New biologically active ionic liquids with benzethonium cation-efficient SAR inducers and antimicrobial agents.

BACKGROUND: An urgent need to find new methods for crop protection remains open due to the withdrawal from the market of the most toxic pesticides and increasing consumer awareness. One of the alternatives that can be used in modern agriculture is the use of bifunctional compounds whose actions towards plant protection are wider than those of conventional pesticides. RESULTS: In this study, we present the investigation of the biological efficacy of nine dual-functional salts containing a systemic acquired resistance (SAR)-inducing anion and the benzethonium cation. A significant result of the presented study is the discovery of the SAR induction activity of benzethonium chloride, which was previously reported only as an antimicrobial agent. Moreover, the concept of dual functionality was proven, as the application of presented compounds in a given concentrations resulted both in the control of human and plant bacteria species and induction of SAR. CONCLUSION: The strategy presented in this article shows the capabilities of derivatization of common biologically active compounds into their ionic derivatives to obtain bifunctional salts. This approach may be an example of the design of potential new compounds for modern agriculture. It provides plants with two complementary actions allowing to provide efficient protection to plants, if one mode of action is ineffective. © 2024 Society of Chemical Industry.

Anti-biofilm effects and healing promotion by silver oxynitrate-based dressings.

Microbial growth within a wound often manifests as biofilms, which can prevent healing and is difficult to eradicate. Novel silver dressings claim to combat wound infection, but anti-biofilm efficacy and effects on healing independent of infection are often unclear. Using in vitro and in vivo S. aureus and P. aeruginosa biofilm models, we report the efficacy of a dressing which produces Ag1+ ions; an Ag1+ dressing containing ethylenediaminetetraacetic acid and benzethonium chloride (Ag1+/EDTA/BC), and a dressing containing silver oxynitrate (Ag Oxysalts) which produces Ag1+, Ag2+ and Ag3+ ions, against wound biofilms, and their effects on healing. Ag1+ dressings had minimal effect on in vitro and murine (C57BL/6j) wound biofilms. In contrast, Ag Oxysalts and Ag1+/EDTA/BC dressings significantly reduced viable bacteria within in vitro biofilms and demonstrated a visible reduction in bacteria and EPS components within murine wound biofilms. The dressings had different effects on the healing of biofilm-infected and uninfected wounds, with Ag Oxysalts dressings having a greater beneficial effect on re-epithelialisation, wound size and inflammation than the control treatment and the other silver dressings. The different physicochemical properties of the silver dressings result in varied effects on wound biofilms and healing which should be considered when selecting dressings to treat biofilm-infected wounds.

Ketamine preservative benzethonium chloride potentiates hippocampal synaptic transmission and binds neurotransmitter receptors and transporters.

Benzethonium chloride (BZT) is an excipient used in numerous products including (R,S)-ketamine (ketamine) drug formulations for human and veterinary use. Emerging evidence indicates BZT is pharmacologically active. BZT may therefore contribute to some of the clinical or preclinical effects observed with ketamine. In the present study, we evaluated: (i) the affinity of BZT for neurotransmitter receptors and transporters, (ii) the effects of BZT on hippocampal synaptic transmission in vitro, and (iii) plasma and brain concentrations of BZT following its intraperitoneal administration to male CD1 mice. Radioligand binding assays determined the affinity of BZT for neurotransmitter targets. Effects of BZT on field excitatory postsynaptic potentials (fEPSPs) were established via electrophysiological recordings from slices collected from male C57BL/6J mice. The binding assays revealed that BZT binds to numerous receptors (e.g., σ2 Ki = 7 nM) and transporters (e.g., dopamine transporter Ki = 545 nM). Bath application of BZT potentiated hippocampal fEPSPs in mouse hippocampal slices with an EC50 of 2.03 nM. Following intraperitoneal administration, BZT was detected in the plasma, but not in the brain of mice. These data highlight that studies measuring peripheral endpoints or directly exposing systems, in vitro, intracerebroventricularly, or intracortically, to BZT-containing formulations should account for the direct effects of BZT. Our findings also suggest that earlier data attributing pharmacological effects to ketamine may be confounded by BZT and that additional investigation into the functional impact of BZT is warranted. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

Impact of benzalkonium chloride, benzethonium chloride and chloroxylenol on bacterial antimicrobial resistance.

