Download The Genus Mycobacterium—Medical

Prokaryotes (2006) 3:919–933
DOI: 10.1007/0-387-30743-5_34
CHAPTER 1.1.19
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The Genus Mycobacterium—Medical
BEATRICE SAVIOLA AND WILLIAM BISHAI
Introduction
The genus Mycobacterium encompasses a number of medically important species that exact an
alarming toll in human morbidity and mortality.
Indeed the World Health Organization (WHO)
estimates that nearly one third of the world’s
population (1.8 billion people) are infected with
Mycobacterium tuberculosis, the cause of
tuberculosis (TB) (http://www.who.org). In 1993,
the WHO declared a global emergency owing to
the fact that TB was epidemic in many areas of
the world. Other mycobacterial diseases continue to plague the world’s populations as well.
Mycobacterium leprae, the cause of leprosy, persists in developing countries, and other mycobacteria (ordinarily nonpathogens) have now
become threats to individuals infected with the
human immunodeficiency virus (HIV).
Mycobacteria have shaped the course of
human history. Indeed until 1900, TB was one of
the chief causes of death in Europe and the
Americas. The nature of the disease, however,
remained poorly understood and as a testimonial
to this fact its contagiousness was the subject of
heated debate well into the 18th and 19th centuries. For many years, psychological and inherited
factors were thought to predispose individuals to
the disease. However, others thought that a contagious agent was the culprit. This debate ended
only after the bacillus Mycobacterium tuberculosis was identified as the causative agent of TB.
Jean Antoine Villemin in 1868 was the first to
transmit TB from man to rabbit (Haas and Haas,
1996). As a military doctor, he had observed that
many young healthy military personnel housed
in barracks eventually succumbed to TB. He also
noted that a disproportionate number of individuals with active TB were prisoners, industrial
workers, members of cloistered religious orders
as well as military personnel; all of them were
housed together with many other people.
Villemin speculated that TB was transmissible.
He used material (gray and soft tubercles) from
humans who had succumbed to TB as well as
blood and sputum from tuberculous patients to
infect rabbits. The inoculated rabbits indeed
developed pathologic evidence of tuberculosis.
Then in 1882, Robert Koch was the first to view
Mycobacterium tuberculosis through a light
microscope. He was then able to grow M. tuberculosis in pure culture, infect guinea pigs with the
bacilli, and reisolate bacteria from these animals
(Adler and Rose, 1996). Thus a new era of mycobacteria research was born.
In Europe and the United States, a sanatorium
movement was underway by the 1890s to isolate
and cure those patients having TB. Edward Livingston Trudeau started one of the first and most
successful of the sanatoria in the United States
(Davis, 1996). After providing hospice care for his
consumptive brother, Dr. Trudeau was stricken
with that same disease. He fled to the Adirondack
Mountains believing that fresh air, nutritious
food and exercise would restore his health. As
hoped, his disease symptoms abated and his
health consequently improved. He chose to move
permanently to Saranac Lake in the Adirondack
Mountains where he bought land, built cottages
and started the Adirondack Cottage Sanatorium.
Dr. Trudeau immersed himself in research and
succeeded in cultivating pure Mycobacterium
tuberculosis, which he provided to other researchers. Out of the Saranac Lake Laboratory, the
Trudeau Institute (http://www.trudeauinstitute.
org) for biomedical research was born in 1954.
Not until the development of successful antimycobacterial chemotherapy did this and other sanitoria lose their function and close their doors to
the public.
In 1944 streptomycin was isolated and identified by Dr. Selman Waksman and his graduate
student Albert Schatz (Harris, 1996). It marked
the first drug in a line of antibiotics having antimycobacterial properties. In 1952 isonicotinic
acid hydrazide (INH) was found to have antimycobacterial properties as well and has since
become a mainstay in the antibiotic therapy of
TB. With the advent of these new advances in the
chemotherapy of TB, victory was declared over
the disease; however, this declaration would
prove premature. Following an extensive period
of decline, the incidence of TB in the United
States began to rise again in the late 1980s. The
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B. Saviola and W. Bishai
HIV epidemic combined with an urban drug
problem provided fertile ground on which M.
tuberculosis could flourish anew. Concomitant
with this alarming trend was detection of an
increasing incidence of drug-resistant M. tuberculosis. To combat these problems, the WHO as
well as government agencies in countries around
the world have increased spending in both the
public health forum as well as biomedical
research. Today, research on M. tuberculosis as
well as other members of the genus is providing
valuable insight into the basic biology of mycobacterial survival and replication inside the host.
Lessons learned through these studies will hopefully provide novel treatments to combat the
renewed threat of this age-old enemy.
Taxonomy
The genus Mycobacterium comprises a number
of Gram-positive aerobic bacteria and is the only
member of the family Mycobacteriaceae within
the order Actinomycetales. The genus shares an
unusually high genomic DNA G+C content (62–
70%) and the production of mycolic acids with
closely related genera, Nocardia and Corynebacterium, within Actinomycetales. Phylogenetic
trees are available which depict genetic relatedness based on homology of the 16S ribosomal
gene sequence. Mycobacteria that have highly
homologous rRNA sequences are closely related
and are on neighboring branches of the tree
(Fig. 1). The mycobacterial phylogenetic tree can
be further subdivided into fast and slow growing
bacteria. The fast growers form colonies on selective media in less than 7 days and the slow growers, in greater than 7 days. In addition, within the
genus Mycobacterium a number of species are
grouped into complexes (e.g., M. avium and M.
tuberculosis complexes) that include bacterial
species that have a high degree of genetic similarity as well as cause similar disease syndromes.
Habitat
Mycobacterium tuberculosis is an obligate pathogen of humans and is rarely identified in other
mammals. It is transmitted from person to person, and it has no significant environmental
reservoirs. Mycobacterium bovis, which causes
tuberculosis in humans and in cattle, has a natural reservoir in ruminants. As a consequence,
foodstuffs (including cheese and milk originating
from these animals) were often contaminated
prior to the introduction of current pasteurization and meat inspection procedures. A number
of other medically important mycobacteria are
found in the environment. Water serves as the
CHAPTER 1.1.19
habitat for a number of mycobacterial species
including M. marinum, M. cheloneae, M. fortuitum, M. kansasii and M. avium. Although not yet
confirmed, stagnant water may be a habitat for
M. ulcerans. Soil may also harbor mycobacteria
including M. cheloneae, M. fortuitum, and M.
avium.
