ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1992,
p.
626-631
Vol. 36, No. 3
0066-4804/92/030626-06$02.00/0
Copyright ©) 1992, American Society for Microbiology
Effect of Glycerol Monolaurate on Bacterial Growth and Toxin Production PATRICK M. SCHLIEVERT,1* JAMES R. DERINGER,1 MICHAEL H. KIM,' STEVEN J. PROJAN,2 AND RICHARD P. NOVICK2
Department of Microbiology, University of Minnesota Medical School, 420 Delaware Street, Minneapolis, Minnesota 55455,1 and The Public Health Research Institute of the City of New
York, Incorporated, New York, New York 100162
Received 23 August 1991/Accepted 2 January 1992
Glycerol monolaurate (GML) is a naturally occurring surfactant that has potential use as an additive to tampons and wound dressings to reduce the incidence of certain bacterial toxin-mediated illnesses. In vitro studies were undertaken to evaluate the effect of GML on the growth of and toxin production by potentially pathogenic bacteria. GML inhibited the growth of clinical isolates of group A, B, F, and G streptococci at concentrations of 10 to 20 ,ug/ml. Exotoxin production, including that of pyrogenic exotoxins and hemolysins, reduced by concentrations of GML that were below those inhibitory for growth as well as growth inhibitory. The growth of Staphylococcus aureus strains from patients with toxic shock syndrome and scalded skin syndrome was inhibited or delayed in the presence of 100 to 300 ig of GML per ml. Growth inhibition by GML could be overcome by the production of lipase. S. aureus elaboration of hemolysin, toxic shock syndrome toxin 1, and exfoliative toxin A was inhibited at GML concentrations below those necessary to inhibit growth. Results similar to those for S. aureus were obtained in tests of S. hominis. Escherichia coli growth and SalmoneUla minnesota growth were unaffected by GML, but an S. minnesota Re mutant was susceptible to growth-inhibitory activity. Endotoxin release into the medium from E. coli cells was also unaffected by GML, but the release or activity of E. coli hemolysin was increased by GML. Streptococcal pyrogenic exotoxin A production by an E. coli clone was not affected by GML. These studies indicate that GML is effective in blocking or delaying the production of exotoxins by pathogenic gram-positive bacteria.
was
Toxic shock syndrome (TSS), caused by Staphylococcus aureus, is characterized by the acute onset of fever, hypotension, rash, a variable multiorgan component, and desquamation of the skin upon recovery (8, 10, 30, 31). Although TSS was initially described in children (31), much attention has focused on the association of TSS with menstruation and the use of certain high-absorbency tampons (10, 19, 30). The illness may occur in nearly any individual, male or female, and is apparently associated with any type of staphylococcal infection in which the causative toxins are made. TSS is induced primarily by TSS toxin 1 (TSST-1) in all menstrual cases and approximately one-half of nonmenstrual cases (3, 5, 24, 29). The remaining cases are associated with the production of enterotoxins, notably, B and C and rarely A (9, 16, 23, 24). All of these toxins have the ability to induce TSS-like symptoms in rabbits experimentally exposed to the
environment surrounding the staphylococci, suggests that it should be possible to add to tampons agents that negatively affect TSST-1 production but do not affect colonization by various bacteria. Such an agent may be useful in reducing the risk of TSS associated with tampons. Glycerol monolaurate (GML) is a compound that has recently been shown to inhibit the production of TSST-1 and staphylococcal enterotoxins in a tampon sac method (7). This compound is routinely used as a surfactant in a variety of products and in various foods for human consumption. Similarly, studies have shown that GML-coated tampons may reduce the risk of the development of TSS in rabbits (17). However, the effect of GML on other potentially pathogenic bacteria has not been investigated. This study was undertaken to evaluate the effect of GML on the growth of various bacteria, both gram positive and gram negative, and on the production of toxins by the organisms.
