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3 ペ ニ シ リ ン耐 性 肺 炎 球 菌 のPBPsとPBP2B遺 NO.11 VOL.42 お,PCR混 合 液AとB(そ ク ス にDNAポ 1227 伝 子 につ いて れ ぞ れの プ ライ マー ミ ッ リ メ ラ ー ゼ 等 を加 え た も の21))用 に,1 菌 株 につ き2本 の マ イ ク ロ チ ュ ー ブ を 準 備 し た チ ュ ー ブ は,Gene Amp PCR System9600-R(日 本 ロ シ ュ )に セ ッ トし,60,20分,続 い て90,10 分 の 溶 菌 操 作 を行 っ た 次 い で,PCR混 合 液 のAと Bと を そ れ ぞ れ の チ ュ ー ブ に 別 々 に5μlず つ加 えた PCRは,①50,30秒,②70,30秒,③94,15 秒 の 条 件 下 で30サ イ ク ル 行 い,反 応 後 の 検 体 は,3% 濃 度 の ア ガ ロ ー ス ゲ ル 板 を用 い て電 気 泳 動 を行 っ て 解 析 した II.結 果 1一 薬 剤 感 受 性 とPCRの Fig.1に は,肺 成 績 の関 係 炎 球 菌3株 に つ い てPCRを 行 い, そ の増 幅 産物 を ゲル 電 気泳 動 した代 表 的 な成 績 を示 す PSSPと 記 し て あ るUB-034株 は,PCGに0.016 μg/ml,mpipcに0.03lμg/mlのmicを 感 受 性 の 上 か らPSSPと ラ ム はlytA遺 DNA分 示 し,薬 剤 判 定 さ れ た 株 で あ る Aカ 伝 子 用 の プ ライ マー で増 幅 され る 子 量 に 相 当 し,Bカ ラ ム はPSSPのPBP2B 検 出 用 プ ラ イ マ ー で増 幅 さ れ る分 子 量 に相 当 した DNAで あ る 以 下,こ の よ う にPCR上 判 定 さ れ る菌 株 をclassSと 中 央 のClassAと でPSSPと 称 す る こ と と した したUB-023株 は,PCGにO.25 μg/ml,mpipcに1.0μg/mlのmicを のPRSPと PCR混 が,分 度 判 定 さ れ た 株 で あ る こ の 菌 株 で は, 合 液Aの み で2本 のDNAが 検 出 され てい る 子 量 の 大 きい 上 の バ ン ドがlytA遺 PBP2BのclassA変 幅 され たDNAの 下,こ 示 し,軽 Fig. 1. Agarose gel electrophotogram of PCR-amplified DNA fragments of the autolysin (lyta) and PBP 2 B (pena) genes. a) penicillin-sensitive Streptococcus pneumoniae strain UB034; b) penicillin-resistant S. pneumoniae strain UB-023; c) penicillin-resistant S. pneumoniae strain UB-008. 伝 子,下 for ly. A and 表現す る contains two sets amplification PBP-2A Tube mers, 分 子 量 に一 致 し た バ ン ドで あ る 以 genes, B contains for of two amplification normal PBP-2B of pri- lyta and respectivesets of genes, of priclass B respec- tively. こと とした 右 側 のUB-008株 A mers, class が 異 を検 出 す る プ ラ イマ ー で 増 の よ う な 菌 株 をclassAのPRSPと Tube は,PCGに4.0μg/ml,MPIPC に8.0μg/mlのMICを 示 し,明 る こ の 菌 株 で はPCR混 当 す るDNA,PCR混 ら か なPRSPで 合 液AでINtA遺 あ 伝 子 に相 め られ る 以 下,こ 分 子 量 に 一 致 し た バ ン ドが 認 の 場 合 に はclassBのPRSPと 成 績 と,β-ラ クタム 系薬感 受性 と 印 で 示 し た4株 で は,変 異 した 検 出 す る た め の プ ラ イ マ ー で はDNAの イ マ ー でDNAが 出用 の プ ラ 増 幅 され た つ ま り,PRSPで あ り 判 定 され て い た こ とで,両 者 の成 の 関 係 をFig.2に 示 す 薬 剤感 受 性成 績 の上 か らの な が ら,classSと PRSPとPSSPの 識 別 は,NCCLSの 績 が 必 ず し も一 致 して い な い菌 株 が み られ た PCGに 対 し て0.1μg/ml以 る い はMPIPCに1.0μg/ml以 PRSPと す る と,こ 勧 告8}に 従 い, 上 のMICを 上 のMICを 示 す 株,あ 示す株 を の 条 件 を 共 に満 た す 菌 株 は 左 上 ま た,PCGに は0.063μg/mlのMICを MPIPCに1.0μg/mlのMICを した が,こ 印 印 で 示 し た1 識別 された し か し な が ら, PBP2Bを で あ っ た そ の うち, はclassBのPRSP, 増 幅 が 認 め られ ず,PSSPのPBP2B検 称 す る こ ととした 各 菌 株 のPCRの で 示 し た10株 株 はclassAのPRSPと 合 液BでPBP2BのclassB 変 異 を検 出 す るDNAの の 図 に 点 線 で 囲 ん だ15株 れ もPRSPで 示 し, 示 し た 株 が1株 あ りな が らclassSと 存在 判定 さ

4 1228 CHEMOTHERAPY NOV Fig. 2. Correlations between the presence of the PBP 2 B (pena) gene and œ normal pena-positive strains by PCR; M: class A pena-positive strains by PCR; A: class B pena-positive strains by PCR. The breakpoints for sensitivity and resistance to penicillin G and oxacillin are based on the recommendations of the NCCLS8).

