Japan. J. Microbiol. Vol. 19(2), 79-86, 1975
Staphylococcal
Footpad
Infection
in Mice
Ken KATAGIRI,Kenji HORI, Takeshi NISHINO1,andTakashi FUKAO ShionogiResearchLaboratory, Shionogi & Co., Ltd., Osaka (Received for publication, July 11, 1974)
ABSTRACT
Local footpad infection in mouse was investigated with 55 clinically isolated strains of Staphylococcus aureus. When 107 viable cells were inoculated into the footpad, local swelling and bacterial growth resulted after 24 hr. With a dose of 106 cells, moderate swelling was observed after a few hours but the reaction had almost disappeared after 24 hr. About 75% of the staphylococcal strains tested caused footpad edema in mice at doses of 107 cells. Astatistical comparison of the virulence of the organisms on intravenous and intraperitoneal injection with that in inducing footpad swelling is also reported.
In many previous reports [7, 10] on the use of experimental infection with Staphylococcus aureus for chemotherapeutic screening, the Smith strain has been used to induce septicemia in mice. The diffuse type of Smith strain of S. aureus is characterized by its ability to kill mice by intraperitoneal injection of 102 with mucin and 106 without mucin [11]. With almost all strains of S. aureus, a dose of more than 107 cells is necessary to cause fatal septicemia in mice when the organism is injected intraperitoneally together with mucin. Compared with systemic infection, there have been relatively few investigations on local infection by S. aureus for chemotherapeutic screening test. Acred and Brown [1] and Harrison and Fuquay [9] have utilized lesions induced by injecting the organism into the thigh muscle for testing antibiotics; Kitamoto and co-workers [13] have studied the effect of demethylchlortetracycline on abscess formation by mouse skin infection; Requests for reprints should be addressed to Dr. Ken Katagiri, Shionogi Research Laboratory, Shionogi & Co., Ltd., 2-42 Sagisu-kami, Fukushima-ku, Osaka 553, Japan. 1 Present address : Department of Microbiology, Kyoto College of Pharmacy, Kyoto 607. 79
Agarwal [2] has studied the production of skin abscesses with the aid of cotton dust; Sokol and colleagues [21] have used S. aureusinduced rat paw edema for evaluation of the anti-inflammatory activities of some drugs; while Beaumont [4] has used this edema to assess the virulence of S. aureus. We reported on a study of local footpad infection produced by inoculation of clinically isolated strains of S. aureus. The swelling produced was assessed quantitatively, and the virulence of the organisms in this respect was compared with that of their intravenous or intraperitoneal challenge in mice. MATERIALSAND METHODS Mice. Groups of five to ten female mice, 5 weeks old, of the DS strain (obtained from Aburahi Laboratories) weighing 17 to 21 g were used for the experiments. All animals were fed on a cube diet and given drinking water ad libitum. Staphylococci. The 55 strains of Staphylococcusaureus tested in this study were isolated in Shionogi's clinical laboratory between January and December 1970. The strains showed positive reactions in the following tests: the tube coagulase test, the detection of deoxyribonuclease activity (DNase test agar,
80
K.
KATAGIRI
BBL), and the mannitol fermentation test. The organisms were cultured on heart infusion agar for 18 hr at 37 C. They were then suspended in heart infusion broth, the suspensions being adjusted to ca. 109 cells/ml by the turbidity at 655 my and stocked at-78 C until used. In vitro tests. Phage typing patterns. Phage typing was done according to the basic techniques described by Weiss and Nitzkin [22]. S. aureusbacteriophages were kindly supplied by Prof. Dr. Mitsuhashi of Gunma University, Japan. Bacteriophage typing was performed at routine test dilutions (RTD) ; those strains that were untypable were further tested at 100 RTDs. Susceptibility. Minimum inhibitory concentrations (MIC) of several antibiotics for the 55 strains of staphylococci were determined using the plate agar streak method. The drug concentrations tested were according to the twofold dilution method. Final inoculum concentrations of 105 bacteria/ml of broth were employed in these tests. The antibiotics used were: penicillin G (PCG), ampicillin (ABPC), erythromycin (EM), streptomycin (SM), kanamycin (KM), tetracycline (TC) and chloramphenicol (CP). In vivo tests. Pathogenicity studies. (1) Production of edema : Animals were inoculated in the left hind paw under ether anaesthesia with 0.05 ml of a heart infusion broth suspension of S. aureuscontaining 106 to 108 organisms. The degree of edema was determined by measuring the increase in footpad volume using an instrument consisting of a glass cylinder filled with water containing 1% detergent and a phototransistor, as described previously by Hakata and Kurokawa [8]. The degree of footpad edema was represented as follows: ratio
treated control
left
footpad
right
footpad
volume
24 hr after
volume
before
ET AL
days. The effect of intravenous inoculation of staphylococci was examined at the same dose described above. Mortality was recorded on the 4th or 7th day after bacterial challenge. Bacterial counts in the footpad. Bacterial counts were performed at 0, 3, 6, 24 and 48 hr after inoculation. Five mice selected at random were sacrificed and the swelling of the footpad was measured. The footpad was then amputated at the ankle joint and the surface of the skin was sterilized for 3 min with 0.1% sodium hypochloride solution. After drying with ethanol, the footpad was cut off with scissors and homogenized in a mortar with 5 ml of heart infusion broth. The homogenized suspension was centrifuged at 1000 g for 5 min and the supernatant was diluted tenfold with the same broth and counts of viable staphylococci were made on heart infusion agar plate. "Zero-hour" counts were made by killing the animal immediately after inoculation with cocci. Histological examinations. For histological examination, the infected footpad was fixed in 10% formalin solution and after decalcification, sections were stained with hematoxylin and eosin, and examined by light microscopic observation. RESULTS Drug-SusceptibilityIn Vitro Susceptibility of the 55 strains of staphylococci to the antibiotics was shown in Table 1. The vertical lines indicated the upper and lower concentrations of the drugs. Among the 55 strains, 27.3 and 61.8% of the staphylococci showed resistance to SM and TC, respectively, but there was a high rate of sensitivity to KM and CP. Such a distribution of drug susceptibility was considered to follow the general pattern for clinically isolated staphylococci except that there was an unusually large number of strains showing resistance to EM.
