Comparison of stool microbiota compositions, stool alpha1-antitrypsin and calprotectin concentrations, and diarrhoeal morbidity of Indonesian infants fed breast milk or probiotic/prebiotic-supplemented formula

Aim The composition of faecal microbiota of babies is known to be influenced by diet. Faecal calprotectin and α1-antitrypsin concentrations may be associated with mucosal permeability and inflammation. We aimed to assess whether there was any difference after consumption of a probiotic/prebiotic formula on faecal microbiota composition, calprotectin and α1-antitrypsin levels, and diarrhoea in comparison with breast milk-fed Indonesian infants. Methods One hundred sixty infants, 2 to 6 weeks old, were recruited to the study. They were either breastfed or formula fed (80 per group). Faecal samples were collected at recruitment and 3 months later. Bacterial groups characteristic of the human faecal microbiota were quantified in faeces by quantitative polymerase chain reaction. Calprotectin and α1-antitrypsin concentrations were measured using commercial kits. Details of diarrhoeal morbidity were documented and rated for severity. Results The compositions of the faecal microbiota of formula-fed compared with breast milk-fed children were similar except that the probiotic strain Bifidobacterium animalis subsp. lactisâ€...DR10 was more abundant after 3 months consumption of the formula. Alpha1-antitrypsin levels were higher in breastfed compared with formula-fed infants. The occurrence of diarrhoea did not differ between the groups of babies. Conclusion Feeding Indonesian babies with a probiotic/prebiotic formula did not produce marked differences in the composition of the faecal microbiota in comparison with breast milk. Detrimental effects of formula feeding on biomarkers of mucosal health were not observed.

Immunomodulatory effects of probiotic bacteria in healthy adults

Probiooteilla kantakohtaisia vaikutuksia ihmisen immuunijärjestelmään terveillä aikuisilla Probiooteilla on kantakohtaisia tulehduksen välittäjäaineita vähentäviä vaikutuksia ja probioottien yhdistelmien vaikutukset eroavat yksittäisten kantojen vaikutuksista selviää TtM Riina Kekkosen tuoreesta väitöstutkimuksesta. TtM Riina Kekkonen on selvittänyt väitöskirjassaan eri probioottikantojen vaikutuksia immuunivasteeseen valkosolumallissa sekä terveillä aikuisilla lumekontrolloiduissa kliinisissä tutkimuksissa. Aikaisemmin probioottien vaikutuksia on tutkittu lähinnä allergian ja erilaisten vatsavaivojen ehkäisyssä ja hoidossa. Probiootteja sisältäviä tuotteita käyttävät kuluttajat ovat kuitenkin useimmiten terveitä aikuisia, ja probioottien vaikutus terveiden aikuisten immuunijärjestelmään on ollut puutteellisesti selvitettyä. Valkosolumallissa probioottikantojen havaittiin poikkeavan toisistaan niiden kyvyssä aktivoida immuunivasteen välittäjäaineiden, sytokiinien, tuotantoa. Anti-inflammatorisia, eli tulehdusta lievittäviä vaikutuksia nähtiin lähinnä Bifidobacterium ja Propionibacterium sukuihin kuuluvilla kannoilla. Streptococcus ja Leuconostoc sukuihin kuuluvat kannat puolestaan aktivoivat Th1 tyyppistä, soluvälitteistä immuunivastetta. Eri probioottien kombinaatiot eivät saaneet aikaan voimakkaampaa aktivaatiota yksittäisiin kantoihin verrattuna, joka viittaa probioottien keskinäiseen kilpailuun niiden ollessa kontaktissa ihmisen solujen kanssa. Probioottikantojen valinta kliinisiin tutkimuksiin tehtiin niiden anti-inflammatoristen ominaisuuksien perusteella. Parhaita anti-inflammatorisia kantoja olivat B. lactis ssp. animalis Bb12 ja P. freudenreichii ssp. shermanii JS, joiden lisäksi tutkimuksiin valittiin myös L. rhamnosus GG (LGG) hyvin tutkittuna referenssikantana. Solutöiden tulokset eivät olleet täysin verrannollisia kliinisen työn tuloksiin, koska LGG näytti omaavan parhaat anti-inflammatoriset ominaisuudet kliinisissä tutkimuksissa vaikka solutyössä sen aikaansaamat vasteet olivat melko vaimeita. Kolmen viikon kliinisessä tutkimuksessa terveillä aikuisilla LGG alensi mm. tulehdusta kuvaavan C-reaktiivisen proteiinin ja inflammatoristen sytokiinien määrää. Pidemmässä kolmen kuukauden pituisessa kliinisessä tutkimuksessa LGG:llä ei ollut vaikutusta terveiden aikuisten infektiosairastavuuteen, mutta LGG lyhensi vatsavaivojen kestoa. Probioottien vaikutukset immuunijärjestelmään näyttävät olevan kantakohtaisia ja erityisesti Lactobacillus rhamnosus GG:llä havaittiin anti-inflammatorisia vaikutuksia. Valkosolumallia ei tulisi käyttää ainoana probioottikantojen skriinausmenetelmänä niiden immunologisia vaikutuksia selvitettäessä, koska solutöiden tulokset eivät olleet täysin verrannollisia kliinisten tutkimusten tuloksiin. Sen sijaan veren perifeeristen lymfosyyttien eristäminen ja niiden aktivoitumisen selvittäminen lyhytaikaisessa kliinisessä tutkimuksessa voisi toimia suhteellisen helppona skiinausmenetelmänä.

