THE JOURNAL OF INFECTIOUS DISEASES. VOL. 139, NO.5. MAY 1979 © 1979 by The University of Chicago. 0022-1899/79/3905-0007$00.75
Methicillin-Resistant Strains of Staphylococcus aureus: Relation between Expression of Resistance and Phagocytosis by Polymorphonuclear Leukocytes From the Infectious Diseases Division, Department of Medicine, Hopital Cantonal, Geneva, Switzerland
Pierre Vaudaux and Francis A. Waldvogel
it has been questioned whether after exposure to methicillin they are still adequate in number and virulence to resist natural host defense mechanisms, when compared with methicillin-sensitive cells of S. aureus [3, 7, 8]. Other investigators [9, 10] have established that virulent strains of S. aureus are not eliminated by polymorphonuclear leukocytes (PMNLs) to the same extent as are avirulent strains. Thus, an in vitro phagocytic-bactericidal assay was used in this study to answer the following clinically relevant questions. (1) Are strains of MRSA eliminated to the same extent by PMNLs as are virulent but methicillin-sensitive strains of S. aureus? (2) Are methicillin-resistant cells of MRSA more easily eliminated by PMNLs than are methicillinsensitive cells? (3) Is methicillin, in conjunction with serum factors and PMNLs, capable of eliminating strains of MRSA as efficiently as it eliminates methicillin-sensitive strains of S. aureus? The answers to these three questions are the basis of the present report.
Although naturally occurring methicillin-resistant Staphylococcus aureus (MRSA) have been demonstrated to be heterogeneously resistant, only a small proportion of the cells are able to resist in vitro destruction by therapeutic levels of methicillin [1-3]. A homogeneous population of methicillin-resistant cells can be selected from the initial population by prolonged in vitro incubation in the presence of methicillin [1-3]. In clinical situations, however, it has not yet been established whether the administration of methicillin to patients with infections due to MRSA selects out the resistant cells [2, 4, 5] as is observed in vitro. Since resistant cells have peculiar growth characteristics and colonial morphology [1-3, 6], Received for publication July 17, 1978, and in revised form December 5,1978. This study was supported in part by a grant from Beecham Pharmaceuticals, Betchworth, Surrey, England, and by a grant from the Societe Academique, Geneva, Switzerland. We thank Dr. Hans Reber, Kantonsspital, Basle, Switzerland, and Dr. Jacques Pillet, Inst.itut Pasteur, Paris, France, for phage-typing and serotyping of the methicillin-resistant strains of Staphylococcus aureus. This paper was presented in part at a meeting of the Swiss Society for Microbiology, Basle, Switzerland, June 16, 1978. Please address requests for reprints to Dr. Pierre Vaudaux, Infectious Diseases Division, Department of Medicine, Hopital Cantonal, 1211 Geneva 4, Switzerland.
Materials and Methods
Organisms and antibiotic resistance. Eight strains of MRSA were tested: two strains isolated at the Hopital Cantonal, Geneva, Switzerland, and six others obtained from other hospitals in
547
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 5, 2015
Virulent strains of staphylococci are known to resist phagocytic destruction better than avirulent strains. In this context, in vitro elimination by human polymorphonuclear leukocytes of eight methicillin-resistant strains of Staphylococcus aureus of unknown virulence was studied. After incubation for I, 3, 5, or 24 hr in a modified phagocytic assay, the methicillin-resistant strains survived as well as other virulent but methicillin-sensitive strains of S. aureus. Highly resistant subpopulations were obtained from three parent strains, and a methicillin-sensitive revertant subpopulation from one resistant parent strain. All subpopulations were eliminated to the same extent as were the moderately resistant parent strains. When the phagocytic assay was performed in the presence of 25 f-Lg of mcthicillin rml, only the methicillin-sensitive strains and the sensitive subpopulation derived from one resistant parent strain were eliminated after incubation for 24 hr. These in vitro data are further evidence against the use of methicillin in infections due to these organisms.
