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A significant number of Enterococcus strains from pristine waters of two brooks in Finland formed a distinct cluster on the basis of whole-cell protein fingerprinting by one-dimensional SDS-PAGE. The strains shared the following characteristics. Cells were ovoid, Gram-positive-staining and non-spore-forming, appearing singly or in pairs or chains. They were facultatively anaerobic and catalase-negative. Growth in broth containing 6.5 % NaCl or at 45 °C was weak or absent. Production of D antigen was variable. The strains tolerated 60 °C for 30 min, 40 % bile and tellurite, hydrolysed aesculin strongly and gelatin weakly, produced no acid from hippurate and did not reduce it, grew weakly at 10 °C, showed a strong reaction for the Voges–Proskauer test and produced acid from methyl α-d-glucoside, mannitol, sorbitol and sucrose, with weak or no production of acid from methyl α-d-mannoside, l-arabinose, gluconate and l-xylose. Several of the strains were selected for identification on the basis of sequencing of almost the whole 16S rRNA gene and partial atpA and pheS genes and of (GTG)5-PCR fingerprints. Partial atpA and pheS gene sequencing was also performed for those type strains of Enterococcus species without available sequences in the database. The pristine brook isolates formed a novel species, for which the name Enterococcus rivorum sp. nov. (type strain S299T = HAMBI 3055T = LMG 25899T = CCM 7986T) is proposed. On the basis of 16S rRNA gene sequence similarity, E. rivorum sp. nov. is related to the Enterococcus faecalis genogoup. It is distinguished from described Enterococcus species on the basis of 16S rRNA, atpA and pheS gene sequences and whole-cell protein and (GTG)5-PCR fingerprints. It is most closely related to E. faecalis , but DNA–DNA hybridization confirms it to represent a novel species.

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.

In the conventional cultivation of potatoes, weed control and the control of potato late blight caused by Phytophthora infestans are carried out by the application of herbicides and fungicides. We investigated the impacts of the herbicides metribuzin and linuron and the fungicide fluazinam on soil microbiota in microcosms, in mesocosms and in the field. The toxicity of each pesticide in solution was assessed using the luminescent bacteria test and in soil by a solid phase modification. In microcosm tests, the microbial activity and biomass were estimated by measuring several soil enzyme activities together with soil ATP content. In the mesocosm tests, the separate addition of each pesticide and the simultaneous use of all the pesticides were investigated. We monitored the impacts on ten different soil enzyme activities and measured soil toxicity with the luminescent bacteria test separately in the 5 cm top layer and in the layer from 5 to about 20 cm below the surface. During one season, the impact of the use of pesticides was monitored in the field in plots receiving pesticides for the third consecutive year and in control plots cultivated without the use of pesticides for the 3 preceding years. The pesticide concentrations were monitored in each experiment. The luminescent bacteria toxicity test revealed a strong inhibition by fluazinam. In microcosms the herbicides increased several enzyme activities but metribuzin inhibited luminescence in the bacterial test. Fluazinam was highly toxic in microcosms. In the mesocosm with combined use of pesticides, decreased activities of some enzymes were observed first in the surface soil and at harvesting also deeper in the soil. In the mesocosm experiment with separate use of pesticides, less impacts were observed. In the field experiment the pesticides decreased seven enzyme activities, when calculated per soil fresh weight but activities of four enzyme decreases if calculated per soil organic matter. Controlling weeds by herbicides decreased weed growth and the decreases in enzyme activities were interpreted to be due to the lack of the stimulating impact of weed roots. A strong inhibition in the soil toxicity test and continuing bioavailability of fluazinam were detected throughout the experiments even after winter in the field.

