Broth Macrodilution Testing Research Articles

Antibacterial Activity of Crude Aqueous Extracts of Tithonia Diversifolia from Chichiri Area in Blantyre District, Malawi.

Background: The global challenge of antimicrobial resistance has spurred scientific research efforts to find alternative sources of new antibiotics. The ethnopharmacological importance of Tithonia diversifolia is well-known. Objective: The present study’s aim was to evaluate the in vitro activity of crude aqueous leaf, stem and root extracts of locally growing Tithonia diversifolia against the clinical bacterial isolates: E. coli, K. pneumoniae, and P. mirabilis. Methods: To obtain antibacterial activity data, the Broth macrodilution testing and Zone of inhibition Kirby-Bauer approaches were used. Results: Estimated diameters of zone of inhibition showed leaf extracts of Tithonia diversifolia had significantly greater antibacterial activity (19.5 ± 3.9 mm, 95% CI: 15.4 - 23.6 mm) than stem (15.2 ± 2.0 mm, 95% CI: 13.0 - 17.3 mm, p = 0.021) or root extracts (15.0 ± 2.1 mm, 95% CI: 12.8 - 17.2 mm, p=0.019). K. pneumoniae was the most susceptible isolate to growth inhibition by extracts from all plant parts. In broth macrodilution testing, leaf extracts exhibited greater potency on all study isolates compared to stem and root extracts. Conclusion: These findings support the traditional use of Tithonia diversifolia decoctions and infusions in infectious processes that are due to these pathogens and further strengthens recommendations for additional work to isolate and characterize the bioactive chemical compounds responsible for the observed antibacterial properties of the plant.

Antimicrobial activity of various extracts of Sargassum glaucescens on the antibiotic resistant organisms

The antimicrobial activity of brown alga methanol, ethyl acetate, hexane, and chloroform extracts on bacteria Gram positive, Gram negative, and fungi was evaluated by using nutrient broth macrodilution test. Sargassum glaucescense was collected around the coastal area of Chabahar (south of Iran) protected marine area of the Oman sea in April and May 2015. Six pathogenic organisms including; Enterococcus faecium ATCC 51299, Streptococcus mutans ATCC 35668, Shigella boydii ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883, Salmonella enteritidi PTCC, 1709, Candida albicans ATCC 10231 and Aspergillus fumigatus PTCC 5009 were investigated by the broth dilution method. Methanolic Extract for six strains showed good activity amongst eight strains. Hexane extract, after methanolic extract has good effect on antimicrobial activity against five strains. All bacteria strain in this survey has showed resistance against ethyl acetate and chloroformic extracts. S. glaucescens using four various solution extract against eight different human pathogens showed an important antimicrobial and antifungal activity. However, more investigation has to be done on separation, purification and detection of the active ingredients in order to recognize their antifungal and antifungal activity.

An alternative disk diffusion test in broth and macrodilution method for colistin susceptibility in Enterobacteriales

Colistin and polymyxin B are old drugs that have been reintroduced to treat Gram-negative infections lacking other treatment options; however, colistin resistance have been reported. To know the correct susceptibility pattern is mandatory for multidrug resistant bacteria. Broth microdilution method is the gold standard to evaluate colistin and polymyxin B susceptibility; nevertheless, it is time consuming and needs expertise to be performed. Disk diffusion method on Müeller-Hinton agar is no longer recommended to evaluate polymyxins susceptibility. In this study we evaluated two methods (disk diffusion in broth and broth macrodilution) as alternative options to identify polymyxin resistance in an easy way. A total of 536 Enterobacteriales isolates were assessed for colistin susceptibility. All non-wild type Enterobacteriales (41) were chosen and 31 wild type bacteria were randomly selected, were used to perform disk diffusion tests in broth and for broth macrodilution tests. We found 100% of concordance between both tested methods and broth microdilution.In conclusion, these two methods are reliable and easier options that complement as initial screening susceptibility for colistin in Enterobacteriales in microbiology laboratories lacking personnel and infrastructure to perform broth microdilution method.

