Campylobacter is the leading cause of human gastroenteritis worldwide. Domestic and wild birds ar... more Campylobacter is the leading cause of human gastroenteritis worldwide. Domestic and wild birds are likely zoonotic vectors of Campylobacter. We demonstrated that American crows, abundant in urban, suburban, and agricultural settings, carry multiple Campylobacter genotypes that are passed on to their own offspring. The capability to distribute to other animals depends on the particular Campylobacter genotype. Contact and proximity to humans and livestock increases the potential spreading of Campylobacter via crows between the environment, livestock, and humans. We observed a critical link between public health and genomic variation of Campylobacter. Using whole genome sequencing (WGS), we evaluated the genotype that enabled zoonotic transfer and pathogenic potential of Campylobacter from crows to other animals in 184 isolates obtained from crow, chicken, cow, sheep, goat, human, and non-human primates. Fig. 1. Model of transmission potential of Campylobacter isolates.
Measurement of heat-stable lipase activity in dairy products relies on methods that are slow or t... more Measurement of heat-stable lipase activity in dairy products relies on methods that are slow or that cannot be used in turbid solutions, which limits their industrial value. A need exists for a rapid, simple, informative assay t o detect lipase activity in dairy products. In this study, we observed that hydrolysis of p-nitrophenyl esters, monitored by reflectance colorimetry, was linearly correlated to spectrophotom-etry (R 2 = 0.93) and release of titratable FFA (R 2 = 0.92 to 0.9'71, indicating that chromogenic substrates were useful in determining lipase activity. However, at the conclentrations reported in milk, FFA inhibited p-nitrophenyl caprylate hydrolysis, which led to an underestimation of lipase activity in milk that had previously undergone lipolysis. Milk fat also significantly reduced hydrolysis of the chromogenic sub-strates tested but could be accounted for by a correction equation. To demonstrate the use of the assay, lipase activity in UHT skim milk inoculated with Pseudomonas fluorescens AFT36 was followed using reflectance colorimetry and tributyrin agar. Lipase activity increased as cell numbers increased during 106 h of incubation. Extracellular lipase activity was detected after 10 h of incubation using reflectance colorimet7, but 28 h were required using tributyrin agar. Reflectance colorimetry and chromogenic sub-strates allowed a rapid, sensitive, and meaningful detection clf esterase and lipase activity in milk. Abbreviation key: DMSO = dimethylsulfoxide, p-NP = p-nitrophenyl, p-NPC = p-nitrophenyl capry-late.
At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to ooc... more At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm–oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm–oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrom-etry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes. Mol. Reprod. Dev. 75: 641–649, 2008.
Increasing emphasis on controlling the uses of chemical fertilizers requires identifying safe Org... more Increasing emphasis on controlling the uses of chemical fertilizers requires identifying safe Organic Soil Amendments (OSA) to use as alternatives. Converting organic waste, such as foodwaste into an OSA can be an option. Such approaches are also an attempt to make beneficial use of the enormous amount of foodwaste generated globally. In this study we conducted a pathogen challenge to determine the inac-tivation of three foodborne pathogens in an OSA derived from a complex foodwaste stream. Further, the physiochemical characteristics of the OSA were assessed at pilot-scale experiments. The inactivation of three most common foodborne pathogens (Escherichia coli O157:H7, Salmonella enterica subspecies enterica sv Typhimurium LT2, and Listeria monocytogenes) was determined using bench-scale tests, simulating the process adopted at a pilot-scale facility. The pilot-scale facility uses three processes (enzyme digestion (55e57 C), pasteurization (75e77 C), and acidification treatments) for producing the OSA. In addition, the yields and nutrient characteristics of the OSA were analyzed using 16 pilot-scale batch tests. The results showed that the process adopted in this study for converting foodwaste to the OSA produced a soil amendment with non-detectable levels of E. coli O157:H7, Salmonella LT2, and L. monocytogenes. The yield of the OSA was 84e96% of the initial foodwaste inputs, and organic matter and C: N ratio of the OSA were 20e25% and 12:1, respectively. We anticipate that the results presented here will help in enhancing agricultural sustainability.
