The Response of Phagocytes to Indoor Air Toxicity.

This perspective presents a viewpoint on potential methods assessing toxicity of indoor air. Until recently, the major techniques to document moldy environment have been microbial isolation using conventional culture techniques for fungi and bacteria as well as in some instances polymerase chain reaction to detect microbial genetic components. However, it has become increasingly evident that bacterial and fungal toxins, their metabolic products, and volatile organic substances emitted from corrupted constructions are the major health risks. Here, we illustrate how phagocytes, especially neutrophils can be used as a toxicological probe. Neutrophils can be used either in vitro as probe cells, directly exposed to the toxic agent studied, or they can act as in vivo indicators of the whole biological system exposed to the agent. There are two convenient methods assessing the responses, one is to measure chemiluminescence emission from activated phagocytes and the other is to measure quantitatively by flow cytometry the expression of complement and immunoglobulin receptors on the phagocyte surface.

Thermal acclimation in the perch (Perca fluviatilis L.) immunity.

Fish immune systems must be able to cope with pathogens over a wide temperature range. Earlier research suggest that fish are more dependent on innate immune responses based on pattern recognition than acquired functions with specific recognition. If this applies to phagocytes, then opsonins (serum factors that augment phagocytosis e.g. immunoglobulins and complement proteins) attached on zymosan (Z) particles should be recognized better at higher temperatures than Z only. Z is recognized by glucan receptor representing pattern recognition. In this study perch were acclimated to 5 °C or 16 °C for 3-5 weeks. The recognition and activation of respiratory burst reaction of peripheral blood phagocytes was examined at seven different measurement temperatures (5, 10, 16, 20, 24 27, and 30 °C) when the cells were stimulated with Z and serum opsonized zymosan (OZ). Respiratory burst was measured as luminol chemiluminescence (CL) from diluted whole blood. OZ-induced CL per volume of blood was on average approximately 4.6 times higher in 16 °C acclimated fish than 5 °C acclimated perch (P<0.0001). Z-induced CL was approximately 3 times higher at lower temperatures in 16 °C acclimated perch than 5 °C acclimated fish and 6-9 times higher at 27 °C and 30 °C (P<0.001), respectively. CL reaction kinetics were faster in perch acclimated to 5 °C than 16 °C -acclimated fish, especially at low temperatures (P<0.001). Thermal acclimation caused a 3-4 °C shift in temperature response curves of CL towards the acclimation temperature (P<0.0001 and P<0.053 in Z and OZ-induced CL, respectively). Serum opsonins activated perch phagocytes substantially better at higher temperatures in both acclimation groups, which is consistent with an earlier study in rainbow trout (O. mykiss). However, opsonin recognition was significantly better in 16 °C acclimated perch than 5 °C acclimated fish, which was seen as higher CLs for OZ compared to Z, especially at higher temperatures. This is opposite to previously reported results in rainbow trout. Differences between rainbow trout and perch in opsonin recognition by blood phagocytes suggest that the living habits of perch, which prefers approximately a 10 °C higher temperature than rainbow trout, may be reflected in immune cell functions. Results of the present examination suggest that also in fish phagocytes pattern recognition is the prevailing system at low temperatures, and specific recognition is more effective at high temperatures.

Repeated dose 28-day oral toxicity study of moniliformin in rats.

Moniliformin is a Fusarium mycotoxin mainly produced by several species infecting grains in different climatic conditions. According to our previous studies, it is acutely toxic to rats, with an LD50 cut-off value of 25mg/kg b.w. To further assess the possible health risks of low dose exposure to moniliformin, a subacute oral toxicity study was conducted in Sprague-Dawley rats, adapting OECD guideline 407. Five dose groups and two satellite groups, each consisting of five male rats, were daily exposed to moniliformin by gavage. Two rats in the highest dose group, showed decreased activity followed by acute heart failure and death. The rats of the lower doses (<9mg/kg b.w.) showed no signs of toxicity. The daily intake of moniliformin strongly reduced the phagocytic activity of neutrophils in all dose groups. The decrease continued in the satellite group during the follow-up period, indicating a severe impact on the immune system and a LOAEL value of 3mg/kg b.w. for moniliformin. Moniliformin was rapidly excreted into urine, ranging between 20.2 and 31.5% daily and showed no signs of accumulation. The concentration of moniliformin in faeces was less than 2%, which suggests efficient absorption from the gastrointestinal tract.

