Dissertations / Theses on the topic 'Bacteria role in bread'

Microbial contamination and survival during storage of bread are a cause of both health concerns and economic losses. Traditional fermentation systems were studied as sources of lactic acid bacteria (LAB) with antagonistic potential against foodborne pathogens and spoilage organisms, with the aim to improve the safety and shelf life of bakery products. The antagonistic activity of four types of buttermilk (BM) products fermented with Lactococcus lactis subsp. lactis was evaluated against a number of pathogenic bacteria to select the best fermented-BM for application as bio-preservatives in bread crumpets, showing up to 9 µg/ml of nisin equivalent antimicrobial activity. These food ingredients could be suitable to be used in crumpet formulations, BM fermented with Lc. lactis subsp. lactis and nisin influenced the quality and shelf life of crumpets; the pH value and firmness of products with fermented BM was lower and the acidity and springiness was higher than for unfermented BM treatment and control withouth additive. The nisin and fermented BM treatment had beneficial effects on the pore size and colour in comparison with the control, and improved microbial shelf life by 2 days. Commercial and traditional sourdough and bread samples (n=18) were collected to assess the diversity of LAB strains and potential properties when applied to dough and bread. DGGE followed by sequencing showed that Lactobacillus was the predominant genus in the studied sourdoughs. Lb. plantarum and Lb. brevis strains accounted for 69% of the 32 isolates, out of which 10 were amylolytic and 12 had proteolytic activity. Most were also good acid producers after 24 h at 30°C. Some LAB strains presented a strong in vitro inhibitory activity against five indicator strains, showing potential as starter cultures to ferment sourdough. In subsequent experiments, the properties of 24 sourdoughs were evaluated, and one of them, fermented with Lb. plantarum (SIN3) yielded low pH value, high lactic acid production, and suitable microbial growth, and was selected for further bread making performance trials. The bread with fast fermentation and high sourdough concentration (FFHSD) had a lower pH, higher acidity and increased the quality attributes with significantly better shelf life comparing to the other treatments during the storage period. Sensory evaluation demonstrated that fast-fermented breads were more acceptable than the slow-fermented counterparts. Bread prepared with high level (18%) of sourdough fast-fermented with the selected culture (SIN3) had a good eating quality and shelf life. The approach of this study is likely to yield feasible improvements of the current methods of preparation of baking goods.

Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: The presence and growth of lactic acid bacteria (LAB) in wine and their influence on wine quality has received much attention in recent years. Lactic acid bacteria are responsible for conducting malolactic fermentation (MLF) in wine. The benefits associated with malolactic fermentation in terms of deacidification of wine and the contribution to wine flavour and complexity have also recently been the topic of research. It is impossible to describe malolactic fermentation as distinctly desirable or undesirable in terms of its influence on the final quality of wine. The benefits and disadvantages are dependent upon viticultural region, grape variety, wine composition, winemaking techniques and the style and objectives of the winemaker. Brandy production is a multi-stage process in which base wine production, distillation technique and wood maturation all have a large influence on the final chemical profile and organoleptic quality of the brandy. The volatile composition of the base wine, which basically undergoes a concentration process during the subsequent double distillation phase, is critical in determining the aroma and flavour quality of the final brandy product. Thus, the brandy is only as good as the base wine it is distilled from. The aims of this study were to determine the effect of lactic acid bacteria and spontaneous malolactic fermentation on the quality of brandy base wine and the resulting distillate, and to determine which LAB species had been responsible for the occurrence of spontaneous MLF. This study showed that LAB are present at high numbers and are able to conduct spontaneous MLF of brandy base wines. It was shown that the incidence of spontaneous MLF varied from year to year. In 1998, 50% of the commercially produced base wines had undergone partial MLF prior to distillation. In 1999 and 2000 respectively, 34% and 45% of the commercial base wines had undergone partial MLF prior to distillation. The occurrence of spontaneous MLF had an influence on the chemical composition and the sensory quality of the base wine and distillate. There was an increase in the concentrations of ethyl lactate, acetic acid and diethyl succinate in samples that had undergone MLF. There was also a decrease in the concentrations of esters, such as iso-amyl acetate, ethyl acetate, ethyl caproate, hexyl acetate and 2-phenethyl acetate in these same samples. Sensory evaluation of the base wines and distillates demonstrated that samples that had undergone MLF differed significantly from samples that had not undergone MLF. It was also shown that distillates that had not undergone MLF had a slightly better aroma profile than those that had. Sweet aromas, like chocolate and caramel, as well as negative aromas, like chemical or solvent, were more prominent in brandy distillates that had undergone MLF. Herbaceous and fruity aromas were more intense in distillates not having undergone MLF. Fifty-four strains, all Gram-positive and catalase negative, were isolated at different stages of brandy production. Seven strains were isolated from the grape juice, 15 strains were isolated from the base wine, 20 strains were isolated during MLF and 12 strains were isolated from the base wine after MLF had been completed. Based on C02 production from glucose and gluconate, 17 strains were classified as facultatively heterofermentative and 37 strains as obligately heterofermentative. Fifteen of the 37 obligately heterofermentative strains were rod-shaped and were regarded as lactobacilli. The remaining 22 strains were oval or cocci-bacilli shaped. The isolates were identified to species level by using numerical analysis of the total soluble cell protein patterns, 16S rRNAsequencing and polymerase chain reaction (PCR) with species-specific primers. The facultative heterofermentative lactobacilli were identified as Lactobacillus paracasei and Lactobacillus p/antarum. The fifteen obligately heterofermentative lactobacilli were identified as members of the species Lactobacillus brevis, Lactobacillus verrniforme, Lactobacillus buchneri and Lactobacillus hi/gardii. The 22 obligate heterofermentative isolates, with a coccoid morphology, could be grouped into two clusters and were identified as Oenococcus oeni. O. oeni was the species responsible for the occurrence of spontaneous MLF in most of the commercial base wines. Lb. brevis, Lb. hi/gardii and Lb. paracasei were also isolated from commercial base wines that had undergone spontaneous MLF. In nine out of 14 experimental base wine samples that had undergone spontaneous MLF, O. oeni was again the predominant species. Lb. brevis, Lb. hi/gardii and Lb. paracasei were identified in the remaining experimental base wine samples. This is the first report of the presence of Lb. perecese! and Lb. vermiforme in brandy base wine. It was shown that the occurrence of spontaneous MLF had a negative effect on the quality of brandy base wine, but that was shown to be due to the different species and strains performing MLF. In the non-preferred distillate samples, Lactobacillus spp. had performed MLF or had developed after or during MLF.
AFRIKAANSE OPSOMMING: Die teenwoordigheid en die vermoë van melksuurbakterieë (MSB) om in wyn te groei, is 'n onderwerp wat al heelwat nagevors is. Melksuurbakterieë is verantwoordelik vir die uitvoering van appelmelksuurgisting (AMG) in wyn. Die voordele verbonde aan appelmelksuurgisting, ten opsigte van die verlaging van die totale suurinhoud en die bydrae tot die verbeterde geur en kompleksiteit van die wyn, is ook al goed bestudeer. Wat die invloed op die finale wynkwaliteit betref, is dit byna onmoontlik om AMG as uitsluitlik gewens óf ongewens te beskou. Die voordele en nadele van AMG is afhanklik van verskeie faktore, nl. wingerdkundige streek, druifkultivar, wynsamestelling, wynmaakpraktyke, asook die styl en doelwitte van die wynmaker. Die produksie van brandewyn is 'n multistapproses waarin die bereidingsmetode van die basiswyn, die distillasietegniek en houtveroudering 'n groot invloed op die finale kwaliteit en chemiese samestelling van die brandewyn het. Die vlugtige verbindings van die basiswyn, wat tydens die dubbele distillasieproses gekonsentreer word, is van wesenlike belang in die bepaling van die aroma en geur van die finale brandewynproduk. Brandewyn is dus inderdaad net so goed soos die basiswyn waarvan dit gestook is. Die doelwitte van hierdie studie was om te bepaal wat die invloed van MSB en die voorkoms van spontane AMG op die kwaliteit van die basiswyn en die distillaat is, asook om die MSB wat vir die voorkoms van spontane AMG verantwoordelik was, te identifiseer. Hierdie studie het bewys dat MSB in hoë getalle teenwoordig was en dat dit in staat is om die spontane AMG van basiswyne uit te voer. Daar is bewys dat die voorkoms van spontaneAMG moontlik van jaar tot jaar kan verskil. In 1998 het 50%, in 1999 het 34% en in 2000 45% van die kommersieel-geproduseerde basiswyn gedeeltelike AMG spontaan voor distillasie ondergaan. Daar is ook gevind dat spontane AMG 'n invloed op die chemiese samestelling en sensoriese kwaliteit van die basiswyn en die distillaat gehad het. Daar was 'n toename in die konsentrasies van etiellaktaat, asynsuur en diëtielsuksinaat in monsters wat spontane AMG ondergaan het. In dieselfde monsters was daar ook 'n afname in die konsentrasies van iso-amielasetaat, etielasetaat, etielkaproaat, heksielasetaat en 2-fenielasetaat. Sensoriese evaluering van die basiswyne en distillate het getoon dat daar betekenisvolle verskille was tussen die monsters wat AMG ondergaan het en dié wat nie AMG ondergaan het nie. Daar is bewys dat die distillate wat nie AMG ondergaan het nie, 'n beter aromaprofiel gehad het as dié wat AMG ondergaan het. Soet geure, soos sjokolade en karamel, en negatiewe geure, soos "chemies" en "oplosmiddel", was prominent in distillate wat AMG ondergaan het. Kruidagtige en vrugtige geure was meer intensief in distillate wat nie AMG ondergaan het nie. Vier-en-vyftig bakteriese rasse, almal Gram-positief en katalase-negatief, is gedurende die verskillende stadia van brandewynproduksie geïsoleer. Sewe rasse is uit druiwesap, 15 rasse gedurende die alkoholiese fermentasie, 20 rasse gedurende AMG en 12 rasse na voltooiing van AMG geïsoleer. Op die basis van koolstofdioksied (C02)-produksie vanaf glukose en glukonaat is 17 rasse as fakultatief heterofermentatief en 37 rasse as obligaat heterofermentatief geklassifiseer. Vyftien van die 37 obligaat-heterofermentatiewe rasse was staafvormig en is as lactobacilli geïdentifiseer. Die oorblywende 22 het ovaal of kokkus-bacillusvormige selmorfologie getoon. Identifikasie tot op spesievlak is gedoen deur van numeriese analise van die totale oplosbare selproteïenprofiele, 16S-rRNAvolgordebepalings en spesie-spesifieke inleiers vir die polimerasekettingreaksie (PKR) gebruik te maak. Die fakultatief-heterofermentatiewe rasse is as Lactobacillus paracasei en Lactobacillus p/antarum geklassifiseer. Die 15 obligaat heterofermentatiewe stafies is as Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus hi/gardii en Lactobacillus vermiforme geïdentifiseer. Die 22 ovaal, obligaat heterofermentatiewe isolate kon in twee groepe ingedeel word en is as Oenococcus oeni geïdentifiseer. Daar is bevind dat O. oeni-isolate vir die voorkoms van spontane AMG in die meeste van die kommersiêle basiswyne verantwoordelik was. Lb. brevis, Lb. hi/gardii en Lb. paracasei is ook uit kommersiêle basiswyne wat spontane AMG ondergaan het, geïsoleer. In nege uit 14 van die eksperimentele basiswyne wat spontane AMG ondergaan het, was O. oeni die dominante spesie. In die oorblywende eksperimentele wyne is Lb. brevis, Lb. hi/gardii en Lb. paracasei aangetref. Hierdie is die eerste vermelding van die teenwoordigheid van Lb. paracasei and Lb. vermiforrne in brandewynbasiswyn. Daar is gevind dat die voorkoms van spontane AMG "n negatiewe invloed op brandewynkwaliteit het, maar dit is as gevolg van die verskeidenheid van MSB-spesies en rasse wat voorkom. In die distillate wat deur die proepaneel afgekeur is, het Lactobacillus spesies die AMG deurgevoer, of het dit tydens of na AMG ontwikkel.

Quorum sensing is a fundamental process to all of microbiology since it is ubiquitous in the bacterial world, where bacterial cells communicate with each other using low molecular weight signal molecules called autoinducers. Despite the fact that quorum sensing regulates numerous bacterial behaviours, very few studies have addressed the role of this phenomenon in foods. The microbial association of beef consists mainly of pseudomonads, Enterobacteriaceae, Brochothrix thermosphacta and lactic acid bacteria as revealed by minced beef samples purchased from retail shops, which fluctuates according to the storage conditions. Certain members of the microbial association, which are considered to produce signal molecules, have been found to be major contributors to meat spoilage. Pseudomonas fragi and Enterobacteriaceae strains, i.e., Hafnia alvei and Serratia liquefaciens are among the most common quorum sensing signal producers recovered from various food environments. Cont/d.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Curriculum of Oral Biology." Discipline: Oral Biology; Department/School: Dentistry.