Isolation
Medically important mycobacterial species can
be isolated and cultured from a number of environmental sources; however, M. tuberculosis is
rarely isolated from nonhuman sources. As pulmonary tuberculosis is the most frequent form of
the disease, sputum is the most common body
specimen submitted for culture—although
blood, gastric aspirates and biopsy specimens
may also be analyzed.
Before sputum samples from patients may be
tested for mycobacterial growth, they must be
decontaminated to remove rapidly growing
upper respiratory flora. Decontamination procedures take advantage of the thick lipid envelope
of mycobacteria and this organism’s natural
resistance to chemical agents. N-Acetyl-Lcysteine (0.5–2.0%; NALC) liquefies sputum
samples. The compound has minor inhibitory
effects on mycobacteria but does allow other
antibacterial agents to access and neutralize contaminating organisms. Sodium hydroxide is used
to inactivate contaminating bacteria with only
modest inhibitory effects against mycobacteria.
Once decontaminated, samples may be used
to inoculate either a solid or liquid growth
medium. The solid growth media are available as
either egg- or agar-based. Both types of solid
media contain malachite green dye, which inhibits the growth of many other bacteria. The commonly used Löwenstein-Jensen (LJ) medium is
egg-based (Fig. 2), whereas Middlebrook 7H10
and 7H11 are agar-based media. Growth can also
be assayed in a liquid medium such as Middlebrook 7H9; however, these media are more
problematic because overgrowth by contaminating bacteria is more difficult to detect.
Growth of mycobacteria from clinical specimens is time-consuming because there is often
a lag of three to four weeks before sufficient
growth is achieved. To shorten growth detection
times, the Bactec (Becton-Dickinson) system
may be used (Fig. 2). Mycobacteria are grown in
7H9 that contains 14C-labeled palmitic acid as a
carbon source. As the bacteria grow they catabolize the radiolabeled carbon source and convert
it to gaseous 14CO2. A specially designed instrument (Becton Dickinson) samples the amount of
14
C above the mycobacterial cultures and converts this information into an index of growth.
CHAPTER 1.1.19
The Genus Mycobacterium—Medical
921
M. fortuitum
M. farcinogenes
M. senegalense
M. chelorne
M. penegrinum
M. neaurum
M. diernhoferi
M. abscessus
M. chitae
M. fullax
M. aurum
M. vaccae
M. confluentis
M. madagascariense
M. flavescens
M. smegmatis
M. thermoresistible
M. phlei
rapid growers
M. triviaic
M. simae
M. genavense
slow growers
M. interjectum
M. intermedium
M. terrar
M. hibernine
M. nonchromogenicum
M. cookii
M. xenapi
M. celatum type 1
M. celatum type 2
M. gordonae
M. asiaticam
M. luberculosis complex
M. marinum
M. leprne
M. scrofmlaceni
M. gratrif, M. kansaii
M. szulgni
M. malmacuse
M. intracellulars
M. paratuberculosis
M. avium
Fig. 1. A phylogenetic tree based on 16S rRNA sequences of the genus Mycobacterium depicts closely related species on
neighboring branches. Redrawn from Shinnick and Good (1994).
The Bactec method reduces detection time in
liquid media to 9–14 days for M. tuberculosis
(Middleton et al., 1997; Pfyffer et al., 1997).
Recently a completely closed system called the
mycobacterial growth indicator tube (MGIT)
system has been introduced. The tube contains a
plastic resin that fluoresces as oxygen levels are
depleted by growing bacteria, and an automated
fluorescence detector reports growth readings at
regular intervals (Saito et al., 1996; Sharp et al.,
1996).
Identification
Once mycobacteria have been isolated from
either an environmental or a human source, the
speciation process can begin. Acid-fast staining
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B. Saviola and W. Bishai
CHAPTER 1.1.19
Fig. 3. Kinyoun acid-fast stain of Mycobacterium smegmatis.
Bacilli appear as small red rods with clumping.
Fig. 2. The growth medium Löwenstein Jensen (left) is used
to culture mycobacteria. Mycobacterium tuberculosis takes 3
to 4 weeks to produce colonies on this growth medium. The
Bactec 12B (right) bottle is used for radiometric detection of
mycobacterial growth. Early growth of mycobacteria is
detected by the conversion of 14C-labeled palmitate to gaseous 14C-labeled CO2. An automated sampling needle
removes a small amount of gas through a rubber septum each
day, and a detector measures the radioactivity obtained.
Using the Bactec system, mycobacterial growth may be
detected in 7–15 days.
in combination with light microscopy permits
an initial rapid diagnosis of a mycobacteriosis.
Mycobacteria are acid fast by nature. They form
stable complexes with arylmethane dyes such as
fuchsin and auramine O. The phenol used in the
primary staining procedure allows the stain to
penetrate. The mycolic acids in the cell wall act
to retain the stain even after the exposure to acid
alcohol or strong mineral acids. The resulting
acid-fast mycobacteria can be identified microscopically. The Ziehl-Neelsen and Kinyoun
methods are acid stains in which the acid-fast
bacilli (AFB) appear red against a blue or green
background (Fig. 3). Fluorochrome-staining procedures can be used as well. In these procedures,
which use auramine O or auramine-rhodamine,
the AFB fluoresce yellow to orange. Fluorescence staining is superior as AFB are more easily
identified at a lower magnification. Initial microscopic identification of bacteria, while rapid, is
not reliable, has a sensitivity that ranges from 22
to 80%, and does not allow species identification
of AFB. Therefore, other methods of mycobacterial identification are necessary (Nolte and
Metchock, 1995).
A host of biochemical tests can be used to
differentiate mycobacterial species. Biochemical
tests that distinguish M. tuberculosis from other
species include the niacin accumulation test, the
68°C catalase test and the nitrate reduction test
(Kent and Kubica, 1985; Nolte and Metchock,
1995). Because M. tuberculosis has a blocked
pathway for the conversion of free niacin to nicotinic acid mononucleotide, the bacterium accumulates niacin and excretes it into the culture
medium. As confirmation, additional biochemical tests (e.g., the nitrate reduction test) must be
performed inasmuch as strains of some mycobacterial species other than M. tuberculosis also
accumulate niacin. Mycobacterium tuberculosis
is a strongly positive nitrate reducer. Because M.
kansasii, M. szulgai and M. fortuitum also reduce
nitrate, an additional test (e.g., heat-stable catalase) should be used. The heat-stable catalase
test assays the amount of catalase present in the
bacterium after it has been resuspended in a
buffer and heated to 68°C. Mycobacterium tuberculosis always loses its catalase activity under
these conditions.
