Med. Microbiol. Immunol. 167, 3 7 4 4 (1979)
~) by Springer-Vedag 91979
Phagocytosis and Intracellular Killing of Coagulase-Negative Staphylococci F. Namavar 1 , J. de Graaff 2 , and D.M. MacLarenl Laboratory of Medical Microbiology 1 and Department of Oral Microbiology 2, Free University, Amsterdam, The Netherlands
Abstract. The phagocytosis and intracellular killing of different coagulasenegative staphylococcal species by polymorphonuclear (PMN) leukocytes from one healthy donor were compared. The uptake of strains belonging to a given species varied from 60 to 80% with an average of 70% after 20 min incubation at 37~ Up to 95% of intracellular bacteria were killed after 10 rain. There was no correlation between uptake capacity and species or biotype. The average rates of phagocytosis and killing strains whether isolated from urinary tract infections or from the skin were virtually the same. Introduction The predominance of certain biotypes o f coagulase-negative Micrococcacaeae in human infections has naturally raised the question whether these are more virulent. S. epidermis biotype 2 predominates in infections associated with atrio-ventricular shunts or artificial heart valves (Holt, 1969). S. sapropbyticus biotype 3 (Sellin et al., 1975; Gillespie et al., 1978) has been shown to be the predominant biotype in acute staphylococcal urinary infections in young women. Very little is known about the pathogenetic mechanisms of coagulase-negative staphylococci, except that a foreign body seems often to provide the niche in which the infection can be established (Holt, 1969). In previous studies (Namavar et al., 1975, 1978) certain biotypes of S. epidermidis and S. sapbropbyticus gave lower LDS0 values after intracerebral inoculation into neonatal mice. Extrapolation from animal studies to the human situation is difficult, but these LDS0 results suggested some differences in virulence. A study of resistance to phagocytosis and intracellular killing, processes of vital importance in the body's defense system, might point to biotypes with potentially enhanced virulence. Such a study might also shed more light on the vexed question of classification. In our previous study (Namavar et al., in press) we found marked heterogeneity when we reclassified BairdParker biotypes (1974) by the Kloos and Schleifer (1975) criteria. The behavior of Address for offprint requests: F. Namavar, Laboratory of Medical Microbiology, Free University, van der Boechorststraat 7, 1007 MC Amsterdam, The Netherlands 0300-8584/79/0167/0037/~gO1.60
38
F. Namavar et al.
biotypes in phagocytosis and leukocyte killing experiments might offer further evidence in assessing the status of the numerous species described by Kloos and Sehleifer. The present study was planned with these objectives in view. Materials and Methods
Bacterial Strains: Coagulase-negative staphylococcal strains were isolated from the healthy human skin and from urinary tract infections. Strains were classified according to the Baird-Parker (1974) and the Kloos and Schliefer schemes (1975). Novobiocin sensitivity was determined by the method described by Mitchell (1968).
Preparation 01'Bacteria: Bacterial suspensions were prepared by adding 0.1 ml of an overnight culture of 10 ml Lab-Lemco broth (Oxoid) containing 2 tzCi of (methyl-3H) thymidine (specific activity 40-60 Ci/mmol; Radiochemical centre, Amersham, UK). After 17 h incubation at 37~ bacteria were washed three times in phosphate-buffered saline (PBS), pH 7.4, and resuspended in 1 ml PBS. Counts of viable bacteria were performed in nutrient agar plates and the bacterial suspension was stored overnight at 4~ When on the following day the viable counts were known the final bacterial concentration was adjusted to give 1 x 108 colony-forming units (CFU)/ml.
Preparation of Polymorpbonuclear (PMN) Leukocytes: Human PMN leukocytes were isolated from fresh defibrinated blood as described by Weening et al. (1974). In every experiment approximately 30 ml blood from the same donor was used. The cells were counted electronically in a Coulter counter (Model ZB1) and the leukocyte pellet was adjusted to a final concentration of 5 x 106 cells/ml of PBS containing 0.5% (w/v) human albumin.
