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302m EFFECTS OF 111 VI’I‘f-LO moms) EMICILLIN masxermcz

ON MICRCCOCCUS PYOGENiS VAR. AUHEUS

by

HOdEnT JAMES COLLINS
v

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfilment of the requirements
for the degree of
MudTEd OP SCIENCi

Menartment of Bacteriology

1954

THESIS

,1. an: .9,4.tfli NIIQ/
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To My Wife

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I
I

INTRODUCTION

HISTORICAL SURVEY

CULTURES USED IN THIS

MATERIALS AND METHODS

RESULTS . .
DISCUSSION .
SUMMARY . .
REFERENCES CITED

ACKNOWLEDGMENT

TABLE OF

STUDY

CONTENTS

Page
. . . . . . . . . 1
. . . . . . . . . 3
. . . . . . . . lb

111

LIST OF TABLES

Table Page
1. Method Used for Determining the M.I.C. of

Sensitive Strains . . . . . . . . .- . 16

2. Results of Direct Cell Counts Calculated
in Bacteria per m1 of Cell Suspension
Standardized to 60 per cent Transmission

on the Ph 0t omet er 0 O O O O O O O O C 2O

3. Results of Drop Plate Counts Calculated in
Bacteria per ml of Cell Suspension Stand-
ardized to 60 per cent Transmission on

the Photoneter . . . . . . . . . . . 20

h. M.L.D. Determinations of Sensitive Parent

strains 0 O O O O O O O O 0 O O O O 23

5. Results of M.I.C. Determinations of Sensi-

tive and Resistant Cultures . . . . . . . 29

6. Results of Direct Cell Counts on Resistant
Cultures Calculated in Bacteria per ml of
Cell Suspension Standardized to 60 per cent

Transmission on the Photoneter . . . . . . 31

iv

Table Page
7. Results of Virulence Tests on Resistant

Cultures . . . . . . . . . . . . . 33

8. Results of Biochemical Reactions of Sen-

sitive and Resistant Strains . . . . . . 37

9. Results of the Measurement of Size of

Sensitive and Resistant Cultures . . . . . 39

FIGURE

Figure Page
1. The Development of Resistance of Four
Strains of 5, ongenes var. aureus

to Penic111m G c s s s s s s s s s s 28

INTRODUCIIMN

Resistance of microorganisns to penicillin is of importance
to the clinician, the geneticist and the liocienist.

Except for penicillin resistant micrococci, the development
of resistant microorganisms in the course of treat ent of Facterial
infections with penicillin has been rare. The steady increase in
these forms among the micrococci has, however, definitely limitei
the usefulness of penicillin in the treatment of micrococeal
diseases. In an effort to iefine the nature of resistance to
penicillin many investigators have successfully produced resistant
.forms by repeated subculture in increasing quantities of the
drug. Authors have reported on the characteristics of these
resistant variants, often with conflicting data. For examnle,
there is lack of agreement as to the pathogenicity, coagulase
production and biochemical and gram stain reactions of micrococci
made resistant in_vitro. Further, because some resistant micro-
cocci have been reported coagulase negative, nonhemolytic and
poorly pigmented, it has been assumed that they are no longer
pathogenic. True, this is indicative of avirulence tut nrcof lies
in pathogenicity tests on suitable laboratory animals with com-
parable numlers of the sensitive and resistant teeteria. However,
the information gained by a study of resistance proiuced in cul-
ture may not be entirely applicable to micrococci which have

acquired.resistance luring treatvent of a patient with penicillin.

 

 

This is because the characteristics of 12.11222 produced resistant
strains may not be the same as those of strains which have acquired
resistance ig’zigg.

Resistance of bacteria to penicillin is important to the
geneticist. Geneticists are concerned with the origin of bacterial
resistance and the changes produced in bacterial populations by
penicillin. biochemists use cultures of bacteria which are resistant
to penicillin to derive information about intermediate steps in
metabolism and mode of action of drugs.

The objectives of this study were to investigate some of the
changes produced in Micrococcus pyogenes var. aureus by repeated
subculture in penicillin. Particular emphasis has been placed on
changes in virulence because the literature is not in agreement as

to whether resistant cultures remain virulent or become avirulent.

 

 

 

HISTORICAL SURVEY

Pasteur and Joubert (1877) made what was perhaps the first
recorded observation of antagonism between microorganisms. They
noted that broth cultures of Bacillus anthracis which recame
contaminated failed to grow.

While investigating staphylococci, Fleming (1929) noted that
a mold growing as a contaminant on a plate caused the staphylo-
coccus colonies to become transparent and then undergo lysis.
When the mold was grown in a liquid medium the inhibiting sub-
stance was found to diffuse into the broth. Further, a broth
.filtrate, even when greatly diluted, would inhibit the growth
of many pathogens. The substance was no more toxic for rabbits
than ordinary broth. Later, the mold was identified as Penicillium
notatum and Fleming called the inhibiting substance penicillin.
Cutterbuck, Lovell and Raistrick (1932) attempted to extract
penicillin from a purely synthetic medium on which they grew the
mold. During the extraction penicillin was found to be unstable
and further work was abandoned.

Penicillin was largely forgotten until 1938 when Florey
and Chain at Oxford reexamined the possibilities of isolation
and concentration of penicillin (Florey 1944). In the next
two years the Oxford group succeeded in purifying, concentrating
and testing bacterial spectra and tissue toxicity of penicillin.

Proof of the efficacy of the antibiotic came when human patients

3

were successfully treated in the winter and spring of 19hO-h1.
Thus, the Oxford group proiuced penicillin as a proved chemother-
apeutic drug. The problem of proiucing penicillin in large quan-
tities still remained. Due to the war, England was unalle to
provide facilities for large-soale production, so Florey and
Heatley came to America. Here development of high-yielding
strains of E. notatum and modification of the culture medium
‘ played an important part in volume production.

Once the new drug became available research was begun on
its mode of action on the bacterial cell. Gardner (19tO) stud-
ied the changes induced in bacteria by growth in concentrations
of penicillin insufficient to cause complete inhibition. En-
largement, pleomorphism and imperfect fission were observed
in micrococci, streptococci and gram positive bacilli. Swelling
and sometimes bursting was noted in gram negative bacilli like
Escherichia coli and some of the salmonella. In contrast to
this, no morphological changes were seen in penicillin sensitive
meningococci. Gardner thought that penicillin might cause in-
complete fission leading to cellular enlargement and, in some
cases, lysis. Hobby, Meyer and Chaffee (1942) subjected susceptible
Lacteria to penicillin and determined the number of surviving
organisms at different time intervals. They found that a straight
{line was obtained if the log of the number of surviving organ-
isms was plotted against time until approximately 99 per cent
«mf‘the organisms were dead. The remaining one per cent were

killxni at a slower rate. Bigger (194A) found that the number of

h

 

 

 

orgarisms able to survive inhibitory concentrations of penicillin
was increased if their rate of growth was slowed down or stooped.
For example, penicillin in a concentration of 10 units per ml
caused death of all micrococci in a culture at 37 C, allowed a
few organisms to survive at room temperature, and caused no re-
duction in the number of viable bacteria at h C. In similar ex-
periments it was found that micrococci, placed in broth diluted
1:800 with distilled water or sutjected to bacteriostatic con-
centrations of boric acid, showed higher survival rates in in-
hibitory concentrations of penicillin than micrococci grown in
plain broth. Bigger stated that these 'persisters' were cocci
with no greater resistance than normal organisms but that they
hermened to be in a non-dividing phase in which they were in-
suscentible to the action of penicillin.

