Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

Impact of the Human Bacterial Environment on Mycobacteriosis and Allergy

My work describes two sectors of the human bacterial environment: 1. The sources of exposure to infectious non-tuberculous mycobacteria. 2. Bacteria in dust, reflecting the airborne bacterial exposure in environments protecting from or predisposing to allergic disorders. Non-tuberculous mycobacteria (NTM) transmit to humans and animals from the environment. Infection by NTM in Finland has increased during the past decade beyond that by Mycobacterium tuberculosis. Among the farm animals, porcine mycobacteriosis is the predominant NTM disease in Finland. Symptoms of mycobacteriosis are found in 0.34 % of slaughtered pigs. Soil and drinking water are suspected as sources for humans and bedding materials for pigs. To achieve quantitative data on the sources of human and porcine NTM exposure, methods for quantitation of environmental NTM are needed. We developed a quantitative real-time PCR method, utilizing primers targeted at the 16S rRNA gene of the genus of Mycobacterium. With this method, I found in Finnish sphagnum peat, sandy soils and mud high contents of mycobacterial DNA, 106 to 107 genome equivalents per gram. A similar result was obtained by a method based on the Mycobacterium-specific hybridization of 16S rRNA. Since rRNA is found mainly in live cells, this result shows that the DNA detected by qPCR mainly represented live mycobacteria. Next, I investigated the occurrence of environmental mycobacteria in the bedding materials obtained from 5 pig farms with high prevalence (>4 %) of mycobacteriosis. When I used for quantification the same qPCR methods as for the soils, I found that piggery samples contained non-mycobacterial DNA that was amplified in spite of several mismatches with the primers. I therefore improved the qPCR assay by designing Mycobacterium-specific detection probes. Using the probe qPCR assay, I found 105 to 107 genome equivalents of mycobacterial DNA in unused bedding materials and up to 1000 fold more in the bedding collected after use in the piggery. This result shows that there was a source of mycobacteria in the bedding materials purchased by the piggery and that mycobacteria increased in the bedding materials during use in the piggery. Allergic diseases have reached epidemic proportions in urbanized countries. At the same time, childhood in rural environment or simple living conditions appears to protect against allergic disorders. Exposure to immunoreactive microbial components in rural environments seems to prevent allergies. I searched for differences in the bacterial communities of two indoor dusts, an urban house dust shown to possess immunoreactivity of the TH2-type and a farm barn dust with TH1-activity. The immunoreactivities of the dusts were revealed by my collaborators, in vitro in human dendritic cells and in vivo in mouse. The dusts accumulated >10 years in the respiratory zone (>1.5 m above floor), thus reflecting the long-term content of airborne bacteria at the two sites. I investigated these dusts by cloning and sequencing of bacterial 16S rRNA genes from dust contained DNA. From the TH2-active urban house dust, I isolated 139 16S rRNA gene clones. The most prevalent genera among the clones were Corynebacterium (5 species, 34 clones), Streptococcus (8 species, 33 clones), Staphylococcus (5 species, 9 clones) and Finegoldia (1 species, 9 clones). Almost all of these species are known as colonizers of the human skin and oral cavity. Species of Corynebacterium and Streptococcus have been reported to contain anti-inflammatory lipoarabinomannans and immunmoreactive beta-glucans respectively. Streptococcus mitis, found in the urban house dust is known as an inducer of TH2 polarized immunity, characteristic of allergic disorders. I isolated 152 DNA clones from the TH1-active farm barn dust and found species quite different from those found from the urban house dust. Among others, I found DNA clones representing Bacillus licheniformis, Acinetobacter lwoffii and Lactobacillus each of which was recently reported to possess anti-allergy immunoreactivity. Moreover, the farm barn dust contained dramatically higher bacterial diversity than the urban house dust. Exposure to this dust thus stimulated the human dendritic cells by multiple microbial components. Such stimulation was reported to promote TH1 immunity. The biodiversity in dust may thus be connected to its immunoreactivity. Furthermore, the bacterial biomass in the farm barn dust consisted of live intact bacteria mainly. In the urban house dust only ~1 % of the biomass appeared as intact bacteria, as judged by microscoping. Fragmented microbes may possess bioactivity different from that of intact cells. This was recently shown for moulds. If this is also valid for bacteria, the different immunoreactivities of the two dusts may be explained by the intactness of dustborne bacteria. Based on these results, we offer three factors potentially contributing to the polarized immunoreactivities of the two dusts: (i) the species-composition, (ii) the biodiversity and (iii) the intactness of the dustborne bacterial biomass. The risk of childhood atopic diseases is 4-fold lower in the Russian compared with the Finnish Karelia. This difference across the country border is not explainable by different geo-climatic factors or genetic susceptibilities of the two populations. Instead, the explanation must be lifestyle-related. It has already been reported that the microbiological quality of drinking water differs on the two sides of the borders. In collaboration with allergists, I investigated dusts collected from homes in the Russian Karelia and in the Finnish Karelia. I found that bacterial 16S rRNA genes cloned from the Russian Karelian dusts (10 homes, 234 clones) predominantly represented Gram-positive taxa (the phyla Actinobacteria and Firmicutes, 67%). The Russian Karelian dusts contained nine-fold more of muramic acid (60 to 70 ng mg-1) than the Finnish Karelian dusts (3 to 11 ng mg-1). Among the DNA clones isolated from the Finnish side (n=231), Gram-negative taxa (40%) outnumbered the Gram-positives (34%). Out of the 465 DNA clones isolated from the Karelian dusts, 242 were assigned to cultured validly described bacterial species. In Russian Karelia, animal-associated species e.g. Staphylococcus and Macrococcus were numerous (27 clones, 14 unique species). This finding may connect to the difference in the prevalence of allergy, as childhood contacts with pets and farm animals have been connected with low allergy risk. Plant-associated bacteria and plant-borne 16S rRNA genes (chloroplast) were frequent among the DNA clones isolated from the Finnish Karelia, indicating components originating from plants. In conclusion, my work revealed three major differences between the bacterial communtites in the Russian and in the Finnish Karelian homes: (i) the high prevalence of Gram-positive bacteria on the Russian side and of Gram-negative bacteria on the Finnish side and (ii) the rich presence of animal-associated bacteria on the Russian side whereas (iii) plant-associated bacteria prevailed on the Finnish side. One or several of these factors may connect to the differences in the prevalence of allergy.

