New bacterial strains isolated from Tunisian biotopes: A sustainable enzymatic approach for decolorization and detoxification of Congo Red and Malachite Green.

Seven bacterial strains, isolated from various Tunisian biotopes, were investigated for Congo Red (CR) and Malachite Green (MG) decolorization. The isolated strains underwent morphological and biochemical tests, including assessments for antibiotic sensitivity as well as biofilm formation. One selected strain, ST11, was partially identified as Paenibacillus sp. strain ST11. The newly isolated crude bacterial filtrates (NICBFs) effectively decolorized CR and MG. Specifically, six and seven NICBFs were found to be effective for degrading CR (150 mg l-1) and MG (50 mg l-1), respectively. Under non-optimized conditions, CR and MG could be decolorized up to 80% within 6-12 h. The degradation products of CR and MG, characterized by UV-visible and FT-IR techniques, demonstrated both decolorization and transformation, highlighting the role of enzymes in dye degradation. Phytotoxicity and cytotoxicity studies evaluated the impact of treated and untreated CR and MG. Some NICBFs showed promise as powerful biological tools, reducing and sometimes detoxifying CR and MG, commonly used as fertilizers. The potential applications of these NICBFs in decolorization and bioremediation of dye-rich textile effluents were explored. The screening also identified environmentally friendly, cost-effective bacterial strains adaptable to various conditions through phytotoxicity and cytotoxicity studies.

Control of Staphylococcus epidermidis biofilm by surfactins of an endophytic bacterium Bacillus sp. 15 F.

The present paper deals with the preparation and annotation of a surfactin(s) derived from a culture of the endophytic bacterium Bacillus 15 F. The LC-MS analysis of the acetonitrile fraction confirmed the presence of surfactins Leu/Ile7 C15, Leu/Ile7 C14 and Leu/Ile7 C13 with [M+H]+ at m/z 1036.6895, 1022.6741 and 1008.6581, respectively. Various concentrations of the surfactin(s) (hereafter referred to as surfactin-15 F) were used to reduce the adhesion of Staphylococcus epidermidis S61, which served as a model for studying antibiofilm activity on polystyrene surfaces. Incubation of Staphylococcus epidermidis S61 with 62.5 µg/ml of surfactin-15 F resulted in almost complete inhibition of biofilm formation (90.3 ± 3.33 %), and a significant reduction of cell viability (resazurin-based fluorescence was more than 200 times lower). The antiadhesive effect of surfactin-15 F was confirmed by scanning electron microscopy. Surfactin-15 F demonstrated an eradication effect against preformed biofilm, causing severe disruption of Staphylococcus epidermidis S61 biofilm structure and reducing viability. The results suggest that surfactins produced by endophytic bacteria could be an alternative to synthetic products. Surfactin-15 F, used in wound dressings, demonstrated an efficient treatment of the preformed Staphylococcus epidermidis S61 biofilm, and thus having a great potential in medical applications.

Unveiling the impact of selected essential oils on MRSA strain ATCC 33591: antibacterial efficiency, biofilm disruption, and staphyloxanthin inhibition.

This work aimed to evaluate the effects of 4 selected essential oils on planktonic cells and microbial biofilms of the Staphylococcus aureus strain (MRSA ATCC 33591). The antibacterial activities of the four essential oils Geranium (Pelargonium graveolens), PgEO, Tea Tree (Melaleuca alternifolia) MaEO, Lemon peel (Citrus limon) ClEO and Peppermint (Mentha piperita) MpEO had MICs ranging from 1.56 to 12.5 µl/ml. The evaluation of the antibiofilm activities of the 4 EOs revealed that they had antiadhesive activities against S. aureus MRSA biofilms; the activity reached 60% (the EO of MpEO peppermint at a concentration of 3.12 µl/ml), and the eradication activity was 80% (the EO of PgEO and MpEO at 3.12 µl/ml). The antibiofilm activity of S. aureus has been explained by the binding of several essential oil bioactive molecules to the SarA protein, the main target protein involved in biofilm formation. The synthesis of the virulence factor staphyloxanthin by S. aureus MRSA ATCC 33591 was significantly inhibited in the presence of PgEO at a concentration of MIC/2. This inhibition was explained by the binding of the main PgEO molecules (ß-citronellol and geraniol) to the CrTM protein involved in the staphyloxanthin synthesis pathway. There is evidence that these essential oils could be used as potential anti-virulents to control Staphylococcus biofilm formation.

A novel perspective on eugenol as a natural anti-quorum sensing molecule against Serratia sp.

