Adult Ossabaw Pigs Prefer Fermented Sorghum Tea over Isocaloric Sweetened Water.

Ossabaw pigs (n = 11; 5-gilts, 6-barrows; age 15.6 ± 0.62 SD months) were exposed to a three-choice preference maze to evaluate preference for fermented sorghum teas (FSTs). After conditioning, pigs were exposed, in four sessions, to choices of white FST, sumac FST, and roasted sumac-FST. Then, pigs were exposed, in three sessions, to choices of deionized H2O (-control; avoidance), isocaloric control (+control; deionized H2O and sucrose), and blended FST (3Tea) (equal portions: white, sumac, and roasted sumac). When tea type was evaluated, no clear preference behaviors for tea type were observed (p > 0.10). When the 3Tea and controls were evaluated, pigs consumed minimal control (p < 0.01;18.0 ± 2.21% SEM), and they consumed great but similar volumes of +control and 3Tea (96.6 and 99.0 ± 2.21% SEM, respectively). Likewise, head-in-bowl duration was the least for -control, but 3Tea was the greatest (p < 0.01; 5.6 and 31.9 ± 1.87% SEM, respectively). Head-in-bowl duration for +control was less than 3Tea (p < 0.01; 27.6 vs. 31.9 ± 1.87% SEM). Exploration duration was the greatest in the area with the -control (p < 0.01; 7.1 ± 1.45% SEM), but 3Tea and +control exploration were not different from each other (1.4 and 3.0 ± 1.45% SEM, respectively). Regardless of tea type, adult pigs show preference for FST, even over +control. Adult pigs likely prefer the complexity of flavors, rather than the sweetness alone.

Investigation of the environmental presence of Salmonella spp. in finishing pigs at commercial swine farms in Kansas (USA).

Salmonellosis remains a major foodborne disease threat to public health worldwide. Swine are considered a reservoir for many Salmonella serotypes affecting humans; however, not all serotypes of concern in food animal products cause clinical signs of infection in swine. The objective of this study was to evaluate the presence and distribution of Salmonella spp. in finishing pigs at commercial farms across Kansas (USA). Five farms were selected and sampled when pigs weighed between 125 and 136 kg. Samples were collected and transported to the laboratory for processing following USDA-FSIS guidelines. Susceptibility and resistance profiles were also studied. Fifty-three percent (100/186) of samples were culture positive for Enterobacteriaceae, and 14% (14/100) were confirmed Salmonella positive by PCR with three of five farms having no PCR-positive samples. Salmonella serotype Braenderup was the most common serovar identified in environmental samples, while Salm. Infantis, Agona, and Montevideo were identified in fecal samples. Multidrug resistance patterns were only found in Farm 3, in fecal samples and in one floor sample. The observations reported in this study highlight areas of concern, such as locations prone to fecal contamination, to be considered when cleaning and sanitizing between groups of pigs to decrease presence of Salmonella spp. in farm environments.

Genotypic and Phenotypic Characterization of Antimicrobial and Heavy Metal Tolerance in Salmonella enterica and Escherichia coli Isolates from Swine Feed Mills.

Antimicrobials and heavy metals are commonly used in the animal feed industry. The role of in-feed antimicrobials on the evolution and persistence of resistance in enteric bacteria is not well described. Whole-Genome Sequencing (WGS) is widely used for genetic characterizations of bacterial isolates, including antimicrobial resistance, heavy metal tolerance, virulence factors, and relatedness to other sequenced isolates. The goals of this study were to i) use WGS to characterize Salmonella enterica (n = 33) and Escherichia coli (n = 30) isolated from swine feed and feed mill environments; and ii) investigate their genotypic and phenotypic antimicrobial and heavy metal tolerance. Salmonella isolates belonged to 10 serovars, the most common being Cubana, Senftenberg, and Tennessee. E. coli isolates were grouped into 22 O groups. Phenotypic resistance to at least one antimicrobial was observed in 19 Salmonella (57.6%) and 17 E. coli (56.7%) isolates, whereas multidrug resistance (resistant to ≥3 antimicrobial classes) was observed in four Salmonella (12%) and two E. coli (7%) isolates. Antimicrobial resistance genes were identified in 17 Salmonella (51%) and 29 E. coli (97%), with 11 and 29 isolates possessing genes conferring resistance to multiple antimicrobial classes. Phenotypically, 53% Salmonella and 58% E. coli presented resistance to copper and arsenic. All isolates that possessed the copper resistance operon were resistant to the highest concentration tested (40 mM). Heavy metal tolerance genes to copper and silver were present in 26 Salmonella isolates. Our study showed a strong agreement between predicted and measured resistances when comparing genotypic and phenotypic data for antimicrobial resistance, with an overall concordance of 99% and 98.3% for Salmonella and E. coli, respectively.

