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STUDIES ON CHROMOGENIC MYCOBACTERIA by Ralph W. Butler, M.Sc., Dip. Bact. A thesis submltted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Department of Bacteriology and lmmunology McGill University Montreal August 1965
Transcript
  • STUDIES ON CHROMOGENIC MYCOBACTERIA

    by

    Ralph W. Butler, M.Sc., Dip. Bact.

    A thesis submltted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

    Department of Bacteriology and lmmunology McGill University Montreal August 1965

  • ACKNOWLEDGEMENTS

    The candidate is grateful to Dr. G. G. Kalz who directed this

    investigation. Her inspiring counsel and guidance helped during the entire

    period of research.

    Gratitude is extended to Dr. R. W. Reed, Chairman of the Depart-

    ment of Bacteriology and lmmunology for permitting this work to be carried

    out in the laboratories of this department.

    Dr. J .E. Josephson, Director of Laboratories, Department of Health,

    Newfoundland, kindly arranged for special leave from duty. His encourage-

    ment and interest is appreciated.

    Sorne of the unclassified strains of mycobacterie investigated were

    kindly provided by Dr. E.H. Runyon, Veterans Administration Hospital, Salt

    LokeCity, Utahi Dr. C.O. Siebenmann, ConnaughtMedical Research Laboro-

    tories, University of Torontoi and Dr. E. Mankiewicz, Royal Edward Lourentian

    Hosp i ta 1, Montree 1 , Q uebec .

    Financiol assistance and leove gronted by the Deportment of Heolth,

    Newfoundland, is gratefully ocknowledged. The project was supported in port

    by a Federal Heolth Grant and by the Canadien Medical Research Council.

  • TABLE OF CONTENTS

    1. INTRODUCTION AND PURPOSE

    Il. HISTORICAL REVIEW

    1. SIGNIFICANCE, GEOGRAPHICAL DISTRIBUTION, INCI-

    DENCE AND EPIDEMIOLOGY OF CHROMOGENIC

    Page

    MYCOBACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

    A. Chromogenic mycobacteria prior to 1950 . . . . . . . . . 6

    B. Clinical significance during the post decade........... 6

    C. Geograph ical distribution and incidence . . . . . . . . . . . . . . 7

    D. Epidemiological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . 9

    a) Soil, water, milk and house dust . . . . . . . . . . . . . . . . . 10

    b} Animais other thon man . . . . . . . . . . . . . . . . . . . . . . . 11

    c) Man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

    E. Origin of chromogenic mycobacteria . . . . . . . . . . . . . . 12

    2. CLASSIFICATION OF CHROMOGENIC MYCOBACTERIA 15

    3. METHODS PROPOSED FOR THE DIFFERENTIATION OF

    CHROMOGENIC MYCOBACTERIA FROM M. TUBERCULO-

    SIS, AND FOR THE DETECTION OF SPECIFIC VARIETIES ---,..-

    AND SUB-GROUPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

    A. Animal virulence studies . . . . . . . . . . . . . . . . . . . . . 21

    B. Antigenic analysis . . . . . . . . . . . . . . . . . . . . . . 22

    C. Resistance to drugs and other agents . . . . . . . . . . . . . . . 25

    D. Bacteriophage typing . . . . . . . . . . . . . . . . . 26

  • Page

    E. Growth at various temperatures . . . . . . . . . . . . . . . . . . . . . 27

    F. 11 Cord11 factor and neutral red test . . . . . . . . . . . . . . . . . 27

    G. Lipid content 28

    H. Tissue culture 29

    1. Morphology and staining . . . . . . . . . . . . . . . . . . . . . . . . . 29

    J. Eh potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

    K. Adansonian classification (Eiectronic computer) . . . . . . . . 31

    l. Biochemical tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

    M. Miscelloneous tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

    N. Characterization of pigments in mycobacterie . . . . . . . . 37

    a) M. tuberculosis 37

    b) Saprophytes (M.phlei, M.smegmatis, M.lacticola,etc.) 37

    c) Unclassified slow growing pathogenic mycobacterie 39

    4. FORMATION OF PIGMENT IN MYCOBACTERIA . . . . . . . . 41

    A. Introduction . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 41

    B. Oxygen tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

    C. Temperature of incubation . . . . . . . . . . . . . . . . . . . . . . . . 42

    D. pH of medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

    E. Composition of medium including the presence of trace elements and drugs . . . . . . . . . . . . . . . . . . . . . . . 44

    F. Exposure to 1 ight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

    G. Exposure to mycobacteriophage . . . . . . . . . . . . . . . . . . . 48

    H. Specifie inhibitors of pigment synthesis . . . . . . . . . . . . . . . 49

  • Ill. MATERIALS AND METHODS

    1 . MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

    A. Strains of mycobacterie . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

    B. Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

    C. Chemicals and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

    2. METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

    A. Pretreatment and culture of clinical specimens . . . . . . . . 54

    B. Identification of M. tuberculosis and chromogenic mycobacterie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

    C . Growth of M. kansasi i for pigment stud i es . . . . . . . . . . . 58

    D. Pigment extraction and partition . . . . . . . . . . . . . . . . . . . . 59

    E. Th in-1 ayer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . 64

    F. Spectrophotometric analysis

    IV. EXPERIMENTAL RESULTS

    PART 1

    STUDIES ON CHROMOGENIC MYCOBACTERIA ISOLATED

    FROM CLINICAL MATERIAL

    71

    1. GENERAL ASPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

    A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

    B. Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

    C. Age and sex of patients . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

    D. Type of specimens yielding chromogenic mycobacterie 75

    E. Rate of growth on primary isolation . . . . . . . . . . . . . . . . . . 77

  • F. Animal virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

    G. Sensitivity to antituberculous agents . . . . . . . . . . . . . . . . . 78

    2. RECOGNITION AND GROUPING OF CHROMOGENIC

    MYCOBACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

    A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

    B. Inconstant pigmentation of individual strains . . . . . . . . . . 80

    a) lsolates from clinical specimens . . . . . . . . . . . . . . . 80

    b) Stock cultures . . . . . . . . . . . . . . . . . . . . . . . . . . 82

    C. A special problem with nonphotochromogens . . . . . . . . . 86

    D. A simple reliable method for the detection of ali chromogenic mycobacterie . . . . . . . . . . . . . . . 89

    3. GROWTH STUD lES .. .. .. . . .. .. .. .. .. .. . .. .. .. . .. .. . 91

    A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

    B. Growth in fluid media .. .. .. . .. . .. . . .. .. .. . .. .. .. .. 92

    a) Dubos medium 92

    b) Sauton medium 97

    C. Effect of pH on growth of M. kansasi i . . . . . . . . . . . . . . 99

    D. Unidentified growth stimulating factor for M. kansasii 103

    4. MORPHO LO GICAL OBSERVATIONS . . . . . . . . . . . . . . . . . 105

    A. Pigmented photochromogens as compared with non-pigmented photochromogens . . . . . . . . . . . . . . . . . . . . . 105

    B. 11 Aibino11 nonphotochromogenic growth of M. kansasii 107

    c. 11 Cord11 formation in chromogen ic mycobacteria . . . . . . . 109

  • PART Il

    PIGMENT STUDIES

    1. INVESTIGATIONS ON FORMATION OF PIGMENT

    IN CHROMOGENJC MYCOBACTERIA . . . . . . . . . . . . 111

    A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1

    B . Moi sture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2

    C. Oxygen ... .. .. .. ..... ... .. .. .. .. ..... ..... .. 118

    D . Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

    E. Age and viabi 1 ity of ce lis .. . . . . . . . . . . . . . . . . . . . . . 128

    F. pH . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . 13:>

    G . Med i u m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lll

    2. 11ALBIN0 11 AND 11 SCOTOCHROMOGENIC11 GROWTH

    OF M. KANSAS li .. . .. . . .. . .. .. .. .. . . . . .. .. .. .. .. . . 131

    3. CHARACTERIZATION OF PIGMENTS . . . . . . . . . . 132

    A. Extraction and partition . . . . . . . . . . . . . . . . . . 132

    B. Quantitative determination of carotenoids . . . . . . . . 135

    C. Thin-layer chromatography and spectrophotometric analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

    V. DISCUSSION AND CONCLUSIONS .. .. .. .. .. .. .. .. .. .. 158

    VI. SUMMARY .. . ... ... .... .. . ... ... .......... ... .. .... .. .. 181

    VIl. CONTRIBUTION TO KNOWLEDGE AND CLAIM TO

    ORIGINALITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

    VIII. APPENDICES ..............................

    A. Media ............................................ .

  • IX.

    B.

    c.

    Rea gents

    Procedures

    BIBLIOGRAPHY

    v

    vi

    ix

  • 1. INTRODUCTION AND PURPOSE

  • INTRODUCTION AND PURPOSE

    The term 11 chromogenic mycobacterie" is but one of severa! commonly

    used to describe ac id-fast bac ill i which differ from M. tuberculosis, and which

    for the past ten years have been isolated from clinical specimens with steadily

    increasing frequency. Additional terms which have been used in recent scientific

    literature and which are usually, but not necessarily synonymous with "chromogenic

    mycobacterie", include: 11atypicaP1 1 11anonymous" ,nparatubercle11 1

    11 unclassified 11 1

    11 nontuberculous acid-fast ba ci Il i" 1 "unidentified mycobacteriar 1 and "MOTT bac i Il i"

    (mycobacteria-other-than tubercle-bacilli). The terms 11yellow bacilli", "orange

    bacilliu, and 11 Battey11 refer to specifie varieties within the general group. The

    current trend in terminology favars 11 unclassified11 as suggested by the American

    Thoracic Society (1961), but the American Review of Respiratory Diseases in recmt

    months {1964} has indicated its intention to use only the term "atypical 11 ln this

    thesis 11 chromogenic mycobacteria 11 may be considered synonymous with '1unclassified 11 1

    11atypi cal" and "anonymousn.

