ABSTRACT
PHAGE TYPING OF STAPHYLOCOCCI OF BOVINE ORIGIN AND
BIOCHEMICAL CHARACTERIZATION OF BASIC PHAGE PROPAGATING STRAINS‘

by John J. Solomon

The present Investigation was concerned with two major objectives.
The first aim was to determine the relationship of staphylococcal strains
found in a sampling of bovine herds in Michigan to those commonly involved
in human infections, as well as the possible occurrence of a widely
distributed strain in the bovine papulation comparable to phage type BOIBl
in human infections. in the second aim, the phage prepagating strains of
staphylococci were used as representative members of the genus Staphylococcus
and a series of biochemical determinations was carried out in order to
characterize these strains more fully.

Milk and cervical samples were obtained from lzh cows located on 23
farms in the lower peninsula of Michigan. One hundred sixty-four
staphylococcal isolates were obtained from the milk of 92 animals. 8y
subsequent phage typing with the International-Blair and Seto-Hilson
phages, it was revealed that there were 30 different phage patterns in
lZS sensitive strains. A widely distributed phage type of staphylococcus
was not found. ‘The most prevalent type, 52, occurred in 2“ samples from
h farms. The number of different patterns in an individual animal
varied from 0 to 3.

The International-Blair phages, normally used to type staphylococci
of human origin. were responsible for 62%.of the total number of lytic
reactions. Phages #28, hZE. and hhA were the most active of this set.

None of the patterns common in human infections was observed, although

John J. Solomon

a closely related pattern was found in 6 isolates from 2 farms. in
addition, I3 isolates from h different herds gave extensive lytic
reactions with this series of phages. This evidence suggested a
possible cross lnfectivity or colonization between man and animal.

Differentiation of representative strains from milk was attempted
by using a tube dilution technique to measure coagulase quantitatively.
Coagulase titers. expressed in reciprocal units, were found to have
divergent values. ranging from less than k to 20MB. Non-typeable
organisms were usually low coagulase producers.

étgphylococcus eggggg was not commonly found in the reproductive
tract samples obtained in this study. Few gram positive cocci were
isolated from the cervical samples. 0f l5 such isolates. none was found
to be coagulase positive and only i was sensitive to the phages used.
The l typeable cervical isolate was not related in phage pattern to the
milk isolates obtained from the same animal.

‘The laCk of uniformity in coagulase production by the staphylococci
obtained from the clinical samples prompted a systematic study of several
biochemical activities of this versatile organism. Using the series of
phage propagating strains. the prOperties measured included quantitative
free coagulase by a tube dilution technique; acid phosphatase by a.
colorimetric procedure using p-nitrophenyi phosphate as a substrate;
extracellular protein by a turbidometric trlchloracetic acid method;
minimum inhibitory concentration of penicillin; growth capacity in a
chemically defined medium; and a qualitative evaluation of mannitol
utilisation, hemolysin and pigment formation. antibiotic sensitivity.

catalase activity. penicillinase production. and gelatin liquefaction.

 

 

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John J. Solomon
These experiments were completed with a manometric determination of
oxygen uptake for each strain, as well as the preparation of a family
of growth curves in brain heart infusion broth.

The most exceptional strain was #73 which was negative with respect
to coagulase, phosphatase, mannitol fermentation, hemolysin formation.
gelatinase and penicillinase; it was also sensitive to the antibiotics
used. and was lysed by most of the phages. This was followed by strain
#70 which not only was catalase negative. and one of the lowest with
respect to oxygen uptake and growth rate, but gave the highest coagulase
titer (#096). Low coagulase producers were 52A/79. 3B. 3C.'55. #7, hZB
and Sh. Other notable strains were 80, 53, Sh. 75. 77. and Bi which were
all penicillinase positive; 44A which gave the highest phosphatase
activity; and 29. 52. #7, 73. and 75 which produced no hemolysis in the
erythrocytes of the three species used. Human red blood cells were
hemolysed only by BA, 53. and hhA of the international-Blair series. and
all but one (52) of the Seto-Hilson strains.‘

in general, no one index measured was found to be uniform. Coagulase
and phosphatase activity. as well as mannitol utilization invariably
occurred together. Variation among the strains was common for all of

the preperties measured.

 

 

 

 

 

PHASE TYPING OF STAPHYLOCOCCI OF BOVINE ORIGIN
AND BIOCHEMICAL CHARACTERIZATION OF

BASIC PHAGE PROPAGATING STRAINS

by ‘
‘0

John JYOSoIomon

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHlLOSOPHY
Department of Microbiology and Public Health

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ACKNOULEOCEHENTS

Sincere thanks are extended to Dr. Charles L. San Clemente for his
many helpful suggestions during the course of this study. His constant
concern for students and colleagues and his ever present willingness to
assist in the solution of any problem were invaluable and very deeply
appreciated.

The author also wishes to thank Dr. Donald D. Mcwade. formerly of
the Department of Veterinary Pathology. and Dr. Albert R. Drury, Depart-
ment of Surgery and Medicine, for their assistance in the clinical phase
of this study. and Dr. Delbert E. Schoenhard. Department of Microbiology
and Public Health, for his assistance during the study, and the valuable

suggestions and criticisms during the preparation of the manuscript.

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TABLE OF

INTRODUCTION . . . . .

H'S'M'W O O O I O 0 O O O O O O O O

Phage Typing . . . . . . . .

Phage Typing of Staphylococci

Mastitis . . . . . .
c0.“ ' .3. O O O O O O O 0
'M‘Pmt.s° O O O O 0 O O

Memolysin . . . . . . . .

Other Physiological Properties

SYI‘ICIIOCIC hd‘m a-e e e e e e

MATERIALS AND METHODS . . . .
Milk Samples .. ... . . .
Reproductive Tract Samples
Phage Preparation . . . .
Phage Typing . ... . . .
Coagulase Determination .
Mannltol Fermentation . .
Pigment Formation . . . .
Memolysin Production . .
Antibiotic Sensitivity .
Penicillinase Production
Extracellular Protein . .
Growth Curves . . . . . .
Phosphatase . . . . . . .

Catalase . . . . . . . .

O O
O O
O O
O 0
O O
O O
O O
O O
O O
C O
O O
O O
O 0

CONTENTS

Associated with

Bovine

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Oxygen Uptake . . . . . . . . . . . . . . . . . . . . . .

Synthetic Medium . . . . . . . . . . . . . . . . . . . .

RESULTS ... . ... .-. . . . . . . . . . . . . . . . . . . . .
Phage Typing and Coagulase Activity of Clinical

isolates . . . . . . . . . . . . . . . . . . ... . .

Characteristics of the Phage Propagating Strains . . . .

Coagulase . . . . . . . . . . . . . . . . . . . ...

Extracellular Protein . . . . . . . . . . . . . . .

Phosphatase . . . . . . . . . . . . . . . . . . . .

Catalase . . . . . . . . . . . . . . . . . . . . . .

Growth Curves and Oxygen Uptake . . . . . . . . . .

Growth in a Chemically Defined Medium . . . . . . .

Antibiograms, Penicillinase Production, and Minimum

inhibitory Concentration of Penicillin . . . .

Pigmentation. Mannitol Fermentation, and Gelatinase

and Memolysln Formation . . . . . . . . . . .

DlSCUSSlOM . . . . . . . . . . . . . . . . . . . . . . . . . .

B'BL'WWH' O O O O O O O O O O O O O O O O O O O O O O O O 0

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LIST OF TABLES

TABLE PAGE

I. The number of phage patterns of strains of
Staphylococcus aureus isolated from the udder of
selected cows from 23 herds ...................... ..... . 30

2. Phage patterns obtained on 23 farms by cow number
.nd w.rt°r .....OOOOOOOOOOIOI......OOOOOOOOIOOOO....... 3|

3. Frequency of the most common phage patterns obtained
from staphylococci of bovine origin .................... 35

h. Frequency of lysis by the most active phages upon
I25 strains of staphylococci ........................... 36

5. Some growth characteristics of the international-Blair
and Seto-Uilson series of phage propagating strains
Of stWhYIOCOCCI 0......00............OOOOOOOOOOOOOO.... 52

6. Antibiograms and some physiological prOperties of
the International-Blair and Seto-Hilson series of
phage propagating strains of staphylococci ........ ..... 56

FIGURE

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LIST OF FIGURES

Significant lytic reactions of the International-Blair
series of phage propagating strains of staphylococci ...

Significant lytic reactions of the Seto-wilson series
of phage pr0pagating strains of staphylococci ..........

Coagulase production by the predominant $2 phage
types of staphylococci isolated from milk ..............

Coagulase production by phage type Sl/S3 and S6
staphylococci isolated from milk .......................

Coagulase production and phage patterns of
staphylococci isolated from two herds ..................

Coagulase production by the International-Blair
and Seto-Hilson strains of staphylococci after five

d.ys St.tlon.ry grmth ......OOOOOOOOOOOOCOO0.0.0.0.0...-

Coagulase production at various intervals of aerated.
growth by high and intermediate coagulase producing
strains of phage propagating staphylococci .............

Coagulase production at various intervals of aerated
growth by low coagulase producing strains of phage
Prmwting Stath'OCOCCI ......OOOOOOOOOIOOOOOOOIOOOCOO

Extracellular protein of the international-Blair and
Soto-Wilson series of staphylococci ....................

Cellular acid phosphatase production of the
international-Blair and Seto-Hiison series of
St.PhYIOCOCCS I.......OOOOOOO.........OOOOO0.0.0.0000...

Catalase activity of the international-Blair and
Seto-Hilson series of staphylococci ....................

Representative growth curves of the international-
Blair and Seto-Hllson series of staphylococci ..........

Oxygen uptake of the International-Blair and Seto-
Hilson series of staphylococci .........................

Growth of the international-Blair and Seto-Hilson
series of staphylococci in a chemically defined

MSW ....0....0......0.00.00.........OOOIOCIOIOOCCOOOO

Minimum inhibitory concentration of penicillin on the
International-Blair and Seto-Uilson series of staphylo-

cocc' ......COOOOOOOOOCOCOOOOI0............OOOOOOCOOCOOO

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INTRODUCTION

Staphylococcus 32393 has become increasingly important as a human
and animal pathogen. In the field of human medicine the staphylococci
are frequently involved in skin lesions. burn and post-operative
infections. pneumonia, septicemia, diarrhea, food poisoning, and osteo-
myelitis. Very painful and disabling! infla-ations are coamon in severe
skin lesions and chronic osteomyelitis caused by this organism. Hospitals
have been plagued with endemic and epidemic outbreaks of staphylococcal
diseases. The causative organisms frequently fall into a few phage
patterns, and often one or two types predominate. The predominating
strains appear to have enhanced powers of lnfectivity and resistance
to the antibiotic drugs in cannon use. One such strain. phage type BO/Bl.
has become known as the epidemic or widely distributed strain because of
its common occurrence in hospital nursery outbreaks.
in veterinary medicine the staphylococci are perhaps primarily
important in the bovine, although occurring in other species. In dairy
animals, _S_. m has become an important cause of mastitis, with a
resultant loss of milk and butterfat production. and permanent damage
or death to the animals. In some herds it may produce a greater incidence
of infection than Streptococcus agglactiae and is often of special concern
in herds in which streptococcal mastitis has decreased. It has been
reported (Easley et al.. I95l; Gunter et al.. 1955) that staphylococci
."0 Present more frequently in the reproductive tract of cows experiencing
diffHmitles in fertilization. The decrease in reproductive capacity may
m“ill". therefore. as a result of infection with these organisms.

