Concentrations Of Benzalkonium Chloride Research Articles

Benzalkonium chloride induced acute toxicity and its multifaceted implications on growth, hematological metrics, biochemical profiles, and stress-responsive biomarkers in tilapia (Oreochromis mossambicus).

This study aimed to investigate the toxic effects of benzalkonium chloride (BAC) on Oreochromis mossambicus, a freshwater fish species. Probit analysis was used to determine the lethal concentration (LC50) of BAC for different exposure periods (24, 48, 72, and 96h). The viability of fish exposed to BAC was assessed using the general threshold survival models (GUTS) and confirmed with relevant datasets to evaluate model accuracy. Experimental groups of fish were exposed to BAC concentrations equivalent to 10% and 20% of the 96-h LC50 for 45days. The study revealed significant alterations in various parameters during sublethal BAC exposure. These effects included decreased specific growth rate (SGR), red blood cell count (RBC), hemoglobin (Hb) concentration, hematocrit (Ht) value, plasma protein, and albumin levels, as well as acetylcholinesterase (AChE) activities in both gills and liver. Additionally, an increase in gastrosomatic index (GSI), feed conversion ratio (FCR), plasma glucose and creatinine concentrations, alanine aminotransferase (ALT), aspartate aminotransferase (AST) enzymatic activities, catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were observed in the exposed fish's gills and liver. Furthermore, the study found that glutathione S-transferase (GST) and glutathione peroxidase (GPx) levels initially increased and then decreased in both gills and liver after exposure to BAC. Correlation matrix analysis, multivariate multiple regression (MMR), canonical correspondence analysis (CCA), integrated biomarker response (IBR), and biomarker response index (BRI) were utilized to assess the impact of BAC on fish, highlighting significant effects on multiple biomarkers in O. mossambicus following surfactant exposure. Thus, the study provides valuable insights into the toxic effects of BAC on this fish species, emphasizing the importance of monitoring such pollutants in aquatic environments.

Sex-Specific Effects of Environmental Exposure to the Antimicrobial Agents Benzalkonium Chloride and Triclosan on the Gut Microbiota and Health of Zebrafish (Danio rerio).

The use of disinfectants containing benzalkonium chloride (BAC) has become increasingly widespread in response to triclosan (TCS) restrictions and the COVID-19 pandemic, leading to the increasing presence of BAC in aquatic ecosystems. However, the potential environmental health impacts of BAC on fish remain poorly explored. In this study, we show that BAC and TCS can induce the gut dysbiosis in zebrafish (Danio rerio), with substantial effects on health. Breeding pairs of adult zebrafish were exposed to environmentally relevant concentrations of BAC and TCS (0.4-40 μg/L) for 42 days. Both BAC and TCS exposure perturbed the gut microbiota, triggering the classical NF-κB signaling pathway and resulting in downstream pathological toxicity associated with inflammatory responses, histological damage, inhibited ingestion, and decreased survival. These effects were dose-dependent and sex-specific, as female zebrafish were more susceptible than male zebrafish. Furthermore, we found that BAC induced toxicity to a greater extent than the restricted TCS at environmentally relevant concentrations, which is particularly concerning. Our results suggest that environmental exposure to antimicrobial chemicals can have ecological consequences by perturbing the gut microbiota, a previously underappreciated target of such chemicals. Rigorous ecological analysis should be conducted before widely introducing replacement antimicrobial compounds into disinfecting products.

Development of a sustainable procedure for smartphone-based colorimetric determination of benzalkonium chloride in pharmaceutical preparations

A sustainable procedure offering green, simple, and rapid analysis was developed to determine benzalkonium chloride (BKC) in pharmaceutical preparations. The determination using smartphones was based on the ion pair colorimetric reaction with bromothymol blue (BTB), which produces a yellow color. The intensity of the product color, which is proportional to the concentration of BKC, was detected and evaluated using a smartphone camera and an image processing application. The procedure was performed in a microliter and was rapidly detected within 1 min after incubation. This offered high throughput at 28 samples per well plate in duplicate. Linear calibration, which was a plot of BKC concentrations and relative red intensities, was in the range of 2.0–24.0 μg/mL with an R2 of 0.997. The limits of detection (LOD) and quantitation (LOQ) were 1.0 and 3.2 μg/mL, respectively. This work was successful in applying it to pharmaceutical materials, disinfectant products, and pharmaceutical products containing BKC. It was discovered that the concentrations of BKC as an active ingredient in pharmaceutical materials were 82% w/v, whereas those in disinfectant products ranged from 0.4 to 2.1% w/v. In pharmaceutical products, ophthalmic drops and nasal sprays contain BKC as preservatives in the 0.01–0.02, and the 0.02% w/v, respectively. The results obtained by the proposed procedure compared with a reference titration method showed no significant differences at a 95% confidence level with 1.2–3.4% RSDs. This promotes the efficiency of pharmaceutical preparations regarding infection prevention and control by ensuring that available disinfectants contain a sufficient concentration of BKC. Additionally, this improves the efficiency of pharmaceutical preparations for quality control of pharmaceutical products by ensuring that the available preservatives maintain a sufficient concentration throughout the lifespan of the products.

Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-β2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-β2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

Understanding Binding of Quaternary Ammonium Compounds with Cellulose-Based Fibers and Wipes for Renewable and Sustainable Hygiene Options

Cellulose-based fibers are desirable materials for nonwoven wipes for their good absorbency, strength, cleaning, and biodegradable properties. However, quaternary ammonium compounds (QACs), being cationic in nature, show electrostatic interactions with anionic cellulosic fibers, reducing the available QACs to efficiently clean surfaces. This research presents sustainable alternative fibers that show better controlled exhaustion than commercial wipes and textile fibers. Textile and lignocellulosic fibers were prepared, soaked in QAC, and a UV–vis spectrophotometer was used to measure their exhaustion percentages. Factors such as immersion time and concentration of the disinfectant were also investigated, which affect the rate of exhaustion of the disinfectant from the fibers. A higher immersion time resulted in better exhaustion, whereas the total exhaustion decreased with an increase in the initial concentration of the disinfectant. The exhaustion of benzalkonium chloride (BAC) from the commercial wipes was also investigated at different immersion times and BAC concentrations. It was found that the wood and non-wood fibers showed more controlled exhaustion than the textile fibers and commercial wipes, and could be considered an alternative option for renewable and sustainable wipes and hygiene products.

Benzalkonium chloride disinfectant residues stimulate biofilm formation and increase survival of Vibrio bacterial pathogens.

Vibrio spp. are opportunistic human and animal pathogens found ubiquitously in marine environments. Globally, there is a predicted rise in the prevalence of Vibrio spp. due to increasing ocean temperatures, which carries significant implications for public health and the seafood industry. Consequently, there is an urgent need for enhanced strategies to control Vibrio spp. and prevent contamination, particularly in aquaculture and seafood processing facilities. Presently, these industries employ various disinfectants, including benzalkonium chloride (BAC), as part of their management strategies. While higher concentrations of BAC may be effective against these pathogens, inadequate rinsing post-disinfection could result in residual concentrations of BAC in the surrounding environment. This study aimed to investigate the adaptation and survival of Vibrio spp. exposed to varying concentrations of BAC residues. Results revealed that Vibrio bacteria, when exposed, exhibited a phenotypic adaptation characterized by an increase in biofilm biomass. Importantly, this effect was found to be strain-specific rather than species-specific. Exposure to BAC residues induced physiological changes in Vibrio biofilms, leading to an increase in the number of injured and alive cells within the biofilm. The exact nature of the "injured" bacteria remains unclear, but it is postulated that BAC might heighten the risk of viable but non-culturable (VBNC) bacteria development. These VBNC bacteria pose a significant threat, especially since they cannot be detected using the standard culture-based methods commonly employed for microbiological risk assessment in aquaculture and seafood industries. The undetected presence of VBNC bacteria could result in recurrent contamination events and subsequent disease outbreaks. This study provides evidence regarding the role of c-di-GMP signaling pathways in Vibrio adaptation mechanisms and suggests that c-di-GMP mediated repression is a potential avenue for further research. The findings underscore that the misuse and overuse of BAC may increase the risk of biofilm development and bacterial survival within the seafood processing chain.

Co-culture of benzalkonium chloride promotes the biofilm formation and decreases the antibiotic susceptibility of a Pseudomonas aeruginosa strain.

