Hiroki Kourai

Correlation between Resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds and Expression of Outer Membrane Protein OprR

ABSTRACT The adaptation mechanism of Pseudomonas aeruginosa ATCC 10145 to quaternary ammonium compounds (QACs) was investigated. A P. aeruginosa strain with adapted resistance to QACs was developed by a standard broth dilution method. It was revealed that P. aeruginosa exhibited remarkable resistance to N-dodecylpyridinium iodide (P-12), whose structure is similar to that of a common disinfectant, cetylpyridinium chloride. Adapted resistance to benzalkonium chloride (BAC), which is commonly used as a disinfectant, was also observed in P. aeruginosa. Moreover, the P-12-resistant strain exhibited cross-resistance to BAC. Analysis of the outer membrane protein of the P-12-resistant strain by two-dimensional polyacrylamide gel electrophoresis showed a significant increase in the level of expression of a protein (named OprR) whose molecular mass was approximately 26 kDa. The actual function of OprR is not yet clear; however, OprR was expected to be an outer membrane-associated protein with homology to lipoproteins of other bacterial species, according to a search of the National Center for Biotechnology Information website with the BLAST program by use of the N-terminal sequence of OprR. A correlation between the level of expression of OprR and the level of resistance of P. aeruginosa to QACs was observed by using a PA2800 gene knockout mutant derived from the P-12-resistant strain. The knockout mutant recovered susceptibility not only to P-12 but also to BAC. These results suggested that OprR significantly participated in the adaptation of P. aeruginosa to QACs, such as P-12 and BAC.

Characterization and photochemical and antibacterial properties of highly stable silver nanoparticles prepared on montmorillonite clay in n-hexanol.

Ag nanoparticles (NPs) of approximately 2.5±0.6 nm in size with high stability were prepared on montmorillonite clay in n-hexanol ([Ag NP/clay]). The aqueous suspensions of Ag NP/clay obtained were colored pale yellow to gray, and an intense yellow surface plasmon band was observed at approximately 397-422 nm at a [Ag(+)] concentration of 0.01-1 M. Fluorescence peaks of these Ag NP/clay suspensions, which were due to interband transitions of Ag NPs, were observed at approximately 447 nm upon excitation at approximately 341 nm. Photoinduced charge separation and electron accumulation in the presence of 2-propanol were observed in the Ag NPs on the clay. The photogenerated holes indicate a reaction with OH(-) because the generation of (·)OH radicals decreased in the presence of 2-propanol, as observed using a spin-trapping electron spin resonance (ESR) method. The amount of H(2) produced depended on the amount of Ag NPs on the clay and the concentration of 2-propanol in the suspension. Antibacterial activity of Ag NPs on clay was also observed against Escherichia coli even after 12 years. This simple method of preparation in n-hexanol led to the successful formation of highly stable and photoactive Ag NPs on montmorillonite clay.

Mutational Upregulation of a Resistance-Nodulation-Cell Division-Type Multidrug Efflux Pump, SdeAB, upon Exposure to a Biocide, Cetylpyridinium Chloride, and Antibiotic Resistance in Serratia marcescens

ABSTRACT Serratia marcescens is an important opportunistic pathogen in hospitals, where quaternary ammonium compounds are often used for disinfection. The aim of this study is to elucidate the effect of a biocide on the emergence of biocide- and antibiotic-resistant mutants and to characterize the molecular mechanism of biocide resistance in Serratia marcescens. A quaternary ammonium compound-resistant strain, CRes01, was selected by exposing a wild-type strain of S. marcescens to cetylpyridinium chloride. The CRes01 cells exhibited 2- to 16-fold more resistance than the wild-type cells to biocides and antibiotics, including cetylpyridinium chloride, benzalkonium chloride, chlorhexidine gluconate, fluoroquinolones, tetracycline, and chloramphenicol, and showed increased susceptibilities to β-lactam antibiotics and N-dodecylpyridinium iodide. Mutant cells accumulated lower levels of norfloxacin than the parent cells in an energized state but not in a de-energized state, suggesting that the strain produced a multidrug efflux pump(s). To verify this assumption, we knocked out a putative efflux pump gene, sdeAB, in CRes01 and found that the knockout restored susceptibility to most quaternary ammonium compounds and antibiotics, to which the CRes01 strain showed resistance. On the basis of these and other results, we concluded that S. marcescens gains resistance to both biocides and antibiotics by expressing the SdeAB efflux pump upon exposure to cetylpyridinium chloride.

Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human‐specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep‐seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep‐seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post‐infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface‐bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein.

Intermedilysin is a pore‐forming cytolysin belonging to the streptolysin O gene family known as the ‘Cholesterol‐binding/dependent cytolysins’ and is unique within the family in that it is highly human‐specific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome‐embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome‐binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human‐specific step of intermedilysin action, that the host cell membrane‐binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human‐specific action step and the cell‐binding site of domain 4 lies within the 56 amino acids of the C‐terminal, excluding the undecapeptide region.

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.