Once AFB have been observed in culture,
most clinical labs use a nucleic acid hybridization
test rather than the more laborious biochemical
tests to speciate the isolate. The Accuprobe
assay is available through Gen-Probe (http://
www.gen-probe.com). This assay uses the
Hybridization Protection Assay (HPA) where a
DNA acridinium-ester-labeled probe hybridizes
to a mycobacterial rRNA target sequence. A
selection agent cleaves the label away from the
unhybridized probe but leaves untouched the
probe that is complexed with its target sequence.
Light (detected with a luminometer) will be
emitted from the DNA probe that is complexed
to the rRNA. The advantage of this technique is
that it can identify small quantities of mycobacteria due to the many copies of the rRNA target
sequence in each mycobacterium. Thus there is
an inherent amplification of the signal sequence.
Gen-Probe has available probes that are specific
for M. avium, M. intracellulare, the M. avium
CHAPTER 1.1.19
complex, M. gordonae, M. kansasii and the M.
tuberculosis complex.
Nucleic Acid Amplification Tests to
Detect and Speciate M. tuberculosis
Directly from Sputum
Even shorter detection times may be achieved
using various commercially available nucleic acid
amplification assays (NAA) designed to work
using sputum. The United States Food and Drug
Administration (USFDA) has approved several
commercially available amplification tests for
use directly with sputum. These tests include the
Mycobacterium tuberculosis Direct Test from
Gene-Probe (MTD; http://www.gen-probe.com)
and the Amplicor (http://www.roche.com/
diagnostics/) Mycobacterium tuberculosis (MTB)
Test. The MTD combines Transcription Mediated Amplification, which can amplify target
rRNA sequences, with the HPA described in the
previous section. This test is approved by the
USFDA for use with sputum samples that are
both positive and negative for AFB. The MTB
test uses the polymerase chain reaction (PCR) to
amplify DNA specific to M. tuberculosis. This
test is approved by the USFDA for use with sputum samples positive for AFB. The LCx M. tuberculosis Test (http://www.abbott.com/research/
diagnostics.htm) (Abbott) utilizes a ligase chain
reaction where two oligos are hybridized to a
target sequence from the mycobacteria and are
subsequently ligated. The ligated oligos then
serve as templates for additional oligos to
hybridize and be ligated, resulting in amplification of the target sequence. At present this test
is not approved for use with sputum within the
United States. A recent study comparing two
NAA methods revealed that MTD and LCx had
sensitivities of 98.6 and 100%, respectively, and
specificities of 99.4 and 99.3% with smear-positive samples (Wang and Tay, 1999). The Amplicor
MTB test was analyzed in clinical trials and had
a sensitivity of 95.0% and a specificity of 100%
with smear-positive sputum samples (Amplicor,
2000). The NAA tests, while costly, are slightly
more sensitive than acid-fast staining procedures. These tests hasten care for infected individuals by quickly identifying mycobacteria
within about 25–50% of sputum samples that are
negative by acid-fast staining but later culture
positive. This aspect of the NAA tests is important especially in view of the recent evidence
supporting the transmission of M. tuberculosis
from patients who did not have acid-fast bacilli
in their sputum (Behr et al., 1999). Additionally,
in contrast to acid-fast staining, NAA tests provide information about species present within a
The Genus Mycobacterium—Medical
923
sputum sample. Thus, M. tuberculosis-infected
individuals can be isolated and treated more rapidly, preventing the spread of the bacteria. The
NAA tests cannot differentiate live from dead
bacteria and therefore may not prove helpful for
monitoring response to therapy.
Once a bacterial isolate has been identified
as M. tuberculosis, it can be further subtyped
to the strain level. The ability differentiate M.
tuberculosis on the strain level has enhanced
our knowledge of the epidemiology of TB. In
fact it has led to the identification of large outbreaks of disease traced back to a single person (Edlin et al., 1992; Valway et al., 1998).
Strain typing also has lead to the discovery of
unsuspected routes of transmission such as
medical equipment (Agerton et al., 1997;
Michele et al., 1997), embalming (Sterling et
al., 2000), or exposure to childhood TB (Curtis
et al., 1999). It has been used, as well, to assess
the amount of recent transmission of M. tuberculosis in the community, thereby redefining
our understanding of the balance between primary active and reactivation TB after a period
of latency. Techniques of DNA fingerprinting
allow the identification of different M. tuberculosis strains. Virtually all M. tuberculosis strains
have 1–26 copies of an insertion sequence
IS6110. When a strain’s chromosomal DNA is
fragmented with restriction enzymes, the number of copies and location of these insertion
sequences within the chromosome produce a
restriction length polymorphism (RFLP) pattern that characterizes the strain. In addition,
the frequency with which IS6110 moves within
the chromosome is low enough to allow identification of specific strains that are the cause of
an outbreak. Reasonable identification of a
strain can be obtained when the bacterium
contains six or more IS6110 elements. In the
event that a strain contains less than six insertion elements, a second probe may be used for
increased specificity. This second probe is generally for the polymorphic GC-rich repetitive
sequence (PGRS). Distinct strains will have a
unique pattern of probe hybridization, thus
facilitating identification (Harrington and
Bishai, 2000).
Current research in diagnostics for TB is
focusing on techniques that are fast, sensitive
and specific. A major challenge is to develop
inexpensive tests, which may be used in developing countries where TB is abundant. These
tests could potentially identify patients with
active TB early, thus permitting the isolation of
such individuals and preventing the spread of
the bacilli. Earlier identification of those individuals that harbor M. tuberculosis could possibly lower TB rates in many parts of the
developing world.
924
B. Saviola and W. Bishai
Physiology
Mycobacteria are straight or slightly curved nonmotile rods (approximately 0.2–0.6 µm wide by
1.0–10 µm long; Fig. 4), aerobic, nonsporeformers that grow at times as filaments. Two remarkable bacteriologic features define mycobacteria.
