Benzalkonium Research Articles

The Development of Disinfectant Tolerance in Klebsiella pneumoniae.

Disinfectants are a critical infection control measure that are relied upon globally in a range of settings including healthcare, food production, and domestic environments. However, bacteria have been shown to survive disinfectant treatments when harboured in dry surface biofilms or when disinfectants are used ineffectively. This provides an opportunity for organisms to develop low-level tolerance to various disinfectants. The capability of bacteria to develop adaptations to non-antibiotic antimicrobial agents is often overlooked. This study investigates the ability of Klebsiella pneumoniae NDM-1 strain NCTC 13443 to adapt to a range of common chemical disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol and bronopol) via serial passage exposure method. After long-term adaptation K. pneumoniae developed tolerance to all tested disinfectants, exhibiting a minimum inhibitory concentration increase of between 30-413% compared to the untreated parent samples. Characterisation of disinfectant cross-tolerance showed that while cross-tolerance can occur, most adapted samples became more susceptible to the second disinfectant treatment, likely because of the fitness cost of adaptation. Observed cross-tolerance/increased susceptibility was not always reciprocated between disinfectant-tolerant samples, indicating that the order of disinfectant exposure is important during tolerance development. This has significant implications for disinfectant cleaning routines, and is likely due to variations in the underpinning tolerance mechanisms, even when the disinfectants display similar mechanisms of action. This study reports the capability and readiness of clinically relevant K. pneumoniae to adapt to common disinfectants that are relied upon every day across the world, delivering much-needed insights into an often-overlooked aspect of antimicrobial resistance.

Variable sensitivity of clinical Candida auris strains to Biocides: implications for infection control in Healthcare Settings

PurposeCandida auris, a multidrug-resistant yeast, poses significant challenges in healthcare settings due to its ability to form biofilms and resistance to common disinfectants. Understanding its susceptibility to biocides used in hospital disinfection practices is crucial for infection control. We investigated the biocide sensitivity of eight clinical C. auris strains from different patients and one reference strain (CDC B11903) using the biocide activity tests.MethodsSpecies identification was confirmed through MALDI-TOF MS, while clade differentiation and phylogenetic classification were determined via whole-genome sequencing. Biofilm formation was assessed using the MTT assay. Antifungal susceptibilities were tested according to CLSI standards. The effectiveness of biocides, including chlorine, chlorhexidine, and benzalkonium chloride, was evaluated through broth microdilution following CLSI standards and quantitative suspension and carrier tests, following EN standards.ResultsAll clinical strains were identified as clade 1, and the reference strain as clade 4, with all exhibiting biofilm formation. Clade 1 strains showed resistance to fluconazole, with MIC values ranging from 8 to 32 µg/ml, while being susceptible to other antifungals. Broth microdilution MIC assays for biocides demonstrated that all strains exhibited resistance to benzalkonium chloride. Chlorine and chlorhexidine showed variable efficacy, dependent on concentration and environmental cleanliness. Alcohol-based hand sanitizers demonstrated effectiveness against C. auris from the first minute of application.ConclusionThe study highlights the variable susceptibility of C.auris to different biocides, underscoring the challenge in eradicating this pathogen from healthcare environments. Our findings advocate for the careful selection of disinfectants in hospital settings, emphasizing the need for high-concentration chlorine and chlorhexidine solutions to combat C. auris, even in especially clean environments.

Listeria monocytogenes in Fruits and Vegetables: Antimicrobial Resistance, Biofilm, and Genomic Insights

