Peppermint (Mentha piperita) inhibits microbial biofilms in vitro

Microbial biofilms have become increasingly problematic in the food processing and medical industries where they cause food and surface contamination. Biofilms have also been implicated as the cause of serious infections in humans as their occurrence makes it difficult to treat common infections and the likelihood of recurrent infections is high. Due to emerging resistance, conventional control methods are fast becoming ineffective. In this study, the use of a selection of commercial plant extracts is investigated. The inhibitory effects of eight herbal extracts on the development of microbial biofilms was investigated against clinical and reference strains of Pseudomonas aeruginosa and Candida albicans. The antimicrobial activity was investigated on the planktonic forms using the minimum inhibitory concentration assay. The extracts that showed the highest antimicrobial activity against the two test organisms were Echinacea angustifolia (cone flower), Mentha piperita (peppermint) and Rosmarinus officinalis (rosemary) with minimum inhibitory concentration values between 0.38 and 2.5 mg/ml. The crystal violet assay was used to assess the effect of pre-treating a surface with plant extracts on cell attachment and the extent of biofilm development following exposure to extracts (biofilm biomass). Most of the extracts reduced microbial colonization by at least 50%. In contrast, preformed biofilms were less responsive to the majority of extracts, thus growth inhibition was more difficult to achieve. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens.     

Sodium chloride affects Listeria monocytogenes adhesion to polystyrene and stainless steel by regulating flagella expression

Aim:  To study the adhesion capability of seven strains of Listeria monocytogenes to polystyrene and stainless steel surfaces after cultivation at various NaCl concentrations.
Methods and Results:  Determination of growth limits indicated that all seven strains were able to grow in up to 11% NaCl in rain heart infusion and 3 g l⁻¹ yeast extract–glucose at 20°C, but no growth was detected at 15% NaCl. Adhesion of L. monocytogenes was estimated after 4-h incubation at 20°C in 96-well microtitre plates. Statistical results revealed no significant difference between adhesion to polystyrene and stainless steel although surface properties were different. Adhesion between 0% and 6% NaCl was not different, whereas adhesion at 11% NaCl was significantly lower. This discrepancy in adhesion was correlated with the down-regulation of flagella at 11% NaCl.
Conclusions:  Only high salinity levels, close to nongrowth conditions, repressed the expression of flagella, and consequently, decreased the adhesion capability of L. monocytogenes .
Significance and Impact of the Study:  Adhesion of L. monocytogenes to inert surfaces depends on environmental conditions that affect flagellum expression. High salinity concentrations would delay biofilm formation.     

Antibiofilm activity of <Emphasis Type="Italic">Andrographis paniculata</Emphasis> against cystic fibrosis clinical isolate <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Respiratory tract and device associated infections caused by biofilm forming Pseudomonas aeruginosa play a primary role in the pathogenesis and prognosis of cystic fibrosis (CF) diseases. The biofilm formed by these pathogens attributes to the antibiotic resistance and protection from host immune response. Once established, the pathogens respond poorly to therapeutic agents. Recently medicinal plants are largely explored as potential source of bioactive agents. In this context the present study reports the antibiofilm activity of the folkloric medicinal plant Andrographis paniculata against biofilm forming CF causative Pseudomonas aeruginosa isolated from CF sputum. P. aeruginosa was also assessed for their growth and development of the biofilm, phylogenetic relationship and antibiotic susceptibility. Antibiogram of the strains indicated that they were resistant to more than one antibiotic. Six extracts of A. paniculata showed significant antibiofilm activity. P. aeruginosa strains, KMS P03 and KMS P05, were found to be maximally inhibited by the methanol extract to an extent of 88.6 and 87.5% respectively. This is the first report on antibiofilm activity of A. paniculata extracts, and our results indicate scope for development of complementary medicine for biofilm associated infections.     

