Opportunistic Gram-negative rods' capability of creating biofilm structures on polivynyl chloride and styrene-acronitrile copolymer surfaces.

Biofilms are highly organized microbial communities displaying high resistance to disinfectants and other external environmental factors. Medical equipment, such as stents and catheters, can be colonized by a variety of bacteria including opportunistic pathogens circulating in the environment and dangerous to immunocompromised patients. Application of materials resistant to biofilm formation will minimize the risk of patients' infection. Hence, the aim of this research was to determine the biofilm growth of environmental bacteria isolates on polyvinyl chloride and styrene-acronitrile copolymer surfaces. Nine strains (Pseudomonas aeruginosa, Burkholderia cepacia and Serratia liquefacies) isolated from cosmetics, and a reference P. aeruginosa strain ATCC 15442, were tested. The ability and dynamics of biofilm formation on intubation catheters (30°C, up to 24 h) in bacterial growth cultures (10(7)-10(8) CFU/ml) was investigated, with subsequent sonication and quantification by agar plate count method. The results indicated that all the tested bacteria expressed a strong ability for the polymer surface adhesion, reaching 4.6 to 6.7 log CFU/cm(2) after 30 minutes. Moreover, for the majority of strains, the level of 24-hour biofilm production was from 6.67-7.61 log CFU/cm(2). This research indicates that the environmental strains circulating between the cosmetics and patients may pose a threat of biofilm formation on medical equipment surfaces, and presumably in the clinical surroundings as well.

Variibacter gotjawalensis gen. nov., sp. nov., isolated from soil of a lava forest.

A novel bacterial strain designated GJW-30(T) was isolated from soil of the lava forest, Gotjawal, located in Aewol, Jeju, Korea. Strain GJW-30(T) was found to be strictly aerobic, Gram-negative and to form pleomorphic, non-motile rods and white colonies on R2A agar. The major fatty acids were identified as C18:1ω7c, C16:0 and C17:0, the predominant isoprenoid quinone as Q-10, the polar lipids as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid and an unidentified lipid. The cell-wall sugar pattern of strain GJW-30(T) was found to be composed of glucose, ribose and rhamnose and meso-DAP as the diagnostic diamino acid in the cell-wall peptidoglycan. The DNA G+C content of strain GJW-30(T) is 62.2 mol%. Phylogenetic analysis, based on 16S rRNA gene sequence similarities, showed that strain GJW-30(T) forms a deep branch within the order Rhizobiales, sharing the highest level of sequence homology with Bradyrhizobium oligotrophicum LMG 10732(T) (93.6 %). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, strain GJW-30(T) is considered to represent a novel genus and species, for which the name Variibacter gotjawalensis gen. nov., sp. nov. (the type strain is GJW-30(T) = KCTC 32391(T) = CECT 8514(T) = LMG 28093(T)) is proposed.

Microbial fouling of a reverse osmosis municipal water treatment system.

Microbial fouling of a municipal water treatment system using reverse osmosis was investigated. From a combination of growth and molecular assays, it was discovered that the prefilter unit concentrated and facilitated microbial growth, and such growth led to microbial fouling of the reverse osmosis unit. Few cells were observed in the prefilter influent, but substantial microbial contamination was observed in the prefilter effluent, and this correlated with increasing headloss across the prefilter. The effluent caused microbial fouling of the leading elements of the reverse osmosis unit, as determined by reduced permeate flow, analysis of the elements, and assays of the membrane foulant. Both the introduction of microorganisms to the reverse osmosis unit from the prefilter unit and headloss across the prefilter could be effectively controlled through cleansing of the prefilter housing unit with sulfuric acid. Such treatments must be performed at appropriate intervals to prevent subsequent microbial growth in the prefilter unit.

Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.

In the present study six assays for the quantification of biofilms formed in 96-well microtiter plates were optimised and evaluated: the crystal violet (CV) assay, the Syto9 assay, the fluorescein diacetate (FDA) assay, the resazurin assay, the XTT assay and the dimethyl methylene blue (DMMB) assay. Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, Propionibacterium acnes and Candida albicans were used as test organisms. In general, these assays showed a broad applicability and a high repeatability for most isolates. In addition, the estimated numbers of CFUs present in the biofilms show limited variations between the different assays. Nevertheless, our data show that some assays are less suitable for the quantification of biofilms of particular isolates (e.g. the CV assay for P. aeruginosa).

