Chloramphenicol is a substrate for a novel nitroreductase pathway in Haemophilus influenzae

Abstract

The p-nitroaromatic antibiotic chloramphenicol has been used extensively to treat life-threatening infections due to Haemophilus influenzae and Neisseria meningitidis; its mechanism of action is the inhibition of protein synthesis. We found that during incubation with H. influenzae cells and lysates, chloramphenicol is converted to a 4-aminophenyl allylic alcohol that lacks antibacterial activity. The allylic alcohol moiety undergoes facile re-addition of water to restore the 1,3-diol, as well as further dehydration driven by the aromatic amine to form the iminoquinone. Several Neisseria species and most chloramphenicol-susceptible Haemophilus species, but not Escherichia coli or other gram-negative or gram-positive bacteria we examined, were also found to metabolize chloramphenicol. The products of chloramphenicol metabolism by species other than H. influenzae have not yet been characterized. The strains reducing the antibiotic were chloramphenicol susceptible, indicating that the pathway does not appear to mediate chloramphenicol resistance. The role of this novel nitroreductase pathway in the physiology of H. influenzae and Neisseria species is unknown. Further understanding of the H. influenzae chloramphenicol reduction pathway will contribute to our knowledge of the diversity of prokaryotic nitroreductase mechanisms.

FIG. 1.

(A) Cm. C₁₁H₁₂Cl₂N₂O₅, M_r = 322 Da. (B) Proposed structure of the proposed metabolites, showing the allylic alcohol (C₁₁H₁₂Cl₂N₂O₂, M_r = 274 Da) gaining (C₁₁H₁₄N₂O₃, M_r = 292 Da) and losing (C₁₁H₁₀Cl₂N₂O, M_r = 256 Da) one molecule of water. Experimental evidence supporting the metabolites’ structures is described in Results.

FIG. 2.

H. influenzae Rd KW20 (Hi) or E. coli 25922 (Ec) were incubated with Cm for 1 h at either 37 or 0°C in sBHI. Ethyl acetate extracts of the culture supernatants were subjected to TLC.

FIG. 3.

H. influenzae Rd KW20 was incubated with Cm at 37°C for 120 min, and aliquots were taken, acidified, and extracted with ethyl acetate at the times indicated (T0 = zero time, T5 = 5 min, etc.). The absorbance between 250 and 350 nm was determined for each extract with the peak absorbance indicated.

FIG. 4.

Production of primary amine-containing material during incubation of H. influenzae Rd KW20 with Cm. Bacteria (5 × 10⁸ CFU/ml) were incubated at 37°C in defined medium with Cm at 100 μg/ml (or media alone), and aliquots were taken at the times indicated. Bacterial cell suspensions and culture supernatants were assayed with the Bratton-Marshall reaction. Symbols: □, cell suspension without Cm; ▵, culture supernatant without Cm; ▪, cell suspension incubated with Cm; ▴, culture supernatant after incubation with Cm.

FIG. 5.

Proposed pathway for the metabolic reduction of chloramphenicol. The nitroso intermediate and the final products were identified by MS analysis. The 1,3-diol was the predominant product observed in the ¹H NMR analysis.

FIG. 6.

Product ion spectra of Cm (A) and its metabolite (B) from the small-scale fermentation showing their [M + H]⁺ precursor ion clusters and proposed structures of major fragment ions (shown as the corresponding neutral species for clarity).

FIG. 7.

ESI spectrum of the ethyl acetate extract of the large-scale production showing the presence of the [M + H]⁺ for the iminoquinone (m/z 257), the allylic alcohol (m/z 275), and the [M + Na]⁺ for the 1,3-diol (m/z 315). For clarity, proposed structures are shown as the corresponding neutral species.

FIG. 8.

One-dimensional ¹H NMR analysis of the 1,3-diol metabolite. The 500 MHz spectrum was obtained in CDCl₃ at a concentration of approximately 6 mM. Peak assignments are as shown; small unassigned peaks are from impurities in the media. See Results for details.