建築物の高層化に伴い，これらの建物における飲料水は水圧の関係から受水槽などの給水用タンクを経由したのちに各戸に配水される，いわゆるタンク水であることが多い。タンク水は塩素消毒が義務づけられており，末端の給水栓で0.1mg/l以上の遊離残留塩素を保持していなければならない。 このようなタンク水にも多数の従属栄養細菌が生息していることが知られつつある。今日，水道水の安全性が種々議論されるなかで，高齢化社会の到来や高度医療の進歩に随伴して生じる易感染者の増加などを考慮すると，これらの細菌に関する検討は公衆衛生上非常に重要である。 本研究では，タンク水中に優占的に生息するMethylobacterium属菌の水環境等における分布状況や，その性状について検討し，分類学的考察を加えた。さらに，わが国で初めてタンク水中での消長を調べるとともに，塩素抵抗性とそのメカニズムを明らかにするための基礎的研究を行った。本研究の概要は次のとおりである。1. Methylobacterium属菌の検出状況とその性状 タンク水では500試料中348試料(69.6%)から検出され，高い検出率であった。また，直結水では100試料中26試料(26%)から，プール水では100試料中31試料(31%)から，実験用精製水でも100試料中37試料(37%)から検出された。ところが，自然環境水からの検出率は非常に低く，井戸水では100試料中5試料(5%)，池水では50試料中2試料(4%)にすぎなかった。なお，河川水からはまったく検出されなかった。 空気環境では室内で100箇所中18箇所(18%)から，屋外ではさらに多く100箇所中34箇所(34%)から検出された。菌数はいずれも約1CFU/シャーレとわずかではあったが，空中浮遊細菌として存在していることが明らかになった。 本菌は標準寒天培地により25℃で7日間培養すると淡紅色で直径1㎜前後のS型集落を形成するグラム陰性の中型桿菌(3.3～8.3×1.7～2.2μm)で，菌体内に空胞を有する特徴があった。また，0.3%半流動寒天培地及び顕微鏡下で活発な運動性が認められ，鞭毛染色によって極単毛が確認された。本菌の酵素活性は非常に弱く，オキシダーゼとカタラーゼのみが陽性率100%であった。炭素源としてはグリセロール，メタノール及びエタノールを利用した。 また，血液寒天などの培地上には発育せず，標準寒天などの比較的栄養価の低い培地にのみ発育した。本菌は15～35℃の範囲内で増殖が認められ，至適温度は30℃であった。pH5.0～9.0の培地で良好に増殖し，塩化ナトリウムに対しては1.5%以上の濃度で増殖抑制がみられた。 抗菌性15薬剤を用いた感受性試験では，ミノサイクリン，カナマイシン，テトラサイクリンなどに高い感受性を示し，リンコマイシン，クリンダマイシン，ナリジキシン酸，コリスチン，ポリミキシンBに対しては抵抗性であった。 さらに，菌体脂肪酸組成の分析ではC_18:1を主成分とする特徴的なパターンを示し，G+C含量は65.8～66.3%であった。2. 分類学的検討 タンク水由来30株(TW-1～TW-30)，井戸水由来9株(WW-1～WW-9)，池水由来1株(PW-1)，空気環境由来28株(A-1～A-6，A-8～A-25，A-27～A-30)，臨床由来7株(C-1～C-7)，基準株M. extorquens JCM 2802(=NCIMB 9399)，M. rhodinum JCM 2811(=ATCC 14821)，M. mesophilicum JCM 2829(=ATCC 29983)，M. radiotolerans JCM 2831(=IAM 12098)，M. organophilum JCM 2833(=ATCC 27886)の5株及び参照株M. rhodesianum JCM 2810(=ATCC 21614)，M. zatmanii JCM 2819(=NCIMB 10606)の2株，合計82株について，炭素源49項目，構成酵素19項目，薬剤感受性20項目の合計88項目の性状試験を行った。これらの成績をもとにクラスター分析を行ってデンドログラムを作製したところ，供試菌株は70%の相似度で12のクラスターに類別され，分離株の表現形質が多様であることが明らかになった。また，分離株は由来によってクラスターが偏る傾向がみられた。 クラスター分析により12群に類別された分離株のうち，1群，4群，7群，11群の各クラスターから1株ずつ(A-8，A-13，TW-18，TW-5)を選び，16S rRNAの塩基配列を決定した。この成績と基準株や参照株の塩基配列とを比較して系統樹を作製した結果，既知菌株との塩基配列相同性は93.8%～99.4%であり，A-8株とA-13株は系統樹では明らかに分岐したことから，両菌株は新菌種であると考えられた。3. タンク水中での消長 タンク水由来T-11株を滅菌タンク水に接種して30℃に放置すると，2.6×10^2 CFU/mlの接種菌数は徐々に増加して4日経過後で最高菌数の3.5×10^5 CFU/mlに達した。このように本菌はほとんど有機物が存在しない水中においても良好に増殖できる特徴があった。このときの増殖曲線から世代時間をもとめたところ5.8時間と長く，増殖速度は著しく遅かったが，培地を用いた場合と大差はなかった。また，一旦タンク水中で10^5 CFU/mlにまで増殖した本菌を室温に放置して，その消長を経日的に調べた結果，500日経過しても1.0×10^5 CFU/mlであり，非常に長期間生存することが明らかになった。4. 分離株の塩素抵抗性とその変化 タンク水由来30株(2群TW-8，3群TW-9，4群TW-2，11，7群TW-10，13，17，18，8群TW-1，10群TW-3，6，11群TW-5，7，12，16，19，20，21，23，25，29，12群TW-14，15，22，26，28，30，末クラスター群TW-4，24，27)，直結水由来10株(DT-1～DT-10)，プール水由来5株(PL-1～PL-5)，精製水由来10株(DW-1～DW-10)，井戸水由来10株(1群WW-4，2群WW-2，3，5，6，9，5群WW-1，9群WW-8，11群WW-7，末クラスター群WW-10)，池水由来1株(5群PW-1)，空気環境由来22株(1群A-8，21，28，2群A-14，16，25，30，3群A-2，3，19，4群A-13，15，18，27，6群A-22，7群A-11，8群A-24，9群A-6，9，10群A-1，4，12群A-12)，臨床材料由来7株(5群C-1，2，3，5，6，6群C-4，8群C-7)，基準株M. extorquens JCM 2802(=NCIMB 9399)，M. rhodinum JCM 2811(=ATCC 14821)，M. mesophilicum JCM 2829(=ATCC 29983)，M. radiotolerans JCM 2831(=IAM 12098)，M. organophilum JCM 2833(=ATCC 27886)の5株及び参照株M. rhodesianum JCM 2810(=ATCC 21614)，M. zatmanii JCM 2819(=NCIMB 10606)の2株，合計102株について塩素抵抗性試験を行った。タンク水やプール水にような常に残留塩素と接触している試料から分離された菌株の塩素抵抗性は非常に強く，分離株の90%以上が抵抗性株であった。 