Effect of xylitol on an in vitro model of oral biofilm.

PURPOSE: The aim of the present study was to examine whether xylitol, at different concentrations, inhibits the formation of an experimental model of oral biofilm. MATERIALS AND METHODS: Biofilms of six bacterial species (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus rhamnosus, Actinomyces viscosus, Porphyromonas gingivalis and Fusobacterium nucleatum) were prepared on hydroxyapatite (HA) discs according to the Zürich Biofilm Model. Xylitol was tested at two concentrations, 1% and 3%. At the end of their designated incubation times, some HA discs were destined for confocal laser scanning microscopy (CLSM) and the others were harvested using a sterile surgical instrument. Aliquots of harvested biofilms were diluted and plated onto specific media. After a 48-h anaerobic incubation at 37 degrees C, the colony-forming units (CFUs) were counted. RESULTS: CLSM images showed that only a small amount of isolated bacteria was observed on the surface of HA discs. Culture of harvested biofilms showed an inhibition in the growth of different species included in the biofilms. CONCLUSIONS: Xylitol has a clear inhibitory effect on the formation of the experimental biofilms. This study shows that xylitol is not only efficient in inhibiting the acid production of cariogenic bacteria, but also in preventing the formation of a multispecies biofilm; it confirms the relevance of the use of this polyol for the prevention of oral diseases caused by dental plaque.

Isolation and characterization of soil bacteria that define Terriglobus gen. nov., in the phylum Acidobacteria.

Bacteria in the phylum Acidobacteria are widely distributed and abundant in soils, but their ecological roles are poorly understood, owing in part to a paucity of cultured representatives. In a molecular survey of acidobacterial diversity at the Michigan State University Kellogg Biological Station Long-Term Ecological Research site, 27% of acidobacterial 16S rRNA gene clones in a never-tilled, successional plant community belonged to subdivision 1, whose relative abundance varied inversely with soil pH. Strains of subdivision 1 were isolated from these never-tilled soils using low-nutrient medium incubated for 3 to 4 weeks under elevated levels of carbon dioxide, which resulted in a slightly acidified medium that matched the pH optima of the strains (between 5 and 6). Colonies were approximately 1 mm in diameter and either white or pink, the latter due to a carotenoid(s) that was synthesized preferentially under 20% instead of 2% oxygen. Strains were gram-negative, aerobic, chemo-organotrophic, nonmotile rods that produced an extracellular matrix. All strains contained either one or two copies of the 16S rRNA encoding gene, which along with a relatively slow doubling time (10 to 15 h at ca. 23 degrees C) is suggestive of an oligotrophic lifestyle. Six of the strains are sufficiently similar to one another, but distinct from previously named Acidobacteria, to warrant creation of a new genus, Terriglobus, with Terriglobus roseus defined as the type species. The physiological and nutritional characteristics of Terriglobus are consistent with its potential widespread distribution in soil.

Isolation and characterization of hydrogen-oxidizing bacteria induced following exposure of soil to hydrogen gas and their impact on plant growth.

In many legumes, the nitrogen fixing root nodules produce H2 gas that diffuses into soil. It has been demonstrated that such exposure of soil to H2 can promote plant growth. To assess whether this may be due to H2-oxidizing microorganisms, bacteria were isolated from soil treated with H2 under laboratory conditions and from soils collected adjacent to H2 producing soybean nodules. Nineteen isolates of H2-oxidizing bacteria were obtained and all exhibited a half-saturation coefficient (Ks) for H2 of about 1 ml l(-1). The isolates were identified as Variovorax paradoxus, Flavobacterium johnsoniae and Burkholderia spp. using conventional microbiological tests and 16S rRNA gene sequence analysis. Seventeen of the isolates enhanced (57-254%) root elongation of spring wheat seedlings. Using an Arabidopsis thaliana bioassay, plant biomass was increased by 11-27% when inoculated by one of four isolates of V. paradoxus or one isolate of Burkholderia that were selected for evaluation. The isolates of V. paradoxus found in both H2-treated soil and in soil adjacent to soybean nodules had the greatest impact on plant growth. The results are consistent with the hypothesis that H2-oxidizing bacteria in soils have plant growth promoting properties.

Ice-active characteristics of soil bacteria selected by ice-affinity.

