RESUMEN
Infection recurrence and antibiotic resistance of bacterial vaginosis-associated pathogenic biofilms underline the need for novel and effective treatment strategies. In this study, we evaluated the antimicrobial, antibiofilm, and quorum sensing inhibitory effects of benzoyl peroxide and salicylic acid against Gardnerella vaginalis ATCC 14018, the predominant pathogen of bacterial vaginosis. While the highest tested concentrations of 250 and 125 µg/mL for both compounds were not sufficient in completely inhibiting the growth of G. vaginalis ATCC 14018, they did prevent biofilm formation by inhibiting the bacterial quorum sensing system in the pathogen. To our knowledge, this report is the first evidence that benzoyl peroxide can have a quorum sensing-mediated biofilm controlling effect, as demonstrated using subinhibitory concentrations of this compound in order to reduce the cost, dosage, and negative side effects associated with current antimicrobial treatments.
Asunto(s)
Antibacterianos/farmacología , Peróxido de Benzoílo/farmacología , Biopelículas/efectos de los fármacos , Gardnerella vaginalis/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , Femenino , Gardnerella vaginalis/fisiología , Humanos , Ácido Salicílico/farmacología , Vaginosis Bacteriana/tratamiento farmacológicoRESUMEN
Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH4)2SO4) precipitation and purified via column chromatography. Subtilosin's antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 µg/mL when compared to Listeria monocytogenes (125 µg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 µg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 µg/mL of subtilosin was applied. Subtilosin with 7.8-125 µg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 µg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95-15.1 µg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.
Asunto(s)
Antibacterianos/farmacología , Bacteriocinas/farmacología , Biopelículas/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Percepción de Quorum/efectos de los fármacos , Bacillus subtilis/química , Bacteriocinas/aislamiento & purificación , Bacterias Gramnegativas/fisiología , Bacterias Grampositivas/fisiología , Péptidos Cíclicos/aislamiento & purificaciónRESUMEN
Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cation-exchange and gel-filtration chromatography. The preparation's purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was tested to be 187.5 AU µg(-1) with 13-fold purification. Purified enterocin LD3 was heat stable up to 121 °C (at 15 psi pressure) and pH 2-6. The activity was lost in the presence of papain, reduced by proteinase K, pepsin and trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role of carbohydrate and lipid moieties in the activity. MALDI-TOF/MS analysis of purified enterocin LD3 resolved m/z 4114.6, and N-terminal amino acid sequence was found to be H2NQGGQANQ-COOH suggesting a new bacteriocin. Dissipation of membrane potential, loss of internal ATP and bactericidal effect were recorded when indicator strain Micrococcus luteus was treated with enterocin LD3. It inhibited Gram-positive and Gram-negative bacteria including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics.