Pyruvate oxidase of Streptococcus pneumoniae contributes to pneumolysin release.

BACKGROUND: Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae's virulence are dependent upon expression and release of the protein toxin pneumolysin (PLY) and upon the activity of the peroxide-producing enzyme, pyruvate oxidase (SpxB). We investigated the possible synergy of these two proteins and identified that release of PLY is enhanced by expression of SpxB prior to stationary phase growth. RESULTS: Mutants lacking the spxB gene were defective in PLY release and complementation of spxB restored PLY release. This was demonstrated by cytotoxic effects of sterile filtered supernatants upon epithelial cells and red blood cells. Additionally, peroxide production appeared to contribute to the mechanism of PLY release since a significant correlation was found between peroxide production and PLY release among a panel of clinical isolates. Exogenous addition of H2O2 failed to induce PLY release and catalase supplementation prevented PLY release in some strains, indicating peroxide may exert its effect intracellularly or in a strain-dependent manner. SpxB expression did not trigger bacterial cell death or LytA-dependent autolysis, but did predispose cells to deoxycholate lysis. CONCLUSIONS: Here we demonstrate a novel link between spxB expression and PLY release. These findings link liberation of PLY toxin to oxygen availability and pneumococcal metabolism.

Lipid accumulation by Rhodococcus rhodochrous grown on glucose.

Biodiesel is an alternative fuel made from costly vegetable oil feedstocks. Some microorganisms can accumulate lipids when nutrients are limited and carbon is in excess. Rhodococcus rhodochrous is a gram-positive bacterium most often used in bioremediation or acrylamide production. The purpose of this study was to investigate and characterize the lipid accumulation capabilities of R. rhodochrous. Shake flasks and a large-scale fermentation were used to cultivate R. rhodochrous in varying concentrations of glucose. R. rhodochrous achieved almost 50 % of dry cell mass as lipid when grown in 20 g/L of glucose. Wax esters and triglycerides were identified in R. rhodochrous lipid extract. The transesterified extractables of R. rhodochrous consisted of mostly palmitic (35 %) and oleic (42 %) acid methyl esters. This study shows R. rhodochrous to be an oleaginous bacterium with potential for application in alternative fuels.

Proteomic analysis of cross protection provided between cold and osmotic stress in Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive, foodborne pathogen responsible for approximately 28% of all food-related deaths each year in the United States. L. monocytogenes infections are linked to the consumption of minimally processed ready-to-eat (RTE) products such as cheese, deli meats, and cold-smoked finfish products. L. monocytogenes is resistant to stresses commonly encountered in the food-processing environment, including low pH, high salinity, oxygen content, and various temperatures. The purpose of this study was to determine if cells habituated at low temperatures would result in cross-protective effects against osmotic stress. We found that cells exposed to refrigerated temperatures prior to a mild salt stress treatment had increased survival in NaCl concentrations of 3%. Additionally, the longer the cells were pre-exposed to cold temperatures, the greater the increase in survival in 3% NaCl. A proteomics analysis was performed in triplicate in order to elucidate mechanisms involved in cold-stress induced cross protection against osmotic stress. Proteins involved in maintenance of the cell wall and cellular processes, such as penicillin binding proteins and osmolyte transporters, and processes involving amino acid metabolism, such as osmolyte synthesis, transport, and lipid biosynthesis, had the greatest increase in expression when cells were exposed to cold temperatures prior to salt. By gaining a better understanding of how this pathogen adapts physiologically to various environmental conditions, improvements can be made in detection and mitigation strategies.

Use of bioluminescent Escherichia coli to determine retention during the life cycle of the housefly, Musca domestica (Diptera: Muscidae, L).

Researchers have documented that the housefly (Musca domestica) can serve as a vector for the spread of foodborne pathogens to livestock, food, and humans. Most studies have investigated Musca domestica as a vector only after the fly comes into contact or consumes the pathogen as an adult. The objective of this study was to determine whether the larvae of Musca domestica could ingest Escherichia coli from bovine manure and whether the E. coli could survive the metamorphosis process and be transmitted. Larvae (n=960) were incubated in sterilized bovine manure inoculated with 0, 3, 5, and 8 log10 colony-forming units (CFU)/mL of bioluminescent E. coli for 24 (larvae stage), 48 (larvae stage), 120 (pupae stage), and 192 h (adult stage). Larvae incubated for 24 h in bovine manure possessed 0.0, 2.7, 2.9, and 3.5 log(10) CFU/mL of E. coli, from inoculated with 0, 3, 5, and 8 log(10) CFU/mL of E. coli, respectively. Concentrations of E. coli within the pupae were 0.0, 1.7, 1.9, and 2.2 log(10) CFU/mL for each inoculation concentration, respectively. Flies that emerged from the pupae stage contained 0.0, 1.3, 2.2, and 1.7 log(10) CFU/mL of E. coli from larvae incubated in manure inoculated with concentrations of E. coli, respectively. These results suggest the housefly can emerge with quantities of E. coli. While this was an enteropathogenic E. coli (EPEC), these data may suggest that if the fly is capable of retaining similar concentrations of an enterohemorrhagic E. coli (EHEC), these concentrations may be capable of initiating illness in humans. Furthermore, the E. coli concentration within and on adult flies is related to environmental exposure. It must be noted that larvae were incubated in sterilized bovine manure, and there was no other bacterial competition for the E. coli. Thus, the rate of positive flies and concentrations present when flies emerged may vary under more realistic conditions.

Survival of Escherichia coli O157:H7 transformed with either the pAK1-lux or pXEN-13 plasmids in in vitro bovine ruminal and fecal microbial fermentations.

The use of luminescent plasmids in bacteria may serve as a viable model for the real-time validation of various pre-harvest interventions on the colonization or shedding patterns of Escherichia coli O157:H7 within cattle. The objective of this study was to determine if the growth characteristics of E. coli O157:H7 in mixed ruminal and fecal microbial fluid cultures would be altered when transformed with one of the two luminescent plasmids: pAK1-lux (PAK) or pXEN-13 (XEN). Transformants harboring the luminescent plasmids were compared to the non-transformed parental strain (wild type [WT]) after incubating in mixed ruminal or fecal microbial fluid media for 6 h in triplicate (n=3). The transformants and WT exhibited similar growth rates. Within mixed ruminal microbial fluid fermentations and mixed fecal microbial fluid, all transformants grew similarly (p=0.28) through the 6-h study. The reflective light unit (RLU; photons/pixel per second) photonic emissions of each plasmid within ruminal fluid differed at 0 h (p=0.002) and 2 h (p=0.02) and within fecal fluid at 0 h (p=0.009) and 2 h (p=0.04). The RLU remained the same within rumen fluid at 4 h (p=0.22) and 6 h (p=0.80) and within fecal fluid at 4 h (p=0.06) and 6 h (p=0.29). Growth of E. coli O157:H7 transformed with the bioluminescent plasmids was not altered in comparison to the WT, suggesting that both plasmids may serve as useful models for in vivo studies.

The influence of the microbiome on aggressive behavior: an insight into age-related aggression.

Aggression is a complex psychological program that is influenced by genetics, environment, and psychological history. Research has shown that the hormonal levels in the body and the development of the brain can be major predictors of aggression. This review highlights recent studies that have connected the gut microbiome to alterations in hormones and brain development and how this can impact aggression. This paper also provides a systematic review on studies that directly assess the connection between the gut microbiome and aggression and reviews these connections in relation to age. We conclude with future directions that are needed to further determine the link between the microbiome and aggression among adolescents.

Survival of O157:H7 and non-O157 serogroups of Escherichia coli in bovine rumen fluid and bile salts.