    Of primary importance is the fact that certain members of this poorly

    defined group can cause disease in man which is clinically, radiologically and patho-

    logically indistinguishable from tuberculosis. A positive diagnosis of such infections,

    however 1 is most difficult because indistinguishable strains are often isolated from

    healthy 1 symptomless persans.

    The types of clinical specimens from which chromogenic mycobacterie

    have been isolated include: sputum, urine, faeces, exudates from various types of

    lesions, skin, tonsils, ear and nasal secretions, mucous membranes, lymph nodes,

  • - 2-

    synovial fluid, bone, spleen, cerebrospinal fluid and blood. Obviously 1 signifi-

    cance is attached to isolations from cerebrospinal fluid, blood,or closed lesions

    and abscesses; however 1 by far the most commonly encountered isolations are from

    pulmonary infections where, in the absence of other recognizable pathogenic organ-

    isms, the presence of chromogenic mycobacterie in sputum cannot be relied upon as

    positive diagnosis. The same situation exists when members of the unclassified my-

    cobacterie are isolated from urine specimens in suspected cases of renal tubercul osis.

    Considerable interest has been aroused in these bacterie, especially with

    regard to the ir exact relation to true human tubercle bacill i 1 the ir pathogenicity 1

    and their sensitivity to antituberculous therapeutic agents.

    The candidate flrst became interested in this particular group of myco-

    bacterie during 1957 when a chromogenic strain was isolated from a sputum specimen

    of a patient with pulmonary disease. On only three previous occasions, ali during

    1953, had pigmented mycobacterie been isolated from clinical specimens in the Pro-

    vince of Newfoundland. A careful search of the literature in 1957 revealed thot

    relatively little was known ab.:>ut slow growin~ chromogenic mycobacterie. Further-

    more, no agreement existed among bacteriologists concerning the cl inical significance

    and the classification of these organisms. lt was apparent thot most workers ignored

    such isolations, especially when made from sputum and urine specimens, on the basis

    of their being 11Something other thon M. tuberculosis". ln instances where microscopie

    examination of cultural growth revealed the presence of acid-fast bacilli, a few

    workers had carried out animal pathogenicity studies. At this level, the conscientious

    few were frequently misled, since these mycobacteria are nonpathogenic for the guinea

  • - 3-

    pig. The old concept that the guinea pig parallels man in susceptibility to patho-

    genic mycobacterie is still difficult for many to reject despite the fact that chromo-

    genie mycobacterie are now known to cause tuberculosis-like disease in man.

    ln Newfoundland 1 following the finding of chromogenic mycobacterie

    in a clinical specimen, the routine procedure for reading TB cultures was modified to

    facilitate a thorough search for these organisms. Almost immediately additional

    strains were found. During the years 1957- 1960 unclassified mycobacterie were

    isolated from 36 of 35,555 clinical specimens cultured for M. tuberculosis in the

    Provincial Public Health Laboratories. The majority of isolations came from patients

    suspected of having tuberculosis and from whom repeated attempts to isolate typical

    tubercle bacilli had failed.

    With increasing experience it became apparent that these organisms could

    be easily recognized. The difficulty then arose concerning their classification and

    clinical significance. Although some workers suggested that certain specifie varietles

    of chromogenic mycobacterie possessed greater clinical significance 1 there were no

    reliable methods available to permit strain recognition.

    A scheme for the grouping of unclassified mycobacterie was suggested by

    Runyon in 1959. lt was based upon the growth rate and the ability (or inability) of

    strains to produce pigment under specified conditions. Runyon introduced the terms

    photochromogen, scotochromogen 1 nonphotochromogen, and rapid growers for his

    Groups 11 11 1 Ill and lV respectively.

    At the time this research was started the author had already isolated over

    50 strains of chromogenic mycobacterie from patients suspected of having tuberculosis.

  • -4-

    Cultural studies had been undertaken in an effort to group these isola tes but it was

    frequently experienced thot repeat 1 or duplicata cultures, under apparently identi-

    cal conditions, displayed such marked differences as to affect their grouping within

    Runyon 's scheme. ldentical experiences had been reported by others and it was

    personally felt that the classification suggested was ineffectual 1 and even potentially

    misleading.

    ln the absence of specifie biochemical, immunological or pathogenicity

    tests to differentiate members of the chromogenic mycobacterie 1 this investigation was

    undertaken to determine the cause 1 or causes responsible for the variation so frequent! y

    encountered in the pigmentation of these bacterie. To facilitate diagnosis it is

    naturally essentiel thot bacteriologists be able to establ ish the identity of strains iso-

    lated from clinical specimens and also to be able to determine whether or not repeat

    isolations from an individuel patient are the same strain. lt was also initially intended

    to extract and characterize the major pigments of these organisms.

    Just prior to these studies the Subcommittee on Mycobacterie 1 American

    Society for Microbiology {1962), decreed thot photochromogens {Runyon 1s Group 1)

    formed a homogenous group regardless of their geographical occurrence. They desig-

    nated the species1 nome M. kansas ii for ali photochromogens previously referred to as

    Group 1, 11Yellow Bacilli 11 , and M. luciflavum. This species was usually associated

    with disease processes and appeared to be a distinct pathogen.

    M. kansasii, because of its clinical significance 1 was chosen for most of

    the present studies. Fortunately 1 chromogenesis is best studied by employing photo-

    chromogens. Pigment formation occurs rapidly 1 following exposure of a mature culture

  • - 5-

    to light. The type species ATCC 12478 (Bostrom) has been used routinely 1 and in

    many instances was paralleled by representatives of photochromogens 1 scotochromo-

    gens and nonphotochromogens kindly supplied by Dr. Runyon.

  • Il. HISTORICAL REVIEW

  • -6-

    H ISTORICAL REVIEW

    1. SIGNIFICANCE, GEOGRAPHICAL DISTRIBUTION 1 INCIDENCE AND

    EPIDEMIOLOGY OF CHROMOGENIC MYCOBACTERIA.

    A. Chromogenic mycobacterie prior to 1950

    ln 1891, Jess thon ten yeors following the isolation of M. tuberculosts

    by Koch 1 Straus and Gamoleyo reported on the chromogenic nature of certain

    tubercle bocilli (cited from Xoloborder, 1961). A review of the literoture

    indicotes thot prior to 1950 at leost 66 investigotors reported the isolation of one

    or more mycobocterio, pothogenic for mon but not for the guinea pig (Xalaborder,

    1961).

    B. Clinical significonce during the past decade

    The year 1952 morked the beginning of a keen ard growing interest in

    chromogeni c mycobacterie. ln thot year Tarshis and Frisch (1952 a, b 1 c) reported

    cultural, pathological and hypersensitivity studies on twenty-six stroins of chromo-

    genie mycobocteria isolated from patients with, or suspected of having tubercu-

    losis. The patients from whom these organisms had been isolated were scattered

    throughout the United States in Minnesota, Washington, Oregon, Californie and

    New York. Also in the same year Prissick and Masson, in Montreal, released a

    report on fourteen stroins of pigmented mycobocterio, ten of which hod been ob-

    tai ned in pure cul ture from pus aspiroted from suppura ting facial, submoxi llory or

    cervical lymph nodes in children (1952i olso 1956 and 1957). ln ali their cases

    the cl inical pic ture suggested a tuberculous infection. Because the orgonisms were

    in pure culture from closed tuberculous-like lesions they were considered to be the

  • -7-

    actual cause of the lymphadenitis. Since then others have demonstrated unclassi-

    fied mycobacterie as the causative agents in cervical adenitis (Weed et al. 1 1956;

    Runyon, 1959a; Davis and Comstock, 1961; Marsdenand Hyde, 1962; Hsu, 1962;

    Chapmanand Guy,1958, 1959; Noufflardetal., 1961; Wolinsky 1 1963; Gerszten

    et al. 1 1964). The role of unclassified mycobacterie in pulmonary tuberculosis-1 ike

    disease has 1 ikewise been esta bi ished sin ce the organisms have frequent! y been iso-

    lated from resected lung tissue following the Isolation of identical strains over long

    periods of time prior to surgery (Buhler and Pollak, 1953; Timpe and Runyon, 1954;

    Nassau and Hamilton, 1957; Huppertetal., 1957; Miyamotoetal., 1959; Corpe

    and lr.Iing, 1960; Lester etal., 1962;and Lawetal., 1963).

    The increased interest in mycobacterie other thon tubercle bacilli has

    also revealed that the prevlously recognized and classified M. fortuitum, considered

    to be a saprophytic mycobacterium, can also cause infection in man (Wells et al. 1

    1955; Gordon and Smith, 1955; Prather, 1960; Corpe et al., 1961; Wa'lle, 1961;

    Hartwig et al., 1962; and Dross et al. 1 1964).

    Three separate reports support the view thot unclassified mycobacterie may

    be etiological agents in cases of sarcoidosis (Chapman, 1961; James, 1961; Mankiewicz

    1963). The atypical acid-fast bacilli have also been implicated as the causative agent

    in cat scratch fever (Boyd and Craig, 1961; Reikesand Washington, 1962} ..

    With present knowledge 1 infection due to unclassified mycobacterie cannot

    be differentiated from tuberculosis by any means other thon bacteriological studies.

    C. Geographical distribution and incidence

    From reports to date 1 a world wide distribution of these organisms is indica-

  • - 8-

    ted. Significant numbers of cases of human disease have been identified in the

    United States, in Wales1 the Netherlands, Spain, Remania, Malta, Australie and

    Jamaica. lsolated cases of pulmonary disease caused by these organisms have been

    reported a Iso from Sweden 1 Finland, France 1 Switzerland, Peru and Canada (Lewis

    et al., 1960). Although there may weil be areas of high or law incidence,

    sufficient valid information is not available upon which to base an opinion.

    Experienced workers, specifically looking for these organisms, have

    reported the isolation of atypical mycobacterie from the sputum of approximately 2

    per cent of admissions to tuberculosis hospitals in Floride (Lewis et al., 1960).