5 PCG: penicillin G, MPIPC: oxacillin Fig. 3. Fluorogram of the penicillin-binding proteins of penicillin-resistant Streptococcus pneumoniae. Membrane fractions were incubated with [3H]-PCG for 10 min at 32 Ž, sarcosyl solubilized, and electrophoresed.

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7 Fig. 6. Correlations between the affinity of penicillin-binding proteins and the presence of normal or altered PBP 2 B genes in penicillin-resistant and -sensitive Streptococcus Pneumoniae. (A) reduction of affinitity of [31-1]-PCG for PBP 2 B; (B) normal affinity of [31-1]-PCG for PBP 2 B.0 and normal pena-positive strains by PCR;

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9 rds. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M 7-A 3. National Committee for Clinical Laboratory Standerds, Villanova, Pa ) Klugman K P: Pneumococcal resistance to antibiotics. Clin Microbiol Rev 3: 171 `196, ) Hakenbeck R, Tarpay M, Tomasz A: Multiple changes of penicillin-binding proteins in penicillin-resistant isolates of Streptococcus Pneumoniae. Antimicrob Agents Chemother 17:364 11) Percheson P B, Bryan L E: Penicillin-binding components of penicillin susceptible and -resistant strains of Streptococcus pneumoniae. Antimicrob Agents Chemother 18: , ) Zighelboim S. Tomasz A: Penicillin-binding proteins of multiply antibiotic-resistant South African strains of Streptococcus pneumoniae. Antimicrob Agents Chemother 17: , ) Hakenbeck R Ellerbrok H Brise T Handwerger S, Tomasz A: Penicillin-binding proteins of penicillin-susceptible and -resistant pneumococci: immunological relatedness of altered proteins and changes in peptides carrying the fi-lactam binding site. Antimicrob Agents Chemother 30: 553 `558, ) Jabes D, Nachman S, Tomasz A: Penicillinbinding protein families: Evidence for the clonal nature of penicillin resistance in chinical isolates of pneumococci. J Infect Dis 159:16-25, ) Hakenbeck R, Briese T, Chalkley L, Ellerbrok H, Kalliokoski R, Latorre C Leinonen M, Martin C: Antigenic variation of penicillin-binding proteins from penicillin-resistant clinical strains of Streptococcus pneumoniae. J Inf Dis 164:313 17) Ubukata K, Yamasita N, Konno M: Occurence of a P-lactam-inducible penisillin-binding prptein in methicillin-resistant staphylococci. Antimicrob Agents Chemother 27: , ) Dowson C G, Hutchison A, Spratt B G: Nucleotide sequence of the penicillin-binding protein 2B gene of Streptococcus pneumoniae strain R 6. Nucleic Acids Res 17: 7518, ) Dowson C G, Hutchison A, Spratt B G: Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2 B of a penicillin -resistant South African isolate of Streptococcus pneumoniae. Mol Microbiol 3: , ) Garcia P, Garcia K L, Garcia E, Lopez R: Nucleotide sequence and expression of the pneunococcal autolysin gene from its own promoter in Escherichia coli. Gene 43: , ) Ubukata K, Nakagami S, Nitta A, Yamane A, Kawakami S, Sugiura M, Konno M: Rapid detection of the meca gene in methicillinresistant staphylococci by enzymatic detection of polymerase chain reaction products, J Clin Microbiol 30: , ) Markiewicz Z, Tomasz A: Variation in penicillin -binding protein patterns of penicillin-resistant clinical isolates of pneumococci. J Clin Microbiol 27: , ) Martin C, Sibold C, Hakenbeck R: Relatedness of penicillin-binding protein la genes from different clones of penicillin-resistent Streptococcus pneumoniae isolated in South Africa and Spain. EMBO J 11: , ) Laible G, Hakenbeck R, Sicard M A, Joris B, Ghuysen J M: Nucleotide sequences of the pbpx genes encoding the penicillin-binding proteins 2 X from Streptococcus pneumoniae R 6 and a cefotaxime-resistant mutant, C 506. Mol Microbiol 3: , ) Laible G, Spratt B G, Hakenbeck R: Interspecies recombinational events during the evolution of altered PBP 2 x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Mol Microbiol 5: , ) Munoz R, Dowson C G, Daniels M. Coffey T J, Martin C, Hakenbeck R, Spratt B G: Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae. Mol Microbiol 6: , ) Potgieter E, Chalkley L: Reciprocal transfer of penicillin resistance genes between Streptococcus pneumoniae, Streptococcus mitior, and Streptococcus sanguis. J Antimicrob Chemother 28: , ) Dowson C G, Hutchison A, Brannigan J A, George R C, Hansman D, Linares J, Tomasz A, Smith J M, Spratt B G: Horizontal transfer of penicillin-binding protein genes in penicillin -resistant clinical isolates of Streptococcus pne , ) Chalkley L, Schuster C, Potgieter E, Hakenbeck R: Relatedness between Streptococcus pneumoniae ana viridans streptococci: transfer of penicillin resistance determinants and immunological similarlities of penicillin-binding proteins. FEMS Microbiol Lett 90:35-42, ) Dowson C G, Coffey T J, Kell C, Whiley R A: Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in

