Multilocus sequence typing supports the hypothesis that Ochrobactrum anthropi displays a human-associated subpopulation

Abstract

Background: Ochrobactrum anthropi is a versatile bacterial species with strains living in very diverse habitats. It is increasingly recognized as opportunistic pathogen in hospitalized patients. The population biology of the species particularly with regard to the characteristics of the human isolates is being investigated. To address this issue, we proposed a polyphasic approach consisting in Multi-Locus Sequence Typing (MLST), multi-locus phylogeny, genomic-based fingerprinting by pulsed-field gel electrophoresis (PFGE) and antibiotyping.

Results: We tested a population of 70 O. anthropi clinical (n = 43) and environmental (n = 24) isolates as well as the type strain O. anthropi ATCC49188T and 2 strains of Ochrobactrum lupini and Ochrobactrum cytisi isolated from plant nodules. A Multi-Locus Sequence Typing (MLST) scheme for O. anthropi is proposed here for the first time. It was based on 7 genes (3490 nucleotides) evolving mostly by neutral mutations. The MLST approach suggested an epidemic population structure. A major clonal complex corresponded to a human-associated lineage since it exclusively contained clinical isolates. Genomic fingerprinting separated isolates displaying the same sequence type but it did not detect a population structure that could be related to the origin of the strains. None of the molecular method allowed the definition of particular lineages associated to the host-bacteria relationship (carriage, colonisation or infection). Antibiotyping was the least discriminative method.

Conclusion: The results reveal a human-associated subpopulation in our collection of strains. The emergence of this clonal complex was probably not driven by the antibiotic selective pressure. Therefore, we hypothesise that the versatile species O. anthropi could be considered as a human-specialized opportunistic pathogen.

Figure 1

Minimum-spanning tree based on MLST data. Colours indicate the source (clinical in blue or environmental in green) of the strains. The number given in the circle corresponds to the sequence type (ST) number. The number given near the circle corresponds to the number of isolates presenting the ST. The size of circles is proportional to the number of isolates representing the ST. MSCC for Minimum Spanning Clonal Clomplex.

Figure 2

ML trees based on concatenated sequences of the seven housekeeping gene fragments. Position of the artificial root (black circle) corresponded to branching of the out-group (B. suis 1330^T) included in the analysis but not shown on the tree. Horizontal lines are scales for genetic distance. Numbers given at the nodes are support values estimated with 100 bootstrap replicates. Only bootstrap values >50% are indicated. For better visualization of the tree, bootstrap values are shown at the terminal nodes. The scale bar indicates the number of substitutions per nucleotide position. The clonal complexes MSCC and eBCC determined by Minimum Spanning and eBurst, respectively are indicated by vertical bold bars. Blue: clinical strains; green: environmental strains. (*) indicated major conflicting phylogenetic positions between the seven genes-based tree and the trpE-based tree in Fig 3.

Figure 3

ML trees based on the trpE gene fragment. Position of the artificial root (black circle) corresponded to branching of the out-group (B. suis 1330^T) included in the analysis but not shown on the tree. Horizontal lines are scales for genetic distance. Numbers given at the nodes are support values estimated with 100 bootstrap replicates. Only bootstrap values >50% are indicated. For better visualization of the tree, bootstrap values are shown at the terminal nodes. The scale bar indicates the number of substitutions per nucleotide position. The clonal complexes MSCC and eBCC determined by Minimum Spanning and eBurst, respectively are indicated by vertical bold bars. Blue: clinical strains; green: environmental strains. (*) indicated major conflicting phylogenetic positions between the seven genes-based tree (Fig. 2) and the trpE-based tree.

Figure 4

SplitsTree decomposition analyses of MLST data for O. anthropi strains. The distance matrix was obtained from allelic profiles of strains. The clonal complexes (MSCC and eBCC) are delineated by bold lines.

Figure 5

Representative PFGE profiles obtained for French clinical strains isolated in the same hospital and belonging to the major clonal complex MSCC4/eBCC4. PFGE clusters at a 60% similarity level are indicated at the bottom of the gel. (*) ADN of the strain ADV77 was deposited twice on the gel to check reproducibility and to help profiles comparison.