Comparison of Colilert-18 with miniaturised most probable number method for monitoring of Escherichia coli in bathing water.

The purpose of this equivalence study was to compare an alternative method, Colilert-18 Quanti-Tray (ISO 9308-2) with the European bathing water directive (2006/7/EC) reference method, the miniaturised most probable number (MMPN) method (ISO 9308-3), for the analysis of Escherichia coli. Six laboratories analysed a total of 263 bathing water samples in Finland. The comparison was carried out according to ISO 17994:2004. The recovery of E. coli using the Colilert-18 method was 7.0% and 8.6% lower than that of the MMPN method after 48 hours and 72 hours of incubation, respectively. The confirmation rate of presumptive E. coli-positive wells in the Colilert-18 and MMPN methods was high (97.8% and 98.0%, respectively). However, the testing of presumptive E. coli-negative but coliform bacteria-positive (yellow but not fluorescent) Colilert-18 wells revealed 7.3% false negative results. There were more false negatives in the naturally contaminated waters than in the samples spiked with waste water. The difference between the recovery of Colilert-18 and the MMPN method was considered not significant, and subsequently the methods are considered as equivalent for bathing water quality monitoring in Finland. Future bathing water method equivalence verification studies may use the data reported herein. The laboratories should make sure that any wells showing even minor fluorescence will be determined as positive for E. coli.

Virulence genes of Aeromonas isolates, bacterial endotoxins and cyanobacterial toxins from recreational water samples associated with human health symptoms.

Exposure to cyanobacterial water blooms has been associated with various kinds of adverse health effects. In addition to cyanobacteria and their toxins, the bacteria associated with cyanobacteria could also be the etiological agents. We isolated Aeromonas strains (n = 176) from water samples (n = 38) taken from sites where cyanobacteria were suspected to have caused human health symptoms, of which fever and gastrointestinal symptoms were the most common. The isolates were screened by PCR for six virulence gene types (12 genes). The majority (90%) of the strains contained at least one of the virulence genes. Most common amplification products were those of genes (act/aerA/hlyA) that encode cytotoxic enterotoxin and haemolytic products. The genes encoding cytotonic enterotoxins (ast and alt), phospholipase (lip/pla/lipH3/alp-1), elastase (ahyB) and flagellin subunits (flaA/flaB) were also present in 5-37% of the Aeromonas strains. Analysed toxins (cyanobacterial hepatotoxins and neurotoxins, and bacterial endotoxins) were not detectable or were present in only low concentrations in the majority of the samples. The results indicated that the toxins were unlikely to be the main cause of the reported adverse health effects, whereas more attention should be paid to bacteria associated with cyanobacteria as a source of health effects.

Endotoxins associated with cyanobacteria and their removal during drinking water treatment.

The aim of this study was to investigate endotoxin concentrations in cyanobacterial water blooms and strains, and to assess the removal of endotoxins during drinking water treatment. Endotoxin concentrations were measured from 151 hepatotoxic, neurotoxic and non-toxic cyanobacterial water blooms by using Limulus amebocyte lysate (LAL) assay, and the results were compared to bacterial data. Endotoxin activities ranged from 20 to 3.8 x 10(4) endotoxin units (EU) per ml. Endotoxicity of the samples correlated with phycobiliprotein concentration that was used to assess cyanobacterial abundance, heterotrophic plate count, and Aeromonas spp. but it did not correlate with the number of coliforms or streptococci. The high endotoxin concentrations occasionally detected in the water bloom samples were probably due to Gram negative bacteria that existed together with cyanobacteria since the 26 axenic cyanobacterial strains from different genera that were studied showed very low endotoxin activity. No differences in endotoxin activity were detected between hepatotoxic, neurotoxic and non-toxic strains. Removal of endotoxins during drinking water treatment was studied at nine waterworks that previously had been associated with high numbers of cyanobacteria and that used different processes for water purification. Endotoxin concentration in raw waters ranged from 18 to 356 EU ml(-1). The treatment processes reduced 59-97% of the endotoxin activity; in the treated water the concentration ranged from 3 to 15 EU ml(-1). The most significant reduction occurred at the early stages of water treatment, during coagulation, settling and sand filtration. Activated carbon filtration either increased or had no effect on endotoxin concentration. Ozonation and chlorination had little effect on the endotoxin concentrations.

Previously uncultured beta-Proteobacteria dominate in biologically active granular activated carbon (BAC) filters.

Bacteria colonizing BAC filters used in drinking water purification from lake water were characterized by morphology, physiological tests, whole cell protein profiles and PLFA (phospholipid fatty acid) composition, and identified by partial 16S rRNA gene sequencing. Epifluorescence revealed prothecate bacteria to dominate in BAC. The majority of the isolates belonged to order Burkholderiales of beta-Proteobacteria, a few to Comamonadaceae but the majority to an undescribed family and the related sequences belonged mainly to uncultured bacteria. Among the less common alpha-Proteobacteria the genus Sphingomonas and the genera Afipia, Bosea or Bradyrhizobium of the Bradyrhizobiaceae family were detected. The majority of cultured bacteria persisting in the BAC biofilter were Burkholderiales, which according to ecological information are efficient in the mineralisation of dissolved organic matter in BAC. The biotechnical potential of the previously uncultured dominant bacteria warrants to be further studied.

High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms.

