CHAPTER 1.1.19
The Genus Mycobacterium—Medical
BEATRICE SAVIOLA AND WILLIAM BISHAI
Prokaryotes (2006) 3:919–933
DOI: 10.1007/0-387-30743-5_34
Introduction
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The genus Mycobacterium encompasses a number of medically important species
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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)
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Mycobacterium leprae ->the cause of leprosy, persists in developing countries.
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Other mycobacteria (ordinarily non-pathogens) have now become threats to
individuals infected with the human immunodeficiency virus (HIV).
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1900,TB was one of the chief causes of death in Europe and the Americas.
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Jean Antoine Villemin in 1868 was the first to transmit TB from man to rabbit.
->Pathologic evidence of tuberculosis.
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In1882, Robert Koch was the first to view Mycobacterium tuberculosis through a
light microscope. -> pure culture of M. tuberculosis
In 1944 streptomycin was isolated and identified by Dr. Selman Waksman and his
graduate student Albert Schatz ->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.
Taxonomy
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Scientific classification
Kingdom:Bacteria
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Phylum: Actinobacteria
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Order: Actinomycetales
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Suborder:Corynebacterineae
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Family:Mycobacteriaceae
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Genus:Mycobacterium
Lehmann & Neumann 1896
Gram-positive aerobic bacteria
An unusually high genomic DNA G+C content
(62–70%)
The production of mycolic acids
The fast growers form colonies on selective
media in less than 7 days
The slow growers, in greater than 7 days.
Homology of the 16S ribosomal gene sequence
Habitat
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Mycobacterium tuberculosis ->humans and in other mammals
Mycobacterium bovis causes tuberculosis in humans and in cattle,
a natural reservoir -> ruminants.
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Other medically important mycobacteria are found in the environment.
Water serves as the habitat for a number of mycobacterial species
including M. marinum, M. cheloneae, M. fortuitum, M. kansasii and M.
avium.
Stagnant water may be a habitat for M. ulcerans
Soil may also harbor mycobacteria including M. cheloneae, M.
fortuitum, and M. avium.
Isolation
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M. tuberculosis is rarely isolated from nonhuman sources.
Pulmonary tuberculosis is the most frequent form (sputum,
blood, gastric aspirates and biopsy specimens)
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N-Acetyl-Lcysteine(0.5–2.0%; NALC) liquefies sputum samples.
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Sodium hydroxide is used to inactivate contaminating bacteria
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Growth of mycobacteria from clinical specimens is timeconsuming because there is often a lag of three to four weeks
before sufficient growth is achieved.
The Bactec method reduces detection time in liquid media
to 9–14 days for M. tuberculosis
Identification
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Mycobacteria have been isolated from either an environmental or a human
source->Acid-fast staining (an initial rapid diagnosis of a mycobacteriosis)
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.
->acid-fast mycobacteria can be identified microscopically.
The Ziehl-Neelsen and Kinyoun methods are acid stains in which the acidfast bacilli (AFB) appear red against a blue or green background.
•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. ->Other methods of
mycobacterial identification are necessary
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As confirmation, additional biochemical tests (the nitrate reduction test)
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Mycobacterium tuberculosis is a strongly positive nitrate reducer.
->M. kansasii, M. szulgai and M. fortuitum reduce nitrate
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Most clinical labs use a nucleic acid hybridization test rather than the more
laborious biochemical tests to speciate the isolate.
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This assay uses the Hybridization Protection Assay (HPA) where a
DNA acridinium-ester-labeled probe hybridizes to a mycobacterial
rRNA target sequence.
Nucleic Acid Amplification Tests to Detect and Speciate M. tuberculosis
Directly from Sputum
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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.
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.
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
【 LIGASE CHAIN REACTION (LCR) 】
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LCR 법은 ligase가 긴주형 DNA에 결합되어있는 두개의 연접한
oligonucleotide를 연결하는 성질을 이용한다. LCR의 유용성은 주형
DNA의 연결 부위의 단일 염기 차이를 구별할 수 있다는 점이다.
6개의 oligonucleotide(2개는 상보적이고 공통적인, 돌연변이 부위 한
쪽 옆에 존재하는 oligonucleotide이고, 나머지 4개는 돌연변이 부위를
한쪽 끝에 포함하고 있는 2개의 정상 염기 서열과 일치하는 상보적인
oligonucleotide와 2개는 돌연변이와 일치하는 염기 서열을 갖는
oligonucleotide)를 이용하여 정상 서열과 돌연변이 서열의 여부를 쉽
게 확인하여 인자형을 알 수 있다.
LCR법은 산물의 유무로 확실한 진단을 내릴 수 있다는 점이 PCR에 비
해 장점이다. 따라서 LCR은 특정 집단 내에서 알려진 돌연변이를 검색
하는데 유용하다.
