PHASE TYPKNG 0F MYCOBACTERM

Thesis fer the Degree of M. S.
MWHIGAN STATE iiNWERSHY
JAMES B. HOELTG‘EN
19517

LIBRARY

Mlcmédx 3 . 3
University

 

PHAGE TYPING OF MYCOBACTERIA

BY

James B. Hoeltgen

A THESIS

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

MASTER OF SCIENCE

Department of Microbiology and Public Health

1967

575733-
8/75/“7

ACKNOWLEDGMENTS

The author wishes to eXpress his sincere thanks
and appreciation to Dr. V. H. Mallmann for her guidance
and assistance throughout this study. Appreciation is
also extended to Dr. W. L. Mallmann for his advice and
counsel.

A special appreciation goes to the author's wife,
Kathy, who gave purpose to the desire for intellectual

enlightenment.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . . . . .

Mycobacteria . . . . . . . . . . . . . .
Bacteriophage . . . . . . . . . . . . .
Bacteriophage Typing . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . . .

Media . . . . . . . . . . . . . . . . .
Strains and Cultivation of Mycobacteria
Source of Phage . . . . . . . . . . . .
Titration of Phage Suspensions . . . . .
Phage Production and Storage . . . . . .
Spot Typing of the Mycobacteria . . . .
Phage Neutralization Tests . . . . . . .

RESULTS . . . . . . . . . . . . . . . . . . .

Confluent Growth on Dubos Agar Plates .
Preliminary Titrations and PrOpagation
of Mycobacteriophage Stock Suspensions
Mycobacteriophage Lysis of Rapid Growers
ATCC 607 or 2118 . . . . . . . . . . .
Phage Production and Storage . . . . . .
Period of Incubation . . . . . . . .
Centrifugation . . . . . . . . . . .
Chloroform Treatment . . . . . . . .
Aerated Broth Cultures . . . . . . .
Phage Typing of the Mycobacteria . . . .

DISCUSSION . . . . . . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . . .

LITERATURE CITED . . . . . . . . . . . . . . .

iii

Page

18

18
18
20
20
22
24
25

26
26
26
26
3O
3O
30
30
33
33
36
43

44

Table

Origin and source of mycobacterial cultures

LIST OF TABLES

The source and prOpagating strains of
mycobacteriophage used in typing . .

Days required for visible growth of phage
propagatigg mycobacteria in 1% dextrose

broth, 35 C .

The amount of mycobacteriophage received
and after prOpagation on the designated

Mycobacterium . .

 

The amount of phage in stock suSpension lytic

for mycobacteria ATCC 607 and the amount of

phage produced by ATCC 607 . . . . .

Bacteriophage typing of the mycobacteria .

Titers of mycobacteriophage suspensions

incubated 24,

36,

and 48 hours at 35°C

iv

Page

. l9

. . 21
27

. 28

. . 29
. 31

. . 32

INTRODUCTION

No single test or set of tests are available with
which many of the mycobacteria isolated from animate and
inanimate sources can be identified easily and conclusively.
Such tests are needed, particularly for epidemiologic
studies of the atypical (anonymous or unclassified) myco-
bacteria and to establish Species identification. In this
regard, phage typing has become important in determining the
strains, species, and genera of some bacteria. This report
constitutes a preliminary study of the susceptibility of

some strains and Species of Mycobacterium to mycobacterio—

 

phage.

LITERATURE REVIEW

Mycobacteria. The genus Mycobacterium contains certain

 

well-defined species which vary from saprophytes to obligate
parasites. Historically, the first mycobacterium associated
with an infectious disease was the causative agent of Han-

sen's disease, M. leprae, in 1868. In 1883, M, tuberculosis

 

was first isolated; with this organism Koch formulated the
Postulates which have been the sound foundation of infec-
tious diseases. In 1896, M, bovis, and in 1901!.M- avium
were reported. Species which have been recognized more
recently are M, ulcerans, M, marinum, M, kansasii.

As the mortality rate of tuberculosis has declined,
an equal decrease in the incidence of disease has not
occurred. There has also been an apparent increase in the
number of cases from which a well-defined species of myco-
bacterium could not be isolated. The increase was not real;

the number of cases due to M, tuberculosis diminished, but

 

the number of cases due to "atypical" or unclassified myco-
bacteria increased relatively. The atypical mycobacteria

are not drug—selected mutants, although they are generally
resistant to therapeutic amounts of streptomycin, paramino—

salicylic acid (PAS) and isoniazid (INH). Runyon divided

the atypicals into the following four groups according to
the rate of growth at 37°C and pigment production (54):

Group I: M, kansasii, a photochromogen: slow growth,
no pigment produced in the dark, yellow pigment pro—
duced after short exposure to light.

Group II: Scotochromogens: slow growth, yellow pigment
produced in light or in the dark, heterogenous
strains.

Group III: Nonphotochromogen: slow growth, no pigment
production, heterogenous strains.

Group IV: Rapid growers: growth in five days, many

strains.

The mycobacteria range from obligate parasites to
saprophytes. Pathogenic mycobacteria are classical intra-
cellular parasites. Many pathogenic mycobacteria are cap—
able of surviving and even multiplying within the phagocytes
after ingestion. The histologic hallmark of tuberculosis is
the presence of acid fast—organisms in giant cells. Patho-
genic mycobacteria vary with respect to the range of hosts
they can infect and the sites of infection in the hosts.

Mycobacterial diseases are characterized by their
chronicity, latency, induction of delayed sensitivity, and
irregular induction of antibody production (2,21). There
seems to be no single virulence factor such as an enzyme or
exotoxin. The cord factor, a waxy fraction of the cell wall,

has been credited with the virulence of the organism, but

this notion is controversial. Lipids undoubtedly contribute
to the organism's resistance to phagocytosis and to the
inflammatory response which the organism incites.

At the present time there is no generally accepted
rapid or even conclusive means for identifying some of the
Species of mycobacteria (14,24,86,87). Serologic methods
are frequently unsatisfactory because of cross reactions,
spontaneous cell aggregation caused by hydrophobic character—
istics, and the unpredictable presence or absence of anti-
bodies in closed or active cases of tuberculosis (53,76).
Chemical tests or combination of tests have been reported as
a means of reliable identification (15,25,27,30,46,68,78,79,
80,84,85). Invariably, this reliability has been questioned

by others. The differential infectivity of M. tuberculosis,

 

‘M. bovis, and M. avium for laboratory animals is the only
method commonly accepted as reliable. Animal testing is
time consuming and eXpensive. The variable virulence of the
atypicals and the unresolved relation of the atypicals to
the classical pathogens have discredited the method.

Perhaps the greatest controversy concerns the Group
III mycobacteria (42,45,96). The Battey bacillus, one strain
of the Group III human pathogens, has little or no virulence
for chickens and rabbits. It is indistinguishable from M,
avium on the basis of colony morphology, growth at 37°C, or
metabolism. There are some differences, although not con-

clusive in certain chemical tests and the optimal temperature

for growth. Runyon haS recently suggested that the name of

M, intracellulare be accepted for the Battey bacillus only,

 

but not for other Group III mycobacteria (56).

Tuberculosis or tuberculosis-like disease may be
caused by a range of mycobacterial organisms in a variety of
domestic animals (33). Some strains of Group III, isolated
from tuberculin positive cattle, produced generalized dis-
ease and hypersensitivity in experimentally infected calves;
others produced limited infections or no infections. Other
Group III strains isolated from swine produced limited or
no disease in eXperimental calves; they caused severe dis—
ease in swine. Strains of Group III isolated from soil and
feed produced neither disease nor hypersensitivity in swine
or calves (33). Differentiation among the Group III myco-
bacteria could not be made on the basis of morphology, cul-

tural characteristics, or cytochemical tests.

Bacteriophage. Bacteriophage, viruses which infect bac-

 

teria, were discovered independently by the British bacteri—
ologist F. W. Twort in 1915, and by the French bacteriolo-
gist Felix d'Herelle in 1917. When phage were found which
were capable of lysing some species or strains of pathogenic
bacteria, a potential therapeutic value was proposed by
d'Herelle and subsequently disproved (32). The book "Mg

bacteriophage; son role dans 1'immunite" was written by

 

d'Herelle in 1921.

Twort and d'Herelle independently developed theories
on the origin of phage. The origin has not been resolved
and is disputed today. One theory, the virus theory, pro-
poses that phage is exogenous and autonomous; the precursor
theory proposes that all phage are endogenous.

The first mycobacteriophage were isolated from soil
by Froman and Bogen in 1953 by a soil enrichment technique
(16). Mycobacterial cells were added to a soil sample, the
mixture was incubated, and, subsequently, bacteriophage were
obtained from filtrates of the soil mixture (9,11,71). It
was recently reported that phage can only be isolated from
soils that have been contaminated with fertilizer or animal
excreta (9,11). Mycobacteriophage have been isolated from
the gut of larva, animal feces, and human feces (12,13,37).

In the light of recent publications, bacteriophage
can be divided into virulent and temperate phage. Both enter
the bacterial cell. The virulent phage induces the lytic
cycle consistently after entering the bacterial cell; the
temperate phage induces lysis infrequently after entering
the bacterial cell. Bacteriophage is adsorbed to the cell
wall of a susceptible bacterium and releases lysozyme which
digests the cell wall. The phage DNA is then injected into
the bacterium. The virulent phage DNA diverts the cell's
activities to make more phage DNA and phage protein. In the

process known as maturation, the phage protein and the phage

DNA combine to form complete phage particles, and the bacte-
rial cell 1yses. The latent period and the burst size for
each phage and host system can be determined by the one-step
growth experiment. The graph below shows a Single-step
growth eXperiment with a typical latent period, rise period,

and plateau period (23).

Plateau

 

Number Phage/ml Rise

Latent

 

 

 

Time

Temperate mycobacteriophage, or some of the compo—
nents of the mycobacteriophage which enter the cell, attach
to the host DNA. The bacterium becomes lysogenized and con-
tains prophage. Prophage only rarely cause the bacteria to
produce more mycobacteriophage. The lytic and lysogenic

cycles are represented in the following diagram (26):

[I]
\.
/'

1
Q

(A

gg—a-

Lytic Cycle

  
  

Lysogenic Cycle

Temperate Phage

\ Virulent Phage \/
1
‘L‘ifl *— [E <—- <-—

Bowman and Redmond reported the first naturally
occurring temperate mycobacteriophage R1, isolated from M,

butyricum (5,6). The mycobacteriophage were induced by

 

ultraviolet light and after a latent period of six hours
yielded 104 mycobacteriophage particles and a burst Size of
20. It was possible to cure, i.e., to free, the bacterial
culture of the prophage by exposure to ultraviolet light (7).
There have been few reports of the isolation of myco—
bacteriophage from naturally occurring lysogenic mycobacteria
(36,57,58,59,60,6l,62, 90,91). Recently two different myco—
bacteriophage were isolated from M, jggMg (43). In addition
to ultraviolet light, streptomycin and hydrogen peroxide
have both been used to induce the prophage of lysogenic myco—
bacteria (50,83). Filtrates of Phage D32 were used to induce

M, tuberculosis (H37Rv), and the surviving colonies were

 

1ySOgenic. Phage induction of the prophage in lysogenic
mycobacteria has also been done with other phage and with

other lysogenic mycobacteria (82).

The number of mycobacteria that have been found to
be lysogenic is relatively few compared to other bacteria.
Mycobacteriophage are essentially the same as other bacte-
riOphage, but the long generation time of many of the myco—
bacteria necessitates some modifications in laboratory tech—
niques (5,9,10,11,17,64,65,66,67,68,69,70). The effect of
the high lipid content in the cell wall is not understood.
The tendency of many of the mycobacteria to clump make
accurate quantitative measurements difficult and, in many
instances, impossible.

Phage D28 propagated on M, smegmatis (9626) had a
latent period of 70 minutes, a rise period of 30 minutes,
and a burst size of 88 (65). The latent period for Phage
D29 was 65 minutes, the rise period was 30 minutes, and the
burst Size was 104 (5). In comparison, many of the T-even
coliphage have a latent period of 25 minutes, a rise period
of 10 minutes, and a burst size of 200 (1).

When M. smegmatis (9033) was the host cell for Phage

 

D32, no morphologic changes were seen by electron microsc0py
during the first 50 minutes after infection with Phage D32
(8,20). Electron dense bodies which closely resembled
mature phage heads began to appear in the host cells 70
minutes after infection.

The adsorption rates of mycobacteriophage were very
slow. The adsorption rate for a given mycophage varied with

the host of the twelve phage; Phage D29 adsorbed fastest to

10

M, ngM_, When the mycobacteriophage were rapidly adsorbed
to the host, they were sometimes aborted. This abortion
occurred when Phage D29 adsorbed to M, pglgi (yoken) (81).

The structure of the mycobacteriophage resemble the
structure of other bacteriophage. It is comparable in size,
shape,and type of structures, although variations are
reported in the length of the tail.

Mycobacteriophage B1 was 2000 R long, the diameter
of the head was 5503, and the tail was 1500 A by 90 A. The
head was icosahedral with six capsomeres on each side. The
tail had a sheath surrounding the inner core and was composed
of 32 subunits. The tail was noncontractile and the promi—
nent base plates were 100 A long. Five tail spikes were
barely visible. The structure of spikes has not yet been
fully described. A comparison of the structure of mycobac-
is represented below (23,73,

teriophage B to coliphage T

 

 

 

 

 

 

 

 

 

 

 

l 2
74,77).
650 A
6—-+
o A __f 4— I—fead
1000 A ~
J DNA'—'
’\ [ Core
1000 R Sheath
J, Tail-

 

 

 

 

 

 

