Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.     

Antibacterial activity of hinokitiol against both antibiotic‐resistant and ‐susceptible pathogenic bacteria that predominate in the oral cavity and upper airways

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin‐resistant and ‐susceptible Staphylococcus aureus, antibiotic‐resistant and ‐susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin‐resistant S. aureus, methicillin‐susceptible S. aureus, antibiotic‐resistant S. pneumoniae isolates, antibiotic‐susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3–1.0, 0.5, and 0.3 μg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9‐22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 μg/mL hinokitiol, which decreased numbers of viable Ca9‐22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.     

Clinically acceptable restorations may be a hotbed for cariogenic microbes

doi: 10.1111/j.1741‐2358.2011.00571.x Clinically acceptable restorations may be a hotbed for cariogenic microbes Objective: The aim of this study was to investigate the cross‐sectional association of dental restorations with salivary cariogenic pathogens among the elderly to establish effective parameters of caries risk for this population. Materials and methods: Stimulated whole saliva was collected from 289 community‐dwelling older adults (66.2 ± 3.9 years old) who had 20 or more teeth. Salivary levels of three cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and lactobacilli) were estimated using quantitative polymerase chain reaction (real‐time PCR) method. Results: The mean number of residual teeth was 26.4, and restored teeth with crowns, inlays and composite resin were 7.35, 3.88 and 0.68, respectively. The number of crowns correlated positively with salivary S. mutans, S. sobrinus and lactobacilli. Multiple linear regression analyses showed that the number of restored teeth with crowns was independently associated with salivary S. mutans, S. sobrinus and lactobacilli after controlling for age, gender, number of residual teeth and salivary flow rate. Salivary flow rate was independently associated with salivary S. mutans and lactobacilli. Conclusion: The number of crowns had an association with salivary levels of cariogenic bacteria, suggesting that this parameter may be a caries risk indicator for the elderly population.     

Antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian medicinal plant Eremophila neglecta

Aims: To determine the antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian plant Eremophila neglecta. Methods and Results: Antimicrobial activities of serrulatane compounds 8,19‐dihydroxyserrulat‐14‐ene ( 1 ) and 8‐hydroxyserrulat‐14‐en‐19‐oic acid ( 2 ) were tested against Gram‐negative and Gram‐positive bacteria including human and veterinary pathogens and some multidrug‐resistant isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the compounds were determined by broth microdilution assay. Both compounds exhibited antibacterial activity against all Gram‐positive test strains. They showed antimycobacterial activity against isolates of Mycobacterium fortuitum and Mycobacterium chelonae. Of the five Gram‐negative bacteria tested, only Moraxella catarrhalis showed susceptibility to the compounds. Cytotoxic activities were tested in the Vero cell line. Compound 1 showed more activity than 2 in both antibacterial and cytotoxicity assays with cytotoxicity at concentrations similar to the MBC. Conclusions: Serrulatane compounds showed significant activity against medically important bacteria, with 1 exhibiting stronger antibacterial activity. However, they also displayed toxicity to mammalian cells. Significance and Impact of the Study: Serrulatanes are of interest as novel antibacterial compounds for use in biomedical applications; this study reports data obtained with a range of bacterial strains and mammalian cells, essential for assessing the capabilities and limitations of potential applicability of these compounds.     

Anti‐biofilm and bactericidal effects of magnolia bark‐derived magnolol and honokiol on Streptococcus mutans

Dental caries affects people of all ages and is a worldwide health concern. Streptococcus mutans is a major cariogenic bacterium because of its ability to form biofilm and induce an acidic environment. In this study, the antibacterial activities of magnolol and honokiol, the main constituents of the bark of magnolia plants, toward planktonic cell and biofilm of S. mutans were examined and compared with those of chlorhexidine. The minimal inhibitory concentrations of magnolol, honokiol and chlorhexidine for S. mutans were 10, 10 and 0.25 µg/mL, respectively. In addition, each agent showed bactericidal activity against S. mutans planktonic cells and inhibited biofilm formation in a dose‐ and time‐dependent manner. Magnolol (50 µg/mL) had greater bactericidal activity against S. mutans biofilm than honokiol (50 µg/mL) and chlorhexidine (500 µg/mL) at 5 min after exposure, while all showed scant activity against biofilm at 30 s. Furthermore; chlorhexidine (0.5–500 µg/mL) exhibited high cellular toxicity for the gingival epithelial cell line Ca9‐22 at 1 hr, whereas magnolol (50 µg/mL) and honokiol (50 µg/mL) did not. Thus; it was found that magnolol has antimicrobial activities against planktonic and biofilm cells of S. mutans. Magnolol may be a candidate for prevention and management of dental caries.     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Copper Oxide Nanomaterials Derived from Zanthoxylum armatum DC. and Berberis lycium Royle Plant Species: Characterization,Assessment of Free Radical Scavenging and Antibacterial Activity

