海水、海泥、カキ及び海産魚類から分離したVibrio vulnificus並びにヒト臨床例由来のV. vulnificusの疫学的検討

Vibrio vulnificus（V.vulnificus）は、海水や汽水を主な生息域とする低濃度好塩性のグラム陰性桿菌である。本菌のヒトへの感染例は、1970年にRolandがヒトの下肢創傷部の皮膚滲出液から本菌分離について報告したのが最初である（Roland, F. P.: New Engl. J. Med., 282:1306, 1970）。本菌は、健康なヒトには感染しないが、肝疾患患者や免疫力の低下したヒトに感染して重篤な感染症を引き起こすことから、近年注目されている。本菌感染症は、臨床症状の違いによって、経口感染型、創傷感染型及び胃腸型の3タイプに分類されている。とくに経口感染型では、発症すると高い割合で敗血症を起し、重篤化して死亡率も高い。本菌の疫学的調査に血清学的手法や分子生物学的手法が用いられているが、詳細な報告はみられない。それ故に、本菌の感染源や感染経路については不明な点が多いのが実状である。また、薬剤感受性については、臨床治療に用いられている各種抗菌剤に対する系統的な検討は行われておらず、その耐性度も明らかにされてないのが実状である。　そこで著者は、本菌の基礎的研究の一環として、自然環境下における本菌の分布状況と汚染菌量の調査を行い、本菌が海水、海泥、カキ及び海産魚類の5.4～54.8%に分布していることを明らかにした（大仲ら：感染症誌、76、528、2002; Oonaka et al.: Jpn. J. Food Microbiol., 25、89、2008）。　本研究の概要は次のとおりである。　最初に、海水、海泥、カキ及び海産魚類から分離した株（環境由来株）とヒト臨床由来株についてShimadaらの方法（Shimada et al.: Jpn. J. Med. Sci. Biol.、37, 241, 1984）に準拠して血清型別を行い、薬剤感受性を検討した。次に、本菌の感染源や感染経路を明らかにする目的で、パルスフィールド・ゲル電気泳動（pulsed-field gel electrophoresis: PFGE）法で解析を行った。I. 環境由来及びヒト臨床由来のV.vulnificus分離株の血清型別と薬剤感受性　本研究には、環境由来の917株及び病院から分与されたヒト臨床由来の62株のV.vulnificus、合計979株を用いた。前者の917株は、千葉県、東京都、徳島県の海水、海泥、カキ（1998年10月～2000年11月に採取、分離した）及び全国9ヵ所の海産魚類（2003年5月～2004年9月に採取、分離した）、後者のヒト臨床由来株は、全国11ヵ所及び海外で分離された株を用いた。Shimadaらの作成したV.vulnificusの血清型（O1～O18：O17、O18は欠番）を用いて血清型別を行ったところ、587株（60.0%）が13血清型に型別されたが、残りの392株（40.0%）については型別不能であった。その型別不能株について、新たにO19～O23の5つの血清型を追加作成して血清型別を行ったところ、122株がこれらの血清型に型別することができた。そして、その結果型別不能株は最終的に270株（27.6%）に減少した。　環境由来の917株では655株（71.4%）が18血清型に型別され、それらの中ではO7が371株（40.5%）と最も多く、続いてO4が58株（6.3%）、O22が45株（4.9%）、O19が37株（4.0%）であった。しかし、残り262株（28.6%）は型別不能であった。一方、ヒト臨床由来の62株では54株（87.1%）が8血清型に型別され、それらの中ではO4が27株（43.5%）と最も多く、続いてO7が8株（12.9%）、O6が7株（11.3%）、O1が5株（8.1%）で、残り8株（12.9%）は型別不能であった。この様に、血清型別に関する解析の結果、両方の由来株には共通してO7とO4が多く認められることから、両血清型と起病性との間には関連性があることが示唆される。　次に採取した地域と血清型の異なる環境由来の191株とヒト臨床由来の57株の計248株について、20薬剤を用いて薬剤感受性試験を行い、各種薬剤に対するMinimum Inhibitory Concentration（MIC）_90を検討した。環境由来株のMIC_90は、Meropenem（MEPM）とCiprofloxacin（CPFX）が各0.05μg/mlと最も抗菌力が高く、次にDoxycycline（DOXY）とMinocycline（MINO）が各0.2μg/ml、Tetracycline（TC）が0.39μg/ml、Chloramphenicol（CP）とNalidixic acid（NA）が各0.78μg/ml、Cefotaxime（CTX）及びErythromycin（EM）が各3.13μg/ml、Gentamicin（GM）が6.25μg/ml、Ampicillin（ABPC）、Cefaloridine（CER）、Cefalotin（CET）及びCefoperazone（CPZ）が各12.5μg/ml、Piperacillin（PIPC）、Cefmetazole（CMZ）、Latamoxef（LMOX）及びAmikacin（AMK）が各25μg/ml、Kanamycin（KM）が50μg/ml、Lincomycin（LCM）が100<μg/mlであった。環境由来株では、リンコマイシン系のLCMに対して耐性株が認められた。一方、ヒト臨床由来株のMIC_90はCPFXが0.05μg/mlと最も抗菌力が高く、続いてMINOが0.1μg/ml、DOXYが0.39μg/ml、TCとNAが各0.78μg/ml、EMが3.13μg/ml、GMが12.5μg/ml、CER、CET、KM及びAMKがいずれも50μg/mlであり、ABPC、PIPC、CPZ、CTX、CMZ、LMOX、MEPM及びLCMのいずれもが100<μg/mlであった。ヒト臨床由来株ではβ-ラクタム系やリンコマイシン系に対して耐性株が多く認められた。　以上の様に、ヒト臨床由来株は環境由来株に比べてβ-ラクタム系やアミノグリコシド系の薬剤に耐性傾向が高いことが明らかになった。II. 環境由来株及びヒト臨床由来株の分子疫学的検討　分子疫学的検討は環境由来の355株とヒト臨床由来の65株の計420株について、Sfi IとNot Iを用いた無傷ゲノムDNA切断後のPFGEパターンで行った。その結果、Not Iでは312株（74.3%）で、Sfi Iでは411株（97.9%）でそれぞれPFGEパターンが得られ、Sfi Iの方がこれらV.vulnificus株の株間識別能において優れていることが明らかになった。また、DNAの切断パターンで、Not Iでは約20～600kilo base pair（kbp）の間に15本から22本のバンドが、Sfi Iでは20～500kbpの間に15本から22本のバンドがそれぞれ確認された。次に、切断が高い割合で認められた制限酵素Sfi Iで切断された411株（環境由来の349株、ヒト臨床由来の62株）のPFGE像の類似度が89%以上の場合を同一群としてUnweighted Pair-Group Method with Arithmatic mean（UPGMA）法によるクラスター解析を行った。その結果、同一群に属する環境由来株とヒト臨床由来株は少なく、18組のみが同一群に位置することが明らかになった。また、一致した18組の中で4組（22.2%）は同一血清型であったが、他の14組（77.8%）は異なる血清型であった。さらに、V.vulnificusの採取場所別に解析したところ、採取場所が同一地点であった18組中10組（55.6%）で同一血清型が認められたが、他の8組（44.4%）では異なっていた。なお、同一血清型が認められた10組中1組（10.0%）のV.vulnificusは東日本地域の海水と西日本地域のカキからそれぞれ分離されたものであった。由来別では18組中15組（83.3%）が環境由来株、2組（11.1%）がヒト臨床由来株、1組（5.6%）が環境由来株とヒト臨床由来株の両方の由来であった。　以上の様に、V.vulnificusの血清型別に関する解析で、既知の血清型に新たに筆者が追加作成した血清型を加えて検討したところ、環境由来株では71.4%が、ヒト臨床由来株では87.1%がそれぞれ血清型別されることが明らかになった。また、両由来株は共通してO7やO4に型別される菌株が多く認められ、両血清型と起病性との間に関連性があることが示唆された。　薬剤感受性試験で、ヒト臨床由来株はβ-ラクタム系やアミノグリコシド系の薬剤に対して耐性傾向にあることが明らかになった。　分子疫学的検討では、Not Iでは76.9%、Sfi Iでは97.9%の切断率がそれぞれ示され、本菌のゲノムDNAの切断にはSfi Iが優れていることが明らかになった。さらに、Sfi IによってDNA切断した菌株のPFGEの泳動像をUPGMA法でクラスター解析を行ったころ、PFGE法が本菌の分子疫学的解析に応用可能であることが示唆された。