Nobuyuki Uehara

Endogenous Reactive Oxygen Species Is an Important Mediator of Miconazole Antifungal Effect

ABSTRACT We investigated the significance of endogenous reactive oxygen species (ROS) produced by fungi treated with miconazole. ROS production in Candida albicans was measured by a real-time fluorogenic assay. The level of ROS production was increased by miconazole at the MIC (0.125 μg/ml) and was enhanced further in a dose-dependent manner, with a fourfold increase detected when miconazole was used at 12.5 μg/ml. This increase in the level of ROS production was completely inhibited by pyrrolidinedithiocarbamate (PDTC), an antioxidant, at 10 μM. In a colony formation assay, the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of PDTC. Moreover, the level of ROS production by 10 clinical isolates of Candida species was inversely correlated with the miconazole MIC (r = −0.8818; P < 0.01). These results indicate that ROS production is important to the antifungal activity of miconazole.

Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction.

MecA, a structural gene located on the chromosome of Staphylococcus aureus, characterizes methicillin-resistant S. aureus (MRSA), and femA and femB(fem) genes encode proteins which influence the level of methicillin resistance of S. aureus. In order to examine effectiveness of detecting mecA and fem genes in identification of MRSA, the presence of these genes in 237 clinically isolated strains of staphylococci was investigated by polymerase chain reaction (PCR). An amplified mecA DNA fragment of 533 base pairs (bp) was detected in 100% of oxacillin-resistant S. aureus, in 16.7% of oxacillin-sensitive S. aureus, in 81.5% of S. epidermidis, and in 58.3% of other coagulase-negative staphylococci (CNS). While the PCR product of femA (509 bp) or femB (651 bp) was obtained from almost all the S. aureus strains except for five oxacillin-resistant strains (2.5%), neither of these genes were detected in CNS. Therefore, the detection of femA and femB together with mecA by PCR was considered to be a more reliable indicator to identify MRSA by differentiating it from mecA-positive CNS than single detection of mecA.

Analysis on Distribution and Genomic Diversity of High-Level Antiseptic Resistance Genes qacA and qacB in Human Clinical Isolates of Staphylococcus aureus

High-level antiseptic resistance of Staphylococcus aureus is mediated by multidrug efflux pumps encoded by qacA and qacB genes. We investigated distribution and genomic diversity of these antiseptic resistance genes in a total of 522 clinical strains of S. aureus isolated recently in a Japanese hospital. The qacA/B gene was detected in 32.6% of methicillin-resistant S. aureus (MRSA) and 7.5% of methicillin-susceptible S. aureus (MSSA), whereas the low-level resistance gene smr, which was examined simultaneously, was detected at lower frequencies in both MRSA (3.3%) and MSSA (5.9%). Epidemiologic typing of S. aureus isolates suggested that higher prevalence of qacA/B in MRSA may be due to spread of a single predominant MRSA strain carrying qacA/B in the hospital. Restriction fragment length polymorphism (RFLP) analysis indicated higher prevalence of the qacB-type gene (59.3%) than the qacA-type gene (40.7%) among the qacA/B genes detected. Nucleotide sequencing analysis revealed the presence of two genetic variants in qacA (V1 and V2) and four variants in qacB (V1-V4) that differ from the qacA prototype in pSK1 by 1-5 nucleotides and 7-9 nucleotides, respectively. Although most strains with qacA-V1, qacA-V2, qacB-V3, and qacB-V4 showed high-level resistance to ethidium bromide (EB)(MIC > 100 microg/ml), all of the S. aureus isolates carrying qacB-V1 and qacB-V2 showed lower MICs of EB and some monovalent cationic antiseptic substances. By analysis of the genomic organization of the qacA/B downstream region, divergent forms of this region rearranged with an insertion of IS256 or IS257 were found primarily for qacB. The downstream region of qacA-V1 was suggested to be an evolutionary origin for other divergent forms. These findings indicated that both qacA and qacB are prevalent in recent clinical isolates, especially in MRSA, and these genes consist of variable genetic variants that may be responsible for different resistance levels against antiseptic substances.

Diversity of staphylocoagulase and identification of novel variants of staphylocoagulase gene in Staphylococcus aureus.

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I–X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp‐nucleotide sequences of SC gene encoding D1, D2, and central regions (N‐terminal half and central regions of SC; SCNC) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I–VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND‐strains). Amino acid sequence identities of SCNC among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56–87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SCNC sequences from the ND‐strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I–X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes.

Analysis of genomic diversity within the Xr -region of the protein A gene in clinical isolates of Staphylococcus aureus

Protein A of Staphylococcus aureus contains a polymorphic Xr-region characterized by a tandem repeat of eight amino acid units. In this study, the diversity of genes encoding the repeat regions and their relatedness among S. aureus strains was analyzed. Ten different protein-A types characterized by repeat numbers 4-13 were identified in a total of 293 clinical isolates. The protein-A type with 10 repeat units (10 repeats) in the Xr-region was most frequently detected in methicillin-resistant S. aureus, whereas the majority of methicillin-susceptible strains were distributed almost evenly into protein-A types with 7-11 repeats. Strains that belonged to a single coagulase type were classified into multiple protein-A types, e.g. strains with the common coagulase types II and VII were differentiated into 7 and 8 protein-A types, respectively. Nucleotide sequence analysis of the Xr-region of 42 representative strains revealed the presence of 37 different genotypes (spa types), which were constituted by a combination of several of 24 different repeat unit genotypes. Based on the similarity in arrangement of repeat unit genotypes, 34 strains with different repeat numbers were classified into 5 genetic clusters (C1-C5). The clusters C1, C2 and C3 consisted exclusively of strains with identical coagulase types II, III, and IV, respectively. These findings suggested that the protein-A gene of S. aureus has evolved from a common ancestral clone in individual clusters independently.

