Tube Dilution Research Articles

Comparison of three methods of antifungal susceptibility testing with the proposed NCCLS standard broth macrodilution assay: Lack of effect of phenol red

Three microtiter plate adaptations of the NCCLS proposed standard for antifungal susceptibility testing were evaluated and compared to the NCCLS broth macrodilution method. Thirteen different fungi, including yeasts and moulds were studied. The first microtiter based method was performed exactly as described for the tube dilution assay, with the exception of performance of the assay in 100 μl in wells of the microtiter plate. The second assay was the same as the first, except for the deletion of phenol red from the RPMI 1640. The third microtiter assay was based on the reduction of the formazan dye, XTT, after only 24 hours of incubation. All three microtiter methods compared favorably with the macrodilution method, when visually read after either 24 or 48 hours of incubation. Minimum inhibitory concentrations obtained by the XTT assay were usually lower than those obtained by the methods requiring a visual end point determination. Results were reproducible and comparable to those obtained with the NCCLS method. We conclude that microtiter plate adaptation of the NCCLS proposed standard is feasible and the presence of phenol red does not alter the results with the drugs tested. A 24 hour assay using XTT may provide a quicker and more quantitative method of susceptibility testing of fungi. Further investigation of this approach is warranted.

Synthesis and antimicrobial activity of thiazolinomethyl-2(3 H)-benzoxazolone derivatives (I)

A number of thiazolinoalkyl-2(3 H )-benzoxazolones have been synthesized by using cyano derivatives of 6-acyl2 (3 H )-benzoxazolones with cysteamine HCl. Their antibacterial and antifungal activities have been evaluated. The chemical structures have been proved by means of their IR, 1 H-NMR and mass spectroscopic data and by elemental analysis. The antimicrobial activity of compounds was investigated by tube dilution and disk techniques using bacteria ( Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Streptococcus faecalis RSKK 10541) and yeast-like fungi ( Candida parapsilosis, C albicans, C pseudotropicalis, C stellatoidea ). Inhibitory effects were observed for many compounds against C albicans eg, compounds 3b and 3c MIC = 25 μg/ml, C pseudotropicalis , eg, compound 3b MIC = 12.5 βg/ml, and P aeruginosa and S faecalis , eg , compound 3c , MIC = 25 μg/ml.

Antifungal susceptibility testing of isolates from a randomized, multicenter trial of fluconazole versus amphotericin B as treatment of nonneutropenic patients with candidemia. NIAID Mycoses Study Group and the Candidemia Study Group

The antifungal susceptibilities of 232 pathogenic blood stream Candida isolates collected during a recently completed trial comparing fluconazole (400 mg/day) with amphotericin B (0.5 mg/kg of body weight per day) as treatment for candidemia in the nonneutropenic patient were determined both by the National committee for Clinical Laboratory Standards M27-P macrobroth methodology and by a less cumbersome broth microdilution methodology. For amphotericin B, M27-P yielded a very narrow range of MICs (0.125 to 1 microgram/ml) and there were no susceptibility differences among species. For fluconazole, a broad range of MICs were seen (0.125 to > 64 micrograms/ml), with characteristic MICs seen for each species in the rank order Candida albicans < C. parapsilosis approximately equal to C. lusitaniae < C. glabrata approximately equal to C. krusei approximately equal to C. lipolytica. The MIC distribution for C. tropicalis was bimodal and could not be ranked. Both microdilution MICs were within one tube dilution of the M27-P MIC for > 90% of isolates with amphotericin B and for > or = 77% of isolates with fluconazole. For both methods, elevated MICs did not predict treatment failure. In the case of amphotericin B, the MIC range was too narrow to permit identification of resistant isolates. In the case of fluconazole, MICs for isolates associated with failure to clear the bloodstream consistently were equivalent to the median MIC for the given species. Successful courses of therapy were seen with four isolates from four patients despite MICs of > or = 32 micrograms/ml. As MICs obtained by M27-P and similar methods correlate with responsiveness to fluconazole therapy in animal models and in AIDS patients with oropharyngeal candidiasis, the lack of correlation in this setting suggests that the MICs for these isolates are at or below the relevant fluconazole breakpoint for this dose of fluconazole and patient setting and that host factors such as failure to exchange intravenous catheters were more important than MIC in predicting outcome.

In vitro activities of metronidazole and its hydroxy metabolite against Bacteroides spp.

