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A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays Hannah B. Pooley, Kumudika de Silva, Auriol C. Purdie, Douglas J. Begg, Richard J. Whittington, Karren M. Plain Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia

ABSTRACT

Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE

Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing.

D

etermining the number of live and dead bacteria in a sample is a key requirement of most in vitro cellular infection/phagocytosis assays. Quantification of live and dead bacteria is generally done by assessing growth in culture (1). However, for fastidious slow-growing bacteria, such as Mycobacterium avium subspecies paratuberculosis, it can take months to obtain culture results (2– 4). This becomes a significant obstacle for in vitro cellular infection assays, which precludes their use in large studies. In vitro cellular infection assays could be a rapid and costeffective alternative to animal trials for assessing M. avium subsp. paratuberculosis interactions with host immune cells. Through examination of interaction outcomes, these assays can provide information relevant to the assessment of vaccine efficacy (5–7), pathogenesis (8), and antimicrobial activity (9). However, if in vitro infection assays are to be used as rapid screening tools, it is essential that they function in a high-throughput capacity for large studies and that the time taken to obtain results does not become rate limiting. Culture is the gold-standard method for confirming the presence of viable bacteria in a sample. Liquid culture is recommended for primary isolation of M. avium subsp. paratuberculosis, because it is more sensitive than culture on solid medium (1). The presence of M. avium subsp. paratuberculosis in liquid culture can be confirmed rapidly by PCR (10). The amount of M. avium subsp. paratuberculosis in a sample can be determined by quantitative PCR (qPCR); however, this method does not discriminate be-

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tween DNA from live bacteria and dead bacteria, making it unable to determine the viability of bacteria based on testing of a sample collected at any single time point. The viability of bacteria can also be determined by examining markers of cell death (11, 12). When a cell dies, there is a loss of cell membrane integrity and a loss of cellular metabolism, and these markers can be used to quantify the numbers of dead and live cells in a population. These marker-associated methods lend themselves to the detection of bacteria, such as M. avium subsp. paratuberculosis, that are difficult or slow to culture and hence may be applicable in in vitro cellular infection assays. The degree of metabolism is a common marker to distinguish between live and dead bacteria. Metabolically active cells can reduce dyes such as resazurin (7-hydroxy-3H-phenoxazin-3-one10-oxide), resulting in a colorimetric and fluorescent change (13).

Received 31 May 2016 Accepted 27 June 2016 Accepted manuscript posted online 1 July 2016 Citation Pooley HB, de Silva K, Purdie AC, Begg DJ, Whittington RJ, Plain KM. 2016. A rapid method for quantifying viable Mycobacterium avium subsp. paratuberculosis in cellular infection assays. Appl Environ Microbiol 82:5553–5562. doi:10.1128/AEM.01668-16. Editor: M. Kivisaar, University of Tartu Address correspondence to Karren M. Plain, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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The degree of reduction is proportional to the number of viable bacteria in suspension. The resazurin viability assay has been used previously with M. avium subsp. paratuberculosis; however, further optimization to enable the detection of low concentrations of viable M. avium subsp. paratuberculosis would be needed for it to be considered a useful tool for determining bacterial viability in in vitro cellular infection assays (14, 15). Membrane integrity also serves as a viability marker, as it decreases as bacterial cells die. Several commercially available bacterial viability kits contain molecules that rely on cell membrane integrity for differentiation of live and dead cells (12). One such kit, the Live/Dead BacLight bacterial viability kit, includes SYTO9, which has an affinity for nucleic acid and stains all cells, and propidium iodide (PI), which enters only cells with compromised membranes. Such kits have been used with mycobacterial species, but in the majority of studies the readout was performed by using either a fluorescence microscope or a microplate reader (16–18). An ideal readout platform for screening assays would be flow cytometry, as it has high-throughput capability, is faster than microscopy, and is more accurate than a microplate reader (19). The few studies that have used flow cytometry as the readout for M. avium subsp. paratuberculosis viability using this method did not report gating strategies and utilized a strain of M. avium subsp. paratuberculosis that was green fluorescent protein (GFP) modified, the staining characteristics of which would be very different (20). Therefore, validation of the Live/Dead BacLight bacterial viability kit for flow cytometry is required, particularly the suggested amount of dye, the cell concentration, and the staining protocol, all of which can vary depending on the readout method. Differentiation of live and dead bacteria based on membrane integrity has been enhanced by coupling DNA-intercalating dyes with qPCR. Dyes such as propidium monoazide (PMA) selectively enter cells with a compromised membrane and upon light exposure irreversibly modify the bacterial DNA (21). Cross-linkage of the dye with DNA irreversibly alters the DNA structure and strongly interferes with qPCR detection (12, 22). However, differentiation of the amount of DNA from treated and untreated M. avium subsp. paratuberculosis samples has proven to be difficult (23, 24). Differentiation of these samples may be enhanced by optimization of the incubation time and dye concentration, which may make this assay suitable for the detection of M. avium subsp. paratuberculosis in an in vitro cellular infection assay. The aim of this study was to develop an assay to quantify the number of viable M. avium subsp. paratuberculosis cells in a sample with sufficient sensitivity at low cell concentrations, to enable application to an in vitro cellular infection model. The initial objective was to screen three different non-culture-dependent viability assays, followed by evaluation of a qPCR-coupled rapidculture protocol. The expected criteria for assessment included accurate discrimination of live/dead cells, assay sensitivity, and the ability for M. avium subsp. paratuberculosis viability to be rapidly quantified. MATERIALS AND METHODS Preparation of mycobacteria. Suspensions of M. avium subsp. paratuberculosis (sheep [S] strain Telford 9.2, IS900 restriction fragment length polymorphism [RFLP] S1 type, and IS1311 S pattern) that were frozen and stored at ⫺80°C were used for all experiments (25, 26). Suspensions were thawed at room temperature and washed by pelleting of the cells in a Microfuge (Beckman Coulter) at 5,000 ⫻ g for 5 min. The supernatant