infection infection
(2) Virulence in intraperitoneal and intravenous challenge : Each strain of S. aureuswas injected intraperitoneally at a dose of 0.2 ml of a heart infusion broth suspension containing 108 cells without mucin in groups of five mice, which were then observed for 7
Relationship between Phage-Typing Pattern and Virulenceto FootpadInfections The results obtained were shown in Table 2. Thirteen out of the 55 strains of S. aureus tested showed a mixed phage pattern of 80/81 and others. Nineteen strains (34.5%) were nontypable. The relationship between phage
STAPHYLOCOCCAL
Table
Table
2.
1.
Distribution
Distribution
FOOTPAD
of drug susceptibility
of bacteriophage
INFECTION
among
groups and virulence of S. aureus
a
inoculation
55 strains of S. aureus
to footpad
swelling
among
55 strains
b
Fig. 1. a, Bacterial counts staphylococci. The arrow after
81
with
doses
at various times after inoculation indicates the initial inoculum size. of 106 and
107 cells
type and the degree of swelling of the footpad induced was investigated. With phage-type 80/81, no relationship was found between the swelling activity of the bacteria and the phage pattern. Staphylococci with phage-type 80/81 are believed to display considerably high resistance against several antibiotics, and in these experiments eight
in the same
with doses of 106 and 107 cells of b, Time course of footpad swelling
experiment
as a.
out of 13 strains of staphylococci showing a mixed phage pattern of 80/81 and others had multiple resistance against various antibiotics tested. RelationshipbetweenInoculumSize and Swelling In order to determine the dose of inoculum needed to produce swelling, S. aureus C-74
82
K.
KATAGIRI
suspensions were diluted tenfold and each dilution was inoculated into mouse footpads. Bacterial counts in the footpad were performed after bacterial inoculation. The results are shown in Fig. 1 a and b. To induce swelling, which was maintained at a significant level after 24 hr, the inoculum had to contain more than 107 cells per 0.05 ml. The degree of swelling induced by the dose of 107 viable cells was 1.7 to 2.0 times the normal size after 24 hr, and slightly greater on the following day. Thereafter, the footpad lesion progressed to abscess and/or necrosis. More severe swelling was produced by an inoculum of 108viable cells. Although a dose of 106 cells induced moderate swelling lasting for 3 hr, the footpad returned to almost normal size by 24 hr. A cell-free filtrate of broth in which staphylococci had been cultured overnight did not produce footpad swelling, nor did a heat-killed cell suspension in a dose of 107 cells elicit any response. We attempted to see if there was any difference in the swelling caused by injection in the plantar region and that by injection into the dorsum of the mouse hind paw. As can be seen in Table 3, the degrees of swelling were almost the same regardless of the site of injection, using subcutaneous injection doses of 107 cells each of ten strains of S. aureus. Bacterial counts performed at 0 (immediately after inoculation), 3, 6, 24 and 48 hr Table 3. Comparison of the footpad swellings caused by injection into the plantar and dorsal regions
ET AL
after inoculation exhibited an initial lag period after which there was a rapid increase in the number of organisms recovered from the lesion at 3 to 6 hr with both inocula of 106 and 107 cells per footpad. Twentyfour hours after injection, however, the counts of viable cocci in the footpad given a dose of 107cells reached a level of more than 107 cells per footpad, while in that given a dose of 106 cells, the number of viable organisms decreased to about 105 cells. The results obtained indicate that the pattern of edema formation induced by S. aureus follows the growth of the organisms. In addition, blood samples were collected aseptically by cardiac puncture and poured onto agar plates, but no organism was present. HistologicalInvestigationof the FootpadLesion As shown in Fig.2 a and b, marked edema formation and cell infiltration in the cutis were observed 24 hr after inoculation with a dose of 107 cells of S. aureus C-74 compared with the normal footpad. Distribution of Swelling-FormingVirulencein the 55 Strains of S. aureus According to the ratio of the size of the swollen footpad to that of the contralateral noninjected footpad, the virulence of each strain of S. aureus was scored as follows: score 1, the ratio was less than 1.2; score 2, 1.3 to 1.4; score 3, 1.5 to 1.6; and score 4, greater than 1.7. The ratio of increase taken for each strain was the mean of the values in five mice. The results obtained showed that 72.2% of the clinically isolated strains had pathogenicity for mice, taking a virulence score of greater than 2 as the criterion (Table 4). However, only six strains of S. aureus (10.9%) could be considered to have high pathogenic activity. RelationshipbetweenFootpadSwellingActivityand Pathogenicityon Intravenousor Intraperitoneal Injection The mortality on the 4th day produced by intravenous injection of each of the 55 strains was compared with the footpad swelling activity. The results were shown in Table 5. A computer was used to analyze the data to
STAPHYLOCOCCAL
FOOTPAD
INFECTION
83
a
b Fig.
