Proposal to transfer Actinomadura carminata to a new subspecies of the genus Nonomuraea as Nonomuraea roseoviolacea subsp. carminata comb. nov.

An anthracycline-producing actinomycete (strain SF2303) was compared with two other anthracycline producers, Actinomadura carminata IFO 15903T and Nonomuraea roseoviolacea IFO 14098T, using morphological, physiological, chemotaxonomic and molecular-genetic criteria. The morphological and cultural characteristics of these three strains are similar. The physiological properties of strain SF2303 and N. roseoviolacea IFO 14098T are very similar, but are different from those of A. carminata IFO 15903T in the utilization of some sugars and the reduction of nitrate. Their chemotaxonomic properties [cell wall chemotype, IIIB; major menaquinone, MK-9 (III, VIIl-H4); phospholipid type, PIV; cellular fatty acids 10M-17:0/17:1 and iso-16:0 as major components and 2-hydroxy fatty acids as minor components; mycolic acid, absent] were identical and indicated that these three strains belong to the family Streptosporangiaceae. On the basis of 16S rDNA sequences and phylogenetic analysis, they were all included in the cluster formed by species of Nonomuraea. The levels of DNA relatedness between strain SF2303 and N. roseoviolacea IFO 14098T ranged from 71 to 78%; however, the levels of relatedness between the two strains and A. carminata IFO 15903T were lower (49-60%). Therefore, strain SF2303 was identified as a member of the species N. roseoviolacea and it is proposed that Actinomadura carminata Gauze et al. 1973 should be transferred to a new subspecies of the genus Nonomuraea Zhang et al. 1998 as N. roseoviolacea subsp. carminata comb. nov.

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

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.

[Typing of group A streptococci by pulsed field gel electrophoresis].

Restriction endonuclease (Sma I) digestion patterns of chromosomal DNAs of T types 1 and 3 (T1 and T3) group A streptococci (GAS), isolated at two hospitals in Toyama Prefecture from 1983 to 1994, were analysed by pulsed-filed gel electrophoresis (PFGE). One hundred twenty seven isolated of T1 GAS were further divided into 5 PFGE types, which were provisionally designated as T1-1 approximately T1-5. Two epidemics of T1 GAS were observed during the 1983 approximately 1994 period, the first of which (1887 approximately 1988) seemed to be caused by T1-2, and the second (1991 approximately 1993) by T1-5. Some differences in biological characteristics between the two PFGE type strains were also observed. On the other hand, eighty one T3 GAS strains were divided into only two PFGE types, provisionally designated as T3-1 and T3-2, most of which belonged to T3-2. Naturally, both epidemics by T3 GAS, first from 1985 to 1986 and second from 1993 to 1994, seemed to be caused by the same PFGE type, T3-2. No differences in biological characteristics were observed between T3-1 and T3-2 strains.

[Molecular epidemiological study on Vibrio cholerae O139].

Genomic DNA from 56 Vibrio cholerae O139 strains isolated in various countries was digested with Sfi I or Not I and analyzed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE patterns were identified. Although the patterns of a large majority of CT-gene-positive epidemic strains isolated in India, Bangladesh and Thailand were the same or similar, but were slightly different from those of two CT-positive strains from India and Nepal. On the other hand, the patterns of CT-negative three strains from Argentine, Sri Lanka and Bangladesh were apparently different not only from each other, but also from those of epidemic CT-positive strains. The pattern of one V. cholerae O1 E1 Tor strain isolated in India and the pattern of epidemic V. cholerae O139 resemble each other in many points.

NAD(+)-glycohydrolase productivity of haemolytic streptococci assayed by a simple fluorescent method and its relation to T serotype.

The ability of haemolytic streptococci to produce NAD(+)-glycohydrolase was investigated by a fluorescent assay. Enzyme production was found in 31 (91%) of 34 group A, 17 (61%) of 28 group C and eight (27%) of 30 group G isolates. The high producers were found in 22 (65%) of group A, one (4%) of group C and none of group G isolates. The high producers of the group A isolates belonged to T-1, T-3, T-4 or T-12 serotype. These results suggest that NAD(+)-glycohydrolase productivity of streptococci is closely related to specific Lancefield's groups or T serotypes.