\ Base Plate
~~~~~~r~Tail Fibres

 

11

The mycobacteriophage head is composed of a protein
coat surrounding an inner core of DNA. The tail also con-
sists of protein surrounding a hollow core. The DNA of
phage is generally double stranded; however, the DNA of
coliphage X174 is single stranded. Recently RNA coliphages
have been isolated which are structurally more similar to
the single stranded DNA phage than to the more common double
stranded DNA phage. In their polygonal shape with no vis-
ible tail structures, they resemble the plant and animal
viruses. They are approximately 225 g in diameter, which
is considerably smaller than the double stranded DNA phage.

Bacteriophage are quite stable, especially in their
own lysates. All are stable in a pH range of 5 to 8, some
are stable at 4, and others at 10. Phage precipitated by
lowering the pH generally retain their infectivity. Free
phage are inactivated by compounds such as urea, urethane,
mustard gas, halogens, permanganate, ozone, hydrogen peroxide,
mercuric ion, thymol, cyanide, and flouride. Some chelating
agents such as citrate and triphosphate increase the rate of
inactivation of phage. Inactivation of phage by formaldehyde
can be reversed by dilution. Glycerine, alcohol, and deter—
gents generally do not inactivate phage.

Phage can be inactivated by sonic vibration and by
osmotic pressure, but different phages vary in their rela—
tive susceptibility. Inactivation can occur in making dilu-

tions and transfers due to breakage of the tails. Without

12

intact tails, the phage do not adsorb to the bacterial host
cell wall.

Certain ions are necessary for the adsorbtion of the
phage to the host cell wall. Divalent cations such as Ca++
or Mg++ and monovalent cations such as K+ and Na+ are
required in low concentrations. The type and concentration
of the cation required depends upon the type of phage.
Mycobacteriophage D28 required Ca++ and Mg++ but not Na+ or
K+. The adsorbtion rate of mycophage D29 was nonspecifi-
cally increased by any of the four cations.

Temperature affects phage in different ways. Heat
can inactivate phage by denaturization of protein and fol—
lows first order kinetics. The rate of heat inactivation is
influenced by the chemical composition of the medium. Phage
in saline solution are readily inactivated by heat; in a
broth they are many times less susceptible. Mycobacterio-
phage are stable for months at 4°C in their lysates. Phage
can be preserved by rapid freezing with liquid nitrogen and
by lypholization (31,93).

Temperature may also influence the infectious pro-
cess. 4M. avium was resistant to infection by phage at 370C,
but it was no longer resistant to the same phage at 420C
(19).

An unusual role in the disease sarcoidosis has been
proposed for mycobacteriophage which lysogenize M, tubercu~

losis (22,38). When the phage are present and an individual

13

does not produce antibodies, the phage affect M, tuberculo—

 

sis so that they do not have cell walls, are not acid fast,
and do not induce delayed sensitivity. Instead of tubercu-

losis, the individual has sarcoidosis (39,40,4l,52).

Bacteriophage Typing, The differential susceptibility of

 

different bacteria to a phage or set of phages has con-
tributed significantly to the identification of several
genera and species of bacteria, and epidemiologic studies.
Many of the procedures used today in phage typing were devel-

Oped with Staphylococus aureus during the 1950's. Serologic

 

methods divided the staphylococi into a few relatively broad
groups, too broad for epidemiological studies (88).

Fish formulated the basis for many of the techniques
with which bacteria are identified by their susceptibilities
to bacteriophage. Many of the bacteriophage which were
first discovered lysed many strains or species of bacteria
and were not Specific. Other phage or combinations of phage
which were more specific were found, either by the isolation
of new phage or by the adaptation of old phage to different
strains of staphylococci. The routine test dilution and the
spot plating method were developed.

Strains of staphylococci retained their original
phage susceptibility after growth in mice or on media.

Phage typing of the staphylococci permitted their classifica—
tion into five groups. This system has made a major contri—

bution to epidemologic studies of staphylococci infections.

14

The classification system of staphylococci by Ripon
has been adOpted by most laboratories and is based on the
serologic relationships and the lytic Spectra of the phage
(88). Approximately 75% of the staphylococci were lysogenic.
The prophage was responsible for the resistance to other
phage, and the resistance was specific for a given serologic
group.

New phage were developed by adaption or conversion.
Adaptions are either mutations which change the host range
or modifications induced by the host. A conversion is due
to the replacement or alteration by the infecting phage of
the prophage initially present in the bacterium. The phage
produced is referred to as converted because it now lyses
a different serologic type of staphylococci. It has been
shown that the plating of concentrated lysates of phage
result in production of characteristic prophage rather than
the original lysate phage.

Variations in the typing occur, 3% with strong lytic
patterns, 50% with partial lytic patterns. There are three
possible results in phage typing, although only two are
recorded: no lysis, incomplete 1ysis,and.complete lysis.

No lysis is recorded as negative; incomplete lysis and
complete lysis are recorded as positive. The partial lytic
variation is due to incomplete lysis and may become complete
lysis or vice versa. Because variation occurs, rigid con-

trols must be employed for exact and reproducible results.

15

Staphylococcal typing generally includes 21 to 24
phage types, five lytic groups and five serologic types (87).
The stock suspensions of phage are obtained after being
prOpagated by the soft agar overlay technique or by the
newer cellophane technique. The phage are titrated to deter—
mine the routine test dilution (RTD), the highest dilution
of phage giving complete lysis. A multiple syringe applica-
tor has been develOped which can apply in one hour, routine
test dilutions of 26 phage to 300 plates seeded with bacte-
ria to form a lawn. The stock phages should not be propo-
gated on a culture that has somehow been altered. Controls
on the typing phage, on its prOpagating host, and its
potency should be included.

The identification of strains and species of the
mycobacteria with mycobacteriophage is in the very early
stages of develOpment (48,72,75). Relatively few mycobacte-
riophage have been isolated, and few exhibit host Specific-
ity (50). This is not unusual since host Specific phage are
rare for any genus of bacteria. Large numbers of phage have
to be isolated and screened to find host specific strains.

Adaptation has recently been described for the myco-
bacteriophage, and it has been suggested as a suitable means
for the procurement of new phage with greater host specific—
ity (28,35). Either the adapted phage has some change in
its genome or it is a new phage which was induced. The

phage typing pattern of Phage 101 became more specific for

16

a Species of mycobacteria after serial passage on that
Species (34).

Another major obstacle in the development of myco-
bacteriophage typing has been the failure of some investiga-
tors to use the routine test dilution (49). If the lysate
is too concentrated, non Specific lysis occurs, perhaps due
to the induction of prophage in the culture or to the pres-
ence of contaminating viruses in the lysate at the higher
concentrations (50,88).

The method used for typing mycobacteria is essen-
tially the same as the methods described for staphylococci
and other general of bacteria. The main difference is that
the soft agar overlay technique has not been satisfactory
for the preparation of stock phage suspensions. The growth
of mycobacteria is inhibited by the soft agar overlay.
Plaques may be formed, but poorly. The method most com-
monly used is to "spot" the routine test dilution of the
phage onto a lawn of the mycobacteria (49,50,51). The lawn
may have been either freshly seeded or have been grown for
