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. 2020 Jul 17;25(14):3255.
doi: 10.3390/molecules25143255.

In Vitro Evaluation of Antimicrobial Peptide Tridecaptin M in Combination with Other Antibiotics against Multidrug Resistant Acinetobacter baumannii

Affiliations

In Vitro Evaluation of Antimicrobial Peptide Tridecaptin M in Combination with Other Antibiotics against Multidrug Resistant Acinetobacter baumannii

Manoj Jangra et al. Molecules. .

Abstract

The rapid emergence of antimicrobial resistance in Acinetobacter baumannii coupled with the dried pipeline of novel treatments has driven the search for new therapeutic modalities. Gram-negative bacteria have an extra outer membrane that serves as a permeability barrier for various hydrophobic and/or large compounds. One of the popular approaches to tackle this penetration barrier is use of potentiators or adjuvants in combination with traditional antibiotics. This study reports the in vitro potential of an antimicrobial peptide tridecaptin M in combination with other antibiotics against different strains of A. baumannii. Tridecaptin M sensitized the bacteria to rifampicin, vancomycin, and ceftazidime. Further, we observed that a tridecaptin M and rifampicin combination killed the bacteria completely in 4 h in an ex vivo blood infection model and was superior to rifampicin monotherapy. The study also found that concomitant administration of both compounds is not necessary to achieve the antimicrobial effect. Bacteria pre-treated with tridecaptin M (for 2-4 h) followed by exposure to rifampicin showed similar killing as obtained for combined treatment. Additionally, this combination hampered the survival of persister development in comparison to rifampicin alone. These findings encourage the future investigation of this combination to treat severe infections caused by extremely drug-resistant A. baumannii.

Keywords: Acinetobacter baumannii; Gram-negative bacteria; Tridecaptin M; antibiotic-resistance; combination therapy; persisters.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures and amino acid sequences of (a) tridecaptin M and (b) unacylated tridecaptin A1. The lipid moiety in natural tridecaptin A1 is 6-methyl-3-hydroxy octanoic acid.
Figure 2
Figure 2
Tridecaptin M shows outer membrane disruption in all Gram-negative bacteria. (a) N-phenyl-1-naphthylamin (NPN) assay with A. baumannii. (b) NPN assay in K. pneumoniae. (c) NPN assay in P. aeruginosa. The data plotted are mean values of three biological replicates. Bars denote the standard deviation. Tri-M—tridecaptin M and Pol B—polymyxin B. (d) Binding kinetics of tridecaptin to bacteria determined by reversed-phase high pressure liquid chromatography (RP-HPLC) using area under the curve. The concentration of tridecaptin M was 16 μg/mL. The data plotted are the mean of three replicates and representative of two biological repeats. Bars denote standard deviation.
Figure 3
Figure 3
Synergy of tridecaptin M with other antibiotics in A. baumannii strains at two different concentrations. The values presented here are the mode of three independent experiments.
Figure 4
Figure 4
In vitro efficacy of tridecaptin M and rifampicin combination against A. baumannii ATCC 19606. (a) Killing of bacteria in nutrient-rich medium in the presence of antibiotics alone or in combination. The data are plotted as the mean of three replicates. Bars indicate the standard deviation. The experiment is representative of two biological repeats. (b) Killing of bacteria in rabbit blood after 4 h treatment. The data represent mean values of three replicates with standard deviation. The dotted line represents the limit of detection.
Figure 5
Figure 5
Time-lag kinetics of tridecaptin M and rifampicin combination to study the effect on bacterial killing. (a) The late-exponential phase bacteria were exposed with rifampicin or tridecaptin M alone and in combination. In the combination panel, both antibiotics were added at the same time. The significance was calculated using the paired Student’s t-test with two-tailed distribution, *** denotes p < 0.0001. (b) The bacteria were initially incubated with rifampicin, and then tridecaptin M was added. (c,d) The bacteria were pre-treated with tridecaptin M for 2 h (c) or 4 h (d), and then tridecaptin was removed by centrifugation followed by the addition of rifampicin. In all the experiments, the data plotted are mean values of three replicates, and bars denote the standard deviation. The experiments are representatives of two biological replicates. In the graphs, the pretreatment bar denotes the initial CFU load before treatment with any of the antibiotics.
Figure 6
Figure 6
Effect of combination therapy on persisters’ survival. (a) The survival of bacterial persisters after 4 h treatment with rifampicin or tridecaptin M alone and in combination. The persisters were confirmed by determining the minimum inhibitory concentration (MIC) of two representatives after subculturing on a fresh agar plate containing no antibiotics. The MIC values were similar, indicating that persisters were not resistant to antibiotics. (b) NPN assay with stationary-phase bacteria indicating the permeabilization of persisters with tridecaptin M at different concentrations. Data plotted are mean values of three replicates. Bars denote the standard deviation.

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