A comparative GC-MS analysis of bioactive secondary metabolites produced by halotolerant Bacillus spp. isolated from the Great Sebkha of Oran.

The reemergence of infectious diseases and resistant pathogens represents a serious problem for human life. Hence, the screening for new or alternative antimicrobial compounds is still urgent. Unusual ecosystems such as saline habitats are considered promising environments for the purposes of isolating bacterial strains able to produce potent natural products. The aim of this study is the identification of bioactive compounds biosynthesized by three halotolerant strains isolated from the Sebkha of Oran (Algeria) using gas chromatography coupled to mass spectrometry. Primary screening investigation of antimicrobial activities were performed against reference bacterial and fungal strains and revealed a broad-spectrum activity. Secondary metabolite extraction was carried out using ethyl acetate and chloroform. Crude extracts were tested for bioactivity using the disc diffusion method and subjected to GC-MS analysis. The extracts showed an important inhibitory effect against all tested strains. Fifty-six compounds were identified; they include tert-butyl phenol compounds, fatty acid methyl esters due to the methylation procedure, hydrocarbons, aldehydes, benzoquinones, pyrrols, and terpenes. Literature reports such compounds to have wide biological and pharmaceutical applications. The molecular identification of the three isolates was achieved using the 16S-23S rRNA gene intergenic spacer region (ITS) and 16S rRNA sequencing. Partial 16S rRNA gene sequencing showed very high similarity with many species of Bacillus. This study provided insights on the potential of halotolerant Bacillus as drug research target for bioactive metabolites. The findings suggest that the Great Sebkha of Oran is a valuable source of strains exhibiting variety of beneficial attributes that can be utilized in the development of biological antibiotics.

Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix.

Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm2) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.

Identification and Characterization of a Newly Isolated Chitinase-Producing Strain Bacillus licheniformis SSCL-10 for Chitin Degradation.

Chitinases or chitinolytic enzymes have different applications in the field of medicine, agriculture, and industry. The present study is aimed at developing an effective hyperchitinase-producing mutant strain of novel Bacillus licheniformis. A simple and rapid methodology was used for screening potential chitinolytic microbiota by chemical mutagenesis with ethylmethane sulfonate and irradiation with UV. There were 16 mutant strains exhibiting chitinase activity. Out of the chitinase-producing strains, the strain with maximum chitinase activity was selected, the protein was partially purified by SDS-PAGE, and the strain was identified as Bacillus licheniformis (SSCL-10) with the highest specific activity of 3.4 U/mL. The induced mutation model has been successfully implemented in the mutant EMS-13 (20.2 U/mL) that produces 5-6-fold higher yield of chitinase, whereas the mutant UV-11 (13.3 U/mL) has 3-4-fold greater chitinase activity compared to the wild strain. The partially purified chitinase has a molecular weight of 66 kDa. The wild strain (SSCL-10) was identified as Bacillus licheniformis using 16S rRNA sequence analysis. This study explores the potential applications of hyperchitinase-producing bacteria in recycling and processing chitin wastes from crustaceans and shrimp, thereby adding value to the crustacean industry.

Engineering a newly isolated Bacillus licheniformis strain for the production of (2R,3R)-butanediol.

Several microorganisms can produce 2,3-butanediol (BDO), an industrially promising chemical. In this study, a Bacillus licheniformis named as 4071, was isolated from soil sample. It is a GRAS (generally recognized as safe) strain and could over-produce 2,3-BDO. Due to its mucoid forming characteristics, UV-random mutagenesis was carried out to obtain a mucoid-free strain, 4071-15. As a result, capabilities of 4071-15 strain in terms of transformation efficiency of bacillus plasmids (pC194, pUB110, and pUCB129) and fermentation performance were highly upgraded compared to those of the parent strain. In particular, 4071-15 strain could produce 123 g/L of 2,3-BDO in a fed-batch fermentation in which the ratio of (2R,3S)- to (2R,3R)-form isomers was 1:1. To increase the selectivity of (2R,3R)-BDO, budC gene was deleted by using temperature-sensitive gene deletion process via homologous recombination. The 4071-15 △budC mutant strain dramatically increased selectivity of (2R,3R)-BDO to 91% [96.3 g/L of (2R,3R)-BDO and 9.33 g/L of (2R,3S)-BDO], which was 43% higher than that obtained by the parent strain. This study has shown the potential of an isolate for 2,3-BDO production, and that the ratio of 2,3-BDO can be controlled by genetic engineering depending on its industrial usage.

Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm.

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.