2.
determine the
a, Infected
whether
footpad
the
calculated
at P=0.01, correlation
tissue
inoculated
showed
differed
from
by the
intravenous challenge of The coefficient of correwas
indicating between
footpad
virulence
swelling
pathogenicity on the staphylococci. lation
mouse
r=0.4668, that
them.
there
significant was
a high
with
S. aureus.
b, Normal
mouse
footpad
tissue.
A similar result was obtained from the data of mortality on the 7th day after intraperitoneal challenge with each strain of S. aureus, a correlation (r=0.3822) being seen between the effect of intraperitoneal injection and the virulence in causing footpad swelling (Table 6).
K. KATAGIRI
84
DISCUSSION Chemotherapeutic screening in vivoagainst S. aureus has mainly been carried out by inducing septicemia in mice by intraperitoneal injection of the organism together with mucin. The role of mucin in the mechanism of this experimental model is still unknown. The technique of using the pathogenicity of staphylococci to produce subcutaneous infection in mice was previously described by Noble [16]. He reported that the virulence of the strains in mice was significantly related to their ability to produce epidemic infections in man, as judged by abscess formation when a dose of about 106 viable organisms was Table
4. on
Distribution the footpad
Table
5.
of virulence of S. aureus infection in mice
Correlation
of pathogenicity
ET AL
injected subcutaneously on a plug of cotton dust. Since then, studies on abscess production by subcutaneous inoculation of staphylococci by Noble's technique have been developed by Foster [6] and Agarwal [3]. Igarashi and his colleagues [12] also studied the abscess formation induced with S. aureus. These authors found 104 cells could produce abscesses in the subcutis of the thigh of mice when 1% Si02 was added to the suspension. These authors used the severity of the lesion as a criterion for scoring on an arbitrary scale. However, since such scoring criterion is subjective, it would seem to be unsuitable for quantitative evaluation. In both experiments using septicemia and abscess formation in the skin, a foreign body such as mucin or cotton dust is needed together with organisms for the production of infection. Since Shepard [19] used subcutaneous footpad inoculation of viable bacteria to study human leprosy bacilli, many authors have used this technique to study various organisms, e.g., Mycobacterium tuberculosisand Mycobacteriumavium [15], five species of mycobacteria includedMycobacterium kansasii, etc. [14] and Cryptococcus neoformans[17] . Concerning S. aureus footpad infections, Sokol and his colleagues [21] have investi-
of S. aureus
in mice
in two
experimental
conditions
of S. aureus
in mice
in two
experimental
conditions
r=0.4668 (significant at P=0.01). Table
6.
r=0.3822
Correlation
of pathogenicity
(significant at P=0.01).
