Colistin potentiation in multidrug-resistant Acinetobacter baumannii by a non-cytotoxic guanidine derivative of silver

Abstract

A novel nano-formulation (NF) that sensitizes Acinetobacter baumannii (AB) to otherwise ineffective colistin is described in the present study. Infections due to multidrug resistant (MDR) AB represent a major therapeutic challenge, especially in situations of pre-existing colistin resistance (colR). Subsequently, boosting the effectiveness of colistin would be a better alternative tactic to treat AB infections rather than discovering a new class of antibiotics. We have previously demonstrated an NF comprising self-assembled guanidinium and ionic silver nanoparticles [AD-L@Ag(0)] to have anti-biofilm and bactericidal activity. We report NF AD-L@Ag(0) for the very first time for the potentiation of colistin in Gram-negative colistin-resistant bacteria. Our results implied that a combination of clinically relevant concentrations of colistin and AD-L@Ag(0) significantly decreased colistin-resistant AB bacterial growth and viability, which otherwise was elevated in the presence of only colistin. In this study, we have described various combinations of minimum inhibitory concentration (MIC) of colistin (MICcol, ^1/2 MICcol, and ^1/4 MICcol) and that of AD-L@Ag(0) [MICAD-L@Ag(0), ^1/2 MICAD-L@Ag(0), and ^1/4 MICAD-L@Ag(0)] and tested them against MDR AB culture. The results (in broth as well as in solid media) signified that AD-L@Ag(0) was able to potentiate the anti-microbial activity of colistin at sub-MIC concentrations. Furthermore, the viability and metabolic activity of bacterial cells were also measured by CTC fluorescence assay and ATP bioluminescence assay. The results of these assays were in perfect concordance with the scores of cultures (colony forming unit and culture turbidity). In addition, quantitative real-time PCR (qRT-PCR) was performed to unveil the expression of selected genes, DNAgyrA, DNAgyrB, and dac. These genes introduce negative supercoiling in the DNA, and hence are important for basic cellular processes. These genes, due to mutation, modified the Lipid A of bacteria, further resisting the uptake of colistin. Therefore, the expression of these genes was upregulated when AB was treated with only colistin, substantiating that AB is resistant to colistin, whereas the combinations of MICcol + MICAD-L@Ag(0) downregulated the expression of these genes, implying that the developed formulation can potentiate the efficiency of colistin. In conclusion, AD-L@Ag(0) can potentiate the proficiency of colistin, further enhancing colistin-mediated death of AB by putatively disrupting the outer membrane (OM) and facilitating bacterial death.

Keywords: Acinetobacter baumannii; antibiotic; colistin resistance; guanidinium; nano-formulation; potentiators; silver.

FIGURE 1

Minimum inhibitory concentration (MIC) calculation: The MIC, ^1/2 MIC and ^1/4 MIC of colistin (MIC_col), of AD-L@Ag(0) nano-formulation [MIC_ADL@Ag(0)] and that of combination [MIC_Col + MIC_ADL@Ag(0)] against Acinetobacter baumannii (AB) was checked using spot assay, CFU count, and by optical density (OD) analysis. The growth of ABin the presence of all the concentrations of colistin confirms resistance. (A) Schematic representation of the checkerboard assay at various MICs of colistin and AD-L@Ag(0) to test their synergy or additive effect. (B) Spot assays for calculation of MICs: (i) Plate showing growth of ABin the presence of all the MICs of colistin with controls; AB-C, AB control; MC, media control (ii) plate showing effect of the combination of all the MICs of colistin (MIC_col, ^1/2 MIC_col, ^1/4 MIC_col) + MIC of AD-L@Ag(0) on the growth of AB (iii) plate showing effect of the combination of all the MICs of colistin (MIC_col, ^1/2 MIC_col, ^1/4 MIC_col) + ^1/2 MIC of AD-L@Ag(0) on the growth of AB (iv) plate showing effect of the combination of all the MICs of colistin (MIC_col, ^1/2 MIC_col, ^1/4 MIC_col) + ^1/4 MIC of AD-L@Ag(0) on the growth of Acinetobacter baumannii. (C) Culture Biomass Growth measured from absorbance (OD₆₀₀) at various combinations of MICs of colistin and AD-L@Ag(0). The X-axis of the histogram represents MIC, ^1/2 MIC, and ^1/4 MIC of AD-L@Ag(0), whereas the Y-axis shows the culture biomass growth at these MICs. The color bars represent different concentrations of colistin (MIC, ^1/2 MIC, and ^1/4 MIC) with controls: CC, colistin control; NF-C, nano-formulation control [AD-L@Ag(0)]; AB-C, Acinetobacter baumannii control. (D) This figure shows the log reduction in CFU count of AB after treatment with different combinations of colistin and AD-L@Ag(0). The Y-axis shows the log reduction of CFU/ml. X-axis shows different colors representing various combinations of colistin (MIC, ^1/2 MIC, and ^1/4 MIC) with multiple concentrations (MIC, ^1/2 MIC, and ^1/4 MIC) of AD-L@Ag(0), along with controls: CC, colistin control; NF-C, nano-formulation control [AD-L@Ag(0)]; AB-C, Acinetobacter baumannii control.