This review examined 3655 articles on benzalkonium chloride (BKC), benzethonium chloride (BZT) and chloroxylenol (CHO) aiming to understand their impact on antimicrobial resistance. Following the application of inclusion/exclusion criteria, only 230 articles were retained for analysis; 212 concerned BKC, with only 18 for CHO and BZT. Seventy-eight percent of studies used MIC to measure BKC efficacy. Very few studies defined the term 'resistance' and 85% of studies defined 'resistance' as <10-fold increase (40% as low as 2-fold) in MIC. Only a few in vitro studies reported on formulated products and when they did, products performed better. In vitro studies looking at the impact of BKC exposure on bacterial resistance used either a stepwise training protocol or exposure to constant BKC concentrations. In these, BKC exposure resulted in elevated MIC or/and MBC, often associated with efflux, and at time, a change in antibiotic susceptibility profile. The clinical relevance of these findings was, however, neither reported nor addressed. Of note, several studies reported that bacterial strains with an elevated MIC or MBC remained susceptible to the in-use BKC concentration. BKC exposure was shown to reduce bacterial diversity in complex microbial microcosms, although the clinical significance of such a change has not been established. The impact of BKC exposure on the dissemination of resistant genes (notably efflux) remains speculative, although it manifests that clinical, veterinary and food isolates with elevated BKC MIC carried multiple efflux pump genes. The correlation between BKC usage and gene carriage, maintenance and dissemination has also not been established. The lack of clinical interpretation and significance in these studies does not allow to establish with certainty the role of BKC on AMR in practice. The limited literature and BZT and CHO do not allow to conclude that these will impact negatively on emerging bacterial resistance in practice.

Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions.

Synergistic Effect of Benzethonium Chloride Combined with Endoxan against Hepatocellular Carcinoma in Rats through Targeting Apoptosis Signaling Pathway.

Combination therapy has been the trendy of care, particularly in cancer remedy, since it is a rational approach to increase response and tolerability and to diminish resistance. Hence, there is a growing interest in combining anticancer drugs to maximizing efficacy with minimum systemic toxicity through the delivery of lower drug doses. Therefore, in the present study, the value of combination between benzethonium chloride (benzo) and endoxan (endo) as anti-tumor drug sensitization of hepatocellular carcinoma HCC treatment were detected both in vitro and in vivo. Crystal violet test was performed to detect the proliferation of HepG2 cells treated with benzo or/and endo. In addition, the HCC rat model was established by diethylnitrosamine (DEN) administration. The antitumor effect was enhanced with the combined treatment of the two drugs, particularly in the group with benzo and endo. The results confirmed that the HCC condition was developed in response to lower expressions of caspase 3 and P53 which, in turn, was due to the overexpression of Bcl-2, and downregulation of cytochrome C. The treatment with benzo combined with endo caused significant activation of caspase-3 mediated apoptotic signals that could be responsible for its anti-HCC potential. Meantime, benzo combined with endo treatments could reduce the hepatocellular carcinogenesis by reducing the expression of MMP-9. Therefore, benzo and endo treatments may be a hopeful therapeutic drug for HCC. Also, more studies are recommended to feat the idea of this research for medical use.
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Drug repurposing approach to target FtsZ cell division protein from Salmonella Typhi.

Drug repurposing is an efficient alternative approach to counter the increasing drug-resistant pathogens to treat infectious diseases. FtsZ is an essential bacterial cytokinesis protein involved in the formation of cell-division complex and targeting FtsZ using FDA approved drugs is a promising strategy to identify and develop a new antibacterial drug. Using in silico pharmacophore-based screening of drug bank, molecular docking and molecular dynamics simulations, we identified six drugs inhibiting the function of stFtsZ from Salmonella Typhi. The selected drugs target stFtsZ at the hydrophobic cleft formed between the C-terminal domain and helix α7 with binding energy better than -8 kcal/mol. Out of these six drugs, benzethonium chloride showed promising results at 8 µM concentration where it inhibits stFtsZ GTPase activity by 80% and prevents polymerization. Benzethonium chloride also possesses an excellent antibacterial activity against the bacterial culture of Salmonella Typhi (ATCC 19430), Staphylococcus aureus (ATCC 43300) and Escherichia coli (ATCC 25922) with the MIC values of 8 µg/mL, 1 µg/mL and 12 µg/mL, respectively. Based on our current study, the scaffold of benzethonium chloride can be used for the development of broad-spectrum antibacterial agents against drug-resistant pathogens.

