Synergistic Hemolysis Exhibited by Species of Staphylococci

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1985, p Vol. 22, No /85/ $02.00/0 Copyright 1985, American Society for Microbiology Synergistic Hemolysis Exhibited by Species of Staphylococci G. ANN HÉBERT* AND GARY A. HANCOCK Hospital Infections Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia Received 29 April 1985/Accepted 5 June 1985 The synergistic hemolysis reactions of 61 reference strains and 189 clinical isolates representing 17 species of staphylococci were examined on plates of Trypticase soy blood agar (BBL Microbiology Systems, Cockeysville, Md.). Some or all of the strains of Staphylococcus aureus, S. epidermidis, S. capitis, S. cohnii, S. haemolyticus, S. hyicus, S. simulans, S. warneri, and S. xylosus produced a delta-hemolysin that gave synergistic, complete hemolysis of washed human, sheep, and ox blood cells in an area of beta-lysin activity from strains of S. aureus and S. intermedius. Strains of the same nine species were positive with a commercial beta-lysin paper disk designed for presumptive identification of group B streptococci; most of these strains also gave synergistic, complete hemolysis with exotoxin from a strain of Corynebacterium pseudotuberculosis. None of the strains of S. auricularis, S. carnosus, S. caseolyticus, S. hominis, S. intermedjus, S. saprophyticus, S. sciuri, or S. lentus were positive by any of these tests for synergistic hemolysis. These results indicate that a synergistic hemolysis test could prove very useful for differentiating these species; they also suggest that one role of some of these organisms in human infections could be that of a synergist. Further studies of synergism may clarify the clinical significance of these results. In the past several years there has been an increasing number of reports of the incidence of coagulase-negative staphylococci in clinical infections, and the pathogenic role of these organisms is now well established (1, 7). Coagulasenegative staphylococci are frequently implicated in infections of the urinary tract, heart valves, eye and ear wounds, cerebrospinal fluid shunts, and vascular and joint prostheses; they are also the implied cause of subacute bacterial endocarditis, peritonitis in patients on continuous peritoneal dialysis, and bacteremia in patients receiving immunosuppressive therapy (1, 7, 17). Since 1975, Kloos and Schleifer (18-21, 28-31) have been systematically reorganizing the coagulase-negative staphylococci into separate species with defined phenotypic characteristics; Devriese and Hajek (6, 13) have defined new species of coagulase-positive and -negative staphylococci that are most often associated with animal infections. All of the named species are quite distinct in DNA homology studies done under restrictive reassociation conditions; the clinical significance of the separate species, however, is still being defined. Before 1975, only three species were recognized in the genus Staphylococcus (2); all of the coagulase-negative staphylococci were either S. epidermidis or S. saprophyticus, and the coagulase-positive strains were S. aureus. The toxins and hemolysins of these staphylococci were the subjects of many extensive reports. Several studies showed that some of the less hemolytic staphylococci (4, 8), some of the coagulase-negative staphylococci (16, 32), and some S. aureus and S. epidermidis strains (3, 5, 23, 25) produced a delta-hemolysin that gave synergistic, complete hemolysis with a beta-toxin-producing Staphylococcus sp. growing on agar with sheep erythrocytes. In one of those studies (32), some coagulase-negative staphylococci also gave synergistic hemolysis with an exotoxin isolated from Corynebacterium pseudotuberculosis. Since 1975, many new species of Staphylococcus have been defined, and the descriptions for S. epidermidis and S. saprophyticus have been amended (31); however, we are not aware of any further studies of hemolysins or synergistic lysis in these defined species. Any * Corresponding author. 