Katsuhiko Machida

An exfoliative toxin A-converting phage isolated from Staphylococcus aureus strain ZM.

Exfoliative toxin A (ETA) causes staphylococcal scalded‐skin syndrome in children. The gene for ETA was believed to be coded by the chromosomal DNA. We isolated temperate phages from an ETA‐producing strain, ZM, using a restriction minus strain, 1039, as an indicator. One of the prophages, designated φ‐ZM‐1 mediated lysogenic conversion of ETA. The polymerase chain reaction assay of the eta gene revealed that phage φ‐ZM‐1 carries the structural gene for ETA.

Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20‐mer, and its PCR product was a 741‐bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20‐mer, and its PCR product was a 629‐bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double‐producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double‐producers. However, positive results were obtained for 5 of the 10 non‐ET‐producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.

Genetic identification of Staphylococcus aureus by polymerase chain reaction using single-base-pair mismatch in 16S ribosomal RNA gene

Staphylococcus aureus is the most predominant and important pathogen in clinical microbiology. A DNA amplification assay using the polymerase chain reaction (PCR) was designed to identify S. aureus through a single‐base‐pair mismatch in the sequences of staphylococcal 16S ribosomal RNA (16S rRNA) genes. It was able to detect and identify S. aureus without requiring additional analytical techniques. Twenty‐eight staphylococcal and non‐staphylococcal strains were tested to verify the specificity of the assay, and only S. aureus strains gave a positive reaction. It may be possible to provide immediate and exact information for the identification of S. aureus.

Mutagenic activity of N-nitrosomethamphetamine and N-nitrosoephedrine

The mutagenicity of N-nitrosomethamphetamine (NMA) and N-nitrosoephedrine (NEP), which were synthesized in our laboratory, was examined by the modified pre-incubation method of Ames assay using Salmonella typhimurium TA100 and TA98. Both nitroso compounds showed significant mutagenic activity in the presence of hamster S9.

Apnea-related heart rate variability in congestive heart failure patients.

Sleep‐disordered breathing (SDB) causes fluctuation of the RR interval. However, the details are uncertain. We studied the characteristics of sleep‐related heart rate variation (HRV) in congestive heart failure (CHF) patients with SDB. Ambulatory electrocardiograms and data on respiration (oronasal flow, trachea sound, abdominal wall movement, and oxygen saturation) were simultaneously recorded by a multi‐channel digital recorder for 13 CHF patients (8 men and 5 women; mean age, 68 ± 4 years). Heart rate variation occurred as a result of cyclical apnea attacks between 0.005 and 0.03 Hz (apnea band). The proportion of the apnea band (% apnea) increased with the number of apnea episodes, and SDB was highly likely when the % apnea was ≥80%. Low‐flow oxygen administration effectively reduced apnea frequency, and the apnea‐related HRV also decreased. We concluded that apnea‐related HRV was useful for detecting and following SDB in CHF cases.

New Evidence That the Tyr-157 and Tyr-159 Residues of Staphylococcal Exfoliative Toxin B Are Essential for Its Toxicity

To determine the active site of exfoliative toxin B (sETB) of Staphylococcus aureus, the etb gene was cloned from an S. aureus SU strain obtained from a patient with impetigo. We prepared a frame shift mutant protein from a recombinant plasmid with a BglII linker inserted into the Tyr‐155 codon of the ETB gene (pETB/BglIIL). The recombinant mutant protein (ETB/BglIIL) obtained from Escherichia coli containing pETB/BglIIL showed no toxicity in neonatal mice and no agglutination activity. The 20‐kDa ETB/BglIIL contained 185 amino acid residues. Site‐directed mutagenesis was used to introduce mutations at either Tyr‐155, Tyr‐157, Tyr‐159, or Tyr‐162. Substitution of any of the Tyr residues decreased exfoliative activity compared with that of native sETB (4,000 EU/ml). Substitution of Tyr‐155 with a Phe (ETB/Y155) decreased activity 5‐fold (800 EU/ml). Substitution of Tyr‐157 with Leu (ETB/Y157) decreased activity 80‐fold (50 EU/ml) and decreased agglutination titer 5‐fold compared with that of native sETB (400,000). Substitution of Tyr‐159 with Leu (ETB/Y159)decreased activity 4‐fold (1,000 EU/ml). When both Tyr‐157 and Tyr‐159 were mutated (ETB/Y157‐159), both toxicity and antigenicity were completely lost. On an immunodiffusion test, ETB/Y157 showed a faint precipitation line, but ETB/BglIIL and ETB/Y157‐159 had no activity, showing that the Tyr‐157 and Tyr‐159 residues are essential for the toxicity and antigenicity of ETB.