Kirby (l9hu), by turiiiimetric readings, found a period
of rapid growth followed by a period of equally rapii lysis at
a concentration of 0.1 unit of penicillin per ml. with concen-
trations of from 1.0 to 100 units per ml a period of slow wrowth
was follow;i by slow lysis. This phercmercn occurred with each
of 100 strains tested. hirny attributed the more rs-id lysis
in small rather than large concentrations to greater initial
growth in the former. Parker and Marsh (1946) and Eriksen (lQAéa)
obtained the same initial growth as did Kirby when using small
concentrations of penicillin but found the lethal effect to be
immediate with large concentrations. Erihsen's technique was
interesting. He added penicillin to a solid medium.and inoculated

5

 

 

it with micrococci. By placing small squares of the meiium on
a slide anl oiserving one area with a microscope it was possible
to tell whether growth or lysis of the bacteria hai occurrei.
'These authors concluded that penicillin was active only against
those bacteria which were iiviiing; that is, it was effective
in stopping some mechanism which the cell needs for division.
However, their results would also support the hypothesis that
penicillin blocks some mechanism involved in food metalolism,
because an active metabolism must arecede growth and division.
Bacteria able to grow in concentrations of penicillin great-
er than can he maintained in the human boiy are termei insensitive
or resistant to the antibiotic. Strains of micrococci which
are resistant to penicillin are occurring more and more frequently.
Spink, Ferris and Vivino (l9hb) reported 12 per cent of 68 strains
of ccagulase positive micrococci to be resistant to between 0.4
and 0.8 unit of penicillin. Bondi and Dietz (19h5) found that
approximately lb per cent of llS recently isolated strains were
able to Withstand O.h5 unit of the antibiotic. The figure had
risen to 21 per cent of 79 cultures as reported by Blair, Carr
and Buchmann (1946). Their strains required from 0.3 to 50
units for inhibition. Roundtree, Barbour and Thompson (1951)
ran sensitivity tests on staphylococci frzm patients at a hos-
pital and reported 53 per cent resistant. Miyahara 22.31- (1953)
reported the amazing total of 76 of 100 coagulase positive micro-
cocci to be resistant. An even more striking example of the

increase in resistant micrococci was that reported by Barber and

\ ‘ '
[';x‘.-.i V. “5.4 s

 

 

Rozwaiowska-Dowzenkc (1948). They found, in the same hospital,
it per cent of the strains to be resistant to nenicillin in lots,
38 per cent in 19h7, and 59 per cent in l9t9.

In an attempt to define the nature of resistance numerous
workers have developed bacteria in culture which are resistant to

penicillin. The first of these were Abraham, Chain 31 §l° (19Al).

 

By subculturing a strain of E. pyogenes var. aureus for 16 weeks
in increasing quantities of penicillin they obtained a culture
1000 times more resistant than the parent strain. Rammelkamp
and Maxon (1942) raised two strains of staphylococci to a 6A ;
fold increase in penicillin resistance in eight weeks. Eriksen
(l9h6b) obtained 12 strains of E. pyggenes var. 235323 which
showed a 500 fold increase in resistance. 0n solid media resis-
tant cultures showed small colonies with poor pigmentation.
Four of five subcultures on media without penicillin brought
normal sized colonies into predominance.

Bellamy (19A8), Bellamy and Klemik (1948), Klimek, Cavallito
and Bailey (l9h8) and McVeigh and Hobiy (1952) reportei on the
morphological and biochemical changes produced in 5. pyogenes

var. aureus by repeated subculture in a medium containing penicillin.

 

All of trese authors observed about the same changes in morpho-
logy. At first, exposure to penicillin resulted in an increase
in size of the cells. After considerable resistance had been
attained the cells became highly pleomorphic, varying from cocci
to cocco—bacilli, rods and diphtheroid forms. As resistance

increased the gram stain reaction became negative, the cells

became smaller and more uniform in size. Increased resistance
was accompanied By a progressive loss in ability to ferment
sugars, reluce nitrate and grow in high salt concentrations. For
example, the resistant micrococci of bellamy and Klimek (19h8)
were unable to grow in broth containing 6.5 per cent sodium
chloride at 1000 times, unable to ferment sucrose and mannite

at 4000 times, and unable to ferment lactose or reduce nitrates
at 60,000 times the resistance of the parent sensitive strain.
Bellamy (19h8) and McVeigh and Hobdy (1952) found no decrease in

resistance of their resistant micrococci on an enriched medium.

However, highly resistant strains could be made much more sensi-
tive by subculture on a deficient medium devised by Bellamy.

In contrast to the complete loss of ability of carbohydrate
fermentation observed by the above investigators, Abraham, Chain
32 E1. (19hl), Spink, Ferris and Vivino (l9hh) and Blair, Carr
and Buchman (19h6) reported a decrease in the rate of fermentation.
Since all authors, who have worked with organisms resistant to
penicillin, have found a decreased rate of growth the fermentation
reactions would be expected to proceed at a slower rate.

Authors also disagree on the coagulase reaction of micrococci
made resistant in culture. Spink, Ferris and Vivino (19hh), and
Blair, Carr and Buchman (l9h6) found their resistant micrococci
to be coagulase positive. Klimek, Cavallito and Bailey (19h8),

Suter 811d Vischer (19A8) and McVeigh and Hobdy (1952) reported

that in vitro produced resistant strains became coagulase negative.

Rake et_al. (l9uh) were the first to report on virulence of
pathogens made resistant in culture. They succeeded in leveloping

resistance in a type III pneumococcus culture, a Streptococcus

 

pyogenes culture ani three cultures of E. pyogenes var. aureus.
Over fifty transfers of a type I and a type II pneumococcus culture
resulted in only a slight increase in resistance. Virulence for
mice of the resistant cultures was reduced. Cultures, which

-7
consistently killed mice at a dilution of 10 before penicillin
passage, killed only irregularly at a dilution of 10.1 after
passage. The types I and II oneunococci, which had acquired
little resistance, were only slightly less virulent.

Miller and bohnhoff (1945) made seven strains of meningococci
resistant to penicillin in vitro. Virulence was determined 1y
inoculating nice intraneritoneally with mucin suspensions of
meningococci. Six of the seven strains, which had acquired
varying degrees of penicillin resistance up to five units per ml,
lost very little or none of their original virulence for mice.
The seventh strain which was able to grow in the presence of 14
units of penicillin remained highly virulent. However, when
the strain had acquired resistance to 18 units per ml, virulence
was lost and could not be restored by mouse passage.

Llair, Carr and Euchman (19A6) ieveloned penicillin resis-
tance in four strains of staphylococci by passage in a solid

meiiuWL containing increasing quantities of the antiliotic.

