GS-2: A Novel Broad-Spectrum Agent for Environmental Microbial Control.

The environmental control of microbial pathogens currently relies on compounds that do not exert long-lasting activity on surfaces, are impaired by soil, and contribute to the growing problem of antimicrobial resistance. This study presents the scientific development and characterization of GS-2, a novel, water-soluble ammonium carboxylate salt of capric acid and L-arginine that demonstrates activity against a range of bacteria (particularly Gram-negative bacteria), fungi, and viruses. In real-world surface testing, GS-2 was more effective than a benzalkonium chloride disinfectant at reducing the bacterial load on common touch-point surfaces in a high-traffic building (average 1.6 vs. 32.6 CFUs recovered from surfaces 90 min after application, respectively). Toxicology testing in rats confirmed GS-2 ingredients were rapidly cleared and posed no toxicities to humans or animals. To enhance the time-kill against Gram-positive bacteria, GS-2 was compounded at a specific ratio with a naturally occurring monoterpenoid, thymol, to produce a water-based antimicrobial solution. This GS-2 with thymol formulation could generate a bactericidal effect after five minutes of exposure and a viricidal effect after 10 min of exposure. Further testing of the GS-2 and thymol combination on glass slides demonstrated that the compound retained bactericidal activity for up to 60 days. Based on these results, GS-2 and GS-2 with thymol represent a novel antimicrobial solution that may have significant utility in the long-term reduction of environmental microbial pathogens in a variety of settings.

Disinfectants facilitate the transformation of exogenous antibiotic resistance genes via multiple pathways.

The prevalence and spread of multidrug-resistant (MDR) bacteria pose a global challenge to public health. Natural transformation is one of the essential ways for horizontal transfer of antibiotic resistance genes (ARGs). Although disinfectants are frequently used during COVID-19, little is known about whether these disinfectants are associated with the transformation of plasmid-borne ARGs. In our study, we assessed the effect of some disinfectants on bacterial transformation using resistance plasmids as extracellular DNA and E. coli DH5α as the recipient bacteria. The results showed that these disinfectants at environmentally relevant concentrations, including benzalkonium bromide (BB), benzalkonium chloride (BC) and polyhexamethylene guanidine hydrochloride (PHMG), significantly enhanced the transformation of plasmid-encoded ARGs. Furthermore, we investigated the mechanisms underlying the promotive effect of disinfectants on transformation. We revealed that the addition of disinfectants significantly increased the membrane permeability and promoted membrane-related genes expression. Moreover, disinfectants led to the boosted bacterial respiration, ATP production and flagellum motility, as well as increased expression of bacterial secretion system-related genes. Together, our findings shed insights into the spread of ARGs through bacterial transformation and indicate potential risks associated with the widespread use of disinfectants.

Exposure to benzalkonium chloride disinfectants promotes antibiotic resistance in sewage sludge microbiomes.

Disinfectants are routinely used in human environments to control and prevent the transmission of microbial disease, and this is particularly true during the current COVID-19 crisis. However, it remains unclear whether the increased disinfectant loadings to wastewater treatment plants facilitate the dissemination of antibiotic resistance genes (ARGs) in sewage sludge microbiomes. Here, we investigated the impacts of benzalkonium chlorides (BACs), widely used disinfectants, on ARGs profiles and microbial community structures in sewage sludge by using high-throughput quantitative PCR and Illumina sequencing. A total of 147 unique ARGs and 39 mobile genetic elements (MGEs) were detected in all sewage sludge samples. Our results show that exposure to BACs disinfectants at environmentally relevant concentrations significantly promotes both the diversity and absolute abundance of ARGs in sludge microbiomes, indicating the co-selection of ARGs by BACs disinfectants. The enrichment of ARGs abundance varied from 2.15-fold to 3.63-fold compared to controls. In addition, BACs exposure significantly alters bacterial and protistan communities, resulting in dysbiosis of the sludge microbiota. The Mantel test and Procrustes analysis confirm that bacterial communities are significantly correlated with ARGs profiles under BACs treatments. The structural equation model explains 83.8 % of the total ARGs variation and further illustrates that the absolute abundance of MGEs exerts greater impacts on the variation of absolute abundance of ARGs than microbial communities under BACs exposure, suggesting BACs may promote antibiotic resistance by enhancing the horizontal gene transfer of ARGs across sludge microbiomes. Collectively, our results provide new insights into the proliferation of antibiotic resistance through disinfectant usage during the pandemic and highlight the necessity to minimize the environmental release of disinfectants into the non-target environment for combating antibiotic resistance.

