Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Prevalence and Heterogeneity of Hemolysin Gene <Emphasis Type="Italic">vhh</Emphasis> Among Hatchery Isolates of <Emphasis Type="Italic">Vibrio harveyi</Emphasis> in India

Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH. The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Antioxidant and antibacterial activities of lichen <Emphasis Type="Italic">Usnea ghattensis</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Various solvent extracts of the lichen Usnea ghattensis showed good antioxidant activity. A methanol extract prevented lipid peroxidation by 87% followed by 65% in Trolox at 20 μg/ml. It also showed superoxide anion scavenging activity and free radical scavenging activity 56% and 73%, respectively. The known antioxidants butylated hydroxytoluene (BHT), butylated hydroxyanisol (BHA) and quercetin at similar concentrations showed superoxide anion scavenging activity of 68, 59 and 47% and free radical scavenging activity 83, 77 and 69%, respectively. In addition, these extracts were inhibitory against Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis and Staphylococcus aureus with MIC values of 5–10 μg/ml.Received after revisions 10 May 2005     

Effect of Organic Acids on Shrimp Pathogen, <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

Shrimp farming accounts for more than 40% of the world shrimp production. Luminous vibriosis is a shrimp disease that causes major economic losses in the shrimp industry as a result of massive shrimp kills due to infection. Some farms in the South Asia use antibiotics to control Vibrio harveyi, a responsible pathogen for luminous vibriosis. However, the antibiotic-resistant strain was found recently in many shrimp farms, which makes it necessary to develop alternative pathogen control methods. Short-chain fatty acids are metabolic products of organisms, and they have been used as food preservatives for a long time. Organic acids are also commonly added in feeds in animal husbandry, but not in aquaculture. In this study, growth inhibitory effects of short-chain fatty acids, namely formic acid, acetic acid, propionic acid, and butyric acid, on V. harveyi were investigated. Among four acids, formic acid showed the strongest inhibitory effect followed by acetic acid, propionic acid, and butyric acid. The minimum inhibitory concentration (MIC) of 0.035% formic acid suppressed growth of V. harveyi. The major inhibitory mechanism seems to be the pH effect of organic acids. The effective concentration 50 (EC₅₀) values at 96 h inoculation for all organic acids were determined to be 0.023, 0.041, 0.03, and 0.066% for formic, acetic, propionic, and butyric acid, respectively. The laboratory study results are encouraging to formulate shrimp feeds with organic acids to control vibrio infection in shrimp aquaculture farms.     

Assay-dependent effect of silver nanoparticles to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

We assess the microbial assay-dependent effect of AgNP on gram-negative Escherichia coli and gram-positive Bacillus subtilis. The experiment was conducted via three different assays: a growth inhibition assay, a colony forming unit assay, and a liquid-to-plate assay. AgNP were exposed either as liquid suspensions or in an agar state. Bacterial sensitivity to AgNP was found to be dependent on the microbial assay employed. E. coli was more sensitive than B. subtilis in the growth inhibition and CFU assays, but B. subtilis was more vulnerable than E. coli in the liquid-to-plate assay, ostensibly owing to the food stress mechanisms of B. subtilis in exposure medium. The dissolution of silver from AgNP could not explain the observed toxicity of AgNP. We detected clear evidence of AgNP uptake by cells. The results of this study showed that the microbial toxicity of AgNP and the effects of dissolved silver ions were influenced profoundly by the microbial test method employed.     

Biological control of root rot fungus <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> and growth enhancement of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> (Sarg.) by rhizosphere competent <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BN1

Bacterial isolates having antifungal and good plant growth-promoting attributes were isolated from chir-pine (Pinus roxburghii) rhizosphere. An isolate, Bacillus subtilis BN1 exhibited strong antagonistic activity against Macrophomina phaseolina, and other phytopathogens including Fusarium oxysporum and Rhizoctonia solani. It was characterized and selected for the present studies. BN1 resulted in vacuolation, hyphal squeezing, swelling, abnormal branching and lysis of mycelia. The cell-free culture filtrate of BN1 inhibited the growth of M. phaseolina. Pot trial study resulted in statistically significant increase in seedling biomass besides reduction in root rot symptoms in chir-pine seedlings. BN1 treatment resulted in 43.6% and 93.54% increases in root and shoot dry weights respectively, as compared to control. Also, 80–85% seed viability was recorded in treatments receiving BN1 either alone or in the presence of M. phaseolina, compared to 54.5% with M. phaseolina. Bioinoculant formulation study suggested that maximum viability of bacteria was in a sawdust-based carrier. B. subtilis BN1 produced lytic enzymes, chitinase and β-1,3-glucanase, which are known to cause hyphal degradation and digestion of the cell wall component of M. phaseolina. In the presence of M. phaseolina, population of B1 was 1.5 × 10⁴ c.f.u. g⁻¹ root after one month, which increased to 4.5 × 10⁴ c.f.u. g⁻¹ root in three months. Positive root colonization capability of B. subtilis BN1 proved it as a potent biocontrol agent.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">polyfermenticus</Emphasis> isolated from <Emphasis Type="Italic">Meju,</Emphasis> Korean soybean fermentation starter

