Surveillance of severe invasive group-G streptococcal infections and molecular typing of the isolates in Japan.

The number of patients with severe invasive group-G streptococcal (Streptococcus dysgalactiae subsp. equisimilis) infections has been increasing in Japan. The emm genotypes and SmaI-digested pulsed-field gel electrophoresis DNA profiles were variable among the strains isolated, suggesting there has not been clonal expansion of a specific subpopulation of strains. However, all strains carried scpA, ska, slo and sag genes, some of which may be involved in the pathogenesis of the disease.

Changing prevalent T serotypes and emm genotypes of Streptococcus pyogenes isolates from streptococcal toxic shock-like syndrome (TSLS) patients in Japan.

We surveyed T serotypes and emm genotypes of Streptococcus pyogenes isolates from streptococcal toxic shock-like syndrome (TSLS) patients. T1 (emm1) remained dominant through 1992 to 2000, but the dominant T3 (emm3.1) strains from 1992 to 1995 disappeared during 1996-2000. Strains of several emm genotypes emerged during 1996-2000, indicating alterations in the prevalent strains causing TSLS.

Distribution of emm genotypes and superantigen genes of Streptococcus pyogenes isolated in Japan, 1994-9.

The purpose of this study was to examine characteristic profiles of Streptococcus pyogenes clinical isolates isolated in Japan during 1994-9. Genotyping of the M protein (emm typing) revealed that emm types 12 and 28 were the most common among 316 isolates. Most of the emm12 isolates were isolated from mucosa, while emm58 and emm89 were from skin. Moreover, the emm3 isolates were dominant in invasive infections. The distribution of 6 superantigen genes showed that all isolates harboured the mf gene and many had the speG gene. Invasive isolates were shown to have the ssa gene at a higher rate (76%) than noninvasive (37%). The distribution of superantigens was significantly different between emm types, but not between isolation sites. These results suggest that the distribution of emm types is related to isolation site, whereas superantigen distribution is related to clinical features of S. pyogenes infections.

[Epidemiological study of microepidemics of tuberculosis by restriction fragment length polymorphism analysis].

Restriction fragment length polymorphism (RFLP) analysis was performed with 13 events considered as microepidemics of tuberculosis occurred in Osaka prefecture from July 1995 to July 1998. In 7 out of 13 events, isolates from patients involved in each event showed identical RFLP patterns, hence these events were verified as microepidemics. Out of 7 microepidemics, three were intrafamily infection, the other three occurred in work places and the remaining one was in a school occurred among students. The total number of patients who were involved in microepidemics was 19 and their ages distributed in the wide range from 18 to 69 years old, but 11 were at twenties or forties. The total delay in case-finding was 2 months in the shortest case, and more than 14 months in the longest. In the remaining 6 of 13 events, the isolates from patients were showed different RFLP patterns, although they were suspected as microepidemics. Furthermore, in the 2 microepidemics, one isolate showed different RFLP pattern from the other isolates involved in each events. These facts suggest that there could be many overlooked sources of tuberculosis infection in Osaka.

Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene.

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, > or =200 microg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.

Serotyping of Streptococcus pyogenes isolated from common and severe invasive infections in Japan, 1990-5: implication of the T3 serotype strain-expansion in TSLS. The Working Group for Group A Streptococci in Japan.

To clarify the relationship between the epidemics of severe invasive group A streptococcal infections (streptococcal Toxic Shock-Like Syndrome: TSLS) and common group A streptococcal infections in Japan, we examined the T serotypes of S. pyogenes strains (group A streptococci) isolated from clinical specimens of the streptococcal infections (17999 cases) in the period 1990-5, including the severe infections (TSLS) (29 cases) in the period 1992-5. Characteristic points of the analyses were: (1) dominant serotypes of the infections in these periods were T12, T4, T1, T28 and TB3264, which were consistently isolated; (2) isolates of T3 rapidly increased through 1990 to 1994 while T6 decreased in the period 1990-3; (3) when Japanese area was divided into three parts, T3 serotype tended to spread out from the north-eastern to the south-western area; (4) strains of T3 and T1 serotypes were dominant in the TSLS. Dominant-serotype strains of streptococcal infections did not always induce severe infections and dominance of T3 serotype in the TSLS seemed to be correlated with the increase of T3 in streptococcal infections. These results may indicate that certain clones of S. pyogenes are involved in the pathogenesis of the TSLS.

Characterization of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan.

Mutations in the rpsL and rrs genes associated with streptomycin resistance in Mycobacterium tuberculosis clinically isolated in Japan were characterized. The rpsL genes of 172 clinical isolates were amplified by PCR and classified into two groups on the basis of MboII restriction digestion. Thirty-three out of 54 (61.1%) streptomycin-highly resistant isolates (MIC > 200 micrograms ml-1) were not digested by MboII. By contrast, the remaining 21 of 54 (38.9%) streptomycin-highly resistant isolates, all of 41 isolates with streptomycin resistance at a lower level (20 micrograms ml-1 < MIC < or = 200 micrograms ml-1), and all of 77 streptomycin-sensitive isolates, were restricted. Thus, all isolates resistant for MboII digestion showed a high level of resistance to streptomycin. Subsequently, the sequence for the rpsL and rrs genes from the 46 isolates were analysed. Eighteen out of 19 (94.7%) streptomycin-highly resistant isolates carried a mutation in any rpsL gene at position 43 or 88, or the rrs gene; 10 out of 17 (58.8%) streptomycin-resistant isolates at a lower level were confirmed to exhibit the mutation of either the mutated rpsL gene at position 88, or the rrs gene. In the total 36 streptomycin-resistant isolates, the mutation of the rpsL or rrs gene was observed in 28 streptomycin-resistant isolates, corresponding to 77.8%, whereas none of the streptomycin-sensitive isolates had mutations in either the rpsL or rrs gene.

Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer.