Exoproducts of the Most Common Achromobacter Species in Cystic Fibrosis Evoke Similar Inflammatory Responses In Vitro

Abstract

Achromobacter is a genus of Gram-negative rods, which can cause persistent airway infections in people with cystic fibrosis (CF). The knowledge about virulence and clinical implications of Achromobacter is still limited, and it is not fully established whether Achromobacter infections contribute to disease progression or if it is a marker of poor lung function. The most commonly reported Achromobacter species in CF is A. xylosoxidans. While other Achromobacter spp. are also identified in CF airways, the currently used Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) method in routine diagnostics cannot distinguish between species. Differences in virulence between Achromobacter species have consequently not been well studied. In this study, we compare phenotypes and proinflammatory properties of A. xylosoxidans, A. dolens, A. insuavis, and A. ruhlandii using in vitro models. Bacterial supernatants were used to stimulate CF bronchial epithelial cells and whole blood from healthy individuals. Supernatants from the well-characterized CF-pathogen Pseudomonas aeruginosa were included for comparison. Inflammatory mediators were analyzed with ELISA and leukocyte activation was assessed using flow cytometry. The four Achromobacter species differed in morphology seen in scanning electron microscopy (SEM), but there were no observed differences in swimming motility or biofilm formation. Exoproducts from all Achromobacter species except A. insuavis caused significant IL-6 and IL-8 secretion from CF lung epithelium. The cytokine release was equivalent or stronger than the response induced by P. aeruginosa. All Achromobacter species activated neutrophils and monocytes ex vivo in a lipopolysaccharide (LPS)-independent manner. Our results indicate that exoproducts of the four included Achromobacter species do not differ consistently in causing inflammatory responses, but they are equally or even more capable of inducing inflammation compared with the classical CF pathogen P. aeruginosa. IMPORTANCE Achromobacter xylosoxidans is an emerging pathogen among people with cystic fibrosis (CF). Current routine diagnostic methods are often unable to distinguish A. xylosoxidans from other Achromobacter species, and the clinical relevance of different species is still unknown. In this work, we show that four different Achromobacter species relevant to CF evoke similar inflammatory responses from airway epithelium and leukocytes in vitro, but they are all equally or even more proinflammatory compared to the classic CF-pathogen Pseudomonas aeruginosa. The results suggest that Achromobacter species are important airway pathogens in CF, and that all Achromobacter species are relevant to treat.

Keywords: Achromobacter dolens; Achromobacter insuavis; Achromobacter ruhlandii; Achromobacter xylosoxidans; Pseudomonas aeruginosa; cystic fibrosis; in vitro inflammatory responses.

FIG 1

Phenotypic characterization of Achromobacter spp. And P. aeruginosa. No difference was observed between species in the swimming motility assay (A). Two of the Achromobacter species, A. xylosoxidans and A. dolens, formed significantly less biofilm than P. aeruginosa, with no significant differences observed between the Achromobacter species (B) (Mann-Whitney U test, *, P < 0.05). Symbols represent the average value of 3 replicate experiments of each bacterial isolate. Bars illustrate the mean.

FIG 2

Achromobacter type strains visualized using scanning electron microscopy at scale 5 μm. A. xylosoxidans (A) appear as short rods with abundant flagellae. A. ruhlandii (B) are shorter rods with very few visible flagellae. A. insuavis (C) have a filamentous appearance without visible flagellae. A. dolens (D) is filamentous similarly to A. insuavis, but with visible flagellae.

FIG 3

Cytokine responses from CFBEo- cell cultures stimulated with 5% (vol/vol) bacterial supernatant. The Figure shows concentrations of IL-6 (A) and IL-8 (B) in the cell culture medium after 24h of stimulation. Each symbol represents a bacterial isolate and shows the mean value of three independent replicates. Bars illustrate the mean. Pairwise comparisons were made using Mann-Whitney U test (*, P < 0.05).

FIG 4

Leukocyte activation by stimulation with 5% (vol/vol) bacterial supernatant in whole blood. CD11b expression (A, B) and HBP release (C) are calculated as fold increase over negative control (HEPES). The solid line represents no increase compared to negative control (1-fold). fMLF is used as positive control. All supernatants caused significant increase of CD11b expression in neutrophils (A) and monocytes (B) compared to LB. The supernatants of all species studied gave rise to a significant HBP release compared to LB (C). Each dot represents one bacterial isolate or control as the average value of 3 repeats. Bars represent the mean with SD. Pairwise comparisons were made using Mann-Whitney U test (*, P < 0.05).

FIG 5

Neutrophils (A) and monocytes (B) from whole blood were stimulated with type strain bacterial supernatants, LB medium or LPS in the absence or presence of 40 μg/mL polymyxin B. Activation was assessed by CD11b upregulation and analyzed with flow cytometry. Bars express the mean with SD of fold change over negative control (HEPES) in 3 repeats. Pairwise comparisons were made using Mann-Whitney U test (*, P < 0.05).