The role of probiotics in Helicobacter pylori infection

Helicobacter pylori (H. pylori) infection is a major cause of chronic gastritis and peptic ulcer disease, and it is also designated as a class-I carcinogen for stomach cancer. The role of probiotics in the treatment of gastrointestinal infections is increasingly documented as an alternative or complement to antibiotics, with the potential to decrease the use of antibiotics or reduce their adverse effects. These studies were conducted to investigate the role of probiotics in the treatment of H. pylori infection. Various aspects included: an investigation of the effects of a probiotic combination consisting of Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 or B. lactis Bb12 as a supplementation to H. pylori eradication therapy, with special reference to tolerability, effectiveness, and microbiota alterations following the treatment; discovering the role of probiotics in vivo with H. pylori infected and uninfected patients, as well as with an in vitro model of H. pylori infection. The probiotic combination therapy was able to reduce significantly the total symptom score, which takes into account both the frequency and the severity of the adverse effects, during the eradication treatment. The supplementation did not improve the success of the eradication treatment significantly, though some difference was seen in the eradication percentages (91% vs. 79%). The quantities of predominant bacterial groups were altered significantly following the triple treatment. Probiotics slightly counteracted the effects of anti-H. pylori treatment, monitored as significantly less alterations in the total numbers of aerobes and lactobacilli/enterococci group bacteria. After probiotic intervention, L. rhamnosus GG adhered to a minority of the patients upper gastrointestinal mucosa, but all of the probiotics survived well through the gastrointestinal tract transit with and without antimicrobial treatment. Probiotic intervention decreased gastrin-17 levels in H. pylori infected patients and appeared to decrease the 13C-urea breath test values. In in vitro Caco-2 cell line experiments, probiotics inhibited H. pylori adhesion to intestinal epithelial cells. Both L. rhamnosus strains, P. freudenreichii ssp. shermanii JS and the combination inhibited the H. pylori-induced acute cell leakage. Simultaneously, both L.rhamnosus strains and the combination transiently improved the epithelial barrier function. The pro-inflammatory effects prevailed when the probiotics were used in combination. According to this series of studies, probiotic combination could have some potential in reducing adverse effects induced by H. pylori eradication treatment and beneficial effects on H. pylori infected subjects.