409 hemolytic activity among these species in synergistic relationships could possibly relate to their virulence and, therefore, their clinical significance. In addition, consistent synergistic reactions among strains within a species could help to differentiate between the species. We have examined reference and clinical strains of defined species of staphylococci for such activities, and the results of our studies are presented in this report. (Part of this work was presented at the 1985 Annual Meeting of the American Society for Microbiology [G. A. Hébert and G. A. Hancock, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, C174, p. 329].) MATERIALS AND METHODS Cultures and growth conditions. A total of 250 strains representing 17 species of staphylococci were examined during this study. The 61 reference strains listed in Table 1 were obtained from W. E. Kloos, Raleigh, N.C., and from the American Type Culture Collection, Rockville, Md. The random set of 189 clinical isolates included many supplied by E. H. Gerlach, Wichita, Kans.; the other clinical strains were from the culture collections of laboratories at the Centers for Disease Control. All ofthe cultures were plated on Trypticase soy agar containing 5% defibrinated sheep blood (TSA II; BBL Microbiology Systems, Cockeysville, Md.) and incubated aerobically for 18 to 24 h at 35 C. For prolonged storage, 24-h growth was harvested in sterile rabbit blood and kept at -70 C. Strains removed from storage were plated on blood agar at least twice before testing; 24-h cells from the second or third plate were used. Identification. All of the strains were gram-positive cocci that produced catalase. All strains were tested for coagulase activity by both the slide and tube tests with EDTA-rabbit plasma. Coagulase-positive strains were tested for acid production from glucose and mannitol anaerobically and from maltose and mannitol aerobically, for DNase and alkaline phosphatase activity, and for pigment production. Coagulase-negative strains were identified by the methods of Kloos (17, 19). Hemolysin definition. Several strains of each of the staphylococcal species studied were grown on plates of

2 410 HÉBERT AND HANCOCK TABLE 1. Reference strains of Staphylococcus species Species Strain designation S. auricularis... B04; ATCC 33750, 33751, 33752, 33753' S. capitis... ATCC 27840T, 27841, 27842, S. carnosus... Hansen, MA S. caseolyticus... ATCC 13548T, S. cohnii... DM224, SH161A; ATCC 29972, 29973, 29974' S. epidermidis... AW269, GH37, ; ATCC 29886, S. haemolyticus... GH59; ATCC 29968, 29969, 29970T S. hominis... JL248; ATCC 27844T, 27845, 27846, S. hyicus subsp. hyicus D; ATCC 11249T S. hyicus subsp. chromogens... 6-IRRb; 93-IRRa S. intermedius... CFDD, RK12; ATCC 29663T S. saprophyticus... KL20, SM295, TW111; ATCC 15305T S. sciuri... ATCC 29059, 29060, 29061, 29062T S. lentus... K-6 S. simulans... 6L31; ATCC 27848T, 27849, 27850, S. warneri... ATCC 27836T, 27837, 27838, S. xylosus... DM37; ATCC 29966, 29967, 29971T Trypticase soy agar containing 5% defibrinated, washed blood cells of five different animal species: rabbit, sheep, horse, ox, and human. A strain of beta-lysin-producing S. intermedius was streaked perpendicular to, but not touching, the test strain on each of the five agars. All of the plates were incubated aerobically at 35 C for 18 to 20 h and then at room temperature for 4 to 6 h. All synergistic hemolysis reactions were recorded at approximately 24 h. A zone of complete hemolysis within the zone of incomplete hemolysis caused by S. intermedius was a positive test (see Fig. 1). These plates were then reincubated overnight, and the hemolysis of the various species of blood cells was recorded at 48 h (9). The generally accepted terminology used by Elek (8, 9) and others to define hemolysins was used: (i) alpha-hemolysin produces a wide zone of complete hemolysis with blurred edges on agar containing rabbit, sheep, or ox erythrocytes, but not horse or human cells; (ài) beta-hemolysin produces a wide zone of incomplete hemolysis with sharp edges on agar containing sheep, ox, or human erythrocytes, but not rabbit or horse cells; (iii) delta-hemolysin produces a narrow zone of complete hemolysis with blurred edges on agars containing erythrocytes of any of the animal species tested. The zone from a delta-hemolysin is poorly developed with the cells of some animal species, and small amounts of deltahemolysin might not show on sheep cells. A delta-hemolysin potentiates the zone of a beta-hemolysin to complete clearing on sheep, ox, and human cells. Selection of beta-lysin and exotoxin-producing strains. Several strains of coagulase-positive