Benzalkonium chloride (BAC) is a disinfectant with broad-spectrum antibacterial properties, yet despite its widespread use and detection in the environment, the effects of BAC exposure on microorganisms remain poorly documented. Herein, the impacts of BAC on a Pseudomonas aeruginosa strain Jade-X were systematically investigated. The results demonstrated that the minimum inhibitory concentration (MIC) of BAC against strain Jade-X was 64 mg L-1. Exposure to BAC concentrations of 8, 16, 32, and 64 mg L-1 significantly augmented biofilm formation by 2.03-, 2.43-, 2.96-, and 2.56-fold respectively. The swimming and twitching abilities, along with the virulence factor production, were inhibited. Consistently, quantitative reverse transcription PCR assays revealed significant downregulation of genes related to flagellate- and pili-mediated motilities (flgD, flgE, pilB, pilQ, and motB), as well as phzA and phzB genes involved in pyocyanin production. The results of disk diffusion and MIC assays demonstrated that BAC decreased the antibiotic susceptibility of ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. Conversely, an opposite trend was observed for polymyxin B and ceftriaxone. Genomic analysis revealed that strain Jade-X harbored eleven resistance-nodulation-cell division efflux pumps, with mexCD-oprJ exhibiting significant upregulation while mexEF-oprN and mexGHI-opmD were downregulated. In addition, the quorum sensing-related regulators LasR and RhlR were also suppressed, implying that BAC might modulate the physiological and biochemical behaviors of strain Jade-X by attenuating the quorum sensing system. This study enhances our understanding of interactions between BAC and P. aeruginosa, providing valuable insights to guide the regulation and rational use of BAC.

An in-depth study on survival mechanism of bacterial isolates in disinfectants within the hospital environment.

The emergence of disinfectant resistance has become a severe threat due to reduced effectiveness. This study was undertaken to determine how bacteria adapt to survive exposure to disinfectants in the busiest section of a tertiary care hospital in Varanasi, India. Four isolates (two Klebsiella pneumoniae, Kp1 and Kp2; two Pseudomonas aeruginosa, Pa1 and Pa2) were obtained from chlorhexidine (CHX)-based handwash during microbiological surveillance of "in-use disinfectants" in hospital. Six disinfectants [4% CHX, 2% glutaraldehyde, 7.5% hydrogen peroxide, 1% sodium hypochlorite and 0.1% benzalkonium chloride (BAC), and 70% ethyl alcohol] were tested against these four isolates to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Antibiotic profile, change in MIC on exposure to disinfectants and biofilm formation in the presence and absence of disinfectants was studied. Whole genome sequencing (WGS) was done to identify the resistance mechanisms. The isolates showed the highest MBC/MIC ratio (4) against glutaraldehyde. Exposure to supra-inhibitory concentration of BAC for 21 days resulted in doubling of MIC/MBC. The majority (75%) of the isolates were multidrug resistant. All the isolates were strong biofilm producers. The reduction rate of biofilm formation decreased with an increase in the concentration of disinfectants (p = 0.05 for BAC). WGS revealed multiple AMR genes including bla DIM-1, disinfectant-resistant gene and efflux pump genes. The study emphasized the various adaptation strategies of these isolates for survival in disinfectant environment, thus posing a huge challenge for their control in the hospital environment.

Optimal effective concentration combinations (OPECCs) for binary application of membrane-targeting antiseptics and TMPyP-mediated antimicrobial photodynamic therapy

The widespread occurrence of multi-resistant bacteria is a health problem of global dimension. Infections caused by multi-resistant pathogens are difficult to treat and often associated with high mortality. Therefore, new treatment strategies are of interest, such as the use of differently acting antibacterial concepts. One of these new concepts is the use of antiseptics in combination with the antibacterial photodynamic therapy (aPDT). Currently, no method has yet been established as a standard procedure for investigating combined effects and evaluating them in a generally valid and unambiguous manner. The focus of this study was on how cationic antiseptics benzalkonium chloride (BAC) and chlorhexidine digluconate (CHX) behave in a combined application with aPDT using the photosensitizer TMPyP. For this purpose, BAC and CHX were applied in combination with the aPDT using TMPyP in non-lethal concentrations to the three bacteria Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis. The results of the combination experiments with sublethal concentrations of BAC or CHX with the aPDT showed that the binary application had a lethal effect. Irrespective of the bacteria, the reduction in concentrations in OPECC, compared to individual concentrations, was more than 50% for TMPyP, 23–40% for BAC, and 18–43% for CHX. Furthermore, the optimal effective concentration combinations (OPECCs) could be determined. The latter showed that the combined application allowed the reduction of both concentrations compared to the single application.