Vaudaux and Waldvogel
548
lution of 6% dextran T 70 (Pharmacia, Uppsala, Sweden). Leukocytes were separated from plasma by two centrifugations at 500 g during a 10-min period. The pellet was suspended after the first centrifugation in isotonic saline, and after the second centrifugation in a Dulbecco's phosphatebuffered saline solution (Gibco Bio-Cult; Glasgow, Scotland), supplemented with 0.1 % glucose (DG). Segmented PMNLs (i.e., 50%-70%) of the leukocyte preparation were adjusted to a final concentration of 107/ml. Autologous serum was collected freshly for each phagocytic assay. Phagocytic assay. Each I-ml incubation mixture included 0.5 ml of the leukocyte preparation (5 X 106 segmented PMNLs), 0.1 ml of serum, 0.1 ml of an overnight culture of MRSA adjusted to an inoculum of 2-3 X 106 cfuyrnl, and 0.3 ml of DG medium, or when methicillin was included in the phagocytic assay, 0.1 ml of a freshly prepared solution of 250 fLg of methicillm rrnl plus 0.2 ml of DG medium. The siliconized tubes used for the phagocytic assay were constantly shaken (240 shakes/min) in a water bath at 37 C ± 0.2 C. After incubation for 0, 1,3,5, and 24 hr, duplicate O.I-ml samples were removed and diluted 100fold in distilled water to obtain adequate lysis of PMNLs and dilution of methicillin (when present) below its MIC; the diluent was plated on
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Figure 1. Survival of four methicillin-resistant strains ( ---------) and two methicillin -sensitive strains (0 0) of Staphylococcus aureus . See Materials and Methods for the phagocytic assay procedure.
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 5, 2015
Switzerland, the United Kingdom, Germany, Australia, and the United States. All strains belonged to phage group III, except for untypable strain 72, and were of either serotype III or 18 [II]. Two methicillin-sensitive strains of S. aureus (phage group I, serotype I) and one penicillinsensitive strain (Ma) of S. aureus (phage type 42 D, serotype 6 [II]) were bacteremic isolates from the Hopital Cantonal. Methicillin resistance was established with use of the three criteria described by Parker and Hewitt [12]. The strains of MRSA were resistant to penicillin, streptomycin, tetracycline, and erythromycin; furthermore, resistance to chloramphenicol was demonstrated in four strains and resistance to kanamycin and neomycin in two strains [13]. Selection of subpopulations of MRSA with modified resistance to methicillin. Three subpopulations were obtained from the parent strain 72: a highly methicillin-resistant subpopulation, a moderately resistant subpopulation, and a sensitive subpopulation. In each of these subpopulations, the percentage of resistant cells was defined as the ratio of cfu counts obtained on MuellerHinton agar containing 25 p.,g of methicillin/rnl to cfu counts obtained on agar without methicillin [I, 2]. The highly resistant subpopulation contained at least 50% resistant cells and was selected by incubation of a preparation of 106 cfu of the parent strainj'ml with 25 p.,g of methicillirr/ml in Mueller-Hinton broth for 48 hr at 37 C. The moderately resistant subpopulation contained 0.01%, resistant cells. The sensitive subpopulation contained less than one resistant cell / 107 cells and was a methicillin-sensitive revertant isolated from the resistant parent strain by repeated subculturing and storage at low temperature (4 C-6 C) [14]. The common origin of parent strain and revertant subpopulation was documented by their identical antibiotic resistance patterns and identical serotype (III). Only phage type and hemolytic properties were modified, the parent strain being nontypable and nonhemolytic, whereas the revertant was lysed by phage 85 and had hemolytic properties. Loss of resistance to methicillin has been shown to be associated with gain of susceptibility to phage 85 [15]. Preparation of leukocytes. Venous blood (30 ml; citrate added) from normal donors was allowed to sediment for 60 min with 15 ml of a so-
549
Methicillin-Resistant S. aUTeUS; Phagocytosis
to the ultraviolet test combination from Boehringer (Mannheim, Germany). Cell viability was determined by the trypan blue exclusion test by mixing 0.5 ml of a trypan blue solution in DG medium with a O.l-ml sample of the phagocytic assay. Statistics. The data were analyzed by Student's unpaired t-test. Results
Description and validation of the phagocyticbactericidal assay extended over 24 hr. Survival of 10 strains of MRSA had been tested previously in a I-hI' phagocytic assay and compared with that of a virulent penicillin-sensitive strain (Ma) of S. aureus [16]. Survival of the strains of MRSA
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Figure 2. Survival and growth of three subpopulations (highly resistant, moderately resistant, and sensitive) of methicillin-resistant Staphylococcus aureus strain 72 during a 24-hr incubation period. See Materials and Methods for moderately resistant, • .; the phagocytic assay procedure. Survival of the subpopulations: highly resistant, and sensitive, . Growth in Mueller-Hinton broth: highly resistant, 0 - - 0; moderately resistant, 0- - - - 0; and sensitive, D - - - - D. Growth in Dulbecco's phosphate-buffered saline solution supplemented with 0.1 % glucose and normal human serum, without polymorphonuclear leukocytes: highly resistant, (> - - - - c: moderately resistant. () - - - - (); and sensitive, II - - - - II. Left, without methicillin. Right. with 25 I-Ig of methicillin/m!.