Background/Objectives: Listeria monocytogenes is a foodborne pathogen that can infect both humans and animals and cause noninvasive gastrointestinal listeriosis or invasive listeriosis. The objectives of this study were to determine the genetic diversity of L. monocytogenes; the genes associated with its resistance to antibiotics, benzalkonium chloride (BC), and cadmium chloride (CdCl2); and its biofilm formation. Methods: A total of 132 fresh fruits (44 samples) and vegetables (88 samples) were selected for this study. The genetic diversity of the isolates and the genes associated with their antibiotic resistance were determined using PCR amplification; meanwhile, their levels of susceptibility to antibiotics were determined using the agar diffusion method. Their levels of resistance to BC and CdCl2 were determined using the minimum inhibitory concentration method, and their capacity for biofilm formation was evaluated using the crystal violet staining method. Results: A total of 17 L. monocytogenes strains were collected: 12.8% (17/132) from fresh fruits and vegetables in this study. The isolates of L. monocytogenes belonged to phylogenetic groups I.1 (29.4% (5/17); serotype 1/2a) and II.2 (70.5% (12/17); serotype 1/2b); strains containing Listeria pathogenicity islands (LIPIs) were also identified at prevalence rates of 100% for LIPI-1 and LIPI-2 (17/17), 29.4% for LIPI-3 (5/17), and 11.7% for LIPI-4 (2/17). The antibiotic susceptibility tests showed that the L. monocytogenes isolates exhibited six different multiresistant patterns, with multiple antibiotic resistance (MAR) index of ≥0.46 (70.5%; 12/17); additionally, the genes Ide, tetM, and msrA, associated with efflux pump Lde, tetracycline, and ciprofloxacin resistance, were detected at 52.9% (9/17), 29.4% (5/17), and 17.6% (3/17), respectively. The phenotypic tests showed that 58.8% (10/17) of cadmium-resistant L. monocytogenes isolates had a co-resistance of 23.5% (4/17) to BC. Finally, all strains of L. monocytogenes exhibited moderate biofilm production. Conclusions: The results of this study contribute to our understanding of the persistence and genetic diversity of L. monocytogenes strains isolated from fresh fruits and vegetables; in addition, their resistance to CdCl2, which is correlated with co-resistance to BC disinfectant, is helpful for the food industry.

Single Method for Determination of Biocides Benzalkonium Chloride, Chloroxylenol and Chlorhexidine Gluconate in Cosmetics, Hygiene and Health Care Products by Using High Performance Liquid Chromatography.

Benzalkonium chloride (BKC), chloroxylenol (PCMX) and chlorhexidine gluconate (CHG) are widely recognized biocidal compounds that require precise control and monitoring in various applications, including cosmetics, hygiene, health and ophthalmic products. In this study, we present a straightforward and highly sensitive high-performance liquid chromatographic (HPLC) method for the detection of these substances. The HPLC method we developed allows for the detection of BKC at concentrations as low as 1.0ppm, PCMX at 1.0ppm and CHG at 2.0ppm, all with a signal-to-noise (S/N) ratio exceeding 3.0. Additionally, the method enables quantification up to 2.0ppm for BKC, 2.0ppm for PCMX and 5ppm for CHG, with an S/N ratio surpassing 10.0. This validated method serves as a versatile tool for the accurate quantification of BKC, PCMX and CHG in cosmetics, hygiene products, health products and ophthalmic products. Its applicability extends across a wide range of industries, ensuring the reliable assessment of these critical biocides.

Oxidation of glyoxal with the Mo-oxime complex in a benzalkonium chloride interface: Raghavan and Srinivasan kinetic model

Gaining an understanding of the glucose oxidation product (glyoxal) interaction with the metal–organic complex in a biological environment is pivotal to its usage. Thus, the glyoxal (Gyx) oxidation with Mo-oxime complex (MC) is studied with the spectrophotometric method, following a pseudo-phase approach. The result highlights the inclusion of hydrolysis, ion catalysis, the neutral primary salt effect, and radical generation as the essential factors in Gyx oxidation, with the exclusion of intermediate species formation. The hydrolysis of Gxy is observed to actively engage MC without charge inhibition as charge-neutral reacting species are involved, thus, implicating neutral primary salt effect where the variation of ionic strength of the system keeps the redox rate unchanged. The involvement of charge-neutral specie at the rate controlling step encouraged electrostatic attraction when Mg2+ ion additive is incorporated into the system, leading to ion catalysis. The generation of free radical from Gxy aids the emergence of formic acid. The zero intercept of Michealis-Menten type plot and the non-appreciable shift in the maximum absorption wavelength of the reaction system and MC rule-out the presence of intermediate species. The contribution of thermodynamic enthalpy is instrumental in the redox process, leading to the formic acid product. The inclusion of benzalkonium chloride (BZC) hastens the Gxy oxidation, which is supported by Raghavan and Srinivasan’s model.

