THE JOURNAL OF INFECTIOUS DISEASES. VOL. 138, NO.6. DECEMBER 1978
© 1978 by The University of Chicago. 0022·1899/78/3806-0026$00.75
Effect of Serum on Gram-Positive Cocci Grown in the Presence of Penicillin Victor Lorian and Barbara Atkinson
From the Section of Microbiology, Department of Pathology, The Bronx-Lebanon Hospital Center, and the Department of Laboratory Medicine, The Albert Einstein College of Medicine, Bronx, New York
Serum, alone and in combination with antibiotics, is known to be bactericidal to various species of Enterobacteriaceae [1, 2]. In contrast, other than enhancing phagocytosis, serum has little effect on gram-positive organisms [3, 4]. Some of the bactericidal effects of serum on Bacillus subtilis and Staphylococcus aureus have been ascribed to a substance released from platelets during the clotting process [5-7]. A normal constituent of serum, l3-lysin, has been shown to exert its bactericidal effect on B. subtilis by damaging the cytoplasmic membrane of the cell [8-
serum on the growth rate and morphology of these filaments, as well as on S. aureus and other streptococci. Materials and Methods
Blood specimens were obtained from healthy, drug-free volunteers by venipuncture with use of a 20-ml plastic syringe. A portion (5 ml) of blood was placed in a tube containing 143 units of heparin, and the remaining blood was allowed to clot in a sterile tube for 3 hr. The tubes were then centrifuged at 150 g for 10 min. The serum or plasma was removed and centrifuged again. The serum was divided, and one part was inactivated at 56 C for 1 hr. Fresh serum, inactivated serum, and plasma were diluted 1:5 in Hanks' balanced salt solution (HBSS) with 0.1 % gelatin (Hanks' gel; Difco Laboratories, Detroit, Mich.) [13]. Ten strains of Streptococcus faecalis isolated from the urine of patients at The Bronx-Lebanon Hospital Center (Bronx, N.Y.) [14], three strains of S. bovis (obtained from the Massachusetts General Hospital, Boston, Mass.), and one strain each of S. aureus 209P (obtained from the Food and Drug Administration, Washington, D.C.), S. aureus Oxford, and the strain of S. bovis that produces filaments after exposure to penicillin [12] were tested. The last strain, pre-
10]. Penicillin, at concentrations below the MIC, has been shown to produce abnormal structures in cocci [11, 12]. One strain of Streptococcus bovis grown in the presence of penicillin at a concentration one-quarter of the MIC became filamentous [12]. This paper reports the effect of human
Received for publication April 10, 1978, and in revised form July 12, 1978. We thank Richard R. Facklam for the identification of the filament-forming Streptococcus and Lawrence J. Kunz for the grouping of this organism and for supplying the strains of Streptococcus bovis. This paper was prepared in honor of Dr. Edward H. Kass on his 60th birthday. Please address requests for reprints to Dr. Victor Lorian, The Bronx-Lebanon Hospital Center, Fulton Avenue at 169th Street, Bronx, New York 10456.
865
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Strains of Staphylococcus aureus, Streptococcus faecalis, Streptococcus boois, and a strain of Streptococcus that produces filaments were grown on agar containing penicillin at concentrations of one-half to 1/1 Oth the minimal inhibitory concentration. These penicillin-exposed organisms as well as untreated control organisms were incubated with human serum or plasma. Both serum and plasma produced a remarkable bactericidal effect on the filament-forming Streptococcus grown in the presence of penicillin, whereas the untreated control was only slightly affected. This response resembled that of gram-negative bacilli rather than that of gram-positive cocci. The growth of staphylococci exposed to penicillin was slightly inhibited in the presence of serum, whereas the growth of untreated staphylococci was stimulated. The streptococci, regardless of whether they were treated with penicillin or whether they were untreated, showed no change in growth pattern in the presence of serum,
866
Lorian and Atkinson
0.1 ml of the I: 10 dilution of the 20-hr TSB culture. The inoculated membranes were incubated at 37 C for 90 min, transferred either to agar containing the antibiotic or to drug-free agar, and incubated for an additional 3.5 hr. The control organisms grown on drug-free agar (six membranes) were eluted with 15 ml of Hanks' gel; the organisms grown on media containing antibiotic (40 membranes) were eluted with 50 ml of Hanks' gel containing the same concentration of antibiotic. Tubes were shaken and volumes were adjusted to give the approximate turbidity of the 0.5 McFarland standard (5 X 107 organisms/ml). These bacterial suspensions were used for the studies with serum or plasma. All tests were done in 15-ml plastic screw-capped tubes (Falcon Plastics, Oxnard, Calif.). Each control tube contained 0.1 ml of normal bacteria (grown on drug-free agar), 0.1 ml of Hanks' gel, and 0.9 ml of serum diluted 1:5, plasma diluted I :5, inactivated serum diluted 1:5, or Hanks' gel. Another set of tubes contained 0.1 ml of bacteria grown on agar containing anti-
Table 2. MICs of oxacillin and penicillin for each strain of Staphylococcus and Streptococcus tested and the fraction of the MIC used in the growth and morphological studies with serum.