Copper oxide nanomaterials were synthesized by a facile sustainable biological method using two plant species (Zanthoxylum armatum DC. and Berberis lycium Royle ). The formation of materials was confirmed by FT‐IR, ATR, UV‐visible, XRD, TEM, SEM, EDX, TGA and PL. The antibacterial activity was evaluated by agar well diffusion method to ascertain the efficacy of plant species extract and extract derived copper oxide nanomaterials against six Gram‐positive bacteria namely Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes, Corynebacterium diphtheriae, Corynebacterium xerosis, Bacillus cereus and four Gram‐negative bacteria such as Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris against the standard drug, Ciprofloxacin for Gram‐positive and Gentamicin for Gram‐negative bacteria, respectively. In both cases, copper oxide nanomaterials were found to be sensitive in all the bacterial species. Sensitivity of copper oxide nanomaterials shows an be higher as compared to plant species extract against different bacteria. Scavenging activity of plant extracts along with nanomaterials have been accessed using previously reported protocols employing ascorbic acid as standard. Scavenging activity of copper oxide nanomaterials shows an increase with increase in concentration. The biological activity (bactericidal and scavenging efficiency) of plant derived copper oxide nanomaterials revealed that these materials can be used as potent antimicrobial agent and DPPH scavengers in industrial as well as pharmacological fields.     

Prevalence of periodontopathic bacteria on the tongue dorsum of elderly people

Objectives: The aim of this study was to analyse the prevalence of oral bacteria on the dorsum of the tongue. In addition, the relationship between the number of teeth and the microflora present on the coating of the tongue in a population of 85‐year‐old people was assessed. Subjects and methods: Two hundred and five individuals (89 males, 116 females) from the same geographical area who were 85 years of age were examined. Five periodontopathic bacteria (Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Treponema denticola) and one cariogenic bacterium (Streptococcus mutans) were analysed using a polymerase chain reaction assay of tongue samples from the population. Results: Periodontal bacteria‐positive individuals have more teeth than that of periodontal bacteria‐negative people. Between the periodontal bacteria‐positive and ‐negative individuals, there were significant differences in the mean number of teeth for P. gingivalis (p < 0.0001), T. denticola (p < 0.001), F. nucleatum (p = 0.002), and T. forsythia (p = 0.005), while there were no significant differences for A. actinomycetemcomitans (p = 0.998) or S. mutans (p = 0.147). Conclusions: A wide range of species, including anaerobes, was detected in 85‐year‐old subjects. It was found that the detection of periodontal bacteria on the tongue coating increased with the number of teeth. There was a positive relationship between the tooth number and periodontopathic bacteria, except for A. actinomycetemcomitans.These results suggest that tongue care is essential for preventing oral disease and needs to be part of any oral care programme in elderly people.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Streptococcus mutans requires mature rhamnose‐glucose polysaccharides for proper pathophysiology,morphogenesis and cellular division

The cell wall of Gram‐positive bacteria has been shown to mediate environmental stress tolerance, antibiotic susceptibility, host immune evasion and overall virulence. The majority of these traits have been demonstrated for the well‐studied system of wall teichoic acid (WTA) synthesis, a common cell wall polysaccharide among Gram‐positive organisms. Streptococcus mutans, a Gram‐positive odontopathogen that contributes to the enamel‐destructive disease dental caries, lacks the capabilities to generate WTA. Instead, the cell wall of S. mutans is highly decorated with rhamnose‐glucose polysaccharides (RGP), for which functional roles are poorly defined. Here, we demonstrate that the RGP has a distinct role in protecting S. mutans from a variety of stress conditions pertinent to pathogenic capability. Mutant strains with disrupted RGP synthesis failed to properly localize cell division complexes, suffered from aberrant septum formation and exhibited enhanced cellular autolysis. Surprisingly, mutant strains of S. mutans with impairment in RGP side chain modification grew into elongated chains and also failed to properly localize the presumed cell wall hydrolase, GbpB. Our results indicate that fully mature RGP has distinct protective and morphogenic roles for S. mutans, and these structures are functionally homologous to the WTA of other Gram‐positive bacteria.     