Investigating the biomass-specific inhibitory effect of benzalkonium chloride on anaerobic granules: A kinetic approach

Benzalkonium Chloride (BAC), a quaternary ammonium compound commonly used in industrial disinfection processes, was studied for its biomass-specific inhibitory effect on the specific methanogenic activity (SMA) of anaerobic granules. Inhibition batch assays were conducted with varying BAC concentrations (5–40 mg/L), resulting in biomass-dependent inhibition. Considering its surface-active nature, a biomass-specific BAC load was a suitable parameter to determine IC50 values, ranging from 4.3 to 6.1 mg BAC/g VS. For predicting the corresponding biomass-specific inhibition constant in ADM1, the inclusion of an additional adsorption-inhibition term was required for a better validation of results. The model yielded a biomass-specific IC50 of 5.3 mg BAC/g VS. The results encourage a change in perspective on IC50 for surfactants by determining a biomass-specific IC50, particularly in scenarios when surfactants accumulate within anaerobic reactors. Hereby, the reliability and practical relevance of IC50 is increased, driving the development of mitigation strategies.

Modification Of Kaolin Capkala With Benzalkonium Chloride (Bkc) Surfactant And Antibacterial Activity Test Against Escherichia Coli

A study was conducted to determine the antibacterial activity of modified benzalkonium chloride (BKC) kaolin Capkala against Escherichia coli (E. coli) bacteria. Kaolin modification begins by melting kaolin with sodium hydroxide (NaOH) and reacting using a reflux device. Furthermore, the sample was reacted with BKC at a concentration variation of 0, 2.5×10-3; 5×10-3, and 2.5×10-2 M for 6 hours with a stirring speed of 150 rpm. The modified kaolin was characterized using Fourier Transformed Infrared (FTIR), and adsorption tests were performed on Cl-. The FTIR spectrum of the modified kaolin showed an absorption peak at 2926.19 cm-1 C-H symmetrical from CH2, which indicated the attachment of surfactant to the kaolin. BKC modified kaolin antibacterial activity test using the halo test method. The adsorption test on Cl- and the antibacterial activity test on kaolin and modified kaolin increased with increasing BKC concentration, namely 3545; 3828.6, 3970.4; 4183.1 mg/L and the inhibitory diameter were 2.1±0.14, 3.3±0.14, 4.5±0.14, and 6±0.14 mm, respectively. The results showed that BKC can increase the antibacterial activity of kaolin

Benzalkonium chloride forces selective evolution of resistance towards antibiotics in Salmonella enterica serovar Typhimurium

BackgroundAlthough food-grade disinfectants are extensively used worldwide, it has been reported that the long-term exposure of bacteria to these compounds may represent a selective force inducing evolution including the emergence of antibiotic resistance. However, the mechanism underlying this correlation has not been elucidated. This study aims to investigate the genomic evolution caused by long-term disinfectant exposure in terms of antibiotic resistance in Salmonella enterica Typhimurium. MethodsS. Typhimurium isolates were exposed to increasing concentrations of benzalkonium chloride (BAC) and variations of their antibiotic susceptibilities were monitored. Strains that survived BAC exposure were analyzed at whole genome perspective using comparative genomics, and Sanger sequencing-confirmed mutations in ramR gene were identified. Next, the efflux activity in ramR-mutated strains shown as bisbenzimide accumulation and expression of genes involved in AcrAB-TolC efflux pump using quantitative reverse transcriptase PCR were determined. ResultsMutation rates of evolved strains varied from 5.82 × 10−9 to 5.56 × 10-8, with fold increase from 18.55 to 1.20 when compared with strains evolved without BAC. Mutations in ramR gene were found in evolved strains. Upregulated expression and increased activity of AcrAB-TolC was observed in evolved strains, which may contribute to their increased resistance to clinically relevant antibiotics. In addition, several indels and point mutations in ramR were identified, including L158P, A37V, G42E, F45L, and R46H which have not yet been linked to antimicrobial resistance. Resistance and mutations were stable after seven consecutive cultivations without BAC exposure. These results suggest that strains with sequence type (ST) ST34 were the most prone to mutations in ramR among the three STs tested (ST34, ST19, ST36). ConclusionsThis work demonstrated that disinfectants, specifically BAC forces S. Typhimurium to enter a specific evolutionary trajectory towards antibiotic resistance illustrating the side effects of long-term exposure to BAC and probably also to other disinfectants. Most significantly, this study provides new insights in understanding the emergence of antibiotic resistance in modern society.

