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Clinical spectrum of infection due to Bacillus species
Clinical Spectrum of Infection Due to Bacillus Species
DANIEL DONALD
C. IHDE,
M.D.*
ARMSTRONG,
M.D. :
New York, New York
From the Department of Medicine, Infectious Disease Service, Memorial Hospital for Cancer and Allied Diseases, and Cornell University Medical College, New York, New York. Requests for reprints should be addressed to Dr. Donald Armstrong, Memorial Hospital, 444 Fast 66th Street, New York, New York 10021. Manuscript accepted July 16, 1973. * Supported in part by a Clinical Fellowship Grant and Clinical Investigation Grant No. 26 from the American Cancer Society.
Bacillus species other than B. anthracis were isolated from rulture material and seen on gram-stained smear in 12 patients over a 6 year period in a hospital for patients with neoplastic diseases. In some cases Bacillus species were recovered repeatedly from an individual patient, occasionally from more than one site. Pulmonary and disseminated infection developed in two patients with acute leukemia Who wert under intensive chemotherapy, and they died following rupture of a brain abscess into the ventricular system. In both patients, Bacillus species were recovered from cultures of blood and sputum antemortern, and in the heart’s blood after death, and seen in histopathologic sections of the lung and brain; These cases were similar to those few previously reported cases in that the usually nonpathogenic Bacillus disseminated in an immunologically compromised host. Nearly all the remaining isolates were from drainage from recent surgical wounds. Rarely was specific treatment given for the Bacillus, and they seemed to exert no definite influence on the patient’s clinical course, although occasionally the character of a wound drainage altered after the Bacillus was no longer recovered on culture. The aerobic, spore-bearing, gram-positive rods comprising the genus Bacillus are widely distributed in nature, being found in air, soil, water, milk, dust, wool and feces [l], and, with the exception of Bacillus anthracis, the cause of anthrax, are usually considered nonpathogenic for man. When injected intraperitoneally into mice, however, sufficient quantities of several Bacillus species can lead to fatal disseminated infection, and intracutaneous injection, particularly of hemolysin-producing strains, into several laboratory animals will produce hemorrhagic, necrotic skin lesions or subcutaneous abscesses [l]. In man as well, under certain situations, pathogenicity of nonanthrax Bacillus species-is well recognized. In 1963, Farrar [2] reviewed 11 previously reported cases of serious, disseminated infections with Bacillus species and added one of his own. The infection was fatal in five cases. Several of these infections were associated with a direct route of entry into the subarachnoid space or the urinary tract; others occurred in patients with severe underlying illnesses such as acute leukemia, subdural hematoma or rheumatic heart disease. Since then several reports have documented the occurrence of Bacillus meningitis, pneumonia and bacteremia both in compromised
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DUE TO BACILLUS
SPECIES-II-IDE,
ARMSTRONG
normal [4] and debilitated [3] and in apparently settings in which Bacillus hosts. Two other for infecspecies have been thought responsible tions were cornea1 ulcerations or panophthalmitis
12-I-71 Figure
1.
monary fever.
Case infiltrate
7. Chest roentgenogram shows associated with hemoptysis
pilland
associated with trauma or surgery [5] and febrile reactions in conjunction with the presence of B. cereus in blood and in dialysis fluid in patients undergoing hemodialysis [6]. Allergic and respiratory symptoms in workers engaged in the manufacture of laundry detergents containing derivatives of B. subtilis, principally proteolytic enzymes, have been noted [7,8]. B. subtilis autolysates were found to be capable of aggravating experimental infection with other organisms [9]. Articles describing outbreaks of food poisoning thought to be due to B. cereus have appeared in the European literature but, to our knowledge, not in the United States. Recently, however, most strains of B. cereus and cell-free culture filtrates of the organism have been found capable of eliciting fluid accumulation in ligated rabbit ileal loops, a model often used to study bacterial enterotoxins [lo]. Probably because of their ubiquitous occurrence, Bacillus species are often recovered from clinical specimens submitted to hospital bacteriology laboratories, and they usually tend to be dismissed as contaminants. Two recent experiences in this institution in which a blood culture, obtained while the patient was still alive, was reported to contain a Bacillus species that was not thought germane until after the patient died with evidence of disseminated Bacillus infection, led to a review of all possibly significant cultural isolates of Bacillus species in Memorial Hospital over a 6 year period. MATERIALS
AND
METHODS
Hospital records from the years 1966 to 1971 were reviewed in all cases in which a bacteriologic specimen revealed large gram-positive or gram-variable sporebearing rods on gram stain and in which a Bacillus species grew on culture. In two instances, speciation was confirmed at the Center for Disease Control, Atlanta, Georgia. The charts of 12 of 13 such patients were available for review. The patient population in this hospital consists mostly of persons with malignant neoplastic disease. Antibiotic sensitivity, when determined, was performed by standard disc sensitivity methods. CASE
1. A 63 year old white man was transferred to Memorial Hospital on November 24, 1971, with a diagnosis of acute myelogenous leukemia. He had presented with fatigue, spontaneous ecchymoses and
Case
Figure 2.