While Shiga toxin-producing Escherichia coli (STEC) reside asymptomatically within ruminants, particularly cattle, these strains pose a serious health risk to humans. Research related to STEC has historically focused upon O157:H7. However, with an increase in foodborne outbreaks of non-O157 origin and recent changes in testing for non-O157 by the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), there is now a critical need to understand the biological activity of non-O157 serogroups. The focus of this study was to determine whether variations exist in the ability of different serotypes of STEC to survive within bovine rumen fluid medium and bile salts. The results of this study demonstrated through viable plate count analysis that the five serotypes tested (O157:H7, O111:H8, O103:K.:H8, O145:H28, and O26:H11) were capable of growing in rumen fluid medium. However, the concentrations of the serotypes O103:K.:H8 and O26:H11 after 24 h were significantly less (p < 0.05) than that observed for the other serotypes tested. A significant decrease (p = 0.03) in the survival of O103:K.:H8 in 50 mg/mL of bovine bile salts in comparison to the other STEC strains tested was also observed. Collectively, these data suggest that non-O157 serogroups of E. coli respond differently to the environment of the bovine gastrointestinal tract. Further research is needed to elucidate how these differential physiological variations correlate with alterations in colonization success within ruminants and how they may impact human illnesses.

Genome sequence of lineage III Listeria monocytogenes strain HCC23.

More than 98% of reported human listeriosis cases are caused by Listeria monocytogenes serotypes within lineages I and II. Serotypes within lineage III (4a and 4c) are commonly isolated from environmental and food specimens. We report the first complete genome sequence of a lineage III isolate, HCC23, which will be used for comparative analysis.

Immunomodulator expression in trophoblasts from the feline immunodeficiency virus (FIV)-infected cat.

BACKGROUND: FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1ß, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF) and anti-inflammatory cytokines (IL-4, IL-5, and IL-10); CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor); the chemokine receptor CXCR4 (FIV co-receptor); SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy. METHODS: We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression. RESULTS: We detected IL-4, IL-5, IL-6, IL-1ß, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens. CONCLUSION: Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in normal tissues. Non-viable pregnancies were associated with decreased expression of immunomodulators which regulate trophoblast invasion in other species. The detection of FIV RNA in trophoblasts was rare, suggesting that the high rate of reproductive failure in FIV-infected queens was not a direct result of viral replication in trophoblasts. The influence of placental immune cells on trophoblast function and pregnancy maintenance in the FIV-infected cat requires additional study.

Transcriptomic Analysis of Listeria monocytogenes in Response to Bile Under Aerobic and Anaerobic Conditions.

Listeria monocytogenes is a gram-positive facultative anaerobic bacterium that causes the foodborne illness listeriosis. The pathogenesis of this bacterium depends on its survival in anaerobic, acidic, and bile conditions encountered throughout the gastrointestinal (GI) tract. This transcriptomics study was conducted to analyze the differences in transcript levels produced under conditions mimicking the GI tract. Changes in transcript levels were analyzed using RNA isolated from L. monocytogenes strain F2365 at both aerobic and anaerobic conditions, upon exposure to 0 and 1% bile at acidic and neutral pH. Transcripts corresponding to genes responsible for pathogenesis, cell wall associated proteins, DNA repair, transcription factors, and stress responses had variations in levels under the conditions tested. Upon exposure to anaerobiosis in acidic conditions, there were variations in the transcript levels for the virulence factors internalins, listeriolysin O, etc., as well as many histidine sensory kinases. These data indicate that the response to anaerobiosis differentially influences the transcription of several genes related to the survival of L. monocytogenes under acidic and bile conditions. Though further research is needed to decipher the role of oxygen in pathogenesis of L. monocytogenes, these data provide comprehensive information on how this pathogen responds to the GI tract.

A dominant, recombination-defective allele of Dmc1 causing male-specific sterility.

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1(Mei11), encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1(Mei11) exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.

Listeria monocytogenes Response to Anaerobic Environments.