Vibrio vulnificus (V. vulnificus) is a low-salt halophile gram-negative rod mainly inhabiting sea and estuarine water. The first reported case of human infection with this bacterium was described by Roland in 1970, in which the bacterium was isolated from skin exudate from a leg wound (Roland, F. P.: New Engl. J. Med. 282:1306, 1970). This bacterium has recently been attracting attention because it infects liver disease patients and humans with impaired immunity, but not healthy humans, and induces severe infectious disease. The disease is divided into 3 types based on the clinical symptoms: oral, wound, and gastrointestinal infection types. In the oral infection type, sepsis occurs after the onset at a high rate and aggravates, leading to a high mortality rate. Serological and molecular-biological techniques are used for epidemiological surveys of this bacterium, but there has been no detailed report. Accordingly, many points regarding the source and route of infection of this bacterium are unclear. We investigated the distribution and contamination level of this bacterium in natural environments as part of a basic study, and found that the bacterium was present in 5.4-54.8% of sea water, sea mud, oysters, and marine fish (Oonaka et al.: kansenshoshi 76:528, 2002; Oonaka et al. Jpn. J. Food Microbiol. 25:89, 2008). A summary of this study is as follows: Serotypes of strains isolated from sea water, sea mud, oysters, marine fish (environmental isolates), and human clinical isolates were determined by the method reported by Shimada et al. (Shimada et al.: Jpn. J. Med. Sci. Biol. 37:241, 1984), and drug sensitivity was investigated. To identify the source and route of infection, the isolates were analyzed by pulsed-field gel electrophoresis (PFGE).I. Drug sensitivity by serotypes of environmental and human clinical isolates of V. vulnificus This study was performed using 917 environmental and 62 human clinical isolates provided by hospitals, with a total of 979 strains of V. vulnificus. The former 917 strains were isolated from sea water, sea mud, and oysters from Chiba and Tokushima Prefectures and the Tokyo Metropolitan area (sampled and isolated in October 1998-November 2000) and marine fish from 9 regions nationwide (sampled and isolated in May 2003- September 2004). The latter were isolated in 11 regions of Japan and overseas. On serotyping following the V. vulnificus serotypes established by Shimada et al. (O1-O18 excluding missing numbers of O17 and O18), 587 isolates (60.0%) were typed into 13 serotypes, but the remaining 392 isolates (40.0%) could not be typed. When 5 new serotypes (O19-O23) were added to typing of the non-typed isolates, the 5 serotypes were identified in 122 isolates, reducing the number of non-serotype identifiable isolates to 270 (27.6%). Of the 917 environmental isolates, 655 (71.4%) were typed into 18 serotypes, and 371 were typed O7 (40.5%), the most common, followed by 58 isolates typed O4 (6.3%), 45 typed O22 (4.9%), and 37 typed O19 (4.0%). The remaining 262 isolates (28.6%) could be not be typed. Of the 62 human clinical isolates, 54 (87.1%) were typed into 8 serotypes: 27 were typed O4 (43.5%), the most common, followed by 8 isolates typed O7 (12.9%), 7 typed O6 (11.3%), and 5 typed O1 (8.1%), and the remaining 8 isolates (12.9%) could not be typed. The frequent identification of O7 and O4 was common to the environmental and clinical isolates (46.8 and 56.4%, respectively), suggesting the presence of an association between the two serotypes and pathogenicity. Drug sensitivity tests involving 20 drugs were performed in 191 environmental strains isolated from various regions and typed serotypes, and 57 human clinical isolates, with a total of 248 strains, and the minimum inhibitory concentrations (MIC)_90 of the drugs were determined. For the environmental isolates, the MIC_90 of meropenem (MEPM) and ciprofloxacin (CPFX) was 