Identification of Luteolin in Henna (Lawsonia inermis) Oil, a Persion Medicine Product, by HPTLC and Evaluating Its Antimicrobial Effects

Background and objectivesPersian scholars such as Avicenna and Zakariya al-Razi have pointed out many uses for henna oil. The present study aimed to propose a method for standardization of this oil.MethodsThe luteolin content has been evaluated quantitively by HPTLC method to standardize the henna oil. The oil sample was applied on silicagel plate and the bands were developed using CHCl3-MeOH (95:5). The plate was scanned at λ 254 nm. The minimum inhibitory concentration was determined through the broth macrodilution test to examine the antibacterial activity of the oil.ResultsThe retention factor of the sample zone of luteolin standard was 0.6±0.01. The concentration of luteolin in henna oil was 56.57±0.66 μg/mL. MICof Henna oil against Gardnerella vaginalis and Neisseria gonorrhoeae was 87 μg/mL and against Streptococcus was 870 μg/mL.ConclusionAccording to the results of this study, henna oil contains the luteolin. Further studies are needed to identify other henna oil compounds and their effects. Gardnerella vaginalis, N. gonorrhoeae, Group B streptococcus are among the pathogenic agents of cervicitis. The application of henna oil for treatment of uterus diseases in Persian medicine can be justified given the antimicrobial effects of henna oil on these three species of bacteria.

Antibacterial activity evaluation of selected essential oils in liquid and vapor phase on respiratory tract pathogens

BackgroundThe increasing number of multidrug-resistant bacteria and the fact of antibiotic resistance is leading to a continuous need for discovering alternative treatments against infections, e.g. in the case of respiratory tract diseases. Essential oils (EOs), because of their volatility, can easily reach both the upper and lower parts of the respiratory tract via inhalation. Therefore, the aim of the present study was the antibacterial evaluation of clove, cinnamon bark, eucalyptus, thyme, scots pine, peppermint, and citronella EOs against respiratory tract pathogens such as Streptococcus pneumoniae, S. mutans, S. pyogenes, Haemophilus influenzae, H. parainfluenzae, and Moraxella catarrhalis. Furthermore, we wanted to compare the antibacterial effect of these EOs in two different test systems to provide data for the development of an appropriate product formulation.MethodsMinimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with in vitro vapor phase test (VPT) and broth macrodilution test (BDT). The chemical and percentage compositions of the EOs were determined by GC-MS and GC-FID analysis.ResultsAmong the EOs, thyme was the most effective against S. mutans (MIC: 0.04 mg/mL in BDT, but cinnamon bark and clove oils also presented high inhibition in liquid medium with MIC values of 0.06 mg/mL and 0.1 mg/mL against S. pneumoniae and S. pyogenes, respectively. M. catarrhalis was the most sensitive to thyme EO (MIC: 0.09 mg/mL). Cinnamon bark EO was the most effective against Haemophilus spp. (MIC: 0.06 mg/mL). In the VPT, cinnamon bark was the most effective oil against all investigated pathogens with MIC values in the range of 15.62–90 μl/L. Surprisingly, the eucalyptus and scots pine showed weak activity against the test bacteria in both test systems.ConclusionsThe EO of thyme, clove and cinnamon bark may provide promising antibacterial activity against respiratory tract pathogens either in liquid medium or in vapor phase. However, their effect is lower than that of the reference antibiotics. The combination of EOs and antibiotics may be beneficial in the alternative treatment of respiratory tract diseases. In vivo studies are necessary to calculate the effective dose of EOs in patients and determine their possible side effects and toxicity.

Antibacterial action of Tropical honey on various bacteria obtained from diabetic foot ulcer

The purpose of study was to evaluate the antibacterial action of Indian honey on bacteria obtained from diabetic foot ulcer. Time kill assay was performed with varying concentration of honey. Broth macro dilution test was used to determine MIC and MB

Comparative Evaluation of Antifungal Activity of Piper Betel Leaf Oil, Origanum vulgare Essential Oil and Fluconazole Suspension on Candida albicans − An In Vitro Study

