Synergistic killing of NDM-producing MDR Klebsiella pneumoniae by two 'old' antibiotics-polymyxin B and chloramphenicol

Abstract

Objectives: Combination therapy is an important option in the fight against Gram-negative 'superbugs'. This study systematically investigated bacterial killing and the emergence of polymyxin resistance with polymyxin B and chloramphenicol combinations used against New Delhi metallo-β-lactamase (NDM)-producing MDR Klebsiella pneumoniae.

Methods: Four NDM-producing K. pneumoniae strains were employed. The presence of genes conferring resistance to chloramphenicol was examined by PCR. Time-kill studies (inocula ∼10(6) cfu/mL) were conducted using various clinically achievable concentrations of each antibiotic (range: polymyxin B, 0.5-2 mg/L; chloramphenicol, 4-32 mg/L), with real-time population analysis profiles documented at baseline and 24 h. The microbiological response was examined using the log change method and pharmacodynamic modelling in conjunction with scanning electron microscopy (SEM).

Results: Multiple genes coding for efflux pumps involved in chloramphenicol resistance were present in all strains. Polymyxin B monotherapy at all concentrations produced rapid bacterial killing followed by rapid regrowth with the emergence of polymyxin resistance; chloramphenicol monotherapy was largely ineffective. Combination therapy significantly delayed regrowth, with synergy observed in 25 out of 28 cases at both 6 and 24 h; at 24 h, no viable bacterial cells were detected in 15 out of 28 cases with various combinations across all strains. No polymyxin-resistant bacteria were detected with combination therapy. These results were supported by pharmacodynamic modelling. SEM revealed significant morphological changes following treatment with polymyxin B both alone and in combination.

Conclusions: The combination of polymyxin B and chloramphenicol used against NDM-producing MDR K. pneumoniae substantially enhanced bacterial killing and suppressed the emergence of polymyxin resistance.

Keywords: K. pneumoniae; New Delhi metallo-β-lactamase; synergy.

Figure 1.

Baseline polymyxin B PAPs of the reference strain and all clinical isolates at an initial inoculum of ∼10⁹ cfu/mL. The y-axis starts from the limit of detection.

Figure 2.

Left: Time–kill curves with various clinically relevant concentrations of polymyxin B and chloramphenicol alone and in combination with an inoculum of ∼10⁶ cfu/mL. Right: PAPs after 24 h of exposure to polymyxin B monotherapy, polymyxin B/chloramphenicol combination therapy or neither antibiotic (control). (a) ATCC BAA-2146 (chloramphenicol resistant, MDR). (b) Isolate 1 (chloramphenicol susceptible, MDR). (c) Isolate S01 (chloramphenicol resistant, MDR). (d) Isolate 129 (chloramphenicol resistant, MDR). The y-axis starts from the limit of detection. For isolates 1 and 129, only three time–kill curves are seen in the PAPs as there were no viable counts above the limit of detection for the two combination regimens. Symbols not seen at certain times are below the limit of detection. PMB, polymyxin B; CHL, chloramphenicol.

Figure 2.

Left: Time–kill curves with various clinically relevant concentrations of polymyxin B and chloramphenicol alone and in combination with an inoculum of ∼10⁶ cfu/mL. Right: PAPs after 24 h of exposure to polymyxin B monotherapy, polymyxin B/chloramphenicol combination therapy or neither antibiotic (control). (a) ATCC BAA-2146 (chloramphenicol resistant, MDR). (b) Isolate 1 (chloramphenicol susceptible, MDR). (c) Isolate S01 (chloramphenicol resistant, MDR). (d) Isolate 129 (chloramphenicol resistant, MDR). The y-axis starts from the limit of detection. For isolates 1 and 129, only three time–kill curves are seen in the PAPs as there were no viable counts above the limit of detection for the two combination regimens. Symbols not seen at certain times are below the limit of detection. PMB, polymyxin B; CHL, chloramphenicol.

Figure 3.

SEM images of K. pneumoniae 1 in the absence of drug therapy (control) or in the presence of polymyxin B at 2 mg/L, chloramphenicol at 32 mg/L or the combination of these. Scale bar = 500 nm.