Chapter 11 The Genus Mycobacterium

Mycobacteria cover the range from saprophytes to opportunists to obligate pathogens, and have close relatives in other genera of pathogenic bacteria (Table 11-1). All members of the genus are aerobic, acid-fast, non–spore-forming, nonmotile gram-positive rods. They often stain irregularly and appear somewhat beaded. They are catalase positive. Many produce pigments, which are a means of classification and differentiation; scotochromogenic organisms produce pigments whether incubated in light or dark, whereas in photochromogenic organisms, light is required for pigment production (Figure 11-1). Most mycobacteria have relatively simple growth requirements. Mycobacterium tuberculosis, for example, can be cultivated in a synthetic liquid medium with only trace metals, asparagine, and glycerol. Rapidly growing mycobacteria produce colonies on solid media in less than 7 days, and slow growers require 7 to 14 days (or longer, if growth must be initiated from low-titer inocula), and, in some cases, as long as several months, to exhibit recognizable colonial growth. To put this in perspective, the generation time of Escherichia coli is usually accepted to be about 15 to 20 minutes, whereas that of Corynebacterium diphtheriae is closer to 60 minutes. Mycobacterium tuberculosis, on the other hand, completes a single round of cell division in about 300 minutes; Mycobacterium avium ssp. paratuberculosis is perhaps the most slow-growing of all the mycobacteria, in that cultures for diagnosis of Johne’s disease are not discarded as negative until they have incubated for greater than or equal to 5 months.

TABLE 11-1 Differentiation of Mycobacteria from Closely Related Genera

FIGURE 11-1 Mycobacterium marinum, cultivated on Middlebrook’s 7H10 agar.

(Courtesy J. Glenn Songer.)

Acid fastness refers to the ability of mycobacterial cells to bind phenol-based dyes (for example, carbol fuchsin in 5% phenol), typically when heated while staining; the dye is retained whenthe smear is subsequently treated with acidified alcohol. Whereas characteristic of mycobacteria, acid-fastness occurs in other genera of bacteria and even in certain life stages of some eukaryotes.

Complex lipids in mycobacterial cell walls include the mycolic acids, which are also found in related genera (Figure 11-2; see Table 11-1); acid fastness relates to the presence of peptidoglycan and glycolipids. This cell wall composition is also responsible for resistance of mycobacteria to drying, extremes of pH, and other environmental stresses. The complex, lipid-rich cell wall alsoprotects the organism in the phagolysosome, and probably plays a major part in mycobacterial survival in macrophages. Furthermore, components of the cell wall are immunostimulatory, and are the basis for adjuvants, including Freund’s complete and N-acetyl-muramyl-L-alanyl-D isoglutamine, or muramyl dipeptide (MDP).

FIGURE 11-2 Schematic representation of mycobacterial cell wall composition.

(Courtesy Ashley E. Harmon.)

Some mycobacteria are major pathogens of domestic animals, whereas others are encountered only occasionally (Table 11-2).

TABLE 11-2 Mycobacteria of Veterinary Significance

MYCOBACTERIUM TUBERCULOSIS COMPLEX

Members of the M. tuberculosis complex differ widely in host preference, phenotype, and virulence, despite 99.9% nucleotide sequence similarity and identical 16S ribosomal ribonucleic acid (rRNA) sequences. They are assumed to be derived from a common ancestor, yet some (Mycobacterium tuberculosis, Mycobacterium africanum, and Mycobacterium canettii) are almost exclusively human pathogens, whereas others (Mycobacterium bovis) are more cosmopolitan in host preference.

Mycobacterial evolutionary dogma has held that M. bovis infecting humans became host adapted, giving rise to what we now know as M. tuberculosis, and that conservation of housekeeping genes within the M. tuberculosis complex suggests that these organisms have been caught in an evolutionary bottleneck since the time of differentiation into species, possibly about 15,000 to 20,000 years ago. However, the availability of whole genome sequences for many of these organisms, and the advent of comparative genomics, promises to bring major changes in our view of evolutionary relationships among pathogenic mycobacteria.

Infection by M. tuberculosis is primarily a problem in humans and subhuman primates, but dogs, canaries, psittacine birds, swine, and many other species are susceptible to human tuberculosis. Feline cutaneous tuberculosis is associated with infection by M. tuberculosis or M. bovis and presents as multiple exudative ulcers and abscesses, in the form of pyogranulomatous dermatitis with caseous necrosis. Mycobacterium tuberculosis should be part of the differential diagnosis in any granulomatous disease of warm-blooded animals.

