TAXONOMY Back to top
Bacillus was defined in 1920 as a genus of gram-positive, aerobic
sporeformers, but since
1995 three strictly anaerobic and seven
asporogenous Bacillus species have been proposed.
This undermining of the definition has occurred
because 16S rRNA gene sequence analysis
has permitted the recognition of genus boundaries,
whereas genera were previously defined
phenotypically, as pragmatic collections of
species sharing key (i.e., diagnostic) features.
Many new species have been delineated largely on
the basis of 16S rRNA gene sequence
similarity and DNA-DNA relatedness—phenotypic
descriptions may be brief, and routine
phenotypic characters for distinguishing some
species are very few and of little practical
value.
With the accumulation of 16S rRNA gene sequence
data, Bacillus has been divided; 14 new
genera containing species originally allocated to Bacillus
have been published since 1990,
and together with Bacillus itself the
species in these genera now number 388. In addition, 38
genera containing aerobic endospore formers not
originally allocated to Bacillus have been
described. Sporosarcina was proposed in
1936, but the other 37 genera have been proposed
since 1990, and together they contain 109 species.
Following some mergers, there are now
53 genera of aerobic endospore formers overall,
and over 520 species. Although seven
named families containing aerobic endospore
formers now lie within the order Bacillales of
the class “Bacilli,” in the phylum Firmicutes,
only the
families Bacillaceae and Paenibacillaceae
contain species that may be of clinical interest.
The clinical bacteriologist need not, therefore,
be greatly concerned at this taxonomic
expansion, because for the medically relevant
species it does not represent taxonomic
upheaval. Bacillus is still the largest
genus, with 162 species (90), and it continues to
accommodate most of the best-known names such as Bacillus
subtilis (the type species), B.
anthracis, B. cereus, B. licheniformis, B.
megaterium, B. pumilus, and
B.
thuringiensis. Relatively few other familiar names, some of which are of
potential clinical
interest, have been transferred to newer genera: Brevibacillus
brevis (91), Geobacillus
stearothermophilus (92; important as a thermophilic contaminant and
autoclave test
organism), Lysinibacillus sphaericus (2),
and Paenibacillus polymyxaand Paenibacillus
macerans. However, several new Bacillus and Paenibacillus species
have been proposed on
the basis of single isolates of unknown
significance from clinical sources: B. idriensis and B.
infantis from neonatal sepsis (82); P. konsidensis, P.
massiliensis, P. sanguinis, and P.
timonensis from blood cultures (83,116);
B. massiliensis and P. provencensis isolated from
cerebrospinal fluid (CSF) (56,
117); P. turicensis from a CSF shunt (14);
and P. urinalis from
urine (117). “Paenibacillus
hongkongensis,” now classified in a new genus of aerobic
endospore formers as Cohnella hongkongensis (74),
was isolated from a case of
pseudobacteremia in a boy with neutropenic fever.
DESCRIPTION OF THE GENERA Back to top
Notwithstanding the handful of strictly anaerobic
and asporogenous members
of Bacillus, those species likely to be
isolated in a clinical laboratory are rod-shaped,
endospore-forming organisms that may be aerobic or
facultatively anaerobic, and young
cultures are usually gram positive but sometimes
gram variable or clearly gram negative.
They are mostly catalase positive and may be
motile by means of peritrichous flagella. Most
species are mesophilic, but there are some
thermophilic and psychrophilic species. A large
number of the other, newer genera comprise small
numbers of species that are unlikely to be
encountered in a clinical laboratory, as many of
them are from exotic and extreme
environments.
EPIDEMIOLOGY AND TRANSMISSION Back to top
Most aerobic endospore formers are saprophytic
organisms living in the natural environment,
and many species are very widely distributed. Some
species, however, are opportunistic or
obligate pathogens of animals, including humans,
other mammals, and insects. The main
habitats are soils of all kinds, ranging from acid
to alkaline, nonsaline to highly saline, hot to
cold, and fertile to desert, and the water columns
and bottom deposits of fresh and marine
waters. They have been isolated from air at high
altitude, from subterranean waters, and
from volcanic and permafrost soils. Endospores
readily survive distribution in soils, dusts,
and aerosols from natural environments to a wide
variety of other habitats such as manmade
environments, and they may be carried across the
globe by wind. The resistance of the
spores to heat, radiation, disinfectants, and
desiccation also results in aerobic endospore
formers being frequent contaminants in the
operating room, on surgical dressings, in
pharmaceutical products, and in foods. Dried foods
such as spices, milk powders, and
farinaceous products are often quite heavily
contaminated with spores, and when water
becomes available during food preparation these
spores may germinate, leading to spoilage
or food poisoning. B. anthracis is, to all
intents and purposes, an obligate pathogen of
animals and humans. Its close relative B.
cereus is well established as an opportunistic
pathogen, and other aerobic endospore formers can
also be opportunistic pathogens
occasionally. Three members of the B. cereus group—B.
anthracis, B. cereus, and B.
thuringiensis—are regarded by many as pathovars of a single species, and there
is evidence
of extensive horizontal gene transfer within the B.
cereus group (18).
Anthrax was the first example of an infection of
humans and animals proven as caused by a
bacterium, and this disease remains the most
widely recognized clinical condition caused by
a Bacillus species. It is primarily a
disease of domestic or wild animals, and prior to the
availability of an effective veterinary vaccine in
the late 1930s, anthrax was one of the
foremost causes worldwide of mortality in cattle,
sheep, goats, and horses. Humans almost
invariably contract anthrax directly or indirectly
from animals. Over the past half century
there has been a marked decline in the incidence
of anthrax in both animals and humans;
the use of veterinary and human vaccines,
improvements in factory hygiene, sterilization
procedures for imported animal products, and the
increased use of man-made alternatives to
animal hides or hair have all contributed to this.
Nevertheless, the disease continues to be
endemic in many countries, particularly those that
lack efficient vaccination policies. It is still
common in several sub-Saharan African countries,
western China, some Mediterranean
countries, small pockets in Canada and the United
States, and certain countries of central
Asia and central and South America. National
vaccination programs have led to a progressive
global reduction in livestock cases over the last
30 years, but there are concomitant
problems of diminishing veterinary experience and
a public ignorance of the disease; the
former problem may lead to individual outbreaks
taking longer to recognize and control, and
the latter to the sale and slaughter of affected
animals (140). Direct animal-to-animal
transmission (i.e., excluding scavengers feeding
on carcasses of animals with anthrax) is
very rare. Because B. anthracis spores
remain viable in soil for many years and their
persistence does not depend on animal reservoirs, B.
anthracis is exceedingly difficult to
eradicate from an area of endemicity, and regions
of nonendemicity must be constantly on
the alert for the arrival of B. anthracis in
imported animal products. Anthrax is not
contagious, and transmission to humans is usually
restricted to direct contact with infected
animals, or products from infected animals;
however, there have been reports of person-toperson
transmission of cutaneous anthrax, including a
case of a mother with a cutaneous
lesion on her finger infecting her 1-year-old
child, and a case of transmission of cutaneous
abdominal lesions from one brother to another
brother who shared a bed (146).
Contaminated fomites, including such oddities as a
communal loofah, or infection of an
injection site by contamination of the skin or by
syringe have also rarely been implicated
(86), as has person-to- person, nosocomial spread
from an umbilical infection (151).
The continued existence of B. anthracis in
the ecosystem appears to depend on a periodic
multiplication phase within an animal host, and so
it is generally regarded as an obligate
pathogen. Its environmental presence thus reflects
contamination from an animal source at
some time and persistence of spores, rather than
replication within the soil. However, some
authorities believe that self-maintenance may
occur within certain soils, and germination
of B. anthracis spores and genetic exchange
between vegetative cells growing in grass
rhizosphere have been demonstrated (119).
In human and animal specimens the organism is
usually sought only when the case history suggests
that it is reasonable to suspect anthrax.
B. anthracis has been subjected to military research, development, and
occasional
deployment in several different countries over
many years, following attacks on livestock
during World War I, and it has remained high on
the list of agents that could be used in
biological warfare or bioterrorism. Few reports of
laboratory-acquired infections exist, but a
major outbreak occurred in April 1979 in the city
of Sverdlovsk, USSR (now Yekaterinburg,
Russia), in the Urals as a result of the
accidental release of spores from a military production
facility. Although the natural disease is readily
controllable, the 2001 bioterrorism-related
anthrax outbreak in the United States increased
the public concern about this disease
(see chapter 12).
CLINICAL SIGNIFICANCE Back to top
The majority of aerobic endospore-forming species
apparently have little or no pathogenic
potential and are rarely associated with disease.
The principal exceptions to this are B.
anthracis, the agent of anthrax, and B. cereus, but a number of other
species, particularly B.
licheniformis and B. pumilus, have been implicated in food poisoning and
other human and
animal infections.
Bacillus anthracis
Human anthrax has traditionally been classified as
either (i) nonindustrial, resulting from
close contact with infected animals or their
carcasses after death from the disease, or (ii)
industrial, as acquired by those employed in
processing wool, hair, hides, bones, or other
animal products. Dependent on the route of
infection, there are three major clinical forms of
anthrax: cutaneous, inhalation, and ingestion or
oral-route anthrax. Anthrax meningitis can
develop as a complication of any of these forms,
or occasionally in the absence of any of
them (57, 121).
Cutaneous anthrax accounts for about 99% of
naturally acquired human anthrax cases
worldwide; an estimated 2,000 cases are reported
annually. Infection typically occurs when
spores are inoculated through a break in the skin,
although case reports and animal studies
suggest that preexisting lesions are not necessary
for infection (60); infections following
insect bites have been reported. Following an
incubation period of usually 2 to 6 days (but
with extremes of a few hours to 3 weeks) a small
papule appears, progressing over the next
24 h to a ring of vesicles, with subsequent
ulceration and formation of a characteristic
blackened eschar. Subsequent eschar formation may
become thick and surrounded by
extensive edema. Fever, pus, and pain at the site
are normally absent; their presence
probably indicates secondary bacterial infection.