Summary in English.
The role of bacteria in the digestive tract of the abalone Haliotis midae was examined to determine whether bacteria aid hydrolysis of polysaccharides present in seaweeds which farmed abalone consume. The enteric bacteria were enumerated using culturable and total (DAPI) counts. The numbers of culturable bacteria fell between 3.5x10⁵ and 2.3x10⁸ cfu/g wet weight tissue. The DAPI counts were between 1.6x10⁹ and 5.1x10⁹ cells per gram of tissue. The numbers of bacteria differed between the crop, stomach and intestine. Electron microscopy showed that bacteria were present on the food and gut wall. No specialised structures, to aid adhesion of bacteria, were apparent on the gut wall. The isolated bacteria were identified to genus level using standard biochemical and morphological tests. The common genera identified were Vibrio, Alcaligenes, Flavobacteria, Pseudomonas and Aeromonas. The bacterial communities in each gut region varied, suggesting that both resident and transient bacterial populations are present in H. midae. Alcaligenes occurred mainly in the crop, while Vibrio species were predominant in the stomach and intestine. The bacterial isolates were tested for their ability to hydrolyse the polysaccharides alginate, laminarin, CMC, carrageenan and agarose. Bacteria able to utilise these polysaccharides belonged to the genera Flavobacteria, Pseudomonas, Vibrio, Alcaligenes, Bacillus and Enterobacteria. Many of the isolated bacteria were capable of utilising two or three of the substrates tested. Quantitative poly saccharase assays using the reducing sugar assays of Nelson (1944) and Somogyi (1952) and Gardner et al. (1988) were employed. These assays showed that bacteria from the crop exhibited the greatest degree of CMC and alginate hydrolysis. Bacteria from the intestine exhibited the greatest carrageenan and agarose hydrolysis. The endogenous enzymes produced by H. midae were examined using extracts of the hepatopancreas as it was found to be bacteria free. It was found that abalone synthesize a CMCase, laminarinase, alginase, carrageenanase and agarase. However, the synthesis of these enzymes was related to the diet of the abalone. Abalone fed Ecklonia (which contains relatively high concentrations of alginate and laminarin) exhibited significantly higher alginase and laminarinase activity than abalone fed Gracilaria. Similarly, abalone fed Gracilaria (which contains relatively high proportions of carrageenan and agar) produced significantly higher carrageenanase and agarase activity. Furthermore, these enzyme activities were found to be similar to those extracted from gnotobiotic abalone (obtained using the antibiotics chloramphenicol (250μg/ml), ampicillin (600μg/ml) and cefotaxime (250μg/ml)), indicating that polysaccharide synthesis occurs in the hepatopancreas of H. midae. Polysaccharase assays on gnotobiotic abalone were compared to assays on untreated abalone. Bacteria were found to significantly enhance the polysaccharase activity of alginase, laminarinase and agarase hydrolysis.

The F[1]F[O]-ATP synthase found in bacteria, mitochondria, and chloroplasts is responsible for synthesizing ATP from the precursors ADP and P[i]. In bacteria, the enzyme is used to synthesize ATP when the electrochemical gradient of protons ([Delta][mu]H⁺) or sodium ions ([Delta][mu]Na⁺) is high, and the phosphorylation potential is low inside the cell. Conversely, the enzyme hydrolyses ATP to generate a [Delta][mu]H⁺ or [Delta][mu]Na⁺ when these gradients are low, and the phosphorylation potential is high inside the cell. This is coupled to intracellular pH homeostasis in some fermentative bacteria. The aim of this project was to determine the role(s) of the F[1]F[O]-ATP synthase in the physiology of two anaerobic extremophiles: Clostridium paradoxum, a thermoalkaliphilic bacterium that grows over the pH range 7.5 to 10, and Thermotoga maritima, a hyperthermophilic bacterium that thrives at neutral to acidic pH. These bacteria represent good models to study membrane-bound energetic processes as a function of pH and temperature. The energetics of C. paradoxum growth was investigated in batch culture. Growth of C. paradoxum was inhibited by the F-type ATP synthase inhibitor N,N�-dicyclohexylcarbodiimide (DCCD) and monensin, suggesting an important role for an F-type ATP synthase and a chemical gradient of sodium ([Delta]pNa⁺) in the growth of this bacterium. Protonophores had no effect on the growth of C. paradoxum. Inverted membrane vesicles contained DCCD-sensitive ATPase activity and this activity was extracted using the detergent Triton X-100. The solubilized enzyme was purified 30-fold by polyethylene glycol-6000 precipitation. The purified enzyme displayed the typical subunit pattern for an F[1]F[O]-ATP synthase, but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The c-ring was purified, crystallized in 2D, and the projection map indicated that C. paradoxum contains an undecameric c-ring. The purified ATPase was stimulated by Na⁺ ions, and sodium provided protection against inhibition by DCCD that was pH-dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed [Delta]pNa⁺ in the presence of a transmembrane electrical potential ([Delta][phi]) and was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q�⁸, E⁶�, and S⁶�). On the basis of these properties, the ATPase is a sodium-translocating enzyme that generates a [Delta][mu]Na⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the [epsilon] subunit that has been shown to be essential for coupled ATP synthesis. To facilitate future work on this enzyme at a molecular level, we developed a heterologous over-expression/production system to produce the ATPase in E. coli DK8 ([Delta]unc) in the presence of the helper plasmid pLysRARE. In future studies, this system will be used for the purification of mutant enzyme complexes. Thermotoga maritima is an anaerobic hyperthermophilic bacterium that grows at 80�C and pH 7.0. Growth is sensitive to DCCD and monensin, but resistant to protonophores. These data suggest a [Delta]pNa⁺ is indispensable for growth and a F-type ATP synthase plays an important role under these conditions. The ATPase activity was extracted from inverted membrane vesicles using the detergent n-dodecyl β-D-maltoside. To investigate the role of the ATPase, the enzyme complex was purified 13-fold by polyethylene glycol-6000 precipitation, anionic exchange, and gel filtration chromatography. The purified enzyme was stimulated in the presence of low concentrations of Na⁺ ions, and ATP-dependent uptake of ��Na⁺ into inverted membrane vesicles was observed to be sensitive to DCCD and monensin. Furthermore, analysis of the published genome sequence revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., E��, E⁶⁵ and T⁶⁶) and analysis of the ATPase by SDS-PAGE revealed the presence of a putative SDS-stable c-ring. On the basis of these properties, the ATPase from T. maritima appears to be a sodium-translocating enzyme. While we were unable to establish a physiological role for the ATPase, we propose that the enzyme generates a [Delta][mu]Na⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). To improve purification and yield, the atp operon was cloned to allow the heterologous over-production of the ATPase. No expression of the ATPase (F[1]F[O]) could be achieved in E. coli BL21(DE3)-CodonPlus, C41(DE3) or E. coli DK8 ([Delta]unc). However, the overproduction of the F₁-ATPase utilizing the helper plasmid pLysRARE was succesful and the enzyme was purified 18-fold by heat precipitation, followed by anionic exchange and gel filtration chromatography, yielding a specific activity of 2.5 U/mg. This complex will be used in the future to investigate the biochemical properties of an F₁-ATPase from a hyperthermophilic anaerobe.

