Biology subjects

Brothers, K. M.

Publications and source records attributed to Brothers, K. M..

4 recordsLinked to original sources

bioRxiv2023

Intra-ocular Predation of Fluoroquinolone-Resistant Pseudomonas aeruginosa and Serratia marcescens by the Predatory Bacteria

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand White rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and S. marcescens. However, it was not able to significantly reduce S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data suggest in vivo predation of Gram-negative bacteria within the intra-ocular environment.

microbiology↗

bioRxiv2022

IgaA protein, GumB, has a global impact on the transcriptome and surface proteome of Serratia marcescens

Bacterial stress response signaling systems, like the Rcs system, can be triggered by membrane and cell wall damaging compounds including antibiotics and innate immune system factors. These regulatory systems help bacteria survive envelope stress by altering the transcriptome resulting in protective phenotypic changes that may also the influence the virulence of the bacterium. This study investigated the role of the Rcs stress response system using a clinical keratitis isolate of S. marcescens with a mutation in the gumB gene. GumB, an IgaA ortholog, inhibits activation of the Rcs system, such that mutants have overactive Rcs signaling. Transcriptomic analysis indicated that approximately 15% of all S. marcescens genes were significantly altered with two-fold or greater changes in expression in the {Delta}gumB mutant compared to the wild type indicating a global transcriptional regulatory role for GumB. We further investigated the phenotypic consequences of two classes of genes with altered expression in the {Delta}gumB mutant expected to contribute to infections: serralysin metalloproteases PrtS, SlpB and SlpE, and type I pili coded by fimABCD. Secreted fractions from the {Delta}gumB mutant had reduced cytotoxicity to a corneal cell line, and could be complemented by induced expression of prtS, but not cytolysin shlBA, phospholipase phlAB, or flagellar master regulator flhDC operons. Proteomic analysis, qRT-PCR, and type I pili dependent yeast agglutination indicated an inhibitory role for the Rcs system in adhesin production. Together these data demonstrate that GumB and the Rcs stress response system control S. marcescens virulence factors beyond the ShlA cytolysin. IMPORTANCEPrevious studies indicate that the bacterial Rcs system is a key regulator of envelope stress. This study demonstrated that activation of the Rcs system had a global impact on the transcriptome of a clinical isolate of S. marcescens including decreased expression of cytotoxic serralysin metalloproteases and biofilm promoting type I pili. These results give mechanistic insight into how the Rcs system contributes to pathogenesis.

microbiology↗

bioRxiv2020

Genotypic diversity between surgical and nasal Staphylococcus aureus isolates

Staphylococcus aureus is a common organism in periprosthetic joint infection (PJI). Little is known about S. aureus genetic diversity in PJI as compared to nasal carriage. We hypothesized PJI S. aureus strains would be associated with increased virulence as compared to those from nasal carriage. Whole genome sequencing and multilocus sequence typing (MLST) was performed to genotype these two populations at high resolution. MLST revealed a variety of genotypes in both populations but many belonged to the most common clonal complexes. In nasal cultures, 69% of strains were of clonal complexes CC5, CC8, and CC30. In PJI cultures, only 51% could be classified in these common clonal complexes. Remaining strains were atypical, and these atypical strains in PJI were associated with poor host status and compromised immune conditions. Mutations in genes involved in fibronectin binding (ebh, fnbA, clfA, clfB) systematically distinguished later PJI isolates from the first PJI isolate from each patient. S. aureus isolated from nasal carriage and PJI specimens differ significantly, with the latter being more diverse. Strains associated with lower pathogenicity tended to be found in immunocompromised patients, suggesting the host immune system plays an important role in preventing PJI. Repeated mutations in S. aureus genes associated with extracellular matrix binding were identified suggesting an adaptive, parallel evolution in S. aureus during the development of PJI.

microbiology↗

bioRxiv2020

Generation of Xylose-inducible promoter tools for Pseudomonas species and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and P. fluorescens. The Pxut promoter derived from the P. flurorescens xut operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. GFP-fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate cloning of toxic genes using E. coli to generate plasmids. The Pxut promoter was expressed at a lower inducer concentration than PBAD in P. fluorescens and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was more leaky than PBAD in the tested Pseudomonas species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of P. aeruginosa or P. fluorescens as a sole carbon source and is less expensive than many other commonly used inducers which could facilitate large scale applications. The efficacy of this system aided in demonstrating a role for the P. aeruginosa type II secretion system gene from xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species. ImportancePseudomonas species are enormously important in human infections, biotechnology, and as a model system for interrogating basic science questions. In this study we have developed a xylose-inducible promoter system and evaluated it in P. aeruginosa and P. fluorescens and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type 2 secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

microbiology↗