Bacterial infection (BI)-INDEX: an improved and simplified rapid flow cytometric bacterial infection marker.

The aim of this study was to develop a rapid and simple flow cytometric bacterial infection marker. In this prospective comparative study, quantitative flow cytometric analysis of CD10, CD35, CD66b, CD282, and MHC Class I molecules on human neutrophils, monocytes, and B-lymphocytes from 141 hospitalized febrile patients with suspected infection and from 50 healthy controls was performed. We developed a flow cytometric marker of local and systemic bacterial infections, designated "bacterial infection (BI)-INDEX", incorporating the quantitative analysis of CD10, CD35, MHCI, CD66b, and CD282 on neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and 96% specificity in distinguishing between microbiologically confirmed bacterial (n = 31) and viral infections (n = 27) within a 1-h time-frame. We propose that our novel rapid BI-INDEX test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.

Resistance to bacterial infectious diseases in rainbow trout (Oncorhynchus mykiss).

Individually tagged rainbow trout representing 15 full-sibling families were sequentially challenged twice with Aeromonas salmonicida causing furunculosis: first as cohabitation and then as injected intraperitoneally. The bleeding procedure prior to challenges caused the outbreak of cold water disease by Flavobacterium psychrophilum. Before and after the outbreak and challenges, 11 immunological parameters were measured from blood samples. The immunological responses predicted the fate of the fish since nearly all the initial responses were lower in individuals which later died from cold water disease than in survivors. Fish died from furunculosis had impaired respiratory burst (RB) response to A. salmonicida. Fish that had initially the highest responses survived in the outbreak and challenges. The outbreak and challenges resulted in these individuals higher and faster responses compared with initial values. Unlike in mammals, the number of monocytes, but not that of granulocytes, in rainbow trout blood correlated well with the whole blood RB activity. The fish families differed markedly from each other in capacity to resist the induced diseases.

Use of complement regulators, CD35, CD46, CD55, and CD59, on leukocytes as markers for diagnosis of viral and bacterial infections.

Several complement regulatory proteins exist on self-cells to prevent damage by the serum complement system. In the present study, we aimed to perform quantitative analysis of membrane-bound complement regulators, CR1 (CD35), MCP (CD46), DAF (CD55), and MIRL (CD59), on peripheral blood neutrophils, monocytes, and lymphocytes from healthy controls (n=36) and febrile patients diagnosed with either bacterial (n=21) or viral (n=26) infections. Our results show that: (a) increased CD35 and CD55 levels on neutrophils and monocytes present potent markers of bacterial infection, (b) increased expression of CD46 on monocytes is an indicator of viral infection, and (c) increased CD59 expression on neutrophils and monocytes is a general infection marker. Additionally, CD19-positive B-lymphocytes represent practically the only lymphocyte population capable of expressing CD35. We further developed two novel clinical flow cytometric markers (indices), specifically, clinical mononucleosis (CM)-INDEX (incorporating CD35, CD55, and CD59 expression on lymphocytes) and clinical bacterial infection (CBI)-INDEX (incorporating CD35 and CD55 expression on neutrophils and lymphocytes), for the effective detection of viral mononucleosis and bacterial infection, respectively. In summary, bacterial and viral infections induce different expression patterns of membrane-bound complement regulators in human leukocytes, which may be effectively exploited in clinical differential diagnosis.

A rapid flow cytometric method for distinguishing between febrile bacterial and viral infections.

Antibiotic resistance due to the inappropriate use of antimicrobials is one of the most critical public health problems worldwide. A major factor underlying the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. Therefore, we aimed to develop a novel rapid flow cytometric method for distinguishing between febrile bacterial and viral infections. In this prospective comparative study, quantitative flow cytometric analysis of FcγRII/CD32, CR1/CD35, MHC Class I receptor (MHCI), and C5aR/CD88 on human phagocytes was performed in 286 hospitalized febrile patients with suspected infection. After using microbiological and serological detection methods, or clinical diagnosis, 205 patients were identified with either bacterial (n=136) or viral (n=69) infection. Receptor data from patients were compared to those of 50 healthy controls. We developed a flow cytometric marker of local and systemic bacterial infections designated "bacterial infection score (BIS)" incorporating the quantitative analysis of FcγRII/CD32, CR1/CD35, C5aR/CD88 and MHCI on neutrophils and/or monocytes, which displays 91% sensitivity and 92% specificity in distinguishing between microbiologically confirmed bacterial (n=77) and serologically confirmed viral infections (n=61) within 1h. The BIS method was effectively applied to distinguish between bacterial and viral (pandemic H1N1 influenza) pneumonia cases with 96% sensitivity and 92% specificity. We propose that the rapid BIS test can assist physicians in deciding whether antibiotic treatment is necessary, thus reducing unnecessary antimicrobial use.

