Aeromonas属の分類学的研究 : 河川土壌・河川水および淡水魚由来運動性Aeromonas属の数値分類およびファージ型別について A taxonomic study on genus Aeromonas : numerical classification and bacteriophage typing of motile Aeromonas isolated from river mud, river water and fresh-water fishes

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著者

    • 福山, 正文 フクヤマ, マサブミ

書誌事項

タイトル

Aeromonas属の分類学的研究 : 河川土壌・河川水および淡水魚由来運動性Aeromonas属の数値分類およびファージ型別について

タイトル別名

A taxonomic study on genus Aeromonas : numerical classification and bacteriophage typing of motile Aeromonas isolated from river mud, river water and fresh-water fishes

著者名

福山, 正文

著者別名

フクヤマ, マサブミ

学位授与大学

麻布大学

取得学位

獣医学博士

学位授与番号

乙第250号

学位授与年月日

1987-03-11

注記・抄録

博士論文

Aeromonas属はVibrio属ときわめて類似した性状を示す細菌で河川,池,湖,沼地の水やその泥土などの自然界に広く分布している淡水性菌である。本菌属に含まれるA.hydrophilaについては,カエルの"red leg"の原因菌として最初に発見されて以来,淡水魚,ハ虫類や両生類に病原性があることで知られていた。なかでも養殖魚に出血性敗血症,鰭赤病や穴あき病などを発病させ養殖業に経済的に大きな被害をもたらしている。 一方,ヒトにおいても本菌は以前から弱病原性を示し,臨床材料(腹膜炎,髄膜炎,肺炎,中耳炎,敗血症など)からしばしば分離され,自発性感染症(日和見感染症)の原因菌として知られている。 ところが,最近,本菌属に含まれるA.hydrophilaやA.sobriaが下痢症患者糞便から分離される症例が次第に多く認められ,ヒトの下痢症の起因菌となることが示唆され,わが国ではCampylobacter jejuni,Vibrio cholerae non-01,Vibrio fluvialisなどとともに食中毒起因菌として新たに認識されるようになり注目されている。しかし,ヒトの下痢症に関与するAeromonas属の自然環境下における分布調査や分類学的研究は必ずしも明らかにされていないのが現状である。 そこで,著者は上述のことを踏まえ,本研究では,第一に自然環境下に存在する本年の分布状況と汚染菌量の調査を行い,現在,本菌の分類に使用されているPopoffら(1984)の方法に従って検討した。第二に分離菌株についてAdansonらの方法に従って数値分類を応用して数値分類とPopoffらの分類について比較検討した。第三に河川水や土壌からファージの分離を試み分離菌株についてファージ型別を検討した。1.Aeromonas属の分離状況 1982年10月から84年7月の2年間に河川水,湖水の132件,河川土壌の514件および淡水魚の511件で計1157件の検査試料についてGenusのレベルで分離状況を検討したところ,河川水や湖水では132件中全例(100.0%)から,河川土壌では514件中304件(59.1%)から,淡水魚では511件中462件(90.4%)から本菌が分離され,自然環境下や淡水魚の腸管や鰓には常在菌として多数分布していることが明らかとなった。2.Aeromonas属の推定菌量 河川水,湖水,河川土壌および淡水魚に分布するAeromonas属菌について汚染菌量を定量的に検討したところ,河川水や湖水では1.0×10^2-2.3×10^4個/lの菌量を示し,平均で1.3×10^3個/lの菌量を,河川土壌では1.0×10^1-1.4×10^8個/gの菌量を示し,平均で1.6×10^6個/gの菌量を,淡水魚では1.0×10^6-4.0×10^7個/gの菌量を示し,平均で1.1×10^6個/gの菌量を示していた。3.Aeromonas属の分類および同定1)Popoffらによる分類学的検討 河川水や湖水由来120株,河川土壌由来176株および淡水魚由来1056株の計1352株について生物学的性状による分類を行ったところ,河川水や湖水由来の120株中17株(14.2%)がA.hydrophilaに,33株(27.5%)がA.sobriaに,35株(29.2%)がA.caviaeに分類されたが,残り35株(29.2%)はAeromonas spp.に分類された。河川土壌由来では176株中38株(21.6%)がA.hydrophilaに,23株(13.1%)がA.sobriaに,41株(23.3%)がA.caviaeに分類されたが,残り74株(42.0%)はAeromonas spp.に分類された。淡水魚由来では1056株中182株(17.2%)がA.hydrophilaに,332株(31.4%)がA.sobriaに,206株(19.5%)がA.caviaeに分類されたが,残り336株(31.8%)はAeromonas spp.に分類された。以上のごとく,河川水,湖水,河川土壌および淡水魚由来株はPopoffらの方法に準拠して分類した成績ではAeromonas spp.が高率に認められ,Aeromonas属についての分類が未解決な点も多数残されていることが確認され,本菌分類については再整理する必要があることが考えられた。