Bacillus species

Authors: Carmelita U. Tuazon, M.D., M.P.H.

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MICROBIOLOGY

Bacillus spp are aerobic spore forming rods that stain gram positive or gram variable. Except for few species the large majority have no pathogenic potential and have never been associated with disease in man or animals. Members of the genus have significant microbiological uses (64). Numerous enzymes, antibiotics and other metabolites have medical, agricultural, pharmaceutical and other industrial applications. Examples of antibiotics formed by Bacillus spp include bacitracin by B. licheniformis or B. subtilis, polymyxin by B. polymyxa and gramicidin by B. brevis. Certain strains of Bacillus have been utilized as biological controls in antibiotics and other assays. Advances in taxonomy have reclassified B. cereus as a group and there are six species included: B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, B. eveihenstephenansis and B. anthracis (12, 20).

EPIDEMIOLOGY

Bacillus organisms are widely distributed in the environment although the primary habitat is the soil. These organisms are usually found in decaying organic matter, dust, vegetable, water, and some species are part of the normal flora. Carriage rate of B. cereus in stools of healthy asymptomatic individuals ranges between 0-43% (62). In the hospital setting, outbreaks and pseudo epidemic have been traced to contaminated ventilator equipment (13), alcohol containing antiseptic wipes (15), disinfectant (ethyl alcohol) (31), hospital linen (10) and dialysis equipment (17). Sources of B. cereus in food borne outbreaks have been described including rice, meat loaf, turkey loaf, mashed potatoes, beef stew, apples and hot chocolate sold in vending machines (19, 39, 45). Epidemiologic studies on the microbiology of street heroin and injection paraphernalia demonstrated that Bacillus spp as the predominant isolates from both specimens (18, 53, 59, 60).

CLINICAL MANIFESTATIONS

Despite the widespread distribution of Bacillus organisms they are rarely implicated with actual infections and are more frequently isolated as a culture contaminant. Careful clinical evaluation is important to determine the significance of the isolation of the Bacillus organism.

The clinical spectrum of infections caused by Bacillus spp. include self-limited food poisoning, localized infections related to trauma (e.g. ocular infections), deep seated soft tissue infections, and systemic infections (e.g. meningitis, endocarditis, osteomyelitis, and bacteremia). Fulminant eye infections are widely recognized complications of non-anthrax Bacillus infections most commonly B. cereus (28, 41). The various species implicated in serious infections include B. cereus, B. subtilis, B. sphaericus, B. alvei, B. laterosporus, B. licheniformis, B.megaterium and B. pumilus (61).

LABORATORY DIAGNOSIS

Although Bacillus spp. have not been recognized as major human pathogens, with recent advances in medical technology and increased number of immunosuppressed patients, they have been increasingly recognized as opportunistic pathogens in the hospitalized patient (5, 16, 23, 26). Most clinical laboratories do not speciate Bacillus spp. because often they are considered as a contaminant. The clinical setting wherein the need for further work-up of the organism is important. Most Bacillus spp grow readily on nutrient agar or peptone media. Growth is sometimes improved by glucose, but not by blood or serum. The optimum temperature for growth varies from 25°C to 37°C (64). In the vegetative form, the bacilli are killed in 1 hour by moist heat at a temperature of 55°C. The spores of B. subtilis may withstand boiling for hours.

In the investigation of food borne related outbreaks, rapid screening of enterotoxin-producing B. cereus the McCoy cell tissue culture system appears to be useful (19). In outbreaks of food poisoning caused by B. cereus, phage typing has been a useful epidemiological tool. A correlation of 80 to 100% between phage types of strains isolated from suspected foods and those of strains isolated from stools of symptomatic patients was observed (1). A commercially available ELISA can be used for the detection of Bacillus diarrheal enterotoxin in a variety of foods and feces(25, 32). Diagnostic real time PCR assays have been used for the detection of B. cereus strains in foods and recent food borne outbreaks (22).