The purpose of this study was to answer several questions. In the
“NW of farms in Michigan, one object was to determine if a con-ion

—____

strain of‘S, 2555!; occurred in bovine herds as in the case of phage
type 80/8! in human infections. A related objective was to learn if
cross infection by identifiable strains of staphylococci occurred
between man and cow. Phage typing offered a convenient tool for
answering both questions. An attempt to achieve an added degree of
differentiation was made by measuring quantitative coagulase.

A preliminary survey indicated the presence of a large number of
repeat breeder cows and in view of the evidence presented by other
workers for the possible incrimination of staphylococci in reproductive
failures. a third objective suggested itself; namely, the sampling and
culturing of the reproductive tracts of a number of repeat breeders.

Finally, the varied results obtained from the phage typing and
coagulase determinations lead to an extensive biochemical character-
ization of Staphylococcug‘ggrggg. The phage prepagating strains. both
the international-Blair and the Seto-Nilson series. appeared to afford
an ideal cross section of the species as indicated by the diversity of
phage patterns obtained during the study of the lytic spectrum of the

phages.

   

 

 

 

HISTORICAL

W

Twort (ISIS) was the first to report lysis of micrococcl by a
transmissible submicroscopic agent. He obtained cultures from calf
veccinia and observed the development of colonies which subsequently
became glassy and transparent. A pure culture of mlcrococcus when
touched with a small portion of one of the glassy colonies soon
became transparent or glassy. The transparent material was found to
pass through porcelain bacteriological filters and to be active in
dilute saline or water solutions. Cultures could be carried for long
periods without showing the transparencies; however, most pure strains
eventually developed such areas. Twort suggested that the phenomenon
might be considered an acute infectious disease of the micrococcl.

In I935 Burnet and Lush presented evidence that certain strains of
_S_. _e_u_r_egs_ could be grouped according to their sensitivity to a series
of phages. Williams/and Tie-ins (I933) used the phages isolated by
Durnet and Lush to differentiate strains. of staphylococcus originating
from patients with osteomyelitis. Phage action was indicated by the
Clearing of broth cultures. , Six phage types were found.

Fisk (I902) described a method for the isolation and propagation
0' bacteriophages carried by Iysogenlc strains of g. m. Lyso-
Senicity was demonstrated in l9 out of #3 coagulase positive staphylo-
°°CCI by a cross-spotting technique. Several different strains were
“raked on a plate and a dr0p of broth culture from a series of
”"IOS was added to each streak. Areas of lysis were harvested and

"PWP-sated on the sensitive cultures until confluent '75“ occurred.

 

 

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Material from the confluent areas was then collected and treated with
a'low concentration of zephiren. Fisk (I9h2a) determined the phage
susceptibility of 95 strains of‘g.‘2g£gg§ using 27 different bacterio-
phages. Cultures to be identified were inoculated on the surface of
agar plates and one leapful of each of the test phage solutions was
spotted on the seeded area. After suitable inoculation, differentiation
of the cultures was accomplished by comparison of their patterns of
susceptibility to the phages. Pathogenic staphylococci could be divided
into groups as a result of the phage action. and strains isolated from
related sources were found to react to the same phages and could be
differentiated from other cultures by this method. Fisk and Mordvin
(l9hh) continued the bacteri0phege typing of staphylococci using 78 strains
of _S_. m obtained from 30 patients. Again it was observed that most
cultures from related sources were of the same bacteriophage type.
Uilson and Atkinson (l9h5) extended Fisk's techniques to permit
the differentiation of staphylococci in a manner comparable to that
used for the phage typing of typhoid bacilli. The phages were isolated
using Fisk's cross-spotting technique and prOpagated initially in young
broth cultures of susceptible host strains. The supernatant fluid was
filtered through a Seitz pad and plated on a susceptible strain of
staphylococcus. A single plaque was taken up in a small volume of
‘water and again plated on the susceptible strain. The fresh plaque
thus obtained was prOpagated in broth until the titer increased and the
resulting phage material was diluted until the highest dilution producing
confluent lysis was obtained. This was the test dilution of the phage
and was used for typing the staphylococci. Four hundred and sixty

strains of'g. aureus were subjected to the phage action and 2i patterns

 

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of lysis noted. Sixty percent of the organisms were successfully typed.
The origin of breast abscesses, pemphigus. and outbreaks of food
poisoning were traced by this procedure.

A valuable contribution to strain identification was made by the
establishment in i9h6 of a center for the routine typing of staphylo-
cocci employing the method and phages of Wilson and Atkinson in the
Central Public Health Laboratories, Colindele. London, England. under
the direction of Dr. V. D. Allison (Blair and Carr. i960). willlams
and Rippon (i952) refined the methods for typing staphylococci and sub-
sequently their techniques were established as a basis for further phage
typing procedures. The Subcommittee on Phage Typing of Staphylococci
of the International Committee on Bacteriological Nomenclature, was
established during the Sixth international Congress for Microbiology in
Rome in I953, and recommended that a basic series of l9 phages be used
for the routine typing of human staphylococci.' The subcommittee felt
that rigid rules should not be established, but formulated certain
recommended basic procedures. Essentially the methods followed those
evolved at the Colindele Laboratories. The l9 basic phages were divided
into A groups as follows:

I. 29, 52, 52A, and 79.
ll. 3A. 3B. 3C, and 55.
Ill. 6, 7, 426, #7. 53. 5#, 70. 73. 75. and 77.
IV. #20.
The broad groups I. ii, and III were so numbered because the phages
comprising each group generally exhibited some degree of specific
action against the staphylococci belonging to the corresponding sero-

logical groups established in i939 by Cowan. In addition to the basic

6 .

series other phages were allowed by the coaleittee if an investigator
felt they would provide useful information.

The techniques of typing were described in further detail by
Blair and Carr (I953). and Jackson et el. (I995). Cultures were assigned
to a broad phage group on the basis of their lysis by one or more of the
phages within that group. it was often found that lysis was produced
by two or more phages from a broad group to give "patterns" of lysis.
although some— cultures were lysed specifically by a single phage.
identical strains of staphylococci gave identical or closely similar
patterns. while unrelated strains showed different lytic reactions.

The Subconmiittee on Staphylococcal Phage Typing of the international
Conittee on Bacteriological Nomenclature at its I958 meeting revised
its list of basic phages. The series included phages originally used
by Wilson and Atkinson (I995) or those isolated in or received at the
Colindele laboratory. The revised groups included:

i. 29. 52. 52A. 79. and 80.

ii. 3A. 3B. 3C. 55. and 7i.
iii. 6, 7. #25. h7. 53. 5h. 73. 75. and 77.
W. 1020.

Miscellaneous. I87.

Additional phages were again allowed as adjuncts to the basic groups.

Phagp Typing of Stpphylococci Associated with Bovine Mastitis
Slanetz and Jawetz (l9lil) isolated staphylococcal bacteriOphage

from the milk of 7 out of 20 cows which contained staphylococci. The
bovine phages resembled those obtained from human sources in many
respects, and attmwts to show specificity of lysis by these phages

on bovine staphylococci failed completely. The broad— lytic spectrum

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of the phages prevented their use in epizootic studies.

Macdonald (Williams-Smith. l9#8) in l9#6 reported on the phage
typing of staphylococci from milk and from cases of bovine mastitis
- using phages provided by Wilson and Atkinson. One hundred twenty-three
of lSO strains isolated from milk were susceptible to phage #20 and
twelve were lysed by other phages. Fifteen strains could not be typed.
All of the strains obtained from cases of mastitis were sensitive, and
all 3# belonged to phage type #20. Villiams-Smith (l9#8) isolated l,Ol6
strains of staphylococci from bovine milk samples and subjected them to
the action of the Wilson series of bacteriOphages. Ninety-three percent
were typeable. In each of 23 herds the predominant phage type was one
of the #20 group. Frequently one phage type predominated in the strains
isolated from any one herd. Phage typing did not aid in distinguishing
between strains isolated from cows with mastitis and those strains from
apparently normal udders.

Price et al.(I95#) found a number of characteristic phage patterns
among their strains of staphylococci isolated from bovine mastitis. The
#20 pattern was again the most prevalent. Staphylococci from heavily
infected herds usually gave several different phage patterns. Only one
type was commonly found in a particular quarter, however.

Seto and wilson (I958) utilized the International-Blair, and
Jackson phages in typing l02 cultures of‘g, 225323 of bovine origin.
Thirty-four cultures were not sensitive to any of the above phages.

Of the sensitive strains. the predominant phage type was ##A. Phage #20
and the six phages isolated by Seto et ai.(l956) were used to type a
larger set of bovine staphylococci. Type 52 was found to be predominant.

accounting for #9%.of the #29 cultures. The organisms isolated from

8 .

small herds tended to be of a single type. while the isolates from a
‘iarge herd consisted of various types or patterns. More than one phage
type of staphylococcus was sometimes obtained from the different
quarters .of the same animal.

Coles and Eisenstark (l959e) found _S_. m strains of bovine
origin to be relatively resistant to the action of phages utilized in
typing cultures of human origin. Two such phages, 8i and #20, however,
did produce lysis in a number of the strains. Using a series of phages
obtained by adaptation and from Iysogenlc staphylococci, Coies and
Eisensterk (I959) found wide variation of phage type strains of _S_. m
within a single herd and more than one type from Individual animals.
Eighteen different patterns of lysis occurred in 50 cultures obtained
from one hard. Not more than‘one strain of g. m was isolated from
any one quarter of a cow.

A nteaber of investigators (Edwards and Rippon. I957; Pagano et ai..
l960; Thorne and Hallmark, I960; and Valiace et al.. I960) have isolated
strains of staphylococci from dairy animals and dairy personnel which
have identical phage patterns of lysis. The widely distributed or
so-caiied epidemic strain, 80/8l. has been isolated from the milk of
cows in a number of herds. (Uallace et al.. I960; Fratto and Mann. I960;

and Pagano et aI.. I960).

