ANTIMICROBIAL AGENTs AND CHEMoTHERAPY, Aug. 1.79, p. 120-122 0066-4804/79/08-0120/03$02.00/0
Vol. 16, No. 2
In Vitro Activity of Sodium Fusidate Against Anaerobic Bacteria G. E. STEINKRAUSt AND L. R. McCARTHY North Carolina Memorial Hospital, and Department of Bacteriology Laboratories, Hospital of Department and Immunology and Department of Pathology, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 11 May 1979
The microtiter broth dilution method was employed to determine the in vitro susceptibility of 525 recent clinical isolates of anaerobic bacteria to sodium fusidate. The minimal inhibitory concentrations of sodium fusidate ranged from -0.06 to 1.0 ,ug/ml for 155 strains of anaerobic gram-positive rods and 130 strains of anaerobic gram-positive cocci. Minimal inhibitory concentrations ranging from s0.06 to 32 ,ug/ml were observed for 240 strains of anaerobic gram-negative rods. Among the latter group a minimal inhibitory concentration of 16 ,ug/ml or greater was encountered with 16% of 45 Bacteroides fragilis strains, 19% of 32 Bacteroides thetaiotaomicron, and all 7 strains of Fusobacterium necrophorum. Minimal inhibitory concentrations for Veillonella parvula, the only gram-negative coccus tested, ranged from 0.5 to 8.0 yig/ml. microdilution modification of the microtiter procedure described by Thornsberry and Swenson (9). The 525 strains of anaerobic bacteria tested were recent clinical isolates recovered from wound, lower respiratory, and genital tract specimens submitted to the Clinical Microbiology Laboratories of the North Carolina Memorial Hospital. Each anaerobic strain was identified by using gas-liquid chromatography and specialized biochemical tests as described by Holdeman and Moore (5). The strains tested included 155 strains of anaerobic gram-positive rods, 240 strains of anaerobic gramnegative rods, 110 strains of anaerobic gram-positive cocci, and 20 strains of anaerobic gram-negative cocci. Sodium fusidate powder was supplied by HoffmanLaRoche Inc., Nutley, N.J. This powder was dissolved in distilled water to yield a concentration of 2,560 Lgg ml. Twelve serial twofold dilutions ranging from 256 to 0.12 gAg/ml were prepared in sterile Schaedler broth supplemented with 5 ,ug of hemin per ml and 0.1 ug of menadione per ml; 50-pd amounts of these dilutions were dispensed aseptically into sterile U-bottom microtiter plates with a Dynatech MIC-2000 dispenser (Cooke Laboratories, Inc., Alexandria, Va.). Plates not used within 24 h of preparation were sealed in polyethylene bags and frozen at -70°C for a maximum of 6 weeks. Before inoculation, microtiter plates were placed in an anaerobic glove box and incubated at ambient temperature in an atmosphere of 85% N2, 10% H2, and 5% CO2 for 24 h. All inocula were prepared and dispensed within the glove box environment. Inocula were prepared by susMATERIALS AND METHODS pending well-isolated colonies of each test strain from The in vitro activity of sodium fusidate against Schaedler sheep blood agar plates in 2 ml of suppleanaerobic bacteria was investigated by employing a mented Schaedler broth. The turbidity of the suspension was adjusted to that of a MacFarland no. 0.5 t Present address: Department of Pathology, Bishop Clark- turbidity standard. A 1:100 dilution of the standardized suspension was prepared in supplemented Schaedler son Hospital, Omaha, NE 68105. 120
Fusidic acid, produced by the fungus Fusidium coccineum, possesses a steroid structure and is chemically related to cephalosporin P1 (3). Unlike the cephalosporins, which inhibit cell wall synthesis, fusidic acid inhibits bacterial protein synthesis by interfering with the translocation enzyme and inhibiting the binding of aminoacyl transfer ribonucleic acid to ribosomes (2). Although a number of derivatives of fusidic acid have been prepared, only the sodium salt, sodium fusidate, possesses a significant degree of antibacterial activity. Sodium fusidate is remarkable for its high activity against both penicillin- and methicillinresistant strains of Staphylococcus aureus (6). Sodium fusidate is also very active against corynebacteria, but is poorly active against streptococci (4). Members of the genus Neisseria are the only aerobic gram-negative bacteria which are susceptible to this antibiotic (4). Other organisms previously shown to be susceptible to sodium fusidate include Clostridium tetani, C. perfringens, and Bacteroides fragilis (4, 7). It was the reported susceptibility of these three anaerobic species which led us to investigate more extensively the activity of sodium fusidate against anaerobic bacteria.
