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 in the skin was inhibited by convalescent serum mixed with the fi1trate betore it was injected, or given intramuscular1y before the test

"as made.

Dochez & Sherman (26) inoculated guinea pige with scarlatina1 strep­ tococci and produced a disease ressembling scar1et tever in its main features. By immunizing a horse with this streptococcus, they obtained an antiserum which possessed the capacity to blanch the rash 10cally in scar1et fever and which, when used therapeutical1y, caused amarked abatement of a11 the s.y.mptoms. Klin10ch, Smith & Taylor (52) made a comprehensive study ot scar1et fever and the relation to it of streptococcus scarlatinae (sic). They

"ere opposed to the current view (1927) of the majority of epidemio10gists who believed "that the virus of scar1et tever is unknown, that those patients who are overwhelmed and prostrated at the very onset are probably intoxicated with the true scar1atinal virus, and that streptococci are -6-

probably the most important bacteria of secondary infections." From 247 cases of scarlet fever, 209 strains of haemolytic streptococci were ob­ tained from throat cultures. These strains could be divided into various groups by means of agglutination and absorption tests. They found that the same type of streptococcus could on occasion originate at least 5 separate cUnically distinguishable diseases, scarlet fever, tonsillitis,

erysipelas, puerperal fever, and bronchopneumonia, and they concluded that so far as the streptococcal infections are concerned, the nature of the disease entity is determined by the toxigenic qualities of the type

of streptococci, by the susceptibility or insusceptibility of the indivi­

dual as determined by the absence or presence of the specifie antibodies

in the blood, and by the site of the infection itself. They further cor­

roborated their view that the haemolytic streptococci are the cause of

scarlet fever in the following findings :

a} The whole clinical picture of scarlet fever going on to marked

desquamination il5 produced by the subcutaneous injection of the exotonn

derived trom haemolytic streptococci, the exotoxin having been heated to

550 C. for 1 hour to destroy any living filterable virus which might he

associated with the filtered exotoxin. b) In the Schultz-Charlton reaction (S9) the blanching of the scarlet

fever rash is caused by the serum of a horse immunized against scarlatinal streptococci and its toxins.

c) Dick-positive and Dick-negative reactions as produced by the intra­

derme.! injection of the streptococcus toxin have the closest correspon­

dence to the degree of susceptibility or immunity to scarlet fever of the

respective reactors.

d) Susceptible nurses and schoolchildren once they are actively immunized -7- by injections of scarlatinal streptococcal toxin have been shown to be immune to the toxin of scarlet fever.

These workers also noted that coincident with the disappearance of scarlet fever in the immunized nurses, there had been a notable in­ crease in the incidence of streptococcal tonsillitis in these immunized nurses. They concluded that immunization protects the individual from the toxic effect of the haemolytic streptococci but not from tonsillitis.

Studying the toxigenic properties ot the haemoly1:.ic streptoeocei,

McLachlan (64) tound, without exception, that 98 strains ot these organ­ isms obtained trom scarlet tever cases produced a toxic principle which in high dilutions was capable ot evoking a cutaneous reaction in suscept­ ible persons. A certain proportion ot the haemolytic streptococci from non-scarlatinal sources possessed a similar toxigenic property, but in the majority ot cases this property was only weakly developed a8 judged by quantitative tests. The tact that on thè whole scarlet tever strains produce more toxin would indicate that these strains torm s group, dif­

terentiated to some extent trom other streptococci b,y their greater power ot producing this extracellular toxic !!ubstance. Griffiths (42) tound that f:l:d> ot strains ot streptococci from acute scarlatina fell into one or the other of types l, 2, 3, and 4 of his classification which by 1934 enlarged to 27 types (43). Support for the

haemoly1:.ic streptococci as the cause ot scarlet tever was provided by bis

tinding in 9 instances the sarne type ot streptococci in 2 or more members

of the same tamily in which scarlet fever had occurred.

Smith (92) reported that his two main serologicsl types conformed with Griffiths' Types 1 and 2. He also determined the agglutinin titre

of scarlet tever patients' serum tor the Btrain of haemolytic streptococei isolated fromthe.throat at the commencement otthe illness (93). Fort y -8-

percent of cases showed a definite increase in the agglutinin titre during the course of the disease, which would indicate that this or­ ganism plays a definite part in the etiology of scarlet fever. Lancefield (55, 56) demonstrated that haemolytic streptococci could he differentiated serologically by means of the precipitin re­ action into distinct and sharply defined groups which were not dis­ closed by the agglutination reaction. The group specifie substance was identified as carbohydrate in nature. She ealled her groups A,

B, C, D, and E. This classification has sinee been added to by

Lancefield and other workers (57, 44). Thus Laneefield has made it possible to identify the strains which are commonly pathogenic to man,

GroupA, presently recognized as constituting the species Streptococcus pYogenes. The serological types described by Griffiths (43) are merely a subdivision of this species into different antigenic types (75). The erythrogenic toxin which was tirst thought to be a distinctive product of those strains of haemolytic streptococci associated with scar1et fever has been shown to be characteristic of streptococcus pxogenes in general and not of scarlatinal strains in particular (64). Returning to the question of the role of haemolytic streptococci in scarlet fever, the high frequency with which these organisms have been isolated from patients with this condition has been reported by many authors. Bliss (12) had tound haemo1ytic streptococci in 100% of patients during the firet week of the disease. Forty cases were examined by stevens & Dochez (96) during the acute stage of the i11nes5, 87% of which h&rboured haemolytic streptococci in their throats. Klinloch et al

(52) studied 247 cases of scarlet fever on admission to hospital. They found 209 positive throats and recovered l strain from pus in a case ot surgical scarlet tever. The percentage of positive throats on admission -9- was def'initely related to the day of' i11ness when the patient came to

hospital, sinee the rate dropped f'rom 91% and 9~ on the let and 2nd days of' il1ness to 76% and 6z:1, on the 3rd and 4th days, and as low as

52% on the 5th and later days. From nasal swabs only 16 of' the 247 were positive. Gasul & Rhoads (31) reported a 100% incidence of haemolytic streptococci in a group of 81 patients, 42 of which were positive in both nose and throat, 34 only in the throat, 1 in the nose onq and 4

positive in surgical lesions. A total of 1875 cases of scarlet fever were examined on admission by Green (41) who recovered haemolytie strep­ tococci from the throats of 84.3% of these patients.

After Lancef'ield developed the method for grouping haemolytic strep­

tococci, many etudies were made testing these organisms with Lancef'ield

grouping sera.

Over a period of one and a half' years, Boisvert (13) grouped by the

Lancef'ield method all available 8trains of haemolytic streptococei f'rom

248 patients with streptococcal disease. All of the 408 etrains tested

fell into Group A. streptococci of Groups B, C, or G were occasionally

encountered, but there was n~ evidence that they were causing disease.

Similar results were obtained by Estrada (29), who round only Group A

organisms in sick chi1dren, and not a single case of' Il streptococcal

diseaseu (scar1et fever, erysipelas, streptococca1 bacteremia, and strep­ tococcal pneumonia) which did not yield Group A organisms.

Boisvert & Bearg (15) &lso had the opportunity to observe a class

of schoolchildren during a scar1et fever epidemic. 18 of the 46 pupils

had Group A streptococci in their throats during the four month study.

Thirteen of the 18 became ill, e1even with scarlet fever, and two with

cervical adenitis, the organisms being of Type 3 in all cases. T hree

chi1dren harbouring Group A streptococci, Type 3, remained healthy but -10-

carried disease to their respective families. The two hea1thy carriers were shown to have a difterent Type of Group A streptococci. Five chi1dren with Groups B, C, or G in their throats did not come down with any i11ness.

The distribution of haemo1ytic streptococci of Groups A, B, or C in human infections was reported by Rantz &.. Keefer (84). They grouped

1159 strains : 95.2% Group A, 1.6% Group B, and 1.2% Group C, with 2% unclassified. 431 of the Group A organisms were obtained from scar1et fever, whi1e Group B were occasiona1ly the etio1ogica1 agent of puer­ peral I~sis, bacteriological endocarditis, suppurative arthritis and urinary infections. Group C were not found to be the cause of infection in any of the cases studied.

Bailey (4) found that of 243 cases of scar1et tever studied, aIl had Group A streptococci, the majority of which were Types 2, 3, or 6.

A smal1 but appreciab1e number of cases of scar1et fever was caused by a type of streptococci frequent1y found in erysipe1as.

In a group of 73 cases of scar1et fever, Boisvert (14) recovered

Group A haemolytic streptococci from aIl throats and from 24 noses.

These organisms were occasionally found in the ear, b100d, pleural fluid and abscesses of the patients. Another 189 patients suffering from other streptococca1 diseases (erysipelas, tonsillitis, pneumonia, otitis media, meningitis, cellulitis, sinusitis, suppurative adenitis, septicemia, pericarditis and mastoiditis) were shown to be harbouring Group A strep­ tococci in aIl cases.

deWaa1 (21) checked the nose and throats of 415 cases of scarlet fever within four hours of admission to hospital. 88.2% had Group A streptococci in either nose or throat. The negative patients were swabbed the fol1owing day, bringing up the total percentage of positive patients to 96.6%. In -11-

9 cases the nose only was positive. The author typed the st.rains and found only two cases where the nose and throat strains were of dif­ ferent Types.

An investigation of the Types of haemolytic streptococci found in tonsillitis without skin rash was conducted in association with studies of cases of scarlet fever during the saroe period (53, 54).

