Journal of Clinical Investigation Vol. 43, No. 11, 1964 Studies of Staphylococcal Infections. I. Virulence of Staphy- lococci and Characteristics of Infections in Embryonated Eggs * WILLIAM R. MCCABE t (From the Research Laboratory, West Side Veterans Administration Hospital, and the Department of Medicine, Research and Educational Hospitals, University of Illinois College of Medicine, Chicago, Ill.) Many of the determinants of the pathogenesis niques still require relatively large numbers of and course of staphylococcal infections remain staphylococci to produce infection (19). Fatal imprecisely defined (1, 2) despite their increas- systemic infections have been equally difficult to ing importance (3-10). Experimental infections produce in animals and have necessitated the in- in suitable laboratory animals have been of con- jection of 107 to 109 bacteria (20-23). A few siderable assistance in clarifying the role of host strains of staphylococci have been found that are defense mechanisms and specific bacterial virulence capable of producing lethal systemic infections factors with a variety of other infectious agents. with inocula of from 102 to 103 bacteria (24) and A sensitive experimental model would be of value have excited considerable interest (25-27). The in defining the importance of these factors in virulence of these strains apparently results from staphylococcal infections, but both humans and an unusual antigenic variation (27, 28) which, the usual laboratory animals are relatively re- despite its interest, is of doubtful significance in sistant. Extremely large numbers of staphylo- human staphylococcal infection, since such strains cocci are required to produce either local or sys- have been isolated only rarely from clinical in- temic infections experimentally. Elek was un- fections (29). The insensitivity of these experi- able to produce local purulent lesions in humans mental systems has been a handicap for compari- with inocula of less than 106 staphylococci (11) son of the relative virulence of strains of staphylo- but subsequently demonstrated that the infectious cocci and evaluation of the effect of antibody and inoculum could be reduced by two to four logs other defense mechanisms in a living system. when the infection was combined with concomitant Preliminary studies of staphylococcal infections insertion of a foreign body (12). Even larger in a variety of animals indicated that embryonated numbers of staphylococci have been required to eggs were readily infected with pathogenic staphy- produce local lesions in mice (13, 14), guinea lococci (30). The present study describes the pigs (15, 16), and rabbits (17, 18). Various sensitivity of embryonated eggs to 68 strains of traumatic procedures or implantation of foreign coagulase positive Staphylococcus aureus and 36 bodies also allows the production of infection with strains of coagulase negative Staphylococcus epi- smaller numbers of bacteria in animals (14, 19), dermidis and defines some of the characteristics but despite such measures many of these tech- of this infection. Subsequent publications will de- * Submitted for publication May 4, 1964; accepted scribe the importance of individual bacterial viru- July 9, 1964. lence factors (31) and the protective effect of anti- Reported in part at the joint sessions of the American toxic and antibacterial antibody in the chick em- Society for Clinical Investigation and the American bryo (32). Federation for Clinical Research, April 28, 1963, Atlantic City, N. J. Methods This work was completed during the tenure of a Veterans Administration Clinical Investigatorship. Strains of staphylococci. Forty-two strains of coagu- t Present address: Evans Memorial Department of lase positive Staphylococcus aureus were obtained from Clinical Research, Massachusetts Memorial Hospitals, blood cultures and specimens of sputum or purulent and the Department of Medicine, Boston University exudates submitted to the routine bacteriology labora- School of Medicine, Boston University Medical Center. tory. In each instance, the patient from whom the 2146 EXPERIMENTAL STAPHYLOCOCCAL INFECTIONS IN EMBRYONATED EGGS 2147 specimen was obtained was examined to insure that the When recoveries were carried out after injection of a staphylococcus isolated was etiologically related to the large inoculum, 12-day-old eggs in which the volume of clinical infection. Additional strains were obtained allantoic fluid is stated to be 6 ml (33, 34) were used from the anterior nares and skin of a group of patients for injection. One-tenth ml samples of allantoic fluid without clinical evidence of infection who were attend- were aspirated aseptically after removal of the shell ing an outpatient clinic for the first time. Specimens over the egg sac, plated, the colonies enumerated, and were obtained only from outpatients who had not re- the numbers corrected for the 1 to 60 dilution. Re- ceived antibiotics within the past 3 months, had not covery with the smaller inoculum was performed simi- been hospitalized during this period, and