Multifrequency STD NMR Unveils the Interactions of Antibiotics With Burkholderia multivorans Biofilm Exopolysaccharide

Abstract

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

Keywords: Burkholderia multivorans; STD NMR; biofilms; exopolysaccharides; multifrequency STD NMR.

SCHEME 1

Repeating unit of the exopolysaccharide produced by B. multivorans C1576 (EpolC1576).

FIGURE 1

Detection and differential-binding epitope characterization of the interaction of ANS with EpolC1576. (A) STD NMR spectra of ANS (5 mM) in the presence of EpolC1576. (B) ANS-binding epitope obtained by selectively saturating EpolC1576 at +1.22 ppm and (C) by selectively saturating EpolC1576 at +3.22 ppm. A comparison of (B) and (C) indicates that the phenyl ring of ANS orients, on average, toward the CH₃ moiety of EpolC1576. Intermediate STD₀ (%) = 50–80% and strong STD₀ (%) = 80–100%.

FIGURE 2

STD NMR does not detect interactions between EpolC1576 and ceftazidime. (A) ¹H STD NMR spectra of ceftazidime in the presence of EpolC1576, suggesting that no interaction occurs. (B) Competition studies of ceftazidime with ANS. STD NMR spectra of ANS (5 mM) in the absence (a) and in the presence of increasing concentrations of ceftazidime in (b) (0.5 mM), (c) (2 mM), and (d) reference spectra.

FIGURE 3

EpolC1576 accelerates the degradation of ceftazidime. (A) ¹H STD NMR spectra at 0 and 48 h of ceftazidime (6 mM) in the presence EpolC1576. Only the degradation product interacts with EpolC1576. (B) ¹H NMR spectra of ceftazidime in the presence and absence of EpolC1576 after 120 h in 20 mM Tris pH 7.5 at 298 K. The degradation product peaks are clearly visible in the presence of EpolC1576 but only slightly noticeable in its absence. (C) DOSY NMR spectra revealing the low molecular weight degradation product compared to ceftazidime. The degradation product was assigned as pyridine. The peak labeled with “?” is another degradation product yet not characterized.

FIGURE 4

Detection and differential binding epitope characterization of the interaction of kanamycin with EpolC1576. (A) (top) reference and (bottom) STD NMR spectra demonstrating the highly sensitive detection of the interaction of kanamycin (2 mM) with EpolC1576 by selectively saturating EpolC1576 at −1.0 ppm, (B) binding epitope mapping of kanamycin by selectively saturating EpolC1576 at −1.0 ppm, and (C) binding epitope mapping by selectively saturating EpolC1576 at +1.2 ppm. The resonances labeled with the asterisk (*) belong to the EpolC1576. Weak STD₀ (%) = 0–49%, intermediate STD₀ (%) = 50–80%, and strong STD₀ (%) = 81–100%.

FIGURE 5

Competition study of kanamycin with ANS. STD NMR spectra of ANS (5 mM) in the absence a) and in the presence of increasing concentrations in b (0.5 mM) and c (2 mM) of kanamycin. The presence of the peaks of kanamycin shows that the antibiotic is interacting with EpolC1576. However, reductions, on average, of 4 and 20% of STD factors for ANS were observed for samples in b and c (see the text) confirming its displacement. The resonances labeled with the (*) symbol belong to the ANS molecule, while those labeled with the (^#) symbol belong to the kanamycin molecule.