Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms.

Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all <96% which is the previously proposed cutoff value for differentiating species within this genus. The in silico DDH values of the three type strains of the new species also showed a similarity<70% with the most closely related species indicating they belong to different taxa. The three groups of strains could be differentiated from each other and from other known Aeromonas species on the basis of several phenotypic characters. This polyphasic study revealed that the 3 groups of strains represent 3 novel Aeromonas species for which the names Aeromonas aquatica sp. nov. (type strain AE235T=CECT 8025T=LMG 26712T), Aeromonas finlandiensis sp. nov. (type strain 4287DT=CECT 8028T=LMG 26709T) and Aeromonas lacus sp. nov. (type strain AE122T=CECT 8024T=LMG 26710T) are proposed.

Sample storage for soil enzyme activity and bacterial community profiles.

Storage of samples is often an unavoidable step in environmental data collection, since available analytical capacity seldom permits immediate processing of large sample sets needed for representative data. In microbiological soil studies, sample pretreatments may have a strong influence on measurement results, and thus careful consideration is required in the selection of storage conditions. The aim of this study was to investigate the suitability of prolonged (up to 16 weeks) frozen or air-dried storage for divergent soil materials. The samples selected to this study were mineral soil (clay loam) from an agricultural field, humus from a pine forest and compost from a municipal sewage sludge composting field. The measured microbiological parameters included functional profiling with ten different hydrolysing enzyme activities determined by artificial fluorogenic substrates, and structural profiling with bacterial 16S rDNA community fingerprints by amplicon length heterogeneity analysis (LH-PCR). Storage of samples affected the observed fluorescence intensity of the enzyme assay's fluorophor standards dissolved in soil suspension. The impact was highly dependent on the soil matrix and storage method, making it important to use separate standardisation for each combination of matrix type, storage method and time. Freezing proved to be a better storage method than air-drying for all the matrices and enzyme activities studied. The effect of freezing on the enzyme activities was small (<20%) in clay loam and forest humus and moderate (generally 20-30%) in compost. The most dramatic decreases (>50%) in activity were observed in compost after air-drying. The bacterial LH-PCR community fingerprints were unaffected by frozen storage in all matrices. The effect of storage treatments was tested using a new statistical method based on showing similarity rather than difference of results.

Stability of the fluorogenic enzyme substrates and pH optima of enzyme activities in different Finnish soils.

Fluorogenic artificial substrates facilitate sensitive enzyme activity measurements for a variety of processes in soil and other environmental samples. It is possible to use in situ pH for measurements on condition that the substrates are chemically stable. We studied the stability of 12 different methyl umbellipherone (MUF) and amino methyl coumarine (AMC) derivatives used as substrates for arylsulphatase, alpha-glucosidase, beta-glucosidase, beta-xylosidase, cellobiosidase, chitinase, phosphomonoesterase (PME), phoshodiesterase (PDE), esterase, lipase and alanine- and leucine aminopeptidases (AP) over the pH range from 4.0 to 8.0 in modified universal buffer (MUB). Stability of the substrates for lipase (4-MUF-heptanoate) and esterase (4-MUF-acetate) measurements was poor, especially at the higher pH values. Chitinase substrate, 4-MUF-N-acetyl-beta-D-glucosamide, was unstable at high pH values whereas the substrate for PME activity measurement (4-MUF-phosphate) disintegrated at low pH. The other substrates and MUF and AMC standard solutions were stable over the pH range studied. The optima between pH 4 and 8 of the 11 different enzyme activities were measured in three forest and two agricultural soil samples and in one activated sludge sample. In soil, for alanine and leucine AP the pH optima were usually 7.5 or higher, for arylsulphatase, beta-glucosidase, beta-xylosidase, esterase and PDE between 4 and 5.5, and for cellobiosidase between 4 and 5. alpha-Glucosidase had an optimum below 5.5 but also exhibited high activity at pH 7. Soil-dependent variation in pH optima were observed for chitinase, esterase, PDE and PME. Enzyme activities were also measured in 0.5 M acetate buffer at pH 5.5. This buffer yielded the highest activities in all soil samples for arylsulphatase, PDE and PME.

Removal of F-specific RNA bacteriophages in artificial recharge of groundwater--a field study.

Artificial recharge of groundwater offers a semi-natural means to produce raw water for drinking-water plants. Surface water works are increasingly being replaced by artificial groundwater works in Finland. Two municipalities, one serving 30,000 and the other 170,000 inhabitants, have considered filtering river water through eskers for the production of potable water. In this study the removal of bacteriophages during infiltration of river water was estimated, for the evaluation of treatment adequacy in a field study. A 5-m-deep column of sand was constructed and used to mimic the percolating phase in infiltration. An artificial esker was constructed on the riverbank by isolating a 2-m-wide, 2-m-deep and 18-m-long bed of coarse sand with plastic. The sand bed represented the saturated zone. River water was pumped at a rate of 40 L/h to the sand column. The river water was spiked with F+ specific RNA phage MS2 by adding phage suspension during one week at an average concentration of 4.3 x 10(9) PFU/mL. Samples for phage assays were taken during one month, from four sampling sites, on the basis of detention time as estimated by a tracer experiment with sodium chloride. The median count of MS2 for percolated water was 2.4 x 10(5) PFU/mL, representing a 96.7% reduction. During the passage of 6 m in the saturated zone, a further reduction of 98.5% occurred. During the passage from 6 m to 12 m the additional reduction was 99.97%. The overall reduction was between 6 and 7 log10 units. The removal of MS2 phages was rather efficient, although the esker material was coarse, mainly sandy, gravel.