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was aspirated, and the cell pellet was resuspended in 1 ml of 0.1 M phosphate-buffered saline (PBS); this process was repeated twice. After the second centrifugation, cells were resuspended at a concentration of 1.08 ⫻ 1010 cells/ml in bacterial liquid culture medium (M7H9C medium without egg yolk) (10). To ensure a single-cell suspension, the solution was passed through a 26-gauge needle three times, and M. avium subsp. paratuberculosis cells were incubated for 24 to 36 h at 37°C in an atmosphere of 5% CO2. Heat-killed M. avium subsp. paratuberculosis was prepared by incubating an aliquot of the resuspended M. avium subsp. paratuberculosis cells at 80°C for 30 min. Resazurin viability dye assay. All of the resazurin experiments were carried out by using the dye as provided by the manufacturer in 96-well flat-bottom plates, and plates were maintained at 37°C in an atmosphere of 5% CO2 between plate readings. To optimize the incubation time, resazurin dye (alamarBlue; AbD Serotec, NC, USA) was added to some wells with live M. avium subsp. paratuberculosis suspensions (106 cells/well) in PBS, to be 10% of the total well volume, at the start of the incubation period. The dye was added to replicate wells 24 h later and then every 48 h thereafter. Heat-killed M. avium subsp. paratuberculosis suspensions (106 cells/well) were analyzed in duplicate on the same plate. Controls on each plate included untreated cells (negative control to account for background absorbance) and PBS with resazurin dye alone. Absorbances at 540 nm and 595 nm were read when the dye was added and then 24 h after each addition of resazurin for 14 days (Multiskan Ascent; Thermo Scientific, Australia). All samples were maintained in PBS for the duration of the assay and were tested in duplicate. To examine assay sensitivity, serial 10-fold dilutions of live cells, heatkilled cells, and a 50/50 mix of live and heat-killed M. avium subsp. paratuberculosis cells (3.43 ⫻ 1010 cells/ml to 3.43 cells/ml) were prepared in bacterial liquid culture medium without egg yolk, and resazurin dye was added. Absorbance readings at 540 nm and 595 nm were taken when the dye was added and at days 1, 2, 7, 8, 9, 10, and 11. Data were analyzed by conversion of the optical density (OD) values obtained into a percent reduction value by using the equation [(O2 ⫻ A1) ⫺ (O1 ⫻ A2)]/[(R1 ⫻ N2) ⫺ (R2 ⫻ N1)] ⫻ 100, where O1 is the molar extinction coefficient (E) of oxidized resazurin at 540 nm (blue), equal to 47,619; O2 is the E value of oxidized resazurin at 595 nm, equal to 117,216; R1 is the E value of reduced resazurin at 540 nm (red), equal to 104,395; R2 is the E value of reduced resazurin at 595 nm, equal to 14,652; A1 is the OD of test wells at 540 nm; A2 is the OD of test wells at 595 nm; N1 is the OD of the negative-control well (medium plus resazurin with no cells) at 540 nm; and N2 is OD of the negative-control well (medium plus resazurin with no cells) at 595 nm (alamarBlue technical data sheet; AbD Serotec). Percent reduction values refer to the proportion of reduction of resazurin in wells with bacteria compared to control wells with dye and medium alone. This serves as a proxy for the concentration of live cells in solution, as only live cells can effectively reduce the dye. Assay using resazurin viability dye for detection of viable M. avium subsp. paratuberculosis cells in macrophages. Murine RAW 264.7 macrophages (European Collection of Cell Culture, Wiltshire, United Kingdom) were seeded at 105 cells/well in cell culture medium (RPMI 1640 with 25 mM HEPES, 10% fetal calf serum [FCS], 58 ␮M ␤-mercaptoethanol, penicillin-streptomycin at 100 ␮g/ml, and 0.2 mM L-glutamine) and were incubated overnight at 37°C in 5% CO2 to allow adherence to the wells. The cell culture medium was discarded, and macrophages were infected at a multiplicity of infection (MOI) of 10:1 with live M. avium subsp. paratuberculosis or heat-killed M. avium subsp. paratuberculosis in cell culture medium without penicillin-streptomycin. Following incubation for 2 h at 37°C in 5% CO2, the supernatant was discarded, and each well was rinsed three times with sterile PBS. Macrophages were lysed for 5 min with 180 ␮l of sterile water to release intracellular M. avium subsp. paratuberculosis. The lysate was transferred to an Eppendorf tube and centrifuged at 5,000 ⫻ g for 5 min, and the supernatant was discarded. Cell pellets from half of the wells that contained either live or heat-killed bac-