STAPHYLOCOCCAL
FOOTPAD
INFECTION
85
there was a good correlation between footpad gated the activities of some anti-inflammatory agents against rat paw edema elicited by the swelling and mortality in septicemia induced organisms, while more recently the patterns with S. aureus. of footpad swelling caused in mice by S. aureus Mortality on the 7th day of septicemia have been described [4], and the virulence of induced by intraperitoneal challenge of each the organisms has been tested by this method strain of staphylococci showed a significant with a dose of 107 viable cells in rats [18]. correlation (r =0.3719) with that on the 4th Prompted by these studies, we used this day, though not with that on the 7th day method to investigate the virulence of 55 (r = 0.1978), of septicemia by intravenous clinically isolated strains of S. aureus. In our challenge. From these results, it is conexperiments, the inoculum size used to ceivable that the cause of death on the 7th produce swelling of mouse footpad, a dose of day of septicemia by intravenous injection 107 cells of staphylococci, is considerably was different from that on the 7th day of larger than that used as a model for experisepticemia by intraperitoneal challenge, and mental local infection by the previous that the animal succumbed owing to pathoauthors. When this technique was applied genic changes of kidney function in the to the footpad of rats, a similar swelling was former case. observed with a dose of 108 cells of staphyloFurthermore, as this method of footpad cocci. infection affords quantitative data with good In comparison with the all-or-none rereproducibility, it may be used to screen sponse of mortality in septicemia, footpad chemotherapeutic activity against clinically swelling can be measured quantitatively by isolated strains of S. aureus. volume, thickness, or edema weight change. ACKNOWLEDGMENT About 75% of the strains of staphylococci The authors expresstheir thanks to Dr. Katsuya used had virulence in edema formation in the Tawara of their laboratoriesfor preparationof the mouse footpad at a dose of 107 cells. In photographs. contrast, only 27.3% of the 55 strains of REFERENCES staphylococci caused 100% mortality in septicemia by intraperitoneal or intravenous [ 1] Acred, P., and Brown,D. M. 1963. Further challenge at a dose of 108 viable cells. On pharmacologyand chemotherapyof cloxacillin. Brit.J. Pharmacol.21: 339-354. the other hand, previous work has shown that [ 2 ] Agarwal,D. S. 1967. Subcutaneousstaphyloan inoculum of 106viable cells was necessary coccalinfectionin mice.I. The roleof cotton-dust to produce abscess or pus formation in normal in enhancinginfection.Brit.J. Exp. Pathol. 48: human skin [20] or in human skin having 436-449. superficial scratches or incisions [5]. Con[ 3 ] Agarwal,D. S. 1967. Subcutaneousstaphylococcal infectionin mice. II. The inflammatory sequently, it seems reasonable to suppose responseto differentstrains of staphylococciand that susceptibility of the human skin to the micrococci.Brit.J. Exp. Pathol.48: 468-482. virulence of staphylococci is very similar to [ 4 ] Beaumont,R. J. 1970.In-vivo experimentswith that of the mouse footpad. ethylenediamine-tetraacetic acid and investigaAlthough the work of these previous tionsinto its actionof penicillin-resistant Staphylococcus aureus.Med.J. Austral.2: 1017-1020. authors greatly advanced our understanding [ 5 ] Elek,S. D., and Conen, P. E. 1957.The viruof the mechanisms responsible for the patholenceof Staphylococcus pyogenes for man. A study of genicity of S. aureus in the mouse, there is the problemsof wound infection.Brit. J. Exp. still much undefined in this test and a lack of Pathol.38: 573-586. information about the correlation between [ 6 ] Foster,W. D. 1971.The relationshipbetween the pathogenicityof Staphylococcus aureusfor the the process of edema formation and the mouse and its pathogenicityfor man and the pattern of bacterial growth. Accordingly, guineapig. Brit.J. Exp. Pathol.52: 307-314. we compared the virulence of footpad infec[ 7 ] Grunberg,E., Berger,J., Beskid,G., Cleeland, tion with that of intraperitoneal and intraR., Prince,H. N., and Titsworth,E. 1967.Studies on the in vitroand in vivochemotherapeutic provenous challenge for 55 strains of S. aureus. perties of the antibioticmyxin. Chemotherapia Our results showed that though virulence in 12: 272-281. causing footpad swelling did not necessarily [ 8 ] Hakata, H., and Kurokawa,K. (Assignersto agree with that in septicemia in the mouse, Shionogi& Co., Ltd.) 1969.A plethysmographic
86
K.
KATAGIRI
apparatus. Japanese Utility Model Registration No. 897,907. (in Japanese) [ 9 1 Harrison, E. F., and Fuquay, M. E. 1964. Evaluation of chemotherapeutic agents as treatment for localized staphylococcal infections in mice. Antimicrob. Agents Chemotherapy 764-766. [10] Holper, J. C., Grundy, W. E., and Sylvester, J. C. 1959. A comparison of the in vitro to in vzvo activity of ristocetin, penicillin, and erythromycin. Antibiotics Ann. 1958-1959: 425-427. [11] Hunt, G. A., and Moses, A. J. 1958. Acute infection of mice with Smith strain of Staphylococcus aureus. Science 128: 1574-1575. [12] Igarashi, I., Hattori, Z., Misawa, H., Fukuda, K., and Okonogi, T. 1965. Experimental infection of Staphylococcus.I. Formation of subcutaneous abscesses in mice. Ann. Sankyo Res. Lab. 17: 148-153. [13] Kitamoto, O., Fukaya, K., Wajima, T., Tadokoro, I., and Morita, S. 1965. Studies on demethylchlortetracycline. Chemotherapy 13: 443-447. [14] Kubica, G. P., Dunbar, F. P., and Kim, T. H. 1973. Response of hypersensitive mice to the footpad injection of living homologous or heterologous mycobacteria : Preliminary report. Appl. Microbiol. 25: 718-723.