Application of Encapsulated and Dry-plated Food Acidulants to Control Salmonella enterica in Raw Meat-based Diets for Dogs.

There is an increasing demand for raw meat-based diets (RMBDs) for dogs, but these foods cannot be heat-pasteurized. Thus, the objective of this study was to evaluate the antimicrobial efficacy of encapsulated and dry-plated glucono delta lactone (GDL), citric acid (CA), and lactic acid (LA) when challenged against Salmonella enterica inoculated in a model raw meat-based diet (RMBDs) for dogs. Nutritionally complete, raw diets were formulated with different levels (1.0, 2.0 and 3.0% (w/w)) of both encapsulated and dry-plated GDL, CA, and LA with both the positive (PC) and the negative controls (NC) without acidulants. The diets were formed into patties of ∼100 g and inoculated with 3-cocktail mixtures of Salmonella enterica serovars, excluding the NC to achieve a final concentration of ∼6.0 Log CFU/patty. Microbial analyses were performed on the inoculated diets and survivors of S. enterica enumerated. Both encapsulated and dry-plated CA and LA had higher log reductions compared to GDL (P < 0.05). However, encapsulated CA and LA at 1.0% (w/w) exhibited higher log reductions (P > 0.05) and preserved product quality compared to the dry-plated acidulants at 1.0%. We concluded that 1.0% (w/w) of encapsulated citric or lactic acids could be successfully applied as an antimicrobial intervention in raw diets for dogs.

The Use of Probiotic Megasphaera elsdenii as a Pre-Harvest Intervention to Reduce Salmonella in Finishing Beef Cattle: An In Vitro Model.

Reducing Salmonella in cattle may mitigate the risk of transmission through the food chain. Megasphaera elsdenii (ME) is a microorganism found naturally in the bovine rumen that can be administered as a probiotic to mitigate ruminal acidosis. Understanding the impact of feeding ME to Salmonella populations in cattle was the objective of this study. Bovine ruminal fluid (RF) and feces were inoculated with antibiotic susceptible or resistant Salmonella and treated with varying concentrations of ME. Salmonella was enumerated at 0, 24, 48, and 72 h using the most probable number (MPN). Volatile fatty acids (VFAs) and pH were recorded from non-inoculated samples. Treating RF with ME did not significantly impact Salmonella concentration or VFA production (p > 0.05). The pH of RF and feces decreased over time (p ≤ 0.05). Salmonella concentration declined in feces, with the largest reduction of 1.92 log MPN/g and 1.05 log MPN/g observed for antibiotic susceptible Salmonella between 0 and 72 h by the 2.5 × 105 CFU/g and control (0.0 CFU/g) concentration of ME, respectively. Treating RF with ME did not impact Salmonella concentration. Salmonella concentration in feces decreased, although ME must be further investigated before a conclusion regarding efficacy in vitro can be determined.

Whole-Genome Sequences of Two Listeria monocytogenes Biofilm Formers.

Listeria monocytogenes strains from different lineages show different biofilm-forming abilities. In this study, two strains of L. monocytogenes were whole genome sequenced using single-molecule real-time (SMRT) technology and characterized.

Inoculation of Weaned Pigs by Feed, Water, and Airborne Transmission of Salmonella enterica Serotype 4,[5],12:i:.