As an initial screen for microorganisms that produce ice-active macromolecules, ice-affinity was used to select microorganisms from soil consortia originating from three temperate regions. Once selected and subsequently purified to single colonies, these microbes were putatively identified by 16S ribosomal RNA sequencing and assayed for various ice-active properties. Ice-affinity selection appeared to select for bacteria with ice-associating activities: inhibition of ice recrystallization; ice nucleation; ice shaping. Although none of these activities were observed in Paenibacillus amyloliticus C8, others such as Chryseobacterium sp. GL8, demonstrated both ice recrystallization inhibition and ice-shaping activities. Pseudomonas borealis DL7 was classified as a type I ice nucleator, Flavobacterium sp. GL7, was identified as a type III ice nucleator and Acinetobacter radioresistens DL5 demonstrated ice recrystallization inhibition. In all, 19 different culturable bacteria were selected from the thousands of microbes in late-summer collected soil samples. Many of the selected microbes have been previously reported in glacial ice cores or polar sea ice, and of five isolates that were further characterized, four showed ice-associating activities. These results indicate the significant potential of ice-affinity selection even with temperate climate soils, suggesting that sampling in more extreme and remote areas is not required for the isolation of ice-active bacteria.

SHR5: a novel plant receptor kinase involved in plant-N2-fixing endophytic bacteria association.

Endophytic nitrogen-fixing bacteria have been isolated from graminaceous plants such as maize, rice, and sugarcane. They are thought to promote plant growth, not only by fixing nitrogen, but also by the production of plant hormones. The molecular mechanisms involved in this interaction are not yet clear. In this work, the identification of a receptor-like kinase (RLK), named SHR5, which may participate in signal transduction involved in the establishment of plant-endophytic bacteria interaction is described for the first time. SHR5 seems to be part of a novel subclass of RLKs present in a wide range of plant species. The expression of this gene is down-regulated in sugarcane plants associated exclusively with beneficial endophytic bacteria and is not a general response caused by micro-organisms or abiotic stress. In addition, more successful sugarcane-endophytic bacteria associations have a more pronounced decrease in SHR5 expression, suggesting that SHR5 mRNA levels in plant cells are inversely related to the efficiency of the association.

Liberation of amino acids by heterotrophic nitrogen fixing bacteria.

Large amounts of amino acids are produced by nitrogen-fixing bacteria such as Azotobacter, Azospirillum, Rhizobium, Mesorhizobium and Sinorhizobium when growing in culture media amended with different carbon and nitrogen sources. This kind of bacteria live in close association with plant roots enhanced plant growth mainly as a result of their ability to fix nitrogen, improving shoot and root development suppression of pathogenic bacteria and fungi, and increase of available P concentration. Also, it has been strongly evidenced that production of biologically substances such as amino acids by these rhizobacteria are involved in many of the processes that explain plant-grown promotion. This paper reviews literature concerning amino acids production by nitrogen-fixing bacteria. The role of amino acids in microbial interactions in the rhizosphere and establishment of plant bacterial association is also discussed.

[Evolution and phylogeny of rhizobia]. / Evolución y filogenia de rhizobia.

Nitrogen fixation an ancient process that may is have originated in the archaean Eon under the primitive atmosphere anoxygenic conditions. Diazotrophy is an exclusive process of prokaryotes, only Euryarchaeota and 6 of 54 Bacteria phyla have diazotrophs lineages. Some of them coevolved with flowering plants for the establishment of molecular bases of a mutualistic symbiosis relationship. In rhizobia, the nitrogen fixation occurs inside the nodules, special structures on the roots or stems of legumes. Nodule organogenesis starts with the bacterial nodulation factors (Nod factors) codified in large plasmids or symbiotic islands in the bacterial genomes. Nodulation genes had more recent origin than the nitrogen fixation ones because the origin of the nod gene is associated with the origin of the hosts. The 16S rRNA phylogeny groups rhizobia in 7 genuses of the alpha-Proteobacteria: Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium, Methylobacterium and Devosia, and two genuses recently described in f-Proteobacteria: Burkholderia and Wautersia. The phylogenies obtained with other chromosomal genes are similar at the genus level, but it is incongruent with the symbiotic gene (nif & nod) phylogeny, because horizontal gene transfer has allowed their evolution in function to the legume host fitness.

Trichloroethylene degradation by toluene-oxidizing bacteria grown on non-aromatic substrates.