Metronidazole is metabolized to two major oxidative products: an acid metabolite and a hydroxy metabolite. While the activity of the acid metabolite is negligible, the activity of the hydroxy metabolite is approximately 65% of the activity of the parent drug. Pharmacokinetic studies of metronidazole and its hydroxy metabolite have shown that the MICs of both compounds remain above the MICs for most anaerobic organisms over an 8-h dosing interval. By a checkerboard assay, the combined activities of metronidazole and the hydroxy metabolite were examined against 4 quality control strains of Bacteroides species. Macrobroth tube dilutions were set up with Wilkins-Chalgren broth. Serial twofold dilutions of each agent were performed to achieve final concentrations ranging from 0.06 to 4.0 micrograms/ml. The MICs for Bacteroides fragilis and B. distasonis were 1.0 microgram/ml for both parent drug and metabolite. For B. thetaiotamicron and B. ovatus, the MICs of metronidazole and the hydroxy metabolite were 1.0 and 2.0 micrograms/ml, respectively. Synergy was determined by calculating the fractional inhibitory concentration (FIC) index. The interpretative criteria for the FIC index were as follows: synergy, FIC < or = 0.5; partial synergy, 0.51 to 0.75; indifference, FIC 0.76 to 4.0; and antagonism, FIC > 4.0. Partial synergy was observed for the four anaerobes tested, with FIC indices ranging from 0.63 to 0.75. On the basis of this data, in vitro susceptibilities to agents such as metronidazole may ultimately require reevaluation to account for active metabolites.

Selection of candidate quality control isolates and tentative quality control ranges for in vitro susceptibility testing of yeast isolates by National Committee for Clinical Laboratory Standards proposed standard methods

The National Committee for Clinical Laboratory Standards has developed a proposed standard method for in vitro antifungal susceptibility testing of yeast isolates (National Committee for Clinical Laboratory Standards, document M27-P, 1992). In order for antifungal testing by the M27-P method to be accepted, reliable quality control (QC) performance criteria must be developed. In the present study, five laboratories tested 10 candidate QC strains 20 times each against three antifungal agents: amphotericin B, fluconazole, and 5-fluorocytosine. All sites conformed to the M27-P standards and used a common lot of tube dilution reagents and RPMI 1640 broth medium. Overall, 98% of MIC results with amphotericin B, 95% with fluconazole, and 92% with 5-fluorocytosine fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). Excellent performance with all three antifungal agents was observed for six strains: Candida albicans ATCC 90028, Candida parapsilosis ATCC 90018, C. parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, and Saccharomyces cerevisiae ATCC 9763. With these strains, 3-log2 dilution ranges encompassing 94 to 100% of MICs for all three drugs were established. Additional studies with multiple lots of RPMI 1640 test medium will be required to establish definitive QC ranges.

Bactericidal properties of crude extracts of Mitracarpus villosus

Mature leaves and inflorescence of Mitracarpus villosus, collected from Niger State, Nigeria, were shade dried over a period of 5 days, ground into fine particles in a Waring blender and extracted individually with hot distilled water and 95% ethanol. The crude extracts obtained were tested for their in vitro antibacterial activities using agar diffusion and tube dilution techniques. The extracts produced zones of inhibition (8–23 mm) against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Streptococcus faecalis, while Pseudomonas aeruginosa and Proteus mirabilis were not inhibited. The minimum inhibitory concentrations (MIC) of the effective extracts were in the range 0.06–8.0 mg/ml, while the minimum bactericidal concentrations (MBC) were in the range 0.06–32.0 mg/ml. The ethanolic extracts appeared to exert more inhibitory action against the bacteria than the hot water extracts.

Synthesis and antimicrobial investigation of thiazolinoalkyl-4(1 H)-pyridones

A number of thiazolinoalkyl-4(1 H)-pyridones have been synthesized using 4-pyrone derivatives with cysteamine HCl, and their antibacterial and antifungal activities have been tested. Their chemical structures have been proved by means of their IR, 1H-NMR, mass spectroscopic data and by elemental analysis. Investigation of antimicrobial activity of compounds was done by tube dilution and disk tecniques using bacteria ( Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Streptococcus faecalis RSKK 10541) and yeast-like fungi ( Candida parapsilosis, C albicans, C pseudotropicalis, C stellatoidea). A significant inhibitory effect was recorded for many compounds against C albicans ( 7a, c, d; minimal inhibitory concentration (MIC) = 12.5-25 μg/ml), S aureus ATCC 25923 ( 4c, 7a; MIC = 25 μg/ml), P aeruginosa ATCC 27923 ( 7a; MIC = 25 μg/ml), S faecalis RSKK 10541 ( 4c; MIC = 25 μg/ml), C pseudotropicalis ( 4d, 6d, 7c; MIC = 25 μg/ml) and C stellatoidea ( 4d; MIC = 25 μg/ml).