Table 1. Biochemical
characteristics
of
filament-
forming Streptococcus.
Characteristic Presence of catalase Production of NH 3 from arginine Growth in 6.5% NaGI broth Growth at 45 G Growth on 40% bile blood agar Utilization of pyruvate Hydrolysis of Sodium hippurate Esculin Bile esculin Fermentation of Trehalose Sorbitol Lactose Mannitol Sucrose Inulin Raffinose Arabinose Salicin NOTE.
Drug, organism, strain Positive (+) or negative (-)
+ + +
+ +
Oxacillin Staphylococcus aureus 209P Oxford Penicillin Filament-forming Streptococcus Streptococcus bovis
3471 9166 8389 StreptococcusfaecaUs 1
7 +
+ + +
+
The MBG of this organism is eight times the MIG.
8 9 11 Williams Boyle Pena Landsky Blair
MIG (J.Lg/ml)
Fraction of MIG used
0.4 0.2
1/3 1/3
0.78
1/4
0.15 0.15 0.078
1/4 1/4 1{4
1.56 1.56 1.56 6.2 1.56
3{4 3{4 1{3
1{2
3.1
1{2 1{10
1.7 1.56 1.56 1.56
1{4 1{2 1{2 1{2
NOTE. See Materials and Methods for procedures used in the growth and morphological studies.
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
viously identified in our clinical laboratory as S. boois, was classified by the Center for Disease Control (CDC), Atlanta, Ga., as an enterococcus without D antigen; it was not speciated but resembled Streptococcus faecium and/or Streptococcus avium. The biochemical characteristics as confirmed by the CDC are listed in table I. This strain was also grouped by the Massachusetts General Hospital as gro~p Q [I5]. In the present report this strain is called filament-forming Streptococcus. The MIC for each strain tested was determined by a twofold agar dilution method [16]. Each organism was grown in trypticase soy broth (TSB; Baltimore Biological Laboratories [BBL], Baltimore, Md.) for 20 hr at 37 C. Trypticase soy agar (TSA; BBL) plates were prepared with penicillin G (Squibb, New Brunswick, N.J.) or oxacillin (Bristol Laboratories, Syracuse, N.Y.). Table 2 shows the MIC determined for each bacterial strain as well as the fraction of the MIC used in the studies of growth and morphology. Filter membranes with a pore size of 3 pm (Millipore Corp., Bedford, Mass.) were placed on drug-free agar and incubated for I hr before inoculation. The membranes were inoculated with
867
Effect of Serum on Cocci Exposed to Penicillin
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Figure I. Bactericidal effect of fresh human serum on Streptococcus faecalis (left) and Streptococcus bovis (right) after incubation for 1 hr and 2 hr. Dashed lines represent organisms not treated with antibiotics, and solid lines represent organisms grown in the presence of penicillin at concentrations below the MIC.
biotics, 0.1 ml of antibiotic (10 times the final concentration desired), and 0.9 ml of serum diluted 1:5, plasma diluted 1:5, inactivated serum diluted 1:5, or Hanks' gel (antibiotic control). This antibiotic control was adjusted so that it would not produce after incubation for 2 hr at 37 C a variation of >25% (increase or decrease) in the cfu count compared with that at zero-time. For each bacterial species, five to 15 different sera were tested. The bactericidal actIVity of serum or plasma was determined by mixing the suspensions on a Vortex mixer and incubating the tubes at 37 C on a multipurpose rotator (Scientific Products, Chamblee, Ga.) at - 6 rpm. After incubation for 0, 0.5, 1, and 2 hr, O.l-ml samples were removed and placed in 9.9 ml of saline at 10 C (to prevent growth or autolysis). The number of cfu was determined by plating of serial dilutions onto TSA and incubation for 24 hr. The morphology of bacteria from membranes and suspensions was studied by interference phase-contrast microscopy. Gram-stained specimens were also studied. Both the filament-forming streptococci exposed to penicillin and incubated for 2 hr with serum and the control incubated with HBSS were ex-
+
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Hours Figure 2. Bactericidal effect of fresh human serum on Staphylococcus aureus after incubation for 1 hr and 2 hr. Dashed lines represent controls not treated with antibiotics, and solid lines represent organisms grown in the presence of oxacillin at concentrations below the MIC.