Before the availability of antimicrobial
therapy, 10 to 20% of untreated cases of cutaneous
anthrax were fatal. Less than 1% of
cases are fatal today, and they are mainly due to obstruction
of the airways by the edema
that accompanies lesions on the face or neck or as
a result of progression of the cutaneous
disease into systemic infection. Eschars take
several days to evolve, and even with effective
antimicrobial therapy they may take several weeks
to resolve.
Ingestion anthrax is not uncommon in regions of
endemicity worldwide where socioeconomic
conditions are poor and people eat raw or
undercooked meat of anthrax-infected animals
that have died suddenly; asymptomatic infections
and symptomatic infections with recovery
may not be uncommon (124).
It takes two forms: oral or oropharyngeal infection, in which
the lesion is in the buccal cavity or on the
tongue, tonsils, or posterior pharyngeal wall, and
gastrointestinal anthrax, in which ulcerations
develop anywhere within the tract, but
primarily in the mucosae of the terminal ileum or
cecum. Oropharyngeal symptoms include
sore throat, dysphagia, and regional
lymphadenopathy, followed by severe edema of neck
and chest. Intestinal anthrax causes nausea,
vomiting, anorexia, abdominal pain, mild
diarrhea, and fever; this can progress to bloody
diarrhea, hematemesis, and massive ascites
(140). Mortality rates range widely, owing to the
nonspecific nature of the early symptoms
and late initiation of antimicrobial therapy.
Between 1900 and 2001, only 18 cases of naturally
acquired inhalation anthrax were
recorded in the United States, with 16 (89%) being
fatal (16), and figures in the United
Kingdom show a similar picture. Recently, however,
anthrax has occurred in persons using
contaminated imported animal hides for drum
making. A case of inhalation anthrax in an
African drum maker in 2006 was the first naturally
occurring case of inhalation anthrax in the
United States since 1976; the hides from west
Africa were contaminated with B.
anthracisspores. Two cases of cutaneous anthrax, in a drum maker and a
family member,
occurred in the United States in 2007 (23),
and there was a fatal case of inhalation anthrax
in a drum maker in the United Kingdom in 2008 (3).
Both cutaneous and inhalation anthrax
have also been associated with the handling or
playing of goatskin drums contaminated with
spores of B. anthracis (40).
Among the 22 cases of the anthrax outbreak in the United States
in late 2001, in which spores were delivered in
mailed letters, early recognition and
treatment of the 11 patients with confirmed
inhalation anthrax resulted in a 65% survival
rate (9).
The inhaled spores are ingested by macrophages,
but the role of macrophages thereafter is
unclear, as they may be bactericidal towards
germinated spores but also be crippled by
lethal and/or edema toxin and so allow bacterial
outgrowth (31). The replacement of the
older name for this form of the disease,
“pulmonary anthrax,” with the newer name
“inhalation anthrax” is a reflection of the fact
that active infection occurs in the lymph nodes
rather than the lungs themselves. Analysis of 10
of the cases associated with the
bioterrorism events of 2001 (71)
revealed a median incubation period of 4 days (range, 4 to
6 days). All of the patients with inhalation
anthrax had severe illness and were hospitalized.
Their clinical presentation included fever or
chills, fatigue or malaise, minimal or
nonproductive cough, dyspnea, and nausea or
vomiting; some had chest pain and sweats. All
patients had abnormal chest radiographic images,
with pleural effusion, infiltrates, or
mediastinal widening.
Regardless of the form of the disease, the
generalized symptoms that may initially be mild
(fatigue, malaise, fever, and/or gastrointestinal
symptoms) can rapidly develop into a
fulminant state following lymphohematogenous
spread from a primary lesion, with symptoms
of dyspnea, cyanosis, severe pyrexia, and
disorientation, followed by circulatory failure,
shock, coma, and death (71).
Depending on the host, there is a rapid buildup of the bacteria
in the blood over the last few hours to terminal
levels of 107 to 109/ml in the most
susceptible species. Enhanced clinical and
laboratory expertise and conducting prospective
surveillance are critical components of rapid
anthrax diagnosis and for response
preparedness, should B. anthracis be used
in bioterrorist attacks (52).
Bacillus cereus Group
Bacillus cereus is a ubiquitous organism, and clinical isolates are phylogenetically
diverse
(68); it is a wide-ranging opportunistic pathogen of
humans and other animals and an
important cause of foodborne illness. B. cereus
caused 21 (2%) mean annual total foodborne
outbreaks and 160 (1%) mean annual total foodborne
illnesses between 2001 and 2005 in
the United States as reported through the
Foodborne Diseases Active Surveillance Network
(FoodNet) of the Centers for Disease Control and
Prevention’s (CDC’s) Emerging Infections
Program. However, this probably underrepresents
the true burden of illness, since outbreaks
involving less commonly identified pathogens, such
as B. cereus, are less likely to have a
confirmed etiology because these organisms are not
always considered in clinical,
epidemiological, and laboratory investigations of
foodborne disease outbreaks (24).
B. cereus is the etiological agent of two distinct food poisoning syndromes.
The diarrheal
type is characterized by abdominal pain and
diarrhea 8 to 16 h after ingestion of
contaminated food; it is associated with a wide
diversity of foods, from meats and vegetable
dishes to pastas, desserts, cakes, sauces, and
milk. The emetic type is characterized by
nausea and vomiting 1 to 5 h after eating the
offending food; oriental rice dishes are
predominant sources, although occasionally other
foods such as pasteurized cream, milk
pudding, pastas, and reconstituted formulas have
been implicated. One emetic outbreak
followed the mere handling of contaminated rice in
a children’s craft activity, and fulminant
liver failure associated with the emetic toxin has
been reported (94). B. cereus spores are
very adherent to surfaces and are widespread in
food preparation environments, and they
can survive many cleaning and normal cooking
procedures. These are key factors for both
syndromes; under conditions of improper food
storage after cooking, the spores germinate
and the vegetative cells multiply.
The toxigenic basis of B. cereus food
poisonings and other B. cereus infections have been
much elucidated over the last three decades. In
diarrheal illness, one or more enterotoxins
are produced by vegetative cells in the small
intestine, following the ingestion of spores or
vegetative cells; the toxins are believed to cause
diarrhea by damaging the integrity of ileal
epithelial cell membranes. B. cereus produces
a range of protein toxins, of which three have
been implicated in diarrheal illness: hemolysin BL
(Hbl) and nonhemolytic enterotoxin (Nhe),
both of which are three-component toxins
restricted to the B. cereus group, and the singlecomponent
β-barrel, pore-forming cytotoxin K (CytK). These
toxins may act synergistically,
but there is evidence that Nhe is the most
dominant in diarrheal illness (128). The emetic
illness is an intoxication caused by a highly
heat-, proteolysis-, and acid-resistant toxin
preformed in the food—hence its rapid onset. The
toxin, cereulide, is a small ring-formed
dodecadepsipeptide produced at the end of
logarithmic growth, and its genetic determinants
are borne on a plasmid related to the pXO1 virulence
plasmid of B. anthracis (41). Cereulide
production by B. cereus has not been
demonstrated at temperatures below 12°C, but the
related and psychrotolerant species Bacillus
weihenstephanensis may produce detectable
cereulide at 8°C (135). Gherlardi et al. (53)
used PCR amplification of toxin genes in
conjunction with randomly amplified polymorphic
DNA (RAPD)-PCR and multiplex RAPD-PCR
to trace the source of two outbreaks of food
poisoning.
Bacillus cereus is an important ocular pathogen, causing a rapidly progressive
endophthalmitis that is refractory to treatment.
Cases are, fortunately, infrequent; they
usually occur after penetrating trauma of the eye
but sometimes follow hematogenous
spread or, occasionally, eye surgery. Significant
loss of vision, and often loss of the eye
itself, can occur within 24 to 48 h (101).
B. cereus keratitis associated with contact lens wear
has also been reported (109).
Other B. cereus infections occur mainly, though not
exclusively, in persons predisposed by neoplastic
disease, immunosuppression, alcoholism
and other drug abuse (including cases associated
with contaminated heroin), the presence of
catheters (65) or implants such as
fluid shunts, or some other underlying condition such as
diabetes, and fatalities occasionally result.
Reported conditions include bacteremia,
septicemia, fulminant sepsis with hemolysis,
meningitis (following allogenic stem cell
transplants in two cases) (58),
brain hemorrhage, ocular infections including
panophthalmitis, ventricular shunt infections,
endocarditis (1), pneumonia (47),
pseudomembranous tracheobronchitis, exacerbation
of bronchiectasis, empyema, pleurisy,
peritonitis in a dialysis patient, lung abscess,
brain abscess, liver abscess, osteomyelitis,
salpingitis, urinary tract infection, and primary
cutaneous infections. El Saleeby et al. (42)
found an association between tea drinking and
invasive B. cereus infection in
immunocompromised children. Ko et al. (84)
reported the emergence of a β-lactamdependent
strain associated with prolonged administration of
β- lactams via an indwelling
catheter in a neutropenic patient. Strains of B.
cereus have been isolated in association with
periodontitis (63). Wound infections, often
of open fractures (38), and mostly in otherwise
healthy persons, have been reported following
surgery (associated, in one report, with
contaminated incontinence pads), road traffic and
other accidents, scalds, burns, plaster
fixation, drug injection, and close-range gunshot
and nail bomb injuries; some became
necrotic and gangrenous (32).
Fatal neonatal meningitis was caused by a blank firearm
injury; blank cartridge propellants are commonly
contaminated with the organism. Neonates
also appear to be particularly susceptible to B.
cereus, especially with umbilical stump
infections, and cases of meningitis have been
associated with the use of catheters and
manual ventilation balloons (44).