ET AL
[15]
Nakamura, M. 1968. Mouse footpad swelling phenomenon by inoculation of mycobacteria. Amer. Rev. Resp. Dis. 97: 24-31. [16] Noble, W. C. 1966. Virulence and the biochemical characters of staphylococci. J. Pathol. Bacteria 91: 181-193. [17] Perceval, A. K. 1965. Experimental cryptococcosis: hypersensitivity and immunity. J. Pathol. Bacteriol. 89: 645-655. [18] Rotter, M., and Stoklaska, E. 1971. Eine einfache Methode zur Virulenz-bestimmung entziindungserzeugender Bakterien. Zbl. Bakt. I. Abt. Orig. 216: 517-526. [19] Shepard, C. C. 1960. The experimental disease that follows the injection of human leprosy bacilli into footpads of mice. J. Exp. Med. 112: 445-454. [20] Singh, G., Marples, R. R., and Kligman, A. M. 1971. Experimental Staphylococcusaureus infections in humans. J. Invest. Dem atol. 57: 149-162. [21] Sokol, G. H., Miller, F. P., and Maickel, R. P. 1968. Studies on rat paw edema induced by S. aureus. Arch. Int. Pharmacodyn. 173: 382-385. [22] Weiss, D. L., and Nitzkin, P. 1969. Phage lysis of Staphylococcusaureus exposed to acridine orange. Amer. J. Clin. Pathol. 51: 379-383.
Staphylococcal
Footpad
Infection
in Mice
Ken KATAGIRI,Kenji HORI, Takeshi NISHINO1,andTakashi FUKAO ShionogiResearchLaboratory, Shionogi & Co., Ltd., Osaka (Received for publication, July 11, 1974)
ABSTRACT
Local footpad infection in mouse was investigated with 55 clinically isolated strains of Staphylococcus aureus. When 107 viable cells were inoculated into the footpad, local swelling and bacterial growth resulted after 24 hr. With a dose of 106 cells, moderate swelling was observed after a few hours but the reaction had almost disappeared after 24 hr. About 75% of the staphylococcal strains tested caused footpad edema in mice at doses of 107 cells. Astatistical comparison of the virulence of the organisms on intravenous and intraperitoneal injection with that in inducing footpad swelling is also reported.
In many previous reports [7, 10] on the use of experimental infection with Staphylococcus aureus for chemotherapeutic screening, the Smith strain has been used to induce septicemia in mice. The diffuse type of Smith strain of S. aureus is characterized by its ability to kill mice by intraperitoneal injection of 102 with mucin and 106 without mucin [11]. With almost all strains of S. aureus, a dose of more than 107 cells is necessary to cause fatal septicemia in mice when the organism is injected intraperitoneally together with mucin. Compared with systemic infection, there have been relatively few investigations on local infection by S. aureus for chemotherapeutic screening test. Acred and Brown [1] and Harrison and Fuquay [9] have utilized lesions induced by injecting the organism into the thigh muscle for testing antibiotics; Kitamoto and co-workers [13] have studied the effect of demethylchlortetracycline on abscess formation by mouse skin infection; Requests for reprints should be addressed to Dr. Ken Katagiri, Shionogi Research Laboratory, Shionogi & Co., Ltd., 2-42 Sagisu-kami, Fukushima-ku, Osaka 553, Japan. 1 Present address : Department of Microbiology, Kyoto College of Pharmacy, Kyoto 607. 79
Agarwal [2] has studied the production of skin abscesses with the aid of cotton dust; Sokol and colleagues [21] have used S. aureusinduced rat paw edema for evaluation of the anti-inflammatory activities of some drugs; while Beaumont [4] has used this edema to assess the virulence of S. aureus. We reported on a study of local footpad infection produced by inoculation of clinically isolated strains of S. aureus. The swelling produced was assessed quantitatively, and the virulence of the organisms in this respect was compared with that of their intravenous or intraperitoneal challenge in mice. MATERIALSAND METHODS Mice. Groups of five to ten female mice, 5 weeks old, of the DS strain (obtained from Aburahi Laboratories) weighing 17 to 21 g were used for the experiments. All animals were fed on a cube diet and given drinking water ad libitum. Staphylococci. The 55 strains of Staphylococcusaureus tested in this study were isolated in Shionogi's clinical laboratory between January and December 1970. The strains showed positive reactions in the following tests: the tube coagulase test, the detection of deoxyribonuclease activity (DNase test agar,
80
K.