Virulence was deterrined by the intravenous injection in mice

’1

weighing lb trans of 0.5 nl c‘ 1 suspension a" nu ‘9 hour slav+

.g 1 r a . - ‘ a . -: v m :- ~ . « ~ ~ . : -. - ' \r 1‘ x n ..
culture, W bbei sires times uni resu» Ohio! 17 all .1 of u.“5

r , J. in '7‘- m, i" . n .,_f in _. \ r. ,.'. .

pe cett ulllth. Lures o: the four ut'~-'n showed a considerible

loss of virulence after the ninth subculture. The four strains
were then maintained at room temperature and transferred to fresh
medium every seven or eight weeks. Their resistance tr penicillin
and virulence for mice was determined at the end of the second and
eighth month. At the end of the second month the three strains
which had previously lost most of their virulence now became
completely avirulent. The fourth strain remained virulent.

Surprisingly enough the resistance of the strains increased con-
siderably in the atsence of penicillin. At the end of the eighth
month all strains showed a loss in resistance to penicillir and
the fourth strain became avirulent. Next, the four strains were
subjected to serial daily transfers in broth without penicillin
with the result that the sensitivity of three of the four strains
was within the 'normal' range while one strain retained a consid-
erable degree of resistance. Virulence for mic- was again tested.
Collectively, the four strains caused death in eight of 36 inocul—
ated mice while at the begining of the experiment the parent
sensitive cultures had killed 30 of 36 mice.

Suter and Vischer (1948) tested the virulence of cultures
of micrococci made resistant to penicillin in zitrg. They in—
jected mice with 16 hour broth cultures of sensitive and resistant

micrococci suspended in mucin. The penicillin resistant cultures

10

remained as virulent as the sensitive ones.
-Matsui (1950) reportei that streptococci male resistant in
vitro became avirulent for mice.
Several authors have reported on pathogenic cocci which
have acquirel resistance in 3212. Rammelkamo and Maxon (19L2)

recovered four strains of penicillin resistant fl. pyogenes var.

 

aureus from fourteen patients treated with the antibiotic. Cul-
tures of the four strains obtained before and after treatment
disclosed increases of from 16 to lhO fold in resistance. Schmidt
and Sesler (19h3) developed two resistant strains of pneumococci
by serial passage in mice treated with penicillin. The resistant
variants appeared stable. Thirty serial passages of one strain in
normal mice produced no loss of resistance. Flair, Carr and
Buckman (l9h6) obtained resistant fl, Byogenes var. aureus

from nine of hl patients undergoing penicillin treatment for

chronic osteomyelitis. As with strains made resistant in culture

these resistant forms showed a tendency toward loss of pigment
and retardation of metabolic processes. The coagulase reaction
was unchanged. No loss of resistance occurred over a period of
from five to sixteen months. Six of the nine strains showed

no change in virulence for mice when compared with the parent
sensitive strain. The other three showed an appreciable loss

of virulence. North and Christie (19h6) obtained three isolates

of E, pyogenes var. aureus at weekly intervals from a leg wound

 

in a patient being treated with penicillin. The first isolate'

TeQUired 0.6 unit per ml for inhibition, the second 2.5 units per

11

ml, and the third 10 units per ml. The three isolates were
believed to be of the same strain because all belongel to the same
phage type and all were culturally identical. Mice were inoculat-
ed with a broth culture of the resistant micrococci diluted in
saline to contain four billion organisms. Twenty-five thousand

units of penicillin failed to protect a group 0” mice injected

 

with the culture resistant to 10 units of penicillin. The same has.

dose of “enicillin protected all mice injected with the culture 3

requiring 0.6 unit for inhibition. i
Some organisms have been shown to be able to destroy pen-

icillin. This is due to the elaboration of an enzyme called

penicillinase. Abraham and Chain (1940) first demonstrated its

presence in Escherichia £21; and Micrococcus lysodeikticus.

A crushed extract of the cells was capable of destroying penicillin

as evidenced by the growth of a sensitive micrococcus in the pre-

sence of enzyme-treated penicillin. Penicillinase producers

have been found widely distributed in nature. Woodrurf and

Foster (194A) found an intracellular enzymatic substance destruc-

tive to penicillin in cultures of spore-forming bacteria, other

bacteria, actinomycetes, fungi and yeasts. They found no corre-

lation between the resistance of a microorganism to penicillin

and its ability to produce penicillinase. Bondi and Dietz (19hha)

found the enzyme amoung coliforms, aerobic-sporeformers and in

the genus Shigella. In another publication (lQhLb) they reported

no penicillinase activity in Salmonella, Pseudomonas or Frucella

although organisms in these genera were not susceptible to the

12

‘1. u? Airy/.-
“

action of penicillin. By sulculturing F. nvogfnes var. aureus,

 

Salmonella typhosa ani Proteus vulgaris in increasing quantities

 

of penicillin, resistance could le increased tut no nericillirsse
activity could be detected. The authors ccncluiei that the in-
ability of ;n orcanism to proiuce penicillinase is not necessarily
a determining factor in its sensitivity to penicillin.

However, in the genus Nicrococcus the ability to produce

 

penicillinase is directly correlated with resistance acquirei
lg XEXQ‘ Kirby (194%) usei Harper's acetcre—ether extract or
method to demonstrate penicillinase activity of micrococci.
Seven resistant strains isolated from patients proiuced the in—
activator while seven sensitive strains showed no penicillinase
activity. Eonli ani Dietz (lQhS) tested 115 strains of micro-
cocci 16 of which were resistant. Only the resistant strains
produced the enzyme.

Apoarently strains cf micrococci made resistant in_vitro
do not produce penicillinase. Spink and Ferris (lQhS) and North
and Christie (lQAb) found no penicillinase activity it micrococci

made resistant by subculture in increasing quantities of penicillin.

13

'-

CULTURES UQED IN THIS ’TU]!

Received in lyonhile from the Michigan Department

IX)
(3
\O
"U

of Health in 1953.

N Isolated from a lesion on the face of a man at the

 

Department of Yacteriology and Public Health, Michigan T-L
a
State College in 19A9. The strain was maintained in i
3
stock culture at room temperature with occasional :
transfers until used in this study. . f
C Isolated from the vagina of a mare during an examination e————-w

at the Department of Bacteriology and Public Health,
Michigan State Collene in 195A.

RF Isolated from a lesion on the arm of a student at the
Department of Bacteriology and Public Health, Kichipan

State College in 195h.

The above organisms were typical of E. pyogenes 223' aureus
in every respect. All produced a golden-yellow pisment, were
coagulase positive and hemolytic.

Throughout this study the letter 3 after the name of an
organism such as N-S was used to designate a parent sensitive
:strain. Any number occurring after the name of a strain indicates
tflrat the strain has been cultured in the presence of penicillin
enui is resistant. The number indicates how many times more

.resisteuit the culture was than its parent sensitive strain.