Effect of culture media on in vitro susceptibility testing of fluconazole against some yeasts

Thirty clinical isolates, comprising six strains of Candida albicans, and four strains each of C. lusitaniae, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Torulopsis glabrata and Trichosporon beigelii were tested against fluconazole, using Sabouraud's dextrose (SD) broth and a high resolution (HR) medium (Pfizer Central Research, Inc.). The procedure was a standard tube (1 ml/tube) dilution, and C. albicans Y01 09 was included as a reference strain to monitor quality and reproducibility. Results indicated that the minimum inhibitory concentrations (MICs) for all isolates of C. albicans, C. lusitaniae, C. tropicalis, and Tr. beigelii were 100 micrograms ml-1 or greater in the SD medium. In the HR medium, however, the MICs for two isolates of C. albicans were 1.56 micrograms ml-1, in other four gave higher values (greater than 100 micrograms ml-1), and the results for C. lusitaniae and Tr. beigelii were in the range 1.56-3.12 micrograms ml-1. The MICs for C. tropicalis were unaffected (100 micrograms ml-1) by the medium used. All Cr. neoformans isolates yielded a uniform value (1.56 micrograms ml-1) in HR medium as compared to somewhat more variable results (MICs 0.39-1.56 micrograms ml-1) in SD broth. The MICs for T. glabrata in the SD and HR media were 3.12-12.5 and 6.25 micrograms ml-1, respectively. The data indicated that the HR medium is preferable for the in vitro susceptibility testing of C. albicans, C. lusitaniae and Tr. beigelii to fluconazole. The MICs for other yeasts were not affected by the culture medium. The reference C. albicans isolate yielded an MIC of 1.56 micrograms ml-1 throughout.

An Assessment of Methods for the Microbiological Analysis of Shellfish

The efficiency of membrane filtration (MF) techniques and most probable number (MPN) tube dilution assays for the bacteriological analysis of homogenized oyster and mussel meat was compared. Tests were done on naturally contaminated shellfish and on homogenates seeded with sewage. Digested homogenates (5% trypsin for 20 min at 37°C) were prefiltered (pore size 5 um) and then filtered (pore size 0.45 µm) for MF counts. Undigested homogenates and dilutions were inoculated into tubes with growth medium for MPN tests. Homogenates (with or without trypsin) were used directly in a toplayer method for the detection of coliphages. Trypsin digestion significantly reduced counts of total coliform bacteria, but not of faecal coliform bacteria, enterococci or coliphages. Prefiltration considerably reduced counts of bacteria, and to a lesser extent, also of coliphages. As a result, MPN tests using minerals-modified-glutamate broth yielded higher counts of coliform bacteria than MF tests using mEndo LES and mTec agar. The latter yielded higher counts of faecal coliforms than mFC agar. In the case of enterococci, the MF procedure yielded lower counts than a spread plate test using undigested homogenates.

Amphotericin B susceptibility testing ofCandida species by flow cytometry

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.

Comparison of the antimicrobial effectiveness of regular and fresh scent Clorox

The antimicrobial properties of two different compositions of sodium hypochlorite were compared in a tube dilution study. Absorbent paper points were contaminated with Streptococcus faecalis or Candida albicans and exposed to 5.25% or 2.62% concentrations of "regular" or "fresh scent" sodium hypochlorite (Clorox) for periods ranging from 15 to 120 s. The points were then removed from the sodium hypochlorite solution, placed into a growth medium, incubated, and the presence or absence of growth recorded. Results showed that formulary changes involved in the manufacture of the "fresh scent" sodium hypochlorite had no apparent effect on its antimicrobial properties, as both compositions proved equally effective against the test organisms at each concentration evaluated.

