Probiotic Potential in Poultry Production.

Performance and efficiency of feed utilization in poultry is highly influenced by gut health, which is dependent on intestinal microbial balance. Probiotics are live microbial feed supplements or viable microorganisms that beneficially affect the host animal by improving its gastrointestinal tract (GIT) microbial balance. However, their mode of action and suitable GIT environment favoring their colonization of the GIT is obscure. The probiotic properties of Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces boulardii were evaluated. These microbes were tested in vitro against gastrointestinal conditions for survivability and their ability to attach to the intestinal mucosa. The ability of the microbes to tolerate and survive varying pH levels and bile concentrations was assessed. The microbes were challenged with a pH of 2 to 7 for 5 h and bile concentrations of 1 to 3% for 6 hrs. The microbes were sampled hourly to evaluate growth or decline in colony-forming units (CFU). B. longum, L. Plantarum, and S. boulardii exhibited significantly higher CFU (p < 0.05) at a pH range of 5 to 7, 4 to 7, and 2 to 7, respectively, when compared with other pH levels. L. plantarum had much higher colony-forming units per mL within each pH level, except at pH 2 where S. boulardii was the only microbe to survive over time. While L. plantarum and S. boulardii were able to tolerate the various bile concentrations, B. longum and L. plantarum showed remarkable ability to attach to the intestinal mucosa and to inhibit pathogenic microbes.

Antibiotic Resistant Enterobacteriaceae in Milk Alternatives.

The consumption of non-dairy milk is on the rise due to health benefits. Although there is increasing inclination towards milk alternatives (MA), there is limited data on antibiotic resistant bacteria in these substitutes. The aim of this study was to investigate antimicrobial resistance of bacteria isolated from MA. A total of 138 extracts from almonds (n = 63), cashew nuts (n = 36), and soybeans (n = 39) were analyzed for Enterobacteriaceae. The identification of the bacteria was based on biochemical and PCR methods. Antibiotic sensitivity was determined by using the Kirby-Bauer disk diffusion technique. Overall, 31% (43 of 138) of extracts were positive for Enterobacteriaceae. Ten bacterial species were identified, of which Enterobacter cloacae (42.7%) and Enterobacter cancerogenus (35.4%) were the most predominant species (p < 0.05). Antibiotic resistance was exhibited to vancomycin (88.3%), novobiocin (83.8%), erythromycin (81.1%), which was significantly higher (p < 0.05) than in tetracycline (59.5%), cefpodoxime (30.6%), and nalidixic acid (6.3%). There was no resistance displayed to kanamycin and imipenem. ERY-NOV-VAN-TET and ERY-NOV-CEP-VAN-TET were the most common resistant patterns displayed by Enterobacter cloacae. The findings of this study suggest that MAs, though considered healthy, may be a reservoir of multidrug resistant opportunist pathogens.

Prevalence of Multidrug-Resistant Foodborne Pathogens and Indicator Bacteria from Edible Offal and Muscle Meats in Nashville, Tennessee.

This study investigated the prevalence of antimicrobial-resistant bacteria in retail edible offal and muscle meats in Nashville, Tennessee. A total of 348 retail meats (160 edible offal and 188 muscle) were analyzed for Salmonella enterica serovar, Campylobacter, Escherichia coli, E. coli O157:H7, and enterococci. Bacteria was identified using biochemical and PCR methods. Salmonella enterica serovar (4.4% and 4.3%), Campylobacter (1.9% and 1.1%), E. coli (79.4% and 89.4%), and enterococci (88.1% and 95.7%) was detected in offal and muscle meats, respectively. Chicken liver (9.7%) was most frequently contaminated with Salmonella enterica serovar, followed by ground chicken (6.9%) and chicken wings (4.2%). No Salmonella enterica serovar was detected in beef liver, beef tripe, and ground beef. The prevalence of Campylobacter was 6.9%, 2.3%, and 1.4% in beef liver, ground beef, and ground chicken, respectively. None of the meats were positive for E. coli O157:H7. Resistance of isolates was significantly (p < 0.05) highest in erythromycin (98.3%; 99.1%), followed by tetracycline (94%; 98.3%), vancomycin (88.8%; 92.2%) as compared to chloramphenicol (43.1%; 53.9%), amoxicillin/clavulanic (43.5%; 45.7%), and ciprofloxacin (45.7%; 55.7%) in offal and muscle meats, respectively. Imipenem showed the lowest resistance (0%; 0.9%). A total of 41 multidrug-resistant patterns were displayed. Edible offal could be a source of antibiotic-resistant bacteria.