several days, depending upon the growth rate of the species

or strain of Mycobacterium used. Phage are harvested from

 

lawns of the prOpagating mycobacteria (previously seeded
with the routine test dilution) by flushing the surface of
the plate or eluting the phage from agar blocks cut from the

area of lysis.

l7

Attempts have been made to type several strains of
mycobacteria with mycobacteriophages (3,4,18,28,29,44,48,50).
Mycobacterium tuberculosis (H37Rv), M, Egy$§,;M..gyiEM, and
the Group III (Battey strain) and a saphrophyte (607) have
been typed using mycobacteriophage with limited Specific
activities (47,54,95). The problem is compounded by the
fact that the present classification of many of the mycobac-
teria is still unresolved. Work has been reported with
phage typing of rapid growers (29). The results are some-
what more encouraging.

There is a great need for a reliable method for the
identification of mycobacterial isolants. If a phage system
can be developed and standardized, it will be a major contri-

bution.

MATERIALS AND METHODS

'Megig. .A 1% dextrose Dubos broth was prepared from Dubos
broth basel without Tween 80 or enrichment, dispensed into
20 m1 screw top tubes in 0.9 ml and 5.0 ml amounts, and
autoclaved (15 minutes, 1210C). Dubos-agar was prepared by
the addition of 1.0% Bacto agar (Difco) to Dubos dextrose
broth and autoclaved (15 minutes, 1210C). A thick layer was
poured into Petri dishes. Dubos-Noble agar plates were pre-
pared as described for the Dubos-agar plates, substituting
Difco-Noble agar for Bacto-agar. Prepared Lowenstein Jensen

medium in tubes was purchased (Difco).

Strains and Cultivation of Mycobacteria. Table 1 lists

 

the mycobacteria used, the laboratory in which they were
isolated, and the source of the specimen from which the

Mycobacterium was isolated. Stock cultures of the Mycobac-

 

terium were maintained on Lowenstein slants at 350C. Rapid

growers, (growth of isolated colonies, 5 days or less) were

transferred every two to four weeks; others were transferred
every four to twelve weeks. Before seeding with phage, a

heavy suspension of cells was used to seed Dubos dextrose

 

1Difco Laboratories, Detroit, Michigan.

18

19

Table 1. Origin and source of mycobacterial cultures

 

 

Source of
Mycobacterium Laboratory Specimen

 

M, tuberculosis

 

 

 

 

 

 

H37Rv Redmond Man
Group I
M, kansasii Redmond Man
P588a Redmond Man
M, Avium
170-2 M.S.U.b Swine
131-4 M.S.U. Chicken
132-4 M.S.U. Chicken
Group III
SOB-0 M.S.U. Cow
51C2-0 M.S.U. Cow
62D-O M.S.U. COW
12100 M.S.U. Man
P39 Redmond Man
15 wet M.S.U. Inanimate
259-1 M.S.U Swine semen
17202—1 M.S.U. Pig
Group II
P17 Redmond Man
P38 Redmond Man
Rapid.Growers
ATCC 607C Redmond Inanimate
2118 Redmond Inanimate

 

aOrganism isolated from surgical Specimens from human tuber-
culosis and identified by Ernest H. Runyon.

bTuberculosis Project at Michigan State University.

cAmerican Type Culture Collection.

20

broth. Rapid growers were incubated at 350C from four to
seven days; others, from three to five weeks. The broth
cultures were used to prepare lawns for the phage or to add

to the phage dilutions for titration.

Source of Phage. Table 2 lists the phage used and the

 

laboratory in which they were isolated, their source, and

the prOpagating Mycobacterium.

 

Titration of Phage Suspensions. Phage received in agar

 

blocks were eluted in 1.0 ml of Dubos broth overnight at
40C. Phage were stored at all times at 40C. The phage were
titrated on their designated prOpagating mycobacteria.
Serial ten fold dilutions of the phage were made using 0.9
ml of Dubos broth per tube as diluent. Each dilution was
mixed by gentle shaking. One-tenth ml of the prOpagating
host bacteria was added to 0.9 ml of the diluted phage sus—
pension. The suspension was incubated for 15 minutes at
hood temperature (approximately 350C), poured, and spread
on a Dubos-agar plate. After being seeded with phage, the
rapid growers were incubated at 350C from 1 to 4 days;
others, at 35°C from 7 to 21 days. After incubation, the
plates with 30 to 300 plaques were counted and the titer
determined by multiplying the number of plaques by the
reciprocal of the dilution.

Each phage was diluted to the routine test dilution

as determined by the preliminary titration of the original

21

Table 2. The source and prOpagating strains of mycobacte-
riophage used in typing

 

 

 

 

 

Individual Original
From Whom PrOpagating
Phage Receiveda Source (Mycobacterium
101A Manion Soilb 2118
G84 Redmond Soil ATCC 607
LEO Mankiewicz Manc ATCC 607
D29 Froman Soil ATCC 607
AGl Redmond ,Adaptedd ‘M, kansasii
GS4E Redmond Adaptede lM, tuberculosis
(H37Rv)
DW Mankiewicz Manf ATCC 607

 

aR. Manion, Veterans Administration Hospital, Minneapolis,
Minnesota. W. Redmond, Veterans Administration Hospital,
Atlanta, Georgia and Microbiology Department, School of
Medicine, Emory University. E. Mankiewicz, Royal Edward
Laurentian Hospital, Montreal, Quebec. S. Froman, Depart—
ment of Infectious Disease, School of Medicine, University
of California, Los Angeles.

bPhage 101 adapted to 2118.

CIsolated from stool Specimen of a sarcoidosis patient.
dFrom a mixture of mycobacteriophage.

eFrom GS4.

fIsolated from a stool Specimen of a tuberculous patient.

22

phage suspension on its propagating host. The routine test
dilution (RTD) is the highest phage dilution giving con-

fluent lysis.

Phage Production and Storagg, The routine test dilution

 

of each phage stock was used to seed its designated propagat-
ing host, as described in Titration of Phage Suspensions.
After incubation, the surface of the plates with complete or
nearly complete lysis were covered with 5.0 m1 of Dubos broth
and refrigerated (40C) overnight. The fluid was removed,
filtered,1 and titrated.

The titer of a Phage G84 suspension was determined
after different periods of incubation. Quadruplicate 0.9 ml
routine test dilutions of phage GS4 were seeded with 0.1 ml
of ATCC 607. After 24, 36, 48, and 96 hours of incubation,
the phage was harvested and titrated.

The phage were titrated on strains ATCC 607 and 2118
(rapid growers). The routine test dilutions were determined
for the phage capable of lysing these mycobacteria. Routine
test dilutions of Phage G84, LEO, D29, AGl, GS4E, and DW
were used to seed ATCC 607: and the routine test dilution of
phage 101A was used to seed 2118 for the production of phage

stock to be used in typing the mycobacteria. The phage

 

lMillipore Filter Corp., HA 0.45u, white grid.

23

suspensions were filtered, chloroform was added, and the
suspension of phage was titrated.

The titer of phage GS4 was determined before and
after filtration, centrifugation, and chloroforming. A sus-
pension of Phage GS4 was divided into 3 samples: one sample
was filtered, one was centrifuged (2010 x g, 20 minutes),
and one was centrifuged (2010 x g, 20 minutes) and then
filtered. The three suSpensions were then titrated on host
ATCC 607. One—tenth ml of chloroform was added to 15 ml of
a filtered suspension of Phage GS4 and titrated. Several
strains of mycobacteria were tested for their susceptibility
to chloroform by adding a heavy inoculum of bacteria to
50 ml of Dubos broth and then adding 0.1 ml of chloroform
and plating 1.0 ml on Dubos—agar plates.

The use of stationery and aerated broth cultures
(aerated by magnetic agitation) for the production of phage
was compared. Six 250 ml Erlenmyer flasks containing 25 ml
of Dubos broth were seeded with 0.1 ml of a heavy inoculum
of host ATCC 607 and 0.9 m1 of the routine test dilution of
Phage GS4. Magnetic stirring bars were added to three of
the flasks: the flasks were sealed in plastic bags and
incubated at 35°C. Those flasks with magnetic stirring bars
were aerated with gentle continuous stirring. Flasks with
magnetic stirring bars and flasks without magnetic stirring

bars were removed from the incubator at 12, 24, and 48 hours.

24

The cultures were centrifuged at 40C (2010 x g, 20 minutes),

filtered, and titrated.

Spot Typing of the Mygobacteria. Stock suspensions of
Phage 101A, GS4, LEO, D29, AGl, GS4E, and DW were used in
typing the mycrobacteria. They were filtered, chloroform
was added, and they were diluted with Dubos broth to the
