General Characteristics

Members of the genus Bacillus are aerobic, grampositive, rod-shaped organisms that form end os pores (Figure 17-2). On Gram stain, however, spores do not stain and appear only as “empty spaces.” The spore stain is used to demonstrate spores (Figure 17-3).

More than 50 species of Bacillus are widely found in the environment. Members of the genus can be

isolated from soil in all climates-the subarctic and desert regions, thermal springs, fresh and salt water, and plant materials. The temperature range for growth depends on the species but includes temperatures as low as _5° C and as high as 75° C. The survival of Bacillus spp. in nature is aided because of the formation of endospores, which are resistant to conditions to which vegetative cells are intolerant.

Bacillus spp. are found as contaminants in specimens from a number of sources. Most species grow well on sheep blood agar (SBA) and other commonly used enriched media. Colony characteristics vary considerably

among the species and are often influenced by the type of media used. Pigment formation is seen in a number of species. Pigment colors range from pink to blue-black and may vary according to growth conditions and substrates. Most species, however, are nonpigmented.

The different species show a wide variety of metabolic characteristics. They are catalase positive and form endospores under aerobic conditions. The Bacillus spp. can be divided into three morphologic groups on the basis of the location and size of the endospore. In group 1, the spores are oval or cylindrical and are located centrally or terminally. The key characteristic placing a species in group I is the spore. Regardless of location, it does not distend the vegetative cell.

Organisms belonging to group II have oval spores that are central or terminal and cause the vegetative cell to swell. Morphologic group III consists of species showing round, terminal, swollen spore. Spore formation takes place aerobically; this feature, along with a positive catalase test, differentiates Bacillus spp. from the clostridia.

B. anthracis was shown to cause anthrax in cattle by Robert Koch in the development of Koch’s postulates

in helping to prove the germ theory of disease.

Historically, B. anthracis, the causative agent of anthrax, has been the most important member of this genus;

however, anthrax is rarely seen in the United States. Other species, most notably B. cereus (Figure 17-4), are seen in the clinical laboratory as pathogens and contaminants. Several species are pathogenic for insects.


 Bacillus anthracis

Virulence Factors

The virulence of B. anthracis depends on a glutamic acid capsule and a three-component protein exotoxin.

The genes that code for the toxin and the enzymes responsible for capsule production are carried on plasmids. If a virulent isolate is repeatedly cultured in vitro, the plasmid can potentially be lost, and the organism is no longer virulent. The capsule, which protects the organism from phagocytosis, is a polypeptide of D-glutamic acid. This particular isomer of glutamic acid is resistant to hydrolysis by host proteolytic enzymes because it is the “unnatural” form of the amino acid. Although the capsule is necessary for virulence, antibodies against the capsule do not confer immunity. Anthrax toxin consists of three proteins called protective antigen (PA), edema factor (EF), and lethal factor (LF), each of which individually is nontoxic but together act synergistically to produce damaging effects.

Protective antigen serves as a necessary binding molecule for EF and LF,permitting their attachment to specific receptors on the host cell’s surface, where the toxins exert their cytotoxic effects. The effect of Efand LF is seen when either is combined with PA. Edema results from the combination of PA with EF, whereas death occurs when PAand LFcombine. Edema factor is an adenylate cyclase that increases the concentration of cyclic adenosine monophosphate (cAMP) in host cells. LFis a protease that kills host cells by an unknown mechanism.

Clinical Infections

Anthrax is a common disease in livestock worldwide when the vaccine is not used. The disease is not spread from animal to animal, but rather by animals feeding on plants contaminated with the spores. Humans are infected primarily as a result of accidental or occupational exposure to animals or animal products. Human anthrax in the United States is rare; generally, less than three cases per year are reported. In 2001, however,11 cases of inhalation and 11 cases of cutaneous anthrax occurred in the United States. Investigations into these cases revealed that they were bioterrorismrelated.

Cases mostly occurred among postal workers

as a result of exposure to spore-tainted material (powder in or on envelopes) sent through the mail, although for some cases, the actual source remains unknown. Refer to Chapter 30 for a discussion of B. anthracis as an agent of bioterrorism.