In vitro studies of anticandidal activity of goniothalamin enantiomers

Aims:  The antifungal activity of ( R )-goniothalamin ( 1 ) and ( S )-goniothalamin ( ent - 1 ) was evaluated against six Candida species. The in vitro effect of these compounds on yeast adhesion to human buccal epithelial cells (BEC) and Candida albicans and C. dubliniensis biofilms progression were also investigated.
Methods and Results:  Yeast susceptibility was evaluated by broth microdilution assay and showed that ent - 1 exhibited higher potency against all fungal clinical isolated when compared to compound 1 . Compounds 1 and ent - 1 were as potent as fluconazole in inhibiting the adhesion of C. albicans and C. dubliniensis to BEC. XTT-reducing assay and scanning electron microscopy revealed that 1 and ent - 1 were twice as potent as fluconazole in the inhibition of yeast biofilms progression.
Conclusions:  Our findings indicate that compounds 1 and ent - 1 are potent anticandidal agents.
Significance and Impact of the Study:  This study highlights goniothalamin enantiomers as promising lead compounds for the design of new antifungal with inhibitory activity on yeast adhesion and biofilm progression.     

Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions

Aims:  To determine whether isolates of Listeria monocytogenes differ in their ability to adsorb and form biofilms on a food-grade stainless steel surface.
Methods and Results:  Strains were assessed for their ability to adsorb to a test surface over a short time period. Although some differences in numbers of bound cells were found among the strains, there were no correlations between the degree of adsorption and either the serotype or source of the strain. The ability of each strain to form a biofilm when grown with the test surface was also assessed. With the exception of a single strain, all strains adhered as single cells and did not form biofilms. Significant differences in adherence levels were found among strains. Strains demonstrating enhanced attachment produced extracellular fibrils, whereas those which adhered poorly did not. A single strain formed a biofilm consisting of adhered single cells and aggregates of cells.
Conclusions:  Significant differences were found in the ability of various L. monocytogenes strains to attach to a test surface. In monoculture, the majority of strains did not form biofilms.
Significance and Impact of the Study:  Differences in attachment and biofilm formation among strains provide a basis to study these characteristics in L. monocytogenes .     

Chelated iron sources are inhibitors of Pseudomonas aeruginosa biofilms and distribute efficiently in an in vitro model of drug delivery to the human lung

Aims:  To determine whether chelated sources of ferric iron were efficient inhibitors of biofilm formation in Pseudomonas aeruginosa and might be suitable for drug delivery to the lungs of cystic fibrosis (CF) patients via nebulization.
Methods and Results:  The response of P. aeruginosa biofilms to elevated iron concentrations in the form of eight structurally varied iron chelators in a microtitre plate assay for biofilm production was examined in the lab. Among these iron chelates, picolinic acid and acetohydroxamic acid-chelated iron were able to effectively thwart biofilm production in P. aeruginosa PA14 and in 20 clinical isolates of P. aeruginosa from a local hospital. The chelated iron sources showed excellent distribution in an Anderson cascade impactor model of particle size distribution in the human lung.
Conclusions:  Ferric picolinate and ferric acetohydroxamate are effective anti-biofilm compounds against both lab and clinical strains of P. aeruginosa and are readily nebulized into particles of suitable size for lung delivery.
Significance and Impact of the Study:  The data herein serve both to solidify the growing base of literature correlating high iron levels with biofilm inhibition in P. aeruginosa and to highlight the potential of these chelators as nebulized agents to combat biofilms of P. aeruginosa in CF patients.     