We measured soil microbiota and enzyme activities in order to compare conventional (CCS: chemical fertilisers, plant rotation with cereals dominating) and organic (OCS: green and farmyard manure, plant rotation including leguminous plants) cropping systems in a long-term field experiment. During the 3-year study period, strawberry was grown on the whole area and peat amendment was applied to a set of plots. Activities of 12 different enzymes, phospholipid fatty acids (PLFA) and microbial biomass (C mic and N mic ) were measured twice each year. Dry weight (dw), water holding capacity (WHC) and pH were also measured. The enzyme activities were generally higher, arylsulphatase, phosphomonoesterase (PME) and esterase activities consistently, in the OCS than in the CCS. Other enzyme activities displayed higher activities either during 1 or 2 years or seasonally. Peat amendment increased PME, phosphodiesterase (PDE), leucine aminopeptidase (AP), chitinase, cellobiosidase, α-glucosidase and esterase activities but decreased arylsulphatase and initially alanine AP activities, whereas β-glucosidase and β-xylosidase activities were increased only during the 3rd year. Microbial biomass was higher in the OCS than in the CCS but peat addition decreased C mic and N mic at least initially. Both the OCS and peat addition increased soil PLFA content. Peat treatment also affected soil microbial structure as revealed by PLFA patterns, whereas the cropping system had no impact.

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 C16 : 0 (9·8–19 %) and C17 : 1 ω7c (14 : 0 3-OH and C16 : 1 iso I, summed feature 4 (54–62 %), which included C16 : 1 ω7c and C15 : 0 iso OH, and summed feature 7 (8·5–28 %), which included ω7c, ω9c and ω12t forms of C18 : 1. A more detailed analysis of two strains indicated that C16 : 1 ω7c 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 2C20T (=DSM 16998T=HAMBI 2767T=VYH 193597T).

A rapid means for restoring soil fertility could be addition of peat to the plough layer. The impact of cultivation of eight different crops (the joint impact of plant and the management tailored for each plant), with and without soil amendment by peat treatment on soil microbiological, physical and chemical properties was assessed for two consecutive growing seasons. As a measure of the functional diversity of soil microbial community we estimated the activity of several different extracellular soil enzymes using the ZymProfiler® test kit. ATP content was measured to yield information on the amount of the active microbial biomass, and phospholipid fatty acid (PLFA) profiles were analysed to reveal the microbial community structure. The enzyme activity patterns of the soil samples indicated several differences due to the different crops and years but ATP content and PLFA profiles were rather stable. However, microbial biomass as total amount of PLFAs depended on the plant and peat treatment and ATP content varied between the years. The effects of the peat treatments were less clearly indicated by the biological parameters one or two years after the amendment, as only arylsulphatase and β-xylosidase activities were affected in both the years. Soil moisture, affecting enzyme activities, depended on the year and crop plant and peat addition increased it.

R. M AARIT N IEMI, J . M ENTU, A . S IITONEN A ND S .I. N IEMELA ¨ . 2003. Aims: In the enumeration of coliform bacteria, confirmation of Escherichia coli has been based upon gas and indole production at the elevated incubation temperature. The test for gas production has recently been questioned. The aim of this study was to investigate the impact of gas production test on the reliability of confirmation of E. coli. Methods and Results: The impact of several media on growth, gas and/or indole formation was tested at 44 and44AE5� C using 547 environmental isolates. These were mainly E. coli, Klebsiella pneumoniae, K. oxytoca and Enterobacter cloacae strains. Another set of 250 faecal and environmental klebsiellae were tested for their maximum temperature for growth (Tmax) and for gas formation. Escherichia coli and even K. pneumoniae grew well in all the media, but gas production was more dependent on the medium used. Growth of the mainly gas negative Ent. cloacae and K. oxytoca strains was still more sensitive to the medium and incubation conditions. Tryptophan salt broth was the most productive medium for the indole test, followed by lauryl tryptose mannitole and tryptone mannitol ricinoleate broth (TRM). Tmax of K. oxytoca was clearly lower than Tmax of K. pneumoniae but a rather high fraction of its isolates produced indole at 44AE5� C. Conclusions: False-positive E. coli confirmation is possible if gas production is not tested for and the confirmation is based on indole test only. Significance and Impact of the Study: Erroneous positive results on routine analysis for E. coli can occur.

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.

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 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.

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.

Ethanol and acetate were examined as potential candidates to enhance denitrification at low temperature in soils contaminated by fur farms. Five pilot-scale sand and gravel columns with a top layer of soil from a fur farm were set up and fed with nitrate-containing water (influent concentration of 100 and 200 mg L−1) for 459 days at 6 ± 2 °C. Two of the columns also received acetate and two other ethanol while one received no additional C-substrate. During the experiment, various C:N-ratios were tested to find the most optimal concentration of the added C-substrates, and effluent concentrations of nitrate, nitrite, and TOC were monitored. At the end of the experiments, soils in the columns were unpacked and the soils were used to measure a pattern of enzyme activities and the rates of denitrification in microcosms. The fur farm contaminated soil appeared to harbour a good intrinsic potential for denitrification, which could be greatly enhanced by the introduction of ethanol or acetate. Consequently, in the C-substrate-fed columns, 95–100% of the influent nitrate was removed after an acclimatization period of some weeks. Ethanol with C:N-ratio of ca. 6 at the nitrate level 200 mg L−1 proved to be the most promising candidate to be used in field trials.

Fifty-seven water samples in all were collected in a northern (latitude 69° N) and in a southern (latitude 61° N) region from waters in the “natural state.” Coliform populations were studied by collecting random isolates of typical sheen colonies from Endo LES 35° C cultivations. The maximum growth temperatures of 607 strains were measured. Identification of 372 isolates was attempted by using the API 20EC and 20E systems. Eleven species were found—seven of them common to both regions. Twenty-nine percent of the strains could not be identified. The most frequent species was Serratia fonticola (26% of all strains tested), the second was Hafnia alvei (14%), and the third Enterobacter cloacae (13%; only encountered in the north). The strains able to grow at or above 44. 5°C were identified as Escherichia coli. In the southern region, environmental coliforms (S. fonticola and H. alvei) so completely outnumbered E. coli that it was met only once among the 438 total coliform isolates, whereas 23% of the isolates from the northern region were E. coli. Typical pipeline/biofilm coliform types were not found, with the exception of numerous E. cloacae strains in the most remote lake samples collected and one Klebsiella oxytoca in a brook. The fecal coliforms (all of them E. coli) were concluded to be of recent animal origin. The standard fecal coliform analysis was estimated to function extremely well in the pristine waters of our subarctic climate. The total coliform analysis has no indicator value under these circumstances.

Fifty-one fast growing rhizobial strains isolated from root nodules of #Acacia senegal$ and #Prosopis chilensis$ in Sudan and Kenya were divided into DNA homology groups using non-radioactive DNA-DNA dot-blot hybridisation. #Rhizobium leguminosarum$ , #R. galegae$, #R. tropici$, #Mesorhizobium loti$, #Sinorhizobium fredii$, #S. meliloti$ used in numerical taxonomy were included in the hybridisation experiments as reference strains and, at a later strage #S. saheli$ and #S. terangae$. Scores given to the intensities of dots detected in the hybridisation experiments were used in principal component analysis, which clustered the majority of the tree rhizobia in two separate DNA-homology groups. The 51 strains were also analysed by genomic fingerprinting using the repetitive sequence-based polymerase chain reaction (rep-PCR) method with REP, ERIC, BOX and GTG5 primers. The resulting genomic fingerprints were compared with strains representing 15 rhizobial species. The relationship of 17 Sudanese strains to established sinorhizobial species was examined using the optical renaturation rates method and the G+C content of nine strains was determined. Results from dot-blot hybridisation and rep-PCR experiments were found to be in close agreement with each other and with the results obtained from spectrophotometric reassociation analysis. We suggest that rep-PCR fingerprinting can be used as a first and dot-blot hybridisation as a second rapid and dependable genomic screening method to classify new rhizobial isolates of unknown taxonomic status and to choose the representative strains for the more laborious DNA-DNA reassociation experiments. (Résumé d'auteur)