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THE SENSITEVITY OF
HEMOPHILUS PERTUSSIS
AND RELATED ORGANISMS
TO VARIOUS ANTIBIOTEC AGENTS

Thesis for the Degree of.
Master of Science
MICHIuAN STATE COLLEGE
fl _ ' , A 1:" . 1
Chester a. fiembaca
19:3

w

THESE

This is to certify that the

thesis entitled
The Sensitivity of
Heme hflus Pertussis
And Belem Organism
To Various Antibiotic Agents
presented by

Chester A. Hombeek

has been accepted towards fulfillment
of the requirements for

Master Wegree in M1087

//flfifM

/Major prof/ssor
Date MUG

0-169

 

q I

m msnmn or memos msxs mm mm 03mm so
' muons mutant: ms

3:
Gheeter A. anuck

1 131818

Submitted to the School of Graduate Studiee of mohigan
State college ot Agriculture and. Applied. Science
in partial fulfillment of the requiremente
for the degree of

mm 01' 801130]

Department of Bacteriolog

1953

THESIS

 

 

 

egmnsmhms

lhe author is grateful for the generous advice and ccuneel of
Dr. G. hearing of the Western Michigan Section of the Michigan
Department of Health, and for the liberal eupply of culturee and
materiale‘ueet.in.thie etudy. rho author is indebted to
Dr. 3. J. Stateeth of Michigan State Gollege for hie helpful comments

and suggestions.

309052

table of Gcntente

Introduction .oo...................o.............................o 1

Part I Seneitivity of L Egtuseis and related organism ee
determined by the nee of antibiotic diece and e eelid
Iediun.
Heteridle and.method eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

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l’art I! Seneitivity of 3‘, mm and related organieme to
varioue antibiotice ueing e liquid media.

Hateriale and method .......................................... 12
Renate ....................................................... 15
Discussion eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 16
Inner: .o..................................................... 27

cou1n.10‘. oeeeeeeeeeeeeeeeeeeeeeeeeeeeOOOcoeooeoooeeoeeeeeooo 27

Part 111 Study of energietic or antagonistic action of varicue
ccnbinatione of antibiotice.

Materials and method eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 29
lfllultl eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 31
DIIGEII1¢I eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 33

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“38111"!!! 01' BIOPHIIMS MSSIB All!) mm
ORGANISMS '10 VARIOUS ANTIBIOTIG AGENTS

MENU! ION

During recent years. new antibiotic agents have been discovered in
rapid succession. their importance can hardly be exaggerated, yet they
are. collectively, not a panacea for all infectious diseases. Bone of
these antibiotic agents are spectacularly effective against a very few
organisms, while others are active against a wide variety. On the
other had. some organisms are acre or less resistant to all of these
agents. Among the infections which have generally resisted treatment
with antibiotics is pertussis. It should be noted. however, tint lost
of the published studies are concerned with streptonycin, chlorcuycetia,
or aureonycin.

In any reports, the primary study consisted of a determination of
the sensitivity in vitro of the organisn to the antibiotic in questien.
has is, of course, desirable since it may provide some basis for pre-
diction of the succeu of the antibiotic in preventing or curing the
disease. hrs met be taken in evaluating any in vitro test since
promising results are not always borne out in clinical trial.

Kegarty. Ehiele. and Verwey (1916) Presented evidence that strepto-
mycin in a concentration of 15 neg/n1 was bactericidal to 3‘ mm
in vitro. Ihen infected mice were treated with 0.5 lg doses, the
survival time averaged approximately twice that of the controls. but
there was still an ultimate mortality of 90 per cent.

who: and Eenans (1951) demonstrated that 0.2 to 0.3 neg/ll of
chlorolycetin inhibited growth of L mgmssis in vitro. In subsequent

"X

in vivo experiments 100 per cent protection was achieved in acne
inetanoes when the intranaeal route of inoculation was used. In intra-
cerebrel infection chloronycetin was much less effective.

sen. Pittman. and Olson (191:9) showed that anreowcin hydrochlor-
ide given subcutaneously to nice in apparently non-toxic doses not only
delayed the tins of death but also prevented deaths due to prior intra-
cerebral infection with L, m. lo in vitro experiments were re-
ported. Preliminary clinical trials with children suggested tint aureo-
min given orally in apparently non-toxic doses shortened the clinical
course of the disease.

a lumber of other investigators (Alexander and BedIan, 1959: Day
and Bradford, 1952; mm than, 1950) have published their findings on the
sensitivity of 3‘ m and sinilar organises to a nunber of anti-
biotics. In the najority of cases. however, only one aethod of testing
was used. In the natter of investigating the possibilities of the
synergistic effect of combinations of antibiotics against L my,
very little. has been done.

his study was planned with three major objectives. as follows:

I. Sensitivity of 3,, m and related organisms

to various antibiotics as detenined by the use
of antibiotic discs and a solid medium.
11. sensitivity of 3. W and related organisns ’
to various antibiotics using a liquid nedium.
III. Synergistic or antagonistic action of various con-

binaticns of antibiotics.

he use of both solid and liquid media was intended to afford a
nore couplets basis for evaluating the antibiotic agents under test.
Also it was hoped that the results would be of sons significance in
conparing the two nethods.

Ihe choice of the related organisms, m W and
the parapertussis bacillus rested largely upon the work of Kendrick and
lldering (1938). and Anderson (1952). m former investigators showed
by agglutination-absorption techniques and cross agglutination reactions
that the three organisms are related but not identical. Anderson (1952)
in a recent report. indicated that these organisms have a cannon '0'
antigen, whereas variations of the antigenic structure of the three
organises were apparently due to slightly different '3' and/or '1'
antigens, based on differentiation according to mm: (1951). Both
he Wand the parapertussis bacillus have been reported as
the cues of respiratory infeetions in hnnan beings (Shih Man. 1950,
and lldering and tendriek. 1951). Because of the sinilarity of these ,
tee organisms to 3., m it seened reasonable to include then in
the study. he parapertussis bacillus has never been given an official
generic designation. Since its taxonomic position has not been clari-
fied, it will be referred to here nerely as the parapertussis bacillus.