Case 1. Pathology slide of material from brain abscess obtained postmortem shows gram-positive spore-bearing rods.
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1973
The American Journal of Medicine
REPORTS
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INFECTION
Figure
3. Case (right).
e&m
fever
responsive
admission. 17,400 mic
2. Chest
to
The
/mm3
94
with
showing
cephalothin;
peripheral
with
therapy
films
per
he
pulmonary
was
afebrile
leukocyte cent
cytosine
blast
count
forms.
arabinoside
Infiltrate
on was
Antileuke-
and
6-thiogua-
nine produced a fall in the peripheral leukocyte count to 400/mm” within 4 days. At that point the patient had a shaking Rales
chill, were
a dry cough heard
and a temperature
in the
left
axilla,
and
of 105°F.
a chest
roent-
genogram revealed a wedge-shaped infiltrate in upper lobe of the left lung, which had been clear admission gentamicin
(Figure therapy
persisted
and,
pectorate
after
copious
following added
1). Intravenous was begun. Fever
day, to the
2 days, amounts
carbenicillin antibiotic
on the
regimen.
spore-bearing tilts on culture. the
patient
following
Postmortem of
the
patient
began
to ex-
sputum.
amphotericin Blood
taken
day
contained
comatose.
examination bone
marrow.
lung was firm in consistency neous appearance. Gross
revealed The
upper
for
cul-
right
noted.
of
Car-
Similar
organisms
tained
postmortem.
Case
2.
An
Memorial fever,
in
sev-
which
blood
old
patient
a
white
acute
leukocyte
mm3.
On the
ductive infiltrate were 103°F
7th
persistent or greater.
seen
on
(Figure
aerobic,
spore-
hospital
was 1970, cough.
was
admitted
to
because
of
She
had
leukemia
administered,
the
daily elevations Kanamycin was distress,
ob-
was and
1,700imm3
and
treatment count fell
day,
tissue
her temperature was negative,
with and
the
vincristine
of her leukebelow 1 ,OOO/
patient
a chest roentgenogram lower lobe of the right
Examination
lung
girl
Prednisone
begun for leukocyte
30, respiratory noted.
were
afebrile.
cough; in the
also
lymphoblastic
count
blast forms. and ampicillin
administration was mia. The peripheral
pleural
abscess
17,
nonproductive
with
became
from
on December
and
were
grew
cultured
since May 1970. On admission, 101.8”F. A chest roentgenogram peripheral
developing
the cerebral
were
relapse
ARMSTRONG
rods thought most similar to B. the Center for Disease Control.
11 year
fatigue
been
um were
revealed
rods,
heart’s
Hospital
cember
left
and had a gray homogesections of the brain re-
of the left lung
of the
SPECIES-IHDE.
rapidly
from
gram-positive on study at
infiltra-
vealed an area of soft, brownish necrosis, replacing the right cingulate gyrus and rupturing into the right lateral ventricle. Gram stain of sections
Culture
bearing cereus
the
leukemic lobe
gram-positive
72 per cent Oxacillin
dilated
were
2).
with
of material
the
gram-positive
A fixed globe
large
sections
On the
rods on smear and colonies of B. subOn the morning of the 6th day of fever,
became
eral
associated
B were
a Bacillus species later reto B. cereus, and sputum
pupil and proptosis of the right dioresprratory arrest followed. tion
oxacillin and and leukopenia
of bloody and
ture at this trme contained ported to be most similar obtained
the
the on
(leff)
DUE TO BACILLUS
had
a pro-
disclosed an lung; and there
in temperature to then given. By De-
hemoptysis
of the
sputum
and deliriand
a tra-
cheal aspirate revealed gram-variable rods on smear and B. subtilis on culture. Blood cultures on the following day contained B. subtilis. Chest films revealed an increase in the pulmonary infiltrate, with a rapidly progressive pleural effusion (Figure 3). The administration
December 1973
of kanamycin
was
discontinued,
The American Journal of Medicine
and carbenicil-
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841
INFECTION
DUE TO BACILLUS
SPECIES-IHDE.