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
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868
Lorian and Atkinson
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Figure 3. Bactericidal effects of fresh human serum (left) and inactivated human serum (right) on filamentforming Streptococcus after incubation for 0.5, I, and 2 hr. Dashed lines represent organisms not treated with antibiotics, and solid lines represent organisms grown in the presence of penicillin at concentrations below the MIC.
amined by electron microscopy as described previously [17,18].
+ Results
Effect of serum or plasma on the growth of bacteria. Curves illustrating the effect of serum
or plasma on the growth of bacteria are expressed as the percentage decrease or increase in the number of C£u compared with that in the appropriate HBSS control (treated or untreated with antibiotic). When the number of C£u for the antibiotic control after incubation for 2 hr showed a variation (increase or decrease) of >25% from the count at zero-time, the results were not included. The growth curves are shown in figures 1-4. Serum had negligible effects on the growth of the 10 strains of S. faecalis and the three strains of S. bovis, whether or not they were exposed to penicillin (figure I). Staphylococci grown in the presence of penicillin and incubated for 1 hr with serum showed decreases in the number of C£u to 40% of control values, whereas all of the untreated control staphylococci showed increases in the number of C£u. After 2 hr two distinct curve patterns were evident (figure 2).
u
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Hours Figure 4. Bactericidal effect of human plasma on filament-forming Streptococcus after incubation for 1 hr and 2 hr. Dashed lines represent organisms not treated with antibiotics, and solid lines represent organisms grown in the presence of penicillin at concentrations below the MIC.
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
0
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869
Effect of Serum on Cocci Exposed to Penicillin
The filament-forming Streptococcus was tested with 15 different sera. Filaments grown with penicillin in the presence of fresh serum showed a 20%-60% decrease in the number of cfu after incubation for 0.5 hr and a 50%-99% decrease after 2 hr. Control cells grown on drug-free agar also showed a slight decrease (30% in some instances) after 2 hr (figure 3, left). The effect of inactivated serum on the filaments was less dramatic; however, two sera had a 99% bactericidal effect after 2 hr, and five other sera produced a 50%-80% decrease in cfu (figure 3, right). The bactericidal effects of plasma were similar to those seen with serum (figure 4). Effect of pencillin and serum on morphology and ultrastructure. The exposure of S. faecalis to subinhibitory concentrations of pencillin resulted in large oval cells. Electron microscopy showed that these cells consisted of two to six organisms held together by extremely thick, cell wall-like material that in some instances appeared to be lamellar. After incubation of bacteria with
serum, no remarkable changes were observed. S. boois showed slight elongation of cells after exposure to subinhibitory concentrations of penicillin; no morphological changes were observed after exposure to serum. As previously described, staphylococci exposed to oxacillin at a concentration one-quarter of the MIC form large cells held together by thick, irregular cross walls [12, 19]. No remarkable changes were observed after incubation of staphylococci with serum. Exposed to penicillin, filament-forming streptococci grow into filaments without cross walls [13]. A gram stain of the filaments incubated with serum for 2 hr showed granular forms; no changes were seen after incubation with HBSS (figure 5). Electron microscopic examination of filaments incubated for 2 hr with serum showed elongated streptococci with detritus sticking to their cell walls, protoplasts, and cell debris (figure 6). Discussion
The effect of serum on the growth and morphol-
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
Figure 5. Filament-forming Streptococcus grown on drug-free agar (normal control) and exposed to fresh human serum (A), grown in the presence of penicillin at a concentration one-fourth of the MIC and exposed to fresh human serum (B), and grown in the presence of penicillin only (C). All forms are shown after incubation for 2 hr (gram stain, X 3,000).