Listeria monocytogenes is a Gram-positive facultative anaerobic bacterium that is responsible for the disease, listeriosis. It is particularly lethal in pregnant women, the fetus, the elderly and the immunocompromised. The pathogen survives and replicates over a wide range of temperatures (4 to 42 °C), pH, salt and oxygen concentrations. Because it can withstand various environments, L. monocytogenes is a major concern in food processing industries, especially in dairy products and ready-to-eat fruits, vegetables and deli meats. The environment in which the pathogen is exposed can influence the expression of virulence genes. For instance, studies have shown that variations in oxygen availability can impact resistance to stressors. Further investigation is needed to understand the essential genes required for the growth of L. monocytogenes in anaerobic conditions. Therefore, the purpose of this review is to highlight the data on L. monocytogenes under known environmental stresses in anaerobic environments and to focus on gaps in knowledge that may be advantageous to study in order to better understand the pathogenicity of the bacterium.

Comparative proteomic analysis of Listeria monocytogenes strains F2365 and EGD.

Listeria monocytogenes is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of L. monocytogenes and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of L. monocytogenes, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37 degrees C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two L. monocytogenes strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins.

Transposon Mutagenesis of Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen that enters the human digestive tract after the consumption of contaminated food. Much research has been done to understand the virulence factors of Listeria monocytogenes. One useful tool to study these virulence factors has been transposon mutagenesis. Many mutants can be generated at a time by performing high-throughput mutagenesis using transposons and later screening these mutants to identify features related to particular functions in the bacteria. Many transposon delivery systems are not ideal for transposon studies in Listeria monocytogenes, as the transposon system is too large, has lower transposition efficiency, and a high rate of plasmid retention. Therefore, a new mariner-based transposition system has been developed for Listeria monocytogenes. This system is an ideal high-throughput transposon mutagenesis as the rate of transposition is high and random, along with very low plasmid retention capacity.

Establishment of Listeria monocytogenes in the Gastrointestinal Tract.

Listeria monocytogenes is a Gram positive foodborne pathogen that can colonize the gastrointestinal tract of a number of hosts, including humans. These environments contain numerous stressors such as bile, low oxygen and acidic pH, which may impact the level of colonization and persistence of this organism within the GI tract. The ability of L. monocytogenes to establish infections and colonize the gastrointestinal tract is directly related to its ability to overcome these stressors, which is mediated by the efficient expression of several stress response mechanisms during its passage. This review will focus upon how and when this occurs and how this impacts the outcome of foodborne disease.

Oxygen deprivation influences the survival of Listeria monocytogenes in gerbils.

Listeria monocytogenes is a facultative anaerobic foodborne pathogen capable of surviving harsh environments. Recent work has indicated that anaerobic conditions increase the resistance capability of certain strains to environmental stressors. The goal of the study was to conduct a preliminary study to determine whether exposure to anaerobic conditions prior to infection increases the ability to survive in vivo. Gerbils were inoculated with one of five doses of the L. monocytogenes strain F2365 by oral gavage: phosphate-buffered saline (control), 5 × 106 colony forming units aerobic culture (low aerobic), 5 × 108 aerobic culture (high aerobic), 5 × 106 anaerobic culture (low anaerobic), or 5 × 108 anaerobic culture (high anaerobic) dose of F2365. Gerbils inoculated with a high aerobic or anaerobic dose exhibited significant weight loss. Gerbils administered either the low or high anaerobic dose had at least 3 log10 of L. monocytogenes present in fecal samples, which contrasted with gerbils that received the low aerobic dose. Animals that received the high anaerobic dose had a significant increase in bacterial loads within the liver. Histologic examination of the L. monocytogenes positive livers exhibited locally extensive areas of hepatocellular necrosis, though the extent of this damage differed between treatment groups. Microbial community analysis of the cecum from gerbils infected with L. monocytogenes indicated that the abundance of Bacteroidales and Clostridiales increased and there was a decrease in the abundance of Spirochaetales. This study suggests that anaerobic conditions alter the localization pattern of L. monocytogenes within the gastrointestinal tract. These findings could relate to how different populations are more susceptible to listeriosis, as oxygen availability may differ within the gastrointestinal tract.

Yeast Pro- and Paraprobiotics Have the Capability to Bind Pathogenic Bacteria Associated with Animal Disease.