Escherichia coli K-12 (pEGFPluxABCDEamp): a tool for analysis of bacterial killing by antibacterial agents and human complement activities on a real-time basis.

Photorhabdus luminescens luxCDABE genes were integrated into E. coli K-12 using a high copy number plasmid containing modified luxABCDE genes under the control of the powerful Lac promoter. This strain emitted 10 times higher bioluminescence (BL) than P. luminescens. BL production under different growth conditions was studied. In both bacterial strains, the increase in BL signal correlated with the increase in optical density (OD) in a rich growth medium. However, at the logarithmic growth phase, the BL signal was roughly constant. By contrast, in minimal growth media, there was no substantial growth and the BL/cell was approximately five times higher than in the rich medium. The dynamic measurement range of BL was 10(2) -10(7) colony-forming units (CFU) in E. coli and 10(3) -10(7) CFU in P. luminescens. Because the decrease in the BL signal correlated with the decrease in CFU and OD, i.e. the number of bacterial cells killed, it proved to be very suitable for assessing the antibacterial effects of different antimicrobial agents. Unlike with plate counting, the kinetics of killing can be monitored on a real-time basis using BL measurements. Complement activities in different samples can be estimated using only one serum dilution. The transformed E. coli strain appeared to be superior to P. luminescens in these applications because E. coli was complement sensitive, the detection limit of E. coli was one order lower and the BL-producing system of P. luminescens appeared to be quite unstable.

Bacterial infections, DNA virus infections, and RNA virus infections manifest differently in neutrophil receptor expression.

Treating viral illnesses or noninfective causes of inflammation with antibiotics is ineffective and contributes to the development of antibiotic resistance, toxicity, and allergic reactions, leading to increasing medical costs. A major factor behind unnecessary use of antibiotics is, of course, incorrect diagnosis. For this reason, timely and accurate information on whether the infection is bacterial in origin would be highly beneficial. In this paper we will present our recent studies on the expression of opsonin receptors on phagocytes. The analysis of the expression levels of FcγRI, CR1, and CR3, along with CRP and ESR data, provides a novel application to the diagnosis of infectious and inflammatory diseases. The best clinical benefit will be obtained when the individual variables are combined to generate the CIS point method for a bacterial infection marker, DNAVS point for differentiating between DNA and RNA virus infections, and CRP/CD11b ratio for a marker of Gram-positive sepsis.

Simultaneous quantitative analysis of Fc gamma RI (CD64) and CR1 (CD35) on neutrophils in distinguishing between bacterial infections, viral infections, and inflammatory diseases.

A flow cytometric quantitative analysis of receptors on neutrophils can be exploited in distinguishing between inflammatory and infectious diseases. In this prospective comparative study, simultaneous quantitative analysis of CD64 and CD35 on peripheral blood neutrophils was performed in febrile patients in order to differentiate between bacterial infections (n=89), viral infections (n=46), and inflammatory diseases (n=21). The patient data was compared to 60 healthy controls. We could divide patients into three groups depending on how they express CD35 and CD64 on neutrophils: (1) patients with a high probability of viral infection (low CD35/low CD64 and low CD35/high CD64), (2) patients with a high probability of inflammatory disease (high CD35/low CD64), and (3) patients with a high probability of bacterial infection (high CD35/high CD64). In summary, simultaneous quantitative analysis of CD64 and CD35 on neutrophils could potentially assist physicians to distinguish between inflammatory and infectious diseases.

CRP/CD11b ratio: a novel parameter for detecting gram-positive sepsis.