ABSTRACT: Salmonella enterica serotype 4,[5],12:i:- (STM) has become an increasing problem for food safety and has been often detected in swine products. Weanling pigs were exposed to STM-contaminated feed, water, or air to determine possible STM transmission routes. A control group of pigs was included. STM was monitored daily in feces and rectal and nasal swabs. STM colonization was most prevalent in tissues from tonsil, lower intestine, and mesenteric lymph nodes. No differences in lesion severity were observed between inoculated and control pigs. Contaminated feed, water, and aerosolized particles caused infection in weaned pigs; however, no STM colonization was observed in skeletal muscle destined for human consumption. Based on the results from this study, STM contamination in pork products most likely results from cross-contamination of meat by digesta or lymph node tissue during processing.

Salmonella enterica 4,[5],12:i:-, an Emerging Threat for the Swine Feed and Pork Production Industry.

ABSTRACT: Salmonella continues to be a significant cause of foodborne illnesses in human medicine. The Centers for Disease Control and Prevention reported Salmonella as the second leading cause of foodborne illness in the United States and the leading cause of both hospitalizations and deaths. Salmonella enterica 4,[5],12:i:- (STM) is a monophasic variant of Salmonella Typhimurium, and it is an emerging threat to both human and animal health. STM was first identified in the 1980s from poultry products and has become increasingly prevalent in meat products including pork. STM has also been identified in swine farms as well as in feed manufacturing environments and feed itself. Similar pulse-field gel electrophoresis profiles have been observed between human clinical cases and the STM samples originating from swine feed. These related profiles suggest a link between ingestion of contaminated feed by swine and the source of foodborne illness in human. The objective of this article was to better understand the history of STM and the possible pathway from swine feed to table. Continued research is necessary to better understand how STM can enter both the feed supply chain and the pork production chain to avoid contamination of pork products destined for human consumption.

Validation of post-harvest antimicrobial interventions to control Shiga toxin-producing Escherichia coli (STEC) on market hog carcass surfaces.

Although swine are less associated with STEC foodborne disease outbreaks, the potential for swine to serve as a source of STEC infections in human beings cannot be disregarded. This study compared eight USDA-approved antimicrobial intervention technologies to quantify their ability to reduce STEC contamination on market hog carcasses. Hogs were harvested to provide skin-on carcass sides, and eight sides (per three replications) were inoculated with a 7-strain STEC cocktail (ca. 5 log CFU/cm2 across all external and body cavity surfaces). Each side was randomly assigned to a final pre-chill wash treatment administered in a commercial Chad carcass cabinet using a low-volume spray [3% lactic acid (lLA; 130 °F), 400 ppm peracetic acid (lPAA), or acidified 400 ppm peracetic acid (laPAA)] or a high-volume wash [ambient water (hAW), 400 ppm PAA (hPAA), 400 or 600 ppm hypobromous acid (hDBDMH), or 71 °C water (hHW)] treatment according to a randomized complete block study design. Post-treatment (after a 10-min hanging drip) and post-chilling (18 h at 2 °C) STEC reductions were compared for external skin-on surfaces and internal body cavity lean surface tissue. Post-treatment color changes were determined for lean, adipose, and skin carcass surfaces before and after chilling. When applied to the external, skin-on surface, the hHW, hPAA, and hDBDMH600 deluge washes were significantly (P ≤ 0.05) more effective than the other intervention technologies, achieving STEC reductions of 3.8, 3.4, and 3.2 log CFU/cm2, respectively. In comparison, the hAW control reduced STEC by 1.7-log CFU/cm2 on the external, skin-on surface. The carcass interventions were less effective at reducing STEC populations attached to interior body cavity (diaphragm region), with post-chill populations reduced by 0.9-2.2 log cycles, while the hAW control wash achieved a 0.6-log reduction. None of the treatments negatively impacted instrumental carcass color. While all market hog carcass interventions reduced STEC populations, larger reductions were observed when applied to the external, skin-on surface, with the largest reductions achieved by the hHW, hPAA, and hDBDMH600 deluge washes. These data equip pork processors with the information necessary to support decision-making when selecting an intervention technology.

Genotypic and Phenotypic Characterization of Antimicrobial Resistance Profiles in Non-typhoidal Salmonella enterica Strains Isolated From Cambodian Informal Markets.

