A novel method to determine antibiotic sensitivity in Bdellovibrio bacteriovorus reveals a DHFR-dependent natural trimethoprim resistance.

Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).

Antimicrobial Sensitivity Assay for Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, an obligate predatory bacterium [i.e., bacteria that kill and feed on other bacteria (prey)], has the potential to be used as a probiotic for the disinfection of surfaces or for the treatment of bacterial infections. One option is to use this organism in combination with antimicrobials to potentiate the effectiveness of treatments. In order to make this approach feasible more has to be known about the ability of B. bacteriovorus to resist antibiotics itself. Standard assays to determine the minimum inhibitory concentration (MIC) are not suitable for B. bacteriovorus, since the small size of this bacterium (0.25-0.35 by 0.5-2 µm) prevents scattering at OD600. Since these predatory bacteria require larger prey bacteria for growth (e.g., E. coli dimensions are 1 by 1-2 µm), the basis for the antimicrobial sensitivity assay described here is the reduction of the OD600 caused by prey lysis during growth. Previous studies on predatory bacteria resistance to antimicrobials employed methods that did not allow a direct comparison of antimicrobial resistance levels to those of other bacterial species. Here, we describe a procedure to determine B. bacteriovorus sensitivity to antimicrobials which can be compared to a reference organism tested as close as possible to the same experimental conditions. Briefly, minimal inhibitory concentration (MIC) values of B. bacteriovorus are determined by measuring the reduction in absorbance at 600 nm of mixed predator/prey cultures in presence and absence of different antimicrobial concentrations. Of note, this method can be modified to obtain antimicrobial MIC values of other predatory bacteria, using different conditions, prey bacteria and/or antimicrobials.