0.05 mg/ml, respectively, showing the most potent antibacterial activity, followed by 0.2 mg/ml for doxycycline (DOXY) and minocycline (MINO), 0.39 mg/ml for tetracycline (TC), 0.78 mg/ml for chloramphenicol (CP) and nalidixic acid (NA), 3.13 mg/m for cefotaxime (CTX), latamoxef (LMOX), and erythromycin (EM), 6.25 mg/ml for gentamicin (GM), 12.5 mg/ml for ampicillin (ABPC), cefaloridine (CER), cefalotin (CET), and cefoperazone (CPZ), 25 mg/ml for piperacillin (PIPC), cefmetazole (CMZ), latamoxef (LMOX), and amikacin (AMK), 50 mg/ml for knamycin (KM), and 100< mg/ml for lincomycin (LCM) . LCM resistance was noted in the environmental isolates. For the human clinical isolates, the MIC_90 of CPFX was 0.05 μg/ml, showing the most potent antibacterial activity, followed by 0.1 μg/ml for MINO, 0.39 μg/ml for DOXY, 0.78 μg/ml for TC and NA, 3.13 μg/ml for EM, 12.5 μg/ml for GM, 50 μg/ml for CER, CET, KM, and AMK, and 100< μg/ml for ABPC, PIPC, CPZ, CTX, CMZ, LMOX, MEPM, and LCM. Many human clinical isolates were resistant to β-lactams and lincomycin antibiotics. The above findings clarified that the human clinical isolates tended to be resistant to β-lactams and aminoglycosides, compared to the environmental isolates.II. Molecular-epidemiology of environmental and human clinical isolates Molecular-epidemiological analysis was performed using 355 environmental and 65 human clinical isolates, with a total of 420 isolates. The PFGE pattern after the cleavage of intact genomic DNA with Sfi I and Not I was investigated. PFGE patterns of digests with Not I and Sfi I were acquired in 312 (74.3%) and 411 (97.9%) isolates, respectively, showing that Sfi I has a more discriminating power among the V. vulnificus strains. Regarding the DNA cleavage pattern, Not I produced 15-20 bands of about 20-600 kilobase pairs (kbp), and Sfi I produced 15-22 bands of 20-500 500 kbp. After treatment with Sfi I, which cleaved the isolates at a high rate, cluster analysis of 411 isolates (349 environmental and 62 human clinical isolates) was performed using the unweighted pair-group method with arithmetic mean (UPGMA), regarding isolates with an 89% or higher similarity in the PFGE profile as belonging to one group. Only a small number of environmental and human clinical isolates belonged to the same clusters: only 18 pairs belonged to the same groups. Furthermore, only 4 of the 18 pairs (22.2%) were serotyped the same, but the serotype was different in the other 14 pairs (77.8%). When analysis was performed by V. vulnificus-sampling sites, the serotype was identical in 10 of the 18 pairs (55.6%) sampled at the same sites, but different in the other 8 pairs (44.4%). In one of the 10 pairs with identical serotypes (10.0%), one was isolated from sea water of the East Japan area and the other from oysters in the West Japan area. By the origin of isolates, 15 (83.3%) of the 18 pairs were environmental isolates, 2 (11.1%) were human clinical isolates, and one (5. 6%) was a pair of environmental and human clinical isolates. When the new serotypes were added to the analysis by serotypes, the serotype was determined in 71.4 and 87.1% of the environmental and human clinical isolates, respectively. In addition, many isolates were typed O7 and O4 in both the environmental and human clinical isolates, suggesting the presence of an association between the 2 serotypes and pathogenicity. The drug sensitivity tests clarified that the human clinical isolates tended to be resistant to β-lactams and aminoglycosides. On molecular-epidemiological analysis, the Not I and Sfi I cleavage rates were 76.9 and 97.9%, respectively, showing that Sfi I is superior regarding the genomic DNA cleavage of this bacterium. In addition, cluster analysis of the PFGE patterns of Sfi I-cleaved strains by the UPGMA method suggested that the PFGE method is applicable for molecular-epidemiological analysis of this bacterium.博士(学術)麻布大

