Comparative studies on the activity of basil--an essential oil from Ocimum basilicum L.--against multidrug resistant clinical isolates of the genera Staphylococcus,Enterococcus and Pseudomonas by using different test methods

The essential oil basil is obtained from the aerial parts of Ocimum basilicum L. After gas chromatographic separation, the following components were identified: linalol (54.95%), methylchavikol (11.98%), methylcinnamat (7.24%) and linolen (0.14%). The activity of basil against multidrug resistant clinical isolates from the genera Staphylococcus, Enterococcus and Pseudomonas has been studied. For this purpose, standard and modified broth macrodilution methods were used and time kill kinetic of basil was studied. The minimum inhibitory concentrations (MICs) were reported between 0.0030% and 0.0007% (v/v). These concentrations were compared with the inhibitory concentrations (ICs) and the logs of the bacterial counts reduction both obtained by basil diluted in 1% Tween (Tw) 80, saline test solution (STS) and spiritus vini (Sv) 95 degrees instead in a broth. The data, obtained after application of different methods of investigation and validated with membrane filtration, showed a strong inhibitory effect of basil on the test bacteria. The chosen bacteria are widespread and pose serious therapeutic difficulties because of their high level of resistance. For this reason, the results obtained were considered encouraging.     

An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora

Adrenomedullin, a novel vasoactive peptide, is known to be expressed by many surface epithelial cells and it was postulated that this peptide may have a protective role. The objective of the study was to assess the antimicrobial activity of adrenomedullin against members of the human skin, oral, respiratory tract and gut microflora using disc diffusion and broth microdilution assays. All strains of bacteria screened in an agar diffusion assay were sensitive; gram-positive and gram-negative bacteria were equally susceptible. No activity against the yeast Candida albicans was observed. In a broth microdilution assay, minimum inhibitory and minimum bacteriocidal concentrations ranged from 7.75 x 10(-1) to 12.5 microg ml(-1) and 0.003 to > 25.0 microg ml(-1), respectively. We propose an antimicrobial role for adrenomedullin. participating in the prevention of local infection, thus contributing to host defence systems.     

Bactericidal action of carbon dioxide laser radiation in experimental dental root canals

The ability of a carbon dioxide laser to sterilize the root canal of human teeth has been investigated. Three oral bacteria, Streptococcus sanguis, Streptococcus mutans, and Actinomyces viscosus, and three other bacteria, Bacillus cereus, Staphylococcus aureus, and Pseudomonas aeruginosa were used as experimental organisms. Exposure of cells on glass slides to laser radiation showed there was little difference in the exposure required to kill these six organisms. Complete recovery of bacteria from the root canal was initially a problem and was only achieved when bacterial manipulations and removal were carried out in rapid succession, within 5 min of inoculation. However, the geometry of the instrumented canal and the laser alignment were major factors in achieving consistent cell death of oral bacteria in the root canals. Using sets of 10 teeth, four repeated exposures of 10 W for 1 s was found to sterilize 4 or more of the teeth.     

Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances

The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16-20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.     

Effect of subinihibitory and inhibitory concentrations of Plectranthus amboinicus (Lour.) Spreng essential oil on Klebsiella pneumoniae

We evaluated the antimicrobial activity and some mechanisms used by subinhibitory and inhibitory concentrations of the essential oil, obtained from leaves of Plectranthus amboinicus, against a standard strain of Klebsiella pneumoniae and 5 multiresistant clinical isolates of the bacteria. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), the rate of kill and the pH sensitivity of the essential oil were determined by microdilution tests performed in 96-well plates. Subinhibitory and inhibitory concentrations of the essential oil were tested in order to check its action on K. pneumoniae membrane permeability, capsule expression, urease activity and cell morphology. The MIC and MBC of the essential oil were 0.09±0.01%. A complete inhibition of the bacterial growth was observed after 2h of incubation with twice the MIC of the essential oil. A better MIC was found when neutral or alkaline pH broth was used. Alteration in membrane permeability was found by the increase of crystal violet uptake when the bacteria were incubated with twice the MIC levels of the essential oil. The urease activity could be prevented when all the subinhibitory concentrations were tested in comparison to the untreated group (p<0.001). Alteration of the bacterial morphology besides inhibition of the capsule expression was verified by atomic force microscopy, and Anthony's stain method, respectively. Our data allow us to conclude that the essential oil of P. amboinicus can be a good candidate for future research.     