1h

MATERIALS AN) iJTHCJS

Sensitivitnyests Ani Development Q£ Resistant Cultures

 

Buffered crystalline penicillin G potassium obtained from
E. H. Squibb and Sons was used throughout this study. The vials
of penicillin were reconstitutei with 0.95 per cent sterile
saline, and stock solutions, also diluted with saline, were
prepared from these. Such solutions were maintained under re-
frigeration and used for a period of no longer than four days.
Penicillin now is a chemically defined, pure product. For this
reason the quantity'pg instead of units of penicillin was used
throughout this study. One unit is equal to 0.667 pg.

The medium chosen for stock cultures, sensitivity tests
and development of resistant cultures was Penassay broth, Difco.
This medium does not inhibit the action of penicillin and pro-
vides the essential growth requirements of g. pyggenes var. aureus.

before attempting to develop resistant strains it was
necessary to determine the smallest Quantity of penicillin which
inhibited parent strains. The lowest concentration of penicillin
‘which completely inhibits the growth of an orwanism will hereafter
be referred to as the minimum inhibitory concentration (M.I.C.),
after McVeigh and Hobdy‘(l952). Table I gives the amount of
broth, and the amount of culture added to sterile four-inch

culture tubes to produce the final concentration of penicillin

shown.

15

F.u

l.’.‘. v

Diluted culture usei is the inoculum in sensitivity tests
was prepared as follows. 1 2; hour culture was st nlvriigel in
a Cenco Iniustrlal type 12 photometer using a rei filter to give
a transnission of 60 per cent. The stariardizei culture wa< then
diluted 1:10 with Penassay lroth. One-tenth ml of the dilutei

6
culture contained approximately 10.6x10 organisms as deter inei
by direct microscopic counts.
TABLE I

METHOD USED FOR DETEHMINING THE M.I.C. CF SENSITIVE STRKIHS

 

 

 

 

 

M1 Penicillin M1 M1 Diluted Final Concentra-
from stock Broth Culture tion Penicillin

solution Added Added in pg/ml
0.02 1.9 0.1 0.01
0.0h 1.9 0.1 0.02
0.06 1.8 0.1 0.03
0.08 1.8 0.1 0.0h
0.10 1.8 0.1 0.05
0.12 1.8 0.1 0.06
0.1h 1.8 0.1 0.07
0.16 1.7 0.1 0.08
0.18 1.7 0.1 0.09
0.20 1.7 0.1 0.10

 

The lowest concentration of the antibiotic in which no
turbidity was evident at the end of 48 hours incutation at 37 C

‘was considered the N.I.C. The M.I.C. of penicillin for resistant

l6

Cultures was determined in the some manner except that more.
concentrated stock solutions were used in order to oltair ir-
hibition.

Preliminary observations revealed that cultures which had
attained considerable resistance lty repeated subculture on a
medium containing penicillin grew very slowly. Eellamy and

k...

Klimek (1948) attributed the slow growth of such resistant cul-

tures to their inability to grow anaerobically. It seemed rea- 1;

1-
sonz—ible that any method that would provide aeration would increase
the rate of growth of resistant cultures. Shaking proved to be
the needed stimulus. No studies of comparative turbi-‘iities of

5......

shaken ani unshaken cultures were undertaken. However, the
rate of growth of shaken cultures was at least doubled over un-
shuken ones for any given incubation period. Shaking cultures
had no effect on resistance attained lgy organisms. l‘enicill in
sensitivity tests on resistant cultures were performed in iupli-
cate. One set was shaken at 37 C while the other set was placel
in the 37 C incubator. The I“..I.C. recorded at either 2A or [,8
hours was invariably the same. Shaking was accomplished with
a Eurrell Moied 012 shaker adjusted to move the culture tubes two
cm in a vertical plane 180 times per minute. This moverrent
agitated the cultures as vigorously as Possible without wetting
the plugs.

The method used for development of resistant cultures was

the same as that used for testi‘ g the M.I.C. of cultures. The

only differences were that the inoculum was not standardizei or

17

 

diluted and shaking was employed. Results were recorded after
2h hours incubation at 37 C. The culture containing the high-
est concentration of penicillin in which good growth occurred
was used to inoculate a new series. When a strain grew in broth
containirg 5, 10, 100, 1000 and 10,000 times more penicillin than
was required to inhibit the sensitive parent strain it was sub-
cultured at that concentration two or three times until maximum
growth was attained. The M.I.C. of penicillin for that culture
was then tested sad the culture served as an inoculum for studies
of virulence, coagulase production, pigment production, hemo-
lytic activity, biochemical reactions, gram stain and morphology.
During frequent subculture any microorganism may undergo
changes in virulence, biochemical reactions, etc. To control
this variable the sensitive parent strain was subcultured on
Penassay broth each time s subculture was made of the resistant
strain. Any differences between sensitive and resistant strains
could then be attributed to growth of the resistant strain in
the presence of penicillin.
Preparation 9; Standard Suspensions

Twenty-four hour cultures grown in Penassay broth were

centrifuged in an International refrigerated Model PR-l centrifuge

equipped with an angle-head for one-half hour at 3500 rpm.
Under'these conditions the centrifuge developed a relative
' centurifugal force of 1670 times gravity. The supernatant broth

was removed and the cells were washed three times by centrifuga-

tion in 0.85 per cent saline. The bowl of the centrifuge was

18

 

maintained at A3 F when the centrifuge was in operation.

To break up clumps of cells after centrifugation saline
suspensions were placed in 250 ml Erlenmeyer flasks containing
about 20 glass beads, six.mm in diameter, and shaken in the Furrell
shaker for one hour. Microscopic examination of shaken suspensions
revealed that all large clumps were broken up but approximately 10
per cent of all bacteria were still in groups of two, three or
four. For the purpose of clarity, saline suspensionscfifmicrococci
after washing and shaking, will hence-forth be referred to as the
crude suspension.

A portion of the crude suspension was diluted with 0.85
per cent saline to give a reading of 60 per cent transmission in
a Cenco Industrial type 82 photometer with a red filter. The
standardized cell suspension was diluted 1:50 in saline and a por-
'tion used to fill a Petroff-Hausser bacteria counting chamber.
Counting was best accomplished with an American Optical Co.
microscope equipped with phase contrast, using the oil immersion
objective and a magnification of 970 diameters. The bacteria
in one-half of the #00 squares of the chamber.were counted and
this number was multiplied by five million to give the number of
bacteria per ml of standardized cell suspension. Two counts were
made on each suspension. From the results of the cell counts the
number of organisms per m1 contained in the crude cell suspen-
sion was calculated. These data are given in Table 2.

The number of viable micrococci contained in cell suspensions

standardized to 60 per cent transmission on the photometer was

19

also calculated using drop plates. One-tenth ml of a 1:1 million
dilution of a standardized cell suspension was pipetted in four
equal portions on the surface of a tryptose agar plate. Colony
counts were made after 18 hours incubation at 37 C. Drop plates
were made in duplicate. The results are shown in Table 3.

