Taxonomically, bacteria with rudimentary filaments rank between classic bacilli and the Actinomycetes, bacteria with branching filaments. They represent an important evolutionary link in microbiology. Among the bacteria with rudimentary filaments, Corynebacterium and Mycobacterium are two important pathogenic genera.
Members of the genus Corynebacterium are aerobic to facultative anaerobic, catalase positive, and do not produce spores. They are common constituents of resident microbiota on human skin and in the mouth and upper respiratory tract. More than 16 species are recognized. Of these, C. diphtheriae is the most important pathogenic species. Like several other causal agents of diseases, asymptomatic carriers of C. diphtheriae are not uncommon.
Disease
Corynebacterium diphtheriae causes diphtheria, an acute, highly infectious upper respiratory tract infection that may eventually involve the heart and nervous system. Diphtheria occurs all over the world, especially in poor countries. But major outbreaks have also occurred in more developed countries. For example, an outbreak in 1994 in what was then the Soviet Union affected more than 50,000 persons and caused nearly 2,000 deaths. Otherwise, the widespread use of vaccines in United States and Western Europe has almost eradicated this disease. However, cases of diphtheria are still recorded each year in the United States. The incubation period can vary from 2 to 7 days. The symptoms include sore throat, malaise, inflammation of pharynx and breathing problems. In advanced cases, the heart and central nervous system (CNS) are often involved. The disease is fatal if untreated. Another species, |
| Table 7.1 Some Differentiating Characteristics of Important Corynebacterium spp |
C. ulcerans, can also cause diphtheria-like disease. Some of the physiological differences among clinically significant species of the genus Corynebacterium are summarized in Table 7.1.
Virulence Factors
An exotoxin that blocks protein synthesis in the host has been recognized as an important virulence factor.
Laboratory Diagnosis
The pathogen can be isolated on Loffler’s agar (a selective medium) and blood agar. Incubation is normally done at 35°C with or without CO2 for 24–48 hours. Since Corynebacterium spp. are common constituents of resident microbiota, it is important to differentiate the isolates. Gram staining of smears prepared from laboratory grown cultures mostly shows long rods, and occasionally some rudimentary filaments (Fig. 7.1).
Antibiotic Sensitivity
Corynebacterium is notorious for variable antibiotic sensitivity; therefore, it is important to perform antibiotic sensitivity test. Depending on the sensitivity, penicillin, erythromycin, tetracycline, or even vancomycin may be required. Vaccines have proven quite effective in preventing diphtheria.
Compared with Corynebacterium, Mycobacterium strains tend to show a greater tendency to form rudimentary branching filaments. Such filaments easily break into bacillary bodies due to their fragility. Therefore, in smears, mostly bacilli are seen. Mycobacteria are theoretically Gram-positive, but are difficult to stain and hard to
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Figure 7.1. A Gram-stained smear showing Gram-positive rods and rudimentary filaments of
C. diphtheriae. |
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| Figure 7.2. A photomicrograph of Mycobacterium fortuitum (grown on tap water agar) showing filaments with rudimentary branching (source: CDC) |
kill because the cells are coated with a thick layer of lipid. The best way to see the rudimentary filaments is to grow them in a slide culture or observe undisturbed growth on a nearly transparent medium using a long working distance (LWD) objec- tive lens (Fig. 7.2). At least 50 species are known; about seven are pathogenic. Ziehl-Nelsen stain, also called acid-fast staining, is very useful in separating Mycobacteria from actinomycetes. The staining involves treating the smear with a boiling Carbolfuchsin solution, decolorizing with acid alcohol, and counterstaining with malachite stain. Because mycobacteria can resist decolorization, they are also called acid-fast bacilli (AFB).
Mycobacterium tuberculosis
Mycobacterium tuberculosis is the most important causal agent of tuberculosis. Other species, such as M. bovis, M. kansasii, M. gordonae, M. avium, M. fortuitum, M. phlei, and M. smegmatis, are also considered pathogenic, but mostly for immunecompromised subjects. Of these, M. fortuitum, M. phlei, and M. smegmatis are fast growers. Mycobacterium avium and M. bovis are primarily known to cause tuberculosis in birds and cattle, respectively. Tuberculosis is perhaps the most dreaded disease of a global significance. The actual number of people suffering from tuberculosis on any given day is anybody’s guess, but the estimates range from nearly two billion cases in 2000, to more than 200 million on other occasions. Perhaps the actual number will never be known but it is a fact that a large number of people suffer from tuberculosis, from a benign subclinical to active fulminating disease. It is also a fact that the disease is curable and there is no reason why it could not be eradicated from the face of the earth. Important physiological differences among clinically significant species of the genus Mycobacterium are noted in Table 7.2.
