A Stenotrophomonas maltophilia Multilocus Sequence Typing Scheme for Inferring Population Structure

Stenotrophomonas maltophilia is an opportunistic, highly resistant, and ubiquitous pathogen. Strains have been assigned to genogroups using amplified fragment length polymorphism. Hence, isolates of environmental and clinical origin predominate in different groups. A multilocus sequence typing (MLST) scheme was developed using a highly diverse selection of 70 strains of various ecological origins from seven countries on all continents including strains of the 10 previously defined genogroups. Sequence data were assigned to 54 sequence types (ST) based on seven loci. Indices of association for all isolates and clinical isolates of 2.498 and 2.562 indicated a significant linkage disequilibrium, as well as high congruence of tree topologies from different loci. Potential recombination events were detected in one-sixth of all ST. Calculation of the mean divergence between and within predicted clusters confirmed previously defined groups and revealed five additional groups. Consideration of the different ecological origins showed that 18 out of 31 respiratory tract isolates, including 12 out of 19 isolates from cystic fibrosis (CF) patients, belonged to genogroup 6. In contrast, 16 invasive strains isolated from blood cultures were distributed among nine different genogroups. Three genogroups contained isolates of strictly environmental origin that also featured high sequence distances to other genogroups, including the S. maltophilia type strain. On the basis of this MLST scheme, isolates can be assigned to the genogroups of this species in order to further scrutinize the population structure of this species and to unravel the uneven distribution of environmental and clinical isolates obtained from infected, colonized, or CF patients.Stenotrophomonas maltophilia is ubiquitous in nature. It has, for instance, been isolated from the rhizosphere of various plants and animals (14, 27, 37). Due to its tolerance against cadmium and its ability to degrade xenobiotic compounds, it has been proposed for remediation of contaminated ground (9, 39). Increasingly, it is being isolated from immunosuppressed individuals and intensive care and cystic fibrosis (CF) patients and has been shown to be resistant to many antimicrobial agents (16, 17, 69). However, the role of this opportunistic pathogen as an innocent bystander or causative agent often remains unclear (30), and little is known about its virulence factors (20, 33).Recently, novel Stenotrophomonas species were described: Stenotrophomonas nitritireducens sp. nov. (24), Stenotrophomonas acidaminiphila (3), Stenotrophomonas rhizophila (73), and Stenotrophomonas africana sp. nov. (21). However, the latter is a synonym of S. maltophilia (10).Using amplified fragment length polymorphism (AFLP) fingerprinting and DNA-DNA hybridizations, remarkable diversity has been shown to exist among S. maltophilia isolates recovered from both the environment and human clinical samples. This species can be subdivided into 10 AFLP genomic groups (35) that comprise to various extents both clinical and environmental isolates. Similarly, different genomic groups of the genus Stenotrophomonas can be distinguished using restriction fragment length polymorphisms (RFLP) in the gyrB gene (11). Surprisingly, 36 out of 40 isolates from CF patients are grouped in just two clusters. However, no such differences were seen in other investigations using pulsed-field gel electrophoresis (PFGE) after DraI digestion, molecular typing by BOX-PCR, or temperature-gradient gel electrophoresis of 16S rRNA PCR fragments (7). Later DNA sequence analyses of the 16S rRNA revealed three clusters, with grouping of the strains according to their sources of isolation and signature sequences in the region V1, which distinguishes clinical from environmental isolates (44).The objective of this study was to develop a multilocus sequence typing (MLST) scheme on the basis of a diverse strain collection comprising isolates from different ecological origins, continents, and DNA hybridization groups (35). We then employed this scheme to start initial analyses of the population structure of this species.(This study was conducted by S. Kaiser in partial fulfillment of the requirements for a diploma thesis in biology from the Faculty of Biology, University of Freiburg, Freiburg, Germany, 2007.)