[Typing of group B streptococci by pulsed-field gel electrophoresis].

Restriction endonuclease (Sma I) digestion patterns of chromosomal DNAs from 459 group B streptococci (GBS) isolated in two hospitals (C and S) in Toyama Prefecture were analysed by pulsed-field gel electrophoresis (PFGE). Results were summarized as follows. 1) One hundred thirty eight isolates of GBS serotype Ia, 174 of serotype III, 102 of serotype JM-9 and 45 of serotype NT-6 were further divided into 56, 41, 36 and 19 PFGE types, respectively. 2) Appreciable differences in the distribution of PFGE types were not observed between C and S hospital isolates, nor among specimens from which GBS strains were isolated. 3) Long-term inspection of 5 patients, from whom the same serotype strains were repeatedly isolated, revealed that some changes in PFGE types were observed in 2 patients, but not in the other 3.

[Enteropathogenicity and enteropathogenic toxin production of Vibrio mimicus].

Enteropathogenicity and enteroreactive-toxins were examined in 66 strains of Vibrio mimicus and the following results were obtained. Frequencies of enteropathogenic strains judged by the result of suckling mouse tests were 11/13 (85%) for clinical isolates and 37/53 (70%) for fish or environmental isolates. Frequencies of preservation of cholera toxin gene and NAG-ST gene were 2 and 15%, respectively, for 48 enteropathogenic strains, and 0 and 6%, respectively, for 18 non-enteropathogenic strains. Frequencies of production of NAG-rTDH, FAF and hemolysin were 4, 63 and 100%, respectively, for 48 enteropathogenic strains, and 6, 50, and 100%, respectively, for 18 non-enteropathogenic strains. No correlation between serovar and enteropathogenicity was observed in the suckling mouse test. Six out of 12 enteropathogenic strains produced hemolysin in ligated rabbit ileal loop, while 1 out of 12 non-enteropathogenic strains did so under the same condition. A significant inhibition of fluid accumulation in the ligated rabbit ileal loop test with viable cells was noted in rabbits immunized with hemolysin of non-O1 V. cholerae. These results suggest that approximately two-thirds of environmental isolates are enteropathogenic and that hemolysin is the most important toxin in the enteropathogenic mechanism of V. mimicus strains.

[Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. IV. Role of hemolysin].

The role of hemolysin in the enteropathogenic mechanism of non-O1 V. cholerae was experimentally investigated, in vitro and in vivo. Results are summarized as follows. 1). A greater majority of enteropathogenic strains produced hemolysin in Eagle MEM medium supplemented with 10% calf serum and in the rabbit ileal loop, while most non-enteropathogenic strains did not under the same conditions. 2). Non-enteropathogenic mutants derived from enteropathogenic parent strains produced much less hemolysin than that of parent strains. 3). A significant inhibition of the fluid accumulation in the ligated rabbit ileal loop test with viable cells was noted in rabbit immunized with purified hemolysin. These results indicate that hemolysin is the most important toxin in the enteropathogenic mechanism of non-O1 V. cholerae.

[Comparison between the aged and children of the normal distribution of pharyngeal and intestinal hemolytic streptococci].

A marked difference between the aged and children has been observed in the group distribution of hemolytic streptococci isolated from various clinical specimens. Group B strains from the urine and sputum, and group G from the sputum, pus and exudate have been predominant in the aged, whereas group A strains from the throat swab have dominated in children. The present study was undertaken to clarify the background for such a marked difference by investigating the normal state of pharyngeal and intestinal carriage of hemolytic streptococci both in the aged and children. 1. As to the pharyngeal carriage, quite a contrast was observed between the aged and children. In the former, the rate of carries was low and the predominant groups among the streptococcus isolates were B (Streptococcus agalactiae) and G (identified as Streptococcus equisimilis), while in the latter, the rate was high and group A (Streptococcus pyogenes) strains comprised approximately 75% of the isolates, most of them being from the throat swab. 2. Both the aged and children showed a similar state of streptococcus carriage in the intestine. The rate of carriers was low and the predominant group among the isolates was B in both populations. Although group G strains were occasionally isolated, group A strains were isolated neither in the aged nor in children. These results explain well the difference between the aged and children in the group distribution of the clinical isolates of hemolytic streptococci.

[Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. III. Production of enteroreactive toxins].

Cholera toxin gene and production of enteroreactive toxins were examined in 134 strains of non-O1 V. cholerae. Results obtained were summarized as follows. Frequencies of cholera-toxin-gene-positive strains were 2/58 (3.4%) from human sources and 2/76 (2.6%) from fish and environment. While, frequencies of production of hemolysin, fluid accumulating factor (FAF) related with protease, fluid accumulating factor in the suckling mouse, NAG-rTDH, NAG-ST and Vero toxin were 100, 72, 31, 2, 0 and 0%, respectively, for 58 strains from human sources, and 100, 57, 24, 0, 1.3 and 0%, respectively, for 76 strains from fish and environment. Among the 31 strains used for the injection of viable cells to the ligated rabbit ileal loop, detection frequencies of these enteroreactive toxins in the accumulated fluids were 100% for hemolysin, 3.2% for both FAF and NAG-rTDH and 0% for cholera toxin, Vero toxin or NAG-ST. Hemolysin and the fluid accumulating factor in the suckling mouse seemed to be identical in most strains. These results suggest that cholera toxin, NAG-ST, NAG-rTDH and Vero toxin may not be very important in the enteropathogenic mechanism of a great majority of non-O1 V. cholerae strains, whereas hemolysin may play an important role in the enteropathogenicity.

[Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. II. Lethality, adhesion, colonization and cytopathogenicity of enteropathogenic strains].

Lethality, adhesion, colonization, hemagglutinable activity, invasiveness and cytopathogenicity of non-O1 V. cholerae were compared between enteropathogenic and non-enteropathogenic strains. The following results were obtained. 1) Minimum lethal doses (MLD) of enteropathogenic strains were significantly lower than those of non-enteropathogenic strains. 2) There were no differences in adhesive and hemagglutinating activities between enteropathogenic and non-enteropathogenic strains. 3) A greater majority of enteropathogenic strains showed cytopathogenic effect on HEp 2 cells, but non-enteropathogenic strains did not. 4) Regardless of enteropathogenicity of viable cells, none of the 13 strains examined were found to be invasive to HEp 2 cells. These results suggest that adhesion and colonization do not draw a clear distinction between enteropathogenic and non-enteropathogenic strains, and that both lethal and cytopathogenic activities of these organisms are correlated with enteropathogenicity.

[Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. I. Enteropathogenicity of clinical and environmental isolates].

Enteropathogenicity and plasmid DNA of clinical and environmental isolates of non-O1 V. cholerae were examined. Results were as follows: 1). The frequencies of enteropathogenic strains judged by the results from both ligated rabbit ileal loop (RIL) and suckling mouse tests were 36/38 (95%) for isolates from overseas travellers, 15/15 (100%) for isolates from food poisoning, 33/44 (75%) for isolates from fish and sea water, and 1/10 (10%) for isolates from river water. 2). Plasmid DNA was detected in eight of the 40 isolates examined, but the presence of plasmid did not correlate with enteropathogenicity. These results indicate that approximately three fourths of the strains isolated from fish and sea water are enteropathogenic, and that the genes controlling the enteropathogenicity of this organism probably exist in chromosomal DNA.

[Surveys on the contamination of marine fish with non-O1 Vibrio cholerae and Vibrio mimicus and food poisoning cases by these organisms].

