Gibbs, K. | bioRxiv Science

bioRxiv2019

Complete genome screening of clinical MRSA isolates identifies lineage diversity and provides full resolution of transmission and outbreak events

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices, but most applications are focused on conserved core genomic regions due to limitations of short-read technologies. In this study we established a long-read technology-based WGS screening program of all first-episode MRSA blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads revealed widespread gain/loss of accessory mobile genetic elements among established hospital- and community-associated lineages impacting >10% of each genome, and frequent megabase-scale inversions between endogenous prophages. We also characterized an outbreak of a CC5/ST105/USA100 clone among 3 adults and 18 infants in a neonatal intensive care unit (NICU) lasting 7 months. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its origins and progression, which was characterized by multiple sub-transmissions and likely precipitated by equipment sharing. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance and persistence. This included a DNA-recognition domain recombination in the hsdS gene of a Type-I restriction-modification system that altered DNA methylation. RNA-Seq profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall our findings demonstrate that long-read sequencing substantially improves our ability to characterize accessory genomic elements that impact MRSA virulence and persistence, and provides valuable information for infection control efforts.

microbiology↗

bioRxiv2017

The Proteus mirabilis rffG gene is essential for swarm-associated cell elongation through an Rcs-mediated pathway

Individual cells of the bacterium Proteus mirabilis can elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarming. How cells regulate this dramatic morphological remodeling remains an open question. In this paper, we move forward the understanding of this regulation by demonstrating that P. mirabilis requires the gene rffG for swarmer cell elongation and subsequent swarm motility. The rffG gene encodes a protein homologous to the dTDP-glucose 4,6 dehydratase protein of Escherichia coli, which contributes to Enterobacterial Common Antigen biosynthesis. Here we characterize the rffG gene in P. mirabilis, demonstrating that it is required for the production of large lipopolysaccharide-linked moieties necessary for wild-type cell envelope integrity. We show that absence of the rffG gene induces several stress-responsive pathways including those controlled by the transcriptional regulators RpoS, CaiF, and RcsB. We further show that in rffG-deficient cells, suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to induce cell elongation and swarm motility. However, loss of RcsB does not rescue cell envelope integrity defects and instead results in abnormally shaped cells, including cells producing more than two poles. We conclude that a RcsB-mediated response acts to suppress emergence of shape defects in cell envelope-compromised cells, suggesting an additional role for RcsB in maintaining cell morphology under stress conditions. We further propose that the composition of the cell envelope acts as a checkpoint before cells initiate swarmer cell elongation and motility.\n\nImportance statementP. mirabilis swarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multi-functional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, the rffG-dependent moieties provide a novel, attractive target for potential antimicrobials.

microbiology↗