Evaluation of several tree species for activity against the animal fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus causes severe problems in poultry production systems. Seven South African tree species were selected from the database of the Phytomedicine Programme based on its antifungal activity against the fungus Cryptococcus neoformans. The acetone leaf extracts of the selected species had minimum inhibitory concentrations (MICs) of 0.16 mg/ml and lower in the preliminary screening. The antibacterial and antifungal activities of hexane, dichloromethane, acetone and methanol extracts of the leaves were determined using a two-fold serial microdilution method against a range of commonly encountered animal pathogenic fungi (A. fumigatus, Candida albicans, C. neoformans, Microsporum canis and Sporothrix schenckii) and four nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa). The plant species investigated were Combretum vendae (A.E. van Wyk) (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae) and Protorhus longifolia (Bernh.) Engl. (Anacardiaceae). All the extracts had activity against at least one of the test organisms over an incubation period of 24 or 48 h. The MIC values of the non-polar and intermediate polarity extracts of O. natalitia, K. anthotheca, C. vendae, C. harveyi, and P. longifolia had MICs as low as 0.08 mg/ml against at least one of the tested bacteria. Furthermore, the acetone extracts of L. alata, K. wilmsii, O. natalitia and C. vendae had antifungal activities with MIC values ranging from 0.04 to 0.08 mg/ml against at least one of the tested fungi. The average MIC values of the plant extracts against the different bacteria ranged from 0.17 to 2.11 mg/ml, while the range was 0.23–1.98 mg/ml for fungi. The Gram-positive bacteria (S. aureus and E. faecalis) were more susceptible to the plant extracts than the Gram-negative bacteria (E. coli and P. aeruginosa). E. faecalis was the most susceptible microbe and C. vendae extracts were the most active against nearly all the bacteria tested. The acetone extract of L. alata was the most active against fungal pathogens, with activity against at least 3 fungal organisms. L. alata was selected for further work to isolate compounds active against A. fumigatus and other fungal pathogens.     

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

32种抗菌药物对临床分离猪源链球菌的体外抗菌活性

采用微量稀释法测定了32种药物对临床分离猪源链球菌的体外最小抑菌浓度(MIC),以美国临床检验标准委员会(NCCL5)的临界浓度做为判断标准,判定了猪源链球菌对32种药物的耐药性。结果表明,临床分离的菌株以耐药菌为主,且96．6％的菌株呈多重耐药,41％菌株为链霉素高耐药菌株(MIC≥2mg／mL);对磺胺类药物(92．3％～98．3％)、氨基糖甙类药物(70．8％～78．5％)、四环素类(72．3％)、林可胺类(66．2％～64．6％)、大环内酯类(53．8％～67．7％)耐药性最为严重,对青霉素类(18．5％～53．8％)、头孢菌素类(18．5％～56．9％)、泰妙灵(21．5％)和喹诺酮类药物(36．9％～78．5％)耐药性次之,而所选菌株对氯霉素类药物氟苯尼考均敏感;检测了所有耐8内酰胺类抗生素菌株是否产β内酰胺酶,结果均为阴性。     

微孔板杂交法测定郴毒假单胞酵米面亚种的ＤＮＡ—ＤＮＡ同源性

采用一种新的ＤＮＡ－ＤＮＡ发校方法－微孔板法对郴毒假单胞菌酵米面亚种及伯克霍尔德氏菌属中１１个的ＤＮＡ－ＤＮＡ同源性进行测定。结果发现郴毒假单胞菌酵米面亚种与唐菖蒲伯克霍尔德氏菌及椰毒伯克霍尔德氏菌的ＤＮＡ－ＤＮＡ同源性均大于７５％,建议这３个种应合并为同一个种,命名为唐菖蒲的伯克霍尔德氏菌（Brukholderia gladioli)。     

In vitro susceptibilities of clinical yeast isolates to three antifungal agents determined by the microdilution method

