Legionnaires' disease associated with habitual drinking of hot spring water.

A 57-year-old man presented with pneumonia, respiratory distress, and myelodysplastic syndrome. A diagnosis of Legionnaires' disease due to Legionella pneumophila (L. pneumophila) was established. The patient had long been drinking tap water via a conduit from a hot spring resource, from which L. pneumophila was also isolated. Both the patient's strain and the water strain of L. pneumophila were identified as serogroup 1, and the genetic relatedness between the two strains as seen by pulsed-field gel electrophoresis was 87%. The patient was successfully treated with erythromycin, fluoroquinolone, and rifampicin. This case raises an important issue on public health represented by legionellosis in Japan.

[A survival case of severe Legionella longbeachae pneumonia].

A 56-year-old Japanese male was admitted to Toyohashi Municipal Hospital because of fever, cough, and dyspnea. Chest X-ray film showed bilateral alveolar infiltrates. He suffered from severe hypoxemia and was given a diagnosis of acute respiratory distress syndrome. He was also complicated with disseminated intravascular coagulation and pseudomembranous colitis. He fully recovered by intensive treatment with antibiotics, mechanical ventilation and endotoxin eliminating therapy. Legionella longbeachae was isolated from his respiratory specimens and was regarded as the etiologic agent of his pneumonia.

Proposal of Sphingomonas wittichii sp. nov. for strain RW1T, known as a dibenzo-p-dioxin metabolizer.

A polyphasic taxonomic study was performed on Sphingomonas sp. strain RW1T. The organism was isolated from water of the River Elbe and has been known as a potent metabolizer of dibenzo-p-dioxin and its relatives. TLC of a mild alkaline hydrolysate of extractable cellular lipids of strain RW1T and type strains of 21 Sphingomonas species gave a spot of sphingoglycolipid (SGL)-1 (glucuronosyl ceramide), which is characteristic of sphingomonads. In addition, strain RW1T and type strains of three Sphingomonas species (Sphingomonas yanoikuyae, Sphingomonas terrae and Sphingomonas macrogoltabidus) showed a second spot of SGL (SGL-1') identified as galacturonosyl ceramide. The presence of SGL-1 in cellular lipids suggested that strain RW1T is a member of the genus Sphingomonas. DNA-DNA reassociation rates between strain RW1T and each type strain of 14 Sphingomonas species including Sphingomonas paucimobilis, type species for the genus, revealed that strain RW1T is independent from these species. Results of phylogenetic analysis of 16S rDNA sequences of strain RW1T and type strains of 21 named Sphingomonas species verified that strain RW1T belongs to the genus Sphingomonas. Strain RW1T could be differentiated from named species of the genus by phenotypic characteristics and has been assigned to a new species, Sphingomonas wittichii sp. nov. The type strain is DSM 6014T (= JCM 10273T = EY 4224T). DNA G+C content is 67 mol %.

Proposal of Sphingomonadaceae fam. nov., consisting of Sphingomonas Yabuuchi et al. 1990, Erythrobacter Shiba and Shimidu 1982, Erythromicrobium Yurkov et al. 1994, Porphyrobacter Fuerst et al. 1993, Zymomonas Kluyver and van Niel 1936, and Sandaracinobacter Yurkov et al. 1997, with the type genus Sphingomonas Yabuuchi et al. 1990.

Based on the results of phylogenetic analysis of the 16S rDNA sequences and the presence of N-2'-hydroxymyristoyl dihydrosphingosine 1-glucuronic acid (SGL-1) and 2-hydroxymyristic acid (non-hydroxymyristic acid in Zymomonas) in cellular lipids, a new family, Sphingomonadaceae, for Group 4 of the alpha-subclass of the class Proteobacteria is herein proposed and a description of the family is given. The family consists of six genera, Sphingomonas, Erythrobacter, Erythromicrobium, Porphyrobacter, Sandaracinobacter and Zymomonas. Thus, all the validly published and currently known genera in Group 4 of the alpha-subclass of Proteobacteria belong to Sphingomonadaceae fam. nov. Among them, type strains of two species, Porphyrobacter and Erythrobacter, Sandaracinobacter sibiricus and Sphingomonas ursincola, respectively, are facultatively photosynthetic due to bacteriochlorophyll (Bchl)-a. The type strains of two subspecies of Zymomonas mobilis are facultative anaerobes. The presence of SGL-1 together with the results of a phylogenetic analysis of the 16S rDNA sequences recommends a new criteria by which to define the new family Sphingomonadaceae. The type genus is Sphingomonas Yabuuchi et al. 1990.

Arguments against the replacement of type species of the genus Salmonella from Salmonella choleraesuis to 'Salmonella enterica' and the creation of the term 'neotype species', and for conservation of Salmonella choleraesuis.