 

Part I
MIT“!!! 01‘ II. PMSSIS AND RELATED ORGANISMS 1'0 VARIOUS
MIDIOTICS A8 WIRED D! m USE 01‘ ANTIBIOTICS D1868.
may;

the medium used in this portion of the study was Bordet-Gengcu
nediun which was made up as follows: the Bordet-Gengcu agar base nediun
(Difco) was hydrated by suspending has an in 100 ll of a one per cent
solution of glycerol in distilled water and heating to boiling to dis-
solve the agar completely. It was then autoclaved for fifteen minutes
at 15 lbs. pressure (1216).- fhe nediun was allowed to cool to approxi-
nately ‘$56 and aseptically enriched with sterile sheep blood to 15 per
cent by velune. After thorough airing plates were poured, incubated at
376 for 2‘!» hours to elininate contaminated plates, and stored in a
refrigerator.

fhe antiuotic discs used were the Bacto Sensitivity Discs, menu-
factured by the Difco Laboratories, Detroit 1, Michigan. has following
antibiotics were tested: bacitracin, chloronycetin, dihydrostreptomin,
penicillin, aureonycin, and terranwcin. lach of these agents was sq-
plied in three concentrations which are indicated in table l.

he lyophilized cultures of the three test organisns were supplied
by the Western nichigan Section Laboratory of the Michigan Departnent
of health. he 6 L mrtgssig and ll parapertuesis cultures were iso-
lated from cough plates collected in Grand Rapids from children with
whoOping cough. lb. bgonchigeptica 22067 was isolated at fhorndyke
Itemrial hospital, Boston, from a child with symptoms of whooping cough.

fhis culture was received in Grand Rapids from Dr. Martha Yard,

Comicable Disease center. Ghanblee, Georgia. he culture
E: W 2361+ was isolated from a ferret by Dr. I. Ferguson,
liehigan Departnent of Health, Lansing.

m

Prior to conducting the tests, the cultures were reconstituted by
suspending the contents of the supine in approximately 0.2 nl of sterile
physiological saline. plating on the Bordet-Gsngou nediun, and incu-_
hating at 35 O. for 2» to 118 hours. After suitable growth had been
established, enough growth was removed by loop and enulsified in 2 d.
of sterile saline to give a suspension of approxinately 1.2 billion
organisns per :1. Previous tests had shown that 0.3 ll of such a sus-
pension, when spread evenly over the surface of the plate. provided the
necessary confluent gowth and the suspending saline was quietly absorbed
by the nsdiun.

After the inoculun had been spread over the plate and had been
absorbed by the nediun, the sensitivity discs were aseptically placed
upon the surface using flaned forceps. lo acre than three discs were
placed on the surface of the nediun to avoid crowding. lines the discs
cone in three concentrations of each antibiotic. one plate was suf-
ficient for deternining the effect of one antibiotic.

After the discs had been placed on the surface of the nediun, the
plates were incubated in an upright position for 21+ hours at 35 C. In
the ease of h m, the incubation period was increased to ’48
hours. he results were read by noting the dimeter of the clear sons

of no growth which surrounded the discs.

1;.

TABLE 1

 

 

Mean diameter in centimeters of inhibition zone with each concentration of antibiotic

Sensitivity g; g. pertussis, parapertussie bacillus, and.Br. bronchiseptica a§_indicated‘21_antibiotig dieoe

 

 

 

 

 

Penicillin Terramycin Dihydrostreptomycin Bacitraoin Chloromycetin Aureomycin
Units Meg Meg Unite Meg Meg
Organism 0.5 1 519_ 10 30 60 _fi;. 10 100 2 _1g_ 20 510, 30 60 .10 .30 60
oertussie
23518“ 0.6 0.8 1.4 1.5 2.0 2.6 0.8 1.1 1.3 0.7 1.5 2 3.4 3.7 3.9 0 1.2 1.6
18323 0.5 0.7 1 1.2 2.1 2.6 0.6 0.8 0.9 2.0 2.4 3.2 3.6 3.8 4 1.0 1.7 2.0
23360 0.5 0.6 1.1 1.6 2.7 2.9 0.5 0.6 0.9 1.8 2.9 3.9 3.3 3.9 3.9 2.0 2.2 2.6
19605 0.7 1.1 1.9 2.1 2.5 2.9 0.8 0.8 1 1 1.3 2.8 3.3 3.8 4 0 1.8 2.3
18560 0 0.9 1.5 3.2 3.6 4 1 0.8 2.2 1.5 2.8 3.1 3.7 3.9 4 0 1.6 3.0
10536 0.8 1 1.2 2.1 2.9 3.4 0.7 1 1.2 2.0 2.8 3.1 3.1 3.5 3.7 0.8 2.1 3.7
parapertuesis bacillus
23788 O 0 2.7 3.1 3.3 0 0 0.9 0.7 1.8 2.2 2.5 3 3.5 0.8 1 1.3
17903 0 0 2.5 2.9 3.6 0 o 0.8 0.9 1.7 2.1 2.7 3.2 3.8 1.1 1.7 2.5
23807 0 0 0.7 2.8 3.1 3.5 0 0 1.1 0 1.5 1.8 3.0 3.3 4 0.8 1.2 1.9
22345 0 o 2.8 2.8 3.2 0 0 0.8 1 0.9 1.6 2.8 3.4 3.8 2.0 2.1 2.8
23456 0 0 0.5 2.7 3.0 3.5 o 0 1.1 0.6 0.9 1.6 2.9 3.5 4 1.2 1.8 2.8
23910 0 0 0 2.6 3.0 3.6 0 0 1.2 0.9 1.7 2.0 2.9 .2 3.5 1 1.6 2.8
23054 0 0 0 2.7 3.1 3.4 0 0 0.7 0.9 1.3 1.7 3.8 4 4 1.3 2.6 2.8
21851 0 0 0 2.2 2.5 3.1 0 0 1.6 0.8 1.1 1.8 2.8 3.2 3.6 0.9 1.3 1.9
23310 0 0 1.1 3.0 3.8 4 0 o 1.2 1.1 1.6 1.8 2.5 3.7 1.4 2.8 2.9
21838 0 0 o 3.2 3.8 4 0 0.7 1.1 0.9 1.8 2.0 3.0 3.4 3.9 1.6 1,8 2
21551 0 0 0.7 3.0 3.7 3.9 0 o 1.6 0.6 1.1 1.4 3.2 3.6 4 2.8 3.2 3.3
Br;_pgonchieegtiqgg -
22067 0 1.5 1.7 1.8 0.6 1.0 1.2 1.5 2.1 2.3 0.6 1.7 1.9
2464 1.7 1.9 2.1 0 1.3 0.5 1.5 1.7 1.8 0.7

 

m

Several duplications of the tests wdth.antibiotio discs were nade,
and the results are given in table 1. 'i'he figures represent the neon
of the recorded diameters of the clear zones, in centineters. These re-
sults are shown in a condensed fan in table 2, where the results are
expressed as an.ayerage of the observatione‘with cultures of each.species.

Wflhw 81: culture- “Ham m-
tested. and these were all inhibited te some degree by each of the anti-
biotics tested. According to the sins of the sons of inhibition, the
greatest effect was observed with.chlcrenycetin.and the least effect
'with.penicillin.and.dihydrostreptonycin. ferranycin, baoitracin.and
aureoqein, all appeared to have the sane general inhibitory effect.

