Cord blood-derived biologics lead to robust axonal regeneration in benzalkonium chloride-injured mouse corneas by modulating the Il-17 pathway and neuropeptide Y.

BACKGROUND: Umbilical cord blood-derived therapeutics, such as serum (UCS) and platelet-rich plasma (UCPRP), are popular treatment options in clinical trials and can potentially be utilized to address a clinically unmet need caused by preservatives, specifically benzalkonium chloride (BAK), present in ophthalmic formulations. As current clinical interventions for secondary injuries caused by BAK are suboptimal, this study will explore the feasibility of utilizing UCS and UCPRP for cornea treatment and investigate the underlying mechanisms associated with this approach. METHODS: Mice's corneas were administered BAK to induce damage. UCS and UCPRP were then utilized to attempt to treat the injuries. Ocular tests were performed on the animals to evaluate recovery, while immunostaining, RNA-seq, and subsequent bioinformatics analysis were conducted to investigate the treatment mechanism. RESULTS: BAK administration led to widespread inflammatory responses in the cornea. Subsequent treatment with UCS and UCPRP led to the downregulation of immune-related 'interactions between cytokine receptors' and 'IL-17 signaling' pathways. Although axonal enhancers such as Ngf, Rac2, Robo2, Srgap1, and Rock2 were found to be present in the injured group, robust axonal regeneration was observed only in the UCS and UCPRP treatment groups. Further analysis revealed that, as compared to normal corneas, inflammation was not restored to pre-injury levels post-treatment. Importantly, Neuropeptide Y (Npy) was also involved in regulating immune responses, indicating neuroimmune axis interactions. CONCLUSIONS: Cord blood-derived therapeutics are feasible options for overcoming the sustained injuries induced by BAK in the cornea. They also have potential applications in areas where axonal regeneration is required.

Microbial biofilm metabolization of benzalkonium compounds (benzyl dimethyl dodecyl ammonium & benzyl dimethyl tetradecyl ammonium chloride).

Benzalkonium chlorides (BACs) are quaternary ammonium compounds (QUATs) that are used as biocides. The degradation of these compounds in wastewater treatment plants is essential to reduce their spread into the environment and thus prevent the development of QUAT-resistant genes. The biodegradation of two BACs (BAC-12 and BAC-14) was investigated in moving bed biofilm reactors (MBBRs). Degradation half-lives of 12 and 20 h for BAC-12 and - 14, respectively, were detected as well as the formation of 42 metabolites. Two new degradation pathways for the BACs were identified in this study: 1) one involving an ω-oxidation, followed by ß-oxidation and 2) one via an ω-oxidation followed by an α-oxidation that was succeeded by ß-oxidation. Similar metabolites were detected for both BAC-12 and BAC-14. Additional metabolites were detected in the study, that could not be assigned to the above-mentioned pathways, revealing even more metabolic pathways in the MBBR which is probably due to the complexity of the microbial community in the biofilm. Interestingly, both TP194 (Benzyl-(carboxymethyl)-dimethylazanium) and TP208B (Benzyl-(2-carboxyethyl)-dimethylazanium) were identified as end products of the ω/ß-pathway and the α/ß-pathway. TP208B, TP152 and TP250 that were identified in this study, as well as the known BDMA were discovered in the effluent of a wastewater treatment plant.

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

BACKGROUND: Although 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. METHODS: S. 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. RESULTS: Mutation 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). CONCLUSIONS: This 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.

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.

Transcriptional insight into the effect of benzalkonium chloride on resistance and virulence potential in Salmonella Typhimurium.

Benzalkonium chloride (BC), a class of quaternary ammonium compounds (QACs), is widely used as a surface disinfectant in food industries and hospitals. To date, little is known about the underlying mechanisms of how bacterial pathogens respond to and develop resistance against QACs. We investigated the genome-wide transcriptional responses of Salmonella enterica serovar Typhimurium to treatment with BC and identified the genetic determinants required for bacterial resistance to BC, including ramRA, phoU-pstBACS, cpxARP, and ugpQCEAB. Remarkably, RamA, a member of the AraC/XylS family transcription regulators, increased its transcription upon treatment with BC and its absence rendered Salmonella susceptible to BC treatment, indicating the positive role of RamA in BC tolerance. The attenuated BC resistance of the ΔramA mutant strain was complemented by the introduction of AcrA in trans, indicating that the AcrAB-TolC efflux system activated by RamA is required for the resistance of Salmonella to BC. Meanwhile, sublethal concentrations of BC downregulated the mRNA expression of genes associated with Salmonella pathogenicity island 1 (SPI-1) and SPI-2, which are critical determinants of Salmonella virulence. In accordance with the downregulation of SPI-1, HilD, the master regulator of SPI-1, also decreased upon treatment with BC; however, the absence of Lon protease nearly nullified the BC-mediated repression of SPI-1 genes. Intriguingly, overexpression of RamA repressed the transcription of SPI-1 genes; however, its negative regulation of SPI-1 expression is likely to be independent of the Lon-mediated regulation of SPI-1. These results demonstrated that treatment with sublethal concentrations of BC not only stimulates Salmonella to develop resistance mechanisms against BC, but also influences Salmonella virulence.