ARMSTRONG
lin and gentamicin therapy was begun, but on the 16th hospital day respiratory distress and delirium worsened, and the patient died. At autopsy, leukemic infiltrates were found in the bone marrow, lymph nodes, liver, kidneys and meninges. The right pleural cavity contained 1,000 cc of cloudy yellow fluid, and areas of consolidation with abscess formation were present in the right lung. Numerous pleomorphic spore-bearing gram-positive rods were seen in the periphery of the abscesses. Multiple, necrotic, green abscess cavities were present in the cerebral cortex and mid-brain. One collection in the right posterior temporal lobe had ruptured into the occipital pole of the lateral ventricle. Clusters of gramvariable rods similar to those in the lung were seen on gram stain. B. subtillis grew from a culture of the heart’s blood, but rio material for culture was obtained from thelung or brain.
Case 3.
A 70 year old white woman underwent right hepatic lobectomy at Memorial Hospital on July 20, 1971, with resection of a hepatocellular carcinoma. A tube was inserted into the right side of her chest at operation. On the 1st postoperative day, bloody drainage was noted from the chest tube and the abdominal drains, and the patient experienced her first elevation in temperature to 101” to 102°F which continued for 5 weeks; cephalothin was administered. The chest tube was removed on the 5th postoperative day, and a right pleural effusion was noted on roentgenograms. Culture of the serosanguineous drainage from the abdominal wound on July 27 revealed moderate numbers of B. subtilis, Esch. coli and enterococci. Several gram-positive spore-bearing rods were seen on smear of the material. On the following day, a right thoracentesis was performed, and moderate numbers of B. subtilis and Esch. coli were cultured from the bloody fluid. Cephalothin therapy was discontinued, and ampicillin and then chloramphenicol therapy was instituted. Culture of fluid from a second thoracentesis on the 9th postoperative day grew B. subtilis, Esch. coli and enterococci, as did drainage from the abdominal wound which still remained serosanguineous, on the 20th postoperative day. Five days later, the abdominal drainage, which had become bile-stained, grew only Esch. coli and enterococci. The patient was discharged with a low grade fever at the end of August, but she returned a few days later with a high fever, persistent drainage from the abdominal wound and a right pleural effusion. A subphrenic pus collection was drained surgically, and revealed Esch. coli and enterococci on culture.
Case 4. A 60 bear old white man had a wide excision of a large malignant melanoma of the right flank on November 30, 1966. Primary wound closure could not be achieved, and pedicle flaps and split thickness skin grafts were placed. Penicillin was given during the first 7 postoperative days; the patient was febrile for the first 4 of these days. On the 2nd postoperative day, musty serosanguineous drainage was noted from the site of the skin flaps. Spore-bearing gram-positive rods later identified as B. macerans were seen on .gram
842
December 1973
The American Journal of Medicine
stain and in cultures of the drainage. They were resistant to penicillin and sensitive to tetracycline. On 3 subsequent days B. macerans was obtained from the drainage, and Staph. aureus began to grow on culture as well. On December 5, tetracycline was added to the drug regimen, and by the following day, the wound drainage was no longer serosanguineous. Many Staph. aureus and few B. macerans grew from the fluid on December 7, but four subsequent cultures revealed only Staph. aureus. The wound began to granulate and eventually healed well. RESULTS
