Population structure of Pseudomonas aeruginosa

Abstract

The metabolically versatile Gram-negative bacterium Pseudomonas aeruginosa inhabits terrestrial, aquatic, animal-, human-, and plant-host-associated environments and is an important causative agent of nosocomial infections, particularly in intensive-care units. The population genetics of P. aeruginosa was investigated by an approach that is generally applicable to the rapid, robust, and informative genotyping of bacteria. DNA, amplified from the bacterial colony by circles of multiplex primer extension, is hybridized onto a microarray to yield an electronically portable binary multimarker genotype that represents the core genome by single nucleotide polymorphisms and the accessory genome by markers of genomic islets and islands. The 240 typed P. aeruginosa strains of diverse habitats and geographic origin segregated into two large nonoverlapping clusters and 45 isolated clonal complexes with few or no partners. The majority of strains belonged to few dominant clones widespread in disease and environmental habitats. The most frequent genotype was represented by the sequenced strain PA14. Core and accessory genome were found to be nonrandomly assembled in P. aeruginosa. Individual clones preferred a specific repertoire of accessory segments. Even the most promiscuous genomic island, pKLC102, had integrated preferentially into a subset of clones. Moreover, some physically distant loci of the core genome, including oriC, showed nonrandom associations of genotypes, whereas other segments in between were freely recombining. Thus, the P. aeruginosa genome is made up of clone-typical segments in core and accessory genome and of blocks in the core with unrestricted gene flow in the population.

Fig. 1.

Flow chart of P. aeruginosa genotyping with the low-resolution microarray.

Fig. 2.

Pulsed-field gel electrophoresis-separated SpeI fragment and microarray hybridization patterns of two closely related strains of the TB clone [Left; isolates from burn wound and CF lungs (29)] and of two distantly related strains of the CHA clone [Right; isolates from river and CF lungs (30)]. Clonal variants share identical SNP genotypes in the core genome (lower part of the array), but they differ in either few (Left) or many (Right) markers of the accessory genome.

Fig. 3.

Widespread geographic distribution of major P. aeruginosa clones. Clones are depicted by uppercase letters and are arranged by decreasing frequency in alphabetical order. Source and origin of strains are listed in SI Table 3. The most abundant clone, A, is represented by the sequenced strain PA14 (27).

Fig. 4.

Clonal complex structures of 240 P. aeruginosa strains collected from diverse habitats and geographic origins (see SI Table 3). Clonal complexes were calculated from the 15-marker genotype of the core genome by the eBurst algorithm (41). Clones represented by two or more isolates in the strain panel are depicted by uppercase letters and are sorted by decreasing frequency in alphabetical order (see SI Table 3). Clones shaded in gray harbor oriC allele 1, and clones encircled by dots are exoU-positive.

Fig. 5.

Nonrandom association of genetic variants in the P. aeruginosa genome. Fourteen two-marker combinations showed significant over- or underrepresentation of marker allele haplotypes (+, P < 0.05; ++, P < 0.01 after Bonferroni correction for multiple testing). The SNPs amp-3, amp-4, amp-6, and amp-7 are located within the ampC gene, but the other six genes are evenly distributed on the chromosome (see Fig. 6).

Fig. 6.

Nonrandom association of the P. aeruginosa SNP genotype of the core genome with elements of the accessory genome. The chromosomal map position of genes of the core and accessory genome is given in boldface and lightface font, respectively. The numbers in parentheses behind the loci of the accessory genome indicate the percentage of the 240 typed strains with a fixed association between the presence or absence of this locus and the clonal frame of the core genome defined by the hexadecimal SNP genotype. The two columns indicate the association between SNP genotype and the presence or absence of members of the abundant PAGI-2/pKLC102 family of genomic islands (7, 10, 11, 40). The extreme values of 50% and 100% indicate no or absolute linkage between the clonal frame of the core genome and the respective element of the accessory genome.