Bicyclomycin Activity against Multidrug-Resistant Gram-Negative Pathogens

Abstract

The growing prevalence of antimicrobial resistance poses a grave threat to human health. Among the most difficult bacterial infections to treat are those caused by multidrug-resistant (MDR) Gram-negative pathogens because few effective regimens are available. One approach to this problem is to find ways to increase the activity of old antimicrobials that had seen limited application. Bicyclomycin, an inhibitor of transcription termination, is an example in which the additional inhibition of protein or RNA synthesis increases bicyclomycin-mediated lethality against Gram-negative bacteria. To examine the potential of bicyclomycin for the treatment of MDR bacterial pathogens, we first measured the MICs of bicyclomycin and other widely used antimicrobials against more than 100 multidrug-resistant Gram-negative clinical isolates. Bicyclomycin showed good coverage of carbapenem-resistant Enterobacteriaceae (CRE) and Escherichia coli (MIC₅₀/MIC₉₀ of 25/50 μg/mL for both bacteria) and moderate activity against Klebsiella pneumoniae (MIC₅₀/MIC₉₀ of 50/200 μg/mL). Bicyclomycin also exhibited synergy (e.g., fractional inhibitory concentration [FIC] index of <0.5) with doxycycline for the inhibition of bacterial growth by a checkerboard assay. Although bicyclomycin exhibited very weak lethality by itself, it showed synthetic lethality with doxycycline against K. pneumoniae: the combination killed 100- to 1,000-fold more bacteria than either agent alone. In a murine model of infection, the bicyclomycin-doxycycline combination showed better efficacy than either agent alone, and the combination treatment largely eliminated histopathological manifestations caused by infection. Thus, bicyclomycin, which has largely been limited to the treatment of Gram-negative digestive tract infections, can now be considered for the combination treatment of systemic multidrug-resistant infections caused by CRE, E. coli, and K. pneumoniae. IMPORTANCE As antimicrobial resistance continues to increase, options for effectively treating multidrug-resistant (MDR) Gram-negative infections are declining. Finding ways to enhance the lethality of old agents that have unique molecular targets is important because developing new antimicrobials is becoming increasingly difficult. The present work showed that the old antibiotic bicyclomycin has good bacteriostatic activity against multiple clinical isolates of three significant types of MDR Gram-negative pathogens frequently encountered in hospital infections, as required for the consideration of expanded indications. More significant is the synergistic growth-inhibitory effect and the enhancement of killing by the additional presence of doxycycline since this increases the in vivo efficacy. These data demonstrate that bicyclomycin-containing regimens have potential as new treatment options for MDR Gram-negative infections such as those caused by CRE, E. coli, and K. pneumoniae.

Keywords: MIC distribution; bicyclomycin; lethal synergy; multidrug-resistant Gram-negative bacteria; murine model of infection.

FIG 1

Bicyclomycin MIC distributions of clinically drug-resistant bacterial strains. (A) CRE clinical isolates (n = 33). (B) E. coli ATCC 25922 and BW25113 reference strains along with 23 clinical isolates. (C) K. pneumoniae ATCC 13883 reference strain along with 22 clinical isolates. (D) A. baumannii ATCC 17978 reference strain along with 20 clinical isolates. (E) Enterobacter cloacae clinical isolates (n = 6). (F) Other drug-resistant clinical isolates (as listed on the x axis) tested (n = 6). The x axis lists strain numbers for each bacterial species tested, except for panel F, which lists bacterial species. Similar results were obtained in a replicate experiment.

FIG 2

Synergistic efficacy of the bicyclomycin-doxycycline combination against K. pneumoniae. (A) Synergistic lethality of bicyclomycin with doxycycline in vitro. Exponentially growing K. pneumoniae 41053 cells were treated with 2× MIC of doxycycline (DC) or the indicated concentrations of bicyclomycin (BCM) alone or with various combinations of bicyclomycin plus doxycycline for the indicated times. The MICs of bicyclomycin and doxycycline were 20 and 4 μg/mL, respectively. The experiments were done in triplicate; error bars indicate standard deviations (SD). (B) Synergistic lethality of bicyclomycin with doxycycline in a murine model of infection. Mice infected with K. pneumoniae strain 41053 were untreated or treated with 5% (wt/vol) Kolliphor HS 15 (no-treatment control), doxycycline alone, bicyclomycin benzoate (BCM-2) alone, or doxycycline plus bicyclomycin benzoate at the indicated concentrations once daily starting at 5 h postinfection. The bacterial burden in the lungs was assessed at the indicated times. Each treatment group included 8 mice, and each time point included samples taken from 4 mice. Error bars indicate means ± SD. The dotted lines indicate the detection limit (33 CFU/g). ***, P < 0.001. (C) A behavioral evaluation was performed for infected mice at the indicated times postinfection. The clinical scores were obtained by observing posture, paralysis, tremor, body weight, fur, and body temperature. Scores are as follows: −5 for death, −4 for dying, −3 for severe illness, −2 for sickness, −1 for mild symptoms, and 0 for asymptomatic. (D to H) Mitigation of lung histopathology by bicyclomycin, doxycycline, and their combination. Representative histological results (hematoxylin-eosin staining) for lungs collected at 5 days postinfection are shown for uninfected mice (D), infected untreated mice (5% [wt/vol] Kolliphor HS 15 solvent control) (E), and infected mice treated 200 mg/kg bicyclomycin benzoate (F), 50 mg/kg doxycycline (G), and 200 mg/kg bicyclomycin benzoate plus 50 mg/kg doxycycline (H). Similar results were obtained in 2 replicate experiments.