Serratia marcescens is commonly noted to be an opportunistic pathogen and is often associated with nosocomial infections. In addition to its high antibiotic resistance, it exhibits a wide range of virulence factors that confer pathogenicity. Targeting quorum sensing (QS) presents a potential therapeutic strategy for treating bacterial infections caused by S. marcescens, as it regulates the expression of various virulence factors. Inhibiting QS can effectively neutralize S. marcescens' bacterial virulence without exerting stress on bacterial growth, facilitating bacterial eradication by the immune system. In this study, the antibacterial and anti-virulence properties of eugenol against Serratia sp. were investigated. Eugenol exhibited inhibitory effects on the growth of Serratia, with a minimal inhibitory concentration (MIC) value of 16.15 mM. At sub-inhibitory concentrations, eugenol also demonstrated antiadhesive and eradication activities by inhibiting biofilm formation. Furthermore, it reduced prodigiosin production and completely inhibited protease production. Additionally, eugenol effectively decreased swimming and swarming motilities in Serratia sp. This study demonstrated through molecular modeling, docking and molecular dynamic that eugenol inhibited biofilm formation and virulence factor production in Serratia by binding to the SmaR receptor and blocking the formation of the HSL-SmaR complex. The binding of eugenol to SmaR modulates biofilm formation and virulence factor production by Serratia sp. These findings highlight the potential of eugenol as a promising agent to combat S. marcescens infections by targeting its virulence factors through quorum sensing inhibition.

Antibacterial, antibiofilm and cytotoxic properties of prodigiosin produced by a newly isolated Serratia sp. C6LB from a milk collection center.

The aim of this work was to produce a red tripyrrole pigment prodigiosin from Serratia sp. C6LB strain, to investigate the promising antimicrobial properties on Gram-positive and Gram-negative bacterial strains. The research was also proposed to evaluate the antibiofilm activity on Staphylococcus epidermidis S61 biofilm and its cytotoxic activity against human cancer cell lines. The production and structural elucidation of prodigiosin was carried out using spectrophotometric scanning, TLC, HPLC, FTIR and NMR analysis. The pigment production was optimized using mannose and peptone as carbon and nitrogen sources, respectively. The study confirmed promising antibacterial properties of prodigiosin on eight Gram-positive and Gram-negative bacterial strains with MICs values ranged from 0.039 to 2.5 mg/mL. Antiadhesive activity test of prodigiosin on Staphylococcus epidermidis S61 biofilm exhibited 99.9% inhibition, whereas maximum biofilm eradication activity reached 65%. Cytotoxic activity showed IC50 of 16 µg/mL and 6.7 µg/mL against breast cancer lines MCF-7 and MDA-MB231, respectively.

Change in some quality parameters and oxidative stability of olive oils with regard to ultrasound pretreatment, depitting and water addition.

Ultrasound pretreatment with different times (0, 4, 8, 10 min) on olive paste previous malaxation of olive oil extraction along with depitting and water application procedures were studied. The effects of these procedures on oil yield, quality parameters and oxidative stability have been studied. Therefore, the olive oil yield increase with increasing time of ultrasound treatment. On the other hand, the application of ultrasound did not adversely affect the quality characteristics, as well as the antioxidant activity, when comparing with untreated paste. Furthermore, the oxidative stability data, we can conclude that ultrasound treatment can affect the olive oil oxidative stability. This study could provide useful information for industry to produce olive oil with high yield and quality.

Antibacterial, anti-adherent and cytotoxic activities of surfactin(s) from a lipolytic strain Bacillus safensis F4.

The bacterial strain F4, isolated from olive oil-contaminated soil, has been found to produce biosurfactants as confirmed by oil displacement test and the emulsification index results. The identification of the strain F4, by 16S ribosomal RNA gene, showed a close similarity to Bacillus safensis, therefore the strain has been termed Bacillus safensis F4. The Thin Layer Chromatography (TLC) and the High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) demonstrated that the biosurfactant had a lipopeptide structure and was classified as surfactin. The present study showed also that the produced biosurfactant has an important antibacterial activity against several pathogen strains as monitored with minimum inhibitory concentration (MIC) micro-assays. In particular, it presented an interesting anti-planktonic activity with a MIC of 6.25 mg mL-1 and anti-adhesive activity which exceeded 80% against the biofilm-forming Staphylococcus epidermidis S61 strain. Moreover, the produced lipopeptide showed an antitumor activity against T47D breast cancer cells and B16F10 mouse melanoma cells with IC50 of 0.66 mg mL-1 and 1.17 mg mL-1, respectively. Thus, our results demonstrated that Bacillus safensis F4 biosurfactant exhibited a polyvalent activity via a considerable antibiofilm and antitumoral potencies.