In-situ immobilization of Fe PBA in the zeolite structure for efficient degradation of benzalkonium chloride: Towards compressed reaction site loss and promoted PMS utilization

In-situ immobilization of Fe PBA in the zeolite structure for efficient degradation of benzalkonium chloride: Towards compressed reaction site loss and promoted PMS utilization

A bifunctional nanocomplex with remineralizing and antibacterial activities to interrupt dental caries

Dental caries result from an imbalance between the demineralization and remineralization of dental hard tissues, primarily caused by biofilm accumulation. According to the theory of interrupting dental caries, effective anticaries materials and techniques should possess both remineralizing and antibacterial properties. However, current anticaries materials cannot replicate amelogenesis, which is crucial for remineralization and for preventing bacterial adhesion and biofilm formation. In this study, silk fibroin (SF) loaded with benzalkonium chloride (BZC) successfully formed an SF-BZC composite. This composite stabilized amorphous calcium phosphate (ACP), creating an ACP@SF-BZC dual-functional nanocomplex with both remineralizing and antibacterial properties. ACP@SF-BZC demonstrated significant anti-adhesion and biofilm inhibitory effects against Streptococcus mutans and Streptococcus sobrinus. Moreover, compared to fluoride, ACP@SF-BZC significantly enhanced the remineralization of demineralized enamel surfaces, forming a stable remineralized layer with improved mechanical properties, both in vitro and in vivo. In summary, the dual-function ACP@SF-BZC nanocomplex, with its remineralizing and antibacterial effects, offers a promising alternative for preventing and arresting enamel caries.

Characterization of Endophytic Streptomyces rhizosphaericola Ahn75 and Its Potential for Biocontrol against Rice Blast.

Plant endophyte Streptomyces are excellent candidates as biocontrol agents against the rice blast fungus, Magnaporthe oryzae. In this study, a novel strain Ahn75 with antifungal activity was isolated from healthy rice stem and identified as Streptomyces rhizosphaericola by phenotypic characterization and phylogenetic analysis based on 16S rRNA, multilocus and genome sequences. Inhibition test using culture filtrate showed that Ahn75 could effectively suppress M. oryzae, with mycelia growth inhibition rate of 80.88% and spore germination inhibition rate of 78.26%. Genome sequence analysis of strain Ahn75 showed 40 gene clusters of secondary metabolites and several genes related to plant growth promotion were predicted in the genome of Ahn75. Several antimicrobial compounds including valinomycin, tetrabutylammonium, and benzalkonium chloride, were also detected in the antifungal fraction from Ahn75 culture filtrate by liquid chromatography and high resolution mass spectrometry. Meanwhile, strain Ahn75 demonstrates UV tolerance under UV irradiation for 60 min, pH tolerance between pH6 and pH9, and a high halotolerance in 7% (w/v) of NaCl. Greenhouse experiments indicated that Ahn75 is able to colonize rice stems, roots, and leaves, which help rice to reduce the rice leaf blast incidence by 59.76%. All these findings suggest that strain Ahn75 could be a potential biocontrol agent for rice blast.

Virucidal Coatings Active Against SARS-CoV-2

Three types of coatings (contact-based, release-based, and combined coatings with both contact-based and release-based actions) were prepared and tested for the ability to inactivate SARS-CoV-2. In these coatings, quaternary ammonium surfactants were used as active agents since quaternary ammonium compounds are some of the most commonly used disinfectants. To provide contact-based action, the glass and silicon surfaces with covalently attached quaternary ammonium cationic surfactant were prepared using a dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride modifier. Surface modification was confirmed by attenuated total reflection infrared spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy, and contact angle measurements. The grafting density of the modifier was estimated by XPS and elemental analysis. To provide release-based action, the widely used quaternary ammonium cationic disinfectant, benzalkonium chloride (BAC), and a newly synthesized cationic gemini surfactant, C18-4-C18, were bound non-covalently to the surface either through hydrophobic or electrostatic interactions. Virus titration revealed that the surfaces with combined contact-based and release-based action and the surfaces with only release-based action completely inactivate SARS-CoV-2. Coatings containing only covalently bound disinfectant are much less effective; they only provide up to 1.25 log10 reduction in the virus titer, probably because of the low disinfectant content in the surface monolayer. No pronounced differences in the activity between the flat and structured surfaces were observed for any of the coatings under study. Comparative studies of free and electrostatically bound disinfectants show that binding to the surface of nanoparticles diminishes the activity. These data indicate that SARS-CoV-2 is more sensitive to the free disinfectants.

