This work comprehensively characterizes lipidomic changes underlying daptomycin resistance in three Gram-positive bacterial species, E. faecalis, S. aureus, and C. striatum, by using a novel three-dimensional lipidomics methodology based on advanced mass spectrometry. We demonstrated a number of advantages of our method in comparison with other methods commonly used in the field, such as high molecular specificity, sensitivity, and throughput. Whole-genome sequencing of the S. aureus and C. striatum strains identified mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, in both resistant strains. Lipidomics revealed significantly decreased levels of lipids downstream of PgsA, as well as accumulation of lipids upstream of PgsA in the resistant strains. Furthermore, we found that changes in individual molecular species of each lipid class depend on the their specific fatty acid compositions. The characteristic changes in individual lipid species could be used as biomarkers for identifying underlying resistance mechanisms and for evaluating potential therapies.

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI. While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.

Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureus genome. We found that EF2132 can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureus strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureus genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds.

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order.

Objectives: Dalbavancin is a long-acting lipoglycopeptide with activity against gram-positives, including methicillin-resistant Staphylococcus aureus (MRSA). The potential for lipoglycopeptides, with half-lives greater than 1 week, to select for resistance is unknown. Here we explore a case of MRSA central lineassociated bloodstream infection in which dalbavancin and vancomycin non-susceptibility emerged in a urine isolate collected after the patient was treated with vancomycin and dalbavancin sequentially. Methods: Isolates from blood and urine underwent susceptibility testing, and whole genome sequencing (WGS). The blood isolate was subjected to successive passage in vitro in the presence of escalating dalbavancin concentrations and the emergent isolate was subjected to repeat susceptibility testing and WGS. Results: The blood isolate was fully susceptible to vancomycin; however, MICs of the urine isolate to dalbavancin, vancomycin, telavancin, and daptomycin were at least fourfold higher than the bloodderived strain. Both strains were indistinguishable by spa and variable number tandem repeat (VNTR) typing, and WGS revealed only seven variants, indicating clonality. Four variants affected genes, including a 3bp in-frame deletion in yvqF, a gene which has been implicated in glycopeptide resistance. Vancomycin and dalbavancin non-susceptibility emerged in the blood isolate after successive passage in vitro in the presence of dalbavancin, and WGS identified a single non-synonymous variant in yvqF. Conclusions: This is the first case in which VISA has emerged in the context of a dalbavancin-containing regimen. The selection for cross-resistance to vancomycin in vitro by dalbavancin exposure alone is troubling. Clinicians should be aware of the possibility for emergence of dalbavancin non-susceptibility and glycopeptide cross-resistance arising following therapy.