29.9.1132
Loeb (I903) first observed in vitro clotting by staphylococci

using goose plasma. lie found that S. aureus had a specific effect in
causing the plasma to coagulate. The phenomenon was independently

described by Much (I908) during an investigation of the bactericidal

behavior of human plasma. Using a small inoculum of _S_. m he noted
a coagulation of the plasma after overnight incubation, while with
larger inocuia a much shorter period of incubation was required for the
plasma to clot. He found that several‘pppppg strains caused coagulation.
whereasiplppp and citreus strains and several other bacterial species
did not. He called the clotting principle a staphylokinase. Gratia
(I920) studied the nature of the coagulating agent and found it to be
thermostable and to resemble the action of thrombin in producing fibrin
from a solution of fibrinogen. lie concluded. however, that the sub-
stance'was not thrombln or any of its precursors. He called the active
material staphylocoagulase. Gross (l93l) showed that the reaction was
readily elicited from culture filtrates of‘S..pp;pp§, and in I933 called
attention to the fact that blood coagulation was frequently accelerated
in staphylococcal Infections. This was observed during surgical inter-
vention of staphylococcal osteomyelitis and carbuncles. The clotting
occurred more quickly than in streoptococcai infections. where secondary
bleeding was sometimes a feared complication. In vitro experiments
confirmed the plasma clotting ability of the staphylococci. Pyogenic
S. £1.22. reacted more slowly and to a lesser extent than _S_. m.

A number of other bacterial species were tested. but all were found to
be negative. Gross' results thus revealed an important characteristic
of the pathogenic staphylococci. Darenyi (I926) had previously demon-
strated that pathogenicity for rabbits and the ability to clot citrated
blood were parallel characteristics, and concluded (l935) that only
staphylococci from pathogenic processes were able to clot plasma. Me
advocated the use of this test for differential diagnosis of staphylo-

coccal pathogenicity. Cruikshank (I937) likewise demonstrated the

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ability of the pathogenic members to coagulate plasma. lie investigated

a nlnber of characteristics associated with staphylococci and found

that the presence of coagulase was the property most closely correlated

with pathogenicity for the rabbit. Me race-landed the use of this

property as a test for pathogenicity because of its constancy and ease

of demonstration. Other workers. Chapman et ai. (l93#) confirmed the

presence of the plasma clotting property in pathogenic staphylococci.

and concluded (Chapman et aI.. I933) that it was the most reliable single 1
test for the differentiation of pathogenic and non-pathogenic staphylo-
cocci. As a result. the coagulase test has been universally accepted -=
for the recognition of pathogenic staphylococci and has replaced the

multitude of other tests used in the past (Elek. I959). however, even

though coagulase has been considered by most to be the best single

criterion in estimating the potential pathogenicity of a staphylococcal

strain. its exact role in the pathogenesis of infection is not clear.

Menkln and Uaiston “93") studied the diffusion of trypan blue‘ in

rabbits after staphylococcal infections and following the injection

of coagulase precipitated from culture filtrates. They found that the

coagulase failed to exert a sufficiently powerful local reaction to

obstruct lymphatic drainage by means of a fibrinous barrier. and con-

cluded that it did not play a role in mechnlcai obstruction to lymph

flow or the 'hlaillng off" reaction induced by staphylococci. Male and

Smith (I9#5) suggested a possible role of coagulase in the inhibition

of phagocytosis by the formation of a fibrin coat around thecocci.

The number of coagulase positive staphylococci ingested by rabbit or

human polymorphonuclear Ieucocytes was reported to be significantly
louver than coagulase negative cocci. Yotis and Ekstedt (i959) showed

 

 

that coagulase positive strains of S. m respired more actively

in huan sen-than did coagulase negative strains. Purified coagliase
increased the respiration of both strains. but affected the negative
strains about twice as much as the positive strains. Ekstedt and

Yotis (l960) also found that the virulence of coagulase negative staphylo-
cocci was increased when the organisms were suspended in solutions of
partially purified coagulase and injected intracerebraliy into mice.
Coccl or coagulase alone were innocuous. IIobeI and Barman (l96l) found
a marked decrease in plasma proteins following the injection-of coagulase
preparations Into rabbits. Fibrinogen was almost cwletely depleted

in‘ some animals. Intravascular clots containing the enzyme were found

in the dilated capillaries and venules of the lungs of rabbits receiving

a lethal dose of the coagulase.

Phospthase
Phosphatase activity was demonstrated In staphylococci in I932 by

Gordon and Cooper who established the presence of a glycerophosphatase
in chloroform killed suspensions of S. _ag;e_u_s_. Bray and King inl9ll2
introduced the use of phenolphthaiein phosphate as a substrate for the
detection of phosphatase activity. and noted that the staphylococci gave
strong reactions. They failed. however. (Isl-3) to find differences
between-S. m and S. 23.22.20 Barber and Kuper (i951) studied the
Phosphatase activity of I00 strains of S. w using phenolphthaiein
Phosnhate In nutrient agar as the substrate. Forty two per cent of the
cultures liberated phenolphthaiein, which was indicated by the presence
0' l pink color after exposure to amnonia. One hundred and sixty
“Presentative colonies were picked from the #2 plates showing phosphatase

P°8ltive staphylococci and were tested for coagulase production. All

 

Abut one were positive. Staphylococcal colonies obtained from phosphatase

negative strains were all found to be coagulase negative. Barber et al.
(l95l) tested a larger series of staphylococci using the same substrate

in a fluid medium. All the coagulase positive strains liberated free
phenolphthaiein in 2# hours or less. Most of the coagulase negative
strains also produced phosphatase; however. a longer period of incubation,
from 2 to 7 days. was required. Twenty seven per cent did not produce
phosphatase at the and of 7 days incubation. Pan and Blumenthal (l96l)
obtained similar results. finding that the average coagulase positive

strain of S, aureus produced almost 5 times more acid phosphatase than

 

the average coagulase negative strain. Occasionally, coagulase negative

strains were more phosphatase active than the coagulase producers.

nails—"l
The importance of the hemolytic activity of staphylococci was dis-

covered early ln the study of these organisms. Krauss and Clairmont
(I900) presented evidence that staphylococci Iaked blood. They used agar
plates smeared with blood and noticed that rabbit cells were lysed by

S. aureus. but human cells were not affected. in the following year,

 

Meisser and Hechsberg (l90l) demonstrated a hemolytic substance in
filtrates of broth cultures. They found that the hemolytic substance
began to appear on the fourth day in alkaline beef broth, and that both
.plppp and‘ppgppg strains produced the iysln. although the virulent
strains produced'greater quantities than.glppg or avirulent strains.
These results were corroborated later by Kutscher and Konrlch (l90h),
who stated that all pyogenlc staphylococci produced hemolysin and that
failure to find it was due to a delayed production by some strains.

Julianelle (I922) found that all the staphylococcal cultures isolated

‘_' - "7E r ' ‘. ,,
._.t‘ ' ,
I".

, .7

4:,

M

,5
(Fr
. .

 

 

 

 

 

ii
ii;
he
Int

COCA

'3

during his investigation were huolytlc. although some strains required

a longer period of Incubation before showing this property. Pathogenicity
studies with mice and rabbits Indicated that hemolytic strains were not
necessarily virulent. Chapman. et al. (l93#) agreed with Julianelle

» after studying some 5,000 strains of staphylococci. They concluded that

a knowledge of hemolytic activity alone was insufficient to confirm
pathogenicity of the staphylococci. Vhen used in conjunction with other
tests. holiever. Chapman et al. (l938) concluded that hemolysis on rabbit
blood agar permitted a simple estimation of the pathogenicity of a strain.
Elek and Levy (I950) examined 359 strains of staphylococci for hemolysin
production, using blood agar plates and specific neutralization with anti-
hemolysin. Three distinct hemolyslns were found to be associated with
coagulase positive strains. They were identified as the alpha. beta, and
delta lysins. Alpha was distinguished by complete lysis of rabbit and
sheep erythrocytes and inhibition by alpha antihemolysin. Beta acted
00W on sheep cells and did not produce coqlete lysis. while delta
caused complete inking of both sheep and rabbit cells. iieither beta nor
delta Iysins were Inhibited by the anti-alpha serum. Delta iysln could
also be distinguished from alpha and beta by its action against horse and
human erythrocytes (Marks and Vaughan, l950)- 0” 0" “I. "If” ”3"“

was invariably present in all the coagulase positive strains. either

alone or in combination. The alpha-delta pattern occurred most frequently

in strains isolated from humans. Some coagulase negath strains were

also lytic for sheep and rabbit cells. but could not be inhibited by

“PM. beta. or delta antilysins. This type was designated as epsilon

probably only three

hemolysin. Elek (I959) concluded that there are
ive staphylo-

entigenlcaily different hamoiyslns produced by coagulase posit

“WI. and that the production of all three is typical for any strain.

J

5‘...-
m
rev—- ...
xvi-r

 

 

.nuh"

 

 

I#.

Losses may occur as a result of mutation. arown (I960) in his study of
#00 coagulase producing strains isolated from human lesions. found 99.8%
of the strains produced alpha lysin in detectable quantities. Strains
from acute lesions in apparently undamaged skin tended to produce more
alpha hemolysin than those from burns and post operative wounds; these.
in turn, were more active than cocci isolated from the urinary tract.

lie concluded that although coagulase may well be necessary for the patho-
genicity of a strain. the degree of virulence might be most conveniently

estimated by its alpha hemolysin production. 1

Ogher Physiolpgical Prpnrties
A number of other characteristics of the staphylococci have been

associated at one time or another with the ability of this genus to estab-
lish pathogenic lesions. The characteristic orange-yellow gralth of
staphylococh was connected to the yellow appearance of pus as far back
as I882 by Ogston (Elek, I959). Dudgeon (I908) observed that suppura-
tive and severe infiamnatory infections were usually caused by the w
strain, whereas mild inflannlatory processes were due to S. 3.ng3.
Julianelle (i922) found no relationship between pipentation and the
production of hemolysin. Chapman et al. 09%). however. found that
the inortance of the hemolysin test depended on an accurate detelgmin-
ation of the color produced by the strain. Memolytic. non-coagulating
.LUL‘Q strains were generally innocuous. whereas hemolytic m strains
were usually pathogenic regardless of their coagalase capacity. Barber
(I955) isolated white variants from 6 of 8 strains of S. m by culti-
Vation in a fluid medium. The variants all, resembled the parent culture
from which they were derived, both In the production, of coagulase and
.lth lysin, and, with one exception. In phage typo. Likewise. she

‘_——+

 

 

. -.?5

W'

Z‘w-

 

 

l5-

obtained golden variants from white strains of coagulase positive
staphylococci which resembled the parent strains.

The use of fenmentation reactions with other species led many
workers to try this approach with the staphylococci. Dudgeon (l908)
tested a number of carbohydrates. but found that most were of little
value. Litmus mannite. however, was acidified by most of the staphylo-
cocci obtained from pathogenic sources. All of the‘gggggg cultures
obtained from bone suppuratlons were found to acidify the medium. About
one-half of the white staphylococci obtained from healing<wounds did not
ferment the mannitol. Julianelle (I937) studied the fermentative ability
of a number of pathogenic and non-pathogenic strains of staphylococci on
mannitol. He found that 97% of 62 pathogenic. and 8% of ‘00 non-pathogenic
strains were able to utilize the mannite. Similar results were obtained
by Thygeson (I938) in a study of staphylococci causing conjunctivitis In
rabbits. He found that 97%.of Il0 strains could utilize the mannitol,
while only 2%.of the nontoxigenic strains possessed this ability. Fahlberg
and Harston (l959) observed that all of the coagulase positive strains
Isolated from human pathological lesions in their study were able to
utilize mannitol.