870
Lorian and Atkinson
ogy of S. faecalis and S. boois, both exposed and unexposed to penicillin, was minimal. Serum slightly inhibited the growth of staphylococci exposed to penicillin but stimulated the growth of untreated controls. The response to human serum exhibited by filament-forming Streptococcus resembled the response of serum-sensitive gram-negative bacilli rather than that of other gram-positive organisms. The growth of these streptococci, exposed to penicillin, was markedly inhibited by fresh human serum. Studies of the morphology and ultrastructure revealed cells characteristic of dying populations. Since heat-inactivated serum also inhibited the growth of filament-forming streptococci, the bactericidal effect is probably not due only to complement. Plasma was similarly bactericidal, a finding indicating that the effect is probably not due to a platelet factor [5, 6].
2.
3.
4.
5.
6.
7.
8. References 1. Wardlaw, A. C. The complement-dependent bacteriolytic activity of normal human serum. The effect
9.
of pH and ionic strength and the role of lysozyme. J. Exp. Med.ll5:1231-1249,l962. Traub, W. H., Sherris, J. C. Studies on the interaction between serum bactericidal activity and antibiotics in vitro. Chemotherapy 15:70-83, 1970. Cohen, Z. A., Morse, S. I. Interactions between rabbit polymorphonuclear leukocytes and staphylococci. J. Exp. Med. llO:419-443, 1959. Li, I. W., Mudd, S. Serum effect on the killing of Staphylococcus aureus by human leukocytic extracts. J. Immunol. 97:41-45,1966. Myrvik, Q. N., Leake, E. S. Studies on antibacterial factors in mammalian tissues and fluid. IV. Demonstration of two nondialyzable components in the serum bactericidin system for Bacillus subtilis. J. Immunol. 84:247-250, 1960. Jago, R., Jacox, R. F. Cellular source and character of a heat-stable bactericidal property associated with rabbit and rat platelets. J. Exp. Med, ll3:701-7ll, 1961. Hirsch, J. G. Comparative bactericidal activities of blood serum and plasma serum. J. Exp, Med. ll2: 15-22,1960. Matheson, A., Donaldson, D. M. Alterations in the morphology of Bacillus subtilis after exposure to ,a-lysin and ultraviolet light. J. Bacteriol. 95:18911902,1968. Matheson, A., Donaldson, D. M. Effect of ,a-lysin on
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
Figure 6. Electron micrograph of the filament of filament-forming Streptococcus grown in the presence of penicillin at a concentration onefourth of the MIC, after incubation for 2 hr. The cell on the left was not exposed to fresh human serum; the cells on the right were incubated in the presence of serum. The bar represents I /-1m (x 30,000).
871
Effect of Serum on Cocci Exposed to Penicillin
10.
II.
12.
14.
15.
16.
17.
18.
19.
Ecology, serology, physiology, and relationship to established enterococci. J. Bacteriol. 94:291-296, 1967. Steers, E., Foltz, E. L., Graves, B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9: 307-311, 1959. Simionescu, N., Simionescu, M., Palade, G. E. Permeability of intestinal capillaries. Pathway followed by dextrans and glycogens. J. Cell. BioI. 53:365-392, 1972. Lorian, V., Atkinson, B. Comparison of the effects of mecillinam and 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus. Antimicrob. Agents Chemother. U:541-552. 1977. Lorian, V. Some effect of subinhibitory concentrations of penicillin on the structure and division of staphylococci. Antimicrob. Agents Chemother. 7:864-870, 1975.
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
13.
isolated cell walls and protoplasts of Bacillus subti/is. J. Bacteriol, 101:314-317, 1970. Rosenthal, S. L., Matheson, A. ATPase in isolated membranes of Bacillus subtilis. Biochim. Biophys. Acta 318:252-261, 1973. Lorian, V., Atkinson, B. Abnormal forms of bacteria produced by antibiotics. Am. J. Clin. Pathol, 64:678688, 1975. Lorian, V., Atkinson, B. Effects of subinhibitory concentrations of antibiotics on cross walls of cocci. Antimicrob. Agents Chemother. 9:1043-1055,1976. Quie, P. G., White, J. G., Holmes, B., Good, R. A. In vitro bactericidal capacity of human polymorphonuclear leukocytes: diminished activity in chronic granulomatous disease of childhood. J. Clin. Invest. 46: 668-679,1967. Lennette, E. H., Spaulding, E. H., Truant, J. P. Manual of clinical microbiology. 2nd ed. American Society for Microbiology, Washington, D.C., 1974. 970 p. Nowlan, S. S., Deibel, R. H. Group Q streptococci. I.
