Membrane-active macromolecules resensitize NDM-1 gram-negative clinical isolates to tetracycline antibiotics

Abstract

Gram-negative 'superbugs' such as New Delhi metallo-beta-lactamase-1 (blaNDM-1) producing pathogens have become world's major public health threats. Development of molecular strategies that can rehabilitate the 'old antibiotics' and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards blaNDM-1 Klebsiella pneumonia and blaNDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards blaNDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.

Fig 1. (A) Structures of the membrane active molecules (MAMs) and (B) Schematic representation of agarose gel (2%) showing the 475 bp amplified product by conventional polymerase chain reaction.

Lane 1, 100 bp DNA ladder; Lane 2, positive control- NDM-1 producing K. pneumoniae (ATCC-BAA-2146); Lane 3, negative control- E. coli (ATCC-25922); Lane 4, E. coli R3336 and Lane 5, K. pneumoniae R3934 confirm the bla _NDM-1 gene; Lane 6, multi-drug resistant (MDR) K. pneumoniae R3421 which was negative for bla _NDM-1 gene.

Fig 2. Synergy, resistance development and in-vivo antibacterial efficacy of MAMs and tetracycline antibiotics against bla _NDM-1 E. coli (R3336).

(A) The combination of MAM1 and minocycline (50 μg mL^-1 + 6.3 μg mL^-1) showed synergistic bactericidal activity whereas MAM1 (50 μg mL^-1) alone and minocycline alone (6.3 μg mL^-1) were devoid of antibacterial activity (star represents < 50 CFU mL^-1, the detection limit of the experiment). (B) Bacteria developed resistance to minocycline alone with increase in MIC up to 400 μg mL^-1 whereas it did not develop resistance to minocycline (6.2 μg mL^-1) in presence of MAM1 (50 μg mL^-1) up to 30 days. Bacteria did not readily develop resistance to MAM1 whereas it developed resistance to colistin with an increase in MIC up to 1000 μg mL^-1. (C) In-vivo antibacterial efficacy of MAM1 and doxycycline in mice models. The bacterial burden in the thighs of the mice (4 mice in each group) were determined and expressed as mean ± S.E.M (standard error of mean). P value was calculated using the unpaired Student’s t test (2 tailed 2 samples assuming equal variances) and a value P < 0.05 was considered significant. *P = 0.03 between the saline treated and combination treated samples (Inset shows the experimental design).

Fig 3. Uptake of tetracycline and membrane-active properties of polymers against bla _NDM-1 E. coli (R3336).

(A) Uptake of tetracycline by increase in its fluorescence in presence of the MAM1 and MAM2 ((both at 20 μg mL^-1). Tetracycline was used at 100 μg mL^-1. (B) Dissipation of membrane potential by increase in the fluorescence of DiSC₃(5) with the treatment of MAM1 and MAM2 (both at 100 μg mL^-1). (C) Uptake of tetracycline in presence of MAM1, MAM2 (both at 20 μg mL^-1) and colistin (30 μg mL^-1) as well as in the presence of nigericin (at 10 μg mL^-1). (D) Membrane permeabilization by increase in the fluorescence of propidium iodide (PI) with the treatment of MAM1 and MAM2 (both at 100 μg mL^-1).