TABLE 2

RESULTS OF DIRECT CELL COUNTS CALCULATED IN BACTERIA PER ML OF CELL
SUSPENSION STANDARDIZED TO 60 PER CENT TRANSMISSION ON THE PHOTOMETER

 

 

 

Strain Cell Count (1108) Average

 

N-S 1 .50 9.65 x 108 ,
3.90 r
209P-S 10.60 9.55 x 103 5;—
3.50
0.3 2.80 9.93 x 108
10.00 s
RF-S 10.30 ‘~ 10.73 x 10
11.15

 

TABLE 3

RESULTS OF DROP PLATE COUNTS CALCULATED IN BACTERIA PER ML OF CELL
SUSPENSION STANDARDIZED TO 60 PER CENT TRANSMISSION ON THE PHOTOMETER

 

Strain Colony Count Average per m1
of Suspension
N-S 1}, 7.2 x 108
71
20919-5 69 6.7 x 108
7h 8
C-3 9!.- 608 x 10
7S 8
RF-S 10. 70" x 10
78

20

Virulence

Rabbits vary greatly in their susceptibility to g. 2*ogenes
13;. 235223. Julianelle (l9hh) inoculated 10 rabbits with 0.5
m1 of a 16 hour broth culture of a highly virulent micrococcus.
Of these, four became ill and recovered, three died of septicemia
in 72 hours and the remaining three died after 12 days. In con-

trast to the results Obtained by Julianelle, it was found possible
to produce death in rabbits quite consistently if relatively

large numbers of micrococci were injected.

Rabbits used for virulence tests on sensitive and resistant
micrococci were approximately two months old and weighed between
two and three kilograms. Before injection the rabbits were
weighed to the nearest 10 grams. The smallest number of micro-
cocci, per kilogram of rabbit weight, required to cause death of
all injected animals in one to six days was considered the minimum
lethal dose. The M.L.D. of each strain was determined seperately
because strains differed markedly in virulence. The number of
cells per m1 contained in the crude cell suspension was calculated
from the results of the direct cell counts. A rabbit could then
be inoculated with any desired number of bacteria from the crude
cell suspension. Rabbits were properly restrained and injected
via the marginal ear vein with a syringe and a 21 gauge hypo-
dermic needle. Observations of injected rabbits were made at

least twice daily and the time of death to the nearest one-forth

day was recorded.

21

The technique outlined above for preparing a standard inoculum
and for testing virulence of sensitive cultures was followed in
detail when testing resistant cultures. The results of M.L.D.
determinations on sensitive strains are shown in Table A.

Coagulase Production
Equal one-half m1 quantities of fresh rabbit plasma and a

21. hour broth culture of the test organism were mixed in three

 

inch tubes. The tubes were incubated in a 37 C water bath for :—
21. hours. Distinct coagulation of the plasma within three hours i
was considered positive.
agent Production and Hemolytic fictivity

,

Staphylococcus medium number 110, Difco, was used to de-

termine the pigment producing ability of both sensitive ans resis-
tant cultures. Petri dishes containing the medium were streaked

with the organisms and incubated at 37 C until colonies were

visible. The medium was then incubated at room temperature for

four days and the color of the colonies was recorded. When
resistant organisms would no longer grow in 6.5 per cent sodium
chloride the staphylococcus medium number 110 was prepared with
(3.5 per cent sodium chloride instead of the 7.5 per cent which
it normally contains.

Blood plates containing 5 per cent sterile defibrinated
horse blood in a tryptose agar base were used to determine
hemolytic activity of sensitive and resistant cultures. The
plates were streaked in such a manner as to obtain isolated
colonies and results were read after two days incubation at 37 C.

22

 

TABLE h

M. L. D. DETERMINATIONS OF SENSITIVE PARENT STRAINS

 

 

 

 

 

 

N0. of Bacteria Wt. of Time in Days
Strain Injected (x109) Rabbit to Death-D or
per Kg Rabbit Wt. in Kg. Survival-S
1.50 2.35 2.75-D
1.50 2.50 1.00-D
0075 2.20 5075'D
N-S
0075 2023 15025.1)
0.38 2.3) 6.25.D
0.38 2.50 30.00-3
[$050 2.10 1.CX)-D
30m 2.00 3050.D
209P-5 3.00 2.30 MOO-D
1.50 2.60 h.506D
0.75 2.90 30.00-3
[$050 2095 0050.1)
3.00 2.50 0.75-D
1050 2060 1000-D
C-S
0075 2030 1075’D
0.75 2.22 2.00-D
0038 2010 30.00-3
L.50 2.15 1.00-D
1.50 2.15 1.75-D
”‘3 0075 2063 1».st
0075 2010 5OSO'D
0.38 2.33 13.00-D

23

Biochemic§1_Reactions

Purple broth base, Difco, containing brom cresol purple as
an indicator was used as a base medium for carbohydrate fermen-
tation studies. The carbohydrate was added to the base medium to
a one per cent concentration tubed in three ml quantities ani

autoclaved for 15 minutes at 121 C. Cultures were tested for

 

their ability to ferment dextrose, lactose, maltose, mannite,
sucrose and glycerol. The tubes were inoculated and incubated at I
37 C for one week. The long period of incubation was necessary

because resistant cultures often fermented the carbohydrates

slowly .

To determine the ability of an organism to reduce nitrate
to nitrite, tubes of Nitrate broth, Difco, were inoculated and
incubated at 37 C for four days. The medium was tested for the
presence of nitrite by adding a few drops each of sulfanilic
acid and a,-naphthy1-amine reagent solution. A distinct cherry

red color indicated the presence of nitrite resulting from re-

duction of nitrate.

Gelatin liquefaction was determined on the staphylococcus
medium number 110 used for chromogenic studies. The organism to
be tested was streaked on a plate of the medium, incubated at 37
Citnrtil colonies were visible and then incutated at room temper-
ature for four days. The plate was then flooded with a saturated
solution of ammonium sulfate, placed in the 37 C incubator for

10 minutes and examined. Any clear zone around colonies indicated

2A

gelatin liquefaction.

Ability of an organism to grow in the presence of 6.5 per
cent sodium chloride was tested by inoculating a tube of Prain
Heart Infusion broth, Difco, to which 6.5 per cent sodium chloride
had been added, and incubating for one week. Any visible tur-
bidity was considered positive.

Reaction £9 Gram's Stain

“up. 7 W”

The organisms to be stained were dried on a slide and fixed

by heat. The slide was flooded with a one per cent aqueous

solution of crystal violet and three or four drops of a five

-x.‘ Jugs-.-me.-m.) -. _.

per cent solution of sodium bicarbonate were mixed with the dye. p
After allowing the preparation to stand two minutes, the slide
was washed with water and flooded with Lugol's iodine solution.
After one minute the slide was washed with water and blotted with
tissue paper. The preparation was decolorized with acetone
for about five seconds, washed and counterstained with a two per
cent solution of safranin O for two minutes.
Morphological Variation

Unstained wet mounts of resistant strains prepared from
a 2L hour Penassay troth culture were examined with the phase
contrast microscope for evidence of pleomorphism caused by repeat-
ed subculture in penicillin. Mounts of the sensitive parent
culture of each resistant strain were also made for the purpose
of comparison. Unstained preparations examined under phase con-

trast were found to be superior to stained smears because the cells

25

 

appeared larger and were not altered 1y fixing and staining.
Measurement of the diameter of a representative sample of

sensitive and resistant cultures was undertaken. A Filar ocular

micrometer equipped with a moving scale was calibrated with the

aid of a stage micrometer. Using the oil immersion objective and

a total magnification of 970 diameters each division on the drum

dial of the Filar micrometer was found to equal to 0.0909 microns.