toxins (20). Several studies have shown that the production of TSST-1 and enterotoxins B and C by S. aureus strains is influenced by factors in the culture medium (6, 21, 22, 26-28, 33). For example, a neutral pH and the presence of oxygen are necessary for toxin production, whereas growth of the staphylococci may occur in their absence (26, 33). Likewise, Schlievert and Kelly (28) showed that clindamycin inhibits the production of TSST-1 at antibiotic concentrations that do not affect bacterial growth. All of these toxins are susceptible to repression by glucose (6, 22, 26). Finally, each of these toxins is regulated by an accessory gene regulatory, agr, a global regulator of exoprotein expression in S. aureus (21). The regulation of exotoxin production, as influenced by the *
MATERIALS AND METHODS Bacteria. S. aureus MN 8 was from a patient with menstruation-associated TSS and makes high levels of TSST-1 (26). S. aureus EV was kindly provided by Mark Kaplan, North Shore University Hospital, Manhassett, N.Y., and makes exfoliative toxin (ET) A (13). Strain MN 8 is weakly hemolytic and strain EV is strongly hemolytic on sheep blood agar plates; both strains are lipase producers, with MN 8 making more lipase than EV (see Results). S. hominis originally came from W. E. Kloos, North Carolina State University, Raleigh, and makes hemolysin and lipase. Group A streptococcal strains 594, 86-858, and T18P were maintained in our laboratory as sources of streptococcal pyro-
Corresponding author. 626
genic exotoxins (SPEs) A, B, and C, respectively (6, 11, 25). Group A streptococcal strain C203U, maintained in our laboratory, was used as the source of streptolysin 0; this strain does not make streptolysin S. Wild-type Salmonella minnesota (wild type with respect to endotoxin production) and an S. minnesota Re mutant (ReS95) were generously provided by Dennis W. Watson, Department of Microbiology, University of Minnesota, Minneapolis. A recent clinical isolate of hemolytic Eschenichia coli was kindly provided by Harriet Lievan, Department of Microbiology, University of Minnesota, Minneapolis. E. coli RR1(pUMN103) was maintained in our laboratory and produces SPE A (12). Antisera and toxins. Hyperimmune rabbit antisera were individually raised against TSST-1, ET A, and SPEs (25). Purified toxins were prepared by ethanol precipitation of culture fluids and then preparative thin-layer isoelectric focusing (29). Purified toxins migrated as homogeneous proteins when 20 ,ug of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (14) and then stained with Coomassie brilliant blue. GML. GML was provided by Personal Products Co., New Brunswick, N.J., and was indicated to have a composition of 95% GML and 5% -glycerol dilaurate. The compound was dissolved in absolute ethanol at a concentration of 100 mg/ml, shown to be sterile by plating of serial 10-fold dilutions on blood agar plates, and diluted into 37°C medium for use. Exoprotein assays. TSST-1, ET A, and SPEs were evaluated by dilution Ouchterlony immunodiffusion and Western blot (immunoblot) analysis (4, 26). For Ouchterlony immunodiffusion, strains to be evaluated were cultured for various times, concentrated 100 times by ethanol precipitation and resolubilization in distilled water, serially diluted in twofold increments, and then reacted against the antisera. The lower limit of toxin detection by this method was determined to be approximately 6 ,ug/ml (in the 100x -concentrated ethanol precipitate), which is 0.06 ,ug/ml for each toxin in the original culture fluid. Toxin concentrations were also estimated by serial dilution and then reactivity in Western blots. The lower limit of toxin detection by this method was approximately 0.013 p.g/ml. Prior to SDS-PAGE, the streptococcal strains were treated with hyaluronidase (10 p.g/ml) for 1 h. When this step was omitted, the streptococcal toxins did not enter the SDS gels. Uninoculated control culture flasks containing known amounts of toxin and GML were treated comparably to evaluate possible interference in the assays by GML. No interference was observed. Staphylococcal lipase was estimated by cleavage of tributyrin (15) or GML. For GML, cleavage assays were run the same as for tributyrin, except that GML was used at 20 p.g/ml and slides were placed at 4°C for 24 h after overnight incubation at 37°C. The 4°C temperature caused intact GML to form an insoluble crystalline matrix, leaving a zone of clearing where GML was cleaved. Staphylococcal hemolysin was assessed as follows. Rabbit erythrocytes (RBCs) were washed twice in phosphate-buffered saline (PBS; 0.005 M sodium phosphate [pH 7.2], 0.15 M NaCl) by centrifugation (500 x g, 10 min). The packed cells were diluted 1:1,000 in PBS-0.75% agarose (Sigma Chemical Co., St. Louis, Mo.), and 4.5 ml was used to coat microscope slides. Four-millimeter wells were punched in the slides after the agarose had solidified, and 20 pul of culture fluid was added to each well. The slides were incubated for 4 h at 37°C, and lysis was measured as clearing in millimeters. When GML alone was added to the slides at concentrations of 0 to 300 ,ug/ml, no lysis of rabbit RBCs was seen.
