Kekkaku Vol. 85, No. 7: 609_614, Mycobacterium gordonae 要旨 Mycobacterium gordnae NHO 46 3 PFGE hsp65pra 16S rrna hsp65pra 13 16
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1 Kekkaku Vol. 85, No. 7: 69_64, 69 Mycobacterium gordonae 4 要旨 Mycobacterium gordnae NHO 46 PFGE hsp65pra 6S rrna hsp65pra 6S rrna M.gordonae PFGE M.gordonae M.gordonae キーワーズ Mycobacterium gordonae 6S rrna hsp65pra PFGE はじめに Mycobacterium gordonae ) ) ) 4) 6S rrna sequevar, 5) rpob 6) hsp65pra 7) 材料と方法 7 8 M.gordonae 8 MDR-TB MDR-TB 8 M.gordonae 8 6 M.gordonae ) μl 7 M. gordonae 9 MDR-TB 4 59 _ dustin@kch.hosp.go.jp Received Dec. 9/Accepted 6 Feb.
2 結核 第 85 巻 第 7 号 年 7 月 6 Table M. gordonae strains which had concordant results by both hsp65pra method and 6S rrna gene sequencing hsp65pra type 6S rrna gene sqvⅠ sqvⅡ sqvⅢ sqvⅣ sqvⅤ new sqv Ⅰ 6 ** Ⅱ Ⅲ Ⅳ ()* 4 () Ⅴ Ⅵ Ⅶ 4 () Ⅷ Ⅸ Ⅹ 6 NP NP NP New pattern () () ()* () strains number in parentheses causes pulmonary infection *strains from a patient with policlonal infection of M. gordonae **three strains from hospital environments ている手洗い場水道水から 株が分離された ちなみに 上記 回の環境調査の結果 細菌検査室の備品 試薬か 結 果 6S rrna 遺伝子 供試された 49 株のうち 6 株の塩基 ら M. gordonae は分離されなかった 6S rrna 遺伝子解析 PCR 反応は岩本らの方法 に 配列は M. gordonae DSM446 基準株と % 一致し sqv 準 じ て 行 っ た 抽 出 し た DNA を テ ン プ レ ー ト と し Ⅰに分類された 5 株は M. gordonae DSM4 株と 9) Takara Ex Taq タカラバイオ を用いて 94 秒 % 一致し sqv Ⅱと分類され そのうち 株は環境分離株 55 秒 7 分を 5 サイクル行った 6S rrna であった sqv Ⅲに分類された 6 株のうち 5 株は M. gor- 遺伝子の超可変部 A と B を含む領域をプライマー 85F donae Borste 4/99 と 塩基の違いが見られた さら 5 -GAG AGT TTG ATC CTG GCT CAG- と 64R 5 に 株が sqv Ⅳに分類され この中には解析期間中に再 -TGC ACA CAG GCC ACA AGG GA- を用いて PCR 発した M. gordonae 症患者からの初発時と再発時の株が 増幅産物を得た PCR 産物を精製した後 BigDye Termi- 含 ま れ て い た こ の 患 者 か ら の 初 発 時 の 株 は M. gor- nator Ready Reaction Cycle Sequencing Kit Applied Bio- donae Borste 68/99 と % 一致したが 年 7 カ月後 systems Japan を用いて 6S rrna 遺伝子の部分配列を に再発した時の分離株では 塩基の違いが見られた 得た 得られた塩基配列は Ribosomal Differentiation of 株 は M. gordonae Borste 94/99 と % 一 致 し sqv Ⅴ に Microorganisms: RIDOM を用いて相同性検索を行い 菌 分類された 株は sqv Ⅴと 5 塩基違いを認め new sqv 種決定をした とした また M. gordonae 症由来 7 株の内訳は sqv Ⅲ Hsp65PRA 解析 Telenti らの方法 に準じて行った 7) Tb 5 -ACC AAC GAT GGT GTG TCC AT- と Tb 5 -CTT GTC GAA CCG CAT ACC CT- のプライマー 株 塩基違い sqv Ⅳ 4 株 株のみ 塩基違い sqv Ⅴ 株 new sqv 株となった Hsp65PRA 解析 M. gordonae における hsp65pra パタ を用い 95 分の熱変性の後 96 4 秒 6 5 秒 ーンは PRANET app.chuv.ch/pls/pranet と従来の 7 分を 45 サイクル行い 最後に 7 7 分間伸張し 報告 7) ) 6) から決定した 今回 hsp65pra のタイプⅡ た 得られた PCR 増幅産物の BstEⅡ切断断片長 6 に属する菌株は検出されなかったが 従来報告されてい 6 分処理 および HaeⅢ切断断片長 7 6 分 を ないタイプ new pattern を含む 種類のタイプに分類 制限酵素失活処理後マイクロキャピラリー電気泳動装置 された M. gordonae 症由来 7 株の hsp65pra タイプはタ Agilent bioanalyzer Agilent Technologies 社 を用い イプⅢが 株 タイプⅦ タイプ NP タイプ NP て解析した タイプ NP new pattern が各 株という結果となり PFGE 解析 抗酸菌の集団感染の解析において PFGE 