ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1990, p. 1762-1765

Vol. 34, No. 9

0066-4804/90/091762-04$02.00/0 Copyright © 1990, American Society for Microbiology

In Vitro Antibacterial Activity of Faeriefungin, a New Broad-Spectrum Polyene Macrolide Antibiotic MARTHA H. MULKS,l* MURALEEDHARAN G. NAIR,2 AND ALAN R. PUTNAM2 Departments of Microbiology and Public Health' and Horticulture,2 Michigan State University, East Lansing, Michigan 48824 Received 12 March 1990/Accepted 6 July 1990

The in vitro activity of faeriefungin, a new pentaene macrolide lactone antibiotic produced by Streptomyces griseus var. autotrophicus, against 263 dinical bacterial isolates was examined. In.contrast to the related fungicidal antibiotics nystatin and amphotericin B, which show no activity against bacteria, faeriefungin showed bactericidal activity against all species of gram-positive bacteria examined. MICs for these species ranged from 8 to 64 Fg/ml, and the MIC for 90% of the isolates tested was 32 Fg/ml. Isolates of some frstidious gram-negative species, including Neisseria gonorrhoeae, N. meningitdis, and Haemophilus influenzae, were slightly susceptible to faeriefungin, with MICs ranging from 16 to 128 ,ug/ml, but all members of the families Enterobacteriaceae and Pseudomonadaceae, with the exception of Pseudomonas cepacia, were completely resistant to faeriefungin at the concentrations tested. Faeriefungin is also active against fungi, nematodes, and mosquito larvae. The mode of action of faeriefungin against both bacteria and fungi is under investigation.

Streptococcus pneumoniae (ATCC 6303) were obtained from the American Type Culture Collection. All other strains tested were either fresh or stock clinical isolates from patients at Ingham Medical Center, Lansing, Mich., or Tufts-New England Medical Center, Boston, Mass. All bacterial strains were stored in 2% tryptone-20%o glycerol at -700C. Faeriefungin. Faeriefungin was isolated from the mycelial biomass of Streptomyces griseus var. autotrophicus (MSU32058; ATCC 53668) grown in A-9 medium as previously described (11). Crystalline faeriefungin was stored in the dark at -20°C for up to 3 months before use. Determination of MICs. Susceptibilities of the bacterial strains to faeriefungin were determined by the agar dilution method (19). Faeriefungin was dissolved in 100lo dimethyl sulfoxide (DMSO) and diluted in 20% DMSO, and serial twofold dilutions at concentrations of 2,560 to 40 ijg/ml were prepared in 20% DMSO. One volume of each stock concentration of faeriefungin was mixed with nine volumes of molten Mueller-Hinton (MH) agar (Difco Laboratories, Detroit, Mich.) which had been autoclaved and cooled to 50°C to prepare agar plates containing 4, 8, 16, 32, 64, 128, and 256 jig of faeriefungin per ml. Control plates containing 20% DMSO were also prepared. Supplements were added to the MH agar as needed to permit growth of the test organisms, i.e., 10 ml of Kellogg supplement (7) per liter of agar for Neisseria sp. and 10 ,ug of hemin per ml plus 10 jig of NAD per ml for Haemophilus sp. Prepared plates were stored in the dark at 4°C and used within 48 h of preparation. To prepare test inocula, an overnight culture of each test organism grown on MH agar (plus supplements if required) at 37°C was inoculated into 5 ml of MH broth (plus supplements if required) and incubated for 2 h at 37°C. Each culture was adjusted to the turbidity of a 0.5 McFarland standard (-0.5 x 108 to 1.0 x 108 CFU/ml). A 1:20 dilution was prepared in warm MH broth, and 2 ,ul of this dilution of bacteria (containing _104 CFU) was used to spot inoculate each antibiotic-containing agar plate and control plate. Plates were incubated overnight at 37°C and scored for growth of each test organism. All plates were incubated in