    Approximately 1 percent of 15,180 patients admitted to tuberculosis hospitals in

    Georgie during the ten year period 1951 - 1961 had lesions associated with these

    organisms (Crow et al., 1961). Chaves in New York City (1960) reported that 8.9

    percent of the specimens positive for acid-fast bacilli were atypical, whlle Keltz

    et al. in Illinois (1958) reviewed 649 culture-positive consecutive cases and found

    164 positive for atypical mycobacterie. This represented an incidence of slightly more

    than 25 percent. Dunbar et al., (1963) released figures from the Floride State Board

    of Health stating that in one year 28 per cent of the cultures isolated at the Central

    Laboratories proved to be mycobacterie ether than M. tuberculosis. Gerszten et al.,

    (1963) after a four year study said that the finding of unclassified mycobacterie in

    Virginie (5%) was midway between that found in the South (10%) and in the North (2%).

    Mercks et al., at the Maya Cl inic (1963) a Iso claimed that 5 per cent of the ir cases

    were caused by unclassified mycobacterie. Youmans (1963) in quoting Lester et al.,

    (1958) cited an interesting epidemiological study of 49 cases due toM. kansasii.

  • -9-

    These workers found thot the incidence of infection with M. kansasii in the Chicago

    suburbs of Cicero, Berwyn, and Oak Park, with a total population of 184,129 was

    1 O. 7 per cent 1 whereas the incidence of infection in the remainder of Cook County

    with a population of 940,000 was only 1.4 per cent. From Texas, LeMaistre (1963)

    reported thot 9 per cent of ali newly discovered mycobacterial disease in Dallas was

    due to M. kansasi i .

    Foreman (1962) of the Central Tuberculosis Laboratory in Wales felt thot 1

    compared with pulmonary tuberculosis due to the tubercle bacillus, disease due to

    anonymous mycobacterie was a rority. ln Wales, in the ten years from 1950- 1960

    only 59 cases could be traced. This figure is fewer thon 6 cases per year in a popu-

    lation of 2 million. Besta {1959) cited the incidence in Jtaly as 0.5 percent.

    Apart from the author's work, figures concerning the incidence of un-

    classified mycobacterie in Canada ore limited to one report. Mankiewicz {1958)

    reported thot over a three year period approximately 4 per cent of the cultures positive

    for acid-fast microorganisms were chromogenic. Her study was performed at the Royal

    Edward Laurentian Hospital, Montreal. The au thor (Butler and J,:>sephson, 1963} 1

    culturing 35,555 specimens during the four year period 1957- 1960, found 2.1 per

    cent of the positive cultures examined at the Newfoundland Provincial Public Health

    Laboratory to be of the unclassified varieties. Figures for the years 1961 - 1964 ore

    included in the body of this thesis.

    D. ~idemiological aspects

    Epidemiological studies carried out by severa! workers have failed to show

    any evidence of contagiousness of infections by the unclassified mycobacterie (Lewis

    et al., 1959; Nassau and Hamilton, 1957; Runyon, 1959a; Kubica et al., 1961;

  • -10-

    Crow et al., 1961). A single instance of conjugal disease has been reported

    (Beek et al. 1 1963).

    What is known concerning the distribution of these organisms is pre-

    sented in the fol lowing paragraphs.

    a) Soil, water 1 milk and house dust

    Kubica et al. 1 {1961) undertook a study of 1200 samples of soil and

    water. Preliminary results released on 452 samples in Georgie revealed thot more

    thon 45 percent of the samples yielded acid-fast bacilli. They identified photo-

    chromogens, scotochromogens, and 11 Battey'' strains, as weil as members of Group IV

    rapid growers. Later (1963) they reported thot the marked similarity of the undassi-

    fied acid-fast bacilli isolated from soil and animais strengthened the belief thot

    these agents of disease in man and lower animais originated in soil. The ubiquity

    of the organisms in nature suggests thot they are relatively nonagressive. ln Austral ia

    large numbers of atypical acid-fast boeil li were found in swimming-pools, beach sand 1

    water tanks, and in mud from dams and creeks (Singer and Rodda 1 1963; Singer, 1964).

    Prather et al. 1 (1961) have isolated unclassified mycobacterie from house dust 1 hospi-

    tal dust 1 soil 1 vegetables and top water. Others a Iso reported isolations from top

    water 1 house dust and soil (Pellman and Runyon, 1964; Jefferies, 1963; Rodda and

    Singer 1 1963).

    Chapman et al., (1965) in Texas found 261 of 270 samples of raw mi lk to

    contain mycobacterie. Representatives of Runyon 1s Groups Il, JI( and IV were identi-

    fied among the iso lotes. Biochemi cal, serological, and biological tests suggested thot

    a small minority of the organisms shared certain characteristics with M. kansasii but the

  • - 11 -

    findings were inconsistent and inadequate for more precise identification. Also in

    Texas, Jones and Jenkins (1965) isolated 101 strains of mycobacteria from 77 of 92

    samples of soli. They failed to isolate photochromogens but did find representatives

    of Groups Il 1 Ill and IV.

    b) Animais other thon man

    Anonymous mycobacteria have been isolated from dogs in Japon (Toda et

    al. 1 1960) and from swine in California (Froman et al., 1961). The strains isolated

    from swine were indistinguishable from the 11Battey" type 1 and it was suggested thot

    swine may serve as potential reservoirs for human infection. Scammon et al. 1 {1963)

    also isolated Group Ill organisms from swine.

    ln a study of the distribution and significance of mycobacteria isolated

    from cattle and swine in the United States, 2,244 tissue specimens of cattle and 172

    tissue specimens of swine were cultured. Ali were negative for Group l organisms,

    but mycobacteria of Groups IJ, Ill and IV 1 as weil as M. avium and M. bovis were

    not uncommon isolates (Ellis and Yoder, 1964). Others had earller isolated members

    of ali four Runyon Groups from cattle, and had found sorne of the Group Ill organisms

    to be more virulent thon those of human origin {Mali man et al. 1 1962). Rodda and

    Singer (1963) reported on anonymous mycobacterla isolated from cattle, sheep, pigs,

    rats, birds, a toad and a fish.

    c} Man

    lt is possible thot man himself may act as a symptomless healthy carrier of

    these organisms occasionally developing clinical disease when body resistance is low-

    ered. ln support of this theory are the reports on the isolation of unclassified myco-

    bacteria from healthy individuals (Edwards and Palmer, 1959i Atwell and Pratt, 1960;

  • - 12 -

    Pra th er et a 1 1 1961; Stewart 1 1962).

    E. Origin of chromogenic mycobacterie

    Several possibilities have been suggested concerning the origin of the

    chromogenic mycobacterie. Thus far epidemiological studies coupled with lobera-

    tory investigations have not yielded satisfactory answers. One must accept the fact

    thot scores of recorded cases of human infection involving mycobacterie thot differed

    from Koch 1s bacillus either in cultural morphology or animal virulence, occurred

    prior to 1950. lndeed sorne of the early isolates were so weil described thot one

    cannet consider them as differing from the chromogens encountered today. We lack,

    however 1 a satisfactory explanation for the apparent increase in the incidence of

    mycobacterial infections caused by these organisms durlng the post decade.

    lt has been suggested thot the chromogenic mycobacterie are variants of

    previously existing forms, dissociation occurring either spontaneously 1 or due to the

    influence of drug therapy or bacteriophage. As yet there is inconclusive evidence to

    faveur either of these as the sole answer 1 however 1 there is sufficient evidence to

    suggest each as possible.

    Dissociation has been observed in mycobacteria by many bacteriologists.

    Petroff reported dissociation of both tubercle ba ci Il i and the BCG strain in 1927 and

    1929 (Petroff 1 1927a 1 b; Petroff et al. 1 1929). Orange colonies were isolated from

    H37 human tubercle boeil li (Petroff and Steenken, 1930). (See also Miller, 1931;

    Pinner, 1935a1 b.) Winn and Petroff {1933) studied dissociated avion bacilli which

    had been accidenta li y exposed to increased temperature. Colonies became chromo-

    genie and were found to have lost their pathogenicity for chickens.

  • - 13-

    A theory upheld by sorne workers (Burrows and Barclay 1 1959; Tarshis,

    1958, 1960, 1962; and Sweany, 1961) is thot antituberculous chemotherapy has

    increased the frequency of isolation of chromogenic mycobacterie. Tarshis has

    produced chromogenic mycobacteria from H37 Rv organisms by culturing them for

    prolonged periods in the presence of either streptomycin or isoniazid. The variants,

    on the basis of cultural, pathological, allergenic and antimicrobial susceptibility

    studies, appeared genetically related to the parent organisms. Other workers have

    reported similar observations {Tirunarayanan et al., 1959). ln a survey, including

    tuberculous patients who had received, or were receiving, streptomycin therapy 1

    Tarshis isolated chromogenic mycobacterie from 19 per cent of 405 patients compared

    to a control series of 203 nontuberculous patients of whom less thon 1 per cent yielded

    chromogens. Runyon {1959b) does not accept the drug theory as a source of chromo-

    gens; however 1 he points out thot because chromogens are drug reslstant1 they are

    favoured by chemotherapy whlch removes other species. This would be a situation

    similar to thot of Candida and drug resistant staphylococci which emerge in large

    numbers following use of broad spectrum antimicrobial agents.

    lt has also been noted thot drug-resistant strains of M. tuberculosis isolated

    from patients receiving para-aminosalicylic acid (PAS) and isoniazid (INH) exhibit

    either a partial or an absolute growth requirement for oleic ac id. ln this respect 1

    Groups 1, Il and Ill of the unclassified mycobacterie resemble drug-resistant strains

    of tubercle bacilli more closely thon normal drug-susceptible strains (Hedgecock, 1958;

    1962).

    Recent studies with mycobacteriophage have proven most interesting, and

  • - 14 -

    speculation concerning the role played by phage is not without support. White

    and Knight (1958} reported thot smooth colonies of mycobacterie could be isolated

    from previously rough strains after exposure to mycobacteriophages. Mankiewicz

    (196la} showed thot a phage immune variant of a strain of chromogenic mycobacterie

    differed from the parent strain in rate of growth, colonial morphology and pigmenta-

    tion, drug sensitivity, Neutral red reaction, animal virulence and in ability to sensi-

    tize guinea pigs to Old Tuberculin. The same yaar (196lb} she was able to demonstrate

    the presence of mycobacteriophages in stool specimens of patients with tuberculous

    and nontuberculous conditions. Up to then mycobacteriophages active on acid-fast

    organisms had been isolated only from ferti 1 ized soil.