The potential of trichloroethylene (TCE) to induce and non-aromatic growth substrates to support TCE degradation in five strains (Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Pseudomonas putida F1, Burkholderia cepacia G4, B. cepacia PR1) of toluene-oxidizing bacteria was examined. LB broth and acetate did not support TCE degradation in any of the wild-type strains. In contrast, fructose supported the highest specific levels of TCE oxidation observed in each of the strains tested, except B. cepacia G4. We discuss the potential mechanisms and implications of this observation. In particular, cells of P. mendocina KR1 degraded significant amounts of TCE during cell growth on non-aromatic substrates. Apparently, TCE degradation was not completely constrained by any given factor in this microorganism, as was observed with P. putida F1 (TCE was an extremely poor substrate) or B. cepacia G4 (lack of oxygenase induction by TCE). Our results indicate that multiple physiological traits are required to enable useful TCE degradation by toluene-oxidizing bacteria in the absence of aromatic cosubstrates. These traits include oxygenase induction, effective TCE turnover, and some level of resistance to TCE mediated toxicity.

Classification of three airborne bacteria and proposal of Hymenobacter aerophilus sp. nov.

Three aerobic, gram-negative, rod-shaped, non-spore-forming, red-pigmented, airborne bacteria (I/26-Cor1T, I/32A-Cor1 and I/74-Cor2) collected in the Museo Correr (Venice, Italy) were investigated to determine their taxonomic status by analysing their biochemical, physiological and chemotaxonomic features and the G+C content of genomic DNA and by comparing their genomic fingerprints. Additionally, the almost complete 16S rRNA gene sequence of strain I/26-Cor1T was analysed. The three strains were nearly identical in their morphological, physiological, biochemical and chemotaxonomic properties. The strains contained a menaquinone system with the predominant menaquinone MK-7 and a fatty acid profile with C15:0 anteiso, C15:0 iso and C16:1 predominant. Phosphatidylethanolamine and several unidentified lipids were detected in the polar lipid profiles. The polyamine pattern consisted of sym-homospermidine as the major compound. meso-Diaminopimelic acid was found as the characteristic cell-wall diamino acid. The DNA base composition of the three strains ranged from 60 to 63 mol% G+C. Phylogenetically, strain I/26-Cor1T was most closely related to Hymenobacter actinosclerus (95.8% 16S rRNA gene sequence similarity). Physiological and genomic characteristics indicated that the two strains I/26-Cor1T and I/32A-Cor1 are representatives of the same species. The phylogenetic distance to any validly described taxon as indicated by 16S rRNA gene sequence similarities demonstrates that I/26-Cor1T and I/32A-Cor1 represent a novel species, for which the name Hymenobacter aerophilus sp. nov. is proposed, with the type strain I/26-Cor1T (= DSM 13606T = LMG 19657T). I/32A-Cor1 (= DSM 13607 = LMG 19658) is another strain of the species Hymenobacter aerophilus. Since the taxonomic status of strain I/74-Cor2 within the genus Hymenobacter was not determined unambiguously, it is designated Hymenobacter sp. I/74-Cor2 (= DSM 13611 = LMG 19659).

Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling disease of rice.

Of 102 rhizoplane and endophytic bacteria isolated from rice roots and stems in California, 37% significantly (P < or = 0.05) inhibited the growth in vitro of two pathogens, Achlya klebsiana and Pythium spinosum, causing seedling disease of rice. Four endophytic strains were highly effective against seedling disease in growth pouch assays, and these were identified as Pseudomonas fluorescens (S3), Pseudomonas tolaasii (S20), Pseudomonas veronii (S21), and Sphingomonas trueperi (S12) by sequencing of amplified 16S rRNA genes. Strains S12, S20, and S21 contained the nitrogen fixation gene, nifD, but only S12 was able to reduce acetylene in pure culture. The four strains significantly enhanced plant growth in the absence of pathogens, as evidenced by increases in plant height and dry weight of inoculated rice seedlings relative to noninoculated rice. Three bacterial strains (S3, S20, and S21) were evaluated in pot bioassays and reduced disease incidence by 50%-73%. Strain S3 was as effective at suppressing disease at the lowest inoculum density (106 CFU/mL) as at higher density (10(8) CFU/mL or undiluted suspension). This study indicates that selected endophytic bacterial strains have potential for control of seedling disease of rice and for plant growth promotion.

Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water.