In Tokyo, from January to March 1935, 65% of cases of tonsillitis and

6~ of scarlet fever were shown to harbour st.rains of Type 2, 4, or 6,

Group A streptococci. While the intra type frequency distribution was not identical in each case, it would appear that epidemics of scarlet fever and tonsillitis occurring simultaneously may be caused by the sarne small group of Types in each case.

Rantz (S3) was of the opinion that the question as to the presence of a specifie tscarlet fever streptococcus' was settled by the observa­ tion that the same types of streptococcus can cause both scarlet fever and tonsillitis without a rash. He felt that the most important factor governing the production of the two diseases is the state of antitoxic immunity in the individual at the onset of the infection. That important di:tferences in the organism must also exist is suggested by the numerous instances in which haemolytic streptococcal pharyngitis without rash has occurred one or more times in individuals who have later acquired scarlet fever. He does not mention, however, whether in this latter instance the streptococci were of the saroe Type in the pharyngitis as in scarlet fever.

Powers & Boisvert (79) list the dif:terent human diseases which can be caused by Group A streptococci. Included are scarlet fever, tonsillitis, pharyngitis, and rnany infections of skin, bone, glands, viscera, meninges,

$inuses and blood. These authors report that the severity of illness was not shown to be related to the serological Type of organism and that no -12-

single Type is regularly associated with a partieular ellnieal syndrome. A v1rus-streptoeoceus combination as etiological agent for scarlet fever was a theory put forward by Bingel (7,8,9,10). He was able to induee non-scarlatogenic haemolytic streptococei to produce a filtrate that would evoke the Dick reaction in a dilution of 1 : 1000. This was demonstrated by adding to cultures of non-toxie strains of haemo1ytic streptococci quantities of a toxie strain before and atter incubation of the former strain. Fi1trates of the subseQuent cultures cou1d evoke the Dick reaction in a dilution of 1 : 1000 only when the toxie strain was added before incu­ bation. Also, if before incubation, sterile searlet fever tonn RS added to any atoxie 5train of haemo1ytic streptococci, the substance deetable in the fi1trate developed anew in the culture. Sub-eultures of this 'secondary' or 'indueed' scarlatina1 streptoeoccus gave rise even in the 4th subculture to full poteney fi1trates, whereas a drop of the cel1-frae fi1trate, after

severa! passages through sterile non-inoeulated broth beeame negative on

such dilution. Evidently the 8carlet fever tonn evoking the Dick reaction, when added to atoxic strains of streptococci before incubation, give to the

subsequent cultures a tox1city of a degree that i8 found in 'primary'

scar1atina1 streptococcie The author conc1udes that it is a virus which converts a non-scarlet tever streptoeoccus into a scar1et fever streptococCU8 and that this virus i8 the necessary precondition for the production of toxin by the bacteria. He found that with the help of scarlet fever anti­

serum, it was possible very quickly to make the 'primary' or 'secondary'

scar1atogenic streptocoeei non-scar1atinal. The cause of this phenomenon

did not appear to be the result of a tox1n-antitox1n union, but the dis­

solution of the virus-streptococeus combination results beeause of the

formation of virus antibodies in the scarlet fever antisera. -13-

2. Mode of Action of Penicilline Hobby et al (48) found that penicillin acts either as a bacterio­ static or bactericidal agent depending on the experimental conditions

and appeared to be effective only when active multiplication took place.

The number of organisms decreased at a constant rate until 9% of the organisms had been destroyed. The rate of ldlling varied with different

organisms. They also noted that no detectable amount of penicillin was

destroyed or absorbed from solution by the bacterial cells.

Experimente were presented by Spicer & Blitz (94) to show that

penicillin was capable of destroying susceptible bacteria, by !yais or

otherw1se, under normal cultural conditions in amounts possible to main­

tain in the body of the patient. The destructive action was not complete,

however, even in high concentrations of the drug, a residuum of viable

cella always remaining, capable of withstanding the destructive action

of the antibiotic, but inhibited from rmùtiplying in its presence. This

inhibitory effect on the residual viable cells extended through a wide

range of concentrations 50 that large and small amounts of the drug had

the eame inhibitory effect on these remaining organisms. The authors

suggest that bacterial cultures do not constitute a homogeneous popula­

tion but that individual members may possess different characteristics as to the manner of reaction under the influence of the antibiotic.

The size of the inocula will influence the sensitivity of the strain

tested. Rasch & Parker (86) found that the less bacteria inoculated on

the plate, the larger was the zone of inhibition around the gutter contain­

ing penicilline

The mode of action of three antibiotics, penicillin, strepto~cin,

and tyrothricin, on cultures of Clostridium was studied by Macheboeuf (61).

Tyrothricin was found to be mainly an agent of cytoplasmic destruction,

accelerating the spontaneous lysis of Clostridium. This action is marked -14- in non-prollferating organisms, where penicillin and streptomycin do not show suèh a lytic effect, and even inhibit noticeably spontaneous lysis. Tyrothricin also prevents the accumulation of ATP during glycol- ysis, and inhibits the formation of acetyl phosphate trom pyruvate, whereas penicillin and streptomycin do not modif.y the phosphoglucocidic metabolism of Clostridium.. Penicillin and streptomycin were found to inhi bi t strongly the catabolism of the mononucleotides, and, as well, inhibit the reactions of deamination coupled with oxidoreduction between pairs of amine acide.

But it was shown that the latter reactions need as an 1 effector' a mono- nucleotide, therefore the inhibition of the latter reaction must be derived fram the primary action of the antibiotic on mononucleotides.

T hi oproli ne , the nucleus of the penicillin mole cule (see fig. 1) was found by Beerstecher (6) to inhibit the growth of Escherichia coli in min- eral salts-glucose medium. This inhibition could be reversed to a limited extent by proline, despite the fact that the inhibitor i5 not isoteric with proline. To a lesser ext.ent, a large number of other amino acids can also reverse the inhibition, but combinations of amino acide exert an even greater reversing ability than any single substance. This would suggest that thio­ proline may inhibit bacterial growth by preventing the conversion of a large number of amino acids to sorne product or producte eBsential for growth.

Penieillin T hioprollne or 4-Thioazolldine carboxyllc acid

FIGURE 1. -15-

3. Bacterial Resistance to Antibiotics The development of resistance to penici1lin has been observed to occur 'in vivo' with strains of viridans streptococci, and StaphYlococcus pyogenes during therapy with this agent (16, 17, 74, 77, S2 & 95). A1- though penicillin has had very wide usage in the treatment of Group A haemolytic streptococcal infections, there have been no reports of the development of strains with lack of sensitivity to this drug which con­

stituted therapeutic problems. SelMe et al (90) has reported a case where Group A streptococci increased resistance l6-fo1d arter penici11in therapy, but it was found that this development of resistance cou1d be overcome with adequate doses of the antibiotic.

streptococcus progenes as encountered in the general population is almost. uniformly highly susceptible to penicillin (0.OO7S - 0.0156 units) on the basis of available information (65, S5, 101). Hirsch et al (47) however, have reported the isolation ot a &train ot this organism which was susceptible only to 0.625 units of penici11in from a patient i11 with

scarlet rever who had not received any antibiotic therapy. Mi1zer et al

(69) round penici11in resistant beta-haemolytic streptococci in the throats

of children with rheumatic rever treated prophylactically with 100,000 units of penicillin oral1y each day for four months. Although these strains were resistant to 10 unite penici11in following the period of prophylU:is,

none of the streptococci present prior to chemotherapy were available for

comparison, nor was the serologica1 group of the 'resistant' strains de­

termined. They 1ater found that 1,000,000 units given orally for five days

was usually enough to rid the throat of haemolytic streptococci and thereby

avoid the possibility ot developing resistant strains.

Hartman & Weinstein (45) isolated str&1ns of beta-haemolytic strep­

tococci from throats and nasopharynx of scarlet rever patients before and

after penicillin therapy. Initial inhibitory concentrations varied from -16-

0.0039 to 0.0313 units, a range that is essentially in agreement with that previously reported for this organism (101). Penici1lin was given over a period of 10 days, 1,200,000 - 3,000,000 units intrarnuscu1ar1y or 3,000,000 - 15,000,000 units orally. Of the 37 different strains is­ olated during therapy, none showed a greater tolerance to the antibiotic than that of the cultures iso1ated at the time of admission. Gt-oup A

streptococci were recovered from 24 casee aiter penici11in treatment was stopped. None of these strains were penici11in resistant, and the

authors were of the opinion that their presence in the throat or naso­

pharynx after a full course of treatment could be exp1a1ned on the basis

of cross-contamination from new patients.

There have been many reports to show that streptococcus pyogenes

cao be made resistant to penici1lin 'in vitro'. McKee and Houck (63)

decreased the sensitivity of 3 strains of staphylococcuspyogenes, one

strain each of types l, II, and III of Dip10coccus pneumoniae, and one stra1n of streptococcus pyogenes to penici1lin, by growing the organisms

in broth containing increasing amounts of the drug. The resistance of

the st~eptococcus was increased 30-fo1d in a period of 3 months, and

there was an accompanying 108s of virulence. Thirty-two rapid transfers in plain broth or two months in the ieebox produced no alteration in the

degree of aequired resistance. Reduced velocity of growth and variation in colonial form were observed during cultivation in media containing in­

creasing amounts of antibiotic, but the organisme grew 1uxuriantly aiter

transfer to plain broth. No change in type of enzyme activity was apparent

a1though fermentation reactions were much delayed.

