Microdilution Wells Research Articles

Novel automated antifungal susceptibility testing system for yeasts based on dual-detection algorithm of turbidimetry and colorimetry.

Introduction. The increasing prevalence and growing resistance of fungi present a significant peril to public health. There are only four classes of antifungal medicines available today, and few candidates are in clinical trials.Hypothesis/Gap Statement. Rapid and sensitive diagnostic techniques are lacking for most fungal pathogens, and those that do exist are expensive or hard to obtain.Aim. This study aimed to evaluate the feasibility of a novel automated antifungal susceptibility testing system, Fungus AST, in comparison to the broth microdilution method (BMD) recommended by the Clinical and Laboratory Standards Institute (CLSI).Methodology. A total of 101 clinical Candida spp. isolates were collected from the Zengcheng Branch of Nanfang Hospital and subjected to antifungal susceptibility testing. Antifungal susceptibility was assessed using the Fungus AST method and the BMD.Results. In this study, we introduce a novel automated antifungal susceptibility testing system, Fungus AST, which detects the turbidity and/or colour intensity of microdilution wells using a four-wavelength detection technology in real time and is designed to match the growth characteristics of strains over time. Based on our analysis, all reportable ranges of Fungus AST were suitable for clinical fungal isolates in PR China. Within ±twofold dilutions, reproducibility was 100 %. Considering the BMD as a referenced method, ten antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole, voriconazole, posaconazole, itraconazole, amphotericin B, 5-flucytosine and nystatin) showed an essential agreement of >95 %. The category agreement of five antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole and voriconazole) was excellent at >90 %. One Candida albicans isolate and voriconazole showed a major error (ME) (1.7 %), and no other ME or very ME agents were found.Conclusion. Given the above, it can be argued that the utilization of Fungus AST is a discretionary automated approach. More improvements are needed in Fungus AST compared to the BMD system for a wider range of clinical isolates, including different types of fungi.

Rapid automated antifungal susceptibility testing system for yeasts based on growth characteristics.

Fungal pathogens are a major threat to public health, as they are becoming increasingly common and resistant to treatment, with only four classes of antifungal medicines currently available and few candidates in the clinical development pipeline. Most fungal pathogens lack rapid and sensitive diagnostic techniques, and those that exist are not widely available or affordable. In this study, we introduce a novel automated antifungal susceptibility testing system, Droplet 48, which detects the fluorescence of microdilution wells in real time and fits growth characteristics using fluorescence intensity over time. We concluded that all reportable ranges of Droplet 48 were appropriate for clinical fungal isolates in China. Reproducibility within ±2 two-fold dilutions was 100%. Considering the Sensititre YeastOne Colorimetric Broth method as a comparator method, eight antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin, micafungin, anidulafungin, amphotericin B, and 5-flucytosine) showed an essential agreement of >90%, except for posaconazole (86.62%). Category agreement of four antifungal agents (fluconazole, caspofungin, micafungin, and anidulafungin) was >90%, except for voriconazole (87.93% agreement). Two Candida albicans isolates and anidulafungin showed a major discrepancy (MD) (2.60%), and no other MD or very MD agents were found. Therefore, Droplet 48 can be considered as an optional method that is more automated and can obtain results and interpretations faster than previous methods. However, the optimization of the detection performance of posaconazole and voriconazole and promotion of Droplet 48 in clinical microbiology laboratories still require further research involving more clinical isolates in the future.

(3-(4-klorofenil)-4,5-dihidroizoksazol-4,5-diil)dimetanol Bileşiği: Antibakteriyel Aktivite, Antifungal Aktivite ve Hesaplanmış Yapısal Parametreler

We intended to quantify the antibacterial and antifungal activity results of 4,5-dihydroisoxazole dimethanol compound (1a) against several bacteria and fungi and also calculate some structural parameters (theoretical descriptors) of compound (1a) with this work. Microdilution broth procedures were studied using microdilution wells for the minimal inhibitory concentrations (MICs) test. Compound (1a) exhibited fair activities against all the bacteria and fungi. Compound (1a) has been a good result (MIC = 50 µg/ml) against particularly P. aeruginosa. The structure of compound (1a) was drawn, and geometrical optimization was done using the Ab initio (RHF/3-21G) level.

