37
Staphylococcus epidermidis Extracted Slime Inhibits the Antimicrobial
Action of Glycopeptide Antibiotics Bruce F. Farber, Mark H. Kaplan, and Arlene G. Clogston
From the Department of Medicine, Division of Infectious Disease and Immunology, North Shore University Hospital, Manhasset, New York; and Department of Medicine, Cornell University Medical College, New York, New York
Coagulase-negative staphylococci have emerged as major pathogens over the past decade [1]. Previously considered contaminants, these organisms are the most common cause of prosthetic device-related infection. [1]. Since methicillin resistance is particularly common among coagulase-negative staphylococci, vancomycin remains the major antibiotic used to treat these infections [1]. In Vitro.. resistance to vancomycin among staphylococci is extremely uncommon [2]; however, foreign body infections due to these organisms frequently do not respond to vancomycin, and removal of the prosthetic device is often required [3]. Certain strains of Staphylococcus epidermidis produce an extracellular slime-like substance that may be an important virulence factor [4-7]. During our studies of the composition of extracellular slime, we observed a unique and unexpected finding: Extracellular slime contains a polysaccharide that interferes with the antimicrobial activity .and may explain why vancomycin is not always successful in eradicating foreign body infections.
Received 20 December 1988; revised Z7 July 1989. Presented in abstract form at the 1989 National Meeting of the American Federation for Clinical Research, Washington, DC. Supported by the Jane and Dayton T. Brown and Family Fund. Reprints and correspondence: Dr. Bruce F. Farber, Division of Infectious Disease and Immunology, North Shore University Hospital, 300 Community Dr., Manhasset, NY 11030. The Journal of Infectious Diseases 1990;161:37-40 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6101-0008$01.00
Materials and Methods Strains. Coagulase-negative staphylococci were collected from the clinical microbiology laboratory at North Shore University Hospital. Three additional strains were obtained from the American Type Culture Collection (Rockville, MO). The isolates were identified using API Staph-Ident (Analytab Products, Plainview, NY) and were maintained on tryptic soy agar slants. Selected isolates identified as S. epidermidis were used in subsequent studies. The bacteria were tested for the production of slime using qualitative methods previously described [6]. Preparation ofslime extracts. Crude slime culture filtrates were prepared from slime-positive strains (91, 95, ATCC 35983, 10, 1) grown in chemically defined media using the method of Johnson et ale [8]. The proteins in this crude culture filtrate were removed by three phenol extractions using the method of Gotschlich et ale [9]. The remaining solution was then extracted with one-half volume of chloroform to remove lipids. The chloroform was vigorously shaken for 30 s and centrifuged for 15 min at 35,000 g. The chloroform layer was then extracted with one-half volume of distilled water to recover polysaccharide lost during extraction. Recovered polysaccharide was added to the original extract. The extracted slime preparation was then dialyzed in cellophane sacks with a 12,000 molecular weight cutoff against distilled water for 24 h at 4°C, lyophilized at -60°C, and reconstituted in distilled water to a concentration of 100 mg/ml. About 1 liter of media was required to produce 100 mg of extracted slime. The S. epidermidis-extracted slime preparations were fractionated using discontinuous polyacrylamide gel electrophoresis (PAGE; Bio-Rad Laboratories, Richmond, CA) [10]; 30 J.11 of the extract (3.0 mg/ml) was combined in an equal volume of lysis buffer (20 ml of 0.05% bromophenol blue, 5 ml of 2-mercaptoethanol, 30 ml of 10% SOS, 10 ml of glycerol, 12.5 ml of 0.5 M Tris [pH 6.8]). Electrophoresis was performed in a protean cell (Bio-Rad) with Tris-glycine running buffer (pH 8.3) overnight at 45 V at room tem-
Downloaded from http://jid.oxfordjournals.org/ at New York University on February 8, 2015
Certain strains of Staphylococcus epidermidis produce a mucoid slime that appears to be an important virulence factor. After crude slime was isolated from selected strains of S. epidermidis, phenol and chloroform were used to remove proteins and lipids. The remaining extract contained a polysaccharide that was seen on SDS gels stained with Stains-all. This extract was an inhibitor of the antimicrobial action of vancomycin, raising the minimum inhibitory concentration (MIC) to vancomycin in all 18 isolates of S. epidermidis. A dose-response curve was seen between the amount of extract added and the degree of resistance, as measured by both MIC and growth curves. A similar effect was noted with MICs of organisms to teicoplanin. Addition of the extract did not change the MIC to LY146032, although a modest effect on growth rate was observed. The extract did not raise the MIC to clindamycin, rifampin, and cefazolin. The extract reversed the synergism seen between vancomycin and gentamicin in the 5 strains tested in time-kill studies. The interference by slime of the antimicrobial effect of vancomycin and teicoplanin may explain why these antibiotics are sometimes ineffective in eradicating foreign body infections due to slimeproducing coagulase-negative staphylococci.