Isolation of a novel poly-γ-glutamic acid-producing Bacillus licheniformis A14 strain and optimization of fermentation conditions for high-level production.

In the present study, bacteria producing poly-γ-glutamic acid were isolated from marine sands, and an efficient producer identified. γ-PGA was rapidly screened by thin-layer chromatography and UV spectrophotometer assay. Media optimization was carried out, and for the cost-effective production of γ-PGA, monosodium glutamate was used as the substrate for the synthesis of γ-PGA instead of glutamic acid. Lastly, Plackett-Buman design (PB) and Response surface methodology (RSM) were used to determine significant media components and their interaction effect to achieve maximum γ-PGA production. With this integrated method, a bacterial strain with a high yield of γ-PGA was obtained rapidly, and the production was increased up to 37.8 g/L after optimization.

Molecular regulation of adhesion and biofilm formation in high and low biofilm producers of Bacillus licheniformis using RNA-Seq.

RNA sequencing was used to reveal transcriptional changes during the motile-to-sessile switch in high and low biofilm-forming dairy strains of B. licheniformis isolated from Chinese milk powders. A significant part of the whole gene content was affected during this transition in both strains. In terms of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, seven metabolic pathways were significantly downregulated in the planktonic state compared to the biofilm state in both strains. Lipid and sugar metabolism seemed to play an important role in matrix production. Several genes involved in adhesion, matrix production and the matrix coating were either absent or less expressed in the biofilm state of the low biofilm producer compared to the high biofilm producer. Genes related to sporulation and the production of extracellular polymeric substances were concomitantly expressed in the biofilm state of both strains. These comprehensive insights will be helpful for future research into mechanisms and targets.

Characterization of bacteria and yeasts isolated from traditional fermentation starter (Fen-Daqu) through a 1H NMR-based metabolomics approach.

Daqu is a traditional fermentation starter for the production of baijiu and vinegar. It is an important saccharifying and fermenting agent associated with alcoholic fermentation and also a determining factor for the flavour development of these products. Bacterial and yeast isolates from a traditional fermentation starter (Fen-Daqu) were examined for their amylolytic activity, ethanol tolerance and metabolite production during sorghum-based laboratory-scale alcoholic fermentation. The selected strains (Bacillus licheniformis, Pediococcus pentosaceus, Lactobacillus plantarum, Pichia kudriavzevii, Wickerhamomyces anomalus, Saccharomyces cerevisiae, and Saccharomycopsis fibuligera) were blended in different combinations, omitting one particular strain in each mixture. 1H nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical analysis was used to investigate the influence of the selected strains on the metabolic changes observed under the different laboratory-controlled fermentation conditions. Principal component analysis showed differences in the metabolites produced by different mixtures of pure cultures. S. cerevisiae was found to be superior to other species with respect to ethanol production. S. fibuligera and B. licheniformis converted starch or polysaccharides to soluble sugars. Lactic acid bacteria had high amylolytic and proteolytic activities, thereby contributing to increased saccharification and protein degradation. W. anomalus was found to have a positive effect on the flavour of the Daqu-derived product. This study highlights the specific functions of S. cerevisiae, S. fibuligera, B. licheniformis, W. anomalus and lactic acid bacteria in the production of light-flavour baijiu (fen-jiu). Our results show that all investigated species deliver an important contribution to the functionality of the fermentation starter Daqu.

Multiplex PCR identification and culture-independent quantification of Bacillus licheniformis by qPCR using specific DNA markers.

Probiotics benefits in fish farming have been usually inferred appraising the effects observed on the host and not through the direct assessment of probiotic dynamics in the host gut microbiota. To overcome this gap, quantitative PCR (qPCR) can be a powerful approach to study the bacterial dynamics in fish gut microbiota. The presented work proposes four B. licheniformis-specific DNA markers and details a qPCR method to track putative probiotics B. licheniformis on fish gut. The four B. licheniformis-specific DNA markers - BL5B (hypothetical protein BL00303), BL8A (serA2), BL13C (rfaB) and BL18A (ligD) - were selected and validated by PCR and multiplex-PCR with 20 B. licheniformis isolates and a broad range of non-target bacteria. To assess the dynamics of B. licheniformis in the digesta of farmed fish, a qPCR was validated using markers BL8A and BL18A and calibration curves obtained for both markers with digesta samples spiked with B. licheniformis cells showed a high correlation (R2 > 0.99) over 6 log units (CFU/reaction), and a limit of detection (LOD) as low as 247 CFUs/reaction. Furthermore, the consistent qPCR repeatability and reproducibility underline the specificity and reliability of the qPCR proposed. Ultimately, the possibility to monitor the dynamics of B. licheniformis probiotics in the gut microbiota of farmed fish might be instrumental to optimize best practices in aquaculture.