In vitro susceptibility testing of dermatophytes with their fragmented mycelia as inoculum

377 clinical isolates and seven dermatophyte culture collection strains were tested in vitro by microdilution against a panel of 18 antifungal agents. A newly introduced method for rapid and reliable inoculum preparation for moulds using the fragmented mycelia of freshly grown isolates as inoculum was firstly applied to dermatophytes. The performance of the method was tested with two different culture media which are recommended by different standardized testing methods for moulds. The standardized fragmented mycelia provided both, countable single colonies (viable units) on solid culture media and distinct readable endpoints (minimum inhibitory concentrations) in the microdilution wells for all tested topical and systemic drugs, including the echinocandins. As with moulds, and as shown by the culture collection strains, reproducible minimum inhibitory concentrations were obtained with an essential agreement (± 1 log2-dilution) of 97% to 100%, by a significant reduction of the overall testing time. As exemplarily shown for the microdilution technique, this inoculum method should also provide a solid basis for improved agar-based susceptibility testing methods such as disc-, tablet-, or strip-tests.

Analysis of Genetic Relationship of Sarothamnus scoparius Microsymbionts and Bradyrhizobium sp. by Hybridization in Microdilution Wells

DNA–DNA hybridization in microdilution wells was successfully used to determine the overall genomic similarity among Sarothamnus scoparius microsymbionts isolated from Poland and Japan and representatives of Bradyrhizobium species including Bradyrhizobium sp. ( Lupinus ) USDA3045. Geographically different S. scoparius rhizobia diverged into two genomospecies at the DNA–DNA similarity level of ∼49%. Polish isolates exhibited high DNA similarity levels to the DNA of Bradyrhizobium japonicum strains (∼78%) and formed a common genomospecies. Japanese S. scoparius rhizobia were allocated into a new genomic species due to the low similarity levels of their DNA to the DNA of representative strains of the Bradyrhizobium genus (from 19% to 52%).

Clinical application of microplate DNA-DNA hybridization procedure for rapid diagnosis of mycobacterial infections

Setting: As an alternative to biochemical analysis, microplate DNA-DNA hybridization was applied for rapid diagnosis of mycobacterial infection. Objective: To assess how rapidly and correctly the microplate hybridization method can progress from clinical sample to final species identification of mycobacteria. Design: Clinical samples (pooled sputa or bronchial lavage fluid) were obtained from patients. Depending on the estimated bacterial amounts, genetic identification was performed either directly or following primary culture. Extracted DNA labeled by photobiotin was hybridized in microdilution wells with type-strain DNAs from 4 species ( Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii), then identified on the basis of genetic relatedness, which was quantitated by colorimetric detection. Results: With samples containing more than 10 8 colony-forming units [CFU](5 cases), species identification was successfully performed on the day of sample preparation. With samples of not more than 10 7 CFU (14 cases), although 4–21 days' primary culture were necessary, species were also correctly identified by the microplate method. Furthermore, M. avium and M. intracellulare were distinctly identified. All the results precisely corresponded to those of biochemical analysis, which took 4–12 weeks to complete identification. Conclusion: We consider that microplate DNA-DNA hybridization is a dependable technique for rapid diagnosis of mycobacterial infection.