KATAGIRI
BBL), and the mannitol fermentation test. The organisms were cultured on heart infusion agar for 18 hr at 37 C. They were then suspended in heart infusion broth, the suspensions being adjusted to ca. 109 cells/ml by the turbidity at 655 my and stocked at-78 C until used. In vitro tests. Phage typing patterns. Phage typing was done according to the basic techniques described by Weiss and Nitzkin [22]. S. aureusbacteriophages were kindly supplied by Prof. Dr. Mitsuhashi of Gunma University, Japan. Bacteriophage typing was performed at routine test dilutions (RTD) ; those strains that were untypable were further tested at 100 RTDs. Susceptibility. Minimum inhibitory concentrations (MIC) of several antibiotics for the 55 strains of staphylococci were determined using the plate agar streak method. The drug concentrations tested were according to the twofold dilution method. Final inoculum concentrations of 105 bacteria/ml of broth were employed in these tests. The antibiotics used were: penicillin G (PCG), ampicillin (ABPC), erythromycin (EM), streptomycin (SM), kanamycin (KM), tetracycline (TC) and chloramphenicol (CP). In vivo tests. Pathogenicity studies. (1) Production of edema : Animals were inoculated in the left hind paw under ether anaesthesia with 0.05 ml of a heart infusion broth suspension of S. aureuscontaining 106 to 108 organisms. The degree of edema was determined by measuring the increase in footpad volume using an instrument consisting of a glass cylinder filled with water containing 1% detergent and a phototransistor, as described previously by Hakata and Kurokawa [8]. The degree of footpad edema was represented as follows: ratio
treated control
left
footpad
right
footpad
volume
24 hr after
volume
before
ET AL
days. The effect of intravenous inoculation of staphylococci was examined at the same dose described above. Mortality was recorded on the 4th or 7th day after bacterial challenge. Bacterial counts in the footpad. Bacterial counts were performed at 0, 3, 6, 24 and 48 hr after inoculation. Five mice selected at random were sacrificed and the swelling of the footpad was measured. The footpad was then amputated at the ankle joint and the surface of the skin was sterilized for 3 min with 0.1% sodium hypochloride solution. After drying with ethanol, the footpad was cut off with scissors and homogenized in a mortar with 5 ml of heart infusion broth. The homogenized suspension was centrifuged at 1000 g for 5 min and the supernatant was diluted tenfold with the same broth and counts of viable staphylococci were made on heart infusion agar plate. "Zero-hour" counts were made by killing the animal immediately after inoculation with cocci. Histological examinations. For histological examination, the infected footpad was fixed in 10% formalin solution and after decalcification, sections were stained with hematoxylin and eosin, and examined by light microscopic observation. RESULTS Drug-SusceptibilityIn Vitro Susceptibility of the 55 strains of staphylococci to the antibiotics was shown in Table 1. The vertical lines indicated the upper and lower concentrations of the drugs. Among the 55 strains, 27.3 and 61.8% of the staphylococci showed resistance to SM and TC, respectively, but there was a high rate of sensitivity to KM and CP. Such a distribution of drug susceptibility was considered to follow the general pattern for clinically isolated staphylococci except that there was an unusually large number of strains showing resistance to EM.
infection infection
(2) Virulence in intraperitoneal and intravenous challenge : Each strain of S. aureuswas injected intraperitoneally at a dose of 0.2 ml of a heart infusion broth suspension containing 108 cells without mucin in groups of five mice, which were then observed for 7
Relationship between Phage-Typing Pattern and Virulenceto FootpadInfections The results obtained were shown in Table 2. Thirteen out of the 55 strains of S. aureus tested showed a mixed phage pattern of 80/81 and others. Nineteen strains (34.5%) were nontypable. The relationship between phage
STAPHYLOCOCCAL
Table
Table
2.
1.
Distribution
Distribution
FOOTPAD
of drug susceptibility
of bacteriophage
INFECTION
among
groups and virulence of S. aureus
a
inoculation
55 strains of S. aureus
to footpad
swelling
among
55 strains
b
Fig. 1. a, Bacterial counts staphylococci. The arrow after
81
with
doses
at various times after inoculation indicates the initial inoculum size. of 106 and
107 cells
type and the degree of swelling of the footpad induced was investigated. With phage-type 80/81, no relationship was found between the swelling activity of the bacteria and the phage pattern. Staphylococci with phage-type 80/81 are believed to display considerably high resistance against several antibiotics, and in these experiments eight
in the same
with doses of 106 and 107 cells of b, Time course of footpad swelling
experiment
as a.
out of 13 strains of staphylococci showing a mixed phage pattern of 80/81 and others had multiple resistance against various antibiotics tested. RelationshipbetweenInoculumSize and Swelling In order to determine the dose of inoculum needed to produce swelling, S. aureus C-74
82
K.
KATAGIRI