Exploring potential applications of a novel extracellular polymeric substance synthesizing bacterium (Bacillus licheniformis) isolated from gut contents of earthworm (Metaphire posthuma) in environmental remediation.

The aim was to isolate, characterize, and explore potentials of gut bacteria from the earthworm (Metaphire posthuma) and imply these bacteria for remediation of Cu(II) and Zn(II). An extracellular polymeric substance (EPS) producing gut bacteria (Bacillus licheniformis strain KX657843) was isolated and identified based on 16S rRNA sequencing and phylogenetic analysis. The strain showed maximum tolerance of 8 and 6 mM for Cu(II) and Zn(II) respectively. It removed 34.5% of Cu(II) and 54.4% of Zn(II) at 25 mg L-1 after 72 and 96 h incubation respectively. The bacteria possessed a great potential to produce indole acetic acid (38.49 µg mL-1) at 5 mg mL-1 L-tryptophan following 12 days incubation. The sterilized seeds of mung beans (Vigna radiata) displayed greater germination and growth under bacterium enriched condition. We observed that the bacterial strain phosphate solubilization ability with a maximum of 204.2 mg L-1 in absence of Cu(II) and Zn(II). Endowed with biosurfactant property the bacterium exhibited 24% emulsification index. The bacterium offered significant potential of plant growth promotion, Cu(II) and Zn(II) removal, and as such this study is the first report on EPS producing B. licheniformis KX657843 from earthworm which can be applied as powerful tool in remediation programs of Cu(II) and Zn(II) contaminated sites.

Biodegradation of chlorpropham and its major products by Bacillus licheniformis NKC-1.

Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] (CIPC), an important phenyl carbamate herbicide, has been used as a plant growth regulator and potato sprout suppressant (Solanum tuberosum L) during long-term storage. A bacterium capable of utilizing the residual herbicide CIPC as a sole source of carbon and energy was isolated from herbicide-contaminated soil samples employing selective enrichment method. The isolated bacterial strain was identified as Bacillus licheniformis NKC-1 on the basis of its morphological, cultural, biochemical characteristics and also by phylogenetic analysis based on 16S rRNA gene sequences. The organism degraded CIPC through its initial hydrolysis by CIPC hydrolase enzyme to yield 3-chloroaniline (3-CA) as a major metabolic product. An inducible 3-CA dioxygenase not only catalyzes the incorporation of molecular oxygen but also removes the amino group by the deamination yielding a monochlorinated catechol. Further, degradation of 4-chlorocatechol proceeded via ortho- ring cleavage through the maleylacetate process. 3-Chloroaniline and 4-chlorocatechol are the intermediates in the CIPC degradation which suggested that dechlorination had occurred after the aromatic ring cleavage. The presence of these metabolites has been confirmed by using ultra-violet (UV), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Fourier transmission-infrared (FT-IR), proton nuclear magnetic resonance (1H NMR) and gas chromatography-mass (GC-MS) spectral analysis. Enzyme activities of CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase were detected in the cell-free-extract of the CIPC culture and are induced by cells of NKC-1 strain. These results demonstrate the biodegradation pathways of herbicide CIPC and promote the potential use of NKC-1 strain to bioremediate CIPC-contaminated environment with subsequent release of ammonia, chloride ions and carbon dioxide.

Purification and Characterization of a recombinant ß-Xylosidase from Bacillus licheniformis ATCC 14580 into E. coli Bl21.

Present research work is aimed to purify and characterize a recombinant ß-xylosidase enzyme which was previously cloned from Bacillus licheniformis ATCC 14580 in to Escherichia coli BL21. Purification of recombinant enzyme was carried out by using ammonium sulphate precipitation method followed by single step immobilized metal ion affinity chromatography. Specific activity of purified recombinant ß-xylosidase enzyme was 20.78 Umg-1 with 2.58 purification fold and 33.75% recovery. SDS-PAGE was used to determine the molecular weight of recombinant purified ß-xylosidase and it was recorded as 52 kDa. Purified enzyme showed stability upto 90°C within a pH range of 3-8 with and optimal temperature and pH, 55ºC and 7.0, respectively. The enzyme activity was not considerably affected in the presence of EDTA. An increase in the enzyme activity was found in the manifestation of Mg+2. Enzyme activity was also increased by 6%, 18% and 22% in the presence of 1% Tween 80, ß-mercaptoethanol and DTT, respectively. Higher concentrations (10 - 40%) of organic solvents did not show any effect upon activity of enzyme. All these characteristics of the recombinant enzyme endorsed it as a potential candidate for biofuel industry.