© 1978 by The University of Chicago. 0022·1899/78/3806-0026$00.75
Effect of Serum on Gram-Positive Cocci Grown in the Presence of Penicillin Victor Lorian and Barbara Atkinson
From the Section of Microbiology, Department of Pathology, The Bronx-Lebanon Hospital Center, and the Department of Laboratory Medicine, The Albert Einstein College of Medicine, Bronx, New York
Serum, alone and in combination with antibiotics, is known to be bactericidal to various species of Enterobacteriaceae [1, 2]. In contrast, other than enhancing phagocytosis, serum has little effect on gram-positive organisms [3, 4]. Some of the bactericidal effects of serum on Bacillus subtilis and Staphylococcus aureus have been ascribed to a substance released from platelets during the clotting process [5-7]. A normal constituent of serum, l3-lysin, has been shown to exert its bactericidal effect on B. subtilis by damaging the cytoplasmic membrane of the cell [8-
serum on the growth rate and morphology of these filaments, as well as on S. aureus and other streptococci. Materials and Methods
Blood specimens were obtained from healthy, drug-free volunteers by venipuncture with use of a 20-ml plastic syringe. A portion (5 ml) of blood was placed in a tube containing 143 units of heparin, and the remaining blood was allowed to clot in a sterile tube for 3 hr. The tubes were then centrifuged at 150 g for 10 min. The serum or plasma was removed and centrifuged again. The serum was divided, and one part was inactivated at 56 C for 1 hr. Fresh serum, inactivated serum, and plasma were diluted 1:5 in Hanks' balanced salt solution (HBSS) with 0.1 % gelatin (Hanks' gel; Difco Laboratories, Detroit, Mich.) [13]. Ten strains of Streptococcus faecalis isolated from the urine of patients at The Bronx-Lebanon Hospital Center (Bronx, N.Y.) [14], three strains of S. bovis (obtained from the Massachusetts General Hospital, Boston, Mass.), and one strain each of S. aureus 209P (obtained from the Food and Drug Administration, Washington, D.C.), S. aureus Oxford, and the strain of S. bovis that produces filaments after exposure to penicillin [12] were tested. The last strain, pre-
10]. Penicillin, at concentrations below the MIC, has been shown to produce abnormal structures in cocci [11, 12]. One strain of Streptococcus bovis grown in the presence of penicillin at a concentration one-quarter of the MIC became filamentous [12]. This paper reports the effect of human
Received for publication April 10, 1978, and in revised form July 12, 1978. We thank Richard R. Facklam for the identification of the filament-forming Streptococcus and Lawrence J. Kunz for the grouping of this organism and for supplying the strains of Streptococcus bovis. This paper was prepared in honor of Dr. Edward H. Kass on his 60th birthday. Please address requests for reprints to Dr. Victor Lorian, The Bronx-Lebanon Hospital Center, Fulton Avenue at 169th Street, Bronx, New York 10456.
865
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Strains of Staphylococcus aureus, Streptococcus faecalis, Streptococcus boois, and a strain of Streptococcus that produces filaments were grown on agar containing penicillin at concentrations of one-half to 1/1 Oth the minimal inhibitory concentration. These penicillin-exposed organisms as well as untreated control organisms were incubated with human serum or plasma. Both serum and plasma produced a remarkable bactericidal effect on the filament-forming Streptococcus grown in the presence of penicillin, whereas the untreated control was only slightly affected. This response resembled that of gram-negative bacilli rather than that of gram-positive cocci. The growth of staphylococci exposed to penicillin was slightly inhibited in the presence of serum, whereas the growth of untreated staphylococci was stimulated. The streptococci, regardless of whether they were treated with penicillin or whether they were untreated, showed no change in growth pattern in the presence of serum,
866
Lorian and Atkinson
0.1 ml of the I: 10 dilution of the 20-hr TSB culture. The inoculated membranes were incubated at 37 C for 90 min, transferred either to agar containing the antibiotic or to drug-free agar, and incubated for an additional 3.5 hr. The control organisms grown on drug-free agar (six membranes) were eluted with 15 ml of Hanks' gel; the organisms grown on media containing antibiotic (40 membranes) were eluted with 50 ml of Hanks' gel containing the same concentration of antibiotic. Tubes were shaken and volumes were adjusted to give the approximate turbidity of the 0.5 McFarland standard (5 X 107 organisms/ml). These bacterial suspensions were used for the studies with serum or plasma. All tests were done in 15-ml plastic screw-capped tubes (Falcon Plastics, Oxnard, Calif.). Each control tube contained 0.1 ml of normal bacteria (grown on drug-free agar), 0.1 ml of Hanks' gel, and 0.9 ml of serum diluted 1:5, plasma diluted I :5, inactivated serum diluted 1:5, or Hanks' gel. Another set of tubes contained 0.1 ml of bacteria grown on agar containing anti-
Table 2. MICs of oxacillin and penicillin for each strain of Staphylococcus and Streptococcus tested and the fraction of the MIC used in the growth and morphological studies with serum.
