Astromicin (Fortimicin A)-Resistant Bacteria in Japan

CHEMOT HERAPY VOL.31NO.3 331

ASTROMICIN (FORTIMICIN A)-RESISTANT BACTERIA IN JAPAN

YASUSHIUEDA (Chief) Jikei University School of Medicine SACHIKOGOTO Department of Microbiology, Toho University, School of Medicine ATSUSHISAITO The Second Department of Internal Medicine, Jikei University School of Medicine KEIICHINAKAGAWA Department of Internal Medicine,Tokyo Kyosai Hospital KIHACHIROSHIMIZU Department of Internal Medicine, Tokyo Women's Medical College KOHEIHARA The Second Department of Internal Medicine, Nagasaki University School of Medicine KEIZOMATSUMOTO Department of Internal Medicine, Institute for Tropical Medicine, Nagasaki University MASAAKIOHKOSHI Department of Urology, Tokai University, School of Medicine TADAONIIJIMA Department of Urology, Faculty of Medicine, University of Tokyo TSUNEONISHIURA Department of Urology, Gifu University School of Medicine SUSUMUMITSUHASHI, SHIZUKO IYOBE, MATSUHISA INOUE*, RYOICHIOKAMOTO, and MITSUYOSHIOKII Department of Microbiology, Laboratory of Drug Resistance in Bacteria*, School of Medicine, Gunma University

(Received Oct. 5, 1982)

The susceptibility of 539 clinical isolates to astromicin (ASTM) and other aminoglycoside antibiotics was determined . Isolation frequency of ASTM-resistant strains among 443 Enterobacteriaceae species (130 Serratia marcescens , 94 Proteus sp., and others) was 2.9%, (13 strains). Thirty-two strains (33.3%) among 96 glucose-nonfermentative gram-negative bacilli (53 Pseudonzonas aeruginosa , and others) were resistant to ASTM. Twenty strains which showed various levels of ASTM resistance among 45 ASTM-resistant strains were selected for further studies . None of these 20 strains was found to produce ASTM-inactivation enzyme [AAC(3)-I or a so far unknown enzyme] . Fifteen among them were shown to possess one , two, or three kinds of aminoglycoside-inactivation 332 CHEMOT HERAPY MAR.1983

enzymes. ASTM resistance in 4 strains of the 20 was decreased in the concomitant presence of subinhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). ASTM resistance of the remaining 16 strains was unchanged even by the presence of EDTA.

INTRODUCTION tone, 8 g of Na2HPO4. 12 H20, 2 g of KH2PO4 , Astromicin (fortimicin, KW-1070) is the most 1.2 g of (NH4)2SO4, 2 g of glucose, 0.4 g of Mg

active member of the fortimicin complex of amino- SO4-7 H20 and 1 liter of distilled water. glycoside antibiotics produced by fermentation of Antibiotics and chemicals. The following

Micromonospora olivoasterospora1). It is a pseudo- drugs were used: gentamicin (GM), GM-C1,

disaccharide-incorporating fortamine, a novel C1a, GM-C2, micronomicin (MCR), sisomicin

aminocyclitol2). This new antibiotic possesses a (SISO), netilmicin (NTL), astromicin (ASTM),

broad spectrum of activity against gram-positive kanamycin (KM)-A, -B, -C, 3', 4'-dideoxykana-

and gram-negative bacteria, but it has relatively mycin-B (DKB), tobramycin, neomycin C, paro-

weak activity against Pseudomonas aeruginosa momycin, lividomycin, amikacin (AMK), ribosta-

strains3). ASTM is effective against many amino- mycin, butirosin B, spectinomycin (SPCM),

glycoside-resistant bacteria capable of producing streptomycin (SM), dihydrostreptomycin (DSM),

am inoglycoside-inactivation enzymes except for and apramycin. Trilithium S-acetyl CoA, disodium