Quantitation of Bacterial Uptake: Phagocytosis mixtures were prepared with 1.2 ml of PMN leukocyte suspension and 0.6 ml of 3H-thymidine-labeled bacterial suspension in plastic tubes (15 x 95 mm; Thoradeo, NewKoop, Netherlands) to give a leukocyte:bacteria ratio of 1:10. Bacterial opsonization was performed by adding normal undiluted serum from the donor at a final concentration of 10% (v/v) to the phagocytosis mixture and incubating in a shaking water bath at 37~ Samples of 0.5 ml were taken from the phagocytosis mixture at 5, 10, and 20 min intervals and placed in 4.5 ml cold gelatin-Locke's solution (GLS: Downey and Diedrich, 1968) in polyethylene vials (Zinsser, Frankfurt, FRG). The vials were centrifuged at 100 g for 10 rain at 4~ and the pellet containing the leukocytes with ingested bacteria was washed twice with 5 ml cold GLS. The supernatants with the extracellular bacteria were combined, the pellet and 0.1 ml of the supernatant were each suspended in 0.5 ml liquid solubilizer (Soluene, Packard Inc., II1., USA) and kept at 60~ for 1-2 h. After the pellet was dissolved, 10 ml scintillation cocktail (Bruno and Christian, 1961) was added. If the vials containing the liquid scintillation cocktail had an alkaline pH due to the liquid solubilizer, they were adjusted to pH 7.0 by adding 2N HCL to prevent false efficiency counting. The number of disintegrations per minute (dpm) was determined in a liquid scintillation counter (Packard Inc. Model 3375. I11., USA). The phagocytosis index (Pl) was calculated according to the formula:
Phagocytosis and Killing of Staphylococci
39
dpm in leukocyte pellet % PI =
X 100 dpm in leukocyte pellet + dpm in supernatant
Quantitation of Bacterial Killing: The number of bacteria, killed after ingestion by the leukocytes, was determined by viable counts of the washed pellet after lysis of the leukocytes. Therefore, after intervals of 5, 10, and 20 min the washed pellet was suspended in 3 ml sterile distilled water and shaken vigorously to lyse the leukocytes. Viable counts were performed to determine the number of surviving bacteria. The total number of bacteria was measured by adding 0.1 ml of the phagocytosis mixture at time zero to 2.9 ml sterile distilled water. The viable counts were made as described above. The percentage of bacteria that survived or were killed was calculated respectively according to the formulas: % ingested bacteria
= % of total viable CFU in leukocyte pellet/% PI
% killing
= % PI - % ingested bacteria
Results
Figure 1 shows the uptake of S. saphropbyticus biotypes 1-3 by PMN leukocytes when arranged according to Baird-Parker's scheme (1974). Each line represents the average of six experiments. The uptake o f each strain from one biotype varied from 60% to 80% after 20 min incubation at 37~ The mean PI of S. sapropbyticus biotypes 1, 2, and 3 was 65, 68, and 70, respectively. No significant differences were found in the uptake of S. sapropbyticus biotypes. Similar results were observed when the same strains were classified according to the Kloos and Schleifer scheme (1975). The uptake of each strain
80
60
4O
20
/ I 5
I 1o
minutes
I 2o
Fig. 1. Phagoeytosis of S. sapropbyticus (Baird-Parker, 1974) biotype l(e), biotype 2(0) and 3(*) by normal human PMN Ieukocytes. Each line r e p r e s e n t s t h e average of six experiments w i t h six different strains of o n e b i o t y p e
40
F. Namavar et al.
from one species varied from 50% to 80%. The mean PI of S. cobnii, S. bominis, S. ~arneri, S. capitis, and S. haemolyticus strains (Fig. 2) was found to be 74, 65, 70, 80, and 66, respectively. No significant differences were observed between these staphylococcal species. In the previous study (Namavar et al., 1978), we noted that novobiocin-resistant S. saprophyticus strains isolated from urinary tract infections and from the skin were more virulent in neonatal mice than strains of other staphylococcal species. We decided, therefore, to study the uptake of novobiocin-resistant and novobiocin-sensitive S, sapropbyticus strains isolated from urinary tract infections and from
80
60
#
20
I 5
I 10
I 20
Fig. 2. Phagoeytosis of S. cobnii (o), S. bominls (r S. ,oarneri (A), S. capitis (o) and S. baemolyticus (*) (Kloos and Schleifer, 1975) by normal human PMN leukocytes. Each line represents the average of six experiments with six different strains of one species
minutes 80
6o
#
Fig. 3. Phagocytosis of novobiocin-resistant
S. sapropbyticus (Kloos and Schleifer, 1975) 20
I 5
minutes
I lO
I 2o
isolated from skin (#) and urinary tract infections (o), and novobiocin-sensitive S. sapropbytieus isolated from the skin (n) by normal human PMN leukocytes. Each line represents ten experiments with ten strains of one species
Phagocytosis and Killing of Staphylococci
41
100 L
=>
80
t~ "0 G)
._r
E ==
0
60
40
JD "10
Fig. 4. Intracellular survival of S. sapropbyticus isolated from skin (e) and from urinary tract infections (o), and S. baemolyticus (*), S. capitis (Q), and S. ~arneri (A) by normal nurnan PMN leukoeytes. Each line represents the average of three experiments with three different strains of one species
Q
.c_ "6 z~
20
L 0
!
!
5
10
20
minutes
the skin. The PI of novobiocin-resistant strains of S. sapropbyticus isolated from urinary tract infections and from the skin was 63% and 70% respectively and the PI of noboviocin-sensitive S. sapropbyticus strains isolated from the skin was 65% (Fig. 3). The average number of bacteria ingested for S. sapropbyticus whether isolated from urinary tract infections or from the skin, was 65% which was not markedly different from the 70% of uptake of other staphylococcal species. Further experiments were performed to study the intraceUular killing of a number of staphylococcal species. The average number of bacteria killed after 5, 10, and 20 rain was found to be 82, 95, and 98%, respectively (Fig. 4). After 10 min incubation with cell species 95% of the bacteria were killed.
