Seven heat-resistant bacterial strains, which grew anaerobically in nitrogen-free medium and aerobically in nitrate-containing medium, were isolated from the rhizosphere soils of wheat, corn and Susuki (Miscanthus sinensis) grown in farms or grasslands in Hiroshima prefecture, western Japan. The cells of the isolated strains were rods, 0.4 to 1.0μm × 1.4 to 3.4μm, and formed ellipsoidal endospores. The guanine plus cytosine contents of these strains were 43.6 to 54.7mol%. The strains produced hydrogen and carbon dioxide from formate under anaerobic conditions, and the enzymes formate dehydrogenase and hydrogenase were rapidly inactivated when crude extracts were shaken under air. Some properties of the isolated strains were compared with those of the strains of Paenibacillus polymyxa, P. macerans and P. azotofixans.
To assess the importance of microbial biomass in determining the litter decomposition rate, we investigated the relation between microbial biomass, respiration rate and chemical properties of the L and F+H layers. Samples were collected from three forests dominated by different tree species: Quercus serrata; Pinus densiflora; and Cryptomeria japonica. A significant correlation was observed between microbial biomass and respiration rate, but metabolic quotients (respiration rate per unit biomass) of samples differed significantly among forests. Microbial biomass was positively correlated with acidsoluble sugar or cellulose content.
Bacterial communities inhabiting the rhizosphere of Lepironia articulata (reedgrass) grown in a strongly acidic sulfate soil distributed in the province of Narathiwat, Thailand were investigated. Fresh roots of L. articulata were cut from the subterranean stem, and the bacteria were isolated from the homogenate on dilution plates of glucose-yeast extract agar medium adjusted to pH 4.5. In or on the roots, 3.3×107 bacteria(cfu)g-1 fresh roots were present. From the dilution plates, 60 bacteria were randomly isolated and their characteristics were examined. All the isolates were Gram-negative rods and grew well at pH 4.5. Analysis of cell fatty acids and the restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rRNA genes indicated that the isolates could be divided into 4 groups (Group I to IV). Group I (65% of total) isolates failed to grow on the agar plate at pH 7. The sequence analysis of 16S rRNA genes of a Group I isolate showed that it resembled Acidocella facilis with 98.5% homology. Growth of Group I bacteria was suppressed by acetate. The next most abundant bacteria, belonging to Group II, grew at pH 7.0 and belonged to Burkholderia. These bacteria accounted for 28% of all the isolates. Bacteria of Group III and IV (identified as Bacillus subtilis) were a minor constituent only. Group III isolates were similar to the genus Xanthomonas, but the similarity of the 16S rRNA gene base arrangement with the nearest species was below 90%.
To understand the molecular basis of the mercury resistance of anaerobic bacteria, twenty-six mercury-resistant bacterial strains were anaerobically isolated from the mercury-polluted sediment of Minamata Bay, Japan. On the basis of taxonomic characteristics, all of the isolates were classified into the genus Clostridium. PCR primers designed from the core sequence of the merA genes that are involved in the inorganic mercury reduction by the aerobic mercury-resistant bacteria were used for the molecular analysis. Southern hybridization analysis confirmed that the PCR products amplified from the chromosomal DNA of the twenty-six Clostridium isolates were highly homologous to the merA gene of an aerobic mercury-resistant Gram-positive bacterium, Bacillus sp. RC607. Furthermore, the DNA sequence of the PCR product amplified from one of the isolates is 99.7% identical to the corresponding region of the merA gene of Bacillus sp. RC607. These results suggest that the same mercury resistance system developed by aerobic mercury-resistant bacteria is acquired by the anaerobic mercury-resistant bacteria.
Adding sodium pyruvate to 1/2 strength PYG medium yielded higher plate counts for a river bacterial community. Maximal counts were obtained when 0.15g liter-1 of sodium pyruvate was added to the medium for both river water and epilithon samples. The degree of magnification of the plate counts on the medium supplemented with sodium pyruvate varied depending on the source of the sample. The findings suggested that the variations in magnification reveal aspects of physiological activities of microbial communities in river environments.
Application of molecular methods to natural ecosystems has revealed the occurrence of many unexpected phylogenetic groups of prokarytoes which have not yet been isolated as cultivable strains. This is true for biological waste water treatment systems. Plate bacterial counts accounted for 1 to 18% of total (DAPI) counts in municipal sewage activated sludge. Microscopic, molecular genetic, and chemical biomarker approaches have suggested that the failure to detect many bacteria as viable counts results from the occurrence of viable but non-culturable bacteria and from the existence and diversity of bacteria undescribed so far.
Verocytotoxin-producing Escherichia coli (VTEC) O157 can cause haemolytic diarrhea and haemolytic uraemic syndrome, and most outbreaks caused by this strain have been food or water related. Cattle are known to be major reservoirs for VTEC including E. coli O157, and this strain has also been isolated from drinking water, treated water and sewage. E. coli O157 was frequently detected in natural river water by immunological and molecular biological methods, even if it was not detected by traditional culture methods. To clarify the physiological state of E. coli O157, flow cytometry was applied with the DVC or the vital staining method. Physiolocially active E. coli O157 was found to be distributed in natural river water. Bacteriophages carrying the sltII gene were also detected in sewage, and they could transform E. coli O157. These results indicate that monitoring not only bacterial cells but also slt genes is necessary to clarify the dynamics of E. coli O157 in natural environments, and examination of the viable but non-culturable state of E. coli O157 is also essential to prevent disease outbreaks.
The pathogenicity of Salmonella was studied in different growth phases to clarify the VBNC state of this bacteria in the environment. Salmonella typhimurium in the logarithmic phase kills BALB/c mice much more efficiently than that in the stationary phase when infected p. o., and similar differences were observed in the binding activity of this bacteria in these growth phases to macrophages and colon epithelial cells. However, logarithmic Salmonella is more sensitive to distilled water than stationary Salmonella and did not survive longer than 2 days. On the contrary, stationary Salmonella survived for more than 8 months in phosphate-buffered saline and was able to regrow in nutrient-rich medium upon incubation at 37°C, showing pathogenic activity to mice. Survey of Salmonella in the Tama River revealed that several colonies were virulent Salmonella enteritidis containing the plasmid encoding invA and enterotoxin, and some of these colonies showed lethality to mice as potently as pathogenic strains isolated from patients. These results suggest that Salmonella is found in river water and might regrow in nutrient-rich medium becoming pathogenic. Furthermore, the growth phase may be an important factor for this bacteria not only to survive in the environment but also to manifest pathogenicity to the host.
Viable but nonculturable (VBNC) bacteria generally inhabit marine environments, especially oligotrophic open ocean, because most marine bacteria which are biologically active can not be cultivated by traditional microbiological protocols. Some researchers argue that VBNC is a temporary state in marine bacteria which are originally cultivated by normal methods, induced by several environmental stresses, for example low temperature, high pressure, and extremely low nutrient concentrations. Other researchers argue that VBNC marine bacteria are novel and unique bacterial groups which have never been isolated by the traditional microbiological techniques. Some phenomenon in marine Vibrio strains support the former and phylogenetic studies on bacterial 16S ribosomal RNA genes directly isolated from seawater samples support the latter view. We introduced here marine oligotrophic bacteria, which were isolated by some low-nutrient media containing only 0.5mg/L of organic nutrients. These bacteria are unculturable in all high-nutrient media containing more than 0.5g/L of organic nutrients, the concentration which is normally used for bacterial cultivation, and their 16S rRNA gene sequences indicate that they are phylogenetically novel.