Non-typhoidal Salmonella enterica is a pathogen of global importance, particularly in low and middle-income countries (LMICs). The presence of antimicrobial resistant (AMR) strains in market environments poses a serious health threat to consumers. In this study we identified and characterized the genotypic and phenotypic AMR profiles of 81 environmental S. enterica strains isolated from samples from informal markets in Cambodia in 2018-2019. AMR genotypes were retrieved from the NCBI Pathogen Detection website (https://www.ncbi.nlm.nih.gov/pathogens/) and using ResFinder (https://cge.cbs.dtu.dk/services/) Salmonella pathogenicity islands (SPIs) were identified with SPIFinder (https://cge.cbs.dtu.dk/services/). Susceptibility testing was performed by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) standard guidelines M100-S22 using the National Antimicrobial Resistance Monitoring System (NARMS) Sensititre Gram Negative plate. A total of 17 unique AMR genes were detected in 53% (43/81) of the isolates, including those encoding tetracycline, beta-lactam, sulfonamide, quinolone, aminoglycoside, phenicol, and trimethoprim resistance. A total of 10 SPIs (SPI-1, 3-5, 8, 9, 12-14, and centisome 63 [C63PI]) were detected in 59 isolates. C63PI, an iron transport system in SPI-1, was observed in 56% of the isolates (n = 46). SPI-1, SPI-4, and SPI-9 were present in 13, 2, and 5% of the isolates, respectively. The most common phenotypic resistances were observed to tetracycline (47%; n = 38), ampicillin (37%; n = 30), streptomycin (20%; n = 16), chloramphenicol (17%; n = 14), and trimethoprim-sulfamethoxazole (16%; n = 13). This study contributes to understanding the AMR genes present in S. enterica isolates from informal markets in Cambodia, as well as support domestic epidemiological investigations of multidrug resistance (MDR) profiles.

Effects of moisture and temperature on Salmonella survivability in beef tallow, white grease, and chicken rendered fat.

High moisture levels introduced to fats after the rendering process can lead to Salmonella presence and growth. Limited research on strategies to eliminate pathogens in these environments are available. Rendered fat characteristics, such as water activity and fatty acids composition, may contribute to Salmonella survivability. The purpose of this research was to evaluate the effects of moisture levels (0%, 0.5%, 1%, and 3%), storage temperatures (48 and 76 ˚C), and fat characteristics on the growth and survival of Salmonella in beef tallow, white grease and poultry fat samples. Samples were inoculated with a high (~108 CFU/mL) and a low (~105 CFU/mL) Salmonella cocktail (S. Senftenberg, S. Newport, S. Thompson and S. Infantis). Samples were stored for up to 5 days at 48 and 76 ºC. Remaining population was evaluated daily with and without enrichment step. Death rates were calculated using Weibull model for each temperature and moisture level. Only temperature had an effect (P < 0.05) on Salmonella inactivation, while no effect between moisture and/or inoculum level were observed. When all products were challenged at 76 ˚C, counts were below detectable limits after 6 hours. At 48˚C a progressive decline in Salmonella population was observed within 3 days for both beef tallow and white grease when high inoculum was used for the challenge study. Salmonella was below detectable limit within 4 days for both fat types when a low inoculum was instead applied. This research identified the effect of moisture and temperature in rendered fat samples contaminated with Salmonella and underlined the need to use time-moisture-temperature data to minimize microbial growth during transportation and storage.

Antifungal Packaging Film to Maintain Quality and Control Postharvest Diseases in Strawberries.

Strawberries are a highly perishable crop with postharvest losses than reach up to 40%. Cost-effective and sustainable technologies in the form of active packing films can provide a solution. Antimicrobial packaging films were produced from pullulan polymer and Solid Lipid Nanoparticles (SLN) containing 1% w/w cinnamaldehyde. Strawberries were stored at 3 °C for 10 days and 12 °C for 6 days. Microbial and physical quality parameters were evaluated during storage. A reduction of approximately 2 Log CFU/g in yeast and mold population was observed for treated strawberries stored at 3 °C as compared to the control (p < 0.05). Yeast and molds counts were significantly lower on day 2 and 4 at 12 °C for treated samples. Strawberries packaged with the active films demonstrated lower respiration rates and the retention of bright red color at both storage temperatures. Active pullulan films were effective in maintaining the desired strawberry quality and reducing fungal decay during refrigerated storage.

The use of a CDC biofilm reactor to grow multi-strain Listeria monocytogenes biofilm.

