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Twenty-eight multidrug-resistant bacterial strains closely related or identical to  Pedobacter cryoconitis ,  Pedobacter lusitanus  and  Pedobacter steynii  were isolated from soil samples by selection for multidrug-resistance. Approximately 3–30% of the selected isolates were identified as  Pedobacter , whereas isolation without antibiotics did not yield any isolates of this genus. Next generation sequencing data showed  Pedobacter  to be on 69th place among the bacterial genera (0.32% of bacterial sequences). The  Pedobacter  isolates produced a wide array of novel compounds when screened by UHPLC-MS/MSMS, and hierarchical cluster analysis resulted in several distinct clusters of compounds produced by specific isolates of  Pedobacter , and most of these compounds were found to be peptides. The  Pedobacter  strain UP508 produced isopedopeptins, whereas another set of strains produced pedopeptins, which both are known cyclic lipodepsipeptides produced by  Pedobacter  sp. Other  Pedobacter  strains produced analogous peptides with a sequence variation. Further strains of  Pedobacter  produced additional novel antibacterial cyclic lipopeptides (ca 800 or 1400 Da in size) and/or linear lipopeptides (ca 700–960 Da in size). A 16S rRNA phylogenetic tree for the  Pedobacter  isolates revealed several distinct clades and subclades of isolates. One of the subclades comprised isolates producing isopedopeptin analogs, but the isopedopeptin producing isolate UP508 was clearly placed on a separate branch. We suggest that the non-ribosomal peptide synthases producing pedopeptins, isopedopeptins, and the analogous peptides, may derive from a common ancestral non-ribosomal peptide synthase gene cluster, which may have been subjected to a mutation leading to changed specificity in one of the modules and then to a modular rearrangement leading to the changed sequence found in the isopedopeptins produced by isolate UP508.

Selective Isolation of Multidrug-Resistant Pedobacter spp., Producers of Novel Antibacterial Peptides