Worldwide, however, cases of anthrax number several thousand. The disease is enzootic in many parts of the world, including Africa, Central America, and South America. The largest outbreak of largely cutaneous anthrax occurred in Zimbabwe in the early 1980s, with approximately 10,000 cases. A number of names have been given to infections with B. anthracis. The majority refer to occupational associations. Terms such as woolsorter’s disease and ragpicker’s disease were used to

describe infection with the spores of B. anthracis as a result of handling contaminated animal fibers, hides, and other animal products. Three forms of anthrax are recognized in humans: cutaneous, inhalation or pulmonary, and gastrointestinal. Allthree forms of infection result from wound contamination, inhalation, or ingestion of spores, which germinate within the host tissue.


Cutaneous Anthrax

Cutaneous anthrax may occur when wounds are contaminated with anthrax spores acquired through skin cuts, abrasions, or insect bites. The overwhelming majority of anthrax cases in the world are cutaneous. In this form of anthrax, a small pimple or papule appears at the site of inoculation 2 to 3 days after exposure. Aring of vesicles develops; the vesicles coalesce to form an erythematous ring. A small dark area appears in the center of the ring and eventually ulcerates and dries, forming a depressed black necrotic central area known as an eschar or black eschar. The lesions are sometimes

referred to as a malignant pustule, even though it is not a pustule and is not malignant. The lesion is painless and does not produce pus, unless it becomes secondarily infected with a pyogenic organism. The eschar is normally 1to 3 cm in diameter, although it may be more extensive. The eschar begins to heal after 1 to 2 weeks.

The lesion dries, separates from the underlying base, and falls off, leaving a scar. Usually the infection remains localized, but regional lymphangitis and lymphadenopathy appear. If septicemia occurs, symptoms of fever, malaise, and headache are seen. Normally, in uncomplicated cases, no systemic symptoms are present.


Inhalation Anthrax

Inhalation anthrax, also called woolsorter’s disease, is acquired when spores are inhaled into the pulmonary parenchyma. The infection begins as a nonspecific illness consisting of mild fever, fatigue, and malaise 2 to 5 days after exposure to the spores. It resembles an upper respiratory tract infection such as that seen with colds and “flu.” This initial, mild form of the disease lasts 2 to 3 days. It is followed by a sudden severe phase in which respiratory distress is common. The severe phase of the disease has a high mortality rate. The respiratory problems (dyspnea, cyanosis, pleural effusion) are followed by disorientation, then coma, and death.

The course of the severe phase (onset of respiratory symptoms to death) may last only 24 hours.


Gastrointestinal Anthrax

Gastrointestinal anthrax occurs when the spores are inoculated into a lesion on the intestinal mucosa following ingestion of the spores. The symptoms of gastrointestinal anthrax include abdominal pain, nausea, anorexia, and vomiting. Bloody diarrhea may also occur. Because this form of the disease is difficult to diagnose, the fatality rate is higher than in the cutaneous form. Gastrointestinal anthrax accounts for less than 1% of the total cases worldwide; it has never been reported in the United States.


Approximately 5%of patients with anthrax (cutaneous, inhalation, gastrointestinal) develop meningitis. The symptoms are typical of any bacterial meningitis and occur rapidly. Unconsciousness and death, if they occur, follow 1to 6 days after initial exposure. Recovery from infection appears to confer immunity. An effective vaccine is available for those who are at risk for occupational exposure. In addition, vaccines are available for veterinary use.

Laboratory Diagnosis


B. anthracis is a large, square-ended, gram-positive or gram-variable rod found singly or in chains. In Gram stain preparations of clinical samples, vegetative cells can appear with clear zones around the cells, representing the presence of a capsule. The presence of large encapsulated gram-positive rods in the blood is strongly presumptive for B. anthracis identification. As the bacteria are subcultured, capsule production will

cease. To stimulate capsule production, cultures can be incubated in an atmosphere containing increased

CO2, This is an important characteristic for laboratory identification.