市販果汁飲料からの耐熱性好酸性菌の検出

To investigate contamination of commercial fruit juice beverages with thermostable acidophilic bacteria in Japan, 208 samples of fruit juice beverages produced in Japan and 162 samples of import beverages (total of 370 samples) purchased in 2005 were investigated according to the `Standard Test Method for Thermostable Acidophilic Bacteria\u27 proposed by the Japan Fruit Beverage Association. TAB was isolated from 9 of the beverages made in Japan (4.3%) and 19 of the import beverages (11.7%), showing about a 3-fold higher isolation rate. The origins of the import beverages from which TAB was isolated were widely distributed : Thailand, Indonesia, Korea, France, Germany, Spain, Australia, America, Mexico, and South Africa. Three of the 9 domestic products from which TAB was isolated were mix juice. As the 2nd most frequently contaminated beverage, 2 orange juice products were contaminated, and 1 sample each of apple, grape, grape fruit, and pineapple juices were also contaminated. Regarding the import beverages, 5 of the 19 samples from which TAB was isolated were mix juice, as in the Japan-made products. The 2nd most frequently contaminated beverages were 3 orange juice products. TAB was also isolated from 2 samples each of grape, mango, and pineapple juices, and 1 sample each of apple, cherry, peach, and prune juices. TAB was isolated from various fruit juice beverages regardless of whether they were domestic products or imports. All of the 9 Japan-made beverages contaminated with TAB were concentrated products, and the bacteria were not detected in any of the pure direct products. The same tendency was noted in the import beverages. Ten of the 19 samples (52.6%) contaminated with TAB were concentrated, and only 1 straight sample (5.3%) was contaminated. Seven of the 9 contaminated domestic products (77.8%) were paper-packed, and 1 sample each was packed in a can or PET bottle. As for the import beverages, 8 beverages (42.1%) were most frequently paper-packed, as in the Japan-made products, and 6 (31.6%), 3 (15.8%), and 2 (10.5%) beverages were packed in a bottle, PET bottle, and can, respectively