Pathophysiological factors of irritable bowel syndrome, and the effects of probiotic supplementation

Gastrointestinal symptoms and impaired quality of life caused by irritable bowel syndrome (IBS) affect up to 20% of the adult population worldwide. The exact aetiology and pathophysiology of IBS are incompletely understood. Clinical studies suggest that supplementation with certain probiotics may be beneficial in IBS, but there is not enough evidence to make general recommendations. The aim of this thesis was to investigate microbiota- and mucosa-associated pathophysiological factors of IBS, and to evaluate the long-term effects of multispecies probiotic supplementation on symptoms, quality of life, intestinal microbiota and systemic inflammatory markers in IBS. The intestinal microbiota composition in IBS patients and healthy control subjects was analysed by quantitative polymerase chain reaction (qPCR). Significantly lower counts for the Clostridium coccoides and the Bifidobacterium catenulatum groups were found in IBS compared to controls. Quantitative differences also appeared in subgroup analysis based on the predominant bowel habit: diarrhoea patients harboured significantly lower numbers of Lactobacillus spp. than the constipation-predominant patients, while higher counts for Veillonella spp. were detected in constipation-predominant patients compared to healthy controls. Analysis of mucosal biopsies by a metabolomic approach revealed multiple differences between patients and controls. The most prominent finding was an upregulation of specific lipid species, principally lysophosphatidylcholines and ceramides, in IBS. The effects of multispecies probiotic supplementation with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium breve Bb99 or Bifidobacterium animalis subsp. lactis Bb12 was evaluated in two, randomised, double-blind, placebo-controlled trials. Compared to placebo, the probiotic supplementation significantly reduced the total symptoms of IBS. No effects on bowel habit were seen. Health-related quality of life (HRQOL) is reduced in patients with IBS in comparison with the Finnish population on the whole. The probiotic supplementation improved one IBS-specific domain of quality of life (bowel symptoms), whereas no other effects on HRQOL were seen. The probiotics had no major effects on the predominant microbiota as measured by qPCR, but a microarray-based analysis suggested that the probiotic consumption stabilised the microbiota. No effects on serum sensitive-CRP or cytokines were detected. In conclusion, alterations in the microbiota composition and in the mucosal metabolite profile are potential pathophysiological factors of IBS. Multispecies probiotic supplementation alleviates the gastrointestinal symptoms of IBS, and improves the bowel symptoms domain of HRQOL. Probiotic supplementation in IBS is associated with a stabilisation of microbiota, but it does not influence systemic inflammatory markers.

Comparative effect of Pediococcus acidilactici and Lactococcus lactis on growth performance, survival and enzyme activity of western white leg shrimp (Litopenaeus vannamei)

This study was done in Shahid Kiani Marine Aquaculture Development Center, Choebde, Abadan in order to evaluate the effects of Pediiococcus acidilactici, Lactococcus lactis and vitamin C on growth performance, survival, enzymatic activities and immune responses of L. vannamei during three months. Treatments were included control group, Pediiococcus and Lactococcus treatments which fed with diet containing 1×10P9P cfu gP_1P bacteria and vitamin C. At the end of the experiment, the growth factors, immune parameters, digestive enzymes, intestinal, histology of intestine, carcasses and microbial flora (bacterial total count and lactic acid count) were evaluated. The results indicated that administration of lactobacillus had significant effects on the growth factors as the highest weight, increase specific growth rate, relative growth rate, feed conversion ratio and protein efficiency in the shrimps received pediococcus and then Lactococcus (P<0.05). The best immune function was also observed in the shrimps fed by probiotics, so that proteins and hemoglobin̛ hemolymph, phenoloxidase activity and challenged with V. parahaemolyticus showed a statistical difference comparing to the control group and the group received vitamin C (P<0.05). Some digestive enzymes, in pediococcus treatment showed a significant increase when compared to other treatments (P<0.05). Significant changes in bacterial intestinal flora were observed in probiotic groups compared with control and vitamin C groups (P < 0.05). Histological results showed the positive effects of probiotics in the gut (P < 0.05). While these supplements cannot caused to significant impacts on the shrimp carcass composition (P ˃ 0.05). As a result pediococcus group had the best performance among treatments.