Detection of benzalkonium chloride phenotypic resistance and its association with qacG, qacH, qacJ resistance genes among methicillin-resistant Staphylococcus aureus clinical isolates

Detection of benzalkonium chloride phenotypic resistance and its association with qacG, qacH, qacJ resistance genes among methicillin-resistant Staphylococcus aureus clinical isolates

Carbon Dots Infused 3D Printed Cephalopod Mimetic Bactericidal and Antioxidant Hydrogel for Uniaxial Mechano-Fluorescent Tactile Sensor.

Cephalopods use stretchy skin and dynamic color-tuning organs for visual communication and camouflage. Inspired by these natural mechanisms, a fluorescent biomaterial for deformation-induced illumination and optical communication is proposed. This is the first report of 3D printed soft biomaterials infused with carbon dots hydrothermally derived from chitosan and benzalkonium chloride. These biomaterials exhibit a comprehensive array of properties, including significant uniaxial stretching, near-instantaneous response to tactile stimuli and pH, UV resistance, antibacterial, antioxidant, noncytotoxicity, and highlighting their potential as mechano-optical materials for biomedical applications. The hydrogel's durability is evaluated by cyclic stretching, folding, rolling, and twisting tests to ensure its integrity and good signal-to-noise ratio. The diffusion mechanism is determined by water imbibition kinetics, network parameters, and time-dependent breathing. Overcoming the common limitations of short lifespans and complex manufacturing processes in existing soft hybrids, this work demonstrates a straightforward method to produce durable, energy-independent, mechano-optical hydrogel. Combined with investigations, molecular dynamic modeling is used to understand the interactions of hydrogel components.

Bactericidal Efficacy of So-Called Sanitizers in Japan.

Commercially available so-called sanitizers in Japan are often touted as having remarkable "sanitizing (jokin)" effect, "virus-removal" capabilities, and "99.99%" removal rate of microbes and pathogens. In this study, we investigated the bactericidal efficacy of these so-called sanitizers for environmental surfaces against Enterococcus faecalis and Pseudomonas aeruginosa. Of note, out of 43 products, 24 (55.8%) did not exhibit bactericidal effects on either Enterococcus or Pseudomonas. Among these 43 products, there were no bactericidal effects in 9 (47.4%) of 19 products that stated as containing "alcohol" as the formula; as well as 4 (80%) of 5 products stating only "benzalkonium chloride"; similarly no effect in 5 (83.3%) of 6 stating only"chlorine chemicals." Furthermore, 6 (46.2%) of 13 products that stated components other than alcohol, benzalkonium chloride, and chlorine chemicals on the product container or with no description of constituent components failed to show any bactericidal effects. Four disinfectants (alcohol for disinfection, 0.1% benzalkonium chloride, 0.05% (500 ppm) hypochlorite, 0.1% (1000 ppm) hypochlorite) as control showed bactericidal effect. The lack of bactericidal activity in nearly half of sanitizers may be explained by the low concentration of the effective ingredient such as alcohol, benzalkonium chloride, and hypochlorite. In sanitizers containing hypochlorite, degradation of hypochlorite with prolongation of time after manufacturing may be another reason.

Managing Ocular Surface Disease in Glaucoma Treatment: A Systematic Review.