Twenty-five bacteria selected at random were measured. The diameter

of the largest and the smallest bacterium measured as well as the

average diameter of all 25 was recorded.

26

.. Lv-_‘_- ._-.. _ a
' 1w

-- ‘w-ncfiupx.

 

RESULTS

Development 93 Resistant Cultures and Sensitivity Tests

 

Figure 1 shows the pattern of development of resistance to
penicillin G of the four strains of E. pyopenes m. M.

Two of the strains, 209? and C, acquired resistance at a more
rapid rate than did strains N and RF. After 50 daily transfers
in broth containing penicillin strain 209? grew well in the
presence of 200 pg while strain N grew in the presence of 1.0 pg
of the antibiotic. After 1.2 transfers strain 0 became resistant
to 200 fig while strain RF was able to grow in only 30 pg of pen-
icillin.

The M.I.C. of penicillin for each resistant culture was
tested at approximately each ten-fold increase in resistance
except for the first variants isolated which were tested after
a five-fold increase in resistance was attained. These results
are shown in Table 5. The number of times the variant culture
was more resistant than the parent strain was found by dividing
the M. LC. of penicillin for the resistant cultures by that of ‘
the parent strains. Resistant cultures will henceforth be iden-
tified by a number after the name of the strain which will indi-
cate how many times more resistant it was than the parent strain.
I Preparation pf Standard Suspensions
It was expected that bacteria grown in broth containing

penicillin would undergo changes in size and morphology. Because

27

Ha}. '. ani.‘ ‘l r~ a_-

 

I 000

10¢)

Micrograls Penicillin per .1

'0

.10

.02

 

 

 

#7’

7d?

F

 

WT

/-’\I

 

 

 

 

 

 

 

FIGURE 1
THE DEVELOPMENT or RESISTANCE
OF FOUR STRAINS or g. PYOGENES
Lip. AUREUS TO PENICIEi'm""c—'

Key

 

209?

—N
--—-RF

-—-—-0

 

 

IO

20

30 '+0 50
Tile in Days

:‘mrmzavgrm—h - - 1
f

TABLE 5

RESULTS OF M.I.C. DETERMINATIONS OF SENSITIVE AND RESISTANT CULTURES

 

 

Strain M.I.C. Number of Times More
Resistant than
Sensitive Parent Culture

 

 

 

 

209P-S 0.03 -
209? 0.15 13.3
1.00 33.0
6.00 ' 200.0
50.00 1666.?
300.00 10,ooo.0
N-S 0.01 -
n 0.30 7.5
0.90 22.0
n.00 100.0
50.00 1250.0
0-3 0.01 -
c 0.20 5.0
2.00 ' 50.0
6.00 150.0
50.00 1250.0
300.00 7500.0
RF-S 0.0a -
as 0.20 5.0 ’
3.00 50.0
7.00 175.0
no.00 1000.0

29

of these changes it seemed reasonable that a saline suspension of
a resistant culture standardized to a reading of 60 per cent trans-
mission would contain a number of bacteria significantly different
from that of a similar standard suspension of the parent culture.
For this reason suspensions of resistant organisms were standard-
ized to a reading of 60 per cent transmission in the photometer
and direct cell counts made of them. Contrary to what was expected
there was no significant increase or decrease in the number of
bacteria in such suspensions when compared to the number of organ-
isms found in suspensions of sensitive micrococci. Results of
direct cell counts on resistant organisms are shown in Table 6.
Virulence 9__f_ Resistant Cultures

As the four strains of £1. plogenes y_§_r_'. m3 became resist-
ance to penicillin there was a rapid and progressive loss in viru-
lence for rabbits. Resistance cultures produced from strains C and
RF failed to cause death in any injected rabbits, even when they
were only five times as resistant as the parent strain and when
five M.L.D. were inoculated. Culture Nx7.5 caused death when two
and five M.L.D. were inoculated but not when l M.L.D. was used.
Cultures of strain N more resistant than Nx7.5 failed to cause
death in any injected rabbits. Resistant cultures produced from
strain 209? maintained their virulencelonger than those of other
8trains. Culture 209Px13.3 killed only the rabbit receiving 5
M.L.D., 209?x33 killed at 1, 5 and 10 M.L.D. and 209?x200 killed
at 10 H.L.D. Cultures of strain 209? more resistant than 209Px-

200 Were avirulent. Factors other than the injected bacteria may

30

 

TM¢E6

RESULTS OF DIRECT CELL CCUFTS CN RESISTANT CULTURES CALCULATED IN
BACTERIA PER ML OF CELL SUSPENSICN STANDARDIZED TC 60 PER CENT
TRANSMISSION ON THE PHOTCMETER

 

 

 

 

 

 

31

Resistant Culture Cell Count (x108) Average
209Px13.3 8.85 9.33 x 108
9.80 R
209Px33 10. 0 10.55 x 10'
10.50 8
209Px200 11.20 11.08 x 10
10.95 8
209Px1667 8.25 8.h8 x 10
8.00
209Px10,000 10.55 10.55 x 108
10.55
Nflos 8.22 90108 X 108
10.00 8
Nx22 8.70 9.15 x 10
9.35 8
leOO 8.25 8.35 x 10
7.75 8
Nx1250 2.00 ' 9.15 x 10
9.30
0x5 11515 10.58 2:108
10.00 8
Cx50 8.50 9.25 x 10
10.00 8
0x150 11.00 11.25 x.10
11.50 8
Cx1250 11.00 11.30 x 10
11.35 8
0x7500 2.60 9.85 x 10
10.15
RFxS 11.0 10.83 x 108
1o. 0 8
Rrxso 11.85 11.13 x.10
11.00 8
RFx175 10,50 11.00 x 10
11.50 8
RFxlOOO 2.80 10.35 x 10
10.90

have contributed to the death of the rabbit receiving one M.L.D.

of 209Px33 since death occurred in only one day.

Each time the virulence of a resistant culture was tested
the sensitive parent strain which had been subcultured a number of
times equal to that of the resistant culture was used as a con-
trol. The only control culture which showed a loss of virulence
was strain N-S. The N-S control on Nx22 required seven days to
cause death of a rabbit while the N-S control on leOO survived.
In the next series of rabbits injected with Nx1250 and N-S as a
control it was necessary to double the number of micrococci con-
tained in a M.L.D. in order for the N-S control to cause death

in from one to six.days. Table 7 shows the results of virulence

tests.

Coagulase Production

The coagulase test was performed on all resistant cultures
produced from the four sensitive parent strains. A coagulase
negative microorganism and a tube containing undiluted plasma
were used as controls. Throughout the experiment all sensitive
parent and all resistant cultures were coagulase positive.
Pigment Production and Hemolxtic Activity

Resistant strains showed little or no loss of pigment pro—
ductixni. The pigment of all resistant cultures appeared identical
‘to that of the sensitive parent strains.