Despite being abundant in the earth's crust, the concentration of Fe in many oceanic regions is so low that it is limiting to the growth of photosynthetic plankton. Heterotrophic bacteria play key roles in the oceanic cycling of carbon and nutrients, but it is unclear whether they can be Fe-deficient in nature, or what possible effects Fe-deficiency might have on their ecology and physiology. In chapter 1, I investigated the response of a natural bacterial community to a mesoscale Fe-enrichment experiment in the northeast subarctic Pacific. The addition of Fe to surface waters caused a rapid stimulation of bacterial growth and production, and induced the organic Fe uptake systems of bacteria. These findings suggest that bacteria responded directly to increased Fe availability, and may be Fe-deficient in situ. In chapter 2, I examined the effects of Fe-deficiency on the coupled processes of carbon catabolism and adenosine triphosphate (ATP) production in cultures of the marine bacterium Pseudoalteromonas haloplanktis. In Fe-limited cells, Fe-dependent oxidative pathways of ATP production were downregulated, leading to an intracellular energy deficit. Thus, by altering carbon metabolism and energy acquisition of heterotrophic bacteria, Fe may affect the cycling of carbon in parts of the sea.

In this thesis sixty-one clinical Pseudomonas aeruginosa isolates were acquired from hospitals within Japan and fifty-one of these strains were resistant to imipenem and/or meropenem (MIC >4mg/l). Neither IMP-1 nor a novel carbapenemase could be detected in any of these strains; instead synergism between a cephalosporinase and lowered outer membrane permeability was found to be the most prevalent mechanism of imipenem resistance. The carbapenem-hydrolysing metallo-β-lactamase produced by members of the genus Aeromonas have in the past few years demanded attention from a clinical and enzymological point of view. Two imipenem-resistant Aeromonas veronii biovar sobria strains 13 and 99 were isolated from a water source in South India. An imipenem-based detection method applied after isoelectric focusing revealed that a β-lactamase with a pI of 5.84 was responsible for carbapenem hydrolysis in strains 13 and 99 and unlike previously reported Aeromonas metallo-β-lactamases this enzyme could be detected with nitrocephin. Purification of this novel enzyme, nominated AVS-1, further demonstrated the unusual properties of this carbapenemase, most notably its insensitivity to EDTA. A metallo-β-lactamase gene was amplified from A. veronii bv. sobria strains 13 and 99 by PCR. Sequencing of the PCR product revealed that these two strains possess a metallo-β-lactamase gene that is closely related to the metallo-β-lactamase gene imiS previously identified in an isolate of A. veronii bv. sobria. Therefore, minor amino acid substitutions may account for the extended substrate specificity and unusual inhibitor profile of AVS-1. Two non-carbapenem-hydrolysing β-lactamases were also cloned from A. veronii bv. sobria strain 13. One of these β-lactamases a clavulanic acid sensitive β-lactamase was found to be an ampS-like penicillinase. The other cloned β-lactamase could unfortunately not be sequenced.

Enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), and Citrobacter rodentium are diarrhoeal pathogens grouped together on the basis of their ability to intimately adhere to host intestinal epithelia and efface brush border microvilli to form the broader category of attaching effacing (A/E) pathogens. While EPEC and EHEC are human pathogens of global health concern, C. rodentium is a natural murine pathogen which serves as an excellent animal model for in vivo studies. A/E pathogens encode a filamentous type 3 secretion system (T3SS) which is used to deliver virulence factors called effectors directly into the host cell. Both in vivo and in vitro studies have demonstrated that the T3SS is integral to the A/E pathogen virulence strategy. Once translocated to the host cell, effector proteins modulate and disrupt a wide range of host cell signalling pathways and processes including the immune response, cytoskeletal dynamics, GTPase signalling pathways, phagocytosis and apoptosis. As a defence strategy, the host responds by activating an immune response and apoptosis. However, several studies have reported that epithelial cells infected with EPEC do not undergo apoptosis despite the presence of early markers of apoptosis. Recently the NleH effectors were identified in a secretomic and genomic analysis, however their function remains unknown. NleH effectors are present in all sequenced A/E pathogen strains and can be found in duplicate copies (nleH1 and nleH2). We verified that EPEC NleH1 and NleH2 are secreted and translocated in a T3SS dependent manner. In this study we demonstrate that NleH effectors have anti-apoptotic function. During infection of cultured cell lines, we found increased nuclear condensation, membrane blebbing, caspase-3 cleavage and cell death and detachment in an nleH1 and nleH2 double mutant in comparison to wild type EPEC. Furthermore, treatment with a global caspase inhibitor abolished cell loss due to cell death or detachment. Using ectopic expression, we showed that NleH1 alone is sufficient to inhibit caspase-3 cleavage in the presence of the general apoptosis inducers staurosporine and the ER stress apoptosis inducers tunicamycin and brefeldin A. Interestingly we found that NleH effectors are kinases, however their kinase activity is not involved in the inhibition of apoptosis. However, an intact C terminus is essential for NleH’s anti-apoptotic activity. To determine the pathway by which NleH effectors inhibit apoptosis a HeLa human cDNA library was used to screen for potential binding partners using a Y2H assay. The ER antiapoptotic protein Bax inhibitor 1 (BI-1) was identified as a putative binding partner and verified using a direct 2 hybrid assay. Knockdown of BI-1 resulted in loss of NleH’s cytoprotective function. As BI-1 plays a role in calcium homeostasis, the effect of NleH effectors on cytosolic Ca2+ levels was assessed. We found that NleH effectors reduced cytosolic Ca2+ levels in a BI-1 dependent manner. Using the C. rodentium animal model we verified the ability of NleH to inhibit apoptosis in vivo by demonstrating that NleH inhibits caspase-3 activation and nuclear condensation at the site of bacterial attachment. Furthermore, we found a reduction in cell exfoliation in the presence of NleH. Additionally we showed that the NleH effectors play a role in competitiveness and also induce a mild but significant increase in NF-KB activation and TNF-[alpha] expression. Together our results demonstrate that NleH effectors are multi-functional proteins that inhibit apoptosis both in vitro and in vivo, and are kinases with an unknown function. Furthermore, NleH effectors induce a local NF-KB and TNF-[alpha] upregulation which could be linked to its anti-apoptotic activity. NleH effectors may provide a competitive advantage by preventing shedding of infected cells to prolong infection.