Listeria monocytogenes is one of the most concerning pathogens for the food industry due to its ability to form biofilms, particularly in difficult-to-clean sites of processing facilities. There is a current industry-wide lack of data to refer to when selecting a strategy to control L. monocytogenes biofilms in the food premises. Many strategies have been developed to study biofilm formation of bacteria; however, few have targeted L. monocytogenes biofilms under dynamic conditions. This study addresses the biofilm formation ability of L. monocytogenes on stainless steel and polycarbonate under dynamic conditions using TSBYE or BHI as media culture at 30 °C or 37 °C. Higher cell counts were recovered at 30 °C in TSBYE on polycarbonate while lower counts were obtained at 37 °C in BHI on stainless steel (P < 0.05). Nonetheless, all factors (temperature, media and material) were statistically significant (P < 0.05) and an interaction between temperature and media was observed (P < 0.05). To our knowledge, this work represents an initial framework to develop L. monocytogenes biofilms under different dynamic conditions. The use of CDC Biofilm Reactor is not widely used yet in the food industry and represent a novel approach to help sanitary control strategies implementation.

Draft Genome Sequences of 81 Salmonella enterica Strains from Informal Markets in Cambodia.

Salmonella enterica is an important global pathogen due to its contribution to human morbidity and death. The presence of S. enterica in Southeast Asian informal markets is amplified by cross-contamination between market surfaces and food products. Here, we describe the draft genome sequences of 81 Salmonella enterica isolates from informal markets in Cambodia.

Characterization of Salmonella enterica Isolates from Selected U.S. Swine Feed Mills by Whole-Genome Sequencing.

Every year salmonellosis is responsible for $2.3 billion in costs to the U.S. food industry, with nearly 6% of the reported cases associated with pork and/or pork products. Several studies have demonstrated the role of pigs as Salmonella reservoirs. Furthermore, this pathogen has been identified as a potential biological hazard in many livestock feeds. The overall objective of this research was to characterize Salmonella enterica isolates in selected U.S. swine feed mills by whole-genome sequencing (WGS) and evaluate isolates in association with the season and feed production stages. Salmonella isolates were collected from 11 facilities during a previous study. Samples were analyzed for Salmonella prevalence following the U.S. Department of Agriculture guidelines and confirmed by PCR. WGS was carried out on either the MiSeq or NextSeq sequencer. De novo genome assemblies were obtained with the Shovill pipeline, version 0.9. ResFinder and SPIFinder were used to identify antibiotic resistance genes and pathogenicity islands. Finally, their phylogenetic relationship and diversity were determined by core genome multilocus sequence typing. Overall, our analysis showed the presence of S. enterica in the feed mill environment. Isolates belonged to 16 different serotypes. Salmonella Agona, Salmonella Mbandaka, Salmonella Senfenberg, and Salmonella Scharzengrund were the most frequently found, and 18 single-nucleotide polymorphism clusters were identified. In silico analysis showed that 40% of the strains carried at least one antimicrobial resistance gene. All isolates in this study could be considered of public health concern and pathogenic potential. Our findings underscore the potential role of the feed mill environment as the pathogen entry route into the human food value chain.

Evaluation of heating effects on the morphology and membrane structure of Escherichia coli using electron paramagnetic resonance spectroscopy.

Bacterial cell characteristics, such as size, morphology, and membrane integrity, are affected by environmental conditions. Thermal treatment results in related structural changes, extent of which is determined by the microorganism's survival skills and inactivation kinetics. The objective of this study was to characterize changes in cell structure of Escherichia coli during heating using the combined analysis of dynamic light scattering (DLS), electron paramagnetic resonance (EPR) spectroscopy, and transmission electron microscopy (TEM) techniques. The size of E. coli cells increased from 2.3 µm to 3.0 µm with heating up to 50 °C followed by a shrinkage with further heating up to 70 °C. The morphological changes were verified using transmission electron microscopy. Related changes in membrane integrity was quantified via the mobility of 16-doxylstearic acid (16-DSA) spin probe using EPR spectroscopy. Two order parameters S1 and S2 defined on x- and y-axes, respectively, decreased with increasing temperature indicating loss of membrane integrity. The combined techniques as in this study can be used to further understand factors that play role in survival behavior of microorganisms.