2.2213222: .91 18.2 W Jeanine. mm B. mm.
the parapertussis bacillus was alnost completely resistant to peni-
cillin and was inhibited by metreptenycin only in the 100 neg an
nount. can one of the 11 cultures tested showed any inhibitive cone
with the 10 neg disc. Chloronyeetin and terranyein were the nest ef-
fective. Dacitracin.and.aureonycin.also were inhibitory in.all three
concentrations although.the tones were not as large.

Wflhhmrahimfla- Only two culture: of this ore-e-
isn were tested and there as sons difference in their sensitivity.
However. both.were resistant to penicillin.and.dihydrostreptcnyctn.
they showed less resistance to baeitraein and sureoqein. Again terrap
nyein.and chloronyoetin.appsared to be the most effective of the anti-

biotics.

W

m results with g. pgrtussi: are qualitatively sinilar to these
with the other two organic-1s. Penicillin had a considerably greater
inhibiting action upon L mrtussig than upon either the parapertussis
bacillus or he. WM. Dihydrostrsptomin showed results
which were very sinilar to that of penicillin. Hearty at 51.. (1945)
showed that E" W was inhibited by 3 ncg of streptonycin, but
nice infected with g, m and treated with streptomycin showed a
90 per cent mortality rate. no observations recorded in table 2 in-
dicate that there is an inhibiting effect with l ncg, but that the
inhibitory effect is not markedly increased by a much greater concen-
tration. Since penicillin has been shown to be of little value in
aninal or clinical tests with L mrtussil as the infecting agent, and
streptomycin but little better, it seens possible that sons sise nay
not be a reliable criterion for choice of a suitable agent in aninal ..
tests. On the other hand, chlcrcnycetin, which displayed a neon inhi-
bition sone disneter of 3.1!- cn in a concentration of 10 neg has been
shown to be active against enperinental pertussis infections. Barber
and Banana (1951). have shown that 0.3 neg of chloruycetin per I]. is
inhibitory to 3., will! and that in treatment of nice infected with
I. m, 100 per cent survived in sons cases.

ferrenycin and bacitracin showed an internediate inhibitory effect
as compared with chloronycetin and dih'drcstreptonycin. inreonycin
gave results showing appreciably nore inhibition than dihydrostreptenycin
er penicillin, though nueh less inhibition than chloronycetin. his is
an interesting finding since Dell gt 51., (19%) have reported. pronising

rssults in aninels with anreow'cin and, to a lesser extent, in clinical
treatment of children. however, in tests with nice, the dosages were
as high as 19 meg, which would produce a relatively high blood level.
he increased inhibiting effect of higher concentrations of aureonyein
as compared with streptenycin or penicillin as shown in table 2 would
give an indication of possible inhibition at high concentrations of the
antibiotic in the blood.

It is of note that Druids and Dockrill, (1952) have reported that
the inhibition none disaster when using enreonycin may be considerably
less than some other antibiotic, yet in a liquid nediun it is mch ncre
effective. successful treatnent of clinical infections due to strains
sensitive to aureonycin in the test tube indicated that the tube method
was nore reliable in their study.

l'ron the results given in table 1. it is evident that penicillin
and dihydrestrsptonycin have little effect upon the parapsrtussis bacillus
in any concentration. fhis result is not surprising since Alexander and
Dodson (19119) found in a recent study that when using Dordet-Gengou
plates containing a concentration of 1,000 neg of streptonycin, a con-
siderable mber of colonies of parapertussis bacilli grew from the
original inoculun. Ehe withers concluded from this result that there were
cone entrsnely resistant organisms initially present in every population
enninsd. One night, therefore, expect to find isolated colonies in the
clear none of inhibition surrounding the antibiotic disc: however, none
was found. a possible explanation is that the occasional clear some
found around the disc containing the highest concentration of dihydro-
streptemin did not include a large enough number of organisms to denou-

10
TABLE 2

Summary of Results with.Antibiotic Discs Showing Mean Diameter Observed
with Several Cultures.

 

Parapertussis Br. bronchi-

 

 

§;_Dertussis bacillus seutica
Penicillin (Units)
0.5 0.5 0 0

1 0.8 (0.1 0

10 1.3 0.2 0
Terramycin (Meg)

10 1.9 2.7 1.6

30 2.6 3.2 1.8

60 3.0 3.6 2.0
Dihydrostreptomycin (Mag)

1 0.8 0 0

10 0.9 <0.1 0

100 1.2 1.1 0.9
Bacitracin (Units)

2 1.5 0.8 0

10 2.3 1.4 0.5

20 3.0 2.1 0.9
Chloromycetin (Mcg)

10 3.“ 2.9 1.5

30 3.7 3.u 1.9

60 309 3.8 2.1
Aureomycin (Meg)

10 0.6 1.“ 0.3

30 1.7 2.1 0.9

60 2.5 2.3 1,3

strate extremely resistant colonies.

In the ease of parapertussis, the most effective antibiotic is
chloronycetin, followed very closely by terranycin. 0f noticeably less
effectiveness are aureonycin and bacitracin. fin W
followed the same pattern, i.e., penicillin and dihydrostreptonycin had
little or no effect, chlorcnycetin and terranycin proved to be the nest
effective and very nearly identical in their effect, and nreonyein was
similar to bacitracin.

the results given in table 2 indicate that as far as relative sen-

sitivity to antibiotics is concerned, pas-apertussis is more sensitive to

all antibiotics used than is k W.
final-29.1.9]!

1. 0f the organisms studied, 1;, W was the nest
resistant, the parapertussis bacillus was of intermediate
resistance, ad 3,. mm the least resistant to the
antibiotics penicillin, terranycin, dihydrostreptonycin,
bacitracin, chlorcnycetin, and euremcin.

2. km was sensitive in some degree to each of the
sin antibiotics.

3. here was very little variation enong the sensitivity of

the sin strains of k W to the antibiotics
studied.

Part II

M11171?! 01‘ H. PIRIUSSIS LED HEATED ORGANISMS 1'0 VARIOUS
AMIBIO'HGS USING A LIWID MEDIUM.

According to plan, when data had been accuulated on the effect of
various antibiotics upon the test organisms using the disc nethod, the
next step was to study the tube dilution method with the sons agents
and cultures. Ihe purpose was to deternine ncre enact endpoints of
inhibition, and also to obtain data for conparison of the two nethods.
Ehis ccnparison seemed especially inportent with mcin, since
Braude (1952) has pointed out discrepant results between test tube and
disc with this antibiotic. He showed that aureonycin was nuch nore
effective in broth cultures than was suggested by the use of aureonycin

discs. However, his observations did not include L m...

m .

