Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the “gold” standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.     

Rapid Bead-Based Antimicrobial Susceptibility Testing by Optical Diffusometry

This study combined optical diffusometry and bead-based immunoassays to develop a novel technique for quantifying the growth of specific microorganisms and achieving rapid AST. Diffusivity rises when live bacteria attach to particles, resulting in additional energy from motile microorganisms. However, when UV-sterilized (dead) bacteria attach to particles, diffusivity declines. The experimental data are consistent with the theoretical model predicted according to the equivalent volume diameter. Using this diffusometric platform, the susceptibility of Pseudomonas aeruginosa to the antibiotic gentamicin was tested. The result suggests that the proliferation of bacteria is effectively controlled by gentamicin. This study demonstrated a sensitive (one bacterium on single particles) and time-saving (within 2 h) platform with a small sample volume (~0.5 μL) and a low initial bacteria count (50 CFU per droplet ~ 10⁵ CFU/mL) for quantifying the growth of microorganisms depending on Brownian motion. The technique can be applied further to other bacterial strains and increase the success of treatments against infectious diseases in the near future.     

解脲脲原体生物膜药敏检测方法的研究进展

解脲脲原体（Uu）生物膜的培养及药敏检测技术是继支原体体外游离药敏试验之后,探讨Uu耐药水平与耐药机制的又一重要手段。然而由于支原体生长的生物学特性,使实验中需要多次更换培养基,增加了污染的概率。培养过程中的杂菌污染由此成为决定Uu生物膜实验成功与否的一大问题。本文将对解脲脲原体生物膜的培养与药敏检测技术,从检测方法、常见污染途径与解决策略3个方面展开,对相关文献作一综述。     

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.Download video file.^{(44M, mov)}     

The Relationship Between Agar Thickness and Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing can be done using solid or liquid-based medium. Solid-based assays are easy and inexpensive; they are limited by not being as quantitative as liquid-based assays. Agar depth can influence the accuracy of plate-based assays and it is assumed the basis of this effect is antimicrobial agent diffusion. We tested this assumption by using ETEST^® to quantitate the relationship between agar depth and minimum inhibitory concentration and zone of inhibition.     

水产病原菌抗菌药物敏感试验标准化探讨

病原菌药物敏感试验是临床微生物学研究的重要手段,相比人类和兽医临床,国内外水产养殖中细菌性病原的药敏试验还未有公认的标准化参考方法。概括近几年国外对水产病原菌药敏试验标准化方法建立的研究进展,探讨了试验中关键因子的影响,指出我国水产药敏试验中存在的不足及对未来发展提出展望。     

Detailed Methodology and Implementation of a Semiautomated Serial Dilution Microtechnique for Antimicrobial Susceptibility Testing

The detailed methodology and implementation of a semiautomatic microtechnique for performing serial dilution antimicrobial susceptibility studies are described. Quantitative susceptibility studies to a battery of antimicrobials are performed routinely on all significant clinical isolates. Results are reported as the minimal inhibitory concentration in micrograms per milliliter of broth. Guidelines relating standard doses of antimicrobials with expected blood and urine levels are presented to facilitate the use of the quantitative data. This microtechnique is used to measure serum and other body fluid levels of antimicrobial agents to document the level attained with a specific course of therapy. This technique is highly reproducible and has a high correlation with, and is at least 10 times faster than, standard glass tube techniques.     

Cryptococcus neoformans: The Model Organism for Yeast Antifungal Drug Susceptibility Testing

We describe an approach to antifungal susceptibility testing of the yeast Cryptococcus neoformans that shows promise for predicting the mycological response in patients to treatment. Quantitative cultures of the cerebrospinal fluid provide a direct measure of the patient's mycological response to treatment and have been used in multiple studies to identify the most promising antifungal drugs for subsequent testing in larger clinical studies. Using these quantitative measures of response, a modified macrobroth dilution assay system shows the potential for predicting the response of an individual patient to treatment with amphotericin B, fluconazole, or the combination of amphotericin B plus flucytosine. We describe this modified macrobroth dilution assay method, the statistical approach for assessing susceptibility, and the clinical decisions that can be guided by this in vitro antifungal drug susceptibility testing.     

Standardized Antimicrobial Disc Susceptibility Testing of Anaerobic Bacteria. I. Susceptibility of Bacteroides fragilis to Tetracycline

A modified Bauer-Kirby-Sherris-Turck method for disc susceptibility testing of anaerobic bacteria is presented. When tetracycline was used against 100 strains of Bacteroides fragilis as a model, reasonably reproducible results were obtained after overnight incubation in both the GasPak atmosphere and an atmosphere achieved by adding 10% CO(2) to a mixture of 10% H(2) and 90% N(2). The minimal inhibitory concentration for the strains determined by the agar dilution technique correlated well with the results of disc tests performed in the GasPak atmosphere with 30-mug tetracycline discs. Among 63 strains isolated from 1970 to the present, only 24 (38.1%) were found to be susceptible to tetracycline.     