38
Farber et al.
Results All, eight crude slime preparations revealed multiple protein bands when they were fractionated on 10% polyacrylamide gels stained with Coomassie brilliant blue. Several common bands were seen in the 25 kDa and 35 kDa regions. All visible protein bands disappeared from the gels after phenol/chloroform extraction of crude slime preparation. These same gels of extracted slime preparation, when stained with Stains-all, demonstrated a broad blue band characteristic of either glycoprotein or complex polysaccharide in all eight strains. This substance was not removed with the multiple chloroform/phenol extractions of crude slime. Tables 1 and 2 list the MICs for isolates of S. epidermidis to several antibiotics. The MICs were run simultaneously with and without the addition of 0.5% (5 mg/ml) extracted slime. All isolates tested had an increase in the MIC to vancomycin with the addition of the extracted slime. In most instances there was a fourfold increase in MIC. Extracts prepared from different strains had an equivalent effect on a weight basis. A similar increase in MIC was seen with teicoplanin. No change in MIC was noted when extracted slime was added to cefazolin, clindamycin, rifampin, and LY146032. MICs to
Table 1. Effect of 0.5 % extracted slime on the minimum inhibitory concentration of Staphylococcus epidermidis to antibiotics. Vancomycin Isolate* 95+ 99+ 6+ 723335983+ 35984+ IM2M5M91+ 36+ 96+ 104+ 3+ 9+ 10+ 1+ Geometric mean S. aureus 25923
Teicoplanin (ug/ml)
LY146032
No extract
Slime extract
No extract
Slime extract
0.8 1.1 1.0 2.0 1.7 2.0 1.0 1.0 0.7 1.0 1.7 2.4 2.2 1.0 1.0 1.0 1.0 2.8
6.3 14.0 6.3 11.0 7.8 7.8 2.7 5.5 3.8 3.8 7.0 14.0 8.8 2.5 7.9 5.6 7.9 7.9
1.9 5.5 1.3 7.9 2.7 5.5 2.7 2.7 0.5 0.5 5.5 2.7 11.0 11.0 1.3 3.9 11.0 15.6
22.0 22.0 11.0 62.5 15.6 15.6 7.9 11.0 3.8 5.5 15.6 15.6 31.3 31.3 7.8 15.6 31.3 44.2
0.2 0.2 0.1 0.4 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.4 0.3 0.3 0.2 0.2 0.5 0.5
0.2 0.4 0.2 0.4 0.1 0.2 0.3 0.4 0.2 0.2 0.2 0.4 0.3 0.3 0.2 0.2 0.5 0.5
1.3
6.6
3.4
15.8
0.24
0.27
1.4
5.5
1.4
7.8
NT
NT
Slime No extract extract
NOTE. Results are expressed as the geometric mean of two determinations and are given in ug/rnl. NT = not tested. * +, slime-positive; -, slime-negative strain.