Disease
Mycobacterium tuberculosis is the principal causal agent of tuberculosis, a slowly progressing disease that may involve any organ including the lungs, brain, kidney,
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| Table 7.2 Comparison of Some Properties of Clinically Important Species of the Genus Mycobacterium (Modified after Howard et al., Clinical and Pathogenic Microbiology, Mosby, St. Louis) |
* Believed to be a common waterborne bacterium. Almost all human cases were noted in AIDS patients.
** Fast-growing bacteria that have been isolated from water and sewage as well.
and the gastrointestinal tract. Bronchopulmonary tuberculosis is the most common form of the disease. Its symptoms include cough, blood-tinged sputum, malaise, weight loss, and mild fever, which may rise in the afternoon. Radiologically, the appearance may range from pulmonary abscess to cavitary or miliary forms of tuberculosis. The incubation period can be as short as a few weeks to several years or decades. Infection can stay dormant for many years but flare up when immunity is diminished. Tuberculosis is a slow killer and it is almost always fatal if not treated. It is a classic example of airborne (bioaerosol borne) disease.
Virulence Factors
No clear-cut virulence factors are recognized except that the pathogen can grow in unactivated alveolar macrophages. Host factors, most importantly cellular immunity, play a critical role in the pathogenesis of tuberculosis. Therefore, those with HIV or on immunosuppressive drugs, as well as those with diabetes and cancer are at increased risk. Also, individuals with genetic or drug-induced defects in the interferon gamma pathway may be particularly susceptible to mycobacterial disease.
Laboratory Diagnosis
In the case of the adults, a first morning sputum every 24 hours for 3 days is examined. For children, a gastric washing is preferred. Occasionally, especially in the case of adults, a sample of bronchial aspirate is more helpful. Sputum is mixed with an equal amount of NALC (N-acetyl L-cysteine): NaOH, mixed vigorously, and centrifuged at 3,000 RPM for about 15 minutes. The sediment is streaked on Lowenstein-Jensen agar or Middle brook agar, which are selective media. Cultures are incubated for up to 3 weeks or longer at 35°C in aerobic conditions. Biopsied tissues do not require NALC : NaOH treatment. The colonies of M. tuberculosis appear rough and nonto slightly pigmented. Other strains may produce smooth colonies with yellowish-orange pigment. Skin test using old tuberculin (OT) or a purified protein derivative (PPD) can be helpful in making a presumptive diagnosis, if supported by clinical and radiological findings. In much of Asia, Africa, and Latin America, a positive skin test merits several considerations. Since a rather large number of subjects are exposed to the pathogen, many are tuberculin positive but devoid of any positive radiological and clinical findings. This may suggest latent tuberculosis infection. However, often these individuals have been previously vaccinated, making interpretation difficult. Newer interferon gamma release assays may help to distinguish latent tuberculosis from merely vaccinated indi- viduals. If a person is suspected to have latent tuberculosis infection, he or she should be evaluated by an expert to determine if treatment is appropriate. Stained smears mostly show acid-fast bacilli. As stated earlier, unlike the slide cultures, in prepared smears mostly bacilli are seen as depicted in an electron micrograph in Figure 7.3.
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| Figure 7.3. A scanning electron micrograph of M. tuberculosis, 15,000× (source: CDC) |
Antibiotic Sensitivity
Rifampin or ethambutol, isoniazid (INH), and pyrazinamide are quite effective in the clinical management of tuberculosis. A large number of strains are now considered streptomycin resistant. In most cases, the clinicians prefer to start with all four drugs, rifampin, ethambutol, INH, and pyrazinamide. Once the sensitivity results are available, the number of drugs may be reduced. Streptomycin, though rarely used these days, can be helpful in drug resistant cases. In order to fully eradicate the infection, a long term therapy, often ranging from 6 to 9 months to more than 1 year is required.
Mycobacterium leprae
Disease
Mycobacterium leprae causes leprosy, a highly infectious/contagious disease that mostly involves skin and the periphery. Daily showering and soap application has almost eliminated this disease in the industrialized world. However, approximately 10 million people still suffer from leprosy globally. Cases of leprosy have been reported, even in Texas, Louisiana, and California, but mostly in prison inmates. Infection is generally acquired through direct skin-to-skin contact or contact with contaminated fomites.
Laboratory Diagnosis
Most strains of M. leprae cannot be cultured on laboratory media. No vaccination is available. Diagnosis is often made on the basis of clinical symptoms and demonstration of acid-fast bacilli in clinical specimens (Fig. 7.4).
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| Figure 7.4. Acid-fast bacilli in a skin biopsy from a leprosy patient (source: CDC). See color insert. |
Antibiotic Sensitivity
The clinical management of leprosy is achieved by therapy with rifampin in combination with dapsone given over a long period of time. Proper personal hygiene and avoiding direct contact with infected patients can be a helpful preventive measure.
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