627
GML INHIBITION OF BACTERIA
VOL. 36, 1992
TABLE 1. Effect of GML on group A streptococci after 24 h of incubation
Bacterium and SPE
GML GM
Lo o
SPE P
Hemolysin"
b
(1±g/ml)
CFU/mla
(ligml)
594 (A)
0 2.5 10.0 20.0
8.6 8.5 8.3 6.0
3.2 0.3 0.3 0.0
7.0 4.0 0.0 0.0
86-858 (B)
0 2.5 10.0 20.0
8.0 7.7 7.7 5.8
0.8 0.0 0.0 0.0
4.0 2.0 0.0 0.0
T18P (C)
0 2.5 10.0
7.9 7.9 6.1
0.4 0.0 0.0
8.0 8.0 0.0
produced
a Inoculum size, 105 to 106 CFU/ml. b Including streptolysins 0 and S; measured as the zone of millimeters, of sheep RBCs.
lysis,
in
E. coli hemolysin was measured comparably, except that sheep RBCs were used. Streptococcal hemolysins were also measured comparably, except that sheep RBCs were used and 0.14% 2-mercaptoethanol was added to the PBS-agarose solution. As controls, GML at concentrations of 1 to 20 ,ug/ml did not inhibit the lysis of RBCs by purified preparations of streptolysin 0 used at concentrations of up to 50
,ug/ml. Endotoxin assay. Gram-negative bacteria were cultured for various times, and culture fluids were clarified by centrifugation (10,000 x g) and filtration (0.45-,um-pore-size filters; Gelman Corp.). Serial twofold dilutions were made in sterile pyrogen-free water, and a Limulus assay was performed by the method recommended by the supplier (Sigma). Control endotoxin was obtained from S. typhimurium (32). Culture media and bacterial quantifications. A dialyzable beef heart medium was used to prepare purified exotoxins, including streptolysin 0 (29). Brain heart infusion broth (Difco) was used to culture all organisms to be tested for toxins. Bacteria were cultured at 370C for various times with shaking (200 rpm; Gyrotory shaker; New Brunswick Scientific, New Brunswick, N.J.). Plate counts on brain heart infusion agar (1.5%) were used to determine CFU per milliliter of culture fluid.
RESULTS All experiments were done a minimum of two times, with those evaluating effects on S. aureus being done several times, and data from representative experiments are presented. The effect of GML on the growth of group A streptococci and the production of exotoxins was evaluated with three strains (Table 1). The growth of strains 594 and 86-858 was inhibited by 20 p.g of GML per ml but not 2.5 and 10 ,ug/ml, and the growth of strain T18P was inhibited by 10 ,ug/ml but not 2.5 ,ug/ml. For all three strains, the production of SPEs was reduced or completely inhibited by GML concentrations of 2.5 ,ug/ml or higher. Except in strain T18P, the production of hemolysin was also reduced or completely inhibited by .2.5 ,ug of GML per ml; strain T18P hemolysin production was inhibited by 10 jig/ml. The effect of GML on the growth of and hemolysin
ANTIMICROB. AGENTS CHEMOTHER.
SCHLIEVERT ET AL.
628
.
I8 a I9
9 vel
4
N
II 11
A
I ; .
=
Ai
a
_
ia
E i
I
e so
"
a
0.
v
4PEj!Ufl)
zwpRi{n) mdl
I.-
E*3_nH
S s
-q
i.
S
_
°
IED 1 r4
0) 3'j cr
(lgs) loLSSJl
II
TABLE 2. Effect of Zn on the lipase activity of S. aureus MN 8a Zn (M)
GML
(jig/ml)
TABLE 3. Effect of GML on E. coli and S. minnesota after 24 h of incubation
CFU/ LogmlLpae Lipase'
0 10 100
9.7 9.8 9.3
15 15 15
0.005
0 10 100
9.2 7.7 4.0
12 0 0
0.0005
0 10 100
9.8 9.3 5.6
12 6 0
0.0
629
GML INHIBITION OF BACTERIA
VOL. 36, 1992
Inoculum size, 5.0 x 107 CFU/ml. 6 Zone of clearing, in millimeters, of GML at 48 h.
Treatment
No inoculum
20 20 20 20
NDC 0.0 0.25 1.0
x 106 ND 1 x 106 2 x 106 2 x 106
2.0 ND 3.0 6.0 12.0
100 500 1,000 2,000
E. coli
0 100 500 1,000 2,000
9.4 9.4 9.4 9.3 9.4
Wild-type S. minnesota
0 100 500 1,000
9.9
0 100 500 1,000
9.4
p.
