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Influence of a Bacillus sp. on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to PEG-induced drought stress.

The effects of bacterial inoculation (Bacillus sp.) on the development and physiology of the symbiosis between lettuce and the arbuscular mycorrhizal (AM) fungi Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe and Glomus intraradices (Schenck and Smith) were investigated. Plant growth, mineral nutrition and gas-exchange values in response to bacterial inoculation after PEG-induced drought stress were also evaluated. In AM plants, inoculation with Bacillus sp. enhanced fungal development and metabolism, measured as succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities, more than plant growth. Under non-stressed conditions, G. intraradices colonization increased all plant physiological values to a higher extent when in dual inoculation with the bacterium. Under stress conditions, the bacterium had an important stimulatory effect on G. intraradices development. Under such conditions, the effects of the bacterium on photosynthetic rate, water use efficiency (WUE) and stomatal conductance of lettuce plants differed with the fungus species. Plant-gas exchange was enhanced in G. intraradices- and reduced in G. mosseae-colonized plants when co-inoculated with Bacillus sp. Thus, the effects of each fungus on plant physiology were modulated by the bacterium. Stress was detrimental, particularly in G. intraradices-colonized plants without the bacterium, reducing intra and extraradical mycelium growth and vitality (SDH), as well as plant-gas exchange. Nevertheless, Bacillus sp. inoculation improved all these plant and fungal parameters to the same level as in non-stressed plants. The highest amount of alive and active AM mycelium for both fungi was obtained after co-inoculation with Bacillus sp. These results suggest that selected free-living bacteria and AM fungi should be co-inoculated to optimize the formation and functioning of the AM symbiosis in both normal and adverse environments.

Bacillus↗

Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH.

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA-DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment.

Alkalies↗

A catalase-peroxidase from a newly isolated thermoalkaliphilic Bacillus sp. with potential for the treatment of textile bleaching effluents.

A new thermoalkaliphilic bacterium was isolated from a textile wastewater drain and identified as a new Bacillus sp. (Bacillus SF). Because of its high pH stability and thermostability, a catalase-peroxidase (CP) from this strain has potential for the treatment of textile bleaching effluents. The CP from Bacillus SF was purified to more than 70.3-fold homogeneity using fractionated ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel-filtration chromatography. The native CP had a molecular mass of 165 kDa and was composed of two identical subunits. The isoelectric point of the protein was at pH 6.0. Peptide mass mapping using matrix-assisted laser desorption ionization-mass spectrometry showed a homology between the CP from Bacillus SF and the CP from Bacillus stearothermophilus. The apparent Km value of the catalase activity for H2O2 was 2.6 mM and the k(cat) value was 11,475 s(-1). The enzyme showed high catalase activity and an appreciable peroxidase activity with guaiacol and o-dianisidine. The enzyme was stable at high pH, with a half-life of 104 h at pH 10 and 25 degrees C and 14 h at 50 degrees C. The enzyme was inhibited by azide and cyanide, in a competitive manner, but not by the catalase-specific inhibitor 3-amino-1,2,4-triazole.

Bacillus↗

Evaluation of Indigenous Bacillus Strains from Asian Fermented Foods for Probiotic Properties.

Bacillus species hold particular importance due to their versatile enzymatic repertoire and ability to synthesize diverse bioactive metabolites. In this study, two fermented food-derived strains, Bacillus siamensis BB3 (from douchi) and Bacillus velezensis TMA10 (from tapai) were evaluated for their probiotic, anti-microbial and functional potentials. Both strains exhibited desirable probiotic characteristics, including desirable tolerance to simulated gastric and intestinal conditions, with BB3 showing greater acid tolerance than TMA10. Safety assessments confirmed the absence of hemolytic activity, virulence factors and antibiotic resistance genes. Whole genome sequencing showed that the strains harbored genomic sequences for a wide range of metabolites, including non-ribosomal peptides and polyketides. Ethyl acetate (EtOAc) extracts from both strains demonstrated broad-spectrum anti-microbial activity against several indicator microorganisms, including Listeria grayi, Bacillus cereus, Serratia marcescens, Escherichia coli, and Pseudomonas aeruginosa, with TMA10 additionally inhibiting MRSA and Streptococcus mutans. Liquid Chromatography-Mass Spectrometry profiling identified key anti-microbial compounds, namely surfactins, macrolactins, bacillaene from BB3 and TMA10. In addition, difficidins were also detected from TMA10. Genomic analysis further indicated diverse carbohydrate utilization capacities; both strains encoded pathways for sucrose, raffinose-family oligosaccharides and lactose metabolism, while TMA10 possessed pathways for trehalose, glucomannan and arabinoxylan degradation. Both strains also showed anti-oxidant activity, with enhanced effects observed in their cell-free supernatants and heat-killed preparations. Overall, these findings highlight BB3 and TMA10 as promising candidates for the development of fermented food-derived Bacillus probiotics and functional cultures with anti-microbial, anti-oxidant, and broad carbohydrate-utilization capabilities.

