Biolog Phenotype Microarrays

Shea, April A.; Wolcott, Mark J.; Daefler, Simon; Rozak, David A.

doi:10.1007/978-1-61779-827-6_12

Cited by 48 publications

(44 citation statements)

References 21 publications

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“…Susceptibility to 240 cell growth-inhibiting chemical compounds (using the Phenotype MicroArray; Biolog, Hayward, CA, USA) was performed using tryptic soy broth (TSB) and tetrazolium dye at a final concentration of 1%, according to the manufacturer's instructions for Gramnegative bacteria (32). Mutant growth at sub-MICs was performed in 96-well microtiter plates sealed with gas-permeable sealing membranes (Breathe-Easy; Diversified Biotech, Boston, MA) and incubated at 37°C for 24 h in a thermostatic kinetic microplate reader; the data were recorded using an OmniLog instrument (VersaMax; Molecular Devices, Sunnyvale, CA).…”

Section: Bacterial Strainsmentioning

confidence: 99%

Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

Curião

Marchi

Viti

et al. 2015

Antimicrob Agents Chemother

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Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRI r ) and triclosan-hypersusceptible (TRI hs ) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRI r mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRI r mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRI r mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRI r mutants had fitness costs, particularly marAoverexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRI r mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits.

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Section: Bacterial Strainsmentioning

confidence: 99%

Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

Curião

Marchi

Viti

et al. 2015

Antimicrob Agents Chemother

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“…With our optimized protocol, gonococci were actively dividing before initiating the screening and showed consistent, reproducible growth kinetics without reaching stationary phase before the end of the experiment. Typically, a tetrazolium dye is added to the suspension used to inoculate the PM plates as a measure of bacterial viability (18); however, we found that this dye prevented gonococcal growth in GW medium, so it was omitted.…”

mentioning

confidence: 99%

“…To increase the number of conditions available for testing, bacterial PMs have been designed to comprehensively screen bacterial strains for unique phenotypes (18). These arrays have been used to assess the fitness cost of antimicrobial resistance (19), to determine whether the increased pathogenicity of sequence type 131 extraintestinal pathogenic Escherichia coli is due to an altered metabolic profile (20), to examine the biological role of two-component systems in E. coli (21), and to identify the physiological functions of genes (22,23).…”

mentioning

confidence: 99%

“…To assess the functions of our six vaccine candidate proteins in the N. gonorrhoeae cell envelope, we chose to use the Phenotype MicroArray (PM) technology developed by Biolog (22). PMs are 96-well microtiter plates formulated with various concentrations of compounds selected to evaluate a wide range of phenotypes, ranging from nutrient requirements and antibiotic sensitivities to responses to osmolytes and chemicals (18). For this study, we have selected PM9, which contains various osmolytes, and PM11 to PM20, which are formulated with a series of compounds to probe cellular membrane integrity, including metals, antimicrobial peptides, small hydrophobic molecules, and dyes.…”

mentioning

confidence: 99%

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Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

et al. 2017

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The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae. A proteomicsdriven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344 -in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae. IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.KEYWORDS Neisseria gonorrhoeae, vaccine antigens, cell envelope, phenotypic microarrays, antibiotics, Biolog I n Gram-negative bacteria, the cell envelope is composed of the outer membrane, the cell wall, and the cytoplasmic or inner membrane, each of which can be further broken down into its constituents (1). The outer membrane is a bilayer composed of a lipopolysaccharide (LPS) or lipooligosaccharide (LOS) outer leaflet facing the extracel-

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“…A digital image of the MicroArray is captured several times each hour by the OmniLog instrument and phenotypes are recorded for comparison [21]. Table S1 gives the nutrients, pH and osmolytes in each well of the PM1, PM2, PM9 and PM10 plates.…”

Section: Name/numbermentioning

confidence: 99%

Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

Quereda¹,

Pucciarelli²

2014

Microbes and Infection

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Biolog Phenotype Microarrays

Cited by 48 publications

References 21 publications

Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

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