The liquefaction of gelatin by staphylococci was also noted early
in the study of these organisms. Dudgeon (l908) found that rarely did
S,‘gg£ggg fall to exhibit this ability, and that most of the'glggg strains
‘were similarly endowed. Stone (I935) used a beef extract gelatin medium
and found that the ability to liquefy gelatin was correlated with entero-
toxin producing capacity. A negative gelatinase test usually indicated
a deficiency in this ability. Cruikshank (I937) found that most of the

strains in his study which were pathogenic for rabbits. and about one-half

 

 

 

 

 

 

 

 

   

l6

of the non-pathogenic strains, were able to llquefy gelatin. Richou,
et al. (I952) however. could find no relationship between pathogenicity
and gelatin liquefaction in staphylococci isolated from the bovine
udder. Elek (I959) likewise indicated that gelatinase production was
not correlated with pathogenicity.

Catalase was probably demonstrated in the staphylococci by Burnet
(I925) when he observed the presence of a diffusible substance which

destroyed hydrogen peroxide formed on agar plates by exposure to sunlight

P:
h

or a carbon arc. Scouller (l9h8) tested lh6 cultures classified as

 

staphylococcus. mlcrococcus. and sarcina for the evolution of gas after
the addition of hydrogen peroxide. All were catalase positive. He
failed to obtain catalase activity from lI6 strains of streptococci and
suggested the separation of these organisms from the micrococcl by the
catalase test. Lucas and Sealey (I955). however, obtained a strain of
‘g..gg£ggs from normal urine which failed to exhibit catalase activity.
Cell suspensions and cell extracts tested manometrically. and colonies on
solid media of low glucose content failed to release oxygen from hydrogen
peroxide, indicating that occasional catalase negative. coagulase
positive staphylococci may be encountered.

Abraham and Chain (l9h0) were the first to show that some bacteria
produce an enzyme capable of destroying penicillin. They called it
penicillinase. The enzyme was obtained from extracts of.§.‘ggli. but
not from a penicillin sensitive strain of‘g.‘gg;gg§. Kirby (l9hh)
obtained the enzyme from 7 strains of coagulase positive‘é, 22522;, all
Of which were naturally resistant to penicillin. Penicillinase was not
Present in extracts of penicillin sensitive strains. Barber (l948)

isolated a number of penicillin resistant strains from clinical samples

 

and-found-that all produced peincillinase. The resistant strains could

not be differentiated from sensitive strains by cultural appearances.
biochemical reactions. or pathogenicity to rabbits. El Ghoroury (I952)
described a plate method for the detection of penicillinase. blood agar
plates were streaked with a penicillin sensitive strain and penicillin
soaked discs placed on the plate surface. A loopful of the strain being
tested was then added to the disc. Subsequent growth of the sensitive
strain around a disc indicated the presence of penicillinase. All the
naturally resistant strains studied were found to produce this penicillin
lnactivator. Gillissen and Rude (I957) noted that a direct relationship
existed between phosphatase activity and penicillin resistance. Resis-
tant strains produced about 2.5 times as much acid phosphatase as sensi-
tive strains. Markov et al. (I958). however. failed to confirm these
results. Resistant and sensitive staphylococci isolated from pathological
processes produced about the same amount of phosphatase. Coagulase pro-

duction was not influenced by the ability to resist penicillin.

S nthetlc Medium
Hughes (I932) Initiated the development of a synthetic medium for

staphylococci. He found that the organisms would not normally grow in
a casein hydrolysate medium. but that the addition of a small amount of
meat extract promoted heavy growth. Heat extract alone proved to be an
adequate medium. but 50 times as much were required. knight (I935) con-
firmed the existence of this essential factor and prepared an active
fraction from yeast extract which allowed aerobic growth of IO strains
of S. aureus on a medium containing acid hydrolysed gelatin. Hemolysin
producers which grew in this medium were able to produce this toxin. It

was then found by Fildes et al. (l936) that the acid hydrolysed gelatin

 

l8

could be replaced by a mixture of "I aino acids and Knight 0937) found

that nicotinic acid or amide could replace the yeast extract In the gela-

tin hydrolysate basal medium, but not in the amino acid medium. Further

work by Knight (l937a), however. demonstrated that vitamin b1 and nico-

tinic acid together would support abundant growth of s. _a;r_e_u_s_ In the

amino acid basal medium previously utilized. Concentrations of 2 ug of

vitamin C. and 200 ug of nicotinic acid per liter were sufficient to

support good growth. Porter and Pelczar (l9hl) used the synthetic medium E‘-
.of Gladstone (I937) and studied, the effect of biotin an the growth of _S_. '
aureu . Certain strains were unable to grow in the synthetic medium “‘-
without the addition of biotin whereas other strains were not stimulated

by this vlt-in. Cretteret al. (I955) found that 90 coagulase positive

strains grew well when thiamin and nicotinic acid were the only vitamins

added to a casein hydrolysate medium. Coagulase negative strains. how-

ever. frequently had an absolute requirement for biotin. Thirty-eight

out of #6 negative strains failed to grow in the absence of biotin. and

the remaining 8 strains attained only 50-60% of maximuagrowth. Surgalla

(I910) described a synthetic meditml for the production of staphylococcal

enterotoxin; Hergenhagen (I958) obtained the synthesis of hyaluronidase

in a defined medium. and Lominski et al. (I950), succeeded in obtaining

the production of coagulase in a medium of known composition. The amount

of coagulase produced was small, however. Szeto and “flick (I958) like-

wise reported low coagulase production in their synthetic medium.

IIarston and Fahlberg (I960). however. using a slight modification of the

defined medium of Ilergenhagen. obtained coagulase production with two

strains of _S. _ag_l_'_e_u_s_.which was comparable to a reference medium prepared

by dialysis of brain heart Infusion broth.

 

 

 

 

 

. HATER IALS AND METHODS

fiilk Sales

Animals with mastitis or reproducth difficulties. or both, were
selected from the herd. Strip cup examination was made of the:mammary
secretion of each quarter prior to collecting the sample for bacterio-
hogical examination. The udder was washed with a bactericidal solution
\eith particular attention centered on washing the teat orifices. Approxi-
mately l0 ml of milk were collected from each quarter in sterile tubes.

A loopful was streaked on tryptose agar (leco) containing 5% bovine
blood from selected animals. and the remainder incubated at 37°C for 2“
hours prior to microscopic examination. The plates were Incubated for

2“ hours at 37°C and hemolytic colonies selected for morphological observ-

ation and coagulase determination.

aggroductlve Tract Sales
The animals were prepared for sampling by scrubbing the area around

the anus and vulva with soap, followed by a rinsing with bactericidal
solution. A sterile glass speculum was then Inserted into the vagina
and the area of the cervix sampled by inserting a sterile swab attached
to a flexible metal rod contained in a stainless steel tube. The swab
was placed in nutrient broth (Difco) and held overnight at 37°C. Both
swab and broth were then streaked on staphylococcus ll0 agar (leco),

and the resulting colonies were selected for further study.

P Pr ration
A set of 2h international phages and their corresponding propagating
“Hills of staphylococcus were obtained from Dr. John E. Blair, Hospital

for Joint Diseases, New York City. New York, and the 6 Seto-Wilson phages

l9.

20

and propagating strains from Dr. J. B. Wilson. University of Hisconsin.
Hadison. Wisconsin. The method used for phage pr0pegatlon was the soft
‘ agar layer procedure recommended by the Communicable Disease Center,
Atlanta. Georgia. The final phage preparation was obtained by freezing
and thawing the agar which contained the phage-staphylococcal mixture.
The free fluid was removed by pipette and assayed by the agar layer
technique described by Adams (I950). it was then filtered through a
.Swinney filter containing approximately one-half thickness of a Seitz
filter pad and again assayed. The critical test dilution (C.T.0.) was
used to determine the lytic pattern of the staphylococci and was the
highest tenfold dilution (sue-3) which caused confluent lysis of the
propagating strain. The phage solutions were stored at h9c.

Phage was also prepared In fluid culture by inoculating flasks of
trypticase soy broth' with equal volumes of concentrated phage and 5-6
hour growth of propagating strain. The flasks were then incubated for
h—6 hours at 37°C on a rotary shaker, and the resulting clear or turbid
fluid assayed for phage activity.. Titers of 103410" particles per milli-

liter were usually obtained by this method.

Phagg Typing
One to 2 mi of a 3-5 hour trypticase soy broth culture of each of the

staphylococcal isolates were flooded over the surface of‘a trypticase soy
agar. plate previously dried at room temperature. The excess inoculum was
removed and the plates again allowed to dry. A drOp of phage (C.T.0.) was
then added to a marked area of the plate using a I ml syringe fitted with e
27 gauge needle. Twenty different phages could be added to one plate. After

‘aaithmore Biological Laboratories, Baltimore. Haryland

 

o.
-.

 

 

 

 

 

2i

overnight incubation. the plates were observed for lytic activity within
the marked areas. - Phages causing more than 20 plaques were reported as
part of the pattern of a strain. Each phage was checked against its own
propagating strain during each experiment. and every phage was checked

against all propagating strains when initially prepared or prepared anew.

Coagulase Determination

The initial coagulase test on the cultures was made by inoculating
0.5 ml of rabbit or human plasma with 0.5 mi of I6-2h hour culture

grown in brain heart infusion broth (Difco). Coagulase was determined

Fantasy-r“ 2-5]

quantitatively by using the tube dilution technique of Teger and Hales
091.8). The isolates were grown for 5 days at 37°C in brain heart
infusion broth. The cells were centrifuged at 2500 rev/min for ID min
and one ml of supernatant fluid diluted serially with an equal volume
of‘z‘x peptone-saline solution containing l:5000 merthiolate. One ml of
plasma. diluted l:5 in physiological saline. was then added to each tube.
After thorough mixing. the tubes were incubated in a 37°C water bath.
Results were recorded after h and 210 hours of incubation and expressed
as the reciprocal of the dilution of the supernatant fluid. The endpoint.
obtained after 2“ hours incubation. was the last tube showing a one plus
reaction. Tubes were graded according to‘ the degree of firmness of the
clot. Tubes in which the entire contents had coagulated were labeled
.5 ti plus and those which contained a large organized clot as 3 plus.
A small organized clot was indicated by a 2 plus reading. and small
unorganized clots as i plus.