Bacillus↗

Postoperative neurosurgical infections due to bacillus species.

The cases of 2 patients with postoperative ventriculitis due to Bacillus species bacteria are presented. Bacillus licheniformis was isolated from one patient following removal of an intraventricular meningioma, and Bacillus cereus from another patient following placement of a ventriculoperitoneal shunt. Both isolates were resistant to a variety of antibiotics, but both were sensitive to gentamicin and chloramphenicol. These cases emphasize several points; (a) Bacillus species, usually thought to be nonpathogenic, may produce intracranial infections; (2) species identification is important for epidemiological purposes and for the selection of appropriate chemotherapeutic agents; and (3) in cases of suspected ventriculitis, chloramphenicol or gentamicin should be considered for Gram's staining revealing gram-positive bacilli. In addition, we recommend that when planning antibiotic prophylactic regimens, consideration should be given to including one of these agents to assure coverage of Bacillus species.

Adult↗

Discrepancy between in vitro and in vivo assays for the susceptibility of Bacillus C.I.P. 5832 to antimicrobial agents.

In vitro, avoparcin, flavomycin, virginiamycin bacitracin, trimethoprim + sulfadimethoxine, colistin, spiramycin, oxolinic acid, lincomycin + spectinomycin and a mixture of organic acids were able to inhibit Bacillus C.I.P. 5832. In vivo, none of these antibacterial agents, at the levels incorporated in feeds, could affect the viable bacillus count in the caecal content of chickens. In another in vitro assay, olaquindox, carbadox and tylosin were also effective in inhibiting Bacillus C.I.P. 5832. But, concomitantly administered with carbadox or tylosin, Bacillus C.I.P. 5832 still promoted growth and feed conversion of fattening pigs. These results, as well as data of the quoted literature, suggest that the current in vitro techniques should not be used to screen the susceptibility of Bacillus C.I.P. 5832 to antimicrobial agents. Only in vivo assays seem to be of value.

Animals↗

Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp.

A newly acquired polyhydroxyalkanoate (PHA) producing Bacillus spp. was identified to be a strain of Bacillus cereus using a range of microbiological and molecular techniques. This strain, named B. cereus SPV, was found to be capable of using a wide range of carbon sources including glucose, fructose, sucrose, various fatty acids and gluconate for the production of PHAs, an advantage for the commercial production of the polymers. The media used for the polymer production was novel in the context of the genus Bacillus. The PHA, once produced, was found to remain at a constant maximal concentration, without any degradation, a great advantage for the commercial production of the PHAs. This particular strain of Bacillus spp. was able to synthesize various PHAs with 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB)-like monomer units from structurally unrelated carbon sources such as fructose, sucrose and gluconate. This is the first report of the incorporation of a 4HB related monomer containing PHA by the genus Bacillus and from structurally unrelated carbon sources. The PHAs isolated had molecular weights ranging between (0.4 and 0.8) x 10(6) and low polydispersity index values (M(W)/M(N)) ranging from 2.6 to 3.4.

Bacillus cereus↗

Bacillus spp. among hospitalized patients with haematological malignancies: clinical features, epidemics and outcomes.