Modification of the Microtiter Reading Mirror for Use in the Standardized Micro Complement Fixation Test

Modification of the Microtiter reading mirror used in the standardized diagnostic complement fixation method permits convenient estimation of the results in per cent hemolysis by direct visual comparison with the hemolytic standards.     

Species Used for Drug Testing Reveal Different Inhibition Susceptibility for 17beta-Hydroxysteroid Dehydrogenase Type 1

Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17β-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17β-HSD types 1, 2, 4, 5 and 7 but also against 17β-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17β-HSDs analyzed were observed. Especially, the rodent 17β-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17β-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.     

An Analytical Method for Assessing Stage-Specific Drug Activity in Plasmodium vivax Malaria: Implications for Ex Vivo Drug Susceptibility Testing

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC(50)) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC(50) for CQ. Our findings indicate that EC(50) values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.     

Comparison of Microtiter Procedures with the Plaque Technique for Assay of Vesicular Stomatitis Virus

This paper describes a microprocedure for the tissue culture assay of vesicular stomatitis virus (VSV) and compares the sensitivity of the method with the conventional plaque assay of viral concentration. Microtiter and plaque assay methods were used in titrations, neutralization tests, and thermoinactivation studies with this virus in chick embryo fibroblasts (CEF) and L, HeLa, and PK cell lines. Titration experiments with VSV by the microtiter procedure on preformed monolayers were significantly more sensitive than the plaque assay (P = 0.05). The results of the neutralization tests and thermoinactivation studies also showed greater ability to detect residual virus by the microtiter procedure (P = 0.10). In addition, the microtiter procedure was simpler, less costly, and more rapid than the plaque assay.     

Tube Dilution Antimicrobial Susceptibility Testing: Efficacy of a Microtechnique Applicable to Diagnostic Laboratories

A microtechnique for determining antibiotic susceptibilities by the serial dilution method was evaluated in a clinical diagnostic microbiology laboratory. As compared with the standard tube method, an agreement of 94% was achieved for determining minimal inhibitory concentration with +/- one tube dilution as the criterion of comparison. The experience with this system suggests that it could easily be incorporated into diagnostic laboratories as a routine procedure.     

Continuous Cultivation and Drug Susceptibility Testing of Plasmodium falciparum in a Malaria Endemic Area

ABSTRACT Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum. This finding will have practical implications in malaria endemic areas where difficulties in obtaining non-immune human plasma or serum limits establishment of continuous culture of P. falciparum and its application in studies on malaria.     

Susceptibility of Genital Mycoplasmas to Antimicrobial Agents

The susceptibility of 11 T-strains, 12 strains of Mycoplasma hominis, and a single strain of M. fermentans to 15 antimicrobial agents was determined by study of inhibition of metabolic activity in a broth dilution system. All three species were inhibited by tetracycline, chloramphenicol, streptomycin, gentamicin, and kanamycin, and were relatively resistant to cephalothin, cephaloridine, polymyxin, vancomycin, and ampicillin. Three antimicrobial agents had significant differential effects on these species. Erythromycin was more active against T-strains than against M. hominis or M. fermentans. Lincomycin, clindamycin, and nitrofurantoin had greater activity against M. hominis and M. fermentans than against T-strains. The activity of the drugs tested was generally uniform over a wide range of inocula. The effect of pH and the difference between minimal inhibiting and minimal mycoplasmacidal concentrations of the drugs tested were consistent with expectations based on the effects of these drugs on bacteria.     

Determination of Antibiotic Susceptibility of Rickettsia by the Plaque Assay Technique

Plaque formation by various rickettsiae was completely inhibited by commercial antibiotic discs impregnated with tetracycline, chloramphenicol, nitrofurantoin, and erythromycin; partial inhibition was observed around discs containing nalidixic acid and sulfisoxazole, but no inhibition was seen around discs containing cephalothin, ampicillin, oxacillin, kanamycin, polymyxin B, streptomycin, or penicillin.     

通过化学结构修饰提高虫草素抗菌活性的试验研究

本文以我国传统药用真菌蛹虫草(Cordyceps militaris)的主要活性成分虫草素为先导化合物,合成了4个虫草素的正构烷酰胺同系物(Cn-AAC),即N-丙酰基虫草素(C3-AAC)、N-正辛酰胺虫草素(C8-AAC)、N-月桂酰基虫草素(C12-AAC)和N-硬脂酰基虫草素(C18-AAC),并通过抑菌试验对比测定了虫草素和4个Cn-AAC的最低抑菌浓度(MIC)。在此基础上,用SPSS10.0对MIC和Cn-AAC烷酰基侧链中碳原子数目(n)之间的关系进行了定量分析。结果表明:通过化学结构修饰的手段将虫草素改变成Cn-AAC同系物后,其抗菌活性会发生有规律的变化。Cn-AAC的MIC和n之间的关系可以用一个一元二次方程进行定量描述,其中抗菌活性最强的为C8-AAC;和虫草素相比,C8-AAC对枯草杆菌的MIC降低5倍,并可对虫草素本身不能产生抗菌活性的金黄色葡萄球菌和白色念珠菌产生抗菌活性。