staphylococci and C. pseudotuberculosis were tested for beta-hemolysin and exotoxin production, respectively, to select strains to use in synergistic hemolysis tests. A technique similar to that described by Zemelman and Longeri (36) was used. A strain of S. epidermidis, known to produce a delta-lysin because of preliminary tests, was streaked down the center of a TSA Il J. CLIN. MICROIBIOL. plate. Strains of S. aureus, S. intermedius, and C. pseudotuberculosis were then streaked perpendicular to, but not touching, this central streak; 8 to 10 strains, 4 or 5 on each half of the plate, were tested per plate. The plates were incubated as described above for the hemolysin tests, and results were recorded at 24 h. A strain of Staphylococcus producing beta-lysin or a strain of C. pseudotuberculosis producing exotoxin gave a wide zone of incomplete hemolysis of sheep erythrocytes all along its line of growth and a large zone of synergistic, complete hemolysis in the area of delta-lysin next to the central line of S. epidermidis growth (see Fig. 2). Synergistic hemolysis growth tests. Strains were tested for synergistic hemolysis by the technique first described by Munch-Petersen et al. (26) for group B streptococci. A strain of S. intermedius (AB 148) that produced beta-lysin in tests described above was streaked down the center of a TSA II plate. A strain of C. pseudotuberculosis (ATCC 19410), selected in the above tests, was streaked down the center of another TSA Il plate. Test strains were streaked perpendicular to, but not touching, the center streaks as described above. The plates were incubated as described for the hemolysin tests, and results were recorded after 24 h. A zone of complete hemolysis, within the zone of incomplete hemolysis caused by the beta-lysin from the S. intermedius growth or the exotoxin from the C. pseudotuberculosis growth, was a positive test (see Fig. 3). Beta-lysin disk test. Paper disks (CAMP disk; Carr- Scarborough Microbiologicals, Inc., Stone Mountain, Ga.), impregnated with partially purified staphylococcal beta-lysin from a strain of S. aureus as described by Wilkinson (34), were placed along the center line of a TSA Il plate. Test strains were streaked in a straight line to within 2 to 4 mm of the disks. The plates were incubated as described for the hemolysin tests, and the results were recorded at 24 h. A zone of complete hemolysis, within the zone of incomplete hemolysis surrounding the disk, was a positive reaction (see Fig. 5). RESULTS The hemolytic reactions of one reference strain each of S. epidermidis and S. intermedius grown for 48 h on agars with a^ FIG. 1. Hemolytic reactions (48 h) of a strain of S. intermedius (streaks of growth along the outer edge in each quadrant) and a strain of S. epidermidis (streaks of growth radiating from the center of the plate in each quadrant) on agar with blood cells of four separate animal species: sheep (upper left), ox (upper right), horse (lower left), and rabbit (lower right).

3 VOL. 22, 1985 sheep, ox, horse, and rabbit blood cells are shown in Fig. 1; the synergistic reactions were more pronounced than those recorded at 24 h, but the hemolysis patterns were typical of those seen on the various species of cells. A very narrow zone of complete hemolysis with a blurred edge developed around the growth of the strain of S. epidermidis on agars with sheep and ox cells, a small clear zone with a blurred edge was seen with horse cells, and a larger clear zone resulted with rabbit cells. A visible zone of incomplete hemolysis diffused from the S. intermedius growth on the agars with sheep and ox cells, but no such activity was seen with horse or rabbit cells. Large zones of complete hemolysis developed around the S. epidermidis growth in the area of incomplete hemolysis caused by the activity of S. intermedius on sheep and ox cells; no synergistic reactions were seen with horse or rabbit cells. The hemolytic reactions on agar with human blood cells are not shown, but they were like those seen with sheep and ox cells, though less intense: the zone of incomplete hemolysis was not as wide, and the zone of complete, synergistic hemolysis was smaller. These same hemolytic reactions on the five species of blood cells were seen with the other strains of S. epidermidis, S. capitis, S. cohnii, S. haemolyticus, S. simulans, S. warneri, and