Rabbit plasma was obtained in the dehydrated form from Di‘fco. Ihlman
plasma was prepared from outdated. citrated hunen blood. Each plasma.

diluted l:5 with saline was first reacted with a coagulase preparation

 

22

obtained from a known high coagulase producing staphylococcus. Comple-

mentary plasmas were then pooled, dispensed in l0 ml amounts. and frozen.

Mannitol Fermentation

 

Phenol red broth containing 1% mannitol was inoculated from stock
slant cultures of staphylococci. The tubes were incubated for #8 hours
at 37°C and the results read at 2“ and #8 hours. The formation of acid

was indicated by a change in color of the indicator from red to yellow.

Piggggt Formation

PigmentatIOn was determined on nutrient agar containing i0% evapo-
rated milk and 0.5% mannitol. The cultures were first grown in brain
heart infusion broth overnight and streaked on the nutrient agar. The
plates were incubated at room temperature for one week and observed

daily.

Hemolysin Production

The hemolytic patterns of the propagating strains were determined
on agar plates with blood from man, rabbit, and sheep. Difco heart
infusion agar was prepared to contain 0.2% KHZPO“ and 0.03% H950“. The
pH was adjusted to 7.“ and the medium autoclaved. After cooling, l2 to
l5 ml of the medium were poured into plates and 0.5 ml of washed red
blood cells of the desired species was dispersed in the agar. The cells
had been collected in Alsever's solution (Jackson and Little. l957).
washed two times in saline. and prepared as a 50% mixture in saline prior
to use. The blood-agar plates were streaked with growth from a Zh-hour
trypticase soy broth tube and incubated for “8 hours at 37°C in an

atmosphere of 30% C02. Results were recorded at 2“ and #8 hours.

 

23

Agtflbhotic Sensitivity

An initial concentration of “0 units per ml of sodium penicillin G
was prepared In sterile pH 6.0 phosphate buffer. Serial two-fold dilutions
were then prepared using penassay broth (Difco) as the diluent, and
0.5 ml of each dilution was dispensed into a set of l3 by l00 mm tubes.
The inoculum was prepared by diluting an overnight trypticase soy broth
culture l:l000 in saline and from this a l:l0 dilution in penassay broth.
One-half ml of the inoculum was then added to tubes containing each concen-
tration of penicillin. The tubes were incubated at 37°C and observed at

2“ and #8 hours for growth.

Penicillinase Production

Penicillinase was determined by the method of Innes (I960). Nutrient
agar was seeded with a penicillin sensitive strain of _S. m, 209p,
and poured into plates. A one-fourth inch ditch was cut Into the agar and
filled with nutrient agar containing i0 units of penicillin per ml. The
test organisms were streaked across the ditch and the plates incubated
overnight. Penicillinase was estimated by the presence of a V-shaped growth
of the sensitive strain near the point where the test strain crossed the

penicillin agar.

. Extracel lulgr: Protein
Extracellular protein (Stadtman et eLT-I9SI) was determined on the

supernatant fluid from a 5-day culture in brain heart infusion booth.
Two ml of 5% trlchloracetic acid were added to a cuvette containing 2 ml
of supernatant fluid. The tubes were shaken and allowed to react for 30
-seconds after which the optical density was determined at 5&0 mu using a
Bausch and Lomb Spectronic 20 spectrophotometer. Supernatant fluid and
water in the same preportions were used to adjust the instrument to zero

optical density.

 

2‘s

Crow urves

A 6-hour culture” in brain heart infusion was adjusted to an
optical density of 0.1i at 650 mu, and 6 ml used to inoculate l00 ml of
brain heart infusion broth. Flask contents were mixed well and dispensed
Into test tubes. The tubes were incubated in a 37°C water bath and at
one-hour Intervals tubes were removed, mixed well, and the optical density
determined after setting the instruent with a sterile brain heart
Infusion broth blank. Optical density was plotted against time, and the

growth curves obtained.

Phosgggtase
Cellular phosphatase was determined using the method of Barnes and

Norris 0957). The cells were grown on brain heart infusion agar slants
for 22 hours and harvested by washing the slants with citric acid-sodium
hydroxide buffer. pH 5.6. The suspension was then adjusted to an optical
density of 0.63 at a wave length of 600 mu with the pil 5.6 buffer. Two
and four-tenths ml of cell suspension were then added to 0.6 ml of 0.5%
p-nitrophenylphosphate disodium solution. The tubes were incubated for
30 minutes In a 37°C water bath, and l ml of 0.0lN Ne0ii added to each to
stop the reaction and to develop the color of the p-nitrophenol. The
samples were centrifuged and the Optical density of the supernatant
fluid determined at W. The measurements were repeated after acidifi-
cation with one drop of concentrated "CI to obtain the Optical density

values not contributed by the p-nitrophenol.

Catalase

Trypticese soy broth tubes were inoculated from stock slants of the

propagating strains and incubated overnight. 0ne loopful was then used

 

25-

to inoculate 20 ml of nutrient broth contained in Smith fermentation
tubes. The fermentation tubes were incubated for 1&8 hours at 37°C.
and I ml of a 3% "202 solution added to each tube. The tubes were
stoppered and the amount of gas formed in the closed end was measured

after an additional l hour incubation period.

Gelatinase
Celantinase activity was measured by two different procedures.
Nutrient gelatin was Inoculated from an overnight broth culture and

incubated #8 hours at 37°C. Liquefaction of the gelatin was then

”ha-\n'm.»
.‘. ‘.‘Ao '.-‘.I ' u"

determined after a l hour period of refrigeration. In the second pro-
cedure. nutrient agar containing 0.10% gelatin was prepared and poured
into petri dishes. An overnight broth culture of the test strain was
then streaked on one-third of the plate. After ‘08 hours incubation. a
mercuric chloride solution (ISg NgClz dissolved in l00 ml of N20 and
20 ml concentrated NCI) was flooded over the surface of the agar plate.
Gelatinase activity was indicated by the presence of a clear zone

around the colonies.

0x take

Oxygen uptake was determined by the conventional manometric technique,
and expressed as mlcroliters of oxygen per milligram dry weight of cells
per hour. The propagating strains were grown in brain heart infusion
broth for two l0-hour periods and washed twice in saline. A suspension
of cells in saline was adjusted to an optical density of l.0. and
l.5 ml added to the main compartment of a Herburg flask containing
. 0.5 ml of D.lli. pH 7.0 phOSphate buffer. Two-tenths ml of 20% RON

‘ was added to the center well, and l.0 ml of 0.2% glucose to the side

 

26

arm. The flasks were placed in a 37°C water bath and allowed to equil-
ibrete to bath temperature. The side arm contents were then tipped into
the main compartment. and manometer readings taken every l0 minutes for
a 2-hour period. Control flasks for endogenous measurement containing
i ml of N20 in the side arm were run for each strain. Dry weights were
determined by drying 5 ml of the cell suspensions overnight at 80°C in

tared containers.

Synthetic Nedium

A synthetic medium was developed by adding mixtures of amino acids
to filter paper discs on the surface of seeded agar plates. The agar
plates contained 0.2% glucose. 0.0005% each of thiamine hydrochloride
and nicotinic acid. 0.02% N950“.7H20. 0.l% NeZNPOu.l2N20. and l.5% agar.
and were seeded by streaking the surface with a I05 dilution of 6 hour
broth cultures of propagating strains 53 and 70. The amino acid mixtures
were prepared by combining individual acids so that each acid in turn
was omitted from a mixture, with the exception of the control. which
contained all l6 amino acids. Individual acids (0.2%) were dissolved
in 0.l5H pH 7.h phosphate buffer and sterilized by autoclaving. Cystine
and tyrosine were first dissolved in a small volume of IN HCl. One-tenth
ml of each mixture was then added to a l cm diameter disc on the surface
of the seeded agar and the plates incubated for #8 hours. letures
which failed to support growth or which allowed poor growth were thus
determined and the omitted amino acids of these mixtures were utilized

for further development of a liquid medium.

 

 

 

 

RESULTS

Phagg Typing and Coagglase Activity of Clinical Isolates

The lytic activity of the typing phages against all the propagating
strains was determined prior to the study of the clinical isolates. The
results, shown in Figures i and 2. compared favorably with those of other
workers (Seto and Hilson. I958; Blair and Carr, i960).

Milk and cervical samples were obtained from l2h cows located on
23 farms in the lower peninsula of Michigan. From the li96'quarters
sampled. l6h (92 cows) yielded staphylococci which were phage typed.
The results are illustration In Tables l-h. Table l indicates the total
number of phage patterns obtained on the 23 farms. The number of
patterns per herd varied from 0 (non-typeable) to h, and the total
number of patterns from all the herds was #2. However. since 3 of the
patterns (Table 3) were common to more than I farm and 2 remained
unchanged during two visits to the same fanm. only 30 of the patterns
were different from one another. The various patterns obtained are
indicated in Table 2 where the lytic spectrum of the l6h isolates is
shown by cow number and quarter. One hundred twenty five of the strains
were sensitive to one or more of the typing phages. giving a total of 6lh
significant lytic reactions. Thirty nine of the strains were not type-
able. lt is evident from these data. as well as in Table i, that a
number of herds contained strains of more than one phage pattern.
included‘within this group were several farms which had only a small
number of infected animals, as in herds D. J, N, and R. Farms‘which
yielded a larger number of infected animals (P. Q, and S) had about the

same number of phage strains as those yielding fewer positive animals.

27

x.-

fi'upsnm4v- ......

PROPAGA TING STRA INS

IV

Misc.

29

29H

52

7'9
3A
38
3C
55

6

7

47

53
54
7O

73
75
77

Fig.

28

PHAGE
| I I III N Misc.

52 4
52A793A383C5567 52475354707375”

ill
“Mill

 

l. Significant lytic reactions of the International-Blair
series of phage propagating strains of staphylococci

 

 

 

FVNWRAGATNW3:5DRAHMS

SI.

82
S3

S4

29

PHAGE
SI 52 53 S4 85 S6

 

 

 

 

 

 

 

 

 

 

 

 

 

....H.