ABSTRACT Lactic acid bacteria are exposed to a series of stresses during environmental transit an... more ABSTRACT Lactic acid bacteria are exposed to a series of stresses during environmental transit and food fermentation processes. Stresses unique to fermented foods are high acid, low temperature, and limited to no free sugar. In fermented foods, lactobacilli and lactococci recently have been recognized to become nonculturable (NC) under these conditions. It has been shown that lactococci become NC under carbohydrate starvation, which leads to additional metabolic changes during 180 days of incubation. Important end products include branched-chain fatty acids from the catabolism of branched-chain amino acids. In addition, sulfur metabolism changes. It has yet to be demonstrated that NC lactococci can be resuscitated with known compounds or peptides. The NC state of lactococci leads to new metabolic end products not produced during log-phase growth.
ABSTRACT Detection of sulfur compounds is difficult in complex samples such as soil and food. The... more ABSTRACT Detection of sulfur compounds is difficult in complex samples such as soil and food. The volatility of these compounds can be an advantage if a trapping method is used. This method must be inert for the highly reactive compounds to avoid artifacts resulting from sample preparation and analysis procedures. This chapter describes the use headspace analysis with cryofocusing to detect low levels (ppb) of volatile sulfur compounds produced via fermentation of milk. This method is relatively simple compared to the use of sophisticated instrumentation for the unique identification tools available. It is also useful for other sample types. Milk is a good sample of a difficult substrate that reacts with volatile compounds and is reflective of the level of difficulty for this analysis. Key WordsMethanethiol–cheddar cheese–sulfur
Summary Gene expression in microbes can be detected with an oligonucleotide-based DNA macroar-ray... more Summary Gene expression in microbes can be detected with an oligonucleotide-based DNA macroar-ray. The DNA macroarray is composed of short (2224-mer) oligonucleotide probes immobilized onto a nylon membrane via polyinosine tails. An indirect high-density ...
Salmonella encounters various stresses in the environment and in the host during infection. The e... more Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H2O2, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes, implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Salmonella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. Increased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-...
Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develo... more Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only. BL2 also converted alpha-ket...
Volatile sulfur compounds such as methanethiol, dimethyl disulfide, dimethyl trisulfide, and hydr... more Volatile sulfur compounds such as methanethiol, dimethyl disulfide, dimethyl trisulfide, and hydrogen sulfide constitute an important fraction of Cheddar cheese flavor. These compounds are products of the catabolism of methionine and cysteine by bacteria in the cheese matrix. The objectives of this study were to examine the levels and types of volatile sulfur compounds produced from methionine by lactic acid bacteria frequently used in cheese making and to investigate cystathionine degrading activity, which may be responsible for the liberation of these compounds. Gas chromatography with headspace sampling was used to determine volatile sulfur compounds (VSC) produced by whole cells of 24 strains of lactobacilli and 13 strains of lactococci incubated with methionine. Total VSC production varied widely in the species and subspecies tested. Nearly all strains produced VSC from methionine, but the enzyme responsible for this activity remains unclear. Cystathionine-degrading ability and...
Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest. Volati... more Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest. Volatile sulfur compounds, specifically methanethiol, are correlated to desirable flavor in Cheddar cheese, but their definitive role remains elusive. Only recently have enzymes been found that produce this compound in bacteria associated with cheese making. Cystathionine beta- and gamma-lyase are found in lactic acid bacteria and are capable of producing methanethiol from methionine. Their primary function is in the metabolism of cysteine. Methionine gamma-lyase produces methanethiol from methionine at a higher efficiency than the cystathionine enzymes. This enzyme is found in brevibacteria, bacilli, and pseudomonads. Addition of brevibacteria containing this enzyme improves Cheddar cheese flavor. Despite recent progress in sulfur metabolism more information is needed before cheese flavor associated with sulfur can be predicted or controlled.
