Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis

Natto, Manal J.; Savioli, Francesca; Quashie, Neils B; Dardonville, Christophe; Rodenko, Boris; Koning, Harry P. de

doi:10.1093/jac/dkr572

Cited by 26 publications

(37 citation statements)

References 43 publications

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“…Until now, it has not been used in broth media for Malassezia spp. because it is not always color stable during prolonged incubation and therefore needs to be buffered (16,17). The careful buffering of OptiMAL with sodium bicarbonate and esculin allowed for the concurrent use of resazurin as a color indicator.…”

Section: Discussionmentioning

confidence: 99%

Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method

et al. 2017

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Malassezia is a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malassezia spp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing of Malassezia that is based on the CLSI and EUCAST assays for Candida and other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of all Malassezia spp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13 Malassezia species to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC 90 values of 0.03 to 1.0 g/ml, 0.06 to 0.5 g/ml, and 0.03 to 2.0 g/ml, respectively. All Malassezia spp. were resistant to echinocandins and griseofulvin. Some Malassezia spp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treat Malassezia skin infections. In summary, our assay enables the fast and reliable susceptibility testing of Malassezia spp. with a large panel of different antifungals.

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Section: Discussionmentioning

confidence: 99%

Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method

et al. 2017

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“…In the second series of experiments, we validated our viability assay based on the reduction of resazurin into resorufin. The rate at which this reduction occurs is indeed dependent on both the biofilm maturity and the number of metabolically active bacteria, but the reaction product may undergo additional enzymatic and nonenzymatic transformation(s) (42,43), causing the fluorescent signal to increase and then decrease over time. Figure 2 shows the change in fluorescence recorded over time upon incubation at room temperature for (i) planktonic cells at different densities, and (ii) sessile cells in (a) young (2 days) and old (11 days) biofilms and (b) naive and induced biofilms.…”

Section: Resultsmentioning

confidence: 99%

“…We showed here that it can be applied to S. pneumoniae biofilms, provided the time after which readings are made is carefully selected to capture the maximal fluorescence signal, which critically depends on both the number of bacteria and the degree of biofilm maturity (since the decrease in the signal can also occur upon too prolonged incubation due to further metabolization in nonfluorescent dihydroresorufin) (42,48). Because of its proportionality with the number of bacteria, this approach may help in avoiding pitfalls inherent in the other more commonly used method to assess bacterial viability in biofilms, namely, CFU counting after sonication (18,23,24,49,50).…”

Section: Discussionmentioning

confidence: 99%

Antibiotic Activity against Naive and Induced Streptococcus pneumoniae Biofilms in an In Vitro Pharmacodynamic Model

Vandevelde

Tulkens

2014

Antimicrob Agents Chemother

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Biofilms play a role in the pathogenicity of pneumococcal infections. A pharmacodynamic in vitro model of biofilm was developed that allows characterization of the activity of antibiotics against viability and biomass by using in parallel capsulated (ATCC 49619) and noncapsulated (R6) reference strains. Naive biofilms were obtained by incubating fresh planktonic cultures for 2 to 11 days in 96-well polystyrene plates. Induced biofilms were obtained using planktonic bacteria collected from the supernatant of 6-day-old naive biofilms. Biomass production was more rapid and intense in the induced model, but the levels were similar for both strains. Full concentration responses fitting sigmoidal regressions allowed calculation of maximal efficacies and relative potencies of drugs. All antibiotics tested (amoxicillin, clarithromycin, solithromycin, levofloxacin, and moxifloxacin) were more effective against young naive biofilms than against old or induced biofilms, except macrolides/ketolides, which were as effective at reducing viability in 2-day-old naive biofilms and in 11-day-old induced biofilms of R6. Macrolides/ketolides, however, were less potent than fluoroquinolones against R6 (approximately 5-to 20-fold-higher concentrations needed to reduction viability of 20%). However, at concentrations obtainable in epithelial lining fluid, the viabilities of mature or induced biofilms were reduced 15 to 45% (amoxicillin), 17 to 44% (macrolides/ketolides), and 12 to 64% (fluoroquinolones), and biomasses were reduced 5 to 45% (amoxicillin), 5 to 60% (macrolides/ketolides), and 10 to 76% (fluoroquinolones), with solithromycin and moxifloxacin being the most effective and the most potent agents (due to lower MICs) in their respective classes. This study allowed the ranking of antibiotics with respect to their potential effectiveness in biofilm-related infections, underlining the need to search for still more effective options.

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“…This application is common in the bio-medical field [38][39][40] and progressively arises in environmental research [41][42][43]. Even in wastewater treatment some investigations were made [34] but knowledge about it is still poor.…”

Section: Detection Of Bacterial Inhibitorsmentioning

confidence: 99%

New outlook in BOD measurement and bioprocess management: the Enverdi® kit

Müller¹,

Bellaton²,

Yoris³

et al. 2017

18th International Congress of Metrology

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Abstract. Knowing the quantity and biodegradability of wastewater organic matter (OM) is really essential for the optimization of modern biological treatment processes, particularly when considering the highly challenging context for sewage treatment plant operators. However, the current standard methods for wastewater characterisation, as for 5-days biochemical oxygen demand (BOD5) measurement, OM fractionation analysis and bacterial inhibitor detection, show very poor suitability for routine operational use. In this paper, the efficiency of the commercial Enverdi® method (fluorimetric respirometry) for such analytical applications is investigated, especially for the BOD5-equivalent measurement in municipal wastewater samples. The result of numerous experiments showed that the BOD5 values obtained from the alternative Enverdi® method and the main standard methods are statistically equivalent. The analytical performances of the Enverdi® method was found similar to the standard BOD5 method specifications and perfectly matched the French regulatory requirements in force. The regulatory acceptance of this method is presently under consideration. Such an outlook would be really valuable for end-users given the main advantages of this analytical solution which is time-and space-saving, user-friendly and financially competitive. In addition to BOD5 measurement, the fluorimetric respirometry displayed very promising abilities for the OM fractionation and the detection of bacterial inhibitors in wastewater.

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Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis

Abstract: The resorufin- and PI-based assays are suitable for routine and high-throughput drug screening, whereas resazurin-based assays are not. These assays constitute a major advance in the current protocols as demonstrated by a successful screen for new antitrichomonal lead compounds.

Cited by 26 publications

References 43 publications

Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method

Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method

Antibiotic Activity against Naive and Induced Streptococcus pneumoniae Biofilms in an In Vitro Pharmacodynamic Model

New outlook in BOD measurement and bioprocess management: the Enverdi® kit

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