Novel effect of plant lectins on the inhibition of Streptococcus mutans biofilm formation on saliva-coated surface

Aims:  Dental caries is caused by the disturbance in oral homeostasis, marked by a notable increase in the population of Streptococcus mutans . Lectins are a group of plant proteins that are capable of recognizing the glycoconjugates present on the bacterial surface. The aim of this study was to evaluate the effect of seven plant lectins on the growth and initial adhesion of S. mutans .
Methods and Results:  Lectins of different carbohydrate specificities were isolated from plant sources by conventional methods of protein purification. The effect on growth of S. mutans was evaluated following CLSI guidelines. None of the lectins used in this study inhibited the bacterial growth and multiplication. The adherence and biofilm formation of bacteria to saliva-coated polystyrene plates was tested in the presence of plant lectins. All the plant lectins tested, inhibited both the adherence and biofilm in a concentration dependent manner. Confocal microscopy and scanning electron microscopy were employed to assess the biofilm formation in the presence of plant lectin (glucose/mannose-specific) at sub-minimal inhibitory concentrations. These evaluations revealed that lectins inhibited the clumping and attachment of S. mutans .
Conclusions:  Lectins tested here inhibited initial biofilm formation by S. mutans. Glucose/Mannose-specific lectin altered the adhesion arrangement of the bacteria on the saliva-coated surfaces.
Significance and Impact of the Study:  The plant lectins used in this study may offer a novel strategy to reduce development of dental caries by inhibiting the initial adhesion and subsequent biofilm formation of S. mutans.     

A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants

Aims:  The aim of this study was to develop an assay system that can quantify the amount of biomass in biofilms formed by different isogenic mutants of an Escherichia coli K-12 strain.
Methods and Results:  The reported assay, which is based on the BacTiter-Glo™ assay from Promega, uses bioluminescence to detect the intracellular concentration of ATP, which correlates with viable bacterial cell numbers. The quantitative data obtained with this ATP assay were compared to those obtained with the conventional crystal violet assay. As a qualitative control, scanning electron microscopy was performed.
Conclusions:  The ATP assay, the crystal violet assay and scanning electron microscopy yielded similar results for six of the eight strains tested. For the remaining two strains, the images from the scanning electron microscopy confirmed the results from the ATP assay.
Significance and Impact of the Study:  The ATP assay, in combination with other quantitative and qualitative assays, will allow us to perform genetic studies on the regulatory network that underlies the early steps in E. coli biofilm formation.     

Factors affecting the attachment of micro-organisms isolated from ultrafiltration and reverse osmosis membranes in dairy processing plants

Aims:  To identify the types of micro-organisms involved in the formation of biofilms on dairy ultrafiltration and reverse osmosis membranes and investigate factors affecting the attachment of those isolates.
Methods and Results:  Micro-organisms isolated from industrial membranes following standard cleaning were identified using the API culture identification system. Thirteen different isolates representing eight genera were isolated and their ability to attach to surfaces was compared using a microtitre plate assay. Three Klebsiella strains attached best, while mixed strains of Pseudomonas and Klebsiella attached better than individual strains. Whey enhanced the attachment of the isolates. The micro-organisms were characterized according to cell surface hydrophobicity using the microbial adhesion to hydrocarbon (MATH) test, and cell surface charge by measuring the zeta potential. These cell surface characteristics did not show a clear relationship with the attachment of our strains.
Conclusions:  A variety of different micro-organisms is associated with dairy ultrafiltration and reverse osmosis membranes after cleaning, suggesting several possible sources of contamination. The cleaning of these membranes may be inadequate. The attachment of the different isolates is highly variable and enhanced in the presence of whey.
Significance and Impact of the Study:  Knowledge of persistent microflora colonizing dairy membrane systems will help develop strategies to mitigate biofilm development in this environment, improving hygiene in membrane processing plants.     