Clinical Features and Microbiology. Bacillus species were responsible for a life-threatening infection in only two patients (Cases 1 and 2). The organism was recovered from surgical wound drainage in 8 of the remaining 10 patients. In one of them (Case 3), B. subtilis was also isolated from a pleural effusion contiguous to a subphrenic abscess. A breast prosthesis, removed because of pain and serous drainage after repositioning, and a necrotic, draining axillary tumor mass in a patient with carcinoma of the breast were the sources of the other two cultures. Characteristics of the cultured material, the age and sex of the patients, and the presence or absence of fever at the time of the first positive culture for a Bacillus species are recorded in Table I. In two cases, organisms were recovered in multiple specimens and typed by the Center for Disease Control B. cereus and
as being
most
closely
related
to
B. macerans. All other isolates were classed as B. subtilis. The organisms were found in four, five or seven specimens in the cases reported in detail, but in the other cases only one or two specimens contained the organism. Discounting blood cultures, other bacteria were recovered from the same specimens which yielded the Bacillus species in all but three cases. These organisms were considered significant only when they were also seen on gram-stained smear. Esch. coli and entercocci in Case 3 and Staph. aureus in Case 4 were present in multiple specimens, and persisted in subsequent cultures after the Bacillus could no longer be isolated. Nine of 12 patients were febrile at the time the Bacillus was cultured. In most instances, the surgical wound or tumor drainage from which Bacillus species were isolated was purulent, bloody or serosanguineous. Other pathogenic bacteria were sometimes present in such culture material as well. In two cases physicians caring for the patients commented on the character of the drainage which changed from bloody to serous; thereafter the Bacillus was no longer recovered on culture. Antibiotic sensitivities on the isolated Bacillus
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INFECTION
TABLE I
Characteristics
of Patients
* $ 0 t
with Bacillus
Source of Bacillus Species Isolate*
AgeW) Case No.
Infected
and Sex
DUE
TO
No. Positive Cultures
+
Serous Serosanguineous
1 2
Blood and purulent Purulent
2
None Gram-positive micrococci Staph. albus
+
1
None
+
Serous
1
None
-
Bile stained
2
+
Serosanguineous
2
Yellow and gelatinous, malodorous
1
Gram-positive micrococcl Gram-positive micrococci Staph. al bus, gram-positive diphtheroides
Hemoptysis, grayish consolidation in lung
4
2
11.
F
Hemoptysis
4
3
70, F
Bloody, serosanguineus
5
4
60,
M
Serosanguineus
5 6
31, 22,
F M
7
11, F
a
50, F
9
43, M
10
52, F
11
59, M
12
39, F
Sputum, tracheal aspirate, blood, postmortem heart’s blood Abdominal wound drainage, pleural fluid Drainage under skin flaps over flank wound3 Breast prosthesis Abdominal wound drainage Abdominal wound drainage Abdominal wound drainage Abdominal wound drainage Abdominal wound drainage Axillary wound drainage Necrotic axillary tumor
a 9 10 11 12 Totals
Recent Surgery
+ + + + 9112
Underlying
Fever
Staph.
Sputum, blood, postmortem lung sections, heart’s blood$
Case No.
Other Bacterlat
7
M
with Infection
ARMSTRONG
+
64,
Factors Associated
IHDE.
None in blood, Pr. mirabilis in sputum, enterococci in lung sections None in blood or sputum, alphastreptococcus in tracheal aspirate Esch. coli, enterococci in both
1
TABLE II
SPECIES
Species Character of Drainage
B. subtilis unless otherwise indicated. B. cereus, see case report. B. macerans see case report. Cultured from same specimen(s) as Bacillus
BACILLUS
+
+
aureus
+
+
and seen on smear.
with Bacillus Species*
Neoplasm
Acute leukemia Acute leukemia Hepatoma Melanoma None Hepatoma Malignant neurilemoma Cancer of breast None Cancer of breast Melanoma Cancer of breast 10/12
Antibiotics
Leukopenia
Recent Radiotherapy
+ -
+ + + +
+
-
+ +
+
+ +
3/12
5112
3112
+ + -
+ + + -
3112
* Measurements of total globulins or total gamma globulins, as determined by total protein electrophoresis or serum immunoelectrophoresis, were normal in all 11 patients
December 1973
Chemotherapy
+ -
+ + + + -
+ + + IO/12
Steroids
serum protein tested.
minus
The American Journal of Medicine
albumin,
Volume 55
serum
843
INFECTION
DUE TO BACILLUS
SPECIES-IHDE.
