Adjunctive Use of Phage Sb-1 in Antibiotics Enhances Inhibitory Biofilm Growth Activity versus Rifampin-Resistant Staphylococcus aureus Strains

Abstract

Effective antimicrobials are crucial for managing Staphylococcus aureus implant-associated bone infections (IABIs), particularly for infections due to rifampin-resistant S. aureus (RRSA). Failure to remove the implant results in persistent infection; thus, prolonged suppressive antibiotic therapy may be a reasonable alternative. However, a high incidence of adverse events can necessitate the discontinuation of therapy. In this scenario, commercial Staphylococcal bacteriophage Sb-1 combined with antibiotics is an option, showing a promising synergistic activity to facilitate the treatment of biofilm infections. Therefore, we evaluated the efficacy of the inhibitory activity of five antibiotics (doxycycline, levofloxacin, clindamycin, linezolid, and rifampin) alone or combined with phage Sb-1 (10⁶ PFU/mL) in a simultaneous and staggered manner, to combat five clinical RRSA strains and the laboratory strain MRSA ATCC 43300 in 72 h by isothermal microcalorimetry. The synergistic effects were observed when phage Sb-1 (10⁶ PFU/mL) combined with antibiotics had at least 2 log-reduction lower concentrations, represented by a fractional biofilm inhibitory concentration (FBIC) of <0.25. Among the antibiotics that we tested, the synergistic effect of all six strains was achieved in phage/doxycycline and phage/linezolid combinations in a staggered manner, whereas a distinctly noticeable improvement in inhibitory activity was observed in the phage/doxycycline combination with a low concentration of doxycycline. Moreover, phage/levofloxacin and phage/clindamycin combinations also showed a synergistic inhibitory effect against five strains and four strains, respectively. Interestingly, the synergistic inhibitory activity was also observed in the doxycycline-resistant and levofloxacin-resistant profile strains. However, no inhibitory activity was observed for all of the combinations in a simultaneous manner, as well as for the phage/rifampin combination in a staggered manner. These results have implications for alternative, combined, and prolonged suppressive antimicrobial treatment approaches.

Keywords: antibiotic-phage combination; biofilm infection; rifampin-resistant Staphylococcus aureus; suppression therapy; synergism.

Figure 1

Microcalorimetry analysis of MRSA ATCC 43300 and five rifampin-resistance S. aureus biofilms inhibited by single antibiotics at different concentrations. Each curve shows the heat produced by viable bacteria presented in the biofilm after 24 h of antibiotic application. Numbers represent concentrations of the antibiotics (in μg/mL) doxycycline (DOX), levofloxacin (LEV), linezolid (LNZ), clindamycin (CLI), and rifampin (RIF). Circled values represent the MBIC, defined as the lowest antimicrobial concentration leading to biofilm metabolism inhibition in the absence of antimicrobials for 72 h. GC, growth control; NC, negative control. Data of a representative experiment are reported.

Figure 2

Microcalorimetry analysis of MRSA ATCC 43300 and five rifampin-resistance S. aureus biofilms inhibited by Sb-1. Each curve shows the heat produced by viable bacteria presented in the biofilm after 24 h of exposure to Sb-1. Numbers represent concentrations of Sb-1 (in PFU/mL). GC, growth control; NC, negative control. Data of a representative experiment are reported.

Figure 3

Microcalorimetry analysis of the reference strain MRSA ATCC 43300 and five rifampin-resistant S. aureus strains treated with phage and sub-inhibitory concentrations of antibiotic in a simultaneous (left column) or staggered (right column) manner. Each curve shows the heat produced by viable bacteria presented in the biofilm after 24 h of phage–antibiotic treatment. Numbers represent concentrations of antibiotic (in μg/mL) of doxycycline (DOX), levofloxacin (LEV), linezolid (LNZ), clindamycin (CLI), and rifampin (RIF). Circled values represent the MBIC, defined as the lowest antimicrobial concentration leading to the biofilm metabolism inhibition in the absence of antimicrobials for 72 h. GC, growth control; NC, negative control. Data of a representative experiment are reported.