The novel cationic cell-penetrating peptide PEP-NJSM is highly active against Staphylococcus epidermidis biofilm.

A cationic cell-penetrating peptide PEP-NJSM was identified in human virus proteomes by a screening of charge clusters in protein sequences generating Cell-Penetrating Peptides (CPP). PEP-NJSM was selectively active against Gram-positive Staphylococcus epidermidis as antibacterial agent with MIC value of 128 µM compared to the Gram-negative Pseudomonas aeruginosa strain with MIC value exceeded 512 µM. The selected peptide exhibited an important anti-biofilm activity even at sub-MIC levels. PEP-NJSM could prevent biofilm formation and increase the mortality of cells inside mature S. epidermidis biofilm. The results demonstrated that PEP-NJSM presented an important anti-adherent activity. It showed a S. epidermidis inhibition of biofilm formation >84% at a concentration of 256 µM (2 X MIC) and remained active even at a concentration of 4 µM with 32% of inhibition. The eradication of the established biofilm was observed at a concentration of 256 µM with 55.7% of biofilm eradication. The peptide was active against mature biofilm even at low concentration of 0.5 µM with approximately 22.9% of eradication. PEP-NJSM exhibited low hemolytic activity and cytotoxicity against mammalian cells. Our results demonstrate that PEP-NJSM could have a potential role in the treatment of diseases related to Staphylococcus epidermidis infection.

Chemical composition, anti-biofilm activity and potential cytotoxic effect on cancer cells of Rosmarinus officinalis L. essential oil from Tunisia.

BACKGROUND: Rosmarinus officinalis L. from Tunisia, popularly known as rosemary, is of a considerable importance for its medicinal uses and aromatic value. The aim of this study was to examine the chemical composition of Rosmarinus officinalis essential oil (ROEO) and to evaluate its antibiofilm activity on biofilm-forming bacterium and its anticancer activity on cancer cell lines. METHODS: The chemical composition of Rosmarinus officinalis essential oil (ROEO) was analyzed by GC-MS and its antibacterial activity was evaluated by micro-dilution method. The antibofilm activity of ROEO was evaluated using the crystal violet test and the cytotoxicity activity was determined by the MTT assay. RESULTS: In this research, thirty-six compounds were identified in ROEO using GC-MS analyses. The main components were 1,8-cineole (23.56%), camphene (12.78%), camphor (12.55%) and ß-pinene (12.3%). The antibacterial activity of ROEO was evaluated by micro-dilution method. The oil exhibited inhibition and bactericidal effect against two strains: Staphylococcus aureus ATCC 9144 and Staphylococcus epidermidis S61. It was found that the minimum inhibitory concentration (MIC) obtained for S. aureus and S. epidermidis ranged from 1.25 to 2.5 and from 0.312 to 0.625 µl ml-1, respectively and the minimum bactericidal concentration (MBC) were in the order of 5 and 2.5 µl ml-1, respectively. Furthermore, this oil showed a S. epidermidis biofilm inhibition more than 57% at a concentration of 25 µl ml-1. The eradication of 67% of the established biofilm was observed at a concentration of 50 µl ml-1 of ROEO, whereas the dose of 25 µl ml-1 removed only 38% of preformed biofilm. ROEO strongly inhibited the proliferation of Hela and MCF-7 cells with IC50 values of 0.011 and 0.253 µl ml-1, respectively. CONCLUSION: Our results demonstrate that ROEO could have a potential role in the treatment of diseases related to infection by microorganisms or proliferation of cancer cells.

Evaluation of biofilm-forming ability of bacterial strains isolated from the roof of an old house.

The bacterial diversity associated with biofilm-forming ability was studied. Eighteen bacterial strains were isolated from a microbial film collected from the roof of an old house located in Sfax, Tunisia. The purity of these microorganisms was confirmed by microscopic observation after repeated streaking on a Tryptic Soy agar medium. Biofilm formation was estimated using preliminary tests including a motility test, microbial adhesion to solvents (MATS), and the Congo Red Agar method (CRA). Since these tests showed no significant result, microplate tests, such as crystal violet and resazurin assays, were used. The results obtained showed that strain S61 was able to form a biofilm within 24 h (OD570 = 4.87). The viability of the S61 biofilm with resazurin assessed with fluorescence measurement was about 1.5 × 103. The S61 strain was identified as Staphylococcus epidermidis. In the biofilm studied here, it was the most biofilm-forming bacterium and will be used as a bacterial model for studying anti-biofilm activity.

Cumin (Cuminum cyminum L.) from traditional uses to potential biomedical applications.