Table 1. Biochemical
characteristics
of
filament-
forming Streptococcus.
Characteristic Presence of catalase Production of NH 3 from arginine Growth in 6.5% NaGI broth Growth at 45 G Growth on 40% bile blood agar Utilization of pyruvate Hydrolysis of Sodium hippurate Esculin Bile esculin Fermentation of Trehalose Sorbitol Lactose Mannitol Sucrose Inulin Raffinose Arabinose Salicin NOTE.
Drug, organism, strain Positive (+) or negative (-)
+ + +
+ +
Oxacillin Staphylococcus aureus 209P Oxford Penicillin Filament-forming Streptococcus Streptococcus bovis
3471 9166 8389 StreptococcusfaecaUs 1
7 +
+ + +
+
The MBG of this organism is eight times the MIG.
8 9 11 Williams Boyle Pena Landsky Blair
MIG (J.Lg/ml)
Fraction of MIG used
0.4 0.2
1/3 1/3
0.78
1/4
0.15 0.15 0.078
1/4 1/4 1{4
1.56 1.56 1.56 6.2 1.56
3{4 3{4 1{3
1{2
3.1
1{2 1{10
1.7 1.56 1.56 1.56
1{4 1{2 1{2 1{2
NOTE. See Materials and Methods for procedures used in the growth and morphological studies.
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
viously identified in our clinical laboratory as S. boois, was classified by the Center for Disease Control (CDC), Atlanta, Ga., as an enterococcus without D antigen; it was not speciated but resembled Streptococcus faecium and/or Streptococcus avium. The biochemical characteristics as confirmed by the CDC are listed in table I. This strain was also grouped by the Massachusetts General Hospital as gro~p Q [I5]. In the present report this strain is called filament-forming Streptococcus. The MIC for each strain tested was determined by a twofold agar dilution method [16]. Each organism was grown in trypticase soy broth (TSB; Baltimore Biological Laboratories [BBL], Baltimore, Md.) for 20 hr at 37 C. Trypticase soy agar (TSA; BBL) plates were prepared with penicillin G (Squibb, New Brunswick, N.J.) or oxacillin (Bristol Laboratories, Syracuse, N.Y.). Table 2 shows the MIC determined for each bacterial strain as well as the fraction of the MIC used in the studies of growth and morphology. Filter membranes with a pore size of 3 pm (Millipore Corp., Bedford, Mass.) were placed on drug-free agar and incubated for I hr before inoculation. The membranes were inoculated with
867
Effect of Serum on Cocci Exposed to Penicillin
+
+
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Figure I. Bactericidal effect of fresh human serum on Streptococcus faecalis (left) and Streptococcus bovis (right) after incubation for 1 hr and 2 hr. Dashed lines represent organisms not treated with antibiotics, and solid lines represent organisms grown in the presence of penicillin at concentrations below the MIC.
biotics, 0.1 ml of antibiotic (10 times the final concentration desired), and 0.9 ml of serum diluted 1:5, plasma diluted 1:5, inactivated serum diluted 1:5, or Hanks' gel (antibiotic control). This antibiotic control was adjusted so that it would not produce after incubation for 2 hr at 37 C a variation of >25% (increase or decrease) in the cfu count compared with that at zero-time. For each bacterial species, five to 15 different sera were tested. The bactericidal actIVity of serum or plasma was determined by mixing the suspensions on a Vortex mixer and incubating the tubes at 37 C on a multipurpose rotator (Scientific Products, Chamblee, Ga.) at - 6 rpm. After incubation for 0, 0.5, 1, and 2 hr, O.l-ml samples were removed and placed in 9.9 ml of saline at 10 C (to prevent growth or autolysis). The number of cfu was determined by plating of serial dilutions onto TSA and incubation for 24 hr. The morphology of bacteria from membranes and suspensions was studied by interference phase-contrast microscopy. Gram-stained specimens were also studied. Both the filament-forming streptococci exposed to penicillin and incubated for 2 hr with serum and the control incubated with HBSS were ex-
+
---- -::::------:: ---- ---- --------------------------
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Hours Figure 2. Bactericidal effect of fresh human serum on Staphylococcus aureus after incubation for 1 hr and 2 hr. Dashed lines represent controls not treated with antibiotics, and solid lines represent organisms grown in the presence of oxacillin at concentrations below the MIC.