The spores are generally not present in clinical samples and can only be seen as unstained areas within the cells. When they are in chains, the ends of the single cells fit snugly together. This, together with the unstained central spore, gives the appearance of bamboo rods. Young cultures are gram-positive; as the cells become older, or if they are under nutritional stress, they become gram-variable.

Cultural Characteristics

On SBA, colonies of B. anthracis are nonhemolytic, large (2 to 5 mm), gray, and flat with an irregular margin because of outgrowths of long filamentous projections of bacteria that can be seen with a dissecting microscope.

The term Medusa head has been used to describe the colony morphology of B. anthracis. Colonies have a tenacious consistency, hold tightly to the agar surface, and when the edges are lifted with a loop, they stand upright without support. This has been described as having the appearance or characteristic of beaten egg whites. While B. anthracis is nonhemolytic on SBA, weak hemolysis may appear under areas of heavy growth after prolonged incubation. This should not be confused with I3-hemolysis. Many other species of Bacillus are hemolytic.

Because B. anthracis would frequently be isolated from normally sterile sites, e.g., blood, lung tissue, and cerebrospinal fluid (CSF), selective media is not usually needed for recovery. However, it should be noted that while most strains of B. anthracis will grow on phenyl ethyl alcohol (PEA) medium, growth is usually weak. The current American Society for Microbiology Anthrax Protocol does recommend that PEA agar be used for stools suspected of containing.

B. anthracis.


B. anthracis is catalase positive and will grow aerobically or anaerobically. It is nonmotile, distinguishing it from most other members of the genus Bacillus.

Although B. anthracis ferments glucose, it fails to ferment mannitol, arabinose, or xylose. B. anthracis produces lecithinase; therefore an opaque zone can be seen around colonies growing on egg-yolk agar.

This species grows in high salt (7% NaCI) and low pH «۶). Unlike B. cereus, B. anthracis is generally susceptible to penicillin (10 U;mL). Characteristics important to differentiate B. anthracis from the closely related B. cereus are listed in Table 17-1.

The Centers for Disease Control and Prevention (CDC) and the Association of Public Health Laboratories established the Laboratory Response Network (LRN) in August 1999 to help the public health system be better prepared for chemical and biological attacks. Currently, the LRN consists of three levels: sentinel, reference, and national laboratories. The role of sentinel laboratories is to recognize, rule out, and refer. Refer to Chapter 30 for a discussion of the LRN.

In order to rule out B. anthracis, the CDC has established basic diagnostic protocols. These protocols include a minimum number of common tests. Caution should always be used in working with an isolate suspected of being B. anthracis. Work should be done in a biologic safety cabinet, and the area should be disinfected when the work is completed. Approved tests to be performed by sentinel laboratories include Gram stain, colony morphology, catalase, motility, and capsule detection. The Gram stain can be performed directly on clinical specimens or on culture isolates.

If the microscopic and colonial morphologies of the isolate are compatible with B. anthracis, additional tests need to be performed. B. anthracis is catalase positive and nonmotile. Motility can be tested for by either wet

mount preparation or by inoculation into motility test medium. Lack of motility is unusual among the Bacillus spp.; B. cereus var mycoides is also nonmotile. Capsule production by B. anthracis can be detected by the India

stain on blood or CSF, or on cells isolated in media supplemented with sodium bicarbonate. As mentioned previously, growth in increased CO2 also stimulates

capsule production.

If the laboratory cannot rule out B. anthracis, then the isolate is sent to a reference laboratory, usually a state laboratory, for confirmation. The reference laboratory will likely perform direct fluorescent antibody assays for a cell wall polysaccharide and a capsule antigen. The presence of both antigens is confirmation for B. anthracis. Antimicrobial susceptibility testing will be performed by reference or national laboratories.

A test of historical importance is performed by inoculating the suspected isolate onto agar containing penicillin (0.05 to 0.5 UjmL). After incubation for 3 to 6 hours at 37° C, the areas of inoculation are examined microscopically for the presence of large spherical bacilli in chains; this phenomenon is referred to as a string of pearls. Species identification of bacilli can be accomplished by the use of the API 20E and 50CH systems (bioMerieux, Inc., Hazelwood, Mo). Nucleic acid amplification tests have been developed, but such applications for diagnosis and identification of B. anthracis remain useful in special situations and at specialized laboratories.

