Flow Cytometric Method for the Assessment of the Minimal Inhibitory Concentrations of Antibacterial Agents to Mycoplasma agalactiae

q 2006 International Society for Analytical Cytology Cytometry Part A 69A:1071 1076 (2006) Flow Cytometric Method for the Assessment of the Minimal Inhibitory Concentrations of Antibacterial Agents to Mycoplasma agalactiae Patrıcia Assunȩcao, 1 * Nuno T. Antunes, 1 Ruben S. Rosales, 1 Christian de la Fe, 1 Carlos Poveda, 1 Jose B. Poveda, 1 and Hazel M. Davey 2 1 Unidad de Epidemiologıa y Medicina Preventiva, Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Arucas, Spain 2 Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, Wales, United Kingdom Received 19 January 2006; Accepted 19 June 2006 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. q 2006 International Society for Analytical Cytology Key terms: cytometry; antibiotics; MIC; Mycoplasma agalactiae; susceptibility Traditional assessment of antimicrobial action involves the incubation of microorganisms in liquid or on solid media for 18 48 h in the presence of the antibacterial agent (1). With mycoplasmas, the minimal inhibitory concentration (MIC) is determined by a broth dilution method preformed in microtitration plates (2). The MIC is defined as the lowest concentration of agents at which no growth occurred (2). The major problem of the assessment of antibiotic sensitivity in mycoplasmas is that incubation periods of up to several days, depending on the growth rate of the mycoplasma species that is being studied, may be required before a result can be obtained. Furthermore, in some mycoplasma species such as Mycoplasma (M.) agalactiae, the causative agent of contagious agalactiae in small ruminants, the common substrates that are added to the medium for a color development purpose like glucose, mannose, etc. do not work so well, since M. agalactiae do not have the capability of utilizing these nutrients. For MIC assessment in M. agalactiae, a combination of glucose and pyruvate are added to the medium (2), but we have experienced that care must be taken because the weak color change can be observed in the medium that tends to disappear after 1 day (Antunes NT, personal observation). An alternative method, based on a low speed centrifugation of the cells after incubation for 48 h with the antibacterial agents, has been developed for M. agalactiae (3). The presence or absence Grant sponsor: Gobierno de Canarias; Grant number: IDT-LP-04/016. *Correspondence to: Patrıcia Assunç~ao, Unidad de Epidemiologıa y Medicina Preventiva, Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Trasmonta~na s/n, 35416 Arucas, Las Palmas, Spain. E-mail: passuncao@becarios.ulpgc.es Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cyto.a.20331

1072 ASSUNȩCAO ET AL. FIG. 1. Validation of the live/dead mycoplasma cell staining. A log culture phase of M. agalactiae was used as a live cell control (A C). Mycoplasma cells were heat-injured at 60 C for 1 h (E G). Artificial mixture of live and dead mycoplasma cells (D). Broth medium as negative control (H). Cells were stained with SYBR or/and PI. Region R1 corresponds to live cells and region R2 corresponds to dead cells when the double stain was used. Region R3 corresponds to dead cells when only PI stain was used. When used alone SYBR can stain both live and dead cells (R1). of growth is determined according to the amount of pellet that is observed (3). The main disadvantage of this method is that the threshold pellet size can sometimes be difficult to establish, since it is dependent on human interpretation. The aim of the work presented was to determine whether a flow cytometric approach could provide a useful alternative technique for the determination of MIC for M. agalactiae. MATERIALS AND METHODS Strains, Antibacterial Agents, and Culture Conditions The reference strain of M. agalactiae was obtained from The National Collection of Type Cultures (NCTC, United Kingdom). Enrofloxacin and ciprofloxacin were obtained from Sigma (MO), and gentamicin, chloramphenicol, oxytetracycline, tylosin, and streptomycin were obtained from Serva (Heidelberg, Germany). Stock solutions of the antibacterial agents were made by standard protocols (2). M. agalactiae was propagated in PH broth medium (4) under aerobic conditions at 37 C for 24 h. Mycoplasma counts were performed by flow cytometry as previously