Master of Science
Food Science
Fadi Aramouni
Due to an increase in awareness of celiac disease, the gluten-free market continues to expand. However, gluten-free breads are still characterized by a poor structure and overall mediocre quality. This research was aimed at determining the impact of egg addition as well as an antistaling agent (DATEM) on the quality of a batter-based gluten-free sorghum bread. Gluten-free bread loaves containing 20, 25, or 30% eggs (as is) on a flour basis were evaluated against a control (no egg). The impact of the antistaling agent, DATEM at 0.5% was also studied for each of these formulations. Quality factors evaluated included water activity, color, specific volume, and cell size. Texture profile analysis was performed to evaluate staling rate based on changes in crumb hardness values and a trained panel evaluated staling attributes by descriptive analysis. Finally, a consumer acceptance test on sorghum bread with and without eggs was also conducted. Results showed that sorghum breads with eggs had higher specific volumes than control (increase from 0.06 cm[superscript]3/g to 0.11 cm[superscript]3/g), while DATEM had a negative effect on the volume of gluten-free bread (decrease of 0.73 cm[superscript]3/g). Eggs also improved cell structure and produced significantly darker crust (p<0.05). Additionally, the addition of eggs reduced bread hardness (from 54 g force to 142 g force on fresh bread) and slowed the rate of staling over the 12 day storage period studied. Descriptive analysis results confirmed the findings of the texture analysis, showing control bread significantly harder (p<0.05) than egg-containing bread at days 0 and 4. The consumer test indicated a significant preference (p<0.05) for sorghum bread with eggs over the control. The overall acceptability score for this bread was above 6 on a 1 to 9 hedonic scale. The score was closer to 7 when the bread was rated by consumers with celiac disease. This research proved that the addition of eggs to a gluten-free sorghum bread formulation resulted in delayed staling and better overall quality and acceptability of the product.

The interaction between microorganisms and the calcite mineral surface in aqueous solutions, under earth surface conditions, was the focus of this study. More specifically, we investigated if bacterial attachment and metabolism increase the dissolution rates of calcite crystals and alter their surfaces in solution. A natural microbial consortium, rather than model organisms, was used in the experiments. Weathered samples from the Trenton carbonates were collected on the flanks of Mount Royal in Montréal (Québec, Canada). The associated bacteria were identified using molecular biology DNA fingerprinting techniques. This information was used to determine the nutrient requirements of suitable growth media. Samples contained typical soil dwelling organisms from the phylum Actinobacteria, gram-positive heterotrophs. Bacteria were combined with cleaved Iceland Spar calcite rhombohedra in a low-ionic strength (10−2 M) NaCl solution at ambient pCO2 , 25°C and 1 atm pressure. The effect of solution chemistry (e.g. the presence of phosphate) on the calcite dissolution kinetics was also investigated. The dissolution rates in the presence of bacteria, did not vary significantly from abiotic conditions, but decreased notably in the presence of phosphate.
Cette étude porte sur les interactions entre des micro-organismes et la surface de la calcite en solution aqueuse sous des conditions équivalentes à celles de la surface de la terre. Plus précisément, nous avons étudié si l'attachement des bactéries et leur métabolisme augmentent la vitesse de dissolution des cristaux de calcite et altérent leur surface en solution. Des communautés microbiennes naturelles ont été privilégiées à des organismes types pour les expériences. Des échantillons altérés provenant de carbonates de Trenton ont été récoltés sur les flancs du Mont Royal à Montréal (Québec, Canada). Les bactéries associées ont été identifiées par des techniques de biologie moléculaire utilisant leurs empreintes génétiques d'ADN. Ces informations ont servi à déterminer les besoins en nutriments des milieux de croissance. Les échantillons contenaient des organismes typiques de sols, hétérotrophes, à gram positif, du phylum Actinobacteria. Les bactéries ont été combinées avec des rhombohèdres clivés de calcite provenant de spaths d'Islande dans une solution de NaCl de faible force ionique (10−2 M) à pCO2 ambiante, 25°C et 1 atm de pression. L'effet de la composition chimique de la solution sur la cinétique de dissolution des calcites (en particulier, la présence de phosphates) a également été étudié. Les vitesses de dissolution augmentent en présence de bactéries ne varient pas de façon significative aux échantillons exposés aux conditions abiotiques. En revanche, la présence de phosphate das le milieu de culture masque l'effet des bactéries sur la vitesse de dissolution.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xvii, 163 p.; also includes graphics (some col.) Includes bibliographical references (p. 149-163). Available online via OhioLINK's ETD Center

The role of bacteria in the decay of building stone from ancient monuments was examined using the framework of Koch's postulates. This involved a stepwise approach to investigate the occurrence, nature and decay potential of bacteria on stone. Prior to investigating the occurrence of bacteria on stonework it was necessary to develop a standardised procedure of high precision for the recovery and enumeration of these bacteria. A number of different methods to remove bacteria from stone were studied including physical agitation, chemical desorption and surfactant treatment. Finally a method was adopted in which stone samples were powdered, homogenised in a dilute solution of surfactant (Tween 80) and counted on an automatic plating system. A range of growth media were used to examine three different bacterial types, namely, sulphur-oxidising, nitrifying and heterotrophic. To investigate the occurrence and distri but10n of bacteria on both sound and decayed stone extensive bacteriological surveys were conducted on stonework at two monuments, Portchester Castle and Tintern Abbey. All types of bacteria were widely distributed on both sandstone and limestone at the monuments. At each monument, significantly more sulphur-oxidising and heterotrophic bacteria were associated with severely decayed stone than undecayed stone. Electron microscopy confirmed that large populations of bacteria could be found predominantly 5-10mm below the surface of decayed stone. Approximately 200 bacteria were isolated into pure culture during the field surveys of the two monuments. All isolates were screened for decay potential using a liquid culture system involving static growth of bacteria in the presence of 1cm stone discs. From the 200 isolates, about 30 were capable of causing substantial weight loss in sandstone discs under heterotrophic conditions. Five isolates were able to cause a large weight loss using only mineral nutrients. Some isolates caused a significant weight gain in the stone discs under these conditions. Statistical analysis of the data from this decay screen indicated that weight loss of stone could be directly correlated to a decrease in pH of the medium and a release of calcium and silicate from the stone. Futher decay studies carried out on selected isolates suggested that under heterotrophic conditions the bacteria secreted quantities of organic acids in to the medium which could attack the stone. However, in the presence of an inorganic nutrient source, the generation of mineral acids may be involved. Under both conditions different stones had varying resistance to bacterial decay and this appeared to be dependent upon the level of calcite in the stone. Specific antibody techniques such as BLISA and FAT were examined and proved very useful in demonstrating the presence of certain principal decay species on samples of decayed stone.

Haptoglobin (HP) is a positive acute-phase serum protein. It is upregulated during infection and is a valuable marker for many inflammatory-related diseases. In serum it is present as a disulphide-linked homodimer, with each subunit being composed of two domains, a complement control protein domain (CCP-domain) and a serine-protease-like domain. During biosynthesis the polypeptides are cleaved into α- (the CCP-domain) and β-chains (the SP domain) and both remain linked together by a disulphide bond. A crucial function of HP is to act as a scavenger of free haemoglobin from plasma, since high quantities of free haemoglobin can be deleterious for the host. More recent work indicates that HP also interacts with lipoteichoic acid (LTA) of Gram-positive bacteria, an important virulence factor, and this is the focus of my thesis. The results of this work demonstrate that HP binds to LTA directly as well as to LTA on a wide range of Gram-positive bacteria (including LTA from S. aureus and S. pneumoniae). The IC50 of the interaction is ~40 nM in competition experiments to immobilised S. aureus. My work has shown that the LTA interaction site of HP is located on the β-chain and that LTA competes for binding with haemoglobin indicating that the binding sites for LTA and haemoglobin overlap. Surprisingly, in vivo studies showed that C57BL/6J HP-/- mice show a significant degree of protection from experimental S. pneumoniae infection. Over the course of the experiments, approximately 90% of HP-/- mice were resistance to the pneumococcal infection compared to 40% in strain, age and sex matched control wild-type mice infected in parallel. In line with the infection study, wild-type mice showed significantly higher levels of bacteraemia than HP-/- mice and the bacterial load in the lung, liver, kidney was significantly higher. These findings demonstrate that HP interacts with bacteria and plays an important role during infection. Surprisingly, the presence of HP appears to give S. pneumoniae a so far unknown advantage during infection.