Plasmid biology of natural Lactococcus lactis strains and molecular mechanisms of bacteriophage-host interaction

Lacticin 3147, enterocin AS-48, lacticin 481, variacin, and sakacin P are bacteriocins offering promising perspectives in terms of preservation and shelf-life extension of food products and should find commercial application in the near future. The studies detailing their characterization and bio-preservative applications are reviewed. Transcriptomic analyses showed a cell wall-targeted response of Lactococcus lactis IL1403 during the early stages of infection with the lytic bacteriophage c2, which is probably orchestrated by a number of membrane stress proteins and involves D-alanylation of membrane lipoteichoic acids, restoration of the physiological proton motive force disrupted following bacteriophage infection, and energy conservation. Sequencing of the eight plasmids of L. lactis subsp. cremoris DPC3758 from raw milk cheese revealed three anti-phage restriction/modification (R/M) systems, immunity/resistance to nisin, lacticin 481, cadmium and copper, and six conjugative/mobilization regions. A food-grade derivative strain with enhanced bacteriophage resistance was generated via stacking of R/M plasmids. Sequencing and functional analysis of the four plasmids of L. lactis subsp. lactis biovar. diacetylactis DPC3901 from raw milk cheese revealed genes novel to Lactococcus and typical of bacteria associated with plants, in addition to genes associated with plant-derived lactococcal strains. The functionality of a novel high-affinity regulated system for cobalt uptake was demonstrated. The bacteriophage resistant and bacteriocin-producing plasmid pMRC01 places a metabolic burden on lactococcal hosts resulting in lowered growth rates and increased cell permeability and autolysis. The magnitude of these effects is strain dependent but not related to bacteriocin production. Starters’ acidification capacity is not significantly affected. Transcriptomic analyses showed that pMRC01 abortive infection (Abi) system is probably subjected to a complex regulatory control by Rgg-like ORF51 and CopG-like ORF58 proteins. These regulators are suggested to modulate the activity of the putative Abi effectors ORF50 and ORF49 exhibiting topology and functional similarities to the Rex system aborting bacteriophage λ lytic growth.

Osmotic stress tolerance mechanisms in Lactococcus lactis

The response of Lactococcus lactis subsp. cremoris NCDO 712 to low water activity (aw) was investigated, both in relation to growth following moderate reductions in the aw and in terms of survival following substantial reduction of the aw with NaCI. Lc.lactis NCDO 712 was capable of growth in the presence of ≤ 4% w/v NaCI and concentrations in excess of 4% w/v were lethal to the cells. The presence of magnesium ions significantly increased the resistance of NCDO 712 to challenge with NaCI and also to challenge with high temperature or low pH. Survival of Lc.lactis NCDO 712 exposed to high NaCI concentrations was growth phase dependent and cells were most sensitive in the early exponential phase of growth. Pre-exposure to 3% w/v NaCI induced limited protection against subsequent challenge with higher NaCI concentrations. The induction was inhibited by chloramphenicol and even when induced, the response did not protect against NaCI concentrations> 10% w/v. When growing at low aw, potassium was accumulated by Lc. lactis NCDO 712 growing at low aw, if the aw was reduced by glucose or fructose, but not by NaCI. Reducing the potassium concentration of chemically defined medium from 20 to 0.5 mM) produced a substantial reduction in the growth rate, if the aw was reduced with NaCI, but not with glucose or fructose. The reduction of the growth rate correlated strongly with a reduction in the cytoplasmic potassium concentration and in cell volume. Addition of the compatible solute glycine betaine, partially reversed the inhibition of growth rate and partially restored the cell volume. The potassium transport system was characterised in cells grown in medium at both high and low aw. It appeared that a single system was present, which was induced approximately two-fold by growth at low aw. Potassium transport was assayed in vitro using cells depleted of potassium; the assay was competitively inhibited by Na+ and by the other monovalent cations NH4+, Li+, and Cs+. There was a strong correlation between the ability of strains of Lc. lactis subsp. lactis and subsp. cremoris to grow at low aw and their ability to accumulate the compatible solute glycine betaine. The Lc. lactis subsp. cremoris strains incapable of growth at NaCI concentrations> 2% w/v did not accumulate glycine betaine when growing at low aw, whereas strains capable of growth at NaCI concentrations up to 4% w/v did. A mutant, extremely sensitive to low aw was isolated from the parent strain Lc. lactis subsp. cremoris MG 1363, a plasmid free derivative of NCDO 712. The parent strain tolerated up to 4% w/v NaCI and actively accumulated glycine betaine when challenged at low aw. The mutant had lost the ability to accumulate glycine betaine and was incapable of growth at NaCI concentrations >2% w/v or the equivalent concentration of glucose. As no other compatible solute seemed capable of substitution for glycine betaine, the data suggest that the traditional; phenotypic speciation of strains on the basis of tolerance to 4% w/v NaCI can be explained as possession or lack of a glycine betaine transport system.