再発患者からの初発時分離株はタイプⅢ 再発時分離株 に準拠して制限酵 は new pattern と分類された Hsp65PRA のタイプと 6S 素 DraⅠを用いて実施し 得られた泳動像を Phoretix D rrna の sqv との比較では sqv Ⅰの 6 株は DSM446 と Pro Nonlinear Dynamics 社 を用いてゲルイメージ解析 同一の hsp65rpa タイプⅠと分類された sqv Ⅱの臨床 処理し Phoretix D Database Nonlinear Dynamics 社 に 分離株 株は hsp65pra タイプ new pattern となり 一方 て 系 統 樹 を 作 成 し た 得 ら れ た 結 果 は Tenover ら の 環境分離株 株は hsp65pra タイプⅠと分類された は有用である 今回吉田らの方法 ) PFGE 電気泳動パターンの評価基準 価を行った ) を用いて疫学的評 Sqv Ⅲの 6 株はタイプⅤとタイプⅦに分類され sqv Ⅳ sqv V は順に 5 種類と 8 種類の PRA タイプを示し 多型 性が見られた Table PFGE 解析 供試菌 49 株から得られた PFGE パターン
3 Molecular Typing in Mycobacterium gordonae/s.yoshida et al. 6 Strains No S rrna gene hsp65pra Characteristics of strains Lambda laddar 5 Environment Water into the nebulizer using at an outpatient booth Environment Tap water at MDR-TB patient bathroom * M.gordonae polyclonal infection: relapse strain at Oct * 5 9 Environment new sqv RRAtype PRAtype NP PRAtype New pattern PRAtype New pattern PRAtype NP PRAtype New pattern PRAtype PRAtype NP PRAtype NP PRAtype NP PRAtype MDR-TB patient MDR-TB patient M.gordonae polyclonal infection: new strain at Apr. 7 Tap water at bringing room of phlegm Fig. Dendrogram based on computer-assisted comparison of the 49 PFGE pattern with genotype in 6S rrna and hsp65pra. The positions of Lambda laddar size marker are indicated on the top. *strains from a patient with polyclonal infection of M.gordonae
4 結核 第 85 巻 第 7 号 年 7 月 6 のうち sqv Ⅲ hsp65pra タイプⅦである臨床分離株 あったと報告している7) 以降 多数の研究データが蓄 株 No.とNo. のパターンが一致した そのほかに 積され 996 年にはアメリカの Taylor らによって 株 同じ M. gordonae 症患者から分離された 株を含む 47 株 中 株 84.6% がタイプⅠであったとする結果が報告 間で一致するパターンは認められなかった Fig. され ) 年にはイタリアの Brunello らにより M. gor- 考 donae のタイプⅡが 5% と最も多く分布していたと報告 察 さ れ た ) 同 時 期 ブ ラ ジ ル か ら つ の 報 告 が あ り Suffys らはタイプⅠが 7.6% タイプⅢが 5.%4) とし 非結核性抗酸菌は自然界のみならず医療施設の環境に も広範囲に分布している 7) 8) M. gordonae の院内におけ Chimara らはタイプⅠが 4.5% タイプⅢが 4.% 他に る環境汚染の報告では 患者と接触する機会の多い気管 4 種類の新しいタイプ X NP NP NP が認め 支鏡 9) や 水道水 ) 冷却水槽 ) に汚染源が見つかって られたという 5) 続いて 年 da Silva Rocha らはブ いる したがって感染対策上 初歩的な衛生管理の不手 ラジルの 6 州から臨床分離株を集め大規模な解析を実 際による院内感染で易感染性患者が犠牲にならないよう 施したところ 9 株中タイプⅠが 4 株.% タイ に 院内感染対策活動での監視を強化し継続することは プⅢが 5 株 6.% 認められ 8 株 4.% が新しい 最も重要である また院内において非結核性抗酸菌の汚 種類のタイプとして認められたとしている 6) わが国 染と思われる事例が発生した場合 疑似アウトブレイク では Itoh らが 988 年に集められた 4 株のうち の可能性を考慮し 汚染源から分離された菌と臨床検体 タイプⅣが 6.4% と最も多く存在し次にタイプⅢ.5 からの分離株との遺伝子型の異同を検証することが求め % が続いたとしている6) 今回われわれの結果から られる 今回 M. gordonae 株が院内の水回り環境に存 最も多かったタイプはタイプⅠとタイプⅩ 各 8.4% 在することが認められたが これらの環境分離株は臨床 であり 次にⅢ.% タイプⅣとⅦがそれぞれ 8.