Key words: Streptococcus agalactiae, Group B Streptococci, Chloramphenicol resistance Chloramphenicol acetyltransferase

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1 Key words: Streptococcus agalactiae, Group B Streptococci, Chloramphenicol resistance Chloramphenicol acetyltransferase

2 Table 1 Properties of Bacterial strains used CP: Chloramphenicol TC: Tetracycline RFP: Rifampicin FA: Fusidic acid EM: Erythromycin

3 Table 2 Serotypes of clinical group B Streptococci isolates (100) Table 3 Susceptibility patterns of clinical group B Streptococci isolates to antimicrobial agents Table 4 Transfer of chloramphenicol and tetracycline resistance determinants CP: Chloramphenicol TC: Tetracycline RFP: Rifampicin -: Not tested

4 452 感 染 症 学雑 誌 Fig. 1 Thin-layer chromatogram and the authentic The solvent BTO22株 system was chloroform: of chloramphenicol methanol= 95: 5. chromatogram 保 有 して い た. のCP TC耐 の 伝 達 頻 度 は10-6レ products は い ず れ も0.5%と 性 因 子 のBMU73株 へ ベ ル で 低 率 で あ っ た も の の, 5.CP不 CPの ア セ チ ル 化 を 証 明 す る こ と に よ りCP不 活 化 酵 素 がCATで びTCの CP不 伝 達 頻 度 が 同 程 度 で あ る こ と か らCP 耐 性 が 連 関 し て い る 可 能 性 が あ る の でCP で 選 択 し た 株 のCP TC耐 のCP TC耐 性,TCで 性 を 調 べ た.そ 択 し て もtransconjugantは も っ て い た.こ 選 択 した 株 の 結 果,ど ち らで 選 両 者 の 耐 性 を 併 せ の こ と か らCP TC耐 性 因 子 は1 低 率 で あ った. 活化産物 の同定 そ の 他 の 株 へ は10-4と 高 頻 度 で 伝 達 さ れ た.CP及 TCの あ る こ とが確 認 され る の で 活 化 産 物 の 同定 を 行 った. Fig.1に 示 す よ うに,BTO株 たtransconjugant株 る1-acetoxy ア セ チ ル 化 機 構 は 各 種 のCAT産 子 の 伝 達 頻 度 はBTO22株 あ っ た.BTO23株 のCP,TC耐 の そ れ と比 べ て 低 率 で 性 は連 関 して い な い も の と 推 定 さ れ る. 4.耐 BTO株3株 μg/ml)及 をethidiumbromide(MICO 生 菌 で 明 らか に さ れ た 経 路 を と る も の と 思 わ れ る, 6.CP耐 性transconjugantのCAT活 donorbto22株 も<0.78μg/mlか 性 の脱落 の こ とか 生 に よ る も の で あ り,そ の 性 無 作 為 に 選 びMICを た.CPのMICは6.25μg/mlか.78 びacrifiavine(MIC3.125μg/ml)で 処 理 し 感 性 株 の 出 現 を 検 し た が,CP耐 性 はCAT産 transconjugantを 性 の脱 落 び1,3-diace- ス ポ ッ トの み が 検 出 さ れ た.こ ら,CP耐 性因 不活化産物 で あ ス ポ ッ トが 検 出 さ れ た がrecipient株 4).BTO21株 のCP,TC耐 及 び耐性 を獲得 し CP,3-acetoxyCP及 toxycpの はCPの に は,CPの つ の 耐 性 因 子 上 に あ る こ と が 推 定 さ れ た(Table 及 びBTO23株 第4号 samples. Thin-layer は す べ て 抗 原IIIを of the inactivated 第61巻 測定 し ら50μg/mlと と 同 程 度 の 耐 性 度 を 得,TC耐 ら25μg/ml 50μg/mlと く上 昇 し て い た(Table5).更 にCATの 性 著 し 活 性を