A novel ophthalmic latanoprost 0.005% nanoemulsion: a cytotoxicity study.

BACKGROUND: Benzalkonium chloride (BAK), the most commonly used preservative in anti-glaucoma eye drops, inflicts damage to the ocular surface. A novel anti-glaucoma formulation that avoids the use of BAK has been developed. The aim of this study was to evaluate the cytotoxicity of this formulation and to compare it with an ophthalmic solution containing BAK. METHODS: Two different latanoprost eye drops were used: one ophthalmic solution (LSc) containing BAK 0.02% and one ophthalmic nanoemulsion (LNe) with a soft preservative (potassium sorbate 0.18%). Human epithelial conjunctival cells were incubated for 15, 30, and 60 min with either LSc or LNe. The cytotoxicity was determined by MTT assay. Cell death was measured by flow cytometry using annexin V-FITC and propidium iodide. RESULTS: The values of cell viability and proliferation obtained from cells exposed to LNe were between 80 and 90% relative to the control group, whereas values obtained from cells exposed to LSc were around 30% at all study times (p < 0.05 at 15 and 30 min; p < 0.01 at 60 min). The percentage of viable cells decreased significantly when cells were incubated with LSc compared with cells incubated with LNe at all the study times, while the percentage of cells in late apoptosis/necrosis increased significantly in cells exposed to LSc compared to LNe. CONCLUSIONS: The new latanoprost nanoemulsion is significantly less cytotoxic on human conjunctival cells than LSc. These results suggest that the new formulation might be gentler on the eye surface than currently available BAK-preserved latanoprost solutions.

[Effects of Benzalkonium Chloride in Ophthalmic Eyedrop Medications on Corneal Epithelium].

Eyedrops often contain additives other than active pharmaceutical ingredients, such as preservatives. The most frequently used preservative is benzalkonium chloride (BAC). When the ocular surface is exposed to eyedrops, the active pharmaceutical ingredients and additives can cause corneal epithelial disorder. Particularly in clinical settings, there is great interest in corneal epithelial disorders resulting from the use of glaucoma eyedrops, which is inevitable when instilled for a long period of time after the onset of disease. At the authors' institute, glaucoma is treated with consideration of reducing corneal epithelial disorder while ensuring the effect of lowering intraocular pressure by the appropriate choice of eyedrops. In this review, we show the examples of the retrospective studies. Sodium hyaluronate eyedrops are prescribed for corneal epithelial disorders such as superficial punctate keratitis associated with dry eye. Prescribable concentrations of sodium hyaluronate in Japan are 0.1% or 0.3%, and the 0.3% formulation does not contain BAC. The authors' study showed that 0.3% sodium hyaluronate pretreatment reduced the cytotoxicity of BAC in cultured corneal epithelial cells, whereas an in vivo study in mice showed that a 0.3% sodium hyaluronate instillation was suggested and that the drug may enhance the cytotoxicity of separately administered BAC. It is suggested that sodium hyaluronate prolonged the retention of BAC on the ocular surface. However, there have been no reports of this problem in the clinical setting. It is important for ophthalmologists to understand the properties of additives other than the active pharmaceutical ingredients in eyedrops.

Kinetics and distribution of benzalkonium compounds with different alkyl chain length following intravenous administration in rats.

