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An evaluation of the mechanism of penicillin resistance in vivo
An Evaluation of the Mechanism of Penicillin Resistance in Vivo PETER
DINEEN,
M . D . , AND H E N R Y
MANNIX,
JR., M . D . , T h e N e w Y o r k H o s p i t a l ~
C o r n e l l 21Iedical C e n t e r
T h e mechanism by which certain strains of staphylococci are resistant to the action of penicillin G is not clear. It has been assumed that staphylococcal penicillinase plays a role in this resistance, but whether or not genetic mutation of the species or some still unknown means is also a factor, as with other antimicrobial drugs, remains unanswered. Tire advent of a synthetic penicillin which apparently is relatively unaffected by the enzymatic action of staphylopenicillinase has opened u p this area for investigation. T h e r e have been many in vitro laboratory studies of 2,6 dimethoxyphenyl penicillin. ~, ~, 7, 10, a5 These were concerned primarily with the efficacy of the drug against various strains of staphylococci in vitro and with comparisons of degrees of degradation of penicillin G and dimethoxyphenyl penicillin by penicillinase. Also there have been several clinical reports of di,nethoxyphenyl penicillin treatment of infections, n, a-~ as, a4 However, these were difficult to evaluate from a longterm standpoint at this early stage of o u r experience with this drug. Therefore, it was believed possible tltat two factors could be studied simtthaneously with the use of tiffs new agent. T h e first, and more basic, was tlte investigation of the role of penicillinase in the in vivo mechanisms of penicillin resistance by certain strains of Staphylococcus aureus. Second, by employing a standard biologic model wltich has been used in tlfis laboratory for several )'ears an in vivo assessment of tlte efficacy of dimethoxyphenyl penicillin could be made. From the Bacteriology Research Laboratory, Departmerit of Surgeo', The New York Hospital--Cornell Medical Center, New York 21, N. Y.
This study was supported in part by U.S. Public Health Service Grant (Allerg)" and Infectious Diseases) #E-3642 (RI). Submitted for publication September 14, 1961; accepted October 24, 1961. JSR - Vol. II, No. 2 - March, 1962
MATERIALS AND BIETHODS T h e experiments were divided into in vitro and in vivo studies. T h e only organism used in these experiments was Staphflococcus aureus (strain Giorgio). This strain is coagulase positive, hemolytic, mannitol positive and resistant to penicillin and susceptible to other antimicrobial drugs. Penicillin susceptibility was determined by tile serial tube dilution method. Tiffs strain muItiplied in the presence of 3.1 units/ml, of penicillin G, but was inhibited by 6.2 units/ml. It was isolated from a brain abscess at Tile New Y o r k Hospital-Cornell Medical Center by one of us (P. D.) in 1950, and has been carried in the laboratory since then. In vivo this strain is lethal to mice and has an SD~0 of 7 days in Swiss Webster albino mice. I t has been shown in this laboratory 4 that this strain produced penicillinase in vivo as well as in vitro. I n Vitro Studies. T h e serial tube dihttion test described by R a m m e l k a m p was employed to determine the susceptibility of Staphylococcus attretts (Giorgio) to penicillin G and dimethoxyphenyl penicillin. I n Vivo Studies. Swiss Webster male albino mice about four weeks old antl between 16 and 20 gm. were used throughout the experiments. T h e y were housed in steel mesh cages, 10 animals in each cage. Tltey were fed commercial mouse pellets, and allowed water ad libitum. Inoculum. Tlte inoculum for each challenge was prepared from an 18 hour undihtted c u h u r e of Staphylococcus aureus (Giorgio) grown in beef heart infusion peptone broth at 37 ~ C. T h e standard infecting dose consisted of 0.1 ml. of c u h u r e diluted in physiologic saline to a final volume of 0.2 ml. and tiffs was injected into one of the dorsal tail veins of the mouse. Tiffs represented inocula in each mouse of from 1.0 X 107 to 5.0 X 10r cuhurable units. T h e resulting infection in mice is predictable 151
152
JSR - Vol. H, No. 2 - March, 1962
PETER DINEEN a n d HENRY ~[ANNIX
and has been reported by McCune et al. s, 9 and Dinccn.4, 5 In brief, the untreated mice develop a scvcrc infection which becomes localized in the kidneys with the appearance of abscesses. About half the mice are dead in 7 days and almost all are dead in from 14 to 21 days. T h e microbial population in the kidneys reaches a peak at about 5 days (approximately 109 organisms/m]. of tissue) and remains constant to death. In all experiments the microbial population of the kidneys of the mice was used as tim index of degree of infection and success or failure of treatment. T o measure the bacterial census in the kidneys the following procedure described by McCune et al.S, 9 was employed. At various periods of time after intravenous challenge in each experiment three to five mice in each experimental group were sacrificed. T h e kidneys were removed aseptically and added to 5.0 ml. of distilled water and then homogenized with a Teflon grinder. T h e resulting homogenate was appropriately diluted and 1.0 ml. aliquots were plated