Comparison of methods for determining the numbers and species distribution of coliform bacteria in well water samples.

AIMS: Enumeration of coliform bacteria and Escherichia coli is the most widely used method in the estimation of hygienic quality of drinking water. The yield of target bacteria and the species composition of different populations of coliform bacteria may depend on the method.Three methods were compared. METHODS AND RESULTS: Three membrane filtration methods were used for the enumeration of coliform bacteria in shallow well waters. The yield of confirmed coliform bacteria was highest on Differential Coliform agar, followed by LES Endo agar. Differential Coliform agar had the highest proportion of typical colonies, of which 74% were confirmed as belonging to the Enterobacteriaceae. Of the typical colonies on Lactose Tergitol 7 TTC agar, 75% were confirmed as Enterobacteriaceae, whereas 92% of typical colonies on LES Endo agar belonged to the Enterobacteriaceae. LES Endo agar yielded many Serratia strains, Lactose Tergitol 7 TTC agar yielded numerous strains of Rahnella aquatilis and Enterobacter, whereas Differential Coliform agar yielded the widest range of species. CONCLUSION: The yield of coliform bacteria varied between methods. Each method compared had a characteristic species distribution of target bacteria and a typical level of interference of non-target bacteria. Identification with routine physiological tests to distinct species was hampered by the slight differences between species. High yield and sufficient selectivity are difficult to achieve simultaneously, especially if the target group is diverse. SIGNIFICANCE AND IMPACT OF THE STUDY: The results showed that several aspects of method performance should be considered, and that the target group must be distinctly defined to enable method comparisons.

Enumeration of intestinal enterococci and interfering organisms with Slanetz-Bartley agar, KF streptococcus agar and the MUST method.

The recovery of intestinal species of enterococci and streptococci and potentially interfering nonfaecal species was measured on KF streptococcus agar, Slanetz-Bartley agar and in a medium based on 4-methylumbelliferyl-beta-D-glucoside (MUST method) using pure cultures. Both of the solid media yielded high recoveries of the target species. Their selectivity was better at elevated incubation temperature but nonfaecal Enterococcus and Staphylococcus species were not eliminated even at the elevated temperature. The MUST method tended to give slightly lower recoveries than the agar cultivation methods with some target species at 44 degrees C but recoveries were better at 41 degrees C.

Growth of Enterococcus, Lactococcus and Streptococcus strains and environmental isolates in liquid media and their reactions on BEEA.

Growth of known species of Enterococcus, Lactococcus and Streptococcus and Aerococcus viridans in selective and nonselective liquid media routinely used to enumerate faecal streptococci was measured optically at different temperatures. Growth of environmental isolates was measured in some of these media. Growth of the reference strains on Bile esculin azide agar at elevated incubation temperatures was tested. The results revealed only minor differences between media but strong influence of incubation temperature. Some media tended to yield higher cell densities than others. For many species the inoculum size affected maximum turbidity. To combine selective media with selective incubation temperatures seems to be necessary to achieve satisfactory reliability in traditional liquid enumeration methods for faecal streptococci. Because of the diversity of this group, optimal selectivity and recovery can hardly be achieved simultaneously.

Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.

Growth of Escherichia coli in a pulp and cardboard mill.

The coliform flora of a pulp and cardboard mill that uses birch as the raw material and ammonium sulphate as the process chemical was studied. Escherichia coli was observed to multiply in the mill. It persisted as the dominant thermotolerant coliform in the effluent. Klebsiellae were encountered among total coliforms only. The E. coli strains isolated had the biochemical characteristics and maximum growth temperatures typical to the species. However, serotyping and hemolysin test differentiated these strains from pathogenic and fecal E. coli.

Actinomycetes and fungi in surface waters and in potable water.

In Finnish lakes and rivers used as water supplies, mesophilic fungi and actinomycetes were common, whereas thermophilic fungi and actinomycetes were present only in low concentrations. Fungi and actinomycetes were more abundant in eutrophic and mesotrophic lakes than in oligotrophic lakes. River water contained more thermophilic actinomycetes and fungi and mesophilic actinomycetes than did lake water. Runoff from soil seemed to be an important factor contributing to the incidence of these microbes in water. Chemical coagulation removed actinomycetes and fungi efficiently, but sand filtration allowed their passage. Disinfection could not prevent actinomycetes and fungi from reaching the distribution system. During infiltration in the production of recharged groundwater, mesophilic actinomycetes could even multiply appreciably.