Genome dynamics of Bartonella grahamii in micro-populations of woodland rodents

Abstract

Background: Rodents represent a high-risk reservoir for the emergence of new human pathogens. The recent completion of the 2.3 Mb genome of Bartonella grahamii, one of the most prevalent blood-borne bacteria in wild rodents, revealed a higher abundance of genes for host-cell interaction systems than in the genomes of closely related human pathogens. The sequence variability within the global B. grahamii population was recently investigated by multi locus sequence typing, but no study on the variability of putative host-cell interaction systems has been performed.

Results: To study the population dynamics of B. grahamii, we analyzed the genomic diversity on a whole-genome scale of 27 B. grahamii strains isolated from four different species of wild rodents in three geographic locations separated by less than 30 km. Even using highly variable spacer regions, only 3 sequence types were identified. This low sequence diversity contrasted with a high variability in genome content. Microarray comparative genome hybridizations identified genes for outer surface proteins, including a repeated region containing the fha gene for filamentous hemaggluttinin and a plasmid that encodes a type IV secretion system, as the most variable. The estimated generation times in liquid culture medium for a subset of strains ranged from 5 to 22 hours, but did not correlate with sequence type or presence/absence patterns of the fha gene or the plasmid.

Conclusion: Our study has revealed a geographic microstructure of B. grahamii in wild rodents. Despite near-identity in nucleotide sequence, major differences were observed in gene presence/absence patterns that did not segregate with host species. This suggests that genetically similar strains can infect a range of different hosts.

Figure 1

Circular representation of the microarray results for the chromosome. The innermost circle shows the genomic islands (BgGI 1-16) in magenta and prophages in yellow. The black lines within this circle indicate the genome position, with 100 kb between each line. Each other circle shows the CGH results for one strain, with the color corresponding to the hybridization signal relative to as4aup (red for lower and blue for higher signal according to the scale bar below the circles). Microarray probes are ordered according to the genomic position in B. grahamii as4aup. The reason why the badA region appears white is that these genes are very long, and therefore the density of probes is lower. The strains are, from inside: 1, mm3up; 2, as4bup; 3, af9up; 4, af30up; 5, af43up; 6, af47up; 7, af66up; 8, af50up; 9, af68up; 10, af156up; 11, cg60up; 12, cg64up; 13, cg90up; 14, af115up; 15, cg147up; 16, as134up; 17, cg120up; 18, af140up; 19, af144up; 20, af163up; 21, af164up; 22, af165up; 23, as224up; 24, as211up; 25, af233up; 26, af206up. Strains 1-15 are of ST1.

Figure 2

Circular representation of the microarray results for the plasmid. The innermost circle shows the plasmid genes (magenta indicates the vbh genes). The coloring and order of strains are the same as in Figure 1.

Figure 3

Schematic illustration of the PFGE results. Geographic origin, sequence type (ST) and the numbers in the strain names are shown on top. Below are the estimated sizes of all bands retrieved with the NotI and SgfI enzymes. Strains with the same pattern are shown in the same color (except grey). At the bottom are estimated genome sizes. Since the SgfI 80 kb band was shown to be a double band in the sequenced strain [20], and these parts of the genome appear to be well conserved in B. grahamii, this band was assumed to be a double band in all strains in the genome size estimation. Strains af164up and cg120up probably have additional double bands, or undetected bands, in the SgfI restriction since the size estimates differ a lot between SgfI and NotI.

Figure 4

Predicted restriction sites of NotI and SgfI in B. grahamii as4aup. The circle represents the genome of B. grahamii strain as4aup, with genomic islands in magenta and prophages in yellow. Restriction sites are shown in black (NotI) and green (SgfI). The predicted size of each band is shown inside the circle for NotI and outside for SgfI.

Figure 5

Distribution of genomotypes across geographic sites. The map shows the locations of the geographic sites where the rodents were collected. The definition of the genomotypes (based on sequence type, presence/absence of the fha-repeat and presence/absence of the plasmid) is shown in the upper left corner. For each genomotype at each site, the different rodent hosts associated with this particular variant are shown, color-coded according to the legend in the upper right corner.

Figure 6

Growth curves of B. grahamii. Growth curves of five B. grahamii strains in supplemented Schneider's medium. Bacterial growth was determined by measuring the OD₆₀₀ in triplicates at 24-h intervals.