Between April 2000 and May 2005, 350 bacteraemic episodes occurred among patients treated in our haematology unit. Two hundred and twenty-eight of these episodes were caused by Gram-positive pathogens, most commonly coagulase-negative staphylococci and Staphylococcus aureus. One hundred and twenty-two episodes were due to Gram-negative pathogens, with a predominance of Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa. Bacillus bacteraemias constituted 12 of these episodes occurring in 12 patients, and accounted for 3.4% of all bacteraemic episodes. Of the 12 strains evaluated, seven were Bacillus licheniformis, three were Bacillus cereus and two were Bacillus pumilus. Seven episodes presented with bloodstream infection, three with pneumonia, one with severe abdominal pain and deterioration of liver function, and one with a catheter-related bloodstream infection. B. licheniformis was isolated from five patients who had been hospitalized at the same time. This outbreak was related to non-sterile cotton wool used during skin disinfection. B. cereus and B. licheniformis isolates were susceptible to cefepime, carbapenems, aminoglycosides and vancomycin, but B. pumilus isolates were resistant to all antibiotics except for quinolones and vancomycin. Two deaths were observed. In conclusion, Bacillus spp. may cause serious infections, diagnostic and therapeutic dilemmas, and high morbidity and mortality in patients with haematological malignancies. Both B. cereus and B. licheniformis may be among the 'new' Gram-positive pathogens to cause serious infection in patients with neutropenia.

Acinetobacter baumannii↗

Undetected Bacillus pseudo-outbreak after renovation work in a teaching hospital.

A 602-bed capacity hospital underwent complete renovation from 1999 to 2004. In April 2005, the Infection Control Team was informed of the occurrence of three consecutive cases of Bacillus cereus bacteremia in a ward for patients with hematologic malignancies. A retrospective analysis of patients with Bacillus isolates was initiated. We found more Bacillus cereus isolates from blood samples in 2004 compare to the preceding years. Swab samples were collected in the particular ward from the surface of a working desk, filter unit of the air-conditioners, entrance of air inlet ducts, exit of the air outlet ducts and three-way valves of the particular ward under the consideration of iatrogenic contamination. Towels and gowns used in the ward were examined. Dens dust was noted in the filter of the air-conditioner and inlets/outlets of the air-ventilation system of the ward. Bacillus cereus was isolated from the dust, and from cleaned towels and gowns. PFGE fingerprinting differed among four patients' sample. We considered the present case as an undetected Bacillus cereus pseudo-outbreak that lasted for about one year after the renovation work of the hospital. We also considered that filters of the HVAC-system and towels and gowns were probable sources of the outbreak.

Bacillus cereus↗

Development of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance.

A group-specific primer pair was designed to amplify the 16S rRNA gene of representative reference strains from environmentally sourced, mesophilic aerobic spore-forming Bacillus taxa. The PCR generated a 1114 bp amplicon but did not do so with DNA extracted from 16 other Eubacterial species. When amplicons were digested with restriction enzymes AluI or TaqI, different profiles containing between 2 and 5 fragments ranging in size from 76 to 804 base pairs were seen with different Bacillus species. This procedure, known otherwise as amplified ribosomal DNA restriction analysis or ARDRA, produced unique and distinguishable patterns to differentiate between 15 ATCC reference strains (10 Bacillus, 3 Paenibacillus and 2 Brevibacillus member species) as well as 3 misidentified Bacillus probiotic strains in a commercial collection. Our simplified PCR-ARDRA protocol provides a facile method for the identification of most environmentally important species of Bacillus.

Animals↗

Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters.

The production of the type I antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner [Kleerebezem M, de Vos WM, Kuipers OP. The lantibiotics nisin and subtilin act as extracellular regulators of their own biosynthesis. In: Dunny GM, Winans SC, editors. Cell-cell signaling in bacteria. Washington, D.C., USA: ASM Press; 1999. p. 159-74; Stein T, Borchert S, Kiesau P, Heinzmann S, Kloss S, Klein C, Helfrich M, Entian KD. Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2002;44:403-16; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Three subtilin-responsive promoter elements within the spaBTCSIFEGRK are controlled by the specific cis-acting sequence element called the spa-box, which represents the binding site of the subtilin regulator SpaR [Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Here, we describe the functional characterization of the spaB, spaS and spaI promoters by transcriptional fusion with a promoterless beta-glucuronidase encoding gusA gene. Within these gusA fusion constructs, transcription initiation start sites of the spaS and spaI promoters were mapped to be located downstream of the spa-box, which is in contrast to previous reports [Banerjee S, Hansen JN. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J Biol Chem 1988;263:9508-14; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Nevertheless, all spa-promoters displayed typical cell-density-dependent activity in a subtilin-producing strain B. subtilis ATCC6633. Moreover, analysis of beta-glucuronidase activities in a spaB mutant of B. subtilis ATCC6633 and a derivative of strain 168 that harbors the spaRK genes integrated in the chromosomal amyE locus, confirmed that these promoters are activated by subtilin-triggered, SpaRK-mediated, quorum-sensing control. Quantitative analysis showed that the spaS promoter strength at a given subtilin concentration appeared to be approximately five-fold higher than the spaB promoter, which in turn is approximately two-fold higher than the spaI promoter. Finally, it is shown that the elementary components involved in subtilin-mediated regulation are the two-component system, SpaRK, and a spa-box containing promoter.