Working with 6 stra1ns of haemolytic streptococci, Erikson (26) was

able to increase resistance to penicillin 100- 400 iold aiter one month

of dai1y transfers in penici11in medium. On blood agar plate resistant -17-

straine grel' slowl1' and without haemolysis. Virulence was complete1y lost. Even atter long transfers on penici11in-free medium the strains retained their resistance.

The same ease of inducing resistance of st.reptococcus pYogenes to penici1lin l'as encountered by Weinstein & Tsao (102). Tl'elve out of 15 strains were made from 2 to .32 times more resistant. The resistance of

6 strains was aboli shed rapidly by culture in drug-free medium. No striking effects on the colonial and cellular morphology, degree of haemolysis, or rate of growth could be demonstrated during the process of acquisition or 108S of sensitivity. The resistant strains, however, grew more slol'1y, produced slightly smal1er colonies and were a little less haemolytic than the highly susceptible ones.

The reversibi1ity of induced resistance of streptococcus pyogenes to penicillin es a1so noted by Todd et al (97). Aiter 35 subcu1tures in penici1lin broth, the resi stance es increased 5-fold, but l'as re­ turned to the original level of sensitivity aiter 76 transfers in plain broth. On the other hand, they found that pneumococci maintained its

'faetness', or acquired resistance, as a permanent character.

Gazon and his associates performed a series of antibiotic etudies on the beta-haemo1ytic streptococci (32, .33, 34, 35, 36, 37 & .38).

Thirteen strains of Group A streptococci were transferred serially on medium containing penicillin, with the maximum increase in resistance being a l7-fo1d rise (.32). Growing the same organisms in seria1 trans­ fers on control medium did not increase resistance. A marked loss of virulence for mice l'as shown by all resistant strains of streptococcie

In some resistant strains, virulence ns restored atter passage in normal mice. The Gi"oup specifie antigen could not be demonstrated in half of the resistant strains, while transient changes in colonial appearances -18-

and changes in haemolysis l'lere shown by oost strains when grown·,on maximal concentrations of penicilline Resi stant strains l'lere restored to their original level of penicillin sensitivity by seriaI subcultures on penicillin-free medium or by seriaI intracerebral passages in mice.

Similar results l'lere obtained with Group Band C streptococci except that acquired resistance l'las maintained even ai'ter serial passages through mice or seriaI transfers on drug-free medium <:33, 34).

These workers a1so reported the ability to induce resistance of

Group B and C organisms by repeated exposures to sublethal amounts of penicillin in embryonated hens' eggs (35). This could not be done with

Group A organisms. Induced resistance of Group A, B, and C organisma to aureomycin and bacitracin l'las obtained after seriaI subculturea with theae drugs (37, 38).

The resultant decrease in sensitivity l'las much higher than with penicillin: for aureomycin up to 6O-fold, and for bacitracin up to 3,750-fold. How­ ever, mou se virulence, group specifi city, colonial characteristics, and temporariness of the resistance was the same as with penicilline The behaviour with streptomycin l'las somewhat different (36). Arter 40 trans­ fers in antibiotic medium the minimum increase in resistance na 40-fold while the maxl.mum l'las 3000-fold. When compared with resistance of menin­ gogocci (68) ot' staphylococci ~ ., ( 91), streptococci acquired resistance to streptomycin relatively slowly. Mouse virulence ns decreased in 8 of 9 resistant strains, but Da partially restored by passage through normal mice. Transient changes in colonial appearances, and changes in haemo­ lysis from beta to alpha or gamma. was demonstrated when gI'own on maximal concentrations of streptomycine Differing from penicillin, aureomycin or bacitracin resistant strains, group specificity l'las maintained by all strep­ tomycin resistant organisms, and resistance DS kept throughout subculti- -19-

vations in antibiotic-free medium, although partial loss of resistance was observed in 5 of 6 strains on serial passages through mice.

No relationship between acquired resistance to sulphonam1des and

to penicillin has been found in the literature.1' Rantz and cOl-workers (S5)

isolated sulpha resistant stra1ns of Group A organisms, which, when tested

with penicillin, were relatively sensitive to this drug. Rake et al, (Sl)

noted that increased resistance to penicillin was not accompanied by an

increase in sulphonamide resistance with strains of staph.ylococcus pYogenes,

streptococcusNogenes, and TYpe III Pneumococcus.

4. Antiobiotic Inhibitors. Bacteria1 cells have been shown to possess the ability to produce

substances which inhibit, inactivate, or even destroy bacteriostic and

bactericida1 substances. Such specifie chemical inhibitors have been

established for a number of antibiotic substances. Yeasts, for instance,

were found to contain a substance which inhibits the action of sulphon­

amides against streptococci and pneumococci. This substance has been

identified as p-amino benzoic acid (100). Pike & Foster (76) found that Streptococcus pyogenes grew as satel­ lites about colonies of Pseudomonas.aeruginosa and staphYlococcus pYogenes

on plates containing sulphonamides. This phenomenon appeared to be due to

the diffusion through the agar of sulphonamide-inhibiting substances

elaborated by the test colony, but not effective on the sensitive strain

on agar without sulphonamides.

Certain bacteria not inhibited by penicillin were found capable of

producing a substance which deBtroyed the growth-inhibiting property of

this antibiotic (1). This substance, designated as penicillinase, was -20-

enzymatic.· The penicillin resistance of some bacteria was ascribed to their ability to elaborate penicillinase, but this explanation did not apply to others.

Bondi & Deitz (16) demonstrated that resistant strains of l!rt.aph­ ylococci produoed penicillinase, whereas other resistant organisms such as Salmonella tyPhoea .and other salmonellas "ere not shown to produce this enzyme. They oonsidered that the penicillinase-type of resistance of the strains they "orked with RS a natural rather than an acquired one. Organisms made resistant to penicillin 'in vivo' or 'in vitro' did not acquire the ability to form penicillinase. Confirmation of these results was given by Spink & Ferris (95) who found that staphylococci made resistant to penicillin did not acquire the ability to make pen­ icillinase, and that such induced resistance was only temporary, while strains of staphylococci which were found to possess a 'permanent' type of resistance produced the enzymatic inhibitor.

Luria (60) found that protection by penicillinase was a mass phen­ pmenon, occurring only when bacteria wereinoculated in large numbers.

When resistance was bt this type, he felt that individual celle may be more sensitive than the strain appears to be as a "hole, and therefore it would eeem unlikely that penicillinase production be secondarily ac­ quired by strains made resistant to penicilline

Woodruff & Foster (103) reported contradictory results concerning the relation of penicillinase to resistance. They showed that resistant bact­ eria such as Bacillus cereus, Bacillus subtilis, and Bacillus mesentericu8

"ere strong penicillin decomposers, while, on the other hand, Pasturella species, one of the gram-negative rods most sensitive to penicillin was found to have considerable capacity for penicillin destruction. -21-

From the foregoing f'indings, it appears to be obvious that other mechanisms are involved which cause bacteria to become resistant to antibiotics.

5. Mechanisms of Acguired Resistance. T here are t'Wo main theories concerning the origin of microbial resistance to toxic agents :

I. The adapl;ation theory explains resistance as the result of sorne interaction between microorganisme and toxic agent, resu1ting in an increased ability of the cel1s to ldthstand the de1eterious effects of the drug. This may come about either by the establishment in the cells of a mechanism alternative to that norma11y in use, or by the quantita­ tive modifications of exl.eting mechanisms.

II. The opponents to this theory are in favour of exp1aining developed resistance as a mutation and biologica1 selection phenomenon. They believe that resistant straine originate independantly of the antibiotic serving merely as a selective agent in the isolation of mutants, by des­ troying sensitive bacteria. In studying the resistance of staphy1ococci to penici1lin, Demerec (19) and Luria (60) found that for a given inoculum (approx. 300 million bacteria) there was a variation in resistance of about lO-fo1d for al1 their strains, that is, if the inoculum required 0.1 unit penici11in for complete inhibi­ tion, many of the bacteria in the inoculum would be inhibited by as 1itt1e as 0.001 unit penicillin. Demerec indicated that the deve10pment of peni­ cil1in resistance resulted from the selection of the most resistant bacteria in concentrations not comp1ete1y inhibiting growth. By continua1 subcu1tures

~n higher concentrations of the antibiotic, resistance gradua1ly developed -22-

through a series or small increments. In other words, at each step of increased concentration of antibiotic, there would be present cells resistant not only to the concentration used, but to even higher doses.

Therefore, in a series of selections highly resistant strains can be developed.

Klein & Kimmelman (51) reported that, if they examined very large numbers (several billion) of bacteria, it was possible to isolate, from ail six strains of the Shigellae studieà, variants resistant to more than

1000 units streptomycine Ordinarily, the inoculum DS resistant to 3 - 7 units of this drug. Presence of these highly resist.ant variants (approx. l / billion) was indicated to be the critical factor for the very rapid development of streptomycin resistance. A year later, Klein (50) developed four strains of bacteria resistant to 5000 unite streptomycin after only 3

subcultures, while penicillin-treated strains showed only slight increased resistance and never reached the eame level of resistance as obtained with

streptomycine A variation in range of resistance, greater in streptomycin than in penicillin, DS thought to account for the difference in levels of resistance. The author did not diseuse nor determine where or ho,.. these

cells originated. In 1948, Demerec (20) published a paper in which he de!ended the

mutational origin of resistance. Using small inocula, to rule out varia­ tion between cells, and inhibiting doses of streptomycin, he isolated resi­

stant cells. Results showed that the average number of resistants from

20 independent subcultures was considerably less than that of multiple

platings from a single sample. There was much less variance in the single

culture platings than in the independent culture platings. If resistance

were caused by the interaction of antibiotic and bacteria while in contact

together, one would expect to find approximately the same number of resistant -23-

cella from a11 plates regardless of origine If the origin were mutat­ ional, similar numbers of resistant colonies would be found only on plates of the same origin, since they represent tests of the same mix­ ture of resistant and sensitive bacteria. The data would appear to

support the mutational theory. The difference between penicillin and streptomycin resistance patterns are explained as a multiple, equally potent gene s.ystem for penicillin, and a multiple, but variably potent gene system for streptomycine In the latter, a single mutation in a

highly potent gene would give rise to a high degree of res1stance, while with penicillin, resistance is step-wise, an accumulative effect of suc­

cessive mutations in equally potent genes.