vancomycin were determined as increasing amounts of extract were added to the media. In two strains tested (91 and 10) there was a direct linear increase in the MIC to vancomycin, which went from 1.56 ug/ml in the absence of extract to 6.25 ug/ml in the presence of 1% (10 mg/ml) extract and to 12.5 ug/ml in the presence of 2 % (20 mg/ml) extract. Figure 1 demonstrates the relationship between growth of an organism and time, in the presence of vancomycin supplemented with two different concentrations of extracted slime and in Mueller-Hinton broth (control) for strain 91. A relationship was again observed between the amount of extract and growth in the presence of vancomycin. Inhibition of the antibiotic effect was greater at 24 than at 4 h. Figure 2 shows the relationship between growth and time in the presence of vancomycin, gentamicin, and a combination of these antibiotics with and without the extracted polysaccharide for strain 91. The addition of a subinhibitory concentration of gentamicin to vancomycin resulted in synergism (>2 log increase in bacterial inhibition). The addition of the extract reversed the increased bacterial killing that was seen in the presence of vancomycin and gentamicin. Figure 3 shows a time-kill curve of teicoplanin and LY149032, with and without the extracted slime for strain 91. Although the extracted slime influenced both curves, the effect was much more pronounced for teicoplanin. Results similar to those shown in figure 1 and 2 were obtained with four ad-
Downloaded from http://jid.oxfordjournals.org/ at New York University on February 8, 2015
perature. The gels were fixed in trichloroacetic acid (Sigma Chemical, St. Louis), stained in 0.5% Coomassie brilliant blue, and destained in 30% methanol-IO% acetic acid. Gels prepared in an identical manner were also stained with Stains-all (Kodak, Rochester, NY). Stains-all identifies proteins (red), lipids (yellow-orange), and glycoproteins and polysaccharides (blue) on the same gel [11]. Susceptibility testing. Antibiotic susceptibility testing was done using a standard microtiter method for the determination of minimum inhibitory concentration (MIC) [12]. Serial twofold dilutions of antibiotics were prepared in Mueller-Hinton broth (Difco, Detroit). The inoculum consisted of organisms grown into log phase, which were then diluted to a concentration of 105 cfu/ml. Each well consisted of 50 J.11 of antibiotic, 50 J.11 of broth or extracted slime, and 100 J.11 of inoculum. Testing was done in Mueller-Hinton media, with or without extracted slime supplementation, at a final concentration of 0.5% (5 mg/ml). The Mueller-Hinton broth was supplemented with calcium chloride and magnesium chloride at concentrations of 50 ug/ml and 25 ug/ml for the teicoplanin and LY146032 studies. Incubations were at 35°C for 24 h. The MIC was defined as the lowest concentration of each antibiotic that inhibited visible growth of the organism. The antibiotics used included: vancomycin, LY146032 (Eli Lilly, Indianapolis), rifampin (Sigma), clindamycin (Upjohn, Kalamazoo, MI), cefazolin (Smith Kline & French Laboratories, Philadelphia), and teicoplanin (Merrell Dow Research Labs, Cincinnati). Selected strains had multiple MICs performed using increasing concentrations of the extract. Staphylococcus aureus strain ATCC 25923 was used as a control organism for vancomycin and teicoplanin. TIme-kill studies. Antibiotic concentrations and combinations were tested in Mueller-Hinton broth using a time-kill method previously described [12]. Samples (0.5 ml) were removed for colony counts at 0,4, and 24 h: after serial dilution in saline, 0.025 ml was inoculated onto Mueller-Hinton agar. Plates were incubated for 24 h at 35°C and colonies were counted.
JID 1990;161 (January)
S. epidermidis Extracted Slime
JID 1990;161 (January)
39
Table 2. Effect of 0.5% extracted slime on the minimum inhibitory concentration of Staphylococcus epidermidis to antibiotics. Rifampin Isolate
Cefazolin
No extract
Slime extract
No extract
Slime extract
No Slime extract extract
0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.003
0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.003
15.6 22.0 15.6 11.0 22.0 22.0 22.0 22.0 31.3 15.6 15.6 22.0
15.6 15.6 15.6 15.6 22.0 15.6 15.6 22.0 22.0 15.6 15.6 22.0
0.2 0.4 0.2 0.4 0.2 0.8 0.4 2.2 0.2 0.2 0.4 6.3
0.3 0.4 0.2 0.4 0.2 0.8 0.4 2.2 0.2 0.2 0.3 6.3
0.014
0.014
18.7
17.5
0.5
0.5
12
8
16
24
20
Time (h)
Figure 1. Time-kill curve of colony-forming units per milliliter (cfu/ml) versus time. Vancomycin (VANCO) 2 ug/ml with and without phenol/chloroform extract (PE), at concentrations of 5 mg/ml and 1 mg/ml, and Mueller-Hinton broth (MHB).
NOTE. Results are expressed as the geometric mean of two determinations and are given in ug/rnl. .0 • • • • • • . • . • • • • • • • • • • • . •
8
ditional strains tested (ATCC 35983, 33, 9, 1). Mueller-Hinton broth with and without extract resulted in essentially superimposable growth curves.