As each strain became more resistant to penicillin its

hemolytic activity was decreased. Two of the most resistant cultures,

32

 

TABLE 7

RESULTS OF VIRULENCE TESTS OR RESISTANT CULTURES

 

Time in Days
to Death-D or

No. of Bacter a No. of Wt. of
Culture Injected (x10 ) M.L.D.s Rabbit

 

 

 

 

 

 

 

 

per Kg Rabtit Wt. Injected in Kg Survival-S
2090-3
control 3.00 1 2.1L 2.00-D
209? 3.00 l 2.2h 30.00-S
x13.3 .

6.00 2 2.70 30.00-S
12.00 5 2.96 3.00-D
209P-S
control 3.00 l 2.3h b.50-D
209? 3.00 l 2.82 1.00-D
XBB
6.00 2 2.16 30.00-S
15.00 5 2.35 11.00-D

209P-S
control 3.00 l 2.h0 3.50-D
209? 30.00 10 2.35 8.00-D
x200
209P-S
control 3.00 l 2.52 2.50-0
209? 30.00 10 2.35 30.00-S
x1667
209P-S 3.00 l 2.30 3.00-D
control
x10,000
N—S
control 0.75 l 2.38 h.50-D

33

TABLE 7 (CONTINUED)

 

 

 

 

 

 

 

 

 

 

3h

No. of Bacter$a No. of Wt. of Time in Days
' Culture Injected (x10 ) M.L.D.s Rabbit to Death-D or
per Kg Rabbit Wt. Injected in Kg Survival-S
Nx7.5 1.50 2 2.63 6T50-D
3.75 5 3.16 1.50-0
N-S
contrOI 0075 1 3 .00 7.00-D
Nx22 0.75 l 3.00 30.00-S
1.50 2 2.20 30.00-S
N-S
control 0.75 1 2.h1 30.00-S
Nx100 7.50 10 2.20 30.00-S
~__ N-S
control 1.50 1 2.h8 3.50-D
le250 15.00 10 2.50 30.00-S
C-S
control 0.75 1 2.9h 3.25-D
0x5 0.75 1 2.25 30.00-S
3075 5 2.65 30.00-S
C-S
control 0.75 l 2.53 2.25-D
1.50 2 2.30 30.00-S
C-S '
control 0.75 1 2.25 1.50-D

TABLE 7 (CONTINUED)

 

 

No. of Bacteria No. of Wt. of :Time in Days

 

 

 

 

 

 

 

 

 

35

Culture Injected (x109) M.L.D.s Rabbit to Death-D or
per Kg Rabbit Wt. Injected in Kg Survival-S
0x150 7.50 10 2.12 30.00-S
C-S
control 0.75 1 2.30 2.00—D
Cx1250 7.50 10 2.65 30.00-S
C-S
control 0.75 l 2.53 b.00-D
Cx7500 7.50 10 2.66 30.00-S
RF-S
control 0.75 l 2.00 2.50-D
RFxS 0.75 l 2.h0 30.00-S
1.50 2 2.01 30.oo-s
3.75 5 2.hh 30.00-S
RF-S
contra]. 0075 1 2089 1.75‘D
RFxSO 0.75 l 2.51 30.00—S
1.50 2 2.30 30.00-S
3075 5 2060 30.00-S
7.50 10 2.37 30.00-S
RF-S
control 0.75 1 2.50 2.25-D
RFx175 7.50 10 2.22 30.00-S
RF-S
control 0.75 1 2.53 h.75-D
RFxlOOO 7.50 10 2.76 30.00-S

produced from strains 209? and C, became completely nonhemolytic.
Resistant cultures developed from strains N and RF still retained
some of their hemolytic activity at the end of this study but they
did not reach the same degree of resistance as did strains 209?
and C.

Resistant cultures produced from strains 209?, C, and RF

 

showed increasingly smaller zones of clear hemolysis as resis- ”__
tance increased. Resistant cultures produced from strain N did 1
not behave in this manner. The size of the zone of hemolysis I
remained about the same but as resistance developed hemolysis {
within the zone became less complete. On a tlood plate the L
hemolytic zone of Nx1250 was just discernible.
0f the sensitive strains that were subcultured in broth
each time a culture was made of a resistant strain only strain
N-S showed a change in hemolytic activity. The zone of hemolysis
it produced became less complete in a manner similar to that of
the resistant cultures produced from strain N but not to the same
degree.
Biochemical Reactions
The biochemical reactions of cultures resistant to penicillin
.are shown in Table 8. All resistant cultures fermented dextrose,
lactose, maltose, mannite and sucrose. The ability of resistant
cultures to ferment glycerol was lost when they became about 1000
times more resistant than the parent. When strains acquired
:resistance, growth in the presence of 6.5 per cent sodium chloride

became scant and finally ceased altogether. The capacity of the

36

TABLE 8

RESULTS OF BIOCHEMICAL REACTIONS OF SENSITIVE AND RESISTANT STRAINS

 

 

 

 

 

 

Culture Dext. Lact. Malt. Mann. Glyc. 3.3: N03 Gelatin
2099-5 ‘ / / ¢ ¢ ‘ / / /
209Px13.3 7‘ / 7‘ 7‘ 7‘ 7‘ 7‘ ¢ 7‘
209Px33 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘
209Px200 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘
209Px1667 7‘ 7‘ 7‘ 7‘ - - 7‘ -
209Px10.ooo 7‘ 7‘ / 7‘ - - 7‘ -
N-3 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ ‘
Nx7.5 7‘ ‘ 7‘ 7‘ 7‘ 7‘ / / 7‘
Nx22 / # 7‘ 7‘ 7‘ 7‘ 7‘ /
leOO 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘

Nx1250 7‘ ‘ 7‘ 7‘ - - 7‘

c-s / / 7‘ 7‘ 7‘ 7‘ I 7‘ 7‘ 7‘
0x5 7‘ ‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘
0x50 7‘ 7‘ 7‘ 7‘ 7‘ ‘ 7‘ 7‘ 7‘
0x1250 7‘ 7‘ 7‘ 7‘ - - 7‘

Cx7500 7‘ / 7‘ / - - %

" RF-5 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ /
RFxS 7‘ / 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ ‘
RFx50 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ 7‘ ‘ 7‘ 7‘
121-“7:175 7‘ 7‘ 7‘ 7‘ 7‘ - 7‘ 7‘ 7‘ 7‘ 7‘
RI-‘xlooo 7‘ 7‘ # 7‘ - - 7‘ K 7‘ 7‘

37

four strains to reduce nitrate to nitrite was unaffected by their
becoming resistant to penicillin. Gelatin liquefaction of resis-
tant cultures was reduced to some extent and cultures 209le667 and
209Px10,000 became gelatin negative.