PATHOGENESIS

The different species of Bacillus produce a variety of extracellular products including antimicrobial substances, enzymes, pigments, and toxins in few species (64). Enzymes that can be found on culture include amylase, collagenase, hemolysin, lecithinase, phospholipase, protease, and urease. Two different types of enterotoxins are produced by B. cereus during exponential growth: the enterotoxin causing diarrhea and the emetic toxin, which causes a vomiting syndrome (58). Recent studies on the ocular virulence factors of B. cereus demonstrated that hemolysin BL, a tripartite dermonecrotic permeability factor and crude exotoxin preparations in vivo caused endophthalmitis clinically characteristic of the organism within 4 hours of inoculation (11).

Bacillus cereus may have three different forms of enzyme activity related to lecithinaselike activity; phosphatidylcholine hydrolase is the most studied form and frequently referred to as phospholipase C. This enzyme may have a secondary role in ocular infections by disrupting host cell membrane phospholipids exposed by the action of other toxins (47).

SUSCEPTIBILITY IN VITRO AND IN VIVO

The selection of antibiotics in the treatment of serious Bacillus infections is based on susceptibility in vitro. More recent studies on the in vitro susceptibilities of Bacillus spp have been reported (4, 24, 38, 63, 65).

A microbiological study of 89 strains of Bacillus spp isolated from clinical blood cultures was performed (65). Species of the isolates were determined by the API 50 CA and API 20E systems. The most common clinically significant isolate was B. cereus. Other strains isolated include B. megaterium, B. polymyxa, B. pumilus, B. subtilis, B. circulans, B. amyloliquifaciens, and B. licheniformis.

All strains of B. cereus were susceptible to imipenem, vancomycin, chloramphenicol, gentamicin and ciprofloxacin using the micro dilution susceptibility method (Table 1). Most strains were resistant to clindamycin, cefazolin and cefotaxime. Disc diffusion susceptibility revealed that B. cereus strains were resistant to all penicillins, oxacillin and cephalosporins with the exception of mezlocillin. There was no enhanced activity of clavulanic acid when combined with ticarcillin. Many strains were susceptible to erythromycin and tetracycycline. Over 95% of non B. cereus strains were susceptible to imipenem, vancomycin, LY 146032 and ciprofloxacin (Table 1). Between 75 and 90% of strains were susceptible to penicillin, oxacillin, cefazolin, cefotaxime and chloramphenicol. B. circulans and B. polymyxa were more likely than other non B. cereus to be resistant to penicillin, oxacillin, cefazolin and cefotaxime. Using the disc diffusion method, many non B. cereus strains were susceptible to penicillin, cephalosporins and trimethoprim-sulfamethoxazole (63). Another study of in vitro susceptibilities of ocular B. cereus isolates demonstrated that clindamycin, gentamicin, and vancomycin are all relatively effective against B. cereus as single agents (24). Using checker board in vitro synergy study, clindamycin-gentamicin combination demonstrated higher rate of bactericidal synergy than vancomycin-gentamicin (60% versus 40%). Such bactericidal effect maybe important clinically in disease states e.g. endophthalmitis in which host immune response to infection results in end organ damage and in immunocompromised hosts who have been shown to be at risk of developing B. cereus infections.

In a more recent study using E test, MIC of selected antibiotics was performed on 69 strains of B. cereus from various sources (63). Resistance to penicillin, ampicillin, cephalosporin and trimethoprim was reported and strains were susceptible to clindamycin, erythromycin, chloramphenicol, vancomycin, aminoglycosides and tetracycline. Ciprofloxacin was active against all isolates. These results were confirmed by another study and extended observation to other class of drugs including quinolones (gatifloxacin, levofloxacin, moxifloxacin) rifampin, daptomycin and linezolid which showed 100% susceptibility of all isolates (38).

In vivo studies are limited to ocular infections in animal models. In experimental rabbit models of posttraumatic endophthalmitis caused by B. cereus intravitreal administration of 100 ug of ciprofloxacin prevented the development of disease when given 1 hour and 6 hours after trauma and inoculation (2). In a swine model of experimental posttraumatic B. cereus endophthalmitis the intravitreal efficacy of ciprofloxacin, vancomycin and imipenem was compared. Vancomycin and imipenem treated animals had less inflammation and tissue destruction than control animals. Ciprofloxacin treated animals showed significantly more intraocular destruction and were indistinguishable from controls (3).