routine test dilution. The phage were spotted onto the

plate according to the following diagram:

 

One-tenth ml of a heavy inoculum of the rapid growers was;
spread on duplicate Dubos-agar plates and immediately
spotted with one drop of each of the seven typing phages
from a 0.5 ml disposable syringe with 26 guage needle.
One-tenth ml of a heavy inoculum of the slow growers was
spread on duplicate Dubos-agar plates, incubated at 35°C for
7 to 14 days, and spotted with the typing phage. The plates
were incubated at 350C. Rapid growers were observed after
three days and slow growers at 7, l4, and 21 days for areas

of lysis.

25

Phgge Neutralization Tests. Mice were inoculated sub-
cutaneously with 0.1 ml of either a 10"3 dilution of phage
GS4 which had been filtered and treated with chloroform
(l x 109 plaque forming units per ml). The mice were
reinoculated after seven days with the same amount of phage.
Blood was collected 14 days post inoculation; the serum was
prepared and inactivated (560C, 30 minutes). Serial two-
fold dilutions of the serum were made. One-hundredth ml of
a 106 or 108 dilution of phage was added to each serum dilu-
tion and the mixtures incubated (350C, 30 minutes). One-
tenth m1 of a suspension of ATCC 607 was added to the sus-

pension and spread on Dubos-agar plates. Normal mouse serum

was used in control tests.

RESULTS

The time required for confluent growth on Dubos—agar
plates differed among the mycobacteria, as listed in Table 3.
The rapid growers ATCC 607 and 2118 had confluent growth
after four days; one Group I (P588), two Group II (P17 and
P38» and Group III (P39L after three weeks; the otherstafter
five weeks.

The results of the preliminary titrations and propa-
gation of mycobacteriophage stock suspensions on the orig-
inal propagating hosts are listed in Table 4. Eleven of
fifteen phages lysed their prOpagating hosts to some degree.

There were 1 x 109 phage/ml of phage LEO with an RTD of 10—5

and 3 x 107 phage/ml of phage D29 with an RTD of 10_4. The
other nine mycobacteriophage which lysed their original
prOpagating hosts to some extent had 1 x 104 phage/ml or
less and RTDs of 100. When the RTD of each of eleven myco-
bacteriophages was seeded onto their corresponding propagat-
ing hosts, six mycobacteriophage, 101A, GS4, LEO, D29, AGl,
and DW;ranged from 1 x 102 to 2.3 x 1010 phage/ml after har—
vesting.

Seven mycobacteriophage 101A, GS4, LEO, D29, AGl,
GS4E, and DW,lysed either of the rapid growers ATCC 607 or

2118 as listed in Table 5. All seven mycobacteriophage

26

 

 

 

27

 

 

 

 

 

 

 

 

Table 3. Days required for visible growth of phage propa at-
ing mycobacteria in Dubos 1% dextrose broth, 35 C
_Mycobacterium 4 Days 3 Weeks 5 Weeks
M, tuberculosis
H37Rv None Very little Good growth
Group I
M, kansasii None Very little Good growth
P388 None Good growth
M, avium
170-2 None Very little Good growth
131-4 None Very little Good growth
132—4 None Very little Good growth
Group III
50B-0 None Very little Good growth
51C2-0 None Very little Good growth
62D-0 None Very little Good growth
12100 None Very little Good growth
P39 None Good growth
15 wet None Very little Good growth
259-1 None Very little Good growth
172C2-l None Very little Good growth
Group II
P17 None Good growth
P38 None Good growth

 

Rapid growers

ATTC 607
2118

Good growth
Good growth

 

28

 

 

 

 

 

 

Table 4. The amount of mycobacteriophage receiveda and
after prOpagation on the designated Mycobacteriumb
a b Number Phage/ml
Phage Host Stock Phage/ml After PrOpagation

101A (R) 2118 1 x 104 3 108

GS4 (R) ATCC 607 1 x 103 1.5 108

LEO (m) ATCC 607 1 x 109 8 109

D29 (M) ATCC 607 3 x 107 2.3 1010
AGl (R) .N- kansasii 1 x 102 1 103

GS4E (R) ‘M, tuberculosis 0

(H37RV)
DW (M) ATCC 607 9 x 102 1 102
DS6A M, tuberculosis O
(H37Rv)

AXl M, kansasii 0

BM4Ps Ps88 (Group I) 10

C3Ps P588 (Group I) 10

LlPs Ps88 (Group I) 100

BGl P17 (Group II) 10

BG2 P39 (Group III) 1

G1 P38 (Group II) 0

 

aStock phage obtained from W. Redmond, (R), Microbiology

Dept.,

Mankiewicz,

Quebec.

School of Medicine,

Emory University and E.
(M), Royal Edward Laurentian Hospital, Montreal,

PrOpagating host designated by laboratory from which phage
were received.

29

Table 5. The amount of phage in stock suspension lytic for
mycobacteria ATCC 607 and the amount of phage
produced by ATCC 607a

 

Original Number b Number Phage/ml

Phage Phage/ml RTD Recovered
lMAC ., ... .u

GS4 1 x 103 100 1.5 x 108
LEO 1 x 109 10‘5 8 x 109
D29 3 x 107 10'4 2.3 x 1010
AGl 4 x 108 10‘3 1 x 108
GS4E 2.4 x 109 10"4 4 x 109
DW 9 x 102 100 l x 103

 

a .
Rapid grower.
bSubsequently propagated on strain 2118.

CRTD: highest dilution of phage giving complete lysis.

30

lysed ATCC 607; and three mycobacteriophage,D29, LEO, and
lOlAvlysed 2118 (Table 6). Titers of stock phage suspen-

sions ranged from 9 x 102 to 2.4 x 109 phage/ml, and RTDs

from 100 to 10-5. When one RTD of the mycobacteriophages
was seeded onto either ATCC 607 or 2118, harvested, and
titrated, relatively large amounts of six phage had been
produced. There were 1 x 108 phage/m1 of Phages 101A, GS4,
LEO, D29, AGl, and GS4E. Mycobacteriophage DW had a titer
of l x 103/ml. The other eight mycobacteriophage tested,

DS6A, AXl, BM Ps, C Ps, BGl, BG2, and.Gl’did not lyse either

4 3

ATCC 607 or 2118.

The titer of phage suspensions increased with the
period of incubation up to 48 hr. The titer of the suspen-
sion remained constant upon further incubation. The highest
titer of a suspension of mycobacteriophage GS4, prOpagated
and titrated on ATCC 607, was obtained after 48 hours of
incubation. The amount of phage recovered increased from
24 to 48 hours where it remained constant upon further incu-
bation (Table 7).

Centrifuging reduced the titer of Phage GS4 suspen-
sion recovered from and titrated on host ATCC 607. The
titer of the centrifuged suspension was 2 x 104 phage/ml,
whereas the non-centrifuged control was 5 x 107 phage/ml.

Chloroform killed all strains of mycobacteria tested

and did not inactivate the mycobacteriophage. There were

Table 6. Bacteriophage typing of the mycobacteria

31

 

 

 

 

 

 

 

 

 

 

Mycophagea

Mygobacterium Source 101A GS4 LEO D29 AGl GS4E DW
'M. tuberculosis

H37RV Man - + + + + +
Group I

'M. Kansasii Man + + — _ + _

P588 Man + - - + + _
M, avium

170-2 Pig - + - — - +

131-4 Chicken — + — — - -

132-4 Chicken - - + — _ -
Group III

50B-0 Cow - + + + + +

51C2-O COW -

62D-0 Cow - + + + + _

12100 Man - - + + + +

P39 Man - + + + + +

15 wet Inanimate - - - + + +

259-1 Swine

semen - + _ + + +

172C2—l Pig — + — + + +
Group II

P17 Man - - + + + +

P38 Man - + + — - -
Rapid growers

ATCC 607 Inanimate + + + + + +

2118 Inanimate + - + + — -

 

aPrOpagating host ATCC 607 for all mycobacteriophage except

101A prOpagated on 2118.

32

 

 

 

 

Table 7. Titers of mycobacteriophage suspensions incubated
24, 36, and 48 hours at 35 C
Propagated and Number of Phage/ml
Phage Titrated On
24 36 48
4 6 8
GS4 ATCC 607 l x 10 2 x 10 1.5 x 10

 

 

 

 

 

33

5 x 107 phage/ml of GS4 propagated and titrated on ATCC 607

7 phage/ml without

when treated with chloroform; 3 x 10
chloroform treatment.
Aerated broth cultures of mycophage GS4 and LEO had

3 x 102 and 1 x 103

phage/ml respectively. Broth cultures
which were not aerated by a spinning magnetic bar had no
detectable phage. All three phage tested were propagated
and titrated on host ATCC 607. Mycobacteriophage D29 did
not prOpagate in either the broth or the aerated broth cul-
tures.

Phage GS4 when adapted to ATCC 607 Showed no signif-
icant difference from phage GS4 which was not adapted when
both were prOpagated and titrated on ATCC 607. The unadapted
mycobacteriophage GS4 could lyse P38, whereas the adapted
mycobacteriophage could no longer lyse P38.

The 18 strains of mycobacteria varied in their sus-
ceptibility to seven of the mycobacteriophages (propagated
and titrated on ATCC 607 except for 101A prOpagated and
titrated on 2118). The results are listed in Table 6. The
three M, EXEEE strains differed in their susceptibility to
mycobacteriophages D29, AGl, and to a certain extent to GS4E.
Mycobacterium tuberculosis, Group I, Group III, Group II,
and the rapid growers were susceptible to mycobacteriophages,
