Bacillus Species and Related Genera Other Than Bacillus anthracis

Microbiology

Bacteria of the genus Bacillus are well adapted to their normal environment of soil. This includes Bacillus anthracis, discussed elsewhere in this text (see Chapter 207 ). These gram-positive or gram-variable, aerobic or facultatively anaerobic, rod-shaped bacilli have rounded or squared-off ends, form endospores, tolerate extremes of temperature and moisture, and are ubiquitous. They are found in superficial lake and ocean sediment, even in deep water. Their hardiness under conditions of desiccation and heat has been used to determine the efficacy of heat sterilization (Bacillus stearothermophilus) and fumigation procedures (Bacillus subtilis). For many members of the genus Bacillus, an association with animals (either saprophytic or pathogenic) has also been noted. These animals range from small insects to large mammals, including humans.

Genomic tools have reshaped the taxonomy of Bacillus spp. Earlier changes included the movement of Bacillus alvei into the genus Paenibacillus and placement of both Bacillus brevis and Bacillus laterosporus into the genus Brevibacillus. Newer changes indicate upwards of 56 genera and 545 total species of aerobic, gram-positive, spore-forming bacteria, although few of them can cause human disease. Recent analyses of Bacillus cereus (sensu lato) have shown it to be essentially a pangenome with clonality throughout B. cereus , B. thuringiensis , B. mycoides , B. weihenstephanensi s, B. pseudomycoides, and B. anthracis . Environmental strains resemble those found in cultures of human clinical material. For practical purposes, we retain the current species because they are fairly easy to distinguish, and because it is useful to make distinctions around the public health threat of B. anthracis and to recognize the commercial role of B. thuringiensis . Another organism in the B. cereus group, Bacillus cytotoxicus, has been associated with foodborne illness. This species can produce a number of toxins and has unusual thermotolerance, being able to grow up to 50°C. Less-related species of Bacillus that may occasionally be encountered in the human clinical microbiology laboratory are B. subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus , and Bacillus sphaericus. Bacillus spp. grow quickly at 25° to 37°C on the usual culture media of the clinical laboratory. By definition, all have the capacity to form spores, but they vary widely in motility, colony morphology, and nutritional requirements. They are fairly large bacteria, with dimensions ranging from 3 × 0.4 to 9 × 2 µm. Although they are usually gram positive in early growth, old cultures can be gram variable or even gram negative. In most clinical laboratories, the most urgent task is to distinguish B. anthracis from other Bacillus spp. B. anthracis is nonhemolytic on sheep or horse blood agar and nonmotile, whereas most other clinical isolates are motile and β-hemolytic. Strains of B. anthracis that are slightly hemolytic have been reported, and some of the less frequently isolated non- anthracis strains are nonmotile and nonhemolytic. For the latter strains, detailed biochemical analysis and toxin testing may be required. It is perhaps surprising, yet fortunate, that the commercial tests to distinguish B. anthracis from its near relatives generally are easily carried out on widely available commercial kits. Some species, such as B. sphaericus and Bacillus badius, are biochemically unreactive and difficult to identify using commercial biochemical kits. Fortunately, newer technologies, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, have been shown to be reliable for species determination.

Epidemiology

The widespread distribution in nature of Bacillus spp. explains its frequent isolation in the laboratory. In many cases the isolation of Bacillus spp. from a clinical specimen raises the possibility of contamination because environmental spores can germinate quickly on various laboratory media. Bacillus spp. are infrequently cultured but commonly present in human intestinal contents when a genomic analysis is performed. They represent the most common members of Actinobacteria, the phylum that also includes mycobacteria, corynebacteria, nocardia, and actinomyces. Using more conventional culture techniques, children and adults were tested for the presence of B. cereus in the stool, and rates of recovery from 0% to 43% were found in the absence of diarrhea. The density of B. cereus in stool is usually low (≈100 viable organisms/g) but can be considerably higher. Strains of B. cereus in the stool are the same as those found in the food supply, and the ubiquity of B. cereus is reflected in a large number of different strains in fecal cultures of healthy people. However, during outbreaks, it can be shown that the strain of B. cereus causing food poisoning is consistent by biotype, serotype, toxin production, and phage type among patients.