 

Fig. 2. Significant lytic reactions of the Seto-Wilson series
of phage prOpagating strains of staphylococci

 

 

 

 

30

TABLE l. The number of phage patterns of strains of Staphylococcus
£35323 isolated from the udder of selected cows
from 23 herds
Herd Cows Quarters No. of No. of
Yielding Yielding Phage Organisms
St lococci Sta h lococci Patterns Mot T able

A 3 6 l 5
B 2 5 l 2
C 2 3 l 0
O h l0 h 3
E 3 3 I 2'
F 3 5 l O
G 2 3 l 2
H 3 5 2 O
I h l2 l 2
J 2 h 2 2
K h ll 2 3
L i l l 0
M 3 6 l i
N 2 2 2 O
0 h 5 l 0
P 9 I7 h 5
Q (l) 8 8 3 I
Q (2) 16 3| 3 7
R h 9 3 I
S 6 l0 3 O
T 3 3 2 l
U l l O l
V 2 3 2 O

_I_ _I_ _o_ _L
Totals 92 l6h #2 39

 

 

TABLE 2. Phage patterns obtained on 23 farms by cow number and quarter

 

 

 

 

 

 

 

 

 

 

 

Nerd Cow # and erter* Pha e Pattern
A 3c 29/hhA/52/52A/80/Sl/Sh/S6
la. ic 2a. 2b, 2d Non-Typeable
B l3a, l3b, l3d 56
la l3c Non-Typeable
C 6a, 6c 7c Sl/S3
D l20c, l20d 52
la, Ic , 55
3a hZB/QZE/Shl55/8l/S6
lb 3b . All numbers except 52. 53.
and 75
hb. kc, hd Non-typeable
E ihd S6
' lc 2c Non-typeable
F lb. ic 2b, 2d 3d HMA/79/Sl/S3/Sfi/SS
C - 2a 52A/80/Sh/S6
ld 2b Non-typeable
H H9d Zha, 29c 28c 52
v9. VA‘i/6/‘iZB/‘i7/5‘f/75/55
I la. lb, lc. ld 2d Sl/S3
3a he. “b, kc. hd
2b 3b Non-typeable
J Id 56
ic #25/55/79/8l/55/S6
2e.2c Non-typeable
R 2b. 2d Si/S3
ld 2a. 2c. 3c, 3d 4d hhA/SZ/SS
3b hb, hc Non-typeable
* e--left front quarter c--left rear quarter

b--right front quarter d--rlght rear quarter

Wrxs «1...

 

 

32.

 

 

 

 

 

 

 

 

 

TABLE 2. (Continued)
Nerd Cow # and anrter Phage Pattern
L lc 6/h2B/hkA/8l/Sl/S3
M la, lc, ld 2d 3c 52/52A/80/5h
2c Non-typeabie
N la “bl/$2
2b 3C/Q2E/h7/70/SZ
0 la 2d 3c he, hb 6/29/“28/92E/hhA/Sl/S3/Sh/S6
2lc, 2ld 72c 56
lSa 18.. i8d Sl/S3 l
4... 5d 29/SIIS3 F
lc 2la. 215 72a All numbers except 33, 3c, 53, i;
' SS, 79. $2. Sh. and S6
2a, 2b. 2d 3a he Non-typeable
Q 5 8 9 S2
(I)s l0 l2 25 h20/h2E/S2
23 All numbers except 3A, 3B. 3C
VA“. “AA. 52. 52A. 53. 55. 70.
73. and 7S
7 Non-typeable
q Sc lOO, lOd l8a. l8b 2h. - sz
(2)** 27¢ 28b 3la 33a 35b h3b
hhe
24 5a. 5b 6b l8c zhc 25d azaIuZE/sz
27b “Se, #Sd
28a 6/7
2c 27d 35a. 35c h2a Non-Typeable
hhd “Sc
R Ia VAh/7/53/52/SS/56
ld 3A/3B/3C/6/7/h25/Sh
lc 2e, 2c, 2d 3b, 3c 6/7/“28/h2E/hhA/k7/5h/70/
73/75/8l/Sl/53
he Non-typeable

 

* First sampling of herd Q,
'** Second sampling of herd Q

 

TABLE 2. (Continued)

 

 

 

 

 

Herd Cow # and Quarter Phage Pattern
S Pd Ta 52
lb. Id 2d Veh/77/55
Pb kc 5e. Sc Tc 3B/h2B/h26/54/79/8i/52/SS/S6
T 56c 56
2b 52/52A/80/Sh/S6
ld Non-typeable
U lb Non-typeable
V 2a VAh/6/h7/53/SS
la, lb hzo/uzelu7/5li/79/8I/sI/szl
_ S3/Sh/SS/S6
H lb

Non-typeable

 

 

3a.

The possible distribution of staphylococci in one animal is
illustrated by the patterns of lysis obtained on several farms. as
shown in Table 2. Two animals. I and h on Farm I, were infected in all
four quarters with the same phage type staphylococcus, while cow'fll F
from Nerd R carried 3 different phage types in 3 quarters. This was

the maximum number of different patterns obtained from any one animal.

Most other possible variations between these two extremes could also

be found on one of the farms.

Samples were obtained from one farm. Q, during two different

-

occasions, about three months apart. Two of the patterns present
during the first sampling were also found to be present during the
second visit as shown in Table 2. No record was made of the quarters
sampled during the first visit. Cows 5 and 25 carried the same type in
the second isolation as in the first. Some of the patterns reported
as 52 also had weak lytic reactions with #28 and 42E; thus. cow #iO
with an 52 pattern from the second sampling may be identical with the
h23/h2E/SZ pattern obtained at the first milking.

Table 3 indicates the frequency of the most common patterns
obtained from the l25 phage sensitive organisms. Each of 3 phage
patterns‘wes found on 2 or more farms.. All were the result of lytic
action by the Seto-Hilson series of phages. 52 was the most prevalent
type and occurred in 2b samples from h farms. This was followed by the
Sl/S3 pattern in l8 samples from h farms and nine S6 patterns in 5 herds.
The most active of the 30 phages was 52 which caused lysis in 56 (h4.8%)
_ of the l25 sensitive staphylococci, as shown in Table h. This finding

was in agreement with that of Seto and Hilson (I953) who found that 52

was the predominant type in their study, lysing h9%.of the sensitive

 

35

TABLE 3. Frequency of the most common phage patterns obtained from

staphylococci of bovine origin

Number of organISms of phage type

 

 

Nerd $2 $6 Sl/S3
8 0 3 0
C 0 0 3
D 2 0 0
E 0 l 0
H h 0 0
l 0 0 l0
J 0 l 0
K 0 O 2
p O 3 3
Q (I) 3 o 0
Q (2) l3 0 o
S 2 0 0
T 0 l 0

63

Totals 2h 9

 

TABLE 9.

Frequency of lysis by the most active phages upon

36

l25 strains of staphylococci

 

 

No. of Percent of

Strains Strains
Phage Lysed Lysed
$2 56 hh.8
SI “7 37.6
$3 #6 36.8
hZE #2 33.6
#23 39 3l.2
55 310 27.2
hhA 3i 2h.8
S6 30 2h.0
5b 2b l9.2
Sh 23 l8.h
BI 23 l8.h

 

.J

r‘ w Aer-AI

- | I b. I - - - i ‘ - T7-

37

cultures tested. Si and 53 were the next most active phages in this
study, followed closely by h2E, #28, and SS. Phages #28, h2E, and hhA
were the most active of the International-Blair series of phages used
in human medicine. Sixty-two percent of the total number of lytic
reactions (6lh) was due to this.series of phages, giving some indication
of a possible cross-lnfectivity or colonization between man and animal.
Further, such evidence was Indicated by the broad lytic pattern evident
in 13 organisms from h different farms, 0, P. Q, and R. as illustrated'
by samples lb and 3b, herd O. and 23 of herd Q. in Table 2. In addition,
6 organisms from 2 farms. M and T, gave a 52/52A/80 pattern with the fie
international phages, closely resembling the pattern 52/52A/80/8l found =
in human infections.I

Isolates obtained from the cervical samples failed to give
significant lyticheactions or phage patterns. From I2h such samples,
2h gave growth on staphylococcus IIO agar. All the isolates were gram
positive organisms, but only l5 were mannitol-fermenting gram positive
cocci. Subsequent coagulase determinations and phage typing failed to
indicate the presence of _S_. _a_u_r_e_u_s_. None of the IS organisms was
coagulase positive, and only one could be phage typed. giving a
3A/75/80/S3 pattern. Milk samples from 3 quarters of the animal yielding
the typeable cervical sample were positive. None of the milk isolates,
however, was related to the cervical isolate. Two gave a hhA/SZ/SS
pattern. and the other was non-typeable. All*were coagulase positive.

To further characterize the staphylococci obtained from milk, a

quantitative coagulase test was carried out using lOO of the l6h isolates.

'Personal communication, Dr. Stanley Read. Michigan Department of

health, Lansing. Michigan

 

38

The results obtained with some of the more common phage patterns are
shown in Figures 3, h, and 5. The phage type S2 cocci (Fig. 3) obtained
from the la herds indicated in Table 3 were quite variable in their
ability to’produce coagulase, ranging from a reciprocal titer of less
than ii to a high of 5l2. Eleven of the 2“ organisms with titers of
l28 or greater produced about the same» amount of coagulase even though
isolated from 3 different farms. The strains isolated from the first
sampling of hard Q (cows 5. 8. and 9) gave about the same coagulase
titer as most of the organisms isolated during the second sampling), and
the $2 organism of cow #5 appears to be the same in each case. The ‘0
staphylococci obtained from herd N also appear to be Identical on the

basis of phage type and coagulase production.

Figure A represents 8 herds with phage patterns Sl/S3 and S6 and
shows uniformly high coagulase producing organisms. Only 5 exceptions
occurred among the 26 samples. The A quarters of 2 cows from hard I
are represented in the Sl/S3 group. Four of these organisms (from cow
'flh) show almost identical titers. whereas staphylococci from cow'#l
range from a titer of less than h.to 5l2. All of the S6 organisms gave
high coagulase production although isolated from h different farms.

In Figure 5. the coagulase titer of some of the different phage
type organisms found in 2 herds is presented. The pattern h28/h2E/8l/S2
in the first hard (second visit to herd Q) compares favorably in
coagulase production with most of the hZB/hZE/SZ organisms in the same
herd (first visit). and thus should probably be considered to be the
same strain. Variability in coagulase production among the same phage
type organisms is again illustrated in the hhAlSZISS patterns of the
second herd, K. although most of the 6 cultures fall in the higher

coagulase range. The primary feature in evidence from Figure 5 is the

uniformly low titer of coagulase by the organisms which were not phage

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(Herds I, C, P. K, and 8, P. E, T)

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isolated from 2 herds, Q2 and K

and phage patterns of staphylococc

Fig. 5. Coagulase production

 

#2.

sensitive. 0f l0 such strains in both herds only 2 had coagulase titers
above h, and these remained In the low titer range. Twelve other non-
typeable organisms, not shown in Figure 5, were also compared in their
coagulase producing ability. Five gave titers of less than k, while h

were high producers (l02h), and 3 were in the medium range (l28).

Characteristics of the Phage Prggaggting Strains

’ Coagulase
The amount of coagulase produced by the propagating strains after 5 days

incubation is indicated in Figure 6, where the reciprocal of the greatest
dilution of culture supernatant fluid causing visible clot formation is
shown. All the cultures except 52A/79 and 73 were coagulase positive
at a dilution of i:h or greater. Strain 52A/79 was active at a lower
dilution (l:2). while number 73 failed to initiate clot formation at any
dilution. it was subsequently tested by direct inoculation into plasma
and proved to be coagulase negative. The remaining strains varied con-
siderably in their ability to produce coagulase. Host fell in the inter-
mediate ranges of 32 to 256 reciprocal titer units (RTU). Three of the
strains--70, Si, and S3--were very active, ranging from Sl2 to #096 RTU.
Strain 70 was the highest producer. giving a titer of #096. Nine strains,
in addition to 52A/79 and 73. gave low coagulase activity, with titers of
32 or less. All the group I organisms were within this range.

