Biomedical subjects

Hazel M Davey

Publications and source records attributed to Hazel M Davey.

8 recordsLinked to original sources

PubMed2006

Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae.

In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MIC) of seven antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, oxytetracycline, and tylosin) on Mycoplasma (M.) agalactiae. Flow cytometry was able to detect M. agalactiae inhibition from 6 h postincubation, although it seems that definitive MIC values determined by flow cytometry were only possible at 12-h postincubation. However, the results obtained by the traditional method were only obtained at 24 h, when a visible change in the medium had occurred. At 24 h, both methods gave the same result for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, and oxytetracycline); whereas flow cytometry gave slightly higher MIC for tylosin. This was attributed to the fact that the M. agalactiae growth that had occurred in the tubes containing tylosin was not enough to visibly change the color of the medium. Futhermore, flow cytometry detected that inhibitory concentrations of oxytetracycline, chloramphenicol, and tylosin as judged at 24 h were not able to inhibit the M. agalactiae growth after 48 h. MIC values of enrofloxacin and ciprofloxacin were sufficient only to maintain the total counts per milliliter throughout the time matched samples, whereas higher concentrations of theses antibacterial agents reduced the total counts per milliliter over the course of the experiment. The main advantage of the flow cytometric method is that MIC results for M. agalactiae can be obtained in a shorter time than is possible with the traditional method. The method presented makes identification of resistant populations of M. agalactiae possible and, unlike the traditional method, allows the effect of each antibacterial agent to be determined in real-time at the single-cell level.

Anti-Bacterial Agents↗

PubMed2006

Flow cytometric determination of the effects of antibacterial agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides large colony type.

Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells.

Antitubercular Agents↗

PubMed2005

Metabolic footprinting and systems biology: the medium is the message.

One element of classical systems analysis treats a system as a black or grey box, the inner structure and behaviour of which can be analysed and modelled by varying an internal or external condition, probing it from outside and studying the effect of the variation on the external observables. The result is an understanding of the inner make-up and workings of the system. The equivalent of this in biology is to observe what a cell or system excretes under controlled conditions - the 'metabolic footprint' or exometabolome - as this is readily and accurately measurable. Here, we review the principles, experimental approaches and scientific outcomes that have been obtained with this useful and convenient strategy.

Animals↗

PubMed2004

A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge.

A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide-mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With-in "sequence spaces" ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane-associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of -1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore-dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.

Amino Acid Sequence↗

PubMed2004

Discrimination of modes of action of antifungal substances by use of metabolic footprinting.

Diploid cells of Saccharomyces cerevisiae were grown under controlled conditions with a Bioscreen instrument, which permitted the essentially continuous registration of their growth via optical density measurements. Some cultures were exposed to concentrations of a number of antifungal substances with different targets or modes of action (sterol biosynthesis, respiratory chain, amino acid synthesis, and the uncoupler). Culture supernatants were taken and analyzed for their "metabolic footprints" by using direct-injection mass spectrometry. Discriminant function analysis and hierarchical cluster analysis allowed these antifungal compounds to be distinguished and classified according to their modes of action. Genetic programming, a rule-evolving machine learning strategy, allowed respiratory inhibitors to be discriminated from others by using just two masses. Metabolic footprinting thus represents a rapid, convenient, and information-rich method for classifying the modes of action of antifungal substances.

Antifungal Agents↗

PubMed2003

High-throughput classification of yeast mutants for functional genomics using metabolic footprinting.

Many technologies have been developed to help explain the function of genes discovered by systematic genome sequencing. At present, transcriptome and proteome studies dominate large-scale functional analysis strategies. Yet the metabolome, because it is 'downstream', should show greater effects of genetic or physiological changes and thus should be much closer to the phenotype of the organism. We earlier presented a functional analysis strategy that used metabolic fingerprinting to reveal the phenotype of silent mutations of yeast genes. However, this is difficult to scale up for high-throughput screening. Here we present an alternative that has the required throughput (2 min per sample). This 'metabolic footprinting' approach recognizes the significance of 'overflow metabolism' in appropriate media. Measuring intracellular metabolites is time-consuming and subject to technical difficulties caused by the rapid turnover of intracellular metabolites and the need to quench metabolism and separate metabolites from the extracellular space. We therefore focused instead on direct, noninvasive, mass spectrometric monitoring of extracellular metabolites in spent culture medium. Metabolic footprinting can distinguish between different physiological states of wild-type yeast and between yeast single-gene deletion mutants even from related areas of metabolism. By using appropriate clustering and machine learning techniques, the latter based on genetic programming, we show that metabolic footprinting is an effective method to classify 'unknown' mutants by genetic defect.

Cells, Cultured↗

PubMed2003

Using flow cytometry to quantify microbial heterogeneity.

Flow cytometry is a powerful technique for the study of single cells, and thus it is of particular utility in the study of heterogeneity in microbial populations. This review seeks to highlight the role of flow cytometric analyses in studies of microbial heterogeneity, drawing wherever possible on recently published research articles. Whilst microbial heterogeneity is well documented in both natural and laboratory environments, the underlying causes are less well understood. Possible sources for the heterogeneity that is observed in microbial systems are discussed, together with the flow cytometric tools that aid its study. The role of flow cytometry in molecular biology is discussed with reference to gene reporter systems, which enable heterogeneity of gene expression to be monitored. With the recent sequencing of a variety of microbial genomes, it is anticipated that flow cytometry will have an increasing role to play in studying the effects of gene expression and mutation on heterogeneity, and in resolving the interactions of genetics and physiology.

Bacteria↗

PubMed2002

Flow cytometric techniques for the detection of microorganisms.

Flow cytometry (FCM) is a technique, which allows one to analyse cells rapidly and individually, and permits the quantitative analysis of distributions of a property or properties in a population. It therefore offers many advantages over conventional measurements for the analysis of biological cells. Historically the technique has been widely applied for the study of mammalian cells, but its use in microbiology has been more limited; this is mainly a consequence of the smaller size of microbes, which results in the smaller optical signals that can be obtained from them. Developments in light sources and optics, together with brighter, spectrally-diverse dyes have reduced this barrier over recent years and the flow cytometer is now an essential tool in many microbiological research establishments. FCM has an increasing role to play in the detection of microbes in both industrial and clinical settings. Environmental monitoring to prevent outbreaks of human diseases such as cryptosporidiosis and Legionnaires' disease and to detect acts of biowarfare or bioterrorism are all amenable to flow cytometric study. This review seeks to highlight the role of the flow cytometer in the detection of microbial cells.

Animals↗