ANTIMICROBIAL THERAPY

Isolation of Bacillus organism requires careful clinical evaluation to determine the significance of the finding. Certain risk factors have been associated with significant Bacillus infections including intravenous drug abuse, sickle cell disease immunosuppression from malignancy, renal transplant, neutropenia and corticosteroid therapy and foreign devices including indwelling intravascular catheters central nervous system shunts, breast implants, and pacemaker (16, 34, 54).

Based on in vitro data, the drug of choice for serious infections caused by Bacillus infections is vancomycin since B. cereus is the most common isolate. Based on clinical data, both vancomycin and clindamycin have been used successfully. The latter should not be used until susceptibility is known due to inducible resistance to clindamycin among clinical isolates of B. cereus (38). Whether monotherapy is adequate or combination therapy is better has not been addressed in in vitro models or clinical trials. There is limited data on use of combination therapy. Synergy has been demonstrated between gentamicin in combination with clindamycin or vancomycin (4). There are few reports of patients treated with vancomycin or clindamycin with or without an aminoglycoside (16,53). Other drugs that are highly active and likely to be bactericidal include imipenem, ciprofloxacin and gentamicin, tetracycline, chloramphenicol, clindamycin and erythromycin have activity against Bacillus species. Most Bacillus strains are resistant to broad spectrum cephalosporins and ticarcillin-clavulanate. Empiric coverage with the latter agents should be avoided in immunocompromised patients whose blood cultures yield gram positive aerobic Bacillus until susceptibility testing is available.

Serious infections caused by Bacillus species include ocular infections, endocarditis, bacteremia and septicemia, pneumonia, meningitis, musculoskeletal infections (48), and infections associated with injuries from motor vehicle accidents associated with road trauma (67) and gunshot injuries (37). Systemic antibiotic therapy is usually required in the treatment of most seriousBacillus infections.

A self-limited illness which presents as food poisoning caused by B. cereus requires no antimicrobial therapy. Treatment is usually symptomatic and fluid replacement may be indicated for patients who are severely dehydrated.

Ocular Infections

B. cereus has been recently recognized as a primary pathogen of ocular infections (1 1, 41, 60). Endophthalmitis is a serious illness that can result in visual compromise within 12-48 hours after inoculation (28, 53). Early diagnosis is important to achieve successful treatment. A high index of suspicion is important in the setting of a patient who presents with ocular infection after trauma or in the setting of drug abuse. Prompt recognition of the infection should allow initiation of appropriate therapy before permanent structural changes occur (47). In patients with post-traumatic endophthalmitis caused by B. cereus, if managed aggressively outcome may be associated with preservation of anatomic integrity and restoration of useful visual acuity (8, 21).

In the drug abuse setting both clindamycin and vancomycin have been used as a single agent. Intravitreal dexamethasone to control the destructive inflammation and early vitrectomy have recently been recommended in the management of sight-threatening endophthalmitis such as that induced by B. cereus (47).

Because of the serious sequelae of panophthalmitis an aggressive approach with early vitrectomy and vitreal instillation of appropriate antibiotic is indicated (47). Both local and systemic antibiotics are used. Antibiotics administered systemically, intravitreally, topically, and via periocular routes are used in conjunction with surgical intervention. An aminoglycoside either gentamicin or tobramycin has been administered locally and systemically but is inadequate to eradicate the infection (35). Clindamycin or vancomycin with or without aminoglycoside is appropriate before the results of culture since B. cereus is the predictable isolate. Clindamycin and gentamicin seem to be favored by the ophthalmologists. Clindamycin has moderate to good activity against B. cereus and when administered subconjunctivally or parenterally reaches therapeutic levels in the iris, choroid and vitreous. Intravitreal administration is favored and a combination of 200-400 µg of gentamicin and 450 µg of clindamycin is recommended (28). In addition, 8 µg/ml gentamicin and 9 µg/ml clindamycin can be added to the vitrectomy infusion fluid (43). Newer drugs (e.g., i mipenem and quinolones) appear active, but more experience is needed in their use (65). In experimental rabbit models of postraumatic endophthalmitis caused by B. cereus intravitreal administration of 100 ug of ciprofloxacin prevented the development of disease when given 1 hour and 6 hours after trauma and inoculation (3). In a swine model of experimental posttraumatic B. cereus endophthalmitis the intravitreal efficacy of ciprofloxacin, vancomycin and imipenem was compared. Vancomycin and imipenem treated animals had less inflammation and tissue destruction than control animals. Ciprofloxacin treated animals showed significantly more intraocular destruction and were indistinguishable from controls (2).