Multidrug Resistance in Enterococci Isolated from Cheese and Capable of Producing Benzalkonium Chloride-Resistant Biofilms.

This study aimed to investigate enterococci recovered from eight Portuguese cheeses made with raw ewe's milk, regarding antibiotic resistance, virulence genes, minimum inhibitory concentration (MIC) of benzalkonium chloride (BAC), biofilm formation capacity, and biofilm eradication (MBEC) by BAC. Antimicrobial resistance against seven antibiotics of five groups was evaluated using the disk diffusion method. The presence of the genes that encode resistance to the antibiotics penicillin (blaZ), erythromycin (ermA, ermB, and ermC), vancomycin (vanA and vanB), aminoglycoside (aac(6')-Ie-aph(2″)-Ia), and β-lactam (pbp5) and the genes that encode virulence factors, frsB, cylA, gelE, esp, and agg, were investigated via multiplex PCR. The susceptibility of planktonic cells to BAC was evaluated by the MIC and MBC values of the isolates, using the broth microdilution method. To assess the biofilm-forming ability and resistance of biofilms to BAC, biofilms were produced on stainless steel coupons, followed by exposure to BAC. The results showed a high resistance to the antibiotics vancomycin (87.5%), erythromycin (75%), tetracycline (50%), and penicillin (37.5%). Multidrug resistance was observed in 68.8% of the isolates. Genes encoding the virulence factors FrsB (frsB) and gelatinase E (gelE) were detected in all isolates. The esp and cylA genes were found in 56.3% and 37.5% of the isolates, respectively. All isolates exhibited a biofilm-forming ability, regardless of incubation time and temperature tested. However, after 72 h at 37 °C, E. faecium and E. faecalis biofilms showed significant differences (p ≤ 0.05). Although most isolates (62.5%) were susceptible to BAC (MIC ≤ 10 mg/L), biofilms of the same isolates were, generally, resistant to the higher concentration of BAC (80 mg/mL) tested. This study using Enterococcus isolates from a ready-to-eat food, such as cheese, reveals the high percentages of vancomycin resistance and multidrug resistance, associated with the presence of virulence genes, in isolates also capable of producing biofilms resistant to BAC, an important active ingredient of many disinfectants. These results emphasize the need for effective control measures to ensure the safety and quality of dairy products.

Effect of benzalkonium chloride adaptation on the tolerance of Cronobacter sakazakii exposed to subsequent lethal stresses

Benzalkonium Chloride (BC) is applied in food processing, clinical settings, and households to control the spread of pathogens. However, BC adaptation may promote bacterial resistance against biocides and physical stresses. In the study, the sensitivity of non-adapted and BC-adapted C. sakazakii to subsequent lethal stresses was assessed. Results indicated that BC adaptation provided C. sakazakii direct protection against high BC concentrations, but no cross-protection against heat, acid, osmotic, desiccative, and ethanol-related stress. BC adaptation reduced the proportion of saturated fatty acids (SFA) and the ratio of SFA to unsaturated fatty acids (SFA/USFA), whereas increasing the proportion of unsaturated fatty acids (USFA) in C. sakazakii. Six genes related to motility, metabolism, transport, and efflux were upregulated under BC adaptation. Some Surface-enhanced Raman spectroscopy (SERS) signal characteristics of the BC-adapted cell were significantly different from that of the non-adapted cell, which reflected the changes of intracellular proteins, nucleic acids, carbohydrates, and other cellular components. Consequently, the ability of BC-adapted C. sakazakii to withstand subsequent stresses should be taken into account when developing microbial control measures. A greater understanding of BC adaptation mechanisms in C. sakazakii will provide important insights for disease prevention.

Rice husk fiber-reinforced starch antimicrobial biocomposite film for active food packaging

Active packaging with antimicrobial properties has the potential to impede the growth of bacteria and extend the storage life of packaged food items. Corn starch (CS) presents an excellent alternative to conventional packaging materials. However, its usage is limited due to its high hydrophilicity and low mechanical properties. Herein, rice husk fiber (RHF), an agricultural waste, was used as reinforcement, and benzalkonium chloride (BKC) was used as an antimicrobial agent to develop a sustainable and biodegradable packaging film exhibiting antimicrobial properties. The content of RHF varied between 10% and 50%, while the concentration of BKC ranged from 0.05% to 0.2%. The impact of these variations on the mechanical properties, thermal properties, moisture content, surface morphology, biodegradability, and optical properties of the biocomposite film (RHF/BKC/CS) was evaluated and analyzed. At 20% fiber content and 0.05% BKC content, the biocomposite film showed 61% improvement in tensile strength, optimum moisture content, transparency, and water solubility. Also, BKC content was optimized to attain good antimicrobial properties against food-spoiling bacteria. The biocomposite film was found to increase the shelf life of packaged strawberries by more than 11 days. The biocomposite film with excellent properties could be used as a potential packaging material in food, cosmetics, and pharmaceutical industries.