S. xylosus. No or only occasional, very weak hemolysis was recorded for strains of S. auricularis, S. carnosus, S. caseolyticus, S. hominis, S. saprophyticus, S. sciuri, and S. lentus. Strains of S. aureus had from one to three of the defined hemolysins, and S. hyicus was not hemolytic but showed some synergism. The patterns of hemolysis seen when coagulase-positive strains of S. aureus and S. intermedius were tested for beta-lysin production to determine which would be best to use in tests for synergistic hemolysis with other staphylococci are shown in Fig. 2. Each of the positive test strains had a wide visible zone of incomplete hemolysis due to beta-lysin activity, which was then enhanced to complete hemolysis in the area adjacent to the growth of a strain of S. epidermidis. The outer edge of the beta-lysin zone was from 6 to 9 mm from the edge of cell growth among the strains tested. Four of 15 S. aureus and 5 of 6 S. intermedius strains had distinct beta-lysin zones, but the other strains had P~~~~~~~~~~~~~~~~~~~~~~~~~~u FIG. 2. Synergistic hemolysis between a strain of S. epidermidis (vertical streak of growth) and six strains of coagulase-positive staphylococci. The top strain on the left and ail three strains on the right are S. intermedius; the left center and bottom strains are S. aureus. Each of these six strains had a wide zonec of incomplete hemolysis that was enhanced to complete hemolysis in the zone of delta-lysin activity from the vertical strain. SYNERGISTIC HEMOLYSIS IN STAPHYLOCOCCI 411 FIG. 3. Synergistic hemolysis between a strain of S. intermedius (vertical streak of growth) and 10 strains of S. epidermidis. The left center strain had a wide zone of incomplete hemolysis that was enhanced to complete hemolysis in the zone of beta-lysin activity from the vertical strain. The right center strain was negative. multiple other hemolysins and zones of complete hemolysis that would have masked or interfered with any test for synergistic hemolysis. Strain AB 148 of S. intermedius was selected to test other strains for synergistic hemolysis; this strain produced a 7- to 8-mm zone of beta-lysin activity and had a very narrow zone of complete hemolysis that did not conflict with tests for enhancement or synergism. The four strains of C. pseudotuberculosis that were tested for exotoxin production were only slightly hemolytic and gave zones of synergistic, complete hemolysis with the test strain of S. epidermidis and a reference strain (ATCC 6939) of C. equi (Rhodococcus equi). For these strains, the outer edge of the exotoxin zone was 6 to 10 mm from the edge of cell growth at 24 h and increased to 7 to 14 mm at 48 h. All four strains gave satisfactory results; the reference strain, ATCC 19410, was selected as the test strain. Ten of the clinical isolates of S. epidermidis tested for synergistic hemolysis with S. intermedius are shown in Fig. 3. The size and shape of the clear zone of synergistic, complete hemolysis varied among strains, but a given strain gave reproducible results; hazy or incomplete zones were recorded as negative. Individual strains of S. epidermidis exhibited no or only weak hemolysis of sheep cells at 24 h, but two of the clinical isolates had moderate zones (4.5 and 5.5 mm) of incomplete hemolysis on sheep blood agar. Beta-lysin activity was enhanced to complete hemolysis in these zones, and these two strains did not give synergistic hemolysis with the strains that were producing delta-lysin; therefore, the moderate-width zones were not beta-lysin reactions and were recorded as atypical delta-lysin zones. One of these strains is shown in Fig. 3. Some or all of the reference and clinical strains of S. aureus, S. epidermidis, S. capitis, S. cohnii, S. haemolyticus, S. hyicus, S. simulans, S. warneri, and S. xylosus gave a distinct, clear zone of synergistic, complete hemolysis when tested against strain AB 148 of S. intermedius. Some of these reactions are shown in Fig. 3 and 4; all of the results are given in Tables 2 and 3. The two reference strains of S. hyicus subsp. hyicus were recorded as positive, but the area of complete hemolysis was unique; a clear area shaped like an arc or a parenthesis developed within a zone of partial hemolysis that was clearer than the outer area of beta-lysin activity. None of the reference or clinical strains of S.

