Evaluation of the effectiveness of the potent bis-quaternary ammonium compound, 4,4'-(α,ω-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) on Pseudomonas aeruginosa.

AIMS: Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) are cationic surfactants and have been used widely as general disinfectants in the medical field due to their strong antibacterial effects and low cytotoxicity to human cells. 4,4'-(α,ω-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) is one of the potent bis-QACs synthesized to improve the antimicrobial activities of mono-QACs such as BAC. This study aimed to assess the effectiveness of 4DTBP-6,8 against Pseudomonas aeruginosa, a prevalent hospital pathogen. METHODS AND RESULTS: The minimum inhibitory concentrations of 4DTBP-6,8, CPC and BAC against P. aeruginosa were measured. 4DTBP-6,8 exhibited strong antibacterial activity. We assessed the bactericidal effects of QACs against P. aeruginosa under certain conditions and their cytotoxicities in human epithelial cells using lactate dehydrogenase (LDH) release. 4DTBP-6,8 exerted excellent bactericidal effects against high concentrations of bacteria, biofilm cells and even in the presence of contaminated proteins. Cellular LDH was not released by the treatment with 4DTBP-6,8. CONCLUSIONS: 4DTBP-6,8 exhibited the strongest bactericidal activity against P. aeruginosa among the three QACs tested without any cytotoxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The potent bis-QAC, 4DTBP-6,8 has the potential to be an effective disinfectant in preventing hospital infections caused by P. aeruginosa.

Evaluation of the cytotoxic effects of bis-quaternary ammonium antimicrobial reagents on human cells.

The cytotoxic effects of newly synthesized bis-quaternary ammonium compounds (bis-QACs) and benzalkonium chloride were investigated on skin and blood cells, namely normal human epidermal keratinocytes of neonatal foreskin, a normal human skin fibroblast cell line NB1RGB, erythrocytes and a lymphoma cell line JM. The bis-QACs tested were 4, 4'-(1,6-hexamethylenedithio)bis(1-octylpyridinium iodide) (4DTBP-6, 8), N,N'-tetramethylenebis(1-dodecyl-4-carbamoylpyridinium iodide) (4BCAP-4,12), N,N'-hexamethylenebis(1-decyl-4-carbamoylpyridinium iodide) (4BCAP-6,10), 4,4'-(1,4-tetramethylenedicarbonyldiamine) bis (1-decylpyridinium iodide) (4DCABP-4,10), 4,4'-(1, 4-tetramethylenedicarbonyldiamine) bis (1-dodecylpyridinium iodide) (4DCABP-4,12), 4,4'-(1, 4-phenyldicarbonyldiamine)bis(1-dodecylpyridinium iodide) (4DCABP-P, 12), and 4,4'-(1, 6-hexamethylenedioxydicarbonyl)bis(1-dodecylpyridinium iodide) (4DOCBP-6,12). All bis-QACs consisted of two identical alkylpyridinium rings and bridge structure linking rings to each other have a methylene bridge but only 4DCABP-P,12 has a phenyl ring as a bridge. Most of the LD(50) values in acute cytotoxic assays of these bis-QACs tended to be lower than those of benzalkonium chloride. However, the comparison of the antibacterial activity against cytotoxic effects on several human cells revealed that bis-QACs, especially 4DTBP-6,8, have wide concentration ranges showing sufficient antibacterial activity and lower cytotoxic effect on human cells (except for 4DOCBP-6,12), although benzalkonium chloride caused significant human cell damage at the concentrations necessary for antibacterial activity. Moreover, judging from the LD(50) value of 4DTBP-6,8 [67 microM, a 6.7-fold higher concentration than the upper value of MIC] obtained in an artificial human skin model TESTSKIN, 4DTBP-6,8 is thought to be a more effective and safer antiseptic reagent for application to the skin surface than benzalkonium chloride. Taken together, bis-QACs are expected to be a promising new generation of antimicrobial QACs.

Distribution of the intermedilysin gene among the anginosus group streptococci and correlation between intermedilysin production and deep-seated infection with Streptococcus intermedius.

The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by Streptococcus intermedius were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in S. intermedius and no homologue to the toxin gene is distributed in S. anginosus and S. constellatus. Thus, the intermedilysin gene is useful as a marker gene of S. intermedius. Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of S. intermedius, such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with S. intermedius.

Synthesis and antimicrobial characteristics of novel biocides, 4,4'-(1,6-hexamethylenedioxydicarbonyl)bis(1-alkylpyridinium++ + iodide)s.

