Research Article: Stress Conditions Triggering Mucoid Morphotype Variation in Burkholderia Species and Effect on Virulence in Galleria mellonella and Biofilm Formation In Vitro

Date Published: December 16, 2013

Publisher: Public Library of Science

Author(s): Inês N. Silva, Andreia C. Tavares, Ana S. Ferreira, Leonilde M. Moreira, Daniela Flavia Hozbor.


Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens causing chronic respiratory infections particularly among cystic fibrosis patients. During these chronic infections, mucoid-to-nonmucoid morphotype variation occurs, with the two morphotypes exhibiting different phenotypic properties. Here we show that in vitro, the mucoid clinical isolate Burkholderia multivorans D2095 gives rise to stable nonmucoid variants in response to prolonged stationary phase, presence of antibiotics, and osmotic and oxidative stresses. Furthermore, in vitro colony morphotype variation within other members of the Burkholderia genus occurred in Bcc and non-Bcc strains, irrespectively of their clinical or environmental origin. Survival to starvation and iron limitation was comparable for the mucoid parental isolate and the respective nonmucoid variant, while susceptibility to antibiotics and to oxidative stress was increased in the nonmucoid variants. Acute infection of Galleria mellonella larvae showed that, in general, the nonmucoid variants were less virulent than the respective parental mucoid isolate, suggesting a role for the exopolysaccharide in virulence. In addition, most of the tested nonmucoid variants produced more biofilm biomass than their respective mucoid parental isolate. As biofilms are often associated with increased persistence of pathogens in the CF lungs and are an indicative of different cell-to-cell interactions, it is possible that the nonmucoid variants are better adapted to persist in this host environment.

Partial Text

The Burkholderia cepacia complex (Bcc) is a group of bacterial species that comprises opportunistic pathogens causing severe chronic infections in cystic fibrosis (CF) and immunocompromised patients. The respiratory tract of CF patients is predominantly colonized with Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenza, and, to a lesser extent, Bcc bacteria. Nevertheless, the infections caused by Bcc bacteria are extremely difficult to eradicate, leading in some cases to necrotizing pneumonia and septicemia [1], [2]. Within the CF lung, colonizing bacteria face an environment with high osmolarity, heterogeneous distribution of oxygen and nutrients, high concentration of antimicrobials, and constant challenge by the host immune defenses [3], [4]. These factors exert a selective pressure in colonizing bacteria and are thought to be the driving force of microevolution during their persistence in the CF lung. Phenotypic and genotypic variation within the CF lung have been well described in P. aeruginosa (reviewed in [5]) and is associated with mutator strains arising after long-term infection [6], [7]. Several studies have been undertaken to evaluate in vitro the effect of specific environmental stressors on the generation of P. aeruginosa variants in the CF lung. For example, under in vitro conditions of suboptimal nutrition, isolates of P. aeruginosa that produced rough lipopolysaccharide and were mucoid have emerged. These two phenotypes have been associated with long-term colonization of CF patients [8]. Similarly, prolonged incubation of nonmucoid P. aeruginosa in minimal medium with acetamide as the sole carbon source triggered the switch to the mucoid phenotype [9]. Mucoid variation of another P. aeruginosa isolate was also shown when cultivated in a chemostat system in a medium with high osmolarity or under carbon and nitrogen limitation [10]. Biofilm communities were also proven to be a source of self-generated diversity since P. aeruginosa was shown to undergo extensive genetic diversification during short-term growth in biofilms [11].

Conversion to the mucoid phenotype is well known in Pseudomonas aeruginosa, with the initial isolates colonizing CF patients airways being nonmucoid, but becoming mucoid during the course of respiratory chronic infections due to overproduction of alginate [25]. An opposite observation was reported for other CF opportunistic pathogens, namely for Bcc bacteria in which morphotype transition is mainly from mucoid-to-nonmucoid [12]. To understand this morphotype transition in Bcc, we have previously reported the characterization of a pair of clonal isolates of B. multivorans (D2095 and D2214) from a chronically infected CF patient where a mucoid-to-nonmucoid transition had occurred [13]. Compared to the mucoid parental isolate D2095, the nonmucoid isolate D2214 exhibited a reduced expression of some virulence factors, it caused a lower mortality rate of G. mellonella larvae, but displayed a higher survival ability under nutrient starvation and increased biofilm formation ability [13]. In this study we have investigated the morphotype stability of the mucoid D2095 and nonmucoid D2214 isolates after exposure to stress conditions. We never observed mucoid morphotype variation when D2095 or D2214 isolates were grown under standard conditions (for instance SM, MM, and YEM liquid media, aerobically at 37°C for 3 days) or by multiple passages onto solid media, confirming their morphotype stability. Nevertheless, during prolonged incubation for at least 7 days, nonmucoid variant colonies arised from the mucoid D2095 culture while no mucoid colonies were ever recovered from nonmucoid D2214 culture even for longer periods of incubation. The nonmucoid variants of D2095 are stable since they maintained the same morphotype in the absence of the stress condition.