このうち，池水由来PW-1株(以後P-0株と称する)について，遊離残留塩素0.1，0.5，1.0mg/lの3段階で一定時間経過毎に菌数の変化を調べたところ，タンク水由来株と比較して塩素に対して感受性であることが確認された。この塩素感受性P-0株を滅菌タンク水に浮遊させ，断続的に塩素と接触させたときの塩素抵抗性の変化を検討した結果，感受性株は耐塩素性株(以後P-45株と称する)に変異することが判明した。こうして実験的に得た塩素抵抗性変異株P-45株と，これを15週間滅菌タンク水中に放置したのちに分離した菌株P-15株の塩素に対する抵抗性は両者ともほとんど差異がなく，一度高度の塩素抵抗性を発現した菌株は不可逆的にその性質を維持することが確認された。5. 塩素感受性株(P-0)と抵抗性株(P-45)の比較 性状タイプを比較すると，P-0株は12群に，P-45株は1群に型別され，まったく異なった性状を示した。 構成酵素は両菌株とも同一の13酵素(L-リシンアリルアミダーゼ，L-アラニンアリルァミダーゼ，β-アラニンアリルアミダーゼ，ボスホーβ-D-ガラクトシダーゼ，α-L-アラビノシダーゼ，N-アセチル-β-D-グルコサミニダーゼ，α-D-マンノシダーゼ，エステラーゼ-C4，エステラーゼ-C5，エステラーゼ-C6，エステラーゼ-C8，エステラーゼ-C9，エステラーゼ-C10)で活性を示し，差異はみられなかった。 15薬剤(ペニシリン，エリスロマイシン，ジョサマイシン，リンコマイシン，クリンダマイシン，クロラムフェニコール，テトラサイクリン，ミノサイクリン，カナマイシン，ゲンタマイシン，セファレキシン，セファゾリン，ナリジキシン酸，コリスチン，ポリミキシンB)に対する感受性試験成績では，テトラサイクリン，ミノサイクリンなどに対しては両菌株とも感受性が高く，ペニシリン，リンコマイシンなどにはともに抵抗性を示した。 菌体脂肪酸組成は両菌株とも不飽和脂肪酸C_18:1が主要な構成成分であった。また，定量分析の結果，P-0株では10.7μg/mg，P-45株では13.4μg/mgで大差はなかった。 フローサイトメトリーによる形態観察では，P-45株の方が原点に近いところに細胞数のピークが認められ，形態学的にはP-0株よりやや均一であった。 電子顕微鏡により細胞表層部を観察したところ，P-0株の外膜は約17nm以内であり，その表面には細繊維状の突起形成が認められた。P-45株では表面部の突起形成は観察されず，外膜の厚さは約25nmとP-0株より厚く，均一な層の様子を呈しており，このことが塩素抵抗性の強弱の一因であると考えられた。 塩素感受性P-0株，抵抗性P-45株及びタンク水由来T-7株の3株についてプラスミドの検出を試みた結果，後二者から共通して12kbのプラスミドが検出され，このプラスミドが塩素抵抗性に関与している可能性が考えられた。しかし，同一のバンドが塩素感受性のP-0株でも認められたため，これらのプラスミドを除去して塩素抵抗性を比較したところ，いずれも塩素抵抗性に差異は認められず，これらプラスミドの関与は否定された。 パルスフィールド電気泳動によりP-0株とP-45株の染色体DNAの制限酵素パターンを検討したところ，Spe I，Xba Iにより処理した両菌株の泳動パターンは明らかに異なった。また，Dra IとSsp Iにより塩素感受性株(A-8，A-19，C-5)と抵抗性株(TW-5，TW-15，TW-18)を比較したが，泳動パターンはすべての株で異なり，塩素抵抗性に共通した泳動パターンは認められなかった。 塩素抵抗性TW-21株とP-45株の染色体DNAを制限酵素Pst I，Hin d IIIで処理し，1kb以上の断片をE. coliに挿入して形質転換を試みた。その結果，塩素抵抗性のE. coliが確認され，塩素抵抗性の発現には染色体DNAが関与している可能性が示唆された。 以上のことから，Methylobacterium属菌は水環境，空気環境に広く分布しており，特に前者では，自然環境水よりも塩素消毒された水において優占していることが明らかになった。この属に共通した特徴的な性状は，増殖速度が遅いこと，淡紅色の非水溶性色素を産生すること，メタノールを分解することであった。分離株の表現形質は非常に多様であり，クラスター分析の結果から12群に類別された。このうちの4株について16S rRNAの塩基配列を決定して既知菌株のそれと比較したところ，2株の塩基配列が明らかに異なり，いずれも新種である可能性が高かった。また，本菌はタンク水のような有機物濃度が低い水中においても良好に増殖できる低濃度栄養性細菌の一種であり，一旦最高菌数にまで増殖すると非常に長期間生残することが知られた。塩素抵抗性に関しては，分離株の由来によって抵抗性が大きく異なること，塩素感受性株が断続的に塩素と接触すると抵抗性株に変異すること，高度の耐塩素性を発現した菌株は不可逆的にその性質を維持することが判明し，これらはいずれも新知見であった。塩素抵抗性の変異にプラスミドは関与しておらず，染色体DNAによる支配が示唆された。また，超薄切片の観察によって，細胞膜，特に外膜が肥厚して強固になっていることが明らかになり，このことが塩素抵抗性の強弱に大いに関連していると考えられた。In connection with a number of constructions of multistoried residential buildings in recent years, it is now very popular in these buildings for drinking water to be once held in a tank, which is so-called tank-water, and then distributed to respective homes. The tank-water for drinking is legally required to be disinfected by chlorine at a residual level higher than 0.1 mg/l as tap water. It is now known that even in disinfected tank-water a variety of heterotrophic bacteria can survive. Since safety of drinking water is sincerely discussed under the recent social circumstances such as the increases of aged people and compromised hosts, study on these bacteria is becoming very important in public health. In the present work, Methylobacterium strains living predominantly in tank-water were studied on their distribution in various environmental waters as well as on their characteristics from the viewpoint of taxonomy. In