First, they have a cell wall that is rich in longchain fatty acid esters known as “mycolic acids”
that are attached to the cell wall through arabinogalactan (Fig. 5). Mycolic acids are chemically related to wax and give the colonies their
characteristic waxy appearance and the cells a
tendency to clump and resist dispersion. The
waxy nature of the coat renders the bacteria not
readily stainable (such as by the Gram method)
with aniline dyes, though the bacteria are considered to be Gram positive. Mycobacterial mycolic
acids do, however, form strong complexes with
some dyes that leave them resistant to decolorization with acid alcohol. Therefore the identification of AFB in a sample is suggestive of
CHAPTER 1.1.19
mycobacteria (Fig. 3). In addition, this lipid coat
renders the mycobacteria impervious to many
aseptic solutions and antibiotics. Second, all
mycobacteria grow slowly with generation times
Fig. 4. Mycobacterium smegmatis colony morphology after
three days of growth from a single bacterium.
Branched and capped
portion of LAM
Free Lipids
Purin
Mycolic acids
Arabinan portion
of LAM
Pentaarbinasyl
Motifs
Arabinan
Linker
Galactan
Pepcidaglyean
LM portion of LAM
PIMs
Plasma membrane
Polyprenyl sugars
Associated plasma
membrane proteins
The Mycobacterium ruberculosis cell envelope
Fig. 5. Schematic representation of
the plasma membrane and outer wall
of Mycobacterium tuberculosis reveals
an abundance of mycolic acids.
Phosphatidyl-inositol
mannosides
(PIMs), lipoarabinomannans (LAMs)
and lipomannan (LM) portion of
LAM are shown as outer wall constituents. From Brennan and Nikaido
(1995).
CHAPTER 1.1.19
Genetics
The Completed Mycobacterium
tuberculosis Genome
Genetic research into the virulent nature of M.
tuberculosis has long been hampered by the
organism’s slow growth. The generation time of
M. tuberculosis is 24 hours in contrast to the 20minute generation time of the common laboratory bacterium Escherichia coli. The tendency for
mycobacteria to form clumps also leads to significant technical challenges in the research laboratory. As a consequence, traditional genetic
screens and selections are difficult to conduct.
Therefore, reverse genetic approaches have been
emphasized in the study of M. tuberculosis. This
technique targets specific proteins homologous
to proteins of known function and importance in
other organisms for study at a molecular level
within a host. As a result, the elucidation and
analysis of the whole genome sequence of
M. tuberculosis have become increasingly
important.
In 1998, the complete genome sequence of the
M. tuberculosis strain H37Rv was elucidated
(Cole et al., 1998). The completion of the
genome sequence was a collaborative effort of
the Wellcome Trust and the Institute Pasteur. A
web site at the Institute Pasteur shows the
completed genome. In addition, The Sanger
Center maintains a web site where DNA segments and predicted proteins from the M. tuberculosis strain H37Rv may be compared to
peptides and DNA from other organisms. The
Institute for Genomic Research (TIGR) has completed the sequence for another M. tuberculosis
strain, CDC1551 (CSU93). At the TIGR web
site, one may also compare the chromosomal
sequence to other known genes and predicted
proteins. In addition, The National Center for
Biotechnology Information offers a web site
where the M. tuberculosis genome may be
accessed and the chromosomal sequence may be
visualized on a map.
The genome sequence is 4.4 Mbp. Researchers
now have at their disposal the information defining every possible drug target, antigen for incorporation into a vaccine, as well as every virulence
determinant from M. tuberculosis. Annotation of
the H37Rv sequence reveals 3,924 open reading
frames (Cole et al., 1998). The genome has no
easily identifiable pathogenicity islands as other
organisms have. However, many repetitive elements were identified, including IS6110, which
have been used as a tool to identify varying
925
strains during outbreaks of M. tuberculosis (Harrington and Bishai, 2000). The mycobacterium
contains within its repertoire genes that could
allow the bacterium to adapt to a range of environmental conditions, from growth in a rich
broth to survival in host macrophages. These
genes include 13 putative σ factors, 140 transcriptional regulators, 32 component regulators that
presumably sense environmental signals, and 14
protein kinases or phosphatases. The genome
also possesses 250 genes involved in a complex
lipid metabolism, which the cell needs to synthesize material for its waxy coat (Cole et al., 1998).
Several new classes of putative proteins termed
“PE” and “PPE” (each with internal repetitive
sequences) were also identified in the genome
sequence. Indeed, ten percent of the genome
is composed of these new potential coding
sequences. The PE or PPE class of proteins has
been proposed to be involved in modulation of
the host immune response (Cole et al., 1998).
Epidemiology
Tuberculosis causes more deaths than any other
infectious organism; 6 million new active cases
and 3 million deaths annually are attributed to
M. tuberculosis. Indeed, rates of TB in the
world are predicted to increase 50% each
decade. However, the annual incidence of TB
in the United States remains low with 8.7 cases/
100,000 people. Prior to 1985, TB rates within
the United States were steadily declining at
approximately 6% per year. In striking contrast
to this trend, from 1986 to 1992 the incidence
of TB cases rose about 3% per year (Fig. 6). A
Reported TB Cases
United States, 1975 - 1997
35,000
30,000
Cases
that range from 2 hours for M. smegmatis to 12
days for M. leprae.
The Genus Mycobacterium—Medical
25,000
20,000
75 77 79 81 83 85 87 89 91 93 95 97
Year
CDC
Fig. 6. After many years of decline, reported TB cases in the
United States began to rise in the late 1980s. Following public
health intervention, TB cases are once again dropping
CDC.
926
B. Saviola and W. Bishai
maximum of 26,673 cases occurred in 1992.
Increased government funding for public health
programs and increased use of directly
observed therapy (DOT) for tuberculosis have
decreased rates since 1992 (Chaisson and
Bishai, 1997). As a result, in 1998 the United
States Centers for Disease Control and Prevention (CDC) reported 18,361 cases. Again, in
1999 the incidence of new TB cases fell to
17,528 cases.
Despite the fact that the number of cases in
the United States is now dropping, there is
the new problem of multidrug-resistant TB.
