Understanding the pathophysiology of the coronavirus disease 2019 (COVID-19) infection remains a significant challenge of our times. The gingival crevicular fluid being representative of systemic status and having a proven track record of detecting viruses and biomarkers forms a logical basis for evaluating the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The study aimed to assess gingival crevicular fluid (GCF) for evidence of SARS-CoV-2 in 33 patients who were deemed to be COVID-19 positive upon nasopharyngeal sampling. An attempt was also made to comparatively evaluate it with saliva in terms of its sensitivity, as a diagnostic fluid for SARS-CoV-2. GCF and saliva samples were collected from 33 COVID-19–confirmed patients. Total RNA was extracted using NucliSENS easyMAG (bioMérieux) and eluted in the elution buffer. Envelope gene ( E gene) of SARS-CoV-2 and human RNase P gene as internal control were detected in GCF samples by using the TRUPCR SARS-CoV-2 RT qPCR kit V-2.0 (I) in an Applied Biosystems 7500 real-time machine. A significant majority of both asymptomatic and mildly symptomatic patients exhibited the presence of the novel coronavirus in their GCF samples. Considering the presence of SARS-CoV-2 RNA in the nasopharyngeal swab sampling as gold standard, the sensitivity of GCF and saliva, respectively, was 63.64% (confidence interval [CI], 45.1% to 79.60%) and 64.52% (CI, 45.37% to 80.77%). GCF was found to be comparable to saliva in terms of its sensitivity to detect SARS-CoV-2. Saliva samples tested positive in 3 of the 12 patients whose GCF tested negative, and likewise GCF tested positive for 2 of the 11 patients whose saliva tested negative on real-time reverse transcription polymerase chain reaction. The results establish GCF as a possible mode of transmission of SARS-CoV-2, which is the first such report in the literature, and also provide the first quantifiable evidence pointing toward a link between the COVID-19 infection and oral health.

The use of efflux pump inhibitors (EPIs) as potentiators along with the traditional antibiotics assists in the warfare against antibiotic-resistant superbugs. Efflux pumps of the resistance-nodulation-cell division (RND) family play crucial roles in multidrug resistance in Escherichia coli and Pseudomonas aeruginosa. Despite several efforts, clinically useful inhibitors are not available at present. This study describes ethyl 4-bromopyrrole-2-carboxylate (RP1) isolation, an inhibitor of RND transporters from the library of 4000 microbial exudates. RP1 acts synergistically with antibiotics by reducing their minimum inhibitory concentration in strains overexpressing archetype RND transporters (AcrAB-TolC and MexAB-OprM). It also improves the accumulation of Hoechst 33342 and inhibits its efflux (a hallmark of EPI functionality). The antibiotic-RP1 combinations prolong the postantibiotic effects and reduce the mutation prevention concentration of antibiotics. Additionally, from Biolayer Interferometry spectra, it appears that RP1 is bound to AcrB. RP1 displays low mammalian cytotoxicity, no Ca2+ channel inhibitory effects, and reduces the intracellular invasion of E. coli and P. aeruginosa in macrophages. Furthermore, the RP1-levofloxacin combination is nontoxic, well-tolerated, and notably effective in a murine lung infection model. In sum, RP1 is a potent EPI and worthy of further consideration as a potentiator to improve the effectiveness of existing antibiotics.

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Efflux pumps are always at the forefront of bacterial multidrug resistance and account for the failure of antibiotics. The present study explored the potential of 2-(2-Aminophenyl) indole (RP2), an efflux pump inhibitor (EPI) isolated from the soil bacterium, to overcome the efflux-mediated resistance in Staphylococcus aureus. The RP2/antibiotic combination was tested against efflux pump over-expressed S. aureus strains. The compound was further examined for the ethidium bromide (EtBr) uptake and efflux inhibition assay (a hallmark of EPI functionality) and cytoplasmic membrane depolarization. The safety profile of RP2 was investigated using in vitro cytotoxicity assay and Ca 2+ channel inhibitory effect. The in vivo efficacy of RP2 was studied in an animal model in combination with ciprofloxacin. RP2 exhibited the synergistic activity with several antibiotics in efflux pump over-expressed strains of S. aureus. In the mechanistic experiments, RP2 increased the accumulation of EtBr, and demonstrated the inhibition of its efflux. The antibiotic-EPI combinations resulted in extended post antibiotic effects as well as a decrease in mutation prevention concentration of antibiotics. Additionally, the in silico docking studies suggested the binding of RP2 to the active site of modeled structure of NorA efflux pump. The compound displayed low mammalian cytotoxicity and had no Ca 2+ channel inhibitory effect. In ex vivo experiments, RP2 reduced the intracellular invasion of S. aureus in macrophages. Furthermore, the RP2/ciprofloxacin combination demonstrated remarkable efficacy in a murine thigh infection model. In conclusion, RP2 represents a promising candidate as bacterial EPI, which can be used in the form of a novel therapeutic regimen along with existing and upcoming antibiotics, for the eradication of S. aureus infections.

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