D29, AGl, and GS4E, to varying degrees and all were lysed by
at least one of the three. The Group III mycobacteria iso-

lated from bovine tissues had the same phage susceptibilities;

34

the Group III mycobacteria from other sources varied in
susceptibility to mycobacteriophages GS4, LEO, and GS4E.
The Group III mycobacteria of bovine origin were similar to

;M. tuberculosis (H37Rv); they were not susceptible to phage

 

101A; Group 1 and rapid growers were susceptible to phage
101A. They could be distinguished from the other mycobacte-
ria in that the Group III mycobacteria were susceptible to
GS4E. Group I, Group II, Group IV, and Group III mycobacte-
ria other than those of bovine origin, were very similar in
phage susceptibility. Mycobacteriophage 101A lysed the
rapid growers and Group I but not Group III or Group II
mycobacteria. Mycobacteriophage LEO did not lyse Group I
mycobacteria, but it did lyse the rapid growers. Mycobac—
teriophage 101A was the least active phage; it lysed only
four mycobacteria. No mycobacteriophage lysed all the
strains tested. ATCC 607 was susceptible to all seven
mycobacteriophages.

Important differential susceptibilities were:

1. Group I mycobacteria (Ps88 and M. kansasii) were the
only slow growers lysed by Phage lOLA.

2. ‘M, avium (170-2, 131-4, 132-4) was not lysed by
Phage D29 or AGl; all Group III mycobacteria were
lysed.by both.

3. SaprOphytic Group III mycobacteria (15 wet and 259-1)
and.Group III mycobacteria of swine origin (l72C-l)
were M93_lysed by Phage LEO; all other Group III
mycobacteria were lysed by Phage LEO.

35

4. Saprophytic Group III mycobacteria (15 wet and 259—1)
were not lysed by Phage GS4E; Group III of swine
origin (172) was lysed by Phage GS4E.

The phage neutralization titer was greater than
1280; normal serum was negative. The phage control, consist-

ing of phage incubated at 370C for 30 minutes, had a reduc—

tion in titer of three log units.

DISCUSSION

Because the original pr0pagating hosts were largely
slow growers and in some instances not very susceptible to
their phage, low phage titers were obtained from them.

Rapid growers ATCC 607 and 2118 were used to prOpagate the
seven mycobacteriophages used for typing. By adaptation to
the rapid growers, considerable time can be saved in obtain-
ing and titrating phage. The host range may be modified,
but this does not necessarily reduce the usefulness of the
differential susceptibility, once established.

Centrifugation of phage lysates was not regularly
used because the titer of the lysate was reduced by centrif—
ugation. Filtration did not reduce the titer, and, in
addition, all cells were removed. The tendency of clumping
of the mycobacterial cells is often a disadvantage; it is a
distinct advantage in membrane filtration.

Chloroform, which is not reportedly used to any
extent with mycobacteriophage, appeared to be very satis-
factory for the inactivation of any bacterial cells in phage
lysates. This was not discussed in any of the pertinent
literature read thus far, and no disadvantage to its use is

known. It is advisable to exercise all feasible methods of

36

37

inactivating the mycobacteria from the standpoint of avoid-
ing possible infections.

More phage was obtained in broth when the medium was
agitated during incubation than without. However, enough of
the mycobacteriophage for typing could be collected by the
Dubos-agar method, and there is less chance of glass break-
age, aerosols or other hazardous technique. The necessity
of using liquids with mycobacteriophage and the mixing and
transferring of those liquids undoubtedly creates aerosols.
The route of infection by mycobacteria is the mucous mem—
branes of the respiratory tract. Very small droplets are
required. Any method which removes or reduces the probabil-
ity of an aerosol of viable organisms is desirable. Mycobac-
teriophage are relatively stable at 4°C for long periods of
time. However, after two and one—half months at 4°C in fil-
tered, chloroform-treated lysates, the mycobacteriophage
used for typing were no longer capable of lysing the myco-
bacteria. The means by which they are inactivated is not
known. Most probably, the tail proteins or tail structure
have been altered or broken, and the phage can no longer
adsorb to bacterial cells.

Many of the phages used in typing other genera of
bacteria probably have their origin as prOphage in those
genera. They can generally be induced by several commonly
used chemical or physical methods, and exposure to infective

phage. The prophage may exist in the host as a defective

38

prophage, lacking genetic information necessary to direct
the formation of mature phage particles. The infecting
phage may supply the necessary information, and the phage
with the genetic material of the prophage is produced (23).
This is one explantion for what appears to be a change in
the phage's capacity to infect (88).

Few of the mycobacteria have been found to be lyso-
genic. This could be more correctly stated, very few of
the prophage of the mycobacteria have been induced. The
lack of lysogeny may be due to the existence of an unusu-
ally high proportion of the prophage as defective prophage
due to improper methods of induction. The unusually high
lipid content in the cell wall of mycobacteria may protect
the cell from infection. Directly or indirectly, the
repressor which is thought to prevent prophage induction
in the bacterial cell may be less readily countermanded.
Inducers probably destroy the repressor which leads to pro-
phage development and eventually lysis. In some preliminary
studies of induction, not included in this thesis, ATCC 607
was successfully induced by ultraviolet light. This area of
research, induction of prophage in mycobacteria, will be
pursued further and may provide more specific phage which
are needed for typing mycobacteria.

In addition to the phages obtained by isolation from
natural sources, phages have been obtained by adaptation

which can be used for typing. Their host range is frequently

39

altered and not necessarily stable. Proper controls must
always be included. There may be a slightly different lytic
pattern after several transfers on the same host, as was
observed with an adapted mycobacteriophage GS4. However,
typing phage generally vary up to 15%, which emphasizes the
necessity of 'host controls' in any set of typing tests.

Due to the long periods of time needed to propagate
an assay mycobacteriophage on slow growers, the work becomes
tedious and time consuming. More importantly, there must
necessarily be a greater length of time required to obtain
results from titration and control plates to determine the
RTD. The procedures can be accomplished more effectively
and reliably if the phage are adapted so that rapid growers
can be used as prOpagating and control hosts. It has been
suggested that this is not adaption due to crossing over
between the phage and host genomes but due to the induction
of closely related preexisting prophage.

The difference in the susceptibility of M, avium and
Group III mycobacteria supports other evidence that M, gyigM
and Group III organisms are not identical. The Group III
mycobacteria of bovine origin appear to differ greatly from
‘M. avium, and to a lesser extent from the Group III myco-
bacteria from other sources. However, the lack of suscepti-
bility of M, avium may be nothing more than the result of

the difference in the Optimum temperatures for growth.

40

M, EXEEE strains are not susceptible at 35°C, but they may
be at 42°C. This will be investigated further. The lack of
susceptibility at 350C of M, gyigM does not negate the value
of differentiation at 35°C of other mycobacteria. If phage
could be isolated from lysogenic M, ayigM_strains which
would only lyse M, EXEEE at 420C and no other mycobacteria
at 35°C it would provide an ideal method for the identifica-
tion of M, gyigM, Such systems for the other mycobacteria
may be possible also.

A large amount of variation does occur in phage
typing performed with different lots of phage at different
times. This variation was noted for those mycobacteria
which were typed over a period of six months. Besides the
inherent variation of 15%, which occurs in most of the phage