The term multidrug-resistant tuberculosis
(MDRTB) is used to describe strains that are
resistant to two or more of the five first-line
anti-TB drugs: isoniazid, rifampin, pyrazinamide, ethambutol and streptomycin. Indeed the
incidence of drug resistance has increased in
urban settings and among HIV-infected populations. In fact, during 1991, 33% of all TB strains
recovered in New York City were MDRTB. The
United States rate of MDRTB, however, has
now fallen with improved control measures, but
the global problem of drug resistance continues.
In an international survey between 1994 and
1997, 12% of incident cases were resistant to at
least one drug and 7.6% of TB strains were
resistant to isoniazid (Pablos-Mendez et al.,
1998).
Risk factors for TB include intravenous drug
abuse, alcoholism, chronic pulmonary disease,
prolonged steroid use, diabetes, renal failure,
malnutrition and organ transplant. Infection
with HIV is also a risk factor for TB: HIVinfected patients have a 25–50 fold increased risk
compared with HIV-negative individuals. A
number of social factors also increase a person’s
risk of TB including institutional living, urban
dwellings, poverty and low educational levels
(Chaisson and Bishai, 1997).
The incidence of other mycobacterial diseases
has increased in recent years. Diseases caused
by the Mycobacterium avium complex (MAC)
were uncommon prior to the AIDS epidemic.
Infection with MAC was mostly seen in individuals that were immunocompromised or had
underlying lung disease. Indeed, prior to 1981
the incidence of MAC cervical lymphadenitis
and MAC pulmonary infection was approximately 300 and 3,000 cases per year, respectively, in the United States. With the onset of the
AIDS epidemic, the incidence of disseminated
MAC infection jumped in the mid-1990s to
approximately 20,000 individuals per year
(Chaisson and Bishai, 1997). The MAC rates,
however, appear to have peaked in 1997 and
have since fallen due to improved antiretroviral
therapy for patients infected with HIV (Palella
et al., 1998).
CHAPTER 1.1.19
Disease
The MYCOBACTERIUM TUBERCULOSIS Complex The
M. tuberculosis complex is comprised of M.
tuberculosis, M. bovis, M. microti and M. africanum. Only M. tuberculosis and M. bovis are a
significant source of human disease (http://
www.hopkins-tb.org). Mycobacterium microti
causes disease in voles and was employed as a
vaccine in the earlier part of the twentieth century (Wells and Oxon, 1937; Birkhaug, 1946;
Wells, 1949; Wells and Wylie, 1954). Mycobacterium africanum causes disease in humans but
makes up only a minority of cases of pulmonary
TB, and its incidence is isolated to Africa.
M. TUBERCULOSIS The main route of infection of
M. tuberculosis is by person-to-person inhalation
of infectious aerosols. Bacilli are transmitted
through speaking, coughing, sneezing and singing. Droplets of infectious M. tuberculosis are
sized in the range of 1–10 µm. After inhalation
of the droplets, the bacteria travel to the terminal
bronchioles and alveoli where they are phagocytosed by alveolar macrophages. Many of these
bacilli are killed in the phagosomes after these
fuse with lysosomes and become acidified. Mycobacterium tuberculosis, however, is an intracellular pathogen and can efficiently inhibit
phagosome-lysosome fusion (Fig. 7). As a result
some of the invading M. tuberculosis bacilli survive these initial host defenses. Surviving bacilli
grow within the macrophages and are released
when the macrophages die. Unactivated macrophages arrive from the blood stream and
ingest the newly liberated bacilli, which grow
symbiotically within the unactivated macrophages for approximately 3 weeks. Eventually the
bacilli lyse the macrophages and spill out into the
host tissue.
Fig. 7. Macrophages infected with M. bovis BCG are visualized by acid-fast staining. Mycobacteria are small red rods
within the blue-stained macrophages.
CHAPTER 1.1.19
The Genus Mycobacterium—Medical
927
Fig. 8. Cavitary pulmonary tuberculosis in a 36-year old man.
Right upper lobe streaky infiltrates and nodules are seen; two
cavities are also present near the right apex.
In resistant individuals, released bacterial
products stimulate strong cell-mediated immunity through Th1 signaling with INF-γ, Il-2, and
Il-12. Activated macrophages that can kill M.
tuberculosis, as well as T cells, are recruited to
the periphery of the infectious focus. The bacteria and cellular debris are contained in a tissue
structure called “a caseous granuloma.” The disease will be halted at the stage where this small
granuloma is formed.
In sensitive individuals, cell-mediated immunity is weak, and bacilli continue to multiply.
Macrophages arrive to engulf the bacilli while T
cells also accumulate. Because the macrophages
are inadequately activated, the M. tuberculosis
bacilli also parasitize the recruited macrophages.
Cytotoxic T-cells produce toxic substances, which
results in damage of host tissues. This cycle is
repeated so that the granuloma enlarges. Eventually, tissue damage may result in cavity formation within the lung as well as in liquefaction of
the granuloma (Figs. 8 and 9). Mycobacterium
tuberculosis bacilli are particularly adept at multiplying in a liquefied cavity from which they
are then expelled and spread through coughing
and sneezing (Dannenberg, 1993; Dannenberg,
1994).
As they have no environmental reservoirs, M.
tuberculosis organisms spread from person to
person. In addition, fomites are not thought to
support the transmission of the disease. Some
(25–50%) of the diseased individual’s close contacts will become infected with M. tuberculosis.
However, only a minority (5% of those exposed)
is susceptible and will develop primary active
disease. In contrast, most humans seem to be
naturally resistant to M. tuberculosis; these indi-
Fig. 9. Lung of a rabbit infected with M. bovis. Cavities are
abundant within the lung.
viduals harbor the bacilli and develop only a
latent infection. Approximately 95% of infected
individuals control the spread of M. tuberculosis
within the body and disease progression is halted
at the stage where a small granuloma is formed.
These individuals may have a lifelong latent
infection with no symptom other than a positive
reaction to the administration of purified protein
derivative (PPD) of M. tuberculosis, indicating
immune memory of M. tuberculosis infection.
Any process that disrupts the immunocompetence of a latently infected individual, however,
may cause active infection to develop. The elderly often have a recrudescence of disease after
decades of a latent infection due to their waning
immunity. Acquiring a viral infection such as
HIV may cause an individual to become immunosuppressed and develop active from latent
disease. In fact, HIV-infected individuals have a
4–8% yearly risk for reactivation TB whereas
immunocompetent individuals have only a 5–
10% lifetime risk (Chaisson and Bishai, 1997).