Discussion In our previous study (Namavar et al., 1976) we studied the factors which influence phagocytosis in order to develop a consistent system of classification. We found that a leukocyte:bacteria ratio of 1:10 was optimal, and that differences in phagocytosis capacity existed between donors. Since we had shown that phagocytosis capacity of a donor remains relatively constant we decided to perform all further experiments with leukocytes o f one donor and with a leukocyte :bacteria ratio of 1 : 10. We further found that S. aureus strains gave a lower LDs0 values (Namavar et al., 1976, 1978) than coagulase-negative staphylococci but seemed to form a homogeneous group so far as LD50 values were concerned; on the other hand, the coagulase-negative staphylococci showed a wider spread of LD50 values, indicating a greater heterogeneity,
42
F. Namavar et al.
The objectives of this study were twofold. In the first place we wished to investigate whether staphylococcal species differ in their resistance to phagocytosis and intracellular killing by human polymorphs. Such differences, if they exist, would explain, at least in part, the prevalence of certain species in published series of infections. In the second place, different responses to phagocytosis and intracellular killing might shed further light on the vexed question of classification which hampers a better understanding of the pathogenesis of these infections. Differences in virulence would be additional evidence in favor of the status of species for staphylococcal isolates, as proposed by Kloos and Schleifer (1975). We (Namavar et al., in press) had also observed a lack of correspondence between the biotypes proposed by Baird-Parker (1974) and the species of Kloos and Schleifer (1975). Strains belonging to one Baird-Parker biotype would be different species in the latter scheme. Moreover, in previous studies (Namavar et al., 1978) we found the novobiocin-resistant S. saprophytieus biotype 3 to be more virulent than novobiocin-sensitive strains, when they were inoculated intracerebrally into neonatal mice. This was intriguing since strains isolated from urinary tract infections were invaribly novobiocin resistant. The phagocytosis technique we used was found to be reproducible in studying both the kinetics and the effect of various factors on the degree of phagocytosis. However, in quantitation of intracellular killing, antibiotics and lysostaphin were not used as recommended by some investigators (Miller et al., 1968; Tan et al., 1971 ; Solberg, 1972) because antibiotics need a definite time to destroy extracellular bacteria (Baughn and Bonventre, 1975) and may modify the physiology and viability of phagoeytosed bacteria (Chang, 1964; Patterson and Youmans, 1970). The addition of lysostaphin might be useful in rapidly destroying extracellular S. aureus without damaging PMN leukocytes (Schaffner et al., 1967; Tan et al., 1971; B iggar, 1975; Baughn and Bonventre, 1975), but it is not applicable to coagulase-negative staphylococci since some of these strains are resistant. Thus so far as coagulase-negative staphylococci are concerned centrifugation is stin the best way of studying phagocytosis and killing by PMN leukocytes as independent cellular events. By means of this technique, the uptake was found to vary from 60% to 80%. The average percentage of bacteria ingested was almost 70%. In spite of the differences in LDs0 values of novobiocin-resistant S. saprophyticus strains compared with other staphylococcal strains (Namavar et al., 1978), there were no marked differences in their uptake and killing by human leukocytes after 20 rain incubation at 37~ In this study when a leukocyte:bacteria ratio of 1:10 was used, an average of 6-8 bacteria were ingested per leukocyte after 20 min, which is slightly lower than the average 8 bacteria ingested per leukocyte in experiments with S. epidermidis strains (Namavar et al., 1976). However, since the number of bacteria ingested per leukocyte ranged from 6 to 8, the differences were not found to be significant. Differences in virulence cannot, therefore, be explained in terms of resistance to phagocytosis and intracellular killing. Most infections caused by coagulase-negative Micrococcaceae are associated either with a general increased susceptibility, e.g., in prematures, or with a local susceptibility, e.g., artificial heart valves. One notable exception is urinary infection caused by S. sapropbyticus biotype 3 which occurs in healthy young women. As with E. coli infections sexual intercourse may be a precipitating factor. Thus it is puzzling that Gillespie (1978) failed to find these organisms on the preiurethral skin or in the bowel. Pead and MaskeU (1977), using selective media, succeeded in isolating S. sapropbyticus biotype 3
Phagocytosis and Killing of Staphylococci
43
from the feces in only 10% of a group of young women. S. sapropbyticus biotype 3 is clearly not a predominant member of the fecal flora. Also, its tissue specificity cannot be explained in terms of a predilection for growth in urine since Anderson et al. (1976) failed to demonstrate this. Why this biotype par excellence should invade the urinary tract of young women is thus unknown. Neither periuretheral colonization, nor enhanced growth in urine, nor resistance to phagoeytosis seem to play a part in the pathogenesis. Further studies might be directed to a study of the superficial antigens of S. saprophyticus biotype 3 in relation to cell adherence. There are great gaps in our knowledge of pathogenesis of S. aureus infections, although a number of important factors have been delineated. Virtually nothing is known as yet about the pathogenesis of coagulase-negative staphylococcal infections. The increased number of patients susceptible to these infections and general resistance of these bacteria to antibiotics makes further research highly desirable.
