Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 1, Num. 1, 1990

In Vitro Susceptibility of Nocardia asteroides and Nocardia brasiliensis to 1-N-derivatives of Aminoglycoside Antibiotics

K. YAZAWA, Y. MIKAMI and K. OTOZAI

Department of Experimental Chemotherapy, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan

Code Number: AC90001
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ABSTRACT.

Four 1-N-substitute aminoglycosides were tested for their in vitro activity against the Nocardia asteroides group and Nocardia brasiliensis by agar dilution method. Among these aminoglycosides, the recently developed arbekacin, l-N-((S)-4-amino-2-hydroxybutyryl)- 3',4'dideoxykanamycin B, had the best activity, showing the respective MIC90 values of 1.25 and 1.55 p.g/ml against the N. asteroides group and N. brasiliensis. The activity of arbekacin is higher than that of amikacin, followed by isepamicin and netilmicin. Recently established Nocardia species, i.e., N. asteroides sensu stricto, N. nova and N. farcinica from the N. asteroides group were sensitive to these aminoglycosides, but showed different sensitivity patterns.

Nocardia an opportunistic pathogen and its infections are encountered with increasing frequency in patients under prolonged treatment with immunosuppressive agents (Bach et al., 1973; Dewsnup et al., 1984).

Sulfonamides are the accepted therapy for many forms of nocardiosis, there are reports of patients being successfully treated with these agents (Gombert, 1982). There are also reported cases of patients who developed side effects, or were allergic to the drugs, necessitating withdrawal of these sulfonamide compounds (Bach et al., 1973). In the past, some useful chemotherapeutic agents have been suggested, but variations in susceptibility testing procedures and undefined factors frequently led to conflicting results (Bach et al., 1973; Gutman et al., 1983; Schaal et al., 1985; Gombert et al., 1987). These contradictory results are often encountered in aminoglycoside antibiotics such as gentamicin, tobramycin and amikacin, or B-lactam antibiotics such as ampicillin and imipenem (Bach et al.; Gutman et al., 1983; Schaal et al., 1985).

Our recent studies have suggested, as the most probable explanation of these conflicting data, that N. brasiliensis and the N. asteroides group have some inactivation enzymes: N. brasiliensis has aminoglycoside 3'- phosphotransferase [APH (3')] while most of the N. asteroides group have other enzymes, aminoglycoside 3-N- acetyl transferase [AAC (3)], 2"-nucleotidyltransferase [(AAD (2)] and APH (3') (Yazawa and Mikami, manuscript in preparation) (Fig. 1).

Various new aminoglycosides, such as arbekacin (HBK), netilmicin (NTL) and isepamicin (HAPA-B) whose 1-NH2 group is acylated or alkylated and which are expected to resist the above inactivation enzymes, have been developed (Kondo et al., 1973; Daniels et al., 1979; Matsuhashi and Yamamoto, 1988). The purpose of this study was to compare the susceptibility of clinically isolated Nocardia, especially N. brasiliensis and the N. asteroides group, to amikacin as compared to these 1-N-substituted aminoglycosides and to determine which antibiotic most closely fits the clinical requirements for the therapy of Nocardia infections.

Materials and Methods

Organisms. Fifty-two human isolates of the N. asteroides group, which were recently divided into the three species of N. asteroides sensu stricto (15 strains), N. farcinica (28 strains) and N. nova (9 strains), were used (Goodfellow and Minnikin, 1981; Lechevalier, 1986), together with thirty-three strains of N. brasiliensis. These strains were isolated from clinical specimens, most of them from immunosuppressive patients in Japan between 1977 and 1988. Isolates were identified by colony morphology, staining characteristics, ability to hydrolyse various organic substances, cell wall composition and sugar fermentation pattern according to Lechevalier (1986) and Goodfellow and Minnikin (1981).

Antimicrobial agents. The following antibiotic powders were supplied by the manufacturers: AMK (Banyu Pharmaceutical Co. Tokyo, Japan), HBK (Meiji Seika Co. Tokyo, Japan), HAPA-B (Toyo Jyozo Co. Shizuoka, Japan) and NTL (Sankyo Co. Tokyo, Japan).