Horizontal gene transfer (HGT) is probably the most important mechanism for functional novelty and adaption in bacteria. However, a robust understanding of the rates of HGT for most bacterial species and the influence of the ecological settings on the rates remain elusive. Four whole-genome comparative studies of free-living bacteria will be described that integrated physiological and ecological data with novel detection bioinformatic pipelines to elucidate the magnitude of HGT at three distinct levels of genetic relatedness: i) the species level, where overlapping ecological niche among co-occurring bacteria in the water column of the Baltic Sea has caused HGT to have been so rampant that it has served as the force of species cohesion; ii) the genus level, where HGT appeared to predominantly mobilize a limited number of genes with ecological/selective advantage (e.g., antibiotic resistance genes) among distinct pathogenic Campylobacter species and hence, did not lead to species convergence; and iii) the phylum level, where HGT was found to be, in general, less frequent than the genus level but, over evolutionary time, has assembled a large part of the metabolic functions of natural microbial communities, especially within organic matter rich, anaerobic habitats. In conclusion, this work advances the methods to link ecological relationships with HGT and suggests that HGT among very divergent organisms may have been more frequent than previously thought and led to successful adaptation.

his study investigated the underlying mechanisms of outer membrane homeostasis in Gram-negative bacteria. Using both the evolved laboratory strain E. coli K12 and the broad host range pathogen Salmonella enterica serovar Typhimurium, we have identified and characterised a series of non-essential genes responsible for the maintenance of the outer membrane barrier function. We have revealed their importance for bacterial pathogenesis suggesting their use as novel targets for drug development. This study has provided the first description of a pathway for phospholipid transport from the inner membrane to the outer membrane via the lipoprotein PlpA, a gene previously of unknown function. As several of the genes highlighted by our initial studies were associated with the biogenesis of virulence factors, we complemented our investigations by characterising the contributions of the S. Typhimurium Type V proteins to virulence in a murine model. These investigations have provided the first peer reviewed characterisation of a trimeric autotransporter from Salmonella, has identified a mechanism by which Salmonella can survive on tomatoes, and has highlighted the functional redundancy of these proteins in Salmonella infection. These findings have significantly advanced our understanding of the mechanisms mediating outer membrane homeostasis and the biogenesis and functions of virulence factors.

In questa tesi di Dottorato, l’obiettivo era valutare l’impatto di diversi ingredienti alimentari sul metabolismo di alcuni batteri intestinali e viceversa, mediante l’applicazione di tecniche omiche. Utilizzando le tecniche di metabolomica e trascrittomica, è stata studiata la risposta all’acido linoleico del ceppo Bifidobacterium breve DSM 20213. Utilizzando un approccio combinato di metagenomica e metabolomica, è stato possibile studiare le modifiche a carico del microbiota intestinale, del profilo fenolico e degli acidi grassi, in biscotti senza glutine (a base di erba medica) durante digestione e fermentazione in vitro. Inoltre, è stato valutato come alcuni batteri potessero interferire negativamente su una terapia farmacologica a base di Diclofenac, un farmaco usato per alcune patologie intestinali. Per questo tipo di studio è stata utilizzata la spettrometria di massa ad alta risoluzione, che ha consentito di ipotizzare un coinvolgimento dell’enzima batterico β-glucuronidasi. Una sola tecnica omica, seppure di ultima generazione, non permette di valutare tutte le modificazioni del microbiota intestinale data la complessità dei fattori coinvolti. Per questa ragione, integrare più approcci omici potrebbe risultare una buona strategia per analizzare il reale impatto del microbiota sulla salute dell’ospite. Questo permetterebbe di valutare le interazioni microbiota-ospite, i principali metabolismi e le interconnessioni tra gruppi batterici coinvolti nella risposta ad uno stimolo esterno come l’assunzione di particolari ingredienti con l’alimentazione.
The aim of the present PhD thesis was to explore the metabolic response of intestinal bacteria to food components by using ‘omics’ approaches. In particular, the first part of this thesis was focused on the effect of linoleic acid on Bifidobacterium breve DSM 20213 strain. Firstly, an untargeted metabolomics-based approach was used to explore the primary changes in metabolic profile of this strain grown in presence of linoleic acid. Secondly, the gene expression of B. breve DSM 20213 induced by linoleic acid exposure was investigated. Integrated use of metabolomics/transcriptomics was applied to better understand the response mechanisms to linoleic acid stress. In the third part of the thesis, using a combination of metagenomics and metabolomics, the in vitro large intestine fermentation of gluten-free rice cookies containing alfalfa seed was investigated. In the last part of my PhD, the negative effect of β-glucuronidase producing bacteria was evaluated by means of qualitative high-resolution mass spectrometry. Based on my experience there is not a gold standard approach for evaluating a complex environment such as the gastrointestinal tract. For this reason, an integrated use of different techniques should be mandatory to have an accurate framework of gut microbiota composition, its potential metabolic network and the impact on the host physiology and health.

