Bacillus and Similar Organisms



Bacillus and Similar Organisms



Objectives



1. Describe the general characteristics of B. anthracis, including colonial morphology and Gram stain appearance.


2. State the location of the organisms in the natural environment, and list the modes of transmission as they relate to human infections.


3. Describe the three forms of B. anthracis infection, including source, route of transmission, signs, and symptoms.


4. Summarize the types of infections associated with B. cereus.


5. Outline the laboratory tests utilized to differentiate B. anthracis from other Bacillus species.


6. State the culture media used to differentiate Bacillus spp., and include the chemical principle and interpretation.


7. Summarize the approach to species differentiation within the genera Bacillus, Brevibacillus, and Paenibacillus.


8. Indicate the appropriate therapy for B. anthracis infection.



Genera and Species to Be Considered






General Characteristics


Bacillus species previously were phenotypically classified. With the development of rapid nucleic acid sequencing, the genus has been reorganized based on 16srRNA sequence analysis. The group now contains 53 genera. Bacillus remains the largest genus within this group and contains the most important medically relevant organisms. Bacillus spp. and related genera Brevibacillus and Paenibacillus are aerobic and facultative anaerobic, gram-positive, spore-forming rods. Only the species most commonly associated with human infections are discussed.



Bacillus Anthracis


Clinical microbiologists are sentinels for recognition of a bioterrorist event, especially involving microorganisms such as B. anthracis. Even though this organism is rarely found, sentinel laboratory protocols require ruling out the possibility of anthrax before reporting any blood, CSF, or wound cultures in which a large gram-positive aerobic rod is isolated. During the 2001 terrorist attacks on the United States, the index case associated with the anthrax distribution was discovered by an astute clinical microbiologist who identified large gram-positive rods in a patient’s cerebrospinal fluid. B. anthracis should be suspected if typical nonhemolytic “Medusa head” or ground glass colonies are observed on 5% sheep blood agar. The Red Line Alert Test (Tetracore, Inc., Gaithersburg, Maryland) is a Food and Drug Administration (FDA)-cleared immunochromatographic test that presumptively identifies B. anthracis from blood agar (Figure 16-1). The sentinel laboratory anthrax protocol was revised in 2005 and again in 2010 to use FDA-cleared tests in order to rule out nonhemolytic, nonmotile Bacillus spp. as potential isolates of B. anthracis.


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Figure 16-1 Red Line Alert Test. A red line appears on the cassette if the culture isolate is presumptive Bacillus anthracis. (Courtesy Tetracore, Inc., Gaithersburg, Maryland.)


Epidemiology


Anthrax remains the most widely recognized bacillus in clinical microbiology laboratories. It is primarily a disease of wild and domestic animals including sheep, goats, horses, and cattle. The decline in animal and human infections is a result of the development of veterinary and human vaccines as well as improvements in industrial applications for handing and importing animal products. The organism is normally found in the soil and primarily causes disease in herbivores. Humans acquire infections when inoculated with the spores, either by traumatic introduction, ingestion, or inhalation during exposure to contaminated animal products, such as hides (Table 16-1). Bacillus anthracis produces endospores, which are highly resistant to heat and desiccation. The spores remain viable in a dormant state until they are deposited in a suitable environment for growth, including moisture, temperature, oxygenation, and nutrient availability. Because of the ability to survive harsh environments, infectiousness, ease of aerosol dissemination, and high mortality rate, the spores may be effectively used as an agent of biologic warfare (see Chapter 80 for additional information).




Pathogenesis and Spectrum of Disease


B. anthracis is the most highly virulent species for humans and is the causative agent of anthrax. The three forms of disease are cutaneous, gastrointestinal (ingestion), and pulmonary (inhalation) or woolsorters’ disease (Table 16-2). The cutaneous form accounts for most human infections and is associated with contact with infected animal products. Infection results from close contact and inoculation of endospores through a break in the skin. Following inoculation and incubation period of approximately 2 to 6 days in most cases, a small papule appears that progresses to a ring of vesicles. The vesicles then develop into an ulceration. The typical presentation is of a black, necrotic lesion known as an eschar. The mortality rate for untreated cutaneous anthrax is low, approximately 1%.



