Evaluation of a Scanner-Assisted Colorimetric MIC Method for Susceptibility Testing of Gram-Negative Fermentative Bacteria

We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within ±1 log₂ dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.     

Use of spectrophotometric reading for in vitro antifungal susceptibility testing of Aspergillus spp

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72 h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.     

Comparative study of in vitro methods to analyse the antifungal activity of propolis against yeasts isolated from patients with superficial mycoses

AIM: To test a total of 15 strains belonging to four species of yeasts by different in vitro methods against propolis and itraconazole (ITC). METHODS AND RESULTS: Three methods were compared for susceptibility testing of yeast isolates to propolis: disc diffusion method, agar dilution method and National Committee for Clinical Laboratory Standards (NCCLS, M27A) broth microdilution method. ITC was selected as the antifungal agent for comparison study. Using the broth microdilution method, the geometric mean for MIC (microg ml(-1)) with regard to all isolates was < or =0.06 for propolis and < or =0.35 for ITC. The broth microdilution and the agar dilution methods were in good agreement (75%) for propolis against yeasts isolated from patients with superficial mycoses. Using the diffusion method, all strains showed a broad zone of inhibition at the first available reading time (24 or 48 h). An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. A favourable correlation was found between MIC and inhibition zone around the disc for propolis sample and the correlation coefficient was: r = -0.626 (P < 0.01). CONCLUSIONS: This study suggests the potential value of the agar dilution and disc diffusion method as a convenient alternative method for testing of yeasts to propolis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that propolis and ITC were very active against yeasts from patients with superficial mycoses. The other prominent finding in this study is that RPMI 1640 with L-glutamine was the available broth for the in vitro susceptibility testing of yeasts.     

Comparison of the WST-8 colorimetric method and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria

The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24h tended to give lower MICs. The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent agreement between the MICs determined after 24h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria.     

Standardization of a broth microdilution susceptibility testing method to determine minimum inhibitory concentrations of aquatic bacteria

A multiple laboratory study was conducted in accordance with the standards established by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), for the development of quality control (QC) ranges using dilution antimicrobial susceptibility testing methods for bacterial isolates from aquatic animal species. QC ranges were established for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 when testing at 22, 28 and 35 degrees C (E. coli only) for 10 different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline and trimethoprim/sulfamethoxazole). Minimum inhibitory concentration (MIC) QC ranges were determined using dry- and frozen-form 96-well plates and cation-adjusted Mueller-Hinton broth. These QC ranges were accepted by the CLSI/NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing in January 2004. This broth microdilution testing method represents the first standardized method for determining MICs of bacterial isolates whose preferred growth temperatures are below 35 degrees C. Methods and QC ranges defined in this study will enable aquatic animal disease researchers to reliably compare quantitative susceptibility testing data between laboratories, and will be used to ensure both precision and inter-laboratory harmonization.     

Colorimetric broth microdilution method for the antifungal screening of plant extracts against yeasts

Screening plant extracts for antifungal activity is increasing due to demand for new antifungal agents, but the testing methods present many challenges. Standard broth microdilution methods for antifungal susceptibility testing of available antifungal agents are available now, but these methods are optimised for single compounds instead of crude plant extracts. In this study we evaluated the standard NCCLS method as well as a modification which uses spectrophotometric determination of the end-points with a plate reader. We also evaluated another standard method, the EUCAST method, which is a similar microdilution assay to the NCCLS method, but uses a larger inoculum size and a higher glucose concentration in the medium as well as spectrophotometric end-point determination. The results showed that all three methods had some drawbacks for testing plant extracts and thus we modified the NCCLS broth microdilution method by including a colorimetric indicator-resazurin for end-point determination. This modified method showed good reproducibility and clear-cut end-point, plus the end-point determination needed no instruments. It enabled us to evaluate the activity of a selection of extracts from six Combretaceous plants against three Candida spp. and thus provided pharmacological evidence for some traditional uses of these plants while assisting the identification of the active ingredients.     

Tetrazolium salts for MIC determination in microplates: why? Which salt to select? How?

We reported evaluation of a colorimetric MIC assessment for routine susceptibility testing of non-fastidious bacteria, with addition of growth indicators (INT and MTT). Our results made us postulate that the use of such indicators was unnecessary for MIC determination in routine microdilution method.     

Development of Reference Procedures for Broth Microdilution Antifungal Susceptibility Testing of Yeasts with Standardized Endpoint Determination

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.     