Pharmacological Characterization of the Novel and Selective α7 Nicotinic Acetylcholine Receptor-Positive Allosteric Modulator BNC375.

Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. SIGNIFICANCE STATEMENT: BNC375 is a novel and selective α7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PAM) that potentiates acetylcholine-evoked α7 currents in in vitro assays with little to no effect on the desensitization kinetics. In vivo, BNC375 demonstrated robust procognitive effects in multiple preclinical models across a wide exposure range. These results suggest that α7 nAChR PAMs have therapeutic potential in central nervous system diseases with cognitive impairments.

Combined Treatment of High Hydrostatic Pressure and Cationic Surfactant Washing to Inactivate Listeria monocytogenes on Fresh-Cut Broccoli.

This study was conducted to examine the inactivation effect of the combined treatment of high hydrostatic pressure (HHP; 400 MPa for 1, 3, and 5 min) and cationic surfactant washing (0.05% benzethonium chloride, BEC) against Listeria monocytogenes inoculated on fresh-cut broccoli (FCB). Washing with BEC at concentrations exceeding 0.05% resulted in 2.3 logreduction of L. monocytogenes counts on FCB, whereas HHP treatment had approximately 5.5- 5.6 log-reductions regardless of the treatment time. Scanning electron microscopy corroborated microbial enumeration, revealing that the combined treatment was more effective in removing L. monocytogenes from FCB than individual treatment with HHP or BEC. Color and total glucosinolate content were maintained after the combined treatment, although the hardness of the FCB slightly decreased. The results clearly suggest that the combined treatment of HHP and BEC washing has potential value as a new sanitization method to improve the microbial safety of FCB.

Biocide susceptibilities and biofilm-forming capacities of Acinetobacter baumannii clinical isolates from Malaysia.

INTRODUCTION: Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has the capacity to develop resistance to all classes of antimicrobial compounds. However, very little is known regarding its susceptibility to biocides (antiseptics and disinfectants) and capacity to form biofilms, particularly for Malaysian isolates. AIM: To determine the susceptibility of A. baumannii isolates to commonly-used biocides, investigate their biofilm-forming capacities and the prevalence of biocide resistance and biofilm-associated genes. METHODOLOGY: . The minimum inhibitory concentration (MIC) values of 100 A. baumannii hospital isolates from Terengganu, Malaysia, towards the biocides benzalkonium chloride (BZK), benzethonium chloride (BZT) and chlorhexidine digluconate (CLX), were determined by broth microdilution. The isolates were also examined for their ability to form biofilms in 96-well microplates. The prevalence of biocide resistance genes qacA, qacE and qacDE1 and the biofilm-associated genes bap and abaI were determined by polymerase chain reaction (PCR). RESULTS: Majority of the A. baumannii isolates (43%) showed higher MIC values (> 50 µg/mL) for CLX than for BZK (5% for MIC > 50 µg/mL) and BZT (9% for MIC > 50 µg/mL). The qacDE1 gene was predominant (63%) followed by qacE (28%) whereas no isolate was found harbouring qacA. All isolates were positive for the bap and abaI genes although the biofilm-forming capacity varied among the isolates. CONCLUSION: The Terengganu A. baumannii isolates showed higher prevalence of qacDE1 compared to qacE although no correlation was found with the biocides' MIC values. No correlation was also observed between the isolates' biofilm-forming capacity and the MIC values for the biocides.

Susceptibility of Methicillin-Resistant and -Susceptible Staphylococcus aureus Isolates of Various Clonal Lineages from Germany to Eight Biocides.