Evaluation of microbial adjuncts and their effect on the ripening of cheddar cheese

A bacteriocin-producing strain of Lactobacillus paracasei DPC 4715 was used as an adjunct culture in Cheddar cheese in order to control the growth of “wild” nonstarter lactic acid bacteria. No suppression of growth of the indicator strain was observed in the experimental cheese. The bacteriocin produced by Lactobacillus paracasei DPC 4715 was sensitive to chymosin and cathepsin D and it may have been cleaved by the rennet used for the cheese manufactured or by indigenous milk proteases. A series of studies were performed using various microbial adjuncts to influence cheese ripening. Microbacterium casei DPC 5281, Corynebacterium casei DPC 5293 and Corynebacterium variabile DPC 5305 were added to the cheesemilk at level of 109 cfu/ml resulting in a final concentration of 108 cfu/g in Cheddar cheese. The strains significantly increased the level of pH 4.6-soluble nitrogen, total free amino acids after 60 and 180 d of ripening and some individual free amino acids after 180 d. Yarrowia lipolytica DPC 6266, Yarrowia lipolytica DPC 6268 and Candida intermedia DPC 6271 were used to accelerate the ripening of Cheddar cheese. Strains were grown in YG broth to a final concentration of 107 cfu/ml, microfluidized, freeze-dried and added to the curd during salting at level of 2% w/w. The yeasts positively affected the primary, secondary proteolysis and lipolysis of cheeses and had aminopeptidase, dipeptidase, esterase and 5’ phosphodiestere activities that contributed to accelerate the ripening and improve the flavor of cheese. Hafia alvei was added to Cheddar cheesemilk at levels of 107 cfu/ml and 108 cfu/ml and its contribution during ripening was evaluated. The strain significantly increased the level of pH 4.6-soluble nitrogen, total free amino-acids, and some individual free amino-acids of Cheddar cheese, whereas no differences in the urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms of the cheeses were detected. Hafia alvei also significantly increased the level of some biogenic amines. A low-fat Cheddar cheese was made with Bifidobacterium animalis subsp. lactis, strain BB-12® at level of 108 cfu/ml, as a probiotic adjunct culture and Hi-Maize® 260 (resistant high amylose maize starch) at level of 2% and 4% w/v, as a prebiotic fiber which also played the role of fat replacer. Bifidobacterium BB-12 decreased by 1 log cycle after 60 d of ripening and remained steady at level of ~107 cfu/g during ripening. The Young’s modulus also increased proportionally with increasing levels of Hi-maize. Hencky strain at fracture decreased over ripening and increased with increasing in fat replacer. A cheese based medium (CBM) was developed with the purpose of mimicking the cheese environment at an early ripening stage. The strains grown in CBM showed aminopeptidase activity against Gly-, Arg-, Pro- and Phe-p-nitroanalide, whereas, when grown in MRS they were active against all the substrates tested. Both Lb. danicus strains grown in MRS and in CBM had aminotransferase activity towards aromatic amino acids (Phe and Trp) and also branched-chain amino acids (Leu and Val). Esterase activity was expressed against p-nitrophenyl-acetate (C2), pnitrophenyl- butyrate (C4) and p-nitrophenyl-palmitate (C16) and was significantly higher in CBM than in MRS.