in duplicate with 9.0 ml. of poured agar. After 48 h o u r s ' incubation the colonies on each plate were counted and, by computing the dilutions used, the n u m b e r of organisms per milliliter of kidney tissue could be computed. Experimental Groups. T h e r e were a series of six separate experiments. T h e following were the experimental groups of the investigation. Control group which was infected with 0.2 ml. of a 1:1 dilution of an 18 hour culture of Staphylococcus aureus (Giorgio) and received no treatment. All other groups were similarly infected but received various modalities of therapy. T h e r e were five simple treatment groups and five groups in which treatment was combined with the use of one of the corticosteroids. T h e treatment groups were: Penicillin G--3.0 mg. intramuscularly once each day for 21 days. Penicillin G--3.0 mg. intramuscularly twice each day for 21 days. 2,6 Dimethoxyplwnyl penicillin-2.5 rag. intramuscularly once each day for 21 days. 2,6 Dimethoxyphenyl penicillin--5.0 mg. intramuscularly once each day for 21 days. 2,6 Dimethoxyphenyl penicillin--5.0 mg. intramnscularly twice each day for 21 days. T h e corticosteroid plus antimicrobial treatment groups were as follows: Hydrocortisone--O.1 mg. intramuscularly once each day for 21 days. Penicillin G--3.0 mg. plus hydrocortisone-O.1 mg. once each day for 21 days.
2,6 Dimethoxyphenyl penicillin--2.5 rag. plus 0.1 mg. of hydrocortisone once each day for 21 days. 2,6 Dimethoxyphenyl penicillin--5.0 mg. plus 0.1 mg. of hydrocortisone once each day for 21 days. 2,6 Dirnethoxyphenyl penicillin-5.0 mg. twice each day and 0.1 rag. hydrocortisone once each day for 21 days. Animals were sacrificed at successive points from 15 minutes after the challenge up to 21 days. T h e microbial census in the kidneys was calculated and plotted logarittmfically. RESULTS
IN VIIRO T h e growth of Staphylococcus aureus (Giorgio) was inhibited by 6.2/.tg./ml. of penicillin G and by 3.1 ptg./ml, of 2,6 dimethoxyphenyl penicillin. IN V~vo
2,6 Dimethoxyphenyl penicillin does not have a striking effect on the microbial population in mouse kidneys. However, there is a definite decrease as compared to the control. In Figure 1 three different dosage schedules are compared to the control and to penicillin G treatment. It is noted that for 24 hours there was multiplication in the kidneys in all groups and the census rose. I n the groups of animals that received 2.5 rag. dimethoxyphenyl penicillin once each day there was a rise in microbial popnlation for three days and then it slowly but steadily fell for the remaining 18 days. In the groups that received twice as much drug (5.0 rag. once each day) there was an initial rise in population. T h e census hovered at this level for about one week and then
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$ Fig. 1. Microbial populations of Staphylococcus aureus (Giorgio) in the kidneys of mice. T h e effect of various dosage schedules of dimethoxyphenyl penicillin as compared to the untreated control and to penicillin G treatment.
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Vol. II, No. 2 - March,
~[ECtlANISM OF PENICILLIN RESISTANCE
1962
a sustained fall began. In the groups tltat received 5.0 rag. twice a day almost an identical picture was presented. It is of interest tlaat the two points of significant difference between the penicillin G groups and the dimethoxyphenyl penicillin groups are (I) at 24 hours after challenge and (2) at two weeks a n d later after challenge. T h e r e was no difference between tlte population levels of those treated with penicillin G 3.0 mg. once a day and twice a day. Hydrocortisone 0.1 mg. intramuscttlarly each day had no significant effect on the microbial populations as compared to the control. (In the experiments of Batten et al. a the steroid effect was not seen for 30 days and this was with Mycobacteria.) T h e censns rose promptly to a peak at four days and remained elevated for two weeks. After that there was a slight decrease in populations of staphylococci in ttte kidneys of the hydrocortisone group compared to the control. However, both groups exhibited renal abscesses from the secoud day onward. As can be noted from Figure 2, penicillin G 3.0 rag. intramuscularly twice a day did reduce tlle bacterial counts in the kidneys as compared to the control. T h e kidneys also exltibited abscesses. T h e effect of the corticosteroid (hydrocortisone) on the various treatment groups is noted in Figures 3 and 4. In general the steroids did not potentiate tlle action of the antimicrobial but tended rather to interfere with it. In Figure 3 it can be seen that tlle gronp that received din,ethoxyphenyl penicillin alone did better (as measured by lower bacterial popnlations) than did the treatment group which was also on hydrocortisone~ From the second day onward the group of animals that received 5.0 mg. of dimethoxyphenyl penicillin twice a day and no
153
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Fig. 3. Microbial populations of Staphylococcus aureus (Giorgio) in the kidneys of mice. The effect on the kidney bacterial census of hydrocortisone and dimethoxyphenyl penicillin are shown singly and together.
