INTRODUCTION

Top Previous Next References

Microbiology in general and clinical microbiology in particular have witnessed important changes during the last few years (82). An issue for microbiology laboratories compared with other clinical laboratories is the relative slowness of definitive reports. Traditional methods of bacteriology and mycology require the isolation of the organism prior to identification and other possible testing. In most cases, culture results are available in 48 to 72 h. Virus isolation in cell cultures and detection of specific antibodies have been widely used for the diagnosis of viral infections (181). These methods are sensitive and specific, but, again, the time required for virus isolation is quite long and is governed by viral replication times. Additionally, serological assays on serum from infected patients are more useful for determining chronic than acute infections. Life-threatening infections require prompt antimicrobial therapy and therefore need rapid and accurate diagnostic tests. Procedures which do not require culture and which detect the presence of antigens or the host's specific immune response have shortened the diagnostic time. More recently, the emergence of molecular biology techniques, particularly those based on nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis (139). These techniques have led to a revolutionary change in many of the traditional routines used in clinical microbiology laboratories. Results are offered quickly, the diagnosis of emerging infections has become easier, and unculturable pathogens have been identified (109).

On the other hand, the current organization of clinical microbiology laboratories is now subject to automation and competition, both overshadowed by increasing costs (282, 339). Increased use of automation in clinical microbiology laboratories is best exemplified by systems used for detecting bacteremia, screening of urinary tract infections, antimicrobial susceptibility testing, and antibody detection. To obtain better sensitivity and speed, manufacturers continuously modify all these systems. Nevertheless, the equipment needed for all these approaches is different, and therefore the initial costs, both in equipment and materials, are high.

Flow cytometry (FCM) could be successfully applied to most of these situations. In bacteremia and bacteriuria, FCM would not only rapidly detect organisms responsible for the infection but would also initially identify the type of microorganism on the basis of its cytometric characteristics. Although FCM offers a broad range of potential applications for susceptibility testing, a major contribution would be in testing for slow-growing microorganisms, such as mycobacteria and fungi (108, 163, 262). Results are obtained rapidly, frequently in less than 4 h; when appropriately combined with the classical techniques, FCM may offer susceptibility results even before the microorganism has been identified. The most outstanding contribution offered by FCM is the detection of mixed populations, which may respond to antimicrobial agents in different ways (331).

This technique could also be applied to study the immune response in patients, detect specific antibodies (27, 133), and monitor clinical status after antimicrobial treatments (58, 244). Moreover, when properly applied, FCM can be adjusted to use defined parameters that avoid subjectivity and aid the clinical microbiologist in the interpretation of specific results, particularly in the field of rapid diagnosis.

FCM is an analytical method that allows the rapid measurement of light scattered and fluorescence emission produced by suitably illuminated cells. The cells, or particles, are suspended in liquid and produce signals when they pass individually through a beam of light (Fig. 1). Since measurements of each particle or cell are made separately, the results represent cumulative individual cytometric characteristics. An important analytical feature of flow cytometers is their ability to measure multiple cellular parameters (analytical flow cytometers). Some flow cytometers are able to physically separate cell subsets (sorting) based on their cytometric characteristics (cell sorters) (Fig. 2). The scattered light (intrinsic parameters) and fluorescence emissions of each particle are collected by detectors and sent to a computer, where the distribution of the population with respect to the different parameters is represented. Scattered light collected in the same direction as the incident light is related to cell size, and scattered light collected at an angle of 90° gives an idea of the particle complexity. This parameter is related to cell surface roughness and the number of organelles present in the cell. Size and complexity are considered intrinsic parameters since they can be obtained without having to stain the sample. To obtain additional information, samples can be stained using different fluorochromes. Fluorochromes can be classified according to their mechanism of action (127): those whose fluorescence increases with binding to specific cell compounds such as proteins (fluorescein isothiocyanate [FITC]), nucleic acids (propidium iodide [PI]), and lipids (Nile Red); those whose fluorescence depends on cellular physiological parameters (pH, membrane potential, etc.); and those whose fluorescence depends on enzymatic activity (fluorogenic substrates) such as esterases, peroxidases, and peptidases (Table 1). Fluorochromes can also be conjugated to antibodies or nucleotide probes to directly detect microbial antigens or DNA and RNA sequences.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Light-scattering and fluorescence signal production at the flow cell analysis point of the flow cytometer. From Purdue Cytometry CD-ROM vol. 1 (adapted with permission of the publisher).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Scheme of optic (dichroic mirrors and bandpass filters) and illumination (laser) systems of a flow cytometer with six parameters detected (size, granularity, and four fluorescences) by separate photomultiplier tubes (except size, which can be detected by photodiode or a PMT tube) and sorting capacity. From Purdue Cytometry CD-ROM vol. 1 (adapted with permission of the publisher).

A typical flow cytometer has several parts. (i) The hydraulic system produces the fluid stream, with a liquid sheath surrounding the cell suspension (hydrodynamic focusing). This sheath is responsible for the passage of the particles through the sensing point at a constant velocity. (ii) The illumination system consists of the light that produces the scatter signals and fluorescence emission when the particles pass through it. There are two types of flow cytometers, depending on the illumination source: those with a laser light source, and those with an arc lamp source. Each has it own advantages and disadvantages, but the main difference lies in their fields of application. Arc lamp cytometers are frequently used in microbiological applications due to their better scatter resolution and versatility. In contrast, laser flow cytometers have wider applications in immunology and hematology because they excite fluorochromes associated with cells. Studies comparing the two types of cytometers have concluded that the selection of one rather than the other depends mainly on the range of wavelengths required for the excitation of the selected fluorescent stains (13, 161). Our personal experience supports this opinion and work should aim at developing protocols according to the type of cytometer available. (iii) The optic system focuses incident light on the crossing particles, recovers the scattered light and the fluorescence produced by the fluorochromes present in the cells, and directs both to the appropriate photomultiplier tubes (Fig. 2). (iv) The electronic system transforms the incident light from fluorescence and light scattered into electric pulses (analogic). The magnitudes of these pulses are distributed electronically into channels, permitting the display of histograms of the number of cells plotted against the channel numbers (digital) (68). If the instrument has the capacity to do so, it also controls the cell-sorting process. Fluorescence-activated cell sorting refers to the ability to select a subpopulation from the whole population, following cytometric classification, and to physically separate this particular population. To do this, the machine produces a uniform stream of droplets; a particular droplet containing a cell can be charged, permitting selection of the droplet when it passes through an electrical field produced by deflection plates (Fig. 2). In this way, two populations can be sorted at the same time (positively and negatively charged droplets). A new high-speed sorter machine has been developed with the possibility of sorting four populations at the same time (MoFlo; Cytomation, Freiburg, Germany). (v) The data analysis system consists of software that allows the analysis of the huge amount of information produced by multiparameter data acquisition. The analytical software permits the study and independent analysis of a particular subpopulation. Besides all the statistical information, the data can be represented in several different ways: monoparametric histograms, biparametric histograms, and three-dimensional representations (Fig. 3). There is a growing market of commercial FCM software. Free software can also be downloaded from the Internet, where it is possible to find information about all the fields related to FCM (cytometry network sites, http://nucleus.immunol.washington.edu/ISAC /network_sites.html; JCSMR flow cytometry software, http://jcsmr.anu.edu.au/facslab/facs.html; ISAC WWW home page, http://www10.uniovi.es/ISAC.html).

View larger version (40K): [in this window] [in a new window]   FIG. 3.   The data obtained from a flow cytometer can be displayed in several ways. The most common are the mono- and biparametric histograms (A and B), which usually include a statistical analysis of the results. (A) Monoparametric histogram showing the selected parameter on the x axis and the relative cell number on the y axis. (B) Biparametric histogram showing cells distributed as a function of their signal intensity with respect to each parameter. Cells located in the upper left quadrant are positive for the parameter represented on the y axis, cells located in the upper right quadrant are positive for both parameters, cells located in the lower left quadrant are double negative, while cells in the lower right panel are positive for the parameters on the x axis. (C and D) Three-dimensional representations. The z axis can represent the relative number of cells (C) or a third parameter (D), such as scattered light on the x and y axes and fluorescence signals on the z axis.