English and McCoy (27), using a single-celled strain of staph.ylococcus pyogenes, grew 100 inoOUla in the presence of a highly inhibitory concen­ tration of streptomycin, and found one tube only showed growth alter 24

hours incubation. They ruled out adaptation due to chemical interaction

between antibiotic and organism, aince on that basis, each culture should

have the sarna opportunity to become resistant. Assumi.ng one resistant

cell as the cause of growth, the authors favour a random mutation explana­

tion because of the low frequency of appearance of resistant cella and

their stability a.rt.er two years of transfers in the absence of antibiotic.

Growing the organisms in the presence of non-inhibitory levels of strepto­

~cin, conditions excluding selection and not affecting the normal growth

curve, did not result in developing resistance. This might he criticised

on the basis that adaptation rnight occur only when cells are faced with

inhibitory levels of the antibiotlc and are forced to adapt to its dele­

terious effects. When they grew the organisms in sub-lethal doses of

streptomycin, altering the normal growt.h curve, they were able to demon­

strate development of resistance, but of a low magnitude. They inferred -24-

that any level ot drug capable ot altering the growth curve would aid in the selection ot mutants which, in the altered conditions, have a growth

advantage over the usual type ot cells. One other argument they put tor­ ward to support the mutational theory was the fact that they were able to

isolate atter seriaI transfers in antibiotic-free media an organism re­

sistant to at least 1000 units / ml. streptomycine

The mutation rate to streptomycin resistance of Escherichia coli was

found by Newcombe & McGregor (73) to he higher when growth took place in

the presence of streptomycin than under antibiotic-free conditions. They

ruled out a speciticaction of the drug when they discovered that mutation

to Phage T-l resistance increased to a similar degree in the presence of

the drug. Two or more gene loci tor streptomycin was postulated because

t'Wo types of mutants were found, simple resistant organisms, and strepto­

mycin dependant ones.

Miller (67) also demonstrated streptomycin-dependant meningococci,

whose morphology, sugar fermentation reactions, and type speciticity did

not difter from normal meningococci. No substance was found to substi­

tute tor streptomycin in supporting growth of dependant organisms. Con­

firmation was given by Rake (80). Supporters oE an adaptation mechanism Eor acquired resistance are

Seligmann & Wassermann (91). They easily acclimatized normal bacteria

to 50,000 ug. streptomycin in the course of one to three weeks, and

maintained this resistance for six monthe. They were also able to re­

cover resistant strains of Pseudomonas aeruginosa trom streptomycin

treated patients, which showed no reaction different from the test-tube

treated ones. As a rule, a11 resistant strains had undergone distinct

alteration of their metabolic activity : -25-

a. their growth rate was slower

b. their reducing power was lowered c. pigment production of Pseudomonas aeruginosa was impaired.

d. enzymatic activi.ties, involving carbohydrate fermentation

and hydrogen sulphide production, were changed. e. 108s ot virulence was observed in strains of Salmonella typhimurium, Salmonella newport, and Salmonella enteritides.

The regularity of the changes was noteworthy especially since aIl original

,sensitive strains retained their original cultural and biological charac­

teristics. These workers suggest that, correlated with other enzymatic

108ses, a strepto~cin 'receptor' of the bacterial cell may become weakened

or lost. The streptomycin 'key' which fits the lock of a sensitive bacteria

does not fit any more in resistant strains where the look has been altered

under the impact of streptomycin treatment.

Barer (5) found that survivors ot cultures of Aerobacteraerogenes, exposed to the bactericidal action of streptomycin, sometimes had a long

lag before growth occurred in liquid or solid media, which would suggest

that modification of the cell protoplasm might have been taking place.

When ditferent samples from one culture ware tested they frequently showed considerable variation in the number of resistant cells present, depending

at least partly on the dilution of the sample, and the condition of spacing

on the plate. In view of the fact that resistance was found to he a con­

ditional property, Barer felt that it was dangerous to construct mutational theories on the evidence of Tariability between different cultures. She

suggests an alternative to the mutational hypothesis in that those cells

survive which possees an extreme degree of sorne variable property, which

might he a continuous variation depending on a polygenic system, rather -26-

than a mutation, and that direct modification of the protoplasm of surv1vors takes place in the presence of streptolIij'"cin.

Much support for the adaptation theory is based on its accept­ ibility from the point of view of the known existence of adaptive enzymes, and the di f'fi cult Y of accepting the vast numbers of mutant types which would appear to be necessary to account for stra1ns adapted to grow on numerable antibiotics, drugs, ·phages, and even a whole spee­ trum of strains adapted to different concentrations of' the agents. There may be less dif'f'erence between the two theories than is apparent, the es­ senti&! point being whether or not adaptation i8 related to a specifie stimulus. -27-

srATEMENr OF PURPOSES OF THIS INVESrIGATION

The etiology of scarlet fever i5 generally attributed to streptoeoceus pYogenes, but sorne doubt still exista as to its primary role in the disease proeess, Binee this organism is not isolated from all cases of 5carlet fever. The author undertook to observe the frequency with which streptocoeeus pyogenes oecurred in the throats of ecarlet fever patients, the relation to it of the day of disease on admission to hospital, and the length of duration of this organism in the throats of patients. The appearance of other pathogenic bacteria in the ear, nose or throat fiaS recorded.

The wide use of penieillin for the treatment of scarlet fever poses the problem of developing resistant strains of bacteria. It waa of interest therefore, to test the sensitivity to penicillin of strains of streptococeus pYogenes i80lated from scarlet fever patients, to see if any change in sensitivity took place where the organism was recovered aiter penieillin therapy.

Since sulphadiazine was administered to sorne patients, sensitivity to this drug Ra determined during the early part of the investigation. -2B-

MATERIALS AND MEl'HODS

1. General Media.

Blood agar plates were made from peptone agar produced in the McGill bacteriology department, to which was added 4% of human blood *. Glucose broth was made by adding 1% glucose to peptone broth.

2. Lancef'ield Grouping Technique.'.

The Lancef'ield antigen was prepared by a method adapted from o Lancef'ield's technique (lB). A 24-hour culture, grown at 37 C in

5 ml. of glucose broth, RS centrifuged, the supernatant removed, and 2 drops of' metacresol purple added to the sediment. Af'ter adding Z1>

HCl until a slight pink colour was obtained (about pH 3.0), the sediment

was heated in boiling water for fifteen minutes, and then cooled in air

f'or ten minutes, and in running water for one minute. 2% NaOH RS added until the c010ur changed t hrough yellow and began to dar ken (about pH 7.5)

but RS not noticeably purple. Af'ter fifteen minutes centrifugation, the resultant supernatant was the antigen.

On a glass petri dish cover a square was marked with a grease penail,

inside of' which space one drop of antigen was added to a drop of group

specifie antiserum. In the bottom of' the dish ns placed a moistened

eircle of' filter paper, over this the top was inverted and 1eft to stand

at room temperature for lB - 24 hours. Results were read microscopically,

with precipitation indicating a positive reaction.

* Obtain.ed from the Canadian Red Cross Blood Transfusion Service. -29-

3. Penicillin Sensitivity Test. Penici11in sensitivity tests were carried out using Difco sensi­ tivity dises of three concentrations : 0.5 units, 1 unit, 10 units. 0 The strains to be tested were grown for 18 - 24 hours at 37 C in glu­ cose broth, and then heavily seeded on a b100d agar plate. One disc of each concentration of penicillin ns p1aced on the plate, which was then incubated for 24 hours at 370 C, atter which the results were noted.

Haemolysis of the b100d, due to the growth ot Streptococcus pYogenes. occurred on the plate except around the dises of penici11in to which the organism was sensitive, giving a red, ha1o-like zone around the disco

C10ser observation of the plate was a1so made to detect any colonies that might have escaped notice by the gross method. A record ns kept of the lowest concentration of penici11in that inhibited the strain.

4. Su1phadiazine Senaitivity Test.

This method i8 given by Gradwho1 (40) as being especia11y suited for

Group! haemolytic streptococcie

Medium. proteose peptone #3 ------10 grams

dextrose ------5 grams sodium chloride ------4 grama

agar ------4 grams disti11ed water ------1000 cc. Adjust the reaction to pH 7.6 to 7.8 beiore steri1ization and before the

addition of the drug solution. Three concentrations of the drug were

used, 2.5 mg. per cent, 5 mg. per cent, and 10 mg. per cent su1phadiazine.

The medium DS tubed in 10 cc quantities and autoc1aved at 15 lbs. pressure for 15 minutes. -30-

A loop of 18 - 24 hour broth culture was transferred to a tube of tryptose-phosphate broth M-2 (Difeo). One loop of this dilution was added to the melted (and cooled) semisolid medium containing sulpha­ diazine and to the control tube. The loop was fiamed before each ino­ culation to remove traces of drug and agar and to keep the size of the inoculum as constant as possible. Results were read ai'ter 24 and 48 hours incubation at 370 c.

