Increased prevalence of group A streptococcus isolates in streptococcal toxic shock syndrome cases in Japan from 2010 to 2012.

Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock, multi-organ failure, and high mortality. In Japan, appropriate notification measures based on the Infectious Disease Control law are mandatory for cases of STSS caused by ß-haemolytic streptococcus. STSS is mainly caused by group A streptococcus (GAS). Although an average of 60-70 cases of GAS-induced STSS are reported annually, 143 cases were recorded in 2011. To determine the reason behind this marked increase, we characterized the emm genotype of 249 GAS isolates from STSS patients in Japan from 2010 to 2012 and performed antimicrobial susceptibility testing. The predominant genotype was found to be emm1, followed by emm89, emm12, emm28, emm3, and emm90. These six genotypes constituted more than 90% of the STSS isolates. The number of emm1, emm89, emm12, and emm28 isolates increased concomitantly with the increase in the total number of STSS cases. In particular, the number of mefA-positive emm1 isolates has escalated since 2011. Thus, the increase in the incidence of STSS can be attributed to an increase in the number of cases associated with specific genotypes.

The detection of toxigenic Corynebacterium ulcerans from cats with nasal inflammation in Japan.

Corynebacterium ulcerans (toxigenic C. ulcerans) produces the diphtheria toxin, which causes pharyngeal and cutaneous diphtheria-like disease in people, and this bacterium is commonly detected in dogs and cats that are reared at home. It is considered dangerous when a carrier animal becomes the source of infection in people. To investigate the carrier situation of toxigenic C. ulcerans of cats bred in Japan, bacteria were isolated from 37 cats with a primary complaint of rhinitis in 16 veterinary hospitals in Osaka. Toxigenic C. ulcerans was detected in two of the cats. By drug sensitivity testing, the detected bacterium was sensitive to all investigated drugs, except clindamycin. It appears necessary to create awareness regarding toxigenic C. ulcerans infection in pet owners because this bacterium is believed to be the causative organism for rhinitis in cats.

Laboratory-based surveillance of pertussis using multitarget real-time PCR in Japan: evidence for Bordetella pertussis infection in preteens and teens.

Between January 2013 and December 2014, we conducted laboratory-based surveillance of pertussis using multitarget real-time PCR, which discriminates among Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii and Mycoplasma pneumoniae. Of 355 patients clinically diagnosed with pertussis in Japan, B. pertussis, B. parapertussis and M. pneumoniae were detected in 26% (n = 94), 1.1% (n = 4) and 0.6% (n = 2), respectively, whereas B. holmesii was not detected. It was confirmed that B. parapertussis and M. pneumoniae are also responsible for causing pertussis-like illness. The positive rates for B. pertussis ranged from 16% to 49%, depending on age. Infants aged ≤ 3 months had the highest rate (49%), and children aged 1 to 4 years had the lowest rate (16%, p < 0.01 vs. infants aged ≤ 3 months). Persons aged 10 to 14 and 15 to 19 years also showed high positive rates (29% each); the positive rates were not statistically significant compared with that of infants aged ≤ 3 months (p ≥ 0.06). Our observations indicate that similar to infants, preteens and teens are at high risk of B. pertussis infection.

Distribution of emm genotypes among group A streptococcus isolates from patients with severe invasive streptococcal infections in Japan, 2001-2005.

We surveyed emm genotypes of group A streptococcus (GAS) isolates from patients with severe invasive streptococcal infections during 2001-2005 and compared their prevalence with that of the preceding 5 years. Genotype emm1 remained dominant throughout 2001 to 2005, but the frequency rate of this type decreased compared with the earlier period. Various other emm types have appeared in recent years indicating alterations in the prevalent strains causing severe invasive streptococcal infections. The cover of the new 26-valent GAS vaccine fell from 93.5% for genotypes of isolates from 1996-2000 to 81.8% in 2001-2005.

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.

Properties of the bimodal fluorescent protein produced by Photobacterium phosphoreum.