Faeriefungin is a recently described polyol polyene macrolide lactone antibiotic produced by a strain of Streptomyces griseus var. autotrophicus (MSU-32058; ATCC 53668) which was isolated from a soil sample collected from the center of a fairy ring in an old lawn (10, 11). Faeriefungin is a crystalline and stable compound with the structure shown in Fig. 1 (11) and is physicochemically similar to the previously reported pentaene macrolide lactones mycoticin (2, 14, 15, 20) and flavofungin (1, 17). Comparison of the antifungal activity of faeriefungin with the activities of known polyene antibiotics currently in clinical use showed that faeriefungin was more active than amphotericin B, nystatin, or pimaricin against Aspergillus fumigatus, Microsporum canis, and Trichophyton rubrum, in addition to several other fungi, with MICs ranging from 3.2 to 12.0 ,ug/ml (11). For Candida albicans, faeriefungin had an MIC of 5.5 ,ug/ml, compared with 3.2 pLg/ml for amphotericin B, 3.2 ,ug/ml for nystatin, and 5.5 ,ug/ml for pimaricin (11). Preliminary cytotoxicity studies of faeriefungin against WB-S cells and human erythrocytes showed that faeriefungin is 10-fold less toxic than amphotericin B (11). However, while the antibiotic activities of other polyene macrolides are primarily antifungal (2), preliminary analysis of the biological properties of faeriefungin showed that it inhibits the growth of several species of bacteria, as well as a wide range of filamentous and yeastlike fungi, and that it has nematocidal and mosquitocidal activities (11). In this study, we examined the in vitro antibacterial activity of faeriefungin against a large number of clinical isolates. MATERIALS AND METHODS Bacterial strains. A total of 263 bacterial isolates were examined in this study. Reference strains of Escherichia coli (ATCC 25922), Neisseria gonorrhoeae (ATCC 19424 and 31426), Proteus mirabilis (ATCC 7002), P. vulgaris (ATCC 13315), Staphylococcus aureus (ATCC 25923 and 29213), Enterococcus (Streptococcus) faecalis (ATCC 29212), and *

Corresponding author. 1762

ANTIBACTERIAL ACTIVITY OF FAERIEFUNGIN

VOL. 34, 1990

1763

TABLE 1. MICs of faeriefungin against gram-positive and selected gram-negative species

3028

26

OH

24

OH

Faeriefungin

22

18

20

OH OH

Organism (no. of isolates)

OH

MIC

Range

(4Lg/ml)a 50%

90%

6

OH

A R=H B R=CH3

FIG. 1. Chemical structure of faeriefungin.

air, except for those containing Neisseria sp., Haemophilus influenzae, and Pseudomonas cepacia, which were incubated in air enriched with 7% CO2. The MIC for each isolate represents the lowest concentration of faeriefungin at which complete inhibition of growth occurred. Determination of MBCs. Bactericidal assays were performed by the method described by Taylor et al. (16), with an inoculum of 5 x 10' CFU/ml. After overnight incubation in MH broth containing twofold serial dilutions of the antibiotic, samples were diluted 10-fold in antibiotic-free MH broth and a subculture volume of 0.1 ml was plated on MH agar and incubated overnight at 37°C. The MBC was defined as the lowest concentration of faeriefungin that produced 99.9% or greater killing of the initial inoculum (12). Killing curves. Killing curves were determined by the method described by Schoenknecht et al. (13) by using a starting inoculum of 1 x 106 to 2 x 106 CFU/ml. At each time point, samples were serially diluted in MH broth and a subculture volume of 0.1 ml was plated on MH agar. Antibiotic broths contained 32 ,ug of faeriefungin per ml and 0.2% DMSO in MH broth (Difco); controls contained 0.2% DMSO in MH broth. The lowest reliably detectable viable cell count was 102 CFU/ml.

RESULTS In contrast to the other polyene macrolides, such as amphotericin B and nystatin, which have no antibacterial activity, faeriefungin inhibited the growth of a variety of bacterial species (Tables 1 and 2). The 263 bacterial isolates tested included the four standard MIC quality control strains recommended for use in clinical laboratories, i.e., S. aureus ATCC 29213, E. coli ATCC 25922, E. faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC 27853 (6, 19), as well as type strains and fresh clinical isolates of 52 species. All 110 isolates of gram-positive bacteria tested, including methicillin-resistant S. aureus strains, were inhibited by faeriefungin; MICs for these isolates ranged from 8 to 64 j.ug/ml, with an MIC%0 (antibiotic concentration at which 90%o of the isolates were inhibited) of 32 jig/ml (Tables 1 and 2). Among the 153 gram-negative strains tested, all members of the families Enterobacteriaceae and Pseudomonadaceae, with the exception of P. cepacia isolates, were completely resistant to faeriefungin at the concentrations tested (Table 3). In contrast, isolates of N. gonorrhoeae, Neisseria meningitidis, and Branhamella catarrhalis, including penicillin-resistant strains, were susceptible to faeriefungin, although generally at slightly higher concentrations than those which inhibited gram-positive bacteria; MICs for these isolates ranged from 16 to 64 jig/ml, with MIC%0s of 64 ,ug/ml (Tables 1 and 2). Isolates of H. influenzae and P. cepacia were marginally