Live yeast probiotics and yeast cell wall components (paraprobiotics) may serve as an alternative to the use of antibiotics in prevention and treatment of infections caused by pathogenic bacteria. Probiotics and paraprobiotics can bind directly to pathogens, which limits binding of the pathogens to the intestinal cells and also facilitates removal from the host. However, knowledge of bacterial binding, specificity, and/or capability is limited with regard to probiotics or paraprobiotics. The goal of this study was to characterize the qualitative and quantitative nature of two Saccharomyces cerevisiae probiotics and three S. cerevisiae paraprobiotics to adhere to thirteen different pathogenic bacteria using scanning electron miscroscopy and filtration assays. On average, the yeast probiotics (LYA and LYB) exhibited overall greater (P < 0.05) adhesion to the pathogenic bacteria tested (41% and 34%) in comparison to paraprobiotics (23%, 21%, and 22%), though variations were observed between pathogens tested. The ability of Salmonella and Listeria to utilize components of the yeast as a nutrient source was also tested. Bacteria were cultured in media with limited carbon and supplemented with cell free extracts of the probiotics and paraprobiotics. Salmonella exhibited growth, indicating these pathogens could utilize the yeast lysates as a carbon source. Listeria monocytogenes had limited growth in only one of the lysates tested. Together, these data indicate that the interaction between probiotics and paraprobiotics occurs in a strain dependent mechanism. Administration of probiotics and paraprobiotics as therapeutics therefore needs to be specific against the bacterial pathogen target.

Listeria Occurrence in Poultry Flocks: Detection and Potential Implications.

Foodborne pathogens such as Salmonella, Campylobacter, Escherichia coli, and Listeria are a major concern within the food industry due to their pathogenic potential to cause infection. Of these, Listeria monocytogenes, possesses a high mortality rate (approximately 20%) and is considered one of the most dangerous foodborne pathogens. Although the usual reservoirs for Listeria transmission have been extensively studied, little is known about the relationship between Listeria and live poultry production. Sporadic and isolated cases of listeriosis have been attributed to poultry production and Listeria spp. have been isolated from all stages of poultry production and processing. Farm studies suggest that live birds may be an important vector and contributor to contamination of the processing environment and transmission of Listeria to consumers. Therefore, the purpose of this review is to highlight the occurrence, incidence, and potential systemic interactions of Listeria spp. with poultry.

The Effect of Oxygen on Bile Resistance in Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive facultative anaerobe that is the causative agent of the disease listeriosis. The infectious ability of this bacterium is dependent upon resistance to stressors encountered within the gastrointestinal tract, including bile. Previous studies have indicated bile salt hydrolase activity increases under anaerobic conditions, suggesting anaerobic conditions influence stress responses. Therefore, the goal of this study was to determine if reduced oxygen availability increased bile resistance of L. monocytogenes. Four strains representing three serovars were evaluated for changes in viability and proteome expression following exposure to bile in aerobic or anaerobic conditions. Viability for F2365 (serovar 4b), EGD-e (serovar 1/2a), and 10403S (serovar 1/2a) increased following exposure to 10% porcine bile under anaerobic conditions (P < 0.05). However, HCC23 (serovar 4a) exhibited no difference (P > 0.05) in bile resistance between aerobic and anaerobic conditions, indicating that oxygen availability does not influence resistance in this strain. The proteomic analysis indicated F2365 and EGD-e had an increased expression of proteins associated with cell envelope and membrane bioenergetics under anaerobic conditions, including thioredoxin-disulfide reductase and cell division proteins. Interestingly, HCC23 had an increase in several dehydrogenases following exposure to bile under aerobic conditions, suggesting that the NADH:NAD+ is altered and may impact bile resistance. Variations were observed in the expression of the cell shape proteins between strains, which corresponded to morphological differences observed by scanning electron microscopy. These data indicate that oxygen availability influences bile resistance. Further research is needed to decipher how these changes in metabolism impact pathogenicity in vivo and also the impact that this has on susceptibility of a host to listeriosis.

RuvAB and RecG are not essential for the recovery of DNA synthesis following UV-induced DNA damage in Escherichia coli.

Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.