ARMSTRONG
were determined in nine cases; unfortunately, Cases 1 and 2 were not included. The strains isolated were uniformly resistant to penicillin, ampicillin, oxacillin, methicillin and colistin, whereas they were uniformly sensitive to tetracyclin, chloramphenicol and kanamycin (and to gentamicin in two instances when tested), and variably sensitive to cephalothin and erythromycin. Only once (Case 4) was a clinical decision to alter antibiotic coverage made on the basis of these sensitivity tests, and, at least coincident with the new antibiotic and the disappearance of the Bacillus from cultures, the character of the drainage changed and wound healing accelerated. Predisposing Factors. Factors possibly predisposing to infection in these cases are presented in Table II. Two patients (Cases 1 and 2) were severely leukopenic and were being treated intensively with antileukemic agents and several antibiotics at the time Bacillus species were first isolated. Nine of the remaining 10 patients had undergone recent surgery which was followed by wound drainage; in 8 of these, the operations were for malignant neoplastic disease. Aside from the postoperative administration of antibiotics, factors often associated with increased susceptibility to infection in our patients [ll], such as leukopenia, diminished levels of gamma globulins, and recent treatment with adrenal corticosteroids, chemotherapeutic agents or radiation, were not often present in these latter 10 cases. Significance of Isolations. It was often difficult to evaluate the contribution of the isolated Bacillus species to the subsequent clinical course of the patient. Two patients (Cases 1 and 2), who had leukemia, bacteremia, pneumonia and brain abscess, obviously died of their infections. In one patient (Case 4), in whom a different antibiotic was administered because of the organism’s drug sensit$!ities, the presence of B. macerans was very possibly exacerbating a wound infection. In the remaining patients, however, no specific therapy other than drainage of an infected space was instituted because of the recovery of the Bacillus organism, although one patient (Case 3) was given chloramphenicol therapy for reasons unrelated to the Bacillus isolation. Shortly thereafter cultures from her pleural and abdominal spaces grew only Esch. coli and enterococci and not B. subtilis. On review of the other cases we found no convincing evidence that the presence of Bacillus species altered the patient’s course for good or ill, except that their presence seemed to indicate infection of the operative wound or infection of a tumor mass. Blood cultures were frequently obtained in febrile or deteriorating patients, but with two exceptions (Cases 1 and 2), no Bacillus bac044
December
1973
The American
Journal
of Medicine
teremia was found in association infection or colonization elsewhere
with a Bacillus in the body.
COMMENTS Farrar [2] divided human infections caused by “nonpathogenic” Bacillus species into three groups: (1) local infections of a closed space, such as the eye, that is usually violated by trauma or manipulation; (2) mixed infections in which the Bacillus is found in company with another organism of recognized pathogenicity; and (3), disseminated infections in which the organism is recovered from multiple sites, usually including the blood, in a seriously ill patient. Examples of the second and third groups, and possibly of the first, are included in this series. The B. subtilis recovered from the breast prosthesis in a setting of pain and drainage shortly after surgical repositioning could be considered to have arisen in a closed space subjected to manipulation, and several of the present isolates from surgical wound drainage were recovered in the company of an organism that is a recognized pathogen. The twelve disseminated infections discussed by Farrar [2] included eight cases of meningitis, often accompanied by bacteremia. Meningitis occurred in three patients following spinal anesthesia, in one patient with a subdural hematoma, in one alcoholic with rheumatic heart disease, in one patient with chronic mastoiditis, in one patient with urinary tract instrumentation and obstruction, and in one infant. The remaining four infections occurred in a patient with nephrotic syndrome and in three patients with bacteremia (one with leukemia, one with hydrocephalis and a ventriculoatrial shunt, and one a newborn infant). In the patient of Allen and Wilkinson [4], an apparently previously healthy man, meningitis, bacteremia and possible Schwartzman reaction developed, and the patient described by Coonrod and colleagues [3], who died of B. cereus pneumonia and bacteremia, had “subacute” lymphatic leukemia. Two of our patients (Cases 1 and 2) had leukemia and profound treatment-induced leukopenia. The tendency of disseminated Bacillus infections to occur in debilitated hosts or in those with compromised resistance to infection has been commented upon [l]. Nevertheless, it should be noted that in at least 7 of the 14 patients recently discussed in the English literature, the infection disseminated, albeit sometimes after instrumentation, in apparently immunologically normal subjects [2-41. Antibiotic sensitivities of these organisms are of interest in that they could be variable in different institutions or could be undergoing a change. Of the six patients discussed by Farrar [2] who reVolume