Campylobacter is the leading cause of human gastroenteritis worldwide. Domestic and wild birds ar... more Campylobacter is the leading cause of human gastroenteritis worldwide. Domestic and wild birds are likely zoonotic vectors of Campylobacter. We demonstrated that American crows, abundant in urban, suburban, and agricultural settings, carry multiple Campylobacter genotypes that are passed on to their own offspring. The capability to distribute to other animals depends on the particular Campylobacter genotype. Contact and proximity to humans and livestock increases the potential spreading of Campylobacter via crows between the environment, livestock, and humans. We observed a critical link between public health and genomic variation of Campylobacter. Using whole genome sequencing (WGS), we evaluated the genotype that enabled zoonotic transfer and pathogenic potential of Campylobacter from crows to other animals in 184 isolates obtained from crow, chicken, cow, sheep, goat, human, and non-human primates. Fig. 1. Model of transmission potential of Campylobacter isolates.
Measurement of heat-stable lipase activity in dairy products relies on methods that are slow or t... more Measurement of heat-stable lipase activity in dairy products relies on methods that are slow or that cannot be used in turbid solutions, which limits their industrial value. A need exists for a rapid, simple, informative assay t o detect lipase activity in dairy products. In this study, we observed that hydrolysis of p-nitrophenyl esters, monitored by reflectance colorimetry, was linearly correlated to spectrophotom-etry (R 2 = 0.93) and release of titratable FFA (R 2 = 0.92 to 0.9'71, indicating that chromogenic substrates were useful in determining lipase activity. However, at the conclentrations reported in milk, FFA inhibited p-nitrophenyl caprylate hydrolysis, which led to an underestimation of lipase activity in milk that had previously undergone lipolysis. Milk fat also significantly reduced hydrolysis of the chromogenic sub-strates tested but could be accounted for by a correction equation. To demonstrate the use of the assay, lipase activity in UHT skim milk inoculated with Pseudomonas fluorescens AFT36 was followed using reflectance colorimetry and tributyrin agar. Lipase activity increased as cell numbers increased during 106 h of incubation. Extracellular lipase activity was detected after 10 h of incubation using reflectance colorimet7, but 28 h were required using tributyrin agar. Reflectance colorimetry and chromogenic sub-strates allowed a rapid, sensitive, and meaningful detection clf esterase and lipase activity in milk. Abbreviation key: DMSO = dimethylsulfoxide, p-NP = p-nitrophenyl, p-NPC = p-nitrophenyl capry-late.
At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to ooc... more At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm–oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm–oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrom-etry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes. Mol. Reprod. Dev. 75: 641–649, 2008.
Increasing emphasis on controlling the uses of chemical fertilizers requires identifying safe Org... more Increasing emphasis on controlling the uses of chemical fertilizers requires identifying safe Organic Soil Amendments (OSA) to use as alternatives. Converting organic waste, such as foodwaste into an OSA can be an option. Such approaches are also an attempt to make beneficial use of the enormous amount of foodwaste generated globally. In this study we conducted a pathogen challenge to determine the inac-tivation of three foodborne pathogens in an OSA derived from a complex foodwaste stream. Further, the physiochemical characteristics of the OSA were assessed at pilot-scale experiments. The inactivation of three most common foodborne pathogens (Escherichia coli O157:H7, Salmonella enterica subspecies enterica sv Typhimurium LT2, and Listeria monocytogenes) was determined using bench-scale tests, simulating the process adopted at a pilot-scale facility. The pilot-scale facility uses three processes (enzyme digestion (55e57 C), pasteurization (75e77 C), and acidification treatments) for producing the OSA. In addition, the yields and nutrient characteristics of the OSA were analyzed using 16 pilot-scale batch tests. The results showed that the process adopted in this study for converting foodwaste to the OSA produced a soil amendment with non-detectable levels of E. coli O157:H7, Salmonella LT2, and L. monocytogenes. The yield of the OSA was 84e96% of the initial foodwaste inputs, and organic matter and C: N ratio of the OSA were 20e25% and 12:1, respectively. We anticipate that the results presented here will help in enhancing agricultural sustainability.