The proposals of Le Minor and Popoff in 1987 and again of Euzéby in 1999 on the type species of the genus Salmonella are in violation of Rule 20a of the Bacteriological Code (1990 Revision) and should be rejected. The introduction of the term 'neotype species' should be rejected. The specific epithet choleraesuis in the binary combination Salmonella choleraesuis should be conserved. The serovar name Choleraesuis should be changed to Hogcholera.

Burkholderia uboniae sp. nov., L-arabinose-assimilating but different from Burkholderia thailandensis and Burkholderia vietnamiensis.

A polar multitrichous gram-negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA-DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, bio-chemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA-DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone-8(Q8). G+C content of DNA is 69.71%.

Proposal of Salmonella paratyphi sp. nov., nom. rev. and request for an opinion to conserve the specific epithet paratyphi in the binary combination Salmonella paratyphi as nomen epitheton conservandum.

We propose Salmonella paratyphi sp. nov., nom. rev., by raising Salmonella choleraesuis subsp. choleraesuis serovar Paratyphi A to species status and request an Opinion to include the specific epithet paratyphi in the binary combination of Salmonella paratyphi in the list of epitheta specifica conservanda.

Recognition of nomenclatural standing of Salmonella typhi (Approved Lists 1980), Salmonella enteritidis (Approved Lists 1980) and Salmonella typhimurium (Approved Lists 1980), and conservation of the specific epithets enteritidis and typhimurium. Request for an opinion.

In 1994, the Judicial Commission of the ICSB announced that Le Minor and Popoff's Request for an Opinion in 1987 to designate Salmonella enterica sp. nov., nom. rev. as the type and only species of the genus Salmonella was denied. Thus, the current species of the genus Salmonella are Salmonella typhimurium, Salmonella enteritidis, Salmonella typhi, Salmonella choleraesuis (including six subspecies) and Salmonella bongori, with the type species, S. choleraesuis (Smith 1894) Weldin 1927 (Approved Lists 1980). Because the decision of the Judicial Commission about the request by Le Minor in 1987 was suspended for 7 years, the non-validated name 'S. enterica' has been used among microbiologists and has caused confusion in the nomenclature of Salmonella. In order to overcome such confusion, and because of their importance as human pathogens, we herein propose to recognize the nomenclatural status of S. typhi, S. enteritidis and S. typhimurium as species and request to issue an Opinion to conserve the specific epithets enteritidis and typhimurium in the species names Salmonella enteritidis and Salmonella typhimurium.

A novel sphingoglycolipid containing galacturonic acid and 2-hydroxy fatty acid in cellular lipids of Sphingomonas yanoikuyae.

A novel sphingoglycolipid was isolated from Sphingomonas yanoikuyae, and its structure was identified as a galacturonosyl-beta (1-->1)-ceramide. This was a characteristic sphingoglycolipid present in S. yanoikuyae and certain other species of Sphingomonas, such as Sphingomonas mali, Sphingomonas terrae, and Sphingomonas macrogoltabidus, but not in the type species of Sphingomonas, Sphingomonas paucimobilis.

[Evaluation of urinary antigen detection methods for rapid diagnosis of Legionella pneumonia].

We have evaluated urine specimens of presumptive cases of legionnaires' disease (110 cases, 173 sample), collected in the past eight years (April, 1990-August, 1998) with the Binax EIA kit which detects the soluble antigen of Legionella pneumophila serogroup (SG) 1, and the Biotest EIA kit which detects Legionella species. Seven cases (19 specimens) were positive for the Binax EIA kit, and nine cases (22 specimens) were positive for the Biotest EIA kit. The sensitivity for culture, PCR, IFA method were 100%, 100%, and 50%, the specificity for these method were 93%, 97.1%, and 90% respectively. Overall agreements for these method were 93.5%, 97.4%, 86.8%, these results suggested that the urinary antigen detection test had high sensitivity and specificity. Our study indicated that concentrated urine samples increase sensitivity. We also evaluated the capabilities of both EIAs to detect soluble antigens were extracted from bacterial suspension of 18 strains of 5 Legionella species by heating. Both assays detected L. pneumophila serogroups 1 to 14, L. bozemanii. The Binax EIA proved to be useful as the Biotest EIA for diagnosis of legionellosis caused by Legionella species and serogroups other than L. pneumophila serogroup 1. Some cases have been shown to excrete antigen for prolonged period of times despite recovery from infection, so that the patient's history should be sought. The urine antigen detection EIA methods proved to be rapid and easy to use, detect antigen in the early stage of the disease with high sensitivity and specificity. Its use for the definition of legionellosis should be considered in Japan.

Proposal of Sphingomonas suberifaciens (van Bruggen, Jochimsen and Brown 1990) comb. nov., sphingomonas natatoria (Sly 1985) comb. nov., Sphingomonas ursincola (Yurkov et al. 1997) comb. nov., and emendation of the genus Sphingomonas.

