Anti-adhesive, anti-biofilm and fungicidal action of newly synthesized gemini quaternary ammonium salts.

Newly synthesized gemini quaternary ammonium salts (QAS) with different counterions (bromide, hydrogen chloride, methylcarbonate, acetate, lactate), chain lengths (C12, C14, C16) and methylene linker (3xCH2) were tested. Dihydrochlorides and dibromides with 12 carbon atoms in hydrophobic chains were characterized by the highest biological activity against planktonic forms of yeast and yeast-like fungi. The tested gemini surfactants also inhibited the production of filaments by C. albicans. Moreover, they reduced the adhesion of C. albicans cells to the surfaces of stainless steel, silicone and glass, and slightly to polystyrene. In particular, the gemini compounds with 16-carbon alkyl chains were most effective against biofilms. It was also found that the tested surfactants were not cytotoxic to yeast cells. Moreover, dimethylcarbonate (2xC12MeCO3G3) did not cause hemolysis of sheep erythrocytes. Dihydrochlorides, dilactate and diacetate showed no mutagenic potential.

Newly synthesized surfactants as antimicrobial and anti-adhesion agents.

Quaternary ammonium salts (QAS) are widely used in medicine, industry and agriculture as disinfectants, biocides, and fungicides. QAS have the ability to coat various surfaces, prevent adhesion of microorganisms to them and inhibit the formation of biofilm. A group of surfactants derived from benzoic acid with different chemical structures was tested: monomeric QAS with different alkyl chain lengths (C12, C14, C16), gemini QAS containing 12-carbon alkyl chains and linkers of various lengths (3,4,6 methylene groups), as well as multifunctional QAS. Among the tested surfactants, monomeric QAS showed the highest bactericidal and fungicidal activity. All three groups of tested compounds inhibited the filamentation of C. albicans. The best antimicrobial activity was demonstrated by the monomeric surfactant C12AA, while the multifunctional equivalent (2xC12AA) was characterized by good anti-adhesive activity. All tested compounds are non-mutagenic and cause low hemolysis of sheep erythrocytes. Multifunctional and gemini surfactants are also non-toxic.

The effectiveness of newly synthesized quaternary ammonium salts differing in chain length and type of counterion against priority human pathogens.

Quaternary ammonium salts (QAS) commonly occur as active substances in disinfectants. QAS have the important property of coating abiotic surfaces, which prevents adhesion of microorganisms, thus inhibiting biofilm formation. In this study, a group of nine monomeric QAS, differing in the structure and length of the aliphatic chain (C12, C14, C16) and the counterion (methylcarbonate, acetate, bromide), were investigated. The study included an analysis of their action against planktonic forms as well as bacterial biofilms. The compounds were tested for their anti-adhesion properties on stainless steel, polystyrene, silicone and glass surfaces. Moreover, mutagenicity analysis and evaluation of hemolytic properties were performed. It was found that compounds with 16-carbon hydrophobic chains were the most promising against both planktonic forms and biofilms. Tested surfactants (C12, C14, C16) showed anti-adhesion activity but it was dependent on the type of the surface and strain used. The tested compounds at MIC concentrations did not cause hemolysis of sheep blood cells. The type of counterion was not as significant for the activity of the compound as the length of the hydrophobic aliphatic chain.

Biofilm eradication and antifungal mechanism of action against Candida albicans of cationic dicephalic surfactants with a labile linker.

Our research aims to expand the knowledge on relationships between the structure of cationic dicephalic surfactants-N,N-bis[3,3_-(dimethylamine)propyl]alkylamide dihydrochlorides and N,N-bis[3,3_-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9H19, n-C11H23, n-C13H27, n-C15H31)-and their antifungal mechanism of action on Candida albicans. The mentioned groups of amphiphilic substances are characterized by the presence of a weak, hydrochloride cationic center readily undergoing deprotonation, as well as a stable, strong quaternary ammonium group and alkyl chains capable of strong interactions with fungal cells. Strong fungicidal properties and the role in creation and eradication of biofilm of those compounds were discussed in our earlier works, yet their mechanism of action remained unclear. It was shown that investigated surfactants induce strong oxidative stress and cause increase in cell membrane permeability without compromising its continuity, as indicated by increased potassium ion (K+) leakage. Thus experiments carried out on the investigated opportunistic pathogen indicate that the mechanism of action of the researched surfactants is different than in the case of the majority of known surfactants. Results presented in this paper significantly broaden the understanding on multifunctional cationic surfactants and their mechanism of action, as well as suggest their possible future applications as surface coating antiadhesives, fungicides and antibiofilm agents in medicine or industry.