626-631
Vol. 36, No. 3
0066-4804/92/030626-06$02.00/0
Copyright ©) 1992, American Society for Microbiology
Effect of Glycerol Monolaurate on Bacterial Growth and Toxin Production PATRICK M. SCHLIEVERT,1* JAMES R. DERINGER,1 MICHAEL H. KIM,' STEVEN J. PROJAN,2 AND RICHARD P. NOVICK2
Department of Microbiology, University of Minnesota Medical School, 420 Delaware Street, Minneapolis, Minnesota 55455,1 and The Public Health Research Institute of the City of New
York, Incorporated, New York, New York 100162
Received 23 August 1991/Accepted 2 January 1992
Glycerol monolaurate (GML) is a naturally occurring surfactant that has potential use as an additive to tampons and wound dressings to reduce the incidence of certain bacterial toxin-mediated illnesses. In vitro studies were undertaken to evaluate the effect of GML on the growth of and toxin production by potentially pathogenic bacteria. GML inhibited the growth of clinical isolates of group A, B, F, and G streptococci at concentrations of 10 to 20 ,ug/ml. Exotoxin production, including that of pyrogenic exotoxins and hemolysins, reduced by concentrations of GML that were below those inhibitory for growth as well as growth inhibitory. The growth of Staphylococcus aureus strains from patients with toxic shock syndrome and scalded skin syndrome was inhibited or delayed in the presence of 100 to 300 ig of GML per ml. Growth inhibition by GML could be overcome by the production of lipase. S. aureus elaboration of hemolysin, toxic shock syndrome toxin 1, and exfoliative toxin A was inhibited at GML concentrations below those necessary to inhibit growth. Results similar to those for S. aureus were obtained in tests of S. hominis. Escherichia coli growth and SalmoneUla minnesota growth were unaffected by GML, but an S. minnesota Re mutant was susceptible to growth-inhibitory activity. Endotoxin release into the medium from E. coli cells was also unaffected by GML, but the release or activity of E. coli hemolysin was increased by GML. Streptococcal pyrogenic exotoxin A production by an E. coli clone was not affected by GML. These studies indicate that GML is effective in blocking or delaying the production of exotoxins by pathogenic gram-positive bacteria.
was
Toxic shock syndrome (TSS), caused by Staphylococcus aureus, is characterized by the acute onset of fever, hypotension, rash, a variable multiorgan component, and desquamation of the skin upon recovery (8, 10, 30, 31). Although TSS was initially described in children (31), much attention has focused on the association of TSS with menstruation and the use of certain high-absorbency tampons (10, 19, 30). The illness may occur in nearly any individual, male or female, and is apparently associated with any type of staphylococcal infection in which the causative toxins are made. TSS is induced primarily by TSS toxin 1 (TSST-1) in all menstrual cases and approximately one-half of nonmenstrual cases (3, 5, 24, 29). The remaining cases are associated with the production of enterotoxins, notably, B and C and rarely A (9, 16, 23, 24). All of these toxins have the ability to induce TSS-like symptoms in rabbits experimentally exposed to the
environment surrounding the staphylococci, suggests that it should be possible to add to tampons agents that negatively affect TSST-1 production but do not affect colonization by various bacteria. Such an agent may be useful in reducing the risk of TSS associated with tampons. Glycerol monolaurate (GML) is a compound that has recently been shown to inhibit the production of TSST-1 and staphylococcal enterotoxins in a tampon sac method (7). This compound is routinely used as a surfactant in a variety of products and in various foods for human consumption. Similarly, studies have shown that GML-coated tampons may reduce the risk of the development of TSS in rabbits (17). However, the effect of GML on other potentially pathogenic bacteria has not been investigated. This study was undertaken to evaluate the effect of GML on the growth of various bacteria, both gram positive and gram negative, and on the production of toxins by the organisms.