Respiratory tract infections have also been associated with
contaminated ventilation systems among neonates
and in a pediatric intensive care unit
(73). Several cases of B. cereus pneumonia in
metal workers and welders have been
reported, including fatal infections (67).
Infection of these metal workers may be related to
welding-fume exposure, demonstrated to suppress
pulmonary defenses in animal studies.
Interestingly, the isolate from one of these cases
was found to harbor a plasmid (pBCXO1)
that was 99.6% similar to the pXO1 virulence
plasmid of B. anthracis, while other isolates
were also found to harbor either pXO1 virulence
genes or both pXO1 and pXO2 virulence
genes (67); the role of these pXO1
or pXO2 plasmid genes, if any, in the virulence of these
isolates is not known. Additional atypical B.
cereus strains, originally identified as B.
anthracis due to the presence of pXO1 and pXO2 plasmids, have also been
isolated in
association with fatal infections of chimpanzees
and a gorilla in Ivory Coast and Cameroon
(79, 85). A nosocomial outbreak of B. cereus infection
related to catheter use was associated
with reused towels (35), and a hospital
pseudo-outbreak was associated with contaminated
ethanol used as a skin disinfectant. B. cereus also
causes infections in domestic animals. It is
a well-recognized agent of mastitis and abortion
in cattle, particularly when animals are in
winter housing, and can cause these conditions in
other livestock.
Strains of B. thuringiensis commonly carry
genes for B. cereus enterotoxins, and assays
have demonstrated that enterotoxin production
occurs, but the cereulide synthetase gene
has not been detected in this species (6,
105). B. thuringiensis is well known as an
insect
pathogen, preparations of certain strains are
widely used as biopesticides, and transgenic
crop plants that express the insecticidal crystal
toxin genes have been developed, but there
is as yet no evidence of infections directly
associated with the use of this organism as an
insecticide. Occupational exposure to the organism
has been connected with presence of the
organism in feces but without gastrointestinal
symptoms. A review of the safety of using B.
thuringiensis as a biopesticide on crop plants found that the main pesticide
strains assayed
produced low titers of enterotoxin (13).
There have been few reports of gastroenteritis
outbreaks in which B. thuringiensis was
implicated (98, 128). However, cases of such illness
caused by B. thuringiensis may have been
attributed to B. cereus, as the former may not
produce its characteristic insecticidal toxin
crystals when incubated at 37°C, owing to the
loss of the plasmids carrying the relevant genes.
There have been reports of wound, burn,
and ocular infections with B. thuringiensis, and
in a fatal case of pulmonary disease and
bacteremia in a neutropenic patient, there was
evidence that membrane-damaging toxins
contributed to the infection (54).
Experimental B. thuringiensisendophthalmitis has been
achieved in rabbits (17).
Other Species
Reports of infections with non-B. cereus group
species are comparatively rare but very
diverse, and there have been several hospital
pseudoepidemics associated with contaminated
blood culture systems. B. licheniformis has
been reported from prosthetic-valve endocarditis,
pacemaker wire infection, ventriculitis following
the removal of a meningioma, brain
abscesses, septicemia following arteriography,
bacteremia associated with indwelling central
venous catheters, bacteremia during pregnancy with
eclampsia and acute fibrinolysis,
peritonitis in patients undergoing continuous
ambulatory peritoneal dialysis (CAPD) and in a
patient with volvulus and small-bowel perforation,
ophthalmitis, and corneal ulcer after
trauma. An outbreak of bacteremia among patients
with blood malignancies was related to
nonsterile cotton wool used during skin
disinfection (106). B. licheniformis bacteremia has
been reported for an immunocompetent man and has
twice been reported in association with
Munchausen’s syndrome: one case followed
self-inoculation with organic drain cleaner (61),
and in another case following self-inoculation
with soil, B. pumilus and Paenibacillus
polymyxa were also isolated (51). B. licheniformis can
cause foodborne diarrheal illness, and
in a case associated with an infant fatality,
lichenilysin A, a heat-stable cyclic lipopeptide, has
been implicated (99). This organism is
frequently associated with bovine abortion and has
been shown to have a tropism for the bovine
placenta; it has also been associated with
abortion in water buffalo and occasionally with
bovine mastitis. As with such B.
cereus infections, these types of B. licheniformis infection are
associated with wet and dirty
conditions during winter housing, particularly
when the animals lie in spilled silage, and in
one outbreak a water tank contaminated with B.
licheniformis was implicated.
The name B. subtilis was often used in the
past for any clinical isolate, but since 1970 there
have been reports of infection in which this
species appears to have been identified
accurately. They include cases of pneumonia,
bacteremia, and septicemia associated with
neoplastic disease; breast prosthesis and
ventriculo-atrial shunt infections; isolations from
surgical wound drainage sites; endocarditis in a
drug abuser; meningitis following a head
injury; bacteremia associated with trauma;
bacteremia in cancer patients; cholangitis
associated with kidney and liver disease; and
isolation from dermatolymphangioadenitis
associated with filarial lymphedema. Two cases of
severe hepatotoxicity followed the
ingestion of nutritional supplements contaminated
with B. subtilis (130). Administration of an
oral probiotic preparation marketed for the
treatment or prevention of intestinal disorders,
and allegedly containing B. subtilis, led
to a fatal septicemia in an immunocompromised
patient; subsequently, the organism concerned was
identified as Bacillus clausii (127). These
authors reported another B. clausii infection,
cholangitis in polycystic kidney disease, in a 15-
year-old French boy who had undergone a renal
transplant, but the source of the organism
was unclear. B. subtilis has also been
associated with cases of bovine mastitis and ovine
abortion. B. subtilis has been implicated
in foodborne illness: vomiting has been the
commonest symptom, but with accompanying diarrhea
frequently reported, the onset
periods have been short (ranging from 10 min to 14
h; median, 2.5 h), the bacterial loads of
the organism were high (105 to 109 CFU/g), and the
implicated foods were often prepared
dishes in which meat or fish was served with
cereal-based components such as bread,
pastry, rice, or stuffing. Bacillus
amyloliquefaciens, a close relative of B. subtilis, is widely
used industrially for enzyme and amino acid
production, but human consumption of Ltryptophan
manufactured in an organism genetically engineered
from a strain of this species
was associated with a large epidemic of
eosinophilia-myalgia syndrome with 37 deaths; the
causative agent has not been identified with
certainty (100). Environmental strains of this
species producing a heat-stable, nonprotein toxin
have been isolated in association with
building-related health problems (100).
Organisms identified as B. circulans have
been isolated from cases of bacteremia in cancer
patients, meningitis, CSF shunt infections,
endocarditis, a wound infection in a cancer
patient, a bite wound, endophthalmitis,
peritonitis in a patient undergoing CAPD (12), and
epidemic endophthalmitis associated with a
contaminated product used during cataract
surgery. It must be noted, however, that many
isolates previously regarded as B.
circulans might have been wrongly identified (see comments on B.
circulans below).B.
coagulans has been isolated from corneal infection, bacteremia, and bovine
abortion. B.
pumilus has been found in cases of cutaneous, pustule, and rectal fistula
infections, central
venous catheter infection in an immunocompetent
child (10), bacteremias in
immunosuppressed patients (106),
bacteremia in a patient with Munchausen’s syndrome
(51), and repeated contamination of a blood platelet
screening procedure; it has also been
found in association with bovine mastitis.
Toxigenic strains of B. pumilus have been isolated
in association with foodborne illness and from
clinical and environmental specimens, and a
heat-stable cyclic lipopeptide, pumilacidin, was
implicated in a rice-associated food poisoning
outbreak (50). Lysinibacillus
sphaericus has been implicated in a fatal lung pseudotumor,
bacteremia, and meningitis. B. megaterium (eight
isolates), B. pumilus (six), Brevibacillus
brevis (five), B. licheniformis (two), and B. subtilis (one)
isolated from chewing tobacco were
found to produce potent exogenous virulence
factors that caused plasma exudation and
tissue dysfunction in an animal model (118).
B. megaterium caused a delayed-onset lamellar
keratitis following laser-assisted eye surgery (114).
Bacillus brevis has been isolated from corneal infection and implicated in several
incidents of
food poisoning; since those reports, the species
was split (see “Taxonomy” above) and
transferred to the new genusBrevibacillus. Strains
of one of the newer species, Brevibacillus
agri, were isolated in association with an outbreak of waterborne
illness in
Sweden; Brevibacillus centrosporus was
isolated from a bronchoalveolar lavage fluid
sample, Brevibacillus parabrevis was found
in a breast abscess, and both species have been
isolated from human blood (93).
Brevibacillus laterosporus has been reported in association
with a severe case of endophthalmitis.
Paenibacillus alvei has been isolated from cases of meningitis,
endophthalmitis, a prosthetic
hip infection in a patient with sickle cell
anemia, wound infections, and, in association
with Clostridium perfringens, a case of gas
gangrene. P. macerans has been isolated from a
wound infection following removal of a malignant
melanoma, from a brain abscess following
penetrating periorbital injury, from a
catheter-associated infection in a leukemic patient, and
from bovine abortion, and P. polymyxa has
been isolated from bacteremia in a patient with
cerebral infarction, a bacteremic case of Munchausen’s
syndrome (51), and ovine
abortion. Paenibacillus popilliae has been
reported from endocarditis, and Paenibacillus
larvae has been reported from infection of a CSF shunt system.
Single isolations of several new Bacillus and
Paenibacillus species from clinical specimens,
but of unknown significance, are mentioned in
“Taxonomy” above.