Susceptibility tests. Isolates to be tested had been stored in ampoules by the liquid-dry method and were passed on Sabouraud dextrose agar (Difco) slants. A 10opful of the heavy growth from the slant was inoculated into a 10 ml Erlenmeyer shake flask containing 5 ml of 3% glucose enriched Brain Heart Infusion broth (Difco) and some sterile glass beads. The flask was placed on a rotary shaker and incubated with shaking for 3 to 4 days at 32 C.

Cultures that showed heavy growth were diluted 1:10 to 1:50 to obtain 1 x 10^8 viable cells/ml suspension. Homogenous suspensions were generally obtained in this manner (Bach et al., 1973).

The resulting culture was transferred to sterile tubes with tight caps and frozen at -70 C until used. Agar dilution susceptibility was performed with twofold dilutions of the antibiotics. The antimicrobial agents were incorporated into sensitivity agar (Nissui Seiyaku Co. Tokyo, Japan) after dilution and were used within 5 days. The agar plates were inoculated with a Multipoint Inoculator A 400 (Sussex, England). The inoculated plates were incubated at 27 C for 7 days and than examined for growth to determine the MIC (minimum inhibitory concentration). The MIC was defined as the lowest concentration of antibiotic on which there was no visible growth; antibioticfree plates showed heavy growth.

Figure 1. Possible sites of inactivation by Nocardia in model aminoglycoside antibiotics.

Results

The antimicrobial activity of four aminoglycosides, HBK, AMK, HAPA-B and NTL against N. brasiliensis was compared and is shown in Fig. 2.

Figure 2. Comparison of cumulative MIC values œor Nocardia brasiliensis against 1-N-derivatives of aminoglycoside antibiotics.

These antibiotics differed in their relative activity against the strains tested: HBK was the most active, followed by AMK. HBK inhibited all strains at a concentration of 3.13 ug/ml and MIC50 and MIC90 were 0.702 and 1.40 ug/ml respectively. AMK was less active than HBK against N. brasiliensis, one half of the strains being susceptible to 2.24 ug/ml and 90% being inhibited by 5.62 ug/ml in these experiments.

MIC50 values of the remaining two aminoglycosides, NTL and HAPA-B, against N. brasiliensis were 3.34 and 4.17 ug/ml respectively.

Figure 3. Comparison of cumulative MIC values for the Nocardia asteroides group against 1-N-derivatives of aminoglycoside antibiotics.

As shown in Fig. 3, the N. asteroides group showed similar susceptibility as that of N. brasiliensis to the four aminoglycosides. Again, HBK was the most active followed by AMK, with the activity of HAPA-B and NTL being lower than either of them.

We also determined the MIC values for each of the three species of the N. asteroides group. MIC ranges, and MIC50 and MIC90 values, are shown in Table 1. Except for HBK, N. nova was the most sensitive, followed by N. asteroides sensu stricto. N. farcinica was found to be more resistant than the other two species to all four aminoglycosides.

Table 1. Comparison of aminoglycoside antibiotic activity against Nocardia

                       MIC values (ug/ml) 
                     -------------------------
Antibiotic Organism  MIC ranges   MIC50  MIC90
----------------------------------------------
AMK        N.B.       0.78-12.5   2.24   5.62
           N.A.       <0.2-6.25   0.41   1.09
           N.F.       0.39-6.25   1.56   3.29
           N.N.       <0.2-0.78   0.32   0.61
HBK        N.B.       0.39-3.13   0.70   1.40
           N.A.       <0.2-6.25  <0.20   0.56
           N.F.       0.39-1.56   0.61   1.33
           N.N.       <0.2-1.56   0.55   1.20
HAPA-B     N.B.       1.56-12.5   4.17   6.04
           N.A.       <0.2-12.5   0.59   4.70
           N.F.       0.78-6.25   2.65   5.38
           N.N.        0.2-1.56   0.98   1.47
NTL        N.B.       0.78-12.5   3.34   6.25
           N.A.       <0.2-6.25   2.78   5.38
           N.F.       0.39-6.25   2.81   9.69
           N.N.       <0.2-0.78  <0.20  <0.20

N.B.: N. brasiliensis, N.A.: N. asteroides, N.F.: N.
farcinica, N.N.: N. nova. AMK. amikacin, HBK: arbekacin, HAPA-B:
isepamicin, NTL: netilmicin.