    Examining 50 stool specimens from tuberculous patients Mankiewicz found

    10 to contain mycobacteriophage. Most interesting was the finding thot only 1 myco-

    bacteriophage 1 isolated repeatedly from one patient was active on pathogenic myco-

    bacterie. Sputum cultures tested in parallel revealed thot the sputum of the patient

    excreting mycobacteriophage active against pathogenic mycobacteria contained atypi-

    cal chromogenic mycobacteria 1 while the sputum of the 9 patients excreting myco-

    bacteriophage active against saprophytic and anonymous mycobacterie contained

    M. tuberculosis.

    More recent studies reported by Mankiewicz and van Walbeek (1962) re-

    vealed further interesting results. Human tubercle bacilli H37 Rv when infected with

    the mycobacteriophage found to be active against virulent tubercle bacilli, showed

    the emergence of phage-resistant bacteria which differed in colonial morphology

    (smooth, rounded colonies) and which reacted to the photochromogenicity test, i.e.

  • - 15 -

    pigmentation increased when1 after 30 minutes exposure to light, incubation

    was resumed. Upon repeated exposure to bacteriophage, bacterie from the

    smooth colonies underwent further changes involving growth rate, nutritional

    requirements, loss of niacin production, and loss of catalase activity. The lyso-

    genic bacterie did not elicit tuberculin reaction in guinea pigs. Cytological

    changes occurred showing elongated and branching bacillary elements, only the

    intracellular granules of which were acid-fast by Ziehi-Neelsen staining.

    White et al., (1962) also reported additional results which they felt were

    evidence suggesting that lysogeny with certain mycobacteriophages altered colony

    morphology.

    lt is also an interesting fact that chromogenic mycobacterie are frequently

    isolated from patients when they are in a clinically improved phase of their disease.

    At times both chromogenic and typical mycobacterie are found together, although

    it is more common to isolate chromogenic mycobacterie when M. tuberculosis no

    longer can be demonstrated (Huppert et al. 1 1957; Nassau et al. 1 1958; Keltz

    et al. 1 1958; Runyon 1 1959b; Tarshis, 1960; Mankiewicz, 196la; Butler and

    Josephson 1 1963).

    2. CLASSIFICATION OF CHROMOGENIC MYCOBACTERIA

    Since the beginning of bacteriology 1 the ability of certain organisms to

    produce pigment has been utilized as a criterion in the identification of several bac-

    terial species. Although, in most instances, bacterial classification is today based on

    combinatlons of morphology, staining reactions, cultural characterlstics, biochemical

    tests, animal pathogenicity and antigenic analysis, the ability of microorganisms to

  • - 16 -

    produce pigment still remains important in the identification of certain species.

    This is particularly true for this group of slow growing chromogenic mycobacteria

    now incriminated as etiological agents of tuberculous-like disease in man. lndeed,

    it was through their chromogenicity thot they were first recognized and subsequently

    named.

    Following the increase in the rate of isolation of chromogenic mycobac-

    teria from clinicat specimens during the early fifties, Timpe and Runy'on {1954) pro-

    posed a tentative grouping for strains isolated from 120 patients, ali of whom were

    thought, or known, to have pulmonary disease. An extended scheme proposed later

    by Runyon (1959a) has served as the basis of classification until the present time.

    Runyon s groups are described as follows:

    GROUP 1, PHOTOCHROMOGENS: ("Yellow Bacilli" of Buhler and Pollak,

    1953; M. kansasii 1 Hauduroy 1 Subcommittee

    on Mycobacteria, American Society for

    Microbiology 1 1962; M. luclflavum of

    Middlebrook, 1956). Pigmentation: Little

    or none if grown in the dark; bright yellow

    ta orange or brick red if grown in continu-

    ous light. Young actively growing non-

    pigmented colonies will become yellow in

    the dark incubator 6 ta 12 hours after expo-

    sure for 1 hour 1 45 cm. from a 30-Watt lam p.

    Photochromogeniclty is a pronounced and

  • GROUP Il, SCOTOCHROMOGENS:

    - 17-

    rapid change. lt is imperative thot tests

    be made with actively growing cultures

    (5 ta 6 days old).

    Growth rate: Almost as for tubercle

    bacilli, or slightly more rapid at 37C.

    (11 0range Boeil li", of Buhler and Pollak,

    1953). Pigmentation: Yellow or orange

    from the b.eginning of growth in the dark;

    more reddish if grown continuously in light.

    Growth rate: About as for tubercle bacilli

    or a little more rapid at 37C.

    GROUP Ill, NONPHOTOCHROMOGENS: ("Battey" type of Crowetal., 1957).

    GROUP IV, RAPID GROWERS:

    Pigmentotion: Usuall y weak or none; if

    present slowly developing and not as des-

    cribed for Groups 1 and Il.

    Growth rate: As described for Groups 1

    and Il.

    Growth rate: Growth within 48 hours at

    20- 25C from invisible small inocula.

    This Group includes described species of

    Mycobacterium, as M. fortuitum, M. phlei,

    M. smegmatis or Nocardia species.

  • - 18 -

    The current classification for recognized members of the genus Myco-

    bacterium is outlined in Table 1. A description of M. kansasii 1 designated in

    1962 by ~he Subcommittee on Mycobacteria, American Society for Microbiology 1

    as a type species for Group 1 photochromogens appears in Table Il.

  • TABLE 1

    GENUS MYC08ACTERIUM

    (8ergey 1s Manual of Determinative Bacteriology, 7th Edition, 1957)

    SAPROPHYTES PARASITES

    including potentiaJ parasites; grow on warm-blooded animais

    rapldly on most media at 28 C. A. GROWTH ON ORDINARY OR SPECIAL MEDIA

    1. M. phJei 7. M. ulcerons

    2. M. smegmatis 8. M. tuberculosis

    3. M. fortuitum 9. M. bovis '()

    4. M, marinum 10. M. microti

    5. M. thamnopheos 11. M. avium

    6. M. platypoecilus 12. M. paratuberculosis

    B. HAVE NOT BEEN GROWN ON NON-LIVING

    CULTURE MEDIA

    13. M. leprae

    14. M. lepraemurium

  • TABLE Il

    MYCOBACTERIUM KANSAS!!

    (Subcommittee on Mycobacterie, American Society for Microbiology, 1962)

    COMMON NAMES:

    GL YCEROL EGG SLANTS:

    PATHOGENICITY:

    DISTINCTIVE CHARACTER:

    TYPE CUL TURE:

    PHOTOCHROMOGEN; 11YELLOW BACILLUS11

    AFTER TWO WEEKS IN DARK INCUBATOR, RAISED WITH IRREGULAR

    SURFACE AND MARGJNS, IVORY, OR OFF-WHITE. IF GROWN IN

    LIGHTED INCUBATOR, LEMON-YELLOW BECOMING ORANGE OR

    EVEN RED-ORANGE WITH AGE.

    IN MAN IT PRODUCES PULMONARY AND EXTRAPULMONARY DISEASE

    VERY SIMILAR TO TUBERCULOSIS. VERY SLJGHT OR NO PATHOGEN-

    ICITY FOR GUINEA PIGS, RABBITS, AND FOWL.

    DEFINITE YELLOW PIGMENTATION WITHIN 24 HOURS AFTER 1 HOUR

    EXPOSURE OF AN ACTIVELY GROWING CULTURE TO BRIGHT LIGHT.

    AMERICAN TYPE CULTURE COLLECTION STRAIN 12478 (Bostrom).

    1

    N 0

  • - 21 -

    3. METHODS PROPOSED FOR THE DJFFERENTIATION OF CHROMOGENIC

    MYCOBACTERIA FROM M. TUBERCULOSIS, AND FOR THE DETECTION

    OF SPECIFIC VARIETIES AND SUB-GROUPS

    A. Animal viru1ence studies

    Very few of the many strains of acid-fast bacilli which occur in nature

    are capable of causing progressive disease in man or animais. Furthermore 1

    virulent forms exhibit a well-marked degree of specificity in host range (Dubos,

    1948). This characteristic is utilized in the current clssification of human,

    bovine and avion strains (Bergey, 1957}.

    The most commonly employed animais for virulence testing of myco-

    bacterie include guinee pigs 1 rabbits, chickens, hamsters and mice. Untll the

    early nineteen fifties much rel iance was placed on the close correlation in

    pathogenlcity of mycobacterie for guinee pigs and man. This no longer con be

    maintained as many INH-resistant strains of tubercle bactlli, as weil as numerous

    unclassified strains of mycobacterie have since been found to possess either very

    low or undetectable levels of virulence for the guinee pig.

    Animal virulence studies, as related to the unclassified mycobacterie,

    have been of little value either in differentlating types within the group, or in

    evaluating the pathogenicity of such strains for man. Runyon (1959a), reported

    thot unclassified mycobacterie 1 in general 1 foi led to produce progressive disease

    in guinee pigs. Photochromogens usually cause disease in mice if 1/100 mg. is

    inoculated intravenously, or 3 mg. lntraperitoneally. Scotochromogens are non-

    pathogenic for laboratory animais, and Group Ill nonphotochromogens ("Battey")

  • -22-

    are variable in pathogenicity for animais, sorne strains resembling Group 1

    by showing limited pathogenicity for mice, and others lacking pathogenicity

    for ali laboratory animais.