typing systems, additional variation undoubtedly occurs due
to variations in the mycobacteria as they are maintained in
the laboratory.

Temperature plays an important role in phage specif-
icity, adsorption, and multiplication. A heated block was
not used to incubate the phage and mycobacterial mixtures at
35°C for one-half hour prior to plating. This may introduce
some variation, although the temperature of the room and
bacteriologic hood is relatively constant.

Phage GS4 and the GS4eATCC 607 system were the most
reproducible system tested. Therefore, it was used in the

adaptation, centrifugation, chloroform treatment, time, and

41

induction studies. The results may or may not be applicable
to other phage.

An examination of the results of phage.typing of the
mycobacteria indicate that such studies should be continued.
There.is no single test or set of tests which are presently
satisfactory for the identification of many of the mycobac-
teria isolated from animate and inanimate sources. If
developed, they can contribute significantly to epidemio-
logic studies. At the present time, the distribution, mode
of transmission, and relationships among many of the atypi-
cal mycobacteria are not known. It is only known that they
can be isolated from a wide range of animate and inaminate
sources, that some do produce disease in man and animals,
and others are undoubtedly saprophytes. There is no usable
method available to differentiate between pathogens and
saprophytes. By inference, those isolated from tuberculosis
tissues are assumed to be pathogens. Some are, but some may
be contaminants. Phage typing may contribute to the present
controversy and conflicts.

One of the primary problems of the Tuberculosis
Research Project at Michigan State University is one con-
cerned with reliability differentiating between M, avium
and Group III mycobacteria, which are potential pathogens
for swine but not for calves. By the use of D29 or AGl and
LEO, and then GS4E, this differentiation was possible as

follows:

42

D29 or AGl LEO GS4E

 

M, avium —‘ + —
Group III -

bOV1ne + .+ +
Group III -

swine + - +

Group III -
saprophytes + - -

Many more strains and species of mycobacteria will
need to be examined for their susceptibility to these and
other phage. If the phage system can be develOped, phage
typing of mycobacteria can be of considerable practical
importance, contribute to epidemiologic studies, and provide
models for studies on the fundamentals of bacterial host-

parasite interactions.

SUMMARY

Mycobacteriophage was prOpagated on nonpathogenic,
rapidly growing mycobacteria. The infectivity of the phage
for mycobacteria, for 18 stains or species of mycobacteria,

representing M, tuberculosis, M, avium, Group III, Group II,

 

and rapid growers was determined.

By the use of four phage, D29 or AGl, LEO, and GS4E,
it is possible to separate M, avium from Group III mycobac-
teria; Group III mycobacteria of bovine origin from Group
III mycobacteria of swine and inanimate origin; Group III
mycobacteria of swine origin from Group III of inanimate

origin.

43

10.

11.

LITERATURE CITED

Adams, M. H. 1959. Bacteriophages. Interscience
Publishers, Inc., New York.

Albertson, J. N. 1965. Changing concepts in tubercu-
losis. The anonymous mycobacteria. Am. Rev. Resp.
Dis. 91: 782.

Bates, J., and J. Fitzhugh. 1967. Subdivision of the
SPECIGSJM. tuberculosis by mycobacteriophage typing.
Am. Rev. Resp. Dis. 96: 7-10.

 

Bonicke, R., and D. Stottmeier. 1965. Identifizerung
von stammen der species M. borstelense. Klinik Und
Er Forschung der Tub. 130: 210-220.

 

Bowman, B. U. 1958. Quantitative studies on some
mycobacterial phage host systems. Amer. Rev.
Resp. Dis. 79: 52-62.

Bowman, B. U., and W. Redmond. 1959. A temperate
bacteriophage from M. butyricum. Am. Rev. Resp.
Dis. 80: 232-239.

 

Bowman, B. U., and W. Redmond. 1960. Studies on M.
butyricum (R1) cured of its prophage. Isolation
and factors contributing to an initial failure to
support growth of the homologous phage. Bact.
Proceed.: 83.

 

Carlberg, D., and G. Jann. 1963. DNA, protein, and
inducible enzyme synthesis in phage infected M.
smegmatis. Am. Rev. ReSp. Dis. 88: 235-239.

 

Cater, J. C., and W. Redmond. 1960. Isolation and
studies of phage lytic for mycobacteria. Bact.
Proceed.: 83.

Cater, J. C., and W. Redmond. 1961. A recombinant of
three phage strains lytic for sprophytic mycobac-
teria. Bact. Proceed.: 164.

Cater, J. C., and W. Redmond. 1961. Isolation of and

studies on bacteriophage active against mycobacteria.
Canad. J. Micro. 7: 697-703.

44

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

45

Cater, J. C., and W. Redmond. 1962. Phage active on
mycobacteria from stool specimens of tuberculosis
patients. Bact. Proceed.: 96.

Cater, J. C., and W. Redmond. 1963. Phage isolated
from stool specimens. Am. Rev. Resp. Dis. 87:
726—729.

Carbon, J., and A. Trujillo. 1963. A comparison of
methods for classification of mycobacteria. Am.
Rev. Resp. Dis. 88: 546-550.

Erlich, H. 1961. Rapid identification of mycobacte-
rial colonies I. The auramine test. Am. Rev.
Resp. Dis. 81: 218-225.

Froman, S., D. Will, and E. Bogen. 1954. Phage activ-
ity against virulent M, tuberculosis. I. Isola-
tion and activity. Am. J. Pub. Health. 44:
1326-1333.

 

Froman, S., M. Lechtman, L- Scammon, D. Will, and B.
Eckmann. 1961. Mycobacteriophage lysates as
serologic antigens. Am. Rev. Resp. Dis. 83:
901-902.

Froman, S., L. Scammon, and C. Hanson. 1961. Phage
susceptibility of Battey type of unclassified acid
fast bacillus and M. avium isolated from fowl and
swine. Am. Rev. Resp. Dis. 84: 118-119.

Froman, S., and L. Scammon. 1964. Effect of tempera—
ture on the phage susceptibility of strains of
M, avium (isolated from fowl). Am. Rev. ReSp. Dis.
89: 236-239.

Fukai, K., and M. Sellers. 1960. A morphologic study
of mycobacteria infected with phage D32. Am. Rev.
Resp. Dis. 81: 52-59.

Gale, D., E. Lockhart, and D. Reynolds. 1966. Studies
of unclassified mycobacteria: II. Survival and
dissemination of typical and unclassified mycobac-
teria in the organs of mice inoculated intraperi-
tonealy or intravenously. Health Lab. Sc. 3:
48-64.

Groman, N. 1961. Phage host relationships in some
genera of medical significance. Am. Rev. Micro.
15: 153-175.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

46

Hayes, W. 1964. The Genetics of Bacteria and their
Viruses. John Wiley and Sons, Inc., New YOrk.

Hobby, G., W. Redmond, E. Runyon, W. Schaeffer, L.
Wayne, and R. Wichelhausen. 1967. A study on
pulmonary disease associated with mycobacteria
other than M, tuberculosis: Identification and
characterization of the mycobacteria. Am. Rev.
Resp. Dis. 95: 954-971.

 

Hok, T. 1962. Nicotinamide test for differentiating
M, tuberculosis from other mycobacteria. Am. Rev.
Resp. Dis. 86: 94.

 

Jawitz, E., J. Melnick, and E. Adelberg. 1964.
Medical Microbiology. 103.

Jones, W., and G. Kubica. 1963. The differential
typing of certain rapidly growing mycobacteria
based on their sensitivity to various dyes. Am.
Rev. Resp. Dis. 88: 355-359.

Juhasz, S. 1963. Phage Specific for some mycobacteria.
Nature. 200: 804-810.

Juhasz, S., and R. Bonicke. ~1965. Possible classifica-
tion of rapidly growing mycobacteria on the basis
of their phage susceptibility. Canad. J. Micro.
11: 235-41.

Karlson, A. J. 1965. Identification of M. tuberculosis.
Am. Rev. Resp. Dis. 91: 801.

 

Meyle, J. S. 1965. Preservation of T2 phage with
liquid nitrogen. Am. Rev. Resp. Dis. 91: 471.

Mallmann, W. L. 1931. Studies on bacteriophage in
relation to salmonella and pullorum. Agr. EXpt.
Sta., Mich. State College, Tech. Bull. 109: 1-14.

Mallmann, W. L., V. H. Mallmann, M. D. McGavin, and
J. A. Ray. 1965. A study of pathogenicity of
Runyon Group III organisms isolated from bovine
and porcine sources. Am. Rev. Resp. Dis. 92: 82-84.

Mallmann, V. H., W. L. Mallmann, and P. Robinson. 1964.
Relationship of atypical bovine and porcine myco-
bacteria to those of human origin. Health Lab. Sc.
1: 11-20.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47

Manion, R., and S. Bradely. 1964. The derivation of
new mycobacteriophage typing reagents by pr0paga-
tion on alternate hosts. Am. Rev. Resp. Dis. 89:
764-766.

Mankiewicz, E. 1961. Morphological and biological