Anti-Bacterial Agents↗

Supplementing Bacillus sp. RS1 with Dechloromonas sp. HZ for enhancing selenate reduction in agricultural drainage water.

Cost and efficiency are two important factors considered in the remediation of Se-contaminated agricultural drainage water through bacterial reduction of soluble Se(VI) to insoluble Se(0). Bacillus sp. RS1 isolated from rice straw was assessed for its ability to use inexpensive molasses to reduce Se(VI) in agricultural drainage water containing NO3- levels of 0, 50, 100, 250, and 500 mg/L. The results showed that Se(VI) (1000 microg/L) was almost entirely reduced to Se(IV) (62.7%) and Se(0) (36.4%) by Bacillus sp. RS1 in synthetic agricultural drainage (SAD) water without the presence of NO3-. The reduction Se(VI) to Se(0) was limited in the SAD water with NO3- levels of 100, 250, and 500 mg/L. The addition of Dechloromonas sp., a NO3- reducer, to the SAD water not only increased NO3- removal, but also enhanced Se(VI) reduction by Bacillus sp. RS1. During an 8-day experiment, 98-99% of the added Se(VI) was reduced to Se(0) with small amounts of Se(IV) and Se(-II) in the SAD water containing 100 and 250 mg/L NO3-. The addition of Dechloromonas sp. HZ to the natural agricultural drainage water also significantly increased the reduction of Se(VI) (748 microg/L) by Bacillus sp. RS1, with a production of Se(0) (65%) and Se(-II) (32%). These results suggest that a combination of Bacillus sp. RS1 with Dechloromonas sp. HZ has great potential with the use of inexpensive molasses to remediate Se-contaminated agricultural drainage water containing relatively high NO3- levels.

Agriculture↗

Bacillus nematocida sp. nov., a novel bacterial strain with nematotoxic activity isolated from soil in Yunnan, China.

An endospore-forming bacterium, designated strain B-16T, was isolated from a forest soil sample in Yunnan, China. The isolate presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The organism was strictly aerobic, motile, spore forming and rod shaped, catalase- and oxidase-positive. The predominant isoprenoid quinone was menaquinone 7 (MK-7). The major cellular fatty acid profiles were anteiso-C15:0 (48.67%), iso-C15:0 (13.45%), C16:0 (9.06%) and anteiso-Cl7:0 (8.29%). The DNA G+C content was 46%. Phylogenetic analyses based on 16S rDNA sequence revealed that isolate belongs to the genus Bacillus. Strain B-16T exhibited high 16S rDNA similarity with its closest neighbors Bacillus vallismortis (99.79%), B. subtilis (99.43%), B. atrophaeus (99.43%), B. amyloliquefaciens (99.36%), B. licheniformis (98.0%) and less than 97.0% with all the other relative type strains in the genus Bacillus. The phenotypic and genotypic characteristics and DNA-DNA relatedness data indicate that strain B-16T should be distinguished from all the relative species of genus Bacillus. Therefore, on the basis of the polyphasic taxonomic data presented, a new species of the genus Bacillus, B. nematocida, with the type strain B-16T ( = CGMCC 1128T) is proposed. The GenBank accession number for the sequence reported in this paper is AY820954.

Aerobiosis↗

Association of cilia-associated respiratory (CAR) bacillus with natural chronic tracheitis in goats.

A histological, histochemical and immunohistological study of the respiratory tract of 83 slaughtered goats (50 adults and 33 kids) is described. Cilia-associated respiratory (CAR) bacillus was detected by means of the Warthin Starry method in the tracheal epithelium of seven (21.2%) of the kids and 16 (32%) of the adult goats. A chronic diffuse tracheitis characterized by mixed lymphocyte and plasma-cell infiltration was found in all seven kids and in 17 adults, including the 16 infected with the CAR bacillus. Although not proved, it is possible that the CAR bacillus caused the chronic tracheitis. Immunohistochemical results suggested that the caprine CAR bacillus was closely related to the rabbit CAR bacillus.