Bacteria of the Bacillus species have been reported as an important microorganism in fermented soybean products. In the present study, thirty Bacillus isolates were screened from Meju, a Korean soybean fermentation starter. The comparative analysis of 16S rDNA sequences, 16S-23S internal transcribed spacer sequences, phenotypic, and biochemical characterizations revealed three phylogenetically distinct groups namely Bacillus atrophaeus, Bacillus polyfermenticus and Bacillus subtilis. The isolates were assayed for poly-γ-glutamate production and fibrinolytic activity. Among the isolates, B. polyfermenticus exhibited maximum poly-γ-glutamate production and fibrinolytic activity. Moreover, the soybean products fermented by B. polyfermenticus have increased the time taken for coagulation and hemorrhage in mice. The results of the present study clearly indicate the functional role of B. polyfermenticus in fermented soybean products.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

Efficient decomposition of shrimp shell waste using <Emphasis Type="Italic">Bacillus cereus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis>

Two bacterial cultures were isolated and tested for degradation of shrimp shell waste. According to morphological examination, physiological tests, and applied molecular techniques, isolates were identified as Bacillus cereus and Exiguobacterium acetylicum. Both strains were cultivated separately in flasks with 100 mL of shrimp shell waste broth (3% of washed, dried and ground shrimp shell waste in tap water, pH 7.0) at 37°C. At determined periods of time, deproteinization and demineralization of residuals were measured. Fermentation of 3% shell waste with B. cereus indicated 97.1% deproteinization and 95% demineralization. For E. acetylicum, the level of deproteinization and demineralization was 92.8 and 92%, respectively. Protein content was reduced from 18.7 to 5.3% with B. cereus and to 7.3% with E. acetylicum. No additional supplements were used during the fermentation of shell waste. B. cereus strain showed higher efficacy in decomposition of shell waste and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth. Incubation of bacteria with shell waste during 14 days at 37°C resulted in 78.6% deproteinization and 73% demineralization. High activity of isolated cultures in decomposition of shrimp shell waste suggests broad potential for application of these bacteria in environmentally friendly approaches to chitin extraction from chitin-rich wastes.     

An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

Root-promoting rhizobacteria in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> cuttings

Cloned Eucalyptus spp. plantations are based in greenhouse production of plants generated by vegetative propagation. Diverse studies have demonstrated that rhizospheric bacteria can stimulate plant growth, and more recently that they can increase rooting in vegetative material. Considering this potential, the objective of this study was to verify the effect of bacterial strains on rooting Eucalyptus globulus. A total of 132 bacterial strains isolated from the rhizosphere of E. globulus and Eucalyptus nitens were studied. The bacterial inoculums in a concentration of 4 × 10⁸ cfu/ml were applied to the rooting substrate at the cutting installation and 45 days after by irrigation. Rooting was evaluated on days 60 and 75 after cutting installation, considering the number of roots as well as their fibrosity and roots biomass. Of the 132 strains evaluated, 26 significantly increased cutting rooting in a range of 191.4–69.4% with respect to the control. Additionally, some strains stimulated the development of fine roots and incremented the roots biomass. The strains identificated that produced a rooting effect were: Bacillus firmus, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, B. subtilis/amyloliquefaciens, Bacillus circulans, Brevibacillus brevis, Paenibacillus lautus and Stenotrophomona maltophilia. These first trials suggest the potential of these bacteria to be used in clonal production programs for E. globulus.     

Phenotypic diversity and technological properties of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> species isolated from <Emphasis Type="Italic">okpehe</Emphasis>, a traditional fermented condiment

The Bacillus subtilis wild strains isolated from okpehe, a traditional fermented condiment used as seasoning in Nigeria, the reference and typed strains were investigated for their phenotypic diversity and their technological parameters with a view to obtain adequate data that would enable selection of appropriated starter cultures for vegetable protein fermentation in West Africa. All the 7 strains studied demonstrated diverse phenotypic characteristics and they were identified as Bacillus subtilis, based on the API 50 CHB combined with API 20E profile. Specific sugars that indicated a good hydrolytic potential of the wild strains were fermented. The highest proteinase activity of 90 AU/ml determined quantitatively was observed in the strain Bacillus subtilis BFE 5372, the proteinase was identified by the APIZYM gallery as chymotrypsin. Highest amylase activity of 13 AU/ml was noticed in strain Bacillus subtilis DSM 347 while only 4 strains produced polyglutamic acid with the strain Bacillus subtilis BFE 5359 producing the highest polyglutamate activity of 2.5 mm. Although strain Bacillus subtilis BFE 5301 did not release detectable polyglutamate, the strain demonstrated antagonism against different bacteria and the antimicrobial substance produced by strain Bacillus subtilis BFE 5301 was confirmed as a bacteriocin since its activities were lost after treatment with chymotrypsin and pepsin. The data generated showed the technological parameters that can aid selection of wild strains such as Bacillus subtilis BFE 5301, BFE 5359 and BFE 5372 for optimization of condiment production.     