Screening and identification of lignin-degrading bacteria in termite gut and the construction of LiP-expressing recombinant Lactococcus lactis.

Lignin, a common natural polymers, is abundant and complex, and termites can break down and utilize the lignin in their food. In this study an attempt was made to isolate and characterize the lignolytic bacteria from termite (Reticulitermes chinensis Snyder) gut. Two strains (PY12 and MX5) with high lignin peroxidase (LiP) activity were screened using the azure B method. By analyzing their 16S rRNA, the strain PY12 was classified as Enterobacter hormaechei; MX5, as Bacillus licheniformis. We then optimized the different conditions of liquid fermentation medium, and obtained LiP activities of 278 U/L and 256 U/L for PY12 and MX5, respectively. Subsequently, we confirmed the LiP activities of the strains by evaluating their decolorizing effects on various dyes. Finally, we cloned the LiP gene of strain PY12 and successfully transferred it to Lactococcus lactis. We believe that our results provide the theoretical and practical basis for the production of genetically engineered bacteria that produce LiP, thus allowing for the utilization of naturally available lignin as an energy resource.

Isolation and characterization of metabolites from Bacillus licheniformis MH48 with antifungal activity against plant pathogens.

In this study, we isolated Bacillus licheniformis MH48 from rhizosphere soil and demonstrated that this strain shows significant antifungal activity against Rhizoctonia solani, Colletotrichum gloeosporioides, and Phytophthora capsici. Our results showed that a 50% concentration of bacterial cell-free culture filtrate of B. licheniformis MH48 shows strong activity against fungal pathogens. Benzoic acid produced by B. licheniformis MH48 was purified by various chromatographic techniques and identified by nuclear magnetic resonance and gas chromatography-mass spectrometry analysis. Benzoic acid displayed antifungal activity against R. solani and C. gloeosporides with minimum inhibitory concentration of 128 µg/mL against mycelial growth. Microscopic examination revealed that benzoic acid (50 µg/mL and 100 µg/mL) transformed C. gloeosporioides conidial morphology and inhibited conidial germination. In addition, benzoic acid (100 µg/mL and 200 µg/mL) degraded R. solani mycelia. Therefore, our results demonstrate that B. licheniformis MH48 strain shows potential for utility as a biological agent for the control of various fungal pathogens of plants.

In situ analysis of Bacillus licheniformis biofilms: amyloid-like polymers and eDNA are involved in the adherence and aggregation of the extracellular matrix.

AIMS: This study attempts to determine which of the exopolymeric substances are involved in the adherence and aggregation of a Bacillus licheniformis biofilm. METHODS AND RESULTS: The involvement of extracellular proteins and eDNA were particularly investigated using DNase and proteinase K treatment. The permeability of the biofilms increased fivefold after DNase I treatment. The quantification of the matrix components showed that, irrespective to the enzyme tested, eDNA and amyloid-like polymers were removed simultaneously. Size-exclusion chromatography analyses supported these observations and revealed the presence of associated nucleic acid and protein complexes in the biofilm lysates. These data suggest that some extracellular DNA and amyloid-like proteins were closely interlaced within the matrix. Finally, confocal laser scanning microscopy imaging gave supplementary clues about the 3D organization of the biofilms, confirming that eDNA and exoproteins were essentially layered under and around the bacterial cells, whereas the amyloid-like fractions were homogeneously distributed within the matrix. CONCLUSION: These results confirm that some DNA-amyloid complexes play a key role in the modulation of the mechanical resistance of B. licheniformis biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights the need to consider the whole structure of biofilms and to target the interactions between matrix components. A better understanding of B. licheniformis biofilm physiology and the structural organization of the matrix will strengthen strategies of biofilm control.

Chitosanase purified from bacterial isolate Bacillus licheniformis of ruined vegetables displays broad spectrum biofilm inhibition.

A number of bacterial species produces chitosanases which has variety of applications because of its high biodegradability, non-toxicity and antimicrobial assets. In the present study chitosanase is purified from new bacterial species Bacillus licheniformis from spoiled vegetable. This novel strain of Bacillus licheniformis isolated from spoilt cucumber and pepper samples has the ability to produce the chitosanase enzyme when grown on chitosan substrate. Study also examined its antibiofilm properties against diverse bacterial species with biofilm forming ability. The purified chitosanase inhibited the biofilm formation ability for all Gram-negative and Gram-positive biofilm-forming bacteria [biofilm producers] tested in this study in congo red agar and microtiter plate's methods. Highly antibiofilm activity of chitosanase was recorded against Pseudomonas aeruginosa followed by Klebsiella pneumoniae with reduction of biofilm formation upto 22 and 29%, respectively compared with [100] % of control. Biofilm formation has multiple role including ability to enhance resistance and self-protection from external stress. This chitosanase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria and has promising application as alternative antibiofilm agents to combat the growing number of multidrug resistant pathogen-associated infections, especially in situation where biofilms are involved.