toxins (20). Several studies have shown that the production of TSST-1 and enterotoxins B and C by S. aureus strains is influenced by factors in the culture medium (6, 21, 22, 26-28, 33). For example, a neutral pH and the presence of oxygen are necessary for toxin production, whereas growth of the staphylococci may occur in their absence (26, 33). Likewise, Schlievert and Kelly (28) showed that clindamycin inhibits the production of TSST-1 at antibiotic concentrations that do not affect bacterial growth. All of these toxins are susceptible to repression by glucose (6, 22, 26). Finally, each of these toxins is regulated by an accessory gene regulatory, agr, a global regulator of exoprotein expression in S. aureus (21). The regulation of exotoxin production, as influenced by the *
MATERIALS AND METHODS Bacteria. S. aureus MN 8 was from a patient with menstruation-associated TSS and makes high levels of TSST-1 (26). S. aureus EV was kindly provided by Mark Kaplan, North Shore University Hospital, Manhassett, N.Y., and makes exfoliative toxin (ET) A (13). Strain MN 8 is weakly hemolytic and strain EV is strongly hemolytic on sheep blood agar plates; both strains are lipase producers, with MN 8 making more lipase than EV (see Results). S. hominis originally came from W. E. Kloos, North Carolina State University, Raleigh, and makes hemolysin and lipase. Group A streptococcal strains 594, 86-858, and T18P were maintained in our laboratory as sources of streptococcal pyro-
Corresponding author. 626
genic exotoxins (SPEs) A, B, and C, respectively (6, 11, 25). Group A streptococcal strain C203U, maintained in our laboratory, was used as the source of streptolysin 0; this strain does not make streptolysin S. Wild-type Salmonella minnesota (wild type with respect to endotoxin production) and an S. minnesota Re mutant (ReS95) were generously provided by Dennis W. Watson, Department of Microbiology, University of Minnesota, Minneapolis. A recent clinical isolate of hemolytic Eschenichia coli was kindly provided by Harriet Lievan, Department of Microbiology, University of Minnesota, Minneapolis. E. coli RR1(pUMN103) was maintained in our laboratory and produces SPE A (12). Antisera and toxins. Hyperimmune rabbit antisera were individually raised against TSST-1, ET A, and SPEs (25). Purified toxins were prepared by ethanol precipitation of culture fluids and then preparative thin-layer isoelectric focusing (29). Purified toxins migrated as homogeneous proteins when 20 ,ug of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (14) and then stained with Coomassie brilliant blue. GML. GML was provided by Personal Products Co., New Brunswick, N.J., and was indicated to have a composition of 95% GML and 5% -glycerol dilaurate. The compound was dissolved in absolute ethanol at a concentration of 100 mg/ml, shown to be sterile by plating of serial 10-fold dilutions on blood agar plates, and diluted into 37°C medium for use. Exoprotein assays. TSST-1, ET A, and SPEs were evaluated by dilution Ouchterlony immunodiffusion and Western blot (immunoblot) analysis (4, 26). For Ouchterlony immunodiffusion, strains to be evaluated were cultured for various times, concentrated 100 times by ethanol precipitation and resolubilization in distilled water, serially diluted in twofold increments, and then reacted against the antisera. The lower limit of toxin detection by this method was determined to be approximately 6 ,ug/ml (in the 100x -concentrated ethanol precipitate), which is 0.06 ,ug/ml for each toxin in the original culture fluid. Toxin concentrations were also estimated by serial dilution and then reactivity in Western blots. The lower limit of toxin detection by this method was approximately 0.013 p.g/ml. Prior to SDS-PAGE, the streptococcal strains were treated with hyaluronidase (10 p.g/ml) for 1 h. When this step was omitted, the streptococcal toxins did not enter the SDS gels. Uninoculated control culture flasks containing known amounts of toxin and GML were treated comparably to evaluate possible interference in the assays by GML. No interference was observed. Staphylococcal lipase was estimated by cleavage of tributyrin (15) or GML. For GML, cleavage assays were run the same as for tributyrin, except that GML was used at 20 p.g/ml and slides were placed at 4°C for 24 h after overnight incubation at 37°C. The 4°C temperature caused intact GML to form an insoluble crystalline matrix, leaving a zone of clearing where GML was cleaved. Staphylococcal hemolysin was assessed as follows. Rabbit erythrocytes (RBCs) were washed twice in phosphate-buffered saline (PBS; 0.005 M sodium phosphate [pH 7.2], 0.15 M NaCl) by centrifugation (500 x g, 10 min). The packed cells were diluted 1:1,000 in PBS-0.75% agarose (Sigma Chemical Co., St. Louis, Mo.), and 4.5 ml was used to coat microscope slides. Four-millimeter wells were punched in the slides after the agarose had solidified, and 20 pul of culture fluid was added to each well. The slides were incubated for 4 h at 37°C, and lysis was measured as clearing in millimeters. When GML alone was added to the slides at concentrations of 0 to 300 ,ug/ml, no lysis of rabbit RBCs was seen.