Animals↗

Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay.

Thirty mercury-resistant (Hg R) Bacillus strains were isolated from mercury-polluted sediment of Minamata Bay, Japan. Mercury resistance phenotypes were classified into broad-spectrum (resistant to inorganic Hg(2+) and organomercurials) and narrow-spectrum (resistant to inorganic Hg(2+) and sensitive to organomercurials) groups. Polymerase chain reaction (PCR) product sizes and the restriction nuclease site maps of mer operon regions from all broad-spectrum Hg R Bacillus were identical to that of Bacillus megaterium MB1. On the other hand, the PCR products of the targeted merP (extracellular mercury-binding protein gene) and merA (intracellular mercury reductase protein gene) regions from the narrow-spectrum Hg R Bacillus were generally smaller than those of the B. megaterium MB1 mer determinant. Diversity of gene structure configurations was also observed by restriction fragment length polymorphism (RFLP) profiles of the merA PCR products from the narrow-spectrum Hg R Bacillus. The genetic diversity of narrow-spectrum mer operons was greater than that of broad-spectrum ones.

Bacillus↗

"Bacillus hackensackii" sp. nov., a novel carbon dioxide sensitive bacterium isolated from blood culture.

An endospore-forming, gram-positive bacillus was isolated from a patient's blood culture. This bacillus did not grow in the presence of 5% carbon dioxide although it grew well in ambient air at 37 degrees C. Although the organism thus is an aerobic bacterium, its sensitivity to increased carbon dioxide concentration places it in a distinct category of gaseous atmospheric requirement: capnophobic. Based on its morphology, growth characteristics, biochemical reactions and a complete 16S rRNA gene nucleotide sequence analysis, this microorganism represents a novel Bacillus species. The clinical significance of this isolate is unknown. It is proposed that the bacterium be classified in the genus Bacillus as "Bacillus hackensackii".

Adult↗

Variation in rRNA operon number as revealed by ribotyping of Bacillus anthracis strains.

Ribotyping of various Bacillus strains with one restriction enzyme (AccI) revealed significant similarity between Bacillus anthracis strains, Bacillus thuringiensis and Bacillus cereus strains, which are all members of the Bacillus cereus group. A further ribotyping study of 10 virulent and 8 attenuated B. anthracis strains, using 4 endonucleases and both 23S and 16S probes independently, was performed. The discrimination index D of Hunter and Gaston showed that the best combination for future large-scale ribotyping studies would be either the combination of AccI and 23S, or that of EcoRI and 16S. Depending on the B. anthracis strain analyzed 10 or 11 rRNA operons were found. In all cases, many strains were grouped into 2 to 3 patterns. Attenuated strains, including a laboratory-cured strain, yielded aberrant patterns.

Bacillus anthracis↗

Three clusters of Bacillus pseudobacteremia related to a radiometric blood culture analyzer.

During a ten-month period from September 1981 to July 1982 three episodes of pseudobacteremia due to Bacillus species occurred at this 550-bed institution. The first involved eight isolates, the second 11, and the third seven isolates of the organism, all with the same antibiogram. The patients involved did not exhibit clinical signs of septicemia, and in only one case was more than one specimen per patient positive when multiple blood samples were obtained. Occasional blood cultures of Bacillus species identified in between clusters revealed a different antibiogram. Extensive epidemiologic investigation of patient locations, phlebotomists, and time of cultures yielded no common source. Components involved in the transport and processing of blood cultures, including the radiometric blood culture processor, were also sampled but without recovery of the organism. After the last episode, a layer of dust was noted inside the machine, and culture of this dust grew Bacillus spp. with the same antibiogram as those found in the blood cultures. The filter from an air conditioning unit in close proximity to the machine grew several species of Bacillus. It is presumed that Bacillus spores in the dust were introduced into the blood culture bottles following the heat sterilization of the gas sampling (inoculation/removal) needles. Modification of the cover of the machine was undertaken to prevent access of dust bearing microbes to the inside of the machine. In addition, maintenance now includes regular disinfection/cleaning of the "floor" of the machine, and more frequent changes of the air conditioner filter.

Autoanalysis↗