5rREPl'OCOCCUS PYOGENES IN SCARLEl' FEVER AND THEIR PENICILLIN SENSITIVITY by Shirley Cahn A Thesis subndtted to the Faculty of Graduate studies and Research of McGill University in partial fulfillment of the requirements for the degree of Master of Science. Department of Baoteriology and Immunology August 1953 MoGill University Montreal, Quebeo. ACKNOWLEDGEMENTS In appreciation of help and guidance, l would like to eJq>ress IllY gratitude to Professor E.G.D. Murray. l wish to thank Dr. O. Morgante for her assistance and encouragement given me throughout the investi­ gation. l gratefully acknowledge the use of laboratory facilities and the co-operation of the staff of the Alexandra Hospital. TABLE OF CONI'ENI' S Page 1. Historical Review. 1. Etiology of scarlet fever ..•••••••••••.••••••••••• l 2. Mode of action of penicillin •.••••••••••••.••••••• 13 3. Bacterial resistance to antibiotics •.••.••••..•••• 15 4. Antibiotic inhibitors ••••.•..•••..•••••.•••..••••• 19 5. Mechanism of acquired resistance ••.•.•••.••••••••• 21 II. Statement of Purposes of this Investigation .••••.••••••••••• 27 III. Materia1s and Methods 1. General media............... .. .. .. .. 28 2. Lancefie1d grouping technique •••••.••••••••••••••• 28 3. Penici11in sensitivity test •••••••••.•••••••.••••. 29 4. Sulphadiazine sensitivity test •••.•...•••••.•••••• 29 5. Routine of cultures .................................... 30 6. Schedule of therapy .•••••..•.•••••••..•.•••..••••• 31 IV. Experimental Resu.lts ..................... ........................ 32 V. Discussion and Conclusions •••...•••••••••••••••••••••••••••• 50 VI. final S'uInma:ry of Resulta ....................................... 53 VII. Bi bliograplly' . .. .. .. .. .. .. .. .. .. .. .. .. .. 54 -1- HIsrœICAL REVIE.W 1. Etio1ogy of Scar1et Fever. As far back as 1884, Loefner (59) had directed attention to the haemolytic streptococci which were more or less constantly present in the throats of acute1y ill scarlet fever patients. Lemoine (58), in 1895, found streptococci alone in 93 cases when examining the mucus trom 117 throats in 8car1et tever, while 14 cases had streptococei in association with other organisme. The absence ot streptoeoeci in only 5 of 701 searlet fever patients was reported by Baginsky and Sommerfeld (:3), while Ruediger (88), examining 75 throats of patients i11 with this disease, found that 64 showed streptoeocei alone, with pneumococci and streptocoeei occurring together in 10 cases. Sueh widespread and genera1 re1ationship of a haemolytie strep­ tococcue (called then Streptoeoccus haemolyticus) to searlet tever naturally gave rise to the view that the streptococci may be the etiologica1 agent of this disease. This idea caused much discussion during the tirst years of the present century, and led to the prep­ aration of a number of antistreptococcic serums for the treatment of this disease (Marmorek (62), Moser (70), Moser & von Pirquet (71), and Aronson (2) ). In 1907, Gabritschewsky (30) active1y immunized children.:-with a scar1atina1 streptococcal vaccine, whose potency was later demonstra­ ted by Polotevkova (78). Since haemo1ytic streptococci were known to he associated with a variety of pathologiesl manifestations, viz., otitis media, adenitis, -2- cellulitis, septicemia, and erysipe1as, controversy concerning its primary significance in scar1et fever large1y revo1ved about the question of the specificity of the streptococcus associated with it. Using the streptococcus of scarlet fever as antigen, immune serums were prepared by Moser (70), Moser & von Pirquet (71), Meyer (66), and Rossiwa1l &. Schick (87). These serums were found to agg1utinate specifica1ly various strains of scar1atinal streptococci, but not the haemo1ytic streptococci from non-scarlatinal sources. However, Hasenknopf & Salge (46), Aronson (2), and Neufe1d (72) fai1ed to confirm these results. They were of the opinion that it was not possible to differentiate between the types of haemolytic strep­ tococci by means of the agglutination reaction, and that these org­ anisms bear ooly a secondary relationship to scar1et fever. This view was supported by the findings of Jochmann (49) who reported his failure to iso1ate streptococci from the blood or tissues of patients dying from a ma1ignant f'orm of the disease. An attempt to divide the 8treptococci into groups on the basis of biochemical reactions was made by Cumpston (18). In over 250 cases of' scarlet fever, he found that 50% of streptococci isolated from the throats of patients gave identical results. St reptococci iso1ated from abcesses in various parts of the body were identical biochemically in 68.