Based on the results of a phylogenetic analysis of 16S rRNA and the presence of sphingoglycolipid in cellular lipids of the type strains, transfer of "Rhizomonas" suberifaciens, Blastomonas natatoria and Erythromonas ursincola to the genus Sphingomonas as Sphingomonas suberifaciens (van Bruggen et al 1990) comb. nov., Sphingomonas natatoria (Sly 1985) comb. nov., and Sphingomonas ursincola (Yurkov et al 1997) comb. nov. are herein proposed together with the emendation of genus Sphingomonas. The type strain of S. suberifaciens is van Bruggen Cal=ATCC 49382=NCPPB 3629=IFO 15211=JCM 8521, that of S. natatoria is ATCC 35951 =DSM 3183=NCIMB 12085=JCM10396, and that of S. ursincola is DSM 9006= KR-99.

Present situation of serotyping of Pseudomonas aeruginosa in Japan and correlation among three kinds of commercially available serotyping kits.

We carried out the serotyping of 196 freshly isolated strains of Pseudomonas aeruginosa with international antigenic typing system (IATS) antibodies from Rougier Bio-Tech (RBT kit) and other two kinds of antibodies, from Meiji Seika Kaisha (Mei assay kit) and Denka Seiken (Seiken kit) to ascertain the relationship among the serotypes obtained by the different methods. All the test strains belonging to the serogroups A, C, E, F, G, H, I, J, and K were well correlated with each other with the Mei assay and the Seiken kits. However, 21 of 26 strains of serogroup M typed with the Mei assay kit were non-typable with the Seiken kit. It was confirmed that serogroups obtained with both these methods were closely related; 171 of the 196 strains of P. aeruginosa tested (87.2%) were classified into identical serogroups. From the serotyping results for P. aeruginosa strains with the Seiken and the RBT kits and with the Mei assay and the RBT kits, it was found that the serotypes of 172 of the 192 strains tested (89.6%) and those of 164 of the 170 strains tested (96.5%), respectively were well correlated. However, the serotyping of P. aeruginosa with the RBT kit may be problematic, as the kit did not contain the antisera agglutinated with the serogroup M strains of P. aeruginosa classified with the Mei assay and Seiken kits.

[Isolation of Legionella and free-living amoebae at hot spring spas in Kanagawa, Japan].

Microbiological contamination of hot spring bath water is a public health concern. A province-wide survey was carried out to determine the extent and distribution of both Legionella and free-living amoebae contamination. Among 30 samples of hot spring bath from 12 sites in Kanagawa, Japan, L. pneumophila was detected in 21 water samples from 11 sites, ranging from 10(1)-10(3) CFU/100 ml. Serogroups 3, 5 and 6 of L. pneumophila were predominantly isolated from the samples. Naegleria (46.7%), Platyamoeba (33.3%), Acanthamoeba (10.0%) and 2 other genera of free-living amoebae were detected in 22 samples from 11 sites. One or more genera of host amoebae of Legionella occurred in 17 samples (56.7%) from 9 sites. Another thing to be noted is that 13 water samples contained N. lovaniensis. Although N. lovaniensis is nonpathogenic, it is considered an indicator organism for places that are suitable for the growth of N. fowleri, a causative agent of primary amoebic meningoencephalitis in man.

Emendation of genus Achromobacter and Achromobacter xylosoxidans (Yabuuchi and Yano) and proposal of Achromobacter ruhlandii (Packer and Vishniac) comb. nov., Achromobacter piechaudii (Kiredjian et al.) comb. nov., and Achromobacter xylosoxidans subsp. denitrificans (Rüger and Tan) comb. nov.

Based on the results of GC content determination and 16S rRNA sequence analysis among the type strains of Achromobacter xylosoxidans, 4 Alcaligenes species, 5 Bordetella species, and 12 species of 4 other genera, the separation of genus Achromobacter Yabuuchi and Yano 1981, with the type species Achromobacter xylosoxidans, is confirmed. Alcaligenes ruhlandii (Packer and Vishniac) Aragno and Schlegel 1992 is a distinct species and not a senior synonym of Achromobacter xylosoxidans. Alcaligenes ruhlandii and Alcaligenes piechaudii Kiredjian et al 1986 are transferred to genus Achromobacter. Thus 2 new combinations, Achromobacter ruhlandii (Packer and Vishniac) and Achromobacter piechaudii (Kiredjian et al) are proposed; their type strains are ATCC 15749 and ATCC 43552, respectively. Alcaligenes denitrificans Rüger and Tan 1983 is also transferred to genus Achromobacter and ranked down to the subspecies of Achromobacter xylosoxidans. Thus a new subspecies name, Achromobacter xylosoxidans subsp. denitrificans (Rüger and Tan) is proposed. The type strain of the subspecies is ATCC 15173. This proposal automatically creates type subspecies, Achromobacter xylosoxidans subsp. xylosoxidans, with type strain ATCC 27061. An emended description of genus Achromobacter and of type species Achromobacter xylosoxidans are given.