Acknowledgements. We would like to thank Mrs. P.C. Kreuningand Mrs. U. Verboom-Sohrnerfor technical assistance. References
Anderson, J.D., Forshaw, H.L., Adams, N.A., Gillespie, W.A., Sellin, M.A.: The relevance of growth rates in urine to the pathogenesis of urinary tract infections due to Micrococcus subgroup 3 (Staphylococcus sapropbyticus 3). J. Med. Microbiol. 9,317-323 (1976) Baird-Parker, A.C.: The basis for the classification of staphylococci and micrococci. Ann. N.Y. Acad. Sci. 236, 7-13 (1974) Baughn, R.E., Bonventre, P.F.: Phagocytosis and intracellular killing of Saphylococcus aureus by normal mouse peritoneal macrophages. Infec. Immun. 12,346-352 (1975) Biggar, W.D.: Phagocytosis in patients and carriers of chronic granulomatous disease. Lancet, 1975 I, 991-995 Bruno, G.A., Christian, J.E.: Determination of C-14 in aqueous bicarbonate solutions by liquid scintillation counting technique. Analyt. Chemis. 33, 1216-1219 (1961) Chang, Y.T.: Long term cultivation of mouse peritoneal macrophages. J. Natl. Cancer Inst. 32, 19-35 (1964) Downey, R.J., Diedrich, B.F.: A new method for assessing particle ingestion by phagocytic cells. Exp. Cell. Res. 50,483-489 (1968) Gillespie, W.A., Selin, M.A., Gill, P., Stephens, M., Tuckwell, L.A., Hilton, A.L.: Urinary tract infection in young women, with special reference to Staphylococcus saprophyticus. J. Clin. Path. 31,348-350 (1978) Holt, R.: The classification of staphylococci from colonized ventriculo-atrial shunts. J. Clin. Path. 22,475-482 (1969) Kloos, W.E., Schleifer, K.H.: Simplified scheme for routine identification of human Staphylococcus species. J. Clin. Microbiol. 1, 82-88 (1975) Miller, M.E., Seals, J., Kay, R., Levitsky, L.C.: A familiar plasma-associated defect of phagocytosis. Lancet, 1968 I, 60-63 Mitchell, R.G.: Classification of Staphylococcus albus strains isolated from the urinary tract. J. Clin. Pathol. 21, 93-96 (1968)
44
F. Namavar et al.
Namavar, F., De Graaff, J., Veldhuizen, R., Verhoef, J.: Virulence of staphylococci in neonatal mice. Antonie van Leeuwenhoek 41,211 (1975) Namavar, F., De Graaff, J., Verhoef, J.: Virulence of staphylococci (with special reference to experimental infection in neonatal mice, and phagocytosis by polymorphonuclear cells). Zentbl. Bakt. I Abt. Orig. A., Suppl. 5, 813-818 (1976) Namavar, F., De Graaff, J., MacLaren, D.M.: Novobiocin resistance and virulence of strains of Stapbylococcus sapropbyticus isolated from urine and skin. J. Med. Microbiol. 11,243-248 (1978) Namavar, F., De Graaff, J., MacLaren, D.M.: Taxonomy of coagulase-negative staphylococci: a comparison of two widely used classification scheme. Antonie van Leeuwenhoek (in press) Patterson, R.J., Youman, G.I.: Multiplication of Mycobacterium tuberculosis within normal and immune mouse macrophages cultivated with and without streptomycin. Infec. Immun. 1, 30-40 (1970) Pead, L., MaskeU, R.: Micrococci and urinary infection (Letter). Lancet 1977 II, 565 Schaffner, W., MeUy, N.A., Hash, J.H., Loeing, M.G.: Lysostaphin: an enzymatic approach to staphylococcal disease. 1. In vitro studies. Yale, J. Biol. Med. 39, 215229 (1967) SeUin, M., Cooke, D.I., Gillespie, W.A., Sylvester, D.G.H., Anderson, J.D.: Micrococcal urinary tract infections in young women. Lancet 1975 If, 570-572 Solberg, C.O.: Protection of phagocytized bacteria against antibiotics. Acta. Med. Scan. 191,383-387 (1972) Tan, J.S., Watanakunakorn, D., Phair; J.P.: A modified assay of neutrophil function: use of lysostaphin to differentiate defective phagocytosis from impaired intracellular killing. J. Lab. Clin. Med. 78, 316-322 (1971) Weening, R.S., Roos, D., Loos, J.A.: Oxygen consumption of phagocytizing cells in human leukocytes and granulocyte-preparation. A comparative study. J. Lab. Clin. Med. 83,570-576 (1974)
Received August 23, 1978
~) by Springer-Vedag 91979
Phagocytosis and Intracellular Killing of Coagulase-Negative Staphylococci F. Namavar 1 , J. de Graaff 2 , and D.M. MacLarenl Laboratory of Medical Microbiology 1 and Department of Oral Microbiology 2, Free University, Amsterdam, The Netherlands