Since we had had considrable enerience with the semi-synthetic
nedinn described by ooh-n and Wheeler (19%). this nediun was chosen for
the sensitivity studies. It contains casein hydrolysate, various salts,
starch, and yeast dialyeate, and is a clear faintly yellowish fluid.
the formla as published by Gohen and Wheeler (191:6) follows.

mmmnmm I I
mwmmmm ate)
caseninc acid (Beets) ............................... 10 ms
Bodiun chloride (i.e.n.) mm...................... 2.5 grans
lionopotassiun phosphate, nzroh(i.c.s.) ............. 0.5 era-
Magnesium chloride, figclz.6320 (i.e.s.) ............. 9.1+ gran

00"...I'.‘

sensecv§ 0

13

Starch, ”lublog Pomred onooeeeeeeeeeeeeeeeeeeeeeeeee 1e5 m
0alciun chloride, c3012 (1.0.8.), 1 per cent solution .. 1 n1

ferrous sulfate, 1e80u.7E20 0.0.8.), 0.5 per cent
solution eeeee 2e5 ‘1

Copper sulfate, cusou.5nzo (i.0.8.), 0.05 per cent

solution ..... 1.5 II
”It'an hydrochloride, 1 per *0 solution eeeeeeeeeeee Boo 111
1.881 dialysate ss...................................... 50 I1

MItHICd water t0 “9 es.............................. 1 u

m casanino acid, salt, phosphate, and magnesiun chloride were
dissolved in part of the water. the starch, dissolved separately by
heat in part of the water, was added and then the reuining ing-ediente.
After staking up to 1 kg, the pH was adjusted to 7.2-7.3. |I‘he nediun
was dispensed in 6 n1 enounts in 175-: 22-nn tubes and autoclaved at
10 lbs. pressure for fifteen ninutes.

fhe yeast dielysate was prepared fron neischnann pure dry yeast,
type 2019, dialyzed against distilled water at 78-80 0 for 7 hours.

!he antibiotics were generously furnished by Dr. 1'. 6. link of
the Ghas. Pfiser 0o., Bahway, how Jersey.

mu

Ewe strains of h, m, 10536 and 18560, one strain of the
parapertussis bacillus, 21838, and one strain of Es. bmnchisgptig,
22067, were chosen after prelininary testing as typical and repre-
sentative of the three species. ipproxinately two weeks before con-
ducting the experiments these organisms were renoved from the dried
state by suspending the pellets from the lyOphile tube in sterile

phsiological saline. fhis suspension was spread over the surface of

A....IOQooo‘oe.soolQO-‘beoQ‘O.

one.-¢e--ee.-eeee.s...se.-s-sI-eselse.6Q

etcosoooae-veenees-d..90.'oooow‘

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herdet-Geneon plates and the plates incubated until growth was es-
tablished, usually ‘18 hours. Incubator temperature was naintained at
35 0 throughout, unless otherwise stated. Several successive transfers
were made, and the cultures studied for typical morpholog and henolysis.
Vhen the criteria for snooth strains had been satisfied, isolated cold-
nies were streaked on Bordet-Gengou agar slants and incubated for he
hours. it the end of this period another transfer was made, incubated
for 21+ hours, harvested by pipetting 2 ml of the liquid nediun into the
test tube and gently rubbing off the surface growth. fhis suspension
was transferred to 125 nl lrlenneyer flasks containing approxinately
10 ml of liquid nediun. Gran stains were made before transferring the
suspension to the flasks to disclose any gross eontenination. After
2‘} hours incubation, portions were renewed and Gran stained for puri-
ty. After purity was established, other portions were inoculated

into the flasks as described above. his procedure we carried out
for three successive days. it this tine, samples of each strain were
both stained and plated on Bordet-Gengou nediun to rule out norpho-
logical changes and loss of henolytic ability. If no change was ob-
served, the culture was considered to be satisfactory for the re-
nainder of the test.

Upon the day of the test, the antibiotic under study was dis-
solved in a neasured amount of the liquid nediun to provide a known
concentration of antibiotic. Dilutions were node, in which the con-
contration was twise that to be tested, to allow for addition of an
equal quantity of the culture.

One ml of each concentration as placed in a 15 n 90 - test

l5

tube. To each tube was added 1 ml of a one billion per m1 Cohen-
lheeler suspension of L pgrflssie. Previous tests showed that a final
density of 0.5 billion per n1 of L pgrtussig was necessary to insure
growth. fhe parapertussis bacillus suspension was also adjusted to one
billion per I1. 111 density determinations were nade with a Genoa
photeloneter using the pyrex glass suspension from the National Insti-
tutes of Health, representing an opacity of 10 billion organisms per
n1. 'fhe he. hmhiseptica suspension was diluted to 2 nillion organ-
isns per nl since this organism is nuch less fastidious than the other
two, and cows more rapidly. Gontrols consisting of 1 ll of Cohen-
Vheeler nediun plus 1 ll of the seed suspension were included. All
tubes were then incubated for 148 hours. it the end of this period,
suitable dilutions were made, and portions plated on berdet-Gengou
plates. Ihe plates were incubated for 96 hours in the ease of
L W and #8 hours for the two other organisms. After the in-
cubation period colonies were counted, norphology and staining proper-
ties observed, and results recorded as percentage of surviving organ-
isms, arrived at by comparing nunber of colonies on test and control
plates.
mil

fhe results of the sensitivity tests are shown as an average of
several tests in tables 3, It and 5. Iron the data on 3,. pggtusgis
in table 3, it my be seen that penicillin, dihydrostreptonycin,
ehlorosweetin and neonycin, in this order, are considerably sure ef-
fective aginst this organisn than are aureom'cin, bacitracin and
terrewcin, with the method used. the latter three antibiotics are

16

very similar to each other in their effectiveness. The data in table 1%
indicate that the parapertussis bacillus is generally more resistant to
all the antibiotics except polynixin and neowcin. Heonycin and polyb
ninin, respectively, were the most effective agents against para»
pertussis. Ihlorowcetin, terrenycin and meonycin were respectively
somewhat less effective, and bacitracin, penicillin and dihydrostrepto-
min much less effective.

fhe data in table 5 show that chloronycetin, aureonycin, poly-
nixin and neonycin were, respectively, the most effective against
1;, W, showing quite comparable results. ferrawcin,
dihydrostreptonycin, baoitracin and penicillin were all much less ef-
fective in their action.

m amt-icon“, m 32],“, am. In order to obtain
further data on the sensitivity of the test organisms, Bordet-Gengcu
agar plates were prepared containing known amounts of the various
antibiotics. the choice of the ccmentrations was governed by results
with the discs since comparison of disc and liquid nadiun methods was
desirable. !he preparation of the nediun has already been described
(lethods, Section 1). Before pouring the plates, the proper con-
centration of antibiotic in 1 n1 was added to 1&9 ml of the blood agar
nodiun, thoroughly nixed and poured. .After the plates had cooled,
and had been incubated to disclose any contamination, they were seeded
with 0.35 ml of a saline suspension containing approxinately 80 organ-
isns per .03 n1. fhe plates were incubated for “8 to 96 hours, colo-
nies counted, and results expressed in per cent of survival, as com--

pared to control plates inoculated in the sane nauner at the seas tine.

Ar

I?