Photochemical property and surface characterization of silver-loaded zirconium phosphate

Abstract Superoxide radicals such as OH · and O 2 − were detected by DMPO as a spin trap reagent in visible light-irradiated silver-loaded zirconium phosphate [Ag 1- x H x Zr 2 (PO 4 ) 3 ] water suspension. It depended on Ag content and irradiation wavelength. When no-silver-loaded zirconium phosphate [HZr 2 (PO 4 ) 3 ]/Nafion-modified Pt electrode in 0.1 mol dm −3 Na 2 SO 4 aqueous solution was irradiated with 500 W superhigh pressure mercury lamp, a 26.1 μA cm −2 photocathodic current was observed at −0.2 V vs. SCE. This indicated that the formation of superoxides was due to the charge separation on the photoirradiated HZr 2 (PO 4 ) 3 . In the loading of Ag + ions to the zirconium phosphate, it was found that Ag + ions were mostly located on the surface and were stabilized by the surrounded oxygen atoms. Furthermore, Raman shifts due to an interaction between O 2 and Ag for Ag 1- x H x Zr 2 (PO 4 ) 3 were observed by FT-Raman technique. The ESR peak at g =2.0036 was detected in vacuo and in the air it was disappeared. This g value was similar to that which is due to the conduction electron in small silver nanocrystallites. Such conduction electron in Ag 1- x H x Zr 2 (PO 4 ) 3 seemed to be due to the increase of the electron density of Ag + by the effects of the octahedrally surrounded oxygen such as − l effects. It was found that the Ag + site worked as an electron pool. Consequently, it was suggested that photogenerated electron reduced O 2 to O 2 − radical via the Ag surface + site and the remaining hole on the zirconium phosphate oxidized OH − in the aqueous solution to OH radical.

Characterization of recombinant Streptococcus mitis‐derived human platelet aggregation factor

Ohkuni H, Nagamune H, Ozaki N, Tabata A, Todome Y, Watanabe Y, Takahashi H, Ohkura K, Kourai H, Ohtsuka H, Fischetti VA, Zabriskie JB. Characterization of recombinant Streptococcus mitis‐derived human platelet aggregation factor. APMIS 2012; 120: 56–71.

Light-induced formation of 2,5-dihydroxy-p-benzoquinonefrom hydroquinone in photoirradiated silver-loaded zirconium phosphate suspension

Silver-loaded zirconium phosphate [Ag1-xHxZr2(PO4)3] has shown photocatalytic activity concerning the generation of OH and the hydroxylation of hydroquinone (HQ) to 2,5-dihydroxy-p-benzoquinone (DHQH2). HQ was initially oxidized to p-benzoquinone (BQ) by Ag+ on the surface of Ag1-xHxZr2(PO4)3 in the dark and by photogenerated OH during visible-light irradiation. As an intermediate, a semiquinone radical BQH was detected by EPR measurements in both cases. Furthermore, under visible-light irradiation, DHQH2 was identified by its absorption spectrum and thin layer chromatography. The amount of DHQH2 increased and BQ decreased with irradiation time. The total amount of BQ and DHQH2 under visible-light irradiation agreed with that of BQ in the dark. Consequently, DHQH2 appeared to form from BQH generated by the oxidation of HQ and the reduction of BQ by photogenerated e-. From the XPS and FT-Raman technique analyses, it was found that the addition of the photogenerated OH to BQH occurred at the surface of Ag1-xHxZr2(PO4)3. This indicated that both the OH and HBQ were stabilized on the surface of Ag1-xHxZr2(PO4)3.

Role of Cl− adsorbed on silver-loaded zirconium phosphate for the photooxidation of OH− to OH·

Abstract When a silver-loaded zirconium phosphate [Ag 1- x H x Zr 2 (PO 4 ) 3 ] suspension was irradiated with a 500 W Xe lamp, the generation of OH · was observed as the DMPO-OH using an ESR spin-trapping technique. The generation rate of DMPO-OH changed by adding various ions to the suspensions. The addition of Fe 3+ , which has a redox potential of +0.771 V vs. NHE, increased the generation rate of DMPO-OH, indicating that the ion acted as an electron acceptor. In the presence of I − , the generation rate of DMPO-OH decreased by oxidizing I − with OH · . However, in the presence of Cl − , the generation rate remarkably increased with the increase in the concentration of Cl. The quantum yield of the generation rate of DMPO-OH was about 10 times larger than that in the absence of Cl − ; at 400 nm from 0.36% to 2.7%, and at 500nm from 0.037% to 0.41%. Such an effect of Cl could not be observed in the absence of Ag 1- x H x Zr 2 (PO 4 ) 3 . Furthermore, the generation rate of DMPO-OH during loading of Cl − into the suspension showed a pH dependence with the maximal value at pH ca. 5.8. From a Lineweaver—Burk plot, K m and V max(DMPO-OH) were determined to be 83.3 μ mol dm −3 and 0.44 μ mol dm −3 min −1 , respectively. These results suggested that the adsorption of Cl − on the surface lead to the formation of an active site like a catalysis center in the enzyme. Cl adsorbed on a Zr atom not on a Ag atom in the surface region, because of the ca. 1 eV shift in binding energy at the peak of only Zr 3author/2 in the XPS spectra of the Cl − -adsorbed Ag 1- x H x Zr 2 (PO 4 ) 3 . Consequently, the Cl − -adsorbed Zr atom on the surface was suggested to act as an electron pool in the photo-induced charge separation.