addition, their growth and survival in tank-water was investigated for the first time in Japan, and their chlorine-resistance and its mechanism were also analyzed. The present work is outlined below.1. Distribution and phenotypic characterization Detection frequencies of Methylobacterium strains in various water samples were as high as 69.6% (348 out of 500 water samples) for drinking tank-water (hereafter briefly called tank-water), 26% (26/100) for directly distributed tap water, 31% (31/100) for swimming pool-water, and 37% (37/100) for purified water for experimental use. For natural environmental waters, however, the frequencies were very low 5% (5/100) for well water, 4% (2/50) for pond water, and none (0/50) for river water. The detection frequencies in the air were 18% (18/100) for indoor air and 34% (34/100) for outdoor air. All air samples had the bacterial count as low as ca. 1 CFU/dish (57cm2), showing that Methylobacterium strains are present in dispersing in the air. Methylobacterium strains tested were Gram-negative bacilli of middle size (3.3 to 8.3 x 1.7 to 2.2 μm), which formed S-type colonies of ca. 1 mm in diameter with pale red color after culture on standard agar plate for 7 days at 250 C. The organisms were characterized by the presence of vacuoles in their cells. Microscopically they showed active movement in a semi-fluid 0.3% agar culture medium, and were confirmed to be polar-monotrichous by flagellar staining. The enzyme activity of these bacteria was 100% positive for oxidase and catalase only. As carbon source were used glycerol, methanol and ethanol. It was also found that Methylobacterium strains can be cultured only on low-nutritious media such as standard agar medium, but not on high-nutritious ones such as blood agar medium. They grew within the temperature range from 15 to 350 ℃ at maximum level at 300 ℃. For pH of culture medium, they grew well in the range from 5.0 to 9.0. Sodium chloride inhibited the growth at a concentration higher than 1.5%. Examination for drug susceptibility to 15 antibiotics gave the result as follows: highly sensitive to minocycline, kanamycin and tetracycline and resistant to lincomycin, clindamycin, nalidixic acid, colistin and polymyxin B. The chemical analysis of fatty acid composition of the organisms gave characteristic pattern consisting mainly of C_18:1 and the G+C content ranged 65.8 to 66.3%.2. Taxonomical investigation A total of 82 strains were examined in their characteristics for 88 items including 49 for carbon source, 19 for constitutive enzyme and 20 for drug susceptibility. The test strains were consisted of 30 strains (TW-1 to 30) from tank-water, 9 (WW-1 to 9) from well water, 1 (PW-1) from pond water, 28 (A-1 to 6, A-8 to 25, A-27 to 30) from air, 7 (C-1 to 7) from clinical source, 5 type strains [M. extorquens JCM 2802 (=NCIMB 9399), M. rhodinum JCM 2811 (=ATCC 14821), M. mesophilicum JCM 2829 (=ATCC 29983), M. radiotolerans JCM 2831 (=IAM 12098) and M. organophilum JCM 2833 (=ATCC 27886)], and 2 reference strains [M. rhodesianum JCM 2810 (=ATCC 21614) and M. zatmanii JCM 2819 (=NCIMB 10606)]. The results were processed for cluster analysis to make