Relationship between Susceptibility and Immune Response to Staphylococcal Exfoliative Toxin A in Mammalian Species

Staphylococcal exfoliative toxin A (ETA) had a splitting effect at the granular layer of skin in humans and neonatal mice, but not in rabbits, guinea pigs, golden hamsters, or rats. Besides its splitting effect, ETA could stimulate productions of neutralizing antibody to ETA in rabbits, rats and B10D2 mice, but not in golden hamsters, guinea pigs, or ICR, HRS/J, and C57BL/10 mice. In our epidemiological investigation of human sera, the percentage of antibody to ETA in sera obtained from patients with impetigo (8%) was lower than those in sera of healthy males (23%) and females (29%). The relationship between susceptibility and immune response to ETA in these mammalians could be divided into three groups: the possession of resistant skin and high production of antibody to ETA; the possession of resistant skin and low production of antibody to ETA; the possession of sensitive skin and various titers of antibody to ETA.

HEART RATE VARIABILITY IN CONGESTIVE HEART FAILURE

Congestive heart failure (CHF) is frequently a complication of sleepdisordered breathing (SDB) such as Cheyne–Stokes respiration. Such cases have a poor prognosis (1–9). New methods of treating these patients, such as the continuous positive airway pressure (CPAP) method, have been developed and offer desirable effect (2,8). However, the diagnosis of SDB places heavy demands on both medical staff and patients because of the need for continuous overnight polysomnographic recording, and this contributes to the difficulty of treatment of SDB patients. SDB effects hemodynamic changes in the cardiovascular system and these cause fluctuation of the RR interval. We studied the characteristics of the heart rate variation of SDB patients and the clinical significance as an index for detecting SDB among CHF patients. CLIN. AND EXPER. HYPERTENSION, 24(1&2), 75–81 (2002)

Oxygen desaturation and heart rate variability due to Cheyne-Stokes respiration in congestive heart failure patients

Cheyne-Stokes respiration is common in congestive heart failure patients and causes cyclical fluctuation of the RR interval. We studied the characteristics of apnea-related heart rate variability (apnea HRV) and the relation between apnea HRV and oxygen desaturation was examined. Ambulatory electrocardiograms and data on respiration (oronasal flow, tracheal sounds, abdominal wall movement and oxygen saturation) were simultaneously recorded by a multi-channel digital recorder for 16 congestive heart failure patients (10 men and 6 women; mean age, 68 +/- 13 years). HRV occurred as a result of cyclical apnea attacks between 0.005 and 0.03 Hz (apnea band). Apnea HRV was obtained as the power ratio of apnea HRV normalized by the very low frequency band (0.003-0.04 Hz). Apnea HRV increased with the number of apnea episodes and the oxygen desaturation index, but no relation between apnea HRV and either mean oxygen density or oxygen desaturation time was observed. We concluded that apnea HRV is a predictor of the number of apnea attacks or oxygen desaturation, but does not reflect the degree of oxygen desaturation in Cheyne-Stokes respiration.

A simple and rapid method for purifying staphylococcal exfoliative toxin A.

A rapid and efficient method of purification of staphylococcal exfoliative toxin A, the causative agent of staphylococcal scalded skin syndrome (SSSS), has been developed. It is based on ammonium sulfate precipitation of the culture supernatant and hydrophobic interaction chromatography on Phenyl‐Sepharose CL‐4B. This procedure results in 87‐fold purification of this toxin, which appears as a single band in sodium dodecyl sulfate‐polyacrylamide gels.