Physiology
Mycobacteria are straight or slightly curved
nonmotile rods
(approximately 0.2–0.6μm wide by 1.0–10μm long)
Aerobic, nonsporeformers & grow at times
as filaments.
Two remarkable bacteriologic features
1.“mycolic acids” ->attached to the cell wall
through arabinogalactan
2.All mycobacteria grow slowly with
generation times that range from 2 hours
for M. smegmatis to 12 days for M. leprae.
Arabinan +Galactan=
Arabinogalactan
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Phosphatidyl-inositol
mannosides(PIMs),
Lipoarabinomannans (LAMs)
Lipomannan (LM)
Genetics
The Completed Mycobacterium tuberculosis Genome
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The generation time of M. tuberculosis is 24 hours
Reverse genetic approaches have been emphasized in the study of M.
tuberculosis.
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.
The genome sequence is 4.4 Mbp.
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.
Genetics
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These genes include 13 putative σ factors, 140 transcriptional regulators,
32 component regulators that presumably sense environmental signals, and
14protein 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
Epidemiology
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Tuberculosis causes more deaths than any other
infectious organism;
6 million new active cases and 3 million deaths
annually are attributed to M. tuberculosis.
Despite the fact that the number of cases in the
United States is now dropping, there is the new
problem of multidrug-resistant TB.
->multidrug-resistant tuberculosis
(MDRTB)
anti-TB drugs: isoniazid, rifampin, pyrazinamide,
ethambutol and streptomycin
Mycobacterium avium complex (MAC)
were uncommon prior to the AIDS epidemic.
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Infection with MAC was mostly seen in individuals
that were immunocompromised or had underlying
lung disease
Epidemiology
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Disease
The MYCOBACTERIUM TUBERCULOSIS Complex
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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
M. TUBERCULOSIS
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The main route of infection of M. tuberculosis is by person-to-person
inhalation of infectious aerosols. (ex:speaking, coughing, sneezing and
singing.)
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.
M. TUBERCULOSIS
->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.(3 weeks)
In resistant individuals, released bacterial
products stimulate strong cell-mediated
immunity through Th1 signaling with INF-, Il-2,
and Il-12.
Activated macrophages can kill M.tuberculosis,
as well as T cells, are recruited to the periphery
of the infectious focus.
M. TUBERCULOSIS
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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.
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.
->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
M. TUBERCULOSIS
M. BOVIS
Isolated from cattle, causes TB in ruminants and occasionally humans.
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.->Milk was an important reservoir of infectious bacilli.
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
M. AFRICANUM
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M. AFRICANUM This slow growing mycobacterium (first described
in 1969)is infrequently associated with human pulmonary disease.
causes a disease similar to M. tuberculosis and M. bovis.
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Most patients with this disease reside or have resided in Africa.
Mycobacterium
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Mycobacteriumafricanum probably spreads by an aerosol route
THE MYCOBACTERIUM AVIUM COMPLEX
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Comprise of three species (M.avium, M. intracellulare and M. paratuberculosis.)
->Categorized as slow growing mycobacteria, grow optimally at 37°C and
achieve growth in approximately 4–6 weeks
All three species are common in the environment, (isolated from soil, water,
food and domestic animals.)
Colonies are nonpigmented and are both opaque and domed or flat and
transparen
MYCOBACTERIUM LEPRAE
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Leprosy is particularly dreaded because it may cause bodily deformities
leading to social stigmatization.
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.
MYCOBACTERIUM KANSASII
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This slow-growing organism is the second most frequent cause of
pulmonary disease by a nontuberculous mycobacteria.
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Mycobacterum kansasii is found in environmental water, and water is
probably the source of human infection.
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The clinical presentations of pulmonary M. kansasii are a chronic cough,
lowgrade fever, malaise and chest pain.
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Although extrapulmonary infections are rare, M. kansasii can cause diffuse
cutaneous disease as well as cervical lymphadenitis, especially in children
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Patients with AIDS or with impaired cellular immunity may also
develop a disseminated infection.
MYCOBACTERIUM MARINUM
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M. marinum causes a cutaneus disease in humans known as swimming pool
granuloma, fish handler’s nodule or surfer’s nodule.
->water is the major environmental reservoir
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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,
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.
MYCOBACTERIUM SCROFULACEUM
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Slowly growing mycobacterium, first isolated in 1956, has an
optimal growth temperature of 37°C and, uponinitial isolation,
can take approximately 4 to 6weeks 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,
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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
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Their mode of transmission to humans remains unknown .
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
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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).
Although no bacteria have been cultivated from
environmental sources, the use of polymerase
chain reaction (PCR) has identified M. ulcerans
in water sources
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.
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Large ulcers require extensive surgery with skin grafting. In
extreme cases where muscle tissue is involved, amputation
of an affected limb may be required.
It has long been suspected that a toxin mediates the
extensive tissue damage associated with the Buruli ulcer.
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