Biological activity of quaternary ammonium salts and resistance of microorganisms to these compounds.

Quaternary ammonium salts (QASs) are ubiquitous in nature, being found in organisms ranging from microorganisms to vertebrates (e.g., glycine betaine, carnitine) where they have important cellular functions. QASs are also obtained by chemical synthesis. These compounds, due to their diverse chemical structure (e.g. monomeric QAS or gemini) and their biological properties, are widely used in medicine (as disinfectants, drugs, and DNA carriers), industry, environmental protection and agriculture (as preservatives, biocides, herbicides and fungicides). Discussed chemical compounds reduce the adhesion of microorganisms to various biotic and abiotic surfaces and cause the eradication of biofilms produced by pathogenic microorganisms. The properties of these chemicals depend on their chemical structure (length of the alkyl chain, linker and counterion), which has a direct impact on the physicochemical and biological activity of these compounds. QASs by incorporation into the membranes, inhibit the activity of proteins (H+-ATPase) and disrupt the transport of substances to the cell. Moreover, in the presence of QASs, changes in lipid composition (qualitative and quantitative) of plasma membrane are observed. The widespread use of disinfectants in commercial products can induce resistance in microorganisms to these surfactants and even to antibiotics. In this article we discuss the biological activity of QASs as cationic surfactants against microorganisms and their resistance to these compounds.

New mild amphoteric sulfohydroxybetaine-type surfactants containing different labile spacers: Synthesis, surface properties and performance.

HYPOTHESIS: The excellent performance of zwitterionic alkylamide-type surfactants may be tuned by slight modifications of their structures, especially concerning linking groups between hydrophilic and hydrophobic moieties and the moderation of strong NH hydrogen bonds via the introduction of a methyl group attached to the nitrogen. EXPERIMENTS: The influence of the structure of alkylamidehydroxysulfobetaine-type surfactants on their adsorption and micellization, lime soap dispersing ability, wettability, as well as antimicrobial and hemolytic activities was studied in this work. We synthesized a series of novel surfactants with labile CON(Me)CH2 or CH2N(Me) CO spacers that were adjacent to the hydrophobic tail CnH2n+1 and separated from the zwitterionic hydroxysulfobetaine headgroup by C3H6 or C2H4 linkers, i.e., [(3-alkanoyilomethyoamine)propyl] dimethylammonium 2-hydroxypropanesulfonates (CnTMDAS) and [3-(alkylmetyloamino)-3-oxopropyl] dimethylammonium 2-hydroxypropanesulfonates (CnDMPAS), respectively. FINDINGS: The CnTMDAS surfactants showed slightly higher surface activity and lower CMC than the respective CnDMPAS with the same number of carbon atoms. Moreover, the CnTMDAS series exhibited significantly higher lime soap dispersing abilities and softer interactions with biological systems than CnDMPAS. The molecular modeling computations revealed that the difference in surface activity originated from lower dipole moment of CnTMDAS, hence, lower polarity, whereas higher free energy of dimerization of those surfactants with oleic acid may account for the favorable formation of mixed micelles.

Activity of gemini quaternary ammonium salts against microorganisms.

Quaternary ammonium salts (QAS), as the surface active compounds, are widely used in medicine and industry. Their common application is responsible for the development of microbial resistance to QAS. To overcome, this issue novel surfactants, including gemini-type ones, were developed. These unique compounds are built of two hydrophilic and two hydrophobic parts. The double-head double-tail type of structure enhances their physicochemical properties (like surface activity) and biological activity and makes them a potential candidate for new drugs and disinfectants. Antimicrobial activity is mainly attributed to the biocidal action towards bacteria and fungi in their planktonic and biofilm forms, but the mode of action of gemini QAS is not yet fully understood. Moreover, gemini surfactants are of particular interest towards their application as gene carriers. Cationic charge of gemini QAS and their ability to form liposomes facilitate DNA compaction and transfection of the target cells. Multifunctional nature of gemini QAS is the reason of the long-standing research on mainly their structure-activity relationship.

Quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride induced alterations in Saccharomyces cerevisiae physiology.

We investigated the influence of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)- N,N,N-trimethyl ammonium chloride) on yeast cells of the parental strain and the IM-resistant mutant (EO25 IMR) growth. The phenotype of this mutant was pleiotropic. The IMR mutant exhibited resistance to ethanol, osmotic shock and oxidative stress, as well as increased sensitivity to UV. Moreover, it was noted that mutant EO25 appears to have an increased resistance to clotrimazole, ketoconazole, fluconazole, nystatin and cycloheximide. It also tolerated growth in the presence of crystal violet, DTT and metals (selenium, tin, arsenic). It was shown that the presence of IM decreased ergosterol level in mutant plasma membrane and increased its unsaturation. These results indicate changes in the cell lipid composition. Western blot analysis showed the induction of Pma1 level by IM. RT-PCR revealed an increased PMA1 expression after IM treatment.

Antibacterial Activity of Alanine-Derived Gemini Quaternary Ammonium Compounds.

The antibacterial activity of alanine-derived gemini quaternary ammonium salts (chlorides and bromides) with various spacer and alkyl chain lengths was investigated. The studied compounds exhibited a strong bactericidal effect, especially bromides with 10 and 12 carbon alkyl chains and 3 carbon spacer groups (TMPAL-10 Br and TMPAL-12 Br), with a short contact time. Both salts dislodged biofilms of Pseudomonas aeruginosa and Staphylococcus epidermidis, and were lethal to adherent cells of S. epidermidis. Bromide with 2 carbon spacer groups and 12 carbon alkyl chains (TMEAL-12 Br) effectively reduced microbial adhesion by coating polystyrene and silicone surfaces. The results obtained suggest that, after further studies, gemini QAS might be considered as antimicrobial agents in medicine or industry.

The influence of biodegradable gemini surfactants, N,N'-bis(1-decyloxy-1-oxopronan-2-yl)-N,N,N',N' tetramethylpropane-1,3-diammonium dibromide and N,N'-bis(1-dodecyloxy-1-oxopronan-2-yl) N,N,N',N'-tetramethylethane-1,2-diammonium dibromide, on fungal biofilm and adhesion.

A group of biodegradable alanine-derived gemini quaternary ammonium salts (bromides and chlorides) with various alkyl chains and spacer lengths was tested for anti-adhesive and anti-biofilm activity. The strongest antifungal activity was exhibited by bromides with 10 and 12 carbon atoms within hydrophobic chains (N,N'-bis(1-decyloxy-1-oxopronan-2-yl)-N,N,N',N'-tetramethylpropane-1,3-diammonium dibromide and N,N'-bis(1-dodecyloxy-1-oxopronan-2-yl)-N,N,N',N'-tetramethylethane-1,2-diammonium dibromide). It was also demonstrated that these gemini surfactants enhanced the sensitivity of Candida albicans to azoles (itraconazole and fluconazole) and polyenes (amphotericin B and nystatine). Gemini quaternary ammonium salts effectively inhibited fungal cell adhesion to polystyrene and silicone surface. These compounds reduced C. albicans filamentation and eradicated C. albicans and Rhodotorula mucilaginosa biofilms, as it was shown in crystal violet and fluorescent staining. None of the tested compounds were cytotoxic against yeast mitochondrial metabolism.

Yeast ABC proteins involved in multidrug resistance.