Abstract. The phagocytosis and intracellular killing of different coagulasenegative staphylococcal species by polymorphonuclear (PMN) leukocytes from one healthy donor were compared. The uptake of strains belonging to a given species varied from 60 to 80% with an average of 70% after 20 min incubation at 37~ Up to 95% of intracellular bacteria were killed after 10 rain. There was no correlation between uptake capacity and species or biotype. The average rates of phagocytosis and killing strains whether isolated from urinary tract infections or from the skin were virtually the same. Introduction The predominance of certain biotypes o f coagulase-negative Micrococcacaeae in human infections has naturally raised the question whether these are more virulent. S. epidermis biotype 2 predominates in infections associated with atrio-ventricular shunts or artificial heart valves (Holt, 1969). S. sapropbyticus biotype 3 (Sellin et al., 1975; Gillespie et al., 1978) has been shown to be the predominant biotype in acute staphylococcal urinary infections in young women. Very little is known about the pathogenetic mechanisms of coagulase-negative staphylococci, except that a foreign body seems often to provide the niche in which the infection can be established (Holt, 1969). In previous studies (Namavar et al., 1975, 1978) certain biotypes of S. epidermidis and S. sapbropbyticus gave lower LDS0 values after intracerebral inoculation into neonatal mice. Extrapolation from animal studies to the human situation is difficult, but these LDS0 results suggested some differences in virulence. A study of resistance to phagocytosis and intracellular killing, processes of vital importance in the body's defense system, might point to biotypes with potentially enhanced virulence. Such a study might also shed more light on the vexed question of classification. In our previous study (Namavar et al., in press) we found marked heterogeneity when we reclassified BairdParker biotypes (1974) by the Kloos and Schleifer (1975) criteria. The behavior of Address for offprint requests: F. Namavar, Laboratory of Medical Microbiology, Free University, van der Boechorststraat 7, 1007 MC Amsterdam, The Netherlands 0300-8584/79/0167/0037/~gO1.60
38
F. Namavar et al.
biotypes in phagocytosis and leukocyte killing experiments might offer further evidence in assessing the status of the numerous species described by Kloos and Sehleifer. The present study was planned with these objectives in view. Materials and Methods
Bacterial Strains: Coagulase-negative staphylococcal strains were isolated from the healthy human skin and from urinary tract infections. Strains were classified according to the Baird-Parker (1974) and the Kloos and Schliefer schemes (1975). Novobiocin sensitivity was determined by the method described by Mitchell (1968).
Preparation 01'Bacteria: Bacterial suspensions were prepared by adding 0.1 ml of an overnight culture of 10 ml Lab-Lemco broth (Oxoid) containing 2 tzCi of (methyl-3H) thymidine (specific activity 40-60 Ci/mmol; Radiochemical centre, Amersham, UK). After 17 h incubation at 37~ bacteria were washed three times in phosphate-buffered saline (PBS), pH 7.4, and resuspended in 1 ml PBS. Counts of viable bacteria were performed in nutrient agar plates and the bacterial suspension was stored overnight at 4~ When on the following day the viable counts were known the final bacterial concentration was adjusted to give 1 x 108 colony-forming units (CFU)/ml.
Preparation of Polymorpbonuclear (PMN) Leukocytes: Human PMN leukocytes were isolated from fresh defibrinated blood as described by Weening et al. (1974). In every experiment approximately 30 ml blood from the same donor was used. The cells were counted electronically in a Coulter counter (Model ZB1) and the leukocyte pellet was adjusted to a final concentration of 5 x 106 cells/ml of PBS containing 0.5% (w/v) human albumin.