Rational Use of Disease-Modifying Antirheumatic Drugs

The currently available, most frequently used disease-modifying antirheumatic drugs (DMARDs) include auranofin, azathioprine, D-penicillamine, gold sodium thiomalate, hydroxychloroquine, methotrexate (amethopterin) and sulphasalazine. Controlled trials of these agents are reviewed to compare their relative efficacy and tolerability. Tender joint counts decreased with all drugs, as did joint swelling (measured as the percentage of patients with greater than or equal to 50% improvement in joint swelling). Tender joint count decreased by 8 to 57% in drug-treated patients, compared with 3 to 30% (1 study exceeded this degree of placebo response) in the placebo groups. The ratio of drug to placebo improvement usually averaged greater than 2. A 50% improvement in joint swelling occurred in between 15 and 65% of drug-treated patients. Time to onset of response varied from 6 weeks (with methotrexate) to as long as 18 months (some patients on hydroxychloroquine). The remission rate was inconsistent and unusual in controlled studies (5 to 7%), but very high in some open studies (e.g. 43%). While up to 8% of patients on DMARDs stopped therapy secondary to unsatisfactory therapeutic response (with 1 exception) up to 43% of placebo patients discontinued therapy for this reason. The ratio of dropouts for unsatisfactory therapeutic response for DMARD compared to placebo was less than 1 in 16 of 22 studies, and it was usually less than 0.5. Laboratory data examined include ESR, rheumatoid factor (RF), immunoglobulins and radiographic data. Ratios of decreases in ESR, comparing drug and placebo, were usually greater than 2. ESRs decreased from 3.6 to 27 mm/h, with gold sodium thiomalate, auranofin and methotrexate being most effective relative to placebo. RF decreased by greater than or equal to 2 tube dilutions in 15 to 53% of the DMARD groups but also decreased in up to 26% of placebo patients, with ratios of drug: placebo usually greater than 2. Immunoglobulins tended to decrease with DMARDs but the data are fragmentary. Radiographic evidence that a drug slows the rate of bony damage is strong evidence that it is a DMARD. These data, however, are not easily available because measurements of bony damage is insensitive and difficult. The best evidence of radiographic efficacy exists for gold, although the data are not uniform even here. Studies with other DMARDs suffer from lack of convincing control populations, methodological failures or small numbers, although trends exist showing that azathioprine and D-penicillamine (and perhaps sulphasalazine and methotrexate) may also slow bony deterioration. The other side of efficacy, of course, is tolerability.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and Antimicrobial Activities of Some New Tetrahydro‐2H‐1,3,5‐thiadiazine‐2‐thione Derivatives of Ampicillin

Compounds having alpha-[dihydro-5-substituted 6-thioxo-2H- 1,3,5-thiadiazine-3(4H)-yl]benzylpenicillin structure were synthesized by the reaction of ampicillin trihydrate, formaldehyde and dithiocarbamic acid salts. The structures were evident from chemical and spectral analysis. The antimicrobial activities of the compounds were investigated against some gram-positive (Staphylococcus aureus and Streptococcus faecalis) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria and some yeast-like fungi (Candida albicans, C. parapsilosis, C. stellatoidea and C. pseudotropicalis) and molds such as Trichophyton rubrum, T. mentagrophytes, Microsporum canis, M. gypseum, Penicillium and Aspergillus by the tube dilution method. In addition to MIC (minimal inhibitory concentration), MBC (minimal bactericidal concentration) and MFC (minimal fungicidal concentration) values were determined using ampicillin trihydrate as standard. The compounds synthesized were usually found as effective as ampicillin trihydrate against S. aureus and S. faecalis and less effective than ampicillin trihydrate against E. coli. Both the compounds synthesized and ampicillin trihydrate are ineffective in the concentrations studied against P. aeruginosa. Compound 10 and 11 are more effective against all the yeast-like fungi than the other compounds and ampicillin trihydrate.

Comparison of the agar dilution, tube dilution, and broth microdilution susceptibility tests for determination of teicoplanin MICs

The purpose of this study was to evaluate the National Committee for Clinical Laboratory Standards agar dilution, tube dilution, and broth microdilution susceptibility tests for the measurement of teicoplanin MICs. The three standardized tests gave equivalent (within a twofold dilution) results with 98.8 to 99.0% of the 508 gram-positive clinical isolates tested, indicating that either method may be used for teicoplanin MIC determination.

