Comparative analysis of partial gyrB, recA, and gltB gene sequences of 84 Pandoraea reference strains and field isolates revealed several clusters that included no taxonomic reference strains. The gyrB, recA, and gltB phylogenetic trees were used to select 27 strains for whole-genome sequence analysis and for a comparative genomics study that also included 41 publicly available Pandoraea genome sequences. The phylogenomic analyses included a Genome BLAST Distance Phylogeny approach to calculate pairwise digital DNA–DNA hybridization values and their confidence intervals, average nucleotide identity analyses using the OrthoANIu algorithm, and a whole-genome phylogeny reconstruction based on 107 single-copy core genes using bcgTree. These analyses, along with subsequent chemotaxonomic and traditional phenotypic analyses, revealed the presence of 17 novel Pandoraea species among the strains analyzed, and allowed the identification of several unclassified Pandoraea strains reported in the literature. The genus Pandoraea has an open pan genome that includes many orthogroups in the ‘Xenobiotics biodegradation and metabolism’ KEGG pathway, which likely explains the enrichment of these species in polluted soils and participation in the biodegradation of complex organic substances. We propose to formally classify the 17 novel Pandoraea species as P. anapnoica sp. nov. (type strain LMG 31117^T = CCUG 73385^T), P. anhela sp. nov. (type strain LMG 31108^T = CCUG 73386^T), P. aquatica sp. nov. (type strain LMG 31011^T = CCUG 73384^T), P. bronchicola sp. nov. (type strain LMG 20603^T = ATCC BAA-110^T), P. capi sp. nov. (type strain LMG 20602^T = ATCC BAA-109^T), P. captiosa sp. nov. (type strain LMG 31118^T = CCUG 73387^T), P. cepalis sp. nov. (type strain LMG 31106^T = CCUG 39680^T), P. commovens sp. nov. (type strain LMG 31010^T = CCUG 73378^T), P. communis sp. nov. (type strain LMG 31110^T = CCUG 73383^T), P. eparura sp. nov. (type strain LMG 31012^T = CCUG 73380^T), P. horticolens sp. nov. (type strain LMG 31112^T = CCUG 73379^T), P. iniqua sp. nov. (type strain LMG 31009^T = CCUG 73377^T), P. morbifera sp. nov. (type strain LMG 31116^T = CCUG 73389^T), P. nosoerga sp. nov. (type strain LMG 31109^T = CCUG 73390^T), P. pneumonica sp. nov. (type strain LMG 31114^T = CCUG 73388^T), P. soli sp. nov. (type strain LMG 31014^T = CCUG 73382^T), and P. terrigena sp. nov. (type strain LMG 31013^T = CCUG 73381^T).

While the epidemiology of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients suggests that Burkholderia multivorans is acquired from environmental sources, this species has rarely been isolated from soil and water samples. Multiple isolation strategies were applied to water and soil samples that were previously shown to be B. multivorans PCR positive. These included direct plating and liquid enrichment procedures and the use of selective media, acclimatizing recovery and co-cultivation with CF sputum. MALDI-TOF mass spectrometry and sequence analysis of 16S rRNA and housekeeping genes were used to identify all isolates. None of the approaches yielded B. multivorans isolates. Other Burkholderia species, several Gram-negative non-fermenting bacteria (including Cupriavidus, Inquilinus, Pandoraea, Pseudomonas and Stenotrophomonas) and rapidly growing mycobacteria (including Mycobacterium chelonae) were all isolated from water and soil samples. The use of Bcc isolation media yielded a surprisingly wide array of rare but often clinically relevant CF pathogens, confirming that soil and water are reservoirs of these infectious agents.

Antimicrobial resistance in Gram-negative pathogens represents a global threat to human health. This study determines the antimicrobial potential of a taxonomically and geographically diverse collection of 263 Burkholderia (sensu lato) isolates and applies natural product dereplication strategies to identify potentially novel molecules. Antimicrobial activity is almost exclusively present in Burkholderia sensu stricto bacteria and rarely observed in the novel genera Paraburkholderia, Caballeronia, Robbsia, Trinickia, and Mycetohabitans. Fourteen isolates show a unique spectrum of antimicrobial activity and inhibited carbapenem-resistant Gram-negative bacterial pathogens. Dereplication of the molecules present in crude spent agar extracts identifies 42 specialized metabolites, 19 of which represented potentially novel molecules. The known identified Burkholderia metabolites include toxoflavin, reumycin, pyrrolnitrin, enacyloxin, bactobolin, cepacidin, ditropolonyl sulfide, and antibiotics BN-227-F and SF 2420B, as well as the siderophores ornibactin, pyochelin, and cepabactin. Following semipreparative fractionation and activity testing, a total of five potentially novel molecules are detected in active fractions. Given the molecular formula and UV spectrum, two of those putative novel molecules are likely related to bactobolins, and another is likely related to enacyloxins. The results from this study confirm and extend the observation that Burkholderia bacteria present exciting opportunities for the discovery of potentially novel bioactive molecules.

Burkholderia bacteria are historically known for their biocontrol properties and have been proposed as a promising and underexplored source of bioactive specialized metabolites. Burkholderia cepacia complex strain R-12632 inhibits various Gram-positive and Gram-negative resistant pathogens and produces numerous specialized metabolites, among which is ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remains unknown.