    Hardy et al. 1 (1958) performed virulence studtes on 73 atypical strains

    of human origln. Not one produced generalized or progressive disease in gulnea

    pigs. They fou nd ali 4 photochromogens tested to be pathogenlc for mi ce 1 as

    were 25 of 47 nonphotochromogens tested. None of the 5 scotochromogens

    studied infected mi ce. ln contrast 1 others found scotochromogenlc mycobacterie

    to be virulent for mice, possessing a special affinity for the kidney and forming

    epithelioid tubercles 8 weeks after inoculation (Matsumoto et al. 1 1963). Sorne

    workers feel thot differences in results of animal virulence studies for unclassified

    mycobacterie are due to too short a period of observation.. Characteristic lesions

    have been observed to develop ln guinea pigs and mice thot have been retained

    under observation for longer periods of time (Xalabarder, 1961; Kertay et al.,

    1962). Chromogenic mycobacterie exhibit limited pathogenicity for the rhesus

    monkey (Schmidt 1 1957). 1 t has recent! y been shown thot dogs may be come in-

    fected with photochromogenlc mycobacteria (M. kansasit) but the infection is not

    serious or progressive (Leon et al., 1964).

    B. ~ntigenic analysls

    Cross-sensitization experiments indicated an immunological relationship

    between hu man tuber cie bac i Il i and the chromogens (Tarsh is and Frisch 1 l952c;

    Nassau and Hamilton 1 1957). lt appears thot they not only shore antigenic pro-

    parties but thot they also possess specifie components. Using a hemolytic modifi-

  • - 23-

    cation of the Middlebrook-Dubos hemagglutination test it was shown thot sera

    from tuberculous patients reacted to antigens prepared from atypical 11yellow11

    mycobacterie, and sera from patients with atypical mycobacterie! disease reacted

    with human Old Tubercul ln antigens (Nassau et al. 1 1958). Sera from guinea pigs

    infected with human strains usually react to Old Tuberculin antigen only 1 whereas

    sera of animais infected with strains of the yellow bocillus type cross react with

    the Old Tuberculin antigen. Absorption of these sera with heat-killed H37 Rv and

    "yellow" bocilli gives a typical absorption pattern. The H37 Rv removes antlbodies

    leaving the "yellow titer unchanged, but the 11yellow'1 bocilli remove both human

    and 11yellow11 antibodies.

    Mankiewicz {1958) using two serological techniques (agar diffusion

    precipitation and complement fixation) attempted to classlfy chromogenic acid-fast

    bacilli, and found both methods revealed evidence of overlapplng of antlgens be-

    tween chromogens, and both typical tubercle boeil li and saprophytic mycobacterla.

    This was also the finding of Beek (1959) using culture filtrates of acid-fast bacilli.

    He showed cross reaction between the unclassified saprophytes and tubercle baclll i

    when performing skln sensitivity tests ln gulnea pigs. There was serologlcal speci-

    ficity to sorne degree on a quantitative basis in thot animais infected with unclassi-

    fied mycobacterie, or wlth tubercle bacilli, showed strongest reactions wlth homolo-

    gous PPD extract (Beek, 1960). The difference ln strength of the reactions wlth

    homologous and htHerologous was not marked and consistent enough to distinguish

    between various mycobacterie (Beek, 1961i Affrontl 1 1959).

  • -24-

    ln a study uslng mammolian tuberculin (PPD-S) and nonphotochromogen

    "Battey'' tuberculin (PPD-B} it was found thot patients infected with tubercle bacilll

    had definite sfrong reactions to PPD-S and weaker or no reactions to PPD-B. Patients

    infected wlth 11Battey11 organisms had lorger reactions to PPD-B thon to PPD-S. Com-

    parative testlng provided a way to separate tubercul in sensitivity produced by the

    11Battey11 organism from thot produced by tubercle bac ill i. Using PPD-Y (yellow

    bacilli) and PPD-S the sensitivity produced by the two organisms was too similar to

    be distingulshed by comparative testing. An interesting epidemiologlcal finding was

    made using PPD-S and PPD-B. lt was observed thot two-thirds of the men recruited

    for novy services from the states of Georgia and Florida were reactors to PPD antigen

    prepared from the 11 Battey11 mycobacterlum while only about 6 per cent were considered

    reactors to PPD-S (Edwards and Palmer r 1958}.

    The study of Magnusson et al. {1961) suggests antigenic differences among

    sfrains isolated in different areas of the world. 11Sensitlns11 prepared from lndian orange-

    pigmented stroins were similar to 11sensitins 11 prepared from Danish orange-pigmented

    sfrains, but differed from those produced from oronge-pigmented African strains.

    lt is proven thot a significant number of non-specifie tuberculin reactions

    results from Infections with unclassified mycobacteria (Kendlg, 1962, 1963; Flynn,

    1962; Smith and Johnston, 1963).

    The amount of protection or immunity conferred upon animais by exposure

    to unclassified mycobacterie is under active study. Guinea pigs and mi ce vaccinated

    wlth unclassified mycobacteria have been observed to possess sorne degree of immunity

    (Wenkle et al. r 1948; Youmans et al. r 1961; Klugh and Pratt r 1962; Larson and

    Wicht, 1963). By means of protection tests in mlce, it has been shawn thot immuni-

  • -25-

    zation with atypical mycobacteria representing Runyon 1s Groups 1, Il and Ill gave

    partial protection against infection with M. tuberculosis. A photochromogenic

    strain equalled or surpassed BCG in protective power. Vaccination of mi ce with

    BCG conferred a significant degree of protection against pathogenic photochrome-

    genie strains (Siebenmann 1 1964). ln other studies the degree of immunity to

    M. tuberculosis H37 Rv induced by a photochromogen in guinee pigs and mlce was

    as good as thot produced by BCG (Satake 1 1963).

    lmmunological cross reactions between mycobacterial organisms may

    eventually be eliminated with purer antigens. Kniker (1961) ln a study of H37 Rv

    as weil as representatives of ali groups of unclassified mycobacterie, employed ion-

    exchange chromatography and was able to demonstrate thot while sorne antigens were

    common to ali or most organisms, others possessed specificlty for lndividual organisms.

    A large number of strains have been studied by agglutination and antibody

    absorption techniques. This approach may have greater application in epidemiologi-

    cal studies thon in cl inical diagnostic laboratorles (Schaefer and Reggiardo, 1963;

    Salto et al., 1964). Using agar diffusion methods Chapman {1961) obtained a sero-

    logical reaction between sera of patients with sarcoidosis and antigens from photochro-

    mogenlc mycobacterie.

    C. Resistance to drugs and other agents

    Primary drug resistance is a characteristic of the unclassified mycobacterie.

    The level of resistance naturally varies with strainsi however, broadly speaking the

    photochromogens 1 scotochromogens and nonphotochromogens show parti a 1 resistance

    to 1 mcg. per ml. streptomycin 1 10 mcg. per ml. PAS and 1 mcg. per ml. INH

    (Runyon, 1959a). Much higher levels of resistance are frequently reported for these

  • - 26 -

    organisms especially when they are isolated from treated cases. Prissick and

    Masson (1957) found sorne strains to be inhibited by PAS only in 1000 mcg. con-

    centration. Tarshis et al. , (1955) testing a series of chromogens, observed thot

    INH failed to inhibit the growth in concentrations up to 100 mcg. per ml. Chro-

    mogens have also been found to grow in medium containing 750 mcg. per ml. of

    streptomycin (Butler and Josephson, 1963). Results of susceptibility tests performed

    on these organisms with the lesser used antituberculous drugs are too scarce and

    variable to provide useful information. Resistance to various agents at specifie

    concentrations has been suggested for the separation of mycobacteria, but division

    on this basis has not been too practical (Gastambide-Odier and S~ith, 1 958; Collins,

    1962; Eidus et al., 1959, 1960; Hedgecock and Faucher, 1961; Tsukamura, 1962;

    Jonesand Kubica,1963, 1965; Minsley,1964).

    D. Bacteriophage typing

    The application of bacteriophages for the classification of mycobacteria

    was suggested in 1956. Hnatko (1956) phage typed 34 classified a cid-fast micro-

    organisms and compared them with 33 unclassified mycobacteria which had been

    isolated from tuberculous patients. Seventy~three per cent of the unclassified organ-

    isms were lysed by one or more of the 9 phages used.

    Mankiewicz (1961b) also studied the phage susceptibil ity of chromogenic

    acld-fast bacteria isolated from patients with tuberculosis-1 ike disease and found

    thot 22 of 98 were affected by exposure to one or more of the six phages used. Other

    workers have since isolated mycobacteriophages from stool specimens of patients with

    pulmonary disease (Coter and Redmond, 1963).

  • - 27-

    lt should be mentioned thot non-specifie clearing occurs when heavy

    concentrations of phage are employed in the typing of mycobacterie. This find-

    ing decreases the value of many results reported from earlier studies in which typing

    was performed wh ile uslng heavy suspensions of phage. The routine test dilution

    method and lts modifications give promise of more reliable results (Tokunaga et al. 1

    1961; T okunaga and Murohash i 1 1961 1 1963; Redmond 1 1963a, b; Red mo nd et a 1. ,

    1963). lt would appear 1 however 1 thot ali the technical problems are not yet solved

    and more remains to be known before rel labie methods are avallable to glve repro-

    ducible results thus permitting the phage typing of mycobacterie on a basis simllar

    to thot employed with other groups of b'Jcteria (White et al., 1963; Tokunaga et al.,

    1964; Manion and Bradley1 1964; Redmond 1 1964).

    E. Growth at various temperatures

    Information of 1 imited value can be obtained by preparing subcultures of

    mycobacterial isolates and incubating them at 45C. 1 22 - 25C. 1 and 3~C.

    Mammalien tubercle bacilli, both human and bovine, grow slowly and only at 37C.

    The avion tubercle bacilli grow slowly at ali three temperatures, while the described

    species of saprophytic mycobacterie (M .. phlei 1 M. smegmotis) grow rapidly at ali

    three temperatures. Most of the unclassified mycobacterie grow slowly at 37C. 1

    and at room temperature, but variable results are obtained at 45 C. 1 (American

    Thora ci c Society 1 1961).