Cumin (Cuminum cyminum L.) is a small annual and herbaceous plant belonging to the Apiaceae family. It is a multipurpose plant species cultivated in the Middle East, India, China, and several Mediterranean countries, including Tunisia. Its fruit, known as cumin seed, is most widely used for culinary and medicinal purposes. It is generally used as a food additive, popular spice, and flavoring agent in many cuisines. Cumin has also been widely used in traditional medicine to treat a variety of diseases, including hypolipidemia, cancer, and diabetes. The literature presents ample evidence for the biological and biomedical activities of cumin, which have generally been ascribed to its content and action of its active constituents, such as terpens, phenols, and flavonoids. The present paper provides an overview of phytochemical profile, biological activities, and ethnomedical and pharmacological uses of Cumin.

Biodegradation of diesel oil by a novel microbial consortium: comparison between co-inoculation with biosurfactant-producing strain and exogenously added biosurfactants.

Bioremediation, involving the use of microorganisms to detoxify or remove pollutants, is the most interesting strategy for hydrocarbon remediation. In this aim, four hydrocarbon-degrading bacteria were isolated from oil-contaminated soil in Tunisia. They were identified by the 16S rDNA sequence analysis, as Lysinibacillus bronitolerans RI18 (KF964487), Bacillus thuringiensis RI16 (KM111604), Bacillus weihenstephanensis RI12 (KM094930), and Acinetobacter radioresistens RI7 (KJ829530). Moreover, a lipopeptide biosurfactant produced by Bacillus subtilis SPB1, confirmed to increase diesel solubility, was tested to increase diesel biodegradation along with co-inoculation with two biosurfactant-producing strains. Culture studies revealed the enhancement of diesel biodegradation by the selected consortium with the addition of SPB1 lipopeptide and in the cases of co-inoculation by biosurfactant-producing strain. In fact, an improvement of about 38.42 and 49.65 % of diesel degradation was registered in the presence of 0.1 % lipopeptide biosurfactant and when culturing B. subtilis SPB1 strain with the isolated consortium, respectively. Furthermore, the best improvement, evaluated to about 55.4 %, was recorded when using the consortium cultured with B. subtilis SPB1 and A. radioresistens RI7 strains. Gas chromatography analyses were correlated with the gravimetric evaluation of the residual hydrocarbons. Results suggested the potential applicability of the selected consortium along with the ex situ- and in situ-added biosurfactant for the effective bioremediation of diesel-contaminated water and soil.

Characterization of the microbial diversity in production waters of mesothermic and geothermic Tunisian oilfields.

The microbial diversity of production waters of five Tunisian oilfields was investigated using Single Strand Conformation Polymorphism (SSCP) technique followed by cloning-sequencing. Dynamics of bacterial populations in production waters collected from four wellheads were also evaluated. For all production water samples collected, DNA from Archaea and Eucarya was not sufficiently abundant to permit detection rRNA genes from these groups by PCR-SSCP. In contrast, the bacterial rRNA genes were detected in all samples, except for samples from DOULEB12 and RAMOURA wells. SSCP profiles attested that two of the studied geothermic wells (ASHTART47 and ASHTART48) had shown a clear change over time, whereas a stable diversity was found with the mesophilic DOULEB well (DL3). PCR amplification of rRNA genes was unsuccessful with samples from DOULEB (DL12) at all three sampling time. The bacterial diversity present in production waters collected from pipelines of SERCINA and LITAYEM oilfields was high, while production waters collected from wellheads (ASHTART and DOULEB) exhibited lower diversity. The partial study of the biodiversity showed a dominance of uncultured bacteria and Pseudomonas genus (class of the Gammaproteobacteria) in three of the studied oilfields (ASHTART47, ASHTART48 and DOULEB3). However, for LITAYEM oilfield, a significant dominance of 5 phyla (Proteobacteria, Thermotogae, Firmicutes, Synergistetes, Bacteroidetes) was shown. Our study gives a real picture of the microbiology of some Tunisian oilfield production waters and shows that some of the sequenced bacterial clones have a great similarity to previous sequenced clones described from other oilfields all over the world, indicating that these ecosystems harbour specific microbial communities. These findings can be considered as an indirect indication of the indigenous origin of these clones.

Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation.

