Generation of ramoplanin-resistant Staphylococcus aureus

Abstract

Ramoplanin is a lipoglycodepsipeptide antimicrobial active against clinically important Gram-positive bacteria including methicillin-resistant Staphylococcus aureus. To proactively examine ramoplanin resistance, we subjected S. aureus NCTC 8325-4 to serial passage in the presence of increasing concentrations of ramoplanin, generating the markedly resistant strain RRSA16. Susceptibility testing of RRSA16 revealed the unanticipated acquisition of cross-resistance to vancomycin and nisin. RRSA16 displayed phenotypes, including a thickened cell wall and reduced susceptibility to Triton X-100-induced autolysis, which are associated with vancomycin intermediate-resistant S. aureus strains. Passage of RRSA16 for 18 days in a drug-free medium yielded strain R16-18d with restored antibiotic susceptibility. The RRSA16 isolate may be used to identify the genetic and biochemical basis for ramoplanin resistance and to further our understanding of the evolution of antibiotic cross-resistance mechanisms in S. aureus.

Figure 1. Effect of ramoplanin on S. aureus viability is delayed and reduced in RRSA16

S. aureus was grown to mid exponential phase (OD₆₂₀ ≈ 0.4) and ramoplanin was added to the final concentrations indicated. No ramoplanin was added to control cultures. The horizontal dotted line indicates the viable count limit of detection of 300 CFU/mL. (A) Effect of ramoplanin on S. aureus NCTC 8325-4 viability. Note the drop in the viable counts of NCTC 8325-4 cultures following the addition of ramoplanin at all concentrations shown at 0.5 hr following antibiotic addition. (B) Effect of ramoplanin on S. aureus RRSA16 viability. Note that the viable counts of RRSA16 cultures were increased at 0.5 hr following ramoplanin addition at all antibiotic concentrations tested. Higher concentrations of ramoplanin and longer times were required to reduce RRSA16 viable counts three orders of magnitude.

Figure 2. Bacteriolytic effect of ramoplanin not observed with RRSA16

S. aureus was grown to mid exponential phase (OD₆₂₀ ≈ 0.4) and ramoplanin was added to the indicated final concentration. No ramoplanin was added to control cultures. The horizontal dotted line indicates the spectrophotometer sensitivity limit of 0.1 AU. (A) Ramoplanin induces lysis of NCTC 8325-4. Optical density recorded at 620 nm of NCTC 8325-4 cultures when treated with all concentrations shown (B) Graph of the optical density at 620 nm of S. aureus RRSA16 as a function of time following addition of ramoplanin. Optical density does not drop rapidly following the addition of ramoplanin to RRSA16 cultures.

Figure 3. Thickened cell wall and reduced cell size of RRSA16

Representative transmission electron microscopy images at ×35,000 of NCTC 8325-4 and RRSA16 prepared as described in the Materials and Methods section. The average cell diameter and cell wall thickness (expressed as mean ± standard deviation of the mean) for each strain are indicated. A P-value of 5.7 × 10^-20 was calculated using a two-tailed Student’s t test comparing the cell diameter measurements of NCTC 8325-4 and RRSA16 indicating that the cell diameters were significantly different. A P-value of 1.4 × 10^-15 was calculated using a two-tailed Student’s t test comparing the cell wall thickness measurements of NCTC 8325-4 and RRSA16 indicating that the cell wall thicknesses were significantly different.

Figure 4. Reduced susceptibility of RRSA16 to Triton X-100 induced lysis

Cultures of S. aureus growing in exponential phase (OD₆₂₀ ≈ 0.6) were resuspended in buffer containing 0.05% Triton X-100 as described in the Materials and Methods section. The decrease in OD₆₂₀ as a function of time is shown for each strain. Note the slower and less complete lysis of RRSA16 in comparison to its progenitor strain NCTC 8325-4. Two-tailed Student’s t tests were performed to determine if the values observed for RRSA16 were significantly different from the values for NCTC 8325-4 and R16-18d at each time point except 0 hours. Error bars indicate the standard deviation of the mean for each value. Values observed for RRSA16 that are labeled with a double asterisk (**) were determined to be significantly different from both NCTC 8325-4 and R16-18d since the calculated P-values for both Student’s t tests at that point were < 0.05. Values observed for RRSA16 that are labeled with a single asterisk (*) were determined to be significantly different from only NCTC 8325-4 since the P-values returned from the Student’s t tests performed comparing RRSA16 and NCTC 8325-4 values were < 0.05 but the P-values returned from the Student’s t tests performed comparing RRSA16 and R16-18d values were > 0.05.