Texture and quality of chicken sausage formulated with woody breast meat.

Woody breast (WB) myopathy is a quality defect, afflicting a large portion of commercial broilers to some degree. The WB myopathy is commonly attributed to rapid bird growth and characterized by excessive fibrosis within the pectoralis major, which is thought to cause the palpably hardened texture observed in the afflicted breast meat. These phenotypically tough breast fillets are not marketed for traditional intact muscle products owing to poor quality and eating experience. Potential avenues for these afflicted breast fillets include their use in formulation of fresh and cooked sausages. Two degrees of WB fillets (moderate and severe) were used as a replacement for normal (unafflicted) breast fillet meat at levels of 25, 50, and 100%, in a sausage formulation with 1.5% salt and 15% chicken fat. All 6 treatments were compared with a control formulation (100% normal breast meat) and analyzed for texture profile, cook loss, color, and proximate composition. Moisture and fat content for all formulations were similar (P = 0.95 and P = 0.33, respectively), but with increase in the inclusion rate of WB meat, lower protein content (P < 0.01) was observed. Raw sausage color indicated a lighter (P < 0.05) color for the control sausage (100% normal) than with both 100% moderate and 100% severe formulations. Similarly, sausages containing 100% severe WB meat were the darkest (L∗; P < 0.05), but they were similar to sausages containing 100% moderate (P > 0.05) WB meat. Texture profile analysis indicated a decrease in hardness, cohesiveness, and springiness with use of 100% severe WB meat, while inclusion of lower proportions of severe WB meat resulted in similar textural characteristics. These results indicate the possibility of using WB fillet meat in a sausage formulation with minimal impact on sausage texture profiles.

Validation of Commonly Used Antimicrobial Interventions on Bob Veal Carcasses for Reducing Shiga Toxin-Producing Escherichia coli Surrogate Populations.

ABSTRACT: Ruminants are natural reservoirs of Shiga toxin-producing Escherichia coli (STEC), and the STEC can be easily transferred to carcasses during the conversion of animals to meat. Three experiments were conducted to validate the efficacy of lactic acid (LA; 4%), peroxyacetic acid (PAA; 300 ppm), and hot water (HW; 80°C) for their individual or combined abilities to reduce STEC surrogates on bob veal carcasses pre- and postchill and through fabrication. In experiment 1, hot carcasses (n = 9) were inoculated with a five-strain cocktail (ca. 8 log CFU/mL) containing rifampin-resistant surrogate E. coli (BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431) and then treated with HW, LA, or PAA. Carcasses were then chilled (0 ± 1°C; 24 h) and split in half, and each side was treated with either LA or PAA. In experiment 2, hot carcasses (n = 3) were inoculated and chilled (24 h). After 24 h, the carcasses were split, and each side was treated with either LA or PAA. For experiment 3, carcasses (n = 3) were chilled for 24 h, split, inoculated, and treated with either LA or PAA. After chilling, carcasses from all three experiments were fabricated to subprimals and the cut surfaces were sampled to determine the translocation of bacteria. Experiment 1 showed that LA+LA was the most effective (P ≤ 0.05) treatment for reducing surrogate E. coli on veal. In experiments 2 and 3, LA and PAA were similar (P > 0.05) in their abilities to reduce E. coli on chilled veal carcasses. In experiments 1 and 2, all antimicrobial treatments resulted in undetectable levels (<0.2 log CFU/cm2) of surrogate E. coli on cut surfaces after fabrication, whereas low levels (1.7 and 1.0 log CFU/cm2 for LA and PAA, respectively) were observed in experiment 3. Of the antimicrobial interventions utilized, LA was more effective for reducing STEC surrogate populations on veal carcasses, pre- and/or postchill.

Warm-season annual forages in forage-finishing beef systems: II. Animal performance and carcass characteristics.

