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.

The scientific basis and development of a matrix metalloproteinase (MMP) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid

Matrix metalloproteinase (MMP) -8, collagenase-2, is a key mediator of irreversible tissue destruction in chronic periodontitis and detectable in gingival crevicular fluid (GCF). MMP-8 mostly originates from neutrophil leukocytes, the first line of defence cells which exist abundantly in GCF, especially in inflammation. MMP-8 is capable of degrading almost all extra-cellular matrix and basement membrane components and is especially efficient against type I collagen. Thus the expression of MMP-8 in GCF could be valuable in monitoring the activity of periodontitis and possibly offers a diagnostic means to predict progression of periodontitis. In this study the value of MMP-8 detection from GCF in monitoring of periodontal health and disease was evaluated with special reference to its ability to differentiate periodontal health and different disease states of the periodontium and to recognise the progression of periodontitis, i.e. active sites. For chair-side detection of MMP-8 from the GCF or peri-implant sulcus fluid (PISF) samples, a dip-stick test based on immunochromatography involving two monoclonal antibodies was developed. The immunoassay for the detection of MMP-8 from GCF was found to be more suitable for monitoring of periodontitis than detection of GCF elastase concentration or activity. Periodontally healthy subjects and individuals suffering of gingivitis or of periodontitis could be differentiated by means of GCF MMP-8 levels and dipstick testing when the positive threshold value of the MMP-8 chair-side test was set at 1000 µg/l. MMP-8 dipstick test results from periodontally healthy and from subjects with gingivitis were mainly negative while periodontitis patients sites with deep pockets ( 5 mm) and which were bleeding on probing were most often test positive. Periodontitis patients GCF MMP-8 levels decreased with hygiene phase periodontal treatment (scaling and root planing, SRP) and even reduced during the three month maintenance phase. A decrease in GCF MMP-8 levels could be monitored with the MMP-8 test. Agreement between the test stick and the quantitative assay was very good (κ = 0.81) and the test provided a baseline sensitivity of 0.83 and specificity of 0.96. During the 12-month longitudinal maintenance phase, periodontitis patients progressing sites (sites with an increase in attachment loss ≥ 2 mm during the maintenance phase) had elevated GCF MMP-8 levels compared with stable sites. General mean MMP-8 concentrations in smokers (S) sites were lower than in non-smokers (NS) sites but in progressing S and NS sites concentrations were at an equal level. Sites with exceptionally and repeatedly elevated MMP-8 concentrations during the maintenance phase were clustered in smoking patients with poor response to SRP (refractory patients). These sites especially were identified by the MMP-8 test. Subgingival plaque samples from periodontitis patients deep periodontal pockets were examined by polymerase chain reaction (PCR) to find out if periodontal lesions may serve as a niche for Chlamydia pneumoniae. Findings were compared with the clinical periodontal parameters and GCF MMP-8 levels to determine the correlation with periodontal status. Traces of C. pneumoniae were identified from one periodontitis patient s pooled subgingival plaque sample by means of PCR. After periodontal treatment (SRP) the sample was negative for C. pneumoniae. Clinical parameters or biomarkers (MMP-8) of the patient with the positive C. pneumoniae finding did not differ from other study patients. In this study it was concluded that MMP-8 concentrations in GCF of sites from periodontally healthy individuals, subjects with gingivitis or with periodontitis are at different levels. The cut-off value of the developed MMP-8 test is at an optimal level to differentiate between these conditions and can possibly be utilised in identification of individuals at the risk of the transition of gingivitis to periodontitis. In periodontitis patients, repeatedly elevated GCF MMP-8 concentrations may indicate sites at risk of progression of periodontitis as well as patients with poor response to conventional periodontal treatment (SRP). This can be monitored by MMP-8 testing. Despite the lower mean GCF MMP-8 concentrations in smokers, a fraction of smokers sites expressed very high MMP-8 concentrations together with enhanced periodontal activity and could be identified with MMP-8 specific chair-side test. Deep periodontal lesions may be niches for non-periodontopathogenic micro-organisms with systemic effects like C. pneumoniae and possibly play a role in the transmission from one subject to another.

Controlling transduction and replication of oncolytic adenoviruses

Despite progress in conventional cancer treatment regimes, metastatic disease essentially remains incurable and new treatment alternatives are needed. Virotherapy is a relatively novel approach in cancer treatment. It harnesses the natural ability of oncolytic viruses to kill the cells they proliferate in and to spread to neighboring cells, thereby amplifying the therapeutic effect of the initial input dose. The use of replicating, oncolytic viruses for cancer treatment necessitates introduction of various genetic modifications to the viral genome, thereby restraining replication exclusively to tumor cells and eventually obtaining selective eradication of the tumor without side effects to healthy tissue. Furthermore, various modifications can be applied to the viral capsid in hope of gaining effective transduction of target tissue. In other words, the entry of viruses into tumor tissue can be augmented by allowing the virus to utilize non-native receptors for entry. Genetic capsid modifications may also help to avoid some major hurdles in systemic delivery that ultimately lead to the rapid clearance of the virus from the blood and virus induced toxicity. In addition to genetic modifications that alter the phenotype of the virus, some pharmacologic agents may be utilized to enhance the virus entry to target site. Liver kupffer cells (KC) are responsible for the majority of viral clearance after systemic viral delivery and they play a major role in adenovirus induced acute toxicity. The therapeutic window could possibly be widened by transiently depleting KCs, allowing smaller viral input doses and diminishing KC related toxicity. The transductional efficacy of various capsid modified viruses was analyzed in vitro and in vivo in murine orthotopic breast cancer model. The effect of capsid modifications on the oncolytic efficacy, i.e. the ability of the viruses to kill cancer cells, was evaluated in vitro and in vivo in murine cancer models. We concluded that capsid modifications result in transductional enhancement, and that enhanced transduction translates into more potent oncolysis in vitro and in vivo. When KC depleting agents were used in vivo prior to viral injections, enhanced tumor transduction was seen, but this effect was not translated into enhanced antitumor activity. Transcriptional regulation of replicative oncolytic viruses is a prerequisite for virotherapy. Tumor or tissue specific promoters can be used to control the transcription of adenoviral early genes to gain cancer specific viral replication. Specific deletions in viral regions essential for virus replication in normal cells can further increase the safety by allowing viral genome replication in cancer cells featuring specific mutations. Genetically modified viruses were shown to be able to kill putative cancer stem cells that are thought to be responsible for post treatment relapses and metastasis. Further, pharmacologic intervention reduced viral replication and thereby might offer an additional safety switch in case viral replication related side effects are encountered.

Jäteveden kemiallisen puhdistuksen teoriaa. Jäteveden kemiallisia puhdistuskokeita ferrikloridilla ja kalkilla

Theory of chemical waste water treatment. Pilot plant tests on chemical precipitation with ferric chloride and lime.