Since polymixin discs in three concentrations, and neonycin in one
concentration, had becone available, plates were prepared as in Part I,
and these data are included with results of the plate method in tables
6 and 7.

new:

It is evident from these tables that in the majority of cases the
results shown by the disc method were in agreement with results given
by the poured plate nethod. fhe results shown by the tube dilution
nethod were not in as good agreement with either the tube dilution or
poured plate nethod if & mrtussig is considered. the results shown
by the tube dilution were more comparable to results of the other two
nethods in the case of the parapertussis bacillus and his. m-
'eptica. With neonycin discs of 10 neg concentration, the inhibition
zones indicated considerable effectiveness against the three organ-
isns. Polymirin 3 discs of 5 neg concentration produced inhibition
zones considerably «taller than neonycin with the organisms tested.

nigcussm

fhe results obtained in using a liquid nediun should be con-
sidered in the light of results with antibiotic discs since it is
especially ilportaut to establish the degree of correlation between
the two methods.

In table 2 penicillin and streptonycin discs at respective
levels of 10 units and 100 ngs produced inhibition zone diameters of
no ncre than half the size of the inhibition sense of the other four
antibiotics tested against L. m. However, in the test tube,

using liquid nediun, table 3, these two antibiotics produced 100

TABLE 3

13

Per cent Survival of‘§;_nertu§§i§.After Incubetion for #8 hours in a

Medium Containing Various Quantities of Antibiotics

 

Units or Nicrograms

 

 

 

 

 

 

 

 

 

Culture

Number 20 15 12.5 10 7.5 5 2.5, 1 0.5 Control
Aureomycin

10536 0 12 15 17 42 63 62 76 91 100

18 560 o 3 4 6 9 10 21 49 73 100
Chloromycetin

10536 0 0 0 0 .0 0 0 41 M6 100

18560 0 0 0. 0 0 0 1 22 30 100
Terramycin

10 536 8. 11 16 21 24 24 24 53 73 100

18560 19 20 20 21 29 38 5o 69 77 100
Polymixin

10536 0 0 0 0 0 0 17 25 95 100

18560 0 0 0 0 0 1 22 36 90 100
Dihydrostreptomycin

10536 0 0 0 0 0 0 0 0 0 100

18560 0 0 0 0 0 0 0 13 61 100
Bacitracin

10 536 o 18 19 20 35 39 47 93 100 100

18560 0 19 24 29 37 43 52 84 96 100
Penicillin

10536 0 0 0 0 0 0 0 0 10 100

18560 0 O 0 0 0 0 0 0 19 100

. Neomycin
10536 0 0 0 0 0 0 0 17 95 100
18560 0 0 0 0 0 0 1 35 39 100

19

.memMMOHUfiE no mpHa5.qu

 

 

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00H mm on No an Nm NN ON mH NH eaoeecHoem
00H emeeHsOHao p02 ma Ha mm eHoanpamnpm
toneagHm
ooH .za mH o o o o o o o aaxHaaHoa
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OCH 0: mm 0H m N H o o o aHhooaSOHOHeo
00H mm mm on d n n n H H aHthooaad
4mmnpeoo m.o H m.N m m.a 0H m.NH mH 0N ‘ oHpoHpHemm

.moHpOHpapaa.ao mmapaneeea maOHeca meannepeoo aeHema
c an endow w: pom nonpaanoeH nopmu wmmam .02 mammdpaomenoe Mc Hc>H>Hnm pace new

3 HHm4a

20

 

 

3.." an mm a m d 4. c c o flag-Hoe:
co." decadence ooh mm 50 55333
SH 33338 can Hm am 52538
8H osmosis can 3 N» on Na 599.
1390535
8H 3 Ha Na $ 3 H e e o aflEHoa
2H sensuous son 3 Nn “N aN sunset;
2: an mN a H o o o o o 5:58.29
8H mm 3 m H H o o o . ans-8.84
H828 m6 H “N n n; S n.NH «H 8 03.33st

 

oacavoupuana. Mo acne»; unease». wading I930:

a «H 55a 3 .Se 3:33: ~33 aeoNN .3 3 «an no H9556 use 3a

“a

(Q‘

21

per cent inhibition at the 2.5 unit level (penicillin) and the 2.5 neg
level (dihydrostreptonycin). Indeed, penicillin produced eonplete in-
hibition at the one unit level. he other antibiotic gave 'eonplete in-
hibition of both strains at the 2.5 unit or neg level. i'hese results
suggest' that both penicillin and dihydrostreptonycin in the fora of
discs used on Bordet-Gengou plates gave inhibition zones nuch enaller
than their effectiveness in liquid nediun would indicate. Ehis dis-
crepancy could be sinilar to that observed by Brands (1952) when
studyiu aureonycin: however, there was good correlation between the
effectiveness of aureonycin in the test tube and the success in clini-
cal cases in his study. lo sinilar correlation has been noted with
either dihydrostreptoswcin or penicillin in animal tests or clinical
cases of infection with L m. In contrast to the foregoing,
anreoayeia at a comparable level of concentration of 10 neg displayed
a somewhat analler sons of inhibition than did either penicillin or
dihydrostreptenycin, yet at the 2.5 neg level, there were survival
rates of 62 and 21 per cent with strains 10536 and 18560, respective-
ly, when tested with anreoayein. It nest also be noted that auras-
nycin is distinctly acre effective in treating experinental and
clinical infections caused by 3,, mm; (Karen 35 al. 1951).
Inhibition sones produced by terrawoin and bacitracin were
sinilar and the results shown in table 3 indicate that there is
reasonably close agreenent between the effects of the two antibiotics
in the liquid nediun. Ionparisen of the cone disaster with the per
cent of survival suggests that there night be a snailer percentage of
L 29.129.12.13 surviving than shown in table 3. Poly-12in produced an

I

C

2!
inhibition zone at the 5 ncg level almost identical with penicillin at
the 10 unit level: however. penicillin allowed no survivors at the 1
unit level while polynixin allowed survivors at the 2.5 ncg level. Of
the antibiotics tested. chlorcnycetin and neonycin (table 7) respective-
ly gave the best correlation in that the disasters of the inhibition
zones were the largest produced by any of the antibiotics at the 10
neg or unit level and inhibition in liquid nedia at the identical
level was complete. Lack of better correlation betnen the solid
nediun disc nethod and the liquid nediun nethod when testing the
& nertussig might be attributed to the slowness of growth of the organ-
isns. After 118 hours in liquid nediun some of the more sensitive anti-
biotics, aureomin (Brands and Dockrill. 1952) for maple, my have
partially lost their effectiveness. Another possible saplanatien is
the large initial inoculun, approximately 0.5 billion per n1 final
density, necessary to establish dependable growth in a stationary
liquid nediun. It is certainly a possibility that this large inoculun
night be of an overwhelning nature and produce erratic results.

