Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis.

AIMS: The biofilm produced by Staphylococcus aureus isolates involved in clinical or subclinical bovine mastitis and the activity of nisin and lysostaphin against the preformed biofilm produced by these strains were investigated. METHODS AND RESULTS: Eighteen strains were tested and all produced biofilm. Eight strains with distinct biofilm composition were selected for the antimicrobial activity assays. The minimal inhibitory concentration of each bacteriocin was determined against the planktonic cells and ranged from 15·6 to 500 µg ml(-1) for nisin, and from 3·9 to 50 µg ml(-1) , for lysostaphin. Lysostaphin treatment (0·4 µg ml(-1) ) for 4 h caused a strong Staph. aureus 4181 biofilm detachment and death of the majority of the sessile cells, while nisin treatment (100 µg ml(-1) ) for the same time caused only a great reduction in cell viability. Additionally, combination of both bacteriocins for 4 h resulted in significant death of the sessile cells but no biofilm detachment. CONCLUSIONS: The treatment with lysostaphin alone or in combination with nisin was effective in killing most biofilm sessile cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved.

The antimicrobial peptide aureocin A53 as an alternative agent for biopreservation of dairy products.

AIMS: The aim of this study was to investigate the potential of aureocin A53, a staphylococcal antimicrobial peptide, for improving food safety. METHODS AND RESULTS: The antimicrobial activity of aureocin A53 against strains of Listeria monocytogenes isolated from food was tested and the bacteriocin proved to be bactericidal and bacteriolytic against the listerial strains. Aureocin A53 was neither toxic to eukaryotic cell lines nor haemolytic against sheep erythrocytes. It also exhibited a remarkable stability during storage at different temperatures and sensitivity to both simulated gastric juice and bile salts. When the antibacterial activity of aureocin A53 (256 AU ml(-1) ) was tested in skimmed milk artificially inoculated with a L. monocytogenes strain (1·0 × 10(4)  CFU ml(-1) ) isolated from food, during storage at 4°C, the bacteriocin reduced the viable counts by 7·7-log10 units up to 7 days of incubation, when compared with the controls not treated with the bacteriocin. CONCLUSIONS: Aureocin A53 exhibited several features considered important for biopreservation and remained fully active in a food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Taken together, the results confirmed that aureocin A53 has potential to be used as a food preservative, representing an alternative to the use of nisin in biopreservation of dairy products.

Antimicrobial activity of Paenibacillus polymyxa SCE2 against some mycotoxin-producing fungi.

AIMS: The purpose of this study was to investigate the antagonistic activity of Paenibacillus polymyxa strain SCE2 against mycotoxigenic fungi and to characterize the antimicrobial compound. METHODS AND RESULTS: Strain SCE2 showed a broad inhibition spectrum against different mycotoxigenic fungi. The crude supernatant obtained from strain SCE2 was filtered with Amicon Diaflo membranes, and the antimicrobial activity was detected in the fraction ranging from 0.5 to 1 kDa. The bioautography of this fraction presented two inhibition zones with both indicator strains (Micrococcus sp. and Aspergillus versicolor), suggesting that more than one substance is produced by SCE2. Based on UV-visible spectral and liquid chromatography/mass spectrometry data, phenazine-1-carboxylic acid (PCA) was characterized as the major compound present in the highest purity active fraction. Drastic alterations in the cytoplasm of A. versicolor were observed by electron microscopy. CONCLUSIONS: One of the antimicrobial substances produced by P. polymyxa SCE2 is PCA. SIGNIFICANCE AND IMPACT OF THE STUDY: The broad antifungal spectrum observed by the compound produced by SCE2 suggests that it has the potential to be used as an alternative or supplementary method to chemical pesticides against mycotoxigenic fungi. This is the first description of a phenazine produced by a member of the genus Paenibacillus.

Hyptis pectinata essential oil: chemical composition and anti-Streptococcus mutans activity.