To commence proper antibiotic treatment in sepsis, timely knowledge of whether the cause of systemic infection is gram-negative (gram(-)) or gram-positive (gram(+)) bacteria in origin would be beneficial for clinicians. In this clinical prospective study, our objective was to develop a method for distinguishing between gram(+) and gram(-) bacterial infection. In gram(-) bacterial infection (n = 21), the average amount of CD11b on neutrophils was significantly higher than in gram(+) bacterial infection (n = 22). On the contrary, serum C-reactive protein (CRP) level was significantly higher in gram(+) than in gram(-) bacterial infection. By dividing the serum CRP value by the amount of CD11b on neutrophils, we derived a novel marker of gram(+) sepsis, CRP/CD11b ratio, which displayed 76% sensitivity and 80% specificity for the detection of gram(+) sepsis (n = 17) among febrile patients with microbiologically confirmed or clinically diagnosed bacterial infection. The detection of gram(+) sepsis is possible after the combination of neutrophil CD11b data and serum CRP level. In conclusion, our findings indicate that the proposed CRP/CD11b ratio test could potentially assist physicians in determining an appropriate antibiotic treatment in patients with severe bacterial infection.

Comparison of degranulation of easily mobilizable intracellular granules by human phagocytes in healthy subjects and patients with infectious diseases.

The aim of this study was to compare degranulation of easily mobilizable secretory vesicles (SVs) or secretory vesicle-like granules (SVLGs) in neutrophils, monocytes, and eosinophils of healthy controls (n = 60) and febrile patients with microbiologically confirmed or clinically diagnosed bacterial (n = 89) and viral (n = 46) infections. For this purpose, flow cytometric immunophenotyping of isolated phagocytes was performed using monoclonal antibodies against the phagocytosis receptors CR1 (CD35) and CR3 (CD11b) that are predominantly stored in the SVs of resting neutrophils. Similar to neutrophils, monocytes contain easily mobilizable SVLGs that constitute the main intracellular reservoir of CD35 and CD11b. In both neutrophils and monocytes, activation mechanisms leading to degranulation of SV and SVLG appeared dependent on both intra- and extracellular calcium levels. The kinetics of degranulation of SVLGs in control monocytes was significantly faster than that of SVs of control neutrophils. We conclude that phagocytes in patients with bacterial infections can be arranged in order of decreasing magnitude of SV or SVLG degranulation as follows (from left to right): neutrophils > monocytes " eosinophils. However, in viral infections, the corresponding degranulation order is monocytes > neutrophils approximately eosinophils.

Bacteriolytic activity of the alternative pathway of complement differs kinetically from the classical pathway.

The interaction between bacterial cells and activated complement components as a kinetic biological event is described. The bacteriolytic activity of complement in human and fish serum was assayed by measuring the decrease of bioluminescence of Escherichia coli transformed with lux genes. From the kinetic curves, the bacteriolytic CB(50)- and AB(50)-units were derived at any desired time point. It was observed that these values were irregular but decreased as a function of incubation time, and reached equal values during prolonged incubation, suggesting that the difference between the classical and alternative pathway activity is kinetic. From the kinetic curves, entirely new parameters could be derived: rate of the activation phase, rate of killing by the lytic phase and rate of killing by the entire pathway in undiluted serum. The rates of human and fish classical pathway were about five and two times higher than those of the alternative pathway respectively.

A novel method for distinguishing between dsDNA and ssRNA virus infections.

BACKGROUND: To commence proper antiviral treatment, timely knowledge of whether the infection is caused by DNA or RNA virus would be beneficial for the clinician. OBJECTIVES: Our objective was to develop a method for distinguishing between DNA and RNA virus infections. STUDY DESIGN: In this prospective study, total and differential count of leukocytes, serum C-reactive protein level, erythrocyte sedimentation rate, and quantitative flow cytometric analysis of FcgammaRI (CD64) on neutrophils and monocytes were obtained from 289 hospitalized febrile patients. After microbiological confirmation, 89 patients (31%) were found to have either bacterial (n=46) or viral (n=43) infection. The patient data was compared to 60 healthy controls. RESULTS: For the first time ever, it was noticed that in dsDNA virus infections (n=21) the average amount of CD64 on neutrophils was over five-fold compared to ssRNA virus infections (n=22). CONCLUSIONS: DNA virus score (DNAVS) point, which incorporates quantitative analysis of CD64 on neutrophils and total and differential count of leukocytes, varied between 0 and 8, and displayed 95% sensitivity and 100% specificity in distinguishing between dsDNA and ssRNA virus infections [average (S.D.); DNAVS points: 5.4 (2.5) vs. 0.3 (0.4); p<0.001].

Simultaneous quantitative analysis of FcgammaRI (CD64) expression on neutrophils and monocytes: a new, improved way to detect infections.

