Genetic diversity and multihost pathogenicity of clinical and environmental strains of Burkholderia cenocepacia

Abstract

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.

FIG. 1.

(A) Phylogenetic relationships among 23 distinct multilocus STs from the BCC (numbers at the branch nodes represent relationships with >50% bootstrap support). Groups are shaded according to which hosts the genotypes that are generally pathogenic. Ag, agricultural source; CF, CF or other clinical source. (B) Neighbor-net analysis revealed extensive recombination among the 21 STs representing the B. cenocepacia isolates (illustrated as parallelograms).

FIG. 2.

Plot of nonsynonymous substitution rate versus synonymous substitution rate for each MLST gene, the concatenated gene sequences (ST), and the average for all seven loci. The line plotted represents a d_N-to-d_S ratio of 1; conserved genes are expected to fall below this line. The relatively high values for gltB and gyrB suggest these genes are not evolving under the same evolutionary pressures as are the other five MLST loci.

FIG. 3.

Pathogenicity of B. cenocepacia and control strains to onions. (A) Rating scale. N indicates a null reaction and no affected tissue, 0 indicates water-soaking without maceration, 1 indicates 1% to 33% water-soaked/macerated tissue, 2 indicates 34% to 66% water-soaked/macerated tissue, and 3 indicates 67% to 100% water-soaked/macerated tissue. Examples of the rating scale are as follows: N, inoculated with AU10575 (no. 54); 0, inoculated with B. cenocepacia CF20 (no. 32); 3, inoculated with B. cenocepacia AU1054 (no. 12). (B) Onion pathogenesis ratings for 56 BCC strains. Strain order (no. 1 to 56) is as listed in Table 1, and the shaded box indicates strains belonging to the ST-40 Midwest epidemic clone lineage. All affected onion tissue exhibited water-soaking symptoms; the presence of an asterisk above the bar for a particular strain indicates positive tissue maceration, and a P indicates a PCR-positive result for pehA.

FIG. 4.

Antibiosis activity of B. cenocepacia and control strains against Rhizoctonia solani AG4. (A) Qualitative rating scale (0 to 4) for fungal inhibition. Examples of the rating scale are as follows: 0, B. cenocepacia AU10310 (no. 2); 1, B. cenocepacia AU0283 (no. 21); 2, B. cenocepacia 6RT131 (no. 11); 3, B. cenocepacia AU1054 (no. 12); 4, B. cenocepacia CF20 (no. 32). (B) Antibiosis activity ratings for 54 B. cenocepacia strains, B. cepacia 25416 (no. 55), and B. ambifaria AMMD (no. 56). The strain order (no. 1 to 56) is as listed in Table 1, the shaded box indicates strains belonging to the ST-40 Midwest epidemic clone lineage, and filled circles indicate the presence of the prnD gene required for pyrrolnitrin biosynthesis. Bars shown depict the standard error of the mean values. If no bars are present, the standard error was either 0 or smaller than the size of the symbol.

FIG. 5.

Pathogenicity of B. cenocepacia and control strains to C. elegans. Survival of early-L4-stage C. elegans at days 3 and 7 after addition to Burkholderia lawns growing on slow-kill (NGM) (A) or fast-kill (PGS) (B) media. (C) CSS calculated by the mixed model analysis. The x-axes in panels A to C indicates the strain numbers listed in Table 1, and the shaded boxes indicate strains belonging to the ST-40 Midwest epidemic clone lineage. Closed diamonds and open circles represent worm survival data at day 3 and day 7, respectively. Bars shown depict the standard error of the mean values. If no bars are present, the standard error was either 0 or smaller than the size of the symbol.