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Mycobacteriology *


Abstract

Background

There are currently approximately 200 recognized species of mycobacteria. Although some species are strictly environmental organisms, several are significant human pathogens, including Mycobacterium tuberculosis . M. tuberculosis is responsible for nearly 1.5 million deaths each year, and nontuberculous mycobacteria (NTM), such as Mycobacterium avium complex (MAC), are also responsible for significant morbidity and mortality.

Content

This chapter describes the laboratory methods used for the detection and identification of Mycobacterium species. Methods used for mycobacteria detection and identification are continually evolving to achieve more rapid, cost-effective, and accurate results. Traditional microbiologic methods such as morphology and biochemical profiling have given way to molecular detection and identification methods; however, acid-fast staining and culture for mycobacteria remain at the core of any diagnostic algorithm. After growth in culture, molecular technologies such as nucleic acid hybridization probes, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and DNA sequencing are used for definitive species identification. Nucleic acid amplification methods allow for culture-independent, direct detection of the M. tuberculosis complex (MTBC) and MAC within respiratory specimens and predict susceptibility to anti-infective therapy, which leads to more rapid diagnoses of appropriate patient care.

Introduction and overview of mycobacteria

Mycobacteria are obligately aerobic, nonmotile, rod-shaped bacilli. Members of the genus Mycobacterium have several unique characteristics compared with other genera of bacteria, primarily due to structural differences in cell wall composition. The cell walls of mycobacteria contain a higher content of complex lipids (>60%, as opposed to approximately 5 and 20% in gram-positive and gram-negative organisms, respectively), including long-chain (C 60 –C 90 ) fatty acids called mycolic acids. Mycolic acids make the cell wall extremely hydrophobic and enhance resistance to desiccation, killing by disinfectants, staining with basic aniline dyes, and penetration by many of the antibiotics used to treat infections caused by other bacteria. These unique features of the mycobacterial cell wall structure provide the basis for distinct laboratory considerations when performing direct stains from specimens, growing organisms in culture, and determining species identification by molecular methods.

Classification of mycobacteria and clinical significance

As discussed in the following paragraphs, mycobacteria can be categorized into groups comprising members of the Mycobacterium tuberculosis complex (MTBC), other slowly growing nontuberculous mycobacteria (those that require >7 days for growth upon subculture), rapidly growing nontuberculous mycobacteria (those that require <7 days for growth upon subculture), and uncultivatable species ( Box 86.1 and Table 86.1 ). A recent comparative genomics study suggested that the genus Mycobacterium consisted of five monophyletic groups and proposed dividing the genus into distinct groups consisting of Mycobacterium species, Mycolicibacterium species, Mycolicibacter species, Mycolicibacillus species, and Mycobacterioides species. This proposal has met with resistance due to the perceived potential for confusion and possible failure to recognize the clinical importance of species previously falling in the Mycobacterium genera. Currently, the genera listed above are considered synonyms of Mycobacterium.

BOX 86.1
Major Groups of Mycobacteria

Slowly growing

  • Mycobacterium tuberculosis complex

    • M. tuberculosis

    • M. bovis (cattle)

    • M. bovis BCG (vaccine strain)