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teria were resuspended in 180 ␮l of sterile PBS. Resazurin (20 ␮l) was added to these suspensions, the suspensions were mixed, and absorbances were read at 540 nm and 595 nm on days 1, 6, 7, 9, and 12. The cell pellets of the other replicates were resuspended in 180 ␮l of bacterial liquid culture medium with egg yolk and incubated at 37°C in 5% CO2 for 1 week before they were resuspended in PBS and treated with resazurin dye as described above. Plate readings were taken for these cell suspensions immediately after staining and at days 2 and 5 of incubation. At all times, other than when absorbance readings were being performed, the plate was incubated at 37°C with 5% CO2. All samples were run in duplicate. Live/Dead BacLight bacterial viability kit. Suspensions of live, heatkilled, and a 50/50 mix of live and heat-killed M. avium subsp. paratuberculosis cells (106 cells/ml) in sterile PBS were stained with the Live/Dead BacLight bacterial viability kit (Molecular Probes, Life Technologies) according to the manufacturer’s instructions. Briefly, equal proportions of dye components A and B were added to each suspension at a rate of 6 ␮l dye mix per 2 ml M. avium subsp. paratuberculosis suspension, and the suspensions were incubated in the dark at room temperature for 15 min prior to flow cytometric data acquisition (Guava easyCyte HT sampling flow cytometer; Merck Millipore). Data were analyzed by using InCyte in Guavasoft 2.7 software, with gating applied to distinguish between unstained and stained events based on a comparison of unstained controls and sample wells. All samples were run in duplicate, and the gates selected were applied across all samples to allow comparisons between different treatments. Propidium monoazide staining and IS900 quantitative PCR method. The protocol for staining with PMA was based on an optimized protocol described previously by Kralik et al. (22). Briefly, a 1 mM working stock solution of PMA dye (Biotium) was prepared in sterile water. Duplicate suspensions of live, heat-killed, and a 50/50 mix of live and heat-killed M. avium subsp. paratuberculosis cells (106 cells/ml) in PBS were added to light-permeable strip tubes (Scientific Specialties, Inc.) in 195-␮l aliquots. PMA was added to half of the M. avium subsp. paratuberculosis suspensions at a final concentration of 25 ␮M. All suspensions were incubated in the dark for 5 min with continuous shaking. Half of the PMA samples and half of the untreated samples were then placed horizontally on ice and exposed to a 700-W halogen lamp for 2 min at a distance of 20 cm. The remainder of the samples were kept in the dark on ice for 2 min. DNA was isolated, and quantification of M. avium subsp. paratuberculosis DNA from the suspensions using an IS900 qPCR assay was done as previously described and as detailed below (27). The entire experiment was repeated twice to examine the reproducibility and consistency of the results. DNA isolation. Briefly, DNA isolation from each sample was done by using a combined method involving mechanical cell disruption followed by magnetic bead-based DNA purification using the BioSprint 96 Onefor-All Vet kit (Qiagen). All M. avium subsp. paratuberculosis solutions were added to 2-ml conical-base screw-cap tubes containing 200 ␮l of RLT buffer and 0.3 g of zirconia-silica beads (BioSpec Products, Inc., Daintree Scientific). The cells were disrupted by using a TissueLyser II system (Qiagen). Bead tubes were then centrifuged at 16,000 ⫻ g for 3 min, and 100 ␮l of the supernatant underwent magnetic bead DNA purification. The samples were transferred to a lysate plate (deep 96-well plate) along with 40 ␮l proteinase K and 600 ␮l magnetic bead mix (preparation per reaction mixture, 300 ␮l isopropanol, 25 ␮l MagAttract suspension, 300 ␮l RLT buffer, and 2.7 ␮l of cRNA, vortexed thoroughly as recommended by the manufacturer). Three additional deep 96-well (S-Block) plates and one standard 96-well plate were labeled and prepared as follows: wash 1 consisted of 700 ␮l AW1 buffer/well in a deep-well plate, washes 2 and 3 consisted of 500 ␮l RPE buffer/well in two deep-well plates, and elution consisted of 75 ␮l AVE buffer/well in a standard 96-well plate. All the plates were then run on an automated magnetic particle processor (MagMAX Express-96; Life Technologies) according to the BS96 Vet 100 instrument protocol (Qiagen). The contents of the individual wells of the elution plate were stored at ⫺20°C.