VOL. 16, 1979
ACTIVITY OF SODIUM FUSIDATE
121
broth. The diluted organism suspension was mixed RESULTS thoroughly, and 50 id of this suspension was added to each antibiotic well and to a growth control well Table 1 presents the results obtained for the containing 50 pl of the supplemented broth. The mi- in vitro activity of sodium fusidate against the crotiter plates were inoculated by using a Minitek 525 anaerobes tested. All 114 clostridial strains dispensing gun (BBL Microbiology Systems, Cockeys- tested MICs which ranged between 0.06 ville, Md.). With inoculation, the final concentration and 1.0yielded pg/ml. Clostridium bifermentans, C. of sodium fusidate contained within the 12 wells ranged between 128 and 0.06 /Ag/ml. The inoculated paraputrificum, and C. sordellii were the most plates were sealed in polyethylene bags and incubated susceptible of the 10 species of Clostridium at 35°C for 48 h in an anaerobic glove box (Coy tested, each species having an MIC of 50.25 ug Manufacturing Co., Ann Arbor, Mich.) with an atmos- of sodium fusidate per ml. All 16 strains of phere of 85% N2, 10% C02, and 5% H2. The minimal Eubacterium lentum were susceptible to 1.0 pAg inhibitory concentration (MIC) was read as the lowest or less of sodium fusidate per ml. Of the 25 concentration of the antibiotic which inhibited visible strains of Propionibacterium acnes tested, 96% growth of each organism tested. were inhibited by 1.0 pg or less per ml, whereas The stability of fusidic acid in the prepared micro- 4% required 2.0 pg/ml for inhibition. titer trays was monitored by testing a strain of B. Bacteroides corrodens, B. melanmnogenicus fragilis (for which the MIC of fusidic acid was known) on each day tests were performed. This quality control subsp. asaccharolyticus, B. melaninogenicus testing indicated that the antibiotic was stable for at subsp. intermedius, B. oralis, and B. ruminicola least 6 weeks under the storage, prereduction, and subsp. ruminicola had MICs comparable to those observed for the anaerobic gram-positive incubation conditions employed. TABLE 1. In vitro activity of sodium fusidate against 525 anaerobes MIC (ag/ml of medium) Organism
No. of strains
Range
Anaerobic gram-positive rods Clostridium bifermentans C. cochlearum C. histolyticum C. innocuum C. paraputrificum C. perfringens C. ramosum C. sordellii C. subterminale C. tertium Eubacterium lentum Propionibacterium acnes Anaerobic gram-negative rods Bacteroides corrodens B. melaninogenicus subsp. asaccharolyticus B. melaninogenicus subsp. intermedius B. oralis B. ruminicola subsp. ruminicola Fusobacterium necrophorum Bacteroides distasonis B. fragilis B. ovatus B. thetaiotaomicron B. vulgatus Anaerobic cocci Gaffkya anaerobia Peptococcus assaccharolyticus P. magnus P. prevotii Peptostreptococcus anaerobius P. micros
Veillonella parvula
For 50% of strains
For 90% of strains
11 4 10 12 5 39 9 6 8 10 16 25
_0.06-0.25 0.25-1.0 _0.06-0.5 0.12-1.0 C0.06-0.25 50.06-1.0 50.06-0.5 0.12-0.25 0.12-0.5 0.12-1.0 C0.06-1.0 _0.06-2.0
0.12 0.5 0.12 0.25 0.12 0.12 0.12 0.12 0.25 0.25 0.12 0.25
0.25
19 31
0.12-0.5 C0.06-1.0
0.25 0.25
0.5 1.0
31
C0.06-1.0
0.25
1.0
0.12-0.5
0.25 0.12 16 1.0 2.0 0.5 2.0 0.5
0.5 0.25 32 2.0 16 4.0 16
0.12 0.25 0.25 0.12 0.25 0.25 2.0
0.12 0.5 0.5 0.25 0.5 1.0 4.0
14 5 7 26 45 9 32 21 5 19 19 18 34 15 20
C0.06-0.25 16-32 0.12-8.0 0.5-16 0.25-4.0 0.5-32 0.12-4.0
_0.06-0.12 C0.06-0.5
Vol. 16, No. 2
In Vitro Activity of Sodium Fusidate Against Anaerobic Bacteria G. E. STEINKRAUSt AND L. R. McCARTHY North Carolina Memorial Hospital, and Department of Bacteriology Laboratories, Hospital of Department and Immunology and Department of Pathology, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 11 May 1979