The present paper describes the relationship between the contamination with non-O1 Vibrio cholerae and Vibrio mimicus of marine fish, with special reference to the seasonal variation and the concentration of contamination, and the actual cases of domestic food poisoning by these organisms. A 10 year survey revealed that non-O1 Vibrio cholerae (non-O1 V. cholerae) strains were frequently isolated from fish during the summer season with some variations from one year to another, and isolates from fish showed similar biological properties to those of isolates from diarrhea cases of over-sea travellers. Experimentally enteropathogenic strains were included among these isolates. Vibrio mimicus (V. mimicus) strains were also isolated from fish, the frequency being not so high as in the case of non-O1 V. cholerae Strains of serovar O-41 which was most predominant among strains from diarrhea cases were also detected among the isolates from fish. The viable cell counts, however, were very small with regard to both non-O1 V. cholerae and V. mimicus From these observations, factors causing food poisoning by non-O1 V. cholerae or V. mimicus seemed to be essentially similar to those by Vibrio parahaemolyticus (V. parahaemolyticus); that is, the food poisoning by non-O1 V. cholerae or V. mimicus is apt to occur in the summer season and is caused by the consumption of raw fish, although the frequency might be significantly low in comparison to that of V. parahaemolyticus. The actual cases of the domestic food poisoning by non-O1 V. cholerae or V. mimicus were retrospectively surveyed by the literature.(ABSTRACT TRUNCATED AT 250 WORDS)

[Ecological studies on hemolytic streptococci in aged population].

The pattern of group distribution of hemolytic streptococci and the source of specimen, when these organisms were isolated from the clinical specimen of the aged, were quite different from that of streptococcosis among children. The isolation frequency from the upper respiratory tract was rather low in comparison to the other specimen, such as sputum, exudate and urine. Both groups B and G were predominant among isolates from sputum, B more prevailing than G from exudate, and the isolates from the urinary tract were consisted almost solely of group B. These trends have been unchanged in the past 3 years. Normal pharyngeal streptococcus flora among the aged also differ from that of healthy children, the carrier rate being rather low, and groups B and G, instead of A in the case of children, being predominant. This pattern of group distribution, among the isolates of normal pharyngeal streptococci of the aged, seemed to be reflected on that of isolates from clinical specimen, especially those from the sputum. The type distribution of group B isolates from normal throat swabs of the aged resemble that of clinical isolates from the same generation, types Ia and III dominating in both cases.

Production and partial purification of a fluid-accumulating factor of non-O1 Vibrio cholerae.

A fluid-accumulating factor (FAF in the ligated rabbit ileal loop test) from a strain of non-O1 Vibrio cholerae not producing cholera toxin-like enterotoxin (CTLT) was partially purified by ammonium sulfate precipitation, gel filtration with Sephadex G-100, and DEAE cellulose column chromatography. The preparation thus obtained showed collagenolytic, cytolytic, necrotic, and hemorrhagic activities, but was not lethal to mice nor hemolytic to sheep erythrocytes. Desquamation of epithelial cells, inflammatory edema, and hemorrhage were observed in sections of rabbit intestine after inoculation of partially purified FAF (PPFAF). Biological and enzymatic activities of FAF were completely neutralized with anti-PPFAF rabbit serum. More than 70% of non-O1 V. cholerae strains from human diarrheal feces produced FAF in the shake culture of heart infusion broth (Difco). A fluid-accumulating factor immunologically similar to FAF of non-O1 V. cholerae was also produced by V. mimicus strains isolated from human diarrheal feces. These results indicate that the FAF produced by CTLT-negative non-O1 V. cholerae strains is an entity closely related to a cytolytic and hemorrhagic substance or the like, and that this FAF may play a role in the enteropathogenicity of CTLT-negative strains.

Response of ligated rabbit ileal loop to Clostridium perfringens type C strains and their toxic filtrates.

The vegetative cells and toxic filtrates of Clostridium perfringens type C strains were injected into ligated rabbit ileal loops and the responses were observed. Out of 12 strains examined, 2 strains showed positive reaction in this test, when the vegetative cells were injected. One of these 2 strains was an enterotoxigenic and beta-toxigenic and the other was beta- and delta-toxigenic but not enterotoxigenic. Culture filtrates containing beta or delta toxin also showed fluid accumulation in the rabbit ileal loop. Histological findings of loops injected with culture filtrates containing beta toxin showed separated and effaced villi, hemorrhage in the mucosa, engorged vessels, inflammatory cell infiltration, and hyperplasia of the lymphoid tissue.