Hospital outbreaks of Bacillus spp. infection include one in which B. cereus was an ongoing cause of positive respiratory cultures and morbidity, including two cases of true bacteremia and one fatal pneumonia, in an intensive care unit. This epidemic was the consequence of inadequate sterilization of respiratory circuits. No other bacterial infections occurred at higher than usual rates during the epidemic period because the degree of sterilization was sufficient to eradicate non–spore-forming bacteria but not B. cereus. Failure to sterilize surgical instruments has been associated with a number of postoperative infections, including those caused by Bacillus spp. In a Scottish outbreak, Bacillus spp. and coagulase-negative staphylococci were isolated from a variety of autoclaved instruments, and a number of patients had infections in operative sites caused by various bacteria, including coliforms, staphylococci, and Bacillus spp. A more dramatic example of a large-scale outbreak occurred at a hospital in Singapore during a period of major construction. A 10-fold increase in isolates of Bacillus spp. (including true infections) was noted in association with drilling and excavation. Linens were heavily contaminated by Bacillus, and more meticulous handling of hospital linens reduced the rate of infections, but a relaxation of linen cleaning standards allowed a resurgence of Bacillus infections.

Other species of Bacillus can persist for a long period and then cause intermittent medical problems, such as 12 cases in 10 years of B. sphaericus bacteremia in a children's cancer hospital in Italy.

Pseudoinfection and Contamination

More common than true outbreaks are pseudoepidemics, in which a strain or strains of Bacillus spp. are recovered from patients with a common source of contamination. In these settings biochemical and molecular studies can show that a single strain is found, even though it was not actually causing disease. Conversely, clusters of Bacillus spp. infection may look like point source outbreaks when they represent a higher than expected rate of infection by environmental organisms. One small cluster of serious Bacillus spp. infections, all of which were accompanied by bacteremia, occurring over 10 days in a children's cancer ward showed that the strains recovered were different from one another and from other isolates submitted for analysis. Because Bacillus spp. is such a common contaminant and such a rare cause of disease, many laboratories do not identify Bacillus to the species level, except to exclude the possibility of B. anthracis. Bacillus spp. can survive in high concentrations of ethyl alcohol, up to 95%, including the sprays of 70% ethanol that are sometimes used for hand hygiene. The failure of alcohol to kill Bacillus spp. led to a serious problem when a manufacturing defect in alcohol prep pads allowed for an outbreak of some serious infections. Fresh prep pads tested positive for Bacillus spp., whereas other sterilizing liquids were, in fact, sterile.

Bacillus spp. contamination has resulted in false-positive rates of up to 0.1% to 0.9% of all blood cultures submitted. Construction on a hospital driveway resulted in a 13-fold increase in the number of blood cultures that tested positive for Bacillus spp., although no true infections were identified. The problem was related to direct contamination of stored blood culture bottles and inadequate cleaning of the bottles before introduction of the specimen. Even in the absence of a pseudoepidemic, it can be difficult to separate true Bacillus spp. infection from contamination. The best indicator of true bacteremia is the presence of multiple positive cultures or recurrent bacteremia. In one study that compared patients for whom both bottles were positive in a set with Bacillus spp. against patients with only a single bottle positive, 29% (5/17) of episodes with both bottles positive were associated with a subsequent positive blood culture, as opposed to 3% (2/59) in patients with only a single bottle positive. This suggests that skin preparation may be less important than specimen handling in false-positive blood cultures for Bacillus spp. In a Japanese hospital 29 patients were noted to have Bacillus spp. Bacteremia; more than half of these were B. cereus . However, these patients were not treated for Bacillus spp. and did well clinically. Review of infection control policies showed suboptimal approaches to handling the catheters—wrong disinfectant, pauses during infusion, and reuse of caps on stopcocks. When these shortcomings were corrected, the Bacillus spp. bacteremia pseudoepidemic ceased. False-positive cultures of cerebrospinal fluid (CSF) for Bacillus spp. have also been reported. A Polish study of microbial contamination of postcollection and postprocessing peripheral blood and bone marrow products found a contamination rate of 0.87% in the postprocessing products, of which 67% were Bacillus spp.

Commercial Uses of Bacillus Species

The toxins of the insect pathogen B. thuringiensis (Bt) have been purified and are among the most widely used “natural” control agents in agriculture. Bt organisms or their purified toxins can be applied to commercially important plants to reduce damage from insect pests, and these can be easily purchased in garden centers to spray or dust in areas of insect activity. Despite its close genetic relatedness to potential pathogens, Bt used for farming and horticulture is very safe for humans. Genetic engineering has allowed the insertion of the toxin gene from Bt into other bacteria that can live closely with plants (e.g., among their roots or even between their cells) and protect them. Bt toxin genes have been inserted into commercially farmed plants, such as tobacco, tomato, and cotton, making them naturally resistant to insects. Bacillus spp. spores have been marketed in nonchemical drain cleaners that work when the spores germinate and enzymatically digest part of the clog. There is a growing practice of including Bacillus spp. in commercial probiotic formulations. In one case a B. subtilis probiotic resulted in infection of an immunocompromised patient.