Few studies have been conducted on the susceptibility of bacteria to biocides. A total of 182 methicillin-resistant and -susceptible Staphylococcus aureus isolates collected from healthy or diseased humans and animals in Germany were included in the present study. Sixty-three isolates of animal origin and 119 human isolates were tested for their MICs to eight biocides or heavy metals by the broth microdilution method. The MIC50 and MIC90 values of human and animal isolates were equal or differed by not more than 1 dilution step, and statistical analysis revealed that differences between MICs of human and animal isolates were not significant. However, when taking into account the multilocus sequence type (MLST), a strong tendency (P = 0.054) to higher MICs of silver nitrate was detected for clonal complex 398 (CC398) isolates from humans compared to those from animals. Furthermore, a comparison of MIC values from isolates belonging to different clonal lineages revealed that important human lineages such as CC22 and CC5 exhibited significantly (P < 0.05) higher MICs for the biocides chlorhexidine, benzethonium chloride, and acriflavine than the main animal lineage sequence type 398 (ST398). Isolates with elevated MIC values were tested for the presence of biocide and heavy metal tolerance-mediating genes by PCR assays, and the following genes were detected: mepA (n [no. of isolates containing the gene] = 44), lmrS (n = 36), norA (n = 35), sepA (n = 22), mco (n = 5), czrC (n = 3), smr (n = 2), copA (n = 1), qacA and/or -B (n = 1), qacG (n = 2), and qacJ (n = 1). However, only for some compounds was a correlation between the presence of a biocide tolerance gene and the level of MIC values detected.IMPORTANCE Biocides play an essential role in controlling the growth of microorganisms and the dissemination of nosocomial pathogens. In this study, we determined the susceptibility of methicillin-resistant and -susceptible S. aureus isolates from humans and animals to various biocides and heavy metal ions and analyzed differences in susceptibilities between important clonal lineages. In addition, the presence of biocide or heavy metal tolerance-mediating genes was investigated. We demonstrated that important human lineages such as CC22 and CC5 had significantly higher MIC values for chlorhexidine, benzethonium chloride, and acriflavine than the main farm animal lineage, ST398. In addition, it was shown that for some combinations of biocides and tolerance genes, significantly higher MICs were detected for carriers. These findings provide new insights into S. aureus biocide and heavy metal tolerance.

Differential Gene Expression of Three Gene Targets among Persistent and Nonpersistent Listeria monocytogenes Strains in the Presence or Absence of Benzethonium Chloride.

Persistence of Listeria monocytogenes strains in food processing environments remains relatively common but is difficult to control. Understanding the basis for such persistence represents an important step in the potential control or eradication of this pathogen from these environments. In this study, reverse transcription PCR was used to determine the relative and absolute expression of selected gene targets (pocR, eutJ, and qacH) among five persistent and four presumed nonpersistent L. monocytogenes strains. The quantification of these genes as markers for the persistent phenotype and the effect of benzethonium chloride (BZT) on their expression was investigated. Although no markers correlated with the ability of strains to persist in food processing facilities were found, expression of pocR was upregulated in three of the five persistent strains, in contrast to the four presumed nonpersistent strains, which showed down-regulation of this gene. These results provide further knowledge of the differential expression of genes of persistent and presumed nonpersistent strains of L. monocytogenes grown in the presence or absence of BZT and identifies upregulation of pocR as a potential response of persistent strains of L. monocytogenes to exposure to BZT.

Traditionally used medicinal plants against uncomplicated urinary tract infections: Are unusual, flavan-4-ol- and derhamnosylmaysin derivatives responsible for the antiadhesive activity of extracts obtained from stigmata of Zea mays L. against uropathogenic E. coli and Benzethonium chloride as frequent contaminant faking potential antibacterial activities?