FIGURE 2

Batch characterization of AD-L@Ag(0): (A) XPS analysis: (a,b) XPS survey spectra of AD-L (blue trace), and AD-L@Ag (red trace) respectively. (c) Represents high-resolution deconvoluted spectra collected in the Ag 3d region for AD-L@Ag. (B) TEM analysis: TEM images of metallogel from AD-L@Ag(0) showing the gel fibers with in situ formed silver nanoparticle at 20 nm and 0.1 μm. (C) FT-IR analysis: FT-IR analysis of AD-L@Ag(0) in powder (blue line) and the gel phase (green line).

FIGURE 3

Determination of potentiating ability of AD-L@Ag(0) by estimation of cell viability: The viability of ABin the presence of various combinations of MICs of colistin and AD-L@Ag(0) was analyzed using ATP assay, flow cytometry, and by confocal laser scanning microscopy (CLSM). (A) ATP assay: The graph represents presence/absence of ATP production at various combinations of colistin + AD-L@Ag(0). The x-axis in the graph represents various concentrations of MICs (MIC, ^1/2 MIC, ^1/4 MIC) of AD-L@Ag(0); whereas the y-axis represents viability by ATP in terms of RLU (relative light unit). The color bars represent different concentrations of colistin (MIC, ^1/2 MIC, and ^1/4 MIC) with controls: CC, colistin control; NF-C, nano-formulation control [AD-L@Ag(0)]; AB-C, Acinetobacter baumannii control; MC, media control. The graph suggested similar results as those obtained from CFU and OD calculation. The RLU of ATP at colistin ^1/2 MIC + AD-L@Ag(0) ^1/4 MIC was found to be zero indicating bactericidal effect on resistant AB strain. (B) Flow cytometry: The viability percentage of ABin various combinations of colistin and AD-L@Ag(0) was analyzed by flow cytometry using DAPI and CTC staining. The x-axis represents the treatment of ABat various combinations and the y-axis represents the percentage of viable cells among total cells. A1: Acinetobacter baumannii control (AB-C), A2: Colistin control (CC), A3: Nano-formulation control [AD-L@Ag(0)] (NF–C), A4: ^1/2 MIC_col + ^1/4 MIC_AD–L@Ag(0), A5: ^1/4 MIC_col + ^1/2 MIC_AD–L@Ag(0), and A6: ^1/4 MIC_col + MIC_AD–L@Ag(0). Flow cytometry analysis indicated that at colistin ^1/2 MIC + AD-L@Ag(0) ^1/4 MIC (A4), the difference between viable cells (CTC stained) to total cells (DAPI stained) was very huge. This means that the percentage of viable cells was very low when compared to total cells; indicating bactericidal effect at this combination. (C) Pseudo-color plot (Flow cytometry): The graph represents four quadrant images with the percentage of cells in each quadrant observed by flow cytometric analysis. Q1: Shows AB cells stained with CTC only, Q2: Shows AB cells stained with both CTC and DAPI, Q3: Shows AB cells stained with DAPI only, Q4: Shows AB cell debris. A1: Acinetobacter baumannii control (AB-C), A2: Colistin control (CC), A3: Nano-formulation control [AD-L@Ag(0)] (NF–C), A4: ^1/2 MIC_col + ^1/4 MIC_AD–L@Ag(0), A5: ^1/4 MIC_col + ^1/2 MIC_AD–L@Ag(0), and A6: ^1/4 MIC_col + MIC_AD–L@Ag(0). ** (0.0081) Shows the significant difference between AB–C and ^1/2 MIC_col + ^1/4 MIC_AD–L@Ag(0) (TOTAL CELLS). **** (<0.0001) Shows the significant difference between AB–C and ^1/2 MIC_col + ^1/4 MIC_AD–L@Ag(0) (VIABLE CELLS).