Endocarditis

Endocarditis caused by Bacillus organisms is a well-recognized complication of intravenous drug abuse (54, 60, 61). Rarely, it has been isolated from patients with underlying valvular disease (55) as well as native valves (46, 57). Since B. cereus is the most common isolate, empirical use of penicillin is usually not effective. Antibiotic therapy with vancomycin or clindamycin has achieved high cure rates in Bacillus endocarditis. Intravenous drug abusers with endocarditis caused by Bacillus species have responded well to clindamycin.

Other patients with underlying valvular heart disease prosthetic valve (14) and pacemaker (9) have required surgery in addition to antibiotic therapy (55). In this series, majority of patients with Bacillus endocarditis were treated with clindamycin.

Bacteremia

Intravascular devices are the common source of positive blood cultures for Bacillus species. In patients with positive blood cultures for Bacillus, a decision has to be made whether the organism is causing disease. In most cases, especially if the patient is asymptomatic, the bacteremia is limited and requires no antimicrobial therapy which emphasizes that the process is relatively benign. A recent report on AIDS patient with Bacillus cereus bacteremia also emphasizes the low morbidity associated with this condition (7). However, there have been case reports of fulminant sepsis complicated by hemolysis in patients with acute leukemia (5), as well as bacteremia and meningitis in immunocompromised children (23). Bacteremia and catheter infection due to B. cereus has been reported in an immunocompetent patient (29). B. cereus can produce biofilm which may play a role in attachment to catheters (29). B. cereus bacteremia in neonates and infants has poor prognosis with mortality rate of 60% (36).

Appropriate therapy can be readily instituted once a decision has been made that a clinically significant infection is present. Usually infections have an indolent course and the institution of antimicrobial therapy can await specific sensitivity results. In the setting of immunosuppressed patients with chronic indwelling catheters, recent experience suggests that catheters should be promptly removed to prevent recurrent bacteremia in addition to administering antibiotic therapy (16). Depending on how ill the patient, antibiotics e.g. vancomycin with or without gentamicin maybe initiated until susceptibility studies become available.

Musculoskeletal Infections

Musculoskeletal infections caused by Bacillus species have been reported. Necrotizing fasciitis has been described in patients with lymphoma, leukemia sickle cell disease, alcoholic/diabetic patients (54, 60, 61). Antibiotic therapy alone is not sufficient, multiple surgical debridement is usually required and rarely amputation maybe necessary.

Depending on the species, antibiotic therapy should be tailored accordingly. Again, if the patient is critically ill, vancomycin plus gentamicin is a reasonable initial regimen.

Acute vertebral osteomyelitis caused by B. cereus has been reported in drug abusers and most likely introduced via blood stream by infection of contaminated heroin and/or paraphernalia (59). Chronic osteomyelitis has usually been related to accidental trauma and infection is usually difficult to eradicate and requires multiple surgical procedures. Cases of vertebral osteomyelitis caused by B. cereus have responded well to prolonged intravenous antibiotic therapy (60).

Therapy for chronic osteomyelitis can be very prolonged as was in a 13 year old girl who developed osteomyelitis of femur caused by S. aureus and subsequently superinfected with B.cereus. She was treated with vancomycin for 6 months (52).

Clinically significant infections by Bacillus spp in patients who are involved in motor vehicle accidents and sustain injury related to road contact have been reported (67). B. cereus is the major isolate from cultures of wound and bone biopsies from infected sites. Patients require extensive surgical debridement and amputation in some cases in addition to intravenous vancomycin with or without gentamicin. B. cereus has also been recovered from patients with severe soft tissue infections after close range gunshot wounds inflicted through clothing (37). The organism was isolated from all three patients and in one patient B. cereus was also recovered from the blood. Cefuroxime and metronidazole used in the treatment of these patients were not effective. The authors emphasized that antimicrobial coverage should include agent (s) with spectrum against B. cereus in the setting of traumatic wounds after gunshot injuries.

