Biology subjects

Shanks, R. M. Q.

Publications and source records attributed to Shanks, R. M. Q..

7 recordsLinked to original sources

bioRxiv2026

A tetracycline-inducible promoter for gene expression in Gram-negative bacteria including Acinetobacter baumannii

Carbohydrate inducible promoters on expression plasmids are widely used for biotechnology and basic science research. However, using carbohydrates for inducers has drawbacks including influencing bacterial metabolism, the inducer being used as a carbon source by the bacteria, high background expression, and only a subset of cells experiencing promoter activation. Furthermore, some bacteria including the pathogen Acinetobacter baumannii currently lack good carbohydrate-inducible promoters. To address this issue, a tetracycline-inducible promoter, PtetB, isolated from A. baumannii was cloned and evaluated in a number of important bacteria: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. A highly sensitive luxCDABE reporter was used to evaluate promoter activity. S. marcescens strains with elevated and reduced cyclic-AMP levels were used to evaluate the impact of cAMP on PtetB function. The PtetB promoter was induced over 800-fold in A. baumannii and 10-750-fold inducible in the other tested species. Background expression was lower from PtetB in the pseudomonads than previously described carbohydrate inducible promoters. Data indicated that the PtetB promoter system may be a useful tool for future basic science research with the tested bacteria.

microbiology↗

bioRxiv2026

Oxazolidinone antibiotics exhibit potent in vitro activity against Gram-positive bacterial endophthalmitis isolates

Purpose: Vancomycin-resistant Gram-positive organisms represent a potential therapeutic challenge in endophthalmitis. To evaluate alternative treatment options, the FDA-approved oxazolidinones linezolid (LZD) and tedizolid (TZD) were tested against a collection of Gram-positive bacterial endophthalmitis isolates. Methods: Minimum inhibitory concentrations (MICs) were determined using epsilometers (Etest strips) for coagulase negative staphylococci (CoNS, n=20), Staphylococcus aureus (n=18), Bacillus species (n=10), Enterococcus species (n=5), and Streptococcus pneumoniae (n=1). Available CLSI and EUCAST breakpoints were applied to determine susceptibility. Results: TZD exhibited a >5-fold reduction in median MIC compared with LZD across all organisms tested (p<0.0001). MIC90 values for CoNS were 2.0 /ml for LZD and 0.25 /ml for TZD. For S. aureus, MIC90 values were 1.5 /ml (LZD) and 0.19 /ml (TZD). For Bacillus spp., MIC90 values were 1.0 /ml (LZD) and 0.25 /ml (TZD). Among Enterococcus species, the MIC range was 0.5-4.0 /ml (LZD) and 0.064-0.75 /ml (TDZ). The single S. pneumoniae strain had a MIC of 0.5 for LZD and 0.064 for TZD. All isolates were susceptible to TZD. One Enterococcus isolate was non-susceptible to LZD; the remaining isolates were susceptible. Conclusions: LZD and TZD demonstrated in vitro activity against Gram-positive bacterial endophthalmitis isolates, with TZD displaying consistently lower MICs than LZD. These findings suggest that oxazolidinones may represent potential alternatives when vancomycin cannot be used, including infections caused by vancomycin-resistant Gram-positive organisms. Given their excellent oral bioavailability, favorable safety profile, and reported anti-virulence effects in certain pathogens, further evaluation of oxazolidinones for the treatment of intraocular infections is warranted.

microbiology↗

bioRxiv2026

C. mastitidis requires the protein Sortase F to colonize the eye

The ocular surface is a mucosal tissue that is constantly exposed to environmental antigens and potential pathogens. Human microbiomes play a critical role in the balance of surveillance and inflammation at sites of colonization. Historically, the investigation of the ocular microbiome has been difficult due to its paucibacterial nature and the inhospitable environment of the ocular surface. Despite this, Corynebacterium mastitidis (C. mast) developed a unique ability to colonize the eye and elicit a protective immune response characterized by induction of IL-17 from {gamma}{delta} T cells and protection from corneal infection. Therefore, we sought to understand the unique bacterial machinery that C. mast utilizes to colonize the eye and how it affects the induction of an eye-specific immune signature. Using a C. mast transposon mutant library, we identified a mutant that completely lacked an ability to form biofilm, colonize the eye, and induce in vivo immunity. Whole genome sequencing revealed a disruption in the sortase F gene, which anchors proteins to the cell wall of C. mast, governing biofilm formation and tethering of adhesins to the cell surface. Additionally, we show that mutation in individual C. mast adhesins does not affect ocular colonization or immune induction. By understanding the molecular mechanism of ocular microbial colonization, this work advances our understanding of how bacteria colonize and induce immune responses on the eye, providing a foundation for developing novel therapeutic strategies against ocular infections.