55
INFECTION
covered, four were treated with penicillin, alone or in combination, and the organism recovered by Allen and Wilkinson [4] was sensitive to penicillin and most other antibiotics tested. Sensitivities were not performed in our Cases 1 and 2, but the results in another nine of our cases of uniform resistance to penicillin and its congeners, and sensitivity to kanamycin, chloramphenicol and tetracycline, parallel those found by Coonrod and colleagues [3] in their patient with leukemia. However, in our Case 1 the patient received gentamitin and in Case 2, kanamycin followed by gentamicin, all without effect. Although patients with dissemination of Bacillus species are relatively infrequent, the pathogenicity of the organisms in these instances is easily recognized in retrospect. Infections of ocular structures by Bacillus species are well accepted [1,5]. More difficult to evaluate are the local and mixed infections. The usual event is recovery of the organism from the site of a wound (in conjunction with bacteria on smear of the cultured material that are morphologically identical with Bacillus species) followed by arousal of suspicion that the isolation may be significant. Forty-eight such isolates over a 2 year period in a general hospital were discussed by Pearson [12]. Various Bacillus species were found in skin ulcers, conjunctival ulcers, traumatic and surgical wounds, burns, bone fracture sites and sites of osteomyelitis, urogenital infection and infection of the biliary tree. In over half, or 25, of the cases, another bacterium was present in the isolate, although it is not stated
DUE TO BACILLUS
SPECIES----ibiDE.
ARMSTRONG
whether the second organism was seen on smear. Bacillus species were grown from more than one culture in six patients, and from more than one site in the same patient twice. The uncertainty of significant pathogenicity of the Bacillus species in most of these cases is similar to that in all but Cases 1 and 2 of the current series. Isolation of the bacteria from multiple sites and/or on multiple occasions in a single patient, as was observed in several instances by Pearson [12] and especially in Cases 3 and 4 by us, could tend to confirm that a significant Bacillus infection, or at least colonization, is present. This hypothesis may be further substantiated when the character of a wound drainage or other material is altered in association with treatment and eradication of the Bacillus from cultures. Bacillus species are known to produce hemolysins [l], and alteration in the sanguineous nature of drainage could be consistent with their eradication. Most recoveries of Bacillus species from clinical material are thought to be due to contamination. But the observation of gram-positive rods on smear of the material, especially in a seriously ill patient with compromised immune status, should raise the possibility of an invasive infection and lead to further cultures, including blood cultures. Much more common, however, is the culturing of a Bacillus from local drainage in the absence of bacteremia or a critical clinical situation. The significance of these isolates, even in large numbers and on repeated cultures, remains to be ascertained by further observations.
REFERENCES 1.
2.
3.
4.
5. 6.
Wilson GS. Miles AA: Topley and Wilson’s Principles of Bacteriology and Immunity, 5th ed, Baltimore, Williams 8 Wilkins Co., 1964. Farrar WE: Serious infections due to “non-pathogenic” organisms of the genus Bacillus. Am J Med 34: 134, 1963. Coonrod JE. Leadley PJ, Eickhoff TC: Bacillus cereus pneumonia and bacteremia. Am Rev Resp Dis 103: 711,197l. Allen BT, Wilkinson HA: A case of meningitis and generalized Schwartzman reaction caused by BacilIus sphaericus. John Hopkins Med J 125: 8.1969. Van Bijsterveld GP, Richards RD: Bacillus infections of the cornea. Arch Ophthal 74: 91, 1965. Curtis JR, Wing AJ, Coleman JC: Bacillus cereus bacteremia: a complication of intermittent haemodialysis. Lancet 1: 136. 1967.
7.
8.
9. 10.
11.
12.
December 1973
Flindt MLH: Pulmonary disease due to inhalation of derivatives of Bacillus subtilis containing proteolytic enzyme. Lancet 1: 1177, 1969. Greenberg M: Survey of workers exposed to dusts containing derivatives of Bacillus subtilis. Br Med J 1: 629, 1970. Dubos R: Toxic factors in enzymes used in laundry products. Science 173: 259, 1971. Spira WM, Goepfert JM: Bacillus cereus-induced fluid accumulation in rabbit ileal loops. Appl Microbial 24: 341,1972. Armstrong D, Young LS, Meyer RD, Blevins AH: Infectious complications of neoplastic disease. Med Clin North Am 55: 729, 1971. Pearson HE: Human infections caused by organisms of the Bacillus species. Am J Clin Pathol 53: 506, 1970.