Short-term exposure to benzalkonium chloride in bacteria from activated sludge alters the community diversity and the antibiotic resistance profile.

The continuous introduction of cleaning products containing benzalkonium chloride (BAC) from household discharges can mold the microbial communities in wastewater treatment plants (WWTPs) in a way still poorly understood. In this study, we performed an in vitro exposure of activated sludge from a WWTP in Costa Rica to BAC, quantified the changes in intI1, sul2, and qacE/qacEΔ1 gene profiles, and determined alterations in the bacterial community composition. The analysis of the qPCR data revealed elevated charges of antibiotic resistance genes in the microbial community; after BAC's exposure, a significant increase in the qacE/qacEΔ1 gene, which is related to ammonium quaternary resistance, was observed. The 16S rRNA gene sequences' analysis showed pronounced variations in the structure of the bacterial communities, including reduction of the alpha diversity values and an increase of the relative abundance of Alphaproteobacteria, particularly of Rhodospseudomonas and Rhodobacter. We confirmed that the microbial communities presented high resilience to BAC at the mg/mL concentration, probably due to constant exposure to this pollutant. They also presented antibiotic resistance-related genes with similar mechanisms to tolerate this substance. These mechanisms should be explored more thoroughly, especially in the context of high use of disinfectant.

Cationic Compounds with SARS-CoV-2 Antiviral Activity and Their Interaction with Organic Cation Transporter/Multidrug and Toxin Extruder Secretory Transporters.

In the wake of the COVID-19 pandemic, drug repurposing has been highlighted for rapid introduction of therapeutics. Proposed drugs with activity against SARS-CoV-2 include compounds with positive charges at physiologic pH, making them potential targets for the organic cation secretory transporters of kidney and liver, i.e., the basolateral organic cation transporters, OCT1 and OCT2; and the apical multidrug and toxin extruders, MATE1 and MATE2-K. We selected several compounds proposed to have in vitro activity against SARS-CoV-2 (chloroquine, hydroxychloroquine, quinacrine, tilorone, pyronaridine, cetylpyridinium, and miramistin) to test their interaction with OCT and MATE transporters. We used Bayesian machine learning models to generate predictions for each molecule with each transporter and also experimentally determined IC50 values for each compound against labeled substrate transport into CHO cells that stably expressed OCT2, MATE1, or MATE2-K using three structurally distinct substrates (atenolol, metformin and 1-methyl-4-phenylpyridinium) to assess the impact of substrate structure on inhibitory efficacy. For the OCTs substrate identity influenced IC50 values, although the effect was larger and more systematic for OCT2. In contrast, inhibition of MATE1-mediated transport was largely insensitive to substrate identity. Unlike MATE1, inhibition of MATE2-K was influenced, albeit modestly, by substrate identity. Maximum unbound plasma concentration/IC50 ratios were used to identify potential clinical DDI recommendations; all the compounds interacted with the OCT/MATE secretory pathway, most with sufficient avidity to represent potential DDI issues for secretion of cationic drugs. This should be considered when proposing cationic agents as repurposed antivirals. SIGNIFICANCE STATEMENT: Drugs proposed as potential COVID-19 therapeutics based on in vitro activity data against SARS-CoV-2 include compounds with positive charges at physiological pH, making them potential interactors with the OCT/MATE renal secretory pathway. We tested seven such molecules as inhibitors of OCT1/2 and MATE1/2-K. All the compounds blocked transport activity regardless of substrate used to monitor activity. Suggesting that plasma concentrations achieved by normal clinical application of the test agents could be expected to influence the pharmacokinetics of selected cationic drugs.

Antimicrobial Face Shield: Next Generation of Facial Protective Equipment against SARS-CoV-2 and Multidrug-Resistant Bacteria.