    F. 11 Cord 11 factor and neutra! red test

    Both the ability to form ''cords11 in fluid medium and the ability to bind

    neutra! red in alkal ine buffer solution have been associated with virulence in tubercle

    bac ill i (Middle brook et a 1. 1 1947; Dubos and Middlebrook, 1948). Unclassified

  • -28-

    mycobocterio give variable results in both tests (Huppert et al. 1 1957; Wayne

    et al. 1 1 957; Hardy et al., 1958i Torshis, 1960b; Xoloborder 1 1961; Butler and

    Josephson, 1963; Korlson et al., 1964).

    G. Lipid content

    Specifie lipids hove been extrocted from mycobocterio and it is possible

    thot this may serve to differentiote species and strains. Each strain possesses severa!

    1 ipid fractions 1 and each fraction has on infrared spectrum sufficlently characteristic

    ta differentiate lt from other fractions. Using this principle H37 Rv and H37 Ra

    strains of M. tuberculosis can be distinguished from eoch other. Bovine and human

    stralns have a Iso been spearated. Extending the method to the unclassified myco-

    bocterio and using a combination of column chromotography and infrored spectroscopy

    it hos been possible to extra ct a specifie glycol ipid from ali 17 photochromogens

    exomined. The fraction hos been identified and hos been found to be entirely inde-

    pendent of pigment formation and photooctivotion {Randell et al., 1951, 1952; Smith

    etal., 1954,1957,1960a,b). ln 1964approximately 150culturesofmycobacterla

    were exomined for their lipid content. Basically the results indicated thot among the

    20 to 30 substances recognized in the lipids of a given culture, ali but one or at most

    two were shared between that culture and most other cultures of mycobacterie regard-

    Jess of species. The substance thot is limited in distribution would appear to permit

    the recognition of the species or sub-group of mycobacterie to whlch the culture

    belongs (Randell and Smith, 1964). lt has a Iso been demonstroted that relative af-

    finities of acid-fast bocillt for the fat solvents moy have taxonomie importance. Par-

    tition offinity for a given strain of bacillus was found reproducible and characteristic

    (Wayne and Jaurez, 1955).

  • -29-

    H Tissue cu 1 ture

    ln Hela cells photochromogenic and 11 Battey11 stroins hove been

    shown to grow intracellulorly in charocterlstic patterns, the most prominent feature

    was a distinct beading which occupied the entire length of the orgonisms {ShepordJ'

    1958). Bronching filaments were also observed.

    Morigi (1959) studied phagocytosis of atypical mycobacterie belonging

    to the chromogenic group. He found thot the chromogens did not show invasive

    capacity comparable to thot of virulent tubercle bacflli 1 and their multiplication

    in the Hela cells did not produce cytopathological changes. Scotochromogens

    were phagocytosed at a faster rote thon photochromogens. From his studies on

    scotochromogens and photochromogens it appeared evident thot intracellular multi-

    plication of the scotochromogen proceeded more rapidly and thot they showed a

    more active invasion. This is contrary to clinical and experimental findings which

    indicate thot scotochromogens are genera li y nonpathogenlc. Others (Brosbe et al.,

    1962) 1 a Iso using Hela ce lis 1 studied avion and "Battey11 mycobacterie and found

    a difference in growth rate, but concluded thot the difference observed did not

    permit differentiation of species. Bronching filaments were observed with variable

    frequency in ali of the stroins studied, the bronching occurred at right angles and

    was seen more often in 11Battey 11 strains. Withfn three to five days, tissue cultures

    showed numerous intracellulor organfsms for ali of 9 11 Batteyu and 4 avium stroins

    tested.

    1. Morphology and stoining

    The morphology of chromogenic mycobacterio studied by Tarshis and

    Frisch (1952a), voried considerably depending upon the medium used and the age of

  • -30-

    the cu 1 ture.

    ln general, most reports agree with Runyon (1959a) in which he

    described his Groups as follows:

    Photochromogens:

    Scotochromogens:

    Nonphotochromogens:

    Average size larger thon tubercle-

    bacill i 1 often qui te long, banded and

    beaded; strongl y a cid -fast.

    Variable in size 1 strongly acid-fast.

    Highly pleomorphic, but often very

    short, characteristically containing a

    single hyperchromie granule.

    Kappler and Janowiec {1963) are of the opinion thot photochromogens

    are not strongly acid-fast. This has a Iso been the experience of the author (un-

    published data). Others reported thot photochromogens were easily differentiated

    from other mycobacteria when stained by the Ziehi-Neelsen method, stating thot

    skilled observers would seldom miss them in direct smears or histological sections

    (Nassau and Hamilton, 1957).

    Periodic acid-Schiff stain has been used to differentiate photochromo-

    genic and scotochromogenic mycobacterla from other varieties (Csillag, 1960).

    The validity of attempts to dlfferentiate photochromogens on a morpho-

    logical basis from other mycobacteria is weakened somewhat by the finding of Gale

    (1961) who showed a dramatic difference in the morphological appearance of these

    organisms before and after exposure to 1 ight. Prior to exposure to light these or-

  • - 31 -

    ganisms were typical of tubercle boeil li in appearance, but following exposure to

    light and subsequent pigment formation the organisms were twice as long, 4- 6

    times as wide, and contained more thon three times as many granules. Similar

    observations are reported by the author in this thesis.

    J. Eh potentiel

    Earl y attempts to separate human, bovine and BCG strains by measurlng

    their Eh potentiel in buffer solutions showed thot there was no distinct difference

    in types (Aksianzew 1 1933; Wilson et al., 1952). Likewise nothing conclusive

    was derived from studies involving methylene-blue reduction (Bloch, 1950a 1 bi

    Aksianzew 1 1933; Desbordes and Fournier, 1950).

    K. Adansonian classification {electronic computer)

    Bojal il et al. 1 (1962) using the electronic computer method of Sneath

    (1957a 1 b, 1958), studied 229 unclassffied strains of mycobacteria and classified

    them into 12 different categories on the basis of physiological properties. Their

    study revealed the existence of intermediates between categories (or branches as

    they cal led them) which formed a continuous metabol ic spectrum and made difficult

    the separation of related categories.

    L. Biochemical tests

    a) Introduction

    For the classification of microorgcmisms, biochemical and metabolic

    differences ore naturally preferred to those of cultural morphology and animal

    pathogenicity. The slow growth rate of mycobacterie, however, presents technicol

    difficulties along these lines.

  • - 32 -

    ln severa! tests thot have been suggested for the detection of bio-

    chemical and metabolic differences in members of the mycobacterie, the differ-

    ences observed have been quantitative rather th an qualitative in nature. Su ch

    tests have limited value in the recognition of specifie types of organisms because

    resu 1 ts depend upon the meta bol ic state of the organism und er test, as weil as the

    degree of sensitivity and specificity of the test employed. Thus far few useful

    quai itative differences for strain recognition have been observed among the myco-

    bacterie. Certain tests which distinguish only between rapidly growing saprophytes

    and M. tuberculosis are not of much practlcal value.

    Biochemical tests which have served to help in dlfferentiating M. tuber-

    culosis from chromogenic mycobacterie, and to detect specifie varieties or sub-groups

    of the unclassified organisms are listed below under appropriate headings.

    b) Niaci n test

    One of the more useful tests available for the differentiation of M. tuber-

    culosis from other members of the mycobacterie is thot of niacin testing, based on the

    principle thot tubercle bacill i con synthesize nicotinic acid from certain ami no ac ids

    (e.g. asparagine and glutamic acld) if they are the only source of nitrogen in the

    medium. Konno (1953) reported on a marked quantitative difference in the niacin

    production of human type tuber cie bacill i and other mycobacterie when grown in a

    synthetic culture medium. These workers (Konno, 1956i Konno et al., 1957, 1958a,b)

    introduced a test designed to differentiate human from ali other mycobacterie. Bovine

    avion, nonpathogenic and unclassified acld-fast badlli were reported to be negative

    while the human type gave positive results irrespective of INH susceptibility, cota-

  • - 33-

    Jase content, or pathogenicity for the guinea pig. Rare exceptions have been

    reported but it is genera fly accepted that only M. tuberculosis var. hominis pro-

    duces enough niacin to give a positive reaction by the Konno test or its modifica-

    tions. ft has recently been reported thot a culture of M. fortuitum was isolated

    which gave a positive reaction for nlacin (Karl son et al., 1964). As this organism

    grows rapidly it is not llkely to be confused with M. tuberculosis. The niacin test

    may be considered as reliable as any other test currently available for differentiating

    human tubercle bacilli from ali other mycobacteria {Konno and Sbarra, 1959; Konno,

    1960).

    c) Catalase activity

    Unclassified mycobacterla as a group show strong catalase activity

    {Runyon, 1959a). ln this respect they differ from strains of INH resistant M. tuber-

    culosls which generally show decreased catalase activity (Middlebrook, 1954). lt

    is known tht~t catalase of human and bovine stralns, regardless of their virulence,

    is inactivated by suspending cultures in a phosphate buffer of pH 7 at 68C for 20

    minutes. Unclassified mycobacterie retain their catalase activity under these condi-

    tions (Kubica and Pool, 1960). A correlation appears to exist between catalase

    activity and animal virulence (Peizer et al. 1 1960). A study of 46 strains of tubercle

    bacill i from INH treated patients showed that the organisms could be grouped as

    follows:

    1. Those of hlgh catalase activity which were virulent for

    guinea pigs;

    2. Those of moderate catalase activity which were or were not

    virulent for guinea pigs;

  • -34-

    3. Those of low catalase activity which were usually non-

    virulent for guinea pigs.

    Thot catalase activity is in sorne way related to virulence was further supported

    by the finding thot the degree of catalase activity in photochromogens paralleled

    the clinical significance of the strains in the patients from whom they were isolated

    (Wayne 1 1962).

    d) Arylsulfatase activity

    Early studies on arylsulfatase activity of mycobacterie revealed variable

    quantitative differences for most members including unclassified varieties (Whitehead

    et al. 1 1953; Engbaek 1 1954; Wayne et al., 1958) 1 but recent reports indicate

    thot this test under control led combination of substrate-concentration and bacterial

    inoculum may be employed in the differentiation of mammal ian and avion tubercle

    bacill i from ali ether mycobacterla. The arylsulfatase test promises to provide a

    method of dlstinguishing M. avium from Group Ill nonphotochromogens (nBattey11).