A A C (3)-I4). adenosine 5'-triphosphate (ATP), and EDTA were

The present studies have been carried out in an all commercial products. The isotope-labeled

attempt to investigate, at the present stage, the compounds, i. e., [ƒÁ-32P]ATP(6. 3 Ci/mmol), [8- isolation frequency of ASTM-resistant gram-nega- 14C ]ATP(54. 2 mCi/mmol), and [1-14C] S-acetyl tive isolates and the presence of aminoglycoside- coenzyme A (54.0 mCi/mmol) were purchased from

inactivation enzymes in certain of these isolates. the New England Nuclear Corp., Boston, Mass.,

Furthermore, we carried out studies on the effects USA.

of subinhibitory concentrations of ethylenediamine- Antimicrobial susceptibility tests. The

tetraacetic acid (EDTA) on minimal inhibitory MICs were determined by the agar dilution method.

concentrations (MICs) of ASTM. Overnight cultures of test strains were diluted to

MATERIALS AND METHODS a final concentration of approximately 5 •~105 cells/

Bacterial strains. A total of 539 gram-nega- ml, and 5 ƒÊl of each culture was inoculated by an

tive clinical isolates were obtained from 63 hospitals inoculator (Microplanter, Sakuma, Tokyo) on

(medical organs of the authors and the related Mueller-Hinton agar plates containing serial two-

hospitals) in Japan between 1979 and 1980. These fold dilutions of the drug. MICs were recorded

were random isolates from urine obtained by after 18 hours of incubation at 37•Ž. The criteria

catheter or mid stream of different patients. Among for resistance to aminoglycoside antibiotics were

45 ASTM-resistant strains (Table 1, 2), 20 strains 12. 5 ƒÊg/ml. The effects of EDTA were estimated

(Table 3) were chosen as the representative ASTM- in the same way (agar dilution method) whether

resistant strains, showing various resistance-levels in the presence or absence of a subinhibitory

to ASTM. Staphylococcus aureus FDA 209 P was concentration (1/2 or 1/4 MIC) of EDTA. also used as a reference for the EDTA test5) . Preparation of cell-free extracts. An organ-

Media. Lac-BTB (lactose-bromothymol blue) ism derived from a single colony was grown at

agar was used as a solid medium. Lac-BTB agar 37•Ž in 1 liter of Medium B or Medium B supple-

consisted of nutrient broth (10 g of beef extract, mented with 0. 4% KNO3. The crude cell-free

10 g of peptone and 3 g of NaCl in 1 liter of extracts were prepared from late logarithmic grow-

distilled water) supplemented with 10 g of lactose, ing cells of tested strains. The cells were harvested

80 mg of bromothymol blue and 15% of agar. For by centrifugation, washed twice with TMK solu- the antibiotic susceptibility tests, Mueller-Hinton tion (0. 01 M Tris-HCI buffer containing 0.01 M medium (Nissui, Tokyo) was used. For the prepa- MgSO4•E6 H2O, 0.06 M KCl, and 6 mM 2-mercapto- ration of cell-free extracts, Medium B was used ethanol, pH 7. 5), and resuspended in the same and consisted of 2 g of yeast extracts, 10 g of pep- buffer. The cell suspension was disrupted sonically CHEMOT HERAPY VOL.31NO.3 333

Table 1 Isolation frequency of ASTM-resistant strains

* Other Enterobacteriaceae species . G-, gram-negative bacteria. † Other glucose-nonfermentative (GNF) gram-negative bacteria.