E ~
~
A-AMHB ----VANCO . - ·VANCO+GENT 0 · · · ,oGENT 0- -00.5% PE+VANCO+GENT
5· 4
,_-_- - -
.3
Staphylococcus epidermidis Extracted Slime Inhibits the Antimicrobial
Action of Glycopeptide Antibiotics Bruce F. Farber, Mark H. Kaplan, and Arlene G. Clogston
From the Department of Medicine, Division of Infectious Disease and Immunology, North Shore University Hospital, Manhasset, New York; and Department of Medicine, Cornell University Medical College, New York, New York
Coagulase-negative staphylococci have emerged as major pathogens over the past decade [1]. Previously considered contaminants, these organisms are the most common cause of prosthetic device-related infection. [1]. Since methicillin resistance is particularly common among coagulase-negative staphylococci, vancomycin remains the major antibiotic used to treat these infections [1]. In Vitro.. resistance to vancomycin among staphylococci is extremely uncommon [2]; however, foreign body infections due to these organisms frequently do not respond to vancomycin, and removal of the prosthetic device is often required [3]. Certain strains of Staphylococcus epidermidis produce an extracellular slime-like substance that may be an important virulence factor [4-7]. During our studies of the composition of extracellular slime, we observed a unique and unexpected finding: Extracellular slime contains a polysaccharide that interferes with the antimicrobial activity .and may explain why vancomycin is not always successful in eradicating foreign body infections.
Received 20 December 1988; revised Z7 July 1989. Presented in abstract form at the 1989 National Meeting of the American Federation for Clinical Research, Washington, DC. Supported by the Jane and Dayton T. Brown and Family Fund. Reprints and correspondence: Dr. Bruce F. Farber, Division of Infectious Disease and Immunology, North Shore University Hospital, 300 Community Dr., Manhasset, NY 11030. The Journal of Infectious Diseases 1990;161:37-40 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6101-0008$01.00
Materials and Methods Strains. Coagulase-negative staphylococci were collected from the clinical microbiology laboratory at North Shore University Hospital. Three additional strains were obtained from the American Type Culture Collection (Rockville, MO). The isolates were identified using API Staph-Ident (Analytab Products, Plainview, NY) and were maintained on tryptic soy agar slants. Selected isolates identified as S. epidermidis were used in subsequent studies. The bacteria were tested for the production of slime using qualitative methods previously described [6]. Preparation ofslime extracts. Crude slime culture filtrates were prepared from slime-positive strains (91, 95, ATCC 35983, 10, 1) grown in chemically defined media using the method of Johnson et ale [8]. The proteins in this crude culture filtrate were removed by three phenol extractions using the method of Gotschlich et ale [9]. The remaining solution was then extracted with one-half volume of chloroform to remove lipids. The chloroform was vigorously shaken for 30 s and centrifuged for 15 min at 35,000 g. The chloroform layer was then extracted with one-half volume of distilled water to recover polysaccharide lost during extraction. Recovered polysaccharide was added to the original extract. The extracted slime preparation was then dialyzed in cellophane sacks with a 12,000 molecular weight cutoff against distilled water for 24 h at 4°C, lyophilized at -60°C, and reconstituted in distilled water to a concentration of 100 mg/ml. About 1 liter of media was required to produce 100 mg of extracted slime. The S. epidermidis-extracted slime preparations were fractionated using discontinuous polyacrylamide gel electrophoresis (PAGE; Bio-Rad Laboratories, Richmond, CA) [10]; 30 J.11 of the extract (3.0 mg/ml) was combined in an equal volume of lysis buffer (20 ml of 0.05% bromophenol blue, 5 ml of 2-mercaptoethanol, 30 ml of 10% SOS, 10 ml of glycerol, 12.5 ml of 0.5 M Tris [pH 6.8]). Electrophoresis was performed in a protean cell (Bio-Rad) with Tris-glycine running buffer (pH 8.3) overnight at 45 V at room tem-
Downloaded from http://jid.oxfordjournals.org/ at New York University on February 8, 2015
Certain strains of Staphylococcus epidermidis produce a mucoid slime that appears to be an important virulence factor. After crude slime was isolated from selected strains of S. epidermidis, phenol and chloroform were used to remove proteins and lipids. The remaining extract contained a polysaccharide that was seen on SDS gels stained with Stains-all. This extract was an inhibitor of the antimicrobial action of vancomycin, raising the minimum inhibitory concentration (MIC) to vancomycin in all 18 isolates of S. epidermidis. A dose-response curve was seen between the amount of extract added and the degree of resistance, as measured by both MIC and growth curves. A similar effect was noted with MICs of organisms to teicoplanin. Addition of the extract did not change the MIC to LY146032, although a modest effect on growth rate was observed. The extract did not raise the MIC to clindamycin, rifampin, and cefazolin. The extract reversed the synergism seen between vancomycin and gentamicin in the 5 strains tested in time-kill studies. The interference by slime of the antimicrobial effect of vancomycin and teicoplanin may explain why these antibiotics are sometimes ineffective in eradicating foreign body infections due to slimeproducing coagulase-negative staphylococci.