糞便汚染指標としての大腸菌検出に関する基礎的検討

各種特定酵素基質培地を用いて環境水から大腸菌の検出を試み,検出精度を中心に比較検討を行った。陽性管数の比較では,4種類の市販培地問に有意差は認められず,いずれの培地でも大腸菌検出に支障はなかった。偽陽性に関しては,C.koseri, E.hermannii, Y.regensburgei, Yersina sp., Aeromonas sp.などの菌種によっても陽性反応が起こることが明らかとなった。また,偽陰性に関しては,大腸菌のなかにもわずかながらβ-グルクロニダーゼ陰性株が存在することが確認された。このように,偽陽性および偽陰性の可能性は否定できないが,水道の分野では安全側に傾斜した評価のため,偽陽性は必ずしも試験法における大きな問題点とはならず,試験法の特性として,こうした事実を十分に認識した上で検査を行うことが重要である。A new water quality regulation came into effect in April 2004, and the microbiological test item of tap water, coliform group, has been changed to Escherichia coli. To investigate the accuracy of the new test method, we evaluated E.coli in environmental water using commercial culture media, and investigated false negativity and positivity. Four types of commercial medium were simultaneously used to detect E.coli. No significant difference was noted in the number of positive tubes, and E.coli was detectable using all media to a similar degree. Regarding false positivity, Citrobacter koseri, Escherichia hermannii, Yokenella regensburgei, Yersinia sp., and Aeromonas sp. showed positive reactions at a level of about 10^0-10^2 CFU. As for false negativity, 2 strains of E.coli were MUG reaction-negative. Therefore, false positivity and negativity may be judged, although the possibility is low