Quantitation of Bacterial Uptake: Phagocytosis mixtures were prepared with 1.2 ml of PMN leukocyte suspension and 0.6 ml of 3H-thymidine-labeled bacterial suspension in plastic tubes (15 x 95 mm; Thoradeo, NewKoop, Netherlands) to give a leukocyte:bacteria ratio of 1:10. Bacterial opsonization was performed by adding normal undiluted serum from the donor at a final concentration of 10% (v/v) to the phagocytosis mixture and incubating in a shaking water bath at 37~ Samples of 0.5 ml were taken from the phagocytosis mixture at 5, 10, and 20 min intervals and placed in 4.5 ml cold gelatin-Locke's solution (GLS: Downey and Diedrich, 1968) in polyethylene vials (Zinsser, Frankfurt, FRG). The vials were centrifuged at 100 g for 10 rain at 4~ and the pellet containing the leukocytes with ingested bacteria was washed twice with 5 ml cold GLS. The supernatants with the extracellular bacteria were combined, the pellet and 0.1 ml of the supernatant were each suspended in 0.5 ml liquid solubilizer (Soluene, Packard Inc., II1., USA) and kept at 60~ for 1-2 h. After the pellet was dissolved, 10 ml scintillation cocktail (Bruno and Christian, 1961) was added. If the vials containing the liquid scintillation cocktail had an alkaline pH due to the liquid solubilizer, they were adjusted to pH 7.0 by adding 2N HCL to prevent false efficiency counting. The number of disintegrations per minute (dpm) was determined in a liquid scintillation counter (Packard Inc. Model 3375. I11., USA). The phagocytosis index (Pl) was calculated according to the formula:
Phagocytosis and Killing of Staphylococci
39
dpm in leukocyte pellet % PI =
X 100 dpm in leukocyte pellet + dpm in supernatant
Quantitation of Bacterial Killing: The number of bacteria, killed after ingestion by the leukocytes, was determined by viable counts of the washed pellet after lysis of the leukocytes. Therefore, after intervals of 5, 10, and 20 min the washed pellet was suspended in 3 ml sterile distilled water and shaken vigorously to lyse the leukocytes. Viable counts were performed to determine the number of surviving bacteria. The total number of bacteria was measured by adding 0.1 ml of the phagocytosis mixture at time zero to 2.9 ml sterile distilled water. The viable counts were made as described above. The percentage of bacteria that survived or were killed was calculated respectively according to the formulas: % ingested bacteria
= % of total viable CFU in leukocyte pellet/% PI
% killing
= % PI - % ingested bacteria
Results
Figure 1 shows the uptake of S. saphropbyticus biotypes 1-3 by PMN leukocytes when arranged according to Baird-Parker's scheme (1974). Each line represents the average of six experiments. The uptake o f each strain from one biotype varied from 60% to 80% after 20 min incubation at 37~ The mean PI of S. sapropbyticus biotypes 1, 2, and 3 was 65, 68, and 70, respectively. No significant differences were found in the uptake of S. sapropbyticus biotypes. Similar results were observed when the same strains were classified according to the Kloos and Schleifer scheme (1975). The uptake of each strain
80
60
4O
20
/ I 5
I 1o
minutes
I 2o
Fig. 1. Phagoeytosis of S. sapropbyticus (Baird-Parker, 1974) biotype l(e), biotype 2(0) and 3(*) by normal human PMN Ieukocytes. Each line r e p r e s e n t s t h e average of six experiments w i t h six different strains of o n e b i o t y p e
40
F. Namavar et al.
from one species varied from 50% to 80%. The mean PI of S. cobnii, S. bominis, S. ~arneri, S. capitis, and S. haemolyticus strains (Fig. 2) was found to be 74, 65, 70, 80, and 66, respectively. No significant differences were observed between these staphylococcal species. In the previous study (Namavar et al., 1978), we noted that novobiocin-resistant S. saprophyticus strains isolated from urinary tract infections and from the skin were more virulent in neonatal mice than strains of other staphylococcal species. We decided, therefore, to study the uptake of novobiocin-resistant and novobiocin-sensitive S, sapropbyticus strains isolated from urinary tract infections and from
80
60
#
20
I 5
I 10
I 20
Fig. 2. Phagoeytosis of S. cobnii (o), S. bominls (r S. ,oarneri (A), S. capitis (o) and S. baemolyticus (*) (Kloos and Schleifer, 1975) by normal human PMN leukocytes. Each line represents the average of six experiments with six different strains of one species
minutes 80
6o
#
Fig. 3. Phagocytosis of novobiocin-resistant
S. sapropbyticus (Kloos and Schleifer, 1975) 20
I 5
minutes
I lO
I 2o
isolated from skin (#) and urinary tract infections (o), and novobiocin-sensitive S. sapropbytieus isolated from the skin (n) by normal human PMN leukocytes. Each line represents ten experiments with ten strains of one species
Phagocytosis and Killing of Staphylococci
41
100 L
=>
80
t~ "0 G)
._r
E ==
0
60
40
JD "10
Fig. 4. Intracellular survival of S. sapropbyticus isolated from skin (e) and from urinary tract infections (o), and S. baemolyticus (*), S. capitis (Q), and S. ~arneri (A) by normal nurnan PMN leukoeytes. Each line represents the average of three experiments with three different strains of one species
Q
.c_ "6 z~
20
L 0
!
!
5
10
20
minutes
the skin. The PI of novobiocin-resistant strains of S. sapropbyticus isolated from urinary tract infections and from the skin was 63% and 70% respectively and the PI of noboviocin-sensitive S. sapropbyticus strains isolated from the skin was 65% (Fig. 3). The average number of bacteria ingested for S. sapropbyticus whether isolated from urinary tract infections or from the skin, was 65% which was not markedly different from the 70% of uptake of other staphylococcal species. Further experiments were performed to study the intraceUular killing of a number of staphylococcal species. The average number of bacteria killed after 5, 10, and 20 rain was found to be 82, 95, and 98%, respectively (Fig. 4). After 10 min incubation with cell species 95% of the bacteria were killed.