5 453 昭 和62年4月20日 Table 5 Susceptibility of transconjugants chloramphenicol and tetracycline to Fig. 2 DNA Agarose from gel CP Bacteriophage ƒé a activity digested weight (M 株 に 無 いCAT活 性 が出現 し も つ 酸 素 活 性 と 同 レベ ル ま で 上 昇 し て い た(Table6). Fig.2に 性 菌 の培 養 液 活 化 作 用 を 認 め 報 告 し て 以 来,Suzukilo) 分子量 伝 達 頻 度 が 高 いBTO22株 につ い て 証 明 と分 子 量 測 定 を 行 っ た. BMU72)及 びBMU74/22(BTO22 BMU74) に つ い て,ア ガ ロ ー ス 電 気 泳 動 を 行 っ た と こ ろ, 量 は で mid(cpr,tcr)をpbto22と ン ドが 検 出 さ れ そ の 分 子 あ った.BTO22株 保 有 のplasし た. 達 もす で に びS.faecalisに つ い てCATの 証 明 を 行 い ア セ チル 作 機 構 の 経 路 を 明 らか に して き た. S.agalactiaeのCP耐 性 機 構 に 関 し て は,す に さ れ て い る がCAT産 (BTO22 BMU71),BMU72/22(BTO22 産 生 に レ ン サ 球 菌 属2)のStreptococcuspyogenes,S に 接 合 伝 達 性plasmid12) 14)に 示 す よ う に,BTO22,BMU71/22 同 一 のplasmidDNAバ 活 化 作 用 はCATの よ る も の で あ る こ と を 報 告 し た.私 pneumoniae及 のplasmidDNAの plasmiddnaの as acetyltransferase 測 定 し た と こ ろ,原 CP TCの III numbers 察 子 保 有CP耐 とShawら11)はCP不 7.BTO22株 Hind with in にCP不 donorの with standard, 考 Chloramphenicol plasmid dal). Miyamura9)がR因 CAT: of transconjugants. DNA molecular shown ~106 Table 6 Levels of enzyme (CAT) donor, recipient and transconjugants electrophoresis resistant よ る ことが 明 らか 生 の有無 につ いては不 明 で あ っ た.本 実 験 で 供 試 し たBTO22株 の 分 子 量 を も つpBTO22を CATを 産 生 し た.BTO21株 い て はplasmidを 生,伝 で は, 保 有 し 及 びBTO23株 検 索 し な か っ た が,CATの 達 実 験 等 か らBTO22株 同 様plasmidが につ 産 関

6 - gative 7) Nakagawa, Y., Nitahara, Y. & Miyamura, S.: Kinetic studies on enzymatic acetylation of chloramphenicol in Streptococcus faecalis. Antimicrob. Agemts Chemother., 16: , ) Kado, C.I. and Liu, ST.: Rapid procedure for detection and isolation of large and small plasmids. J. Bactriol., 145: , ) Miyamura, S.: Chloramphenicol inactivation by dysentery bacill, wituh special reference to chloramphenicol resistance. (In Jpanese.). Jpn. J. Bacteriol., 16: , ) Suzuki, Y. & Okamoto, S.: The enzymatic acetylation of chloramphenicol by the multiple drug-resistant Escherichia coli carrying R factor. J. Biol. CHem., 242: , ) Shaw, W.V.: The enzymatic acetylation of chloramphenicol by extracts of R factor-resistant Esherichia coli. J. Biol. Chem., 242: 687 1) Okamoto, S., Suzuki, Y., Mise, K. & Nakaya, R.: Occurrence of chloramphenicol-acetylat- 12) Clewell, D.B.: Plasmid, drug resistance, and ing enzymes in various gram negative bacilli. J. Bacteriol., 94: , ) Miyamura, S., Ochiai, H., Nitahara, Y., Nakagawa, Y. & Terao, M.: Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis. Microbiol. Immunol., 21: 69 3) Terao, M., Morishita, H. & Miyamura, S.: Mechanism of chloramphenicol resistance in Serratia macrosecence recently isolated from a clinical meterial. (In Japanese.). Jpn. J. Bacteriol., 36: , ) Yow, M. Group B streptococci: A serious threat to the neonate. J. Am. Med. Assoc., 230: gene tranfer in the genus sterptococcus. Microbiol. Rev., 45: , ) Hershfield, V.: Plasmids mediating multiple drug resistance in group B Straptococcus: Transferability and molecular properties. Plasmid, 2: , ) Horodniceanu, T., Bougueleret, L., Elsolh, N., Bouanchaud, D. and Chabbert, Y.: Conju R plasmids in Streptococcus agalactiae group B). Plasmid, 2: , ) Clewell, D.B. and Brown, B.: Sex pheromone cad1 in Streptococcus faecalis: Induction of a function related to plasmid transfer. J. Bacteriol., 143: , 1980.

7 Mechanism of Chloramphenicol Resistance in Group B Streptococci (Streptococcus agalactiae) Michinori TERAO & Yuuichi OKAO Niigata Prefectural Research Laboratory for Health and Environment Shuzo OKETANI Department of Oral Microbiology School of Dentistry, Niigata University One hundred group B Streptococci (Streptococcus agalactiae) isolated from clinical specimens investiaged for serotyping and drug susceptibility. Of these, 3 donor strains designated BTO 21, BTO 22 and BTO 23, transferred chloramphenicol (CP) resistance marker and chloramphenicol acetyltransferase (CAT) producing activity by conjugation into group B Streptococci recipient strains. The CP resistance appeared to be due to the presence of CAT mediated by a plasmid. The molecular weight of a streptococcal conjugative R plasmaid, designanted pbto 22, was investigated after transfer into recipient strains and was found to be similar to that the wild-type group B Streptococcus host (19-20 ~106). The products of chloramphenicol acetylation obtained by inactivation were identified as 1- acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives, respectively, by thin-layer chromatography.

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