Mycoplasma spp. are facultative pathogens that contribute to the pathogenesis of multiple bovine diseases including the bovine respiratory disease complex and that have been shown to form biofilms. Biofilm formation is associated with increased antibiotic resistance in many organisms, but accurate determination of antimicrobial susceptibility in biofilms is challenging. In Mycoplasma spp., antimicrobial susceptibility is routinely determined using metabolic pH-dependent color change. However, biofilm formation generally leads to reduced metabolism, making interpretation of metabolic readouts difficult. Therefore, we developed and optimized a new flow-cytometry-based method for antimicrobial susceptibility testing in biofilm-forming Mycoplasma, termed the live-dead antimicrobial susceptibility test (LD-AST). The LD-AST measures the proportion of dead bacteria upon exposure to antibiotics, works robustly with both planktonic and biofilm cultures, and enables the determination of the minimum bactericidal concentration (MBC) for a given antibiotic. We used two strains of M. bovis (Donetta PG45 and Madison) and two clinical bovine Mycoplasma spp. isolates (MVDL1 & MVDL2) to determine the impact of biofilm growth on antimicrobial susceptibility for gentamicin, enrofloxacin, or tetracycline. All Mycoplasma strains were susceptible to all antibiotics when cultured as planktonic cells, with MBCs in the expected range. However, three out of four strains (Donetta PG45, MVDL1 and MVDL2) were completely resistant to all three antibiotics when newly adhered biofilms were analyzed, whereas M. bovis Madison gave variable results. For mature biofilms that were cultured for 4- 5 days before antibiotic exposure, results also were variable, with some strains showing an increased resistance with certain antibiotics and a decreased resistance with others. Overall, these results are consistent with earlier reports that biofilms can exhibit increased antimicrobial resistance.

README: Data from: Innovative methodology for antimicrobial susceptibility determination in M. bovis biofilms

https://doi.org/10.5061/dryad.m905qfv8m

Description of the data and file structure

2_4_Confluence_and_Structure_Size.csv was generated using the methods described in section 2.4

Isolate: Mycoplasma bovis or Mycoplasma bovoculi isolate name.
Percent Area: The confluence of the biofilm on the field of view expressed as percent area.
Day: The time point post inoculation expressed in days.
Well: The well of the 96-well plate that the reading corresponds to.
plate.well: The concatenated individual IDs for both the well within individual plates.
measurement: The measurement in micrometers of the largest observed structure.

The data points where there are NAs for measurement are due to the absence of large structures observed at the time points.

2_5_pH_Experiment_Compiled_Data.csv was generated using the methods described in section 2.5 and shown in Figure 4.

well: The well for the 96-well plate that the reading corresponds to.
plate: The 96-well plate ID that the readings are from.
reading: The time-point (Baseline or Endpoint), that the readings are from.
biofilm.percent: The confluence of the biofilm on the field of view expressed as percent area.
strain: Mycoplasma bovis or Mycoplasma bovoculi isolate name.
antibiotic.mic: The concentration of gentamicin at each of the observations.
absorbance.620nm: The absorbance observed for 620 nm.
absorbance.700nm: The absorbance observed for 700 nm.
Ratio 620.700: The ratio of the absorbances at 620nm and 700nm.
Estimated pH: The estimated pH of the observation based on the absorbances.
contamination: Observed contamination in the well (0 for no contamination, 1 for contamination).
debris: Observed noncellular debris observed in the well (0 for no debris, 1 for debris).

The NAs observed at the baseline readings are due to this time point only being observed for biofilm formation rather than pH.

2_7-8_Disruption_Imaging_Cytometry_*.csv files were generated using the methods described in section 2.7 and 2.8 and shown in Figure 5

Object Number: The individual observation number.
Area_M02: This corresponds to the calculated area in square micrometers for the green fluorescence channel.
Intensity_M02: This corresponds to the fluorescent intensity of the green fluorescence channel (Relative fluorescent intensity).
Width_M02: This corresponds to the particle width in micrometers calculated for the green fluorescence channel.

2_9_Small_Particle_Analysis.csv was generated using the methods described in section 2.9 and shown in Figure 6

FileName: This corresponds to the original data file that the observations are from.
Treatment: This corresponds to the biofilm disruption treatment
Replicate: This corresponds to the replicated sample number.
FSC: This is the fluorescent intensity for the forward scatter channel of the flow cytometer (Relative fluorescent intensity).

2_11_Disruption_Survival.csv was generated using the methods described in section 2.11 and shown in Figure 8A.

sample: This corresponds to the individualized sample ID
group: This corresponds to the treatment group.
live.percent: This corresponds to the percentage of live cells in the sample.
dead.percent: This corresponds to the percentage of dead cells in the sample.
method: This corresponds to the method that the live and dead cell percentages were collected with.

2_12_Cell_Adhesion.csv was generated using the methods described in section 2.12 and shown in Figure 8B.

Time: This corresponds to the time in hours that the cell counts were collected at, as well as the planktonic inoculum used to start the assay.
Count: This is the cell concentration in cells/mL observed.

2_14_LD-AST_Combined_Data_Mbovis.csv was generated using the methods described in section 2.14 and shown in Table 3.

Well ID: This is the well number of the 96-well plate for each of the observations.
Organism: Mycoplasma bovis or Mycoplasma bovoculi isolate name.
Group: This corresponds to the organism state (Planktonic, newly adhered biofilm, mature biofilm).
Sample Name: This is the output of the sample name from the flow cytometer.
R2_Count: This is the count of live cells observed.
R3_Count: This is the count of the dead cells observed.
live percent: This is the calculated percentage of live cells observed.
dead percent: This is the calculated percentage of dead cells observed.
total count: This is the count of total cells (live and dead) observed.
antibiotic: This is the antibiotic that the organism was exposed to for this observation.
concentration: This is the antibiotic concentration in micrograms/mL for the antibiotic at this observation.
antibiotic length: This is the antibiotic exposure length in days.
incubation length: This is the length of incubation of the organism on the plates in hours or days (h or d).
BR: This is the biological replicate name for the individual observation
Notes: This is any additional notes for the observation.

Code/Software

The code and software used for this analysis is outlined in the associated publication section 2.15

Data from: Innovative methodology for antimicrobial susceptibility determination in Mycoplasma biofilms

Data files

Abstract