Comparative genomics of Bifidobacteria

Chapter 2 of this thesis describes the sequence analysis of 14 bifidobacterial genomes from various species of the genus Bifidobacterium, and the determination of their open pan-genome trend. This analysis first determined the total number of genes to be considered as the reservoir of functions available to representatives of this genus. Many identified genes are still uncharacterized, but may be involved in the adaptation to the gut environment. This comparative genomic analysis also determined a pool of ortholog functions used to infer their phylogenetic relationship, thereby providing a more robust approach compared to that based solely on the16S rRNA-encoding gene. The genome sequence of an isolate from the insect hindgut Bifidobacterium asteroides PRL2011 was fully characterized in Chapter 3, surprisingly revealing a putative respiratory metabolism, which was also found to be present in other insect isolates, suggesting that respiration was an ancient feature of this genus, but also an adaptative trait to different atmosferic oxygen levels. Chapter 4 of this thesis outlines a comparative study which focused on the analysis of representatives of the Bifidobacterium breve species, revealing that the genetic variability among members of this species principally consists of genes with a role in adaptation to host environment and gut colonization. Finally, Chapter 5 describes the analysis of the genome sequence of Bifidobacterium animalis subsp. lactis BLC1, a probiotic bacterium widely used in food industries as an ingredient of functional foods, providing information that will allow future investigations of this species.

Characterisation of bacteriophage-host interactions in Lactococcus lactis

Lactococcus lactis is used extensively world-wide for the production of fermented dairy products. Bacteriophages (phages) infecting L. lactis can result in slow or incomplete fermentations, or may even cause total fermentation failure. Therefore, bacteriophages disrupting L. lactis fermentation are of economic concern. This thesis employed a multifaceted approach to investigate various molecular aspects of phage-host interaction in L. lactis. The genome sequence of an Irish dairy starter strain, the prophage-cured L. lactis subsp. cremoris UC509.9, was studied. The 2,250,427 bp circular chromosome represents the smallest among its sequenced lactococcal equivalents. The genome displays clear genetic adaptation to the dairy niche in the form of extensive reductive evolution. Gene prediction identified 2066 protein-encoding genes, including 104 which showed significant homology to transposase-specifying genes. Over 9 % of the identified genes appear to be inactivated through stop codons or frame shift mutations. Many pseudogenes were found in genes that are assigned to carbohydrate and amino acid transport and metabolism orthologous groups, reflecting L. lactis UC509.9’s adaptation to the lactose and casein-rich dairy environment. Sequence analysis of the eight plasmids of L. lactis revealed extensive adaptation to the dairy environment. Key industrial phenotypes were mapped and novel lactococcal plasmid-associated genes highlighted. In addition to chromosomally-encoded bacteriophage resistance systems, six functional such systems were identified, including two abortive infection systems, AbiB and AbiD1, explaining the observed phage resistance of L. lactis UC509.9 Molecular analysis suggests that the constitutive expression of AbiB is not lethal to cells, suggesting the protein is expressed in an un/inactivated form. Analysis of 936 species phage sk1-escape mutants of AbiB revealed that all such mutants harbour mutations in orf6, which encodes the major capsid protein. Results suggest that the major capsid protein is required for activation of the AbiB system, although this requires furrther investigations. Temporal transcriptomes of L. lactis UC509.9 undergoing lytic infection with either one of two distinct bacteriophages, Tuc2009 and c2, was determined and compared to the transcriptome of uninfected UC509.9 cells. Whole genome microarrays performed at various time-points post-infection demonstrated a rather modest impact on host transcription. Alterations in the UC509.9 transcriptome during lytic infection appear phage-specific, with a relatively small number of differentially transcribed genes shared between infection with either Tuc2009 or c2. Transcriptional profiles of both bacteriophages during lytic infection was shown to generally correlate with previous studies and allowed the confirmation of previously predicted promoter sequences. Bioinformatic analysis of genomic regions encoding the presumed cell wall polysaccharide (CW PS) biosynthesis gene cluster of several strains of L. lactis was performed. Results demonstrate the presence of three dominant genetic types of this gene cluster, termed type A, B and C. These regions were used for the development of a multiplex PCR to identify CW PS genotype of various lactococcal strains. Analysis of 936 species phage receptor binding protein phylogeny (RBP) and CW PS genotype revealed an apparent correlation between RBP phylogeny and CW PS type, thereby providing a partial explanation for the observed narrow host range of 936 phages. Further analysis of the genetic locus encompassing the presumed CW PS biosynthesis operon of eight strains identified as belonging to the CW PS C (geno)type, revealed the presence of a variable region among the examined strains. The obtained comparative analysis allowed for the identification of five subgroups of the C type, named C1 to C5. We purified an acidic polysaccharide from the cell wall of L. lactis 3107 (C2 subtype) and confirmed that it is structurally different from the CW PS of the C1 subtype L. lactis MG1363. Combinations of genes from the variable region of C2 subtype were amplified from L. lactis 3107 and introduced into a mutant of the C1 subtype L. lactis NZ9000 (a direct derivative of MG1363) deficient in CW PS biosynthesis. The resulting recombinant mutant synthesized a CW PS with a composition characteristic for that of the C2 subtype L. lactis 3107 and not the wildtype C1 L. lactis NZ9000. The recombinant mutant exhibited a changed phage resistance/sensitivity profile consistent with that of L. lactis 3107, which unambiguously demonstrated that L. lactis 3107 CW PS is the host cell surface receptor of two bacteriophages belonging to the P335 species as well as phages that are member of the 936 species. The research presented in this thesis has significantly advanced our understanding of L. lactis bacteriophage-host interactions in several ways. Firstly, the examination of plasmidencoded bacteriophage resistance systems has allowed inferences to be made regarding the mode of action of AbiB, thereby providing a platform for further elucidation of the molecular trigger of this system. Secondly, the phage infection transcriptome data presented, in addition to previous work, has made L. lactis a model organism in terms of transcriptomic studies of bacteriophage-host interactions. And finally, the research described in this thesis has for the first time explicitly revealed the nature of a carbohydrate bacteriophage receptor in L. lactis, while also providing a logical explanation for the observed narrow host ranges exhibited by 936 and P335 phages. Future research in discerning the structures of other L. lactis CW PS, combined with the determination of the molecular interplay between receptor binding proteins of these phages and CW PS will allow an in depth understanding of the mechanism by which the most prevalent lactococcal phages identify and adsorb to their specific host.