Gram-positive species (92) Enterococcus Spp.b (18) Staphylococcus aureus (28) Staphylococcus epidermidis (14) Other coagulase-negative

8-32 16-32 16-32 16-32 32

32 32 32 16 32

staphylococcic (10) Streptococcus pyogenes (8) Streptococcus agalactiae (7) Streptococcus pneumoniae (7)

8-32 16 16-32

16 16 16

Gram-negative species (50) Haemophilus influenzae (12) Neisseria gonorrhoeae (10) Neisseria meningitidis (10) Neisseria Spp.d (11) Pseudomonas cepacia (7)

16->128 64->128 16-64 32-64 64->128 64-128

32 32 32 32 32

64 128 32 64 64 128

.128 >128 32 64 >128

For species of which more than nine isolates were tested, MIC50s and are given; for species of which six to nine isolates were tested, approximate MIC50s are given. b Includes 15 E. faecalis and 3 E. faecium isolates. c Includes three S. capitis, two S. cohnii, one S. haemolyticus, two S. simulans, and two S. warneri isolates. d Includes two N. cinerea, two N. flava, two N. mucosa, two N. perflava, one N. sicca, and two N. subflava isolates. a

MIC90s

susceptible to faeriefungin; MICs for these isolates ranged from 64 to >128 ,uglml, with MIC%0s of -128 ,ug/ml. MBC determinations performed on selected isolates of S. aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and E. faecalis demonstrated that the MBCs were equal to the MICs for the isolates tested and that faeriefungin is bactericidal rather than bacteriostatic. Killing curves were determined to ascertain the rates of killing of two representative isolates of gram-positive bacteria, S. aureus ATCC 29213 (MIC, 32 ,ugIml) and E. faecalis ATCC 29212 (MIC, 16 ,ug/ml), in MH broth containing 32 ,ug of faeriefungin per ml. The number of viable E. faecalis cells decreased by 3 orders of magnitude (99.9% killing) within 15 TABLE 2. MICs of faeriefungin against species represented by five or fewer clinical isolates each

Organism (no. of isolates)

MICs for individual isolates

(p.g/ml)a

Gram-positive species (18) .............................. Bacillus cereus (1) ...................................... Corynebacterium sp. (4) .............................. Listeria monocytogenes (1) .......................... Micrococcus sp. (1) ....................................

168, 328, 642 16 323, 641 32 32 Staphylococcus saprophyticus (2) .................. 32, 64 Streptococcus sp. group C (2) ....................... 16 Streptococcus avium (1) .............................. 32 Streptococcus bovis (1) ............................... 16 Streptococcus constellatus (1) ............ .......... 16 Streptococcus intermedius (1) ....................... 16 Streptococcus mitis (1) ................................ 32 Streptococcus salivarius (1) .......................... 16 Streptococcus sanguis (1) ............................ 16

Gram-negative species (3) ............................... 641, 1282 Branhamella catarrhalis (1) .......................... 64 Flavobacterium sp. (2) ................................ 128 a

The inferior number is the number of isolates with the indicated MIC.

1764

ANTIMICROB. AGENTS CHEMOTHER.

MULKS ET AL.

TABLE 3. Gram-negative bacterial species not susceptible' to faeriefungin No. of

Species

isolates tested

Enterobacteriaceae Citrobacter diversus .......................................

Citrobacterfreundii ....................................... Enterobacter aerogenes ..................................... Enterobacter cloacae ....................................... Escherichia coli .......................... ............. Klebsiella oxytoca ....................................... Klebsiella ozaenae ....................................... Klebsiella pneumoniae ....................................... Proteus mirabilis ....................................... Proteus vulgaris ....................................... Serratia marcescens .......................................

2 5 6 6 18 3 2 13 1 1 12

Pseudomonadaceae Pseudomonas aeruginosa ................................... Pseudomonas maltophilia ...................................

25 2

Other gram-negative species Acinetobacter calcoaceticus ................................ Aeromonas hydrophila ....................................... a MIC, >128 ,g/ml.

3 1

min, while 99.9% killing of S. aureus was achieved within

In vitro antibacterial activity of faeriefungin, a new broad-spectrum polyene macrolide antibiotic.

The in vitro activity of faeriefungin, a new pentaene macrolide lactone antibiotic produced by Streptomyces griseus var. autotrophicus, against 263 cl...
662KB Sizes 0 Downloads 0 Views