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

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.