Cyanobacterial mass occurrences (water blooms) cause ecological, economic and health problems worldwide. Still, little is known about heterotrophic bacteria associated with cyanobacteria and the interactions between those organisms. We isolated 460 bacterial strains from more than 40 lakes and rivers (151 samples), Baltic Sea (32 samples) and treated drinking water of seven treatment plants (29 samples). The water bodies and the raw water of the treatment plants were frequently dominated by high numbers of cyanobacteria. Various growth media were used to isolate the strains. Analysis of partial 16S rRNA gene fragments (701-905 bp for 358 strains and 413-497 bp for 102 strains) classified the isolated bacteria as Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Deinococcus-Thermus. Some of these isolates represented possible new bacterial orders, families, genera or species. We isolated various potentially pathogenic bacteria, such as Aeromonas, Vibrio, Acinetobacter and Pseudomonas, that may cause adverse health effects in humans and animals and should be taken into consideration when assessing the risks caused by cyanobacterial blooms. Several strains also inhibited or enhanced the growth of cyanobacteria. Most of such strains had an enhancing effect on the cyanobacterial growth. Other isolates were affiliated with genera such as Sphingomonas or Flavobacterium, which include strains that are capable of degrading cyanobacterial toxins or other recalcitrant and problematic organic compounds. The isolated strains provide a large group of bacteria that could be used in assessing and controlling the harmful effects of cyanobacteria.

First observation of cylindrospermopsin in Anabaena lapponica isolated from the boreal environment (Finland).

The cyanobacterial cytotoxin cylindrospermopsin has been mostly associated with cyanobacteria present in tropical and subtropical regions. Cylindrospermopsin has recently been found in cyanobacterial samples in central and southern Europe but the possible presence of the toxin in northern Europe has been unknown. Fifty-eight field and laboratory culture samples of Finnish cyanobacteria were analyzed by high-performance liquid chromatography combined with UV diode-array detection, multiple reactant monitoring in a triple-quadrupole mass spectrometer (MS), and accurate mass measurements using a time-of-flight MS instrument. Cylindrospermopsin was confirmed by all three techniques in a culture sample of Anabaena lapponica at a concentration of 242 microg cylindrospermopsin per g freeze-dried cyanobacterial material.

Paucibacter toxinivorans gen. nov., sp. nov., a bacterium that degrades cyclic cyanobacterial hepatotoxins microcystins and nodularin.

Thirteen bacterial isolates from lake sediment, capable of degrading cyanobacterial hepatotoxins microcystins and nodularin, were characterized by phenotypic, genetic and genomic approaches. Cells of these isolates were Gram-negative, motile by means of a single polar flagellum, oxidase-positive, weakly catalase-positive and rod-shaped. According to phenotypic characteristics (carbon utilization, fatty acid and enzyme activity profiles), the G+C content of the genomic DNA (66.1-68.0 mol%) and 16S rRNA gene sequence analysis (98.9-100% similarity) the strains formed a single microdiverse genospecies that was most closely related to Roseateles depolymerans (95.7-96.3% 16S rRNA gene sequence similarity). The isolates assimilated only a few carbon sources. Of the 96 carbon sources tested, Tween 40 was the only one used by all strains. The strains were able to mineralize phosphorus from organic compounds, and they had strong leucine arylamidase and chymotrypsin activities. The cellular fatty acids identified from all strains were C(16:0) (9.8-19%) and C(17:1)omega7c (<1-5.8%). The other predominant fatty acids comprised three groups: summed feature 3 (<1-2.2%), which included C(14:0) 3-OH and C(16:1) iso I, summed feature 4 (54-62%), which included C(16:1)omega7c and C(15:0) iso OH, and summed feature 7 (8.5-28%), which included omega7c, omega9c and omega12t forms of C(18:1). A more detailed analysis of two strains indicated that C(16:1)omega7c was the main fatty acid. The phylogenetic and phenotypic features separating our strains from recognized bacteria support the creation of a novel genus and species, for which the name Paucibacter toxinivorans gen. nov., sp. nov. is proposed. The type strain is 2C20(T) (=DSM 16998(T)=HAMBI 2767(T)=VYH 193597(T)).

First report of saxitoxin in Finnish lakes and possible associated effects on human health.

This study is the first report of saxitoxin in cyanobacterial blooms in Finland. Bloom samples (n = 50) were collected from Finnish freshwater sites during summer months of 2002 and 2003. These samples were screened for the presence of paralytic shellfish toxins (PSTs) using the Jellett rapid PSP screening test. Samples testing positive for PSTs (n = 7) were further analyzed with saxiphilin- and voltage-gated sodium channel [(3)H]-STX-binding radioreceptor assays and liquid chromatography using fluorescence and mass spectrometric analysis. The results indicated that saxitoxin (STX) was the only PST analogue in the samples and that it was present in high concentrations, as much as 1 mg L(-1). Microscopic analysis revealed that 95%-100% of the phytoplankton in the positive samples consisted of Anabaena lemmermannii. The trophic status of lakes in which STX-containing blooms were found varied from oligotrophic to hypertrophic. All the lakes had high nitrogen-to-phosphorus ratios. In some instances, samples had been collected from sites where swimmers had reported adverse health effects, and in three such cases, reported adverse health effects were associated with sites from which samples testing positive for STX had been received. Symptoms of fever, eye irritation, abdominal pains, and skin rash were reported in children aged 2-10 years after exposure to the water. These were not the adverse human symptoms typical of STX poisoning; rather, they represented acute effects often reported following recreational exposure to cyanobacterial blooms.