Discussion In our previous study (Namavar et al., 1976) we studied the factors which influence phagocytosis in order to develop a consistent system of classification. We found that a leukocyte:bacteria ratio of 1:10 was optimal, and that differences in phagocytosis capacity existed between donors. Since we had shown that phagocytosis capacity of a donor remains relatively constant we decided to perform all further experiments with leukocytes o f one donor and with a leukocyte :bacteria ratio of 1 : 10. We further found that S. aureus strains gave a lower LDs0 values (Namavar et al., 1976, 1978) than coagulase-negative staphylococci but seemed to form a homogeneous group so far as LD50 values were concerned; on the other hand, the coagulase-negative staphylococci showed a wider spread of LD50 values, indicating a greater heterogeneity,
42
F. Namavar et al.
The objectives of this study were twofold. In the first place we wished to investigate whether staphylococcal species differ in their resistance to phagocytosis and intracellular killing by human polymorphs. Such differences, if they exist, would explain, at least in part, the prevalence of certain species in published series of infections. In the second place, different responses to phagocytosis and intracellular killing might shed further light on the vexed question of classification which hampers a better understanding of the pathogenesis of these infections. Differences in virulence would be additional evidence in favor of the status of species for staphylococcal isolates, as proposed by Kloos and Schleifer (1975). We (Namavar et al., in press) had also observed a lack of correspondence between the biotypes proposed by Baird-Parker (1974) and the species of Kloos and Schleifer (1975). Strains belonging to one Baird-Parker biotype would be different species in the latter scheme. Moreover, in previous studies (Namavar et al., 1978) we found the novobiocin-resistant S. saprophytieus biotype 3 to be more virulent than novobiocin-sensitive strains, when they were inoculated intracerebrally into neonatal mice. This was intriguing since strains isolated from urinary tract infections were invaribly novobiocin resistant. The phagocytosis technique we used was found to be reproducible in studying both the kinetics and the effect of various factors on the degree of phagocytosis. However, in quantitation of intracellular killing, antibiotics and lysostaphin were not used as recommended by some investigators (Miller et al., 1968; Tan et al., 1971 ; Solberg, 1972) because antibiotics need a definite time to destroy extracellular bacteria (Baughn and Bonventre, 1975) and may modify the physiology and viability of phagoeytosed bacteria (Chang, 1964; Patterson and Youmans, 1970). The addition of lysostaphin might be useful in rapidly destroying extracellular S. aureus without damaging PMN leukocytes (Schaffner et al., 1967; Tan et al., 1971; B iggar, 1975; Baughn and Bonventre, 1975), but it is not applicable to coagulase-negative staphylococci since some of these strains are resistant. Thus so far as coagulase-negative staphylococci are concerned centrifugation is stin the best way of studying phagocytosis and killing by PMN leukocytes as independent cellular events. By means of this technique, the uptake was found to vary from 60% to 80%. The average percentage of bacteria ingested was almost 70%. In spite of the differences in LDs0 values of novobiocin-resistant S. saprophyticus strains compared with other staphylococcal strains (Namavar et al., 1978), there were no marked differences in their uptake and killing by human leukocytes after 20 rain incubation at 37~ In this study when a leukocyte:bacteria ratio of 1:10 was used, an average of 6-8 bacteria were ingested per leukocyte after 20 min, which is slightly lower than the average 8 bacteria ingested per leukocyte in experiments with S. epidermidis strains (Namavar et al., 1976). However, since the number of bacteria ingested per leukocyte ranged from 6 to 8, the differences were not found to be significant. Differences in virulence cannot, therefore, be explained in terms of resistance to phagocytosis and intracellular killing. Most infections caused by coagulase-negative Micrococcaceae are associated either with a general increased susceptibility, e.g., in prematures, or with a local susceptibility, e.g., artificial heart valves. One notable exception is urinary infection caused by S. sapropbyticus biotype 3 which occurs in healthy young women. As with E. coli infections sexual intercourse may be a precipitating factor. Thus it is puzzling that Gillespie (1978) failed to find these organisms on the preiurethral skin or in the bowel. Pead and MaskeU (1977), using selective media, succeeded in isolating S. sapropbyticus biotype 3
Phagocytosis and Killing of Staphylococci
43
from the feces in only 10% of a group of young women. S. sapropbyticus biotype 3 is clearly not a predominant member of the fecal flora. Also, its tissue specificity cannot be explained in terms of a predilection for growth in urine since Anderson et al. (1976) failed to demonstrate this. Why this biotype par excellence should invade the urinary tract of young women is thus unknown. Neither periuretheral colonization, nor enhanced growth in urine, nor resistance to phagoeytosis seem to play a part in the pathogenesis. Further studies might be directed to a study of the superficial antigens of S. saprophyticus biotype 3 in relation to cell adherence. There are great gaps in our knowledge of pathogenesis of S. aureus infections, although a number of important factors have been delineated. Virtually nothing is known as yet about the pathogenesis of coagulase-negative staphylococcal infections. The increased number of patients susceptible to these infections and general resistance of these bacteria to antibiotics makes further research highly desirable.