In the early 1800s, tuberculosis was an epidemic disease in the United States and Europe, with an annual death rate approaching 1% of the population in some cities. The incidence and prevalence of disease declined with improved living conditions, screening, and aggressive case studies to identify and deal with contacts; the availabilityof antimicrobials in the early 1950s provided another tool for dealing with the disease.

However, even at the times when the incidence and prevalence were lowest, tuberculosis made a major impact on human health worldwide: about 2 billion people were infected, with 8 millionnew cases per year. Tuberculosis caused more than 5% of the infant deaths, nearly 20% of adult deaths, and, perhaps worst of all, more than 25% of avoidable deaths.

Regardless, success in decreasing incidenceof tuberculosis pushed it into the shadows ofthe public health enterprise in the United States; research, screening, and drug discovery were essentially halted in favor of topics considered more important. Dismantling of antituberculosis programs left physicians and public health officials unprepared to deal with the disease, in large part because of an erosion of diagnostic capabilities. In this environment of neglect, the incidence of tuberculosis has made well-publicized increases, and is once again a major focus of public health concern and infectious disease research. Part of this is a result of failures in management of screening programs for tuberculosis in immigrants from endemic countries; at one time, fully one quarter of new tuberculosis cases in United States werein immigrants. The increase in prevalence was encouraged by crowded conditions in homeless shelters and prisons, and by the increased sus-ceptibility of substance abusers and patients with acquired immunodeficiency syndrome (AIDS). In developing countries, the incidence of tuberculosis is higher in AIDS patients than in the noninfected population, and the time from exposure to shedd-ing is greatly shortened, facilitating rapid spread of infection. Drug resistance had been a problem since the early days of streptomycin use, but this became worse with the emergence of multidrug resistance. The case fatality rate of untreated tuberculosis can be as high as 50%, and the arising of untreatable strains through mutation to drug resistance presents a serious public health concern. Other infectious diseases have received significantly greater press coverage and political attention, but few have had the sustained negative impact on human health of M. tuberculosis infection.

Pathogenesis

Mycobacterium tuberculosis can infect any area of body, including bones, joints, liver, spleen, gastrointestinal tract, and brain. In these sites its preferred residence is within cells of the reticuloendothelial system. Tuberculosis is not normally a rapidly developing disease, but is rather, if untreated, associated with years of declining health, culminating in death. The pace of the clinical course is more rapid in the immunocompromised, often lasting only a few months, and with a case fatality rate of nearly 80%. The disease is also not highly contagious, but transmission occurs with prolonged contact between susceptible individuals and an active case. Transmission is usually by the airborne route, and bacteria-bearing droplets must be of a size and mass to allow them to penetrate deep into the respiratory tree and impinge into mucus overlying the epithelium. Infection can occur by other routes, as well; ingestion of the organism can lead to infection through cervical or mesenteric lymph nodes. Individuals developing tuberculosis experience fever, cough (often with bloody sputum), malaise, and anorexia, with progressive, irreversible lung destruction.

Regardless of the route of infection, the organism is phagocytosed by macrophages, probably following complement-mediated opsonization. Defense of the lungs centers, of course, onthe work of pulmonary alveolar macrophages;survival and multiplication of M. tuberculosis in phagocytes is the key factor in development of disease. Phagosome-lysosome fusion occurs, but the organisms either escape to the cytoplasm or simply multiply within the phagolysosome, eventually causing it to burst. Part of the success of M. tuberculosis in this endeavor lies in its abilityto prevent acidification of the phagosome, which decreases the killing capacity of phagocytes. Mycobacteria in general are relatively resistantto the bactericidal mechanisms of professional phagocytes, with resistance to reactive oxygen inter-mediates associated with catalase, peroxidase, and alkyl hydroperoxidase reductase production; the last also mediates resistance to reactive nitrogen intermediates. Mycobacterial sulfolipids may inhibit phagosome:lysosome fusion and potentiate the cord factor–induced inhibition of oxidative phosphorylation in mitochondria. Phenolic glycolipids in the cell wall scavenge and detoxify oxygen radicals. Furthermore, M. tuberculosis produces compounds that may interfere with T-cell activation. Lipoarabinomannan, a mycobacterial cell wall glycolipid, suppresses T-cell proliferation, blocks transcriptional activation of interferon (IFN)-γ–inducible genes in macrophages cell lines, and might prevent macrophage activation by IFN-γ. A fibronectin-binding protein may be involved in fibronectin depletion, making it unavailable for binding and stimulation of T cells and interfering with the activated macrophage response.