TABLE 16-2


Pathogenesis and Spectrum of Disease




















Species Virulence Factors Spectrum of Diseases and Infections
Bacillus anthracis Capsule exotoxins (edema toxin and lethal toxin) swelling and tissue death Causative agent of anthrax, of which there are three forms:
Cutaneous anthrax occurs at site of spore penetration 2 to 5 days after exposure and is manifested by progressive stages from an erythematous papule to ulceration and finally to formation of a black scar (i.e., eschar); may progress to toxemia and death
Pulmonary anthrax, also known as woolsorters’ disease, follows inhalation of spores and progresses from malaise with mild fever and nonproductive cough to respiratory distress, massive chest edema, cyanosis, and death
Gastrointestinal anthrax may follow ingestion of spores and affects either the oropharyngeal or the abdominal area; most patients die from toxemia and overwhelming sepsis
Bacillus cereus Enterotoxins and pyogenic toxin Food poisoning of two types: diarrheal type, characterized by abdominal pain and watery diarrhea, and emetic type, which is manifested by profuse vomiting; B. cereus is the most commonly encountered species of Bacillus in opportunistic infections including posttraumatic eye infections, endocarditis, and bacteremia; infections of other sites are rare and usually involve intravenous drug abusers or immunocompromised patients
Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, other Bacillus spp., Brevibacillus sp., and Paenibacillus spp. Virulence factors unknown Food poisoning has been associated with some species but is uncommon; these organisms may also be involved in opportunistic infections similar to those described for B. cereus

Ingestion anthrax results from ingestion of spores and is presented in two forms: oral or oropharyngeal with the lesion in the buccal cavity, on the tongue, tonsils, or pharyngeal mucosa and gastrointestinal anthrax with the lesions developing anywhere in the gastrointestinal tract. Oropharyngeal symptoms may include sore throat, lymphadenopathy, and edema of the throat and chest. The initial symptoms on gastrointestinal anthrax may be nonspecific with progression to abdominal pain, bloody diarrhea, and hematemesis. The mortality rate is much higher than that of cutaneous anthrax and usually attributed to toxemia and sepsis.


Pulmonary (inhalation) anthrax is due to inhalation of the spores. The endospores are ingested by macrophages and taken to the lymph nodes where the infection develops into a systemic infection. The disease develops from flulike symptoms to respiratory distress, edema, cyanosis, shock, and death. Patients typically demonstrate abnormal chest x-rays with pleural effusion, infiltrates, and mediastinal widening. Woolsorters’ disease and ragpickers’ disease are used to describe respiratory infections that result from exposure to endospores during the handling of animal hides, hair, or fibers and other animal products.


Complications often follow all three forms of anthrax disease. Patients often develop meningitis within 6 days after exposure. Recovery results in long-term immunity to subsequent infections.


Virulence is attributed to the production of anthrax toxin. The toxin consists of three proteins. One of these proteins, protective antigen (PA), facilitates the transport of the other two proteins into the cell. Edema factor, EF, is responsible for edema, whereas lethal factor, LF, is primarily responsible for death.



Bacillus Cereus


B. cereus is another clinically relevant species worthy of identification. It is penicillin resistant, beta-hemolytic, and motile, and it produces a wide zone of lecithinase on egg yolk agar (Figure 16-2).


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Figure 16-2 Lecithinase production by Bacillus cereus on egg yolk agar. The organism has been streaked down the center of the plate. The positive test for lecithinase is indicated by the opaque zone of precipitation around the bacterial growth (arrows).



Pathogenesis and Spectrum of Disease


B. cereus “food poisoning” is associated with the ingestion of a wide variety of foods including meats, vegetables, deserts, sauces, and milk. A higher incidence is seen following the ingestion of rice dishes. Following ingestion patients present with one of two types of symptoms: diarrhea and abdominal pain within 8 to 16 hours or nausea and vomiting within 1 to 5 hours. B. cereus produces several toxins implicated in the diarrheal symptoms, including hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe), and cytotoxin K (CytK). The three toxins are believed to act synergistically, with Nhe responsible for the major symptoms in the diarrheal presentation of the infection. The emetic form of illness is associated with a heat-stable, proteolysis, and acid resistant toxin, cereulide, produced in food.