OBJECTIVES: The aim of the present study was to evaluate the anti-Streptococcus mutans activity of Hyptis pectinata essential oil, and present its promising potential against oral diseases. MATERIALS AND METHODS: The essential oil of H. pectinata was obtained by hydrodistillation from dried leaves and analyzed by GC / MS. The effectiveness of this essential oil regarding the antimicrobial activity against several S. mutans strains was investigated by the agar diffusion and microdilution methods, and chlorohexidine was used as a standard control. RESULTS: The H. pectinata essential oil exhibited considerable inhibitory effect against either all the clinical isolates obtained from patients' saliva or the ATCC strains tested, with minimum inhibitory and bactericidal concentrations of 200 microg ml(-1). The study also compared the efficiency of the emulsifying agents Tween 20, Tween 80, dimethyl sulfoxide and propylene glycol in H. pectinata essential oil when tested against S. mutans. The data obtained confirmed the better inhibitory effect of the oil when using all tested diluents, although Tween 80 seemed to be more suitable for emulsification. CONCLUSION: According to our results, H. pectinata essential oil can be considered a promising alternative to chlorhexidine for the control of oral bacteria-related diseases and hygiene.

Antifungal activity of Brazilian medicinal plants involved in popular treatment of mycoses.

A survey of medicinal plants used to treat common mycoses was done in the Curituba district, Sergipe State, Brazil. One hundred inhabitants were interviewed by health agents and traditional healers. Four different plants were the most cited (more than 50% of the citations): Ziziphus joazeiro, Caesalpinia pyramidalis, Bumelia sartorum and Hymenea courbaril. The aqueous extracts obtained following traditional methods and using different parts of these plants, were submitted to drop agar diffusion tests for primary antimicrobial screening. Only the water infusion extract of Ziziphus joazeiro and Caesalpinea pyramidalis presented a significant antifungal activity against Trichophyton rubrum, Candida guilliermondii, Candida albicans, Cryptococcus neoformans and Fonsecaea pedrosoi, when compared to the antifungal agent amphotericin B. The minimal inhibitory concentration (MIC) of the bioactive extracts was evaluated by the microdilution method. Best activity with a MIC of 6.5 microg/ml for both extracts was observed against Trichophyton rubrum and Candida guilliermondii. Ziziphus joazeiro and Caesalpinea pyramidalis extracts presented also low acute toxicity in murine models. The present study validates the folk use of these plant extracts and indicates that they can be effective potential candidates for the development of new strategies to treat fungal infections.

Identification of sialic acids on the cell surface of Candida albicans.

The cell-surface expression of sialic acids in two isolates of Candida albicans was analyzed by thin-layer and gas chromatography, binding of lectins, colorimetry, sialidase treatment and flow cytofluorimetry with fluorescein-labeled lectins. N-acetylneuraminic acid (NANA) was the only derivative found in both strains of C. albicans grown in a chemically defined medium. Its identification was confirmed by mass spectrometry in comparison with an authentic standard. The density of sialic acid residues per cell ranged from 1. 6x10(6) to 2.8x10(6). The surface distribution of sialic acids over the entire C. albicans was inferred from labeling with fluorescein-Limulus polyphemus and Limax flavus agglutinins and directly observed by optical microscopy with (FITC)-Sambucus nigra agglutinin (SNA), abrogated by previous treatment of yeasts with bacterial sialidase. Sialidase-treated yeasts generated beta-galactopyranosyl terminal residues that reacted with peanut agglutinin. In C. albicans N-acetyl-neuraminic acids are alpha2,6- and alpha2,3-linked as indicated by yeast binding to SNA and Maackia amurensis agglutinin. The alpha2,6-linkage clearly predominated in both strains. We also investigated the contribution of sialic acids to the electronegativity of C. albicans, an important factor determining fungal interactions in vivo. Adhesion of yeast cells to a cationic solid phase substrate (poly-L-lysine) was mediated in part by sialic acids, since the number of adherent cells was significantly reduced after treatment with bacterial sialidase. The present evidence adds C. albicans to the list of pathogenic fungi that synthesize sialic acids, which contribute to the negative charge of fungal cells and have a role in their specific interaction with the host tissue.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates.

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Plant extracts: search for new alternatives to treat microbial diseases.