5. Routine of cultures. Patients with scarlet fever had throat swabs taken routinely on admission. Nose swabs were also taken in some cases. On the 6th and llth days ai'ter admission, throat swabs were again taken. In sorne cases, due to technical difficulties, throat swabs were not repeated aiter ad­ mission. The swabs were cultured aerobically on blood agar plates, and any pathogenic bacteria appearing on the culture media were identified. All strains of beta haemolytic streptococci were grouped by Lancefield' s pre­ cipitin technique with Group A antiserum. If the test was negative, a repètition of the grouping was made using the antisera of Group A, B, C,

D, G, and H. AIl Btrains of streptococcus pyogenes (Group A) were teBted for their sensitivity to penicillin and, in the early part of the investigation, were tested as weIl for their sensitivity to sulphadiazine.

Patients were divided into three groups, for Btatistical purposes, on the basis of therapy : 1) those receiving penici11in, 2) those receiving sulphadiazine, and 3) those receiving neither drug (control group). -31-

6. Schedule of Therapy.

Penici11in was administered intramuscular1y, 400,000 units / day for five days.

Sulphadiazine doses were given on the basis of l grain/pound/24 hours for five days.

• -32-

EXPERlMEN1' AL RESULT S

TABLE 1.

Incidence of Streptococcus pyogenes in throat, nose and ears of Scarlet rever patients. a) • Throat swab taken only on admission.

Therapy Groups Control Penicillin Sulphadiazine Total

No. patients pos- 25 Il 5 41 itive No. patients neg- Il 9 4 24 ative Total 36 20 9 65 b). Throat swabs taken on admission and one or more times subsequently.

Therapy Groups Control Penicillin Sulpha. Pen. T Sulpha. Total lst T .S.* positive only 27 63 14 l 105 lst & later T .S. pos- itive 93 23 40 0 156 lst T .S. negative and 1ater T .S. positive fJJ 11 21 0 92 Ooly nose S. positive 2 3 1 0 6 Ooly ear S. positive 0 2 1 0 3

Total positive patients 182 102 77 l 362

AlI swabs ~egative 60 44 26 4 134

Total patients 242 146 103 5 496

* T.S. =Throat swab -33- Table l continued c). Nose swabs taken on admission.

Therapy Groups Control Penici11in Su1pha. Pen. +Su1pha Total

No. Patients positive 6 10 1 1 lS No. Patients negative 10 5 o 29

Total 16 24 6 1 47 -34-

A total of 561 scarlet fever patients were observed during the pariod from June 1951 to February 1953. The results of throat, nose and ear cultures are shown in Table I.

Patients were divided into groups on the basis of therapy, 166 receiving penicillin, 112 receiving sulphadiazine, 5 treated with both drugs, while 278 were given neither.

Throat cultures were taken only on admission in 65 cases. Fort y­ one, or 63.0%, of these cases harboured streptococcus pyogenes. The remainder of the patients in this study had two or more throat swabs taken, as well as the occasional nose or ear culture. In this group the incidence of streptococcus pyogenes ns 72.9%.

Only 23.2% of penicillin treated patients showed the organism in their throats after the course of treatment as compared w1th 59.2% for sulphadiazine treated cases, while in the control group, 63.2% "are positive on cultures taken on the 6th or llth day after admission.

Of the 47 patients with nose swabs taken on admission, in 18 cases streptococcus pYogenes could be demonstrated. -35-

TABLE II

Relation of day of disease to incidence of streptococcus pyogenes.

Day of Patients positive on lat day Patients negative on lst Disease day & thereatter. No. patients Total days No. patients Total days

1 1 1 2 2 2 12 24 15 30 3 13 39 21 63 4 7 28 28 112 5 8 40 18 90 6 2 12 Il 66

7 4 28 13 9l. 8 3 24 6 48 9 1 9 2 18 10 0 0 4 40

il 0 0 1 Il 15 0 0 1 15

20 0 0 l 20

Total 51 205 123 606

Average day of disease 4.0 4.9 -36-

TABLE III

Duration of streptococcus ~ogenes in the throats of scarlet fever patients

Therapy Groups Control Penicillin Sulphadiazine Total

Positive on let day Sr negative later 27 63 14 104 Positive on 6th day & negative later 53 11 18 82 Positive on llth day 89 28 41 158

Positive on 14th day 3 0 l 4 " " 17th " 1 0 0 l II Il 21st " 3 1 0 4 Il fi 23rd " l 2 0 3 " fi 24th II l 0 1 2

Total 178 105 75 358

N.B. The patients positive from 14th to 24th days were the only ones

who had throat swabs taken at'ter the Ilth day. -37-

Penicillin treated patients became negative after therapy in

6CJ% of cases, whereas oo1y lS.6% of those given sulphadiazine were cleared of streptococcus pyogenes d'ter treatment. The control patients became negative in 15.2% of cases after the lat throat swab.

The group in whose throats the streptococcus pyogenes lingered longest l'fas the sulphadiazine treated cases where 54.7% still har­ boured the organism on the llth day of hospitallzation, while 50% of untreated patients and 26.6% of those receiving penicillin l'fere positive at this stage. In a few untreated cases, the streptococci remained in their throats until the 21st to 24th days, 'While even t'Wo patients, after having recelved penicillin, were positive on the

23rd day. -38-

TABLE IV Occurrence of pathogenic bacteria, other than streptococcus pyogenes, in ear, nose, or throat of scarlet fever patients.

Therapx Gi"ou~s Or gani sm Control Penicillin Sulphadiazine Totals pos. neg. pos. neg. pos. neg. pos. neg. combined staphylococcus 2E lE 2E lE 3E pyogenes 5I' lT 3T zr IT gr Jf 121' lN 4N lN 2N lN 7N 2N 9N l(E&T) l(ElT) l(F&T)

Haemophilus lT zr 21' IT 3T Jr 6T influenza lN lN lN lN 2N

Diplococcus IT IT IT 3T Jr pneumoniae l(N&T) l(N&T) l(N&T)

Aerobacter IT lT lT aerogenes

Paracolobactrum lT lT lT intermedium

Pseudomonas 1 (T&..E) l(T&E) l(T&E) aeruginoas

Escherichia lN lN lN coli i ; . N.B. pos. - cases in which Streptococcuspyogenes was isolated neg. - " "" " "was not isolated. T - organi 8ms isolated from throat N -" " "nose E- Il Il "eu -39-

Complete data on the occurrence of other pathogens is given in

Table IV. By far the most frequently occurring pathogen was Staph.ylo­ coccus QYogenes, appearing in the throats of 12 patients, in the noses of 19, in the ears of 3 and in both ear and throat of 1 patient. Haemophilusinfluenzae .occurred in 8 instances, and Diplococcus pneumoniae in 4 cases. other pathogens isolated less frequently were

Aerobacter aerogenes, Paracolobactrumintermedium, Pseudomonas aeruginosa, and Escherichia coli.

There is no significant difference among the penicillin, sulphadia­ zine, or control groups as to the frequency with which these pathogens occurred. However, other pathogens appeared three times as often in patients who also harboured Streptococcus pYogenes than in those who did not. -40-

TABLE V

Penicillin sensitivity of strains of streptococcus pyogenes isolated from patients receiving peniei11in therapy. a) One test. per patient

Day of hospital- Concentration of penici11in, in units Total ization when to which strain is sensitive. No.of strain isolated. Patients 0.5 1 10

l 65 0 0 65 6 2 0 0 2 il 4 0 0 4 21 2 0 0 2 Total No. of Patients 73 0 0 73

b) Two tests per patient

Days of hospitalization Concentration of penici1lin, in units when strains isolated to which strains are sensitive.

0.5 - 0.5

l and 6 7 1 and 11 9

6 and 11 2 Total No. of Patients 18 -41-

Table V continued. c) Three test s per patient

Days of hospita1- Concentration of penici11in,in units Total ization when to which strains were sensitive No. of strains iso1ated. Patients 0.5-0.5-0.5 0.5-1-1

l, 6 and 11 4 0 4 1, Il and 23 0 1 1

Total No. of Patients 4 1 5 -42-

'TABLE VI

Su1phadiazine sensitivity of strains of streptococcus pyogenes isolated from patients receiving penicillin therapy. a) One test per patient

Day of hospita1- Concentration of su1phadiazine, in mg.%, Total i zation when to which strain is sensitive or resistant~R} No. of strain isolated. Patients 2.5 5 10 (R} 10 l 5 4 5 4 18

6 0 0 0 0 0

11 0 0 0 2 2

21 0 1 0 0 1

Total No. of Patients 5 5 5 6 21 b) Two tests per patient

Day of hospital- ,Total ization when No. of strains iso1ated. Patients

1 and 6 0 1 0 1

l and 11 1 0 l 2

6 and 11 l 0 0 1

Total No. of Patients 2 1 l 4 -43-

Table VI continued. c) Three tests per patient

Days of hospital­ Concentration of sulphadiazine, ization "hen in meg.%, to "hich strains are strains isolated sensitive.

2. 5 -- 10 2.5

1, 11 and 23 1 -44-

TABLE VII

Penicillin sensitivity of strains of Streptococcus pYogenes isolated from patients receiving sulphadiazine therapy. a) One test per patient.