described (5) in order that appropriate dilutions of the mycoplasma cultures could be produced. Final concentrations of 5 3 10 5 cells/ml were placed into tubes with PH broth medium (4) with the appropriate concentration of the different antibacterial agents. A tube of M. agalactiae without antibacterial agents was retained as a positive control. The minimal inhibitory concentration (MIC) for each antibacterial agent was estimated by the traditional method at 24 h (when the control tube, without antibacterial agents, changed color from orange to smooth yellow, because of glucose-pyruvate metabolization by the growing M. agalactiae), and was defined as the lowest concentration of antibiotic that completely inhibited visible growth of the organism (6). With respect to flow cytometry, samples were analyzed at 6, 12, 24, 48, and 72 h. Preliminary Experiments To establish the regions in the flow cytometric analysis that corresponded to live and dead mycoplasma cells, M. agalactiae cells were heat-injured at 60 C for 1 h and used as a dead control, whereas an early logarithmic phase LC culture (24 h) was used as a live control. Cells were stained (15 min at room temperature, in the dark) with the cell-permeant double stranded DNA-fluorochrome Sybr green-i (SYBR; Amresco, OH) at a final concentration (1:10,000, vol/vol) of the commercial stock solution (neither the molecular weight nor the chemical formula are provided by the manufacturer), and/or with propidium iodide (PI; Sigma, MO) at a final concentration of 10 lg/ml. SYBR stains nucleic acid in all cells, while PI stains nucleic acids in cells with damaged membranes. Fluorescence Labelling and Flow Cytometry Analysis M. agalactiae cultures (10 ll) were diluted to 1 ml with sterile-filtered saline solution (0.85% NaCl) and stained with SYBR and PI as described above. Sample analysis was performed in a Coulter Epics Elite flow cytometer (Coulter; Miami, FL) equipped with an air-cooled 488-nm argon-ion FIG. 2. Dot plots (FL1 vs. SSC) of M. agalactiae cells stained with SYBR. M. agalactiae (A); broth medium (B). Region R1 corresponds to M. agalactiae cells.

FLOW CYTOMETRIC ASSESSMENT OF MIC TO M. AGALACTIAE 1073 FIG. 3. Total counts (cells per milliliter) of the M. agalactiae cultures in time-matched samples, with the antibacterial agents at the indicated concentrations. Enrofloxacin (ENR), ciprofloxacin (CIP), gentamicin (GEN), chloramphenicol (CHL), oxytetracycline (OXT), tylosin (TYL), and streptomycin (STR). The concentration of the antibacterial agents (microgram per milliliter) is shown in the key table. In all cases, the standard errors are less than the data symbol drawn. laser. Each cell was characterized by four optical parameters: side-angle-scatter (SSC), forward angle scatter (FSC), green fluorescence for SYBR (525 nm), and red fluorescence for PI (675 nm). Data were acquired in a four-decade logarithmic scale. Green fluorescence from SYBR was collected combining a 550-nm dichroic long filter and a 525- nm long pass filter. PI was collected combining a 600-nm dichroic long filter and a 675-nm long pass filter. All filters were supplied with the instrument. Optical alignment was based on an optimized signal from Immuno-Check Epics alignment fluorospheres (Epics Devison; FL). For absolute counts, we used 5.5-lm beads with a known concentration as a reference (Optoflow, Oslo, Norway). The number of cells counted was then converted to cells per milliliter. Data Analysis Data were collected with software supplied by Beckman-Coulter (Coulter Elite Software) and further analyzed using WinMDI Software Version 2.8 (Joseph Trotter, The Scripps Research Institute, La Jolla, CA). RESULTS The fluorochromes SYBR and PI were able to distinguish between live and dead mycoplasma cells (Fig. 1). PI only stained the cells in the samples that were subjected to the heat treatment (Fig. 1). Regions corresponding to dead cells were defined for when PI was used alone (Fig. 1F, region R3), or in combination with SYBR (Fig. 1G, region R2). Red fluorescence (PI), derived from dead cells, was higher when used in combination with SYBR than when used alone (Fig. 1, dot plots F and G). SYBR stained both live and dead cells when used alone; however, when it was used in combination with PI, two different populations corresponding to live and dead cells could be easily distinguished (Fig. 1D).