We performed simultaneous quantitative flow cytometric analysis of neutrophil and monocyte FcgammaRI (CD64) in 289 hospitalized febrile patients. Microbiological evaluation or clinical diagnosis confirmed bacterial (n=89) or viral (n=46) infection in 135 patients. Patient data were compared with data from 60 healthy controls. The average number of FcgammaRI on the surfaces of both neutrophils and monocytes was significantly increased in patients with febrile viral and bacterial infections, compared to healthy controls. Furthermore, we describe a novel marker of febrile infection, designated 'CD64 score point', which incorporates the quantitative analysis of FcgammaRI expressed on both neutrophils and monocytes, with 94% sensitivity and 98% specificity in distinguishing between febrile infections and healthy controls. By contrast, analysis of FcgammaRI expression on neutrophils and monocytes displayed poor sensitivity (73% and 52%) and specificity (65% and 52%) in distinguishing between bacterial and viral infections, and the levels did not differ significantly between systemic (sepsis), local, and clinically diagnosed bacterial infections. In summary, our results clearly show that the increased number of FcgammaRI on neutrophils and monocytes is a useful marker of febrile infection, but cannot be applied for differential diagnosis between bacterial and viral infections or between systemic and local bacterial infections.

Distinction between bacterial and viral infections.

PURPOSE OF REVIEW: To commence proper treatment as rapidly as possible and to reduce unnecessary antibiotic treatments, timely knowledge of whether the infection is bacterial or viral in origin would be beneficial for the clinician. As a reliable prediction of the causative agent of bacterial infection is not possible based on clinical features, there is an ongoing need for sensitive and specific markers of bacterial infection. RECENT FINDINGS: The most common differential diagnosis methods are reviewed here. It is also demonstrated that the measurement of the expression of complement receptors, particularly CR1 (CD35), on neutrophils can be a useful preliminary test to differentiate between bacterial and viral infections. In addition, a novel marker of local and systemic bacterial infections designated 'clinical infection score (CIS) point', which incorporates quantitative analysis of complement receptors on neutrophils and standard clinical laboratory data and displays 98% sensitivity and 97% specificity in distinguishing between bacterial and viral infections, is presented. SUMMARY: We conclude that the diagnostic yield of measured individual variables in distinguishing between bacterial and viral infections increases upon combination.

Promethazine renders Escherichia coli susceptible to penicillin G: real-time measurement of bacterial susceptibility by fluoro-luminometry.

Gram-negative bacteria are resistant to many hydrophobic antibiotics (such as penicillin G) owing to the highly hydrophilic saccharide part of lipopolysaccharide in the cell membrane, whilst most hydrophilic antibiotics (such as ampicillin) are more freely diffused into cells through aqueous porins. In this study, we investigated the possibility of altering the membrane permeability of Escherichia coli with tricyclic cationic compounds, such as the non-antibiotic drug promethazine. We also established the activity of these compounds as modifiers of antibiotic resistance in bacteria by a fluoro-luminometric approach. According to the results, promethazine has no bacteriostatic effect on E. coli at concentrations <64 microg/mL. However, promethazine at these concentrations in combination with penicillin G produced a significant synergistic activity against E. coli. Specifically, a constant promethazine concentration of 32 microg/mL in combination with penicillin G concentrations of 16-128 microg/mL suppressed the growth and viability of bacteria and converted penicillin G-resistant cells to being susceptible to this antibiotic with a minimum inhibitory concentration of 128 microg/mL. In contrast to penicillin G, the efficacy of ampicillin was apparently not increased in the presence of promethazine, suggesting that promethazine directly affects the membrane permeability of bacteria or alternatively inhibits the function of efflux pumps. In conclusion, we conclude that exposing E. coli cells to a given antibiotic in combination with promethazine can increase the susceptibility of bacteria; this effect is reliably assessed on a real-time basis using kinetic fluoro-luminometric measurements.

Multiple whole bacterial antigens in polyvalent vaccine may result in inhibition of specific responses in rainbow trout (Oncorhynchus mykiss).