Assessment of cell surface properties and adhesion potential of selected probiotic strains

Aim:  To evaluate the physicochemical cell surface and adhesive properties of selected probiotic strains for human use.
Methods and Results:  Probiotic strains, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus paracasei , Lactobacillus rhamnosus GG, Lactobacillus brevis , Lactobacillus casei , Leuconostoc mesenteroides and Pediococcus acidilactici were tested for the physicochemical properties of cell surfaces and the adhesion abilities against foodborne pathogens. Bif .  longum B6 (53·6%) and Lact .  rhamnosus GG (46·5%) showed the highest hydrophobicity, while the least affinity to xylene was observed in Ped .  acidilactici (10·4%). Bifidobacterium longum B6 showed the strongest coaggregation phenotype with Listeria monocytogenes (53·0%), Shigella boydii (42·0%) and Staphylococcus aureus (45·9%). Lactobacillus rhamnosus GG had the strong binding ability to Caco-2 cells and effectively inhibited the adhesion of L .  monocytogenes , Salmonella Typhimurium, Sh .  boydii and Staph .  aureus to Caco-2 cells. The hydrophobicity was highly correlated with coaggregative abilities and competitive inhibition, suggesting a good relationship between in vitro adhesion and in vivo colonization.
Conclusion:  The results suggest that Bif .  longum B6 and Lact .  rhamnosus GG can be candidate probiotics available for human consumption.
Significance and Impact of the Study:  Because the use of probiotic strains has been more concerned with their beneficial effects in the GI tract, it is essential to examine the potential of probiotic strains based on the physicochemical properties in terms of bacterial-binding and adhesion capabilities.     

Adhesion of food-borne bacteria to stainless steel is reduced by food conditioning films

Aims:  Preconditioning of stainless steel with aqueous cod muscle extract significantly impedes subsequent bacterial adhesion most likely due to repelling effects of fish tropomyosin. The purpose of this study was to determine if other food conditioning films decrease or enhance bacterial adhesion to stainless steel.
Methods and Results:  Attachment of Pseudomonas fluorescens AH2 to stainless steel coated with water-soluble coatings of animal origin was significantly reduced as compared with noncoated stainless steel or stainless steel coated with laboratory substrate or extracts of plant origin. Coating with animal extracts also decreases adhesion of other food-relevant bacteria. The manipulation of adhesion was not attributable to growth inhibitory effects. Chemical analysis revealed that the stainless steels were covered by homogenous layers of adsorbed proteins. The presence of tropomyocin was indicated by appearance of proteins with similar molecular weight based in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in several extracts that reduced adhesion but also extracts not containing this protein reduced bacterial adhesion, indicating that several molecular species may be involved in the phenomenon.
Conclusions:  It is a common perception that food materials facilitate bacterial adhesion to surfaces; however, this study demonstrates that aqueous coatings of food origin may actually reduce bacterial adhesion.
Significance and Impact of the Study:  Compounds from food extracts may potentially be used as nontoxic coatings to reduce bacterial attachment to inert surfaces.     

Microbiology and performance of a methanogenic biofilm reactor during the start-up period

Aims:  To understand the interactions between anaerobic biofilm development and process performances during the start-up period of methanogenic biofilm reactor.
Methods and Results:  Two methanogenic inverse turbulent bed reactors have been started and monitored for 81 days. Biofilm development (adhesion, growth, population dynamic) and characteristics (biodiversity, structure) were investigated using molecular tools (PCR–SSCP, FISH-CSLM). Identification of the dominant populations, in relation to process performances and to the present knowledge of their metabolic activities, was used to propose a global scheme of the degradation routes involved. The inoculum, which determines the microbial species present in the biofilm influences bioreactor performances during the start-up period. FISH observations revealed a homogeneous distribution of the Archaea and bacterial populations inside the biofilm.
Conclusion:  This study points out the link between biodiversity, functional stability and methanogenic process performances during start-up of anaerobic biofilm reactor. It shows that inoculum and substrate composition greatly influence biodiversity, physiology and structure of the biofilm.
Significance and Impact of the Study:  The combination of molecular techniques associated to a biochemical engineering approach is useful to get relevant information on the microbiology of a methanogenic growing biofilm, in relation with the start-up of the process.     