From the beginning of FCM (68), the ancestor of modern flow cytometers has been identified with an aerosol particle counter designed to analyze mine dust (124). This apparatus was used in World War II by the U.S. Army in experiments for the detection of bacteria and spores. Gucker et al. (124) reported that the instrument could be used with biological samples (bacteria), as well as particles in air suspension or aerosols. Thus, FCM with an application to microbiology originated many years before the use of flow cytometry as a tool for studying mammalian cells. The original device incorporated a sheath of filtered air to limit the air sample stream to the central portion of the flow chamber. The detector used was a then recently developed device called a photomultiplier tube. Particle counters based on the Coulter orifice principle, in which the difference in electrical conductivity between the cells and the medium in which they are suspended is measured by the change in electrical impedance produced as they pass through an orifice, were later developed. These instruments were widely applied in hematology studies. However, the first real flow cytometer was built by Kamentsky et al. (154), using spectophotometric techniques to detect and measure nucleic acids and light scattering of unstained cervical cells in a flow stream. At the same time, Fulwyler, working at the Los Alamos Scientific Laboratory, described the first flow cytometer with sorting capability (104). This machine worked by measuring cell volumes obtained by the Coulter orifice principle. Fulwyler adapted the ink jet printer principle, using electrostatic deflection of charged droplets, as a cell-sorting mechanism. In fact, sorting capability was introduced to demonstrate the accuracy of the signals obtained by the machine and to ascribe a given distribution of cell volume detected by an electronic signal to a specific cell type. During the 1970s, applications of FCM to research into mammalian cells advanced rapidly, but at that time few instruments were developed for microbiological studies. The subsequent applications to microbiology of FCM techniques that were initially developed to study mammalian cells were due to optical improvements in flow cytometers and newly developed fluorochromes. The development of an arc lamp-based instrument by Steen's group in 1979 (301, 303) allowed the use of FCM for basic research on bacteria. Because of the design of the flow chamber and the use of photomultiplier tubes for detecting scattered light, this instrument was ideal for studying microorganisms (7, 37). The promising tool described by Boye and Steen in 1983 became a "potent illuminating light" in the 1990s (38), as was stated in the book edited by David Lloyd, Flow Cytometry in Microbiology (186a), from which most microbiological cytometrists have learned their trade. In the last years of the 1990s, the applications of FCM in microbiology have significantly increased (9, 28, 103, 148, 291).

The applications of FCM to microbiology have been so widespread that discussion of all of them is beyond the scope of this review. For more information, see the excellent reviews by Davey and Kell (68), Porter et al. (263), and McSharry (211) and the "Bible" of flow cytometry by Howard Shapiro, Practical Flow Cytometry (291). Below, we briefly describe some of the applications of FCM in the field of microbiology, focusing on present or future applications in clinical microbiology.

Earlier works by Steen had demonstrated the applicability of dual-parameter analysis to discriminate among different bacteria in the same sample (301, 302, 306). One parameter was light scattered (size), and the other was either fluorescence emission from fluorochromes coupled to cellular components (protein and DNA) or autofluorescence (Fig. 4), or light scattered acquired from another angle (42, 151, 277, 293, 301, 302, 306, 316). However, the use of several fluorochromes for direct staining or through antibody or oligonucleotide conjugates plus size detection is the simplest way to visualize or identify microorganisms by FCM (6, 7, 10, 11, 257, 317, 332).

View larger version (21K): [in this window] [in a new window]   FIG. 4.   (A) Dual-parameter analysis of forward light scatter (size) and red fluorescence signals allowed the discrimination between two species of Candida, based on different fluorochrome staining backgrounds. These yeast species are indistinguishable by monoparametric analysis of forward light scatter or red autofluorescence. However, after addition of PI, they show different basal levels, and if this is plotted against size, it is possible to discriminate them. This kind of analysis permits quantification of both species in mixed cultures. (B) Quantification of different protein amounts (measured as FITC fluorescence) can be used to distinguish different microorganisms such as those represented in the histogram (from Purdue Cytometry CD-ROM, vol 2., ISSN 1091-2037, provided by Hazel M. Davey [adapted with permission of the publisher]). (C) Dual-fluorescence discrimination of fungal spores. Spores from Aspergillus, Mucor, Cladosporium, and Fusarium were fixed and stained with Calcofluor, which binds to chitin in the spore wall, and PI, which stains nucleic acids. As shown, the spores have different amounts of chitin and nucleic acids, permitting their segregation by FCM. Samples shown in panel A were run on a FACScan (Becton-Dickinson) flow cytometer, the ones shown in panel B were run on an EPICS Elite (Coulter) flow cytometer, and those shown in panel C were run on a Bryte-HS (Bio-Rad) flow cytometer.