Day of hospital­ Concentration of penicillin, in units Total ization "hen to which strain is sensitive No. of strain isolated 0.5 l 10 Patients

l 17 l o lS 6 S o o a 11 6 o o 6

Total No. of Patients 31 l o 32 b) Two tests per patient

Day of hospital- Concentration of penicillin, in units Total ization when to which strains are sensitive No.of strains isolated 0.5 - 0.5 1-1 0.5 - l Patients

l and 6 9 0 l 10

1 and 11 9 0 l 10 6 and 11 6 0 0 6 11 and 24 0 1 0 1

Total No. of Patients 24 l 2 27 c) Three tests per patient

Day of hospital­ Concentration of penicil1in, in units Total ization when to which strains are sensitive No. of strain isolated 0.5 - 0.5 - 0.5 0.5 - 0.5 - 1 Patients

l, 6 and 11 11 2 13 -45-

TABLE VIII

Sulphadiazine sensitivity of strains of Streptococcus pYogenes i solated from patient s receiving sulphadiazine therapy. a) One test per patient

Day of hospital­ Concentration of sulphadiazine in mg. % Total ization when to which strain is sensitivè or resistant R No. of strain isolated 2.5 5 10 R 10 Patients

1 2 2 2 6 12

6 o o o 3 3 11 o l o 2 3 Total No. of Patients 2 3 2 11 18

b) Two tests per patient

Day of hotSpital­ Concentration of sulphadiazine in mg.% Total ization when to which strains are sensitive or resi5tant(R) No.of 5train isolated Patients (R) 10 (R)10 10 --- (R) 10

1 and 6 3 o 3 land 11 1 o l

6 and 11 1 o 1

11 and 24- 0 l l

Total No. of Patients 5 1 6 c) Three tests per patient Day of hospital­ Concentration of sulphadiazine, in mg.% Total ization when to which stra1ns are sensitive or resistant R No.of 5train i solated Patients (R) 10 - (R) 10 - (R) 10 10-10-(R) 10

l, 6 and 11 1 l 2 -46-

TABLE IX

Penicillin sensitivity ot strains ot streptococcus pYogenesisolated trom patient s receiving no drug therapy

a) One test per patient

Day ot hospital­ Concentration ot penici11in, in units, Totà1 ization when to which strain i8 sensitive No. ot strain isolated 0.5 1 10 Patients

1 46 1 50

6 29 1 o 30 11 15 o o 15

Total No. ot Patients 90 4 1 95

b) Two tests per patient

Day ot hospita1- Concentration of penicil1in, in units Total ization when to which straine are sensitive No. of strains i solated 0.5-0.5 1-1 0.5-1 0.5-10 1~0.5 Patiente

1 and 6 20 l 2 0 1 24 l and 11 18 l 0 l l 21

6 and 11 14 0 0 0 1 15

6 and 23 1 0 0 0 0 1

Total No. ot Patients 53 2 2 l 3 61

c) Three tests per patient

Day of hospita1- Concentration ot penicillin, in unite Total ization when to which strains are sensitive No. of strains iso1ated 0.5-0.5-0.5 1 0.5-1-0.5 1 1-1-0.5 Patiente

l, 6 and Il 36 1 0 37

6, 11 and 21 0 0 l l

Total No. of Patients 36 1 1 3S -47-

TABLE X

Sulphadiazine sensitivity of strains of streptococcus pYogenes iso1ated from patients receiving no drug therapy. a) One test per patient

Day of hospital­ Concentration of Total ization when to which etrain i8 No. of strain iso1ated 2.5 5 Patients

1 2 6 14

6 1 o 1 3 5 il o o o 2 2 Total No. of Patients 4 11 21 b) Two tests per patient

Day of hospital- Concentration of su1phadiazine, in mg.% i zation when to which strains are sensitive or resistant(R) Total strains isolated 2.5- (R)10- 2.5- 2.5- 5- 10.. 5- 10- I(R)lO- No. of 2.5 (R)10 10 (R)10 (R)10 (R) 2.5 2.5 2.5 Patients 10

1 and 6 0 1 1 0 1 1 1 0 1 6

1 and 11 2 2 1 1 1 1 0 0 0 S

6 and il 0 1 0 1 0 0 0 0 0 2

1 and 23 0 0 0 0 0 0 0 1 0 1 Total No. of 2 4 2 2 2 2 1 1 1 17 Patients c) Three tests per patient

Day of hospital- ization when Total strains iso1ated No. of Patients

1, 6 and 11 1 6 7 -48-

Penicillin treated patients The sensitivity to penicillin of strains of Streptococcus pyogenes i solated from penicillin-treated patients is shown in Table V. In a11

cases where only one strain l'faS isolated per patient, the strains were uniformly sensitive to 0.5 units penicillin, the lowest test concentra­ tion of the drug. In the group where two or more strains were isolated from each patient, there was only one instance of decreased sensitivity of the strain isolated after therapy with this drug.

The sensitivity to sulphadiazine of strains of Streptococcus pyogenes isolated from penicillin-treated patients is shown in Table VI. The range of sensitivity of single strains, isolated before penicillin thérapy, varied from 2.5 mg.% to complete resistance to 10 mg.% of sulphadiazine. In two cases there was a.n increase in resistance to sulphadiazine in strains isolated after penicillin, while in another the sensitivity l'faS increased.

In the one case where the penicillin sensitivity l'faS decreased, there was also a similar change in sulphadiazine sensitivity.

Sulphadiazine treated patients

The sensitivity of strains of Streptococcus pyogenes to penicillin from patients given sulphadiazine is recorded in Table VII. Except in one case, single strains were sensitive to 0.5 unite penicilline Out of

40 cases, 4 showed an increase in resi stance to penicillin after the patient

had received sulphadiazine therapy. Results of sulphadiazine tests of this gr-oup of patients are shown in Table VIII. Sensitivity of single strains varied from 2.5 mg.% to resistance to 10 mg.% sulphadiazine. In two out of 8 cases there was an increased resistance to sulphadiazine in strains isolated after therapy with that drug. -49-

Control patients Results of penicillin sensitivity of strains of streptococcus pyogenes isolated from patients receiving neither penicillin nor sulphadiazine are given in Table IX. The majority of single strains were sensitive to 0.5 units penicillin. Where strains l'fere isolated on the 6th or llth day of hospitalization, corresponding to the post therapeutic period of penicillin or sulphadiazine treated patients,

4 of 99 patients showed increased resistance to penicillin, while 4 showed an increased sensitivity to this antibiotic.

Sulphadiazine sensitivity tests are recorded in Table X. Single strains varied in sensitivity from the lowest to the highest test con­ centration. In eight patients there was an increased resistance to sulphadiazine in strains i solated in the hypothetical post therapeutic period, while there was an increased sensitivity to the drug in 3 cases. -50-

DISCUSSION AND CONCLUSIONS

1. Streptococcus pyogenes wae isolated in 6'J.C1J, of scarlet fever patients where throat swabs ?lere taken only on admission. Where throat swabs were repeated after the aOmission specimen, the incidence rose to 72.%. This would indicate that multiple cultures improve the frequency of occurrence of this organisme That this improvement is not more marked might be explained by the fact that the organism disappears quite rapidly in throats of patients recelving penicillln or sulphadia­ zine, and thereby causes a lowering of the recovery rate of the bacteria.

Although only 38.3% of nose swabs were positive for this organism, nasal cultures can be of diagnostic value in scarlet fever, aince 6 patients showed streptococcus pYogenes only in the nose.

2. The average duration of illness of patients positive, on admission, for Streptococcus pyogenes was 4.0 days, while negative patients showed 4.9 days. Although there la a small disparity between the average day of disease on admission, the majority of negative cases came to hospital a few days later than the majority of positive ones. These results do not point conclusively to a time factor in the ability to isolate

Streptococcus pyogenes from scarlet fever patients, but they indicate that the day of illness when the throat swabs are first taken will influence the cultures. -51-

3. Streptococcus pyogenes disappeared more rapidly from the throats of patients receiving penicillin therapy than from those reeeiving sulphadiazine or no drugs. This indicated the importance, from a bac­ teriological point of view, of obtaining swabs before the administration of penicilline Streptococcus pyogenes lingered longest in the throats of eulphadiazine treated patients.

4. The most frequent pathogen, other than Streptococcus pyogenes, oc­ curring in the ear, nose or throat of scarlet fever patients, ns Staphy­ lococcus pyogenes. Other pathogens round were Haemophilus innuenzae,

Diplococcusq pneumoniae, Aerobacter . aerogenes, Paracolobactrum intermedium,

Pseudomonas aeruginosa, and Escheriehiacoli.

These organisms can only be considered as secondary invaders judging by the relative rarity of occurrence. Although streptococcus progenes was not found in the throats of every scarlet fever patient, its close re1ation­

ship to the development of symptoms of the disease is known. It ls either a primary cause, or p1ays a secondary ro1e to an, as yet, undiscovered agent.

5. Strains of streptococcus pyogenes isolated from patients bafore therapy were generally sensitive to penici11in. Only 13 of the 360 strains ls01ated were sensitive to 1 unit or more of penicilline There ne a very 10w in­ cidence of decreased sensitivity to this antibiotic after either penicl11in or su1phadiazine therapy. The fact that the control group showed a similar rate of increased resistance indicates that neither drug may have a specifie

effect in producing penici1lin resistant strains. It would seem that there ls no major clinical problem in the treatment of cases of scarlet fever with -52- penicillin insofar as the development of highly resistant strains of

Streptococcus pyogenes i5 concerned.

Strains of streptococcus pyogenes were, to begin with, not very sensitive to sulphadiazine, for one-half of the strains were resistant to the highest test concentration. The fact that the initial sensitiv­ ities varied over the whole test range makes it difficult to assay the significance of the few cases where there was an increase in resistance to sulphadiazine in the sulphadiazine and penicillin treated groups as weIl as in the control group.