A fluorescent protein isolated from the deep-sea luminous bacterium Photobacterium phosphoreum strain bmFP has been purified, cloned and sequenced. The protein is 96.5% identical in amino acid sequence to FP390, the weakly fluorescent flavoprotein encoded by the luxF gene characteristic of Photobacterium species. Similar to FP390, bmFP is a dimer of two homologous subunits binding four FMN-myristate chromophores but has the distinctive feature of emitting a bimodal fluorescence with maxima at about 488 and 517 nm, hence the name bmFP. For both bands of this fluorescence, the excitation spectrum exhibits a peak at 336 nm, not corresponding to its flavin-like absorption spectrum. Heating of bmFP in urea resulted in a decrease in the intensity of the 488 nm band along with the appearance of a new fluorescence peaking at 423 nm, partially reversible upon the removal of the urea. Upon complete denaturation, either by heat or guanidium chloride at 65 degrees C, fluorescence characteristic of both free flavin and this 423 nm species appears. It is speculated that chromophores in different states of protonation, associated with a single protein, are responsible for the unusual spectral properties of bmFP.

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.

Mutations in rpoB gene of rifampicin resistant clinical isolates of Mycobacterium tuberculosis in Japan.

In Mycobacterium tuberculosis, involvement of alterations of the RNA polymerase beta subunit in resistance to rifampicin has been described by Telenti et al. To determine if the same correlation could be observed between the mutation of the rpoB gene and clinically isolated M. tuberculosis of the rifampicin-resistant phenotype in Japan, 47 strains of M. tuberculosis of the rifampicin-resistant phenotype, 17 of the rifampicin-susceptible phenotype, and 4 type strains were examined. A 411-base pair (bp) rpoB fragment was amplified by the polymerase chain reaction and subjected to solid phase direct sequencing. By comparing the nucleotides, mutation involving 8 conserved amino acids were identified in 44 of the 47 (93.6%) rifampicin-resistant isolates, but in none of the 17 sensitive isolates and 4 type strains. All mutations found were clustered within a region of 23 amino acids. Thus, similar to the results reported by Telenti et al., substitution of a limited number of highly conserved amino acids encoded by the rpoB gene appears to be the molecular mechanism responsible for resistance to rifampicin in Japanese clinical isolates of M. tuberculosis. Our results suggest that direct DNA sequencing of the rpoB gene may be a reliable method for identifying rifampicin-resistant M. tuberculosis strains among Japanese clinical isolates.

Cloning and nucleotide sequence of type 3 M protein gene (emm3) consisting of an N-terminal variable portion and C-terminal conserved C repeat regions: relation to other genes of Streptococcus pyogenes. Streptococcal Diseases Study Group.

The structural gene for type 3 M protein of Streptococcus pyogenes, which consists of an N-terminal variable portion and C-terminal conserved repeat regions, has been cloned by the polymerase chain reaction (PCR) with two primers (K-1 and K-2). They were selected from the best conserved region of the leader sequences and of the C-terminal portion near the Hexapeptide (LPSTGE) sequence found in different M proteins. From the nucleotide sequence of the product, 1645 base pairs were determined, including 32 amino acids of the leader sequences, the complete N-terminal variable region and the conserved C repeat region. Analysis of the deduced amino acids of the sequence revealed the existence of two major repeat regions, the B and C repeat regions. Comparison of the C-repeat regions among M3 and other M proteins showed them to be more than 90% identical. The two B repeat blocks in M3 protein are also similar to those in M12 protein. Predictive secondary structure analysis of M3 protein reveals a strong alpha-helical potential. The algorithm also shows that the beta-sheet and turn potential for region 23-42 in M3 protein are similar to those for region 28-50 in M12 protein. The results indicate that M3 protein is closely related to M12 protein.

[M-typing of Streptococcus pyogenes by latex agglutination using monoclonal antibody. Streptococcal Diseases Study Group].