Determination of lead in whole blood by electrothermal atomisation atomic absorption spectrometry using tube and platform atomisers and dilution with Triton X-100

The determination of lead in whole blood by electrothermal atomisation atomic absorption spectrometry using a Varian Model AA875 atomic absorption spectrometer coupled to a Varian Model GTA-95 graphite furnace is reported. An evaluation was undertaken to determine whether the use of (NH4)2HPO4 was beneficial when using tube and platform atomisers. Optimisations of the furnace heating conditions and of the best absorbance mode and sensitivity in both systems were also undertaken. The analysis of a target standard showed that it was possible to determine lead in blood by simple dilution with Triton X-100 using platform atomisation and the peak-area calculation mode. Calibration was carried out by direct comparison with linear working graphs prepared from aqueous standards.

人工股関節置換術後感染例に対するＲｅｖｉｓｉｏｎの経験

Recently we treated two cases of infected hip prosthesis. At the first stage, surgical treatment consisted of removal of prosthesis, debridement and curettage, irrigation with saline solution and antibiotics, changing operative clothes and tube dilution method. At the second stage, we used bone graft for severe bone loss and cementless hip prosthetic implantation. Our cases have been asymptomatic.

In vitro susceptibility of Listeria monocytogenes isolated from human blood and cerebrospinal fluid

The in vitro susceptibility of 156 strains of Listeria monocytogenes isolated since 1958 from human cerebrospinal fluid or blood to twelve antibiotics was determined by an agar dilution technique. Erythromycin (0.05), trimethoprim (0.2), netilmicin (0.2), and penicillin (0.2) were the most active drugs on weight basis (MIC90 0.05-0.2 micrograms/ml). Ampicillin and imipenem had MICs for 90% of the strains of 0.4 micrograms/ml. Ceftazidime was inactive (MIC90 greater than 100 micrograms/ml). Comparison of susceptibility pattern between strains isolated in different years showed that the antimicrobial susceptibility of L. monocytogenes has not changed during the last 25 years. The minimal bactericidal concentration (MBC) of penicillin was determined by a macro tube dilution method in ten recent isolates. Penicillin was bactericidal for all the strains with a MBC of 0.4-3.1 micrograms/ml, i.e. one to three two-fold dilutions above the MIC of 0.2-0.8 micrograms/ml, which means that no tolerant strains were found.

Ceftriaxone treatment of multidrug-resistant salmonella osteomyelitis

Empiric treatment of serious Salmonella infections has been complicated by the emergence of strains resistant to multiple antimicrobial agents, including ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole. Recent reports suggest that the third-generation cephalosporins may be effective therapy for Salmonella infections. This report describes a case of antibiotic-resistant Salmonella heidelberg prosthetic hip infection successfully treated with prosthesis removal and once-daily ceftriaxone. Tube dilution sensitivity testing of the organism demonstrated minimal inhibitory and minimal bactericidal concentrations of 0.12 μg/ml. Serum bactericidal activity, 30 minutes after infusion, was inhibitory and bactericidal at 1:512. It is concluded that the favorable preliminary results reported in the literature and the outcome in this patient suggest that the third-generation cephalosporins may be effective therapy for Salmonella infections and should undergo clinical trials.

Effects of long-chain fatty acids and fatty alcohols on the growth of Streptococcus mutans.

Minimal inhibitory concentrations (MICs) of a series of fatty acids and fatty alcohols against a cariogenic bacterium, Streptococcus mutans, were determined by a tube dilution technique. Among saturated fatty alcohols, tetradecanol and pentadecanol had the highest activity (MIC, 1.56 μg/ml), while among monounsaturated fatty alcohols, 10Z-pentadecenol had the strongest activity (MIC, 0.78 μg/ml). Saturated fatty acids showed relatively weak activity; tridecanoic acid had the highest activity among them (MIC, 12.5 μg/ml). Among unsaturated fatty acids, 10Z-heptadecenoic, 6Z-octadecenoic, 11 Z-octadecenoic and 9Z, 12Z-octadecadienoic acids had potent activity (MIC, 3.13 μg/ml). The antibacterial activities of methyl-branched and hydroxyl fatty acids as well as long chain dicarboxylic acids were also investigated.

The sensitivity to antimicrobial agents of species of yersinia isolated from cattle and pigs in Nigeria

Fourteen isolates of yersiniae belonging to three species isolated from cattle and pigs were tested for their sensitivity to 12 antimicrobial agents by the tube dilution technique. All the isolates were sensitive to gentamicin and 93.0%, 93.0%, 85.7% and 85.7% sensitive to tetracycline, nitrofurantoin, chloramphenicol and sulphamethoxazole-trimethoprim, respectively. There were no consistent differences in the rates and patterns of resistance based on source, species and serotypes of organisms.