a dendrogram, where the strains were divided in to 12 cluster groups at a similarity level of 70%. The analysis made it clear that these strains revealed a variety of phenotypes and that they showed a tendency to concentrate in to a certain cluster groups, depending on their origins. Out of the isolated strains classified into 12 groups by cluster analysis, each representative strain was chosen from 1st, 4th, 7th and 11th cluster groups (A-8, A-13, TW-18 and TW-5) to determine their 16S rRNA sequences. The result was compared with the known sequences of the type and reference strains to make a neighbor-joining phylogenetic tree. As a result, the sequence similarity between the test strains and the reference strains was 93.8 to 99.4% and two strains, A-8 and A -13, were confirmed to be new species, because they diverged clearly in this phylogenetic tree.3. Growth and survival in tank-water When the strain T-11 from tank-water was inoculated into a sterilized tank-water and allowed to stand at 300 C, the bacterial count increased gradually from the initial value of 2.6 x 102 CFU/ml to the maximum value of 3.5 x 105 CFU/ml after a 4-day incubation. This indicates that the organisms can grow well even in a water that contains nearly no organic substance. From the growth curve the generation time was determined to be as long as 5.8 hrs, which was not different so greatly from the value in culture medium. T-11 was also let to grow to the level of bacterial count of 105 CFU/ml in tank water, and then observed in its growth and survival in the water according to time. The bacterial count was 1.0 x 105 CFU/ml 500 days later, which means that the bacterium can keep living for a very long time. 4. Chlorine-resistance and its change A total of 102 strains were tested for chlorine-resistance. The strains were 30 strains from tank-water (TW-8, 2nd group; TW-9, 3rd group; TW-2 and 11, 4th group; TW-10, 13, 17 and 18, 7th group; TW-1, 8th group; TW-3 and 6, 10th group; TW-5, 7, 12, 16, 19, 20, 21, 23, 25 and 29, 11th group; TW-14, 15, 22, 26, 28 and 30, 12th group; TW-4, 24 and 27, unclustered group), 10 strains from directly distributed tap water (DT-1 to 10), 5 strains from swimming pool-water (PL-1 to 5), 10 strains from purified water for experimental use (DW-1 to 10), 10 strains from well water (WW-4, 1st group; WW-2, 3, 5, 6 and 9, 2nd group ; WW-1, 5th group; WW-8, 9th group; WW-7, 11th group; WW-10, unclustered group), one strain from pond water (PW-1, 5th group), 22 strains from air (A-8, 21 and 28, 1st group; A-14, 16, 25 and 30, 2nd group; A-2, 3 and 19, 3rd group; A-13, 15, 18 and 27, 4th group; A-22, 6th group; A-11, 7th group; A-24, 8th group; A-6 and 9, 9th group; A-1 and 4, 10th group; A-12, 12th group), 7 strains from clinical source (C-1, 2, 3, 5 and 6, 5th group; C-4, 6th group; C-7, 8th group), 5 type strains [M. extorquens JCM 2802 (=NCIMB 9399), M. rhodinum JCM 2811 (=ATCC 14821), M. mesophilicum JCM 2829 (=ATCC 29983), M. radiotolerans JCM 2831 (=IAM 12098), M. organophilum JCM 2833 (=ATCC 27886)], and 2 reference strains [M. rhodesianum JCM 2810 (=ATCC 21614), M. zatmanii JCM 2819 (=NCIMB 10606)]. The strains isolated from