Treatment Most isolates of B. anthracis are susceptible to penicillin, but resistance can occur due to B-Iactamase production. The organism is also susceptible to many broad-spectrum antimicrobial agents, including gentamicin, erythromycin, tetracycline, and chloramphenicol.

In 2000, ciprofloxacin was approved by the Food and Drug Administration for management of postexposure inhalation anthrax based on data from animal models. According to the CDC recommendations and based on studies in nonhuman primates and other animal and in vitro data, ciprofloxacin or doxycycline should be used for initial intravenous (IV) therapy until antimicrobial susceptibility results are known. Current recommendations for initial therapy of pulmonary and cutaneous anthrax include ciprofloxacin or doxycycline plus one or two additional antimicrobial agents depending on disease severity.


Other Bacillus Species

Bacillus cereus

Bacillus cereus is a relatively common cause of food poisoning and opportunistic infections in susceptible

hosts. Food poisoning caused by B. cereus takes two forms: diarrheal and emetic. The diarrheal syndrome, usually associated with ingestion of meat or poultry, is characterized by an incubation period of 8 to 16 hours.

Afflicted individuals suffer abdominal pain and diarrhea.

About 25% of individuals experience vomiting; fever is uncommon. The average duration of the illness is 24 hours. The diarrheal form is clinically indistinguishable from the diarrhea caused by Clostridium perfringens. The emetic form has the predominant symptoms of abdominal cramps and vomiting. Diarrhea is present in about one third of those affected. This form has been associated with ingestion of fried rice, particularly when prepared in Oriental restaurants. The average duration of the illness is 9 hours. For both the diarrheal and emetic forms of B. cereus food poisoning, the illness is usually mild and self-limiting. The two forms of illness are caused by two distinct enterotoxins produced by B. cereus. A comparison of the enterotoxins is shown in Table 17-2.

B. cereus is similar to B. anthracis in many waysmorphologically and metabolically. Differentiation between B. cereus and B. anthracis is outlined in Table 17-1. B. cereus can be grown aerobically at 37° C on SBA. A B hemolytic frosted glass-appearing colony containing spore-forming, gram-positive bacilli that are motile, able to ferment salicin, and lecithinase positive is likely B. cereus.

Culture of the suspected food may be done to quantitate and isolate B. cereus. If more than 105 B. cereus cells per gram of food are present and other pathogens are absent, then food poisoning by this organism is

confirmed. The stool of patients with food poisoning may also be examined for B. cereus, although the organism can be part of the normal fecal flora. To confirm the organism as the cause of the disease, viable counts from the stool should also be at least 105cells per gram.

Because B. cereus may be found in small numbers in a significant proportion of healthy people, quantitative

cultures must be done.

B. cereus involvement in infections of the eye is the most common type of nongastrointestinal infections with this organism and these include endophthalmitis, panophthalmitis, and keratitis with abscess formation.

B. cereus has been documented as a cause of meningitis, septicemia, osteomyelitis, and a number of other types of infections. Although rare, these serious, nongastrointestinal infections due to B. cereus do occur, particularly in drug addicts, the immunosuppressed, neonates, and postsurgical patients.

Unlike B. anthracis, B. cereus is resistant to penicillin and many of the other cell wall antibiotics (e.g., ampicillin, cephalothin, methicillin). Treatment with c1indamycin and gentamicin combination has been successful.


Bacillus subtilis

Infections by other members of the genus are rare. They occasionally cause gastrointestinal illness. They are seen, however, as contaminants. One of the more commonly seen species is Bacillus subtilis. The colonies of B. subtilis are large, hemolytic on SBA, and may be pigmented. Pigment colors range from pink and yellow to orange or brown. Identification can be made using the API 20E and 50CH systems (bioMerieux

Vitek, Inc.).

table 17-2 figure 17-1 figure 17-2 figure 17-3 figure 17-4 table 17-1