Ocular surface disease (OSD) is a frequent disabling challenge among patients with glaucoma who use benzalkonium chloride (BAK)-containing topical glaucoma medications for prolonged periods. In this comprehensive review, we evaluated the prevalence of OSD and its management, focusing on both current and future alternatives. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria were used to assess a) the impact of active ingredients and preservatives on the ocular surface and b) the efficacy of preservative-free (PF) alternatives and adjunctive therapies. BAK-containing glaucoma medications were found to significantly contribute to OSD by increasing corneal staining, reducing tear film stability, and elevating ocular surface disease index (OSDI) scores. Transitioning to PF formulations or those with less cytotoxic preservatives, such as Polyquad® and SofZia®, demonstrated a marked improvement in OSD symptoms. In particular, the use of adjunct cyclosporine A, through its anti-inflammatory and enhanced tear film stability actions, was shown to be very beneficial to the ocular surface. Therefore, the most effective management of OSD is multi-factorial, consisting of switching to PF or less cytotoxic medications, adjunct use of cyclosporine A, and early incorporation of glaucoma surgical treatments such as laser trabeculoplasty, trabeculectomy, glaucoma drainage devices, or minimally invasive glaucoma surgery (MIGS).

Evaluation of ten plant-derived biocides for the inhibition of photosynthetic organisms on the karst surfaces of heritage buildings

Biodeterioration is a significant problem in the conservation of stone heritage buildings. In this study, 10 plant essential oils were assessed for their effectiveness in biofilm inhibition on stone heritage building surfaces under laboratory and in situ conditions, and were compared with traditional biocides such as benzalkonium chloride. The plant extracts were tested against algae and mosses. The effect on algae removal was evaluated by measuring the color of the surface before treatment, after 24 h and after 1 month of treatment. The effect on mosses was assessed by measuring the photosynthetic pigment content of mosses after 24 h of treatment. The results showed that the different plant extracts exhibited different levels of antibiotic activity. Benzalkonium chloride, S. aromaticum and C. cassia extracts showed strong antibiotic activity against all algae and mosses tested. T. mongolicus extracts showed antibiotic activity against only some of the algae and mosses, while the application of P. cablin extracts increased chlorophyll b content in the mosses. The other plant extracts were less effective at inhibiting the growth of algae and mosses. GC–MS compositional analysis further indicated that the higher antibiotic activity of S. aromaticum and C. cassia extracts was related to the high content of eugenol and cinnamaldehyde.

The Optejet Technology Minimizes Preservative-Mediated Cytotoxicity of Conjunctival Epithelial Cells Treated with Latanoprost In Vitro.

Purpose: Benzalkonium chloride (BAK) is a commonly used preservative to maintain sterility for multiuse eye drops such as latanoprost. One option to minimize the deleterious effects of BAK in eye drops may be to reduce the volume administered. The aim of this study was to assess the response of cells from the ocular surface to latanoprost+BAK administered by the Optejet technology, which dispenses a microdose (∼8 µL) ophthalmical spray. Methods: Cultured human conjunctival epithelial cells were exposed to the following treatments: (1) no treatment, (2) drop form of latanoprost without BAK (∼35 µL), (3) drop form of latanoprost with 0.01% BAK (∼35 µL), (4) ophthalmical spray form of latanoprost with 0.01% BAK delivered by the Optejet technology (∼8 µL). After 5 h, cells were assessed for changes in cytotoxicity, morphology, and inflammatory marker expression. Results: Latanoprost+BAK delivered by a drop induced cytotoxicity, cytoplasmic shrinkage, and loss of cell-cell contact, and expression of chemokine (C-C motif) ligand 2 and interleukin-6. In contrast, latanoprost+BAK delivered by the Optejet technology was both well tolerated and similar to no treatment controls and BAK-free latanoprost treatment. Conclusions: A microdose of latanoprost+BAK ophthalmical spray administered with the Optejet technology prevented the cytotoxicity associated with larger volumes found in eye drops. Precision dosing by the Optejet technology has the potential to decrease ocular surface disorder typically associated with eye drops containing preservatives.