Introduction: Oropharyngeal candidiasis is an opportunistic mucosal infection caused by Candida albicans. It usually responds to topical treatments such as clotrimazole troches, topical fluconazole, chlorhexidine mouthwash and nystatin suspension. Piper betel leaf oil and Origanum vulgare essential oil have shown some topical antifungal activity. Aim: To determine and compare the antifungal efficacy of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension against C. albicans. Materials and Methods: The zone of inhibition was measured by the cup–plate diffusion method using 100 μl volume of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension, which were pipetted into the wells of the inoculated Sabouraud’s dextrose agar plates. The zone of inhibition was measured in millimetres using Vernier calliper. Minimum inhibitory concentration (MIC) was determined by the broth macro-dilution test by pouring 1 ml of the respective concentrations of the test material to the individual test tubes along with 10 μl of the diluted test organism inoculum. Finally, MIC was calculated using a reflective viewer. Results: The zone of inhibition for O. vulgare essential oil (>40 mm) was more than fluconazole suspension (>35 mm). MIC of O. vulgare essential oil, piper betel leaf oil and fluconazole suspension was 1.6%, 0.4% and 0.8%, respectively. Conclusion: O. vulgare essential oil was found to be a more effective antifungal agent than piper betel leaf oil and fluconazole suspension.

Chemical Composition and in Vitro Antibacterial Activity of Native Men‌tha longifolia Essential Oil from Ardabil, Iran on Streptococcus mutans, Lactobacillus rhamnosus and Actinomyces viscosus

Background: Most of the population in the different areas of world is affected by bacterial infections responsible for dental caries. Due to the importance of traditional medicines derived from herbs used for dental problems, this study investigated in vitro antibacterial activity of Mentha longifolia essential oil from Ardabil, Iran, on Streptococcus mutans, Lactobacillus rhamnosus and Actinomyces viscosus, bacteria that cause tooth decay. Materials and Methods: The volatile oil of Mentha longifolia leaves was extracted by hydrodistilla tion method using a Clevenger-type apparatus and analyzed by GC and GC/MS system.The antibacterial activity was evaluated by the disk diffusion susceptibility in dilutions of 62.5, 125, 250, 500, 1000 and 2000�� g/�� l and broth macrodilution test methods. Results: The oil was particularly rich in Pulegone (31.78%), 1,8-cineole (15.99%), menthoforan (11.25%), cis-isopulegon (10.5%) and paramenth-3-n-8-l (6.85%). The medicinal plant essential oil could prevent the growth of the bacteria, and the rates of MIC and MBC of native pennyroyal essential oil on Streptococcus mutans, Lactobacillus rhamnosus and Actinomyces viscosus were 110, 165, 80, 120, 450 and 650�� g/�� l, respectively. The maximum inhibition zone diameter was about 12.2, 27.2 and 4.8 mm, for Streptococcus mutans, Lactobacillus rhamnosus and Actinomyces viscosus respectively, at the concentration of 500��J�� l -1 . Conclusion: In this work, the essential oil of medicinal plant containing effective ingredients could prevent the growth of bacteria and may be used as an affordable and available source for medicinal purposes.

Characterization, activity, and computer modeling of a molecular inclusion complex containing rifaldazine

BackgroundThe purpose of this study was to develop, characterize, and investigate a molecular inclusion complex containing rifaldazine with good solubility and antibacterial activity.MethodsRifaldazine, a lipophilic molecule, was encapsulated into the hydrophobic cavity of β-cyclodextrin to form a molecular inclusion complex (RAABCD) with good solubility. RAABCD was prepared in a short time using a solid-state grinding method. The inclusion ratio, binding constant, and change in Gibbs free energy were determined by a phase solubility diagram and/or ultraviolet-visible spectroscopy. Differential scanning calorimetry and Fourier transform infrared spectroscopy of RAABCD were performed. Morphological features of RAABCD were observed by photomicroscopy. The most likely optimal configuration for RAABCD was simulated by computer modeling. Broth macrodilution testing was done to investigate the antibacterial activity of RAABCD.ResultsThe inclusion ratio, binding constant, and change in Gibbs free energy, determined by a phase solubility diagram and/or ultraviolet-visible spectroscopy were 1:1, 288.33/261.33 L/mol, and 32.29/31.73 kJ/mol, respectively. Differential scanning calorimetry and Fourier transformed infrared spectra of RAABCD confirmed the molecular interaction between rifaldazine and β-cyclodextrin. The morphological difference between irregular and amorphous-shaped RAABCD and columnar-shaped rifaldazine further confirmed the molecular encapsulation of rifaldazine. The most likely optimal configuration for RAABCD was confirmed by computer modeling. Broth macrodilution testing indicated that RAABCD had good antibacterial activity.ConclusionRAABCD had improved solubility and good activity, and might be a promising alternative for treatment of a range of bacterial infections.