4 412 HÉBERT AND HANCOCK FIG. 4. Synergistic hemolysis between a strain of S. intermedius (vertical streak of growth) and strains of coagulase-negative staphylococci: three S. epidermidis (upper left), two S. simulans (lower left), one S. capitis (top right), and four S. haemolyticus (others right). auricularis, S. carnosus, S. caseolyticus, S. hominis, S. intermedius, S. saprophyticus, S. sciuri, or S. lentus were positive. Many of the reference and clinical strains representing most of the 17 species tested were checked against several other beta-lysin-producing strains of S. intermedius and S. aureus; the results were the same as those obtained with AB 148. The same pattern of results was obtained when our reference and clinical strains of staphylococci were tested with the commercial beta-lysin disks which were designed TABLE 2. Synergistic hemolysis exhibited by reference strains of staphylococci No. of strains exhibiting a zone of syn- Staphylococcus No. of ergistic, complete hemolysis with: species strains S. inter- C. tested S. aureus tuberculomedius b pseudo- tbruo (growthïa (disky sis" S. capitis S. cohnii S. epidermidis S. haemolyticus S. simulans d S. warneri S. xylosus S. hyicus subsp chromogens S. hyicus subsp. 2 (2)e 0 0 hyicus S. auricularis 5 O O O S. carnosus S. caseolyticus S. hominis 5 O O O S. intermedius S. saprophyticus S. sciuri S. lentus a S. intermedius strain AB 148 on TSA II sheep blood agar. b Paper disks impregnated with beta-lysin from a strain of S. aureus. C C. pseudotuberculosis strain ATCC on TSA II sheep blood agar. d A very faint zone at 24 h and a definite clear zone at 48 h. e A clear, arc-shaped area developed within a zone of partial hemolysis that was clearer than the surrounding area of beta-lysin activity (atypical reaction). TABLE 3. Synergistic hemolysis exhibited by clinical isolates of staphylococci No. (%) of strains exhibiting a zone of Staphylococcus No. of synergistic, complete hemolysis with: species strains C. tested S. inter- pseudo- S. aureus" tuberculomedius' S. haemolyticus (100) 23 (100) 21(91) S. simulans (100) 16 (94) 15 (88) S. capitis 5 5 (100) 4 (80) 4 (80) S. cohnii 1 1 (100) 1 (100) 1 (100') S. epidermidis (81) 58 (74) 54 (69) S. warneri (79) 11 (79) 9 (64) S. xylosus 7 5 (71) 5 (71) 5 (71)' S. hominis S. saprophyticus O S. sciuri S. intermedius S. aureus 15 5 (33) 5 (33) 5 (33) a Strain AB 148. bpaper disk impregnated with beta-lysin. C Strain ATCC d Partial at 24 h but clear at 48 h. J. CLIN. MICROBIOL. for the presumptive identification and grouping of streptococci. The results are listed in Tables 2 and 3; some of the reactions are shown in Fig. 5 and 6. All but three of the reference strains that gave synergistic hemolysis with strain AB 148 of S. intermedius also gave a positive beta-lysin disk test. The three disk-negative strains were a strain of S. warneri and both strains of S. hyicus subsp. hyicus (Table 2); however, the two S. hyicus reactions with S. intermedius were atypical and should be considered negative, so the only real discrepancy was a single strain of S. warneri. All but 7 of the 189 clinical isolates gave the same results with S. intermedius and the beta-lysin disk (Table 3). The seven strains that were positive with S. intermedius but negative with the disk were five strains of S. epidermidis and one strain each of S. simulans and S. capitis. Repeated tests with these strains gave consistent results. None of the reference or clinical strains tested were negative with S. intermedius and positive with the paper disk containing S. aureus betalysin. The beta-lysin pattern of results was again seen when the FIG. 5. Reactions of a commercial beta-lysin disk with reference strains of coagulase-negative staphylococci: two S. haemolyticus (upper and center left), one S. hominids (lower left), and three S. epidermidis (right). is

5 VOL. 22, 1985 SYNERGISTIC HEMOLYSIS IN STAPHYLOCOCCI FIG. 6. Reactions of a commercial beta-lysin disk with reference strains of coagulase-negative staphylococci: two S. epidermidis (upper and center left), one S. simulans (lower left), and three S. capitis (right). reference and clinical strains of staphylococci were tested against the exotoxin of C. pseudotuberculosis (Tables 2 and 3). Only two of the reference strains, an S. cohnii and an S. hyicus strain, were positive with S. intermedius and then negative with C. pseudotuberculosis (Table 2). All but 16 of the 189 clinical isolates gave the same results with S. intermedius and C. pseudotuberculosis. The 16 strains that were positive with the beta-lysin but negative with the exotoxin included nine strains of S. epidermidis, one strain of S. capitis, and two strains each of S. haemolyticus, S. simulans, and S. warneri (Table 3). None of the strains tested were negative with S. intermedius and positive with C. pseudotuberculosis. Strains of S. aureus, S. capitis, S. epidermidis, S. haemolyticus, and S. warneri gave strong positive reactions at 24 h, but strains of S. cohnii, S. simulans, and S. xylosus were very weak at 24 h and then distinct at 48 h. These weak reactions at 24 h were recorded as negative. DISCUSSION