hydrocortisone had consistently lower populations. A further study of the effect of hydrocortisone on various treatment groups again showed that the steroid tended to interfere with the action of the antimicrobial agent. As shown in Figure 4, lower doses of dimethoxyphenyl penicillin were used (2.5 mg. each day and 5.0 mg. each day respectively). In the first week of the experiment there was only one significant difference between any of the groups. This was at day 4 when the groups treated witlt 5.0 mg. of dimethoxypheny] penicillin had populations much lower (Fig. 4) than the same group which received hydrocortisone. After the first week a more pronotmced difference was apparent between tlte hydrocortisone groups and those that received only dimethoxyphenyl penicillin. 8.
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Fig. 2. Microbial poptflations of Staphylococcus attreus (Giorgio) in the kidneys of nfice. The effect of hydrocortisone and penicillin separately is shown.
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Fig. 4. Microbial populations of Staphylococcus attreus (Giorgio) in tile kidneys of mice. Tile effect of various dosage schedules of dimethoxyphenyl penicillin used singly or with hydrocortisone are compared.
154
PETERDINEEN and HENRY ~IANNIX DISCUSSION
T h e results of this experiment shed some light on the mechanism of bacterial resistance to penicillin in vivo. There has always been the question as to how large a part penicillinase plays in this resistance. In previous studies from this laboratory* it has been demonstrated for the first time that penicillin resistant staphylococci do produce penicillinase in vivo. In fact with a model exactly similar to that used in this series of experiments the actual amounts of staphylopenicillinase produced were measured. B u n n et a13 have shown that dimethoxyphenyl .penicillin was not blocked in its antimicrobial activity unless there was an excessive a m o u h t of penicillinase (B. cereus) in the in vitro system. This work has been confirmed by other observers as well. n Therefore, it is reasonable to expect that if penicillinase is the mechanism by which staphylococci are resistant to the action of penicillin and if dimethoxyphenyl penicillin is not blocked by penicillinase (except in very large amounts), then the fall in microbial population in the kidneys should be similar to that of a penicillin susceptible population under penicillin treatment. However, when a penicillin susceptible strain is treated with penicillin G in the animal model described there is a precipitous fall in the bacterial population in the kidneys from the time of first treatment. T h e Organisms are reduced in number to about I0 p e r nfilliliter of kidney tissue in five days. This study has been reported in detail by McCune et al. 8 Wompsett a6 Ires raised a doubt as to the importance of the in vivo production of penicillinase in the determination of penicillin resistance. T h e experiments reported here tend to favor t h i s latter concept. Although some degree of effectiveness was shown by dimethoxyphenyl penicillin (Fig. 1) as compared to penicillin G in this penicillin resistant infection, it did not compare favorably with previous resuhs obtained by penicillin G in a penicillin susceptible infection. This leads to one of two conclusions: (I) Penicillinase is not an important factor in penicillin resistance, or (2) dimethoxyphenyl penicillin is affected seriously by staphylopenicillinase in vivo. Since it is known that the production of penicillinase in vivo reaches a peak about two or three days after the inception of infection, it would follow tlmt the importance of penicillinase in any infection should increase as time progresses. Since dimethoxyphenyl penicillin is least effective (Fig. 1) in the first few days, one must conclude from these facts that dimethoxyphenyl penicillin is not
J S R - Vol. 11, No. 2 - March, 1962
significantly affected by staphylopenicillinase and tlmt resistance to penicillin is much more complex. In this penicillinase is only one facet. Further investigation into the mechanism of penicillin resistance must be done. However, the Staphylococcus aureus (Giorgio) apparently does have another means of resistance to penicillin independent of penicillinase. This is evidcnt because of the lack of strong responses to treatment despite massive doses of dimethoxyphenyl penicillin (5.0 mg. twice a day). Because the rapid excretion of dimethoxyphenyl penicillin might have explained its mediocre action, experiments were done with both once a day and twice a day dosage. There was not a significant difference between these two groups. If resistance to penicillin cannot be wholly explained by penicillinase or by genetic mutation, then a third mechanism must exist. One may only speculate as to what this may be. It seems likely, however, that some other enzyme system must be operative which is less potent than the penicillinase system. This may be intracellular, as comparcd to the apparently extracelhdar position (or at least surface location) of penicillinase. If this is so it may