+--+;
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 5, 2015
Mueller-Hinton agar for counting of residual colonies. Other bactericidal tests. Bactericidal actrvity of methicillin was tested at 37 C by addition of 0.1 ml of saline solution containing 250 p,g of methicillinjml to 0.1 ml of an overnight culture of MRSA (diluted 20-fold in distilled water) and 0.8 ml of Mueller-Hinton broth, or to 0.1 ml of serum and 0.7 ml of DG medium (DGS medium). These samples were processed as above for the phagocytic assay. Viability tests of PMNLs. Lactate dehydrogenase (LDH) activity was determined according
Vaudaux and Waldvogel
550
as those shown in figure 2, left. At 1,3, 5, and 24 hr, there was no significant difference (P > 0.05) in the number of residual du. Effect of methicillin on survival of sub-populotions of MRSA with modified resistance. The three subpopulations with different degrees of resistance, derived from parent strain 72, were tested comparatively in the phagocytic-bactericidal assay supplemented with 25 fLg of methicillinj ml. After incubation for 24 hr, the highly resistant subpopulation survived better than did the sensitive one (figure 2, right). Intermediate results were obtained at 24 hr with the moderately resistant subpopulation. Repeated determinations (n = 5) of residual du of each subpopulation at 24 hI' showed increased survival for the highly resistant subpopulation only, as compared with the moderately resistant and the sensitive subpopulations (a logio for the highly resistant subpopulation, -0.76 ± 0.16; for the moderately resistant, -2.33 ± 0.33; for the sensitive, -2.37 ± 0.23; P < 0.001). Shorter incubation times, up to 5 hr, did not reveal any significant difference in survival rate (P> 0.05) between the three subpopulations. No significant modification was noted in the resistance of each subpopulation during incubation for 24 hr in the presence of methicillin and PMNLs (table I). Finally, assay with methicillin in the medium containing active PMNLs demonstrated that a high concentration of methicillin (15 fLgjml) was still present after incubation for 24 hr with all three subpopulations (data not shown). Table 1. Percentage of methicillin-resistant cells in the highly resistant and the moderately resistant subpopulations of methicillin-resistant Staphylococcus aureus strain
72. Percentage of subpopulation resistant* Incubation period (hr)
0 3 5 24
Highly resistant
Moderately resistant
100 59 67 100
NTt NT
0.4
Methicillin-Resistant Strains of Staphylococcus aureus: Relation between Expression of Resistance and Phagocytosis by Polymorphonuclear Leukocytes From the Infectious Diseases Division, Department of Medicine, Hopital Cantonal, Geneva, Switzerland
Pierre Vaudaux and Francis A. Waldvogel
it has been questioned whether after exposure to methicillin they are still adequate in number and virulence to resist natural host defense mechanisms, when compared with methicillin-sensitive cells of S. aureus [3, 7, 8]. Other investigators [9, 10] have established that virulent strains of S. aureus are not eliminated by polymorphonuclear leukocytes (PMNLs) to the same extent as are avirulent strains. Thus, an in vitro phagocytic-bactericidal assay was used in this study to answer the following clinically relevant questions. (1) Are strains of MRSA eliminated to the same extent by PMNLs as are virulent but methicillin-sensitive strains of S. aureus? (2) Are methicillin-resistant cells of MRSA more easily eliminated by PMNLs than are methicillinsensitive cells? (3) Is methicillin, in conjunction with serum factors and PMNLs, capable of eliminating strains of MRSA as efficiently as it eliminates methicillin-sensitive strains of S. aureus? The answers to these three questions are the basis of the present report.
Although naturally occurring methicillin-resistant Staphylococcus aureus (MRSA) have been demonstrated to be heterogeneously resistant, only a small proportion of the cells are able to resist in vitro destruction by therapeutic levels of methicillin [1-3]. A homogeneous population of methicillin-resistant cells can be selected from the initial population by prolonged in vitro incubation in the presence of methicillin [1-3]. In clinical situations, however, it has not yet been established whether the administration of methicillin to patients with infections due to MRSA selects out the resistant cells [2, 4, 5] as is observed in vitro. Since resistant cells have peculiar growth characteristics and colonial morphology [1-3, 6], Received for publication July 17, 1978, and in revised form December 5,1978. This study was supported in part by a grant from Beecham Pharmaceuticals, Betchworth, Surrey, England, and by a grant from the Societe Academique, Geneva, Switzerland. We thank Dr. Hans Reber, Kantonsspital, Basle, Switzerland, and Dr. Jacques Pillet, Inst.itut Pasteur, Paris, France, for phage-typing and serotyping of the methicillin-resistant strains of Staphylococcus aureus. This paper was presented in part at a meeting of the Swiss Society for Microbiology, Basle, Switzerland, June 16, 1978. Please address requests for reprints to Dr. Pierre Vaudaux, Infectious Diseases Division, Department of Medicine, Hopital Cantonal, 1211 Geneva 4, Switzerland.