waters including tank-water and swimming pool-water that were in contact with residual chlorine were highly resistant to chlorine; 90% of them were found to be chlorine-resistant. For chlorine resistance, the strain PW-1 (hereafter called P-0) from pond water was examined on survival at three different residual chlorine concentration of 0.1, 0.5 and 1.0 mg/l. It was found that the strain was more sensitive to chlorine than the strains from tank-water. When this chlorine-sensitive strain P-0 was supended in a sterilized tank-water and then subjected to intermittently contact with chlorine to investigate the change in chlorine-resistance, the strain transformed itself into chlorine-resistant variant (hereafter called P-45). The chlorine-resistant variant P-45 had nearly the same chlorine-resistance as the strain P-15 isolated from a sterilized tank-water which was inoculated with P-45 and allowed to stand for 15 weeks. This suggests that a strain, when once expresses high chlorine-resistance, would maintain it nature irreversibly.5. Comparison between chlorine-sensitive (P-0) and chlorine-resistant (P-45) strains P-0 and P-45 were quite different from each other in phenotypic characterization, and classified into 12th and first cluster groups of the dendrogram, respectively. The two strains showed the same enzyme activity for 13 enzymes including L-lysineallylamidase, L-allanineallylamidase, β-allanineallylamidase, phospho-β-D-galactosidase, α-L-arabinosidase, N-acetyl-β-glucosaminidase, α-D-mannosidase, esterase-C4, esterase-C5, esterase-C6, esterase-C8, esterase-C9, and esterase-C1O. As to drug-susceptibility, both strains were tested for the fo11owing 15 antibiotics of penicillin, erythromycin, josamycin, lincomycin, clindamycin, chloramphenicol, tetracycline, minocycline, Kanamycin, gentamycin, cefalexin, cefazoline, nalidixic acid, colistin, and polymyxin B. Both showed high sensitivity to tetracycline and minocycline and resistance to penicillin and lincomycin. For fatty acid composition, both strains were constituted mainly from an unsaturated fatty acid, C_18:1 with a small difference in the content between them, being 10.7 μg/mg for P-0 and 13.4 μg/mg for P-45. Morphological observation with flow cytometry revealed that P-45 showed a peak of cell counts nearer the origin and was morphologically more uniform than P-0. Electronmicroscopic observation of the outer layer of the cell gave the results as follows: For P-0, the outer membrane was less than l7 nm in thickness and formation of fine fibrous processes was observed on its surface. For P-45, it was ca. 25 nm in thickness, thicker than that of P-0, and no formation of processes was shown on its surface, giving uniform appearance of the cell outer layer. These differences in the outer layer would be partly the cause for the difference in chlorine-resistance between P-0 and P-45. Plasmid detection was tested for three strains of chlorine-sensitive P-0, chlorine-resistant P-45 and T-7 from tank-water. In the latter two a 12 kb plasmid was detected, suggesting the possibility of participation of the plasmid in the expression of chlorine-resistance. However, chlorine-sensitive