A multilocus sequence typing scheme implies population structure and reveals several putative novel Achromobacter species

Abstract

The genus Achromobacter currently is comprised of seven species, including Achromobacter xylosoxidans, an opportunistic and nosocomial pathogen that displays broad-spectrum antimicrobial resistance and is recognized as causing chronic respiratory tract infection in persons with cystic fibrosis (CF). To enable strain typing for global epidemiologic investigations, to clarify the taxonomy of "Achromobacter-like" strains, and to elucidate the population structure of this genus, we developed a genus-level multilocus sequence typing (MLST) scheme. We employed in silico analyses of whole-genome sequences of several phylogenetically related genera, including Bordetella, Burkholderia, Cupriavidus, Herminiimonas, Janthinobacterium, Methylibium, and Ralstonia, for selecting loci and designing PCR primers. Using this MLST scheme, we analyzed 107 genetically diverse Achromobacter isolates cultured from biologic specimens from CF and non-CF patients, 1 isolate recovered from sludge, and an additional 39 strains obtained from culture collections. Sequence data from these 147 strains, plus three recently genome-sequenced Achromobacter strains, were assigned to 129 sequence types based on seven loci. Calculation of the nucleotide divergence of concatenated locus sequences within and between MLST clusters confirmed the seven previously named Achromobacter species and revealed 14 additional genogroups. Indices of association showed significant linkage disequilibrium in all of the species/genogroups able to be tested, indicating that each group has a clonal population structure. No clear segregation of species/genogroups between CF and non-CF sources was found.

Fig 1

Nucleotide divergence of concatenated MLST loci. Shaded bars represent divergence within a species/genogroup; open bars represent the divergence between species/genogroup and its closest neighbor (listed to the right), as defined by percentage similarity. Error bars represent the standard errors of the shaded and open bars. The solid line represents maximum intraspecies divergence; the dashed line represents the average interspecies divergence.

Fig 2

Dendrogram of concatenated MLST loci sequences containing two strains of each Achromobacter species or genogroup (unless only a single strain was available) and rooted with Bordetella species.