10 the formation of a law affinity PBP 2 B In S. pneumoniae. Mol Microbiol 9: 635 `643, ) Coffey T J, Dowson C G, Daniels M, Zhou J, Martin C, Spratt B G, Musser J M: Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae. Mol Microbiol 5: 2255 `2260, ) Munoz R, Musser J M, Crain M, Briles D E, Marton A Parkinson A J Sorenson U, Tomasz A: Geographic distribution of penicillinresistant clones of Streptococcus pneumoniae: charcterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis. Clin Infect Dis 15: , ) Figueiredo A M, Connor J D, Severin A, Vaz Pato M V, Tomasz A: A pneumococcal clinical isolate with high-level resistance to cefotaxime and ceftriaxone. Antimicrob Agents Chemother 36: , ) Bradley J S. Connor J D: Ceftriaxone failure in meningitis caused by Streptococcus pneumoniae with reduced susceptibility to bata-lactam antibiotics. Pediatr Infect Dis J 11:871^-873, ) Sloss M M, Barrett F F, Chesney J P, English B K, Hill B C, Tenover F C, Leggiadro R: Cephalosporin treatment failure in penicillinand cephalosporin-resistant Streptococcus Pneumo- 666, ) Kleiman M B, Weinberg G A, Reynolds J K, Allen S a Meningitis with beta-lactam resistant Streptococcus pneumoniae; the need for early repeat lumbar puncture. Pediatr Infect Dis j 12: 782 `784, ) Viladrich P F, Gudiol F, Linares J, Pallares R, Sabate I, Rufi G, Ariza J: Evaluation of vancomycin for therapy of adult pneumococcal meningitis. Antimicrob Agents Chemother 35: 2467 `2472, ) Rudolph K M, Parkinson A J. Black C M, Mayer L W: Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia. J Clin Microbiol 31: , 1993

11 Mechanisim of resistance in penicillin-resistant clinical isolates of Streptococcus pneumoniae: Analysis of penicillin-binding proteins and the PBP 2 B gene Kimiko Ubukata, Mutumi Sugiura and Masatoshi Konno Department of Clinical Pathology, Teikyo University, School of Medicine, Tokyo, Japan Shigeru Ohnari Department of Pediatris, Saiseikai Hiroshima Hospital, Hiroshima, Japan Akio Yamane Wakunaga Pharmaceutical Co., Ltd., Hiroshima, Japan Susceptibility to fl-lactam antibiotics, affinity of penicillin-binding proteins (PBPs), and the detection of the PBP 2 B gene (pena) by the polymerase chain reaction (PCR) were studied using 30 clinical strains of pneumococci isolated in The following results were obtained: 1) Fifteen of strains were identified as penicillin (PCG) -rsistant strains of pneumococcus (PRSP), with minimum inhibitory concentrations (MIC) of PCGa0.125pg/ml. 2) The MICs of oxacillin, ampicillin, cefotaxime, cefdinir, and imipenem clearly differed in PRSP and PCG-sensitive strains of pneumococcus (PSSP). 3) PCR was performed by preparing three sets of primers for amplification of part of the PBP 2 B gene in order to differentiate between PRSP and PSSP. DNA from 15 strains with a PCG MIC <0.125 jig/m1 was amplified using primers for detection of sensitive strains. The strains for which DNA was amplified with primers for PRSP detection all had MICs a yg/ml. These strains consisted of one class A strain and ten class B strains. However, DNA was amplified by primers for PSSP even in four PRSP strains. 4) In PRSP strains, the affinity of [3H]-PCG for PBP 2 B was reduced, and affinity for PBP-1 A or PBP-1 B was also reduced in many strains. Some PSSP strains showed reduced affinity for PBP-1 A or PBP 1 B, and PBP-2 X newly appeared in some strains. These results suggest that many PRSP in Japan are variable strains with an altered PBP 2 B gene belonging to class B, that there are few class A strains,and that there are PCGresistant strains with changes in the PBP-2 B gene that have not yet beem reported.

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