FIGURE 4

Confocal laser scanning microscopy (CLSM): Morphological changes when using the combination of AD-L@Ag(0) + colistin were observed using CLSM and compared with the controls. Here, (A1): Acinetobacter baumannii control (AB-C), (A2): Colistin control (CC), (A3): Nano-formulation control [AD-L@Ag(0)] (NF–C), (A4): ^1/2 MIC_col + ^1/4 MIC_AD–L@Ag(0), (A5): ^1/4 MIC_col + ^1/2 MIC_AD–L@Ag(0), and (A6): ^1/4 MIC_col + MIC_AD–L@Ag(0). The results were consistent with our previous observations. Colistin MIC concentration showed visible growth of AB (A2) and MIC of AD-L@Ag(0) shows no visible bacteria due to bactericidal effect (A3). Whereas, the combination of two at ^1/2 MIC_col + ^1/4 MIC_AD–L@Ag(0) (A4) bacterial growth was inhibited. That is why there is negligible number of bacteria visible under confocal microscope.

FIGURE 5

Quantitative Real-time PCR (qRT–PCR) analysis: The graph shows gene expression of GyrA, GyrB, and dacB (presented in y-axis) upon different treatments (presented in x-axis). Here, A1: Acinetobacter baumannii control A–C, A2: Colistin control (CC), A3: Nano-formulation control [AD-L@Ag(0)] (NF–C), A4: ^1/2 MIC_col + ^1/4 MIC_AD–L@Ag(0), A5: ^1/4 MIC_col + ^1/2 MIC_AD–L@Ag(0), and A6: ^1/4 MIC_col + MIC_AD–L@Ag(0). A decrease in gene expression when colistin and AD-L@Ag(0) were used in combination was observed in comparison to when only colistin was used.

FIGURE 6

Cytotoxicity assay: A comparison between untreated Vero E6 Cells against treatment with different combinations of AD-L@Ag(0) and colistin is shown here. The X-axis represents various combinations of colistin and AD-L@Ag(0), along with colistin control (CC), AD-L@Ag(0) control (NF–C), untreated Vero E6 cells, positive control (Docetaxel),and the Y-axis represents the OD values at 570 nm. Results show that there is no significant cytotoxicity found in the case of colistin ^1/2 MIC and AD-L@Ag(0) ^1/4 MIC concentration. This is the best combination for potentiation because, at this concentration, it shows less or negligible cytotoxicity to mammalian cells and also demonstrates significant bactericidal effect on colistin-resistant Acinetobacter baumannii. **** Shows the significant difference between Live Vero E6 Control and Cytotoxic Drug treated Vero E6 cells (Cytotoxicity Control).

FIGURE 7

Schematic representation of the induction of colistin resistance as well as reversal of colistin resistance on Acinetobacter baumanni. (A) Representation of the gene expression in colistin resistant cell: Here green color shows the upregulation of genes, e.g., GyraseA/B and pmrHIJKLM operon. pmrHIJKLM operon codes for phosphoethanolamine (PEtn) or 4-amino-4-deoxy-L-arabinose (L-Ara4N). PEtn gets deposited in the Lipid A layer, and this leads to a decrease in net negative charge, which ultimately leads to colistin resistance. (B) Representation of the gene expression in the reversal of colistin resistance when colistin + AD-L@Ag(0) combination is used: Here, it is shown how AD-L@Ag(0) affects GyraseA/B activity, which leads to inhibition of pmrHIJKLM operon. Red color shows the downregulation of genes GyraseA/b genes when the combination was used. GyraseA/B are important for negative supercoiling during processes like DNA replication, transcription, and translation. Hence, its downregulation ultimately hinders the expression of pmrHIJKLM operon. So, there is no PEtn or L-Ara4N production and there is no change in Lipid A composition and net charge present on OM. This leads colistin and AD-L@Ag(0) to exert their bactericidal effect by disrupting the cell membrane, which leads to cell death.