Benzalkonium chloride is widely used in disinfectants. Several toxicological and fatal cases have been reported; however, little is known about its kinetics and distribution. We investigated the kinetic characteristics and distribution of benzalkonium cation (BZK) based on the length of the alkyl chains C12, C14, and C16. Rats were treated intravenously with BZK solution (dose, 13.9 mg/kg) containing equal amounts of the three homologues. Kinetic parameters in the blood were assessed, and BZK distribution in the blood and tissues was examined both in rapid intravenous (IV) and drip intravenous (DIV) administrations. BZK concentrations were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). BZK with longer alkyl chains showed lower elimination tendencies and remained in the blood for a longer duration. Concentrations of BZK were higher in the heart, lung, spleen, and kidney than those in the blood, and lower in the brain and fat. In both the IV and DIV groups, the lung, liver, spleen, and fat samples showed higher concentrations of the longer alkyl chains (BZK-C12 < -C14 < -C16), and the opposite trend was observed in the kidney (BZK-C16 < -C14 < -C12). Only the heart and muscle samples displayed the homologues in ratios comparable to the original administered solutions. Differences between IV and DIV groups could be identified by comparing concentrations of BZK homologues in the heart, lung, spleen, and kidney samples. We found that the kinetics and distribution of BZK were influenced by the alkyl chain length, and analysing each BZK homologues in blood and tissue samples may provide useful information.

Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450.

Benzalkonium chlorides (BACs) are widely used as disinfectants in cleaning products, medical products, and the food processing industry. Despite a wide range of reported toxicities, limited studies have been conducted on the metabolism of these compounds in animal models and none in human-derived cells or tissues. In this work, we report on the metabolism of BACs in human liver microsomes (HLM) and by recombinant human hepatic cytochrome P450 (CYP) enzymes. BAC metabolism in HLM was NADPH-dependent and displayed apparent half-lives that increased with BAC alkyl chain length (C10 < C12 < C14 < C16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. Tandem mass spectrometry fragmentation of BAC metabolites confirmed that oxidation occurs on the alkyl chain region. Major metabolites of C10-BAC were identified as ω-hydroxy-, (ω-1)-hydroxy-, (ω, ω-1)-diol-, (ω-1)-ketone-, and ω-carboxylic acid-C10-BAC by liquid chromatography-mass spectrometry comparison with synthetic standards. In a screen of hepatic CYP isoforms, recombinant CYP2D6, CYP4F2, and CYP4F12 consumed substantial quantities of BAC substrates and produced the major microsomal metabolites. The use of potent pan-CYP4 inhibitor HET0016, the specific CYP2D6 inhibitor quinidine, or both confirmed major contributions of CYP4- and CYP2D6-mediated metabolism in the microsomal disappearance of BACs. Kinetic characterization of C10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ω-hydroxylation (Vmax = 380 pmol/min/mg, Km = 0.69 µM) and (ω-1)-hydroxylation (Vmax = 126 pmol/min/mg, Km = 0.13 µM) reactions. This work illustrates important roles for CYP4-mediated ω-hydroxylation and CYP2D6/CYP4-mediated (ω-1)-hydroxylation during the hepatic elimination of BACs, an environmental contaminant of emerging concern. Furthermore, we demonstrate that CYP-mediated oxidation of C10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.

Adaptation of Pseudomonas aeruginosa clinical isolates to benzalkonium chloride retards its growth and enhances biofilm production.

The increasing percentage of Pseudomonas aeruginosa strains that are resistant to multiple antibiotics is a global problem. The exposure of P. aeruginosa isolates to repeated sub lethal concentrations of biocides in hospitals and communities may be one of the causes leading to increased antibiotic resistance. Benzalkonium chloride (BAC) is widely used as disinfectant and preservative. This study investigated the effect of exposure of P. aeruginosa clinical isolates to sub lethal concentrations of BAC on their antibiotic resistance, growth process and biofilm formation. The collected 43 P. aeruginosa clinical isolates were daily subjected to increasing sub lethal concentrations of BAC. The effect of adaptation on antibiotic resistance, growth process, cell surface hydrophobicity and biofilm formation of P. aeruginosa isolates were examined. Interestingly, Most P. aeruginosa isolates adapted to BAC showed an increase in antibiotic resistance and 66% of the isolates showed retardation of growth, 63% showed increased cell surface hydrophobicity and 23.5% exhibited enhanced biofilm formation by crystal violet assay. To define whether the effect of BAC adaptation on biofilm production was manifested at the transcriptional level, quantitative RT-PCR was used. We found that 60% of the tested isolates showed overexpression of ndvB biofilm gene. More efforts are required to diminish the increasing use of BAC to avoid bacterial adaptation to this biocide with subsequent retardation of growth and enhanced biofilm formation which could lead to antibiotic resistance and treatment failure of infections caused by this opportunistic pathogen.

Evaluation of pulmonary toxicity of benzalkonium chloride and triethylene glycol mixtures using in vitro and in vivo systems.

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray-form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1-10 µg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 µg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC-promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N-acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 µg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 µg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 µg/kg) and TEG (1000 µg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in BALF. These results suggest that TEG can potentiate BAC-induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray-form products containing this chemical combination is potentially harmful to humans.