!he parapertussis bacillus, table it, gave results showing in
general a better correlation with the disc method. Penicillin and di-
hydrcstreptmcin discs gave very ans-11 inhibition zones, and when
tested in the liquid nediun the nnnber of survivors was greater than
50 per cent at the 10 unit or ncg level. Again the best correlation
between the results with the two methods was with chloronycetin.

Using the disc nethod, bacitracin showed inhibition sones nuch
smaller than terrancin against paraperhissis, and in the test tube,

at any level of concentration, the per cent of survivors was greater

6

til

TAEL

Sensitivity'of‘gL'nertussis and Related Organisms to Various
Antibiotics Incorporated in Bordet-Gengou Medium.

 

Per cent Survival

 

 

Concentration Br.bron£2i~

Antibiotic per ml §L_;§g§g§§i§ Parapertussis sentica
Chloro-

mycetin 10 mcg 0 O O
Neomycin 10 mcg O O 0
Disc

MethOd* 2e2 2 1.8
Terra-

mycin 10 meg 0 0 0
Poly~

mixin B 5 mcg 0 O 23
Disc

I-.ze’c:‘r-loc1"I 1.1+ 1.2 0.9
Baci-

tracin 2 units 22 100 100
Peni-

cillin 10 units 0 100 100
Aureo-

mycin 10 meg O O O
Dihydro-

streptomycin 1 meg O 100 100

*Zone diameter (cm) of inhibition.

25

TABLE 7

Sensitivity of B. pertussis and.Related Organisms to Various
Antibiotics. A Comoarison of Three Methods.

 

 

 

 

 

 

 

 

 

 

Antibiotic Concentration 22;
and Method lg; nertussis Paranertussis bronchisentica

Chloromycetin 10 mcg
Disc(cm inhibition zone) 3.4 2.9 1.5
Test Tube(Per cent

Survivals) O O O
Poured P12te(Per cent

Survivals) O 0 O
Neomycin 10 mcg
Disc 2.2 2 1.8
Test Tube 0 O O
figured Plate 0 O O
Terramycin 10 meg
Disc 1.9 2e? 1e6
Test Tube 6 l 1
Poured Plate 0 O 0
Polymixin B 5 meg
Disc 1.4 1.2 0.9
Test Tube 0 O 69
Pgured.Plate O O 23
Bacitracin 2 units
Disc 1e5 Oe8 0
Test Tube 24 28 91
‘Pgured Plate 22 100 100
Penicillin 10 units
Disc 1.3 . 0.2 0
Test Tube ' O 62 100
Poured Plate 0 100 100
Aureomycin 10 meg
Disc 0.6 l.# 0.3
Test Tube 12 4 O
Poured.Plate O O O
Dihydro—

streptomycin l mcg

Disc 0.8 O 0
Test Tube 0 100 100
Poured Plate O 100 100

 

25

with bacitracin than with terranycin. dureomcin gave results quite
sinilar to those obtained when using the L portage g, i.e. a reason-
ably large zone of inhibition using discs but survivors in the highest
concentrations in the test tube. The correlation betwaen the disc and
test tube nethod was thought to be due prinarily to the sonewhat better
growth of the parapertussis bacillus compared to h We

1h W, tables 2 and 5. gave results sinilar to those
observed with the parapertussis bacillus. Penicillin and dihydrc-
streptomin discs produced very sill inhibition zones, and these
antibiotics in the test tube showed but little inhibition. lhlerc-
nycetin and terranycin discs gave slnost identical inhibition zones.
1.5 and 1.6 on respectively at the 10 neg level. In the test tube there
was conplete inhibition of the organisn at this level with both anti-
biotics. There was evidence of the call inhibition zone of the nurse—
nycin disc being nisleading, as Brande (l952)reported. it the 10 ncg
level the inhibition zone disaster was 0.3 centineters as compared with
a dianeter of 1.5 centineters at the sons level of chloronycetin, yet
there was complete inhibition with both agents in the test tube.
Bacitracin gave results sinilar to those of penicillin and dihydro-
streptomn in that the discs produced snall inhibition zones and
the survival rate was more than 75 Per cent with the highest concen~
tration of antibiotic.

The general correlation of the two methods when using lb. bronchi-
m as the test organism was of the cane order as with parapertussis.

The agreenent was nuch closer with these organisms than with
3.. mo

TABLE 8

26

Decreasing.Aetivity of a Number of Antibiotics Against g; nertussis

and Related Organisms.

A Comparison of Two Methods.

 

Test Tube Method.Using a

Liquid Medium*

Disc Method Using a
Solid Medium"I

 

 

 

 

Penicillin Chloromycetin
Dihydrostrentomycin Neomycin
Neomyein Terramyein
H. nertussis Chloromycetin Baeitraein
""’“’““"‘ Polymixin B Polymixin B
Aureomycin Aureomycin
Terramyein Dihydrostrentomycin
Baeitracin Penicillin
Neomyein Chloromycetin
Polymixin B Terramycin
Chloromycetin Neomycin
Terramycin Aureomyein
Parapertussis Aureomycin Polymixin B
Bacitraein Baeitraein
Penicillin Dihydrostreptomycin
Dihydrostreptomyein Penicillin
Chloromycetin Neomyein
Aureomyein Chloromyeetin
Neomycin Terramyein
Br.bronehisentica Polymixin B Polymixin B
Terramyein Aureomyein
Dihydrostrentomyein Dihydrostrentomycin
Baeitracin Bacitracin
Penicillin Penicillin

*Denotes decreasing inhibition zone size in disc
method and decreasing survival nercentsre in the

test tube method.

m

Table 8 presents a summarized comparison of the overall results
of the disc and dilution methods. The results with the test tube
nethod were based on per cent of survival at comparable levels when de-
termining the order of activity of the antibiotic in question. It is
evident fmm table 8 that there is no correlation between the results
of the two methods in the case of the L pertussi . However, if anti-
biotics shown to be the most efficacious in the treatment of experi-
mental pertussis infections in animals or clinical human infections
are considered, Karen 21 5], (1951). the disc method is much more
reliable. Vith either of the other two organisms used in this study.
the results of the two methods are similar. There was a marked
similarity of results betwun the poured plates and the disc nethod.
Comparative results between the poured plate method and the test tube
nethod were not as striking. It is interesting that with bacitracin
the results with the two methods were nearly identical. is previ-
ously stated, bacitracin using the disc method gave an inhibition sons
which might indicate a greater inhibiting action than is actually
present.

ssh-mm

Using the tube dilution method. the various antibiotic agents
were tested for inhibitoiv aetihn against L, m, the para-
pertussis bacillus, and EB. bgnchilgp. tica. Based upon the data, the
conclusions were as follows:

I. Is. pgrtugg‘s was most susceptible to penicillin,

dibydrostrepteucin, neonycin and chloronycetin,

2.

3.