The simple and rapid assessment of the viability of a microorganism is another important aspect of FCM. The effect of environmental stress or starvation on the membrane potential of bacteria has been studied by several groups using fluorochromes that distinguish among nonviable, viable, and dormant cells (155-157, 188; see references 68, 71, 78, 89, 135, 150, 200-203, 265, and 328 for reviews). Other authors have demonstrated the use of PI as a viability marker in yeasts (72), Pneumocystis carinii (177), and bacteria (89, 230, 264).

FCM has also been used in metabolic studies of microorganisms. This was first accomplished by Thorell (316), using autofluorescence due to NADPH and flavins as metabolic status markers. Other authors studied DNA, protein, peroxide production, and intracellular pH (3, 5, 39, 140, 324, 345). Recently developed fluorochromes and kits (Sytox Green and Live/Dead kits; Molecular Probes, Eugene, Oreg.) have been used for the FCM-based counting of live and dead bacteria and yeasts (176, 178, 311), simplifying staining protocols and making data interpretation easier. Other kits are available for detecting gram-positive and gram-negative bacteria or for studying yeast organelles (127). Recently, Mason et al. (204) described a method which enables the discrimination of gram-positive from gram-negative bacteria on the basis of the fluorescence emitted when the organisms are stained with two fluorochromes. These authors correctly predicted the Gram stain reaction of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, which are normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.

FCM also offers the possibility of studying gene expression using reporter genes in yeasts (56, 258, 297, 338) and bacteria (8, 55). The development of gene expression systems based on green fluorescent proteins facilitates this kind of study due to the simplicity of the technique (60, 77, 229, 320).

The sensitivity of FCM allowed Philips and Martin (255) to detect Bacillus spores (254). Using a similar approach, Griffiths et al. (121) and Challier et al. (49) were able to sort spores from Dictyostelium discoideum and Enterocytozoon bieneusi, respectively. These examples show the potential of FCM in the investigation of small microbes.

The interaction between pathogens and phagocytic cells has also been studied by FCM (22, 23). The development of fluorochromes to detect oxidative bursts due to phagocytosis (17, 251, 281) increased the number of studies with different microbes such as Borrelia burgdorferi (17), Staphylococcus spp. (128, 196), Escherichia coli (70, 271), Bordetella pertussis (299), Cryptococcus neoformans (48), Salmonella (272), and yeasts (90, 96, 114).

FCM has been extensively used for studying virus-cell interactions (172, 180, 334). This topic was reviewed in depth by McSharry in 1994 (211). Modulation of the expression of cellular proteins due to viral infection has been studied by FCM for cytomegalovirus (CMV) (116), herpes simplex virus (HSV) (149), adenovirus (168), human immunodeficiency virus (HIV) (53), and hepatitis B virus (HBV) (346). Perturbation of the cell cycle and DNA replication in virus-infected cells have also been studied by FCM for papillomavirus (25), CMV (80) and human HIV-1 (273). This technique has been also used to study the effect of viral infection on intracellular Ca²⁺ levels ([Ca²⁺]_i) by Irurzun et al. (145) and by Miller et al. (221). FCM has also permitted the demonstration of apoptosis associated with viral infection, including HSV (144, 146), Epstein-Barr virus (EBV) (4), influenza virus (267), measles virus (93), papillomavirus (340), and HIV-1 (129, 194) infections. Furthermore, by means of biotinylated or directly FITC-labeled virus, interactions of EBV (142, 159, 182, 335), echovirus (206), adenovirus (217), influenza virus (228), simian virus 40 (SV40) (20), human T-cell leukemia virus type 1 (HTLV-1) (110), measles virus (226, 232), bovine herpesvirus (326), papillomavirus (268), bunyaviruses (249), poliovirus (102), and HIV-1 (14, 308, 318) with their putative cell receptors have been described. These investigations show that FCM is able to provide solutions to problems arising when working with microorganisms.