A biosurfactant-producing bacterium (Staphylococcus sp. strain 1E) was isolated from an Algerian crude oil contaminated soil. Biosurfactant production was tested with different carbon sources using the surface tension measurement and the oil displacement test. Olive oil produced the highest reduction in surface tension (25.9 dynes cm(-1)). Crude oil presented the best substrate for 1E biosurfactant emulsification activity. The biosurfactant produced by strain 1E reduced the growth medium surface tension below 30 dynes cm(-1). This reduction was also obtained in cell-free filtrates. Biosurfactant produced by strain 1E showed stability in a wide range of pH (from 2 to 12), temperature (from 4 to 55 °C) and salinity (from 0 to 300 g l(-1)) variations. The biosurfactant produced by strain 1E belonged to lipopeptide group and also constituted an antibacterial activity againt the pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Phenanthrene solubility in water was enhanced by biosurfactant addition. Our results suggest that the 1E biosurfactant has interesting properties for its application in bioremediation of hydrocarbons contaminated sites.

Isolation and characterization of Klebsiella oxytoca strain degrading crude oil from a Tunisian off-shore oil field.

A facultatively anaerobic, Gram-negative, mesophilic, moderately halotolerant, non-motile, and non-sporulated bacterium, designated strain BSC5 was isolated from an off-shore "Sercina" oil field, located near the Kerkennah island, Tunisia. Yeast extract was not required for growth. Phenotypic characteristics and phylogenetic analysis of the 16S rRNA gene sequence of strain BSC5 revealed that it was related to members of the genus Klebsiella, being most closely related to the type strain of K. oxytoca (99% sequence similarity). Strain BSC5 was capable of using aerobically the crude oil as substrate growth. The growth of strain BSC5 on crude oil was followed by measuring the OD(600 nm) and by enumeration of viable cells at different culture's time. GC-MS analysis showed that strain BSC5 was capable of degrading a wide range of aliphatic hydrocarbons from C(13) to C(30) . The biodegradation rate for n -alkanes reached 44% and 75%, after 20 and 45 days of incubation, respectively. Addition of the synthetic surfactant, Tween 80, accelerated the crude oil degradation. The biodegradation rate for n -alkanes reached 61% and 98%, after 20 and 45 days of incubation, respectively. Moreover, three aromatic compounds, p -hydroxybenzoate, protocatechuate and gentisate, were metabolized completely by strain BSC5 after 24 h, under aerobic conditions.

Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain.

BACKGROUND: Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. RESULTS: This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C). The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry), was used as the sole carbon source. CONCLUSION: A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment.

A newly high alkaline lipase: an ideal choice for application in detergent formulations.

BACKGROUND: Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. RESULTS: A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1) were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents. CONCLUSIONS: These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations.

Anaerobic membrane bioreactor for the treatment of leachates from Jebel Chakir discharge in Tunisia.

Landfill leachate (LFL) collected from the controlled discharge of Jebel Chakir in Tunisia was treated without any physical or chemical pretreatment in an anaerobic membrane bioreactor (AnMBR). The organic loading rate (OLR) in the AnMBR was gradually increased from 1 g COD l(-1)d(-1) to an average of 6.27 g COD l(-1)d(-1). At the highest OLR, the biogas production was more than 3 volumes of biogas per volume of the bioreactor. The volatile suspended solids (VSSs) reached a value of approximately 3 g l(-1) in the bioreactor. At stable conditions, the treatment efficiency was high with an average COD reduction of 90% and biogas yield of 0.46 l biogas per g COD removed.

Isolation of a thermophilic and halophilic tyrosol-degrading Geobacillus from a Tunisian high-temperature oil field.

An aerobic, thermophilic, halotolerant and Gram-positive bacterium, designated strain C5, was isolated from a high-temperature oil field, located in Sfax, Tunisia, after enrichment on tyrosol. Strain C5 grew between 25 and 70 degrees C and optimally at 50 degrees C. It grew in the presence of 0-12% (w/v) NaCl, with optimum growth at 3% (w/v) NaCl. Strain C5 was able to degrade tyrosol aerobically, in the presence of 30 g L(-1) NaCl and under warm conditions (55 degrees C). The degradation of tyrosol proceeded via p-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids. The products were confirmed by HPLC and GC-MS analyses. Strain C5 was also found to degrde a wide range of other aromatic compounds, including benzoic, p-hydroxybenzoic, protocatechuic, vanillic, p-hydroxyphenylacetic, 3,4-dihydroxyphenylacetic, cinnamic and ferulic acids, phenol and m-cresol. Moreover, strain C5 was grown on diesel and crude oil as sole carbon and energy sources. Strain C5 was also able to utilize several carbohydrates. Phenotypic characteristics and phylogenetic analysis of the 16S rRNA gene sequence of strain C5 revealed that it was related to members of the genus Geobacillus, being most closely related to the type strain of G. pallidus (99% sequence similarity). In addition, we report on growth of the type strain of G. pallidus on different aromatic compounds and hydrocarbons.