2)数値分類学的検討 河川水,湖水,河川土壌および淡水魚からの分離菌株がPopoffらの分類でAeromonas sppに該当する菌株が多数認められた。そこで,Adansonらの方法に従って数値分類法を応用し,河川水由来72株,河川土壌由来82株および淡水魚由来441株の計595株を任意に選び73種類の各種生化学的性状試験成績を利用して検討した。 河川水,湖水及び河川土壌由来は75%の相似度を持ってI群からX群に,淡水魚由来は70%の相似度を持ってI群からIX群にそれぞれ群別された。 供試菌株595株中103株(17.3%)がA.hydrophilaに,292株(49.1%)がA.sobriaに,148株(24.8%)がA.caviaeの各グループの中に該当していた。しかし,残り52株(8.7%)は上記3s種のグループに該当しなかった。以上のごとく数値分類を行うことによりPopoffらの分類でAeromonas spp.が42.2%と高率に認められたものが8.7%に減少したことからも理解できる様に現在の分類基準では,本菌の分類が困難であり,Aeromonas属の分類には数値分類や菌体脂肪酸による分類あるいはDNAによる遺伝学的な分類を応用し,菌種レベル,亜種レベルあるいは生物型レベルで再整理を行う必要があろう。3)ファージの分離とファージ型別 河川水195件と土壌90件の計285件よりファージの分離を試み,105件(36.8%)から溶菌班をみい出し,それぞれの溶菌班よりファージの分離にわが国で最初に成功し,A.hydrophila,A.sobria,A.caviaeに対しA1-A26型を,Aeromonas spp.に対しAM1-AM22のファージ型別を確立した。 48種のファージ型を用い河川水由来71株,河川土壌由来82株および淡水魚由来441株の計594株についてファージ型別を行ったところ,河川水由来11株(15.5%),河川土壌由来29株(35.4%)および淡水魚由来89株(20.2%)が型別された。 A.hydrophila,A.sobria,およびA.caviaeに対する26種のファージを用い543株についてファージ型別(A型)を行ったところ,4型が最も多く7株,続いて12型が6株,18型が3株,1型および22型が2株,8型および13型が各1株が型別されたが,残り68株は混合型に該当した。 Aeromonas spp.に対する22種のファージを用い51株についてファージ型別(AM型)を行ったところ,7型に3株,続いて9型と16型に各2株,2型,3型,4型,8型,10型,12型,14型,15型,17型および22型に各1株が型別されたが,残り25株は混合型に該当した。4.河川水,湖水,河川土壌および淡水魚における検体別の本菌分類状況1)Popoffらによる分類学的検討 河川水や湖水48件,河川土壌200件および淡水魚414件の計662件からの分離菌株について検体別に生物学的性状による分類を行ったところ,河川水や湖水由来では48件中15件(31.3%)がA.hydrophilaに,各21件(43.8%)がA.sobriaとAeromonas spp.に,22件(45.8%)がA.caviaeにそれぞれ認められた。河川土壌由来では200件中33件(16.5%)がA.hydrophilaに,21件(10.5%)がA.sobriaに,39件(19.5%)がA.caviaeに,60件(30.0%)がAeromonas spp.にそれぞれ認められた。淡水魚由来では414件中149件(36.0%)がA.hydrophilaに,204件(49.3%)がA.sobriaに,159件(38.4%)がA.caviaeに,210件(50.7%)がAeromonas spp.にそれぞれ認められた。2)数値分類学的検討 河川水や湖水24件,河川土壌100件および淡水魚161件からの分離菌株について検体別に生物学的性状による分類を行ったところ,河川水,湖水由来では24件中3件(12.5%)がA.hydrophilaに,各18件(75.0%)がA.sobriaとA.caviaeに,4件(16.7%)がAeromonas spp.にそれぞれ認められた。河川土壌由来では100件中15件(15.0%)がA.hydrophilaに,25件(25.0%)がA.sobriaに,20件(20.0%)がA.caviaeに,13件(13.0%)がAeromonas spp.にそれぞれ認められた。淡水魚由来では161件中59件(36.6%)がA.hydrophilaに,100件(62.1%)がA.sobriaに,71件(44.4%)がA.caviaeに,34件(21.1%)がAeromonas spp.にそれぞれ認められた。3)ファージ型別状況 48種類のファージ型を用い河川水や湖水24件,河川土壌100件および淡水魚161件の計285件からの分離菌株について検体別に型別を行ったところ,河川水,湖水由来に9件(37.5%)が,河川土壌由来に30件(30.0%)が,淡水魚由来に78件(48.4%)が型別された。 上述の研究成績から河川土壌,河川水,湖水及び淡水魚から本菌が高率に分離され自然環境下や淡水魚に高頻度に分布する菌種であることが明らかになった。また,本菌の分類においてPopoffらの分類ではAeromonas spp.が42.2%と高率に認められたが著者が応用した数値分類を用いることによりAeromonas spp.の多くはA.sobriaやA.caviaeに移行し,Aeromonas spp.は8.7%に減少が認められPopoffらの分類基準では分類が困難であることが示唆され数値分類を応用することにより本菌を再整理することが妥当であることが考えられる成績であった。ファージ型別についてはわが国で最初に分離することに成功し,本菌のファージ型別を確立し本菌の生態学的調査の解析に応用できることが示唆された。また,将来ヒト下痢症由来と自然界由来の溶菌パターンを比較することにより疫学調査にも応用できるものと思われる。