Exposure of Salmonella enterica serovar 1,4, [5],12:i:- to benzalkonium chloride leads to acquired resistance to this disinfectant and antibiotics.

Disinfectants such as benzalkonium chloride (BC) extensively used in animal farms and food-processing industries contribute to the development of adaptive- and cross-resistance in foodborne pathogens, posing a serious threat to food safety and human health. The purpose of this study is to explore whether continuous exposure of Salmonella enterica serovar 1,4, [5],12: i:- (S. 1,4, [5],12: i:-) to sublethal concentrations of BC could result in acquired resistance to this agent and other environmental stresses (e.g. antibiotics, heat, and acid). BC tolerance increased in all tested strains after exposure to gradually increasing concentrations of BC, with increases in minimum inhibitory concentrations between two and six-fold. The survival rate of BC-adapted strains was significantly (P<0.05) higher than that of their wild-type (non-adapted) counterparts in lethal concentrations of BC. In addition, significant reductions (P<0.05) in zeta potential were observed in BC-adapted strains compared to wild-type ones, indicating that reduction in cell surface charge was a cause of adaptative resistance. More importantly, two BC-adapted strains exhibited increased antibiotic resistance to levofloxacin, ceftazidime, and tigecycline, while gene mutations (gyrA, parC) and antibiotic efflux-related genes (acrB, mdsA, mdsB) were detected by genomic sequencing analysis. Moreover, the tolerance of BC-adapted strains to heat (50, 55, and 60°C) and acid (pH 2.0, 2.5) was strain-dependent and condition-dependent. Repeated exposure to sublethal concentrations of BC could result in the emergence of BC- and antibiotic-resistant S. 1,4, [5],12: i:- strains.

The Impact of Low-Level Benzalkonium Chloride Exposure on Staphylococcus spp. Strains and Control by Photoinactivation.

Exposure of bacteria to low concentrations of biocides can facilitate horizontal gene transfer, which may lead to bacterial adaptive responses and resistance to antimicrobial agents. The emergence of antibacterial resistance not only poses a significant concern to the dairy industry but also adds to the complexity and cost of mastitis treatment. This study was aimed to evaluate how selective stress induced by benzalkonium chloride (BC) promotes antibiotic non-susceptibility in Staphylococcus spp. In addition, we investigated the efficacy of photodynamic inactivation (PDI) in both resistant and susceptible strains. The study determined the minimum inhibitory concentration (MIC) of BC using the broth microdilution method for different Staphylococcus strains. The experiments involved pairing strains carrying the qacA/qacC resistance genes with susceptible strains and exposing them to subinhibitory concentrations of BC for 72 h. The recovered isolates were tested for MIC BC and subjected to disc diffusion tests to assess changes in susceptibility patterns. The results demonstrated that subinhibitory concentrations of BC could select strains with reduced susceptibility and antibiotic resistance, particularly in the presence of S. pasteuri. The results of PDI mediated by toluidine blue (100 µM) followed by 60 min irradiation (total light dose of 2.5 J/cm2) were highly effective, showing complete inactivation for some bacterial strains and a reduction of up to 5 logs in others.

Environmental concentrations of antibiotics, biocides, and heavy metals fail to induce phenotypic antimicrobial resistance in Escherichia coli