Ultraviolet inactivation of bacteria and model viruses in coconut water using a collimated beam system.

This study investigated the effect of ultraviolet-C irradiation on the inactivation of microorganisms in coconut water, a highly opaque liquid food (1.01 ± 0.018 absorption coefficient). Ultraviolet-C inactivation kinetics of two bacteriophages (MS2, T1UV) and three surrogate bacteria (Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes) in 0.1% (w/v) peptone and coconut water were investigated. Ultraviolet-C irradiation at 254 nm was applied to stirred samples, using a collimated beam device. A series of known ultraviolet-C doses (0-40 mJ cm-2) were applied for ultraviolet-C treatment except for MS2 where higher doses were delivered (100 mJ cm-2). Inactivation levels of all organisms were proportional to ultraviolet-C dose. At the highest dose of 40 mJ cm-2, three surrogates of pathogenic bacteria were inactivated by more than 5-log10 (p < 0.05) in 0.1% (w/v) peptone and coconut water. Results showed that ultraviolet-C irradiation effectively inactivated bacteriophage and surrogate bacteria in highly opaque coconut water. The log reduction kinetics of microorganisms followed log-linear and exponential models with higher R2 (>0.95) and low root mean square error values. The D10 values of 3, 5.48, and 4.58 mJ cm-2 were obtained from the inactivation of E. coli, S. Typhimurium, and L. monocytogenes, respectively. Models for predicting log reduction as a function of ultraviolet-C irradiation dose were found to be significant (p < 0.05). Fluid optics were the key controlling parameters for efficient microbial inactivation. Therefore, the ultraviolet-C dose must be calculated not only from the incident ultraviolet-C intensity but must also consider the attenuation in the samples. The results from this study imply that adequate log reduction of vegetative cells and model viruses is achievable in coconut water and suggested significant potential for ultraviolet-C treatment of other liquid foods.

A comparative analysis of microbial profile of Guinea fowl and chicken using metagenomic approach.

Probiotics are live microbial feed supplements that promote growth and health to the host by minimizing non-essential and pathogenic microorganisms in the host's gastrointestinal tract (GIT). The campaign to minimize excessive use of antibiotics in poultry production has necessitated development of probiotics with broad application in multiple poultry species. Design of such probiotics requires understanding of the diversity or similarity in microbial profiles among avian species of economic importance. Therefore, the objective of this research was to establish and compare the microbial profiles of the GIT of Guinea fowl and chicken and to establish the microbial diversity or similarity between the two avian species. A metagenomic approach consisting of the amplification and sequence analysis of the hypervariable regions V1-V9 of the 16S rRNA gene was used to identify the GIT microbes. Collectively, we detected more than 150 microbial families. The total number of microbial species detected in the chicken GIT was higher than that found in the Guinea Fowl GIT. Our studies also revealed phylogenetic diversity among the microbial species found in chicken and guinea fowl. The phylum Firmicutes was most abundant in both avian species whereas Phylum Actinobacteria was most abundant in chickens than Guinea fowls. The diversity of the microbial profiles found in broiler chickens and Guinea fowls suggest that the design of effective avian probiotics would require species specificity.

Atmospheric Cold Plasma Inactivation of Salmonella and Escherichia coli on the Surface of Golden Delicious Apples.