explain the less dramatic action of this substance, but also it would explain why dimethoxyphenyl penicillin is not so effective as one would expect theoretically. T h e effect of corticosteroids in infections-particularly experimental myobacterial infections-has gradually become more clear by virtue of the basic work of Batten et alA The use of corticosteroids (particularly cortisone or hydrocortisone) enhances bacterial multiplication both in vivo and in vitro. While certain factors of host resistance may also be increased, the overall picture both experimentally and clinically is deleterious to the patient unless the infection is also treated with appropriate antimicrobial dn~gs. In the experiments reported here the infection-enhancing properties of hydrocortisone were not completely neutralized by dimethoxyphenyl penicillin. However, the difference in microbial populations between the two groups (Fig. 3) while statistically significant was not considered to be a serious drawback. In this study two questions have been considered. One has been the mechanism of penicillin resistance in vivo. Dimethoxyphenyl penicillin served as a tool to study this. Second, an investigation was made into the efficacy of dimethoxyphenyl penicillin itself, and an evahmtion of it in terms of a well established model infection was undertaken. Based on previous experience
JSR
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Vol. II, No. 2 - March, 1962
witll this biologic system, dimethoxyphenyl penicillin has only a moderate effectiveness. It should be employed with these reservations in mind. SUMMARY
Ttle i n v i v o mechanism of penicillin resistance was studied by the use of 2,6 dimethoxyphenyl penicillin. Despite its apparent impunity to staphylopenicillinase 2,6 dimethoxyphenyl penicillin is not as effective against a penicillin resistant staphylococcal infection as penicillin G is against a penicillin susceptible infection. Thus the mechanism of penicillin resistance is more complex than the mere presence of peiaicillinase in the enzyme substrate system. Dinaethoxyphenyl penicillin is only moderately effective in the experimental model as compared to other antimicrobial agents. The use of hydrocortisone concomitantly interferes moderately with the effectiveness of dimethoxyphenyl penicillin. REFERENCES 1. Batten, J. C., and McCune, R. M., Jr.: T h e influence of corticotrophin and certain corticosteroids on populations of Mycobacterium tuberculosis in tissues of mice. Brit. J. Exper. Path., 38:413, 1957. 2. Bunn, P. A., Editor: A symposium on the new dimethoxyphenyl penicillin. Early laboratory and clinical experiences with particular reference to resistant staphylococcal disease. State 9 University of New York Upstate Medical Center, Syracuse, N. Y. September 7, 1960. 3. Cronk, G. A., and Morigi, E. M. E.: Absorption, excretion, and distribution of dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 58 (I960). 4. Dineen, P.: The in vivo production of penlcillinase by hemolytic staphylococcus aureus. J. hmnunol., 86:496, 1961. 5. Dineen, P.: A period of unusual microbial susceptibility in an experimental staphylococcal infection. J. Infect. Dis., 108:174, 1961.
~[ECtlANISM OF PENICILLIN RESISTANCE
155
6. Finegold, S. M., Mouzon, O. T., Sweeney, E. E., Dangerfield, H. G., Blackman, B. T., and Hewitt, W. L.: Laboratory experiences with dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 26 (1960). 7. Gourevitch, A., Hunt, G. A., Pursiano, T. A., Moses, A. J., and Lein, J.: Microbiological studies on dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y, p. 4 (1960). 8. McCune, R., Dineen, P., and Batten, J. C.: The influence of antimicrobial agents on total populations of staphylococci in animal tissues. J. Immunol., 85:447, I960. 9. McCune, R. M., Jr., Dineen, P., and Batten, J. C.: The effect of antimicroblal drugs on an experimental staphylococcal infection in mice. Ann. N. Y. Acad. Sc., 65:91, 1956. I0. Pindell, M. H., Tisch, D. E., and Reiffenstein, J. C.: Pharmacological properties of dimethoxyphenyl penicillin. Dimetlmxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 50 (1960). 11. Rifkind, D., and Knight, V.: Treatment of staphylococcal and streptococcal infections with dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 160 (1960). 12. Roberts, C. E., Jr., Allen, J. D., and Kirby, ~V. M. M.: Laboratory and clinical stndies of dimcthoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 109 (1960). 13. Rutenburg. A , Greenberg, It. L., and Schweinburg, F. B.: Clinical experiences with dimethoxyphenyl penicillin in staphylococcal infections. Dimethoxy9 phenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 101 0960). 14. Smith, I. M., and Counts, G. W.." Laboratory and clinical studies of dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 83 (1960). 15. Steinman, H. G.: Biochemical studies on 6-aminopenicillanic acid, benzyl penicillin, and 2,6-dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposimn, State University of New York College of Medicine, Syracuse, N. Y., p. 17 (1960). 16. Tompsett, Ralph: Personal communication.