Evaluation of the Performance of the MicroScan Pos Breakpoint Combo Panel Type 28 for Susceptibility Testing of Staphylococcus aureus: Low-range Minimum Inhibitory Concentration of Vancomycin, Cefoxitin Screening, and Inducible Clindamycin Resistance Detection

Susceptibility testing of Staphylococcus aureus often requires cumbersome supplementary tests. MicroScan Pos Breakpoint Combo Panel Type 28 (PBC28) (Siemens, USA) includes cefoxitin screening to detect methicillin-resistant Staphylococcus aureus (MRSA), inducible clindamycin resistance detection (ICD), and determination of low-range minimum inhibitory concentration of vancomycin (0.5-16 µg/mL). The purpose of this study was to evaluate the performance of PBC28 in comparison with that of Pos Combo Type 1A (PC1A) (Siemens). From December 2009 to March 2010, 500 non-duplicate clinical isolates of S. aureus were tested with PC1A and PBC28. Categorical agreements (CA) between the interpretations of the 2 panels were estimated. The presence of the mecA gene was determined by PCR, and double-disk diffusion test (D-test) was performed on the isolates resistant to erythromycin but susceptible or intermediately resistant to clindamycin. Ninety-six isolates representing various vancomycin minimum inhibitory concentrations (MICs) were tested in parallel with repeat PBC28, broth macrodilution, and epsilometer test (E test). The CA was 99.3% with a very major error (VME) of 0.2%, major error (ME) of 0.1%, and minor error (mE) of 0.4% in total. PBC28 showed 100% CA for 1 isolate with vancomycin MIC of 4 µg/mL and 35 isolates (7.0%) with MIC of 2 µg/mL. However, only 15, 27, and 35 isolates with vancomycin MIC of 2 µg/mL showed 100% CA in repeat PBC28, broth macrodilution, and E test, respectively. PC1A and PBC28 detected all 314 mecA-positive isolates. Among the 63 isolates tested with the D-test, 58 (92.1%) were positive, and the results were 100% concordant with those of ICD. PBC28 can be appropriate susceptibility testing of S. aureus, including MRSA detection and ICD. However, the lower-range vancomycin MIC test was not reproducible enough to reliably differentiate MIC of 2 µg/mL from MIC ≤ 1 µg/mL.

Antifungal activity of synthetic di(hetero)arylamines based on the benzo[ b]thiophene moiety

The antifungal activity of several di(hetero)arylamine derivatives of the benzo[ b]thiophene system was evaluated against clinically relevant Candida, Aspergillus, and dermatophyte species by a broth macrodilution test based on CLSI (formerly NCCLS) guidelines. The most active compound showed a broad spectrum of activity (against all tested fungal strains, including fluconazole-resistant fungi), with particularly low MICs for dermatophytes. Results from the inhibition of the dimorphic transition in Candida albicans and flow cytometry studies further confirmed their biological activity. With this study it was possible to establish some structure–activity relationships (SARs). The hydroxy groups proved to be essential for the activity in the aryl derivatives. Furthermore, the spectrum of activity in the pyridine derivatives was broadened by the absence of the ester group on position 2 of the benzo[ b]thiophene system.