Evaluating cholinesterases inhibition by BAC and DDAC biocides: A combined experimental and theoretical approach

Disinfectant biocides are chemicals that are heavily used for disinfection purposes in households, hospitals, and agrifood industry. The most common type of biocides are quaternary ammonium compounds (QAs), notably benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC), which have been shown to inhibit cholinesterases. This study aims to evaluate the effect of these biocides towards different cholinesterases using both enzyme inhibition and molecular docking experiments. Acetylcholinesterase (AChE) from Drosophila melanogaster (DM-AChE), Electrophorus electricus (EE-AChE), bovine erythrocytes (BE-AChE) and butyrylcholinesterase from horse serum (BChE) were selected for this study. Using a colorimetric assay, all these enzymes were shown to be inhibited in a competitive form by both biocides, BAC and DDAC, with the exception of DM-AChE, which was inhibited in a non-competitive manner by BAC. Molecular docking experiments enabled to identify structural determinants involved in the different modes of inhibition observed. More particularly, our results suggest that non-competitive inhibition of DM-AChE by BAC could be related to the binding of the inhibitor into a more extended active site compared to other cholinesterases.

Mesenchymal Stem Cells-Derived Exosomal miR-223-3p Alleviates Ocular Surface Damage and Inflammation by Downregulating Fbxw7 in Dry Eye Models.

Our previous study indicated that exosomes derived from mouse adipose-derived mesenchymal stem cells (mADSC-Exos) alleviated the benzalkonium chloride (BAC)-induced mouse dry eye model. However, the specific active molecules in mADSC-Exos that contribute to anti-dry eye therapy remain unidentified. In this study, we aimed to investigate the efficacy and mechanisms of miR-223-3p derived from mADSC-Exos in dry eye models. Enzyme-linked immunosorbent assay (ELISA) experiments were conducted to determine miR-223-3p derived from mADSC-Exos that exerted anti-inflammatory effects on hyperosmolarity-induced mouse corneal epithelial cells (MCECs). The therapeutic efficacy of miR-223-3p was evaluated in mice with dry eye induced by either BAC or scopolamine (Scop). Mice were randomly assigned to 5 groups: sham, model, miR-223-3p overexpression, miR-223-3p knockdown, and 0.1% pranoprofen (positive group). Post-treatment, the severity of dry eye symptoms, and the pro-inflammatory cytokine levels were assessed. The effect of miR-223-3p on silencing the target gene was verified using ELISA and dual luciferase reporter assays. The mADSC-Exos that knocked out miR-223-3p did not reduce interleukin (IL)-6 content. Supplementing with miR-223-3p could restore the reduction of IL-6. The miR-223-3p effectively ameliorated ocular surface damage and decreased pro-inflammatory cytokines or chemokines in both BAC- and Scop-induced mouse dry eye models. Furthermore, miR-223-3p inhibited cell apoptosis. F-box and WD repeat domain-containing 7 (Fbxw7) was the potential direct target of miR-223-3p. The miR-223-3p suppressed the 3'-untranslated region of Fbxw7. The Fbxw7 knockdown suppressed hyperosmolarity-induced inflammation in MCECs. The mADSC-derived exosomal miR-223-3p mitigates ocular surface damage and inflammation, indicating its potential as a promising treatment option for dry eye.

Effects of preservatives on the activities of salivary enzymes

ObjectivesTo investigate the effects of common preservatives used in oral health care products on the enzymatic activities of lysozyme, peroxidase, and α-amylase in-solution and on-hydroxyapatite surface phases. DesignThe preservatives used in this study were sodium benzoate, methylparaben, propylparaben, and benzalkonium chloride. Hen egg-white lysozyme, bovine lactoperoxidase, and α-amylase from Bacillus sp. served as sources of purified enzymes. Human unstimulated whole saliva was used as a source of salivary enzymes. Hydroxyapatite beads were used as the surface phase. The preservatives were incubated with purified enzymes or saliva samples in-solution or on-hydroxyapatite surface phases, respectively. Enzymatic activities of lysozyme, peroxidase, and α-amylase were measured by hydrolysis of fluorescein-labelled Micrococcus lysodeikticus, oxidation of fluorogenic 2′,7′-dichlorofluorescin, and hydrolysis of fluorogenic starch, respectively. ResultsThe effects of the preservatives on the enzymatic activities of lysozyme and peroxidase were more distinct in the saliva samples than purified substances, and in the in-solution phase than on-hydroxyapatite surface phase, and the opposite was true for α-amylase. The most significant result was apparent decrease in peroxidase activities caused by the parabens in the in-solution phase (P<0.05). Sodium benzoate and parabens inhibited lysozyme activity in the in-solution phase, but differently for the purified and salivary lysozymes. Parabens and benzalkonium chloride inhibited the enzymatic activity of α-amylase from Bacillus sp., not saliva samples, only on-hydroxyapatite surface (P<0.05). ConclusionsEach preservative affected the enzymatic activities of lysozyme, peroxidase, and α-amylase differently. Based on the effects on salivary enzymes, sodium benzoate or benzalkonium chloride was recommended as preservatives rather than parabens.