Vibrio vulnificus感染症に関する疫学的検討

Vibrio vulnificus感染症の感染経路や感染源を解明する一環として,環境由来株とヒト臨床由来株について血清型別,各種抗菌剤の薬剤感受性試験およびPFGE法によりDNA解析を行い分子疫学的検討を行ったところ,以下の成績が得られた。1.由来別に血清型を検討したところ,環境由来では72.5%が18菌型に型別され,O7が43.1%と最も多く,次にO4が6.1%などであった。ヒト臨床由来では87.1%が8菌型に型別され,O4が43.5%と最も多く,次にO7が12.9%などであった。その地域別において,東日本地域では69.2%が18菌型に型別され,O7が44.6%,04が5.7%などに,西日本地域では64.8%が8菌型に型別され,O7が20.4%と最も多く,次にO4が11.1%などにそれぞれ型別された。2.由来別に薬剤感受性をMIC_で比較検討したところ,環境由来ではABPC,PIPC,CPZ,CTX,LMOX,MEPM,GM,EM,TC,DOXY,MINO,CP,NAおよびCPFXに感受性を示したが,CER,CET,CTX,CMZ,KMおよびLCMに対して耐性株が認められた。ヒト臨床由来ではEM,TC,DOXY,MINO,CP,NAおよびCPFXに感受性を示したが,ABPC,PIPC,CER,CET,CPZ,CTX,CMZ,LMOX,MEPM,KM,GM,AMKおよびLCMに対して耐性株が認められた。3.Not IとSfi Iの2種類の酵素を用いてそれぞれDNA切断を行い,PFGE法でDNA解析を検討したところ,酵素別のDNAパターン判読率は,Not I では76.9%,sfi Iでは97.9%を示し本菌のDNAパターン判読率はSfi Iが優れていた。4.Sfi IによってDNAパターンが判読された菌株をUPGMA法で解析を行ったところ,類似度が低く多岐のクラスターを示し,本菌感染症は複数のクローンから発生していることが明らかとなった。また,由来等が異なるが類似度が89%以上の類似度で,臨床株と環境株の組合せが認められることから,環境からヒト,ヒトから環境への感染の可能性が考えられた。As a part of elucidation of the route and source of Vibrio vulnificus infection, antimicrobial drug sensitivity tests and molecular-epidemiological investigation by DNA analysis using PFGE of each serotype of environmental and human clinical strains were performed, and the following findings were obtained. 1. On serotyping of the strains from each source, 72.5% of the environmental strains belonged to 18 types : the most frequent type was O7, accounting for 43.1%, followed by O4 accounting for 6.1%. As for the human clinical strains, 87.1% belonged to 8 types : the most frequent type was O4, accounting for 43.5%, followed by O7 accounting for 12.9%. 3. On determination of MIC_ for the strains from each source as drug sensitivity, the environmental strains were sensitive to ABPC, PIPC, CPZ, CTX, LMOX, MEPM, GM, EM, TC, DOXY, MINO, CP, NA, and CPFX, but there were strains resistant to CER, CET, CTX, CMZ, KM, and LCM. The human clinical strains were sensitive to EM, TC, DOXY, MINO, CP, NA, and CPFX, but there were strains resistant to ABPC, PIPC, CER, CET, CPZ, CTX, CMZ, LMOX, MEPM, KM, GM, AMK, and LCM. 4. DNA was cleaved with 2 enzymes, Not I and Sfi I, and DNA was analyzed by PFGE. The DNA pattern reading rate was 76.9% on cleavage with Not I and 97.9% with Sfi I, showing that Sfi I was superior in reading the DNA patterns. 5. When strains whose DNA patterns were read on cleavage with Sfi I were analyzed by the UPGMA method, the similarity was low, and various clusters were present, clarifying that this infectious disease is caused by multiple clones. Moreover, combinations of clinical and environmental strains with 89% or higher similarity were noted, although the sources were different, showing a possibility of transmission from the environment to humans and vice versa

ヒト下痢症由来および鶏肉由来Campylobacter属の疫学的研究

Campylobacter感染症に関する基礎的研究の一環として,2002～2006年にかけてヒト下痢便および鶏肉,鶏糞便から分解した株を用いて,血清型別,RAPD法による分子疫学的解析,薬剤感受性試験およびキノロン系薬剤に対する耐性株の遺伝子変異について検討を行ったところ,以下の成績が得られた。1)ヒト臨床由来53株と鶏由来102株の計155株を用いて血清型別を行ったところ,85株(54.8%)が16菌型に型別されたが,残りの70株(45.2%)は型別不能であった。型別された85株の内訳はY群に20株(12.9%)と最も多く,次にB群18株(11.6%),D群8株(5.2%)であった。由来別において,ヒト臨床由来ではB群に,鶏由来ではY群にそれぞれ多く型別された。2)RAPD法による分子疫学的検討を行ったところ,供試した155株中124株(80.0%)が類似度89%≦において1～30群に分類され,22群に22株(14.8%)と最も多く,次に18群10株価7%),16群8株(5.4%)などであった。由来別においては同一由来別に分類される傾向が強く認められ,由来の異なる株が共通した群へ分類される株は少数であった。3)ヒト下痢症由来53株の各種薬剤に対するMICの分布状況をMIC_で比較したところ,GMが0.5μg/mlで最も感受性が高く,次にSMとLMが各2μg/ml,EMが4μg/ml,KMとCPが各8μg/ml,RXMが16μg/ml,MINOとCPFXが各32μg/ml,NAとNFLXが各128μg/ml,ABPC,PIPC,CEXおよびTCが各128. The MIC_ value of GM was 0.5 mg/ml, showing the highest sensitivity, followed by: SM and LM, 2 mg/ml; EM, 4 mg/ml; KM and CP, 8 mg/ml; RXM, 16 mg/ml; MINO and CPFX, 32 mg/ml; NA and NFLX, 128 mg/ml; and ABPC, PIPC, CEX, and TC, 128 mg/ml <. 4) There were isolates resistant to 10 of the 15 drugs tested: 99.4 % of the isolates were resistant to CEX, 59.4 % to ABPC, 40.6 % to NA, 40.0 % to NFLX, 39.4 % each to TC and CPFX, 38.1 % to PIPC, 30.3 % to MINO, 3.2 % to KM, and 2.6 % to SM. 5) Regarding the resistance patterns of the 155 isolates, 28 isolates showed single drug resistance (18.1 %), and 127 isolates showed multidrug resistance (81.9 %), revealing that many isolates were multidrug resistant. The most frequent multidrug resistance pattern was ABPC/PIPC/CEX, followed by ABPC/PIPC/CEXATC/MINO/NA/NFLX/CPFX. 6) GryA mutation (Thr86 → Ile) was detected in 43 (97.7 %) of 44 quinolone-resistant isolates, but no amino acid mutation was noted in the other regions