Coagulase production was also determined during aeration of the
cultures by continuous mechanical shaking. Representative strains of
the high, low and intermediate producers were tested. The results are
indicated in Figures 7 and 8 for samples obtained at different time
intervals. The titers compared favorably with those obtained during

stationary growth, and in most instances coagulase activity was slightly

 

 

 

 

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PHAGE PROPAGATING STRAINSB

Fig. 6. Coagulase production by the international-Blair and Seto-Hilson strains of staphylococci
after 5 days stationary growth

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enhanced. All the strains attained their maximum capacities early in
the growth period. The high and intermediate producers maintained
their maxima throughout the test period. while the low producers were

markedly less active near the end of this period.

Extracellular_froteln

The results of the extracellular protein determinations are shown
in Figure 9. host of the strains gave fairly low values for this
determination, and four strains (55. hzfi, #23. and SI) failed to show a
significant amount. Strain 7 gave the highest reading at an Optical
density of 0.58. Slx strains fell in an lntenmediate range, showing

optical density values between 0.3 and 0.“.

Phosphatase
The amount of phosphatase produced by a fixed amount of cells is

indicated for each strain in Figure l0. All strains except 73 were
significantly positive. Strain hhA produced the greatest amount of
phosphatase. Five other strains were also high producers. but only
about one-half as active as hhA. Almost one-half of the cultures gave

an optical density reading of less than 0.3.

Catalase

Catalase production is indicated graphically in Figure ll. The
significant result to be noted here is the ability of most of the
organisms to decompose hydrogen peroxide to about the same extent, with
the exception of strain number 70. This strain, which produced the
greatest amount of coagulase, failed to show catalase activity.
Growth Curves and Oxygen Uptake

The results of the growth curve determinations are indicated in

 

 

 

 

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79

Extracellular protein of the International-Blair and Seto-Wilson series of
staphylococci

Fig. 9.

 

   
       
    

  

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PHAGE PROPAGATI NG STRAIN

Fig. l0. Callular acid phosphatase production of the International-Blair and Seto-Wilson series
of staphylococci

3 "\_"

 

 

 

 

 

PHAGE PROPAGATING STRAINS

 

Catalase activity of the international-Blair and Seto-Hilson series of staphylococci

Fig. ii.

 

figure l2.. where three representative curves are drawn to illustrate

the data shown in Table 5. Strains 30. $6, 70, 73. 75. and 77 are exempli-
fied by the lower curve (A), and strains 6, SI, SW79, 53, 80. and 8|

by the top curve (0). The remaining strains tend to fall on the middle
sigmoid curve (a). The apparent short lag in the growth curves world

have. disappeared if a semi-logarithmic plot had been used. and the
differences among the growth slopes (Table 5) would be lea masked.

Oxygen uptake during the utilization of glucose by washed cells was
determined for all the strains and is. expressed as microliters of oxygen
per as dry weight of cells perhour in Figure l3. 0,02 values ranged frat
23 to 82. Eight strains had a fairly high oxygen consumption. ranging from
70 to 82 mlcroliters. These included 29, 52, 52A/79. 30. 6. 7. 53. and ‘620.
Five strains, 55.470, 75, 77 and 1628, gave low values of 23 to 32 micro-

liters. The Seto-Uilson strains were fairly uniform in this msurement.

Growth in a Chemically Defined Media:

A synthetic medium was developed which supported the growth of

most of the strains of staphylococci studied. it contained the following
nutrients: 0L aspartic acid, L cysteine, L giutamic acid. glycine. 0L
leucine. L lysine hydrochloride. DL phenylalanine, L proline, and DL
valine, 0.08% each; glucose, 0.10%; nicotinic acid and thiamine hydro-
chloride, 0.00051 each; HgSOu'7H20, 0.02%; and NazHPOg'IZI-lzo, 0. 5%.

One ml of a mineral solution containing boric acid. 0.069; molybdic acid.
0.029; Mason-H20. 0.039; mason-5:120. 0.049; FeCl3-6H20, 0.259; ZnSOh,
QJg; Kl. 0.lg-; and H20, l000 ml was added' per liter of synthetic medium.
The pH was adjusted to 7.1. with IN Maori prior to autoclaving. All the
Propagating strains gave less growth in the synthetic medium than in the

cantrol brain heart infusion brotl'r. Plate counts indicated a lo to NO

 

 

 

 

OPTICAL DENSITY - 650

SI

 

 

 

 

 

 

0.0% I I I I I I I I
0 I 2 3 4 5 6 7 8

TIME - HOURS

Fig. l2. Representative growth curves of the International-

Blair and Seto-Wilson series of staphylococci

9

IO

 

52

TABLE 5. Some growth characteristics of the international-Blair and
Seto-Wilson series of phage propagating strains of staphylococci.

Time required to

 

Logarithmic Maximum reach maximum density
Strain No. Growth Slope Density (Hours)

29 2.0 0.88 6.5
52 l.9 ' 0.95 8.0
52A/79 2.7 0.95 5.5
80 3.2 0.98 6.0
3A 2.2 0.8I 5.0
33 2.9 0.80 6.5
30 .2.2 0.73 6.5
55 2.6 0.89 V 9.5
6 2.3 0.92 5.0

7 2.0 0.82 6.0
42E 2.0 0.86 6.5
#7 2.l 0.80 6.0
53 2.8 0.90 ' 5.0
5“ 2.2 0.85 7.0
70 l.2 0.78 9.0
73 |.2 0.78 9.0
75 l.6 0.65 6.0
77 l.5 0.76 6.5
“23 2.] 0.88 7.0
VA“ l.6 0.85 9.0
“20 l.6 ‘ 0.93 ‘ l0.0
94A 2.“ 0.83 7.0
8| 3.0 ' 0.98 6.0
Sl 3.0 0.98 7.0
$2 2.l ' 0.88 7.5
$3 l.6 0.95 8.0
$0 l.“ ' 0.93 l0.0
$5 2.2 0.85 7.0

$6 l.2 0.76 9.0

weir“!-

 

 

 

 

53

 

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Fig. l3. Oxygen uptake of the international-Blair and Seto-Ullson series of staphylococci

 

w W \‘i‘f't

 

Sh.

fold decrease in numbers in the defined medium. Coagulase was produced

by a number of strains in this medium, but the titer was much less than
that obtained with the control broth. Host of the low coagulase producers
grew very poorly, as indicated in Figure lh by strains 29. BIA/79. 3C, 73,
- and #23. Strain $6. an intermediate coagulase producer. also failed to
grow'well in this medium. Subsequent studies with a semi-synthetic casein
hydrolysate medium revealed a marked enhancement in growth of this strain
rafter the addition of L tryptophane to the medium. All other strains grew
well in the hydrolysate medium*without added tryptOphane.

AntibiogramsI Penicillinase ProductionI and Hinimum Inhibitory Concen-
tration of Peniciilin

 

The antibiograms and penicillinase activity are shown in Table 6.
Six strains (53. 5h. 75. 77. 80, and 8i) were resistant to penicillin.
and produced penicillinase. Strain 8i was also resistant to most of the
other antibiotics tested. The remaining strains had almost identical
antibiograms. The minimumiinhibitory concentration of penicillin is
shown for all the strains in Figure l5. Five of the 6 resistant strains
grew in the highest concentration of penicillin used in the experiment
(20 units/ml) while the sixth strain. 53. was inhibited by a concentration
of 0.63 units per ml.
Eiggggtation. figgnitol Fermenggglgg. and figlgtiggse gag Hemolysin
Fonmatlon V

The remaining results are grouped in Table 6. Pigmentation of the
strains varied from white to orange. Host of the strains were white or
yellow; only two strains. 3A and 6, showed a marked orange pigmentation.
All of the cultures except number 73 were able to utilize mannitol. host

caf the strains were also able to hemolyze the blood of one of the species

 

55

   
 

   

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PHAGE PROPAGATING STRAINS

Fig. I#. Growth of the international-Blair and Seto-Wilson series of staphylococci in a

chemically defined medium

 

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used in the experiment. Only four strains failed to demonstrate lysis:

29, 52, 73, and 75. All of these were also low or negative in coagulase
producing ability. Few of the strains caused lysis of human red blood
cells. Only three (3A, 53, and AHA) of the International-Blair series
and five of the Seto-Wilson strains were capable of laking the human
cells. The Seto-Wilson strains showed almost Identical lytic spectrums.
Strain 52 was slightly less active than the remaining five strains.

Less than one-half of the strains showed the ability to liquefy
gelatin. Both of the 80 and Bi strains failed to exhibit this property,

as did the two highest coagulase producers.

wuw'

—.‘ a uni.

 

 

TU,“ ’2

 

 

'—

DISCUSSION

A number of workers (Coles s Eisenstark, I959:.Edwards S Rippon.
l957; Williams-Smith, l9#8) have found staphylococci of phage type #20 ‘
to be the predominant type among bovine staphylococci.. Our study, using
phage at routine test dilution, failed to yield #20 as a significant
phage type or as an active phage. Out of a total of l25 sensitive
strains only 8 were lysed by phage #20. On the other hand, phage 52,
which was originally derived by propagating phage #20 on staphylococci
obtained from milk, was the most active of the 30 phages used, lysing 56
(#5%) of the sensitive organisms. This finding compared favorably with
that of Seto and Hilson (I953) who found phage type 52 to be the pre- 8 ~
dominant strain In their study, lysing #9%.of the sensitive cultures
tested. Phage type 52 staphylococcus was also our most predominant
strain, appearing in 2# cows on # farms. Strain Sl/S3 was the second
most common organism but was not reported by Seto and Wilson, whereas
the 56 strain which appeared in 7%.of the Michigan animals also appeared
in the Wisconsin study but with a higher frequency (l9%).

Seto and Wilson also utilized the international phages to type
staphylococci of bovine origin. Over one-half of l02 cultures were lysed
by phage ##A, with 36 being lysed by this phage alone. Equivalent results
were not obtained in our study of bovine staphylococci, although phage
##A was one of the more active of the international phages. Twenty-five
percent of the staphylococci were sensitive to this phage. Only phages
#26 and #28 of the international set were more active. However, phage
##A alone, considering the international set separately, lysed only 7
strains, or 5.6%.of the sensitive cultures. This value was much lower

than the 35%.obtained by Seto and Wilson in their study.
59.

 

60.