Synthesis and Properties of Novel Antifungal Gemini Compounds Derived from N‐Acetyl Diethanolamines

AbstractA series of new N‐acetylated non‐ionic and cationic gemini surfactants (3a–f) having dimeric structures derived from primary and tertiary amines with variably long tails (C8–C12–C18) were synthesized. In addition, N‐acetylated monomeric analogues 6a and 6b were prepared and their antifungal potency and surface properties were also determined. Critical micelle concentration (CMC), effectiveness of surface tension reduction (γCMC), surface excess concentration (Γ), and area per molecule at the interface (A) were also determined and the resulting values indicate that the cationic series is characterized by good surface‐active and self‐aggregation properties. For the first time, all surfactants were tested to evaluate their antifungal properties using the method for the broth macrodilution test (M27‐A2, NCCLS). Four microbial strains were used to perform the study: Candida parapsilosis (ATCC 22019), C. albicans (ATCC 64548), and a wild‐type strain of C. parasilosis and Saccharomyces cerevisiae (ATCC 9763). The antimicrobial activity was measured by yeast growth inhibition expressed as minimum inhibitory concentration (MIC) values. Results were compared to those obtained for their monomeric analogues and for a commercially available reference compound (Fluconazole). Gemini 3b, 3e and 3f were found to be the most potent compounds. The results show S. cerevisiae as the most sensitive strain. In contrast, the wild strain of C. parapsilosis was resistant.

EUCAST Definitive Document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts: Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)∗

INTRODUCTIONAntifungal susceptibility tests are performed onfungi that cause disease, especially if they belongto a species exhibiting resistance to commonlyused antifungal agents. Antifungal susceptibilitytesting is also important for resistance surveil-lance, for epidemiological studies and forcomparing the in-vitro activity of new and exist-ing agents.Dilution methods are used to establish theMICs of antimicrobial agents. These are thereference methods for antimicrobial susceptibilitytesting, and are used mainly to establish theactivity of a new antifungal agent, to confirm thesusceptibility of organisms that give equivocalresults in routine tests, and to determine thesusceptibility of fungi where routine dilution testsmay be unreliable. Fungi are tested for theirability to produce visible growth in microdilutionplate wells containing broth culture media andserial dilutions of the antifungal agents (brothmicrodilution). The MIC is defined as the lowestconcentration (in mg⁄L) of an antifungal agentthat inhibits the growth of a fungus. The MICprovides information concerning the susceptibil-ity or resistance of an organism to the antifungalagent and can help in making correct treatmentdecisions.The method described in this document isintended for testing the susceptibility ofyeasts that cause clinically significant infections(primarily Candida spp.). The method encom-passes only those yeasts that are able to fermentglucose. Thus, the susceptibility of non-fermenta-tive yeasts, e.g., Cryptococcus neoformans, cannotbe determined by the current procedure, and themethod is not suitable for testing the yeast formsof dimorphic fungi.SCOPEThe standard method described in this documentprovides a valid method for testing the suscepti-bility of glucose-fermenting yeasts to antifungalagents by determination of the MIC. MICs indi-cate the activity of a given antifungal drug underthe described test conditions, and can be used inmaking decisions concerning patient manage-ment after taking into account other factors, e.g.,pharmacokinetics, pharmacodynamics and resis-tance mechanisms. The MIC also allows fungi tobe categorised as ‘susceptible’ (S), ‘intermediate’(I) or ‘resistant’ (R) to a drug. In addition, MICdistributions can be used to define wild-type ornon-wild-type fungal populations.This method is intended primarily to facilitatean acceptable degree of conformity, i.e., agree-ment within specified ranges among laboratories,in measuring the susceptibility of yeasts to anti-fungal agents. The method is designed to be easyto perform, rapid, economical, and suitable forreading by microdilution plate readers in order toallow direct transfer, storage and manipulation ofthe data by computer. The method is alsointended to yield results that are concordantwith those obtained using the procedure

Isolation and Antimicrobial Susceptibility Testing of Fecal Strains of the Archaeon Methanobrevibacter smithii

Background: The archaeon Methanobrevibacter smithii is regarded as part of the indigenous microflora of the human intestine but may be connected with pathological conditions. The microbe is extremely oxygen intolerant and is not detectable by anaerobic culture techniques for bacteria. Accordingly, to date quantitative antimicrobial susceptibility data of human isolates are missing. Methods: The anoxic Hungate technique and a three-step culture procedure using media supplemented with antibiotics were applied to isolate M. smithii from randomly selected human feces. The minimum inhibitory concentrations (MICs) of 15 isolates and the reference strain DSM 861 were determined using a broth macrodilution test resembling the procedure for testing anaerobic bacteria. Results: The 16 strains were highly resistant (MICs >64 mg/l) against penicillin G, cephalothin, vancomycin, streptomycin, gentamicin, ciprofloxacin, and clindamycin. Metronidazole inhibited the strains at MICs between 0.5 and 64 mg/l. Conclusions: Multiple-antibiotic-resistant Methanoarchaea occur in the human gut. They may be selected during therapy with common antibacterial agents and may be eliminated by the application of metronidazole.