Reaction £2 §5§313_§t§ig

Burke's modification of the gram stain was used to stain
preparationscfi'sensitive and resistant cultures. Only cultures
Cx7500 and 209Px10,000 showed a change in reaction to the stain.
In culture Cx7500 about 25 per cent of the cells stained gram
negative. Many of the gram positive cells seemed to retain the'
crystal violet sparingly. Gram stained slides of culture 209Px-
10,000 revealed that approximately 75 per cent of the cells were
negative while 25 per cent were positive. In this culture the
individual cells appeared either distinctly gram positive or
gram.negative with no intermediate forms.

Morphological Variation

‘Wet mount preparations of resistant micrococci were examined
.for evidence of pleomorphism. In most wet mounts a small number of
cells, usually less than one per cent, could be found which were
liot cocci. These forms varied from coccobacilli to short rods,
diplococci and diphtheroid forms.

Penicillin also affected a change in the size of resistant
cultures. These changes are shown in Table 9. As resistance to
penicillin increased there was an increase in size of the cells
in the culture. This continued until each culture became approxi-

:mately 1000 times more resistant than its parent strain. At this

38

Q.-. L-a 7 7.7...115'
4 a

I . ”4.1.14

.7 i

TM¢E9

RESULTS OF THE MEASUREMENT OF SIZE OF SENSITIVE
AND RESISTANT CULTURES

 

 

Culture Average Diameter Diameter of Largest Diameter of Smallest

 

 

 

 

 

39

in Microns in Microns in Microns
209P-S 1.33 1.6 1.2
209Px13.3 1.3h 1.6 0.8
209PXB3 1.3h 1.7 1.1
209Px200 1.63 1.9 1.2
209Px1667 1.63 1.9 1.1
209P110,000 1.h6 1.8 1.2
N-S 1.23 1.h 1.0
Nx7.5 1.15 1.h 0.9
Nx22 1.89 2.6 0.9
leOO 1.83 2.1 1.1
N11250 1.33 1.5 1.2
“(ifs 1.58 1.4‘“ 1.1
0x5 1.26 1.5 1.1
Cx50 1.h9 1.9 1.2
0x1250 1.75 2.0 1.h
Cx7500 1:23 __7 1.6 019“
RF-S 1.25 1.L 1.1
RFxS 1.28 1.6 1.2
RPx50 1.62 1.8 1,1
RFx175 1.61. 2.0 1.2
RFxlOOO 1453 1.8 0.9

time the average diameter of the cells of the resistant cultures
was akout 0.4A microns larger than that of the sensitive cells.

On further increase in resistance the average diameter of the cells
tecame smaller. Table 9 also shows that resistant cells are more
variable in size than are the parent sensitive cells. While the
average difference between the largest and smallest cells measured
was 0.3 micron for the four sensitive strains, the value was 0.7
micron for the four resistant cultures which had attained a 1000

fold increase in resistance.

40

DISCUSSION

The present investigation indicates that cultures of E.
pyogenes 335. 225535, when subcultured in the presence of penicil-
lin, became increasingly tolerant to it. All the strains appeared
capable of acquiring a higher degree of resistance than they at-
tained. One can only speculate on how resistant an organism could
become if subcultured in the presence of penicillin for an extended
period of time. As a culture acquires resistance, growth becomes
less luxuriant. Eventually, this would be a limiting factor in its
ability to acquire further tolerance to penicillin. Nevertheless,
it is a tribute to the versatility of a bacterium that the descend-
ants of a culture are capable of living and growing in the presence
of a comparatively large amount of a toxic substance which, a few
weeks previously, would have been lethal in a very high dilution.

There are two theories concerning the mechanism whereby
bacteria acquire resistance to penicillin‘ig'ziggg. The first is
that acquired resistance is only an adaptive change on the part of
the organism, that is, a result of interaction between the antibi-
otic and the bacterium. The second, as first presented by Demercc
(l9h8), postulates that resistant bacteria arise by.a process of
spontaneous mutation, the penicillin acting only as a selective
agent by the destruction of sensitive cells. According to Demeree's
hypothesis, resistance to penicillin acquired EBHXEEES could be

expected to proceed in a stepwise fashion. Figure l, which shows
hl

the pattern of development of resistance of the four strains

used in this study demonstrates no such stepwise increase in
resistance. However, a strain would often grow in the presence
of a certain concentration of penicillin through several sub-
cultures, then suddenly become tolerant to much larger concentra-
tions.

When comparing the virulence of sensitive and resistant
bacteria it was necessary to consider all the factors which affect
virulence. Factors such as volume of inoculum, suspending medium,
route of inoculation, size, age, weight and species of animal
can be easily controlled. To a less extent the log phase of the
organism can be regulated. However, in zitgg produced resistant
micrococci exhibit a slower rate of growth than do sensitive
cultures. This slower rate of growth naturally affects virulence.
An organism that divides slowly will have less chance to produce
a fatal infection because the host's defensive mechanism will
have more time to destroy the parasite. Certainly some, and possi-
bly a considerable part, of the decreased virulence of resistant
micrococci is due to this decreased rate of growth. Then, loss
of virulence may not be due entirely to dissociation phenomena
brought on by the organisms having acquired a resistance to penicil-
lin.

On the other hand, the argument mdght be presented that this
slowed rate of growth should not be considered in evaluating the
results of virulence tests on resistant micrococci. It might be

said that this is one of the ways in which the organism was changed

42

by subculture in penicillin and that this was one of the character-
istics of the resistant organism which was altered.

Micrococcal infections are usually localized. In a localized
infection the slow rate of division of a resistant micrococcus
might not be important from the point of view of virulence because
the organism is usually comparatively well isolated from the
defensive mechanisms of the host. However, when the virulence of
a resistant micrococcus is tested by intravenous inoculation of
rabbits whose defensive mechanisms have ready access to the
injected bacteria the results can be far different. It is difficult
to control or to evaluate the effects of this slow rate of growth
when comparing the virulence of sensitive and resistant organisms

but it must be taken into consideration.

#3

S UMMA RY

In vitrc induced resistance of g, pyogenes var. aureus

 

to penicillin is accompanied by changes in virulence, hemolytic
activity, some biochemical reactions, gram stain reaction and
morphology. The most striking change was in virulence. All
four strains lost their virulence for rabbits as penicillin re—
sistance was acquired. Two of the resistant cultures became non-
hemclytic, while the other two still retained some hemolytic
activity. All highly resistant strains failed to ferment glycerol
and to grow in the presence of 6.5 per cent sodium chloride while
only one strain lost the ability to liquefy gelatin. Many of the
cells in the two most resistant cultures became negative to
Gram's stain. On continued exposure to penicillin the cells of
the cultures enlarged. After becoming highly resistant the size
of the cells returned to near normal.

Resistant cultures showed no change in ccagulase production,
chromogen production, nitrate reduction or in fermentation of

dextrose, lactose, maltose, mannite or sucrose.

LL

 

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#5

 

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A6

 

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Je BaCts, 51, [$25-4260

47

I wish to express my gratitude to Dr. Jack
J. Stockton assistant professor of bacteriology
at Michigan State College for suggesting this
problem, for guidance in the laboratory work and

for assistance in the preparation of this thesis.

 

Son .3“ '1'!

Oct] 5 3

 

   

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