% 分離株とは異なった PFGE パターンを有していた Fig と続いた 残り 6.6% は新しいタイプを含む 8 種類のタ また院内では患者に対して採痰時に水道水を用いたうが イプに分類され hsp65pra タイプの高い多様性が認め いの指導は行っていないため あらゆる環境に生息して られた Table これらの結果は M. gordonae の地域 いるであろう M. gordonae を起因とする検体採取時の検 的分布の差を反映しているのか もしくは 99 年代か 体への混入も考えにくい したがって院内における疑似 ら 年代後半にかけての M. gordonae の時間的な変動 ダイナミクス を表しているのか 今後 臨床分離株 アウトブレイクの可能性は低いと考えられた 99 年 スウェーデンの Telenti らにより遺伝子内の 環境分離株での遺伝子型別に関するデータが蓄積される 抗酸菌特異的部位を PRA 法で分別し遺伝子内の多様性 ことで その臨床的意義 地域特異性などとの関連性が の違いから同定する方法が報告され 5 種類の M. gor- 明確になることが期待される donae の菌種内変異が分別可能となった 7) 彼らは 99 M. gordonae 症分離株は 6S rrna 遺伝子解析によって 年に収集した臨床分離株の 8% が hsp65pra タイプⅠで sqv Ⅲ Ⅳ Ⅴ New sqv に分類され 多様な菌種内変異 Table hsp65pra type I II III IV V VI VII VIII IX X NP NP NP New Pattern a Frequency of hsp65pra types in M. gordonae strains from different geographic regions Sweden (99)7) (n 4) 8 7 United States (996)) (n ) Italy ()) (n 8) PRA typing in geographic region Brazil Brazil Brazil ()4) ()5) ()6) (n 7) (n 44) (n 9) Japan (998_)6) (n 4) three of nine strains were isolated from hospital environments Japan (7_9) n a
5 Molecular Typing in Mycobacterium gordonae/s.yoshida et al. 6 hsp65pra 6 M.gordonae hsp65pra M.gordonae sqv hsp65pra M.gordonae Prince 6 M. gordonae ) M.gordonae M. gordonae M.gordonae, M.chelonae 8) M.gordonae 6.% M.gordonae M.gordonae PFGE No. No. No. No. 6 M.gordonae 6S rrna hsp65pra PFGE 7 No.6 No. Fig MAC 文 Barber TW, Craven DE, Farber HW: Mycobacterium gordonae: a possible opportunistic respiratory tract pathogen in patients with advanced human immunodeficiency virus, type 献 infection. Chest. 99 ; : 76 _ 7. Lessnau KD, Milanese S, Talavera W: Mycobacterium gordonae: a treatable disease in HIV-positive patients. Chest. 99 ; 4 : 779 _ 785.,,, Mycobacterium gordonae.. ; 75 : 69 _ Weinberger M, Berg SL, Feuerstein IM, et al.: Disseminated infection with Mycobacterium gordonae: report of a case and critical review of the literature. Clin Infect Dis. 99 ; 4 : 9 _ 9. 5 Kirschner P, Böttger EC: Microheterogeneity within rrna of Mycobacterium gordonae. J Clin Microbiol. 99 ; : 49 _ 5. 6 Itoh S, Kazumi Y, Abe C, et al.: Heterogeneity of RNA polymerase Gene (rpob) sequences of Mycobacterium gordonae clinical isolates identified with a DNA probe kit and by conventional methods. J Clin Microbiol. ; 4 : 656 _ Telenti A, Marchesi F, Balz M, et al.: Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 99 ; : 75 _ 78. 8, ; 8 : 55 _ 56. 9,,, Mycobacterium lentiflavum.. 8 ; 8 : 47 _ 4.,,, Mycobacterium kansasii.. 7 ; 8 : _. Tenover FC, Arbeit RD, Goering RV, et al.: Interpreting chromosomal DNA restriction patterns produced by pulsedfield gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 995 ; : _ 9. Taylor TB, Patterson C, Hale Y, et al.: Routine use of PCRrestriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media. J Clin Microbiol. 997 ; 5 : 79 _ 85. Brunello F, Ligozzi M, Cristelli E, et al.: Identification of 54 Mycobacterial Species by PCR-Restriction Fragment Length Polymorphism Analysis of the hsp65 Gene. J Clin Microbiol. ; 9 : 799 _ 86.