The hemolysin produced by strains from 8 of 15 species of coagulase-negative staphylococci displayed the reactivity of a delta-hemolysin; it was active, though variable, on the blood cells of all five animal species, and it gave synergistic, complete hemolysis with a beta-lysin on the sheep, ox, and human cells but not the horse or rabbit cells. We did not see evidence of the epsilon-hemolysin discussed by Elek and others (8, 9); it was described as a wide-zone hemolysin causing complete hemolysis of both sheep and rabbit cells, which clearly distinguished the coagulase-negative skin strains of staphylococci. Our findings agree with other studies (11, 16, 22, 24), which state that the typical hemolysin produced by cultures of coagulase-negative staphylococci is delta-hemolysin. Our coagulase-positive strains exhibited the hemolytic activity described by Hajek (13) and Levy (as quoted in reference 8). The strains of S. intermedius produced a beta-hemolysin, and one strain also had an alpha- or deltahemolysin that gave a clear zone; the strains of S. aureus displayed multiple hemolysins, including delta-hemolysin. One strain of S. hyicus subsp. hyicus gave a late-positive tube-coagulase test result; both strains of this subspecies and one strain of S. hyicus subsp. chromogens produced a delta-like hemolysin that was only detected because of its c synergistic reaction. No hemolysins were reported in a previous study of S. hyicus (6). Kloos and Schleifer (18, 31) tested strains for hemolysis on bovine, human, sheep, and rabbit blood agar plates after incubation for 24, 48, and 72 h. Their simplified scheme (19) for differentiating species of coagulase-negative staphylococci includes a test for hemolysis with 5% bovine blood in P agar that is read at 72 h; hemolysis is reported as strong (+) or moderate-to-weak (±) on the basis of zone width from the culture streak. The zones are not described as clear or incomplete or as having either distinct or blurred edges. In defining the hemolysis of a species, they sometimes used the words partial, weak, moderate, or good but did not use the terms alpha, beta, delta, or epsilon to define the hemolysin. In their studies of nine species (18, 31), some strains of each had no hemolytic activity, some strains of each gave weak hemolysis, and some strains of S. haemolyticus, S. cohnii, S. xylosus, and S. warneri gave strong hemolytic reactions. No hemolytic reactions were reported for other coagulasenegative species (6, 20, 21, 28-30). During our studies, some or all of the strains tested from nine different species of staphylococci gave positive synergistic hemolysis tests. Many times the positive synergistic reactions were completely unexpected, because the 24- and even 48-h growth of the strains on TSA II plates had shown little or no visible hemolytic activity with sheep cells. These seemingly nonhemolytic strains were apparently producing small amounts of hemolysin that did not show on sheep cells unless the cells were also attacked by a beta-lysin or an exotoxin. The results obtained with the two staphylococcal betalysin test systems were comparable. The S. intermedius growth and disk of S. aureus beta-lysin produced positive synergistic-hemolysis test results with strains of the same nine species of staphylococci. The negative strains of S. warneri and S. xylosus gave weak reactions at 24 h with both the growth and disk tests, but the zones were not completely cleared and barely extended beyond the growth of the strains. The positive strains of both of these species gave very strong reactions at 24 h. The clear, arc-shaped areas seen with S. hyicus subsp. hyicus in the S. intermedius growth tests were also seen with the disk tests, but only after prolonged incubation; at 24 h, the zones were hazy, but the arcs had not yet developed. These diminished reactions and the strain of S. warneri that was negative only in the disk test were the only differences seen in the reactivity of reference strains in the two staphylococcal beta-lysin test systems. A few more differences in reactivity were seen with clinical isolates; seven strains from three other species were negative with the disk test and positive in growth tests. It is unlikely that these differences are due to the different species sources of the beta-lysins, since the beta-lysins from several strains each of S. aureus and S. intermedius gave identical reactions in growth tests. The differences we observed were probably because of less reactive beta-lysin in the paper disks. The growth test has a continuous supply of fresh beta-lysin diffusing from the growing organism, but the reactivity in the disk test is limited by the measured quantity and strength of the partially purified beta-lysin prepared by the commercial supplier. The reactive zone around a disk soon reaches its limit, but the zone from a growing organism will grow stronger with continued incubation. Despite this limitation, the beta-lysin disk is a reasonable alternative to a viable Staphylococcus strain as a source of beta-lysin for synergistic-hemolysis tests. The differences seen with the third test system, C.