38
Farber et al.
Results All, eight crude slime preparations revealed multiple protein bands when they were fractionated on 10% polyacrylamide gels stained with Coomassie brilliant blue. Several common bands were seen in the 25 kDa and 35 kDa regions. All visible protein bands disappeared from the gels after phenol/chloroform extraction of crude slime preparation. These same gels of extracted slime preparation, when stained with Stains-all, demonstrated a broad blue band characteristic of either glycoprotein or complex polysaccharide in all eight strains. This substance was not removed with the multiple chloroform/phenol extractions of crude slime. Tables 1 and 2 list the MICs for isolates of S. epidermidis to several antibiotics. The MICs were run simultaneously with and without the addition of 0.5% (5 mg/ml) extracted slime. All isolates tested had an increase in the MIC to vancomycin with the addition of the extracted slime. In most instances there was a fourfold increase in MIC. Extracts prepared from different strains had an equivalent effect on a weight basis. A similar increase in MIC was seen with teicoplanin. No change in MIC was noted when extracted slime was added to cefazolin, clindamycin, rifampin, and LY146032. MICs to
Table 1. Effect of 0.5 % extracted slime on the minimum inhibitory concentration of Staphylococcus epidermidis to antibiotics. Vancomycin Isolate* 95+ 99+ 6+ 723335983+ 35984+ IM2M5M91+ 36+ 96+ 104+ 3+ 9+ 10+ 1+ Geometric mean S. aureus 25923
Teicoplanin (ug/ml)
LY146032
No extract
Slime extract
No extract
Slime extract
0.8 1.1 1.0 2.0 1.7 2.0 1.0 1.0 0.7 1.0 1.7 2.4 2.2 1.0 1.0 1.0 1.0 2.8
6.3 14.0 6.3 11.0 7.8 7.8 2.7 5.5 3.8 3.8 7.0 14.0 8.8 2.5 7.9 5.6 7.9 7.9
1.9 5.5 1.3 7.9 2.7 5.5 2.7 2.7 0.5 0.5 5.5 2.7 11.0 11.0 1.3 3.9 11.0 15.6
22.0 22.0 11.0 62.5 15.6 15.6 7.9 11.0 3.8 5.5 15.6 15.6 31.3 31.3 7.8 15.6 31.3 44.2
0.2 0.2 0.1 0.4 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.4 0.3 0.3 0.2 0.2 0.5 0.5
0.2 0.4 0.2 0.4 0.1 0.2 0.3 0.4 0.2 0.2 0.2 0.4 0.3 0.3 0.2 0.2 0.5 0.5
1.3
6.6
3.4
15.8
0.24
0.27
1.4
5.5
1.4
7.8
NT
NT
Slime No extract extract
NOTE. Results are expressed as the geometric mean of two determinations and are given in ug/rnl. NT = not tested. * +, slime-positive; -, slime-negative strain.
vancomycin were determined as increasing amounts of extract were added to the media. In two strains tested (91 and 10) there was a direct linear increase in the MIC to vancomycin, which went from 1.56 ug/ml in the absence of extract to 6.25 ug/ml in the presence of 1% (10 mg/ml) extract and to 12.5 ug/ml in the presence of 2 % (20 mg/ml) extract. Figure 1 demonstrates the relationship between growth of an organism and time, in the presence of vancomycin supplemented with two different concentrations of extracted slime and in Mueller-Hinton broth (control) for strain 91. A relationship was again observed between the amount of extract and growth in the presence of vancomycin. Inhibition of the antibiotic effect was greater at 24 than at 4 h. Figure 2 shows the relationship between growth and time in the presence of vancomycin, gentamicin, and a combination of these antibiotics with and without the extracted polysaccharide for strain 91. The addition of a subinhibitory concentration of gentamicin to vancomycin resulted in synergism (>2 log increase in bacterial inhibition). The addition of the extract reversed the increased bacterial killing that was seen in the presence of vancomycin and gentamicin. Figure 3 shows a time-kill curve of teicoplanin and LY149032, with and without the extracted slime for strain 91. Although the extracted slime influenced both curves, the effect was much more pronounced for teicoplanin. Results similar to those shown in figure 1 and 2 were obtained with four ad-
Downloaded from http://jid.oxfordjournals.org/ at New York University on February 8, 2015
perature. The gels were fixed in trichloroacetic acid (Sigma Chemical, St. Louis), stained in 0.5% Coomassie brilliant blue, and destained in 30% methanol-IO% acetic acid. Gels prepared in an identical manner were also stained with Stains-all (Kodak, Rochester, NY). Stains-all identifies proteins (red), lipids (yellow-orange), and glycoproteins and polysaccharides (blue) on the same gel [11]. Susceptibility testing. Antibiotic susceptibility testing was done using a standard microtiter method for the determination of minimum inhibitory concentration (MIC) [12]. Serial twofold dilutions of antibiotics were prepared in Mueller-Hinton broth (Difco, Detroit). The inoculum consisted of organisms grown into log phase, which were then diluted to a concentration of 105 cfu/ml. Each well consisted of 50 J.11 of antibiotic, 50 J.11 of broth or extracted slime, and 100 J.11 of inoculum. Testing was done in Mueller-Hinton media, with or without extracted slime supplementation, at a final concentration of 0.5% (5 mg/ml). The Mueller-Hinton broth was supplemented with calcium chloride and magnesium chloride at concentrations of 50 ug/ml and 25 ug/ml for the teicoplanin and LY146032 studies. Incubations were at 35°C for 24 h. The MIC was defined as the lowest concentration of each antibiotic that inhibited visible growth of the organism. The antibiotics used included: vancomycin, LY146032 (Eli Lilly, Indianapolis), rifampin (Sigma), clindamycin (Upjohn, Kalamazoo, MI), cefazolin (Smith Kline & French Laboratories, Philadelphia), and teicoplanin (Merrell Dow Research Labs, Cincinnati). Selected strains had multiple MICs performed using increasing concentrations of the extract. Staphylococcus aureus strain ATCC 25923 was used as a control organism for vancomycin and teicoplanin. TIme-kill studies. Antibiotic concentrations and combinations were tested in Mueller-Hinton broth using a time-kill method previously described [12]. Samples (0.5 ml) were removed for colony counts at 0,4, and 24 h: after serial dilution in saline, 0.025 ml was inoculated onto Mueller-Hinton agar. Plates were incubated for 24 h at 35°C and colonies were counted.
JID 1990;161 (January)
S. epidermidis Extracted Slime
JID 1990;161 (January)
39
Table 2. Effect of 0.5% extracted slime on the minimum inhibitory concentration of Staphylococcus epidermidis to antibiotics. Rifampin Isolate
Cefazolin
No extract
Slime extract
No extract
Slime extract
No Slime extract extract
0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.003
0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.003
15.6 22.0 15.6 11.0 22.0 22.0 22.0 22.0 31.3 15.6 15.6 22.0
15.6 15.6 15.6 15.6 22.0 15.6 15.6 22.0 22.0 15.6 15.6 22.0
0.2 0.4 0.2 0.4 0.2 0.8 0.4 2.2 0.2 0.2 0.4 6.3
0.3 0.4 0.2 0.4 0.2 0.8 0.4 2.2 0.2 0.2 0.3 6.3
0.014
0.014
18.7
17.5
0.5
0.5
12
8
16
24
20
Time (h)
Figure 1. Time-kill curve of colony-forming units per milliliter (cfu/ml) versus time. Vancomycin (VANCO) 2 ug/ml with and without phenol/chloroform extract (PE), at concentrations of 5 mg/ml and 1 mg/ml, and Mueller-Hinton broth (MHB).
NOTE. Results are expressed as the geometric mean of two determinations and are given in ug/rnl. .0 • • • • • • . • . • • • • • • • • • • • . •
8
ditional strains tested (ATCC 35983, 33, 9, 1). Mueller-Hinton broth with and without extract resulted in essentially superimposable growth curves.
E ~
~
A-AMHB ----VANCO . - ·VANCO+GENT 0 · · · ,oGENT 0- -00.5% PE+VANCO+GENT
5· 4
,_-_- - -
.3