DINEEN,
M . D . , AND H E N R Y
MANNIX,
JR., M . D . , T h e N e w Y o r k H o s p i t a l ~
C o r n e l l 21Iedical C e n t e r
T h e mechanism by which certain strains of staphylococci are resistant to the action of penicillin G is not clear. It has been assumed that staphylococcal penicillinase plays a role in this resistance, but whether or not genetic mutation of the species or some still unknown means is also a factor, as with other antimicrobial drugs, remains unanswered. Tire advent of a synthetic penicillin which apparently is relatively unaffected by the enzymatic action of staphylopenicillinase has opened u p this area for investigation. T h e r e have been many in vitro laboratory studies of 2,6 dimethoxyphenyl penicillin. ~, ~, 7, 10, a5 These were concerned primarily with the efficacy of the drug against various strains of staphylococci in vitro and with comparisons of degrees of degradation of penicillin G and dimethoxyphenyl penicillin by penicillinase. Also there have been several clinical reports of di,nethoxyphenyl penicillin treatment of infections, n, a-~ as, a4 However, these were difficult to evaluate from a longterm standpoint at this early stage of o u r experience with this drug. Therefore, it was believed possible tltat two factors could be studied simtthaneously with the use of tiffs new agent. T h e first, and more basic, was tlte investigation of the role of penicillinase in the in vivo mechanisms of penicillin resistance by certain strains of Staphylococcus aureus. Second, by employing a standard biologic model wltich has been used in tlfis laboratory for several )'ears an in vivo assessment of tlte efficacy of dimethoxyphenyl penicillin could be made. From the Bacteriology Research Laboratory, Departmerit of Surgeo', The New York Hospital--Cornell Medical Center, New York 21, N. Y.
This study was supported in part by U.S. Public Health Service Grant (Allerg)" and Infectious Diseases) #E-3642 (RI). Submitted for publication September 14, 1961; accepted October 24, 1961. JSR - Vol. II, No. 2 - March, 1962
MATERIALS AND BIETHODS T h e experiments were divided into in vitro and in vivo studies. T h e only organism used in these experiments was Staphflococcus aureus (strain Giorgio). This strain is coagulase positive, hemolytic, mannitol positive and resistant to penicillin and susceptible to other antimicrobial drugs. Penicillin susceptibility was determined by tile serial tube dilution method. Tiffs strain muItiplied in the presence of 3.1 units/ml, of penicillin G, but was inhibited by 6.2 units/ml. It was isolated from a brain abscess at Tile New Y o r k Hospital-Cornell Medical Center by one of us (P. D.) in 1950, and has been carried in the laboratory since then. In vivo this strain is lethal to mice and has an SD~0 of 7 days in Swiss Webster albino mice. I t has been shown in this laboratory 4 that this strain produced penicillinase in vivo as well as in vitro. I n Vitro Studies. T h e serial tube dihttion test described by R a m m e l k a m p was employed to determine the susceptibility of Staphylococcus attretts (Giorgio) to penicillin G and dimethoxyphenyl penicillin. I n Vivo Studies. Swiss Webster male albino mice about four weeks old antl between 16 and 20 gm. were used throughout the experiments. T h e y were housed in steel mesh cages, 10 animals in each cage. Tltey were fed commercial mouse pellets, and allowed water ad libitum. Inoculum. Tlte inoculum for each challenge was prepared from an 18 hour undihtted c u h u r e of Staphylococcus aureus (Giorgio) grown in beef heart infusion peptone broth at 37 ~ C. T h e standard infecting dose consisted of 0.1 ml. of c u h u r e diluted in physiologic saline to a final volume of 0.2 ml. and tiffs was injected into one of the dorsal tail veins of the mouse. Tiffs represented inocula in each mouse of from 1.0 X 107 to 5.0 X 10r cuhurable units. T h e resulting infection in mice is predictable 151
152
JSR - Vol. H, No. 2 - March, 1962
PETER DINEEN a n d HENRY ~[ANNIX
and has been reported by McCune et al. s, 9 and Dinccn.4, 5 In brief, the untreated mice develop a scvcrc infection which becomes localized in the kidneys with the appearance of abscesses. About half the mice are dead in 7 days and almost all are dead in from 14 to 21 days. T h e microbial population in the kidneys reaches a peak at about 5 days (approximately 109 organisms/m]. of tissue) and remains constant to death. In all experiments the microbial population of the kidneys of the mice was used as tim index of degree of infection and success or failure of treatment. T o measure the bacterial census in the kidneys the following procedure described by McCune et al.S, 9 was employed. At various periods of time after intravenous challenge in each experiment three to five mice in each experimental group were sacrificed. T h e kidneys were removed aseptically and added to 5.0 ml. of distilled water and then homogenized with a Teflon grinder. T h e resulting homogenate was appropriately diluted and 1.0 ml. aliquots were plated in duplicate with 9.0 ml. of poured agar. After 48 h o u r s ' incubation the colonies on each plate were counted and, by computing the dilutions used, the n u m b e r of organisms per milliliter of kidney tissue could be computed. Experimental Groups. T h e r e were a series of six separate experiments. T h e following were the experimental groups of the investigation. Control group which was infected with 0.2 ml. of a 1:1 dilution of an 18 hour culture of Staphylococcus aureus (Giorgio) and received no treatment. All other groups were similarly infected but received various modalities of therapy. T h e r e were five simple treatment groups and five groups in which treatment was combined with the use of one of the corticosteroids. T h e treatment groups were: Penicillin G--3.0 mg. intramuscularly once each day for 21 days. Penicillin G--3.0 mg. intramuscularly twice each day for 21 days. 2,6 Dimethoxyplwnyl penicillin-2.5 rag. intramuscularly once each day for 21 days. 2,6 Dimethoxyphenyl penicillin--5.0 mg. intramuscularly once each day for 21 days. 2,6 Dimethoxyphenyl penicillin--5.0 mg. intramnscularly twice each day for 21 days. T h e corticosteroid plus antimicrobial treatment groups were as follows: Hydrocortisone--O.1 mg. intramuscularly once each day for 21 days. Penicillin G--3.0 mg. plus hydrocortisone-O.1 mg. once each day for 21 days.