Multicenter evaluation of commercial frozen plates for microdilution broth antifungal susceptibility testing of yeasts and comparison of MIC limits recommended in NCCLS M27-A2

A commercial kit, Frozen Plate for Antifungal Susceptibility Testing of Yeasts, Eiken (Eiken Chemical Co., Ltd., Tokyo), was tested in a multi-institute study to evaluate the agreement between interinstitute MICs and National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 recommendation limits of MIC value. The kit was reported as a method equivalent to the standardized guidelines for antifungal susceptibility testing by the Committee for Clinical Laboratory Standards-1994, the Japanese Society for Medical Mycology, and which is widely used in Japan for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole. The degrees of inter-institute and NCCLS agreements were good to excellent especially with 48-hr incubation for all antifungal agents. However, the percent agreements to NCCLS recommendations against itraconazole were poor. Overall, MIC values obtained using the frozen plate antifungal susceptibility testing kit, with 48-hr incubation, were thought to be reliable and convenient alternatives to the data obtained by the NCCLS M27-A2 reference macrodilution and microdilution method. This kit will allow matching of results between international laboratories. However, the MIC value for itraconazole requires careful interpretation.     

Susceptibility testing of anaerobic bacteria: A review of current methods and future prospects

The current NCCLS document, M11 A2, describes two methods for susceptibility testing of anaerobic bacteria. The reference method utilizes an agar dilution procedure, which is labor intensive and not convenient for testing individual patient isolates. The broth microdilution method does not support the growth of 15–40% clinical isolates and demonstrates poor correlation with the reference method for some members of the Bacteroides fragilis group with β-lactam agents and clindamycin. Etest is a new technique that incorporates an antibiotic gradient onto a plastic strip and utilizes agar media. This method is easily performed, permits growth of all anaerobes, and provides quantitative MICs for rapidly growing strains after overnight (20 hr) incubation. This method is convenient and reliable and enables the laboratory to provide the clinician with MIC data for individual patient isolates within a clinically relevant time period.     

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log(2) dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (+/-1 log(2) dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.     

Importance of biofilm in<Emphasis Type="Italic">Candida parapsilosis</Emphasis> and evaluation of its susceptibility to antifungal agents by colorimetric method

The ability of C. parapsilosis (an important cause of nosocomial infections) to produce biofilm was evaluated in 32 bloodstream isolates and 85 strains isolated from skin. The biofilm formation was found in 19 (59%) blood isolates and only in 33 (39%) isolates from skin. The antifungal susceptibility was assessed for amphotericin B, itraconazole and voriconazole in planktonic and biofilm form of the 19 biofilm-positive bloodstream strains by broth microdilution method according to NCCLS standards. The method was modified by the use of resazurin as a colorimetric indicator of the metabolically active cells which makes the determination of the effect of antifungal agents easier. Biofilm forms of all strains were more resistant than their planktonic form.     

Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts

The aim of this study was to evaluate diffusion and dilution methods for determining the antibacterial activity of plant extracts and their mixtures. Several methods for measurement of the minimal inhibitory concentration (MIC) of a plant extract are available, but there is no standard procedure as there is for antibiotics. We tested different plant extracts, their mixtures and phenolic acids on selected gram-positive (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter jejuni, Campylobacter coli) with the disk diffusion, agar dilution, broth microdilution and macrodilution methods. The disk diffusion method was appropriate only as a preliminary screening test prior to quantitative MIC determination with dilution methods. A comparison of the results for MIC obtained by agar dilution and broth microdilution was possible only for gram-positive bacteria, and indicated the latter as the most accurate way of assessing the antimicrobial effect. The microdilution method with TTC (2,3,5-triphenyl tetrazolium chloride) or INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to indicate the viability of aerobic bacteria was found to be the best alternative approach, while only ATP determination was appropriate for microaerophilic Campylobacter spp. Using survival curves the kinetics of bacterial inactivation on plant extract exposure was followed for 24 h and in this way the MIC values determined by the microdilution method were confirmed as the concentrations of extracts that inhibited bacterial growth. We suggest evaluation of the antibacterial activity of plant extracts using the broth microdilution method as a fast screening method for MIC determination and the macrodilution method at selected MIC values to confirm bacterial inactivation. Campylobacter spp. showed a similar sensitivity to plant extracts as the tested gram-positive bacteria, but S. Infantis and E. coli O157:H7 were more resistant.     

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium,monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log₂ dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (± 1 log₂ dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.     

Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi

The aim of this study was to establish a broth microdilution method for antimicrobial susceptibility testing of Helicobacter cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified Levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The minimum inhibitory concentrations obtained by these two methods were almost the same for all the antibiotics tested, demonstrating the broth microdilution method to be a suitable and reliable technique for antimicrobial susceptibility testing. A broth microdilution method for antimicrobial susceptibility test for H. cinaedi was established. This method is expected to help improve treatment.     

Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods

A modified fluorescein diacetate (FDA) assay has been compared with standard NCCLS broth macrodilution and broth microdilution methods for the detection of antifungal activity. The FDA assay was performed in a medium containing bacteriological peptone, NaCl, yeast extract and glucose (0.2%, 0.1%, 0.1% and 1% w/v, respectively) and buffered with 10 mM BES buffer. The MICs of amphotericin B, fluconazole, miconazole and flucytosine (representing three major classes of antifungal agents) obtained by the three methods were compared. The results obtained with the FDA assays correlated well with the NCCLS macrodilution method for MICs of amphotericin B, miconazole and fluconazole, but not for flucytosine. However, the MIC values of flucytosine obtained with the FDA assay were well within the quality control range for the two reference strains recommended by the NCCLS. The FDA assay described is an attractive alternative to the NCCLS methods for screening for antifungal agents, with the added advantage of objectivity of fluorescence measurement.     