1074 ASSUNȩCAO ET AL. Table 1 MIC Values Determined by Flow Cytometry and by the Traditional Method a Flow cytometry (lgml 21 ) Traditional method (lgml 21 ) 12 h 24 h 48 h 72 h 24 h ENR 0.12 0.12 0.12 0.12 0.12 CIP 0.12 0.12 0.12 0.12 0.12 GEN 128 128 128 128 128 STR 128 128 128 128 128 CHL 2 2 4 4 2 OXT 0.5 0.5 1 1 0.5 TYL 0.12 0.12 0.25 0.25 0.06 Enrofloxacin (ENR), ciprofloxacin (CIP), gentamicin (GEN), chloramphenicol (CHL), oxytetracycline (OXT), tylosin (TYL), streptomycin (STR). a The assessment of MIC by traditional methods was only performed at 24 h, when a change of color from orange/red to smooth yellow in the medium was visible. The values given by flow cytometric analysis correspond to the minimal concentration of the antibacterial agents where mycoplasma growth did not occur. In this study, the MIC of seven antibacterial agents to M. agalactiae were determined by flow cytometry. Total counts were assessed by the SYBR stained cells (Fig. 2), and Figure 3 shows the growth rate of M. agalactiae when incubated with the different concentrations of the antibacterial agents tested throughout the time matched samples. The assessment of MIC by traditional methods was only performed at 24 h, when a change of color in the medium of the control tube (culture without any antibacterial agent) was observed, whereas flow cytometry analysis was performed from 6 to 72 h. The values given by flow cytometry analysis correspond to the minimal concentration of the antibacterial agents where no mycoplasma growth was observed (Table 1). At 24 h, the same MIC was recorded in both methods for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and streptomycin). Flow cytometry gave slightly higher MIC than traditional method for tylosin (Table 1). MICs obtained by flow cytometric analysis at 12, 24, 48, and 72 h were the same for four antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, and streptomycin). On the other hand, oxytetracycline, chloramphenicol, and tylosin gave slightly higher MIC values after 48 h (1, 4, and 0.25 lg/ml, respectively) with respect to the results obtained at 12 and 24 h (0.5, 2, and 0.12 lg/ml, respectively, for both time matched samples, Table 1). Interestingly, the MIC determined by flow cytometry for enrofloxacin and ciprofloxacin (0.12 lg/ml) resulted only in a maintenance of the total counts throughout the time matched samples, whereas use of higher concentrations of these antibacterial agents (>0.25 ll/ml) resulted in a reduction in the total counts per milliliter (Fig. 3). Concentrations of 0.5, 2, and 0.12 lg/ml of oxytetracycline, chloramphenicol, and tylosin, respectively, were only able to inhibit the M. agalactiae growth until 24 h, after which the cell number began to increase (Fig. 3). Figure 4 shows dot plots of the analysis at 24 h for each of the antibacterial agents at 2 MIC, 1 MIC and 0.5 MIC. Two different populations of cells can be observed. Those in region R3 have taken up only the SYBR stain and from control samples we deduce that these are alive. The cells in region R4 have also taken up PI and are therefore permeabilized, dead cells. It is interesting to note that a higher percentage of cells stained only with SYBR (region R3) were detected in the dot plots of the MIC value of oxytetracycline, chloramphenicol, tylosin, and streptomycin, than in the enrofloxacin, ciprofloxacin, and gentamicin dot plots (Fig. 4). DISCUSSION One of the advantages of flow cytometry is the visualization of the heterogeneity of the response of the cells to the antimicrobial agent. This heterogeneity is manifested in the presence of subpopulations that are less susceptible to the antimicrobial agent treatment (7 11). In this study, we have tested seven antibacterial agents, three known to be bactericidal (enrofloxacin, ciprofloxacin, and gentamicin) and four known to be bacteriostatic (streptomycin, chloramphenicol, and oxytetracycline and tylosin) for the assessment of MIC in M. agalactiae by flow cytometry. The accurate determination of the MIC of M. agalactiae for each antibacterial agent has been accomplished by the enumeration of SYBR stained mycoplasma cells throughout the different time matched samples. The detection of growth inhibition of M. agalactiae by flow cytometry was possible at the earliest time matched sample performed in this study (6 h). However, at this point, MIC values were difficult to interpret because of the fact that sub-inhibitory concentrations of each antibacterial agent resulted in some inhibition of mycoplasma growth at this time point. The same MIC values were obtained for six antibacterial agents for both methods at 24 h (enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and streptomycin). A higher MIC value was obtained for tylosin in the flow cytometric analysis with respect to the traditional method. This is more likely to be due to the fact that the minimum number of mycoplasma organisms required to cause a color change in the medium is higher than the minimum change in mycoplasma number that can be detected by flow cytometry.