Transparent materials used for facial protection equipment provide protection against microbial infections caused by viruses and bacteria, including multidrug-resistant strains. However, transparent materials used for this type of application are made of materials that do not possess antimicrobial activity. They just avoid direct contact between the person and the biological agent. Therefore, healthy people can become infected through contact of the contaminated material surfaces and this equipment constitute an increasing source of infectious biological waste. Furthermore, infected people can transmit microbial infections easily because the protective equipment do not inactivate the microbial load generated while breathing, sneezing or coughing. In this regard, the goal of this work consisted of fabricating a transparent face shield with intrinsic antimicrobial activity that could provide extra-protection against infectious agents and reduce the generation of infectious waste. Thus, a single-use transparent antimicrobial face shield composed of polyethylene terephthalate and an antimicrobial coating of benzalkonium chloride has been developed for the next generation of facial protective equipment. The antimicrobial coating was analyzed by atomic force microscopy and field emission scanning electron microscopy with elemental analysis. This is the first facial transparent protective material capable of inactivating enveloped viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one minute of contact, and the methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Bacterial infections contribute to severe pneumonia associated with the SARS-CoV-2 infection, and their resistance to antibiotics is increasing. Our extra protective broad-spectrum antimicrobial composite material could also be applied for the fabrication of other facial protective tools such as such as goggles, helmets, plastic masks and space separation screens used for counters or vehicles. This low-cost technology would be very useful to combat the current pandemic and protect health care workers from multidrug-resistant infections in developed and underdeveloped countries.

Disinfectant effectiveness against SARS-CoV-2 and influenza viruses present on human skin: model-based evaluation.

OBJECTIVES: Disinfection effectiveness against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on human skin remains unclear because of the hazards of viral exposure. An evaluation model, which has been previously generated using human skin obtained from forensic autopsy samples, accurately mimics in vivo skin conditions for evaluating the effectiveness of disinfection against the virus. Using this model, we evaluated disinfection effectiveness against viruses on human skin. METHODS: Ethanol (EA), isopropanol (IPA), chlorhexidine gluconate (CHG) and benzalkonium chloride (BAC) were used as target disinfectants. First, disinfectant effectiveness against SARS-CoV-2 and influenza A virus (IAV) was evaluated in vitro. Disinfectant effectiveness against SARS-CoV-2 and IAV on human skin was then evaluated by titrating viruses present on the skin after applying each disinfectant on the skin for 5-60 seconds. RESULTS: Both, SARS-CoV-2 and IAV on human skin were completely inactivated within 5 seconds by 40%-80% EA and 70% IPA (log reduction values (LRVs) were >4). However, SARS-CoV-2 and IAV were barely inactivated by 20% EA (LRVs were <1). In vitro evaluation showed that, compared with EA and IPA, CHG and BAC were significantly inferior in terms of disinfection effectiveness. Conversely, the disinfection effectiveness of CHG and BAC against SARS-CoV-2 was higher on human skin than in vitro, and increased with increases in their concentration and reaction time (LRVs of 0.2% CHG/0.05% BAC were >2, and LRVs of 1.0% CHG/0.2% BAC were >2.5). CONCLUSIONS: Proper hand hygiene practices using alcohol-based disinfectants such as EA/IPA effectively inactivate SARS-CoV-2 and IAV on human skin.

Alcohol-free hand sanitizer and other quaternary ammonium disinfectants quickly and effectively inactivate SARS-CoV-2.

BACKGROUND: SARS-CoV-2 is the virus responsible for the current global pandemic, COVID-19. Because this virus is novel, little is known about its sensitivity to disinfection. METHODS: We performed suspension tests against SARS-CoV-2 using three commercially available quaternary ammonium compound (Quat) disinfectants and one laboratory-made 0.2% benzalkonium chloride solution. FINDINGS: Three of the four formulations completely inactivated the virus within 15 s of contact, even in the presence of a soil load or when diluted in hard water. CONCLUSION: Quats rapidly inactivate SARS-CoV-2, making them potentially useful for controlling SARS-CoV-2 spread in hospitals and the community.