Characterization of lipopeptides produced by Bacillus licheniformis using liquid chromatography with accurate tandem mass spectrometry.

RATIONALE: The plant endophyte Bacillus licheniformis, isolated from leaves of Vitis vinifera, was studied to individuate and characterize the presence of bioactive lipopeptides having amino acidic structures. METHODS: Crude extracts of liquid cultures were analyzed by ultra-high-performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight (QTOF) mass analyzer. Chromatographic conditions were optimized in order to obtain an efficient separation of the different isobaric lipopeptides, avoiding merged fragmentations of co-eluted isomeric compounds and reducing possible cross-talk phenomena. Composition of the amino acids was outlined through the interpretation of the fragmentation behavior in tandem high-resolution mass spectrometry (HRMS/MS) mode, which showed both common-class and peculiar fragment ions. Both [M + H](+) and [M + Na](+) precursor ions were fragmented in order to differentiate some isobaric amino acids, i.e. Leu/Ile. Neutral losses characteristic of the iso acyl chain were also evidenced. RESULTS: More than 90 compounds belonging to the classes of surfactins and lichenysins, known as biosurfactant molecules, were detected. Sequential LC/HRMS/MS analysis was used to identify linear and cyclic lipopeptides, and to single out the presence of a large number of isomers not previously reported. Some critical issues related to the simultaneous selection of different compounds by the quadrupole filter were highlighted and partially solved, leading to tentative assignments of several structures. Linear lichenysins are described here for the first time. CONCLUSIONS: The approach was proved to be useful for the characterization of non-target lipopeptides, and proposes a rationale MS experimental scheme aimed to investigate the difference in amino acid sequence and/or in the acyl chain of the various congeners, when standards are not available. Results expanded the knowledge about production of linear and cyclic bioactive compounds from Bacillus licheniformis, clarifying the structures of isomeric forms, and enabling the use of selected endophytes to produce fungicides for eco-friendly biocontrol. Copyright © 2016 John Wiley & Sons, Ltd.

In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine Thermophilic Bacillus licheniformis T14.

The development of antibiofilm strategies is of major interest in contrasting bacterial pathogenic biofilms. A novel fructose and fucose rich exopolysaccharide (EPS1-T14) produced by the recently described thermophilic Bacillus licheniformis T14, isolated from a shallow hydrothermal vent of Panarea Island (Eolian Island, Italy), was evaluated for its effects on biofilm formation by multiresistant clinical strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The antibiofilm activity of EPS1-T14 was assessed by microtiter plate assays and visualized by confocal laser scanning microscopic images. EPS1-T14, with molecular weight of 1000 kDa, reduced biofilm formation on abiotic surfaces without affecting bacterial vitality. The novel EPS1-T14 is a water-soluble, noncytotoxic exopolymer able to prevent biofilm formation and its use may represent a promising therapeutic strategy for combating bacterial biofilm-associated infections. EPS1-T14 as antiadhesive biomolecule could be useful for novel prospective in medical and nonmedical applications.

pH modulates arsenic toxicity in Bacillus licheniformis DAS-2.

The toxic characteristics of arsenic species, As(V) and As(III) result in ecological risks. Arsenic tolerant bacterium was isolated and identified as the Bacillus licheniformis DAS-2 through 16SrDNA sequencing. B. licheniformis DAS-2 was efficient to tolerate and remove both the As(V)[MIC 8mM] and As(III)[MIC 6mM] from the growth medium. The potential for the removal/uptake of arsenic from the 3, 5 and 7mM As(V) enriched growth media was 100%, 60% and 35% respectively and from the 1, 3 and 5mM As(III) enrichment it was 100%, 99% and 58% respectively at neutral pH. 80% of uptake As(V) was reduced to As(III) in 3mM As(V) enrichment which was gradually decreased to only 17% at 7mM As(V) enrichment at neutral pH. The arsenic toxicity in B. licheniformis DAS-2 was found modulated by pH and was examined through alteration in growth, uptake/removal, reduction and measurement of chemical toxicity.

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs.

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.