Bacteria. Antibodies are currently changing the way in which we identify microbes, making it easier and faster. Their specificity and the possibility of using fluorochrome-labeled antibodies to specific antigens render them one of the most powerful tools in the identification of pathogens. The main disadvantage of this method is still the limited availability of antibodies directed against particular microbes. Other advantages of using antibodies are that the cells do not need to be cultivable and that the method is simple and fast. In an early work from 1983, Groschel (122) explained somewhat prophetically the use of antibodies in clinical microbiology. FCM in conjunction with fluorescent antibodies has been used to detect surface antigens in Haemophilus (298), Salmonella (57, 207), Mycobacterium (238), Brucella (35), Branhamella catarrhalis (29), Mycoplasma fermentans (50), Pseudomonas aeruginosa (134), Bacteroides fragilis (191, 239) and Legionella (143), among other microorganisms. These examples illustrate the sensitivity and specificity of using antibodies that allow the detection of particular cell types (of as few as 100 cells per ml in 30 min) in heterogeneous populations (57).

Fungi. With regard to yeasts, the work by Groshen et al. (122), Chaffin et al. (47), and Han et al. (126) has shown that surface antigens of Candida albicans can be detected by flow cytometry in conjunction with available specific antibodies. As discussed below, this approach can be used for clinical samples.

Parasites. The first applications of FCM to parasites involved a study of the cell cycle and the amounts of DNA of Physarum polycephalum myxamoebae (319) and the characterization of monoclonal antibodies against membrane antigens from Leishmania (337). Specific clinical applications came later, when Flores et al. (99) used monoclonal antibodies, FCM, and immunofluorescence microscopy for the direct identification of Naegleria fowleri and Acanthamoeba spp. in clinical specimens.

(i) Detection and quantification of viral antigens. FCM can detect viral antigens either on the surface of or within infected cells (172). It can rapidly detect and quantify virus-infected cells using antibodies that specifically recognize surface or internal antigens (91, 211, 213, 334); in the latter case, permeabilization of the cells is required. A thorough review of different permeabilization methods, including the advantages and disadvantages of each, for viral antigen and nucleic acid detection has been written by McSharry (211). Direct and indirect fluorescent-antibody methods are used. Direct detection involves the use of fluorescently labeled antibody (labeled with FITC or phycoerythrin). In the indirect fluorescent-antibody method, unlabeled antibody is bound to infected cells, which are then incubated with fluorescence-labeled anti-Ig that binds to the first viral antibody.

(ii) Detection and quantification of viral nucleic acids. The emergence of PCR and rPCR RT-PCR techniques has allowed the highly sensitive detection of specific viral nucleic acids (DNA or RNA) in virus-infected cells. These methods are indeed the most sensitive for the detection and characterization of viral genomes, especially in the case of rare target viral sequences (123). However, the association between the viral nucleic acid and an individual cell is lost, and therefore no information about productively infected cell populations is obtained by this method. FCM analysis of fluorescent in situ hybridization in cell suspension overcomes this problem (33, 173), since this assay can be coupled with simultaneous cell phenotyping (by using specific antibodies to different cell markers).

Bacteria. The identification of pathogens by microsphere immunoassays using FCM offers the specificity provided by antibodies coupled with the speed and multiparametric analysis provided by FCM. Although these assays can be used to directly detect microbes, they are more useful for detecting antibodies against microbes in sera obtained from patients. Generally, either a bacterial antigen preparation or the whole organism is attached to polystyrene microspheres with a uniform diameter. The antigen-coated microspheres are incubated with the sera, the putative human antibodies recognize the antigen, and, in a second step, a fluorescence-conjugated antibody against human Igs is used for detection. FCM allows this assay to be completed in a short time with excellent sensitivity and reliability. Using this approach, Best et al. (27) detected the presence of antibodies against Helicobacter pylori in sera from 55 patients. These authors demonstrated that this method was as sensitive and reliable as ELISA but faster and cheaper. The simplicity of the technique and the stability of the coated microspheres make the FCM immunofluorescence assay highly practical for serodiagnosis.

Fungi. The use of FCM and the antibodies present in patient sera to detect fungal pathogens was first described by Libertin et al. (183) in 1984. Pneumocystis carinii cysts in lung homogenates from biopsy specimens were detected by these authors using sera from patients and experimentally infected rats. Bergbrant (26), using FCM to monitor antibodies against Pityrosporum ovale in sera from patients with seborrheic dermatitis, demonstrated that there was no relationship between this microorganism and the illness.