Some cross-infection mayhave occurred in the wards, but it w&s not possible, within the limits of this investigation, to determine whether the strains of streptococcuspyogenes isolated alter therapy were of the same serological Type as those isolated on admission. This might explain why in some cases an increased sensitivity to the drugs w&s found.

Taking into account that penicillin was found to rid the throat s of Streptococcus pyogenes more quickly than sulphadiazinedti!d, the former

May he considered the better therapeutic agent. -53-

FINAL SUMMARY OF RESULT S

1. Multiple throat swabs from scarlet fever patients improved the rate of recovery of streptococcus pyogenes from cases of this disease from 63% to 73%. Nasal swabs Ware positive in 6 cases where the throat swabs were constantly negative.

2. The average day of disease on admission for patients negative for

Streptococcus pyogenes on the day of admission was 1 day later than the average day for positive cases.

3. streptococcus pyogenes disappeared more rapidly from throats of patients receiving penicillin than from those treated with sulphadiazine or with no drugs. The organism remained longest in the sulphadiazine treated patients.

4. The most frequent pathogen, other than Streptococcus pyogenes, re­ covered from ear, nose, or throat of scarlet fever patients, was staph­ ylococcus pYogenes. Others found were Haemophilus infiuenzae, Diplococ­

~ pneumoniae, Aerobacter aerogenes, Paracocobactrum intermedium,

Pseudomonas aeruginosa, and Escherichia coli.

5. Strains of Streptococcus pYogenes isolated from scarlet fever patients were generally very sensitive to penicilline There was no significant lowering of the sensitivity to this antibiotic in strains isolated from patients at"t,er being treated with it.

These organisms were not very sensitive to the action of sulpha­ diazine. Sorne resistant strains were developed af't.er both sulphadiazine and penicillin therapy, as well as in patients receiving neither drug. -54-

BI BLI OŒ.APHY

1. Abraham, E.P., and Chain, E. " An enzyme from bacteria able to destroy

penici11in." Nature, 146: 837, 1940.

2. Aronson, H. Deutsch med. Wchnschr. ~ : 439, 1903.

3. Baginsky, A. and Sommerfeld, P. Il Uber einen constanten Bacterienbefund

bei Schar1ach." Berl. klin. Wchnschr. 21 : 558, 1900.

4. Bailey, J.H. Il The types of haemolytic streptococci found in scarlet

fever patients and in the throats of grammar school childreU."

Am. J. Hyg. ~ : 107, 1939.

5. Barer, G.R., " The action of streptomycin on Bacterium lactis aerogenes."

J. Gen. Microbiol. 2 : l, 1951.

6. Beerstecher, E. fi Bacterial inhibition by a penici1lin moi ety."

Proc. Soc. Exper. Biol. & Med. 12. : 200, 1950. 7. Bingel, K.F., " Experimentelle Beitrage zur Scharla.chatiologie."

Zeitschr. Hyg. v. Infecktionskrankh. 127 : 216, 1947.

8. Bingel, K.F., " Vergleichende Untersuchungen uber die Warmeemp­

findlichkeit des Streptokokken-Begleitvirus und der unmittelbar ery­

throgenen Noxe beim Scharlach. 1I Ibid. 127 : 280, 1947.

9. Bingel, K.F. Il Uber das atoxischwerden von Scharlachstreptokken als

Folge der Trennung einer Virus-Streptokokken-Kombination:""

Ibid. 127 : 286, 1947.

10. Bingel, K.F. Il Uber die Entstehhung scharlachspezifischer und

unspezifischer Noxen bei hamolysierenden Streptokokken."

Ibid. 127 : 434, 1947.

11. Bliss, W.P. " A biologieal study of hemolytic streptoeocci from throats

of patients suffering from searlet fever. Preliminary report. Il

Bull. Johns Hopkins Ho spital 21 : l73, 1920. -55-

12. Blias, W.P. "studiea on the bio1ogy of Streptococcus. II. Antigenic relationships between straina of streptococcus haemolyticu8 iaolated from scarlet fever." J. Exp. Med. 22 : 575, 1922.

13. Boisvert, P .L. Il The grouping of hemolytic streptococci asaoeiated with disease in chi1dhood." J. Bact. 1§ : 333, 1938. 14. Boisvert, P.L. " Human hemo1ytic streptococci from diseases of chi1d­

ren. n Am. J. Dis. Child. 2.2 : 281, 1940.

15. Boisvert, P.L. and Bearg, P.A. Il Hemolytic streptococci recovered

during an epidemic in a kindergarten." Yale J. Biol. & Med. 14 519,1942.

16. Bondi, A. and Deitz, C.C. "Penici1lin resistant staphylococcie"

Proc. Soc. Exper. Biol. & Med. €fJ : 55, 1945.

17. Clark, W.H. Bryner, S., and Rantz, L.A. Am. J. Med. l:! : 671, 1948. 18. Cumpston, H. " The relative frequency of various types of streptococci in scarlatina. " J. Hyg. 1 : 599, 1907. 19. Demerec, M. "Production of staphy1ococca1 strains resistant to various

concentrations of penici1lin,1l Proc. Nat. Acad.Sc. U.S. 2!. : 16, 1945.

20. Demerec, M. "Origin of bacterial resistance to antibiotics."

J. Bact. ~ : 63, 1948.

21. deWaa1, H.L. " The sero1ogical types of haemo1ytic streptococci in

relation to the epidemio1ogy of scarlet fever and its complications."

J. Hyg. ~ : 172, 1940.

22. Dick, G.F. and Dick, G.H. " Experimental scar1et fever./I

J.A.M.A. 81 : 1166, 1923.

23. Dick, G.F. and Dick, G.H. " A skin test for susceptibility to scarlet

fever. 1I J.A.M.A. 82 : 265, 1924.

24. Dochez, A.R., Avery, O.T. and Lancefie1d, ft.C. "Studies on the bio1ogy

of Streptococcus. 1. Antigenic re1ationshipa between stra1ns of Strep­

tococcus haemolyticus." J. Exp. Med. 2Q : 179, 1919. -56-

25. Dochez, A.R. and Bliss, W.P. Il Bio1ogic study of hemo1ytic strepto­

cocci from throat s of patient s suffering from scar let fever."

J.A.M.A. ~ : 1600, 1920.

26. Dochez, A.R. and Sherman, L. Il The significance of Streptococcus haem­

o1yticus in scarlet fever and the preparation of a specific antiscar-

1atina1 serum by immunization of the horse to streptococcus haemo1y­

ticus-scar1atinae. IIJ.A.M.A. 82 : 542, 1924.

27. English, A.R. and McCoy, E. liA study of streptomycin resistance in

Micrococcus pYogenes var. aureus. 11 J. Bact. 61 : 51, 1951.

28. Eriksen,K.R. " Acquired penici1lin resistance in pathogenic cocci."

Acta Path. et ~icrobio1. Scand. ~ : 249, 1948.

29. Estrada, E. Il The Lancefie1d groups of hemo1ytic streptococci found

in chi1cirert. Il Yale J. Biol. & Med. 12 : 407, 1940.

30. Gabritschewsky, Berl. k1in. Wochenschr. ~ : 556, 1907.

31. Gasul, B.M. and Rhoads, P.S. Il Dick test and b100d agar cultures as

aide in diagnosis of scarlet fever." AM. J. Dis. Child. 49: 603,1935.

32. Gezon, H.M. "Antibiotic studiee on beta haemolytic streptococci

1. Penicillin resistance acquired by Group A organism."

Proc. Soc. Exper. Biol. & Med. ~ : 208, 1948.

33. Gezon, H.M. " Antibiotic studies on beta haemo1ytic streptococcie

II. Penici1lin resistance acquired by Group B organi sm. "

Ibid. 67 : 212, 1948.

34. Gezon, H.M ... Antibiotic studies on beta haemolytic streptococcie

III. Penicillin resistance acquired by Group C organisms."

Ibid. 67 : 215, 1948.

35. Gezon, H.M. and Collins, G.:t. Il Antibiotic studies on beta haemolytic

:Streptococci. IV. Penicillin resistance induced in mice and embryon­

ated eggs. Il Ibid. É.2 : 314, 1948. -57-

36. Gezon, H.M. and Cryst, E.E. " Antibiotic studies on beta haemo1ytic

streptococcie V. Streptomycin resistance acquired by Group A, B, and

C. organisms." Ibid. 68 : 653, 1948.

37. Gezon, H.M. and Fa san , D.M. " Antibiotic studies on beta haemo1ytic

streptococci. VI Acquired 'in vitro' and 'in vivo' resistance to

aureomycin." Ibid. 1.2 : 10, 1950.

3B. Gezon, H.M. Fa san, D.M. and Collins, G.R. " Antibiotic studies of

beta haemo1ytic streptococcie VII Acquired 'in vitro' resistance to

bacitrac1n." Ibid. 74 : 505, 1950.

39. Gordon, M.H. "A sero10gica1 study of haemo1ytic streptococc1:

Differentiation 01' streptococcus pyogenes, from streptococcus scar1atinae."

Brit. Med. J. ! : 632, 1921.

40. Gradwohl, R.B.H. "C1inical laboratory methods and diagnosis. ri

Mosby, 4th edition. p.1610.

41. Green, C.A. " Serological types of haemolytic streptococci in an

epidemic of scar1atina. Il J. Hyg. Il.: 31B, 1937.

42. Griffith, F. " Types of haemolytic streptococci in relation to scar1et

fever." J. Hyg. 26 : 363, 1927.