A comparative evaluation of the in vitro susceptibilities of 597 clinical yeast isolates to amphotericin B, fluconazole, and 5-fluorocytosine (5FC) was conducted. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. Microdilution endpoints for amphotericin B were scored as the lowest concentration in which a score of 0 (complete absence of growth) was observed and for 5FC and fluconazole as the lowest concentration in which a score of 2 (prominent decrease in turbidity; MIC-2) was observed compared to the growth control. The MIC values were read after 24 and 48 h incubation. A broad range of MIC values was observed with each antifungal agent. Amphotericin B was very active (MIC₉₀1.0 µg/ml) against all of the yeast isolates with the exception ofC. lusitaniae (MIC₉₀2.0 µg/ml). Fluconazole was most active againstC. parapsilosis (MIC₉₀ of 1.0 µg/ml) and least active againstC. krusei (MIC₉₀ of 32 µg/ml). 5FC was most active againstC. albicans, C. parapsilosis, C. tropicalis, andT. glabrata (MIC₉₀1.0 µg/ml) and was least active againstC. krusei andC. lusitaniae (MIC₉₀16 µg/ml). These data indicate that the microdilution method, performed in accordance with M27-P, provides a means of testing larger numbers of yeast isolates against an array of antifungal agents and allows this to be accomplished in a reproducible and standardized manner. Given these results, it appears that the microdilution method may be a useful alternative to the macrodilution reference method for susceptibility testing of yeasts.     

Trypanosoma rhodesiense: semiautomated microtesting for quantitation of antitrypanosomal activity in vitro

A rapid, semiautomated microdilution method was developed to measure the antitrypanosomal activity of large numbers of compounds against blood form Trypanosoma rhodesiense at 37 C in vitro. Parasites harvested from infected rats were incubated for 3 hr in microtitration plates with serial dilutions of test compounds. Inhibition of uptake of radiolabeled thymidine and l-leucine by the parasites served as indicators of antitrypanosomal activity. Repeated measurements of the activity of ethidium bromide demonstrated the sensitivity and precision of the method. Several additional antitrypanosomal drugs were tested in vitro and all except tryparsamide (a pentavalent arsenical) and suramin (a complex polyanion) were consistent with in vivo activity. Antimicrobial agents which are not effective antitrypanosomal drugs failed to show activity in vitro. Other compounds active in vitro included allopurinol and a number of aromatic diamidines, carbamodithioic acid derivatives, and 2-acetylpyridine thiosemicarbazones. This in vitro microtest system was developed as a primary screen for the selection of active compounds in a new Antitrypanosomal Drug Development Program.     

Dihydrophenanthrenes and other antifungal stilbenoids from Stemona cf. pierrei

Three new dihydrophenanthrenes, stemanthrenes A-C, along with the new dihydrostilbene stilbostemin G were isolated and identified from the underground parts of Stemona cf. pierrei together with the known pinosylvin, 4'-methylpinosylvin, dihydropinosylvin, stilbostemins B, D, and E as well as the pyrrolo[1,2-a]azepine alkaloids protostemonine and stemonine. The structures of all new stilbenoids, elucidated by NMR analyses, showed a common substitution pattern for aromatic ring A and characteristic C-methylations for ring B. The trivial name racemosol, previously reported for S. collinsae, was renamed to stemanthrene D due to its priority for another compound. Bioautographic tests on TLC plates with Cladosporium herbarum displayed high antifungal activity for compounds with an unsubstituted aromatic ring A, e.g. pinosylvin, but only weak effects for the higher substituted stilbostemin G and stemanthrenes A-C. Similar results were obtained by germ tube inhibition of five microfungi using 2-fold serial broth dilutions determined by a microplate reader. Because of weak inhibition and chemical instability of stemanthrenes, no EC(50) and EC(90) values could be calculated.     

Menadione (vitamin K) enhances the antibiotic activity of drugs by cell membrane permeabilization mechanism

Menadione, vitamin K3, belongs to the class of lipid-soluble vitamins and lipophilic substances as menadione cause disturbances in the bacterial membrane, resulting in damage to the fundamental elements for the integrity of the membrane, thus allowing increased permeability. Accordingly, the aim of this study was to evaluate in vitro the antibiotic-modifying activity of menadione in multiresistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, with a gradual increase in its subinhibitory concentration. In addition, menadione was compared with cholesterol and ergosterol for similarity in mechanism of drug modulatory action. Antibiotic-modifying activity and antibacterial effect were determined by the broth microdilution assay. Menadione, cholesterol and ergosterol showed modulatory activity at clinically relevant concentrations, characterizing them as modifiers of bacterial drug resistance, since they lowered the MIC of the antibiotics tested. This is the first report of the antibacterial activity of menadione and its potentiation of aminoglycosides against multiresistant bacteria.