Factors affecting adhesion of bacterial to a tooth in vitro

The initial adhesion of oral bacteria to a tooth in vitro was examined. The organisms were grown in broth with and without sucrose, and suspensions made either in broth or a modified Ringer's solution. The tooth used was either dry or coated with natural or synthetic saliva. Adhesion was determined by counting organisms removed from the tooth surface by simple washing or by sonication. It was found that the firmest bonding occurred when a dry tooth was immersed in a suspension of bacteria in Ringer's solution; the prior growth of the organisms in the presence of sucrose did not affect adhesion. It was concluded that instantaneous irreversible adhesion of bacteria to a tooth occurs without the need for active metabolism, and that this process is inhibited by the presence of competing organic substances which probably produce a surface-conditioning film.     

罗氏乳杆菌无细胞上清培养液移除胆固醇能力的研究

本文探讨了罗氏乳杆菌DSM122460无细胞上清培养液(Cell-Free Supernatant,CFS)移除胆固醇的能力。采用邻苯二甲醛法测定DSM122460和对照菌株ST-III发酵过程中及其CFS对胆固醇的移除能力,并研究不同CFS浓度下的移除能力。并采用HPLC法测定CFS对照、热处理组和pH7.0组的胆盐水解酶活力,同时测定其移除胆固醇能力。结果显示,DSM122460不仅在发酵过程中具有较高的移除胆固醇能力,其CFS也表现出较高的移除能力,CFS中含有除胆盐水解酶以外的可移除胆固醇的蛋白类成分。这提示可能存在一种乳酸菌移除胆固醇的新机制。     

Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.     

Dual mode of action of Bac7, a proline-rich antibacterial peptide

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.     

Comparison of the WST-8 colorimetric method and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria

The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24h tended to give lower MICs. The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent agreement between the MICs determined after 24h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria.     

Ice-nucleating bacteria from the guts of two sub-antarctic beetles, hydromedion sparsutum and perimylops antarcticus (Perimylopidae)

The site of ice nucleation in the freeze-tolerant, sub-Antarctic beetle Hydromedion sparsutum has been investigated. Ice+ bacteria, active at above -2.0 degrees C, were isolated from the guts of beetles and identified as a fluorescent Pseudomonas species. Other possible sites of nucleation, including the hemolymph, were examined but had a lower activity. Ice+ bacteria were isolated from mixed populations, isolated from the guts of adult beetles, and grown on nutrient agar plates and in nutrient broth. Nucleation activity of the broth culture peaked after only 2 days although the number of live cells continued to increase until day 6. These cultures were used to determine the maximum nucleation activity of a bacterial suspension in sterile distilled water (-3.4 degrees C) and the dilution factor required to cause a 50% reduction in activity (10(4)). The original bacterial suspension had an absorbance of 0.5 measured at 660 nm and contained 6 x 10(11) bacteria per milliliter. From this it is estimated that only 1 in 10(6) bacteria possessed the highest levels of ice-nucleating activity. Other insect species, including Perimylops antarcticus, which are found in habitats similar to that of H. sparsutum, were examined for the presence of ice+ bacteria. All contained ice-nucleating bacteria in their guts but with a lower level of activity than in H. sparsutum. Copyright 1999 Academic Press.     

Antimicrobial activity and effects of resveratrol on human pathogenic bacteria

Resveratrol (3,4′,5-trihydroxistilbene) is a phytoalexin commonly found in food and drinks, which is thought to possess antimicrobial activity. These effects together with its well known antioxidant properties are beneficial for the prevention of some diseases, e.g. cancer. In this study we have verified that resveratrol has antibacterial activity against all tested Gram-positive bacteria using both the disk diffusion and broth microdilution methods. Time kill assays of this compound against Gram-positive bacteria showed that its effects on the growth of bacterial cells were due to bacteriostatic action. The addition of resveratrol has allowed the identification of changes in cell morphology and DNA contents, which have been assessed through microscopic analysis and flow cytometry; this suggests that the cell cycle is affected by resveratrol. This study indicates that this compound may have potential as a natural antibacterial agent for both food preservation and medicinal use.     