COLLECTION, TRANSPORT, AND STORAGE OF
SPECIMENS Back to top
Bacillus species normally survive transport in freshly collected specimens
or in a standard
transport medium. Local transport of specimens
(for no longer than a few hours) can be
done at room temperature or at 2 to 8°C for most
specimens, including serum. Generally, if
specimens such as stool, sputum, pleural fluid,
blood, and material on swabs are to be
shipped overnight or longer, they should be sent
at 2 to 8°C, while fresh tissue and serum
samples should be shipped frozen. Formalin-fixed
tissues can be sent at room temperature
primarily for detection using immunohistochemistry
and (although they are much less
suitable) PCR. For blood specimens with which PCR
will be used to detect B. anthracis DNA,
collection tubes containing EDTA or citrate as an
anticoagulant are preferable to those
containing heparin.
All the clinically significant isolates reported
to date are of species that grow, and often
sporulate, on routine laboratory media at 37°C. It
seems unlikely that many clinically
important but more fastidious strains are being
missed for the want of special media or
growth conditions. Maintenance is simple if spores
can be obtained, but it is a mistake to
assume that a primary culture or subculture on
blood agar will automatically yield spores if it
is stored on the bench or in the incubator. It is
best to grow the organism on nutrient agar
(or Trypticase soy agar) containing 5 mg/liter
manganese sulfate for a few days, and
refrigerate when microscopy shows that most cells
have sporulated. For most species,
sporulated cultures on slants of this medium,
sealed after incubation, can survive in a
refrigerator for years. Alternatively, cultures
(preferably sporulated) can be frozen or
lyophilized.
Safety Aspects
Clinical specimens for isolation of Bacillus species
other than B. anthracis can be handled
safely on the open bench without special
precautions. Efforts should be made to avoid
methods that produce aerosols. Any procedures that
have the potential to generate aerosols
should be done in a microbiological safety
cabinet. Biosafety level 2 practices, containment
equipment, and facilities are recommended for activities
using clinical materials and
diagnostic quantities of infectious cultures.
Isolation and presumptive identification of B.
anthracis can be performed safely in the
routine clinical microbiology laboratory, provided
that normal good laboratory practice is
observed. Preexposure vaccination is not
recommended for laboratory personnel doing
routine processing of clinical or environmental
specimens in general diagnostic laboratories
(26); however, biosafety level 3 facilities are
recommended for all such work (140). When
working with pure cultures of B. anthracis, direct
and indirect contact of broken skin with
cultures and contaminated laboratory surfaces,
accidental parenteral inoculation, and, rarely,
exposure to infectious aerosols are the primary hazards
to laboratory personnel. Laboratories
that frequently centrifuge B. anthracissuspensions
should use an aerosol-tight rotor that can
be repeatedly autoclaved (26).
Human infectious doses have not been established
for B. anthracis; the U.S. Department of
Defense estimates that a 50% lethal dose for
humans is 8,000 to 10,000 spores, but this is
largely based on data from nonhuman primate
studies. When collecting clinical specimens for
suspected anthrax, appropriate personal protective
equipment should be used, such as
disposable gloves, disposable apron or overalls,
and boots which can be disinfected after
use; for dusty samples that might contain many
spores, the use of a face shield and/or a
respirator should be considered. Full details of
personal protective equipment and of
disinfection and decontamination are given in
Annexes 1 and 3 of the WHO Anthrax in
Humans and Animals guidelines (140). It should be noted that
although hand washing with
soap and water or with chlorhexidine gluconate,
and the use of hypochlorite- releasing
towels, may reduce endospore contamination of the
skin, waterless rubs containing ethanol
are ineffective at removing endospores.
Preexposure vaccination recommendations by
Advisory Committee on Immunization Practices
(ACIP) for laboratory exposures are
addressed in “Vaccination” below.
Bacillus anthracis is defined as a select agent and is included on
both the Department of
Health and Human Services/CDC and U.S. Department
of Agriculture/APHIS select agent
lists. Thus, possession of the agent in the United
States requires registration of the
laboratory with either the CDC or APHIS. When B.
anthracis is identified by a laboratory, the
identification of this agent must be reported to
the CDC or APHIS immediately and a
APHIS/CDC Form 4 (Report of the Identification of
a Select Agent or Toxin) submitted within
7 days. Other authorities should be notified as
required by federal, state, or local laws.
When B. anthracis is isolated in an
unregistered laboratory, the organism must either be
destroyed on-site by a recognized sterilization or
inactivation process or be transferred to a
registered laboratory within 7 days. Shipping of
this agent requires completion of the
APHIS/CDC Form 2 (Request to Transfer Select
Agents and Toxins) and prior approval from
either the CDC or APHIS (see http://www.selectagents.gov for additional information of
select agent regulation in the United States).
Specimens from Patients Suspected To Have Anthrax
In all cases, specimens from possible sources of
infection (carcass, hides, hair, bones, etc.)
should be sought in addition to patient specimens.
Leakproof containers, to be placed in
secondary containers for “double-bagging,” and
then secure, outer containers for carriage
are needed. A blood smear may reveal the
capsulated rods or, if treatment has started,
capsule “ghosts.” Postmortem blood collected by
venipuncture (a characteristic of anthrax is
nonclotting blood at death) should be examined by
smear (for capsule) and culture.
Guidelines for clinical evaluation of persons with
possible anthrax can be found
athttp://www.cdc.gov/mmwr/preview/mmwrhtml/mm5043a1.htm.
Cutaneous Anthrax
The edge of an eschar should be lifted and two
specimens of vesicular fluid collected
(preferably prior to initiation of antimicrobial
therapy) by rotating swabs beneath it; one is
for a smear for visualizing the capsule, Gram
stain, and culture, and the other is for PCR. For
immunohistochemical (IHC) analysis of cutaneous
lesions, a full-thickness punch biopsy
specimen fixed in 10% buffered formalin from a
papule or vesicle lesion and including
adjacent skin should be taken. Biopsies should
also be taken from both vesicle and eschar if
present (122).
Inhalation Anthrax
Anthrax will be suspected only if the patient’s
history suggests it. Chest radiographs and
chest computed tomography scans are recommended.
In addition to imaging studies, obtain
blood cultures prior to antimicrobial therapy.
Pleural fluid, if present, should be obtained for
Gram stain, culture, and PCR. Serology is also
useful for the diagnosis of cases when culture
fails owing to previous treatment. Collect acute-
and convalescent-phase serum samples 2 to
4 weeks apart for serologic testing. Pleural
and/or bronchial biopsy samples can be tested by
immunohistochemistry. Postmortem, the approach
given for ingestion anthrax should be
followed (see below).
Ingestion Anthrax
As with the inhalation form, anthrax is suspected
only if an adequate history of the patient is
known. Specimens from oral lesions may be collected
in the same way as for the cutaneous
disease (140). Premortem, obtain blood
cultures before antimicrobial therapy. Ascites fluid, if
present, should be collected for Gram stain,
culture, and PCR. A stool or rectal swab, and
material from any oropharyngeal lesions, should
also be collected for Gram stain, culture,
and PCR. Acute- and convalescent-phase serum
samples, with the first obtained within 7
days of onset and the convalescent-phase sample
obtained 2 to 3 weeks later, should be
collected for serologic testing. Postmortem blood
collected by venipuncture should be
examined by smear (for observation of capsule) and
culture. Any hemorrhagic fluid from the
nose, mouth, or anus should be cultured. If these
are positive, no further specimens are
needed. Again, if negative, specimens of
peritoneal or ascitic fluid, spleen, and/or mesenteric
lymph nodes, aspirated by techniques avoiding
spillage of fluids, may be collected for smear
and culture.
Anthrax Meningitis
Collect CSF and blood specimens for cultivation,
capsule staining, and PCR; specimens
should also be subjected to antigen detection
testing, if available.
Specimens from Animals Suspected To Have Died of
Anthrax
Although the organism is rapidly destroyed by
putrefaction in the intact carcass, the carcass
of an animal that died of anthrax still generally
yields positive cultures from appropriate
specimens, such as blood from a peripheral vein,
or from snips of tissue such as from the tip
of an ear, as sporulation will still occur in some
tissues; however, hemorrhagic exudate is
preferred to aspirated blood or tissue specimens
for direct demonstration of the capsulated
organism using Gram, Giemsa, or polychrome
methylene blue (M’Fadyean) staining.
DIRECT EXAMINATION Back to top
The first examination of smears and cultures in
many clinical laboratories is done with the
Gram stain. In the past, it was regarded as of
limited value in anthrax diagnosis, because the
capsule is not seen. Notwithstanding this, if
large numbers of gram-positive bacteria are
observed in a patient’s blood at death, then B.
anthracis should be suspected. As in the 2001
bioterrorism incident in the United States, such
preparations can be very useful (Fig. 1a). In
other circumstances and in animals in particular,
the blood or other specimen may not be
collected soon after death and before putrefactive
organisms appear; B. anthracismay then
be indistinguishable without the use of the proper
capsule stain.
In
addition to culturing B. anthracis, there are molecular and antigen-based
detection
methods
available for direct detection in clinical and environmental samples. These
detection
methods
may provide additional information. Several methods, including a B.
anthracisspecific
Laboratory
Response Network (LRN) PCR assay, IHC assays, and serology, were
useful
for confirmation of cases during the 2001 bioterrorism-associated outbreak and
more
recent
cases associated with drum making using B. anthracis-contaminated hides
(23, 66,112, 113, 122).
The
IHC assay, as performed at the CDC, uses the same antibodies as the direct
fluorescentantibody
(DFA)
assay (specific to cell wall antigen or the capsule) to detect B. anthracis in
formalin-fixed,
paraffin-embedded tissues. This method was particularly useful in the
diagnosis
of cutaneous cases during the 2001 bioterrorism-associated outbreak. Skin
biopsy
samples
from cutaneous lesions from 8 of 10 patients were positive for both the capsule
and
cell
wall antigens (122). The most widely used and available detection method in the U.S.
public
health system is the LRN PCR (66). In addition to being
widely used in 2001, this
assay
and the IHC assay were used in 2007 to diagnose two cutaneous anthrax cases in
a
drum
maker and his child (23).