Materials and Methods
Organisms and antibiotic resistance. Eight strains of MRSA were tested: two strains isolated at the Hopital Cantonal, Geneva, Switzerland, and six others obtained from other hospitals in
547
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 5, 2015
Virulent strains of staphylococci are known to resist phagocytic destruction better than avirulent strains. In this context, in vitro elimination by human polymorphonuclear leukocytes of eight methicillin-resistant strains of Staphylococcus aureus of unknown virulence was studied. After incubation for I, 3, 5, or 24 hr in a modified phagocytic assay, the methicillin-resistant strains survived as well as other virulent but methicillin-sensitive strains of S. aureus. Highly resistant subpopulations were obtained from three parent strains, and a methicillin-sensitive revertant subpopulation from one resistant parent strain. All subpopulations were eliminated to the same extent as were the moderately resistant parent strains. When the phagocytic assay was performed in the presence of 25 f-Lg of mcthicillin rml, only the methicillin-sensitive strains and the sensitive subpopulation derived from one resistant parent strain were eliminated after incubation for 24 hr. These in vitro data are further evidence against the use of methicillin in infections due to these organisms.
Vaudaux and Waldvogel
548
lution of 6% dextran T 70 (Pharmacia, Uppsala, Sweden). Leukocytes were separated from plasma by two centrifugations at 500 g during a 10-min period. The pellet was suspended after the first centrifugation in isotonic saline, and after the second centrifugation in a Dulbecco's phosphatebuffered saline solution (Gibco Bio-Cult; Glasgow, Scotland), supplemented with 0.1 % glucose (DG). Segmented PMNLs (i.e., 50%-70%) of the leukocyte preparation were adjusted to a final concentration of 107/ml. Autologous serum was collected freshly for each phagocytic assay. Phagocytic assay. Each I-ml incubation mixture included 0.5 ml of the leukocyte preparation (5 X 106 segmented PMNLs), 0.1 ml of serum, 0.1 ml of an overnight culture of MRSA adjusted to an inoculum of 2-3 X 106 cfuyrnl, and 0.3 ml of DG medium, or when methicillin was included in the phagocytic assay, 0.1 ml of a freshly prepared solution of 250 fLg of methicillm rrnl plus 0.2 ml of DG medium. The siliconized tubes used for the phagocytic assay were constantly shaken (240 shakes/min) in a water bath at 37 C ± 0.2 C. After incubation for 0, 1,3,5, and 24 hr, duplicate O.I-ml samples were removed and diluted 100fold in distilled water to obtain adequate lysis of PMNLs and dilution of methicillin (when present) below its MIC; the diluent was plated on
8
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4 5 HOURS
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Figure 1. Survival of four methicillin-resistant strains ( ---------) and two methicillin -sensitive strains (0 0) of Staphylococcus aureus . See Materials and Methods for the phagocytic assay procedure.