More information on expected animal performance and carcass traits of forage-finished steers grazing warm-season annual forages is needed. To achieve this objective, a grazing trial was conducted in 2014, 2015, and 2016 (70, 63, and 56 d, respectively), with variation in length of grazing based on forage availability. Sixteen pastures (0.81 ha) were assigned to 1 of 4 forage treatments in a randomized complete block design. Forage treatments were brown midrib sorghum × sudangrass (BMR; Sorghum bicolor var. bicolor*bicolor var. sudanense), sorghum × sudangrass (SS), pearl millet [PM; Pennisetum glaucum (L.)R.Br.], or pearl millet planted with crabgrass [PMCG; Digitaria sanguinalis (L.) Scop.]. Each year, British-cross beef steers (n = 32; 3 y average: 429 ± 22 kg) were stratified by weight and randomly assigned to 1 of the 16 pastures for forage finishing. Each pasture was subdivided into two 0.405-ha paddocks for rotational stocking and a put-and-take stocking method was used to maintain a forage allowance of 116 kg forage dry matter/100 kg body weight (BW). Shrunk body weight and ultrasonically measured carcass composition were recorded at the initiation, middle, and end of each grazing season. Steers were harvested once forage availability became limited and chilled carcasses (24 h) were evaluated for yield grade and quality grade attributes. Statistical analysis was conducted using the GLIMMIX procedure in SAS 9.4 (Cary, NC) with main effects of treatment, year, and the interaction. Pasture and block were considered random effects while date was assessed as a main effect when applicable. Daily stocking densities were greater (P < 0.04) for SS than PMCG in the first 20 d of 2014 and 2015. Forage treatment did not affect (P > 0.17) total gain, total average daily gain, or body weight at any time point. Ultrasound composition traits of loin muscle area, 12th rib fat thickness, intramuscular fat, and rump fat were impacted (P < 0.01) by scanning date. No differences (P > 0.08) in forage treatments were observed for carcass characteristics associated with yield grade or quality grade. The findings suggest that forage-finished cattle during the summer months on BMR, SS, PM, and PMCG perform similarly, giving producers the option to use the most economical or practical forage type for their production system.

Validation of Antimicrobial Interventions for Reducing Shiga Toxin-Producing Escherichia coli Surrogate Populations during Goat Slaughter and Carcass Chilling.

Demand and consumption of goat meat is increasing in the United States due to an increase in ethnic populations that prefer goat meat. As ruminant animals, goats are known reservoirs for Shiga toxin-producing Escherichia coli (STEC) and proper handling, especially during slaughter, is imperative to reduce the likelihood of carcass and meat contamination. However, the majority of antimicrobial intervention studies during the slaughter of ruminant species have focused on beef, highlighting the need for validation studies targeting small ruminants, such as goats, during slaughter and chilling procedures. The objective of this research was to evaluate 4.5% lactic acid (LA; pH 2.1), peroxyacetic acid (PAA; 400 ppm; pH 4.7), a hydrochloric and citric acid blend (Citrilow [CL]; pH 1.2), 5% levulinic acid plus 0.5% sodium dodecyl sulfate (LVA+SDS; pH 2.60), and a nontreated control (CON) for their efficacy in reducing STEC surrogates and their effect on carcass color from slaughter through 24-h chill. Fifteen goat carcasses across three replications were inoculated with a five-strain cocktail (ca. 5 log CFU/cm2 attachment), containing rifampin-resistant surrogate E. coli (BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431) and were randomly assigned to an antimicrobial treatment. Antimicrobials were applied prechill and 24 h postchill. Mean log reductions achieved after prechill treatment with LA, PAA, CL, and LVA+SDS were 2.00, 1.86, 2.26, and 1.90 log CFU/cm2, respectively. Antimicrobial treatment after the 24-h chilling, resulted in additional reductions of surrogate E. coli by 0.99, 1.03, 1.94, and 0.47 log CFU/cm2 for LA, PAA, CL, and LVA+SDS, respectively. Antimicrobial treatments did not impact goat carcass objective color (L* and a*), except for b*. The antimicrobials tested in this study were able to effectively reduce surrogate STEC populations during slaughter and subsequent chilling without compromising carcass color.

Warm-season annual forages in forage-finishing beef systems: I. Forage yield and quality.

The demand for a year-round supply of fresh, locally grown, forage-finished beef products has created a need for forage-finishing strategies during the summer months in the southeast. A 3-yr study was conducted to evaluate four warm-season annual forages in a southeastern forage-finishing beef production system. Treatments were four forage species and included brown-midrib sorghum × sudangrass (Sorghum bicolor var. bicolor*bicolor var. sudanense; BMR), sorghum × sudangrass (SS), pearl millet [Pennisetum glaucum (L.) R. Br.; PM], or pearl millet planted with crabgrass [Digitaria sanguinalis (L.) Scop.; PMCG]. Treatments were distributed in a randomized complete block design with four replicates. Pastures (0.81 ha, experimental unit) were assigned to one of four forage treatments, subdivided, and rotationally stocked with a variable stocking density. British-cross beef steers (n = 32; 3-yr average: 429 ± 22 kg) grazed for 70, 63, and 56 d in 2014, 2015, and 2016, respectively. Put-and-take animals were used to maintain a forage allowance of 116 kg forage dry matter /100 kg body weight. Forage mass was measured by clipping a 4.3-m2 area in triplicate on d 0 and on 14-d intervals. Hand grab samples for forage nutritive value determination and quadrat clippings for species compositions were measured on d 0 and on 34-d intervals until termination of the trial. Forage mass was lowest (P < 0.01) for PMCG at the initiation of the grazing trial, whereas BMR was greater (P < 0.01) than SS at wk 6. Total digestible nutrients in 2014 were greater for SS compared to BMR and PM at the middle harvest (P < 0.01) and BMR, PM, and PMCG at the final harvest (P < 0.01). At the middle and final harvests in both 2015 and 2016, PM and PMCG contained greater (P < 0.01) concentrations of crude protein than SS. These results suggest that BMR, SS, PM, and PMCG may all be used in southeastern forage-finishing beef production systems, as long as the producer strategically accounts for the slight growth and nutritive value differences throughout the season.

Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in Clostridium difficile.

The intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that in Clostridium difficile (also called Clostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP in C. difficile, employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formation in vitro A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as "off" switches in response to c-di-GMP, while class II riboswitches acted as "on" switches. Transcriptional analyses of genes 3' of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network in C. difficile and emphasizes the role of the riboswitches in controlling known and putative virulence factors in C. difficileIMPORTANCE In Clostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP in C. difficile Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functional in vivo and displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression in C. difficile.

Type IV Pili Promote Clostridium difficile Adherence and Persistence in a Mouse Model of Infection.

Cyclic diguanylate (c-di-GMP) is a second messenger that regulates the transition from motile to sessile lifestyles in numerous bacteria and controls virulence factor production in a variety of pathogens. In Clostridium difficile, c-di-GMP negatively regulates flagellum biosynthesis and swimming motility and promotes the production of type IV pili (TFP), biofilm formation, and surface motility in vitro Flagella have been identified as colonization factors in C. difficile, but the role of TFP in adherence to host cells and in colonization of the mammalian gut is unknown. Here we show that c-di-GMP promotes adherence to epithelial cells in vitro, which can be partly attributed to the loss of flagella. Using TFP-null mutants, we demonstrate that adherence to epithelial cells is partially mediated by TFP and that this TFP-mediated adherence requires c-di-GMP regulation. In a mouse model of colonization, the TFP-null mutants initially colonized the intestine as well as the parental strain but were cleared more quickly. Moreover, compared to the parent strain, C. difficile strains lacking TFP were particularly deficient in association with the cecal mucosa. Together these data indicate that TFP and their positive regulation by c-di-GMP promote attachment of C. difficile to the intestinal epithelium and contribute to persistence of C. difficile in the host intestine.

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase.

The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile.

UNLABELLED: The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called "hypervirulent" epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile. We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile. The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCE: How Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by C. difficile. TFP are also required for surface motility, which has not previously been demonstrated for C. difficile. Furthermore, an epidemic-associated C. difficile strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae.

BACKGROUND: The second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence. RESULTS: Complementary in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization. CONCLUSIONS: These results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of V. cholerae.

The second messenger cyclic Di-GMP regulates Clostridium difficile toxin production by controlling expression of sigD.

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ(28)) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile.

Clostridium difficile-associated disease is increasing in incidence and is costly to treat. Our understanding of how this organism senses its entry into the host and adapts for growth in the large bowel is limited. The small-molecule second messenger cyclic diguanylate (c-di-GMP) has been extensively studied in gram-negative bacteria and has been shown to modulate motility, biofilm formation, and other processes in response to environmental signals, yet little is known about the functions of this signaling molecule in gram-positive bacteria or in C. difficile specifically. In the current study, we investigated the function of the second messenger c-di-GMP in C. difficile. To determine the role of c-di-GMP in C. difficile, we ectopically expressed genes encoding a diguanylate cyclase enzyme, which synthesizes c-di-GMP, or a phosphodiesterase enzyme, which degrades c-di-GMP. This strategy allowed us to artificially elevate or deplete intracellular c-di-GMP, respectively, and determine that c-di-GMP represses motility in C. difficile, consistent with previous studies in gram-negative bacteria, in which c-di-GMP has a negative effect on myriad modes of bacterial motility. Elevated c-di-GMP levels also induced clumping of C. difficile cells, which may signify that C. difficile is capable of forming biofilms in the host. In addition, we directly quantified, for the first time, c-di-GMP production in a gram-positive bacterium. This work demonstrates the effect of c-di-GMP on the motility of a gram-positive bacterium and on aggregation of C. difficile, which may be relevant to the function of this signaling molecule during infection.