6 Suffys PN, da Silva Rocha A, de Oliveira M, et al.: Rapid Identification of Mycobacteria to the Species Level Using INNO-LiPA Mycobacteria, a Reverse Hybridization Assay. J Clin Microbiol. ; 9 : 4477 _ Chimara E, Ferrazoli L, Misuka Ueky SY, et al.: Reliable identification of mycobacterial species by PCR-restrictionenzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRAhsp65 patterns. BMC Microbiol. 8 ; 8 : da Silva Rocha A,Werneck Barreto AM, Dias Campos CE, et al.: Novel allelic variants of Mycobacteria isolated in Brazil as determined by PCR-restriction enzyme analysis of hsp65. J Clin Microbiol. ; 4 : 49 _ Vaerewijck MJM, Huys G, Palomino JC, et al.: Mycobacteria in drinking water distribution systems: ecology and significance for human health. FEMS Microbiol Rev. 5 ; 9 : 9 _ 94. 8,,, Mycobacterium chelonae.. 9 ; 4 : 9 _. 9 Gubler JG, Salfinger M, von Graevenitz A: Pseudoepidemic of nontuberculous mycobacteria due to a contaminated bronchoscope cleaning machine. Report of an outbreak and review of the literature. Chest. 99 ; : 45 _ 49. Stine TM, Harris AA, Levin S, et al.: A pseudoepidemic due to atypical Mycobacteria in a hospital water supply. JAMA. 987 ; 58 : 89 _ 8. Lalande V, Barbut F, Varnerot A, et al.: Pseudo-outbreak of Mycobacterium gordonae associated with water from refrigerated fountains. J Hosp Infect. ; 48 : 76 _ 79. Prince KA, Costa AR, Malaspina AC, et al.: Isolation of Mycobacterium gordonae from poultry slaughterhouse water in Sao Paulo State, Brazil. Rev Argent. Microbiol. 5 ; 7 : 6 _ 8. Original Article DETECTION OF MOLECULAR EPIDEMIOLOGY OF MYCOBACTERIUM GORDONAE ISOLATES Shiomi YOSHIDA, Katsuhiro SUZUKI, 4 Tomotada IWAMOTO, Kazunari TSUYUGUCHI, Motohisa TOMITA, Masaji OKADA, and Mitsunori SAKATANI Abstract [Objects] To analyze the molecular epidemiology of Mycobacterium gordonae strains from patients and environments in the hospital. [Subjects] A total of 46 clinical strains were obtained from patients registered at the NHO Kinki-chuo Chest Medical Center and strains from hospital environments. [Methods] By using genetic data from the 6S rrna gene and hsp65pra, pulsed-field gel electrophoresis (PFGE) assessment of their intraspecies variability and epidemiology was carried out. [Results] Strains from six patients and environmental cultures exhibited the different genotypes of 6S rrna gene sequencing and the hsp65pra type. The PFGE analysis suggested no pseudo-outbreak and showed a polyclonal infection in one patient. [Conclusion] These findings suggest that we should maintain effective surveillance of environments in the hospital and continuously perform molecular epidemiological investigations for infection control of M.gordonae. Key words: Mycobacterium gordonae, 6S rrna gene sequence, hsp65pra, PFGE, Polyclonal infection Clinical Research Center, Department of Clinical Laboratory, Department of Respiratory Medicine, National Hospital Organization Kinki-chuo Chest Medical Center; 4 Department of Microbiology, Kobe Institute of Health Correspondence to: Shiomi Yoshida, Clinical Research Center, National Hospital Organization Kinki-chuo Chest Medical Center, 8 Nagasone-cho, Kita-ku, Sakai-shi, Osaka 59 _ 8555 Japan. ( dustin@kch.hosp.go.jp)
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