P-0 also showed the same band. For this reason, the two strains were again tested for chlorine-resistance after removal of plasmid. They showed no difference in chlorine-resistance before and after removal of plasmid, denying the participation of the plasmid in the expression of chlorine-resistance. By using pulsed field gel electrophoresis, two strains of P-0 and P-45 were examined on the restriction enzyme patterns of their chromosomal DNA. The DNA of P-0 treated with ,Spe I and Xba I showed different electrophoretic patterns from that of P-45. When fingerprint patterns with the treatment with Dra I and Ssp I were compared between chlorine-sensitive strains (A-8, A-19 and C-5) and chlorine-resistant strains (TW-5, TW-15 and TW-18), all strains showed different patterns from each other, indicating that there is no restriction pattern specific to chlorine-resistance. As to the two chlorine-resistant strains of TW-21 and P-45, their chromosomal DNA fragments of larger than 1 kb treated with Pst I and Hin d III, were inserted in to E. coli to cause transformation. As a result, emergence of chlorine-resistant E. coli was confirmed, suggesting the possibility of chromosomal DNA is involved in the expression of chlorine-resistance. The results of the present work are summarized as described below. Methylobacterium strains were widely distributed in environmental water and in air as well. It should be noted that they were predominant in water disinfected by chlorine rather than in natural environ mental water. The characteristics of these strains were low growth rate, production of water-insoluble pigment with pale red color, and ability to utilize methanol. The isolates showed a variety of phenotypes and were classified taxonomically into 12 groups by means of cluster analysis. Four out of these isolated strains were determined for 16S rRNA sequence and compared with known type and reference strains. Two of the isolates were possibly new species, because they were different in the level of sequence similarity from the known species. It was made clear that Methylobacterium strains are oligotrophic bacteria that can grow well even in such water low in organic content as tank-water and that the organisms can maintain living for avery long time after reaching the maximum growth. Followings are new findings as to the resistance to chlorine: The isolates showed differences in chlorine-resistance depending on their origins; chlorine-sensitive strains showed transformation to chlorine-resistant ones through intermittent contact with chlorine; and strains that once expressed the chlorine-resistance maintained the nature irreversibly. As to the chlorine-resistance, while plasmid was not found to play a role, the participation of chromosomal DNA was suggested in terms of transformation. In addition, electronmicroscopic observation of extra-thin sections made it clear that a chlorine-resistant variant was thicker and rougher in cell surface layer, especially in outer membrane, than its original chlorine-sensitive strain. This suggests that these structures in surface membrane would be related with the chlorine-resistance a great deal.博士(獣医学)麻布大