Disease development due to M. tuberculosis
infection can be either pulmonary or extrapulmonary. Inasmuch as infection is mainly through
the aerosol route, immunocompetent individuals
manifest primarily pulmonary disease. Symptoms include cough, chest pain, sputum production, fever, night sweats and hemoptysis in
advanced disease. About 15% of TB cases in
immunocompetent individuals occur at extrapulmonary sites. Such cases are attributed to reactivation of a latent extrapulmonary focus of
infection, because during the initial infection
928
B. Saviola and W. Bishai
process transient bacteremia may occur. Occasionally hospital and laboratory workers infect
soft tissues through accidental injection with
M. tuberculosis-contaminated syringes. In those
cases infection is extrapulmonary but may
spread by the blood to the lungs and other
organs if the immune system cannot check the
growth of the bacilli. Immunocompromised individuals (such as the very young, old and those
infected with HIV) are more likely to develop
extrapulmonary tuberculosis.
M. BOVIS Isolated from cattle, M. bovis causes
TB in ruminants and occasionally humans. This
organism prefers to grow at 37°C and growth of
a colony from a single bacillus usually requires
3–4 weeks of culture. Before routine pasteurization was practiced, humans could be infected by
drinking M. bovis-contaminated milk. In fact
milk was an important reservoir of infectious
bacilli. However, since pasteurization became
commonplace in the United States and in much
of the world, the incidence of M. bovis infection
has decreased considerably. Mycobacterium
bovis is spread among cattle by an aerosol route
and from cattle to humans by either a gastrointestinal or an aerosol route. In humans, M.
bovis may cause intra-abdominal TB, cervical
lymphadenitis (scrofula) or pulmonary TB
(Adler and Rose, 1996). Mycobacterium bovis
was used to derive the well-known attenuated
vaccine strain, bacille Calmette Guerin (BCG),
which is still used in many parts of the world.
M. AFRICANUM This slow growing mycobacterium, first described in 1969, is infrequently associated with human pulmonary disease. It causes
a disease similar to that caused by M. tuberculosis and M. bovis. Most patients with this disease
reside or have resided in Africa. Mycobacterium
africanum probably spreads by an aerosol route
(Adler and Rose, 1996).
THE MYCOBACTERIUM AVIUM COMPLEX The M.
avium complex is comprised of three species, M.
avium, M. intracellulare and M. paratuberculosis.
All three species are common in the environment, as they are isolated from soil, water, food
and domestic animals. Categorized as slow growing mycobacteria, these species grow optimally
at 37°C and achieve growth in approximately 4–
6 weeks (Havlir and Ellner, 2000). Colonies are
nonpigmented and are both opaque and domed
or flat and transparent (Toosi and Ellner, 1998).
Mycobacterium avium causes a tuberculosis-like
infection in chickens, pigeons and other birds,
whereas M. paratuberculosis causes Johne’s disease in ruminants, which is a contagious enteritis
resulting in progressive wasting and eventual
death (Fraser et al., 1986). Mycobacterium
CHAPTER 1.1.19
paratuberculosis has not been shown to cause
human disease.
In humans, M. avium and M. intracellulare can
cause pulmonary disease, regional lymphadenitis
or disseminated disease. Those individuals who
are coinfected with HIV are prone to disseminated disease, whereas those who are not coinfected are likely to have pulmonary disease.
Predisposing factors to infection with M. avium
include underlying lung disease, HIV infection,
chronic obstructive pulmonary disease, chronic
bronchitis, healed or active TB, pulmonary
mycosis and malignancy. Infection occurs by
either inhalation or ingestion of the infectious
bacilli. Diagnosis in those affected individuals
without HIV is made by radiographic evidence
of disease, positive sputum cultures and an acidfast smear. Symptoms are a productive cough,
hemoptysis, fever and weight loss (Chaisson and
Bishai, 1997).
Disseminated M. avium is a late opportunistic
infection in those individuals coinfected with
HIV. In fact, disseminated MAC generally
occurs only in AIDS patients with CD4 cell
counts less than 100/mm3 (Chaisson and Bishai,
1997). Symptoms of infection are fever, drenching night sweats and weight loss. The hallmark of
a disseminated infection is the high circulating
levels of MAC bacteremia, which can reach 104
bacteria/ml. Organ involvement may be widespread and the tissue burden may reach 106 bacteria/g. The gastrointestinal tract is frequently
infected, leading to symptoms including nausea,
vomiting, watery diarrhea and abdominal pain.
Left untreated, the disease will cause progressive
clinical deterioration (Havlir and Ellner, 2000).
MYCOBACTERIUM LEPRAE Leprosy is particularly
dreaded because it may cause bodily deformities
leading to social stigmatization. In centuries past,
persons suffering from leprosy were ostracized
and forced to live away from the general population, in colonies or leprosaria. Today many
patients suffering from leprosy are still concerned about the social stigma associated with
leprosy. As of 1999, there were 719,332 leprosy
cases registered for treatment. Almost all of
these individuals were receiving multidrug therapy. The new case detection rate stayed the same
or was increasing with 747,369 new cases
detected during 1998 (http://www.who.int/lep).
The prevalence of leprosy is not evenly distributed, as much of the disease seen today occurs in
the third world. Most cases (72%) are from Asia
and Oceania, whereas 18% are from Africa and
only 7% are from the Americas (Lockwood and
McAdam, 1998).
The genome of the 1–8 µm long and 0.3–
0.5 µm wide M. leprae bacillus is approximately
3.2 MB (Gelber and Rea, 2000). The sequence
CHAPTER 1.1.19
has been partially determined and is available
for
inspection
(http://www.sanger.ac.uk/
DataSearch). It is speculated that many of the
genes essential for the survival of M. leprae in the
environment have been deleted, and the bacillus
has become an obligate parasite of its human
host. Indeed to date no one has been able to
continuously culture the bacillus in either in vitro
conditions or cell culture. It may be true that the
mycobacterium has lost genes essential for survival in these ex vivo conditions and thus has an
absolute requirement for host factors (Gelber
and Rea, 2000). In fact, the only methods to
propagate M. leprae bacilli are via animal infections with either 9-banded armadillos or severe
combined immunodeficiency (SCID) mice.
Growth of sufficient bacilli for laboratory manipulation takes approximately one year, inasmuch
as the generation time for this organism is
approximately 14 days in animal tissues.