Bacterial species being classified into genus Aeromonas are known to have been having properties quite resembled with those of the ones in genus Vibrio, and extensively inhabited in natural environment, such as in water and/or mud of river, pond, lake or marsh. Among the species, A.hydrophila has been known as a pathogene for fresh-water-fishes, reptiles and amphibians, since the first recognition of the organisms as a causal agent of red leg disease of frogs. The evidence has been accumulated on the bacterium, that it is also capable of producing hemorrhagic septicemia or red pest in fresh-water eel(s) and perforation disease in artificially breeded fresh-water fishes, and often giving much economic losses to the fish breeders. On the other hand, a weak pathogenisity of the bacterium against humanbeings is also reported. Namely, a frequent isolation of the bacterium from the clinical specimens, obtained from peritonitis, meningitis, pneumonia, otitis interna, arose thesuspicion, and now regarded as a causal agent of oppotunistic infection or autogenous infection. In addition to the above mentioned information, a frequent isolation of A.hydrophila and A.sobria, from the feces of diarrheal disease patients, has also been reported. It is suggesting that the presence of some pathogenic factor(s) in these two organism s affecting on the occurrance of human diarrhea. At present, however, little is known about Aeromonas which mightbe involve in human diarrheal diseases. Thus, in the first part of this study, a survey was conducted on the incidence of Aeromonas in natural environment, and the taxonomic investigation of the isolates was made by the method described by Popoff et al. (1984). In the second part, Adansonian (or numerical) classification was performed on the 73 items of biochemical characteristics of the isolates, and results obtained were compared with those of the ones obtained by the conventional Popoff's classification. In the last part, isolation of 105 bacteriophages from river water and river mud and establishment of a new phage set, consisted of 48 phage types, were reported. Followed by the results on the phage typing made on the isolates and its advantageous point were discussed.1.Incidence of Aeromonas in natural environment During the period from October 1982 through July 1984, a total of 1,157 specimens, consisted of 132 river- and lake- waters, 514 river- and lake-muds, and 511 fresh-water fishes caught at both River-Tama and River-Sagami were examined for the presence of the organisms. Of them, 132 (100%) river- and lake-waters, 304 (59.1%) river- and lake-muds, and 462 (90.4%) intestinal contents of fresh -water fishes were found to had been harboring a mean concentration of 1.3 x 10^3/l, 1.6 x 10^6/g, and 1.1 x 10^6/g of Aeromonas respectively. However, no seasonal variation was observed on the incidence of the organisms throughout the investigation period. When attempts were made to classify the isolates by the method described Popoff et al., 17(14.2%) of 120 water-isolates were typed as A.hydrophila, 33 (27.5%) as A.sobria and 35 (29.2%) as A.caviae respectively. And rest of the 35 (29.2%) were remained as untypable. As for the 176 mud-isolates, 38 (21.6%) were typed as A.hydrophila 23 (13.1%) as A.sobria and 43 (24.4%) as A.caviae respectively. And rest of the 74 (42.0%) were remained untypable. Same efforts were made on the 1,056 strains obtained from fresh-water fishes, and 182 (17.2%) were typed as A.hydrophila, 332 (31.4%) as A.sobria and 206 (19.5%) as A.caviae respectively. And rest of the 336 (31.8%) were remained as untypable.2.Adansonian classification of the isolates Since many strains remained as untypable by the conventional Popoff's criteria, further studies were conducted by themethod of Adansonian classification (or numerical classification), on the 595 representative strains, consisted of 72 water-isolates,82 mud-isolates and 441 fish-isolates, on the 73 items of their biochemical characteristics. Through the analysis made on the results obtained, water- and mud-isolates were classified into groups I to X, and fish-isolates into groups I to IX by the similarity value of greater than 70% respectively. Among the above mentioned 595 strains, 103 (17.3%) were classified into A.hydrophila, 292 (49.1%) into A.sobria and 148 (24.8%) into A.caviae respectively. And rest of the 52 (8.7%) were remained as untypable. The reduction of untypable strains from 42.2% to 8.7% by the adoption of Adansonian classification reveals that some inaccuracy