Metal-ion susceptibility of oral bacterial species

Aims: This study aimed to evaluate the effect of lead (Pb) on growth of bacterial species related to dental diseases in vitro. Methods and Results: The effects of lead acetate on representative species of the oral flora were examined at 0·1–10 mmol l^?1 and compared with the effect of silver nitrate and ferrous sulfate. The minimal inhibitory concentration of lead acetate was between 0·15 and 5 mmol l^?1 for the bacterial strains tested. The minimal bactericidal concentration of lead acetate for most oral species was detected in the range of 5–10 mmol l^?1. Silver nitrate at a concentration of 1·25 mmol l^?1 was sufficient to exhibit antibacterial activity against almost all bacteria tested. Ferrous sulfate had the lowest effect. Conclusions: The study indicated a general antimicrobial effect of lead on oral bacterial species in the range of 0·15–10 mmol l^?1. The toxicity of silver nitrate was the highest, whereas that of ferrous sulfate was the lowest. Gram‐positive species had a tendency to be less susceptible for metals than Gram‐negatives. Significance and Impact of the Study: The study shows that it is possible that microbiological changes may occur in the dental plaque in children because of toxic exposure of environmental lead.     

Comparison of bacterial communities in faeces of beef cattle fed diets containing corn and wet distillers' grain with solubles

Aim: The mammalian intestinal microflora has been shown to impact host physiology. In cattle, intestinal bacteria are also associated with faecal contamination of environmental sources and human illness via foodborne pathogens. Use of wet distillers’ grains with solubles (WDGS) in cattle feed creates a gastrointestinal environment where some bacterial species are enriched. Here, we examine if a diet containing 40% WDGS results in fundamentally different microbial community structures. Methods and Results: The 20 002 16S r‐RNA gene sequences from 20 beef cattle were analysed using Sanger sequencing methods. At the genus level, Prevotella (Gram negative) and Anaerobacter (Gram positive) were the most frequently occurring bacteria in our beef cattle faecal samples. Diet‐associated differences in prevalence were noted for Prevotella but not Anaerobacter. Conclusions: Diet affects community structure. Faecal communities of co‐housed beef cattle are not identical. Significance and Impact of the Study: It is known that a diet of 40% corn‐based WDGS increases the generic Escherichia coli in the faeces and enriches E. coli O157:H7. The results from the current study suggest that in addition to previously observed changes in E. coli, the entire bacterial community structure is different for animals fed 40% corn‐based WDGS compared to a traditional corn‐finishing diet.     

Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions

This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity.     

Antibacterial activity of naringin derivatives against pathogenic strains

Aims: To study the antimicrobial activity of naringin (NAR), a flavonoid extracted from citrus industry waste, and NAR derivatives [naringenin (NGE), prunin and alkyl prunin esters] against pathogenic bacteria such as L. monocytogenes, E. coli O157:H7 and S. aureus. The relationship between the structure of the chemical compounds and their antagonistic effect was also analysed. Methods and Results: The agar dilution technique and direct contact assaying were applied. NGE, prunin and NAR showed no antimicrobial activity at a concentration of 0·25 mmol l^?1. Similarly, fatty acids with a chain length between C2 and C18 showed no antimicrobial activity at the same concentration. However, prunin‐6″‐O‐acyl esters presented high antibacterial activity, mainly against Gram‐positive strains. This activity increased with increasing chain length (up to 10–12 carbon atoms). Alkyl prunin esters with 10–12 carbon atoms diminished viability of L. monocytogenes by about 3 log orders and S. aureus by 6 log orders after 2 h of contact at 37°C and at a concentration of 0·25 mmol l^?1. The compounds examined were not effective against any of the Gram‐negative strains assayed, even at the highest concentration. Conclusions: Addition of sugars to the aglycone did not enhance its antimicrobial activity. Attachment of a saturated aliphatic chain with 10–12 carbon atoms to the A ring of the flavonoid (or to sugars attached to this ring), seems to be the most promising modification. In conclusion, alkyl prunin esters with a chain length of C10–C12 have promising features as antimicrobial agents because of their high antilisterial and antistaphylococcal activity. Significance and Impact of the Study: This study shows that it is possible to obtain NAR derivatives with important antimicrobial activity, especially against Gram‐positive pathogenic bacteria. It also provides guidelines on the structural modifications in similar molecules to enhance the antimicrobial activity.     