I}.

and least to terramycin, bacitracin and aureomycin.

i'his was in disagreement with the results with discs.

Ihe parapertussis bacillus was most susceptible to
neoswcin and polymixin, least susceptible to dihydro-
streptomycin, penicillin and bacitracin, and of inter-
nediate susceptibility to chloromycetin, terramycin,

and anreonwcin. his was in agreement with the re-

sults shown by discs in a najority of the antibiotics
tested.

he. ngnchimtica was most susceptible to chloromycetin,
anreomycin, neonycin and polymixin, and least susceptible
to penicillin. bacitracin, dihydrostreptonycin and terra-
min. This was in general agreement with the results
with the discs in a majority of the antibiotics tested.
Ehe results of both the antibiotic disc method and anti-
biotic containing !Bordet-Gengou medium method were in
close agreement; further both of these methods were nere
reliable than the tube dilution method using Gohen-
Wheeler nediun in predicting the most successful agents
for treatment of infections as shown by clinical studies

by other investigators.

29

Part 1!!
scan! of SYHERGISTIG on ANTAGONISTIC ACTION or VARIOUS

COMBIRATIONS OT mmxorxcs.

In recent studies Jawets and coworkers (1952), studied the effect
of a number of antibiotic combinations against both gram positive and
gram negative organisms. lo reports on synergistic action upon
L mmssis have been found in the literature. It was decided to
determine if there were combinations of antibiotics which would dis-
play synergisn or antagonism in their action on the three organisms
under study.

new

The concentrations of the antibiotics to be used in combination
were based upon the results of each used alone in liquid medium,
shown in tables 3, b and 5. he concentration of each antibiotic
chosen was such that the surviving population was approximately 25
per cent of the control without antibiotic. Cohen-lheeler medium was
nade up to contain 8 times the desired concentrations. to allow for
dilution in the tests.

In each combination tested the set-up consisted of four
15 x 90 I- test tubes containing the following:

tube 1. One half ll of each antibiotic in concentrations

8 times the final concentration required per ml.
hbe 2. One ml of one of the antibiotics appearing in
the combination.

Tube 3. One ll of the other antibiotic present in the

combination.

I‘ube 1}. One ml of plain Gohen-lheeler medium. this
tube served as a control.
L mrtussis culture suspension prepared as in Part II was added,
1 ml to each tube. Ihe procedure was repeated for the parapertussis
bacillus and 1;... W. After a. suitable incubation period,
#8 hours for 3,, m and the parapertussis bacillus and 2!} hours
for El; mngisgptica. suitable dilutions were plated on hordet-Gengou
plates, the plates incubated and colonies counted. The results were
expressed according to the following scheme:
Antagonism (Antag.) Indicates the surviving population
equals the control within normal
dilution error.
Indifference (1nd.) Indicates the surviving population
is from 50 to 90 per cent of the
control.
amt synergism (4-) Indicates a surviving population of
from 25 to ‘19 per cent as compared
to the control.
llederate synergism (H) Indicates a surviving population
of from 10 to 21$ per cent.
Marked synergism («4-) Indicates a surviving population
of less 10 per cent.
Complete (Comm) Indicates no surviving population.

fhe above description of the synergistic activity is emperical

particularly when defining “Indifference“. It would probably be

fallacious to assume that one antibiotic in the combination would
inhibit one portion of the microbial population and the other antibiotic
of the combination an entirely different portion even though these
agents are chemically different. It is more reasonable to presume that
both agents, initially, exert to some degree an overlapping inhibitory
effect upon the same organisms but may then act soth more selective-
ly and separately upon different, more resistant survivors. The results
from the various combinations are given in table 9.
am

L m“. Of the combinations in table 9, those containing
bacitracin exhibit the most synergistic activity. either marked or
moderate. Oombinations containing dihydrostreptomycin also displayed
synergistic activity though not marked. Penicillin combinations were of
less activity. leoswcin combinations showed variable activity, i.e.,
indifference, complete inhibition, and moderate activity.

figspertuggis bacillgg. All bacitrecin combiuaions, and neomycin
combinations, table 9, showed very similar and slightly synergistic
activity in the majority of combinations. One bacitracin combination
and one neomycin combination exhibited moderate activity. Dihydro—
streptomycin combinations showed slight activity except that of dihydro—
streptomycin and aureonycin which completely inhibited the parapertussis
bacillus.

he. W Dacitracin combinations exhibited indifferent
synergistic activity with the exception of bacitracin and terramycin,
which was completely inhibitory. Neonycin combinations showed slight

activity except with terrawcin with which it was completely inhibitory.

31

ems

Synergistic Effects of Antibiotic Oombinations upon 3‘ pertusgis and
gated Organisms

 

 

 

 

 

 

 

L parapertussis 32s.
Antibiotic combinations mrtussis bacillus bronchiseptica
Penicillin plus
lecwcin e «I- +
Penicillin plus
Dihydrostreptomycin Ind. Ind. Ind.
Penicillin plus
' Egitracg e «o- e
Dihydrostreptomycin plus . . .
hacitracin «H ' e Ind.
Dihydrostreptomycin plus
remain «H» + 1-
Dihydrostreptomycin plus.
Ohlommycetin #- + intag.
Dihydrostreptonycin plus
dureomycin «H- Oomp. e
Dihydrostreptomycin plus ' '
renaming A. 3* j" I”
hacitracin plus . . . . .
leoswcin see + - Ind.
hacitraoin plus
Ohleronycetin «- + Ind.
Bacitracin plus
Lureewcin «H4- - «H- Ind.
Bacitracin plus ' ' ' ' ’
m 11+ + Ms.—
Ieomycin plus . . . ”
Ohleromyeetin Ind. e +
leomycin plus
dureomycin Comp. e e
leomycin plus

Dihydrostreptonycin combinations ranged from antagonism to moderate
activity. Penicillin combinations were of slight or indifferent syner-
gistic activity.

scussi

i'he remnts shown in table 9 are what might be expected i.e..

L w, generally the most sensitive to the antibiotics tested,
being the most susceptible appearing to be synergistic action, and the
parapertussis bacillus, of intermediate sensitivity, displaying the
same general sensitivity to the combinations but of a noticeably lower
order. 33., brgnchiseutica, which is the most resistant of the three
organisms to single antibiotics also appeared to be the most resistant
to the combinations. Of interest too is the fact that both

1s. pertussig and the parapertussis bacillus showed a airly uniform
degree of sensitivity, the former being of eather moderate or mrhed
sensitivity, and the latter of generally slight sensitivity to the
madcrity of combinations. On the other hand, the activity of the com-
binations against he. W was quite variable, ranging from
antagonism to moderate effectiveness in the combinations which included
dihydrostreptomycin. fhis variance was not as marked in the other com-
binations.