The contamination of fruits with human pathogens is a reoccurring concern in the fresh produce industry. Atmospheric cold plasma (ACP) is a potential alternate to customary approaches for non-thermal decontamination of foods. In this study, the efficacy of a dielectric barrier discharge ACP system against Salmonella (Salmonella Typhimurium, ATCC 13311; Salmonella Choleraesuis, ATCC 10708) and Escherichia coli (ATCC 25922, ATCC 11775) was explored. For each bacteria, a two-strain mixture at 8 log10 CFU/ml was spot inoculated on the surface of Golden Delicious apples, air dried, and exposed to ACP at a fixed distance of 35 mm, input power of 200 W for 30, 60, 120, 180, and 240 s. Bacterial inactivation was achieved in all treatment times with highest reduction of 5.3 log10 CFU/cm2 for Salmonella and 5.5 log10 CFU/cm2 for E. coli. Our results showed that reductions were interrelated to exposure time and ranged from 1.3 to 5.3 and 0.6 to 5.5 log10 CFU/cm2 for Salmonella and E. coli, respectively. Salmonella and E. coli significantly decreased (>5.0 log) at 180 and 240 s as compared to 30, 60, and 120 s exposure. Microbial inactivation data was modeled by using Weibull distribution. These findings demonstrate the potential of ACP as a postharvest technology to effectively reduce pathogens on apples, with reference to Salmonella and E. coli.

Prevalence of Multidrug-Resistant Bacteria from U.S.-Grown and Imported Fresh Produce Retailed in Chain Supermarkets and Ethnic Stores of Davidson County, Tennessee.

The aim of this study was to determine whether U.S.-grown and imported fresh produce retailed in ethnic stores and chain supermarkets was a reservoir of antibiotic-resistant bacteria. A total of 360 (129 imported and 231 U.S.-grown) samples of fresh produce were purchased from retail stores and analyzed for Enterobacteriaceae , including three pathogenic bacteria ( Escherichia coli O157:H7, Shigella , and Salmonella ), using standard methods. Presumptive pathogenic isolates were confirmed using PCR. The mean Enterobacteriaceae counts for imported produce were 6.87 ± 0.15 log CFU/g and 7.16 ± 0.11 log CFU/g in ethnic stores and chain supermarkets, respectively. For U.S.-grown produce, the contamination levels were at 8.35 ± 0.17 log CFU/g and 7.52 ± 0.13 log CFU/g in ethnic stores and chain supermarkets, respectively. Salmonella (0 and 0.3%), Shigella (1.7 and 0.6%), E. coli (3.1 and 1.4%), Enterobacter (9.4 and 8.6%), Klebsiella (6.7 and 0.6%), and Serratia (5.8 and 1.4%) were detected in produce from ethnic stores and chain supermarkets, respectively. None of the samples were positive for E. coli O157:H7. Regarding distribution by produce type, leafy vegetables had a significantly (P < 0.05) higher prevalence of Enterobacteriaceae (19.2%) than the other types, followed by root vegetables (6.4%), tomatoes (5.6%), and fruits (3.9%). Antibiotic-resistant Salmonella , Shigella , E. coli , Enterobacter , Klebsiella , and Erwinia bacteria were also isolated from fresh produce. The frequencies of vancomycin resistance (98.1 and 100%) were significantly higher (P < 0.05) than the frequencies of ampicillin resistance (42.3 and 72.9%) for imported and U.S.-grown produce, respectively. Despite the increased attention to the role of imported produce as a source of antimicrobial resistance, this study indicates that U.S.-grown produce is also contaminated with antibiotic-resistant bacteria. Good agricultural practices on the farms and washing of fresh produce before consumption are greatly recommended to avoid possible public health hazards.

Occurrence of Listeria and Enterobacteriaceae in domestic refrigerators.