6 414 HÉBERT AND HANCOCK pseudotuberculosis exotoxin, were much greater. Most strains gave reactions comparable to those seen with the two staphylococcal test systems, but 48 h was required for the positive strains of three species to produce clear zones, and several more strains were nonreactive with this test than with either of the beta-lysin tests. C. pseudotuberculosis exotoxin should not, therefore, be used to test staphylococci for delta-lysin production, but the data we obtained demonstrated the synergistic relationships between these organisms. Synergistic hemolysis has been the subject of many previous studies of a variety of microorganisms. In 1944, Christie et al. (4) described a lytic extracellular agent produced by group B streptococci (Streptococcus agalactiae) that gave complete hemolysis when the streptococcus was grown in a zone of staphylococcal beta-lysin on sheep or ox blood agar plates. Murphy et al. (27) later called the lytic phenomenon the CAMP reaction and defined the CAMP test as a way to determine the ability of streptococci to produce a lytic agent (CAMP factor) which gave a hemolytic zone with staphylococcal beta-lysin. The acronym CAMP was thus established, and since then a number of organisms have been reported as CAMP positive or said to produce the CAMP reaction with S. aureus beta-lysin; Listeria monocytogenes, Pasteurella haemolytica, Loefflerella pseudomallei, Corynebacterium equi, and C. renale (10); Mobiluncus mulieris and M. curtisii (33); and Propionibacterium acnes (15). In another study (32), the purified beta-toxin of S. aureus and an isolated exotoxin of C. pseudotuberculosis gave synergistic lysis with the delta-hemolysin of S. aureus and epsilon-hemolysin of coagulase-negative staphylococci (species not determined). Under anaerobic conditions, the alpha-toxin of Clostridium perfringens gave synergistic lysis on sheep (35), human, and guinea pig blood agars (12) with groups A, B, C, and G streptococci and on sheep and human blood agars with P. acnes (15). A reverse CAMP test was proposed (14) with group B streptococci in a synergistic hemolysis reaction for presumptive identification of C. perfringens. Darling (5), however, argues that the acronym CAMP should only apply when testing strains of streptococci against staphylococci and that the phrase "synergistic hemolytic effect" or some similar one should be used to describe the potentiation of hemolysis by any other bacteria in a zone of staphylococcal beta-lysin or similar bacterial exosubstance; we agree. We have demonstrated three of the synergistic relationships that some species of coagulase-negative staphylococci have with other organisms; on agar plates containing ox, sheep, or human erythrocytes, strains of many of the species that we tested exhibited weak or no hemolysis alone but then showed strong, complete hemolysis in a synergistic reaction with the beta-lysins of S. aureus and S. intermedius and the exotoxin of C. pseudotuberculosis. In light of the reports mentioned above, it is likely that the same pattern of reactivity would occur among these species of staphylococci if they were tested with the alpha-toxin of C. perfringens. Perhaps one role of some of these organisms in human infections has been that of a synergist, but when some other more recognized pathogen was also isolated from a clinical specimen, the coagulase-negative staphylococcus was labeled a contaminant. Our data suggest that (i) the coagulasenegative staphylococci, which may act as synergists, should never be discounted as irrelevant when isolated from clinical material; and (ii) further studies of synergism may help clarify their clinical significance. In addition, if the same pattern of results seen in our study of reference and clinical J. CLIN. MICROBIOL. strains of 17 species of staphylococci is obtained with still larger groups of strains, a synergistic hemolysis test might prove helpful in differentiating the species. ACKNOWLEDGMENTS We thank P. B. Smith and Clyde Thornsberry for their critical reviews of this manuscript, Robert E. Weaver and Dannie G. Hollis for providing the strains of Corynebacterium species, and Carol G. Crowder for technical assistance with staphylococcal identifications. LITERATURE CITED 1. Aldridge, K. E Coagulase-negative staphylococci. Infect. Control 3: Baird-Parker, A. C Gram-positive cocci. Genus Il. Staphylococcus, p In R. E. Buchanan and N. E. 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