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
0 "0
868
Lorian and Atkinson
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Figure 3. Bactericidal effects of fresh human serum (left) and inactivated human serum (right) on filamentforming Streptococcus after incubation for 0.5, I, and 2 hr. Dashed lines represent organisms not treated with antibiotics, and solid lines represent organisms grown in the presence of penicillin at concentrations below the MIC.
amined by electron microscopy as described previously [17,18].
+ Results
Effect of serum or plasma on the growth of bacteria. Curves illustrating the effect of serum
or plasma on the growth of bacteria are expressed as the percentage decrease or increase in the number of C£u compared with that in the appropriate HBSS control (treated or untreated with antibiotic). When the number of C£u for the antibiotic control after incubation for 2 hr showed a variation (increase or decrease) of >25% from the count at zero-time, the results were not included. The growth curves are shown in figures 1-4. Serum had negligible effects on the growth of the 10 strains of S. faecalis and the three strains of S. bovis, whether or not they were exposed to penicillin (figure I). Staphylococci grown in the presence of penicillin and incubated for 1 hr with serum showed decreases in the number of C£u to 40% of control values, whereas all of the untreated control staphylococci showed increases in the number of C£u. After 2 hr two distinct curve patterns were evident (figure 2).
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1
2
Hours Figure 4. Bactericidal effect of human plasma on filament-forming Streptococcus after incubation for 1 hr and 2 hr. Dashed lines represent organisms not treated with antibiotics, and solid lines represent organisms grown in the presence of penicillin at concentrations below the MIC.
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Effect of Serum on Cocci Exposed to Penicillin
The filament-forming Streptococcus was tested with 15 different sera. Filaments grown with penicillin in the presence of fresh serum showed a 20%-60% decrease in the number of cfu after incubation for 0.5 hr and a 50%-99% decrease after 2 hr. Control cells grown on drug-free agar also showed a slight decrease (30% in some instances) after 2 hr (figure 3, left). The effect of inactivated serum on the filaments was less dramatic; however, two sera had a 99% bactericidal effect after 2 hr, and five other sera produced a 50%-80% decrease in cfu (figure 3, right). The bactericidal effects of plasma were similar to those seen with serum (figure 4). Effect of pencillin and serum on morphology and ultrastructure. The exposure of S. faecalis to subinhibitory concentrations of pencillin resulted in large oval cells. Electron microscopy showed that these cells consisted of two to six organisms held together by extremely thick, cell wall-like material that in some instances appeared to be lamellar. After incubation of bacteria with
serum, no remarkable changes were observed. S. boois showed slight elongation of cells after exposure to subinhibitory concentrations of penicillin; no morphological changes were observed after exposure to serum. As previously described, staphylococci exposed to oxacillin at a concentration one-quarter of the MIC form large cells held together by thick, irregular cross walls [12, 19]. No remarkable changes were observed after incubation of staphylococci with serum. Exposed to penicillin, filament-forming streptococci grow into filaments without cross walls [13]. A gram stain of the filaments incubated with serum for 2 hr showed granular forms; no changes were seen after incubation with HBSS (figure 5). Electron microscopic examination of filaments incubated for 2 hr with serum showed elongated streptococci with detritus sticking to their cell walls, protoplasts, and cell debris (figure 6). Discussion
The effect of serum on the growth and morphol-
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Figure 5. Filament-forming Streptococcus grown on drug-free agar (normal control) and exposed to fresh human serum (A), grown in the presence of penicillin at a concentration one-fourth of the MIC and exposed to fresh human serum (B), and grown in the presence of penicillin only (C). All forms are shown after incubation for 2 hr (gram stain, X 3,000).