Differential Selection for Survival and for Growth in Adaptive Laboratory Evolution Experiments With Benzalkonium Chloride.

Biocides are used to control microorganisms across different applications, but emerging resistance may pose risks for those applications. Resistance to biocides has commonly been studied using adaptive laboratory evolution (ALE) experiments with growth at subinhibitory concentrations linked to serial subculturing. It has been shown recently that Escherichia coli adapts to repeated lethal stress imposed by the biocide benzalkonium chloride (BAC) by increased survival (i.e., tolerance) and not by evolving the ability to grow at increased concentrations (i.e., resistance). Here, we investigate the contributions of evolution for tolerance as opposed to resistance for the outcome of ALE experiments with E. coli exposed to BAC. We find that BAC concentrations close to the half maximal effective concentration (EC50, 4.36 μg mL-1) show initial killing (~40%) before the population resumes growth. This indicates that cells face a two-fold selection pressure: for increased survival and for increased growth. To disentangle the effects of both selection pressures, we conducted two ALE experiments: (i) one with initial killing and continued stress close to the EC50 during growth and (ii) another with initial killing and no stress during growth. Phenotypic characterization of adapted populations showed that growth at higher BAC concentrations was only selected for when BAC was present during growth. Whole genome sequencing revealed distinct differences in mutated genes across treatments. Treatments selecting for survival-only led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ), while treatments selecting for growth and survival led to mutations in genes related to stress response (hslO and tufA). Our results demonstrate that serial subculture ALE experiments with an antimicrobial at subinhibitory concentrations can select for increased growth and survival. This finding has implications for the design of ALE experiments to assess resistance risks of antimicrobials in different scenarios such as disinfection, preservation, and environmental pollution.

Antibacterial Efficacy of Benzalkonium Chloride Gel with Tongue Brush on S. Mutans and L. Acidophilus and Tongue Coating – A Randomized Controlled Trial

Introduction: The tongue microbiota, like other parts of the oral cavity, plays a significant role in the development of dental caries. S. mutans and L. acidophilus species, are associated with the initiation and progression of dental caries.  Aim: To assess the efficacy of benzalkonium Chloride gel along with tongue cleaning aids in the reduction of tongue coating and bacterial count on dorsal surface of the tongue.  Materials and Methods: A double blinded randomized control trial was conducted among 17-25 years old college students. A total of 60 participants were randomly divided into 4 groups according to their use of tongue cleaning aids as follows: Group A, Tongue Brush Alone; Group B, Tongue Brush with Chlorhexidine Mouthwash (Clohex ADS); Group C, Tongue Brush with Chlorhexidine Gel (Hexigel); and Group D, Tongue Brush with Benzalkonium Chloride Gel. The Winkel tongue coating index (WTCI) was used to measure the tongue coating at baseline and at 14th day and the participant’s tongue coatings on dorsal surface were sampled using swab method, and the number of S. mutans and L. acidophilus colonies were counted before and after intervention.  Results: Both the Hexigel group and Benzalkonium chloride gel group reduced both the bacterial load and tongue coating at 14th Day compared to the baseline data.  Conclusion: This study suggests that usage of Tongue brush along with Benzalkonium Chloride gel decreased the tongue coating and the number of bacteria on the dorsal tongue surface.