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IS900 quantitative PCR. qPCR for M. avium subsp. paratuberculosis IS900 was performed by using an Mx3000P real-time PCR instrument (Stratagene, Agilent). The reaction mixtures each contained 5 ␮l template DNA, 250 nM each forward and reverse primers (MP10-1 [5=-ATGCGC CACGACTTGCAGCCT-3=] and MP11-1 [5=-GGCACGGCTCTTGTTG TAGTCG-3=]), and SensiMix SYBR LowROX qPCR master mix (Bioline). A 5-step standard curve of M. avium subsp. paratuberculosis genomic DNA was included (10-fold serial dilutions over the range of 10 to 0.001 pg/reaction). The cycling parameters were an initial denaturation step at 95°C for 8 1/2 min and 40 cycles of denaturation at 95°C for 15 s, annealing at 68°C for 30 s, and extension at 72°C for 1 min, with fluorescence acquisition at the end of the extension step. This was followed by a melt curve analysis from 55°C to 95°C. The threshold was set in each run based on the M. avium subsp. paratuberculosis genomic DNA standard curve using qPCR analysis software (MxPro; Stratagene) and then applied across the samples. Controls comprised an M. avium subsp. paratuberculosis positive control, a negative buffer control included in the extraction plate, and a negative qPCR no-template control. In addition, the amplification efficiency of the M. avium subsp. paratuberculosis genomic DNA standard curve including at least 4 of the 5 standards needed to be between 90 and 110% (calculated by using the inbuilt algorithm in the qPCR instrument software), with at least one replicate of standard 5 (0.001 pg) giving a positive amplification, for the qPCR experiment to be accepted. A positive result for a single replicate, i.e., one reaction in a single well in a qPCR plate, required a fluorescence curve with an appropriate exponential phase confirmed by visual examination and a correct dissociation peak (melting temperature [Tm]) in melt curve analysis. M. avium subsp. paratuberculosis culture and IS900 quantitative PCR method. Detection of live M. avium subsp. paratuberculosis cells was performed by using a modification of the M7H9C liquid culture method described previously (10, 27). Briefly, multiple 10-fold dilution series of M. avium subsp. paratuberculosis were prepared in PBS. An aliquot (25 ␮l) of these suspensions was then inoculated into M7H9C liquid culture medium with egg yolk (225 ␮l), and all samples were incubated at 37°C. An aliquot from each of the serial dilutions was collected at the time of culture inoculation (T0) and then stored at ⫺80°C, and subsequent aliquots from each serial dilution were taken weekly for 3 weeks and stored at ⫺80°C. The frozen aliquots were then thawed and subjected to DNA extraction and IS900 qPCR using a high-throughput 96-well-plate DNA extraction method, which included the magnetic bead-based DNA isolation method and IS900 qPCR, as previously validated and detailed above (27). This viability curve was repeated 20 times over 5 different experiments to ensure the reliability of the results. Four serial dilutions of heat-killed M. avium subsp. paratuberculosis cells were also subjected to the same culture and quantification protocols and were found to have no growth at 2 weeks (data not shown). The M. avium subsp. paratuberculosis growth rate was measured by subtraction of the total M. avium subsp. paratuberculosis DNA quantity (picograms) at T0 from the total M. avium subsp. paratuberculosis DNA quantity after 2 weeks of culture. This value was plotted against the initial concentration of M. avium subsp. paratuberculosis cells inoculated into culture (initial concentration) to obtain a standard growth curve. Effects of medium and incubation time on growth rate of M. avium subsp. paratuberculosis in culture. M. avium subsp. paratuberculosis suspensions (5 ⫻ 106 cells/ml to 50 cells/ml) were subjected to one of four treatments prior to culture. Bacteria were inoculated into M7H9C culture medium with (treatment 1) or without (treatment 2) the inclusion of egg yolk. M. avium subsp. paratuberculosis suspensions were also incubated at 37°C for 2 weeks prior to the generation of serial dilutions and inoculation (treatment 3) in M7H9C culture medium with egg yolk or were thawed on the day of inoculation (treatment 4). Serial dilution calculations were based on the starting concentration of M. avium subsp. paratuberculosis bacteria, and bacteria in samples that had been cultured for 2 weeks were not recounted prior to inoculation into medium. Culture of M. avium

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subsp. paratuberculosis and construction of the growth rate curve at 2 weeks for each treatment are described above. Detection of viable M. avium subsp. paratuberculosis cells in macrophages by culture/qPCR. Murine macrophages at 104 cells/well in cell culture medium were incubated overnight at 37°C in 5% CO2 to allow adherence to the wells. The cell culture medium was discarded, and macrophages were infected at an MOI of 1:1 or 0.1:1 with live M. avium subsp. paratuberculosis in cell culture medium without penicillin-streptomycin. Following incubation overnight at 37°C in 5% CO2, the medium was discarded, and each well was rinsed three times with sterile PBS. Macrophages were lysed with 50 ␮l of lysis buffer (1% Triton X-100 in sterile H2O) for 2 min, after which the lysate was split into two 25-␮l aliquots and inoculated into 225 ␮l of bacterial liquid culture medium with egg yolk. Culture of M. avium subsp. paratuberculosis and construction of the growth rate curve at 2 weeks for each treatment are described above. All samples were run in duplicate. Statistical analysis. One-way analysis of variance (ANOVA) was performed to assess the effects of the duration of incubation and the limit of detection of the resazurin assay and to examine the ability of the Live/ Dead BacLight viability dye to determine the viability of and to quantify M. avium subsp. paratuberculosis cells. Two-way ANOVA was used to evaluate if the resazurin assay could be used to determine the viability of M. avium subsp. paratuberculosis after macrophage infection and for the analysis of the results from the PMA dye experiment. Linear regression was used to examine the effect of the starting concentration on the growth rate in culture and to produce an equation that could be utilized to determine the starting concentration from the growth rate in an unknown sample. To analyze if killing or growth inhibition could be detected by the culture assay, one-way ANOVA was used. All statistical analyses were performed by using Genstat (16th edition; VSN International Ltd., Hemel Hemstead, United Kingdom), with a P value of ⬍0.05 being considered significant.

RESULTS

Effects of incubation time and limit of detection of the resazurin assay. To allow identification of the ideal incubation time needed for M. avium subsp. paratuberculosis to achieve maximal dye reduction in the shortest duration, resazurin viability dye was added to wells at different times throughout the 14-day incubation period. The length of incubation of M. avium subsp. paratuberculosis prior to the addition of the dye significantly influenced the rate of dye reduction, as determined by one-way ANOVA [F(6,7) ⫽ 121.37; P ⬍ 0.01 (values in parentheses are the degrees of freedom between groups and within a group, in that order)], and the final dye reduction percentage at the conclusion of the experiment [F(6,7) ⫽ 15.25; P ⫽ 0.001] (Fig. 1A). Treatments of live M. avium subsp. paratuberculosis cells to which dye was added in the initial week of the experiment reduced the dye at a much lower rate than did live cells that were dyed after at least a week of incubation (Fig. 1A). Likewise, live cells that were dyed early in the experiment had lower final percent reductions than did cells that were dyed later. Peak dye reduction was seen in cells that were incubated for 11 days prior to the addition of resazurin. Throughout the 14-day experiment, limited dye reduction was observed for the heatkilled M. avium subsp. paratuberculosis treatments, but it gradually increased as the incubation time increased. The limit of detection was assessed in two different ways. Initially, the lowest M. avium subsp. paratuberculosis concentration that can be quantified was assessed. Various starting concentrations of live M. avium subsp. paratuberculosis cells were incubated with the dye, and the percent dye reduction was monitored over an 11-day period. Similarly to the incubation time, there was a trend observed between the starting live M. avium subsp. paratu-