Discussion

Throughout this study, no strains resistant to the aminoglycosides HBK, NTL, HAPA-B and AMK were detected among the N. asteroides group and N. brasiliensis strains tested. This would indicate that, at the present time, these Nocardia strains have no inactivation enzymes for these 1-N-substituted aminoglycosides, such as ADD(4") whose existence was reported in Staphylococcus aureus (Kenneth et al., 1977). Although susceptibility differed depending on the species and N. brasiliensis generally showed slightly higher resistance that any member of the N. asteroides group, the MICs for all strains were below the limit of an achievable concentration of each antibiotic in the serum. It is therefore expected that these 1-N-substituted aminoglycosides could be used as a first choice in treatment of nocardiosis, even before the pathogenic Nocardia species has been identified.

In vitro and in vivo activity of AMK against N. asteroides was reported by Dalovisio and Pankey (1984) and by Gutman et al. (1983). However, in the present experiments, HBK was found to be more active than either AMK or NTL. Therefore, although further detailed in vivo studies are necessary, HBK is expected to be a potentially useful therapeutic agent for the treatment of any type of nocardiosis caused by N. brasiliensis or the N. asteroides group.

As shown in Fig. 4, HBK is 3', 4'-dideoxy type aminoglycoside and has no 3'-OH group which is easily attacked by inactivation enzymes of APH(3') from N. brasiliensis or certain strains of N. asteroides sensu stricto. In addition, it has been reported that the introduction of acyl- or alkyl-groups to the 1-NH2 aminoglycosides can protect the 3-NH2 or 2"-OH group of gentamicin or kanamycin A from inactivation enzymes like AAC(3) of AAD(2") of N. farcinica (Kenneth et al.,1977). This may be the reason why HBK is more active against both the N. asteroides group and N. brasiliensis. Further enzymatic and genetic studies on Nocardia resistance mechanisms to aminoglycosides are now in progress in our laboratory.

Figure 4. Chemical structure of arbekacin (HBK).

Acknowledgment.

The authors wish to thank Emeritus Prof. Tadashi Arai of the Department of Experimental Chemotherapy, Chiba University, for his constant help and encouragement.

References

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Dalovisio, J.R. & G.A.Pankey (1978). In vitro susceptibility of Nocardia asteroides to amikacin. Antimicrob.Ag. Chemother., 13:128-129

Daniels, P.J.L., A.B.Cooper, S.W.Mc Combie, T.L.Nagabhushan, D.F.Rane & J.J.Wright (1979). Some recent advances in the chemistry of antibiotics of the gentamicin series. Jap. J. Antibiot., 32: 195-204.

Dewsnup, D.H. & D.N.Wright (1984). In vitro susceptibility of Nocardia asteroides to 25 antimicrobial agents. Antimicrob.Ag. Chemother., 25:165-167

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Gombert, M.E., T.M.Aulicino, L.du Bouchet & L.R.Berkowitz (1987) Susceptibility of Nocardia asteroides to new quinolones and beta-lactams. Antimicrob.Ag. Chemother., 31:2013-2014

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Lechevalier, H.A. (1986). Nocardioforms. In: Sneath, P.H.A. (ed.) Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, vol. 2, pp. 1458-1506

Matsuhashi, Y. & H.Yamamoto (1988). The enzymatic mechanisms of resistance to aminoglycoside antibiotics in methicillin- cephem-resistant bacteria. Jap. J.Antibiot., 41: 523- 529

Schaal, K.P., H.Schutt-Gerowitt & A. Goldmann (1985). In vitro and in vivo studies on the efficacy of various antimicrobial agents in the treatment of human nocardiosis. In: Szabo, G., S.Bird & M.Goodfellow (eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes. Akademiai Kiado, Budapest, part B, pp. 619- 633.

Copyright 1990 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia

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