suspensions were diluted tenfold and each dilution was inoculated into mouse footpads. Bacterial counts in the footpad were performed after bacterial inoculation. The results are shown in Fig. 1 a and b. To induce swelling, which was maintained at a significant level after 24 hr, the inoculum had to contain more than 107 cells per 0.05 ml. The degree of swelling induced by the dose of 107 viable cells was 1.7 to 2.0 times the normal size after 24 hr, and slightly greater on the following day. Thereafter, the footpad lesion progressed to abscess and/or necrosis. More severe swelling was produced by an inoculum of 108viable cells. Although a dose of 106 cells induced moderate swelling lasting for 3 hr, the footpad returned to almost normal size by 24 hr. A cell-free filtrate of broth in which staphylococci had been cultured overnight did not produce footpad swelling, nor did a heat-killed cell suspension in a dose of 107 cells elicit any response. We attempted to see if there was any difference in the swelling caused by injection in the plantar region and that by injection into the dorsum of the mouse hind paw. As can be seen in Table 3, the degrees of swelling were almost the same regardless of the site of injection, using subcutaneous injection doses of 107 cells each of ten strains of S. aureus. Bacterial counts performed at 0 (immediately after inoculation), 3, 6, 24 and 48 hr Table 3. Comparison of the footpad swellings caused by injection into the plantar and dorsal regions
ET AL
after inoculation exhibited an initial lag period after which there was a rapid increase in the number of organisms recovered from the lesion at 3 to 6 hr with both inocula of 106 and 107 cells per footpad. Twentyfour hours after injection, however, the counts of viable cocci in the footpad given a dose of 107cells reached a level of more than 107 cells per footpad, while in that given a dose of 106 cells, the number of viable organisms decreased to about 105 cells. The results obtained indicate that the pattern of edema formation induced by S. aureus follows the growth of the organisms. In addition, blood samples were collected aseptically by cardiac puncture and poured onto agar plates, but no organism was present. HistologicalInvestigationof the FootpadLesion As shown in Fig.2 a and b, marked edema formation and cell infiltration in the cutis were observed 24 hr after inoculation with a dose of 107 cells of S. aureus C-74 compared with the normal footpad. Distribution of Swelling-FormingVirulencein the 55 Strains of S. aureus According to the ratio of the size of the swollen footpad to that of the contralateral noninjected footpad, the virulence of each strain of S. aureus was scored as follows: score 1, the ratio was less than 1.2; score 2, 1.3 to 1.4; score 3, 1.5 to 1.6; and score 4, greater than 1.7. The ratio of increase taken for each strain was the mean of the values in five mice. The results obtained showed that 72.2% of the clinically isolated strains had pathogenicity for mice, taking a virulence score of greater than 2 as the criterion (Table 4). However, only six strains of S. aureus (10.9%) could be considered to have high pathogenic activity. RelationshipbetweenFootpadSwellingActivityand Pathogenicityon Intravenousor Intraperitoneal Injection The mortality on the 4th day produced by intravenous injection of each of the 55 strains was compared with the footpad swelling activity. The results were shown in Table 5. A computer was used to analyze the data to
STAPHYLOCOCCAL
FOOTPAD
INFECTION
83
a
b Fig.
2.
determine the
a, Infected
whether
footpad
the
calculated
at P=0.01, correlation
tissue
inoculated
showed
differed
from
by the
intravenous challenge of The coefficient of correwas
indicating between
footpad
virulence
swelling
pathogenicity on the staphylococci. lation
mouse
r=0.4668, that
them.
there
significant was
a high
with
S. aureus.
b, Normal
mouse
footpad
tissue.
A similar result was obtained from the data of mortality on the 7th day after intraperitoneal challenge with each strain of S. aureus, a correlation (r=0.3822) being seen between the effect of intraperitoneal injection and the virulence in causing footpad swelling (Table 6).
K. KATAGIRI
84
DISCUSSION Chemotherapeutic screening in vivoagainst S. aureus has mainly been carried out by inducing septicemia in mice by intraperitoneal injection of the organism together with mucin. The role of mucin in the mechanism of this experimental model is still unknown. The technique of using the pathogenicity of staphylococci to produce subcutaneous infection in mice was previously described by Noble [16]. He reported that the virulence of the strains in mice was significantly related to their ability to produce epidemic infections in man, as judged by abscess formation when a dose of about 106 viable organisms was Table
4. on
Distribution the footpad
Table
5.
of virulence of S. aureus infection in mice
Correlation
of pathogenicity
ET AL
injected subcutaneously on a plug of cotton dust. Since then, studies on abscess production by subcutaneous inoculation of staphylococci by Noble's technique have been developed by Foster [6] and Agarwal [3]. Igarashi and his colleagues [12] also studied the abscess formation induced with S. aureus. These authors found 104 cells could produce abscesses in the subcutis of the thigh of mice when 1% Si02 was added to the suspension. These authors used the severity of the lesion as a criterion for scoring on an arbitrary scale. However, since such scoring criterion is subjective, it would seem to be unsuitable for quantitative evaluation. In both experiments using septicemia and abscess formation in the skin, a foreign body such as mucin or cotton dust is needed together with organisms for the production of infection. Since Shepard [19] used subcutaneous footpad inoculation of viable bacteria to study human leprosy bacilli, many authors have used this technique to study various organisms, e.g., Mycobacterium tuberculosisand Mycobacteriumavium [15], five species of mycobacteria includedMycobacterium kansasii, etc. [14] and Cryptococcus neoformans[17] . Concerning S. aureus footpad infections, Sokol and his colleagues [21] have investi-
of S. aureus
in mice
in two
experimental
conditions
of S. aureus
in mice
in two
experimental
conditions
r=0.4668 (significant at P=0.01). Table
6.
r=0.3822
Correlation
of pathogenicity
(significant at P=0.01).