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 5, 2015
Switzerland, the United Kingdom, Germany, Australia, and the United States. All strains belonged to phage group III, except for untypable strain 72, and were of either serotype III or 18 [II]. Two methicillin-sensitive strains of S. aureus (phage group I, serotype I) and one penicillinsensitive strain (Ma) of S. aureus (phage type 42 D, serotype 6 [II]) were bacteremic isolates from the Hopital Cantonal. Methicillin resistance was established with use of the three criteria described by Parker and Hewitt [12]. The strains of MRSA were resistant to penicillin, streptomycin, tetracycline, and erythromycin; furthermore, resistance to chloramphenicol was demonstrated in four strains and resistance to kanamycin and neomycin in two strains [13]. Selection of subpopulations of MRSA with modified resistance to methicillin. Three subpopulations were obtained from the parent strain 72: a highly methicillin-resistant subpopulation, a moderately resistant subpopulation, and a sensitive subpopulation. In each of these subpopulations, the percentage of resistant cells was defined as the ratio of cfu counts obtained on MuellerHinton agar containing 25 p.,g of methicillin/rnl to cfu counts obtained on agar without methicillin [I, 2]. The highly resistant subpopulation contained at least 50% resistant cells and was selected by incubation of a preparation of 106 cfu of the parent strainj'ml with 25 p.,g of methicillirr/ml in Mueller-Hinton broth for 48 hr at 37 C. The moderately resistant subpopulation contained 0.01%, resistant cells. The sensitive subpopulation contained less than one resistant cell / 107 cells and was a methicillin-sensitive revertant isolated from the resistant parent strain by repeated subculturing and storage at low temperature (4 C-6 C) [14]. The common origin of parent strain and revertant subpopulation was documented by their identical antibiotic resistance patterns and identical serotype (III). Only phage type and hemolytic properties were modified, the parent strain being nontypable and nonhemolytic, whereas the revertant was lysed by phage 85 and had hemolytic properties. Loss of resistance to methicillin has been shown to be associated with gain of susceptibility to phage 85 [15]. Preparation of leukocytes. Venous blood (30 ml; citrate added) from normal donors was allowed to sediment for 60 min with 15 ml of a so-
549
Methicillin-Resistant S. aUTeUS; Phagocytosis
to the ultraviolet test combination from Boehringer (Mannheim, Germany). Cell viability was determined by the trypan blue exclusion test by mixing 0.5 ml of a trypan blue solution in DG medium with a O.l-ml sample of the phagocytic assay. Statistics. The data were analyzed by Student's unpaired t-test. Results
Description and validation of the phagocyticbactericidal assay extended over 24 hr. Survival of 10 strains of MRSA had been tested previously in a I-hI' phagocytic assay and compared with that of a virulent penicillin-sensitive strain (Ma) of S. aureus [16]. Survival of the strains of MRSA
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Figure 2. Survival and growth of three subpopulations (highly resistant, moderately resistant, and sensitive) of methicillin-resistant Staphylococcus aureus strain 72 during a 24-hr incubation period. See Materials and Methods for moderately resistant, • .; the phagocytic assay procedure. Survival of the subpopulations: highly resistant, and sensitive, . Growth in Mueller-Hinton broth: highly resistant, 0 - - 0; moderately resistant, 0- - - - 0; and sensitive, D - - - - D. Growth in Dulbecco's phosphate-buffered saline solution supplemented with 0.1 % glucose and normal human serum, without polymorphonuclear leukocytes: highly resistant, (> - - - - c: moderately resistant. () - - - - (); and sensitive, II - - - - II. Left, without methicillin. Right. with 25 I-Ig of methicillin/m!.
+--+;
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 5, 2015
Mueller-Hinton agar for counting of residual colonies. Other bactericidal tests. Bactericidal actrvity of methicillin was tested at 37 C by addition of 0.1 ml of saline solution containing 250 p,g of methicillinjml to 0.1 ml of an overnight culture of MRSA (diluted 20-fold in distilled water) and 0.8 ml of Mueller-Hinton broth, or to 0.1 ml of serum and 0.7 ml of DG medium (DGS medium). These samples were processed as above for the phagocytic assay. Viability tests of PMNLs. Lactate dehydrogenase (LDH) activity was determined according
Vaudaux and Waldvogel
550
as those shown in figure 2, left. At 1,3, 5, and 24 hr, there was no significant difference (P > 0.05) in the number of residual du. Effect of methicillin on survival of sub-populotions of MRSA with modified resistance. The three subpopulations with different degrees of resistance, derived from parent strain 72, were tested comparatively in the phagocytic-bactericidal assay supplemented with 25 fLg of methicillinj ml. After incubation for 24 hr, the highly resistant subpopulation survived better than did the sensitive one (figure 2, right). Intermediate results were obtained at 24 hr with the moderately resistant subpopulation. Repeated determinations (n = 5) of residual du of each subpopulation at 24 hI' showed increased survival for the highly resistant subpopulation only, as compared with the moderately resistant and the sensitive subpopulations (a logio for the highly resistant subpopulation, -0.76 ± 0.16; for the moderately resistant, -2.33 ± 0.33; for the sensitive, -2.37 ± 0.23; P < 0.001). Shorter incubation times, up to 5 hr, did not reveal any significant difference in survival rate (P> 0.05) between the three subpopulations. No significant modification was noted in the resistance of each subpopulation during incubation for 24 hr in the presence of methicillin and PMNLs (table I). Finally, assay with methicillin in the medium containing active PMNLs demonstrated that a high concentration of methicillin (15 fLgjml) was still present after incubation for 24 hr with all three subpopulations (data not shown). Table 1. Percentage of methicillin-resistant cells in the highly resistant and the moderately resistant subpopulations of methicillin-resistant Staphylococcus aureus strain
72. Percentage of subpopulation resistant* Incubation period (hr)
0 3 5 24
Highly resistant
Moderately resistant
100 59 67 100
NTt NT
0.4
