Burkholderia bacteria produce multiple potentially novel molecules that inhibit carbapenem-resistant gram-negative bacterial pathogens

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

Network analysis based on unique spectral features enables an efficient selection of genomically diverse operational isolation units

Culturomics-based bacterial diversity studies benefit from the implementation of MALDI-TOF MS to remove genomically redundant isolates from isolate collections. We previously introduced SPeDE, a novel tool designed to dereplicate spectral datasets at an infraspecific level into operational isolation units (OIUs) based on unique spectral features. However, biological and technical variation may result in methodology-induced differences in MALDI-TOF mass spectra and hence provoke the detection of genomically redundant OIUs. In the present study, we used three datasets to analyze to which extent hierarchical clustering and network analysis allowed to eliminate redundant OIUs obtained through biological and technical sample variation and to describe the diversity within a set of spectra obtained from 134 unknown soil isolates. Overall, network analysis based on unique spectral features in MALDI-TOF mass spectra enabled a superior selection of genomically diverse OIUs compared to hierarchical clustering analysis and provided a better understanding of the inter-OIU relationships

Comparative genomics of Pandoraea, a genus enriched in xenobiotic biodegradation and metabolism

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))

Burkholderia cepacia complex taxon K : where to split?

The objective of the present study was to provide an updated classification for Burkholderia cepacia complex (Bcc) taxon K isolates. A representative set of 39 taxon K isolates were analyzed through multilocus sequence typing (MLST) and phylogenomic analyses. MLST analysis revealed the presence of at least six clusters of sequence types (STs) within taxon K, two of which contain the type strains of Burkholderia contaminans (ST-102) and Burkholderia lata (ST-101), and four corresponding to the previously defined taxa Other Bcc groups C, G, H and M. This clustering was largely supported by a phylogenomic tree which revealed three main clades. Isolates of B. contaminans and of Other Bcc groups C, G, and H represented a first clade which generally shared average nucleotide identity (ANI) and average digital DNA-DNA hybridization (dDDH) values at or above the 95–96% ANI and 70% dDDH thresholds for species delineation. A second clade consisted of Other Bcc group M bacteria and of four B. lata isolates and was supported by average ANI and dDDH values of 97.2 and 76.1% within this clade and average ANI and dDDH values of 94.5 and 57.2% toward the remaining B. lata isolates (including the type strain), which represented a third clade. We therefore concluded that isolates known as Other Bcc groups C, G, and H should be classified as B. contaminans, and propose a novel species, Burkholderia aenigmatica sp. nov., to accommodate Other Bcc M and B. lata ST-98, ST-103, and ST-119 isolates. Optimized MALDI-TOF MS databases for the identification of clinical Burkholderia isolates may provide correct species-level identification for some of these bacteria but would identify most of them as B. cepacia complex. MLST facilitates species-level identification of many taxon K strains but some may require comparative genomics for accurate species-level assignment. Finally, the inclusion of Other Bcc groups C, G, and H into B. contaminans affects the phenotype of this species minimally and the proposal to classify Other Bcc group M and B. lata ST-98, ST-103, and ST-119 strains as a novel Burkholderia species is supported by a distinctive phenotype, i.e., growth at 42°C and lysine decarboxylase activity

Introducing SPeDE : high-throughput dereplication and accurate determination of microbial diversity from matrix-assisted laser desorption-ionization time of flight mass spectrometry data

The isolation of microorganisms from microbial community samples often yields a large number of conspecific isolates. Increasing the diversity covered by an isolate collection entails the implementation of methods and protocols to minimize the number of redundant isolates. Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry methods are ideally suited to this dereplication problem because of their low cost and high throughput. However, the available software tools are cumbersome and rely either on the prior development of reference databases or on global similarity analyses, which are inconvenient and offer low taxonomic resolution. We introduce SPeDE, a user-friendly spectral data analysis tool for the dereplication of MALDI-TOF mass spectra. Rather than relying on global similarity approaches to classify spectra, SPeDE determines the number of unique spectral features by a mix of global and local peak comparisons. This approach allows the identification of a set of nonredundant spectra linked to operational isolation units. We evaluated SPeDE on a data set of 5,228 spectra representing 167 bacterial strains belonging to 132 genera across six phyla and on a data set of 312 spectra of 78 strains measured before and after lyophilization and subculturing. SPeDE was able to dereplicate with high efficiency by identifying redundant spectra while retrieving reference spectra for all strains in a sample. SPeDE can identify distinguishing features between spectra, and its performance exceeds that of established methods in speed and precision. SPeDE is open source under the MIT license and is available from https://github.com/LM-UGent/SPeDE. IMPORTANCE Estimation of the operational isolation units present in a MALDI-TOF mass spectral data set involves an essential dereplication step to identify redundant spectra in a rapid manner and without sacrificing biological resolution. We describe SPeDE, a new algorithm which facilitates culture-dependent clinical or environmental studies. SPeDE enables the rapid analysis and dereplication of isolates, a critical feature when long-term storage of cultures is limited or not feasible. We show that SPeDE can efficiently identify sets of similar spectra at the level of the species or strain, exceeding the taxonomic resolution of other methods. The high-throughput capacity, speed, and low cost of MALDI-TOF mass spectrometry and SPeDE dereplication over traditional gene marker-based sequencing approaches should facilitate adoption of the culturomics approach to bacterial isolation campaigns

Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity

In many countries, Burkholderia multivorans is the most prevalent species within the Burkholderia cepacia complex (Bcc) found infecting the lungs of patients with cystic fibrosis (CF). Its positive identification is of immediate concern to the health of the patient as it is notoriously hard to eradicate using antibiotics and can cause necrosis of the lung tissues (cepacia syndrome). Infection control measures reduced the prevalence of B. cenocepacia in CF wards, but patients continue to acquire infections by B. multivorans from environmental sources. In most reported cases, the infecting strains are unique except in rare cases in which cross-infection is observed between patients. We report here an endemic strain of B. multivorans with sequence type ST-742 that has been infecting multiple patients, without evidence for cross-infection. We investigated the epidemiology and genomics of this ST-742 strain and show that it is microdiverse, as isolates between-patients exhibit numerous genomic differences, at scales that have not been observed previously when looking at evolutionary trajectories within-patients. Additionally, we found that the specific genomic background of a given strain may dictate the strategy of adaptation within the CF lung. Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred Kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung

Paraburkholderia gardini sp. nov. and Paraburkholderia saeva sp. nov. : novel aromatic compound degrading bacteria isolated from garden and forest soil samples

Three forest and four botanical garden top soil isolates with unique MALDI-TOF mass spectra were identified as Paraburkholderia strains closely related to Paraburkholderia sartisoli through recA gene sequence analysis. OrthoANIu, digital DNA-DNA hybridization analyses and phylogenomic analyses demonstrated that the five strains represented two new Paraburkholderia species closely related to P. sartisoli. The genome of strain LMG 31841(T) had a cumulative size of 6.3 Mb and a G + C content of 62.64 mol%; strain LMG 32171(T) had a genome size of 5.8 Mb and a G + C content of 62.91 mol%. Hemolysis on horse blood agar, beta-galactosidase and phosphoamidase activity, and assimilation of adipic acid and trisodium citrate allowed phenotypic differentiation of strains LMG 31841(T), LMG 32171(T) and P. sartisoli LMG 24000(T). An analysis of the genomic potential for aromatic compound degradation yielded additional differences among strains representing these three species, but also highlighted some discrepancies between the presence of genes and pathways, and the phenotype revealed through growth experiments using a mineral salts medium supplemented with single aromatic compounds as carbon sources. We propose to classify all isolates from the present study into two novel Paraburkholderia species, for which we propose the names Paraburkholderia gardini with LMG 32171(T)(=CECT 30344(T)) as the type strain, and Paraburkholderia saeva with LMG 31841(T) (=CECT 30338(T)) as the type strain. (C)& nbsp;2022 Elsevier GmbH. All rights reserved

Genomewide screening for genes involved in biofilm formation and miconazole susceptibility in Saccharomyces cerevisiae

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms

Antibacterial activity of a lectin-like Burkholderia cenocepacia protein

Bacteriocins of the LlpA family have previously been characterized in the γ-proteobacteria Pseudomonas and Xanthomonas. These proteins are composed of two MMBL (monocot mannose-binding lectin) domains, a module predominantly and abundantly found in lectins from monocot plants. Genes encoding four different types of LlpA-like proteins were identified in genomes from strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. A selected recombinant LlpA-like protein from the human isolate Burkholderia cenocepacia AU1054 displayed narrow-spectrum genus-specific antibacterial activity, thus representing the first functionally characterized bacteriocin within this β-proteobacterial genus. Strain-specific killing was confined to other members of the Burkholderia cepacia complex, with mostly Burkholderia ambifaria strains being susceptible. In addition to killing planktonic cells, this bacteriocin also acted as an anti-biofilm agent.status: publishe