Acknowledgements. We would like to thank Mrs. P.C. Kreuningand Mrs. U. Verboom-Sohrnerfor technical assistance. References
Anderson, J.D., Forshaw, H.L., Adams, N.A., Gillespie, W.A., Sellin, M.A.: The relevance of growth rates in urine to the pathogenesis of urinary tract infections due to Micrococcus subgroup 3 (Staphylococcus sapropbyticus 3). J. Med. Microbiol. 9,317-323 (1976) Baird-Parker, A.C.: The basis for the classification of staphylococci and micrococci. Ann. N.Y. Acad. Sci. 236, 7-13 (1974) Baughn, R.E., Bonventre, P.F.: Phagocytosis and intracellular killing of Saphylococcus aureus by normal mouse peritoneal macrophages. Infec. Immun. 12,346-352 (1975) Biggar, W.D.: Phagocytosis in patients and carriers of chronic granulomatous disease. Lancet, 1975 I, 991-995 Bruno, G.A., Christian, J.E.: Determination of C-14 in aqueous bicarbonate solutions by liquid scintillation counting technique. Analyt. Chemis. 33, 1216-1219 (1961) Chang, Y.T.: Long term cultivation of mouse peritoneal macrophages. J. Natl. Cancer Inst. 32, 19-35 (1964) Downey, R.J., Diedrich, B.F.: A new method for assessing particle ingestion by phagocytic cells. Exp. Cell. Res. 50,483-489 (1968) Gillespie, W.A., Selin, M.A., Gill, P., Stephens, M., Tuckwell, L.A., Hilton, A.L.: Urinary tract infection in young women, with special reference to Staphylococcus saprophyticus. J. Clin. Path. 31,348-350 (1978) Holt, R.: The classification of staphylococci from colonized ventriculo-atrial shunts. J. Clin. Path. 22,475-482 (1969) Kloos, W.E., Schleifer, K.H.: Simplified scheme for routine identification of human Staphylococcus species. J. Clin. Microbiol. 1, 82-88 (1975) Miller, M.E., Seals, J., Kay, R., Levitsky, L.C.: A familiar plasma-associated defect of phagocytosis. Lancet, 1968 I, 60-63 Mitchell, R.G.: Classification of Staphylococcus albus strains isolated from the urinary tract. J. Clin. Pathol. 21, 93-96 (1968)
44
F. Namavar et al.
Namavar, F., De Graaff, J., Veldhuizen, R., Verhoef, J.: Virulence of staphylococci in neonatal mice. Antonie van Leeuwenhoek 41,211 (1975) Namavar, F., De Graaff, J., Verhoef, J.: Virulence of staphylococci (with special reference to experimental infection in neonatal mice, and phagocytosis by polymorphonuclear cells). Zentbl. Bakt. I Abt. Orig. A., Suppl. 5, 813-818 (1976) Namavar, F., De Graaff, J., MacLaren, D.M.: Novobiocin resistance and virulence of strains of Stapbylococcus sapropbyticus isolated from urine and skin. J. Med. Microbiol. 11,243-248 (1978) Namavar, F., De Graaff, J., MacLaren, D.M.: Taxonomy of coagulase-negative staphylococci: a comparison of two widely used classification scheme. Antonie van Leeuwenhoek (in press) Patterson, R.J., Youman, G.I.: Multiplication of Mycobacterium tuberculosis within normal and immune mouse macrophages cultivated with and without streptomycin. Infec. Immun. 1, 30-40 (1970) Pead, L., MaskeU, R.: Micrococci and urinary infection (Letter). Lancet 1977 II, 565 Schaffner, W., MeUy, N.A., Hash, J.H., Loeing, M.G.: Lysostaphin: an enzymatic approach to staphylococcal disease. 1. In vitro studies. Yale, J. Biol. Med. 39, 215229 (1967) SeUin, M., Cooke, D.I., Gillespie, W.A., Sylvester, D.G.H., Anderson, J.D.: Micrococcal urinary tract infections in young women. Lancet 1975 If, 570-572 Solberg, C.O.: Protection of phagocytized bacteria against antibiotics. Acta. Med. Scan. 191,383-387 (1972) Tan, J.S., Watanakunakorn, D., Phair; J.P.: A modified assay of neutrophil function: use of lysostaphin to differentiate defective phagocytosis from impaired intracellular killing. J. Lab. Clin. Med. 78, 316-322 (1971) Weening, R.S., Roos, D., Loos, J.A.: Oxygen consumption of phagocytizing cells in human leukocytes and granulocyte-preparation. A comparative study. J. Lab. Clin. Med. 83,570-576 (1974)
Received August 23, 1978