2,6 Dimethoxyphenyl penicillin--2.5 rag. plus 0.1 mg. of hydrocortisone once each day for 21 days. 2,6 Dimethoxyphenyl penicillin--5.0 mg. plus 0.1 mg. of hydrocortisone once each day for 21 days. 2,6 Dirnethoxyphenyl penicillin-5.0 mg. twice each day and 0.1 rag. hydrocortisone once each day for 21 days. Animals were sacrificed at successive points from 15 minutes after the challenge up to 21 days. T h e microbial census in the kidneys was calculated and plotted logarittmfically. RESULTS
IN VIIRO T h e growth of Staphylococcus aureus (Giorgio) was inhibited by 6.2/.tg./ml. of penicillin G and by 3.1 ptg./ml, of 2,6 dimethoxyphenyl penicillin. IN V~vo
2,6 Dimethoxyphenyl penicillin does not have a striking effect on the microbial population in mouse kidneys. However, there is a definite decrease as compared to the control. In Figure 1 three different dosage schedules are compared to the control and to penicillin G treatment. It is noted that for 24 hours there was multiplication in the kidneys in all groups and the census rose. I n the groups of animals that received 2.5 rag. dimethoxyphenyl penicillin once each day there was a rise in microbial popnlation for three days and then it slowly but steadily fell for the remaining 18 days. In the groups that received twice as much drug (5.0 rag. once each day) there was an initial rise in population. T h e census hovered at this level for about one week and then
0
=
NOr
i Day I
.
9 Dtr penicillin, $.Omgm bid organisms 9 116
214 9 215 ~
&214
9 314 & 314
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!
I I
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7
14 15
21
4
$ Fig. 1. Microbial populations of Staphylococcus aureus (Giorgio) in the kidneys of mice. T h e effect of various dosage schedules of dimethoxyphenyl penicillin as compared to the untreated control and to penicillin G treatment.
]SR
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Vol. II, No. 2 - March,
~[ECtlANISM OF PENICILLIN RESISTANCE
1962
a sustained fall began. In the groups tltat received 5.0 rag. twice a day almost an identical picture was presented. It is of interest tlaat the two points of significant difference between the penicillin G groups and the dimethoxyphenyl penicillin groups are (I) at 24 hours after challenge and (2) at two weeks a n d later after challenge. T h e r e was no difference between tlte population levels of those treated with penicillin G 3.0 mg. once a day and twice a day. Hydrocortisone 0.1 mg. intramuscttlarly each day had no significant effect on the microbial populations as compared to the control. (In the experiments of Batten et al. a the steroid effect was not seen for 30 days and this was with Mycobacteria.) T h e censns rose promptly to a peak at four days and remained elevated for two weeks. After that there was a slight decrease in populations of staphylococci in ttte kidneys of the hydrocortisone group compared to the control. However, both groups exhibited renal abscesses from the secoud day onward. As can be noted from Figure 2, penicillin G 3.0 rag. intramuscularly twice a day did reduce tlle bacterial counts in the kidneys as compared to the control. T h e kidneys also exltibited abscesses. T h e effect of the corticosteroid (hydrocortisone) on the various treatment groups is noted in Figures 3 and 4. In general the steroids did not potentiate tlle action of the antimicrobial but tended rather to interfere with it. In Figure 3 it can be seen that tlle gronp that received din,ethoxyphenyl penicillin alone did better (as measured by lower bacterial popnlations) than did the treatment group which was also on hydrocortisone~ From the second day onward the group of animals that received 5.0 mg. of dimethoxyphenyl penicillin twice a day and no
153
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Fig. 3. Microbial populations of Staphylococcus aureus (Giorgio) in the kidneys of mice. The effect on the kidney bacterial census of hydrocortisone and dimethoxyphenyl penicillin are shown singly and together.