Measurement of biofilm formation by clinical isolates of Escherichia coli is method-dependent

Aims:  In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays.
Methods and Results:  The assays were performed in microtitre plates with two minimal and two enriched broths, with one- or two-steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one- and two-steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathematical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm.
Conclusions:  Escherichia coli forms biofilms on static assays in a method-dependent fashion, depending on strain, and it is strongly modulated by culture conditions.
Significance and Impact of the Study:  As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, considering all variables in the experimental settings.     

Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC)

Aims:  The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp-2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases.
Methods and Results:  The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea.
Conclusions:  Low iron availability may modulate biofilm formation and adhesive properties of EAEC strains to HEp-2 cells.
Significance and Impact of the Study:  The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains.     

Antibiofilm activity of coral-associated bacteria against different clinical M serotypes of Streptococcus pyogenes

Streptococcus pyogenes is the frequent cause of purulent infections in humans. Formation of a biofilm is one of the important aspects of its pathogenicity. Streptococcus pyogenes biofilm communities tend to exhibit significant tolerance to antimicrobial challenge during infections. Exploring novel targets against biofilm-forming pathogens is therefore an important alternative treatment measure. We attempted to screen marine bacteria, especially coral-associated bacteria (CAB), for antibiofilm activity against streptococcal biofilm formation. The bacterial biofilms were quantified by crystal violet staining. Of 43 CAB isolates, nine clearly demonstrated antibiofilm activity. At biofilm inhibitory concentrations (BIC), biofilm formation was reduced up to 80%, and sub-BIC (0.5 and 0.25 BIC) significantly reduced biofilm formation by up to 60% and 40–60%, respectively. Extracts of Bacillus horikoshii (E6) displayed efficient antibiofilm activity. As quorum sensing (QS) and cell surface hydrophobicity (CSH) are crucial factors for biofilm formation in S. pyogenes , the CAB were further screened for QS inhibition properties and CSH reduction properties. This study reveals the antibiofilm and QS inhibition property of CAB.     

Lipopeptides from Bacillus strain AR2 inhibits biofilm formation by Candida albicans

The ability of the human fungal pathogen Candida albicans to reversibly switch between different morphological forms and establish biofilms is crucial for establishing infection. Targeting phenotypic plasticity and biofilm formation in C. albicans represents a new concept for antifungal drug discovery. The present study evaluated the influence of cyclic lipopeptide biosurfactant produced by Bacillus amyloliquefaciens strain AR2 on C. albicans biofilms. The biosurfactant was characterized as a mixture of iturin and fengycin by MALDI-TOF and amino acid analysis. The biosurfactant exhibited concentration dependent growth inhibition and fungicidal activity. The biosurfactant at sub-minimum growth inhibition concentration decreased cell surface hydrophobicity, hindered germ tube formation and reduced the mRNA expression of hyphae-specific gene HWP1 and ALS3 without exhibiting significant growth inhibition. The biosurfactants inhibited biofilm formation in the range of 46–100 % depending upon the concentration and Candida strains. The biosurfactant treatment dislodged 25–100 % of preformed biofilm from polystyrene plates. The biosurfactant retained its antifungal and antibiofilm activity even after exposure to extreme temperature. By virtue of the ability to inhibit germ tube and biofilm formation, two important traits of C. albicans involved in establishing infection, lipopeptides from strain AR2 may represent a potential candidate for developing heat stable anti-Candida drugs.     

Influence of phosphorus on biofilm formation in model drinking water distribution systems