The Lancefield's serotyping system of Streptococcus pyogenes is based on the M protein. The typing sera used for this system are prepared in rabbits immunized with whole organisms of specific serotypes. To remove cross reactive antibodies, the sera should be extensively absorbed with organisms of selected heterologous serotypes. In this study, the possibility of using monoclonal antibody (MAb) as a type specific reagent was discussed. MAb which specifically reacted to M type 12 protein of S. pyogenes were produced by cell fusion. This specific MAb reacted to hot acid extracted M type 12 protein in ELISA but didn't react in agglutination and precipitation. Latex beads were sensitized with MAb and examined by the coagglutination method. The latex reagent could detect a very small amount of M type 12 protein, so it could be used for M typing of S. pyogenes which produced a small amount of M 12 protein.

[Treatment of gonococcal urethritis].

Results of the treatment of 5 cases of males with uncomplicated gonoccocal urethritis using levofloxacin (LVFX, DR-3355), the L-type optical isomer of ofloxacin (OFLX), were compared with those treated with OFLX itself. Three hundred mg/day of LVFX or 600 mg/day of OFLX was given to each patient for 5 days. Both drugs showed excellent clinical results in all the patients. When MICs of the 2 drugs were compared against 57 isolated strains of Neisseria gonorrhoeae including 3 penicillinase-producing N. gonorrhoeae, it was found that MICs of LVFX were approximately one half of those of OFLX.

[Serotype and antibiotic susceptibilities of group A hemolytic streptococci isolated in Osaka, 1988-1989].

A total of 386 strains of Group A hemolytic Streptococci isolated from the patients in Osaka in 1988 and 1989 were type-classified by both T-agglutination and M-precipitin methods and were examined for drug-sensitivity. The results were summarized as follows. 1. From T-typing result, T-1 (28.2%) was revealed as the most dominant serotype in 1988, followed by T-4 (24.9%), and T-12 (23.2%), although not as much difference was found in the isolation rate among these three types. A similar tendency was observed in the results of 1989. The order was T-4 (30.3%), T-1 (24.8%), and T-12 (22.1%). 2. In the isolated Group A hemolytic Streptococci, 177 out of 241 strains (73.4%) in 1988 and 126 out of 145 strains (86.9%) in 1989 were M-typable with seven kinds of M-typing sera (Anti-M-1, 3, 4, 6, 12, 13, 18). The result of M-typing was similar to that of T-typing, because the coincidence rate between T and M types was very high among the most prevalent serotypes such as 1, 4, 6, and 12 (type-1: 98.1%, type-4:89.4%, type-6:91.7%, type-12:94.3%). 3. The number of antibiotic-resistant strains decreased. It was especially prominent in the resistant strains to erythromycin, lincomycin and chloramphenicol. While the incidence of tetracycline resistant strains in type-4, 11 and 13 remained at a high level, it decreased in type-12 and 1. All strains were sensitive to the beta-lactam antibiotics. 4. No resistant strains were detected to enoxacin (ENX) and ofloxacin (OFLX), new quinolone. MIC90 values of ENX and OFLX were 16 micrograms/ml and 2 micrograms/ml, respectively, although difference of MIC90 was observed among some strain types: MIC90 of ENX against type-6 strain was 8 times higher than that against type-12 strain.

[Epidemiological survey of group A hemolytic streptococci isolated in Osaka during the 21-year period between 1967 and 1987. Part I. Annual change of epidemic serotype].

During the 21-year period between 1967 and 1987, a total of 6157 strains of group A streptococci isolated in Osaka were type-classified by T-agglutination method. The results were summarized as follows. 1. Sixteen serotypes were differentiated by the T-method during the course of investigation. The most common serotype was T-12 (37.1%), followed by T-4 (13.4%) and T-1 (10.4%). 2. The most dominant serotype was T-12 during 1968-1978, and other relatively frequent serotypes were T-4 in 1971, T-1 in 1972 and T-3 in 1974 throughout the period. Though T-12 was the main serotype in the isolates thereafter, other serotypes such as T-4 in 1979 and T-3 in 1985 also became prevalent, and they occupied the highest percentage. But they declined rapidly, and their duration period was relatively short. After 1976, no definite serotype showed the long-term epidemics, while several types (T-3, T-4 and T-12) showed short-term epidemics lasting a few years. 3. Group A streptococci were isolated most frequently from the patients aged 4-8 years, whereas the isolation rates were extremely lower from the 0- to 2-year-old children. Therefore, younger children seemed to have something which made it difficult to be infected with group A streptococci.

