Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and 15 N 2 gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.

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We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N 2 -fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants. A major feature of ANFICOs is that N 2 fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture. In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture. ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations. These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N 2 fixation arises in association with nondiazotrophic endophytes.Microbes are not always culturable even though their biological activities may be detectable (1,15,20). This is true for some N 2 -fixing bacteria associated with plants, such as Azoarcus endophytes (16) and rhizobial bacteroids (22). Although the functional significance of microbial consortia in biofilms, for example (1, 3), has been emphasized, there are few concrete examples of their specific functions.The availability of fixed nitrogen limits primary productivity in plant ecosystems. During their evolution, legumes have acquired a symbiotic relationship with rhizobia that fix atmospheric nitrogen. Among nonleguminous plants, several diazotrophs have been isolated and characterized as nitrogenfixing endophytes, including Acetobacter (18), Azoarcus (11,16), and Herbaspirillum (6,8). Endophytes are microorganisms that spend most of their life cycles inside plant tissues without causing symptoms of plant damage (16). We still do not know whether these diazotrophic endophytes contribute substantially to the nitrogen economy of grasses (11,12). It is possible that we have overlooked the real contributors to nitrogen fixation in nonleguminous plants. Indeed, nitrogenase transcript analysis has indicated that endophytes, such as Azoarcus sp. and others in an apparently unculturable state, fix nitrogen in plants (11).Wild grasses can often grow in nitrogen-deficient soils, suggesting that functioning diazotrophic bacteria are associated with them. We therefore tried to isolate and characterize diazotrophic bacteria associated with wild rice species in situ and pioneer plants growing on a devastated lahar area with volcanic eruptions. For this work, we used mainly the aerial parts of plants as isolation materials to avoid bacterial contamination from soils. During efforts to isolate endophytic diazotrophs from these plants, we faced problems with unculturable diazotrophic bacteria and found an anaerobic nitrogen-fixing consortium (ANFICO) consisting of N 2 -fixing clostridia and diverse nondiazotrophic bacteria. The objective of this work was to clarify the members of ANFICOs and their interactions. MATERIALS AND METHODSIsolation of nitrogen-fixing bact...

Using a quantitative culture of sputum, the incidence of pathogenic bacteria in respiratory infection in our laboratory between 1990 and 1993 were investigated. While Haemophilus influenzae, Streptococcus pneumoniae and Moraxella (Branhamella) catarrhalis were isolated at high rates (67-78%) from the specimens of outpatients throughout the study period, the incidence of S pneumoniae has increased gradually. The antimicrobial susceptibilities of these three pathogens were examined with the agar dilution method. A marked increase of penicillin (PC) resistant S. pneumoniae (MIC > or = 0.1 microgram/ml) was observed with a resistance rate of 2.1% in 1990 and 25% in 1993. Resistance to erythromycin (EM, MIC > or = 1.56 micrograms/ml) was 8.5% in 1990 but then increased to 34% in 1992. Most of the PC resistant isolates were resistant to multidrugs such as EM, minocycline and clindamycin. The MICs of all beta-lactams examined for S. pneumoniae increased along with the MICs of PC, though the level varied between drugs. The rates of beta-lactamase positive H. influenzae gradually decreased, being 14.3% in 1990 and 7.4% in 1993, whereas those of M. (B) catarrhalis were consistently high (> 90%) every year. In addition to beta-lactamase production, the emergence of strains of H. influenzae and M. (B) catarrhalis resistant to new quinolone drugs should be noted.

Ciliated epithelial cells were obtained from nasal polyps. Bacterial adherence to these cells was compared for the ability to bind Hemophilus influenzae, Pseudomonas aeruginosa, and Branhamella catarrhalis in the presence of 10–5 M erythromycin, which was comparable with a physiologically attainable concentration in the nasal secretion and the maxillary sinus mucosa. Quantification of bacterial adherence showed the strongest ability of P. aeruginosa to the cells. Erythromycin has an inhibitory effect on adherence of P. aeruginose and B. catarrhalis to the nasal epithelial cell. Our findings suggest that the reduced adherence to the host cell is one of the underlying mechanisms to account for efficacy of erythromycin treatment in respiratory disorders.