Pleiotropic drug resistance is a complex phenomenon that involves many proteins that together create a network. One of the common mechanisms of multidrug resistance in eukaryotic cells is the active efflux of a broad range of xenobiotics through ATP-binding cassette (ABC) transporters. Saccharomyces cerevisiae is often used as a model to study such activity because of the functional and structural similarities of its ABC transporters to mammalian ones. Numerous ABC transporters are found in humans and some are associated with the resistance of tumors to chemotherapeutics. Efflux pump modulators that change the activity of ABC proteins are the most promising candidate drugs to overcome such resistance. These modulators can be chemically synthesized or isolated from natural sources (e.g., plant alkaloids) and might also be used in the treatment of fungal infections. There are several generations of synthetic modulators that differ in specificity, toxicity and effectiveness, and are often used for other clinical effects.

Antibacterial activity of gemini quaternary ammonium salts.

A series of gemini quaternary ammonium salts (chlorides and bromides), with various hydrocarbon chain and spacer lengths, were tested. These compounds exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria and were not mutagenic. The strongest antibacterial effect was observed for TMPG-10 Cl (against Pseudomonas aeruginosa ATCC 27853) and TMPG-12 Br (against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 11229 and clinical ESBL(+) isolate 434) surfactants. These compounds inhibited the adhesion of Staphylococcus epidermidis ATCC 35984 to a polystyrene surface and eradicated biofilm formed by P. aeruginosa PAO1. The activity of studied compounds was dependent on hydrocarbon chain length.

Antifungal activity of gemini quaternary ammonium salts.

A series of gemini quaternary ammonium chlorides and bromides with various alkyl chain and spacer lengths was synthesized. The most active compounds against fungi were chlorides with 10 carbon atoms within the hydrophobic chain. Among these compounds were few with no hemolytic activity at minimal inhibitory concentrations. None of the tested compounds were cytotoxic and mutagenic. Cationic gemini surfactants poorly reduced the adhesion of microorganisms to the polystyrene plate, but inhibited the filamentation of Candida albicans. One of the tested compounds eradicated C. albicans and Rodotorula mucilaginosa biofilm, what could be important in overcoming catheter-associated infections. It was also shown that gemini surfactants enhanced the sensitivity of C. albicans to azoles and polyenes, thus they might be potentially used in combined therapy against fungi.

[Mechanisms of yeast resistance to environmental stress]. / Mechanizmy opornosci drozdzyna stres srodowiskowy.

Changes in environmental conditions might be a stress factor for yeast cells. There are several mechanisms of stress tolerance, developed by the cell, which activate when the stress appears. Different transcription factors coordinate the expression of stress response genes. Msn2/4p regulate the expression of the general stress response. Heat shock defense involves heat shock proteins (Hsp), controlled by Hsf1p. Osmotic shock induces the MAP kinase cascade (HOG), whereas the oxidative stress response requires the YAP network. Fungicide resistance is mediated mainly by the activity of membrane transporters and changes in the structure of the plasma membrane. 

[Efflux-mediated antimicrobial multidrug resistance]. / Opornosc wielolekowa zwiazana z aktywnym usuwaniem leków z komórek drobnoustrojów.

Multidrug resistance is a major problem in the treatment of infectious diseases caused by bacteria and fungi. One of the basic mechanisms of resistance is active efflux of distinct drugs from cells. Export of toxic compounds from bacterial cells is mediated by proteins of 5 distinct families: MF, SMR, ABC, RND and MATE. The substrate spectrum of efflux pumps includes antibiotics, chemotherapeutics and detergents. Genes that determine resistance can be located on chromosomes or mobile elements (plasmids, transposons, integrons). The presence of resistance genes on mobile elements enables bacteria to transfer those genes between cells and spread the multidrug resistance phenotype. There are several inhibitors of efflux pumps that are currently in the experimental phase. Proteins that mediate multidrug resistance are also present in fungal cells. They belong mainly to the ABC superfamily of transporters and PDR subfamily. These efflux pumps are widely investigated in Saccharomyces cerevisiae.

[The biological activity of lysosomotropic agents]. / Biologiczna aktywnosc zwiazków lizosomotropowych.