Lantibiotic Nisin and Its Detection Methods

The type A lantibiotic nisin produced by several Lactococcus lactis strains, and one Streptococcus uberis strainis a small antimicrobial peptide that inhibits the growth of a wide range of gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus species. It is nontoxic to humans and used as a food preservative (E234) in more than 50 countries including the EU, the USA, and China. National legislations concerning maximum addition levels of nisin in different foods vary greatly. Therefore, there is a demand for non-laborious and sensitive methods to identify and quantify nisin reliably from different food matrices. The horizontal inhibition assay, based on the inhibitory effect of nisin to Micrococcus luteus is the base for most quantification methods developed so far. However, the sensitivity and accuracy of the agar diffusion method is affected by several parameters. Immunological tests have also been described. Taken into account the sensitivity of immunological methods to interfering substances within sample matrices, and possible cross-reactivities with lantibiotics structurally close to nisin, their usefulness for nisin detection from food samples remains limited. The proteins responsible for nisin biosynthesis, and producer self-immunity are encoded by genes arranged into two inducible operons, nisA/Z/QBTCIPRK and nisFEG, which also contain internal, constitutive promoters PnisI and PnisR. The transmembrane histidine kinase NisK and the response regulator NisR form a two-component signal transduction system, in which NisK autophosphorylates after exposure to extra cellular nisin, and subsequently transfers the phosphate to NisR. The phosphorylated NisR then relays the signal downstream by binding to two regulated promoters in the nisin gene cluster, i.e the nisA/Z/Qand the nisF promoters, thus activating transcription of the structural gene nisA/Z/Q and the downstream genes nisBTCIPRK from the nisA/Z/Q promoter, and the genes nisFEG from the nisF promoter. In this work two novel and highly sensitive nisin bioassays were developed. Both of these quantification methods were based on NisRK mediated, nisin induced Green Fluorescent Protein (GFP) fluorescence. The suitabilities of these assays for quantifica¬tion of nisin from food samples were evaluated in several food matrices. These bioassays had nisin sensitivities in the nanogram or picogram levels. In addition, shelf life of nisin in cooked sausages and retainment of the induction activity of nisin in intestinal chyme (intestinal content) was assessed.

Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.

Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Oral Actinomyces Species in Health and Disease: Identification, Occurence and Importance of Early Colonization

The genus Actinomyces consists of a heterogeneous group of gram-positive, mainly facultatively anaerobic or microaerobic rods showing various degrees of branching. In the oral cavity, streptococci and Actinomyces form a fundamental component of the indigenous microbiota, being among initial colonizers in polymicrobial biofilms. The significance of the genus Actinomyces is based on the capability of species to adhere to surfaces such as on teeth and to co-aggregate with other bacteria. Identification of Actinomyces species has mainly been based on only a few biochemical characteristics, such as pigmentation and catalase production, or on the use of a single commercial kit. The limited identification of oral Actinomyces isolates to species level has hampered knowledge of their role both in health and disease. In recent years, Actinomyces and related organisms have attracted the attention of clinical microbiologists because of a growing awareness of their presence in clinical specimens and their association with disease. This series of studies aimed to amplify the identification methods for Actinomyces species. With the newly developed identification scheme, the age-related occurrence of Actinomyces in healthy mouths of infants and their distribution in failed dental implants was investigated. Adhesion of Actinomyces species to titanium surfaces processed in various ways was studied in vitro. The results of phenotypic identification methods indicated a relatively low applicability of commercially available test kits for reliable identification within the genus Actinomyces. However, in the study of conventional phenotypic methods, it was possible to develop an identification scheme that resulted in accurate differentiation of Actinomyces and closely related species, using various different test methods. Genotypic methods based on 16S rRNA sequence analysis of Actinomyces proved to be a useful method for genus level identification and further clarified the species level identification with phenotypic methods. The results of the study of infants showed that the isolation frequency of salivary Actinomyces species increased according to age: thirty-one percent of the infants at 2 months but 97% at 2 years of age were positive for Actinomyces. A. odontolyticus was the most prominent Actinomyces colonizer during the study period followed in frequency by A. naeslundii and A. viscosus. In the study of explanted dental implants, Actinomyces was the most prevalent bacterial genus, colonizing 94% of the fixtures. Also in the implants A. odontolyticus was revealed as the most common Actinomyces species. It was present in 84% of Actinomyces -positive fixtures followed in frequency by A. naeslundii, A. viscosus and A. israelii. In an in vitro study of titanium surfaces, different Actinomyces species showed variation regarding their adhesion to titanium. Surface roughness as well as albumin coating of titanium had significant effects on adhesion. The use of improved phenotypic and molecular diagnostic methods increased the accuracy of the identification of the Actinomyces to species level. This facilitated an investigation of their occurrence and distribution in oral specimens in both health and disease.

Microbes in the tailoring of barley malt properties

Microbes have a decisive role in the barley-malt-beer chain. A major goal of this thesis was to study the relationships between microbial communities and germinating grains during malting. Furthermore, the study provided a basis for tailoring of malt properties with natural, malt-derived microbes. The malting ecosystem is a dynamic process, exhibiting continous change. The first hours of steeping and kilning were the most important steps in the process with regard to microbiological quality. The microbial communities consisting of various types of bacteria, yeasts and filamentous fungi formed complex biofilms in barley tissues and were well-protected. Inhibition of one microbial population within the complex ecosystem led to an increase of non-suppressed populations, which must be taken into account because a shift in microbial community dynamics may be undesirable. Both bacterial and fungal communities should be monitored simultaneously. Using different molecular approaches we showed that the diversity of microbes in the malting ecosystem was greater than expected. Even some new microbial groups were found in the malting ecosystem. Suppression of Gram-negative bacteria during steeping was advanategous for grain germination and malt brewhouse performance. Fungal communities including both filamentous fungi and yeasts significantly contributed to the production of microbial beta-glucanases and xylanases, and were also involved in proteolysis. Well-characterized lactic acid bacteria (Lactobacillus plantarum VTT E-78076 and Pediococcus pentosaceus VTT E-90390) proved to be an effective way of balancing the microbial communities in malting. Furthermore, they had positive effects on malt characteristics and notably improved wort separation. Previously the significance of yeasts in the malting ecosystem has been largely underestimated. This study showed that yeast community was an important part of the industrial malting ecosystem. Yeasts produced extracellular hydrolytic enzymes with a potentially positive contribution to malt processability. Furthermore, several yeasts showed strong antagonistic activity against field and storage moulds. Addition of a selected yeast culture (Pichia anomala VTT C-04565) into steeping restricted Fusarium growth and hydrophobin production and thus prevented beer gushing. Addition of P. anomala C565 into steeping water tended to retard wort filtration, but the filtration was improved when the yeast culture was combined with L. plantarum E76. The combination of different microbial cultures offers a possibility to use ther different properties, thus making the system more robust. Improved understanding of complex microbial communities and their role in malting enables a more controlled process management and the production of high quality malt with tailored properties

Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Lipid-Containing Icosahedral dsDNA Bacteriophages: Entry, Exit and Structure

In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.