FLOW CYTOMETRIC ASSESSMENT OF MIC TO M. AGALACTIAE 1075 FIG. 4. Dot plots (SYBR vs. PI) of M. agalactiae cells at 24-h postinoculation with different antibacterial agents at concentrations of 2 MIC, 1 MIC, and 0.5 MIC [the concentrations (in units of microgram per milliliter) of the different antibacterial agents are written in left bottom corner of each dot plot]. The population in region R3 corresponds to live cells and those in region R4 are dead cells that had incorporated PI. The number of events shown result from analysis of a fixed volume, low numbers of events are indicative of inhibition of growth in the treated cells when compared with the control sample (top left). Enrofloxacin (ENR), ciprofloxacin (CIP), gentamicin (GEN), chloramphenicol (CHL), oxytetracycline (OXT), tylosin (TYL), and streptomycin (STR).

1076 ASSUNȩCAO ET AL. A higher percentage of cells that had incorporated PI were seen in the samples with enrofloxacin and ciprofloxacin treatments (at inhibitory concentrations), throughout the time matched samples. PI staining indicates that the membrane permeability of the cells has been compromised and is indicative of cell death (10). However, a percentage of SYBR stained cells that have not incorporated PI was always present. This effect is especially noted in the dot plots corresponding to the MIC value of gentamicin, oxytetracycline, chloramphenicol, tylosin, and streptomycin treatments. This may be due to the fact that these antibacterial agents do not cause any damage in the membrane permeability of M. agalactiae. As observed by other authors, (6,12 14) our results show that the most effective antibacterial agents seem to be the quinolones, enrofloxacin, and ciprofloxacin, since inhibitory concentration (0.06 lg/ml) resulted in a reduction in the total counts per milliliter throughout the time matched samples, indicating that these antibacterial agents are indeed killing the mycoplasma cells. Although we were expecting the same behavior with gentamicin, since it is known to be also bactericidal, this was not observed in the present study. It is possible that this antibacterial agent has a different effect on mycoplasmas. With the other antibacterial agents, which are known to have a bacteriostatic effect, MIC concentrations were only able to prevent cell growth resulting in maintenance of cell numbers at the inoculated level. In some cases, a slight increase of total cell counts could be detected over the course of the experiment. These observations may indicate that M. agalactiae growth was just reversibly inhibited or that some resistant populations were emerging during the incubations performed. It is a well recognized fact that although antibiotic therapy of mycoplasma infections can bring about clinical improvements, it rarely eliminates the organism altogether (15). Although in vitro MIC values do not directly relate to an antimicrobial s effectiveness in vivo, they clearly indicate the antimicrobials that are most likely to be effective (3). This study demonstrates that flow cytometry can give definitive MIC values for M. agalactiae at 12-h postincubation with each antibacterial agent. The results obtained also provide information about how M. agalactiae is responding toward each antibacterial agent. For example, concentrations of 0.12, 0.5, and 2 lg/ml of tylosin, oxytetracycline, and chloramphenicol, respectively, were only able to inhibit the M. agalactiae growth until 24 h after which cell numbers increased (Fig. 2). Flow cytometry offers the opportunity to monitor emergence and growth of resistant subpopulations that can offer further information about the potential effectiveness of candidate antibiotic treatment regimes. The major disadvantage of this method is that each sample has to be analyzed individually, in comparison to the traditional methods where the assays are performed in parallel in a 96-well plate with human input limited to reading the results at the end of the experiment. However, since flow cytometric analysis takes 1 min for each sample, this disadvantage is not considered to be a major inconvenience. In addition, a number of commercially-available instruments are now available with automated sample handling devices that reduce the necessity for operator input. Most importantly, the detection of inhibition of growth of mycoplasmas by flow cytometry is not dependent on a change of color or the increment of turbidity in the medium as traditional methods, which often generate problems in interpreting the results. Rather the flow cytometric method provides data on actual changes in cell number during the incubations. We, therefore, conclude that the flow cytometric method of determining MICs for antibiotics targeted against mycoplasma cells offers an attractive alternative to the traditional method. LITERATURE CITED 1. Comas J, Vives-Rego J. Assessment of the effects of gramicidin, formaldehyde, and surfactants on Escherichia coli by flow cytometry using nucleic acid and membrane potential dyes. Cytometry 1997;29:58 64. 2. Hannan PC. 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