The dried stigmata from Zea mays L. are used traditionally for the treatment of uncomplicated urinary tract infections. A recent screening has indicated that hydroalcoholic extract of the herbal material inhibits the adhesion of uropathogenic Escherichia coli (UPEC) to T24 bladder cells. For verification of these data EtOH-water (1:1) extracts from 4 different batches of Maydis stigmata were investigated. Within an in vitro adhesion assay (UPEC strain 2980 and human T24 bladder cells) a dose-dependent antiadhesive activity against UPEC was verified (IC50 1040µg/mL). Bioassay guided fractionation of M. stigmata, batch S1, by EtOH-water extraction, followed by chromatography on Sephadex LH20 revealed two active fractions (I and XI). Further purification of fraction I and structure elucidation of the isolated compound revealed the presence of significant amounts of the biocide benzethonium chloride as contaminant. Benzethonium chloride was also identified in subsequent investigations in 2 different batches of M. stigmata. The presence of such nondeclared and illegal contaminants in the herbal raw material market has to be discussed intensively. From benzethonium-free raw material (batch S2) as well as from batch S1 fraction XI was further fractionated by MPLC and preparative HPLC, leading to a still complex subfraction XIG, which was analyzed by UHPLC/+ESI-QTOF-MS analysis. Advanced data processing and species-metabolite relationship database revealed the tentatively existence of the unusual C-glycosidic flavones derhamnosylmaysin (6), 3'-deoxyrhamnosylmaysin (4), 3'-O-methylderhamnosylmaysin (3), apiferol (2) and alternanthin (8) which might be related to the antiadhesive activity of this subfraction against UPEC.

An in vitro test of the efficacy of an anti-biofilm wound dressing.

Broad-spectrum antimicrobial agents, such as silver, are increasingly being formulated into medicated wound dressings in order to control colonization of wounds by opportunistic pathogens. Medicated wound dressings have been shown in-vitro to be effective against planktonic cultures, but in-vivo bacteria are likely to be present in biofilms, which makes their control and eradication more challenging. Recently, a functional wound dressing (AQUACEL(®) Ag+ Extra™ (AAg + E)) has been developed that in addition to silver contains two agents (ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride (BC)) designed to disrupt biofilms. Here, the efficacy of AAg + E is demonstrated using a biofilm model developed in an isothermal microcalorimeter. The biofilm was seen to remain viable in the presence of unmedicated dressing, silver-containing dressing or silver nitrate solution. In the presence of AAg + E, however, the biofilm was eradicated. Control experiments showed that neither EDTA nor BC alone had a bactericidal effect, which means it is the synergistic action of EDTA and BC disrupting the biofilm with silver being bactericidal that leads to the product's efficacy.

Methicillin-susceptible and -resistant Staphylococcus aureus with high-level antiseptic and low-level mupirocin resistance in Malaysia.

The prevalence and spread of mupirocin and antiseptic resistance among colonizing and infectious Staphylococcus aureus were determined. S. aureus isolated from anterior nares and infection sites of patients hospitalized in the largest tertiary care referral hospital in Malaysia was investigated for mupirocin and antiseptic susceptibility testing, and for PCR detection of mupA, qacA/B, and smr genes. Twelve isolates showed resistance to mupirocin by disk diffusion, of which 10 (3.8%) harbored the mupA gene. Minimum inhibitory concentrations (MICs) ranged from 64 to 768 µg/ml for mupA positive and below 46 µg/ml for negative isolates. The mupA was more common among ST239 isolates (70%). The qacA/B was carried in 67 out of 95 methicillin-resistant Staphylococcus aureus (MRSA) (70.5%) and 3 out of 164 methicillin-susceptible Staphylococcus aureus (MSSA) (1.8%), while smr was carried in 6 out of 95 MRSA (6.3%) strains. MICs ranged from 3.9 to 15.6 µg/ml for benzethonium chloride (BTC) and benzalkonium chloride (BKC), and from 10.3 to 20.7 µg/ml for chlorhexidine digluconate (CHG). Isolates with qacA/B and smr or qacA/B alone showed higher MIC (20.7 µg/ml for CHG and 15.6 µg/ml for BTC and BKC) than the isolates that lacked antiseptic resistance genes (10.3 µg/ml for CHG and 3.9 µg/ml for BTC and BKC). In 16 cases, ST239 was isolated from the infection site and the nares simultaneously, and shared identical resistance patterns (qacAB or qacAB+smr), suggesting possible endogenous infection. Spread of low-level mupirocin resistance expressing ST239 MRSA and high-level resistance expressing emerging ST1, co-existing with antiseptic-resistant genes showing elevated MICs, should be monitored for effective infection control.