[Epidemiological survey of group A hemolytic streptococci isolated in Osaka during the 21-year period between 1967 and 1987. Part II. Antibiotic susceptibility].

During the 21-year period between 1967 and 1987, a total of 4678 strains of group A hemolytic streptococci isolated in Osaka were examined for drug sensitivity. The results were summarized as follows. 1. All strains were sensitive to the beta-lactam group of antibiotics (benzylpenicillin and cephaloridine). Resistant strains (M.I.C. greater than or equal to 25 micrograms/ml) to tetracycline (TC), chloramphenicol (CP) and erythromycin (EM) were isolated in this study period. The incidence of TC-resistant strains in the whole isolates has not much varied through the whole period, in spite of the variation among several T-type. But those of CP- and EM-resistant strains varied greatly by year. 2. Before 1971, a small number of resistant strain was found in 1969 and 1970. Since 1972, increase of such strains had been seen and the incidence of CP-resistant strains reached the highest peak (32.5%). It showed a gradual decrease since 1976 and was 1.1% in 1987. 3. None of EM-resistant strains was isolated before 1972. The incidence of EM-resistant strains exhibited a steep rise in percent since 1973 and reached the highest value showing a two-peak-pattern in 1975 (36.1%) and in 1980 (36.0%). After reaching its peak it decreased gradually and was 2.9% in 1987. 4. Remarkable increase of the multiple drug resistant strains began to be observed since 1972 to 1973, and mainly T-12 and T-4 showed EM. TC. CP- and TC. CP-resistance, respectively. The phenomenon of multiple drug-resistance culminated during 1975-1977, followed by gradual decrease thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunopharmacological activities of a synthetic counterpart of a biosynthetic lipid A precursor molecule and of its analogs.

Synthetic lipid A analogs (compounds 404 through 406) were examined for their immunopharmacological activities. These compounds had two amide-bound and two ester-bound (R)-3-hydroxytetradecanoyl groups at the C-2 and C-2' and the C-3 and C-3' positions, respectively, of beta (1-3)glucosamine disaccharide. In all of the in vitro assays, these synthetic compounds exhibited high activities comparable to those of a reference lipid A prepared from Escherichia coli O8:K27 Re-mutant strain F515. The compounds activated the clotting enzyme cascade of the horseshoe crab, activated the human complement via the classical pathway, caused polyclonal B-cell activation, stimulated the phagocytosis of sheep erythrocytes by murine peritoneal macrophages, and enhanced the migration of human polymorphonuclear leukocytes. They also increased the thymidine uptake of splenocytes of BALB/c nu/nu and C3H/HeN mice but not those of C3H/HeJ (a nonresponder to lipopolysaccharide). A dephosphorylated derivative, compound 403, was barely active in all of the above assays except for the enhancement of polymorphonuclear leukocyte migration. However, compounds 404 through 406 were feeble in pyrogenicity and could not prepare the local Shwartzman reaction, although they were very lethal to galactosamine-loaded mice. Therefore, synthetic lipid A analogs described here were fully immunopharmacologically active in in vitro assays, but all of them were far less active than natural E. coli F515 lipid A regarding the biological activities characteristic of endotoxic lipopolysaccharides and lipid A's. The high lethal toxicity of compound 406 (1,4'-bisphosphate) to the galactosamine-loaded mice may not reflect its real toxicity to normal mice. In all activities examined, compound 406 was quite comparable to a biosynthetic lipid A precursor, a natural counterpart of compound 406. The immunopharmacological activities of these newly synthesized lipid A analogs, especially compound 406, were much stronger than those of compounds that had been synthesized earlier by using the originally proposed model of the lipid A structure. The findings described in this report justify the acylation pattern of a disaccharide backbone of lipid A, revised on the basis of recent analytical studies. The low in vivo endotoxic activities of the present lipid A analogs are most probably due to the fact that the kinds of acyl groups were different from those of the complete lipid A from E. coli, although there were no differences in the acylation positions on the disaccharide backbone.