No abstract

Several strains of a strictly anaerobic, vibrio-shaped or sigmoid, sulfate-reducing bacterium were isolated from deep marine sediments (depth, 80 and 500 m) obtained from the Japan Sea (Ocean Drilling Program Leg 128, site 798B). This bacterium was identified as a member of the genus Desulfovibrio on the basis of the presence of desulfoviridin and characteristic phospholipid fatty acids (is0 17:lw7 and is0 15:0), the small number of growth substrates utilized (lactate, pyruvate, and hydrogen), and 16s rRNA gene sequence analysis data. Based on data for 16s rRNA sequences (1,369 bp), all of the Japan Sea strains were identical to each other and were most closely related to Desulfovibrio salexigens and less closely related to Desulfuvibrio desulfuricans (levels of similarity, 91 and 89.6%, respectively). There were, however, considerable phenotypic differences (in temperatures, pressures, and salinities tolerated, growth substrates, and electron donors) between the Japan Sea isolates and the type strains of previously described desulfovibrios, as well as important differences among the Japan Sea isolates. The Japan Sea isolates were active (with sulfide production) over a wide temperature range (15 to 65°C) and a wide sodium chloride concentration range (0.2 to 10%) (moderate halophile), and they were barophiles that were active at pressures up to about 40 MPa (400 atm). The optimum pressures for activity corresponded to the calculated pressures at the depths from which the organisms were isolated (for isolates obtained at depths of 80 and 500 m the optimum activities occurred at 10 and 15 MPa, respectively [lo0 and 150 atm, respectively]). This confirms that the organisms came from deep sediments and indicates that they are well-adapted for deep sediment conditions, which is consistent with other characteristics (utilization of hydrogen, fermentation, and utilization of ferric iron and organic sulfonates as electron acceptors). We propose that Japan Sea isolate 500-1 is the type strain of a new species, Desulfovibrio profundus.Bacterial sulfate reduction is the dominant anaerobic terminal oxidation process in marine sediments, and in coastal sediments this process can be responsible for more than 50% of the organic matter degradation (21). The importance of sulfate reduction decreases in deeper-water sediments, where the diagenetic zones are greatly extended (16,22), and as a result sulfate can be present to a depth of several hundred meters in some pelagic sediments (4). Thus, there is the potential for active sulfate reduction to continue at great depth within marine sediments. Recently, this was demonstrated for Japan Sea sediments, where sulfate reduction was shown to be present to a depth of at least 425 m together with significant bacterial populations (30). Viable sulfate-reducing bacteria were also present to a depth of 80 m, and other viable bacterial types were present at even greater depths. These findings significantly extend the environment for sulfate-reducing bacteria and are consistent with report...

In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r ‫؍‬ 0.85, n ‫؍‬ 25) and for oocysts exposed to ozone and UV light (r ‫؍‬ 0.89, n ‫؍‬ 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences.

No abstract

The diversity of bacterial communities in deep marine sediments, up to 503 metres below the sea floor of the Japan Sea, was investigated by sequence analysis of amplified 16S rRNA genes. The use of different sample handling procedures greatly affected the types and diversity of sequences obtained. DNA from sediment samples stored aerobically for up to 24 h before freezing was dominated by sequences belonging to the β‐ and γ‐proteobacteria, many of which appeared to originate from aerobic bacteria. Sub‐samples equilibrated anaerobically at 16°C, were then injected with a radiotracer and immediately frozen, to simulate the conditions of a typical control sample from a radiotracer based activity assay, contained mostly α‐proteobacterial sequences. Pristine sediment samples taken anaerobically and frozen within 2 h contained the widest diversity of sequences from α‐, γ‐, δ‐proteobacteria and Gram‐positive bacteria, which appeared to have originated from predominantly anaerobic or facultative bacteria. It was clear that both samples that were not frozen immediately (within 2 h) showed signs of enrichment of specific bacterial groups. Our results strongly suggest that immediate freezing should always be employed when sediment samples are to be used to assess bacterial diversity by molecular methods.

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.

Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log,, molecular size against log,, relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates ( I 3.0 & 1.5 %). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.

Enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were compared for analyzing microcystins in water. ELISA results of microcystin‐LR spiked into raw water samples were close to the spike concentrations, but method variability was ±25%. However, ELISA‐derived microcystin‐LA concentrations were two to three times higher than the spike concentrations obtained using the kit‐provided microcystin‐LR standards, indicating the need for variant‐appropriate ELISA standards. LC/MS/MS results agreed with spike concentrations for all variants in reagent water, but matrix suppression was observed in some raw waters. In bench‐scale studies, ozonated microcystins generated low‐level positive responses by ELISA and a protein phosphatase inhibition assay, even though microcystins were not detected by LC/MS/MS. These findings indicate that ELISA results—particularly in treated water—should be interpreted with caution because of the possibility of false‐positives, relatively high variability, and differential detection of some variants.