Most anthropogenically affected environments contain mixtures of pollutants from different sources. The impact of these pollutants is usually the combined effect of the individual polluting constituents. However, how these stressors contribute to the development of antimicrobial resistance in environmental microorganisms is poorly understood. Thus, a 30-day exposure experiment to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC (benzalkonium chloride) 10 and DADMAC (diallyldimethylammonium chloride) 12, was conducted using fully susceptible E. coli ATCC 25922 to ascertain any development of phenotypic or genotypic resistance. Furthermore, wild-type isolates were collected from the same aquatic environment as the stressors, analysed for phenotypic resistance using the disk diffusion method and genotypically through whole genome sequencing. Exposure to the various concentrations and combinations of the stressors did not trigger phenotypic resistance in the experimental bacteria. Furthermore, genotypic analysis of the WGS on the exposed isolates only found the macrolide resistance mdf(A) gene (also present in the control strain) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes that encoded for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations except for the robA, a transcription protein subset of the ArcC/XylS family of proteins, which confer multidrug resistance in E. coli. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues in the aquatic environmental concentrations of the stressors identified in the current study could not induce phenotypic or genotypic resistance but encoded for genes responsible for the development of persistence and tolerance in bacteria, which could be a precursor to the development of resistance in environmental bacteria.

Analyze the toxicities of benzalkonium chloride as a COVID-19 disinfectant in physiological goldfish (Carassius auratus).

Coronavirus disease-2019 (COVID-19) impacts increasing the use of disinfectants (benzalkonium chloride), which indirectly accumulate in water. The disinfectant accumulation in waters has been studied, but there has been no study of its impact on aquatic commodities, especially fish with a high sensitivity, one of which is goldfish (Carassius auratus). Benzalkonium chloride can potentially affect several body proteins, including the cytoskeleton, nervous and endocrine systems, and fish physiology. This study aimed to determine the impact of benzalkonium chloride as a disinfectant on the level of color brightness, growth, gill histopathology, and mortality in goldfish. This investigation provides input into the impact of using disinfectants to prevent COVID-19 on aquatic commodities. This study utilized goldfish specimens sourced from Tulungagung, East Java, Indonesia. The experiment involved different concentration levels of benzalkonium chloride: (T1) 0 mg/L, (T2) 0.03 mg/L, (T3) 0.06 mg/L, (T4) 0.09 mg/L, and (T5) 0.12 mg/L. The research data were subjected to an analysis of variance for analysis. In cases where significant differences were observed, Duncan's test was conducted for color brightness, growth, and mortality data. Furthermore, if the gill histopathological data yielded significant differences, additional tests were applied (Kruskal-Wallis and Mann-Whitney test). The findings of this study demonstrated significant differences (p < 0.05) in the level of color brightness, growth, gill histopathology, and mortality in goldfish in response to varying concentrations of benzalkonium chloride. The relationship between the length and weight of the goldfish was analyzed using regression coefficients (b values), which were determined as 4.86, -0.04, -0.2, 0.8, and -0.07, respectively. Notably, the brightness level in the T2 group exhibited positive color results with a hue value of 11.55°, while optimal growth was observed in the T4 group, as evidenced by b value of 0.8. The gill histopathological data showed significant differences (p < 0.05). The scoring of histopathological damage in the goldfish gills ranged from 0 to 10, with higher scores indicating more severe damage. The highest total score of 10 was observed in the T5 group exposed to a concentration of 0.12 mg/L, resulting in an 85% mortality rate. This indicates that benzalkonium chloride, with its toxic compounds, can disrupt the respiratory system of fish and lead to death. The effects of benzalkonium chloride were evident even at a concentration of 0.03 mg/L. With increasing concentration, there was an increase in mortality rate, a decrease in growth, and a rise in histopathological damage to the gills. These findings highlight the negative impact of using conventional disinfectants on water and its organisms, emphasizing the need for further research on environmentally friendly alternatives.

A fish perspective on SARS-CoV-2: Toxicity of benzalkonium chloride on Danio rerio

SARS-CoV-2 outbreak led to an increased marketing of disinfectants, creating a potential environmental problem. For instance, pre-pandemic environmental levels of the disinfectant benzalkonium chloride (BAC) ranging from 0.5 to 5 mgL−1 in effluents were expected to further increase threatening aquatic life. Our aim was to characterize potential adverse effects after an acute exposure of zebrafish to different concentrations of BAC. An increase in the overall swimming activity, thigmotaxis behavior, and erratic movements were observed. An increase in CYP1A1 and catalase activities, but inhibitions of CY1A2, GSTs and GPx activities were also noticed. BAC is metabolized by CYP1A1, increasing the production of H2O2, thereby activating the antioxidant enzyme CAT. Data also showed an increase of AChE activity. Our study highlights adverse embryonic, behavioral, and metabolic effects of noteworthy environmental significance, especially considering that the use and release of BAC is most likely to increase in a near future.