ABSTRACT Lactic acid bacteria are exposed to a series of stresses during environmental transit an... more ABSTRACT Lactic acid bacteria are exposed to a series of stresses during environmental transit and food fermentation processes. Stresses unique to fermented foods are high acid, low temperature, and limited to no free sugar. In fermented foods, lactobacilli and lactococci recently have been recognized to become nonculturable (NC) under these conditions. It has been shown that lactococci become NC under carbohydrate starvation, which leads to additional metabolic changes during 180 days of incubation. Important end products include branched-chain fatty acids from the catabolism of branched-chain amino acids. In addition, sulfur metabolism changes. It has yet to be demonstrated that NC lactococci can be resuscitated with known compounds or peptides. The NC state of lactococci leads to new metabolic end products not produced during log-phase growth.
ABSTRACT Detection of sulfur compounds is difficult in complex samples such as soil and food. The... more ABSTRACT Detection of sulfur compounds is difficult in complex samples such as soil and food. The volatility of these compounds can be an advantage if a trapping method is used. This method must be inert for the highly reactive compounds to avoid artifacts resulting from sample preparation and analysis procedures. This chapter describes the use headspace analysis with cryofocusing to detect low levels (ppb) of volatile sulfur compounds produced via fermentation of milk. This method is relatively simple compared to the use of sophisticated instrumentation for the unique identification tools available. It is also useful for other sample types. Milk is a good sample of a difficult substrate that reacts with volatile compounds and is reflective of the level of difficulty for this analysis. Key WordsMethanethiol–cheddar cheese–sulfur
Summary Gene expression in microbes can be detected with an oligonucleotide-based DNA macroar-ray... more Summary Gene expression in microbes can be detected with an oligonucleotide-based DNA macroar-ray. The DNA macroarray is composed of short (2224-mer) oligonucleotide probes immobilized onto a nylon membrane via polyinosine tails. An indirect high-density ...
Salmonella encounters various stresses in the environment and in the host during infection. The e... more Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H2O2, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes, implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Salmonella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. Increased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-...
Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develo... more Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only. BL2 also converted alpha-ket...
Volatile sulfur compounds such as methanethiol, dimethyl disulfide, dimethyl trisulfide, and hydr... more Volatile sulfur compounds such as methanethiol, dimethyl disulfide, dimethyl trisulfide, and hydrogen sulfide constitute an important fraction of Cheddar cheese flavor. These compounds are products of the catabolism of methionine and cysteine by bacteria in the cheese matrix. The objectives of this study were to examine the levels and types of volatile sulfur compounds produced from methionine by lactic acid bacteria frequently used in cheese making and to investigate cystathionine degrading activity, which may be responsible for the liberation of these compounds. Gas chromatography with headspace sampling was used to determine volatile sulfur compounds (VSC) produced by whole cells of 24 strains of lactobacilli and 13 strains of lactococci incubated with methionine. Total VSC production varied widely in the species and subspecies tested. Nearly all strains produced VSC from methionine, but the enzyme responsible for this activity remains unclear. Cystathionine-degrading ability and...
Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest. Volati... more Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest. Volatile sulfur compounds, specifically methanethiol, are correlated to desirable flavor in Cheddar cheese, but their definitive role remains elusive. Only recently have enzymes been found that produce this compound in bacteria associated with cheese making. Cystathionine beta- and gamma-lyase are found in lactic acid bacteria and are capable of producing methanethiol from methionine. Their primary function is in the metabolism of cysteine. Methionine gamma-lyase produces methanethiol from methionine at a higher efficiency than the cystathionine enzymes. This enzyme is found in brevibacteria, bacilli, and pseudomonads. Addition of brevibacteria containing this enzyme improves Cheddar cheese flavor. Despite recent progress in sulfur metabolism more information is needed before cheese flavor associated with sulfur can be predicted or controlled.
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Papers by Bart C Weimer