In Vitro and In Vivo Activity of AS101 against Carbapenem-Resistant Acinetobacter baumannii

Abstract

The increasing trend of carbapenem-resistant Acinetobacter baumannii (CRAB) worldwide has become a concern, limiting therapeutic alternatives and increasing morbidity and mortality rates. The immunomodulation agent ammonium trichloro (dioxoethylene-O,O'-) tellurate (AS101) was repurposed as an antimicrobial agent against CRAB. Between 2016 and 2018, 27 CRAB clinical isolates were collected in Taiwan. The in vitro antibacterial activities of AS101 were evaluated using broth microdilution, time-kill assay, reactive oxygen species (ROS) detection and electron microscopy. In vivo effectiveness was assessed using a sepsis mouse infection model. The MIC range of AS101 for 27 CRAB isolates was from 0.5 to 32 µg/mL, which is below its 50% cytotoxicity (approximately 150 µg/mL). Bactericidal activity was confirmed using a time-kill assay. The antibacterial mechanism of AS101 was the accumulation of the ROS and the disruption of the cell membrane, which, in turn, results in cell death. The carbapenemase-producing A. baumannii mouse sepsis model showed that AS101 was a better therapeutic effect than colistin. The mice survival rate after 120 h was 33% (4/12) in the colistin-treated group and 58% (7/12) in the high-dose AS101 (3.33 mg/kg/day) group. Furthermore, high-dose AS101 significantly decreased bacterial population in the liver, kidney and spleen (all p < 0.001). These findings support the concept that AS101 is an ideal candidate for further testing in future studies.

Keywords: AS101; Acinetobacter baumannii; carbapenem resistance; drug repurposing.

Figure 1

Time kill curve for A. baumannii TSP-AB-03 with antibiotics. Bacteria were grown with the indicated concentrations of minocycline (a), rifampin (b) or AS101 (c). Cell densities were measured for three independent cultures by counting the number of colony-forming units of culture for 24 h. Dotted-dashed lines indicate 99.9% reduction in beginning inoculum. Dotted lines indicate detection limits. Error bars are the standard deviation from the mean.

Figure 2

Detection of reactive oxygen species (ROS) generation after the treatment of TSP-AB-03 with AS101. ROS were significantly produced when compared with the control group (0 μg/mL). Data are normalized to viable bacterial counts and expressed as mean ± SD. **, p < 0.01.

Figure 3

SEM image of TSP-AB-03 strain untreated or treated with AS101. The morphology of untreated group was observed under a magnification of 5000× and a scale bar of 10 μm (a) and a magnification of 10,000× and a scale bar of 5 μm (b). Those treated with 1× MIC (2 μg/mL) of AS101 were captured under a magnification of 5000× and a scale bar of 10 μm (c) and a magnification of 10,000× and a scale bar of 5 μm (d). The collapsed appearance (red arrows in (c) and (d)) indicated the AS101 might attack the bacterial outer membrane.

Figure 4

TEM image of TSP-AB-03 strain untreated or treated with AS101. The morphology of untreated group was observed under a magnification of 12,000× and a scale bar of 1 μm (a) and 20,000× and a scale bar of 1 μm (b). Those treated with 1× MIC (2 μg/mL) of AS101 were captured under a magnification of 12,000× and a scale bar of 1 μm (c) and a magnification of 20,000× and a scale bar of 1 μm (d). The irregular peri-sphere and vacuole inside bacterial cell (red arrows in (c) and (d)) implied the bacterial cell lysis.

Figure 5

Mice infected with TSP-AB-03 show improved survival rate after treatment with AS101. (a), CD-1 mice were intraperitoneally injected with lethal challenges of TSP-AB-03 and treated with 1× PBS (vehicle), meropenem (MEM, 200 mg/kg/day), colistin methanesulfonate (CMS, 40 mg/kg/day) or AS101 at various dosages. Infected mouse viabilities were observed as 16.7% (1/6) for the vehicle treatment; 16.7% (1/6) for MEM; 33.3% (4/12) for CMS; 41.7% (5/12) for AS101 (1.67 mg/kg/day) and 58.3% (7/12) for AS101 (3.33 mg/kg/day). Bacterial clearance was determined using plating counts from the liver (b), kidney (c) or spleen (d) of different treated mice. Placebo: n = 3; CMS and AS101: n = 4. Data expressed as mean ± SD. ns: no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001.