    Avion and "Battey'' mycobacteria have been indistinguishable except on the basis

    of pathogenicity of the former for the chicken (Kubica and Vesta!, 1961i Kubica

    and Bearn, 1961). Arylsulfatase testlng has also been used to separate M. fortuitum

    from ether rapidly growing saprophytes (Kubica and Rigdon, 1961; Wayne,1961).

    M. fortuitum is the only "rapid grower11 which is capable of causing infection in man.

    e) Hydrolysis of Tween uao"

    lt was recently noted that certain mycobacteria when grown in media

    containing 11Tween 80 11 produced a turbidity out of proportion to the actual number

    of viable ce lis present {Wayne, 1962). Sorne organisms could attack "Tween 8011 and

  • - 35-

    form opalescent degradation products. Using this principle as the basis of a test 1

    M. tuberculosis and M. bovis con be differentiated from Group 1 photochromogens

    (M. kansasii). Photochromogens have the abllity to attack 11 Tween 8011 whereas the

    tubercle bacilli and M. bovis as weil as ali Group Ill nonphotochromogens which

    produce disease in man, birds or other animais give negative results. Group Il

    strains are variable. This principle has been used by Wayne et al., (1964) in a

    scheme for the classification and identification of mycobacteria.

    f) Growth in the presence of thioglycollate

    A useful observation made by Tarshis and Frisch (1952a) (also Tarshis,

    1959, 1960b; Potuznik and Matejka, 1962) was thot chromogens would grow slowly

    in fluid thioglycollate-containing medium. The saprophytes grew rapidly and luxur-

    iantly within 24-48 hours forming thick wrinkled pellicles. M. tuberculosis was

    unable to multiply in such a medium. A modified sol id thioglycollate medium con-

    taining methylene blue has been used to separate Group 1 and Il organisms from those

    of Group Ill, the latter failing to grow on the modified medium. (Smith and Steenken,

    1961.)

    M. Miscellaneous tests

    ln attempts to fi nd rel iable criteria for the recognition and identification

    of slow growing mycobacterie a great variety of tests have been proposed. Conven-

    tional differentiai ut il ization of carbohydrates 1 al though suggested from ti me to ti me 1

    has not proven useful. Recently Sweeny and Jann (1961, 1962) developed a new

    technique which may be of possible value. They use a massive inoculum in various

    carbohydrates and polyhydric alcohols superimposed on a solid basal medium contain-

  • - 36 -

    ing phenol red indicator. Without multiplication of the organisms specifie metabo-

    1 ic activities can be detected. The au thors have used the term "non-growth" test-

    ing for this procedure. The organism being tested must first be grown in a rich

    medium and harvested in its logarithmic phase of growth to provide cells of high

    meta bol ic activity.

    Differentiation based on carbon utillzation has been investigated by

    sorne workers (Karlson and Ulrich, 1962i Cerbon and Trujillo, 1963) without leading

    to practical results. Severa] workers have had partial success in differentiating

    groups, species and types of mycobacteria on the basis of amidase activity (Juhlin,

    1960i Bonicke, 1960i Halpern and Grossowicz 1 1957i Cerbon and Trujillo, 1963i

    Satake, 1963). On the basls of ribonucleic acid-desoxyribonucleic acld ratio,

    mycobacteria can be divided lnto two sub-groups (Tsukamura 1 1960).

    A test designed to differentiate M. tuberculosis from ali other mycobac-

    teria was recently described by Karlson et al., (1964). lt is based upon the appear-

    ance of growth within a modified Proskauer and Beek liquid medium. Their test, in

    principle 1 is sim il ar to a procedure developed by the candidate in the present research

    (Butler and Josephson 1 ] 963).

    An indirect approach has been used by McCuiston and Hudglns (1960).

    They compared the electrophoretic patterns of sera from patients with sarcoidosis,

    tuberculosis and disease due to unclassified mycobacteria. The sera from patients

    with sarcoidosis and infection caused by unclassified mycobacteria showed certain

    similarities whereas those from patients with tuberculosis gave dissimilar patterns.

    The authors studied the sera of seventy-four cases suffering from infections caused by

  • - 37-

    unclassified mycobacterie and in ali instances the changes observed were more

    like those in sarcoidosis than tuberculosis. Electrophoresis showed a decrease in

    serum albumin and an increase in gamma globulin without an increase in alpha-2

    globulin.

    N. Characterization of pigments in mycobacteria

    a) M. tuberculosis

    Anderson and Newman (1933a, b) isolated a yellow crystalline pigment

    from saponified acetone-soluble fat of M. tuberculosis var. hominis. They identified

    the pigment as a naphthoquinone and named it phthiocol.

    Cold acetone extracts of M. tuberculosis var. avium cultivated on

    synthetic media containing trace element supplements are often heavily pigmented

    (Patterson 1 1960). The pigment involved was isolated and identified as copropor-

    phyrin Ill.

    M. tuberculosis BCG 1 sensitive to 1 NH 1 produces pigment when exposed

    to the drug whereas a resistant strain of the organism &)es not (Youatt 1 1962).

    Pterin-1 ike pigments were isolated from tubercle bacill i by Crow and

    Walker (1949).

    b) Saprophytes- (M. ph1ei 1 M. smeg:natis, M. locticoto, etc.)

    Chargaff (1930, 1933) isolated beta and gamma carotenes from M. phlei

    by separa ting fractions on columns of al uminum oxide. The same pigments were found

    in the ac id-fast organisms 1 Bacillus lombardo pellegrini and Baclllus grassberger,

    however the latter also contained lycopene (Chargaff and Lederer, 1935).

    From M. phlei lngraham and Steenbock (1935) isolated alpha and beta

    carotenes 1 cryptoxanthin, esters of lutein, zeaxanthin and azafrin. They also reported

  • - 38 -

    a fraction resembling the naphthoquinone phthiocol. The ratio of the various

    pigments end their absolute amounts varied with age and cultural conditions.

    Leprotin 1 a carotenoid hydrocarbon similar to beta carotene was found

    by Grundmann and Takeda (1937) in an acid-fast organism isolated from a leprous

    lesion. This organism was most likely a saprophyte. Takeda and Ohta {1939 1 1940 1

    1944) found leprotin in M. phlei. These workers questioned the flndings of Chargaff 1

    1 ngraham and Steenbock and they fel t that what had been reported as be ta carotene

    was really leprotin.

    M. lacticola (M. smegmatis) when grown on mineral oil media produced

    the acidic carotenoid astacin as weil as carotene, but no xanthophylls (Haas and

    Bushnell 1 1944). The same organism grown on nutrient agar produced carotenes,

    xanthophylls, but not asta ci n.

    Turion (1950b) described a yellow pigment 1 acid in character 1 produced

    by M. phlei. lt differed slightly from astacin and he proposed the name chrysoflein.

    La ter (1951 1 1953) T uri an reported be ta caro te ne 1 xanthophyll 1 leprotene 1 gamma

    carotene 1 a 11rhodopin-like11 lycopenoid, an oxycarotenoid, phytofluene and chryso-

    flein. The pigments differed quantitatively depending on whether growth occurred

    at 30C or 3r>C. Uslng diphenylamine to lnhibit carotenoid biosynthesis in M. phlei 1

    Turion and Haxo (1952) isolated two fractions not detectable in uninhibited normal

    bacterie. One fraction was a visible yellow and the second was colorless 1 but

    possessed a greenish-grey fi uorescence.

    Fisher et al. 1 (1955) found leprotene and beta carotene as epiphasic frac-

    tions when p:~rtitioning extracted pigments of M. phlei between petroleum ether and

  • - 39-

    90 per cent methanol. A hypophasic red pigment was also observed but not iden-

    tified. Goodwin and Jamikorn (1956) investigated three strains of M. phlei and

    found thot they ali produced the sorne carotenoids in the sorne relative amounts.

    lncluded were beta carotene, zeta caratene, leprotene, lycopene and two uniden-

    tified xanthophylls. The xanthophylls were pigments earl ier considered to be cryp-

    toxanthin and zeaxanthin. These workers were of the opinion thot Turian's chryro-

    flein was a mixture of these two unidentified fractions.

    Also working with M. ph lei Schlegel (1959) found phytoene, phyto-

    fluene, beta carotene,zeta carotene,gamma carotene, neurosporene, lycopene and

    myxoxanthophyll. Quantitatively the oxycarotenoid myxoxanthophyll accounted for

    approximately 95 per cent of the total carotenoids.

    A group of mycobacterie isolated from the soil and presumed to be sap-

    rophytes, was found to forma red product from PAS (Tsukamura, 1961). The colored

    matter was not identified. lt could be produced if ce lis of mycobacteria were in-

    cubated at 37 C for 12 hours in a phosphate buffer sol ut ion contai ni ng 2 mg. of PAS

    per ml.

    Rilling (1964) extracted and identified phytoene and phytofluene from

    M. lacticola. Other fractions were obtained which were not identified but which

    were similar to. zeta carotene, be ta carotene and leprotene. A hypophasi c fraction

    was observed but not identified.

    c) Unclassified slow growing pathogenic mycobacterio

    Pigments of a photochromogenic mycobacterium (Runyon P-8) and a scoto-

    chromogenic mycobacterium (Runyon P-6) were investigated by Ebina et al. (1962).

  • -40-

    By chromotography on an alumina column these workers observed four colored

    fractions. Near the top of the column was a very narrow brown band. A very

    narrow red '::~and was immediately below. These were not identified. A broad

    orange band occurred midway down the column, and just below it was a narrow

    yellow band. These were identified as beta and alpha carotene respectively.

    lt was reported thot exposure of nonpigmented photochromogenic mycobacterie

    to light resulted in an increase in the beta carotene content. The appearance

    of the chromatogram for P-6 (scotochromogen) was similar to thot for the "light11

    culture of P-8. By ultraviolet Illumination five fluorescent bands were observed

    but only one was investigated. lt exhiblted the absorption maxima at 348 and

    368 mu in petroleum ether and was considered to be phytofluene. Since this

    band was broader in the 11dark11 culture of P-8 thon in the "light11 culture, the

    workers proposed it as a precursor for beta carotene.