The changing faces of Streptococcus antigen I/II polypeptide family adhesins

Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall‐anchored adhesins identified in Gram‐positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C‐terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate‐binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N‐terminal α‐helix and a C‐terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram‐positive surface proteins that have adhesin domains flanked by α‐helical and proline‐rich regions.     

Inhibitory effect of methyl gallate and gallic acid on oral bacteria

This study examined the ability of methyl gallate (MG) and gallic acid (GA), the main compounds of gallo-tannins in Galla Rhois, to inhibit the proliferation of oral bacterial and the in vitro formation of Streptococcus mutans biofilms. The antimicrobial activities of these compounds were evaluated in vitro using the broth microdilution method and a beaker-wire test. Both MG and GA had inhibitory effects on the growth of cariogenic (MIC<8 mg/ml) and periodontopathic bacteria (MIC=1 mg/ml). Moreover, these compounds significantly inhibited the in vitro formation of S. mutans biofilms (MG, 1 mg/ml; GA, 4 mg/ml; P<0.05). MG was more effective in inhibiting bacterial growth and the formation of S. mutans biofilm than GA. In conclusion, MG and GA can inhibit the growth of oral pathogens and S. mutans biofilm formation, and may be used to prevent the formation of oral biofilms.     

Synthesis of short cationic antimicrobial peptidomimetics containing arginine analogues

Worldwide efforts are underway to develop new antimicrobial agents against bacterial resistance. To identify new compounds with a good antimicrobial profile, we designed and synthesized two series of small cationic antimicrobial peptidomimetics (1–8) containing unusual arginine mimetics (to introduce cationic charges) and several aromatic amino acids (bulky moieties to improve lipophilicity). Both series were screened for in vitro antibacterial activity against a representative panel of Gram‐positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram‐negative (Escherichia coli and Klebsiella pneumoniae) bacterial strains, and Candida albicans. The biological screening showed that peptidomimetics containing tryptophan residues are endowed with the best antimicrobial activity against S. aureus and S. epidermidis in respect to the other synthesized derivatives (MIC values range 7.5–50 µg/ml). Moreover, small antimicrobial peptidomimetics derivatives 2 and 5 showed an appreciable activity against the tested Gram‐negative bacteria and C. albicans. The most active compounds (1–2 and 5–6) have been tested against Gram‐positive established biofilm, too. Results showed that the biofilm inhibitory concentration values of these compounds were never up to 200 µg/ml. The replacement of tryptophan with phenylalanine or tyrosine resulted in considerable loss of the antibacterial action (compounds 3–4 and 7–8) against both Gram‐positive and Gram‐negative bacterial strains. Furthermore, by evaluating hemolytic activity, the synthesized compounds did not reveal cytotoxic activities, except for compound 5. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A study of the efficacy of ultrasonic waves in removing biofilms