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ogy of S. faecalis and S. boois, both exposed and unexposed to penicillin, was minimal. Serum slightly inhibited the growth of staphylococci exposed to penicillin but stimulated the growth of untreated controls. The response to human serum exhibited by filament-forming Streptococcus resembled the response of serum-sensitive gram-negative bacilli rather than that of other gram-positive organisms. The growth of these streptococci, exposed to penicillin, was markedly inhibited by fresh human serum. Studies of the morphology and ultrastructure revealed cells characteristic of dying populations. Since heat-inactivated serum also inhibited the growth of filament-forming streptococci, the bactericidal effect is probably not due only to complement. Plasma was similarly bactericidal, a finding indicating that the effect is probably not due to a platelet factor [5, 6].
2.
3.
4.
5.
6.
7.
8. References 1. Wardlaw, A. C. The complement-dependent bacteriolytic activity of normal human serum. The effect
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of pH and ionic strength and the role of lysozyme. J. Exp. Med.ll5:1231-1249,l962. Traub, W. H., Sherris, J. C. Studies on the interaction between serum bactericidal activity and antibiotics in vitro. Chemotherapy 15:70-83, 1970. Cohen, Z. A., Morse, S. I. Interactions between rabbit polymorphonuclear leukocytes and staphylococci. J. Exp. Med. llO:419-443, 1959. Li, I. W., Mudd, S. Serum effect on the killing of Staphylococcus aureus by human leukocytic extracts. J. Immunol. 97:41-45,1966. Myrvik, Q. N., Leake, E. S. Studies on antibacterial factors in mammalian tissues and fluid. IV. Demonstration of two nondialyzable components in the serum bactericidin system for Bacillus subtilis. J. Immunol. 84:247-250, 1960. Jago, R., Jacox, R. F. Cellular source and character of a heat-stable bactericidal property associated with rabbit and rat platelets. J. Exp. Med, ll3:701-7ll, 1961. Hirsch, J. G. Comparative bactericidal activities of blood serum and plasma serum. J. Exp, Med. ll2: 15-22,1960. Matheson, A., Donaldson, D. M. Alterations in the morphology of Bacillus subtilis after exposure to ,a-lysin and ultraviolet light. J. Bacteriol. 95:18911902,1968. Matheson, A., Donaldson, D. M. Effect of ,a-lysin on
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Figure 6. Electron micrograph of the filament of filament-forming Streptococcus grown in the presence of penicillin at a concentration onefourth of the MIC, after incubation for 2 hr. The cell on the left was not exposed to fresh human serum; the cells on the right were incubated in the presence of serum. The bar represents I /-1m (x 30,000).
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II.
12.
14.
15.
16.
17.
18.
19.
Ecology, serology, physiology, and relationship to established enterococci. J. Bacteriol. 94:291-296, 1967. Steers, E., Foltz, E. L., Graves, B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9: 307-311, 1959. Simionescu, N., Simionescu, M., Palade, G. E. Permeability of intestinal capillaries. Pathway followed by dextrans and glycogens. J. Cell. BioI. 53:365-392, 1972. Lorian, V., Atkinson, B. Comparison of the effects of mecillinam and 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus. Antimicrob. Agents Chemother. U:541-552. 1977. Lorian, V. Some effect of subinhibitory concentrations of penicillin on the structure and division of staphylococci. Antimicrob. Agents Chemother. 7:864-870, 1975.
Downloaded from http://jid.oxfordjournals.org/ at Thomas Jefferson University, Scott Library on August 19, 2015
13.
isolated cell walls and protoplasts of Bacillus subti/is. J. Bacteriol, 101:314-317, 1970. Rosenthal, S. L., Matheson, A. ATPase in isolated membranes of Bacillus subtilis. Biochim. Biophys. Acta 318:252-261, 1973. Lorian, V., Atkinson, B. Abnormal forms of bacteria produced by antibiotics. Am. J. Clin. Pathol, 64:678688, 1975. Lorian, V., Atkinson, B. Effects of subinhibitory concentrations of antibiotics on cross walls of cocci. Antimicrob. Agents Chemother. 9:1043-1055,1976. Quie, P. G., White, J. G., Holmes, B., Good, R. A. In vitro bactericidal capacity of human polymorphonuclear leukocytes: diminished activity in chronic granulomatous disease of childhood. J. Clin. Invest. 46: 668-679,1967. Lennette, E. H., Spaulding, E. H., Truant, J. P. Manual of clinical microbiology. 2nd ed. American Society for Microbiology, Washington, D.C., 1974. 970 p. Nowlan, S. S., Deibel, R. H. Group Q streptococci. I.
