Automated detection of bacterial growth on 96-well plates for high-throughput drug susceptibility testing of Mycobacterium tuberculosis

Fowler, Philip; Cruz, Ana Lúıza Gibertoni; Hoosdally, Sarah; Jarrett, Lisa; Borroni, Emanuele; Chiacchiaretta, Matteo; Rathod, Priti; Lehmann, Sarah; Molodtsov, Nikolay; Walker, Timothy M; Robinson, Esther; Hoffmann, Harald; Peto, Tim E. A.; Cirillo, Daniela María; Smith, Grace; Crook, Derrick W.

doi:10.1099/mic.0.000733

Cited by 24 publications

(29 citation statements)

References 15 publications

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“…In addition, a photograph was taken at each time point by use of the Vizion instrument, and this was retrospectively analyzed using some bespoke plate-reading software, AMyGDA (Fig. S2) ( 18 ). For more information on the above, please see Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

“…MICs measured from photographs of the UKMYC5 plates after 14 days of incubation by use of AMyGDA software (Fig. S2) ( 18 ) were then compared to MICs measured by the laboratory scientist by use of the Vizion system. As before, we consider the MICs to be in agreement if they are within a doubling dilution of one another.…”

Section: Resultsmentioning

confidence: 99%

“…MIC results and additional data were recorded locally on paper and in a shared Web-enabled database (Table S17). An image of each plate was captured using the Vizion system and was stored and subsequently analyzed by AMyGDA (automated mycobacterial growth detection algorithm) software ( 18 ). AMyGDA analysis was performed at the University of Oxford, using default settings (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Validating a 14-Drug Microtiter Plate Containing Bedaquiline and Delamanid for Large-Scale Research Susceptibility Testing of Mycobacterium tuberculosis

Rancoita

Cugnata

et al. 2018

Antimicrob Agents Chemother

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The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical Mycobacterium tuberculosis isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Validating a 14-Drug Microtiter Plate Containing Bedaquiline and Delamanid for Large-Scale Research Susceptibility Testing of Mycobacterium tuberculosis

Rancoita

Cugnata

et al. 2018

Antimicrob Agents Chemother

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“…Images were deduplicated by checking the MD5SUM was unique. The remaining images were first read by bespoke software, AMyGDA (55, 56), which detects the locations of the wells and, by measuring the growth in each well, estimates an MIC for all drugs. For 54.7% of all measurements the MICs measured by the laboratory scientist and AMyGDA were identical (Fig.…”

Section: Methodsmentioning

confidence: 99%

Epidemiological cutoff values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M. tuberculosis

Fowler

2021

Preprint

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Drug susceptibility testing of M. tuberculosis is rooted in a binary susceptible/resistant paradigm. There are considerable advantages in measuring the minimum inhibitory concentrations (MICs) of a panel of drugs for an isolate, including quantifying the magnitude of effect conferred by genetic variants and being able to identify isolates with elevated MICs that can still be treated with standard therapy. It is necessary, however, to measure the epidemiological cutoff values (ECOFF/ECVs) to permit comparison with qualitative data. Here we present ECOFF/ECVs for 13 anti-TB compounds, including bedaquiline and delamanid, derived from 20,637 clinical isolates collected by 14 laboratories based in 11 countries on five continents. Each isolate was incubated for 14 days on a dry 96-well broth microdilution plate and then read. Resistance to the majority of the drugs due to prior exposure is expected and the MIC distributions for many of the compounds are complex and therefore a phenotypically wild-type population could not be defined. Since a majority of samples also underwent genetic sequencing, we defined a genotypically wild-type population and measured the MIC of the 99th percentile by direct measurement and via fitting a Gaussian using interval regression. The proposed ECOFF/ECV values were then validated by comparing to the MIC distributions of high-confidence genetic variants that confer resistance and to qualitative drug susceptibility tests obtained via Mycobacterial Growth Indicator Tube and the Microscopic-Observation Drug-Susceptibility assay.

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“…inconspicua library fraction activity was assessed with a simple 8 straight-channel microfluidic chip (Figure 1) to explore the merits of a microfluidic bioassay format. Whereas traditional "bulk" assays (e.g., 96-well plate) often require a minimum of 50 µL of sample mixture in each well (Sarker et al, 2007;Teh et al, 2017;Fowler et al, 2018) (10 µL extract, 40 µL of media and cells for each replicate) (Kirkpatrick et al, 2017;Parsley et al, 2018Parsley et al, , 2019Moyer et al, 2019), microfluidic devices utilize sample sizes in the nanoliter/picoliter range. However, connectors and interfaces between microfluidic channels/chambers and injection sites can generate significant dead volume, requiring excess sample to ensure complete filling of microfluidic compartments.…”

Section: Bioactivity In Microfluidic Assaysmentioning

confidence: 99%

Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements

Parsley

Smythers

Hicks

2020

Front. Cell. Infect. Microbiol.

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Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.

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Automated detection of bacterial growth on 96-well plates for high-throughput drug susceptibility testing of Mycobacterium tuberculosis

Cited by 24 publications

References 15 publications

Validating a 14-Drug Microtiter Plate Containing Bedaquiline and Delamanid for Large-Scale Research Susceptibility Testing of Mycobacterium tuberculosis

Validating a 14-Drug Microtiter Plate Containing Bedaquiline and Delamanid for Large-Scale Research Susceptibility Testing of Mycobacterium tuberculosis

Epidemiological cutoff values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M. tuberculosis

Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements

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