doi:10.1111/j.1741‐2358.2009.00325.x
A study of the efficacy of ultrasonic waves in removing biofilms Objective: The removal of adherent biofilms was assessed using ultrasonic waves in a non‐contact mode. Materials and Methods: In in vitro experiments, Streptococcus mutans (S. mutans) biofilms were exposed to ultrasonic waves at various frequencies (280 kHz, 1 MHz, or 2 MHz), duty ratios (0–90%), and exposure times (1–3 minutes), and the optimal conditions for biofilm removal were identified. Furthermore, the effect of adding a contrast medium, such as micro bubbles (Sonazoid^®), was examined. The spatial distribution and architecture of S. mutans biofilms before and after ultrasonic wave exposure were examined via scanning electron microscopy. The biofilm removal effect was also examined in in vivo experiments, using a custom‐made oral cleaning device. Results: When a 280 kHz probe was used, the biofilm‐removing effect increased significantly compared to 1 and 2 MHz probes; more than 80% of the adherent biofilm was removed with a duty cycle of 50–90% and a 3 minutes exposure time. The maximum biofilm‐removing effect was observed with a duty cycle of 80%. Furthermore, the addition of micro bubbles enhanced this biofilm‐removing effect. In in vivo experiments, moderate biofilm removal was observed when a 280 kHz probe was used for 5 minutes. Conclusions: This study demonstrated that ultrasonic wave exposure in a non‐contact mode effectively removed adherent biofilms composed of S. mutans in vitro.     

Interference of Seasonal Variation on the Antimicrobial and Cytotoxic Activities of the Essential Oils from the Leaves of Iryanthera polyneura in the Amazon Rain Forest

The essential oils (EOs) obtained from the leaves of Iryanthera polyneura Ducke trees was chemically Assessed and tested for the ability of inhibiting the growth of Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans and S. sanguinis. The oil was also tested against breast (MCF‐7) and prostate (PC‐3) cancer cell lines. Minimum bactericidal concentrations (MBCs) and 50 % inhibition concentrations (IC₅₀) values were obtained. EOs were active against Gram‐positive bacteria. Spathulenol, α‐cadinol and τ‐muurolol were major components of EOs. The oils showed a higher cytotoxicity against PC‐3 than MCF‐7 cells, although the oils were active against both cell types. Oils obtained from leaves collected in the dry season were more active against E. faecalis, S. aureus and PC‐3, while the oils obtained from leaves collected in the rainy season were more active against S. mutans, S. sanguinis and MCF‐7. The antibacterial and cytotoxic activities of the essential oils from the leaves of I. polyneura are related to the seasonal climate variation and are influenced by compounds that are minor components of the oils.     

Detection, cellular localization and antibacterial activity of two lytic enzymes of Pediococcus acidilactici ATCC 8042

Aim: In Pediococcus acidilactici ATCC 8042, two activities of peptidoglycan hydrolase (PGH) with lytic effect against Micrococcus lysodeikticus and Staphylococcus aureus have been detected. This work intends to elucidate the growth phase of maximum lytic activity, the localization and the effectiveness of the activity against pathogenic Gram‐negative and Gram‐positive bacteria. Methods and Results: Cells were grown in MRS medium and collected at different growth stages, and the proteins were extracted. The highest PGH activity was found during the logarithmic growth phase in the protein fraction bound to the cell membrane. From this fraction, two distinct proteins bands (110‐ and 99‐kDa) in SDS–PAGE were partially purified with a three‐step procedure. Both bands showed lytic activity against M. lysodeikticus. Mass spectrometry analysis (LC/ESI‐MS/MS) indicated that the 110‐kDa band corresponded to a protein of unknown function. The 99‐kDa band corresponded to a N‐acetylmuramidase that harboured catalytic sites with N‐acetylmuramoyl‐l ‐alanine amidase and N‐acetylglucosaminidase activities. Both proteins are reported in the Ped. acidilactici 7_4 genome. The fraction containing the concentrated proteins (110 and 99 kDa) inhibited the growth of several pathogenic strains as: Bacillus cereus, Listeria monocytogenes and Salmonella typhimurium. The growth of S. aureus was diminished by 3 logarithmic units as early as 0·5 h of growth, while inhibition of Escherichia coli and Ped. acidilactici was observed after 18 and 8 h, respectively (both in one logarithmic unit). The minimum inhibitory concentration against S. aureus was 10 μg ml^?1. Conclusion: Pediococcus acidilactici harbours at least two lytic enzymes, one of them recognized as PGH for the first time, which exert antibacterial activity against several bacterial strains. Significance and Impact of the Study: Both PGH activities have a broad growth inhibition spectrum and could be used to control pathogenic bacteria. Because this activity comes from a lactic acid bacterium, it could be safely used in manufacturing processes of fermented foods.