Heterotrophic Bacteria Associated with Cyanobacteria in Recreational and Drinking Water

Cyanobacterial mass occurrences, also known as water blooms, have been associated with adverse health effects of both humans and animals. They can also be a burden to drinking water treatment facilities. Risk assessments of the blooms have generally focused on the cyanobacteria themselves and their toxins. However, heterotrophic bacteria thriving among cyanobacteria may also be responsible for many of the adverse health effects, but their role as the etiological agents of these health problems is poorly known. In addition, studies on the water purification efficiency of operating water treatment plants during cyanobacterial mass occurrences in their water sources are rare. In the present study, over 600 heterotrophic bacterial strains were isolated from natural freshwater, brackish water or from treated drinking water. The sampling sites were selected as having frequent cyanobacterial occurrences in the water bodies or in the water sources of the drinking water treatment plants. In addition, samples were taken from sites where cyanobacterial water blooms were surmised to have caused human health problems. The isolated strains represented bacteria from 57 different genera of the Gamma-, Alpha- or Betaproteobacteria, Actinobacteria, Flavobacteria, Sphingobacteria, Bacilli and Deinococci classes, based on their partial 16S rRNA sequences. Several isolates had no close relatives among previously isolated bacteria or cloned 16S rRNA genes of uncultivated bacteria. The results show that water blooms are associated with a diverse community of cultivable heterotrophic bacteria. Chosen subsets of the isolated strains were analysed for features such as their virulence gene content and possible effect on cyanobacterial growth. Of the putatively pathogenic haemolytic strains isolated in the study, the majority represented the genus Aeromonas. Therefore, the Aeromonas spp. strains isolated from water samples associated with adverse health effects were screened for the virulence gene types encoding for enterotoxins (ast, alt and act/aerA/hlyA), flagellin subunits (flaA/flaB), lipase (lip/pla/lipH3/alp-1) and elastase (ahyB) by PCR. The majority (90%) of the Aeromonas strains included one or more of the six screened Aeromonas virulence gene types. The most common gene type was act, which was present in 77% of the strains. The fla, ahyB and lip genes were present in 30 37% of the strains. The prevalence of the virulence genes implies that the Aeromonas may be a factor in some of the cyanobacterial associated health problems. Of the 183 isolated bacterial strains that were studied for possible effects on cyanobacterial growth, the majority (60%) either enhanced or inhibited growth of cyanobacteria. In most cases, they enhanced the growth, which implies mutualistic interactions. The results indicate that the heterotrophic bacteria have a role in the rise and fall of the cyanobacterial water blooms. The genetic and phenotypic characteristics and the ability to degrade cyanobacterial hepatotoxins of 13 previously isolated Betaproteobacteria strains, were also studied. The strains originated from Finnish lakes with frequent cyanobacterial occurrence. Tested strains degraded microcystins -LR and -YR and nodularin. The strains could not be assigned to any described bacterial genus or species based on their genetic or phenotypic features. On the basis of their characteristics a new genus and species Paucibacter toxinivorans was proposed for them. The water purification efficiency of the drinking water treatment processes during cyanobacterial water bloom in water source was assessed at an operating surface water treatment plant. Large phytoplankton, cyanobacterial hepatotoxins, endotoxins and cultivable heterotrophic bacteria were efficiently reduced to low concentrations, often below the detection limits. In contrast, small planktonic cells, including also possible bacterial cells, regularly passed though the water treatment. The passing cells may contribute to biofilm formation within the water distribution system, and therefore lower the obtained drinking water quality. The bacterial strains of this study offer a rich source of isolated strains for examining interactions between cyanobacteria and the heterotrophic bacteria associated with them. The degraders of cyanobacterial hepatotoxins could perhaps be utilized to assist the removal of the hepatotoxins during water treatment, whereas inhibitors of cyanobacterial growth might be useful in controlling cyanobacterial water blooms. The putative pathogenicity of the strains suggests that the health risk assessment of the cyanobacterial blooms should also cover the heterotrophic bacteria.

Identification of Isoflavonoid Metabolites in Humans

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases, such as breast and prostate cancers, osteoporosis, and cardiovascular disease. Soy is a rich source of isoflavones, diphenolic plant compounds that have been shown to possess several biological activities. Soy is not part of the traditional Western diet, but many dietary supplements are commercially available in order to provide the proposed beneficial health effects of isoflavones without changing the original diet. These supplements are usually manufactured from extracts of soy or red clover, which is another important source of isoflavones. However, until recently, detailed studies of the metabolism of these compounds in humans have been lacking. The aim of this study was to identify urinary metabolites of isoflavones originating from soy or red clover using gas chromatography - mass spectrometry (GC-MS). To examine metabolism, soy and red clover supplementation studies with human volunteers were carried out. In addition, the metabolism of isoflavones was investigated in vitro by identification of metabolites formed during a 24-h fermentation of pure isoflavones with a human fecal inoculum. Qualitative methods for identification and analysis of isoflavone metabolites in urine and fecal fermentation samples by GC-MS were developed. Moreover, a detailed investigation of fragmentation of isoflavonoids in electron ionization mass spectrometry (EIMS) was carried out by means of synthetic reference compounds and deuterated trimethylsilyl derivatives. After isoflavone supplementation, 18 new metabolites of isoflavones were identified in human urine samples. The most abundant urinary metabolites of soy isoflavones daidzein, genistein, and glycitein were found to be the reduced metabolites, i.e. analogous isoflavanones, a-methyldeoxybenzoins, and isoflavans. Metabolites having additional hydroxyl and/or methoxy substituents, or their reduced analogs, were also identified. The main metabolites of red clover isoflavones formononetin and biochanin A were identified as daidzein and genistein. In addition, reduced and hydroxylated metabolites of formononetin and biochanin A were identified; however, they occurred at much lower levels in urine samples than daidzein or genistein or their reduced metabolites. The results of this study show that the metabolism of isoflavones is diverse. More studies are needed to determine whether the new isoflavonoid metabolites identified here have biological activities that contribute to the proposed beneficial effects of isoflavones on human health. Another task is to develop validated quantitative methods to determine the actual levels of isoflavones and their metabolites in biological matrices in order to assess the role of isoflavones in prevention of chronic diseases.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.