Bemse of the unexpected results obtained when using sings
antibiotics in liquid medium, there seemed to be adequate Justification
for testing several of the combinations in Bordet-Gengcu plate for
confirmation of the effect observed in liquid medium.

the combination of penicillin and dihydreetreptomin was se-
lected since with all three organisms the synergistic activity

3"

appeared to be indifferent. Ihree combinations which.gave complete
inhibition, one for each organism, were also tested.

Ihe plates were prepared as described previously except that the
two antibiotics were mixed together'inmediately before being added to
the measured amount of Bordet-Gengou medium. the inoculum used was the
same as described.in.Pnrt II. !he plates were incubated.for either "8
or 96 hours depending on the organism, and survivors expressed as a per
cent of the colonies appearing on the control plates. fhese results
are given.in.table lo.

Bani-lie

Iith both combinations tested, L mum exhibited indifferent
sensitivity; the parapertussis bacillus exhibited the same general
type of’indiffbrent sensitivity although there appeared to be evidence
that the combination of dihydrostreptonycin.and.aureomyoin.was slight-
ly more active. he W showed indifferent sensitivity in
the case of the penicillin.and.dihydrostreptomyoin combination, and
complete sensitivity'to the combination of hacitracin and terranycin.
Of the combinations tested, the only definite evidence of'synergisn
noted was in the case of terranwcin and neoswcin tested with h m-
miles-

W

fhe results in table 10 indicate again the lack of agreement
between the two methods, particularly with 3‘ mg and to a
lesser extent with the parapertussis bacillus. In contrast, the re-
sults confirm the agreement of the two methods withwflzguhgggghiggptigg

as the test organism.

 

 

-... on...

35

um: 10

Synergistic Activity of Antibiotic combinations Using a Solid Medium
: Per cgnt g; Survival

 

 

 

 

 

 

 

L paraportussis

Antibiotic Combinations rtuss bacillus W
Penicillin 88 57 85
Penicillin plus

Dihydrostreptomycim 88 85 92
Dimes-o streptomycin 82 77 68
control 100 109 no
Aureomycin 70
Aureomin plus .

leonycin 78
Neomin 75
control 1043

his combination with 3,, m only.

Aureomycin 78
Aureomin plus

Dihydro streptomycin 55
Dihydrostreptomycin 7O
Oontrol IOO

Ehis combination with the parapertussis

: w
ferramycin 52
ferramyoin plus

leoswcin ' O
leomycin ‘ 83
Control 100

his combination with he. W only.

 

Ehe combination of dihydrostreptowcin and penicillin which
exhibited “indifferent" synergistic activity in the liquid nediun was
indifferent in all cases when the Bordet-Gengou mediun was used. It
only this combination were used as a basis of comparison between the
two nethods, it would suggest good agreement. however. when the re-
sults of the other conbinations previously shown to give complete in-
hibition are observed, distinct disagreement is seen with both
3,. mm; and the parapertussis bacillus. In ‘0 onchi t , on
the other hand. is completely inhibited regardless of the nethod. On
the basis of these and. some of the results shown in Part II, it
appeared that synergistic activity of combinations of antibiotics against
both L mm and the paraperhissis bacillus in the liquid nediun
method as described and the Bordet-Gengou medium nethod would have to be
supported by further a m testing as well as animl tests before
reliability or either nethod could be established.

m1. the results in table 10 disagree insofar as the two nethods
are concemed. the discrepucy between percentages of survival is not
great with the single antibiotics. In all cases, the unit level,
chosen as a result of the testing in liquid nediun, was found to allow
nore than fifty per cent survival as was planned. In nest cases, more
than seventy per cent survival was noted. Ibis being the case, the
results of testing single antibiotics in a liquid nediun nay be con-
sidered reliable in terns of survivors, i.e.. the concentration of
an antibiotic necessary to inhibit a certain percentage range of less
than fifty per cent of the three organisms may be relied on to in-
hibit. within limits, a like percentage on Bordet-Gengou nedium. Ihe

37

Activity of antibiotic combinations in liquid medium is not wholly pre-
dictable with respect to the activity found using Bordet-Gengou medium
if & mgtusgis and the parapertussis bacillus are to be considered.
It In. bronchisgptiga is employed as the test organism. the activity
ef antibiotic combinations is more likely to be comparable with both
methods.
no i
1. Synergistic activity of various combinations of
antibiotics against the three organisms in this
study was demonstrated by use of cohen-lheeler
liquid medium containing the various combinations.
2. !he synergistic activity of certain antibiotic
combinations against the pertussis and para-
pertussis organisms shown in the liquid medium
were not duplicated by use of Berdet-Gengou
medium.
3. he synergistic activity or certain other con-
binations of antibiotics in the liquid medium
was duplicated when using Bordet-Gengou medium.
1}. the percentage of surviving organisms resulting
from the activity of a single antibiotic in the
liquid medium remained comparable with Bordet-
Gengou medium at the same antibiotic concentration
with the three organisms tested.

1.

3e

7.

References

Alexander, H. E., and Pedmen, R. 1949 Mechanism of resistance to
strentomycin of‘g; pertussis and g; neranertussis during treatment
with this antibiotic. Pediatrics,.&, 461—467.

Anderson, E. K. 1952 Serologicel studies on.§;'nertussis

‘g;gp§rauertussis, end g; bronchiseoticus. Preliminary reports.

 

Actn. Pathologicia,‘3g, 5h—58.

Bell, J. A., Pittman, M., and Olson, B. l9h9 Pertussis and
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Braude, A. I., end Dockrill, M. 1952 Inaccuracies in the disc
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Cohen, S. M., and Wheeler, M.W. 1946 Pertussis vaccine prepared
with phase I cultures grown in fluid medium. Am. Jour. Pub.
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Bay, E., and Bradford, W. L. 1952 Susceptibility of Hemouhilus

 

pertussis to certain antibiotics. Pediatrics,‘2, No. Q, 320—326.
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A species resembling both Bacillus pertussis and Bacillus bronchi-
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1‘1. NO- 1. (Sect. B), 1-7.

9.

10.

11.

12.

13.

14.

15.

16.

39

Eldering, G., and Kendrick, P. L. 1952 Incidence of pareoertussis
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Essen, L. N., Jackson, G. G., Shih Man, C., and Plac , E. H. 1951

Antibiotic treatment of oertussis. Jour. Ped., 32, No. l, l-lS.

Hegarty, C. P., Thiele, E., and Verwey, W. F. 1945 The in vitro

 

r—-n
and in vivo activity of streotomycin against g;.oertussis.
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1952 Studies on antibiotic synergism and antagonism among seven
g J

antibiotics against various bacteria in vitro. Jour. Bact.,
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Jawetz, B., and Gunnison, J. B. 1952 An exocrimental basis of
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Snin Nan, Chang. 1950 Pertussis due to Brucella bronchisentica.

 

Pediatrics, _6_, No. 2, 227-229.
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B. pertussis. J. Infect. Dis., gs, 50-53.

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