Alkalotolerance of Yersinia enterocolitica as a basis for selective isolation from food enrichments.

Alkalotolerance of Yersinia enterocolitica measured in solutions of potassium hydroxide with 0.5% sodium chloride was influenced by the cell suspension medium, temperature, and growth phase. The rate of cell destruction (delta log N per minute) was five times greater at 30 degrees C than at 20 degrees C. Differences in the degree of cell destruction at various concentrations of potassium hydroxide were related to pH and not to osmolarity. The addition of peptones to potassium hydroxide provided a protective effect that was greater for cells suspended in Trypticase soy broth than for those suspended in phosphate-buffered sorbitol-bile salts broth. Log-phase cells were less alkalotolerant than cells in the stationary phase of growth. A modified procedure for alkali treatment, using peptone-supplemented 0.5% potassium hydroxide-0.5% sodium chloride and the addition of a pH 6.6 buffer after treatment to prevent further cell destruction, was used to observe a marked difference in alkalotolerance between Y. enterocolitica and other gram-negative bacteria. Despite this difference, alkali treatment was not highly successful for recovery of Y. enterocolitica from enrichments of seeded foods in comparison with selective enrichment in bile-oxalate-sorbose broth.     

Susceptibility of Fusobacterium nucleatum to killing by peroxidase-iodide-hydrogen peroxide combination in buffer solution and in human whole saliva

Some Gram-negative anaerobic bacteria have been associated with the infection of tooth supporting tissues, i.e. periodontitis. Of these bacteria, Fusobacterium nucleatum is sensitive to lactoperoxidase/myeloperoxidase-iodide-hydrogen peroxide system in vitro, but salivary concentrations of thiocyanate abolishes the bactericidality. These bacteria are located in periodontal pockets, on oral mucosa and in saliva. Although F. nucleatum most probably does not belong to the group of main periodontal pathogens, it sustains its proportion in the periodontal flora when gingivitis progresses to periodontitis. In this study, the sensitivity of F. nucleatum to different horseradish peroxidase-iodide-hydrogen peroxide combinations was tested both in buffer and in sterilized human whole saliva. Horseradish peroxidase was chosen because it does not bind thiocyanate at pH > or = 6. After 1h incubation at 37 degrees C, the cell viability was estimated by plate count and with flow cytometer using LIVE/DEAD BacLight kit (Molecular Probes, USA). In saliva, the horseradish peroxidase (50 microg/mL)-iodide (2.5 mM)-hydrogen peroxide (2.5 mM) combination decreased the amount of viable bacteria to 37% compared to 85% in the control without any of the components when measured with flow cytometer. Replacement of buffer by saliva decreased the bactericidality of the peroxidase system. However, in buffer less iodide and hydrogen peroxide was needed to produce significant decrease in the number of viable bacteria when measured by plate count than with flow cytometer. Our study shows that horseradish peroxidase-iodide-hydrogen peroxide combination is able to kill F. nucleatum cells in saliva. Horseradish peroxidase-iodide-hydrogen peroxide combination may be useful to diminish the degree of re-colonization of periodontitis-associated bacteria after periodontal therapy and to inhibit the transmission of these bacteria via saliva.     

Antimicrobial properties of diacetyl.