Anaerobic susceptibility testing slight differences in inoculum size can make a difference in minimum inhibitory concentrations

In the present study, we compared the actual inoculum density from inoculated broth microdilution wells to the targeted inoculum size (10 5 CFU/well) when the inoculum was prepared using a McFarland nephelometer to achieve the standard density. Three target inoculum sizes (10 5, 5 × 10 5, and 10 6 CFU/well) were used to compare the effect of slight inoculum size increases of both ATCC and clinical strains of anaerobes on MICs of various antimicrobials. Actual colony counts of Bacteroides fragilis, Bacteroides thetaiotaomicron, Eubacterium lentum, and Veillonella parvula ranged from 0.7 × 10 5 to 1.4 × 10 5 CFU/well. As the inoculum size rose above the desired 10 5 CFU/well level, the MICs of certain antimicrobials became elevated. Ceftizoxime, cefotaxime, and ceftriaxone MICs rose 4- to 16-fold with as little as 0.5 log 10 increase in inoculum size. Other increases were also noted with E. lentum and Clostridium perfringens, but were primarily between the low and high inoculum sizes. Results with cefoxitin, cefotetan, mezlocillin, and imipenem did not show an appreciable increased inoculum effect. This study demonstrates that the variation in organism size among anaerobes (both between species and within species) does make a difference in actual inoculum size and certain anaerobes may require special adjustment to ensure proper MIC results from susceptibility testing.

Identification to the Genospecies Level of Aeromonas Strains Isolated from Raw Meat and Carp, and Evaluation of Their Biological Activities

During the period from January to October 1992, a total of 214 samples (100 raw pork, 94 raw chicken and 20 carp) obtained in Ibaraki prefecture were examined for the presence of Aeromonas. It was found that 42 (42%) of 100 raw pork samples, 30 (32%) of 94 raw chicken samples and 13 (65%) of 20 carp were contaminated by some Aeromonas. The population of Aeromonas in contaminated samples was mostly below 102cfu/g. Attempts to identify the isolates by the biochemical method (phenospecies) revealed that 26 (19%) of 137 isolates belonged to Aeromonas hydrophila groups, 5 (2.3%)to A. caviae, 59 (27.5%) to A. sobria groups, and 4 (1.9%) to A. jandaei; the rest 10 (4.7%) were untypable.On the basis of genetic classification by DNA-DNA hybridization in microdilution wells, 13 of 26 A. hydrophila group isolates were reidentified as A. hydrophila. Eight of 87 A. sobria group isolates were identified as A. sobria and 74 as A. veronii biotype sobria. All of 7 A. jandaei isolates hybridized with the type strain. One each of the 10 untypable isolates hybridized with A. sobria and A. veronii biotype sobria.All of the isolates were investigated for hemolytic activity to rabbit and sheep erythrocytes, and cytotoxicity to HeLa 229 cells. Many strains showed both activities in A. hydrophila groups, A. veronii biotype sobria and A. jandaei, suggesting that these activities were associated. In A. caviae and A. sobria, many strains lacked these activities.

DNA reassociation and electrokaryotype study of some Candida species and synonymy of Candida terebra, Candida entomaea, and Candida veronae

Some Candida species that were similar in phenotypic characteristics were studied by molecular taxonomic comparisons including electrophoretic karyotypes, DNA base composition, and DNA relatedness. Among the species studied, Candida terebra, Candida entomaea, and Candida veronae showed similar electrophoretic karyotypes and a G + C range from 39.7 to 40.2 mol% in DNA base composition, and demonstrated 85–104% DNA similarity on the basis of flourometric hybridization in microdilution wells. As C. terebra has priority, we suggest that C. entomaea and C. veronae should be synonyms of C. terebra. Other species in this study, Candida dendronema, Candida diddensiae, Candida naeodendra, and Candida atmospherica, had low DNA relatedness. Thus, these species should be maintained in their original separate taxa.Key words: DNA reassociation, electrokaryotype, Candida species, synonym.

改良マイクロプレートハイブリダイゼーション法による<I>Legionella</I>属25菌種の同定と分離菌株を用いた評価

Modified microdilution plate hybridization was used for genetic identification of 25 Legionella species. The cell wall of the isolate was broken down by glass beads. And the DNA was extracted, labeled photo-reactive biotin, and hybridized with reference DNAs immobilized in microdilution wells. Hybridized DNAs were detected by colormetric method. Each type strain of 25 Legionella species was clearly differentiated by this method. Among 103 clinical and environmental Legionella strains, 97 strains were genetically identified by this method. 60 strains were identified as the same species as by the conventional method. Concerned with ten strains that had been serologically identified as L. bozemanii, two strains isolated from the human lung were genetically identified as L. bozemanii, but the remaining environmental 8 strains were identified as L. anisa. Among the 35 strains of legionellae that had been unidentified at species level, by physiological and serological tests, 27 strains were genetically identified as L. pneumophila, 1 strains as L. feeleii, and another, as L. anisa, but 6 strains were not identified. We found that three strains among the 6 strains belong to a single species of genus Legionella other than the 25 reference species used in this study.