At
the CDC, a positive PCR result on any clinical specimen from a patient
collected from a
normally
sterile site (such as blood or CSF) or a lesion of other affected tissue (skin,
pulmonary,
reticuloendothelial, or gastrointestinal) is regarded as a supportive or
presumptive
diagnostic test. It is considered sufficient to provide a probable diagnosis
but is
not
confirmatory in itself. The principal reason for such stringent guidelines on
the use of
PCR
approaches and the value of their results towards providing a confirmatory
diagnosis is
based
on the possibility that environmental contamination of a non-anthrax-related
lesion
could
result in a positive result; this is especially the case with the use of some
previously
published
PCR primers for capsule and chromosomal genes that can produce false positives
with
reactions to soil microbiota. This is quite similar to the recommendations that
are
included
in the 2008 WHO guidelines (140), in which PCR can be used
for identification of an
isolate
but is not recommended for testing of specimens.
There
are numerous reports on the use of alternative technologies for the detection
of B.
anthracis,
such as mass spectrometry, flow cytometry, time-resolved
fluorescent assays,
high-performance
liquid chromatography, and even the use of engineered B cells to detect B.
anthracis
(5, 69, 115, 149, 152). Two recent studies focused on detecting the anthrax
toxins,
which are highly expressed during infection, in specimens instead of directly
detecting
the
bacilli (15, 133). One reported on an immunoassay using highly fluorescent
europium
nanoparticles
to detect protective antigen (PA), while the other reported detection of lethal
factor
(LF) by taking advantage of its metalloprotease activity, which cleaves
specific
peptides.
The LF protein was captured by monoclonal antibodies and then incubated with a
synthetic
peptide substrate whose cleavage products were detected by matrix-assisted
laser
desorption
ionization–time of flight (MALDI-TOF) mass spectrometry (15).
These and other
novel
approaches may allow for detection of B. anthracis infection earlier in
the course of the
disease
and thus allow for more successful treatment.
ISOLATION Back to top
Isolation from Human Specimens
Fresh
specimens from patients should be inoculated onto plates of blood agar in the
normal
way.
Enrichment procedures are generally inappropriate for isolations from clinical
specimens.
However, when seeking B. cereusin stools several days after a food
poisoning
episode,
nutrient or tryptic soy broth with polymyxin (100,000 U/liter) may be added to
a
heat-treated
specimen. Heat treatments at 70°C for 30 min or 80°C for 10 min are widely
used
for aerobic endospore formers in general, but 62 to 65°C for 15 to 20 min is
more
suitable
for B. anthracis; temperatures above 70°C are not recommended for this
species
(142).
There is no effective enrichment method for B. anthracis in old animal
specimens or
environmental
samples, and isolation from these is best attempted using polymyxinlysozyme
EDTA-thallous
acetate (PLET) agar (80; see also chapter 17). Aliquots (0.1 ml) of
the
undiluted suspension and 1:10 and 1:100 dilutions of a heat-treated suspension
of the
specimen
are spread across PLET plates, which are read after incubation for 36 to 40 h
at
37°C.
Creamy white, domed, circular colonies, 1 to 3 mm, are subcultured onto (i)
blood
agar
plates to test for gamma phage and for hemolysis and (ii) directly or
subsequently in
blood
to look for capsule production using either the M’Fadyean stain or India ink
negative
stain
(the latter is less reliable, because the ink coagulates the blood and makes
interpretation
difficult); 2.5 ml of blood (preferably defibrinated horse blood, but horse or
fetal
calf serum is satisfactory) is inoculated with a pinhead quantity of growth
from the
suspect
colony, incubated statically for 6 to 18 h at 37°C, and then stained. A
differential/selective
chromogenic medium is marketed by R&F Laboratories, Downers Grove,
IL
(R&F Anthracis chromogenic agar), and it has undergone some
independent evaluation
(72),
but another study (97) found PLET to be more sensitive and selective.
Several
media have been designed for the isolation, identification, and enumeration of B.
cereus
organisms. They exploit the organism’s egg yolk reaction
(phospholipase C) positivity
and
acid-from-mannitol negativity; pyruvate and polymyxin may be included for
selectivity.
Three
satisfactory formulations are MEYP or MYP (mannitol, egg yolk, polymyxin B
agar;
MEYP,
Oxoid, Basingstoke, United Kingdom; MYP, Difco, BD, Franklin Lakes, NJ), PEMBA
(polymyxin
B, egg yolk, mannitol, bromthymol blue agar; Oxoid), and BCM (Bacillus
cereusmedium;
LabM, Bury, United Kingdom) (see chapter 17).
There are more recent
formulations
that reveal phospholipase C positivity using specific chromogenic substrates
rather
than natural egg yolk: Bacillus cereusgroup plating medium (Biosynth
Chemistry and
Biology,
Staad, Switzerland, also available as Cereus-Ident-Agar from Heipha, Eppelheim,
Germany)
and Bacillus cereus/Bacillus thuringiensis chromogenic plating medium
(R&F
Laboratories).
There
are no selective media for other Bacillus species, but spores can be
selected for by
heat
treating part of the specimen as described above; however, this is not
appropriate for
fresh
clinical specimens, where spores are usually sparse or absent. Vegetative cells
of both
sporeformers
and non-sporeformers are killed by heat treatment, but the heat-resistant
spores
not only survive but also may be heat shocked into subsequent germination;
another
part
of the specimen is cultivated without heat treatment in case spores are very
heat
sensitive
or absent.
In
specimens submitted for food poisoning investigations, or for isolation of B.
anthracis from
old
carcasses, animal products, or environmental specimens, the organisms will
mostly be
present
as spores. Heat treatment, as described above, will both heat shock the spores
and
effectively
destroy non-spore-forming contaminants. A variety of approaches are used to
process
dry or solid samples prior to heat treatment. Direct plate cultures are then
made on
blood,
nutrient, or selective agars, as appropriate, by spreading up to 0.1-ml volumes
from
undiluted
sample and 10- and 100-fold dilutions of the treated sample.
Isolation from Animals Suspected To Have Died of
Anthrax
Anthrax
should be considered as the possible cause of death in herbivorous animals that
have
died suddenly and unexpectedly, especially if hemorrhage from the nose, mouth,
or
anus
has occurred, and if death has taken place at a site with a history of
anthrax—perhaps
even
several decades before. PCR-based approaches are becoming more widely used for
direct
detection of B. anthracis in veterinary specimens.
Carcasses 1 to 2 Days Old
Due
to the nonclotting nature of blood in animals that have died of anthrax
infection, it is
usually
possible to aspirate a few drops of blood from a peripheral vein for (i) a
M’Fadyeanstained
smear
and (ii) direct plate culture on blood agar.
In
pigs, the enormous terminal bacteremia seen in herbivores may not develop, and
the
capsulated
rods may not be visible in blood smears. When cervical edema is present, smears
and
cultures should be made of fluid aspirated from the enlarged mandibular and
suprapharyngeal
lymph nodes. Intestinal anthrax of pigs may be obvious only at necropsy,
when
rods are usually visible in stained smears made from mesenteric lymph nodes.
Older Putrefying Carcasses
The
organism may not be visible in smears 2 to 3 days following death, as it
competes poorly
with
putrefactive organisms; culture is then necessary for diagnostic confirmation.
Sections
of
tissue, or any blood-stained material, should be collected, and spleen or lymph
node
specimens
should be taken if the animal has been opened. With putrefied and very old
carcasses,
swabs of the nostrils, nasal turbinates, and eye sockets are likely to yield B.
anthracis,
but the best specimens may be samples of contaminated soil taken
from beneath
the
head and tail.
Isolation of B.
anthracis from Bioterrorism-Related
Specimens
In
1999, an LRN was established in the United States by the CDC in partnership
with the
Association
of Public Health Laboratories, Federal Bureau of Investigation, and Department
of
Defense
to provide the public health laboratory response to acts of bioterrorism (103).
This
network
links local (sentinel) laboratories to laboratories with more specialized
testing and
increased
biosafety capacity at the state (reference) and federal (national) levels.
There are
reference
level laboratories in all 50 states able to detect agents, includingB.
anthracis,
rapidly. State public health laboratories are part of the LRN and
are able to
provide
guidance, or the LRN can be accessed using the Internet
(http://www.bt.cdc.gov/lrn/).
If unable to reach the state public health laboratory during
nonbusiness
hours, LRN consultations may be requested by calling the CDC Emergency
Operations
Center at 770-488-7100 . State and territorial public health
laboratory
contact information and sentinel laboratory guidelines are also available on
the
American
Society for Microbiology website at www.asm.org. For
general questions there is a
24-h
hotline number, 800-CDC-INFO ( 800-232-4636 ), and an e-mail
address,
cdcinfo@cdc.gov. Although initially limited to the United States, there are now
over
150
national and international locations, including laboratories within Canada,
Australia,
Germany
(U.S. military base), Japan (U.S. military base), South Korea (U.S. military
base),
and
the United Kingdom, capable of providing a rapid response to acts of biological
terrorism,
emerging infectious diseases, and other public health threats and emergencies.
Isolation of B.
anthracis from Environmental and
Animal
Product Specimens
Tests
for the presence of B. anthracis may be requested for diverse specimens,
such as
animal
products (e.g., wool, hides, hair, and bonemeal) from regions of endemicity,
soil or
other
materials from old burial sites or tannery or laboratory sites due for
redevelopment, or
other
environmental materials associated with outbreaks (e.g., sewage sludge).
Detection in
such
specimens may mean searching for rather few spores of B. anthracis among
those of
many
other species, especially other members of the B. cereus group. Some
environmental
specimens
may contain substances that inhibit germination and growth of B. anthracis (140).