hydrocortisone had consistently lower populations. A further study of the effect of hydrocortisone on various treatment groups again showed that the steroid tended to interfere with the action of the antimicrobial agent. As shown in Figure 4, lower doses of dimethoxyphenyl penicillin were used (2.5 mg. each day and 5.0 mg. each day respectively). In the first week of the experiment there was only one significant difference between any of the groups. This was at day 4 when the groups treated witlt 5.0 mg. of dimethoxypheny] penicillin had populations much lower (Fig. 4) than the same group which received hydrocortisone. After the first week a more pronotmced difference was apparent between tlte hydrocortisone groups and those that received only dimethoxyphenyl penicillin. 8.
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Fig. 2. Microbial poptflations of Staphylococcus attreus (Giorgio) in the kidneys of nfice. The effect of hydrocortisone and penicillin separately is shown.
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Fig. 4. Microbial populations of Staphylococcus attreus (Giorgio) in tile kidneys of mice. Tile effect of various dosage schedules of dimethoxyphenyl penicillin used singly or with hydrocortisone are compared.
154
PETERDINEEN and HENRY ~IANNIX DISCUSSION
T h e results of this experiment shed some light on the mechanism of bacterial resistance to penicillin in vivo. There has always been the question as to how large a part penicillinase plays in this resistance. In previous studies from this laboratory* it has been demonstrated for the first time that penicillin resistant staphylococci do produce penicillinase in vivo. In fact with a model exactly similar to that used in this series of experiments the actual amounts of staphylopenicillinase produced were measured. B u n n et a13 have shown that dimethoxyphenyl .penicillin was not blocked in its antimicrobial activity unless there was an excessive a m o u h t of penicillinase (B. cereus) in the in vitro system. This work has been confirmed by other observers as well. n Therefore, it is reasonable to expect that if penicillinase is the mechanism by which staphylococci are resistant to the action of penicillin and if dimethoxyphenyl penicillin is not blocked by penicillinase (except in very large amounts), then the fall in microbial population in the kidneys should be similar to that of a penicillin susceptible population under penicillin treatment. However, when a penicillin susceptible strain is treated with penicillin G in the animal model described there is a precipitous fall in the bacterial population in the kidneys from the time of first treatment. T h e Organisms are reduced in number to about I0 p e r nfilliliter of kidney tissue in five days. This study has been reported in detail by McCune et al. 8 Wompsett a6 Ires raised a doubt as to the importance of the in vivo production of penicillinase in the determination of penicillin resistance. T h e experiments reported here tend to favor t h i s latter concept. Although some degree of effectiveness was shown by dimethoxyphenyl penicillin (Fig. 1) as compared to penicillin G in this penicillin resistant infection, it did not compare favorably with previous resuhs obtained by penicillin G in a penicillin susceptible infection. This leads to one of two conclusions: (I) Penicillinase is not an important factor in penicillin resistance, or (2) dimethoxyphenyl penicillin is affected seriously by staphylopenicillinase in vivo. Since it is known that the production of penicillinase in vivo reaches a peak about two or three days after the inception of infection, it would follow tlmt the importance of penicillinase in any infection should increase as time progresses. Since dimethoxyphenyl penicillin is least effective (Fig. 1) in the first few days, one must conclude from these facts that dimethoxyphenyl penicillin is not
J S R - Vol. 11, No. 2 - March, 1962
significantly affected by staphylopenicillinase and tlmt resistance to penicillin is much more complex. In this penicillinase is only one facet. Further investigation into the mechanism of penicillin resistance must be done. However, the Staphylococcus aureus (Giorgio) apparently does have another means of resistance to penicillin independent of penicillinase. This is evidcnt because of the lack of strong responses to treatment despite massive doses of dimethoxyphenyl penicillin (5.0 mg. twice a day). Because the rapid excretion of dimethoxyphenyl penicillin might have explained its mediocre action, experiments were done with both once a day and twice a day dosage. There was not a significant difference between these two groups. If resistance to penicillin cannot be wholly explained by penicillinase or by genetic mutation, then a third mechanism must exist. One may only speculate as to what this may be. It seems likely, however, that some other enzyme system must be operative which is less potent than the penicillinase system. This may be intracellular, as comparcd to the apparently extracelhdar position (or at least surface location) of penicillinase. If this is so it may explain the less dramatic action of this substance, but also