Other patterns of lysis, as indicated in Table 2, were irregular
and varied and no particular relationship existed between the farms.
Within a particular farm, however, frequently one pattern was found in
all the animals which were infected. The most notable example perhaps
occurred in Herd l, where # animals carried the same organism, and 2 of
these were infected in all # quarters. while it was possible to have a
different type present in each of the # quarters of one animal, this was
not observed in our study. However, a different type was obtained from
each of 3 quarters of l cow, and 2 different types were found in 2 or
more quarters of 8 animals. Only l typical isolate from each quarter was
typed. Hilliams-Smith (l9#8) and Price et al. (l95#) have shown that
infection is nearly always due to one type of staphylococcus.

As previously indicated, the same phage type staphylococcus has
been isolated both from dairy animals and dairy personnel. In a number
of cases the widely distributed or so-called epidemic strain, phage type
80/8l, has been obtained from the animal or from dairy products, indicat-
ing possible cross-lnfectivity between human and bovine. The results
obtained in the present study also tend to substantiate this possibility.
Strain 52/52A/80, similar to a common pattern found in human infections
(52/52A/80/8l), was isolated from 6 samples obtained from # cows on 2
different farms. In addition, the 2# phages used to type staphylococci
from human infections were responsible for a large percentage of the
lytic reactions occurring in the bovine staphylococci. Sixty-two
percent of the total was a result of lysis by this series of phages.

The 6 Seto-Wilson phages caused only 38%. This high frequency of lysis
by the human series against the bovine staphylococci suggested a possible
inter-relationship between the organisms isolated from human and bovine

sources. The extensive lytic patterns of a number of the isolated

 

6i.

organisms also supported this conclusion. Thirteen of the bovine
staphylococci were lysed by a large number of the human phage series,
ranging from ii to 2i of the 2# phages. ‘

Thirty-nine of the l6# cultures obtained from milkIwere not sensi-
tive to the action of the 30 phages used in this study. This percentage,
2#, compares well with the results of Seto and Hilson (l958), where 2r:
of l02 cultures were non-typeable. Coles and Eisenstark (l959) reported
#9.7%.of bovine cultures resistant to the action of the human set of
typing phages, but reduced this figure to l8.8%.when 8 adapted phages
were used. in the present study, the use of the adapted or newly iso-
lated phages of Seto and Wilson gave results comparable to those of
Coles and Eisenstark. ‘

A much larger percentage of the organisms isolated from the
reproductive tract were not typeable. The high salt content of the
isolating medium inhibited the gram negative organisms and few gram
positive cocci were obtained. All were coagulase negative. and only
one was sensitive to the phages utilized in these experiments. The“
results suggested, therefore, that _S_. m was not involved in the
etiology of the breeding problems encountered in the present study. in
addition, the differences in phage sensitivity and coagulase production
indicated little or no relationship between the cervical cocci and those
obtained from milk.

Further characterization of a number of the organisms isolated
from milk was obtained by quantitative coagulase determinations.
Particular phage patterns were selected, along with non-typeable organisms.
The non-typeable strains for the most part appeared to be low coagulase

producers, while there was some variability among the other organisms,

 

‘1!

.v_w —rv

 

 

 

62

even among the same phage type obtained from one animal. Normal strain
variation could explain the difference in coagulase production, since
high, low, and negative coagulase producing variants have been found
within the same strain (Smith et aL, l952). in addition, the use of
routine test dilutions of phage may result in the loss of lytic reactions
and phage types, causing an incomplete differentiation of strains. Such
strains would therefore appear to be identical by the phage typing pro-
cedure, but would be expected to show differences in other characteristics.
The phage patterns of the prepagating strains indicated a variety
of phage types and further biochemical studies were carried out on this
group as representative of the genus Staphylococggg. Coagulase activity
differed markedly among the strains. .All were coagulase positive
except number 73. This strain has been reported previously as coagulase
negative (Rippon, l956), although at one time consisting of a mixture
of coagulase positive and negative staphylococci. Only the coagulase
negative strain was lysed by phage 73 and hence became the propagating
strain for this phage. Coagulase production in the remaining cultures
ranged from less than # to #096 RTU, suggesting a need for quantitative
studies of this preperty in pathogenic strains. This was further
indicated in the determination of the plasma clotting ability during
‘timed intervals of growth. Low producing strains attained their highest
titers during the early growth periods but failed to maintain maximum
activity for long periods. in contrast, the high coagulase producers
gave maximum titers throughout the growth period, after reaching this
maximum. Low coagulase producing strains. therefore, might easily—be
reported as coagulase negative if tested at sub-Optimal periods of

production.

63

Coagulase production was correlated in all cases with the ability
to ferment mannitol. Coagulase negative 73 was the only strain which
failed to utilize this carbohydrate. Alpha, or rabbit cell, hemolysin
also correlated well with plasma clotting activity. .Seven strains
failed to lyse rabbit erythrocytes, Including number 73 and three low
coagulase producers. Beta, or sheep cell, hemolysin was occasionally
found alone, but usually in combination with the alpha lysin, or, as in
the case of most of the Seto-Wilson strains, in combination with the
lytic capacity for both human and rabbit cells, an alpha-beta-delta
pattern. Elek and Levy (l950) reported this as_the most common pattern
among staphylococci pathogenic for animals. Host of the Seto-Hilson
strains, originally obtained from milk, showed this pattern. Hemolysis
of human red cells was rare except in the Seto-Wilson strains and never
occurred alone.

Phosphatase activity likewise was correlated with coagulase pro-
duction, though not quantitatively. All strains except number 73 were
positive for both tests. Six of the strains were resistant to penicillin.
Four of these, however, were low phosphatase producers. The results of
Gillison and Rude (l958), who found higher phosphatase activity in peni-
cillin resistant strains, were therefore not corroborated in the present
study. Harkov et aL.(l953) found essentially no differences in the
average amounts of acid phosphatase produced by penicillin sensitive
and resistant strains.

Extracellular protein was low in most strains and appeared to bear
no relationship to the other parameters studied. High coagulase pro-
.ducing strains 70, SI, and S3 were low in extracellular protein while

some of the low'or intermediate coagulase producers were high in protein,

n—Lw- , ,g

 

 

 

 

 

 

-F_" ' .1:

“v.3.

 

 

6#

as in strains 3A, 7, #7, and 75. Rogers (l95#) observed that the forma-
tion of extracellular protein did not reflect the formation of coagulase,
and Bernheimer and Schwartz (l96l) could not relate most of their extra-—
cellular protein species to well known products of staphylococcal growth.

Host of the strains grew in the synthetic medium utilized in these
studies, but growth was always sub-optimal. Low coagulase producing
strains gave the poorest growth, indicating a potential method for
differentiating between high and low coagulase producers.

The remaining characteristics, for the most part, did not appear
to be correlated with the more common indices of pathogenicity, primarily
coagulase, phosphatase and alpha hemolysin activity. Some of the
characteristics were fairly constant within the various groups of the
propagating strains. 'All the organisms of group i, for example, had
a high oxygen uptake, and no gelatinase activity. Two of the strains
possessed hemolytic activity and had high growth rates while the other
two were non-hemolytic and average in rate of growth. The Seto-Wilson
strains were largely average or low in growth rate and gave almost
identical patterns of hemolysis. 002 values were fairly high for three
of the strains (Si, S# and S6) and about average for the Other three
(52, S3 and SS). Host were able to liquefy gelatin and were pigmented,
though not a characteristic orange-yellow. Only two of the 29 strains
possessed this pigmentation and neither was high in coagulase or phos-
phatase activity. All the strains except 70produced catalase.

From the results, it is obvious that several of the strains possess
one or more characteristics which are significantly different from
those of the remaining strains. The outstanding strain in this respect

was 73 which gave negative tests for hemolysin, mannitol, gelatinase,

 

 

 

 

 

65

coagulase, and phosphatase, and yet was lysed by most of the phages

normally specific for coagulase producing strains. Strain ##A, on the
other hand, was positive for these tests and gave fairly high coagulase
titer and exceptionally high phosphatase activity. Strain number 70 was
also unique, being the highest coagulase producer and the only strain
not giving catalase activity. Such marked strain differences suggest

the need for further quantitative studies on the staphylococci.

 

SUHHARY

Hilk and cervical samples were obtained from l2# cows on 23 farms
in the lower peninsula of Hichigan. Four hundred and ninety-six
quarters were sampled and l6# (92 cows) yielded staphylococci which
were typed with 2# international-Blair and 6 Seto-Wilson phages..
Thirty different phage patterns were obtained. Ho one phage type was
found to be widely distributed. 52 was the most prevalent type, occurring

in 2# samples from # farms. This was followed by the Sl/S3 pattern in

 

i8 samples from # farms and nine S6 patterns in 5 herds. The most active
of the 30 phages was 52 which caused lysis in ##.8%.of the l25 sensitive
staphylococci. This finding was in agreement with that of Seto and
Wilson (l958) in their study of bovine staphylococci in Wisconsin.

There was some indication of possible cross lnfectivity or coloni-
zation between man and animal. Phage patterns found in human infections
were not obtained with the bovine staphylococci; however, six organisms
from two farms gave a pattern of lysis which closely resembled a pattern
common in human infections. In addition, the phages used to type staphylo-
cocci of human origin were quite active against the bovine isolates.
Sixty-two percent of the total number of lytic reactions were due to this
series of phages. Thirteen strains were lysed by a large number of the
human phage series, ranging from ii to 2i of the 2# phages.

Cervical samples failed to yield a significant number of staphylo-
cocci. None of l5 isolates was coagulase positive, and only one could
be phage typed. Staphlococcus‘gggggg,apparently was not involved in the
etiology of the breeding problems encountered in the present study.

Coagulase production in the milk isolates varied from a titer of

less than # to 20#8. in some instances there was a marked difference

66

g ~~ 3"!—

4-.

 

 

 

67

in coagulase activity in strains giving the same phage pattern. Hon-
typeable organisms were usually low coagulase producers.

The propagating strains were utilized as representative of the
genus Staphylococcus and studied more intensely. Coagulase production
was similar to that found in the clinical isolates. All were positive
except strain 73. Low producers did not maintain maximum titers during
extended incubation periods.

Hannitol utilization, and alpha hemolysin and phosphatase activity
correlated well with coagulase activity. Pigmentation, gelatinase
production, and extracellular protein content did not.. All the strains
gave evidence of catalase activity except #70. This strain, however,
was the highest coagulase producer.

A synthetic medium was prepared which supported the growth of most
strains. Low coagulase producers failed to growiweli in this medium,
suggesting a possible method for distinguishing such strains.

Six strains (53, 5#, 75, 77, 80 and Bi) were resistant to penicillin
and all produced penicillinase. There appeared to be no correlation
between penicillin resistance and high phosphatase activity.

002 values varied from 23 to 82. Group l organisms gave uniformly
high results. The high coagulase producers usually gave low to average
002 values as did the highest phosphatase producer.

The study of growth rates yielded three representative curves for
the 30 strains. Six strains attained a high maximum density in a short
period of time, and 6 strains required a longer period of incubation to
reach a lower maximum. The remaining strains were represented by a

normal or average growth curve.

 

E

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