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berculosis cell concentration, the rate of dye reduction, and the final percent dye reduction (Fig. 1B). The two highest concentrations (3.43 ⫻ 109 cells/ml and 3.43 ⫻ 1010 cells/ml) had percent dye reduction values above 100% at the commencement of the assay as a result of the high M. avium subsp. paratuberculosis concentrations clouding the medium and impacting the OD values obtained (data not shown). Lower starting cell concentrations resulted in slower reduction of the dye over the course of the experiment and ultimately a lower final dye reduction value. The ability to differentiate between live and dead M. avium subsp. paratuberculosis cells was also assessed by examination of differences in final percent dye reductions of live, heat-killed, and a 50/50 mix of live and heat-killed M. avium subsp. paratuberculosis cells at 106 M. avium subsp. paratuberculosis cells/ml (Fig. 1C). The final percent dye reduction was significantly affected by the viability of the cells, as determined by one-way ANOVA [F(2,6) ⫽ 51.38; P ⬍ 0.001], where final dye reduction percentages in live and mixed live/dead samples were significantly higher than those in heat-killed M. avium subsp. paratuberculosis cells at the same concentration. The rate of dye reduction was also significantly different between live and heat-killed M. avium subsp. paratuberculosis cells, as determined by one-way ANOVA [F(2,6) ⫽ 15.26; P ⫽ 0.017]. The starting concentration of M. avium subsp. paratuberculosis was also examined and found to impact the time taken to observe a difference in dye reduction between live and heat-killed samples, although this could not be statistically analyzed (Fig. 1C). At a higher concentration (3.43 ⫻ 106 M. avium subsp. paratuberculosis cells/ml), there was a greater difference between live and heat-killed cells from day 7 of incubation onwards. At the lower concentration (3.43 M. avium subsp. paratuberculosis cells/ml), there was no observable difference between the live cells and the mix of live/heat-killed cells. Detection of viable M. avium subsp. paratuberculosis cells within macrophages by resazurin. Resazurin was evaluated for its ability to differentiate and quantify live and dead M. avium subsp. paratuberculosis cells in samples from in vitro cellular infection assays. A concentration of 106 M. avium subsp. paratuberculosis cells/well was chosen as the infectious dose, as this was the smallest amount that could be reliably quantified for the optimization of resazurin dye. Two-way ANOVA showed a significant interaction between viability before infection and whether lysates were incubated prior to the dye being added [F(1,4) ⫽ 66.62; P ⫽ 0.001]. Even at the high bacterial concentration chosen for infection, only lysates from macrophages that were infected with live M. avium subsp. paratuberculosis cells and then subsequently incubated for a week in medium prior to treatment with resazurin dye showed a detectable reduction of the dye (Fig. 1D). Live/Dead BacLight viability dye. The ability of Live/Dead BacLight viability dye to differentiate live, dead, and a 50/50 mix of live and dead M. avium subsp. paratuberculosis cells using flow cytometry to detect cellular fluorescence was evaluated. Dot plots of SYTO9 (green fluorescence indicating nuclear staining of all M. avium subsp. paratuberculosis cells) and propidium iodide (red fluorescence indicating staining of M. avium subsp. paratuberculosis cells with compromised cell walls) are shown in Fig. 2. Propidium iodide staining alone was not detected in any of the samples. SYTO9 staining was detected at higher percentages in samples containing heat-killed and, to a lesser degree, live M. avium subsp. paratuberculosis cells. Very few cells were double stained with SYTO9 and propidium iodide. In all suspensions, the

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D u r a tio n ( d a y s )

FIG 1 Optimization and validation of resazurin dye. (A) The incubation time for resazurin dye was optimized by dying suspensions of Mycobacterium avium subsp. paratuberculosis at different time points. Series names refer to the time points when resazurin was added to the sample. (B) Serial dilutions of live M. avium subsp. paratuberculosis. (C) Differentiation of live M. avium subsp. paratuberculosis and heat-killed M. avium subsp. paratuberculosis at high (3.43 ⫻ 106 cells/ml) and low (34 cells/ml) concentrations. (D) M. avium subsp. paratuberculosis (106 cells/well) was used to infect murine macrophages, and the viability of the lysate was determined at lysis or after a week of incubation. For all experiments, readings were taken on a plate reader immediately after dyeing and then at 24 h and other times depending on the specific experiment. Error bars represent the standard errors of the means (n ⫽ 2). HK refers to cells that were heat killed prior to the addition of resazurin.

majority of cells were unstained, as shown in Table 1. There were no significant differences in the percentages of live cells detected between the live, heat-killed, and the 50/50 mix of live and heatkilled samples of M. avium subsp. paratuberculosis by using this method, as determined by one-way ANOVA [F(2,3) ⫽ 2.07; P ⫽ 0.272]. Propidium monoazide viability dye for detection of viable M. avium subsp. paratuberculosis. PMA viability dye was evaluated for its ability to differentiate and quantify live and heat-killed M. avium subsp. paratuberculosis cells based on a previously optimized protocol (21). The graph in Fig. 3 shows that similar DNA quantities between the live and heat-killed treatment groups were obtained by using M. avium subsp. paratuberculosis IS900 qPCR. Two-way ANOVA showed no significant main effect for treatment [F(3,12) ⫽ 0.24; P ⫽ 0.866], no significant main effect for sample viability [F(2,12) ⫽ 0.07; P ⫽ 0.933], and no significant interaction of the two [F(6,12) ⫽ 0.23; P ⫽ 0.960] (Fig. 3). Interestingly, the amount of DNA recovered from samples that were treated with PMA and exposed to light was the same as the amount that was recovered from their untreated and unexposed counterparts.