627
GML INHIBITION OF BACTERIA
VOL. 36, 1992
TABLE 1. Effect of GML on group A streptococci after 24 h of incubation
Bacterium and SPE
GML GM
Lo o
SPE P
Hemolysin"
b
(1±g/ml)
CFU/mla
(ligml)
594 (A)
0 2.5 10.0 20.0
8.6 8.5 8.3 6.0
3.2 0.3 0.3 0.0
7.0 4.0 0.0 0.0
86-858 (B)
0 2.5 10.0 20.0
8.0 7.7 7.7 5.8
0.8 0.0 0.0 0.0
4.0 2.0 0.0 0.0
T18P (C)
0 2.5 10.0
7.9 7.9 6.1
0.4 0.0 0.0
8.0 8.0 0.0
produced
a Inoculum size, 105 to 106 CFU/ml. b Including streptolysins 0 and S; measured as the zone of millimeters, of sheep RBCs.
lysis,
in
E. coli hemolysin was measured comparably, except that sheep RBCs were used. Streptococcal hemolysins were also measured comparably, except that sheep RBCs were used and 0.14% 2-mercaptoethanol was added to the PBS-agarose solution. As controls, GML at concentrations of 1 to 20 ,ug/ml did not inhibit the lysis of RBCs by purified preparations of streptolysin 0 used at concentrations of up to 50
,ug/ml. Endotoxin assay. Gram-negative bacteria were cultured for various times, and culture fluids were clarified by centrifugation (10,000 x g) and filtration (0.45-,um-pore-size filters; Gelman Corp.). Serial twofold dilutions were made in sterile pyrogen-free water, and a Limulus assay was performed by the method recommended by the supplier (Sigma). Control endotoxin was obtained from S. typhimurium (32). Culture media and bacterial quantifications. A dialyzable beef heart medium was used to prepare purified exotoxins, including streptolysin 0 (29). Brain heart infusion broth (Difco) was used to culture all organisms to be tested for toxins. Bacteria were cultured at 370C for various times with shaking (200 rpm; Gyrotory shaker; New Brunswick Scientific, New Brunswick, N.J.). Plate counts on brain heart infusion agar (1.5%) were used to determine CFU per milliliter of culture fluid.
RESULTS All experiments were done a minimum of two times, with those evaluating effects on S. aureus being done several times, and data from representative experiments are presented. The effect of GML on the growth of group A streptococci and the production of exotoxins was evaluated with three strains (Table 1). The growth of strains 594 and 86-858 was inhibited by 20 p.g of GML per ml but not 2.5 and 10 ,ug/ml, and the growth of strain T18P was inhibited by 10 ,ug/ml but not 2.5 ,ug/ml. For all three strains, the production of SPEs was reduced or completely inhibited by GML concentrations of 2.5 ,ug/ml or higher. Except in strain T18P, the production of hemolysin was also reduced or completely inhibited by .2.5 ,ug of GML per ml; strain T18P hemolysin production was inhibited by 10 jig/ml. The effect of GML on the growth of and hemolysin
ANTIMICROB. AGENTS CHEMOTHER.
SCHLIEVERT ET AL.
628
.
I8 a I9
9 vel
4
N
II 11
A
I ; .
=
Ai
a
_
ia
E i
I
e so
"
a
0.
v
4PEj!Ufl)
zwpRi{n) mdl
I.-
E*3_nH
S s
-q
i.
S
_
°
IED 1 r4
0) 3'j cr
(lgs) loLSSJl
II
TABLE 2. Effect of Zn on the lipase activity of S. aureus MN 8a Zn (M)
GML
(jig/ml)
TABLE 3. Effect of GML on E. coli and S. minnesota after 24 h of incubation
CFU/ LogmlLpae Lipase'
0 10 100
9.7 9.8 9.3
15 15 15
0.005
0 10 100
9.2 7.7 4.0
12 0 0
0.0005
0 10 100
9.8 9.3 5.6
12 6 0
0.0
629
GML INHIBITION OF BACTERIA
VOL. 36, 1992
Inoculum size, 5.0 x 107 CFU/ml. 6 Zone of clearing, in millimeters, of GML at 48 h.
Treatment
No inoculum
20 20 20 20
NDC 0.0 0.25 1.0
x 106 ND 1 x 106 2 x 106 2 x 106
2.0 ND 3.0 6.0 12.0
100 500 1,000 2,000
E. coli
0 100 500 1,000 2,000
9.4 9.4 9.4 9.3 9.4
Wild-type S. minnesota
0 100 500 1,000
9.9
0 100 500 1,000
9.4