Parasites. The presence in patient sera of antibodies to any particular parasite can permit an FCM-based diagnosis of parasitism. Martins-Filho et al. (199), using FCM on serum from patients chronically infected with Trypanosoma cruzi, developed a sensitive method for the immunodetection of anti-trypomastigote membrane-bound antibodies. They were also able to monitor the treatment in order to establish its effectiveness. A similar assay was developed by Cozon et al. (64) with Toxoplasma gondii, using fixed tachyzoites and specific conjugates for different human Ig heavy chains. They were able to quantify the amounts of IgM, IgG, and IgA antibodies in patient sera by measuring the amount of fluorescence bound to tachyzoites. The authors stated that the method might offer a major improvement in cost-effectiveness per sample (especially when a large number of tests is used) compared with routine immunofluorescence assays by fluorescence microscopy and further stressed that it could be fully automated.

Viruses. Some methods have been routinely used to detect specific antibodies to viral antigens. Among these techniques are ELISA, complement fixation, indirect immunofluorescence microscopy, and Western blotting. In addition, the detection and quantification of antibodies to viral antigens can be carried out by FCM. This technique has been used to detect and quantify antibodies to CMV (209), HSV-1 and HSV-2 (46, 209), HCV (187, 210, 279), and HIV-1 (100, 118, 133, 290, 294).

The first experiments in which FCM was used to study the effects of antimicrobial agents in prokaryotes were carried out at the beginning of the 1980s (136, 302, 304, 306). In the 1990s, there were interesting advances in this field from microbiology laboratories, and the number of scientific articles addressing the antimicrobial responses of bacteria (including mycobacteria), fungi, and parasites to antimicrobial agents increased considerably. The development of FCM in combination with fluorochromes permits the assessment of individual viability and functional capacity (membrane potential and metabolic pathways) within microbial populations. This allows investigators to explore the possibility of performing susceptibility testing within the applications of FCM. Also, the introduction of this technique in clinical laboratories for routine susceptibility testing has been proposed, since this approach can be performed reliably in just a few hours. Several examples of the study of the antimicrobial effect and susceptibility testing by FCM are shown in Table 2.

Available data clearly demonstrate the utility of this technique to also assay viral susceptibility. Since the pioneer works of Rosenthal et al. (280) and Pauwels et al. (247) on the use of FCM to study the effect of antiviral agents on herpesvirus and HIV, FCM has been used by several groups, particularly those studying these viruses and CMV. The detection and quantification of viral antigens and viral nucleic acids in combination with antibodies or nucleic acid probes, respectively, or any other cellular parameter associated with viral infection have been used in FCM protocols for the in vitro evaluation of antiviral-drug activities. In addition, the possibility of on-line monitoring of the antiviral treatments ex vivo has also been explored. The latter approach has also been applied to bacterial and fungal infections; rapid and sensitive protocols for the evaluation of microbial responses to antimicrobial agents are also available.

In the following sections, the possible uses of FMC in antimicrobial susceptibility testing are discussed according to the type of pathogen involved.

Standardized methods for performing in vitro susceptibility testing of bacteria in clinical microbiology laboratories are widespread (98). Qualitative and/or quantitative results are given on the basis of the size of the zone of inhibition or MIC. Despite the automation of MIC-based broth microdilution systems, an 18- to 24-h incubation period is usually needed before antimicrobial activity can be quantified. Recently, fluorogenic, turbidometric, and colorimetric technology has reduced susceptibility testing to 4 to 6 h (83, 98). However, unless the bacterial inoculum is quantified, only the bacteriostatic effect, i.e., the MIC, is generally tested by clinical laboratories. The MBC, which reflects the bactericidal effect of antimicrobial drugs, is rarely determined. This requires calculation of bacterial counts, generally expressed as CFU, which involves bacterial culture dilutions and subcultures. In addition, neither MIC nor MBC determinations consider the heterogeneity of bacterial populations. Similarly, postantibiotic and subinhibitory concentration effects (106), which offer pharmacodynamic data based on the antimicrobial activity, are rarely determined in clinical laboratories since these determinations (190) are tedious and time-consuming.

FCM has proved to be very useful for studying the physiological effects of antimicrobial agents on bacterial cells due to their effect on certain metabolic parameters (membrane potential, cell size, and amount of DNA). In addition, FCM is a reliable approach for susceptibility testing, offering results in terms of the bactericidal or bacteriostatic effect (86, 108, 198, 262, 331).

