Detection of Escherichia coli in blood using flow cytometry

Abstract

A rapid method for the detection of Escherichiu coZi in blood has been developed. The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry. The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth. This method has been applied to human donor blood specimens seeded with various E. coli concentrations and to two rabbit model systems. Bacterial detection is evident from the in vitro human blood studies at levels of 10 E. coZi/ml and from in vivo rabbit model studies at < 100 E. coli/ml. Key terms: Flow cytometry, ethidium bromide, E. coli, bacterial detection in blood, blood cell lysis Bacteremia, the presence of bacteria in human blood, is a life-threatening situation requiring early detection and appropriate therapy (12). All current methods of detecting bacteria in blood rely on the inoculation of the suspect human blood into or onto a bacterial growth medium (1). Following incubation periods ranging from 8 h, at the very least, to 7 days, signs of bacterial growth may appear, indicating that bacteria were present in the initial sample. The time required for this detection, however, is clearly a problem. A technique that could reduce or obviate the need for prolonged incubation to achieve measurable quantities of bacteria would be of great benefit.Flow cytometry may be particularly well suited to the problem of detecting bacteria in blood. Previous applications of flow cytometry to microbial measurements have taken primary advantage of the high cell analysis rates (> lo3 celldsec) and high specificity afforded by multiple quantitative measurements on each cell (6,7,9,11,13,15,16). A less utilized advantage of flow cytometry is the ability to analyze sample volume at a significant rate typically 0.05-0.1 ml/min. This rate of fluid analysis makes it possible to detect particles present in samples at very low numbers per milliliter in a reasonable period of time. The sensitivity of the technique is limited therefore by the electronic and hydrodynamic "noise" of the system, and by the ability of quantitative multiparameter measurements to differentiate 'Yarget" signals, like bacteria, from all other signals present (e.g., autofluorescence, cellular debris, and the like). Previous experiments in our facility have shown that intrinsic "noise" is sufficiently low, and multiparameter measurements sufficiently specific, to allow the detection of bacteria in sterile-filtered buffers at a level of lO/ml (B. Sage, unpublished experiments). In this report we have developed a flow cytometry-based system to detect E. coli at similar levels in whole blood. The sensitivity of this flow cytometry based system is 100-to 1,000-fold greater than that attainable by microscopy (10). MATERIALS AND METHODS Preparation of In Vitro SamplesE. coli (ATCC 11775) were routinely grown in Trypticase Soy Broth (TSB, BBL Microbiology Systems, Cockeysville, MD) at 37" C to mid-logarithmic phase. They were then harveste...

Applications of Flow Cytometry to Clinical Microbiology

“…The first study detecting of microbes in blood by using FCM was done with ethidium bromide as the detecting fluorochrome (195). Blood cells were lysed, and the remaining bacteria were stained with ethidium bromide; as few as 10 E. coli cells/ml were detected.…”

Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers

“…These techniques have been used for the analysis of bacteria in the environment, including sea water (Monfort and Baleux 1992 ;López-Amorós et al 1995), lake water (Porter et al 1993 ;Diaper and Edwards 1994), sewage effluent (Völsch et al 1990 ;Wallner et al 1995), and soil (Christensen et al 1995). Medical applications include bacterial responses to antibiotics (Gant et al 1993 ;Mason et al 1995), analysis of blood (Mansour et al 1985) and other clinical samples (Humphreys et al 1994 ;van der Waaij et al 1994). Interest in food analysis has centred around pathogenic bacteria and spoilage organisms (Donnelly and Baigent 1986 ;Patchett et al 1991 ;Laplace-Builhé et al 1993).…”

Nouvelles approches d'étude des réseaux microbiens

“…A l'heure actuelle dans les milieux aquatiques, elles sont surtout utilisées pour les organismes photosynthétiques dont les pigments chlorophylliens constituent d'excellents marqueurs. Les premières applications de cette technique aux dénombrements bactériens ont concerné des bactéries présentant un rapport direct avec les actions humaines (Steen et al 1982, Mansour et al 1985. Depuis peu, cette technique a commencé à être appliquée aux comptages bactériens dans les échantillons naturels (Montfort & Baleux 1992, Trousselier et al 1993.…”