it would explain why dimethoxyphenyl penicillin is not so effective as one would expect theoretically. T h e effect of corticosteroids in infections-particularly experimental myobacterial infections-has gradually become more clear by virtue of the basic work of Batten et alA The use of corticosteroids (particularly cortisone or hydrocortisone) enhances bacterial multiplication both in vivo and in vitro. While certain factors of host resistance may also be increased, the overall picture both experimentally and clinically is deleterious to the patient unless the infection is also treated with appropriate antimicrobial dn~gs. In the experiments reported here the infection-enhancing properties of hydrocortisone were not completely neutralized by dimethoxyphenyl penicillin. However, the difference in microbial populations between the two groups (Fig. 3) while statistically significant was not considered to be a serious drawback. In this study two questions have been considered. One has been the mechanism of penicillin resistance in vivo. Dimethoxyphenyl penicillin served as a tool to study this. Second, an investigation was made into the efficacy of dimethoxyphenyl penicillin itself, and an evahmtion of it in terms of a well established model infection was undertaken. Based on previous experience
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Vol. II, No. 2 - March, 1962
witll this biologic system, dimethoxyphenyl penicillin has only a moderate effectiveness. It should be employed with these reservations in mind. SUMMARY
Ttle i n v i v o mechanism of penicillin resistance was studied by the use of 2,6 dimethoxyphenyl penicillin. Despite its apparent impunity to staphylopenicillinase 2,6 dimethoxyphenyl penicillin is not as effective against a penicillin resistant staphylococcal infection as penicillin G is against a penicillin susceptible infection. Thus the mechanism of penicillin resistance is more complex than the mere presence of peiaicillinase in the enzyme substrate system. Dinaethoxyphenyl penicillin is only moderately effective in the experimental model as compared to other antimicrobial agents. The use of hydrocortisone concomitantly interferes moderately with the effectiveness of dimethoxyphenyl penicillin. REFERENCES 1. Batten, J. C., and McCune, R. M., Jr.: T h e influence of corticotrophin and certain corticosteroids on populations of Mycobacterium tuberculosis in tissues of mice. Brit. J. Exper. Path., 38:413, 1957. 2. Bunn, P. A., Editor: A symposium on the new dimethoxyphenyl penicillin. Early laboratory and clinical experiences with particular reference to resistant staphylococcal disease. State 9 University of New York Upstate Medical Center, Syracuse, N. Y. September 7, 1960. 3. Cronk, G. A., and Morigi, E. M. E.: Absorption, excretion, and distribution of dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 58 (I960). 4. Dineen, P.: The in vivo production of penlcillinase by hemolytic staphylococcus aureus. J. hmnunol., 86:496, 1961. 5. Dineen, P.: A period of unusual microbial susceptibility in an experimental staphylococcal infection. J. Infect. Dis., 108:174, 1961.
~[ECtlANISM OF PENICILLIN RESISTANCE
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6. Finegold, S. M., Mouzon, O. T., Sweeney, E. E., Dangerfield, H. G., Blackman, B. T., and Hewitt, W. L.: Laboratory experiences with dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 26 (1960). 7. Gourevitch, A., Hunt, G. A., Pursiano, T. A., Moses, A. J., and Lein, J.: Microbiological studies on dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y, p. 4 (1960). 8. McCune, R., Dineen, P., and Batten, J. C.: The influence of antimicrobial agents on total populations of staphylococci in animal tissues. J. Immunol., 85:447, I960. 9. McCune, R. M., Jr., Dineen, P., and Batten, J. C.: The effect of antimicroblal drugs on an experimental staphylococcal infection in mice. Ann. N. Y. Acad. Sc., 65:91, 1956. I0. Pindell, M. H., Tisch, D. E., and Reiffenstein, J. C.: Pharmacological properties of dimethoxyphenyl penicillin. Dimetlmxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 50 (1960). 11. Rifkind, D., and Knight, V.: Treatment of staphylococcal and streptococcal infections with dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 160 (1960). 12. Roberts, C. E., Jr., Allen, J. D., and Kirby, ~V. M. M.: Laboratory and clinical stndies of dimcthoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 109 (1960). 13. Rutenburg. A , Greenberg, It. L., and Schweinburg, F. B.: Clinical experiences with dimethoxyphenyl penicillin in staphylococcal infections. Dimethoxy9 phenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 101 0960). 14. Smith, I. M., and Counts, G. W.." Laboratory and clinical studies of dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposium, State University of New York College of Medicine, Syracuse, N. Y., p. 83 (1960). 15. Steinman, H. G.: Biochemical studies on 6-aminopenicillanic acid, benzyl penicillin, and 2,6-dimethoxyphenyl penicillin. Dimethoxyphenyl Penicillin Symposimn, State University of New York College of Medicine, Syracuse, N. Y., p. 17 (1960). 16. Tompsett, Ralph: Personal communication.