Seasonal Presence of Salmonella spp., Salmonella Typhimurium and Its Monophasic Variant Serotype I 4,[5],12:i:-, in Selected United States Swine Feed Mills.

This study evaluated the seasonal prevalence and distribution of Salmonella spp., Salmonella enterica serovar Typhimurium (ST) and its monophasic variant 4,[5],12:i:- (STM), in selected swine feed mills across the United States. Eleven facilities were selected for this study and 12 sites were sampled within each mill during fall 2016, early spring 2017, and summer 2017. Samples were evaluated following the USDA-FSIS guidelines for Salmonella isolation and culture positive samples were analyzed by polymerase chain reaction (PCR). A multiplex real-time PCR was used to differentiate ST and STM from other serotypes. Associations between season, mill, and sample site with Salmonella presence were investigated using generalized linear mixed models. Both season (p < 0.007) and mill (p < 0.005) were significantly associated with Salmonella spp. presence. Fall months were associated with a higher Salmonella prevalence (13.2%) compared with early spring and summer. A total of five isolates, among the 383 samples were serotyped as ST and STM. These two serotypes showed a similar seasonal presence throughout the study, being found during fall and summer seasons. These findings demonstrated the seasonal presence of Salmonella spp. in feed mills and the role of these environments as potential pathogen entry route into the human food chain.

Effects of different moisture and temperature levels on Salmonella survival in poultry fat.

Fat products have been historically thought to have too low water activity to harbor pathogens. However, it has been recently reported that high moisture levels in fats may lead to Salmonella presence and growth. Limited research on strategies to eliminate pathogens in these environments is available, and the mechanisms contributing to microbial presence and growth are not yet well understood. The purpose of this research was to evaluate the effects of moisture levels and storage temperatures on the growth and survival of Salmonella in poultry fat. Samples were stored for 7 d at 48°C or 76°C and remaining Salmonella was evaluated. When poultry fat was challenged with a wet high inoculum, more than a 4 log CFU/mL difference in Salmonella population was observed with 1% and 3% moisture levels at 48°C after 5 d (P < 0.05). No differences between moisture levels (P > 0.05) were observed when samples were tested with a wet low inoculum. Counts below detectable limits were observed after 24 h in samples challenged at 76°C, regardless of inoculum level. When poultry fat was stored at 48°C and inoculated with low levels of Salmonella spp., bacterial growth was influenced only by time (P < 0.05) and not affected (P > 0.05) by moisture level. However, when poultry fat was stored at 48°C and inoculated with high levels of Salmonella spp., bacterial decrease was easier (P < 0.05) in samples containing greater moisture. This research suggests that residual moisture in containers during transportation of poultry fat largely does not affect Salmonella spp. growth.

Evaluation of Salmonella presence in selected United States feed mills.

Salmonella is a pathogen of public health concern. Each year, Salmonella infections cost to the food industry approximately $2.3 billion and 33% of the reported cases are associated with beef, poultry, or pork. Pathogen presence in feed mills can represent one of the many potential routes for entry and transmission into the food production chain. Nevertheless, little is known about Salmonella incidence and association with these types of environments. The objective of this study was to investigate Salmonella presence in different feed mills across the United States. Eleven facilities were selected in eight states and 12 sites were sampled within each feed mill. Samples were analyzed following the FSIS guidelines for isolation and identification of Salmonella. Positive isolates were further investigated by a PCR analysis targeting the invA gene to differentiate for Salmonella enterica. The total number of environmental samples collected was 237: 66% resulted culture positive and 13.1% were PCR positive. All sampled feed mills had at least one culture positive site and following production flow the number of positive samples decreased from ingredient receiving to final product. These preliminary results demonstrate the presence of Salmonella in selected United States feed mills and suggest their potential role as vehicle for pathogen transmission and spread into the food production chain.

Draft Genome Sequences of 57 Salmonella enterica Strains from Selected U.S. Swine Feed Mills.

The number of Salmonella infection cases linked to pork products has increased. Pathogen presence in the feed mill environment is one of the many potential transmission routes into the food production chain. Here, we describe the draft genome sequences of 57 Salmonella enterica isolates from selected U.S. swine feed mills.