Consumers' refrigeration practices have a significant impact on the safety and quality of foods. To determine the prevalence and the identity of microorganisms in domestic refrigerators, swab samples were taken from various locations in the refrigerators from 137 households in middle Tennessee. The swabs were inoculated into different media, and standard procedures were used to characterize the isolates. API 20E and API Listeria were used for identification of Enterobacteriaceae and Listeria spp., respectively. The Kirby-Bauer technique was used to test resistance of the isolates. Actual counts for aerobic and Enterobacteriaceae ranged from not detected to 8.53 and 8.39 log CFU per sample, respectively. Klebsiella pneumoniae (23.4%), Klebsiella oxytoca (6.8%), Klebsiella terrigena (4.0%), Enterobacter sakazakii (2.2%), and Yersinia enterocolitica (0.7%) were some of the bacteria of concern that were isolated from domestic refrigerators. Resistance to antibiotics was most common in erythromycin (39.9%), followed by ampicillin (33.8%), cefoxitin (12.8%), tetracycline (5%), streptomycin (4.0%), nalidixic acid (2.1%), kanamycin (1.4%), and colistin (0.7%). None of the isolates tested was resistant to ciprofloxacin or gentamycin. Listeria spp. were also detected in six refrigerators. These findings underline the need for greater consumer education regarding proper refrigerator cleaning and safe food handling practices.

Kinetics of tropomyosin denaturation as a predictive model for verifying thermal processing of beef products.

An enzyme-linked immunosorbent assay (ELISA) was developed to study thermal denaturation of tropomyosin (Tm) using the time-temperature requirements for cooked beef. The ELISA employed a monoclonal antibody (MAb 2C9) raised against bovine Tm for quantifying residual Tm in muscle extracts. The specificity of MAb 2C9 to bovine Tm was demonstrated by Western blot and the analytical validity of ELISA was confirmed by dot blot. Thermal denaturation of Tm, in the temperature range between 54.4 and 70.0 degrees C, showed first-order dependency. Kinetic parameters of Tm denaturation were derived from isothermal heating of beef muscle extract at 54.4, 57.2, 60.0, and 62.8 degrees C. Temperature dependency of the rate constant (k) was demonstrated by Arrhenius plot; the activation energy (E(a)) of Tm denaturation was determined to be 484 kJ x mol(-1). A mathematic model describing the impact of the heating time-temperature on Tm denaturation was developed. Predicted Tm from the integrated time-temperature model agreed closely with the measured Tm in dynamically heat-processed beef samples. Percent errors between the measured and the predicted values ranged from -5.1 to 5.3%. The kinetic model provides an accurate and reproducible prediction of the impact of actual heating time-temperature on residual Tm in cooked beef. The MAb-based ELISA and kinetic model developed in this study have the potential to be adapted by the meat industry as a quality control tool.

Efficacy of home washing methods in controlling surface microbial contamination on fresh produce.

Much effort has been focused on sanitation of fresh produce at the commercial level; however, few options are available to the consumer. The purpose of this study was to determine the efficacy of different cleaning methods in reducing bacterial contamination on fresh produce in a home setting. Lettuce, broccoli, apples, and tomatoes were inoculated with Listeria innocua and then subjected to combinations of the following cleaning procedures: (i) soak for 2 min in tap water, Veggie Wash solution, 5% vinegar solution, or 13% lemon solution and (ii) rinse under running tap water, rinse and rub under running tap water, brush under running tap water, or wipe with wet/dry paper towel. Presoaking in water before rinsing significantly reduced bacteria in apples, tomatoes, and lettuce, but not in broccoli. Wiping apples and tomatoes with wet or dry paper towel showed lower bacterial reductions compared with soaking and rinsing procedures. Blossom ends of apples were more contaminated than the surface after soaking and rinsing; similar results were observed between flower section and stem of broccoli. Reductions of L. innocua in both tomatoes and apples (2.01 to 2.89 log CFU/g) were more than in lettuce and broccoli (1.41 to 1.88 log CFU/g) when subjected to same washing procedures. Reductions of surface contamination of lettuce after soaking in lemon or vinegar solutions were not significantly different (P > 0.05) from lettuce soaking in cold tap water. Therefore, educators and extension workers might consider it appropriate to instruct consumers to rub or brush fresh produce under cold running tap water before consumption.