The ability to mount a rapid and effective activated macrophage response determines the outcome of an encounter with M. tuberculosis. The immune system effectively contains the infection, and less than 10% of those infected developdisease. In healthy adults exposed to relatively low numbers of mycobacteria, the immune response stops the infection before appreciable damage to the lung, and, although the patient will likely become skin-test positive, symptomatic tuberculosis does not develop. In many cases M. tuberculosis is not eradicated but is contained in discrete lesions, and disease may develop through reactivation when resistance is weakened.

The immune response to M. tuberculosis isT-cell dependent, but the immune mechanisms of acquired resistance are associated with activation of macrophages by cytokines and direct cytolytic activity. The initial interaction between M. tuberculosis and macrophages elicits a T-helper (CD4) cell response; the Th2 subset mediates antibody production that has been repeatedly shown to have no role in protection against or recovery from infection. The main contribution of CD4 T cellsis by those of subset Th1, which release IFN-γ, stimulating activation of macrophages, which then ingest and kill mycobacteria. IFN-γ also stimulates endothelial binding and emigration of T cells, allowing them to converge on the infected area; transgenic mice incapable of producing IFN-γare much more susceptible than the wildtype to M. tuberculosis infection. The CD8 response yields cytotoxic T cells that kill and disrupt infected phagocytes. In addition, gamma-delta T cells recognize phospholigands and CD1-restricted T cells recognize glycolipids, both of which are plentiful in the mycobacterial cell wall.

If the immune response is delayed or nonex-istent, viable M. tuberculosis may reach regional lymph nodes and even pass farther by way of lymphatics and the bloodstream, to distant tissues, nearly always within macrophages. Most bacteria, however, are contained locally, in a specific typeof granuloma called a tubercle (Figure 11-3); layers of T cells, neutrophils, macrophages, multinucleated giant cells, and a thick fibrincoat form around growing foci of necrosisand sometimes wall off the lesion. Calcified tubercles appear as lesions in chest radiographs. The walled-off lesions may contain viable bacteria, leaving open the possibility of reactivation tuberculosis.

FIGURE 11-3 Hematoxylin and eosin–stainedsection of a tubercle in a case of bovine tuberculosis.

(Courtesy J. Glenn Songer.)

Mechanisms of tissue destruction in tuberculosis are not fully understood, but certainly involve local and systemic inflammatory responses. Cord factor (trehalose 6,6’-dimycolate), a component of the mycobacterial cell wall, is systemically lethal, inactivates phagocyte mitochondria, and inhibits macrophage chemotaxis. Lung damage may also result from the action of TNF-α, which accumulates in response to mycobacterial cell wall components; injection of TNF-α into lungs causes damage suggestive of tuberculosis, and administration of antibodies against TNF-α reduces lung damage in infected animals without affectingbacterial growth.

Laboratory Diagnosis of Tuberculosis

Diagnosis is based in part on microscopic exami-nation of acid-fast–stained sputum smears. Fluo-rochrome stains (auramine O-acridine orange) are also useful, and evidence suggests that they are more sensitive than traditional acid-fast stains. Bacteriologic culture of appropriate specimens is imperative, both to confirm the etiology and allow for sensitivity testing. Isolation is facilitated by the resistance of the organism to disinfectants and extremes of pH. Steps in the procedure may include treatment with N-acetyl L-cysteine (to liquefy sputum samples), exposure to high pH, or disinfection with quaternary ammonium compounds. Culture is often on inspissated egg-based media (such as Lowenstein-Jensen), agar-based egg media (such as Herrold’s medium), or non-egg media (such as Middlebrook’s 7H10), prepared as slants to allow humidity control. Mature colonies become apparent after 14 to 21 days’ incubation.

The diagnostic key to rapid surveillance has been the intradermal skin test. Tuberculin (a crude extract of the M. tuberculosis cell wall)or purified protein derivative (PPD) injected intradermally stimulates cytokine secretion by preprimed CD4 T-helper cells, which in turn recruits neutrophils, mononuclear cells, and macrophages to the site. If erythema and induration follow, the test is positive. Conversion to positive occurs within about 1 month of exposure to M. tuberculosis, and is associated with immunization. Many with active tuberculosis, especially disseminated disease, convert to skin-test negative, in an immune phenomenon that can be specific for reactivity to tuberculin or PPD, rather than a general decrease in immune competence. However, any general decrease in immune competence can convert a positive skin test to negative. The current recommendation is that recent converters take the full course of therapy for tuberculosis.

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