[Determination of cutoff value of serum anti-Legionella antibody titer--microplate agglutination test (MPAT)].

In order to promote the serological diagnosis of legionellosis in the clinical laboratory, the cutoff values of serum anti-Legionella antibody titers for microplate agglutination (MPAT) test were determined. Antibody levels were tested for 178 serum specimens including 98 healthy persons, 22 ordinally workers having either metabolic or renal failure, definitely diagnosed patients of 17 mycoplasmal and 9 chlamydial pneumonia, 32 patients of other bacterial pneumonia. Heat killed unstained cell suspension of each strain of Legionella pneumophila serogroup (SG) 1a, 1b, SGs 2 to 6, L. bozemanii, L. dumoffii, L. gormanii, and L. micdadei were used as antigens. Strains of L. pneumophila SG 1b were mainly isolated from environmental specimens. However, in some Legionella pneumonia cases, etiologic agents were determined as L. pneumophila SG 1 b. Thus the representative strain of SG 1b was used as an antigen for the determination of the patient's antibody titer. Quantitative agglutination was performed by using a 96-well U-bottom microplate for each antigen. Test sera were diluted from 1:16 to 1:256. Results were read after 20 h at 25 degrees C. Cutoff values for 11 antigens were determined, at this moment, as 4-fold or greater increase in level to > or = 1:128 in paired sera, and > or = 1:256 in single serum. However, final diagnosis should be given by over-all coordination of serological results and clinical symptoms together with other laboratory findings. Two culture-positive Legionella pneumonia cases due to either SG3 or 6 in which significant rise of serum antibody titers against organisms of corresponding SG estimated by MPAT method were discussed.

Molecular determination of infection source of a sporadic Legionella pneumonia case associated with a hot spring bath.

To determine the infection source of a sporadic Legionella pneumonia case associated with a hot spring bath, we used five molecular methods, including repetitive element polymerase chain reaction (rep-PCR), arbitrarily primed PCR (AP-PCR), ribotyping, restriction endonuclease analysis (REA), and macrorestriction endonuclease analysis (MREA) by pulsed-field gel electrophoresis. L. pneumophila serogroup (SG) 3 strain EY 3702, isolated from an intratracheal specimen of a 71-year-old Japanese female who developed pneumonia after nearly drowning in a hot spring spa bath, produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 3 strains EY 3768 and EY 3769 isolated from the bath water. Four epidemiologically unrelated L. pneumophila SG 3 strains showed different rep-PCR or AP-PCR fingerprints from those of the three EY strains (EY 3702, 3768, and 3769). The three EY strains were also genotypically indistinguishable by ribotyping with EcoRI and PstI, by REA with EcoRI or HindIII, and by MREA with NotI. Based on these results, we identified the bath water of the hot spring spa as the source of infection of this patient, even though the viable number of the organisms in the bath water was low (3 CFU/100 ml) when determined 27 days after her nearly drowning.

[Legionella pneumophila serogroup 3 isolated from a patient of pneumonia developed after drowning in bathtub of a hot spring spa].

A 71-year-old Japanese female, was found unconscious by drawing, in a hot spring spa, at around noon of 20 October 1994. She recovered by emergency cardiopulmonary resuscitation, and admitted to the Takinomiya General Hospital, with adult respiratory distress syndrome (ARDS). Although she recovered from ARDS within 4 days after her admission, she developed severe pneumonia accompanied with the second attack of ARDS. Ordinary bacteriological culture of her respiratory specimens failed to yield any significant pathogen for her pneumonia, and neither cefazolin nor imipenem/cilastatin was effective. Thus minocyclin was given on the 7th hospital-day and this was effective for blood gas and C-reactive protein (CRP) levels. Intratracheal exsudate inoculated on BCYE alpha agar plate yielded grayish white colonies. Cells of the colonies were clearly agglutinated by anti-Legionella pneumophila serogroup (SG) 3 serum. Antibody titers of patient's paired sera against the strain L. pneumophila SG3 Bloomington-2 and the patient's strain (Y-1) were determined by microplate agglutination test, and a significant rise from 1:20 to 1:320 was demonstrated. Patient recovered by erythromycin treatment and was discharged on the 59th hospital day. L. pneumophila SG3 organisms were again isolated from the spa water where the patient drawn. From these findings described above, we diagnosed the patient as pneumonia due to L. pneumophila SG3, and the spa water was the most probable source of infection.