microbiology↗

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↗

bioRxiv2023

Cefiderocol: A new and effective topical monotherapy treatment for experimental extensively-drug resistant Pseudomonas aeruginosa keratitis

PurposeTo test cefiderocol, a siderophore-cephalosporin antibiotic for topical monotherapy treatment of experimental extensively drug resistant (XDR) Pseudomonas aeruginosa keratitis. DesignPreclinical study. Subjects and ControlsDeidentified P. aeruginosa keratitis isolates, XDR P. aeruginosa from eye drop outbreak, rabbits, saline, cefiderocol 50 mg/ml, ciprofloxacin 0.3%, and tobramycin 14 mg/ml. Methods, Intervention, or TestingCefiderocol antibacterial activity against P. aeruginosa keratitis isolates (n=135) was evaluated by minimum inhibitory concentration (MIC) testing. Ocular toxicity/tolerability and antibacterial efficacy were tested in vivo with experimental rabbit models. Corneal concentrations and stability were assessed using a bioassay. Main Outcome MeasuresMIC analysis for susceptibility, graded tests for ocular toxicity/tolerability, CFU analysis for bacterial burden, corneal cefiderocol concentrations. Results100% of P. aeruginosa keratitis isolates were susceptible to cefiderocol (n=135), the MIC90 was 0.125 {micro}g/ml including the XDR isolate (MIC = 0.125 {micro}g/ml). Topical cefiderocol 50 mg/ml was minimally toxic to the ocular surface and was well tolerated. For the XDR P. aeruginosa isolate, topical cefiderocol 50 mg/ml, significantly decreased corneal CFU compared to ciprofloxacin 0.3%, tobramycin 14 mg/ml, and saline. In addition, tobramycin 14 mg/ml was more effective than the saline control. Mean cefiderocol corneal concentrations were 191x greater than the MIC90 of the P. aeruginosa keratitis isolates. Refrigerated cefiderocol maintained antimicrobial activity over a one-month period. ConclusionsThese results demonstrate that cefiderocol is well tolerated on rabbit corneas and is effective against P. aeruginosa keratitis isolates in vitro and was effective in vivo against an XDR isolate in a rabbit keratitis model. Given the recent outbreak of keratitis caused by this XDR P. aeruginosa, cefiderocol is a promising additional antibiotic that should be further evaluated for topical treatment of keratitis caused by antibiotic resistant P. aeruginosa.

microbiology↗

bioRxiv2023

Rise in frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021

Pseudomonas aeruginosa causes severe vision threatening keratitis. LasR is a transcription factor that regulates virulence associated genes in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study indicated a high proportion (22 out of 101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive and had mutations in the lasR gene, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. A significant increase in the frequency of isolates with the sheen positive phenotype was observed in isolates between 1993 and 2021. Extracellular protease activity was lower among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Insertion elements were present in a subset of independent isolates and may represent an endemic source from some of the isolates. Retrospective analysis of patient information suggested significantly better visual outcomes for patients with infected by sheen positive isolates. Together, these results indicate an increasing trend towards lasR mutations among keratitis isolates at a tertiary eye care hospital in the United States.

microbiology↗

bioRxiv2023

Predatory Bacteria can Reduce Pseudomonas aeruginosa Induced Corneal Perforation and Proliferation in a Rabbit Keratitis Model

PurposePseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. MethodsRabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic {Delta}lasR mutant and co-injected with PBS or B. bacteriovorus. After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. ResultsWe observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n=24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n=25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The {Delta}lasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus. ConclusionThese studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.

microbiology↗