Gold nanoparticles capped with benzalkonium chloride and poly (ethylene imine) for enhanced loading and skin permeability of 5-fluorouracil.

OBJECTIVE: To enhance 5-fluorouracil (5-FU) permeability through the skin by loading onto gold nanoparticles (GNPs) capped with two cationic ligands, benzalkonium chloride (BC) or poly (ethylene imine) (PEI). Whereas 5-FU has excellent efficacy against many cancers, its poor permeability through biological membranes and several adverse effects limit its clinical benefits. BC and PEI were selected to stabilize GNPs and to load 5-FU through ionic interactions. METHODS: 5-FU/BC-GNPs and 5-FU/PEI-GNPs were prepared at different 5-FU/ligand molar ratios and different pH values and were evaluated using different techniques. GNPs stability was tested as a function of salt concentration and storage time. 5-FU release from BC- and PEI-GNPs was evaluated as a function of solution pH. Ex vivo permeability studies of different 5-FU preparations were carried out using mice skin. RESULTS: 5-FU-loaded GNPs size and surface charge were dependent on the 5-FU/ligand molar ratios. 5-FU entrapment efficiency and loading capacity were dependent on the used ligand, 5-FU/ligand molar ratio and solution pH. Maximum drug entrapment efficiency of 59.0 ± 1.7% and 46.0 ± 1.1% were obtained for 5-FU/BC-GNPs and 5-FU/PEI-GNPs, respectively. 5-FU-loaded GNPs had good stability against salinity and after storage for 4 months at room temperature and at 4 °C. In vitro 5-FU release was pH- and ligand-dependent where slower release was observed at higher pH and for 5-FU/BC-GNPs. 5-FU permeability through mice skin was significantly higher for drug-loaded GNPs compared with drug-ligand complex or drug aqueous solution. CONCLUSION: Based on these results, BC- and PEI-GNPs might find applications as effective topical delivery systems of 5-FU.

Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread. IMPORTANCE: Benzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.

Similar Microbial Consortia and Genes Are Involved in the Biodegradation of Benzalkonium Chlorides in Different Environments.

Benzalkonium chlorides (BACs) are emerging pollutants. Identification of microorganisms and the genes involved in the biodegradation of BACs is crucial for better understanding the fate of BACs in the environment and developing treatment strategies. Four microbial communities degrading BACs were developed from sewage (SEW), activated sludge (AS), soil (SOIL) and sea sediment (SEA) samples. According to 16S rRNA pyrosequencing and shotgun metagenome sequencing analyses, the most abundant species represented uncharacterized members of the Pseudomonas and Achromobacter genera. BAC biotransformation rates of the enriched microbial communities were 2.8, 3.2, 17.8, and 24.3 µM hr(-1) for SEA, AS, SOIL, and SEW, respectively, and were positively correlated with the relative abundance of a particular Pseudomonas sp. strain, BIOMIG1. The strain BIOMIG1 mineralizes BACs at a rate up to 2.40 µmol hr(-1) 10(-11) cells. Genomes of four BAC degrading and nondegrading BIOMIG1 phenotypes were sequenced and differentially compared with each other. As a result, a gene cluster encoding for transporters, an integrase and a dioxygenase were involved in BAC biotransformation. Our results suggest that BIOMIG1 plays a key role on the fate of BACs in the environment and genes, other than those reported to date, are involved in BAC biotransformation in various habitats.

Biodegradation of benzalkonium chlorides singly and in mixtures by a Pseudomonas sp. isolated from returned activated sludge.

Bactericidal cationic surfactants such as quaternary ammonium compounds (QACs) are widely detected in the environment, and found at mg kg(-1) concentrations in biosolids. Although individual QACs are amenable to biodegradation, it is possible that persistence is increased for mixtures of QACs with varying structure. The present study evaluated the biodegradation of benzyl dimethyl dodecyl ammonium chloride (BDDA) singly and in the presence of benzyl dimethyl tetradecyl ammonium chloride (BDTA) using Pseudomonas sp., isolated from returned activated sludge. Growth was evaluated, as was biodegradation using (14)C and HPLC-MS methods. BDTA was more toxic to growth of Pseudomonas sp. compared to BDDA, and BDTA inhibited BDDA biodegradation. The benzyl ring of [U-(14)C-benzyl] BDDA was readily and completely mineralized. The detection of the transformation products benzyl methyl amine and dodecyl dimethyl amine in spent culture liquid was consistent with literature. Overall, this study demonstrates the antagonistic effect of interactions on biodegradation of two widely used QACs suggesting further investigation on the degradation of mixture of QACs in wastewater effluents and biosolids.

Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi.

Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials.

Genetic characterization of plasmid-associated triphenylmethane reductase in Listeria monocytogenes.

The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (bcrABC) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (tmr) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream of bcrABC as part of a putative IS1216 composite transposon. To confirm the role of tmr in triphenylmethane dye detoxification, we introduced various tmr-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning bcrABC and tmr were CV resistant, and in such constructs tmr transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only tmr and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed that all those harboring bcrABC and adjacent pLM80 sequences, including tmr, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of L. monocytogenes.

Microbial community degradation of widely used quaternary ammonium disinfectants.

Benzalkonium chlorides (BACs) are disinfectants widely used in a variety of clinical and environmental settings to prevent microbial infections, and they are frequently detected in nontarget environments, such as aquatic and engineered biological systems, even at toxic levels. Therefore, microbial degradation of BACs has important ramifications for alleviating disinfectant toxicity in nontarget environments as well as compromising disinfectant efficacy in target environments. However, how natural microbial communities respond to BAC exposure and what genes underlie BAC biodegradation remain elusive. Our previous metagenomic analysis of a river sediment microbial community revealed that BAC exposure selected for a low-diversity community, dominated by several members of the Pseudomonas genus that quickly degraded BACs. To elucidate the genetic determinants of BAC degradation, we conducted time-series metatranscriptomic analysis of this microbial community during a complete feeding cycle with BACs as the sole carbon and energy source under aerobic conditions. Metatranscriptomic profiles revealed a candidate gene for BAC dealkylation, the first step in BAC biodegradation that results in a product 500 times less toxic. Subsequent biochemical assays and isolate characterization verified that the putative amine oxidase gene product was functionally capable of initiating BAC degradation. Our analysis also revealed cooperative interactions among community members to alleviate BAC toxicity, such as the further degradation of BAC dealkylation by-products by organisms not encoding amine oxidase. Collectively, our results advance the understanding of BAC aerobic biodegradation and provide genetic biomarkers to assess the critical first step of this process in nontarget environments.

Flexible probe for in vivo quantification of corneal epithelium permeability through non-invasive tetrapolar impedance measurements.

Studies concerning the functional status of the corneal epithelium are of special interest due to its key role in preventing ocular surface disease and corneal infections. In particular, quantitative measurements of the epithelium permeability translayer electrical resistance (TER) have been proven as a sensitive in vitro test for evaluation of the corneal barrier function. In a recent work from the authors (Guimera et al. Biosens. Bioelectron. 31:55-61, 2012), a novel method to non-invasively assess the corneal epithelial permeability by using tetrapolar impedance measurements, based on the same TER theoretical principles, was presented and validated using a rigid sensing device. In this work, the usability of this method has been dramatically improved by using SU-8 photoresist as a substrate material. The flexibility of this novel sensing device makes no need to apply pressure on the cornea to ensure the electrical contact between the electrodes and the corneal surface. The feasibility of this flexible sensor has been evaluated in vivo by increasing the permeability of rabbit corneal epithelium. For that, different concentrations of benzalkonium chloride (BAC) solution were instilled on different rabbit corneas. The obtained results have been compared with measurements of the permeability to sodium fluorescein of different excised corneas, a well-known method used to evaluate the corneal barrier function, to demonstrate the feasibility of this novel flexible sensor for quantifying the corneal epithelium permeability in vivo in a non-invasive way.

Modeling the fate and effect of benzalkonium chlorides in a continuous-flow biological nitrogen removal system treating poultry processing wastewater.

The fate and effect of the antimicrobial compounds benzalkonium chlorides (BACs) on the biological nitrogen removal (BNR) processes for a continuous-flow, three-stage laboratory-scale BNR system were modeled. Three kinetic sub-models, corresponding to each reactor, were developed and then combined in a comprehensive ASM1-based model. Kinetic parameters for the three sub-models were evaluated using experimental data obtained from independent batch assays. The biodegradation of BACs was modeled with a mixed-substrate Monod equation. The inhibitory effect of BACs on the utilization of degradable COD and denitrification was modeled as competitive inhibition, whereas non-competitive inhibition was used to model the effect of BACs on nitrification and inhibition coefficients were evaluated. The model simulated well the long-term performance of the BNR system treating a poultry processing wastewater with and without BACs. Enhanced BAC degradation by heterotrophs and increased resistance of nitrifiers to BACs, reflecting acclimation/enrichment over time, is a salient feature of the model.