properties of a strain of chromogenic acid fast
bacteria and its phage immune variant. J. Gen.
Micro. 24: 63-68.

Mankiewicz, E. 1961. Mycobacteriophage isolated from
persons with tuberculosis and non tuberculosis
conditions. Nature. 191: 1416-1417.

Mankiewicz, E. 1963. On the etiology of sarcoidosis.
Canad. Med. Assoc. J. 88: 593-595.

Mankiewicz, E., and J. Beland. 1964. The role of
mycobacteriophages and of cortisone in experimental
tuberculosis and sardoidosis. Am. Rev. Resp. Dis.
89: 707-720.

Mankiewicz, E. 1965. Phage that lyse mycobacteria
and corynebacteria and Show CPE on tissue culture
of renal cells of Cercgpithecus aethiop_, Canad.
Med. Assoc. J. 92: 31-33.

Mankieicz, E. 1965. Antigenic components shared by
bacteriophages and phage hosts: mycobacteria,
corynebacteria, and Hela cells. Growth. 29:
125-139.

Mercky,.J. 1964. Disease in man associated with the
unclassified acid fast bacteria. Am. Rev. Resp.
Dis. 89: 460.

Mizuguchi, Y. 1965. Lysogenization of Mycobacteria:
I. Lysogeny of M, jucho by phage 413-L and 413-S.
Biol. Abs. 46: 30323.

Murohashi, T., T. Tokunaga, Y. Mizuguchi, and Y.
Maruyama. 1963. Phage typing of slow growing
mycobacteria. Am. Rev. Resp. Dis. 88: 664-669.

Phillips, S. 1965. Atypical pulmonary tuberculosis
caused by unclassified mycobacteria. Am. Rev.
Resp. Dis. 91: 782.

Potriznik, V. 1964. Thioglycollate medium used for
the differentiation of unclassified mycobacteria.
Am. Rev. Resp. Dis. 90: 160.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

48

Redmond, W., and J. Cater. 1960. A phage specific for
M, tuberculosis, varieties hominis and bovis. Am.
Rev. Resp. Dis. 82: 781-786.

 

Redmond, W., J. Cater, and D. Ward. 1963. Spotting
method of phage typing mycobacteria. Am. Rev.
Resp. Dis. 87: 257-263.

Redmond, W. 1963. The spotting method for phage
typing mycobacteria. Am. Rev. Resp. Dis. 87:
294-295.

Redmond, W. 1963. Bacteriophages of the mycobacteria.
A review. Adv. Tuberc. Res. 12: 191-229.

Redmond, W. 1965. Methods used in phage typing the
mycobacteria. Personal communication.

Redmond,‘W., and D. Ward. 1965. Relationships between
.M. tuberculosis (H37Rv) and phage isolated from a
sarcoid patient. National Tuberc. Assoc. Annual
Meeting - Med. Abs.: 59.

 

Reggiardo, Z. 1964. Antigenic relationships among
mycobacteria by agglutination and agglutinin
reactions. Amer. Rev. Resp. Dis. 90: 800-803.

Rodda, R. 1965. Phage patterns of fast growing myco-
bacteria. Bil. Abs. 46: 30325.

Runyon, E. H. 1965. Pathogenic mycobacteria. Adv.
Tuberc. Res. 14: 235-287.

Runyon, E. H. 1967. Mycobacterium intracellulare.
Amer. Rev. Resp. Dis. 95: 861-865.

 

Russell, R., G. Jann, and S. Froman. 1958. Lysogeny
in the mycrobacteria. Bact. Proc.: 39.

Russell, R., G. Jann, and S. Froman. 1959. The induc-
tion of the lysogenic state in M, tuberculosis
(H37Rv). Am. Rev. Resp. Dis. 78: 839.

 

Russell, R., G. Jann, and S. Froman. 1960. Lysogeny
in the mycrobacteria. Am. Rev. Resp. Dis. 82:
384-393.

Russell, R., G. Jann, and S. Froman. 1963. Lysogeny
of the mycobacteria. Am. Rev. Resp. Dis. 88:
528-538.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

49

Russell, R., W. Richards, L. Scammon, and S. Froman.
1964. Isolation of a lysogenic Mycobacterium
fortuitum from soil. Am. Rev. Resp. Dis. 89:
287-288.

 

 

Segawa, J., K. Takeya, and M. Sasaki. 1960. Isolation
of lysogenic acid-fast bacilli from the soil. Amer.
Rev. Resp. Dis. 81: 419-420.

Sellers, M., K. Tokuyasu, Z. Price, and S. Froman.
1957. Electron microsc0pe studies of mycobacterio-
phage. Am. Rev. Resp. Dis. 76: 964-969.

Sellers, M., and H. Runnals. 1961. Mycobacteriophage:
I. Physiochemical characterization. J. Bact. 81:
442-446.

Sellers, M., W. Baxter, and H. Runnals. 1962. Growth
characteristics of mycobacteriophage D28 and D29.
Canad. J. Micro. 8: 389-399.

Sellers, M., and T. Tokunaga. 1966. Further studies
of infectious DNA extracted from mycobacteriophages.
J. Exp. Med. 123: 327-340.

Shadomy, S., S. Froman, and M. Prickett. 1959.
Cytology of phage host interactions in M, tuberculo-
sis. Am. Rev. Resp. Dis. 79: 839.

 

 

Smith, M., and W, Steenken. 1961. Group differentia-
tion of mycobacteria on thioglycolate medium. Am,
Rev. Resp. Dis. 84: 447-450.

Sula, L, and J. sulova. 1964. The tellurite zonal
phenomenon of mycobacterial lysis observed in a
BCG strain exposed to mycobacteriophage My F2/59.
Am. Rev. Resp. Dis. 88: 77-79.

Sula, L., and J. Sulova. 1965. The phage tellurite
zonal phenomenon as a criterion for the differentia-
tion of slowly growing mycobacteria. Bull. Wld.
Hlth. Org. 33: 413-417.

Takeya, K., and T. Yoshimura. 1957. Isolation of
phage active against all types of M. tuberculosis.
J. Bact. 74: 540-541.

Takeya, K., T. Yoshimura, K. Yamaura, and T. Toda.
1959. Studies on the biological properties of
mycobacteriophage. Am. Rev. Resp. Dis. 80: 543-53.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

50

Takeya, K., R. Mori, N. Nakashima, M. Koike, and T.
Toda. 1960. Light and electron microscope studies
of mycobacteriophages and their interactions.I. . J.
Bact. 78: 307-312.

Takeya, K., M. Koike, R. Mori, Y. Yuda, and T. Toda.
1960. Light and electron microsc0pe studies of
mycobacteriophage interactions II. J. Bact. 78:
313-319.

Takeya, K., Y. Zinnaka, K. Yamaura, and T. Toda. 1961.
Bacteriophage susceptibility and tuberculin specifi—
city of unclassified mycobacteria. Am. Rev. Resp.
Dis. 81: 674-682.

Takeya, K., R. Mori, K. Hisatsune, and T. Toda. 1963.
A simple hemolysis test for the classification of
the mycobacteria. Am. Rev. Resp. Dis. 87: 773-774.

Takeya, K., and K. Amako. 1964. The structure of
mycobacteriophages. Virology. 24: 461-466.

Tarshis, M. 1962. A combined neutral red, serpentine
cord, and differential growth test. Am. Rev. Resp.
Dis. 86: 749-754.

Tarshis, M. 1962. Further investigation on the
arylsulfatase activity of mycobacteria. Am. Rev.
Resp. Dis. 88: 847-851.

Tarshis, M. 1964. ,A rapid 24 hour micro phenolphtha-
1ein sulfatase test (PST) for distinguishing M.
avium from Battey bacilli. Am. Rev. Resp. Dis.
90: 455-457.

 

Tokunaga, T., Y. Mizuguchi, and T. Morohashi. 1964.
Abortive infection and host killing of mycobacterio—
phage. Am. Rev. Resp. Dis. 89: 929-932.

Tokunaga, T., Y. Mizuguchi, and T. Murchashi. 1964.
Phage induction of lysogenic mycobacteria. .Am. Rev.
Resp. Dis. 90: 431-446.

Tokunaga, T., and M. Sellers. 1965. Streptomycin
induction of premature lysis of phage infected
mycobacteria. J. Bact. 89: 537-538.

Urabe, K., and H. Saito. 1964. The urease activity of
mycobacteria. Am. Rev. Resp. Dis. 90: 266-277.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

51

Wayne, L. 1962. Differentiation of mycobacteria by
their effect on Tween 80. Am. Rev. Resp. Dis.
86: 579-581.

Wayne, L., J. Doubek, and R. Russell. 1964.
Classification and identification of mycobacteria.
Am. Rev. Resp. Dis. 90: 588-597.

Wayne, L., and J. Doubek. 1965. Classification and
identification of the mycobacteria. II. Tests
employing nitrate and nitrite as substrate. Am.
Rev. Resp. Dis. 91: 738-745.

Wentworth, B. B. 1963. Bacteriophage typing of the
staphylococci. Bact. Rev. 27: 253-269.

White, A., and J. Knight. 1958. Effect of serum and
Tween 80 on phage. Am. Rev. Resp. Dis. 77: 134-
145.

White, A., F. Foster, and L. Lyons. 1961. Alterations
of colony morphology of mycobacteria associated
with lysogeny. Amer. Rev. Resp. Dis. 84: 118.

White, A., F. Foster, and L. Lyons. 1962. Alterations
of colony morphology of mycobacteria associated
with lysogeny. J. Bact. 84: 815-818.

White, A., L. Lyons, and F. Foster. 1963. The effect
of serum and Tween 80 on several mycobacterial
phages and susceptible mycobacteria. Am. Rev.
Resp. Dis. 87: 730-733.

Will, D., S. Froman, I, Kransnow, and E. Bogen. 1957.
Cultural characteristics and drug and bacteriophage
resistance of avian tuberculosis. Am. Rev. Resp.
Dis. 76: 435-450.

Will, D., S. Froman, Y, Akiyama, and L. Scammon. 1961.
Preservation of acid fast bacilli and mycobacterio—
phage by rapid vacuum drying. Am. Rev. Resp. Dis.
84: 739-743.

Vandra, E. 1963. Studies on phage typing the myco-
bacteria. Acta. Micro. 9: 61-67.

Youmans, G. 1963. The pathogenic atypical mycobacteria.
Ann. Rev. Micro. 17: 473-494.

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