Potency of extract contents from selected tropical chewing sticks against Staphylococcus aureus and Staphylococcus auricularis

Two strains of enteropathogenic Gram-positive bacteria (Staphylococcus auricularis, NIAH 11484 and Staphylococcus aureus, IFO 12732) were tested in vitro for their resistance to extracts from tropical chewing stick species, namely, Garcinia mannii Heck, Masularia accuminata (G. Don) Benth, Zanthoxylum gilletii (DeWild) Waterman, Terminalia glaucescens Plauch ex Benth, Azadiracta indica A. Juss, Anogeissus leiocarpus Guill & Perr and Pseudocedrela kotschyi (Schweinf.). The chewing sticks are normally used for oral hygiene and this is felt to be related to their ability to attack bioagents that could invade the mouth during normal nutrition. Results from our study showed that the antibiotic properties of test chewing stick species vary and are target-microbe-specific. Of the species examined, only T. glaucescens showed appreciable broad antibiotic effect against S. aureus and S. auricularis. Intense antibiotic activity against S. aureus occurred when using a 2.0 g/l extract concentration and a 30-h incubation. T. glaucescens also showed intense activity against S. auricularis at 2.0 g/l concentration, 30-h incubation when all other extracts had lost their potency. A. indica is, however, most effective against S. aureus, showing appreciable antibiotic activity at 0.4 g/l concentration, 30-h incubation. Z. gilletii has no antibiotic activity against any of the test bacteria. The antistaphylococcus compounds in T. glaucescens and A. indica are worthy of isolation and further analyses.     

Studies on a new marine streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus

A new actinomycete strain designated as BT-408 producing polyketide antibiotic SBR-22 and showing antibacterial activity against methicillin resistant Staphylococcus aureus has been characterized and found to be a novel strain of Streptomyces psammoticus. Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been optimized. Glucose and ammonium nitrate were found to be best carbon and nitrogen sources respectively for growth and antibiotic production. Similarly initial medium pH of 7.2, incubation temperature of 30 degrees C and incubation time of 96 h were found to be optimal. Optimization of medium and cultural conditions resulted in 1.82-fold increase in antibiotic yield.     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

黑大蒜提取物联合抗生素对金黄色葡萄球菌和肠埃希菌的体外抗菌作用研究

评价黑大蒜提取物分别与头孢唑林或庆大霉素联合应用,对金黄色葡萄球菌和大肠埃希菌的体外抗菌效应。采用液体稀释法分别测定黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的最低抑菌浓度（MIC）。采用棋盘法设计,微量肉汤稀释法测定黑大蒜提取物联合头孢唑林或庆大霉素对金黄色葡萄球菌和大肠埃希菌的MIC,并计算部分抑菌浓度（FIC指数）。测定黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的时间-杀菌曲线。黑大蒜提取物对金黄色葡萄球菌的MIC为256μg/mL,黑大蒜提取物对大肠埃希菌的MIC为256μg/mL。时间-杀菌曲线结果显示黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的抑菌作用呈现较强的浓度依赖性。黑大蒜提取物联合头孢唑林后对金黄色葡萄球菌的FIC指数为0.75;黑大蒜提取物联合庆大霉素后对大肠埃希菌的FIC指数为0.5。黑大蒜提取物与头孢唑林或庆大霉素联合用药,可明显降低抗生素对金黄色葡萄球菌和大肠埃希菌的MIC,表现为相加和协同效应。     

Adaptational change in proline and water content of Staphylococcus aureus after alteration of environmental salt concentration

Adaptation of Staphylococcus aureus to a change in salinity was studied by estimating the intracellular content of water and proline after alteration of the salt concentration of the culture medium. The intracellular water content of S. aureus cultured in normal broth was 1.70 g/g (dry weight). After transfer to 1.8 M NaCl-containing broth, the water content decreased to 0.80 g/g (dry weight) within 1 min. After changing the salt concentration of the medium, intracellular free proline (assumed to be one of the osmoregulators in S. aureus) increased gradually from 0 to 1,400 mumol/g (dry weight) during 30 min of incubation at 37 degrees C. The water content rose to 0.88 g/g (dry weight) in 30 min. Proline was not taken up at 0 to 4 degrees C, suggesting that the process was one of active transport. The salt tolerance of S. aureus, therefore, appears to occur initially by dehydration of the cell after transfer from a medium of low salinity to one of high salinity and then by accumulation of proline, which carries water into the cell with it.     