Lysosomotropic agents have antitumor, antifungal and antimicrobial properties. These small, amphiphilic compounds, as weak bases, readily penetrate the lipid bilayer and diffuse into acidic subcellular compartments such as lysosomes or vacuoles. The mechanism of action of lysosomotropic compounds can be distinct, depending on their chemical structure and/or the kind of cells influenced. Our investigations of the influence of lysosomotropic agents on Saccharomyces cerevisiae have lead to a partial explanation of their mechanism of action. The amphiphilic character enables the compounds to penetrate the lipid bilayer and disturb its structure and functions and can inhibit the activity of plasma membrane H+ATPase. The accumulation of the compounds in yeast vacuoles, disrupting them and killing yeast cells, is another mechanism of action. Lysosomotropic agents can be substrates for ABC transporters in Saccharomyces cerevisiae cells.

[Gemini surfactants as gene carriers]. / Surfaktanty gemini jako nosniki genów.

Gemini surfactants are a new class of amphiphilic compounds built from two classic surfactant moieties bound together by a special spacer group. These compounds appear to be excellent for creating complexes with DNA and are effective in mediating transfection. Thanks to their construction, DNA carrier molecules built from gemini surfactants are able to deliver genes to cells of almost any DNA molecule size, unattainable when using viral gene delivery systems. Moreover, they are much safer for living organisms.

[The biological activity of quaternary ammonium salts (QASs)]. / Biologiczna aktywnosc czwartorzedowych soli amoniowych (CSA).

Quaternary ammonium salts (QASs), especially those of cationic surfactant character, are applied as antibacterial and antifungal disinfectants. QASs affect lipid-enveloped viruses, including human immunodeficiency virus (HIV) and hepatitis B virus (HBV), but not non-enveloped viruses. These compounds are extensively used in domestic (as ingredients of shampoos, hair conditioners), agricultural (as fungicides, pesticides, insecticides), healthcare (as medications), and industrial applications (as biocides, fabric softeners, corrosion inhibitors). The extensive use of quaternary ammonium disinfectants in recent years has led to the development of resistance in microorganisms to these drugs. Thus Staphylococcus aureus strains contain the plasmid-carrying genes qacA and qacB encoding resistance to quaternary ammonium compounds and acriflavine. The membrane proteins QacA and QacB confer multidrug resistance by exporting the compound by the proton motive force which is generated by the transmembrane electrochemical proton gradient.

The physiological and morphological phenotype of a yeast mutant resistant to the quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride.

We investigated the action of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride) on Saccharomyces cerevisiae yeast cells. Changes in the yeast cell ultrastructure were confirmed by electron microscopy. We treated resistant mutant cells with QAS, and confirmed destruction of the mutant cytoplasm, an increase in the thickness of the cell wall, separation of the cell wall from the cytoplasm, and the accumulation of numerous lipid droplets. We also observed a relatively high production of lipids in the cells of the parental wild-type strain Sigma1278b and in its IM-resistant (IM(R)) mutant in the presence of the QAS. The IM(R) mutant showed increased sensitivity to CaCl(2) and SDS, and resistance to ethidium bromide, chloramphenicol, erythromycin and osmotic shock. It also tolerated growth at low pH. We suggest that the resistance to IM could be connected with the level of permeability of the cell membrane because the IM(R) mutant was sensitive to this compound in vivo in the presence of SDS and guanidine hydrochloride, which cause increased permeability of the cell plasma membrane.

The aminoesters as inhibitors of plasma membrane H+-ATPase in the yeast Saccharomyces cerevisiae.

A set of oxalates of alpha-dimethylamino fatty acids n-alkyl esters (MEM-ns and n-MEM-8s) and n-dodecyl-N,N-dimethylalaninate (DMAL-12s) were synthesized. Their activities on the growth, transport, and ATPases from the yeast Saccharomyces cerevisiae were compared. The compounds differ in the number of carbon atoms in their aliphatic chain and in the position of that chain in their molecular structure. The tested aminoesters with twelve carbon atoms (MEM-10s and DMAL-12s) appeared to have the highest level of activity. These drugs inhibited plasma membrane H+-ATPase, but no inhibition of mitochondrial ATPase was observed. Under nitrogen-derepressed conditions, the aminoesters inhibited amino acid uptake by yeast cells.