Rapid susceptibility testing of Candida albicans by flow cytometry.

The emerging magnitude of human fungal infections has renewed interest in developing rapid and standardized methods for susceptibility testing. We demonstrated that susceptibility testing of Candida albicans can be accomplished rapidly by using flow cytometry. Test results were available within 8 to 24 h after C. albicans isolates were incubated with amphotericin B, itraconazole, and flucytosine. This is an improvement of 24 to 60 h in the time to availability of susceptibility test results compared to the time to availability of National Committee for Clinical Laboratory Standards-recommended broth macrodilution test results. In addition, the flow cytometric endpoints, mean channel fluorescence, and number of fluorescence-labeled C. albicans cells were easy to interpret for greater sensitivity and reliability. Flow cytometry provides a more accurate means of obtaining antifungal susceptibility test results.

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests for the filamentous fungi

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, fluconazole, ketoconazole, 5-fluorocytosine, miconazole, and itraconazole against representative species of opportunistic hyphomycetes (Fusarium spp. and Cladosporium [Cladophialophora] spp.) and ascomycetes (Chaetomium spp.). A total of 78 strains were tested, the majority of them twice and some three times on different days. Both methods were performed according to the recommendations of the National Committee for Clinical Laboratory Standards (Document M27-P), with the exception of the temperature of incubation, which was 25 degrees C in our case. A spectrophotometric method for inoculum preparation, RPMI 1640 medium buffered with morpholinepropanesulfonic acid (pH 7.0), and an additive drug dilution procedure were used. The MICs obtained by the two methods were read after 48, 72, and 96 h of incubation for Fusarium spp. and after 72, 96, and 120 h for the remaining isolates. The kappa test was used to calculate the degree of agreement. Considering the three fungal groups together, a good agreement between the results of both tests was observed with almost all the drugs at the different incubation times. There were no cases of poor agreement. The highest level (kappa index = 1) was observed with ketoconazole at the second-day reading. These results support the further evaluation of the broth microdilution test as an alternative to the reference broth macrodilution susceptibility test.

Improved medium for fluconazole susceptibility testing of Candida albicans

We have compared fluconazole susceptibilities of 92 clinical isolates of Candida albicans by broth microdilution in two different media: standard RPMI 1640 (RPMI) and the same medium supplemented with 18 g of glucose per liter (RPMI-glucose). Preparation of media, drugs, and inocula, as well as incubation conditions, followed the preliminary recommendations of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) antifungal agent working group for broth macrodilution tests with antifungal agents, adapted to microdilution. Microtiter plates were agitated for 5 min before spectrophotometric readings were performed with an automatic plate reader set at 405 mm. The MIC endpoint was defined as an inhibitory concentration calculated from the turbidimetric data as a function of the turbidity in the drug-free control wells. The mean absorbances in the drug-free wells in RPMI and RPMI-glucose were, respectively, 0.38 (41.6% transmission) and 0.99 (10.2% transmission) (P < 0.001; Student's t test). Despite the increased growth in RPMI-glucose, 98.9% of the C. albicans strains tested for fluconazole susceptibility yielded similar MICs (+/- 1 dilution) in both media. Moreover, strains with decreased susceptibility to fluconazole displaying similar MICs in both media are easier to detect in RPMI-glucose because of the greater differences between turbidimetric readings in wells with grown or fluconazole-inhibited cultures. This objective turbidimetric method, with an easy-to-read improved medium (RPMI with glucose), together with previous experience of the National Committee for Clinical Laboratory Standards antifungal agent subcommittee, could overcome some of the present problems associated with lack of reproducibility of azole susceptibility testing.