points of the present classification criteria of Popoff et al.3.Establishment of a new phage set and its apprication In order to establish a new phage set for the first time in Japan, a total of 285 specimens, consisted of 195 river waters and 90 river muds, was examined for the presence of bacteriophages against Aeromonas, and obtained 105 strains of bacteriophages. Then, established types A1 through A26 for A.hydrophila, A.sobria and A.caviae, and types AM1 through AM22 for the rest of Aeromonas spp. When newly established phage set was applied on the 71 water-, 82 mud- and 441 fish-isolates, 11 (15.5%), 29 (35.4%) and 89 (20.2%) were typed into some of the 48 types respectively. Namely, when a total of 543 strains which had been tagges as A.hydrophila, A.sobria and A.caviae by the conventional bio-typing methods (including Adansonian classification) were tested by A-type phages, and it was found that 7 strains were types as type A4, 6 strains as type A12, 3 as type A18, 2 as type A1, 2 as type A22, 1 as type A8, 1 as type A13, and rest of 64 strains as mixed A-types respectively. As for the rest of the 51 strains, those being failed to tag by the conventional bio-typing methods, 3 strains were types as type AM7, 2 strains as type AM9, 2 as type AM16, 1 each as types AM2, AM3, AM4, AM8, AM10, AM12, AM14, AM15, AM17, AM22, and rest of 25 strains as mixed AM-types respectivly.4.Distribution of genus Aeromonas and classification from river mud, river water and fresh-water fishes.1)Distribution of genus Aeromonas and classification of its species by the method of Popoff et al. A total of 662 specimens, consisted of 48 river- and lake-waters, 200 river- and lake-muds, and 414 fresh-water fishes caught at bith River-Tama and River-Sagami were examined for the presence of the organisms. When attempts were made to classify the isolates by the method described Popoff et al., 15 (31.3%) of 48 water-isolates were typed as A.hydrophila, 21 (43.8%) as A.sobria and 22 (45.8%) as A.caviae respectively. And rest of the 21 (43.8%) were remained as untypable. As for the 200 mud-isolates, 33 (16.5%) were typed as A.hydrophila, 21 (10.5%) as A.sobria and 39 (19.5%) as A.caviae respectively. And rest of the 60 (30.0%) were remained as untypable. Same efforts were made on the 414 obtained from fresh -water fishes, and 149 (36.0%) were typed as A.hydrophila, 204 (49.3%) as A.sobria and 159 (38.4%) as A.caviae respectively. And rest of the 210 (50.7%) were remained as untypable.2)Adansonian classification (Numerical classification) A total of 285 specimens, consisted of 24 river- and lake-water, 100 river- and lake-muds, and 161 fresh-water fishes caught at both River-Tama and River-Sagami were examined for the presence of the organisms. When attempts were made to classify the isolates by the method of Adansonian classification., 3 (12.5%) of 24 water-isolates were typed as A.hydrophila, 18 (75.0%) as A.sobria and A.caviae respectively. And rest of the 4 (16.7%) were remained as untypable. As for the 100 mud-isolates, 15 (15.0%) were typed as A.hydrophila, 25 (25.0%) as A.sobria and 20 (20.0%) as A.caviae respectively. And rest of the 13 (13.0%) were remained as untypable. Same efforts were made on the 161 obtained from fresh-water fishes, and 59 (36.6%) were typed as A.hydrophila, 100 (62.1%) as A.sobria and 71 (44.4%) as A.caviae respectively. And rest of the 34 (21.1%) were remained as untypable.3)Bacteriophage typing of Aeromonas When newly established phage set was applied on the 24 water-, 100 mud- and 161 fish-isolates, 9 (37.5%), 30 (30.0%) and 78 (48.4%) were typed into some of the 48 types respectively. On the basis of the above mentioned results, it can be concluded that: 1) Extreamely high incidence of Aeromonas in river waters, river muds and fresh-water fishes, suggest us that some of the organisms in the genus may be constituting a normal intestinal flora of fresh-water fishes; 2) The present conventional bio-typing method for the organisms, described by Popoff et al., is likely to have some defective points, since there were many strains failed to tagges at the begining, and later some of these could be confirmed as A.hydrophila, A.sobria or A.caviae by the Adansonian classification; and 3) Application of the newly developed phagetyping method will be a good tool for the clarification of the ecology of Aeromonas in natural environment, and may contribute in future for the epidemiologicalsurveys of unknown human diarrheal diseases.

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