育児用調整粉乳におけるEnterobacter sakazakii等の汚染に関する疫学的検討

Enterobacter sakazakii感染症に関する基礎的研究の一環として感染経路や感染源を解明するため，乳幼児への感染源として密接に関連性が考えられる育児用調整粉乳（以下粉乳）における本菌の汚染状況と各種抗菌剤の薬剤感受性試験を行ったところ，以下の成績が得られた。1. 粉乳から本菌の分離を試みたところ，149例9例（6.6％）から本菌が分離された。その内訳において，国産では61例中4例（6.6％）から，外国産では88例中5例（5.7％）からそれぞれ分離された。2. 本菌が分離された粉乳の推定菌量は，国産では4例とも3.6MPN/Kgであった。外国産では5例中4例が国産と同様に3.6MPN/Kgであったが，残りの1例は9.1MPN/Kgと高い菌量であった。3. 薬剤感受性試験におけるMIC_90値の比較では，CTX，CTRX，CPZ，CAZ，CPR，CZOP，GM，MEPMおよびCPFXの9薬剤に高い感受性を，PIPC，EM，MINO，CPおよびRFPの5薬剤に中間の感受性をそれぞれ示したが，ABPCとLCMの2薬剤は耐性を示すことが明らかになった。To clarify the route and source of infection as a basic study of Enterobacter sakazakii infection, we investigated the state of contamination with this type of bacteria in modified milk powder for infants, which may be closely associated with infantile infection, and conducted drug sensitivity tests using various antimicrobial agents. The following results were obtained:1. In 9 (6.6%) of 149 milk powder samples, this type of bacteria was isolated. They consisted of 4 manufactured in Japan (6.6%, 4/61) and 5 manufactured in other countries (5.7%, 5/88).2. The estimated bacterial amount in milk powder ranged from less than 3 to 3.6 MPN/Kg in the Japanese 4 samples. In the foreign 5 samples, it ranged from less than 3 to 9.1 MPN/Kg.3. The MIC_90 values on drug sensitivity tests were compared. Enterobacter sakazakii was highly susceptible to CTX, CTRX, CPZ, CAZ, CPR, CZOP, GM, MEPM, and CPFX. It showed an intermediate susceptibility to PIPC, EM, MINO, CP, and RFP. However, it was resistance to ABPC and LCM

病院内水道水における貧栄養細菌の生息状況

To investigate the distribution of oligotrophic bacteria inhabiting hospital tap water, tap water samples were subjected to pour culture at 25℃ for 7 days using R2A agar medium. Bacteria were detected in 222 of 271 samples (81.9 %) collected nationwide, mainly in the Kanto district, revealing that oligotrophic bacteria are present at a very high frequency in hospital tap water. The distribution was investigated in relation to the geography or hospital management form, but no marked tendency was noted. The number of isolated oligotrophic bacteria varied from 1.0 × 10^0 CFU/ml to 2.8 × 10^4 CFU/ml among the samples, but the number was less than 1.0 × 10^1 CFU/ml in 54 samples accounting for the highest ratio (24.3 %). Of 538 oligotrophic isolates, 457 isolates (84.9 %) were gram-negative rods, and 108 isolates (23.6 %) were Methylobacterium, ranked the highest, followed by 71 isolates of Pseudomonas (15.5 %). However, identification of 274 isolates (60.0 %) was not possible. Gram-positive rods, Bacillus and Corynebacterium, and cocci, Micrococcus and Staphylococcus, were also isolated, although the frequencies were low. Based on these findings, the presence of oligotrophic bacteria which may cause opportunistic infections in tap water should be noted, when patients use tap water in hospitals, being susceptible to the infection