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M. avium subsp. paratuberculosis culture and IS900 quantitative PCR method for detection of viable M. avium subsp. paratuberculosis. Initial experiments were conducted to evaluate the growth of M. avium subsp. paratuberculosis at different starting concentrations in liquid culture. An increase in the total DNA quantity was observed after 3 weeks of culture with starting bacterial concentrations ranging from 1 ⫻ 103 cells/ml up to 1 ⫻ 106 cells/ml (data not shown). An increase in the total M. avium subsp. paratuberculosis DNA quantity was observed in some samples after as early as 1 week of culture (5 ⫻ 103 cells/ml and 5 ⫻ 105 cells/ml), with all concentrations of ⬎5 ⫻ 103 cells/ml showing a distinct increase in total DNA by 2 weeks of culture (Fig. 4). Replication of the experiment 20 times showed that there was a linear relationship between the starting concentration and the growth rate at 2 weeks of culture, with a very low level of variability between replicates (Fig. 4). A simple linear regression was calculated to predict the growth rate at 2 weeks based on the starting concentration. A significant regression equation was found [F(1,111) ⫽ 227.52; P ⬍ 0.001], with an adjusted R2 value of 0.669. The predicted growth rate of M. avium subsp. paratuberculosis cultures is equal to (⫺1.298 ⫹ 0.7117y) pg of M. avium subsp. paratubercu-

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FIG 2 Flow cytometric dot plots of BacLight-stained M. avium subsp. paratuberculosis cells. Heat-killed (A), 50% heat-killed–50% live (B), and live (C) M. avium subsp. paratuberculosis cell suspensions (106 cells/ml) were stained with Live/Dead BacLight viability dye.

losis DNA, where “y” is the starting concentration of M. avium subsp. paratuberculosis measured as cells per milliliter. The growth rate of samples increased by a factor of 0.7117 for each 10-fold increase in the number of cells per milliliter of live M. avium subsp. paratuberculosis at the start of culture. The effects of egg yolk and preincubation of M. avium subsp. paratuberculosis on growth rate detection using M7H9C medium were examined in a pilot trial. The 2-week growth rate was higher in samples cultured in the presence of egg yolk (Fig. 5). The growth rate was found to be marginally higher in the frozenthawed samples than in those that were preincubated (37°C with 5% CO2) for 2 weeks prior to inoculation (Fig. 5). To determine the significance of these results, further replication of the experiment would be required. Detection of viable M. avium subsp. paratuberculosis cells within macrophages by culture/qPCR. A murine macrophage infection model was used to evaluate the liquid culture/qPCR method that had been developed to assess and quantify the viability of M. avium subsp. paratuberculosis. Growth of M. avium subsp. paratuberculosis was detected in all samples after 2 weeks of culture, indicating that after lysis of infected macrophages, live M. avium subsp. paratuberculosis was present. The concentration of live M. avium subsp. paratuberculosis cells recovered from infected macrophages was significantly lower than the concentration used to infect these macrophages, as determined by one-way ANOVA [F(3,40) ⫽ 10.07; P ⬍ 0.001]. The concentration of live M. avium

subsp. paratuberculosis recovered from infected macrophages was almost halved in samples at an MOI of 1:1 (Fig. 6). DISCUSSION

In this study, we have shown that the viability of M. avium subsp. paratuberculosis in macrophages can be quantified by using a modified, rapid liquid culture method coupled with qPCR to detect the growth rate in culture. This method was the most sensitive of the viability assays assessed and may be directly applicable to samples from an in vitro infection assay. The use of resazurin viability dye was the only commercial method that was able to reproducibly differentiate between live and heat-killed M. avium subsp. paratuberculosis suspensions over a range of concentrations. Unlike the other techniques examined, the resazurin dye assay uses metabolism as a marker of difference between live and dead cells, and this may be the reason for its superior performance over the other commercial kits. The utility of resazurin dye for the detection of intracellular viable M. avium subsp. paratuberculosis was also assessed. Without preincubation of the bacteria after macrophage lysis for a week in bacterial liquid culture medium, resazurin did not detect viable M. avium subsp. paratuberculosis. The inability of M. avium subsp. paratuberculosis to metabolize resazurin immediately after the in vitro infection assay may be due to exposure to stressors during bacterial internalization by macrophages that can transform M. avium subsp. paratuberculosis into a metabolically inactive form (28–30). Cul-

TABLE 1 M. avium subsp. paratuberculosis staining with Live/Dead BacLight viability dyea M. avium subsp. paratuberculosis treatment group 100% HK 50/50 live/HK mix 100% live Unstained control, 100% live Unstained control, 100% HK Unstained control, 50/50 live/HK mix a

% of cells SYTO9⫺ PI⫹

SYTO9⫹ PI⫹ (dead bacteria)

SYTO9⫺ PI⫺ (unstained)

SYTO9⫹ PI⫺ (live bacteria)

0 0 0 0 0 0

0 0.05 0 0 0 0

22.75 8.1 9.1 0.25 0 0.25

56.95 35.55 14 0.25 0 0.25

PI, propidium iodide; HK, heat killed.