Table 2 Resistance pattern of ASTM-resistance strains

* Drugs tested were GM, MCR, SISO, NTL, ASTM, KM-A, DKB, AMK, SPCM, and DSM. 334 CHEMOT HERAPY MAR. 1963

at 20 kHz for 5•`10 minutes and then centrifuged of 0.8 mM drug, 0.01 ml of [ƒÁ-32P]ATP (50 uCi/ at 30, 000 x g for 30 minutes at 4•Ž. The super- ml) (APH system), [8-14C]ATP (20ƒÊCi/ml) (AAD natant thus obtained (S-30 fraction) was used as system), or [1-14C] S-acetyl coenzyme A (10ƒÊCi/ the crude enzyme solution; its protein content was ml) (AAC system), 0.01 ml of 0.8 mM unlabeled adjusted to 10 mg of protein per ml. Protein in ATP or 0.8 mM unlabeled S-acetyl coenzyme A, the S-30 fraction was determined by the method and 0.03 ml of 0.2 M buffer in a total volume of of LOWRY et al. 6), using bovine serum albumin as 0.1 ml. The experimental procedure followed was a standard. as described previously8). Enzyme assay. We examined the existence of Classification of aminoglycoside-inactiv- enzymatic inactivation of aminoglycoside antibiotics ation enzymes. Classification of aminoglycoside- by the method of OZANNE et al.7), with three inactivation enzymes was estimated by the substrate different buffers [acetate buffer (pH 6. 0), Tris- specificities9.10 and the representative inactivation maleate buffer (pH 7. 0), or Tris-HCl buffer (pH products shown below. Inactivated products of the 8. 0)]. Inactivation of each aminoglycoside was representative aminoglycoside antibiotics (SISO, attempted in each buffer for each extract. The KM-A, DKB, and SM) were purified by column reaction mixture consisted of 0. 03 ml of S-30 fraction, 0. 01 ml of 0.02 M MgCl2. 6 H2O, 0. 01 ml Table 4 Aminoglycoside-inactivation enzymes of representative ASTM-resistant strain Table 3 MICs of ASTM and GM for the representative ASTM-resistant strains (20 strains)

* For details, see Materials and Methods; VOL.31 NO.3 CHEMOT HERAPY 335

Table 5 Effect of EDTA on the MICs of ASTM against ASTM-resistant strains

* The ratio of MICs of ASTM in the absence of EDTA to those in the presence of EDTA. 336 CHEMOT HERAPY MAR, 1983 chromatography. The N-acetylated11.12), O-adeny- GN 12799), all strains were found to possess amino- lated8 13),or O-phosphorylated14.15) position of glycoside-inactivation enzymes. APH (3')-II was aminoglycoside antibiotics was estimated by paper detected in every instance. electrophoresis and thin-layer chromatography in Effect of EDTA on the MICs of ASTM. Table comparison with the following authentic samples: 5 shows the MICs of ASTM against 20 ASTM- 3-N-acetylated SISO"), 6'-N-acetylated KM-A12), resistant strains and S. aureus FDA 209 P in the 2"-adenylated DKB13), 3'-phosphorylated KM-A"), presence of subinhibitory concentrations (1/2, or 3"-adenylated SM and 6-adenylated SM81, 3"-phos- 1/4 MIC) of EDTA. ASTM resistance in 4 strains phorylated SM and 6-phosphorylated SM14). of the 20 was decreased by 4- or 16-fold in the RESULTS presence of subinhibitory concentration of EDTA. Isolation frequency of ASTM-resistant On the other hand, ASTM resistance of the strains. The MICs of 539 gram-negative clinical remaining 16 strains was scarcely decreased. isolates to aminoglycoside antibiotics were deter- DISCUSSION mined. The isolation frequency of ASTM-resistant ASTM inhibited the growth of more than 90% strains (MIC,•†12. 5 ug of ASTM per ml) and their of the clinical isolates of Escherichia, Klebsiella, resistance patterns to aminoglycoside antibiotics Enterobacter, Citrobacter, Proteus, Serratia, and are shown in Tables 1 and 2. As shown in Table other Enterobacteriaceae species tested, at a con-

1, 45 strains (8. 3%) among 539 isolates were centration of 8ƒÊg/ml16). In our study, the similar found to be ASTM-resistant. Except for glucose- ratio was obtained in the clinical isolates of the nonfermentative strains, only 13 strains (2.9%) above-mentioned Enterobacteriaceae species by among 443 isolates were resistant to ASTM. Anti- ASTM at a concentration of 12. 5ƒÊg/ml. It was bacterial activity of ASTM against P. aeruginosa reported that AAC(3)-I was produced probably by and other glucose-nonfermentative gram-negative R plasmids in multiple-resistant clinical isolates17). bacilli was weak. Thirtytwo strains (33.3%) among According to the results of our study, however, them were resistant to ASTM. AAC(3)-I does not seem to be widespread in Japan.