whose household larly, except as much of the allantoic fluid as possible contacts had not been hospitalized recently. A third was aspirated and 4 ml of PS was injected, mixed, and group of 36 strains of coagulase negative Staphylococ- removed. The latter procedure was repeated nine times, ctus epidermidis was obtained from a variety of speci- and the pooled allantoic fluid and saline washings were mens submitted to the hospital laboratory and from the filtered through a Millipore H.A. filter (0.45 Ah). After nares and skin of outpatients. filtration, the filter was removed, placed on a blood Tube and slide coagulase determinations and mannitol agar plate, incubated for 48 hours, and the number of fermentation were performed on all isolated strains to bacterial colonies determined. insure their identification. Strains of staphylococci were In vitro growth curves. Allantoic fluid was collected maintained on tryptose phosphate (TP) agar slants aseptically from 15 eggs, pooled, and 4.9 ml dispensed at 0° C after isolation and identification until egg viru- into sterile capped tubes for determination of growth lence studies were performed the following week. curves. One-tenth ml of a 10' dilution of the organism Immediately before use, a single colony selected from to be tested was added and the allantoic fluid incubated a subculture of the original agar slant was inoculated for 18 hours. Duplicate determinations were made at into TP broth for the infectious inoculum. Several 0.1- each sampling time. Concomitant growth curves in TP ml samples of the latter were transferred to 2.5 ml of broth were performed in a similar fashion. TP broth in screw-capped vials and stored in a - 200 C In vivo growth curves. Lots of 64 10-day-old embry- deep freeze for subsequent use. onated eggs were injected with 0.1 ml of a 10' and 10' Preparation and injection of inocula. Serial tenfold dilution of coagulase positive staphylococci and 0.1 ml of dilutions of overnight TP broth cultures of staphylococci a 10' dilution of coagulase negative staphylococci. were made to provide inocula of approximately 1,000 to Four eggs from each of the three groups were sacrificed 99,999 (10' to 10') and 1 to 100 colony forming units at 4, 8, 18, and 24 hours and daily thereafter for 10 days. (cfu). The smaller inocula consisted of less than 10 Only eggs containing living embryos were sacrificed to cfu in over one-half the studies. The size of the inoculum insure that acceleration of growth and invasion of the was determined by plating 0.1 ml of 10-', 10', and 10- embryo "post-mortem" did not obscure the results. dilutions of the overnight culture on TP agar and enu- The shell over the air sac was removed; allantoic fluid merating the number of colonies after overnight incu- was aspirated aseptically and serially diluted for plate bation. Lots of 20 10-day-old fertile eggs were injected counts. The remainder of the allantoic fluid was re- intra-allantoically with 0.1 ml of 10-s and 10' dilutions moved, and 1.5% saponified cresylic acid' was added to of each strain studied, and the eggs were candied daily the shell containing the intact yolk sac, amnion, and to determine viability. A group of ten controls was in- embryo. After 10 minutes, the cresylic acid solution jected with a similar volume of sterile 0.85% saline and the yolk were removed. The amnion and embryo (PS) in each experiment. Subcultures were obtained were extracted with sterile forceps and placed in a from allantoic fluid, yolk, and embryonic blood in the sterile petri dish. The amnion was removed and the first sets of experiments and, when indicated, in sub- embryo washed in the cresylic acid solution and rinsed sequent studies to insure that maternal transmission of with sterile PS. The embryo was opened by a ventral infection or subsequent contamination had not occurred. incision, and the heart and liver were removed asepti- Eggs. Fertilized eggs were obtained from a com- cally. These were ground in a glass tube with a Teflon mercial source that did not use an antibiotic dietary tissue grinder, and 0.2 ml of the bloody supernatant supplement. Tube dilution assays for antibacterial ac- fluid was removed. Bacterial titers were determined by tivity were performed on allantoic and amniotic fluid on plating 0.1 ml of undiluted fluid and serial tenfold several occasion with negative results. Eggs were incu- dilutions of the fluid. bated for 10 davs at 370 C in a rocker incubator before infection and- then incubated upright at 38.5° C with Results 60% relative humidity for an additional 10 days after infection. Lethality of coagulase positive and coagulase Recovery of inoculum. Studies to determine the num- negative staphylococci. The 10-day fatality rates ber of cfu that could be recovered immediately after in- for 10-day-old eggs injected intra-allantoically with jection were carried out by two techniques. Eggs were inocula of two different sizes of 68 strains of co- injected as described above, allowed to stand for 15 min- utes, sealed with paraffin, shaken gently, and sacrificed.