In vitro studies of a new antifungal triazole, D0870, against Candida albicans, Cryptococcus neoformans, and other pathogenic yeasts

We investigated the effects of various assay conditions on the activity of D0870 against seven species of fungi in the broth macrodilution testing procedure proposed by the National Committee for Clinical Laboratory Standards (NCCLS). Multivariate analysis demonstrated that endpoint definition, starting inoculum size, medium composition, type of buffer, and length of incubation, but not pH or temperature, had significant effects on results. Increasing the inoculum from 10(2) to 10(5) yeast cells/ml raised the MICs for all isolates up to > 75,000 fold. This effect was greatest when endpoints corresponded to a 90% reduction in visually determined turbidity (MIC90), was less prominent with an 80% inhibition visual endpoint (MIC80), and was nearly absent with a 50% endpoint measured by a spectrophotometer (IC1/2). Differences due to medium composition were attributable to antibiotic medium 3 with RPMI and yeast nitrogen base media performing nearly identically. Under standardized conditions as specified in NCCLS document M27-P (Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Proposed Standard, 1992), 79 strains (5 to 25 strains for each species) demonstrated median MIC80s of 0.0037 and 0.0075 microgram/ml for Candida albicans and Cryptococcus neoformans, respectively. In contrast, Candida krusei and Torulopsis glabrata had a median MIC80 of 1.0 microgram/ml. Our studies indicate that the pathogenic yeasts C. albicans and C. neoformans are more susceptible to D0870 than other pathogenic yeasts.

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, flucytosine, fluconazole, ketoconazole, and cilofungin against 38 isolates of Candida albicans, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Torulopsis glabrata. The following preliminary antifungal working group recommendations of the National Committee for Clinical Laboratory Standards for broth macrodilution tests with antifungal agents were used: inocula standardized to 1 x 10(4) to 5 x 10(4) CFU/ml with a spectrophotometer, RPMI 1640 medium buffered with morpholinopropanesulfonic acid (pH 7.0), incubation at 35 degrees C for 24 to 48 h, and an additive drug dilution procedure. Broth microdilution MICs were higher (two or more dilutions) than broth macrodilution MICs for all isolates tested with amphotericin B and for most isolates tested with ketoconazole, fluconazole, and cilofungin. MICs of flucytosine were the same by both techniques or lower by the broth microdilution test except in tests with C. neoformans. However, the only statistically significant differences between the two tests were observed with amphotericin B against all isolates (P = 0.01 to 0.07), ketoconazole against C. neoformans (P = 0.01 to 0.02), and cilofungin against C. albicans (P = 0.05 to 0.14). Tests performed with less dense inocula (1 x 10(3) to 5 x 10(3] produced similar results.

WIN 57273 is bactericidal for Legionella pneumophila grown in alveolar macrophages.

The in vitro antimicrobial activity of WIN 57273, a new quinolone antimicrobial agent, was determined for 21 Legionella strains, using broth macrodilution and agar dilution testing methods; ciprofloxacin and erythromycin were tested as well. Three different buffered yeast extract media were used for the agar dilution studies, two of which were made with starch rather than charcoal. Broth macrodilution susceptibility testing was performed with buffered yeast extract broth and two Legionella pneumophila strains. Antimicrobial inhibition of L. pneumophila growth in guinea pig alveolar macrophages was also studied, using a method able to detect bacterial killing. The MICs for 90% of the 21 strains of Legionella spp. grown on buffered charcoal yeast extract medium were 0.125 microgram/ml for WIN 57273, 0.25 microgram/ml for ciprofloxacin, and 1.0 micrograms/ml for erythromycin. These MICs were falsely high, because of inhibition of drug activity by the medium used. Use of less drug-antagonistic, starch-containing media did not support good growth of the test strains. The broth macrodilution MICs for two strains of L. pneumophila serogroup 1 were less than or equal to 0.03 microgram/ml for WIN 57273 and ciprofloxacin and 0.125 microgram/ml for erythromycin. WIN 57273, ciprofloxacin, and erythromycin all inhibited growth of L. pneumophila in guinea pig alveolar macrophages at concentrations of 1 microgram/ml, but only WIN 57273 prevented regrowth or killed L. pneumophila after removal of extracellular antimicrobial agent.