Assay system for detecting bacteriocin in microdilution wells

Bacteriocin production can be easily detected by using microdilution wells. The method is very convenient for screening bacteria for bactericidal activity.

Spiral gradient endpoint method compared to standard agar dilution for susceptibility testing of anaerobic gram-negative bacilli

More efficient and reproducible alternative methods of performing agar dilution susceptibility testing are desirable, particularly for anaerobic bacteria. Anaerobes generally grow more reliably on solid media than they do in broth microdilution wells. A new method, the revised spiral gradient endpoint (SGE) method, was evaluated against the standard agar dilution (SAD) method by using a wide variety of anaerobic gram-negative bacilli (161 strains) and eight antimicrobial agents. For the SGE method, a spiral plater was used to set up a concentration gradient of an antimicrobial agent within an agar plate across which bacterial strains were inoculated as radial streaks. After incubation, the MIC of the antimicrobial agent was calculated from the radial endpoint location where bacterial growth ceased along the streak. The MICs for 90% of strains tested (in micrograms per milliliter) and the cumulative percentages of susceptible strains at the breakpoints for the SGE and SAD methods, respectively, and for all 161 strains were as follows: for metronidazole, 2 and 100 versus 2 and 100; for imipenem, 1 and 99 versus 0.5 and 98; for ampicillin-sulbactam, 8 and 97 versus 8 and 98; for clindamycin, 4 and 90 versus 4 and 91; for cefoxitin, 32 and 95 versus 32 and 95; for mezlocillin, 256 and 88 versus greater than 128 and 86; for ampicillin, greater than or equal to 256 and 51 versus greater than 64 and 51; and for penicillin (in units per milliliter), greater than or equal to 512 and 71 versus greater than 64 and 65. The excellent agreement of these data and the greater sensitivity reproducibility, and efficiency of the revised SGE method warrant further evaluations. Assuming that these advantages are confirmed, the revised SGE method should be a useful alternative test method when detailed susceptibility data are desired.

Colorimetric detection of DNA-DNA hybridization in microdilution wells for taxonomic application on bacterial strains

Colorimetric detection of hybridization in microdilution wells without radioisotope was designed and established for rapid and routine identification of taxonomic relatedness among bacterial species. Total chromosomal DNA for hybridization reactions was labeled with photoreactive biotin (photobiotin). The biotinylated DNA was hybridized with unlabeled single-stranded DNAs which were immobilized on the surfaces of microdilution wells. After hybridization, the biotinylated DNA was quantitatively detected by using an enzyme, streptavidin-conjugated peroxidase, and a dye, 3,3′,5,5′-tetramethylbenzidine, and analyzed with a microplate reader. The homology values among Bacillus spp. with biotinylated DNA of B. subtilis (natto) were in good agreement with those obtained by the membrane filter method with radioisotope. The method presented here is simple, rapid, and safe, and may be applicable routinely for the identification of bacterial species.

Evaluation of the microplate hybridization method for rapid identification ofLegionella species

Rapid genetic identification of 23 Legionella species was performed using microdilution plate hybridization. When an isolate appeared to be a strain of the Legionella species, its DNA was quickly extracted, labelled with photobiotin and distributed to microdilution wells where 23 different DNAs had previously been immobilized. After hybridization for 90 min at 45 degrees C, the microdilution well which emitted the maximum fluorescence was determined. The criterion for interpreting results of this genetic identification method is given.