Potent new biocides against both bacteria and fungi, 4,4'-(1,6-hexamethylenedioxydicarbonyl)bis(1-alkylpyridinium iodide)s (4DOCBP-6,n) (alkyl chain length, n = 8, 10, 12, 14, 16 and 18) were synthesized, 4DOCBP-6,n is a bis-quaternary ammonium compound (bis-QAC) and has a symmetrical dimeric structure which is composed of two alkylpyridinium iodides connected with a hexamethylenedioxydicarbonyl chain. 4DOCBP-6,10 and 4DOCBP-6,12 exhibited wide and effective antimicrobial spectra, compared with those of typical bactericides, N-dodecylpyridinium iodides (P-12) and benzyldimethyldodecylammonium chloride (BAC), or a popularly-used fungicide, 2-(4-thiazolyl)benzimidazole (TBZ). Their superior properties would be due to the unique dimeric structure which contains two active moieties in a molecule.

Bactericidal action of 4,4'-(alpha,omega-polymethylenedithio)bis-(1-alkylpyridinium iodide)s.

Bactericidal action of novel bis-quaternary ammonium compounds (bis-QACs), 4,4'-(alpha,omega-polymethylenedithio)bis(1-alkylpyridinium iodide)s (4DTBP-m,n) was studied. The bactericidal activity of 4DTBP-m,n in water was not affected by the molecular hydrophobicity unlike general mono-QAC, N-dodecylpyridinium iodide (P-12), while the bacteriostatic activity in the medium was reduced with their hydrophobicity. This result suggested that the hydrophobic materials in the medium interact with 4DTBP-m,n and cover their active moiety. Since the bactericidal activity using the measurement system supplemented with peptone was influenced by the molecular hydrophobicity, this speculation was supported. The plots of the bacteriostatic activities of 4DTBP-m,n against the surface hydrophobicities of various bacteria accord to the straight line as in the case of P-12. The slope of the line of 4DTBP-6,12 was comparatively smaller than that of 4DTBP-6,8, indicating that the compounds having longer alkyl group tend to reduce their activities against the bacteria with hydrophobic cell surface because of their interaction with the hydrophobic materials. The novel bis-QACs have an ability to liberate rapidly and abundantly the turbid materials from cells, that is, a bacterioclastic activity. The bacterioclastic activity of P-n was influenced by the length of alkyl group, while 4DTBP-6,n had almost the same activity regardless of its length. Observation by scanning electron microscope (SEM) revealed that 4DTBP-6,8 fatally damaged Escherichia coli cells, and that the morphological alteration of the cells caused by the bis-QAC was greatly different from that of the usual QAC. Therefore, the effective bacterioclastic action, and excellent bactericidal action is due to the unique dimeric structure of 4DTBP-m,n.

Synthesis and antimicrobial characteristics of 4,4'-(alpha,omega-polymethylenedithio)bis(1-alkylpyridinium iodide)s.

Bis-quaternary ammonium compounds (bis-QACs), 4,4'-(alpha,omega-polymethylenedithio)bis(1-alkylpyridinium iodide)s (4DTBP-m,n), which have 3 to 10 carbon atoms in the connecting methylene chain (m) and 8 to 18 carbon atoms of the N-alkyl chain (n), were synthesized. 4DTBP-6,12 exhibited a wide antimicrobial spectrum against gram-positive and gram-negative bacteria and fungi. The activity was stronger than those of N-dodecylpyridinium iodide (P-12), benzyldodecyldimethylammonium chloride and 2-(4-thiazolyl)benzimidazole. The bactericidal activities of 4DTBP-m,n were scarcely affected by the lengths of the alkyl chain and methylene chain. The bis-QAC that showed the highest activity was 4DTBP-6,8 (minimum inhibitory concentration (MIC) = 1.6 microM, minimum bactericidal concentration (MBC) = 2.6 microM), and its activity was about 10 times that of N-hexadecylpyridinium iodide (P-16), which was the most active in the P-n series. In addition, 4DTBP-6,12 showed a high bactericidal activity in the ranges of pH 5 to 8.5 and 10 to 40 degrees C, in contrast to mono-QACs. The bis-QACs synthesized in this study have excellent bactericidal properties.

Effects of ethanol on the growth and elongation of Escherichia coli under high pressures up to 40 MPa.

The effects of ethanol on growth, viability and cell length were studied in Escherichia coli cultured under pressures up to 40 MPa (400 bar). A pressure of 10 MPa reversed the effect of ethanol in retarding cell growth. Cells cultured in the absence of ethanol became about seven times longer at 40 MPa than at atmospheric pressure, and some cells showed incomplete cell division. Ethanol also increased cell length but these effects were not seen at pressures of 20 MPa or more.

Syntheses of 5,6,7- and 5,7,8-trioxygenated 3',4'-dihydroxyflavones having alkoxy groups and their inhibitory activities against arachidonate 5-lipoxygenase.

