Hydroquinine Enhances the Efficacy of Contact Lens Solutions for Inhibiting Pseudomonas aeruginosa Adhesion and Biofilm Formation.

P. aeruginosa is one of the most common bacteria causing contact lens-related microbial keratitis (CLMK). Previous studies report that disinfecting solutions were ineffective in preventing biofilm formation. Solutions containing novel natural agents may be an excellent alternative for reducing the risk of CLMK. Here, we investigate the disinfecting properties of hydroquinine in combination with multipurpose solutions (MPSs) to prevent P. aeruginosa adhesion and biofilm formation. We examined the antibacterial, anti-adhesion, and anti-biofilm properties of hydroquinine-formulated MPSs compared to MPSs alone. Using RT-qPCR, hydroquinine directly affected the expression levels of adhesion-related genes, namely, cgrC, cheY, cheZ, fimU, and pilV, resulting in reduced adhesion and anti-biofilm formation. Using ISO 14729 stand-alone testing, hydroquinine met the criteria (>99.9% killing at disinfection time) against both P. aeruginosa reference and clinical strains. Using the crystal violet retention assay and FE-SEM, MPSs combined with hydroquinine were effective in inhibiting P. aeruginosa adhesion and destroying preexisting biofilms. This report is the first to highlight the potential utility of hydroquinine-containing formulations as a disinfecting solution for contact lenses, specifically for inhibiting adhesion and destroying biofilm. These findings may aid in the development of novel disinfectants aimed at combating P. aeruginosa, thereby potentially reducing the incidence of CLMK.

A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa.

The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as "effluxR detection assay" using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04-34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.

Hydroquinine Inhibits the Growth of Multidrug-Resistant Pseudomonas aeruginosa via the Suppression of the Arginine Deiminase Pathway Genes.

Hydroquinine has antimicrobial potential with demonstrated activity against several bacteria, including multidrug-resistant (MDR) P. aeruginosa reference strains. Despite this, there is limited evidence confirming the antibacterial activity of hydroquinine against clinical isolates and the underlying mechanism of action. Here, we aimed to investigate the antibacterial effect of hydroquinine in clinical P. aeruginosa strains using phenotypic antimicrobial susceptibility testing and synergistic testing. In addition, we examined the potential inhibitory mechanisms against MDR P. aeruginosa isolates using informatic-driven molecular docking analysis in combination with RT-qPCR. We uncovered that hydroquinine inhibits and kills clinical P. aeruginosa at 2.50 mg/mL (MIC) and 5.00 mg/mL (MBC), respectively. Hydroquinine also showed partial synergistic effects with ceftazidime against clinical MDR P. aeruginosa strains. Using SwissDock, we identified potential interactions between arginine deiminase (ADI)-pathway-related proteins and hydroquinine. Furthermore, using RT-qPCR, we found that hydroquinine directly affects the mRNA expression of arc operon. We demonstrated that the ADI-related genes, including the arginine/ornithine antiporter (arcD) and the three enzymes (arginine deiminase (arcA), ornithine transcarbamylase (arcB), and carbamate kinase (arcC)), were significantly downregulated at a half MIC of hydroquinine. This study is the first report that the ADI-related proteins are potential molecular targets for the inhibitory effect of hydroquinine against clinically isolated MDR P. aeruginosa strains.

High-Throughput Transcriptomic Profiling Reveals the Inhibitory Effect of Hydroquinine on Virulence Factors in Pseudomonas aeruginosa.

Hydroquinine is an organic alkaloid compound that exhibits antimicrobial activity against several bacterial strains including strains of both drug-sensitive and multidrug-resistant P. aeruginosa. Despite this, the effects of hydroquinine on virulence factors in P. aeruginosa have not yet been characterized. We therefore aimed to uncover the mechanism of P. aeruginosa hydroquinine-sensitivity using high-throughput transcriptomic analysis. We further confirmed whether hydroquinine inhibits specific virulence factors using RT-qPCR and phenotypic analysis. At half the minimum inhibitory concentration (MIC) of hydroquinine (1.250 mg/mL), 254 genes were differentially expressed (97 downregulated and 157 upregulated). We found that flagellar-related genes were downregulated by between −2.93 and −2.18 Log2-fold change. These genes were consistent with the analysis of gene ontology and KEGG pathway. Further validation by RT-qPCR showed that hydroquinine significantly suppressed expression of the flagellar-related genes. By analyzing cellular phenotypes, P. aeruginosa treated with ½MIC of hydroquinine exhibited inhibition of motility (30−54% reduction) and pyocyanin production (~25−27% reduction) and impaired biofilm formation (~57−87% reduction). These findings suggest that hydroquinine possesses anti-virulence factors, through diminishing flagellar, pyocyanin and biofilm formation.

Hydroquinine Possesses Antibacterial Activity, and at Half the MIC, Induces the Overexpression of RND-Type Efflux Pumps Using Multiplex Digital PCR in Pseudomonas aeruginosa.

Hydroquinine is an organic compound that is closely related to quinine-derivative drugs and contains anti-malarial and anti-arrhythmia activities. It has been also found in abundance in some natural extracts that possess antibacterial properties. However, there is little evidence demonstrating the antibacterial effect of hydroquinine. Therefore, we aimed to investigate the antibacterial properties of hydroquinine using broth microdilution methods. In addition, we evaluated the transcriptional responses of P. aeruginosa to hydroquinine-induced stress using RNA sequencing with transcriptomic analysis and validated the results using PCR-based methods. The MIC and MBC values of hydroquinine against all eight bacterial strains investigated ranged from 650 to 2500 and from 1250 to 5000 µg/mL, respectively. Transcriptomic analysis demonstrated that RND efflux pump transcripts were overexpressed (4.90−9.47 Log2 fold change). Using mRT-dPCR and RT-qPCR, we identified that mRNA levels of mexD and mexY genes were overexpressed in response to just half the MIC of hydroquinine in P. aeruginosa. In conclusion, we uncover the antimicrobial potential of hydroquinine as well as identify changes in gene expression that may contribute to bacterial resistance. Further work will be required to explore the efficacy and potential use of hydroquinine in the clinic.