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FIG 3 M. avium subsp. paratuberculosis DNA recovered from PMA-treated suspensions of M. avium subsp. paratuberculosis. Suspensions of live, heatkilled, and a 50/50 mix of live and heat-killed cells of M. avium subsp. paratuberculosis (106 cells/ml) underwent four treatments: M. avium subsp. paratuberculosis cells were stained with or without PMA and then exposed to a light source or left unexposed. Results are grouped by cell type (live, heat-killed, and a 50/50 mix of live and heat-killed cells) and show the DNA quantity (picograms) after extraction and PCR. Error bars show the standard errors of the means (n ⫽ 2).

ture of M. avium subsp. paratuberculosis after the infection assay alleviated the apparent metabolic suppression seen with resazurin. Therefore, although resazurin dye is a useful tool for determining the viability of pure cultures of M. avium subsp. paratuberculosis, the prolonged incubation required for obtaining results at lower M. avium subsp. paratuberculosis concentrations (⬍104 cells/ml) and the potential issues with metabolic inhibition of M. avium subsp. paratuberculosis in macrophages make it an unsuitable method for in vitro infection trials. The impermeable cell wall of M. avium subsp. paratuberculosis is most likely the reason why the Live/Dead BacLight viability dye and PMA/qPCR methods were unable to differentiate live and dead M. avium subsp. paratuberculosis cells. Presumably, although the M. avium subsp. paratuberculosis cells were not viable, the degree of membrane damage was not sufficient to allow dye penetration; this was seen previously with Escherichia coli, Mycobacterium bovis bacillus Calmette-Guerin (BCG), and Pseudomonas aeruginosa (17, 31, 32). The presence of an intact cytoplasmic membrane most likely accounts for the inability of propidium iodide and PMA to enter and stain the cells. In our hands, neither Live/Dead BacLight viability dye nor PMA in combination with qPCR is a suitable method to discriminate between live M. avium subsp. paratuberculosis and dead M. avium subsp. paratuberculosis in an in vitro cellular infection assay. By using the modified liquid culture method, the growth rate of M. avium subsp. paratuberculosis showed a strong linear trend when plotted against the initial starting quantity of live bacteria. This could be used to determine the initial concentration of live M. avium subsp. paratuberculosis cells in a sample based on the growth rate at 2 weeks. This approach could detect as few as 10 M. avium subsp. paratuberculosis cells in suspension, and there was limited variation between replicates at both high and low concentrations of M. avium subsp. paratuberculosis. Both criteria are essential for methods intended for use in in vitro infection assays.

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FIG 4 Growth of M. avium subsp. paratuberculosis at different starting concentrations. Tenfold dilution series of Mycobacterium avium subsp. paratuberculosis suspensions (106 to 10 cells/ml) were cultured for 2 weeks, and replicate samples were collected and frozen at the start and end of culture. DNA was extracted, and IS900 qPCR was used to quantify total M. avium subsp. paratuberculosis DNA. (A) The total DNA at the start of culture and at 2 weeks is shown in relation to the starting concentration of cells per milliliter. Linear regression trend lines are y ⫽ 1.164 ⫻ x ⫺ 6.811 for the start of culture and y ⫽ 1.273 ⫻ x ⫺ 5.148 for 2 weeks of culture. (B) The 2-week growth rate was calculated by subtracting the total DNA quantity at the initial inoculation from the total quantity at 2 weeks. This was plotted against the initial concentration of M. avium subsp. paratuberculosis cells inoculated into culture. Means ⫾ standard errors of the means are shown (n ⫽ 20, over 5 different experiments). The linear regression trend line equation is y ⫽ 0.5142 ⫻ x ⫺ 0.3203.

The sensitivity of this assay is attributed to the combined use of culture, which is the gold standard for detecting viable M. avium subsp. paratuberculosis cells, and the DNA extraction/qPCR detection method (27). The linearity of the growth curve confirms that the growth rate in relation to the starting inoculum concentration is relatively constant for M. avium subsp. paratuberculosis. As a result of the accuracy and sensitivity of the method developed, it is well suited to in vitro cellular infection assays where

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FIG 6 Two-week growth rates and starting viable numbers of M. avium subsp. FIG 5 Comparison of M. avium subsp. paratuberculosis 2-week growth rates under different treatment conditions. Duplicate 10-fold dilution series of Mycobacterium avium subsp. paratuberculosis were created and subjected to four treatments. Bacteria were inoculated into M7H9C culture medium with (treatment 1 [“Yolk”]) or without (treatment 2 [“no Yolk”]) the inclusion of egg yolk. M. avium subsp. paratuberculosis suspensions were also incubated at 37°C for 2 weeks prior to the generation of serial dilutions and inoculation (treatment 3 [“2WI”]) in M7H9C culture medium with egg yolk or were thawed on the day of inoculation (treatment 4 [“FT”]). Linear regression trend line equations are y ⫽ 0.5733 ⫻ x ⫺ 0.9133 for yolk, y ⫽ 0.7384 ⫻ x ⫺ 2.482 for no yolk, y ⫽ 0.5699 ⫻ x ⫺ 0.8456 for FT, and y ⫽ 0.6083 ⫻ x ⫺ 1.633 for 2WI.