43. Griffith, F. J. Hyg. ~ : 542, 1934.

44. Hare, R. J. Path. & Bact. 41 : 499, 1935.

45. Hartman, T.L. and Weinstein, L. " Drug sensitivity of haemo1ytic strep­

tococci isolated from cases of scarlet fever treated with penici1lin."

Proe. Soc. Exper. Biol. & Med. ~ : 314, 1948.

46. Hasenknopf and Sa1ge. Jahrb. f. Kinderhei1k. ~: 218, 1903.

47. Hirsch, H.L., Rotrnan-Kavka, G., Dowling, H.F. and Sweet, L.K.

J.A.M.A. ~ : 657, 1947.

4B. Hobby, G.L., Meyer, K. and Chaffee, E. " Observations 01'1 the mechanism

of action of penici1lin." Proc.Soc.Exper. Biol.&Med. iQ : 2Bl, 1942. -58-

49. Jochmann, G. Z. klin. Med. ~ : 316, 1905. 50. Klein, M. "A mechanism for the development of resistance to streptomycinandpenicillin." J. Bact • .,22: 463, 1947. 51. Klein, M. and Kimmelman, L.J. "The r01e of spontaneous variants in the acquisition of streptornycin resistance by the Shigellae."

J. Bact. ~ : 471, 1946. 52. Klinlock, J.P., Smith, J. and Taylor, J.S. " The newer knowledge of diphtheria and scarlet fever and its application in hospital practice

and in community immunization." J. Hyg. 26 : 327, 1927.

53. Kodama, T., Tiku, Y., Kodaira, T. and Kodama, A. Il The serological grouping and typing of the haemolytic streptocci isolated in Tokyo. 1.

Kitasato. Arch. Exper. Med. ~ : 245, 1937.

54. Kodama, T., Ozaki, M., Nisyama, S. and Tiku, Y. Il The ser010gical grouping and typing of the haemolytic streptococci isolated in Tokyo. II. Ibid. 12 : 162, 1938.

55. Lancefield, R.C. J. Exper. Med. ~ : 481, 1928. 56. Lancetield, R.C. "A serological differentiation of human and other

groups of haem01ytic streptococci." J. Exp. Med. 21 : 571, 1933. 57. Lancefield, R.C. and Hare, R. J. Exp. Med. 61 : 335, 1935. 58. Lemoine. "Etude bacteriologique de 117 cas d'angines scarlatineuses." Bull. de la Soc. Med. des Hopitauz, p. 847, 1895.

59. Loeffler. Arb. a. d. k. Gesundh. ~ : 421, 1884. 60. Luria, S.E. " A test for penicillin sensitivity and resistanee in

staphy1ococcus'e" Proc. Soc. Exper. Biol. & Med. 61 : 46, 1946. 61. Macheboeuf, M. " Recherches biochemiques sur le mode d'action des antibiotique : penicilline, streptomycine, tyrothricine." Bull. Soc. Chim. Biol. 2Q : 161, 1948. -59-

62. Marmorek. Ann. Inst. Pasteur .2: 592, 1895.

63. McKee, C.M. and Houck, C.L. Il Induced resistance to penicillin of

cultures of st aphy10coc ci , Pneumoeoeci and streptococei." Proc. Soc. Exper. Biol. & Med. 22 : 33, 1943. 64. MeLachlan, D.G.S. " The specifie toxigenie porperties of haemolytic

streptocoeci from scarlatina and other sources. 1I J. Hyg. 26: 84,1927.

65. Meads, M., Ory, E.M., Wilcox, C. and Finland, M.

J. Lab. & Clin. Med • .JQ : 725, 1945.

66. Meyer, F. Deutsch. med. Wochenschr. ~ : 751, 1902.

67. Miller, C.P. 11 Bacterial resistance to antibiotics. "

Ann. InternaI Med. 29 : 765, 1948.

68. tliller, C.P. and Bohnho!f, M. J.A.M.A. 130: 485, 1946.

69. Mi1zer, A., Kohn, K.H. and MaeSears, R. Il Oral prophy1axis of rheumatic

lever with penicilline " J.A.M.A. 136 536, 1948.

70. Moser, P. Wien. klin. Wochenschr. 12: 1053, 1902.

71. Moser, P. and von Pirquet, C. Wien. k1in. Wochenschr., 12 : 1086, 1902.

72. Neufeld, F. Ztschr. f. Hyg. v. Infektionskr. ~: 161, 1903.

73. Newcombe, H.B. and McGregor, J. 11 On the nonadaptive nature of change

to full streptomycin resistance in Escherichia colL"

J. Bact. ~ : 539, 1951.

74. North, E.A. and Christie, R. "Observations on the sensitivity of

Staphylococci to penicillin." M.J. Australia ~ : 44, 1945.

75. Pauli and Coburn. J. Exp. Med. ~ : 595, 1937.

76. Pike, R.M. and Foster, A.Z. "Demonstration of su1phonamide inhibitor-

production by bacteria on agar containing sulphonamide. 1l

J. Bact. ~ : 97, 1944.

77. P10ugh, H.H. Il Penicil1in resistanee of staphyloeoecus aureus and

clinieal implications. Il Am. J. Clin. Path. 12 : 446, 1945. 78. Polotevkova. Russk. Wratch. 11 1183, 1921.

79. Powers, G.F. and Boisvert, P.L. " Age as a factor in Streptococcosis."

J. Pediat. ~: 481, 1944.

80. Rake, G. Il Streptomycin as an essential nutrilite."

Proc. Soc. Exper. Biol. & Med. 67 : 249, 1948. 81. Rake, G., McKee, C.M., Hamre, D.M. and Houck, C.L. "Studies on

penicillin. II Observations on therapeutic activity and toxicity.1I

J. Immunol. ~ : 271, 1948.

82. Ranmelkamp, C.H. and Moxon, T. "Resistance of Staphvlococcus aureus to the action of penicillin." Proc. Soc. Exper. Bio1.& Med • .21 : 386,1942.

83. Rantz, L.A. Il The serological typing of hemolytic streptococci of the

Lancetield Group A." J. Clin. Invest. 21 : 217, 1942.

84. Rantz, L.A. and Keeter, C.S. Il The distribution of hemolytic strepto­

cocci Group A, B, and C in human infections."

J. Infect. Dis. 68 : 128, 1941.

85. Rantz, L.A. Randal, E., Spink, W.W. and Boisvert, P.L. Il Sulphonamide

and penicillin resistance of Group A hemo1ytic streptococci."

Proc. Soc. Exper. Biol. & Med. 62 : 54, 1946.

86. Rasch, C. and Parker, R.F. Il Action of penicillin on streptococci:

t Enhancement of sensitivity 'in vivo ." Science 116 : 16, 1952. 87.Rossiwa1, E. and Schick, B. Wien. kiln. Wochenschr. 18 : 3, 1905.

88. Ruediger, G.F. "Streptococci from scarlatinal and normal throats." J. Inf. Dis. 2 : 755, 1906. 89. Schultz and Charlton. Zeitschr. f. Kinderhei1k. 11: 328, 1918.

90. Selbie, F.R., Simon, R.D. and Mclntosh, J. "Bacteriological aspects

of penicillin therapy." J. Path. & Bact. 21 : 47, 1945.

91. Seligmann, E. and Wa8Sernta:nnt.M."Induced resistance to streptomycin."

~. Immunol. .27.: 351, 1947. -61-

92. Smith, J. n Further studies on the serologieal classification of

haemolytic streptococcLIt J. Hyg. 26 : 420, 1927.

93. Smith, J. "The agglutination of haem01ytic streptococci by sera

from cases of scarlet fever. 1I J. Hyg. ~ : 434,1927.

94. Spicer, S. and Blitz, D. "A study of the response of bacteria1

populations to the action of penicil1in; a quantitative determination

of its effects on the organisms." J. Lab.&Clin.Med. ~ : 417, 1948.

95. Spink, W.W. and Ferris, V. Il Penicillin-resistant staphylococci:

Mechanisms invo1ved in the deve10pment of resistance. 1l

J. Clin. Invest. 26 : 279, 1947.

96. stevens, F .A. and Dochez, A.R. Il The study of hemo1ytic streptocoeci

associated with scarlet fever." Proc.Soc.Exper.Biol.& Med. 21:39,1923.

97. Todd, E.W., Turner, G.S. and Drew, L.G.W. "FaBtness of staphylococci,

haemo1ytic streptococci, and pneumococci to penicillin."

Brit. Med. J. ~ : 603, 1945.

98. Tunnicliff, R. "On the group specificness of antibodies in anti­

streptococcus serum." J. Inf. Dis. 2! : 373, 1922.

99. Tunnie1iff, R. "Specifie nature of the hemo1ytic streptococcus of scarlet fever." J.A.M.A. 1!:J:: 1386, 1920.

100. Waksman, S.A. Il Microbia1 antagonisms and antibiotic substances."

The Commonwealth Fund, 1947.

101. Watson, R.F. Il Sensitivity of various serological (Lancefie1d)

Groups of streptococci to penici11in. 1l

Proc. Soc. Exper. Biol. & Med. 21 : 65, 1944.

102. Weinstein, L. and Tsao, C.C.L." 'In vitro' deve10pment of temp­

orary penici1lin resistance in streptococcus pyogenes."

Proc. Soc. Exper. Biol. &: Med. 66 : 598, 1947.

103. Woodruff, H.B. and Foster, J .W. "Y..ici'Obial aspect s of penicilline VII. Bacterial penicillinase." J. Bact. k2 : 7, 1945.