Metabolism of host-derived carbohydrates by Bifidobacterium breve UCC2003

Bifidobacteria are Gram positive, anaerobic, typically Y-shaped bacteria which are naturally found in the digestive tract of certain mammals, birds and insects. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. The prototypical B. breve strain UCC2003 has previously been shown to utilise numerous carbohydrates of plant origin. Various aspects of host-derived carbohydrate metabolism occurring in this bacterium will be described in this thesis. Chapter II describes B. breve UCC2003 utilisation of sialic acid, a nine-carbon monosaccharide, which is found in human milk oligosaccharides (HMOs) and the mucin glycoprotein. B. breve UCC2003 was also shown to cross-feed on sialic acid released from 3’ sialyllactose, a prominent HMO, by the extracellular sialidase activity of Bifidobacterium bifidum PRL2010. Chapter III reports on the transcriptional regulation of sialic acid metabolism in B. breve UCC2003 by a transcriptional repressor encoded by the nanR gene. NanR belongs to the GntR-family of transcriptional regulators and represents the first bifidobacterial member of this family to be characterised. Chapter IV investigates B. breve UCC2003 utilisation of mucin. B. breve UCC2003 was shown to be incapable of degrading mucin; however when grown in co-culture with B. bifidum PRL2010 it exhibits enhanced growth and survival properties. A number of methods were used to investigate and identify the mucin components supporting this enhanced growth/viability phenotype. Chapter V describes the characterisation of two sulfatase-encoding gene clusters from B. breve UCC2003. The transcriptional regulation of both sulfatase-encoding gene clusters was also investigated. The work presented in this thesis represents new information on the metabolism of host-derived carbohydrates in bifidobacteria, thus increasing our understanding of how these gut commensals are able to colonise and persist in the gastrointestinal tract.

Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

Reacciones positivas falsas de bacterias coliformes : debidas a la levadura Kluyveromyces lactis

p.139-143

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.