  • M. avium complex

  • M. genavense

    • Requires Mycobactin J supplement and prolonged incubation

  • M. gordonae

    • Tap water mycobacterium

    • Generally not a human pathogen

  • M. haemophilum

    • Requires X-factor supplement and incubation at lower temperature

  • M. marinum

    • Fish tank mycobacterium

    • Requires incubation at lower temperature

Rapidly growing

  • M. abscessus complex

    • Resistant to many antimycobacterial agents

    • Some subspecies have an inducible erm gene that conveys macrolide resistance

  • M. chelonae

  • M. fortuitum

  • M. smegmatis

Uncultivatable in the laboratory

  • M. leprae

TABLE 86.1
Major Clinical Syndromes Associated With Nontuberculous Mycobacterial Infection
From Brown-Elliott B, Wallace R Jr. Infections caused by nontuberculous mycobacteria other than mycobacterium avium complex. In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett’s principles and practices of infectious diseases. 8th ed. Philadelphia: Saunders; 2015:2844–52.
Syndrome Most Common Causes Less Frequent Causes
Chronic nodular disease (adults with bronchiectasis; cystic fibrosis) MAC, M. kansasii, M. abscessus M. xenopi , M. malmoense , M. szulgai , M. smegmatis , M. celatum , M. simiae , M. goodii , M. asiaticum , M. heckeshornense , M. branderi , M. lentiflavum , M. triplex , M. fortuitum , M. arupense , M. abscessus subsp. bolletii , M. phocaicum , M. aubagnense , M. florentinum , M. massiliense , M. nebraskense , M. saskatchewanense , M. seoulense , M. senuense , M. paraseoulense , M. europaeum , M. sherrisii , M. kyorinense , M. noviomagense , M. mantenii , M. shinjukuense , M. koreense , M. heraklionense , M. parascrofulaceum , M. arosiense
Cervical or other lymphadenitis (especially children) MAC M. scrofulaceum , M. malmoense (northern Europe), M. abscessus , M. fortuitum , M. lentiflavum , M. tusciae , M. palustre , M. interjectum , M. elephantis , M. heidelbergense , M. parmense , M. bohemicum , M. haemophilum , M. europaeum , M. florentinum , M. triplex , M. asiaticum , M. kansasii , M. heckeshornense
Skin and soft tissue disease M. fortuitum group, M. chelonae, M. abscessus, M. marinum, M. ulcerans (Australia, tropical countries only) M. kansasii , M. haemophilum , M. porcinum , M. smegmatis , M. genavense , M. lacus , M. novocastrense , M. houstonense , M. goodii , M. immunogenum , M. mageritense , M. abscessus massiliense , M. arupense , M. monacense , M. bohemicum , M. branderi , M. shigaense , M. szulgai , M. asiaticum , M. xenopi , M. kumamotense , M. setense , M. montefiorense (eels), M. pseudoshottsii (fish), M. shottsii (fish)
Skeletal (bone, joint, tendon) infection M. marinum, MAC, M. kansasii, M. fortuitum group, M. abscessus, M. chelonae M. haemophilum , M. scrofulaceum , M. heckeshornense , M. smegmatis , M. terrae/chromogenicum complex, M. wolinskyi , M. goodii , M. arupense , M. xenopi , M. triplex , M. lacus , M. arosiense
Disseminated infection MAC M. genavense , M. haemophilum , M. xenopi
HIV-seropositive host M. avium , M. kansasii M. marinum , M. simiae , M. intracellulare , M. scrofulaceum , M. fortuitum , M. conspicuum , M. celatum , M. lentiflavum , M. triplex , M. colombiense , M. sherrisii , M. heckeshornense ,
HIV-seronegative host M. abscessus , M. chelonae M. marinum , M. kansasii , M. haemophilum , M. chimaera , M. conspicuum , M. shottsii (fish), M. pseudoshottsii (fish)
Catheter-related infections M. fortuitum, M. abscessus, M. chelonae M. mucogenicum , M. immunogenum , M. mageritense , M. septicum , M. porcinum , M. bacteremicum , M. brumae
Hypersensitivity pneumonitis M. avium (hot tub) M. immunogenum (metal working fluid)
Too little information is available for selected pathogens such as M. xenopi, M. malmoense, M. szulgai, M. celatum, and M. asiaticum and the newly described species.
MAC, Mycobacterium avium complex.

Mycobacterium tuberculosis complex

The MTBC includes six accepted Mycobacterium species: M. tuberculosis , M. bovis , M. africanum , M. caprae , M. microti , and M. pinnipedii , in addition to three proposed species, M. canettii , M. mungi, and M. orygi s. M. bovis bacillus Calmette-Guérin (BCG) is an attenuated strain of M. bovis used as a vaccine in some parts of the world. Of these, M. tuberculosis , M. bovis , and M. bovis BCG account for most human incidences of disease. Despite a wide diversity of host ranges, the species within the MTBC are genetically homogeneous, with nucleotide variation rates of 0.01 to 0.03%. Genetic variation over time and the sharing of genetic information among species are rare. Clinically, identifying MTBC members to the species level is essential due to varying susceptibility patterns among commonly used antituberculous treatments.

Mycobacterium tuberculosis

First discovered by Robert Koch in 1882, M. tuberculosis is the most crucial member of the MTBC, causing 93 to 97% of pulmonary tuberculosis disease worldwide. , Unlike other members of the MTBC, humans are the definitive host of M. tuberculosis.