Comparison of broth microdilution, Etest, and agar disk diffusion methods for antimicrobial susceptibility testing of Lactobacillus acidophilus group members

In recent years, the absence of acquired antimicrobial resistance has become an important criterion to evaluate the biosafety of lactobacilli used as industrial starter or probiotic cultures. At present, however, standards for susceptibility testing of Lactobacillus strains or approved guidelines for interpreting the test results are not available. Hence, this study was carried out to contribute to the establishment of a standardized procedure for antimicrobial susceptibility testing of lactobacilli. The results obtained by testing 104 strains of the Lactobacillus acidophilus group were compared based on broth microdilution, disk diffusion, and Etest. Except for some specific agent-related effects, agreement between MICs resulting from the broth microdilution method and the Etest was good. In addition, inhibition zone diameters determined with disk diffusion correlated well with MICs from Etest and broth microdilution.     

One step forward to improve the latest method of antibacterial susceptibility testing of vitro-cultured bacteria: an implication for antibacterial efficacy of Enrofloxacine on <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Recently a new method of antibacterial susceptibility testing has been developed, which based on the reduction of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by bacteria. This study aimed to improve the latest developed method in terms of the period of time, concentration of bacterium, accuracy of assay and minimizing the toxic effect of the MTT solution. The commonly used broth microdilution method was compared in this study, as well. The minimum inhibition concentration (MIC) of the enrofloxacine against Aeromonas hydrophila as an opportunistic and ubiquitous bacterium was tested by using the improved MTT method. Using both methods showed that enrofloxacine exerts an excellent antibacterial effect against A. hidrophyla with MIC value of 62.5 ng/ml. The simplicity of the improved MTT method was shown with performing all steps of the assay in one plate, using rather low concentration of the bacterium (500 CFU/well) and shortening of the incubation time to 9 h. Moreover, 5–30 min incubation of bacteria with MTT solution excludes any toxic effect of tetrazolium salt against bacteria. Thus, our results suggest that enrofloxacine might be considered as a potent antibacterial agent against A. hydrophila induced contaminations. Moreover, MTT method with current improved characteristics such as short incubation time, low concentration of bacteria, and high sensitivity could be more practical alternative for the broth microdilution method in antibacterial susceptibility testing protocol.     

Evaluation of a new method for antifungal susceptibility testing for azoles

The interpretation of the end points in azole antifungal drug susceptibility testing is challenging, in part due to incomplete growth inhibition of Candida species. Since the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method have limitation with azoles, a new modification of the CLSI microdilution protocol was evaluated. We measure the decrease in growth rate (μ) of exponentially growing cultures in accordance to different azole concentrations at time intervals up to 10 h. Using 15 different Candida strains, an overall agreement within ± 2 dilutions by the CLSI method at 24 h in RPMI and the μ-dependent method for three antifungal agents (fluconazole- itraconazole and voriconazole) was achieved. MIC measurement by the new method was less sensitive to the medium used or the inoculum size applied. The presented data suggested that, measuring the in vitro inhibition kinetics at the logarithmic phase could have advantages for addressing susceptibility testing toward azoles.     

评估VITEK2-COMPACT GN13与纸片扩散法测定粘质沙雷菌对亚胺培南药敏结果的可靠性研究

目的评估法国生物梅里埃VITEK2-COMPACT GN13药敏系统(VITEK2法)和纸片扩散法(K-B法)检测粘质沙雷菌对亚胺培南药敏结果的可靠性。方法本研究选取了50株金华市中心医院2014年6月至11月临床标本中分离出的非重复的粘质沙雷菌,分别采用VITEK2法、K-B法和微量肉汤稀释法测定其对亚胺培南的体外敏感性,以微量肉汤稀释法作为参考方法,评估VITEK2法、纸片扩散法与参考方法的分类一致率(CA%)。结果K-B法与参考方法的一致率为94.0%(47/50),仅有6.0%的小错误(MIE),未出现大错误(ME)和极大错误(VME);而VITEK2法与参考方法的一致率仅为48.0%(24/50),其中VITEK2法测定为敏感的菌株与参考方法的一致率达95.6%,但VITEK2法测定为耐药或中介的菌株与参考方法的一致率仅为7.4%,小错误率(MIE%)和大错误率(ME%)分别为55.6%和37.0%,未出现极大错误(VME)。结论 K-B法检测粘质沙雷菌对亚胺培南的药敏结果是比较可靠的;VITEK2法检测粘质沙雷菌对亚胺培南敏感的结果也是可靠的,但对亚胺培南非敏感结果的错误率(ME+MIE)高达到92.6%,实验室日常工作中若发现此类结果应采用K-B法或微量肉汤稀释法重新复核。