Central Nervous System Infections

Central nervous system invasion by B. cereus includes meningitis, meningoencephalitis, subarachnoid hemorrage and brain abscess seen in pediatric and adult patients (12, 23, 40, 44, 49).

A wide variety of Bacillus species have been isolated from cerebrospinal fluid of patients who had spinal anesthesia, subdural hematoma, ventricular shunts and parameningeal foci of infections e.g. otitis and mastoiditis. Removal of any foreign body such as a ventricular shunt is necessary to eradicate the infection. Initial therapy is with intravenous vancomycin with or without aminoglycoside and can be adjusted when susceptibility result becomes available. Carbapenems may also be used as an alternative agent.Supplemental intraventricular or intrathecal instillation of vancomycin may be required in patients with poor response. Intrathecal doses of 3-5 mg of Vancomycin may be used in addition to parenteral antibiotics (56).

In the treatment of central nervous system (CNS) infections one has to take into consideration the ability of the antibiotic to penetrate the CNS. Clindamycin does not reach significant levels in the cerebrospinal fluid (CSF) even with meningitis. Vancomycin appears to be the preferred drug regimen in the setting of serious CNS infections. In certain settings, e.g. brain abscesses, prolonged antibiotic treatment may be required. A 3 year old patient with acute lymphocytic leukemia who developed multiple brain abscesses and meningitis was treated successfully with antibiotics alone (33). Surgical excision of the abscesses was precluded by the number and location of the multiple abscesses. The patient was treated with intravenous vancomycin and gentamicin for 3 weeks and then rifampin was substituted to gentamicin for a total treatment of over 8 weeks. The abscesses completely resolved on follow up CT scan studies.

Pneumonia

In previously healthy adult welders rapidly progressive pneumonia and bacteremia caused by B. cereus which resembled Bacillus anthracis has been reported (42). In a more recent series two metal workers with fatal pneumonia caused by B. cereus were also reported (6). Neither patient had any predisposing comorbidities. Interestingly, isolates tested positive for B. anthracis virulence toxins.

Dosage and Duration of Therapy

For deep seated infections caused by Bacillus cereus optimal dosages of clindamycin and vancomycin are indicated. In patients with Bacillus endocarditis vancomycin 1gm every 12 hours maybe used and dosage maybe adjusted to achieve peak therapeutic levels of 30-40 µg/ml and trough levels of 5-10 µg/ml. Gentamicin at 3-5 mg/kg/day in divided dosages adjusted to peak therapeutic levels of 4-10 µg/ml and trough levels of 1-2 µg/ml. Gentamicin maybe used in combination with vancomycin especially in patients with left sided endocarditis. In the setting of drug abuse, vancomycin or clindamycin can be used since both have been proven efficacious with successful outcome. Dosage of clindamycin may vary from 600 to 900 mg every 6 to 8 hours depending on the severity of the illness.

Similarly for osteomyelitis, the same antibiotics may be used and the duration of treatment maybe longer depending on the adequacy of surgical debridement. Treatment can be as long as 6 months depending on healing of wound and fractures (52). Clindamycin maybe preferred in the setting of osteomyelitis because of higher levels of drug achieved in bone tissues.

For ocular infections, doses of clindamycin or vancomycin in combination with gentamicin will be similar to the treatment of deep seated infections as outlined above. In addition, as equally important is the intraocular administration of antibiotics as described earlier. It appears that clindamycin is preferred by ophthalmologists because of good penetration in ocular tissues and also based on limited in vitro synergy data.

In patients with uncomplicated bacteremia dosages for either vancomycin or clindamycin will be as stated above. Duration of treatment can vary from 7-14 days depending on the severity of the illness and underlying host defense impairment.