In a world where the human population is increasing, new innovations to produce enough food are required. Aquaculture’s part of the global animal protein production has increased in recent years and could be a possible solution. However, if aquaculture is poorly managed, it can result in negative consequences and one such consequence is the development of antibiotic resistance. In this review, I examine how aquaculture affect antibiotic resistance by studying what the literature says on accumulation of antibiotics in different organisms and sediment, if antibiotics can be transferred to humans through consumption of antibiotic treated products, and if human pathogens in aquaculture farms may acquire antibiotic resistance. Furthermore, I examine what factors are contributing to irresponsible antibiotic use and how such use is managed. The result of this review indicate that antibiotics are able to accumulate in organisms and sediment. It is not clear however how consumption of these affect human microbiomes. In contrast, it is clear that antibiotic resistance can be transferred from antibiotic-resistant bacteria to human pathogens. Regarding antibiotic use, irresponsible use foremost exists in low-income countries and the main drivers behind such use are socioeconomic ones, such as lack of knowledge, poverty and food security. Finally, I propose possible solutions that might improve future management.
I en värld där den mänskliga befolkningen ökar krävs nya innovationer för att producera tillräckligt med mat. Vattenbrukets andel av den globala animaliska proteinproduktionen har ökat de senaste åren och kan ses som en potentiell lösning. Om vattenbruk dock hanteras ansvarslöst kan det uppstå negativa konsekvenser. En sådan konsekvens är utveckling av antibiotikaresistens hos skadliga bakterier. I denna litteraturstudie undersöker jag vattenbrukets påverkan på antibiotikaresistens genom att studera vad litteraturen säger om ackumulation av antibiotika i olika organismer och sediment, om antibiotika kan överföras till människor genom konsumtion av antibiotikabehandlade produkter, och om mänskliga patogener i vattenbruksodlingar kan förvärva antibiotikaresistens. Jag undersöker också vilka faktorer som bidrar till ansvarslös antibiotikaanvändning och hur den hanteras ur ett hållbarhetsperspektiv. Resultaten i denna studie tyder på att antibiotika kan ackumuleras i organismer och sediment men att det råder oklarheter huruvida konsumtion av antibiotikabehandlad mat påverkar mänskliga bakteriekulturer. Antibiotikaresistens kan dock överföras från antibiotikaresistenta bakterier till mänskliga patogener. Ansvarslös antibiotikaanvändning sker huvudsakligen i fattigare länder och det är förmodligen i stor utsträckning till följd av socioekonomiska faktorer som okunskap, fattigdom och livsmedelstrygghet. Slutligen föreslår jag lösningar som möjligen kan bidra till bättre hantering av framtida antibiotikaanvändning.

Cyanide was rapidly removed when added to culture supernatants of seven different Pseudomonas. The ability to remove cyanide was correlated with the accumulation of α-keto acids (pyruvate and α-ketoglutarate). These compounds react with cyanide forming less toxic cyanohydrins, thus conferring a mechanism for bacterial cyanide tolerance. When added to growth media the α-keto acids were shown also to serve as effective cyanide antagonists. While all bacteria tested accumulated α-keto acids, only those capable of utilizing cyanide as a nutritional nitrogen source were able to metabolize cyanohydrins. In P. fluorescens NCIMB 11764, the same enzyme (cyanide oxygenase) shown previously to be involved in cyanide metabolism appears responsible for cyanohydrin transformation. Keto acid excretion is believed to represent a new mechanism of bacterial cyanide detoxification with further enzymatic metabolism of the cyanohydrins helping to explain how cyanide can satisfy the nitrogen requirement in cyanide-utilizing bacteria.

La sicurezza e la qualità degli alimenti sono tutt’ora un problema critico per i paesi in via di sviluppo. Le diete a basso contenuto di acido folico, per esempio, possono causare gravi problemi di salute, soprattutto nei bambini. Gravi disturbi legati al tubo neurale (DTN) nei neonati possono derivare infatti da madri che hanno insufficiente apporto di acido folico (400-600 g / giorno) durante il periodo di gravidanza. Inoltre, se non adeguatamente protetti o trattati, I prodotti alimentari possono essere vettori di funghi e batteri patogeni rappresentando una fonte potenziale di malattie per l’uomo e una perdita economica per le industrie agro-alimentari. Nella seguente tesi si è quindi quindi studiato il ruolo di batteri lattici selezionati (LAB) in grado di aumentare il valore nutrizionale del latte attraverso la produzione di acido folico durante il processo di fermentazione. Inoltre, ci si è concentrati sul loro uso come "bio-conservanti" contro funghi e batteri, attraverso la sintesi di composti antimicrobici (batteriocine) in grado di inibire la crescita di funghi filamentosi e/o batteri patogeni.
The safety and quality of food are still a critical issue in developing countries. Diets with a low content of folic acid, for example, may cause serious health problems, especially in children. Severe disorders related to neural tube (NTD) in infants may arise from mothers having inadequate intakes of folic acid (400-600 g/dia) during the mother pregnancy period. Moreover foods, when not properly protected or treated, can be vectors of pathogenic fungi and bacteria thereby representing a potential source of human diseases and an economical loss for the food industry. In the following thesis we have therefore investigated the role of selected lactic acid bacteria (LAB) in increasing the nutritional value of milk through the production of folic acid during the fermentation process. In addition, we focused on their use as “bio-preservatives” against fungal and bacterial spoilage, through the synthesis of antimicrobial compounds (bacteriocins) able to inhibit the growth of filamentous fungi and /or pathogenic bacteria.

Pseudomonas aeruginosa ATCC 9027 grew in a chemically defined medium with n-hexadecane or glucose, 0.5% (v/v or w/v, respectively), as the sole carbon source, and the K$ sb2$HPO$ sb4$, concentration was either 0.6 mM or 2.3 mM with both carbon sources. Only cells grown on n-hexadecane and 0.6 mM K$ sb2$HPO$ sb4$ produced pyocyanine. The variable lag period associated with cells grown on n-hexadecane was regulated by the state of the inoculating culture, grown on glucose and 2.3 mM K$ sb2$HPO$ sb4$. It was found that organic phosphate was more prevalent in cells grown on both phosphate concentrations with glucose and the high phosphate concentration with n-hexadecane, than it was for the low phosphate, n-hexadecane grown cells. The inorganic phosphate levels remained low under all conditions, decreasing as the cell culture became older. The inorganic polyphosphate level remained stable for all conditions, except in the high phosphate, n-hexadecane grown cells, where there was an increase.

