The cystic fibrosis pathogen Achromobacter xylosoxidans inhibits biofilm formation of Pseudomonas aeruginosa

Abstract

Background. Achromobacter xylosoxidans and Pseudomonas aeruginosa are two pathogens that cause persistent airway infections in individuals with cystic fibrosis (CF). The persistence of P. aeruginosa is partly due to a high capacity to form biofilms and the ability to exert antagonism against other bacteria. Loss of microbial diversity in conjunction with chronic P. aeruginosa colonization is strongly correlated with low lung function in CF. A. xylosoxidans and P. aeruginosa are frequently co-isolated in CF airway cultures. This study aims to investigate the reciprocal effects on growth inhibition and biofilm formation between P. aeruginosa and A. xylosoxidans in vitro.Method. Six isolates of A. xylosoxidans, isolated from three CF patients in early and late stages of a chronic infection, were cultured together with a CF isolate of P. aeruginosa. Biofilm formation was assessed using a microtiter assay and crystal violet staining. Quantitative PCR was used to quantify species proportions in biofilms. Growth curves were performed to compare planktonic growth rates.Results. Three A. xylosoxidans isolates, all of which were from early-stage infections, inhibited biofilm formation of P. aeruginosa. The inhibition was concentration-dependent and required the interaction of live bacteria during the early stages of biofilm development. The inhibitory effect was not caused by nutrient depletion of the planktonic cells. The selected A. xylosoxidans isolate had a stronger capacity to adhere to plastic surfaces compared to the P. aeruginosa isolate.Conclusions . A. xylosoxidans can inhibit P. aeruginosa biofilm formation in vitro. The observed effect requires active interactions between live cells during the attachment stage of biofilm formation, possibly due to differences in adhesion capacity.

Keywords: Achromobacter xylosoxidans; Pseudomonas aeruginosa; biofilm; cystic fibrosis; polymicrobial interactions.

Fig. 1.. Biofilm formation of CF pathogens in mono- and co-culture. (a) Crystal violet quantification of biofilm formation of P. aeruginosa and A. xylosoxidans isolates in monoculture. (b) Biofilm formation of P. aeruginosa isolate PsA9 grown in monoculture or together with three pairs of early (AX-1A, AX-3A and AX-5A) and late (AX-1B, AX-3B and AX-5B) A. xylosoxidans isolates. (c) PsA9 was co-cultured with increasing proportions of A. xylosoxidans AX-3A, followed by quantification of biofilm formation. Each dot in the graphs represents the average value of 3 replicates, and bars represent the mean. *P<0.05, **P<0.01.

Fig. 2.. Mechanisms of interaction between P. aeruginosa and A. xylosoxidans during biofilm formation. (a–c) PsA9 and AX-3A were grown in dual- or single-species biofilms in 96-well microtiter plates for 48 h. (a) Addition of AX-3A to an established PsA9 biofilm after 24 h does not affect biofilm formation, and PsA9 cannot establish a biofilm in wells already colonized by AX-3A. (b) Co-culturing of live PsA9 with heat-killed AX-3A. (c) PsA9 was allowed to form biofilm in the presence of 0%, 10% or 50% of cell-free culture supernatant from AX-3A. (d) Growth curves of seven A. xylosoxidans and three P. aeruginosa isolates grown as planktonic monocultures in 96-well microtiter plates during 24 h with continuous hourly OD measurement. (e) Comparison of bacterial adhesion to microtiter plates. Equal volumes of PsA9 and AX-3A were incubated in microtiter plates for 90 min to allow bacterial adhesion to the wells. Monocultures were incubated in parallel for comparison. After washing steps, attached bacteria were detached, and A. xylosoxidans and P. aeruginosa DNA were quantified using qPCR. The graph shows bacterial DNA from adhered bacteria in co-cultures compared to monocultures in three replicates. All statistics are unpaired t-tests, *P≤0.05, ****P≤0.0001, ns, not significant.