comparisons may need to be made across multiple experiments and for high-throughput screening assays. The use of multiplication or growth in culture is the gold standard for determining M. avium subsp. paratuberculosis viability. It has been well documented that M. avium subsp. paratuberculosis growth in culture is related directly to inoculum size. Lambrecht and Carriere (33) showed that growth in culture, detected by radiometric means, is directly influenced by the inoculum size and can be used to accurately predict the inoculum size in an unknown sample. Data from further studies using the Bactec radiometric culture system were also in agreement (33–35). The same principles have been applied and validated in our assay, with growth indices being based on the changes in M. avium subsp. paratuberculosis DNA concentrations determined by qPCR rather than 14 CO2 release. The viability culture method developed is intended for use in a research setting, in particular for the identification or assessment of potential vaccine candidates or to examine specific facets of disease pathogenesis. This assay was validated and optimized with a specific strain of M. avium subsp. paratuberculosis and would need to be validated for use with other strains or sample types, as growth dynamics in culture may vary (35, 36). Furthermore, macrophages may not kill bacteria but may cause growth suppression/ inhibition, which may not be distinguishable in the assay (28). Growth suppression or dormancy has been seen with M. avium subsp. paratuberculosis in previous work using other sample types such as feces (37). Whittington et al. showed that key genes that are related to bacterial dormancy in other mycobacterial species are also present in M. avium subsp. paratuberculosis, which confirms the possibility that dormancy is indeed a factor to consider (37). However, as this method was designed to be used as a rapid screening tool and not to entirely replace vaccine efficacy trials,

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paratuberculosis cells in lysates from macrophage infection trials. Murine macrophages were infected with M. avium subsp. paratuberculosis suspensions at two MOIs (1:1 or 0.1:1 ratio of bacteria/macrophages), and lysates were cultured for 2 weeks to determine the growth rate. Culture of a serial dilution of live M. avium subsp. paratuberculosis cells allowed the creation of a viability curve relating the growth rate to the initial inoculum concentration of live cells. Estimation of the initial viability of samples from infected macrophages was done by using a linear regression trend line based on the viability curve equation y ⫽ 0.7087 ⫻ x ⫺ 1.287. For lysates, means ⫾ standard errors of the means are shown (n ⫽ 2). Growth curve means ⫾ standard errors of the means are shown (n ⫽ 20).

this issue would be addressed in animal trials or other assays that would further explore the results from viability screening. Preincubation prior to the start of culture and the inclusion of egg yolk in bacterial liquid culture medium were found to impact the 2-week growth rate of M. avium subsp. paratuberculosis. Egg yolk is commonly included in M. avium subsp. paratuberculosis culture media in Australia to ensure that both C (cattle) strains and S strains can be identified; however, its role during culture is not well characterized (10, 38). Initial experiments have shown that the inclusion of egg yolk in bacterial liquid culture medium increases the growth rate of M. avium subsp. paratuberculosis after 2 weeks. The inability to include egg yolk in the experiments undertaken with resazurin dye, due to its negative impact on optical density readings, could have been one of the factors contributing to the extended incubation times required to obtain readings that may relate to slowing of the metabolism. Interestingly, it was also found that the 2-week growth rate was increased in freshly thawed bacteria compared to preincubated bacteria. This was an unexpected result, and further investigation into the mechanisms responsible for this effect is needed. The use of an in vitro murine macrophage infection model showed the utility of the culture method to determine M. avium subsp. paratuberculosis viability after infection. The viability of M. avium subsp. paratuberculosis recovered from infected macrophages could be determined at two different MOIs. Interestingly, the MOI used had an effect on the killing ability of the macrophages, and this finding could be further explored. In conclusion, the adaptation of a validated liquid culture protocol for M. avium subsp. paratuberculosis coupled to qPCR for incremental analysis of the growth rate was found to provide the best method for rapid determination of M. avium subsp. paratuberculosis viability. This method is ideal for in vitro cellular infection trials, as it is relatively rapid, high throughput, and suffi-

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ciently sensitive to detect low intracellular concentrations of M. avium subsp. paratuberculosis. ACKNOWLEDGMENTS We thank Anna Waldron, Nicole Carter, and Ann-Michele Whittington for laboratory support. We have no conflict of interest to declare.

FUNDING INFORMATION This work was supported by Meat and Livestock Australia, by the Cattle Council of Australia, Sheep Meat Council of Australia, and Wool Producers Australia through Animal Health Australia (grant no. P.PSH.0576), and by an Australian postgraduate award and scholarship from Meat and Livestock Australia (grant no. B.STU.0292 to H.B.P.).

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growth indices from radiometric culture for quantification of sheep strains of Mycobacterium avium subsp. paratuberculosis. Appl Environ Microbiol 69:3510 –3516. http://dx.doi.org/10.1128/AEM.69.6.3510-3516 .2003. 36. Abendaño N, Sevilla I, Prieto JM, Garrido JM, Juste RA, Alonso-Hearn M. 2012. Quantification of Mycobacterium avium subsp. paratuberculosis strains representing distinct genotypes and isolated from domestic and wildlife animal species by use of an automatic liquid culture system. J Clin Microbiol 50:2609 –2617. http://dx.doi.org/10.1128/JCM.00441-12. 37. Whittington RJ, Marshall DJ, Nicholls PJ, Marsh IB, Reddacliff LA. 2004. Survival and dormancy of Mycobacterium avium subsp. paratuberculosis in the environment. Appl Environ Microbiol 70:2989 –3004. http: //dx.doi.org/10.1128/AEM.70.5.2989-3004.2004. 38. Whittington R, Marsh I, McAllister S. 1999. Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep. J Clin Microbiol 37: 1077–1083.

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