Enterolignans (enterodiol and enterolactone) exhibit structural similarity to estradiol and have therefore been hypothesized to modulate hormone related cancers such as breast cancer. The bioactivation of the plant lignan secoisolariciresinol diglucoside (SDG) requires the transformation by intestinal bacteria including the deglycosylation of SDG to secoisolariciresinol (SECO) followed by demethylation and dehydroxylation of SECO to enterodiol (ED). Finally, ED is dehydrogenated to enterolactone (EL). It is unclear whether the bacterial activation of SDG to ED and EL is crucial for the cancer preventing effects of dietary lignans. The possible protective effect of bacterial lignan transformation on a 7,12 dimethylbenz(a)anthracene (DMBA)-induced breast cancer in gnotobiotic rats was investigated. Germ-free rats were associated with a defined lignan-converting consortium (Clostridium saccharogumia, Blautia producta, Eggerthella lenta, and Lactonifactor longoviformis). The rats colonized with lignan-converting bacteria consortium (LCC) were fed a lignan-rich flaxseed diet and breast cancer was chemical induced. Identically treated germ-free rats served as control. All bacteria of the consortium successfully colonized the intestine of the LCC rats. The plant lignan SDG was converted into the enterolignans ED and EL in the LCC rats but not in the germ-free rats. This transformation did not influence cancer incidence but significantly decreased tumor numbers per tumor-bearing rat, and tumor size. Cell proliferation was significantly inhibited and apoptosis was significantly induced in LCC rats. No differences between LCC and control rats were observed in the expression of the genes encoding the estrogen receptors (ERα and ERβ) and G-coupled protein receptor 30 (GPR30). Similar findings were observed for both insulin-like growth factor 1 (IGF-1) and epidermal growth factor receptor (EGFR) genes involved in tumor growth. Proteome analysis revealed that 24 proteins were differentially expressed in tumor tissue from LCC and germ-free. RanBP-type and C3HC4-type zinc finger-containing protein 1 (RBCK1) and poly(rC)-binding protein 1 (PBCP1) were down-regulated by 3.2- and 2.0-fold, respectively. These proteins are associated with cell proliferation. The activity of selected enzymes involved in the degradation of oxidants in plasma and liver was significantly increased in the LCC rats. However, plasma and liver concentrations of reduced glutathione (non-enzymatic antioxidant) and malondialdehyde (oxidative stress marker) did not differ between the groups. In conclusion, the bacterial conversion of plant lignan to enterolignans beneficially influences their anti-cancer effect. However, the mechanisms involved in these effects remain elusive.
Enterolignanen (Enterodiol ED und Enterolacton EL) wird aufgrund ihrer strukturellen Ähnlichkeit zu Estradiol ein modulierender Einfluss auf hormonell bedingte Krebserkrankungen wie Brustkrebs nachgesagt. Das pflanzliche Lignan Secoisolariciresinoldiglucosid (SDG) wird durch Darmbakterien zum Enterolignan aktiviert. Dies erfolgt über dessen Deglykosylierung zu Secoisolariciresinol (SECO) gefolgt durch die Demethylierung und die Dehydroxylierung zu Enterodiol (ED). Schließlich wird ED zu Enterolacton (EL) dehydrogeniert. Es ist allerdings noch nicht bewiesen, dass die bakterielle Aktivierung von SDG zu ED und EL für die antikanzerogenen Wirkungen verantwortlich ist, die für dieses in der menschlichen Ernährung vorkommende Lignan beschrieben wurden. Um dies zu klären, wurde der Einfluss der bakteriellen Lignan-Transformation auf die Protektion gegenüber einem durch 7,12-Dimethylbenz(a)anthracen (DMBA)-induzierten Brustkrebs im gnotobiotischen Rattenmodell untersucht. Keimfreie Ratten wurden hierfür mit einem Konsortium aus vier Bakterienstämmen (Clostridium saccharogumia, Blautia producta, Eggerthella lenta, und Lactonifactor longoviformis) besiedelt, das die Umsetzung von SDG zu ED und EL katalysiert (LCC-Ratten). Ratten, die über den gesamten Versuchszeitraum keimfrei blieben, dienten als Kontrolle. Die Tiere wurden über 16 Wochen mit einer Leinsamen-Diät gefüttert, die reich an pflanzlichen Lignanen war. Während der Fütterung wurde bei allen Tieren Brustkrebs chemisch induziert. Das pflanzliche Lignan SDG wurde nur in den LCC Ratten zu den Enterolignanen ED und EL umgewandelt. Keimfreie Ratten zeigten keine Transformation von SDG. Die bakterielle Transformation von SDG hatte zwar keinen Einfluss auf die Inzidenz von Brustkrebs, jedoch verringerten sich durch die Besiedlung der Ratten mit SDG-transformierenden Bakterien die Anzahl von Tumoren pro tumortragender Ratte und die Tumorgröße deutlich. Zudem wurde die Zellproliferation in den LCC-Ratten deutlich gehemmt und die Apoptose induziert. Unterschiede in der Genexpression der Östrogenrezeptoren (ERα und ERß) und G-Protein-gekoppelte Rezeptoren (GPR30) wurden zwischen den LCC-Ratten und den Kontrolltieren nicht beobachtet. Ebenso verhielt es sich für die Gene des Insulinähnliche Wachstumsfaktoren 1 (IGF-1) und Epidermale Wachstumsfaktor rezeptoren (EGFR), welche in das Tumorwachstum involviert sind. Die Analyse des Proteoms des Tumorgewebes ergab 24 differentiell exprimierte Proteine zwischen keimfreien und LCC-Ratten. So wurden zum Beispiel die Proteine RanBP-type and C3HC4-type zinc finger-containing protein 1 (RBCK1) und poly(rC)-binding protein 1 (PBCP1), die mit der Zellproliferation assoziiert sind, in LCC-Ratten um das 3,2 bzw. 2,0-fache herunterreguliert. Die Aktivität ausgewählter antioxidativer Enzyme in Plasma und Leber war in den LCC-Ratten im Vergleich zu den keimfreien Tieren deutlich erhöht. Allerdings unterschieden sich die Konzentrationen von reduziertem Glutathion (nichtenzymatisches Antioxidans) und Malondialdehyd (oxidativer Stress-Marker) in Plasma und Leber nicht zwischen den beiden Besiedlungs-Gruppen. Zusammenfassend zeigen die Ergebnisse, dass die bakterielle Umwandlung von pflanzlichen Lignanen zu Enterolignanen deren antikanzerogene Wirkung entscheidend beeinflusst. Allerdings bleiben die zugrunde liegenden Mechanismen weiterhin ungeklärt.

The mammalian gastrointestinal tract is home to a complex microbial community engaged in a dynamic interaction with the immune system. Mucus is the first point of contact of the microbiota with the host, acting as a first line of defence. Furthermore γδ intraepithelial lymphocytes (IELs) respond to the invading bacteria that circumvent the mucus barrier. In this study two approaches were used to investigate the role of mucus in intestinal homeostasis; firstly the impact of γδ IELs on the mucus layer, and secondly the adhesion properties of the gut symbiont Lactobacillus reuteri to mucus. To study the impact of IELs on mucus properties, a γδ T cell-deficient (TCRδ-/-) mouse model was used. TCRδ-/- mice showed increased susceptibility to dextran sodium sulphate (DSS)-induced colitis, alterations in mucin expression, glycosylation and goblet cell numbers, but maintained an intact mucus layer in vivo. Moreover, TCRδ-/- mice showed reduced levels of interleukin-33 mRNA, a mediator of mucosal healing. An ex vivo SI organoid model using input cells from TCRδ-/- mice showed, upon addition of keratinocyte growth factor, increases in crypt length, and both goblet cell numbers and redistribution. These findings provide novel mechanisms by which γδ IELs may modulate mucus properties, explaining the increased susceptibility of TCRδ-/- mice to chemically-induced colitis. L. reuteri strains protect against DSS-induced colitis in mice. To investigate the importance of L. reuteri adhesion to the intestinal mucus layer, the mucus-producing HT29-MTX cell line as well as murine and human intestinal tissues were used in conjunction with chemical treatments. The mucus-binding protein MUB of L. reuteri ATCC 53608 was found to promote L. reuteri adhesion to mucins in a host and tissuespecific manner and display sialic acid-binding specificities. Together, these data provide insights into L. reuteri-mucus interactions; a key factor in influencing host response and exerting health effects.