Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity

Abstract

Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.

FIG. 1.

Variations in (A) and light microscopy (B) of biofilm formation produced by genomically diversified clinical and environmental isolates of P. aeruginosa in an in vitro biofilm assay system. For each isolate within each 96-well plate in panel A, the assay was repeated for a total of eight times (vertically). Biofilm formation was analyzed along with PAO1 (far-right vertical lane of each plate). (C) Relationship between CV-stained materials (black bars) and biofilm viable counts (grey bars). (D) Relationship between alginate production in PAO1 and its derivatives and biofilm formation.

FIG. 2.

Motility and twitching activity of P. aeruginosa and the relationship with biofilm formation and planktonic growth rate. Shown are motility (A) and twitching (B) zones between six isolates and PAO1. The first number under the strain designation indicates the relative fold increase in biofilm formation versus PAO1; the number in parentheses indicates the doubling time in hours.

FIG. 3.

Comparison of planktonic growth rate (A) and sensitivity to hydrogen peroxide (B) between CF and non-CF isolates. Specific growth rates and sensitivity to H₂O₂ of each isolate in comparison with PAO1 (stars) were determined as described in Materials and Methods. The horizontal bar in each group represents the median values of growth rates (P = 0.008, t test) and growth inhibition zones in millimeters (P = 0.008, t test).

FIG. 4.

Nonmucoid CF isolates of P. aeruginosa are cleared more efficiently from the mouse lungs than non-CF isolates in an acute aerosol infection mouse model. A group of C57BL/6 mice were exposed to aerosols of different P. aeruginosa isolates as labeled on the x axis. For each exposure, five mice each were included for determination of bacterial deposition to the lungs at t = 0 and 6 h.

FIG. 5.

Relationship between genome diversity and genome size among clinical and nonclinical isolates of P. aeruginosa. (A) PFGE separation of SpeI-digested chromosomal DNA from a selected group of P. aeruginosa isolates. An arrowhead indicates the largest SpeI fragment of PAO1 (SpeA) (57). (B and C) PFGE separation of genomic DNA digested with I-CeuI. Arrowheads indicate the largest PAO1 fragments, 4,064 and 950 kb, respectively. The running conditions were 18 h, 6 V/cm, 120° angle, 1% agarose, and a switch time of 15 to 40 s with a gradient of a = 0.35741 in panel A; 48 h, 2 V/cm, 106° angle, 0.8% agarose, and a switch time of 20 s to 30 s with a linear gradient in panel B; and 22 h, 6 V/cm, 120° angle, 0.8% agarose, and a switch time of 90 s in panel C. The molecular size standards were Schizosaccharomyces pombe (B) and Hansenula wingei (C) (Bio-Rad).

FIG. 6.

Identification of novel DNA sequences from CF isolates of P. aeruginosa. (A) Randomly amplified PCR products using CF005 as a template with primers as indicated above the gel were separated in a 1% agarose gel. After blotting, filters were hybridized with random primer-labeled total PAO1 genomic DNA. (B) Southern blot of the gel shown in panel A with arrows indicating two major bands amplified by primers 272 and 275, respectively, but which failed to be bound by PAO1 DNA.