STAPHYLOCOCCAL
FOOTPAD
INFECTION
85
there was a good correlation between footpad gated the activities of some anti-inflammatory agents against rat paw edema elicited by the swelling and mortality in septicemia induced organisms, while more recently the patterns with S. aureus. of footpad swelling caused in mice by S. aureus Mortality on the 7th day of septicemia have been described [4], and the virulence of induced by intraperitoneal challenge of each the organisms has been tested by this method strain of staphylococci showed a significant with a dose of 107 viable cells in rats [18]. correlation (r =0.3719) with that on the 4th Prompted by these studies, we used this day, though not with that on the 7th day method to investigate the virulence of 55 (r = 0.1978), of septicemia by intravenous clinically isolated strains of S. aureus. In our challenge. From these results, it is conexperiments, the inoculum size used to ceivable that the cause of death on the 7th produce swelling of mouse footpad, a dose of day of septicemia by intravenous injection 107 cells of staphylococci, is considerably was different from that on the 7th day of larger than that used as a model for experisepticemia by intraperitoneal challenge, and mental local infection by the previous that the animal succumbed owing to pathoauthors. When this technique was applied genic changes of kidney function in the to the footpad of rats, a similar swelling was former case. observed with a dose of 108 cells of staphyloFurthermore, as this method of footpad cocci. infection affords quantitative data with good In comparison with the all-or-none rereproducibility, it may be used to screen sponse of mortality in septicemia, footpad chemotherapeutic activity against clinically swelling can be measured quantitatively by isolated strains of S. aureus. volume, thickness, or edema weight change. ACKNOWLEDGMENT About 75% of the strains of staphylococci The authors expresstheir thanks to Dr. Katsuya used had virulence in edema formation in the Tawara of their laboratoriesfor preparationof the mouse footpad at a dose of 107 cells. In photographs. contrast, only 27.3% of the 55 strains of REFERENCES staphylococci caused 100% mortality in septicemia by intraperitoneal or intravenous [ 1] Acred, P., and Brown,D. M. 1963. Further challenge at a dose of 108 viable cells. On pharmacologyand chemotherapyof cloxacillin. Brit.J. Pharmacol.21: 339-354. the other hand, previous work has shown that [ 2 ] Agarwal,D. S. 1967. Subcutaneousstaphyloan inoculum of 106viable cells was necessary coccalinfectionin mice.I. The roleof cotton-dust to produce abscess or pus formation in normal in enhancinginfection.Brit.J. Exp. Pathol. 48: human skin [20] or in human skin having 436-449. superficial scratches or incisions [5]. Con[ 3 ] Agarwal,D. S. 1967. Subcutaneousstaphylococcal infectionin mice. II. The inflammatory sequently, it seems reasonable to suppose responseto differentstrains of staphylococciand that susceptibility of the human skin to the micrococci.Brit.J. Exp. Pathol.48: 468-482. virulence of staphylococci is very similar to [ 4 ] Beaumont,R. J. 1970.In-vivo experimentswith that of the mouse footpad. ethylenediamine-tetraacetic acid and investigaAlthough the work of these previous tionsinto its actionof penicillin-resistant Staphylococcus aureus.Med.J. Austral.2: 1017-1020. authors greatly advanced our understanding [ 5 ] Elek,S. D., and Conen, P. E. 1957.The viruof the mechanisms responsible for the patholenceof Staphylococcus pyogenes for man. A study of genicity of S. aureus in the mouse, there is the problemsof wound infection.Brit. J. Exp. still much undefined in this test and a lack of Pathol.38: 573-586. information about the correlation between [ 6 ] Foster,W. D. 1971.The relationshipbetween the pathogenicityof Staphylococcus aureusfor the the process of edema formation and the mouse and its pathogenicityfor man and the pattern of bacterial growth. Accordingly, guineapig. Brit.J. Exp. Pathol.52: 307-314. we compared the virulence of footpad infec[ 7 ] Grunberg,E., Berger,J., Beskid,G., Cleeland, tion with that of intraperitoneal and intraR., Prince,H. N., and Titsworth,E. 1967.Studies on the in vitroand in vivochemotherapeutic provenous challenge for 55 strains of S. aureus. perties of the antibioticmyxin. Chemotherapia Our results showed that though virulence in 12: 272-281. causing footpad swelling did not necessarily [ 8 ] Hakata, H., and Kurokawa,K. (Assignersto agree with that in septicemia in the mouse, Shionogi& Co., Ltd.) 1969.A plethysmographic
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K.
KATAGIRI
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ET AL
[15]
Nakamura, M. 1968. Mouse footpad swelling phenomenon by inoculation of mycobacteria. Amer. Rev. Resp. Dis. 97: 24-31. [16] Noble, W. C. 1966. Virulence and the biochemical characters of staphylococci. J. Pathol. Bacteria 91: 181-193. [17] Perceval, A. K. 1965. Experimental cryptococcosis: hypersensitivity and immunity. J. Pathol. Bacteriol. 89: 645-655. [18] Rotter, M., and Stoklaska, E. 1971. Eine einfache Methode zur Virulenz-bestimmung entziindungserzeugender Bakterien. Zbl. Bakt. I. Abt. Orig. 216: 517-526. [19] Shepard, C. C. 1960. The experimental disease that follows the injection of human leprosy bacilli into footpads of mice. J. Exp. Med. 112: 445-454. [20] Singh, G., Marples, R. R., and Kligman, A. M. 1971. Experimental Staphylococcusaureus infections in humans. J. Invest. Dem atol. 57: 149-162. [21] Sokol, G. H., Miller, F. P., and Maickel, R. P. 1968. Studies on rat paw edema induced by S. aureus. Arch. Int. Pharmacodyn. 173: 382-385. [22] Weiss, D. L., and Nitzkin, P. 1969. Phage lysis of Staphylococcusaureus exposed to acridine orange. Amer. J. Clin. Pathol. 51: 379-383.