As shown in Table 2, there were no strains By any of our ASTM-resistant strains, ASTM was resistant to ASTM alone among the ASTM-resist- not inactivated, although various enzymes inactiv- ant strains. All were resistant to KM-A, SPCM, ating other aminoglycosides were detected. and DSM except for one strain of A. anitratus. In S. marcescens and P. aeruginosa strains,

More than one-half of the ASTM-resistant strains resistance levels to aminoglycoside antibiotics were highly and multiply resistant to the 10 except ASTM were generally associated with the aminoglycoside antibiotics tested. presence of inactivation enzymes.

Aminoglycoside-inactivation enzymes of the It was reported that the addition of EDTA representative ASTM-resistant strains. Since reduced the MICs of antibiotics in drug-resistant

6'-epi-purpurosamine B residue of ASTM is similar gram-negative bacteria due to damage of the per- to purpurosamine residue of GM-C2, we examined meability barrier of the outer membrane by chela- the resistance correlation between ASTM and GM. tion of the divalent cations5).

Among 45 ASTM-resistant strains, we selected We could not detect an ASTM-inactivation the 20 strains which showed different levels of enzyme(s) among the 20 ASTM-resistant strains resistance to ASTM and GM (from low- to high tested. ASTM resistance in 4 strains was decreased level). The MICs of the 20 strains are shown in in the presence of subinhibitory concentrations of

Table 3. EDTA. ASTM resistance in the remaining 16

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本邦におけるAstromicin(Fortimicin,KW-1070)耐性菌について

KW-1070菌感受性小委員会

上田泰委員長東京慈恵会医科大学

五島瑳智子東邦大学・微生物

斎藤篤東京慈恵会医科大学・第二内科

中川圭一東京共済病院・内科

清水喜八郎東京女子医科大学・内科

原耕平長崎大学・第二内科

松本慶蔵長崎大学・熱研内科

大越正秋東海大学・泌尿器科

新島端夫東京大学・泌尿器科

西浦常雄岐阜大学・泌尿器科

三橋進・伊予部志津子・井上松久・岡本了一・沖井三孔群馬大学・微生物

アミノグリコシド系抗生物質(AGs)であるAstromicin(ASTM)は,その化学構造中に新規な fertamine残基を有し,細菌学的特徴として АAC(3)-I以外のAGs不活化酵素に対し安定であ

る。今回,われわれはASTMの現在時における細菌学的特徴を臨床分離株(1979年から1980年に分離した539株)を用いて調べた。 (1)443株の腸内細菌(S.marcescens,130株;Proteussp.,94株;その他,219株)のうち 13株(2.9%)がASTM耐性菌(MIC,≧12.5μg/ml)であり,ASTM耐性菌の出現頻度は低かった。 (2)96株のブドウ糖非発酵グラム陰性桿菌(P.aerPtginOSa,53株;その他,43株)のうち32 株(33.3%)がASTM耐性菌であった。この点,従来の結果とほぼ一致した。 (3)上記45株(8.3%)のASTM耐性菌の中から代表20株を選びAGs不活化酵素の有無を調べた。その結果,15株にASTM以外のAGs不活化酵素を認めたが,ASTMを不活化する酵素の存在は認められなかった。 (4)上記代表20株のASTMに対する感受性に及ぼすEDTAの同時併用効果を調べたところ,4株において感受性が増大したが,残り16株ではほとんど変化しなかった。