Arachidonate 5-lipoxygenase plays a pivotal role in the biosynthesis of leukotrienes. Cirsiliol (3',4',5-trihydroxy-6,7-dimethoxyflavone), a selective inhibitor of the enzyme, was derivatized by introducing alkyl groups of various chain lengths at positions 5, 6, 7, and 8 of the A ring of the flavone skeleton. Modification of the positions 5 and 6 with an alkyl group of 5-10 carbons markedly decreased the IC50 values for 5-lipoxygenase inhibition to the order of 10 nM. As tested with 5- or 6-hexyloxy derivatives, a relatively selective inhibition of 5-lipoxygenase was shown. Inhibition of 12-lipoxygenase required much higher concentrations of these compounds, and cyclooxygenase was not inhibited. Modification of positions 7 and 8 did not increase the inhibitory effect of most flavone compounds.

A possibility to recognize chirality by an excitable artificial liquid membrane.

Studies were made on oscillations across a liquid membrane consisting of an oil layer, nitrobenzene containing picric acid, between two aqueous layers: a solution of 1.5 M ethanol and 5 mM optically-active cationic detergent, the D- or L-form of N-alpha-methylbenzyl-N,N-dimethylmyristylammonium bromide, on the left and 0.1 M D- or L-form of various ligands, such as glucose, arabinose, alanine, glutamic acid, threonine, leucine, proline, or phenylalanine on the right. This system showed sustained rhythmic oscillations of electrical potential of 200-300 mV with intervals of the order of 1 min. The frequency of oscillations depended on the combination of chiralities of the detergent and ligand. This means that the two forms (D and L) of chiral ligands can be distinguished by differences in the electrical response of the liquid membrane.

Disinfection of water with quaternary ammonium salts insolubilized on a porous glass surface.

Insoluble quaternary ammonium salts bound to porous glass showed antibacterial activity. An agent designated as G(12), which had a dodecyl alkyl chain, was selected for some antibacterial tests on comparison of it with the agent reported previously. The antibacterial activity of G(12) toward Escherichia coli was mainly due to the adsorption of cells and therefore gradually decreased during continuous treatment of a cell suspension. The lost G(12) activity was completely recovered by washing with ethanol, and the activity of refreshed G(12) decreased in the same manner as that of fresh G(12). The lost activity was, however, always recovered only by ethanol treatment. This indicated that G(12) might interact with cells more strongly by means of a hydrophobic force than an electrostatic one. The antimicrobial spectrum showed that G(12) was effective against not only bacteria but also yeasts.

Adsorption of Escherichia coli onto insolubilized lauryl pyridinium iodide and its bacteriostatic action.

Insoluble lauryl pyridinium iodide [C12(50)] was synthesized as an antimicrobial agent. Escherichia coli cells were not killed by C12(50) but only adsorbed onto it. Though cells on C12(50) could not grow in nutrient agar, they possessed the ability to develop once they were liberated from C12(50). The adsorption of cells onto C12(50) was inhibited by iodide anions released from C12(50) itself. The ability of C12(50) to adsorb was decreased by the adsorbed cells, but C12(50) could be reactivated by washing with alkaline solutions. It was, therefore, suggested that this adsorption was mainly due to the electrostatic interaction between cells and C12(50). The adsorption of cells onto C12(50) was confirmed by scanning electron microscopy.

Antimicrobial characteristic of insoluble alkylpyridinium iodide.

Insoluble and soluble alkylpyridinium iodides (C8 to C18) were synthesized. The insoluble agents were quaternized 4-vinylpyridine-divinylbenzene copolymers. The insoluble agent [C12(50)] that contained 50% divinylbenzene and had a C12 alkyl chain was selected as the most suitable insoluble agent. C12(50) showed poor durability of the antibacterial activity, but C12(50), which had lost the activity, was refreshed by washing with ethanol. This washing became ineffective after a few cycles of antibacterial treatment and refreshment. Such C12(50) recovered the activity upon 1.0 N NaOH treatment. The antibacterial activity of C12(50) depended on its surface area. It showed high antimicrobial activity against gram-positive bacteria and also showed activity against gram-negative bacteria and yeasts. But the activities of C12(50) and laurylpyridinium iodide solution were different against some microbes. The antibacterial activities of the agents were investigated against Escherichia coli and Micrococcus luteus under various conditions. The activity of C12(50) was higher at a higher temperature or at a lower cell concentration. The activity of C12(50) decreased on addition of NaCl, glucose, or bovine albumin to the cell suspension or in 0.01 M sodium-potassium phosphate buffer. C12(50) showed less activity when cells were mixed with dead cells or the supernatant of dead cells killed in an autoclave. The mode of action of the laurylpyridinium iodide solution against E. coli and M. luteus was similar to that of C12(50) except for the influence of E. coli cell concentration.