Adaptational change in proline and water content of Staphylococcus aureus after alteration of environmental salt concentration.

Adaptation of Staphylococcus aureus to a change in salinity was studied by estimating the intracellular content of water and proline after alteration of the salt concentration of the culture medium. The intracellular water content of S. aureus cultured in normal broth was 1.70 g/g (dry weight). After transfer to 1.8 M NaCl-containing broth, the water content decreased to 0.80 g/g (dry weight) within 1 min. After changing the salt concentration of the medium, intracellular free proline (assumed to be one of the osmoregulators in S. aureus) increased gradually from 0 to 1,400 mumol/g (dry weight) during 30 min of incubation at 37 degrees C. The water content rose to 0.88 g/g (dry weight) in 30 min. Proline was not taken up at 0 to 4 degrees C, suggesting that the process was one of active transport. The salt tolerance of S. aureus, therefore, appears to occur initially by dehydration of the cell after transfer from a medium of low salinity to one of high salinity and then by accumulation of proline, which carries water into the cell with it.     

乳杆菌抑制金黄色葡萄球菌对HeLa细胞的粘附

对弯曲乳杆菌Lactobacillus crispatus T79-3和T90-1、詹氏乳杆菌Lactobacillus jensenii T118-3和T231-1四株乳杆菌对金黄色葡萄球菌生长的抑制效果以及抑菌成分进行了分析,比较乳杆菌排除、竞争、置换3种不同作用方式对金黄色葡萄球菌粘附HeLa细胞的抑制作用。结果表明4株乳杆菌皆能抑制金黄色葡萄球菌的生长及其粘附HeLa细胞的能力,分析发现4株乳杆菌发挥抑制作用的主要成分是有机酸,同时比较分析乳杆菌3种不同作用方式发现它们对金黄色葡萄球菌粘附HeLa细胞的抑制效果不同,其中,排除作用方式效果最好。另外,乳杆菌对金黄色葡萄球菌粘附HeLa细胞的抑制作用具有浓度依赖性,随着乳杆菌浓度增大,抑制作用增强并逐渐达到饱和。4株乳杆菌中,T79-3粘附能力最强,对金黄色葡萄球菌的抑制作用最强,排除作用方式抑制金黄色葡萄球菌粘附HeLa细胞作用效果较好,提示乳杆菌T79-3有可能作为益生菌防治妇女泌尿生殖道感染。     

Interaction of methylxanthines and gentamicin against Staphylococcus aureus and Pseudomonas aeruginosa: role of phosphodiesterase inhibition

Previous studies showed that methylxanthines increased the antimicrobial activity of gentamicin against Staphylococcus aureus and Pseudomonas aeruginosa. In this study, the effect of non-selective phosphodiesterase (PDE) inhibitors (methylxanthines: aminophylline and caffeine) and partially selective PDE inhibitors, dipyridamole and sildenafil, was evaluated on the antimicrobial activity of gentamicin using checkerboard method. Aminophylline at concentrations of 0.5 and 1 mg/ml reduced the minimal inhibitory concentration (MIC) of gentamicin (2 μg/ml) 2 and 4 times against S. aureus, and at concentrations of 0.5 and 2 mg/ml reduced the MIC of gentamicin (4 μg/ml) 2 and 4 times, respectively, against P. aeruginosa. Caffeine at concentrations of 1 and 2 mg/ml reduced the MIC of gentamicin (2 μg/ml) 4 and 32 times against S. aureus, and at concentrations of 0.12 and 2 mg/ml reduced the MIC of gentamicin (4 μg/ml) 2 and 4 times, respectively, against P. aeruginosa. However, dipyridamole and sildenafil (32 μg/ml) did not show any effect on MIC of gentamicin against S. aureus and P. aeruginosa. These results suggest that methylxanthines could increase gentamicin effects against S. aureus and P. aeruginosa but this effect is not mediated by inhibition of PDE 5, 6, 8, 10 and 11.     