The goal of fish vaccination today is to protect fish against multiple bacterial fish pathogens simultaneously using polyvalent vaccines. However, many immunological processes such as antigenic cross-reaction, antigenic competition, affinity maturation and antigen-induced suppression may affect the specificity, avidity and level of antibodies. Consequently, the biological function of antibodies may be markedly different from that predicted by conventional serologic tests. Here, we investigated the effects of vaccination and composition of vaccine on the plasma antibody levels, biological function of antibodies in opsonophagocytosis as well as the effects of vaccination on the blood leucocyte counts. Rainbow trout were vaccinated with saline or with two different polyvalent, mineral oil-adjuvanted vaccines. Vaccine 1 contained Aeromonas salmonicida, Listonella anguillarum and both Th and Fd serotypes of Flavobacterium psychrophilum antigens and vaccine 2 contained A. salmonicida, L. anguillarum and only Fd serotype of Fl. psychrophilum. The antibody-mediated opsonophagocytosis was determined as the respiratory burst (RB) activity of blood monocytes and granulocytes against the tested bacterial antigens. Three weeks after vaccination both vaccine groups and the control group showed increased RB activity against all bacterial strains. However, the increase in RB activities was non-specific and originated from the increased number of circulating granulocytes and monocytes. On the other hand, at 6 weeks post-vaccination both specific antibodies and antibody-dependent opsonophagocytosis appeared in both vaccine groups. However, the composition of the vaccine had a marked effect on the magnitude of specific responses. The Fd+Th vaccine enhanced the target specific opsonophagocytosis, to a lesser extent than the Fd vaccine. Both polyvalent vaccines appeared to mainly affect the numbers of circulating monocytes and our results suggest that the monocytes play a more significant role than the granulocytes in antibody-dependent opsonophagocytosis. Our results also suggest that the presented opsonophagocytic assay is an advantageous method to predict vaccine efficiency and that the number, and properties, of bacterial antigens in polyvalent vaccines should be carefully selected in order to avoid inhibitory effects of antigens on the specific response of fish.

Quantitative analysis of complement receptors, CR1 (CD35) and CR3 (CD11b), on neutrophils improves distinction between bacterial and viral infections in febrile patients: comparison with standard clinical laboratory data.

There is an ongoing need for sensitive and specific markers of bacterial infection. In this prospective study, standard clinical laboratory data (neutrophil count, serum C reactive protein level, erythrocyte sedimentation rate) and quantitative flow cytometric analysis of neutrophil complement receptors, CR1 and CR3, were obtained from 289 hospitalized febrile patients. After microbiological confirmation or clinical diagnosis, 135 patients were found to have either bacterial (n = 89) or viral (n = 46) infection. The patient data was compared to 60 healthy controls. In bacterial infections, all measured variables were significantly increased, particularly the average amounts of CR1 and CR3 on neutrophils were over three-fold and two-fold higher, respectively, compared to viral infections and controls. We described a novel marker of local and systemic bacterial infections designated 'clinical infection score (CIS) point', which incorporates quantitative analysis of complement receptors on neutrophils and standard clinical laboratory data. CIS point varied between 0 and 8, and displayed 98% sensitivity and 97% specificity in distinguishing between bacterial and viral infections [average (S.D.); CIS points: 6.2 (1.7) vs. 0.6 (1.0); p < 0.001]. These findings suggest that the proposed CIS-based diagnostic test could potentially assist physicians in deciding whether antibiotic treatment is necessary.

Real-time monitoring of antimicrobial activity with the multiparameter microplate assay.

Kinetic measurements of the bacteriostatic, bactericidal and bacteriolytic activities of six model antibiotics (ampicillin, erythromycin, nalidixic acid, polymyxin B, tetracycline, and trimethoprim) against Escherichia coli as target bacteria were performed by bioluminescence, fluorescence, and optical density based real-time assay. Additionally, plate counting was used as a control measurement. The gfp and insect luciferase (lucFF) genes were cloned into cells used for measurements to enable fluoro-luminometric detection. Bacteria were exposed to antibiotics for 10 h, and the effects of antimicrobial agents were established. Inhibitory concentration of 50% (IC(50)), minimum bactericidal concentration (MBC), and bactericidal concentration of 50% (BC(50)) of each antibiotic were calculated from these procedures. Bacteriostatic, bactericidal or bacteriolytic actions of each antibiotic, as well the time interval from exposure to visible effect, were readily observed from kinetic data. No significant differences were observed between data obtained with the different methods employed. Ampicillin and polymyxin B were clearly bacteriolytic, nalidixic acid and tetracycline showed bactericidal effects, and erythromycin and trimethoprim were bacteriostatic drugs. The assay has the advantage of speed and accurately discerns between lytic, cidal and static compounds. Thus, this reliable and fully automated novel kinetic assay with high sample capacity offers new possibilities for real-time detection, making it suitable for diverse high throughput screening (HTS) applications.