The microtiter broth dilution method was employed to determine the in vitro susceptibility of 525 recent clinical isolates of anaerobic bacteria to sodium fusidate. The minimal inhibitory concentrations of sodium fusidate ranged from -0.06 to 1.0 ,ug/ml for 155 strains of anaerobic gram-positive rods and 130 strains of anaerobic gram-positive cocci. Minimal inhibitory concentrations ranging from s0.06 to 32 ,ug/ml were observed for 240 strains of anaerobic gram-negative rods. Among the latter group a minimal inhibitory concentration of 16 ,ug/ml or greater was encountered with 16% of 45 Bacteroides fragilis strains, 19% of 32 Bacteroides thetaiotaomicron, and all 7 strains of Fusobacterium necrophorum. Minimal inhibitory concentrations for Veillonella parvula, the only gram-negative coccus tested, ranged from 0.5 to 8.0 yig/ml. microdilution modification of the microtiter procedure described by Thornsberry and Swenson (9). The 525 strains of anaerobic bacteria tested were recent clinical isolates recovered from wound, lower respiratory, and genital tract specimens submitted to the Clinical Microbiology Laboratories of the North Carolina Memorial Hospital. Each anaerobic strain was identified by using gas-liquid chromatography and specialized biochemical tests as described by Holdeman and Moore (5). The strains tested included 155 strains of anaerobic gram-positive rods, 240 strains of anaerobic gramnegative rods, 110 strains of anaerobic gram-positive cocci, and 20 strains of anaerobic gram-negative cocci. Sodium fusidate powder was supplied by HoffmanLaRoche Inc., Nutley, N.J. This powder was dissolved in distilled water to yield a concentration of 2,560 Lgg ml. Twelve serial twofold dilutions ranging from 256 to 0.12 gAg/ml were prepared in sterile Schaedler broth supplemented with 5 ,ug of hemin per ml and 0.1 ug of menadione per ml; 50-pd amounts of these dilutions were dispensed aseptically into sterile U-bottom microtiter plates with a Dynatech MIC-2000 dispenser (Cooke Laboratories, Inc., Alexandria, Va.). Plates not used within 24 h of preparation were sealed in polyethylene bags and frozen at -70°C for a maximum of 6 weeks. Before inoculation, microtiter plates were placed in an anaerobic glove box and incubated at ambient temperature in an atmosphere of 85% N2, 10% H2, and 5% CO2 for 24 h. All inocula were prepared and dispensed within the glove box environment. Inocula were prepared by susMATERIALS AND METHODS pending well-isolated colonies of each test strain from The in vitro activity of sodium fusidate against Schaedler sheep blood agar plates in 2 ml of suppleanaerobic bacteria was investigated by employing a mented Schaedler broth. The turbidity of the suspension was adjusted to that of a MacFarland no. 0.5 t Present address: Department of Pathology, Bishop Clark- turbidity standard. A 1:100 dilution of the standardized suspension was prepared in supplemented Schaedler son Hospital, Omaha, NE 68105. 120
Fusidic acid, produced by the fungus Fusidium coccineum, possesses a steroid structure and is chemically related to cephalosporin P1 (3). Unlike the cephalosporins, which inhibit cell wall synthesis, fusidic acid inhibits bacterial protein synthesis by interfering with the translocation enzyme and inhibiting the binding of aminoacyl transfer ribonucleic acid to ribosomes (2). Although a number of derivatives of fusidic acid have been prepared, only the sodium salt, sodium fusidate, possesses a significant degree of antibacterial activity. Sodium fusidate is remarkable for its high activity against both penicillin- and methicillinresistant strains of Staphylococcus aureus (6). Sodium fusidate is also very active against corynebacteria, but is poorly active against streptococci (4). Members of the genus Neisseria are the only aerobic gram-negative bacteria which are susceptible to this antibiotic (4). Other organisms previously shown to be susceptible to sodium fusidate include Clostridium tetani, C. perfringens, and Bacteroides fragilis (4, 7). It was the reported susceptibility of these three anaerobic species which led us to investigate more extensively the activity of sodium fusidate against anaerobic bacteria.
