Efficacy of rifampicin combination therapy against MRSA prosthetic vascular graft infections in a rat model.

Staphylococcus aureus is a major cause of prosthetic vascular graft or endograft infections (VGEIs) and the optimal choice of antibiotics is unclear. We investigated various antibiotic choices as either monotherapy or combination therapy with rifampicin against MRSA in vitro and in vivo. Fosfomycin, daptomycin and vancomycin alone or in combination with rifampicin was used against MRSA USA300 FPR3757. Each antibiotic was tested for synergism or antagonism with rifampicin in vitro, and all antibiotic regimens were tested against actively growing bacteria in media and non-growing bacteria in buffer, both as planktonic cells and in biofilms. A rat model of VGEI was used to quantify the therapeutic efficacy of antibiotics in vivo by measuring bacterial load on grafts and in spleen, liver and kidneys. In vitro, rifampicin combinations did not reveal any synergism or antagonism in relation to growth inhibition. However, quantification of bactericidal activity revealed a strong antagonistic effect, both on biofilms and planktonic cells. This effect was only observed when treating active bacteria, as all antibiotics had little or no effect on inactive cells. Only daptomycin showed some biocidal activity against inactive cells. In vivo evaluation of therapy against VGEI contrasted the in vitro results. Rifampicin significantly increased the efficacy of both daptomycin and vancomycin. The combination of daptomycin and rifampicin was by far the most effective, curing 8 of 13 infected animals. Our study demonstrates that daptomycin in combination with rifampicin shows promising potential against VGEI caused by MRSA. Furthermore, we show how in vitro evaluation of antibiotic combinations in laboratory media does not predict their therapeutic effect against VGEI in vivo, presumably due to a difference in the metabolic state of the bacteria.

A high-throughput assay identifies molecules with antimicrobial activity against persister cells.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Activation of the Two-Component System LisRK Promotes Cell Adhesion and High Ampicillin Tolerance in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen which can survive in harsh environmental conditions. It responds to external stimuli through an array of two-component systems (TCS) that sense external cues. Several TCS, including LisRK, have been linked to Listeria's ability to grow at slightly elevated antibiotic levels. The aim of this study was to determine if the TCS LisRK is also involved in acquiring the high antibiotic tolerance that is characteristic of persister cells. LisRK activates a response that leads to remodeling of the cell envelope, and we therefore hypothesized that activation of LisRK could also increase in the cells' adhesiveness and initiate the first step in biofilm formation. We used a ΔlisR mutant to study antibiotic tolerance in the presence and absence of LisRK, and a GFP reporter strain to visualize the activation of LisRK in L. monocytogenes LO28 at a single-cell level. LisRK was activated in most cells in stationary phase cultures. Antimicrobial susceptibility tests showed that LisRK was required for the generation of ampicillin tolerance under these conditions. The wildtype strain tolerated exposure to ampicillin at 1,000 × inhibitory levels for 24 h, and the fraction of surviving cells was 20,000-fold higher in the wildtype strain compared to the ΔlisR mutant. The same protection was not offered to other antibiotics (vancomycin, gentamicin, tetracycline), and the mechanism for antibiotic tolerance is thus highly specific. Furthermore, quantification of bacterial attachment rates and attachment force also revealed that the absence of a functional LisRK rendered the cells less adhesive. Hence, LisRK TCS promotes multiple protective mechanisms simultaneously.