A Novel Triple Combination To Combat Serious Infections with Carbapenem-Resistant Acinetobacter baumannii in a Mouse Pneumonia Model

Abstract

The ongoing crisis of antimicrobial resistance demands novel combinations between antimicrobials and nonantimicrobials to manage infections caused by highly resistant pathogens. This study aimed to evaluate the effect of combining sodium ascorbate and/or apo-transferrin with imipenem, forming double and triple combinations, against 20 multiple-carbapenemase-producing Acinetobacter baumannii strains using the checkerboard test, time-kill assay, and disc diffusion test. The results of the checkerboard assay revealed that all double combinations showed indifference, while only triple combination recorded a synergistic effect (fractional inhibitory concentration index [FICI] < 0.8) in 95% the test isolates. Moreover, the MIC of imipenem (MIC_imp) was strongly reduced (up to 128-fold reduction) after treatment with the triple combination against highly resistant isolates and reached the susceptible range. The time-kill assay revealed that the triple combination led to a 4-log₁₀ reduction in the CFU at 8 h compared with the initial bacterial count, and no viable count was recorded at 10 h. The mouse pneumonia model showed restoration of lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following administration of the triple rescue therapy. Additionally, normal lungs with normal patent alveoli were detected 72 h following treatment. Accordingly, sodium ascorbate and apo-transferrin are promising adjunct biological agents with the potential to restore the effectiveness of critically essential antibiotics like imipenem, commonly used for the treatment of A. baumannii infections. IMPORTANCE Combination therapy provides a perspective to threat multidrug-resistant (MDR) strains. The present study sheds light on a novel and effective triple combination against carbapenem-resistant A. baumannii. Our in vitro results showed that combining imipenem with apo-transferrin and sodium ascorbate yielded synergism in 95% of test isolates, and this was associated with a marked reduction in imipenem MIC, shifting it below the breakpoint. Furthermore, a bactericidal effect was recorded, with no viable count detected at 10 h. An in vivo murine model of pneumonia was induced to mimic human disease. The triple combination therapy restored lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following the initiation of therapy. Therefore, our findings suggest novel insights about a promising new combination therapy against highly resistant carbapenemase-producing A. baumannii to restore the effectiveness of imipenem.

Keywords: A. baumannii; carbapenemases; pneumonia; synergism; triple combination.

FIG 1

Disc diffusion test showing inhibition zones of imipenem, ascorbate, and apo-transferrin of representative A. baumannii isolates: Ac 136 (a), Ac 20 (b), Ac 33 (c), Ac 8 (d), Ac 30 (e), Ac 36 (f), Ac 42 (g), Ac 83 (h), and Ac 139 (i). Clear synergism was detected as common inhibition zones at the centers of the three test agents. White arrows point to the growth present between the two-test-drug-containing discs, which means absence of synergism between double combinations.

FIG 2

Assessment of the triple combination of imipenem (4 μg/mL), 1/4 MIC of both apo-transferrin, and sodium ascorbate against A. baumannii representative test isolates—Ac30 (a), Ac36 (b), Ac42 (c), and Ac83 (d)—using a time-kill assay, showing zero viable count after 10 h of incubation with the triple combination.

FIG 3

Histopathology of pulmonary tissues, H&E stained, representing different groups of the animal model. (a) Negative-control group showing normal lung with thin wall alveoli. (b) Bacterium-infected positive-control group; heavy inflammatory infiltrate was detected with intrabronchial heavy suppurative exudate (yellow arrows) and marked surrounding congested vessels (green arrows). Emphysematous changes or compensatory emphysema in alveoli was seen (asterisk). (c) Imipenem-treated group showing failure of therapy represented in dilated bronchioles filled with inflammatory exudate, markedly congested blood vessels, and few remaining patent alveoli. (d) A representative double combination-treated group showing absence of improvement represented by heavy cellular inflammatory infiltrate within lung parenchyma in addition to dilated congested vessels. (e) Triple-combination rescue group showing within normal lung with mild to moderate residual inflammation (arrow) and moderately congested vessels after 8 h of administration. (f) Triple-combination rescue group after 24 h of treatment showing mild residual inflammation and mildly congested vessels. (g) Triple-combination rescue group following 72 h of treatment showing normal lung with normal patent alveoli. Magnification, ×100.

FIG 4

Giemsa-stained lung tissues for histopathological examination showing prebronchial heavy inflammatory infiltrate and rods (arrows) of the A. baumannii challenge strain in an infection-positive control (a) and normal lung free from bacterial cells following treatment with triple combination rescue therapy (b). Magnification, ×200.

FIG 5

Bacterial burdens of A. baumannii challenge microorganism in lung were determined quantitatively by viable count at 24 h postinoculation. Data are presented as means ± SD, and the experiment was carried out in triplicate. Significance of results was determined when the P value was <0.001 (***).

FIG 6

Histopathological images of spleen section stained with H&E (magnification, ×40) (a), spleen section stained with Giemsa (magnification, ×100) (b), liver section stained with H&E (magnification, ×200) (c), and liver section stained with Giemsa (magnification, ×200) (d), obtained from A. baumannii-infected mice, showing normal histology, indicating absence of bacterial dissemination outside the lungs.