At
present, there is no enrichment method for B. anthracis, and culture by
the selective agar
techniques
described above is the best approach. PCR-based methods are being used
increasingly
for the direct detection of B. anthracis in clinical and environmental
samples
(125),
but it is still advisable to confirm positive results by conventional methods.
IDENTIFICATION Back to top
Remember
that these organisms do not always stain gram positive; some are gram variable,
gram
positivity is readily lost in older cultures, and some species or strains are
frankly gram
negative.
Before attempting to identify to the species level, it is important to
establish that
the
isolate really is an aerobic endospore former and that other inclusions are not
being
mistaken
for spores. Phase contrast (at a magnification of ×1,000) should be used if
available,
as it is superior to spore staining and more convenient. Spores are larger,
more
phase
bright, and more regular in shape, size, and position than other kinds of
inclusions
such
as polyhydroxybutyrate (PHB) granules (Fig. 2d),
and sporangial appearance is valuable
in
identification (Fig. 2). For spore staining, flood a heat-fixed smear with 10% aqueous
malachite
green for up to 45 min (without heating), followed by washing and counterstaining
with
0.5% aqueous safranin for 30 s; spores are green within pink-red cells at a
magnification
of ×1,000 (Fig. 1d). A Gram-stained smear showing cells with unstained areas
suggestive
of spores can be stripped of oil with acetone-alcohol, washed, and then stained
for
spores.
Bacillus
and related genera contain facultative anaerobes as well as strict
aerobes, which can
be a
valuable characteristic in identification. For example, B. licheniformis and
B.
subtilis,
which have very similar colonial (Fig. 3j)
and microscopic (Fig. 2e) morphologies,
are
facultatively anaerobic and strictly aerobic, respectively. Likewise, the two
large-celled
species
B. cereus and B. megaterium (Fig. 2b and d)
are facultatively anaerobic and strictly
aerobic,
respectively.
The
most widely used diagnostic schemes use traditional phenotypic tests, or
miniaturized
tests
of the API 20E and 50CHB kits used together (90)
(bioMerieux, Marcy l’Etoile, France).
The
API 20E and 50CHB kits can be used for the presumptive distinction of B.
anthracis from
other
members of the B. cereus group within 48 h. bioMerieux also offers
identification cards
for Bacillus
and related genera for the VITEK and VITEK Compact automated identification
systems.
As many new species have been proposed since these schemes were established,
updated
API and VITEK databases have been prepared. Biolog Inc. (Hayward, CA) also
offers
aBacillus
database. The effectiveness of such kits can vary with the genera and
species of
aerobic
endospore formers concerned, but they are improving with continuing development
and
enlarged databases. The many proposals for new species, often on the basis of
single
isolates,
make the satisfactory expansion of such databases problematic; for a database
to
be
effective in identifying a particular species, its entry for that species needs
to reflect the
characterization
of at least 10 authentic strains from a range of sources, but this requirement
can
be very difficult, even impossible, to fulfill. It is stressed that the use of
these kits should
always
be preceded by the basic characterization tests described below.
Other
approaches include chemotaxonomic fingerprinting by fatty acid methyl ester
profiling,
pyrolysis
mass spectrometry, and Fourier transform infrared spectroscopy. All these
approaches
have been successfully applied either across the genera or to small groups. As
with
genotypic profiling methods, large databases of authentic strains are
necessary; some
of
these are commercially available, such as the Microbial Identification System
software
(Microbial
ID Inc., Newark, DE) database for fatty acid methyl ester analysis.
For
diagnostic purposes, the aerobic endospore formers comprise two groups: the
reactive
ones,
which give positive results in various routine biochemical tests and which are
therefore
easier
to identify, and the nonreactive ones, which give few, if any, positive results
in such
tests.
Nonreactive isolates tend to dominate the identification requests sent to
reference
laboratories.
Table 1 shows reactions for some species belonging to the former group,
and
the
phenotypic test scheme outlined above may be used in conjunction with it.
Identification and Detection of B. anthracis
It is generally easy to distinguish virulent B.
anthracis from other members of the B.
cereus group. B. anthracisisolates are characterized by typical
microscopic appearance (Fig.
1b and
2a) and colonial morphology (Fig. 3a): colonies are white or gray, nonhemolytic or
only weakly hemolytic, susceptible to the diagnostic
gamma phage (inquiries about gamma
phage should be addressed to the Diagnostics
Systems Division, USAMRIID, Fort Detrick,
Frederick, MD), generally susceptible to
penicillin, nonmotile, and able to produce the
characteristic capsule as shown by M’Fadyean
staining (Fig. 2a) or India ink staining (Fig.
1b).
As an alternative to culture in blood, the capsule of virulent B. anthracis can
be
demonstrated on nutrient agar containing 0.7%
sodium bicarbonate, incubated overnight
under 5 to 7% CO2 (candle jars perform well).
Colonies of the capsulated organism appear
mucoid, and the capsule can be visualized by
M’Fadyean or India ink staining of smears or by
DFA staining (Fig. 1c; see below).
In addition to phenotypic analysis, molecular and
antigenic detection assays are available for
the rapid identification of B. anthracis. The
LRN PCR (restricted to LRN laboratories; see
above) targets three distinct loci on the B.
anthracis chromosome, pXO1 virulence plasmid,
and pXO2 virulence plasmid. Using several loci
increases specificity and allows for the
detection of avirulent strains (lacking pXO1 or
pXO2). The anthrax toxin genes (pagA, lef,
and cya) are located on pXO1, while the
genes required for capsule
biosynthesis (capBCA) are located on pXO2.
Isolates lacking pXO2 or both plasmids are
mostly found in the environment and are frequently
mistaken for B. cereus, due to the lack
of a capsule, and discarded. These genes have been
widely used as B. anthracis-specific
gene targets; however, there have been recent reports
of these genes in species other
than B. anthracis (8,
55, 67). Recently several laboratories have developed
specific PCR
assays for B. anthracis that target
chromosomal genes such
as rpoB, gyrA, and plcR (39,
70, 111).
A two-component DFA assay has been and is
currently used to identify encapsulated
vegetative cells of B. anthracis (33,
45). This assay uses two different monoclonal
antibodies
specific for a B. anthracis cell wall
antigen and the B. anthracis capsule (Fig. 1c). Neither
antigen is 100% specific for B. anthracis; however,
onlyB. anthracis has been found to be
positive for both antigens, and thus, the assay is
100% specific when both cell wall and
capsule components are used together. It was
heavily used at the CDC during the 2001
bioterrorism-associated outbreak for the rapid
(<4-h) identification of isolates (33).
Tetracore Inc. (Gaithersburg, MD) has produced a
rapid (yielding a result within 15 min)
immunochromatographic test (RedLine Alert)
utilizing an antibody specific for one of the B.
anthracis S-layer proteins. This assay has been approved by the Food and
Drug
Administration (FDA) for use on nonhemolyticBacillus
species colonies cultured on sheep
blood agar plates. Manufacturer’s data suggest
that the test was 98.6% sensitive when
tested on 145 B. anthracis isolates and 45
nonhemolytic, non-B. anthracis isolates; however,
such identification of B. anthracis is only
considered presumptive, and this test should not be
used as a stand-alone test.
An immunochromatographic field assay has been
developed by the U.S. Naval Medical
Research Center (NMRC), Silver Spring, MD, to
detect PA in samples of blood or tissue
exudates (Bacillus anthracisimmunochromatographic
field assay). Inquiries from veterinary
and public health authorities should be directed
to the NMRC by calling 301-319-
7409 or in writing to the Naval Medical Research
Center, 503 Robert Grant Ave., Silver
Spring, MD 20910 (J. Czarnecki, NMRC, personal
communication, 2007). The assay has been
used to detect B. anthracis in animals,
even several days after death. The assay has a high
sensitivity for the detection of B. anthracis in
an infected animal and has a high specificity
(regarded as 100%; 95% confidence interval, 98.5
to 100%) for detection of the organism in
cattle (104). Among 10 recently
vaccinated bovines in one study, the assay yielded no falsepositive
reactions.
Identification of Bacillus cereus and B.
thuringiensis
Colonies of B. cereus and relatives are
very variable but readily recognized (Fig. 3a to c):
they are characteristically large (2 to 7 mm in
diameter) and vary in shape from circular to
irregular, with entire to undulate, crenate or
fimbriate edges; they have matte or granular
textures. Smooth and moist colonies are not
uncommon, however. The optimum growth
temperature is about 37°C, with minima and maxima
of 15 to 20°C and 40 to 45°C,
respectively. Although colonies of B. anthracis
and B. cereus can be similar in appearance,
those of the former are generally smaller and
nonhemolytic, may show more spiking or
tailing along the lines of inoculation streaks,
and are very tenacious compared with the
usually more butyrous consistency of B. cereus and
B. thuringiensis colonies, so that they
may be pulled into standing peaks with a loop. Bacillus
mycoides produces characteristic
rhizoid or hairy-looking, adherent colonies which
readily cover the whole agar surface.
The key characteristics for recognizing and
distinguishing the B. cereus group are colonial
morphology (Fig. 3ato c);
large cells often in chains, producing ellipsoidal spores not swelling
the sporangia (Fig. 2b and c),
usually within 48 h and often apparent after 24 h; facultative
anaerobes; and positive egg yolk reaction (i.e.,
lecithinase). Negative or very weak
hemolysis and lack of motility distinguish B.
anthracis and B. mycoides fromB. cereus and B.
thuringiensis. Bacillus cereus, B. mycoides, B.
thuringiensis, and, to a lesser extent, B.
anthracissynthesize lecithinases, forming opaque zones of precipitation around
colonies on
egg yolk agar as the colonies grow (i.e., usually
after overnight or perhaps 24 h of
incubation). Recognition of B. thuringiensis is
largely dependent on observation of its cuboid
or diamond-shaped parasporal crystals in
sporulated cultures (after 2 to 5 days) by phasecontrast
microscopy (Fig. 2c), or by staining with
malachite green counterstained with carbol
fuchsin or safranin.