    Tsukamura (1962) studied two photochromogenic mycobacterie.

    From one strain he isolated beta carotene and lycopene. Only beta carotene

    was isolated from the second strain.

    Costello et al., (1962) working with representatives of Runyon s

    Groups 1, Il and Ill isolated a fraction for wh i ch the absorption spectrum was

    a bell shaped curve with a single broad peak at 452 mu in petroleum ether and

    462 mu in ethanol. This was similar to Turian s chrysoflein. A second fraction

    was considered to be leprotin. Costello (persona) communication, 1963) felt thot

    Group 1 and Il organisms contained essentially similar pigments, however they only

    studied two strains of each Group. Studies on three Group Ill strains revealed thot

    one developed pigments similar to those of Group 1 organisms, but two Group Ill

    strains were markedly different.

  • - 41 -

    4. FORMATION OF PIGMENT IN MYCOBACTERIA

    A. Introduction

    Previous observations reported in the literature concerning pigment

    formation in mycobacterie relate to a variety of species and strains. Just how

    applicable earlier studies are, in terms of the pathogenic chromogens being

    encountered today, is unknown. lt is unfortunate thot practically ali detailed

    chromogenicity and pigment studies on mycobacterie, including those involving

    pigment extraction and characterization, have been performed on saprophytes.

    Pathogens do not lend themselves to the extensive manipulations necessary for

    detailed analysis. Furthermore, the association of chromogenic mycobacterie

    with human disease has only recently been establ ished.

    The factors which have in the post been observed to influence pigment

    production in various acid-fast bacilli are reviewed below under appropriate

    headings.

    B. Oxygen tension

    Schwabacher in 1933, when studylng saprophytic chromogenic acid-fast

    bacilll concluded thot the most important factor influencing pigment production

    appeared to be the oxygen supply. ln tightly sealed tubes little or no pigment

    was produced even after incubation for severa! weeks, while in llghtly corked

    tubes pigment formation was often weil marked within a few days. Petroff and

    Steenken (1935) two years later cultured M. phlei in two series of flasks, one

    series plugged with cotton and capped with perforated rubber nipples, and the

    second series tightly sealed with melted paraffin. Growth in the sealed tubes

    was much smaller and less chromogenic, and they concluded thot chromogenicity

  • -42-

    of M. Ehlei was an unstable characteristic influenced by several factors including

    that of available oxygen supply. The following year Reid {1936- 1937) investi-

    gated seventy-six strains representing twenty-four genera of chromogenic bacterie.

    His collection included M. Ehlei and M. butyricum. No pigment was produced

    when bacterie were grown under low oxygen tension. Abundant growth occurred

    in freshly inoculated tubes sealed with special tube closures, but lack of oxygen

    was reported to have prevented pigmentation. Similarly Baker (1938), using con-

    trol led experiments 1 studied pigment formation in a mycobacterium pathogenic for

    small tropical killifish. He likewise found thot pigment would not form in the

    absence of molecular oxygen. The same has recently been reported for the sapro-

    phyte M. lacticola (Rilling, 1962) and it has been postulated within recent months

    that a compound capable o inducing a carotenogenic enzyme is formed in the same

    organism as a result of exposure to light and oxygen (Rilling 1 1964). At the same

    time, and following release of results in this present study (Butler 1 1964) 1 Wayne

    and Doubek (1964) reported on the role of air on the photochromogenic behavior

    of M. kansasii.

    M. tuberculosis BCG has been found to produce pigment when exposed

    to isoniazid in the presence of oxygen. This may, however 1 be a straight chemical

    reaction since it was not determined whether the pigments were derived from the iso-

    niazid, or whether they were a product of disordered cell metobolism. Indirect evi-

    dence favoured the view that the pigments were derived from the lls (Youatt,1961).

    C. TemEerature of incubation

    As in other bacterial genera, the effect of temperature on pigment forma-

    tion has been examined in mycobacterie. Petroff and Steenken (1935) reported

  • -43-

    M. phlei deepened in pigment content when left at room temperature. Turion

    (1953) has shown quai itative differences in pigment formation by M. ph lei when

    grown in glycerol broth at 30C and 3~C. Decrease in pigment production with

    increase in temperature was recorded by Reid (1936 - 1937) and a Iso Darzins(l939),

    while Baker {1938) found the opposite to be true. He observed greoter chromo-

    genicity at 3~C1 a temperature at which the strain studied did not grow. Still

    another pecul iar flnding was thot of Winn and Petroff {1933). They accidently

    subjected a culture of M. avlum to a high temperature and obtained a dissociated

    form having ali the properties of the original, except it became ochre in color and

    lost its pathogenlcity. Buhler and Pollak (1953) when first describing their "Yellow

    Bacillus", stated thot cultures grown at a temperature of 3~C. could Jose chromo-

    genicity and be cream colored. Their "Orange Baclllus11 retained its deep orange

    irrespectlve of the temperature of incubation.

    D. pH of medium

    There is little agreement in the observations recorded concerning the

    effect of pH o'1 chromogenesis in mycobacterial species. One study indicated thot

    carotenogenesis in a saprophyte, (M. lacticola) was favoured by an alkaline pH

    over a very broad maximum, a pH of 8 being considered satisfactory (Rilling, 1962).

    ln another study 1 pigment production of M. tubereolosis BCG was diminished at pH 6

    compared with thot obtained at pH 7 (Youatt, 1961). Steenken {1935), in contrast,

    reported tubercle baci Il i grew with chromogenicity at pH 6 but lacked col or at pH 7 .6.

    ln a large survey involving 76 cultures of chromogenic bacteria, includlng representa-

    tives of mycobacterie, the reaction of several media was adjusted over a pH range

    from 6.0- 8.4. lt was found thot below pH 6.6 or above pH 8.0 pigmentation be-

  • -44-

    came less marked. Within this range differences in reaction had 1 ittle effect upon

    the production of pigments although changes in shade or tint were detectable (Reid,

    1936- 1937). Bovine tubercle bacilli have been observed to produce definite pig-

    ment below pH 8.0, while a more alkaline reaction resulted in growth without pig-

    ment (Reed and Rice 1 1929). lngraham and Steenbock (1935), determined the

    optimum reaction for pigmentation of M. phlei to be between pH 6.0 and 7.0.

    When the pH was above 8.6 pigmentation was very poor in any medium.

    E. Composition of medium including the presence of trace elements and drugs

    Many sporadic reports have been made on the effect of various media 1

    or specifie chemical substances in a particular medium 1 on the pigmentation of myco-

    bacterla. lt is of no advantage to cite observations related to cultural systems in

    which several variable factors existed. Without controlled pH, temperature, and

    oxygen tension, it is obvlous from the foregoing, thot depending upon various com-

    binations of variables, pigment may or may not be formed. This was the finding of

    Reid (1936- 1937) when 96 media of different composition were investigated to deter-

    mine which factors and substances favoured 1 and which inhibited pigment formation.

    ln the study of 76 species of chromogenic bacterie 1 results led hlm to conclude thot

    pigmentation was governed by a variety of factors, the presence of one or severa! of

    which may be adequate to produce sufficient pigment so thot the absence of sorne

    other factor or factors would not be noticed. The study indicated 1 however 1 thot

    the principal element essential for production of pigment in bacterla was nJtrogen.

    Carbohydrates increased pigmentation probably by virtue of stimulating the amount of

    growth 1 but ln the absence of nitrogen they were not able to support pigmentation.

  • -45-

    Others studying M. phlei also demonstrated the importance of nitrogen, but further

    found that the type of compound which served as a source of nitrogen had little

    effect on pigmentation if the hydrogen ion concentration was maintalned around

    neutrality {lngraham and Steenbock, 1935). Striking changes were noted, however,

    when glycerol was substltuted for glucose. The rate of growth was slower on gly-

    cerol than on glucose-containing medium, but cells grown on glycerol became yellow

    earl ier in the growth period and pigment during the la ter stage was many times that

    obtained on glucose.

    Strains of tubercle bacilli have been noted to produce green pigment in

    Sauton medium. Kolle {1932) stated that this was first observed by Lange and Pis-

    catore at the time of the tragedy in Lubeck caused by contamination of BCG vaccine

    with the "KieP' strain of tubercle bacilli. The strain could be readily recognized

    by its abundant pigment production after recovery from organs of children who had

    died from administration of the contaminated vaccine.

    Cultures of M. phlei, M. leprae (?),and M. smegmatis were pigmented

    when grown on ordinary media but failed to yield pigments when grown on media con-

    taining n-decane, light mineral oil, heavy mineral oil, or paraffin wax. ln contrast

    M. lacticola when grown in mineral oil media produced a bright orange pigment

    {Hass and Bushnell, 1944).

    For certain mycobacteria the enhancement of pigment production ln serum-

    containing medium is recognized. The human form of M. tuberculosis generally de-

    velops a creamy to yellow or faint red pigment especially on media-containing serum,

    while the bovine and murine forms are not pigmented (Bergey, 1957). Griffith {1916) 1

    almost 50 years ago, described the effect of bovine serum on pigment formation in

  • -46-

    cultures of tuber cie ba ci Il i.

    Reed and Rice {1929) 1 demonstrated the influence of iron on the pig-

    mentation of acid-fast bacilli. When grown in the absence of iron, human, bovine,

    avion, and saprophytic strains were colorless. When iron-containing medium was

    employed, ali strains produced pigment. ln contrast, pigmented non-acid-fast

    bacterie tested produced approximately equal amounts of pigment on both iron-free

    and iron-containing media. M. tuberculosis var. avium when grown on synthetic

    media containing trace-element supplements (cobalt, copper, zinc) formed pigments

    which could be extracted from the cells with cold acetone {Patterson, 1960).

    Pinner (1935a 1 b) 1 one of


Recommended