Aims:  This study investigated the effects of phosphorus on biofilm formation via annular reactor systems in terms of biofilm cell growth, exopolysaccharide (EPS) production, biofilm structure and cell metabolic potential.
Methods and Results:  Drinking water biofilms were developed in annular reactors with supplement of carbon and different levels of phosphorus. The biofilm formation was monitored over a period of 30 days. Biofilm related parameters were examined by various methods, which included heterotrophic plate count, total carbohydrate content, confocal laser scanning microscopy and GN2 microplate assay. Our results showed that phosphorus addition can promote the biofilm cell growth (cell count increased about 1 log with addition of 30 and 300 μg l⁻¹ of phosphorus). However, the addition of 30 and 300 μg l⁻¹ of phosphorus caused 81% and 77% decrease in EPS production, respectively. The results of biofilm structure analysis showed that the addition of 30 and 300 μg l⁻¹ of phosphorus can induce thicker and less homogeneous biofilms with more biomass. Furthermore, the addition of 30 and 300 μg l⁻¹ of phosphorus dramatically increased the biofilm cell metabolic potential. The addition of 3 μg l⁻¹ of phosphorus was found to have minor effects on the parameters examined.
Conclusions:  The results indicate phosphorus addition to drinking water distribution system (DWDS) has a complicated effect on the biofilm formation.
Significance and Impact of the Study:  As the addition of phosphorus at certain levels can affect the biofilm growth in DWDS, care should be taken when phosphate-based corrosion inhibitors are used in the DWDS.     

Tetrazolium reduction methods for assessment of substrate oxidation and strain differentiation among mycoplasmas, with particular reference to Mycoplasma bovigenitalium and some members of the Mycoplasma mycoides cluster

Aims:  To apply a rapid nitroblue tetrazolium (NBT) reduction assay of substrate metabolism by mycoplasmas that would help to differentiate Mycoplasmas .
Methods and Results:  Growth, substrate preferences and tetrazolium reduction were assessed for 18 strains of Mycoplasma bovigenitalium and Mycoplasma ovine serogroup 11. NBT reduction was detectable in 1 h with 10⁸ CFU ml⁻¹. Use of α-ketobutyrate, lactate and pyruvate to support growth and NBT reduction were correlated: pyruvate was preferred and lactate was used by only four of the 18 strains. Selected members of the Mycoplasma mycoides cluster were also assessed and monotetrazoles tested as alternatives to NBT. The NBT method was applied to a further 19 species.
Conclusions:  This simple and reproducible method requires only small amounts of cells, enabling routine assessment of substrate use within 1 h, and the rapid assignment of numerous mycoplasmas to one of six physiological groups. The four physiological groups of M. bovigenitalium and Mycoplasma serogroup 11 strains were indistinguishable from each other, which supports the view that these belong to the same species.
Significance and Impact of the Study:  Strain-specific substrate-utilization patterns by mycoplasmas can be obtained rapidly and reliably. The method has potential as a large-scale semi-automated procedure to monitor numerous strains and substrates simultaneously.     

In vitro antibiofilm and anti‐adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic‐resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X‐ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram‐negative bacteria. Cell adherence assay indicated 25.3–49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time‐dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non‐toxic to HeLa cells at concentrations of 15–120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug‐resistant bacteria.

     

Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp

Aim:  To investigate the effects of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium in terms of toxin and growth of pathogenic Vibrio harveyi .
Methods and Results:  Three Bacillus probionts were isolated from probiotic BZT aquaculture and identified using a 16S rDNA sequence. Growth inhibition assay showed that supernatants from the 24-h culture of three Bacillus species were able to inhibit the growth of V. harveyi (LMG 4044); B. subtilis was the most effective based on the well diffusion method. Results of a liquid culture model showed that B. subtilis was also widely effective in inhibiting three strains of V. harveyi (isolated from Thailand, the Philippines and LMG 4044), and that both B. licheniformis and B. megaterium inhibit the growth of V. harveyi isolated from the Philippines. Moreover, a haemolytic activity assay demonstrated that V. harveyi (IFO 15634) was significantly decreased by the addition of B. licheniformis or B. megaterium supernatant.
Conclusions:  Bacillus subtilis inhibited Vibrio growth, and both B. licheniformis and B. megaterium suppressed haemolytic activity in Vibrio .
Significance and Impact of the Study:  The cell-free supernatants produced by Bacillus probionts inhibit Vibrio disease, and Bacillus probionts might have an influence on Vibrio cell-to-cell communications.