#% of cases. Both groups were of the sarne type. Bliss (11) noted certain diff'erences in cultural characteristics, particularly in the fermentation of' carbohydrates, among strains of scarlatinal streptococcie The use of fermentation tests failed, therefore, to establish a distinct group which was constantly present in scarlet fever. -3- Renewed interest in the antigenic analysis of the haemolytic streptococci led Dochez and co-workers (24) in 1919 to differentiate haemolytic streptococci from human sources into four biologie groups by means of agglutation and protection reactions. Working with 25 strains of Streptiococcus haemolyticus (sic) isolated from throats ot scarlet fever patients, Dochez & Bliss (11 & 25) performed eross­ agglutination tests with 4 antistreptocoeeie serums ol searlatinal origin and 5 antiserums ot non-scarlatinal sources. Twenty of the straine tested were agglutinated by the scarlatinal antiserums, while none reaeted with the non-scarlet lever antiserums. Tunnicliff (9S & 99) lound that the serum ot sheep immun1zed with haemolytic streptococci isolated trom the acute stages of scarlet fever contained opsonins and agglutinine for the haemolytie strep­ tococci that prevailed in the throat and complieating lesions early in this dieease, but not tor those tound in erysipelas, mastioditis, measles, intluenza, diphtheria and the normal throat. Results ot absorption tests upheld these tindings. His immune serum retained its specifie antibodies tor Il months in the icebox. Similar results were obtained by Blies (12) using these methods, although on the basis ot morphological and cultural characteristics he could not demonstrate any homogeneity among scarlatinal streptococcie stevens & Dochez (96) recovered haemolytic streptococci trom S7.5% of scarlet lever throats, and the lDa.lority ot these strains were agglu­ tinated by 2 antiscarlatinal streptococcal serums. Cross-agglutinations were pertormed with strains obtained trom scarlet tever occurring in various large cities in U.S.A., and Europe, the results of which were consistent, and strains obtained from conditions other than scarlet lever were not agglutinated. -4- A differentiation of haemo1yt.ic streptococci into 3 distinct groups on the basi e of absorption ot agglutinins wae made by Gordon (39). Type l wae the 1argest serologica1 group to which be10nged the vast majority ot straine from localized or genera1 streptococca1 infections, from cases of puerperal sepsis and trom infections of the respiratory passages other than ec~r1et fever. He said this type represented the species streptococcus pxogenes. Type II occurred very rarely. Type III RS chieny to be tound on the tonsi1 and tauces in scar1et tever. He ca11ed this type streptococcus scar1atinaeand claimed it to be quite distinct sero- 10gica1ly from streptococcus pyogenes. Gordon fe1t that the prevalent view at that time, that the streptococcus represented ooly a secondary intection in scar1et fever, based main1y on the point that it was in­ distinguishable from streptococcus pyogenes recovered trom non-scarla­ tinal /Sources, shou1d be revised in the light ot his findings, as weIl as those ot Bliss (11 & 12), Tunniclitf (98 & 99), and stevens & Dochez (96). Dick & Dick (22) attacked the problem ot the relationship of haemolytic streptococci to scarlet tever by an attempt to reproduce the disease experimental1y in man. A group ot 5 vo1unteers were inoculated with a 4-day old culture of haemo1ytic streptococci, but only one develop­ ed typical, mild, scar1et tever, while another showed only sore throat, fever, but no rash. Five more volunteers were inoculated with a Berketeld V tiltrate ot a streptococcal culture, and all remained welle On the llth day after this inoculation, all were injected with unfiltered culture. This time one developed typical Bcarlet fever, another had tever and sore throat, while a third had just sore throat. Although they con­ c1uded that the two cases of experimental scarlet fever were probably -5- caused by the haemo1ytic streptococci or b,y some unrecognized organism c10se1)" associated with it in cultures, the)" did not go 50 far as to c1aim that a11 cases of scar1et fever are caused by the haemo1ytic streptococci. A l'ear 1ater they performed a sldn test (23) that bore a specific relationship to immunity to scarlet tever. Using the Berkefe1d V fi1trate that had fai1ed to produce scar1et tever (22), a 1:1000 dilution "as made and filtered through a Berketeld W fi1ter. An injection of 0.1 cc. of this di1ution"as made into the skin, the sma11 resultant whea1 disappeared in a tew minutes. Results of many skin tests showed that convalescent scar1et tever patients showed nega­ tive or slightly positive skin tests, whi1e in 2 cases where tests were pertormed bafore and after an attack ot the disease, the test was posi­ tive before, and negative during convalescence. The action of the ti1trate