VOL. 16, 1979
ACTIVITY OF SODIUM FUSIDATE
121
broth. The diluted organism suspension was mixed RESULTS thoroughly, and 50 id of this suspension was added to each antibiotic well and to a growth control well Table 1 presents the results obtained for the containing 50 pl of the supplemented broth. The mi- in vitro activity of sodium fusidate against the crotiter plates were inoculated by using a Minitek 525 anaerobes tested. All 114 clostridial strains dispensing gun (BBL Microbiology Systems, Cockeys- tested MICs which ranged between 0.06 ville, Md.). With inoculation, the final concentration and 1.0yielded pg/ml. Clostridium bifermentans, C. of sodium fusidate contained within the 12 wells ranged between 128 and 0.06 /Ag/ml. The inoculated paraputrificum, and C. sordellii were the most plates were sealed in polyethylene bags and incubated susceptible of the 10 species of Clostridium at 35°C for 48 h in an anaerobic glove box (Coy tested, each species having an MIC of 50.25 ug Manufacturing Co., Ann Arbor, Mich.) with an atmos- of sodium fusidate per ml. All 16 strains of phere of 85% N2, 10% C02, and 5% H2. The minimal Eubacterium lentum were susceptible to 1.0 pAg inhibitory concentration (MIC) was read as the lowest or less of sodium fusidate per ml. Of the 25 concentration of the antibiotic which inhibited visible strains of Propionibacterium acnes tested, 96% growth of each organism tested. were inhibited by 1.0 pg or less per ml, whereas The stability of fusidic acid in the prepared micro- 4% required 2.0 pg/ml for inhibition. titer trays was monitored by testing a strain of B. Bacteroides corrodens, B. melanmnogenicus fragilis (for which the MIC of fusidic acid was known) on each day tests were performed. This quality control subsp. asaccharolyticus, B. melaninogenicus testing indicated that the antibiotic was stable for at subsp. intermedius, B. oralis, and B. ruminicola least 6 weeks under the storage, prereduction, and subsp. ruminicola had MICs comparable to those observed for the anaerobic gram-positive incubation conditions employed. TABLE 1. In vitro activity of sodium fusidate against 525 anaerobes MIC (ag/ml of medium) Organism
No. of strains
Range
Anaerobic gram-positive rods Clostridium bifermentans C. cochlearum C. histolyticum C. innocuum C. paraputrificum C. perfringens C. ramosum C. sordellii C. subterminale C. tertium Eubacterium lentum Propionibacterium acnes Anaerobic gram-negative rods Bacteroides corrodens B. melaninogenicus subsp. asaccharolyticus B. melaninogenicus subsp. intermedius B. oralis B. ruminicola subsp. ruminicola Fusobacterium necrophorum Bacteroides distasonis B. fragilis B. ovatus B. thetaiotaomicron B. vulgatus Anaerobic cocci Gaffkya anaerobia Peptococcus assaccharolyticus P. magnus P. prevotii Peptostreptococcus anaerobius P. micros
Veillonella parvula
For 50% of strains
For 90% of strains
11 4 10 12 5 39 9 6 8 10 16 25
_0.06-0.25 0.25-1.0 _0.06-0.5 0.12-1.0 C0.06-0.25 50.06-1.0 50.06-0.5 0.12-0.25 0.12-0.5 0.12-1.0 C0.06-1.0 _0.06-2.0
0.12 0.5 0.12 0.25 0.12 0.12 0.12 0.12 0.25 0.25 0.12 0.25
0.25
19 31
0.12-0.5 C0.06-1.0
0.25 0.25
0.5 1.0
31
C0.06-1.0
0.25
1.0
0.12-0.5
0.25 0.12 16 1.0 2.0 0.5 2.0 0.5
0.5 0.25 32 2.0 16 4.0 16
0.12 0.25 0.25 0.12 0.25 0.25 2.0
0.12 0.5 0.5 0.25 0.5 1.0 4.0
14 5 7 26 45 9 32 21 5 19 19 18 34 15 20
C0.06-0.25 16-32 0.12-8.0 0.5-16 0.25-4.0 0.5-32 0.12-4.0
_0.06-0.12 C0.06-0.5