Leprosy is a chronic disease with a long incubation period. This fact is affirmed by the observation that children younger than two years do
not have leprosy symptoms. In addition, people
residing in nonendemic countries who have visited a site with endemic leprosy may develop the
disease many years after the initial exposure.
Hence the incubation period is estimated to
range from 2–12 years. Like those infected with
TB, only about 10% of those infected with leprosy go on to develop the disease (Lockwood
and McAdam, 1998). Being a human disease,
leprosy has no established natural reservoir for
infection. Mycobacterum leprae is probably
transmitted from person to person although the
reservoirs and their role in transmission remain
controversial issues. In 1898 Schaffer noted that
leprosy patients discharged large numbers of
acid-fast bacilli (AFB) when coughing, sneezing
or speaking normally. In fact, if left untreated a
lepromatous leprosy patient may dispel 6.8 × 1010
AFB in a single nose blow. Thus leprosy may to
be transmitted via an aerosol route where it goes
on to infect the lining of the nose (Lockwood and
McAdam, 1998). Though M. leprae produces
lesions that often involve the extremities and
skin, bacilli probably reach these locations by
hematogenous spread. In fact people who have
leprosy may have granulomata in the lymph
nodes, liver, kidney, spleen, bone marrow,
adrenals, testes and eyes. Thus the bacilli can
spread to remote portions of the body to cause
lesions. Although M. leprae spreads through the
body via a systemic route, it prefers to grow at
temperatures below 37°C and therefore has a
tropism for the extremities and skin where its
growth can flourish.
The outcome of the disease is strongly dependent on the host’s immune response. Upon infection, bacilli are engulfed by macrophages. If the
The Genus Mycobacterium—Medical
929
macrophages are sufficiently activated to kill
the invading bacilli, then the infection will be
cleared. Otherwise, the bacilli will go on to replicate within macrophages, lyse the cells, and
infect other macrophages (Gelber and Rea,
2000). The host’s immune system can take one of
two pathways. First, the disease is controlled by
strong cell-mediated immunity employing helper
T cells of the Th1 lineage. These helper T-cells
induce the macrophages to kill the bacilli, and
INF-γ and IL-2 are found at the site of infection
(Lockwood and McAdam, 1998). Organized
granulomata are composed of the macrophages
surrounded by the T-cells, resulting in what is
known as tuberculoid or paucibacillary leprosy.
It is known from the days before antibiotic therapy that tuberculoid leprosy often spontaneously
heals (Lockwood and McAdam, 1998). However, tuberculoid leprosy also may result in tissue
damage from continuous lymphocyte infiltration
into the area of infection. Tuberculoid leprosy
has a shorter incubation time, generally 2–5
years, and causes few skin lesions. (Lesions when
manifest are hypopigmented and asymmetric.)
Nerve damage can occur when granulomata are
near small dermal sensory and autonomic nerve
fibers (Lockwood and McAdam, 1998). Tuberculoid leprosy is not associated with the presence
of stainable M. leprae in skin and does not produce upper respiratory signs and symptoms. The
reason is probably that strong cell-mediated
immunity controls the infection, though the control is at the expense of significant tissue damage.
In general, tuberculoid leprosy has a good prognosis with treatment.
The other path of infection occurs in patients
who do not have good cell-mediated immunity
and good initial killing of the M. leprae. In this
expression of the disease, named “lepromatous”
or “multibacillary” leprosy, bacilli multiply out of
control. This form of the disease has a somewhat
longer incubation period, 8–12 years. The bacilli
may invade Schwann cells, resulting in demyelination of nerves and nerve damage (Lockwood
and McAdam, 1998). Granulomata that do form
are poorly organized, with high bacterial loads.
Bacilli may replicate within dermal cells as evidenced by AFB in the skin. Most (80% of) lepromatous patients have some nasal symptoms
due to invasion of the nasal mucosa by M. leprae.
Occasionally, without treatment, the bacilli may
cause so much destruction that the nasal septum
collapses. In addition, denervation and loss of
pain sensation results in repetitive trauma and
injury to limbs. Destruction can occur over vast
areas of limbs that in some instances may necessitate amputation.
In between the two extremes or poles, tuberculoid and lepromatous, there is a continuum.
These disease states, which are defined patholog-
930
B. Saviola and W. Bishai
ically after review of skin biopsy specimens, are
referred to as borderline tuberculoid, borderline
leprosy and borderline lepromatous. Borderline
tuberculoid has some aspects of lepromatous
leprosy, whereas borderline lepromatous leprosy
has some aspects of tuberculoid leprosy. Borderline leprosy refers to a state in between lepromatous and tuberculoid. Borderline states are
extremely unstable and may shift spontaneously
between mild and severe disease.
Until recently not much was known about the
mechanism by which M. leprae interacts with and
causes damage to cells of the nervous system.
Recent evidence has revealed that M. leprae
binds to the basal lamina surrounding Schwann
cells through an interaction between a 21-kDa
surface protein on M. leprae and laminin-2
within the basal lamina (Rambukkana et al.,
1997; Shimoji et al., 1999). Additionally, M. leprae can interact directly with Schwann cells by
binding to α-dystroglycan on the Schwann cell
surface. This interaction, however, occurs only in
the presence of laminin-2 (Rambukkana et al.,
1998). These findings have the promise of providing mechanistic information that may aid in the
development of improved therapeutics against
leprosy.
slow-growing
MYCOBACTERIUM KANSASII This
organism is the second most frequent cause of
pulmonary disease by a nontuberculous mycobacteria (Brown and Wallace, 2004). Mycobacterum kansasii is found in environmental water,
and water is probably the source of human infection (Iseman, 1998). The clinical presentations of
pulmonary M. kansasii are a chronic cough, lowgrade fever, malaise and chest pain. Though clinical manifestations are similar to those caused
by M. tuberculosis, a chest x-ray usually reveals
upper-zone disease with fewer fibronodular
regions than found with either M. tuberculosis or
MAC. Predisposing factors for infection with M.
kansasii are smoking-induced chronic bronchitis,
chronic inorganic dust exposure, and chronic
obstructive pulmonary disease (Toosi and Ellner,
1998). Although extrapulmonary infections are
rare, M. kansasii can cause diffuse cutaneous disease as well as cervical lymphadenitis, especially
in children. Patients with AIDS or with impaired
cellular immunity may also develop a disseminated infection.