Fluorometric Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane Filter Hybridization in which Radioisotopes Are Used To Determine Genetic Relatedness among Bacterial Strains

Fluorometric hybridization in microdilution wells was developed to determine genetic relatedness among microorganisms. Total chromosomal deoxyribonucleic acid (DNA) for hybridization reactions was labeled with photoreactive biotin (photobiotin). The biotinylated DNA was hybridized with single-stranded unlabeled DNAs which had been immobilized on the surfaces of microdilution wells. After hybridization, biotinylated DNA was quantitatively detected with beta-D-galactosidase and a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactopyranoside. Homology values obtained with this fluorometric direct binding method were compared with values obtained with two membrane filter methods, one in which photobiotin labeling was used and one in which radioisotope labeling was used. The results showed that the fluorometric direct binding method in which microdilution wells are used could be an alternative to radioisotope and membrane filter hybridization methods.

Inadequate mixing in microdilution wells during preparation of antimicrobial dilutions for susceptibility testing.

An equation representing a twofold series of antibiotic dilutions was derived. The theoretical results were then validated with experimental observations of piperacillin against Pseudomonas aeruginosa. Insufficient mixing leads to an erroneously low MIC. It is concluded that maintaining antibiotic concentration equilibrium during microdilution is required for accurate microdilution susceptibility tests.

Simple genetic method to identify viridans group streptococci by colorimetric dot hybridization and fluorometric hybridization in microdilution wells.

Simple dot hybridization and fluorometric hybridization methods in microdilution wells were designed and established for rapid and routine genetic identification of viridans group streptococci. Reference DNA extracted from each strain of 24 reference Streptococcus species was fixed both on a nitrocellulose filter and in a microdilution well. A 1-ml portion of the bacterial suspension which matched the turbidity of McFarland no. 2 standard was prepared when a streptococcal strain was isolated. It was lysed with achromopeptidase, and the DNA was quickly labeled with photobiotin under a sunlamp for 15 min. Dot hybridization and fluorometric hybridization were then carried out between the labeled DNA of the unknown organism and 24 unlabeled reference DNAs. Hybridized fragments on a nitrocellulose filter were detected by using alkaline-phosphatase-conjugated streptavidin and analyzed with a color graphic analyzer. Hybridized fragments in microdilution wells were quantitatively detected by using an enzyme, streptavidin-conjugated beta-D-galactosidase, and a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactoside. Strains belonging to each genetically distinct species could be identified by this dot blot hybridization test. However, some clinical strains cross-hybridized with two or more reference species, and then they were difficult to differentiate by dot blot hybridization. In such a case, fluorometric identification provided reliable results because the fluorometric method was more quantitative than dot blot identification. By these methods, it was possible to determine species assignment within the viridans group.

MBCs for Staphylococcus aureus as determined by macrodilution and microdilution techniques.

MBC testing of clindamycin, methicillin, cephalothin, gentamicin, and vancomycin with 67 clinical isolates of Staphylococcus aureus was examined by both standard macrodilution tubes and commercial microdilution trays. Standard macrodilution failed to give reproducible (99.9% killing) MBC results, even when a strictly defined protocol was followed. Continuous shaking during incubation resulted in regrowth of more colonies than did stationary incubation. Vortexing of incubated tubes before subculture resulted in regrowth of more colonies than did careful transfer of the contents to sterile tubes before vortexing and subculture. No significant difference in MBCs was demonstrated by the use of log-phase versus stationary-phase inocula. Use of the multiprong inoculator for subculture from commercial microdilution trays was unsatisfactory because, although antibiotics evaluated were inactivated by subculture to a pH 5.5 agar plate coated with a beta-lactamase solution, the volume of broth transferred by the prongs was small and inconsistent, ranging from 0 to 3 microliter. Subcultures of commercial microdilution panels with a 1-microliter loop, 10-microliter pipette, and 100-microliter pipette were also evaluated. Results of MBC testing were most reproducible when the entire 100-microliter volume was aspirated from commercial microdilution wells after stirring and the contents of each well were spread over a separate sheep blood agar plate.