Synergistic effect of surfactin from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C4 and <Emphasis Type="Italic">Achyrocline satureioides</Emphasis> extracts on the viability of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 μg ml⁻¹ and the HE concentration from 32 to 4 μg ml⁻¹, values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.     

Antimicrobial Activity and Biodiversity of Endophytic Fungi in <Emphasis Type="Italic">Dendrobium</Emphasis><Emphasis Type="Italic">devonianum</Emphasis> and <Emphasis Type="Italic">Dendrobium thyrsiflorum</Emphasis> from Vietman

Endophytic fungi are rich in orchids and have great impacts on their host plants. 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) were isolated, respectively, from roots and stems of Dendrobium species. All the fungi were identified by way of morphological and/or molecular biological methods. 30 endophytic fungi in D. devonianum were categorized into 11 taxa and 23 fungal endophytes in D. thyrsiflorum were grouped into 11 genera, respectively. Fusarium was the dominant species of the two Dendrobium species in common. Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6 pathogenic microbes (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma displayed strong inhibitory activity (inhibition zones in diameter >20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens. The study reinforced the assumption that endophytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.     

Purification and characterization of a potential antifungal protein from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> E1R-J against <Emphasis Type="Italic">Valsa mali</Emphasis>

In order to identify the antagonistic substances produced by Bacillus subtilis E1R-J as candidate of biocontrol agents for controlling Apple Valsa Canker, hydrochloric acid precipitation, reverse phase chromatography, gel filtration, and ion exchange chromatography were used. The purified fraction EP-2 showed a single band in native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fraction EP-2 was eluted from native-PAGE and showed a clear inhibition zone against V. mali 03-8. These results prove that EP-2 is one of the most important antifungal substances produced by B. subtilis E1R-J in fermentation broth. SDS-PAGE and Nano-LC–ESI–MS/MS analysis results demonstrated that EP-2 was likely an antifungal peptide (trA0A086WXP9), with a relative molecular mass of 12.44 kDa and isoelectric point of 9.94. The examination of antagonistic mechanism under SEM and TEM showed that EP-2 appeared to inhibit Valsa mali 03-8 by causing hyphal swelling, distortion, abnormality and protoplasts extravasation. Inhibition spectrum results showed that antifungal protein EP-2 had significantly inhibition on sixteen kinds of plant pathogenic fungi. The stability test results showed that protein EP-2 was stable with antifungal activity at temperatures as high as 100 °C for 30 min and in pH values ranging from 1.0 to 8.0, or incubated with each 5 mM Cu²⁺, Zn²⁺, Mg²⁺, or K⁺. However, the antifungal activity was negatively affected by Proteinase K treatment.     

Serotyping of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolates,their distribution in different Jordanian habitats and pathogenicity in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The investigation of Bacillus thuringiensisin 40 different samples collected from 12 different Jordanian habitats involved the isolation of 80 Bacillus thuringiensis isolates. Out of these isolates, 47 were pathogenic to the third instar larvae of Drosophila melanogaster. The highest viable count of Bacillus thuringiensis was estimated among soil samples contaminated with decomposed animal bodies (14.25 × 10⁷ c.f.u./g), and the lowest viable count was obtained from soils contaminated with engine oil (0.17 × 10⁷c.f.u./g). Serotyping of the 80 isolates against 55 antisera indicated the presence of 13 serotypes, 12 were identical or cross-reacted withaizawai, higo,israelensis, kenyae, kumamotoensis, kurstaki, malaysiensis, morrisoni, pakistani,sooncheon,tohokuensis, andthuringiensis, whereas the remaining one reacted negatively with the 55 tested antisera indicating the presence of an unknown serotype. Israelensis was the dominant serotype among all the samples except those from decomposed animal and olive-cultivated soils. The pathogenic isolates were found to be in 11 of the 13 serotypes. Spherical parasporal crystals were the most common and toxic crystal types.     

Heterospecific Expression of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cell Shape Determination Genes <Emphasis Type="Italic">mreBCD</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>*

The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD. TnphoA mutagenesis was utilized to analyze a topological model for MreC. MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane. Expression of the B. subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E. coli. Immunolocalization of the MreC protein in B. subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis. RID= ID= <E5>Correspondence to: </E5>G.C. Stewart; <E5>email:</E5> stewart&commat;vet.ksu.edu Received: 5 August 2002 / Accepted: 7 October 2002