Mycobacterium bovis

M. bovis is the causative agent of tuberculosis in a wide range of mammals, including humans, dogs, cattle, cats, pigs, and deer. Infection with M. bovis accounts for 1 to 2% of human tuberculosis cases, and human disease caused by M. bovis is similar to that caused by M. tuberculosis. Importantly, M. bovis is intrinsically resistant to pyrazinamide, a first-line treatment agent for tuberculosis disease.

Mycobacterium bovis bacillus calmette-guérin

M. bovis BCG is an attenuated subtype of M. bovis, first distributed in 1921 to prevent M. tuberculosis infection. Strains of M. bovis BCG have been independently maintained and serially passaged in vitro, leading to global heterogeneity and strain-dependent vaccine efficacy. Intravesicular BCG instillation is used to treat bladder cancer and selected other cancers, and in rare instances, M. bovis BCG may disseminate after treatment or vaccination. ,

Mycobacterium africanum

M. africanum is most prevalent in West Africa, representing over half of tuberculosis cases in this region. Reports of M. africanum detected outside of Africa, including cases in the United States, are often associated with individuals who have lived in West Africa. This organism closely resembles M. tuberculosis in the pathogenesis and severity of the disease.

Mycobacterium caprae

Historically associated with tuberculosis in goats, M. caprae may also cause disease in sheep, pigs, wild boars, red deer, and foxes. Human disease is rare and often associated with animal exposure. Notably, M. caprae is susceptible to pyrazinamide, an important criterion used to differentiate M. caprae from M. bovis .

Mycobacterium microti

M. microti is classically associated with the infection of rodents. However, cases of infection of rabbits, llamas, cats, and meerkats have been reported. M. microti displays a classic croissant-like morphology in a direct smear, which is notably different from common acid-fast bacilli (AFB) morphologies. Despite detection on direct smear, M. microti often fails to grow in culture. ,

Mycobacterium pinnipedii

Defined in 2003, M. pinnipedii sp. nov. is most frequently detected in pinnipeds such as seals and walruses. However, infection has also been reported in guinea pigs, rabbits, and larger animals residing near pinnipeds. Transmission from sea lions to humans has been noted in case reports, causing granulomatous lesions in the lymph nodes, pleura, spleen, and lungs.

Proposed taxa

Mycobacterium canettii

First described in 1969, M. canettii is most abundant in Africa. Interestingly, genetic evidence suggests M. canettii may be the source species for M. tuberculosis. In culture, M. canettii more closely resembles other slow-growing nontuberculous mycobacteria, appearing as smooth, round, glossy colonies.

Mycobacterium mungi and mycobacterium orygis

M. mungi has been detected as a causative agent of tuberculosis in the banded mongoose, and outbreaks among mongoose troops are associated with high mortality rates. Transmission dynamics between infected mongooses and humans are currently unknown. M. orygis is the causative agent of tuberculosis in larger African mammals, including oryxes, gazelles, antelopes, waterbucks, and cows and rhesus monkeys of South Asian origin. Tuberculosis in humans caused by infection with M. orygis has been reported in South Asia.

Global public health burden

Tuberculosis is a significant cause of mortality due to infectious disease worldwide. The incidence of tuberculosis declined from the early 20th century through the 1970s; however, in the 1980s, numbers of new infections began steadily climbing, and this climb has been attributed to complacency, decreased funding of anti-tuberculosis public health efforts, the beginning of the HIV epidemic, and the emergence of drug-resistant strains of tuberculosis. Global incidence peaked in 2003, but with coordinated global efforts, it has begun to decline once more. Currently, more than 2 billion individuals, representing roughly one-quarter of the world’s population, are estimated by the World Health Organization (WHO) to be infected with M. tuberculosis . , In 2018, an estimated 10 million people became ill with M. tuberculosis , and 1.5 million died due to the disease. The highest incidence rates occur in India, China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh, and South Africa. These nations account for 87% of new cases of tuberculosis disease. Coinfection with HIV is common, with HIV-positive individuals reported to be 19 times more likely to develop active tuberculosis than non–HIV-infected individuals. A stark difference exists in the epidemiology of disease in industrialized countries versus developing countries, where 80% of disease cases in industrialized countries occur in the elderly population, whereas 80% of disease cases in developing countries occur in individuals aged 15 to 50 years. In the United States, incidence rates have been declining since 1992, falling to a historic low of 2.8 per 100,000 in 2018. , Despite this, 13 million people in the United States have latent M. tuberculosis infection, most of whom are foreign-born immigrants from endemic countries.

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