Antibacterial activity of honey and propolis from Apis mellifera and Tetragonisca angustula against Staphylococcus aureus

AIMS: The antibacterial activity against Staphylococcus aureus of honey and propolis produced by Apis mellifera and Tetragonisca angustula was evaluated. Secondary aims included the study of the chemical composition of propolis and honey samples and its relationship with antibacterial activity against S. aureus. METHODS AND RESULTS: The antibacterial activity of honey and propolis was determined by the method of macrodilution. The minimum inhibitory concentration (MICs) of A. mellifera honey ranged from 126.23 to 185.70 mg ml(-1) and of T. angustula from 142.87 to 214.33 mg ml(-1). For propolis, the MIC ranged from 0.36 to 3.65 mg ml(-1) (A. mellifera) and from 0.44 to 2.01 mg ml(-1) (T. angustula). Honey and propolis were evaluated by high-performance liquid chromatography. Some typical compounds of Brazilian propolis were also identified in honey samples. Principal component analysis revealed that the chemical composition of honey and propolis samples was distinct based on the geographical location of the samples. CONCLUSIONS: Propolis samples had higher antibacterial activity against S. aureus when compared with honey. However, both propolis and honey samples had antibacterial against S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: These antimicrobial properties would warrant further studies on the clinical applications of propolis and honey against S. aureus.     

Antibacterial activity of ethanolic and aqueous extracts of Acacia aroma Gill. ex Hook et Arn

The purpose of the present study was to investigate the antibacterial activity of seven ethanolic extracts and three aqueous extracts from various parts (leaves, stems and flowers) of A. aroma against 163 strains of antibiotic multi-resistant bacteria. The disc diffusion assay was performed to evaluate antibacterial activity of the A. aroma crude extracts, against several Gram-positive bacteria (E. faecalis, S. aureus, coagulase-negative stahylococci, S. pyogenes, S. agalactiae, S. aureus ATCC 29213, E. faecalis ATCC 29212) and Gram-negative bacteria (E. coli., K. pneumoniae, P. mirabilis, E. cloacae, S. marcescens, M morganii, A. baumannii, P. aeruginosa, S. maltophilia, E. coli ATCC 35218, P. aeruginosa ATCC 27853, E. coli ATCC 25922). All ethanolic extracts showed activity against gram-positive bacteria. Among all obtained extracts, only leaf and flower fluid extracts showed activity against Gram-negative bacteria. Based on this bioassay, leaf fluid extracts tended to be the most potent, followed by flower fluid extracts. Minimal inhibitory concentration (MIC) values of extracts and antibiotics were comparatively determined by agar and broth dilution methods. Both extracts were active against S. aureus, coagulase-negative stahylococci, E. faecalis and E. faecium and all tested Gram-negative bacteria with MIC values from 0.067 to 0.308 mg/ml. In this study the minimal bactericidal concentration (MBC) values were identical or twice as high than the corresponding MIC for leaf extracts and four or eight times higher than MIC values for flower extracts. This may indicate a bactericidal effect. Stored extracts have similar antibacterial activity as recently obtained extracts. The A. aroma extracts of leaves and flowers may be useful as antibacterial agents against Gram- negative and Gram-positive antibiotic multi-resistant microorganisms.     

Antibiotic resistance and probiotic properties of dominant lactic microflora from Tungrymbai, an ethnic fermented soybean food of India

The present investigation was conducted to assess lactic acid bacteria present in traditionally fermented food of ethnic tribes in India for probiotic properties, antibacterial activity, and antibiotic tolerance behavior. Enterococcus sp., Lactobacillus sp., and Lactococcus sp. showed antibacterial activity against Bacillus cereus MTCC 430, Staphylococcus aureus subsp. aureus MTCC 740, and Salmonella enterica ser. paratyphi A MTCC 735. Lactococcus sp. and Lactobacillus sp. could tolerate acidic conditions (pH 2) and high bile salt concentration (4000 ppm). The lactic microflora were found to be sensitive to most common antibiotics, except for cloxacillin (5 μg), cephalexin (30 μg), and cephalothin (30 μg).     