Effect of channel blockers on the smooth muscle of the adult crop of the queen blowfly, Phormia regina.

Few studies have examined the various factors affecting the rate of contraction of the supercontractile muscles of the crop lobes of adult Phormia regina Meigen (Diptera: Calliphoridae). Using an in situ bioassay of the crop organ, various ion channel blockers were tested and it was demonstrated that in all cases the blockers (i.e., against the following conductances: Cl⁻, Ca²âº, Na⁺, and a FMRF-amide action) significantly reduced the contraction rates of the crop lobes, which were filled with 4.5 µL of 1.0 M sucrose containing 10 mM of the dye amaranth. Benzyltrimethylammonium chloride, never before reported for its effect on insect muscle, was as effective in suppressing crop muscle contraction as benzethonium chloride, which is a reported agonist of dromyosuppressin.

High-throughput screening of a collection of known pharmacologically active small compounds for identification of Candida albicans biofilm inhibitors.

Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.

Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria.

Bacteria from the Burkholderia cepacia complex (Bcc) are encountered as industrial contaminants, and little is known about the species involved or their mechanisms of preservative resistance. Multilocus sequence typing (MLST) revealed that multiple Bcc species may cause contamination, with B. lata (n = 17) and B. cenocepacia (n = 11) dominant within the collection examined. At the strain level, 11 of the 31 industrial sequence types identified had also been recovered from either natural environments or clinical infections. Minimal inhibitory (MIC) and minimum bactericidal (MBC) preservative concentrations varied across 83 selected Bcc strains, with industrial strains demonstrating increased tolerance for dimethylol dimethyl hydantoin (DMDMH). Benzisothiazolinone (BIT), DMDMH, methylisothiazolinone (MIT), a blend of 3:1 methylisothiazolinone-chloromethylisothiazolinone (M-CMIT), methyl paraben (MP), and phenoxyethanol (PH), were all effective anti-Bcc preservatives; benzethonium chloride (BC) and sodium benzoate (SB) were least effective. Since B. lata was the dominant industrial Bcc species, the type strain, 383(T) (LMG 22485(T)), was used to study preservative tolerance. Strain 383 developed stable preservative tolerance for M-CMIT, MIT, BIT, and BC, which resulted in preservative cross-resistance and altered antibiotic susceptibility, motility, and biofilm formation. Transcriptomic analysis of the B. lata 383 M-CMIT-adapted strain demonstrated that efflux played a key role in its M-CMIT tolerance and elevated fluoroquinolone resistance. The role of efflux was corroborated using the inhibitor l-Phe-Arg-ß-napthylamide, which reduced the MICs of M-CMIT and ciprofloxacin. In summary, intrinsic preservative tolerance and stable adaptive changes, such as enhanced efflux, play a role in the ability of Bcc bacteria to cause industrial contamination.

Mechanism of HERG potassium channel inhibition by tetra-n-octylammonium bromide and benzethonium chloride.

Tetra-n-octylammonium bromide and benzethonium chloride are synthetic quaternary ammonium salts that are widely used in hospitals and industries for the disinfection and surface treatment and as the preservative agent. Recently, the activities of HERG channel inhibition by these compounds have been found to have potential risks to induce the long QT syndrome and cardiac arrhythmia, although the mechanism of action is still elusive. This study was conducted to investigate the mechanism of HERG channel inhibition by these compounds by using whole-cell patch clamp experiments in a CHO cell line stably expressing HERG channels. Tetra-n-octylammonium bromide and benzethonium chloride exhibited concentration-dependent inhibitions of HERG channel currents with IC(50) values of 4nM and 17nM, respectively, which were also voltage-dependent and use-dependent. Both compounds shifted the channel activation I-V curves in a hyperpolarized direction for 10-15mV and accelerated channel activation and inactivation processes by 2-fold. In addition, tetra-n-octylammonium bromide shifted the inactivation I-V curve in a hyperpolarized direction for 24.4mV and slowed the rate of channel deactivation by 2-fold, whereas benzethonium chloride did not. The results indicate that tetra-n-octylammonium bromide and benzethonium chloride are open-channel blockers that inhibit HERG channels in the voltage-dependent, use-dependent and state-dependent manners.