In addition to the food poisoning associated with B. cereus, it is a serious pathogen of the eye, causing progressive endophthalmitis. Identification of B. cereus from a patient’s eye can cause permanent damage and should be reported to the physician immediately.




Bacillus Subtilis, Brevibacillus Sp., and Paenibacillus Spp.


B. subtilis has been identified in clinical specimens in a variety of cases including pneumonia, bacteremia, septicemia, surgical wounds, meningitis following head trauma, and other surgical infections. Rare human infections have been associated with a variety of Bacillus spp., including B. clausii, B. licheniformis, B. circulans, B. coagulans, B. pumilus, Paenibacillus polymyxa, and Brevibacillus sp. Many of these organisms are common environmental contaminants. Identification of these organisms is not recommended unless isolated from a sterile site (e.g., blood) or found in large numbers in pure culture. Therefore, identification and interpretation should be closely evaluated in conjunction with the patient’s signs and symptoms and consultation with the attending physician.





Laboratory Diagnosis


Specimen Processing


With few exceptions, special processing considerations are not required. The organisms are capable of survival in fresh clinical specimens and standard transport medium. Refer to Table 5-1 for general information on specimen processing.


Specimens collected from patients suspected of having anthrax should be placed in leak-proof containers and placed in a secondary container. Cutaneous anthrax specimens should be collected from underneath the eschar. Two specimens of the vesicular fluid should be collected from underneath the lesion with a swab. For histochemical testing, the physician may collect a punch biopsy. Inhalation anthrax specimens should include blood cultures, pleural fluid, and a serum specimen for serology. Again, the physician may collect biopsy of bronchial or pleural tissue. Specimens required for inhalation anthrax include blood cultures, ascites fluid, and material from any lesions as well as serum for serologic testing. Preferred collection of specimens from patients suspected of infection with B. anthracis should be accumulated prior to antibiotic therapy.


Clinical specimens for the isolation of Bacillus species other than B. anthracis and B. cereus may be handled safely under normal standard laboratory practices. The exceptions are processing procedures for foods implicated in B. cereus food poisoning outbreaks and animal hides or products, and environmental samples, for the isolation of B. anthracis. These specimens may contain spores posing an aerosolization and inhalation risk to the laboratory professional and requiring the use of personal protective equipment including a proper respiratory mask.


Specimen processing may include heat or alcohol shock prior to plating on solid media. The pretreatment removes contaminating organisms, and only the spore-forming bacilli survive. This technique is considered an enrichment and selection procedure designed to increase the chance for laboratory isolation of the organisms.


Despite the publicity associated with B. anthracis as a potential agent of biologic warfare, the organism is not highly contagious. However, disinfection with formaldehyde, glutaraldehyde, or hydrogen peroxide and peracetic acid should be performed before the disposal of specimens suspected of containing a large number of spores. B. anthracis is classified by the Department of Health and Human Services/Centers for Disease Control and Prevention (CDC) and the U.S. Department of Agriculture/Animal and Plant Health Inspection Service (APHIS) as a select agent. Any laboratory in possession of the organism must register with one of these agencies and notify the organization within 7 days upon identification of the organism. If the organism is identified in an unregistered laboratory, the isolate must be shipped, using the request to transfer select agents and toxins approval from CDC or APHIS, to a registered laboratory for proper disposal.



Direct Detection Methods


The Gram stain is the only specific procedure for the direct detection of Bacillus spp. in clinical specimens. Microscopically the organisms appear as large gram-positive rods in singles, pairs, or serpentine changes (Figure 16-3).


image
Figure 16-3 Gram stain of Bacillus cereus. The arrow is pointed at a spore, the clear area inside the gram-positive vegetative cell.

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Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on Bacillus and Similar Organisms

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