Diacetyl preparations from three commercial sources were found to be essentially similar when tested primarily against a set of 40 cultures, including 10 of lactic acid bacteria, 4 of yeasts, 12 of gram-positive non-lactic acid bacteria, and 14 of gram-negative bacteria. The compound was effective at pH less than or equal to 7.0 and progressively ineffective at pH greater than 7.0. The lactic acid bacteria were essentially unaffected by concentrations between 100 and 350 micrograms/ml over the pH range of 5.0 to 7.0. Of the 12 gram-positive non-lactic acid bacteria, 11 were inhibited by 300 micrograms/ml at pH less than or equal to 7.0. The three yeasts and the 13 gram-negative bacteria that grew at pH 5.5 were inhibited by 200 micrograms/ml. Diacetyl was ineffective against four clostridia under anaerobic conditions. It was lethal for gram-negative bacteria and generally inhibitory for gram-positive bacteria. Nongrowing cells were not affected. The effectiveness of diacetyl was considerably less in brain heart infusion broth, Trypticase soy agar, and cooked-meat medium than in nutrient broth or plate count agar. The antimicrobial activity was antagonized by glucose, acetate, and Tween 80 but not by gluconic acid. As an antimicrobial agent, diacetyl was clearly more effective against gram-negative bacteria, yeasts, and molds than against gram-positive bacteria.     

GROWTH RESPONSE OF SALMONELLA SPP. TO CYCLOHEXIMIDE AMENDMENT IN MEDIA

The present study was designed to evaluate cycloheximide as a potential media amendment to prevent fungal overgrowth on selective media for salmonellae enumeration. The objectives were to determine the effect of cycloheximide on Salmonella spp growth rates and to determine the effect of cycloheximide addition on Salmonella enumeration in selective media. The bacteria tested included two strains of Salmonella typhimurium (NO/NA and LT2) and one strain of Salmonella arizonae. All strains were grown in tryptic soy broth containing cycloheximide to determine the effect of cycloheximide on bacterial specific growth rates. The growth rate of all strains grown in tryptic soy broth were not significantly influenced by addition of cycloheximide at concentrations up to 1,000 mg/L. Growth rates of S. typhimurium NO/NA in minimal media were significantly decreased by addition of cycloheximide aerobically (300 mg/L) and anaerobically (600 mg/L). However, S. typhimurium NO/NA populations on brilliant green agar, MacConkey agar, and from selenite cysteine broth and tetrathionate broth were not affected by cycloheximide additions at concentrations up to 1,000 mg/L. Cycloheximide has potential as a fungistat additive for salmonellae selective media.     

A rapid method for determination of in vitro susceptibility to antibiotics with a bulk acoustic wave bacterial growth biosensor

A novel bulk acoustic wave (BAW) bacterial growth biosensor was developed to study in vitro susceptibility by continuous monitoring of disturbances of bacterial growth at low antibiotic concentrations, followed by the accurate and rapid estimation of growth kinetic parameters and minimum inhibitory concentrations (MICs). The susceptibilities of bacteria, e.g. Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pr. morganii and Pr. mirabilis , to various antibiotics, e.g. penicillin, streptomycin, gentamicin and cefotaxime, were investigated, respectively, and the MICs were rapidly determined with a higher reproducibility than the conventional broth micro-dilution technique (BMDT). The effects of cell constant of conductivity electrode, pH and temperature on bacterial growth and biosensor signals were discussed in detail. The proposed method offers an effective alternative to the conventional methods.     

土茯苓提取物抗细菌活性的研究

通过测定土茯苓提取物对革兰氏阳性菌和革兰氏阴性菌的抑菌活性,来更全面的评价和综合利用土茯苓资源,实验结果表明土苓955乙醇和乙酸乙酯的提取物抑菌范围广,服抑菌活性强,这两种提取物的MIC和MBC值显示了土茯苓作为抗细菌资源的可利用价值。     

Antimicrobial susceptibility tests for anaerobic bacteria. Comparison of Wilkins-Chalgren agar reference method and a microdilution method,and determination of stability of antimicrobics frozen in broth

Susceptibility results for anaerobic bacteria obtained with a microdilution method with either Wilkins-Chalgren broth or Brain Heart Infusion broth were compared to results obtained with the reference agar dilution method proposed by the National Committee for Clinical Laboratory Standards. Results were generally comparable, but the minimal inhibitory concentrations obtained with the microdilution method were slightly lower than those obtained with the agar dilution method. Antibiotics stored in these broths in microdilution trays at-20°C were stable for up to 130 days.