The studies by Martinez et al. (198) and Steen et al. (302) in 1982 on the antimicrobial effects on bacteria assayed by FCM are now considered classic. They demonstrated that the effect of -lactam antibiotics on E. coli can be detected by measurements of light scattering and DNA content after 10 min of incubation with the drug. Using FCM, Steen et al. (302) showed the effect of several antibiotics that inhibit protein synthesis (chloramphenicol, erythromycin, doxycycline, and streptomycin) on E. coli with the fluorescent DNA probe mithramycin (305). Since then, FCM has been used to measure the effects of different antimicrobial agents on bacteria (Table 2).

Realizing the potential of FCM in this field, several companies have developed new products to rapidly perform antibiotic susceptibility testing in clinical microbiology laboratories. We have used one of these products (Bac-light; Molecular Probes) to analyze the reliability of this test in clinical microbiology. We worked with two strains of Enterococcus, one sensitive to vancomycin (E. faecalis ATCC 29212) and other resistant (E. faecium U2A1), with a vanA element responsible for its vancomycin resistance phenotype. In addition, as a control we included an E. coli isolate showing intrinsic resistance to vancomycin. The above-mentioned products are kits with two fluorochromes that permit the detection of live and dead cells. Bacterial cells were incubated with and without vancomycin for 2 h at the respective MICs of this drug (Fig. 5). Antimicrobial effects were detected by measuring the variations in fluorescence due to dead cells within 3 h of incubation with the antibiotic. Another advantage of FCM (Fig. 5), as mentioned above, is the visualization of the heterogeneity of the response of the cells to the antimicrobial agent. This heterogeneity is detected by determining the presence of subpopulations that are less susceptible to the antimicrobial agent treatment. Davies and Hixson have recently reported similar results (69). Therefore, it is feasible to use FCM and fluorescence probes to perform rapid testing of the susceptibility of bacteria to a panel of antimicrobial agents before choosing the treatment. Moreover, resistant subpopulations can be effectively detected, representing an interesting advantage of FCM that can be suitably exploited in clinical studies (331).

View larger version (48K): [in this window] [in a new window]   FIG. 5.   Antimicrobial susceptibility testing by FCM using the Bac/live kit (Molecular Probes). Antimicrobial susceptibility is shown by the decrease in green fluorescence (live cells) and the increase in red fluorescence (dead cells). (A) Distribution of E. coli cultures without antimicrobial agent incubation; therefore, the cells appear mainly in quadrant 4 (positive for green fluorescence). (B) An E. coli culture was incubated for 2 h with vancomycin at 1.024 g/ml. The distribution is similar to that seen in panel A. Accordingly, this strain was not affected by antimicrobial agent treatment, although a small percentage of cells was positive for the red fluorescence, meaning that the cells were sensitive to vancomycin. The same protocol was applied to E. faecium (C) and E. faecalis (D) cultures. As described in the text, E. faecium is vancomycin resistant and E. faecalis is vancomycin sensitive. (C) One subpopulation of E. faecium lies in quadrant 1 (positive for red fluorescence only), another is in quadrant 2 (positive for red and green fluorescence), and the majority is in quadrant 4 (positive for green fluorescence only). This means that the behavior of the E. faecium population is not homogeneous in the presence of vancomycin, perhaps due to the loss of the element responsible for vancomycin resistance. (D) After 2 h of incubation, most E. faecalis cells appear in quadrant 1 and the rest appear in quadrant 2. Therefore, almost all cells are positive for red fluorescence and are dead. However, a small population (0.5%) is still present in quadrant 4, meaning that this population is less sensitive to vancomycin than the rest (over 99% of the cells).

Measurement of bacterial susceptibility. By using fluorescence probes, FCM measures the light scattering of cells in suspension and fluorescence of cellular contents or metabolic activity. Thus, the activity of antimicrobial agents can also be described in terms of changes in cell shape and in fluorescence (262, 329, 330). In this sense, Walberg et al. (329, 330), and others (108, 200) showed that light scattering is a useful parameter of antimicrobial effects, irrespective of their mechanisms of action. This was demonstrated with two different drugs: ceftazidime, which acts on the bacterial envelope, and ciprofloxacin, whose target is a gyrase. Nevertheless, the same authors (329, 330) used both fluorescence detection and light-scattering analysis when susceptibility testing was performed directly in clinical samples in order to distinguish bacterial cells from debris and other components.