Toxin Detection
The enterotoxin complex responsible for the
diarrheal type of B. cereus food poisoning has
been increasingly well characterized (128).
Two commercial kits are available for its
detection in foods and feces, the Oxoid BCET-RPLA
(Oxoid Ltd.) and the TECRA VIA (TECRA
Diagnostics, Roseville, New South Wales,
Australia). However, these kits detect different
antigens, and there is some controversy about
their reliabilities. Other assays, based on
tissue culture, have also been developed. The
emetic toxin of B. cereus, cereulide, has been
identified as a dodecadepsipeptide, and it may be
nonspecifically assayed in food extracts or
culture filtrates using HEp-2 cells (46),
boar semen (4), and rat mitochondria (75).
Specific
detection requires high-performance liquid
chromatography-mass spectrometry (59), and the
synthetic apparatus may be detected by real-time
PCR (49).
TYPING SYSTEMS Back to top
Genotyping of B. anthracis
B. anthracis is a genetically monomorphic species and has been shown to be
clonal by
multilocus sequence typing (MLST). The first
method described that could differentiate
strains with any useful resolution was a
multiple-locus variable-number tandem-repeat
analysis (MLVA) assay targeting eight loci, MLVA-8
(76). This method was relied on during
the 2001 bioterrorism-associated outbreak in the
United States, which implicated the Ames
strain, and continues to be useful today (76,
132). There are now multiple MLVA schemes
available for the genotyping of B. anthracis, including
expanded versions of the original
MLVA-8. These include schemes which employ agarose
electrophoresis, capillary
electrophoresis, or mass spectrometry for fragment
analysis (76, 87, 89, 143). An online
database, MLVAbank, is also now available at http://minisatellites.u-psud.fr/MLVAnet/, which
can accept data entry for 25 variable-number
tandem repeats. There are also several reports
on the analysis of single nucleotide repeats to
differentiate very closely related isolates
(77, 78, 87,89, 131).
The sequencing of multiple B. anthracis genomes
has also led to additional approaches to the
genotyping of B. anthracis, such as the use
of single nucleotide polymorphisms and DNA
microarrays. Single nucleotide polymorphisms have
been identified and used for the
differentiation of B. anthracis lineages
and for detection of specific strains such as the Ames
strain (144, 145).
This approach may continue to become more powerful as more genome
data become available and are analyzed.
Microarrays have also been used for strain
characterization and comparisons (36,
153). However, they have not been widely used and
seem to be less attractive now that newer de novo
sequencing technologies have become
more affordable and allow for the rapid generation
of complete or almost complete genome
sequence data. As technologies continue to improve
and the costs decrease, the complete
genomic sequence will increasingly be used for
isolate characterization.
Genotyping of Other Species
The majority of work on molecular typing (i.e.,
genotyping) of Bacillus spp. has focused on
members of the B. cereus group due to their
clinical importance and the value of genotyping
for molecular epidemiology. In the past, this
group was differentiated into serovars based on
flagellar antigen variations; however, this is not
commonly used today and has largely been
replaced by molecular methods. Numerous molecular
methods have been attempted to some
degree for the differentiation of Bacillus isolates.
Recently, MLST, which compares the partial
sequences of seven housekeeping genes to
differentiate strains, has become the favored
approach due to decreasing costs, portability of
data, and availability of public databases on
the World Wide Web. Several MLST schemes targeting
different sets of genes have been
reported and generally show similar clusters of
isolates into three distinct clades
(64, 81, 110, 126, 136). Pathogenic isolates are distributed throughout
clades 1 and 2, while
no clinical isolates have been identified as
belonging in clade 3 (34, 68). With the exception
of B. anthracis and emetic isolates of B.
cereus, which are largely clonal, B. cereusgroup
isolates are represented by a large number of
distinct sequence types, and clustering does
not correlate with classic microbiological species
identification. Simpson’s index of diversity
(measure of the likelihood of two isolates from
epidemiologically distinct events having the
same sequence type) was calculated to be 0.989
(1.0 is absolute discrimination) in one study
of clinical isolates, which suggests that in
addition to being a powerful phylogenetic tool, it
may be useful for molecular epidemiology (68).
The most powerful aspect of MLST is the
availability of online databases for several of
the MLST schemes, which allows for worldwide
access to view and deposit isolate data. The
Priest scheme is available
athttp://pubmlst.org/bcereus/, and an optimized version of the Tourasse scheme,
including a
multischeme database (SuperCAT), is available at mlstoslo.uio.no/ (110, 136, 137).
SEROLOGIC TESTS Back to top
Serologic assays for the detection of antibody
response against the anthrax toxin protein, PA,
have been used in combination with PCR or IHC
assay results to confirm anthrax cases when
culture failed. A quantitative human anti-PA
immunoglobulin G (IgG) enzyme-linked
immunosorbent assay was performed at the CDC
during the 2001 outbreak and was positive
only with sera from individuals with anthrax or
vaccinated with anthrax vaccine adsorbed
(AVA; see “Vaccines” below) (112).
An FDA-approved, qualitative kit (QuickELISA Anthrax-
PA kit) from Immunetics (Boston, MA) is not
currently available, but production could be
initiated if needed for the detection of anti-PA
IgG and IgM antibodies in human serum.
Serologic assays aided in the effort to confirm
cases in the 2001 attack, particularly
cutaneous ones; however, the time to
seroconversion after infection limits the usefulness of
this approach, given the rapid diagnosis necessary
for treatment and public health response.
The three protein components of anthrax toxin (PA,
LF, and edema factor [EF]), and
antibodies to them, can be used in enzyme
immunoassay systems. For routine confirmation
of anthrax infection or for monitoring response to
anthrax vaccines, antibodies against PA
alone appear to be satisfactory; they have proved
useful for epidemiological investigations
with humans and animals. In human anthrax,
however, early treatment sometimes prevents
development of a detectable rise in antibody titer
(113). PA, LF, and EF are available
commercially from List Biological Laboratories,
Inc., Campbell, CA (http://www.listlabs.com).
In countries of the former USSR, a skin test
utilizing anthraxin, a heat-stable extract from a
noncapsulate strain of B. anthracis that
has been licensed for human and animal use since
1962, is widely acclaimed for retrospective
diagnosis (123). The delayed-type
hypersensitivity is interpreted as indicating
cell-mediated immunity to anthrax and can be
used to evaluate the vaccine-induced immune status
after periods of several years, as well
as to diagnose anthrax retrospectively. Anthraxin
does not contain highly specific anthrax
antigens and relies on the fact that the only Bacillus
species likely to proliferate within and
throughout an animal is B. anthracis. This
is also true of the Ascoli test, which, dating from
1911, must be one of the oldest antigen detection
tests in microbiology. It is a precipitin test
using hyperimmune serum raised to B. anthraciswhole-cell
antigen to provide rapid
retrospective evidence of anthrax infection in an
animal from which the material being tested
was derived. The test is still in use in Eastern
Europe and central Asia.
ANTIMICROBIAL SUSCEPTIBILITIES Back to top
Bacillus anthracis
Most strains of B. anthracis remain
susceptible to penicillin (30, 102, 141). Of 25 genetically
diverse isolates from around the world, three strains
were resistant to penicillin but were
negative for β-lactamase production. Most strains
give variable susceptibility results for
cephalosporins; in vitro results, even if
susceptible, may not predict clinical efficacy,
particularly for expanded- and broad-spectrum
cephalosporins (30). In a study of 50
historical isolates from humans and animals and 15
clinical isolates from the 2001
bioterrorism attack in the United States, the
majority of strains could be regarded as not
susceptible to the broad-spectrum cephalosporin
ceftriaxone, and 3 were resistant to
penicillin (102). Tetracyclines,
fluoroquino lones, and chloramphenicol are suitable for the
treatment of patients allergic to penicillin; most
strains in the previously mentioned study
showed only intermediate susceptibility to
erythromycin (102). Ciprofloxacin and the newer
quinolone gatifloxacin had good in vitro
activities against 40 Turkish isolates, but for another
new quinolone, levofloxacin, it was observed that
MICs were high for 10 strains (43). Other
in vitro studies have shown novel fluoroquinolones
and a ketolide to be of potential
therapeutic value (37, 48).
Standards for antimicrobial susceptibility testing of B.
anthracis have been recently adopted (28), and a DNA microarray for
detecting antimicrobial
resistance determinants in B. anthracis and
B. cereus has been developed (7).
Postexposure prophylaxis (PEP) is needed for the
prevention of inhalation anthrax following
exposure to aerosols containing B. anthracis spores;
the recommended regimen is 60 days of
antimicrobial therapy and three doses of AVA (17),
and recommended antimicrobial agents
include ciprofloxacin, doxycycline, and
levofloxacin. PEP was, for example, recommended for
four persons who had been in the unventilated work
space during procedures that generated
aerosols from untreated animal hides used for
making drums. Amoxicillin is recommended as
an option in cases where the B. anthracis strain
has been demonstrated to be susceptible to
penicillins, and when other antimicrobial agents
are not considered safe, as in the treatment
of children and pregnant or lactating women (20,
21). The use of penicillins for PEP or for
treatment of inhalation anthrax following the use
of B. anthracis as a bioweapon gives cause
for concern, owing to the presence of β-
lactamases in B. anthracis isolates and the poor
penetration of β-lactams into macrophages, the
site of spore germination (9). Combination
intravenous antimicrobial therapy with two or more
antimicrobial agents, begun early, such
as with ciprofloxacin and one or more other
antimicrobial agents to which the organism is
sensitive, appeared to improve survival rates
during treatment of cases in the 2001 event in
the United States (71). Following that event,
the recommendation for initial treatment of
inhalation anthrax is intravenous ciprofloxacin or
doxycycline along with one or more agents
to which the organism is normally susceptible (19);
ciprofloxacin is favored over doxycycline
as the primary antimicrobial agent due to its
bactericidal action and central nervous system
penetration in the event of meningeal involvement
(129). A mouse aerosol challenge model
has been developed for determining antimicrobial
agent efficacy in the treatment of
inhalation anthrax (62). In a case of inhalation
anthrax that was naturally acquired when
processing untreated animal hides for drum
construction, the patient’s therapy included
adjunctive use of human anthrax immunoglobulin (147);
however, the use of anthrax
immunoglobulin in the treatment of a subsequent
inhalation anthrax case in 2008 did not
prevent a fatal outcome (3).
Bacillus cereus
There have been rather few studies of the
antimicrobial susceptibility of Bacillus cereus, and
most information has to be gleaned from reports of
individual cases or outbreaks. Bacillus
cereus and B. thuringiensis produce a broad-spectrum β-lactamase
and are thus resistant to
aminopenicillins and cephalosporins; they are also
resistant to trimethoprim. An in vitro
study of 54 isolates from blood cultures by disk
diffusion assay found that all strains were
susceptible to imipenem and vancomycin and that
most were sensitive to chloramphenicol,
ciprofloxacin, erythromycin, and gentamicin (but a
small number of strains showed moderate
or intermediate susceptibility), while 22 and 37%
of strains showed only moderate or
intermediate susceptibilities to clindamycin and
tetracycline, respectively (148). Although
strains are almost always susceptible to clindamycin,
erythromycin, chloramphenicol,
vancomycin, and the aminoglycosides, and are
usually sensitive to tetracycline and
sulfonamides, there have been several reports of
treatment failures with some of these
drugs: a fulminant meningitis which did not respond
to chloramphenicol (96); a fulminant
infection in a neonate which was refractory to
treatment that included vancomycin,
gentamicin, imipenem, clindamycin, and
ciprofloxacin (139); failure of vancomycin to
eliminate the organism from CSF in association with
a fluid shunt infection (11); vancomycin
resistance in strains from respiratory specimens
from pediatric intensive care patients (73);
and persistent bacteremias with strains showing
resistance to vancomycin in two
hemodialysis patients (A. von Gottberg and W. van
Nierop, personal communication). Oral
ciprofloxacin has been used successfully in the
treatment of B. cereus wound infections,
bacteremia, and pulmonary infection, and in vitro
activity has been shown for daptomycin
(27). Clindamycin with gentamicin, given early,
appears to be the best treatment for
ophthalmic infections caused by B. cereus, and
experiments with rabbits suggest that
intravitreal corticosteroids and antimicrobials
may be effective in such cases. CLSI document
M45-A (29) tabulates antimicrobial
breakpoints for aerobic, endospore-forming species.
Other Species
Information is sparse on treatment of infections
with other species. Gentamicin was effective
in treating a case of B. licheniformis ophthalmitis,
vancomycin was successful in cases of B.
licheniformis peritonitis in a CAPD patient (107) and a pacemaker wire
infection, meropenem
succeeded in a case of brain abscess, and
cephalozin was effective against B.
licheniformis prosthetic aortic valve endocarditis. Resistance to macrolides
appears to occur
naturally in B. licheniformis. B. subtilis endocarditis
in a drug abuser was successfully treated
with a cephalosporin, and gentamicin was
successful against B. subtilis septicemia. A B.
pumilus central venous catheter infection treated with several drugs,
including flucloxacillin,
clindamycin, and vancomycin, failed to clear, and
the catheter had to be replaced with a new
one (10). Two cases of cutaneous
infections with B. pumilus initially diagnosed as anthrax
were treated successfully with
amoxicillin-clavulanate, while a third such case was treated
with ciprofloxacin (134). Daptomycin and
ciprofloxacin show in vitro activity against strains
of B. pumilus, B. subtilis, and some other
species (27). Penicillin, or its derivatives, or
cephalosporins probably have long been the first
choices for treatment of infections
attributed to other Bacillusspecies.
However, in a study by Weber et al. (148), over 95% of
isolates of B. megaterium, B. pumilus, B.
subtilis, B. circulans, B. amyloliquefaciens, and B.
licheniformis, along with strains of B. (now Paenibacillus)polymyxa
and three unidentified
strains from blood cultures, were susceptible to
imipenem, ciprofloxacin, and vancomycin,
and only between 75 and 90% were susceptible to
penicillins, cephalosporins, and
chloramphenicol. Isolates of “B. polymyxa”
and B. circulans were more likely to be resistant
to the penicillins and cephalosporins than strains
of the other species—it is probable that
some or all of the strains identified as B.
circulans might now be accommodated
in Paenibacillus, along with “B.
polymyxa.” An infection of a human bite wound with an
organism identified as B. circulans did not
respond to treatment with amoxicillin and
flucloxacillin but was resolved with clindamycin,
while peritonitis in a CAPD patient was
resolved with vancomycin. However, a strain
identified as B. circulans and showing
vancomycin resistance has been isolated from an
Italian clinical specimen (88). Vancomycin
resistance has been reported for P. popilliae, a
biopesticide, and isolates of this species have
been shown to carry genes resembling those
responsible for high-level vancomycin
resistance in enterococci (108).
Of two South African vancomycin-resistant clinical isolates,
one was identified as Paenibacillus
thiaminolyticus and the other was unidentified but
considered to be related toBacillus lentus. The
latter was isolated from a case of neonatal
sepsis and has been shown to have inducible
resistance to vancomycin and teicoplanin; this
is in contrast to the B. circulans and P.
thiaminolyticus isolates mentioned above, in which
expression of resistance was found to be
constitutive (von Gottberg and van Nierop, personal
communication).
VACCINATION Back to top
AVA, the current human vaccine in the United
States, is a cell-free filtrate (formalin treated),
in an aluminum hydroxide-adsorbed gel, from a
noncapsulated, nonproteolytic derivative of
strain V770-NP1_R grown under microaerobic conditions.
The FDA has approved a new route
and schedule for preexposure immunization with
AVA, which calls for a series of five
intramuscular injections administered at 0 and 4
weeks and at 6, 12, and 18 months (95).
To maintain immunity, an annual booster injection
is recommended. Anthrax vaccine
precipitated, the current human vaccine in the
United Kingdom, is an alum-precipitated cellfree
filtrate of Sterne strain (34F2) cultured, under
static batch conditions, with activated
charcoal to increase PA production. Both anthrax
vaccine precipitated (150) and AVA contain
PA as well as trace amounts of LF, EF, and cell
wall proteins. In October 2008 the ACIP voted
to accept provisional recommendations for the
pre-event use of anthrax vaccine among
persons considered to be at risk for exposure to
aerosolized B. anthracis spores; these
provisional recommendations included new language
to address emergency responders, in
addition to the previously approved recommended
occupational and laboratory populations.
The ACIP recommends routine preexposure
vaccination with AVA for persons engaged in
work involving (i) production of high
concentrations or pure cultures of B. anthracis spores,
(ii) activities with a high potential for
production of aerosolized spores, (iii) handling of
environmental samples associated with anthrax
investigations (especially powders) and
performance of confirmatory testing for B.
anthracis in the U.S. LRN for bioterrorism level B
laboratories or above, (iv) making repeated
entries into known B. anthracis sporecontaminated
areas or settings in which repeated exposure to
aerosolized B. anthracis spores
might occur, and (v) engaging in environmental
investigations or remediation efforts of
spore-contaminated areas or other settings with
aerosol exposure. Immunization is not
routinely recommended for emergency and other
responders but may be offered on a
voluntary basis as part of a comprehensive
occupational health and safety program.
Laboratory workers using standard biosafety level
2 practices in the routine processing of
clinical or environmental specimens in general
diagnostic laboratories are not at increased
risk for exposure to B. anthracis spores (22,
25). The development of anthrax vaccines,
including second- and third-generation products, and
that of several human monoclonal and
polyclonal antibody products currently in their
early development and testing phases are the
subjects of recent reviews (120,
138).
EVALUATION, INTERPRETATION, AND REPORTING OF
RESULTS Back to top
While, of course, the isolation of B. anthracis
is always significant and requires urgent
reporting, the majority of other aerobic
endospore-forming species are environmental
organisms, and so they are frequent laboratory
contaminants. Therefore, isolation from a
single clinical specimen is generally not a
sufficient basis for incriminating one of these
organisms as the etiological agent; however, any
such organism should be considered of
potential clinical significance if it is isolated
in pure culture or at least apparently dominating
the microbiota, or if it is isolated in large
numbers or isolated more than once. Opportunistic
infections with Bacillusspecies other than B.
anthracis have been reported since the late 19th
century, and in the last 30 years the clinical importance
of aerobic endospore formers (most,
but not all, of them Bacillus species) has
become widely accepted. It is most important to
assess any clinical isolation of such an organism
in the light of any other species cultured and
the clinical context and to be wary of dismissing
it as a mere contaminant. Moderate or
heavy growth of aerobic endospore formers from
wounds is usually significant, and B.
cereusinfections of the eye are serious emergencies that should always
be reported to the
physician immediately.
Low-level contamination of foodstuffs by aerobic
endospore formers is commonplace, as is
asymptomatic transient fecal carriage. Therefore,
in foodborne illness investigations,
qualitative isolation tests are insufficient. The
ideal criteria for establishing that an aerobic
endospore former is the etiological agent are (i)
isolation of significant numbers
(>105 CFU/g) of the organism from the
epidemiologically incriminated food (and, in the case
of suspected B. cereus food poisoning,
detection of emetic toxin and/or enterotoxin) and (ii)
recovery of the same strain (biovar, plasmid type,
etc.) in significant numbers from acutephase
specimens (feces or vomitus) from the patients but
not from healthy controls.
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