Medicinal plants constitute the base of health care systems in many societies. The recovery of the knowledge and practices associated with these plant resources are part of an important strategy linked to the conservation of biodiversity, discovery of new medicines, and the bettering of the quality of life of poor rural communities. Research in phytosciences, an emerging multidisciplinary science, is almost unlimited, with several aspects to be discussed. Therefore, the focus of the present review is mainly on the antimicrobial and antioxidant properties of bioactive phytocompounds resultant of our research with crude plant extracts and essential oils of medicinal plants belonging to different families, used in various infectious disorders. The results obtained in the last years warrant the present review, discussing not only the use of several medicinal plants against bacteria, yeast, filamentous fungi and protozoa, but also their mechanisms of action, interactions with macromolecules and potential for toxicity in mammalian cells. Problems related to the efficacy of the isolation techniques and stability of bioactive compounds are also commented on. In addition, this review aims to emphasize the greatest importance to investigate plant species that have not been the subject of pharmacological studies, although their popular uses have been reported.

The husk fiber of Cocos nucifera L. (Palmae) is a source of anti-neoplastic activity.

In the present study, we investigated the in vitro anti-tumoral activities of fractions from aqueous extracts of the husk fiber of the typical A and common varieties of Cocos nucifera (Palmae). Cytotoxicity against leukemia cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (2 x 10(4)/well) were incubated with 0, 5, 50 or 500 microg/mL high- or low-molecular weight fractions for 48 h, treated with MTT and absorbance was measured with an ELISA reader. The results showed that both varieties have almost similar antitumoral activity against the leukemia cell line K562 (60.1 +/- 8.5 and 47.5 +/- 11.9% for the typical A and common varieties, respectively). Separation of the crude extracts with Amicon membranes yielded fractions with molecular weights ranging in size from 1-3 kDa (fraction A) to 3-10 kDa (fraction B) and to more than 10 kDa (fraction C). Cells were treated with 500 microg/mL of these fractions and cytotoxicity was evaluated by MTT. Fractions ranging in molecular weight from 1-10 kDa had higher cytotoxicity. Interestingly, C. nucifera extracts were also active against Lucena 1, a multidrug-resistant leukemia cell line. Their cytotoxicity against this cell line was about 50% (51.9 +/- 3.2 and 56.3 +/- 2.9 for varieties typical A and common, respectively). Since the common C. nucifera variety is extensively cultured in Brazil and the husk fiber is its industrial by-product, the results obtained in the present study suggest that it might be a very inexpensive source of new antineoplastic and anti-multidrug resistant drugs that warrants further investigation.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Characterization of Gordonia sp. strain F.5.25.8 capable of dibenzothiophene desulfurization and carbazole utilization.

A dibenzothiophene (DBT)-degrading bacterial strain able to utilize carbazole as the only source of nitrogen was identified as Gordonia sp. F.5.25.8 due to its 16S rRNA gene sequence and phenotypic characteristics. Gas chromatography (GC) and GC-mass spectroscopy analyses showed that strain F.5.25.8 transformed DBT into 2-hydroxybiphenyl (2-HBP). This strain was also able to grow using various organic sulfur or nitrogen compounds as the sole sulfur or nitrogen sources. Resting-cell studies indicated that desulfurization occurs either in cell-associated or in cell-free extracts of F.5.25.8. The biological responses of F.5.25.8 to a series of mutagens and environmental agents were also characterized. The results revealed that this strain is highly tolerant to DNA damage and also refractory to induced mutagenesis. Strain F.5.25.8 was also characterized genetically. Results showed that genes involved in desulfurization (dsz) are located in the chromosome, and PCR amplification was observed with primers dszA and dszB designed based on Rhodococcus genes. However, no amplification product was observed with the primer based on dszC.

Antimicrobial activity of Croton cajucara Benth linalool-rich essential oil on artificial biofilms and planktonic microorganisms.

We have previously demonstrated that a linalool-rich essential oil from Croton cajucara Benth presents leishmanicidal activity. In the present study, we demonstrate that this essential oil inhibits the growth of reference samples of Candida albicans, Lactobacillus casei, Staphylococcus aureus, Streptococcus sobrinus, Porphyromonas gingivalis and Streptococcus mutans cell suspensions, all of them associated with oral cavity disease. The purified linalool fraction was only inhibitory for C. albicans. Microbes of saliva specimens from human individuals with fixed orthodontic appliances, as well as the reference strains, were used to construct an artificial biofilm which was exposed to linalool or to the essential oil. As in microbial suspensions, the essential oil was toxic for all the microorganisms, while the purified linalool fraction mainly inhibited the growth of C. albicans. The compounds of the essential oil were separated by thin layer chromatography and exposed to the above-cited microorganisms. In this analysis, the proliferation of the bacterial cells was inhibited by still uncharacterized molecules, and linalool was confirmed as the antifungal component of the essential oil. The effects of linalool on the cell biology of C. albicans were evaluated by electron microscopy, which showed that linalool induced a reduction in cell size and abnormal germination. Neither the crude essential oil nor the purified linalool fraction is toxic to mammalian cells, which suggests that the essential oil or its purified components may be useful to control the microbial population in patients with fixed orthodontic appliances.