Simultaneous Amplification of Bordetella Repeated Insertion Sequences and Toxin Promoter Region Gene by Polymerase Chain Reaction

A polymerase chain reaction was devised to simultaneously detect repeated insertion sequences and the pertussis toxin promoter gene for the diagnostic identification of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. The sensitivity of this method was sufficient to detect one B. pertussis organism using the following cycles and temperatures: 95 degrees C for 15 min, followed by 32 amplification cycles (1 min at 95 degrees C, 1 min at 66 degrees C, 1 min at 72 degrees C), and finally 5 min at 72 degrees C. Using the primers as a combined set did not affect sensitivity, but required an increased temperature for optimal annealing compared with a single-sequence assay. As nasopharyngeal aspirate and swab materials sometimes contain hemoglobin, we also tested the inhibitory effect of hemoglobin on this assay, which was inhibited completely when using DNA extracts from samples containing hemoglobin at a final concentration >0.015 g/L: this inhibition was reversed by addition of bovine serum albumin to the buffer. Our assay shows promising sensitivity and specificity for clinical use.

Pyrazinamide resistance associated with pncA gene mutation in Mycobacterium tuberculosis in Japan

Thirty Japanese clinical isolates of Mycobacterium tuberculosis were analysed by pyrazinamide susceptibility testing and pyrazinamidase assay, as well as polymerase chain reaction for single-strand conformational polymorphism and direct sequencing of the gene encoding pyrazinamidase (pncA). All sensitive isolates showed pyrazinamidase activity and a wild-type pncA gene, but three resistant isolates had pncA gene mutations and lacked pyrazinamidase activity. The latter isolates showed a minimum inhibitory concentration of at least 100 mg/l by the 7H10 agar proportion method and 400 mg/l by the 7H9 liquid medium method. Isolate 28 showed T-to-C change at position 11, leading to Leu4 --> Ser substitution; isolate 29 had an 8-bp deletion from position 382; and isolate 30 had A-to-C change at position 29, leading to Gln10 --> Pro substitution. The deletion has not been described previously. This is the first demonstration of pncA gene mutations in PZA-resistant M. tuberculosis strains isolated from Japanese patients.

Genetic Diversity of the Low-Level Vancomycin Resistance Gene vanC-2/vanC-3 and Identification of a Novel vanC Subtype (vanC-4) in Enterococcus casseliflavus

An intrinsic low-level vancomycin resistance (VanC phenotype) in Enterococcus casseliflavus is conferred by either of two subtypes of vanC genes, that is, vanC-2 or vanC-3, which are genetically closely related. To know genetic diversity of vanC-2/C-3 genes among E. casseliflavus, nucleotide sequences of vanC-2/C-3 and other genetic components in vanC gene cluster (vanXYc, vanTc, vanRc, and vanSc) were analyzed for nine clinical isolates and four standard strains that showed low-level vancomycin resistance. While the vanC-2/C-3 gene sequences showed 93-100% identities among the strains examined, two genetic groups were discriminated by phylogenetic analysis: one closely related to the previously reported vanC-2 or vanC-3 genes (vanC-2/C-3 genotype) with 98-100% identity, and the other distinct from the vanC-2/C-3 genotype (93-95% identity). The latter group found in three clinical isolates was considered as a new subtype of vanC and tentatively designated as vanC-4. Between strains with the vanC-2/C-3 genotype and those with vanC-4, vanXYc genes were also genetically discriminated with 92-93% identity. Similar sequence diversity was observed for vanTc, vanRc, and vanSc (88-93% identity). Clonal relatedness among the E. casseliflavus strains was investigated by phylogenetic analysis of atpA gene. While among E. casseliflavus strains with vanC-2/C-3 genotype, extremely high sequence identities of atpA were found (98.7% or higher), these strains showed slightly lower identity to those with vanC-4 (94-96%). These two groups of E. casseliflavus strains were also discriminated by genotyping with arbitrarily primed PCR. These findings indicated that among E. casseliflavus there are at least two genetic lineages with the distinct vanC genes, that is, a single subtype including previously known vanC-2/C-3, and a novel subtype vanC-4.

Three point mutations of human butyrylcholinesterase in a Japanese family and the alterations of three-dimensional structure.

Three different mutations at codons 330 (TTA to ATA), 365 (GGA to AGA) and 515 (CGT to TGT) of human butyrylcholinesterase (hBChE) were identified in a Japanese family. We correlated alterations in in the patients hBChE activity with possible structural alterations in the three-dimensional structure of hBChE caused by the point mutations. This study was performed using the published computer-generated three-dimensional structure of hBChE based on the structure of acetylcholinesterase. The amino acid substitution at L330I was adjacent to hydrophobic residues that form the channel domain of the active center. This side chain faced the side opposite the active center. The amino acid substitution at G365R was located at the position most remote from the active center, and this substitution site was exposed to the surface of the BChE protein. Alpha-helical structure was present to the active center, and the guanidyl residue of native Arg 515 was hydrogen-bonded to the carboxyl group of Asp 395 in the alpha-helix. These point mutations may cause steric effects on the present patients hBChE activity. This is the first report of three-dimensional structural analysis performed on the L330I, G365R, and R515C mutations of hBChE.

Comparison of arbitrarily primed-polymerase chain reaction and pulse-field gel electrophoresis for characterizing methicillin-resistant Staphylococcus aureus

Aims:  The aim of this study was to analyse genotypes for clinical isolates of methicillin‐resistant Staphylococcus aureus (MRSA), including hetero‐vancomycin‐resistant Staph. aureus (VRSA), at a Japanese university hospital.