The fate of Ralstonia solanacearum bv. 2, the causative agent of brown rot in potato, in aquatic habitats of temperate climate regions is still poorly understood. In this study, the population dynamics and the physiological response of R. solanacearum bv. 2 were tested in sterile pure water and in agricultural drainage water obtained from waterways near potato cropping fields in The Netherlands. The behaviour of five different biovar 2 isolates in drainage water at 20 degrees C was very similar among strains. One typical isolate with consistent virulence (strain 1609) was selected for further studies. The effects of temperature, light, canal sediment, seawater salts, and the presence of competing microorganisms on the survival of strain 1609 were assessed. Moreover, the impacts of the physiological state of the inoculum and the inoculum density were analyzed. The population dynamics of strain 1609 in sterile pure water were also characterized. In sterile pure water, the fate of R. solanacearum 1609 cells depended strongly on temperature, irrespective of inoculum density or physiological state. At 4 degrees C and 44 degrees C, strain 1609 CFU numbers showed declines, whereas the strain was able to undergo several cell divisions at 12 degrees C, 20 degrees C, and 28 degrees C. At 20 degrees C and 28 degrees C, repeated growth took place when the organism was serially transferred, at low inoculum density, from grown water cultures into fresh water devoid of nutrients. Both at low and high cell densities and regardless of physiological state, R. solanacearum 1609 cells persisted as culturable cells for limited periods of time in drainage water. A major effect of temperature was found, with survival being maximal at 12 degrees C, 20 degrees C, and 28 degrees C. Temperatures of 4 degrees C, 36 degrees C, or 44 degrees C induced accelerated declines of the culturable cell numbers. The drainage water biota had a strong effect on survival at 12 degrees C, 20 degrees C, and 28 degrees C, as the persistence of strain 1609 was significantly enhanced in sterile drainage water systems. Furthermore, there was a negative effect of incident light, in a light:dark regime, on the survival of R. solanacearum 1609 in natural drainage water. Also, levels of seawater salts realistic for drainage water in coastal areas were detrimental to strain survival. Ralstonia solanacearum 1609 showed considerable persistence in canal sediment saturated with drainage water, but died out quickly when this sediment was subjected to drying. Evidence was obtained for the conversion of R. solanacearum 1609 cells to nonculturable cells in water microcosms kept at 4 degrees C, but not in those kept at 20 degrees C. A substantial fraction of the cells found to be nonculturable were still viable, as evidenced by the direct viable count and by staining with the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. The potential occurrence of viable-but-nonculturable cells in natural waters poses a problem for the detection of R. solanacearum by cultivation-based methods.

Characterization of nonfermenters from clinical samples.

OBJECTIVE: Nonfermenters are a group of aerobic non sporing gram-negative bacilli found primarily free in nature and as commensals, whose pathogenic potentials are well established. The current study was conducted to assess the role of these nonfermenters in various infections and to characterize these isolates. METHODS: One hundred nonfermenters isolated from various clinical specimens were grouped according to Weaver-Hollis scheme based on growth on MacConkeys agar, oxidase activity and oxidation/fermentation of glucose. Species level identification was attempted based on a battery of biochemical tests. All isolates were then subjected to antimicrobial sensitivity. RESULTS: Majority of the isolates were encountered from pus and urine (50%). These isolates belonged to six of the seven Weaver-Hollis groups. Fifty six per cent of the isolates belonged to genus Pseudomonas. Multidrug resistance with resistance to more than three antimicrobials was frequently seen. Amikacin and ciprofloxacin were found to be most effective. CONCLUSION: Nonfermenting gram negative organisms are responsible for variety of infective conditions. Amongst them genus Pseudomonas and Acinetobacter calcoaceticus were more frequently encountered. Amikacin or ciprofloxacin (for nonfermenters other than Pseudomonas) appears to be the drug of choice for treatment of such infections.

Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations.

The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp. L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2. Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants. Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings. The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd. Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly. The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A. lipoferum 137. Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots. The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements.

Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance.

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortus LPS, but not of Escherichia coli LPS, into Ochrobactrum OM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropi and E. coli-O. anthropi LPS hybrid aggregates. Although Ochrobactrum was sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity. Ochrobactrum and B. abortus LPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, Ochrobactrum LPS, but not B. abortus LPS, contained galacturonic acid. B. abortus and Ochrobactrum smooth LPS aggregates had similar size and zeta potential (-12 to -15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrum smooth LPS while remaining negative (-5 mV) for B. abortus smooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrum and Brucella share a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution of B. abortus to pathogenicity.