MYCOBACTERIUM MARINUM M. marinum causes a
cutaneus disease in humans known as swimming
pool granuloma, fish handler’s nodule or surfer’s
nodule. As implied above, water is the major
environmental reservoir for this mycobacterial
species (Toosi and Ellner, 1998). The bacterium
and therefore most cases of infection are mainly
found in the coastal areas of North America (Ise-
CHAPTER 1.1.19
man, 1998). Humans contract the disease from
environmental water, the handling of domestic
and wild fish, or the cleaning and maintenance
of fish tanks. Mycobacterium marinum lesions
appear on the extremities of affected individuals,
as the bacillus is most adept at growing at a temperature of 32°C (Brown and Wallace, 2004).
Interestingly, M. marinum is closely related to M.
tuberculosis on a genetic level. Because M. marinum is not transmitted to humans through an
aerosol route and is not considered a biosafety
level 3 (BSL3) organism, it has been used as a
model system for the study of M. tuberculosis
pathogenesis (Ramakrishnan et al., 2000). In animal tissues, M. marinum bacilli infect macrophages and this leads to granuloma formation. As
this species of mycobacterium causes disease in
both fish and frogs, the nature of its pathogenesis
has been investigated in these animal models.
Thus, although M. marinum is not a major public
health problem, research into its basic biology is
ongoing due to its genetic similarity with M.
tuberculosis.
MYCOBACTERIUM SCROFULACEUM This slowly growing mycobacterium, first isolated in 1956, has an
optimal growth temperature of 37°C and, upon
initial isolation, can take approximately 4 to 6
weeks to achieve growth. It is a leading cause of
scrofula or cervical lymphadenitis in children 1
to 5 years of age. Infrequently it may cause progressive pulmonary disease as well as bone and
soft tissue disease and, in some instances, disseminated disease.
MYCOBACTERIUM FORTUITUM, M. CHELONAE and M.
ABSCESSUS These organisms comprise a group of
rapidly growing mycobacteria that are found in
soil samples as well as in water; however, their
mode of transmission to humans remains
unknown (Iseman, 1998). These mycobacteria
cause localized infection of the skin, soft tissues
and bones. They also may cause pulmonary disease typified by a persistent hacking cough, lowgrade fever, chills, malaise and mucopurulent
secretions.
MYCOBACTERIUM ULCERANS During the first half of
the 20th century this mycobacterial disease was
recognized in Bairnsdale, Australia. The syndrome produced an ulcer, subsequently named
the “Bairnsdale ulcer,” or “Buruli ulcer,” that
was characterized by a chronic progressive ulceration of the skin (Fig. 10). Acid-fast bacilli, later
identified as M. ulcerans, were eventually cultivated from lesions of infected individuals.
Although no bacteria have been cultivated from
environmental sources, the use of polymerase
chain reaction (PCR) has identified M. ulcerans
in water sources (Ross et al., 1997). Thus it is
CHAPTER 1.1.19
The Genus Mycobacterium—Medical
931
the Buruli ulcer (Johnson et al., 1999). The fact
that tissue necrosis occurs at sites distal to areas
of colonization by M. ulcerans seems to imply
that a factor that can diffuse to distant tissues is
involved. It has recently been discovered that a
polyketide toxin named “mycolactone” is
responsible for histopathologic changes distal to
the site of infection (George et al., 1998; George
et al., 1999). This toxin was present in the culture
filtrates of growing M. ulcerans, indicating that
it is secreted into the milieu of the bacterium.
When this toxin is injected into guinea pig skin,
ulcers similar to those caused by M. ulcerans are
formed. In addition, exposure of L929 murine
fibroblasts to the toxin arrests their cell cycle.
Thus, as a toxinogenic microorganism, M. ulcerans has a mode of pathogenesis that is very different from that of other members of the genus
Mycobacterium.
6-2-A2. About 1/10 of the ulcers are on the trunk. The
advancing edge of the ulcer is frequently hyperpigmiented as
seen here. AFIP 74-4472.
Fig. 10. A young boy infected with M. ulcerans has a buruli
ulcer covering a portion of his torso. From Connor et al.
(1976).
hypothesized that M. ulcerans is acquired from
the environment via water. Indeed it has been
noted that people inhabiting areas close to a
body of stagnant water in tropical regions are
more at risk for developing a Buruli ulcer (Ross
et al., 1997; Johnson et al., 1999). The hallmark
of the disease is a skin ulcer that begins as a small
nodule but (if left untreated) enlarges to encompass a large surface area. The ulcers are themselves painless, suggesting that there is some
nerve involvement. Inflammatory response at
the site of colonization by the AFB or the site of
ulceration appears to be minimal, even though
necrosis of the subcutaneous fat tissue is extensive. In contrast to M. tuberculosis, M. ulcerans
grows as microcolonies extracellularly during
human infection. In addition, treatment of the
disease is not trivial. Antibiotic therapy has variable efficacy, and therefore the best treatment is
surgical excision after the ulcer has been identified at an early nodule stage. Large ulcers require
extensive surgery with skin grafting. In extreme
cases where muscle tissue is involved, amputation of an affected limb may be required
(Iseman, 1998).
It has long been suspected that a toxin mediates the extensive tissue damage associated with
Prevention Vaccination with the attenuated M.
bovis strain bacille Callmette Guerin (BCG) is
common in many parts of the world; however,
protection rates due to this vaccine vary from 0–
80%. Whereas BCG probably provides little protection against adult pulmonary TB, it does
appear to protect against childhood disseminated TB (Colditz et al., 1994). An additional
problem of vaccination is that (once vaccinated
with BCG) individuals will have a positive reaction to the administration of purified protein
derivative (PPD) from M. tuberculosis, excluding
its use as a diagnostic tool. Therefore once a
person has been vaccinated, it is impossible to
know if they have been infected with M. tuberculosis subsequently and therefore if their risk of
developing active disease is substantial. Thus in
the United States vaccination is not recommended as a measure to control M. tuberculosis
spread. In the United States and many European
nations, it is preferable to periodically administer
PPD to persons at high risk of becoming infected
with M. tuberculosis and to treat prophylactically
with antibiotics. Vaccination, however, is useful
in reducing miliary and meningeal disease in
children in third world countries where tuberculosis rates are high and mass screening is
impractical.
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