Inhibitory activity of Paenibacillus polymyxa SCE2 against human pathogenic micro-organisms.

Paenibacillus polymyxa strain SCE2 was shown to inhibit the growth of different potential human pathogenic bacterial strains and fungi in vitro. To determine the genetic characterization of this antimicrobial substance, strain SCE2 was transformed with plasmid pTV32(Ts), a delivery vector for Tn917-lac. After transposition, four mutants were shown to have lost their capability to inhibit Micrococcus sp. and Staphylococcus aureus RN450, but they continued to inhibit the growth of Corynebacterium fimi NCTC7547 and Escherichia coli HB101. Hybridization experiments using the DNA of the four mutants digested with different endonucleases and pTV32(Ts) as a probe showed that the place of insertion of Tn917-lac in the chromosome was the same in mutants 4 and 36 and in mutants 31 and 59, but different between these pairs. It is thought possible that more than one antimicrobial substance is being produced by strain SCE2.

Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi.

AIMS: To investigate the potential antagonistic activity of Paenibacillus peoriae strain NRRL BD-62 against phytopathogenic micro-organisms and to determine the physiological and biochemical characteristics of the antimicrobial compound produced by this strain. METHODS AND RESULTS: Strain NRRL BD-62 showed a broad inhibition spectrum with activity against various phytopathogenic bacteria and fungi. Physico-chemical characterization of the antimicrobial activity showed that it was stable during heat treatment and was retained even after autoclave at 121 degrees C for 10 min. The compound was also stable after the treatment with organic solvents, hydrolytic enzymes and its activity was preserved at a wide range of pH. The partial purification carried out by Sephadex G25 gel filtration showed two profiles of inhibition against the indicator strains tested, suggesting at least two different substances with distinct molecular weight. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of antimicrobial substances in P. peoriae. Besides the antimicrobial inhibition capability, the strain NRRL BD-62 is also able to effectively fix molecular nitrogen, and produce chitinases and proteases as well, suggesting that further studies should be addressed to use P. peoriae strain NRRL BD-62 as a plant growth promoter and/or as a biocontrol agent in field experiments.

The husk fiber of Cocos nucifera L. (Palmae) is a source of anti-neoplastic activity

In the present study, we investigated the in vitro anti-tumoral activities of fractions from aqueous extracts of the husk fiber of the typical A and common varieties of Cocos nucifera (Palmae). Cytotoxicity against leukemia cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (2 x 104/well) were incubated with 0, 5, 50 or 500 æg/mL high- or low-molecular weight fractions for 48 h, treated with MTT and absorbance was measured with an ELISA reader. The results showed that both varieties have almost similar antitumoral activity against the leukemia cell line K562 (60.1 ± 8.5 and 47.5 ± 11.9 percent for the typical A and common varieties, respectively). Separation of the crude extracts with Amicon membranes yielded fractions with molecular weights ranging in size from 1-3 kDa (fraction A) to 3-10 kDa (fraction B) and to more than 10 kDa (fraction C). Cells were treated with 500 æg/mL of these fractions and cytotoxicity was evaluated by MTT. Fractions ranging in molecular weight from 1-10 kDa had higher cytotoxicity. Interestingly, C. nucifera extracts were also active against Lucena 1, a multidrug-resistant leukemia cell line. Their cytotoxicity against this cell line was about 50 percent (51.9 ± 3.2 and 56.3 ± 2.9 for varieties typical A and common, respectively). Since the common C. nucifera variety is extensively cultured in Brazil and the husk fiber is its industrial by-product, the results obtained in the present study suggest that it might be a very inexpensive source of new antineoplastic and anti-multidrug resistant drugs that warrants further investigation.