Depending on the clinical setting, one may prefer to use vancomycin over clindamycin such as nosocomially acquired infection if there is a concern for C. difficile colitis as a complication. Newer drugs e.g. i mipenem and quinolones appear active but more experience is needed in their use. There has been limited experience with the use of oral antibiotics in the treatment of B. cereus infections. A patient who developed severe wound infection with bacteremia caused by B. cereus was treated successfully with ciprofloxacin 750 mg every q 12 hours for almost 3 months (36). Doxycycline has been used in the treatment of bacteremia associated with Hickman catheters caused by B. cereus in a patient with non-Hodgkins lymphoma (50).

After completion of recommended parenteral antibiotics treatment, the clinician may choose to extend treatment with oral antibiotics in such settings as chronic osteomyelitis and soft tissue infections. Oral ciprofloxacin or clindamycin are reasonable choices.

ADJUNCTIVE THERAPY

In the management of serious infections caused by B. cereus surgical procedures e.g. drainage of abscesses or debridement maybe required in addition to appropriate antimicrobial therapy (49). In the setting of neutropenia, granulocyte stimulating factor may be necessary as adjunctive therapy. Exchange transfusion has been utilized in a septic patient with B. cereus infection complicated by massive hemolysis (5).

ENDPOINTS FOR MONITORING THERAPY

To monitor response to therapy, clinical parameters can be followed such as resolution of fever and feeling of well-being. Examination of sites involved such as skin and soft tissues with decreased tenderness, swelling or erythema are useful signs to monitor. In addition, blood cultures should be repeated to ensure clearance of bacteremia. In the setting of endocarditis, follow-up echocardiography to assess size of vegetation and/ or function of valve including prosthetic valve may be useful in the management of these patients. CT scans maybe helpful in detecting recurrent emboli in cases of endocarditis or in detecting loculated infections. Although, a non-specific parameter, sedimentation rate or C-reactive protein maybe helpful especially in the setting of endocarditis, osteomyelitis, or other deep seated infections.

In certain settings wherein adequate debridement has been performed, persistent isolation of Bacillus species is an indication for repeat susceptibility testing to ensure that the organism remains sensitive to the antibiotic being administered.

PREVENTION

The main preventative measure for gastroenteritis caused by B. cereus is proper food handling. The heat-resistant spores of B. cereus survive boiling and germinate when boiled rice is left unrefrigerated (25). Flash frying or brief rewarming of rice before serving is not adequate to destroy the preformed, heat-stable toxin. The food should either be maintained at a temperature higher than 60°C, or if it is going to be stored, should be cooled rapidly to a temperature below 8-10° to prevent growth or greatly reduce its rate(39). Hand hygiene is important especially in the setting of hospital outbreaks, alcohol based products are ineffective. Hand washing with soap and water and 2% chlorhexidine gluconate are effective (51, 66).

COMMENTS

Biocidal agents to decontaminate Bacillus are being investigated. Two non toxic anti microbial nano emulsions BCTP and BCTP 40 have been developed (27). Animal studies demonstrated the protective and therapeutic effect of BCTP in vivo. Mixing BCTP with B. cereus spores before injecting the spores into mice prevented the pathologic effect of B. cereus. Decontamination of Bacillus spores prior to or after exposure effectively reduce the morbidity and mortality from B. cereus infection in mice experiments.

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Tables

Table 1: Antimicrobial Susceptibility of Bacillus spp Isolates

ORGANISM	DRUG	MIC (µg/ml)
Bacillus cereus	Mezlocillin Cefazolin Cefotaxime Imipenem Vancomycin LY146032 Ramoplanin Teicoplanin Chloramphenicol Clindamycin Gentamicin Trovafloxacin Levofloxacin Ciprofloxacin Norfloxacin Ofloxacin Sparfloxacin	1 -32 0.5 - > 64 16 - > 128 0.25 - 4 0.25 - 2 0.25 – 4 0.06 –0.25 0.12 – 1. 2 - 4 0.25 - 2 0.25 – 2 .03 – 0.12 0.06 – >16 0. 0> 0.0> 0.0
Non - B. cereus	Penicillin Mezlocillin Oxacillin Cefazolin Cefotaxime Imipenem Vancomycin Chloramphenicol Clindamycin Ciprofloxacin	0.03 - >8 1- > 128 0.125 - > 16 0.5 - 32 1 - > 128 0.25 - 16 0.25 - 4 1 - 32 0.125 - > 16 0.25 - 1