The Staphylococcus aureus surface protein IsdA mediates resistance to innate defenses of human skin

Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.     

Ranatuerin 1T: an antimicrobial peptide isolated from the skin of the frog, Rana temporaria.

A peptide, termed ranatuerin 1T, with growth-inhibiting activity toward Staphylococcus aureus, was isolated from an extract of the skin of the European brown frog, Rana temporaria. The primary structure of the peptide was established as: GLLSGLKKVG10 KHVAKNVAVS20LMDSLKCKIS30GDC. In common with other anti-microbial peptides from Ranid frogs, (e.g., ranalexin, ranatuerins, gaegurins, brevinins, esculetins, rugosins), ranatuerin IT contains an intramolecular disulfide bridge forming a heptapeptide ring but there is little structural similarity outside this cyclic region. The minimum inhibitory concentration (MIC) of ranatuerin 1T was 120 microM against the Gram-positive bacterium S. aureus and 40 microM against the Gram-negative bacterium Escherichia coli, but the peptide was not active against the yeast Candida albicans.     

Synthesis, molecular modeling and preliminary biological evaluation of a set of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole as potential antibacterial, anti-Trypanosoma cruzi and antifungal agents

A series of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives was synthesized and their activity screened in vitro against Staphylococcus aureus, Trypanosoma cruzi, and Candida albicans. The bioactivity was expressed as minimum inhibitory concentration (MIC) for S. aureus strains, and as fifty-percent inhibitory concentration (IC(50)) of parasite population growth for T. cruzi. A molecular modeling approach was performed to establish qualitative relationships regarding the biological data and the compounds' physicochemical properties. The 5-(4-OC(4)H(9)Ph, 5l), and 5-(4-CO(2)CH(3)Ph, 5o) derivatives were the most active compounds for S. aureus ATCC 25923 (MIC=1.95-1.25 μg/mL) and T. cruzi (IC(50)=7.91 μM), respectively. Also, a preliminary evaluation against C. albicans involving some compounds was performed and the 5-(4-CH(3)Ph, 5e) derivative was the most active compound (MIC=3.28-2.95 μg/mL). In this preliminary study, all synthesized 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives were active against all microorganisms tested.     

Antibacterial and bactericidal activities of tea extracts and catechins against methicillin resistant Staphylococcus aureus]

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antibacterial and bactericidal activities against methicillin resistant Staphylococcus aureus (MRSA) and food poisoning strains of S. aureus. Twenty percent tea extract (50 microliters), EGCg (63 micrograms) and TF3 (125 micrograms) added to one ml of culture medium each inhibited the growth of all strains of MRSA and food poisoning S. aureus tested. Tea extract showed also a bactericidal activity against MRSA even at the same concentration of as in ordinarily brewed tea. EGCg at a concentration of 250 micrograms/ml showed a bactericidal activity against MRSA but not against food poisoning S. aureus, but at 500 micrograms/ml reduced markedly the viable number within 48h. These results suggest that tea and catechin can be used as prophylactic agents against MRSA infection.     

Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus.

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.     

Antibacterial activity of chalcones, hydrazones and oxadiazoles against methicillin-resistant Staphylococcus aureus

The increase in antibiotic resistance due to multiple factors has encouraged the search for new compounds which are active against multidrug-resistant pathogens. In this context, chalcones, dihydrochalcones, hydrazones and oxadiazoles were tested against Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus (MRSA) isolates, which were obtained from clinical laboratories and were characterized as MRSA using traditional and molecular methods. Among 65 tested compounds, two chalcones, one dihydrochalcone and two hydrazones were active against MRSA. Based on the minimal inhibitory concentration and cytotoxicity, hydrazones provided a better selectivity index than chalcones. Active hydrazones are promising antibiotic-like substances and they should be the subject of further microbiological studies.     

Intracellular localization of Staphylococcus aureus within primary cultured mouse kidney cells.

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity-tolerant (HOT) cells were obtained. The rate of cocci-ingesting cells varied from 16.9% to 93.4% among these of the HOT cells at the end of 3-hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK-1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK-1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast-like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast-like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30-min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.