The American Journal of Medicine
Volume 55
845
DANIEL DONALD
C. IHDE,
M.D.*
ARMSTRONG,
M.D. :
New York, New York
From the Department of Medicine, Infectious Disease Service, Memorial Hospital for Cancer and Allied Diseases, and Cornell University Medical College, New York, New York. Requests for reprints should be addressed to Dr. Donald Armstrong, Memorial Hospital, 444 Fast 66th Street, New York, New York 10021. Manuscript accepted July 16, 1973. * Supported in part by a Clinical Fellowship Grant and Clinical Investigation Grant No. 26 from the American Cancer Society.
Bacillus species other than B. anthracis were isolated from rulture material and seen on gram-stained smear in 12 patients over a 6 year period in a hospital for patients with neoplastic diseases. In some cases Bacillus species were recovered repeatedly from an individual patient, occasionally from more than one site. Pulmonary and disseminated infection developed in two patients with acute leukemia Who wert under intensive chemotherapy, and they died following rupture of a brain abscess into the ventricular system. In both patients, Bacillus species were recovered from cultures of blood and sputum antemortern, and in the heart’s blood after death, and seen in histopathologic sections of the lung and brain; These cases were similar to those few previously reported cases in that the usually nonpathogenic Bacillus disseminated in an immunologically compromised host. Nearly all the remaining isolates were from drainage from recent surgical wounds. Rarely was specific treatment given for the Bacillus, and they seemed to exert no definite influence on the patient’s clinical course, although occasionally the character of a wound drainage altered after the Bacillus was no longer recovered on culture. The aerobic, spore-bearing, gram-positive rods comprising the genus Bacillus are widely distributed in nature, being found in air, soil, water, milk, dust, wool and feces [l], and, with the exception of Bacillus anthracis, the cause of anthrax, are usually considered nonpathogenic for man. When injected intraperitoneally into mice, however, sufficient quantities of several Bacillus species can lead to fatal disseminated infection, and intracutaneous injection, particularly of hemolysin-producing strains, into several laboratory animals will produce hemorrhagic, necrotic skin lesions or subcutaneous abscesses [l]. In man as well, under certain situations, pathogenicity of nonanthrax Bacillus species-is well recognized. In 1963, Farrar [2] reviewed 11 previously reported cases of serious, disseminated infections with Bacillus species and added one of his own. The infection was fatal in five cases. Several of these infections were associated with a direct route of entry into the subarachnoid space or the urinary tract; others occurred in patients with severe underlying illnesses such as acute leukemia, subdural hematoma or rheumatic heart disease. Since then several reports have documented the occurrence of Bacillus meningitis, pneumonia and bacteremia both in compromised
December 1973
The American Journal of Medicine
Volume 55
639
INFECTION
DUE TO BACILLUS
SPECIES-II-IDE,
ARMSTRONG
normal [4] and debilitated [3] and in apparently settings in which Bacillus hosts. Two other for infecspecies have been thought responsible tions were cornea1 ulcerations or panophthalmitis
12-I-71 Figure
1.
monary fever.
Case infiltrate
7. Chest roentgenogram shows associated with hemoptysis
pilland
associated with trauma or surgery [5] and febrile reactions in conjunction with the presence of B. cereus in blood and in dialysis fluid in patients undergoing hemodialysis [6]. Allergic and respiratory symptoms in workers engaged in the manufacture of laundry detergents containing derivatives of B. subtilis, principally proteolytic enzymes, have been noted [7,8]. B. subtilis autolysates were found to be capable of aggravating experimental infection with other organisms [9]. Articles describing outbreaks of food poisoning thought to be due to B. cereus have appeared in the European literature but, to our knowledge, not in the United States. Recently, however, most strains of B. cereus and cell-free culture filtrates of the organism have been found capable of eliciting fluid accumulation in ligated rabbit ileal loops, a model often used to study bacterial enterotoxins [lo]. Probably because of their ubiquitous occurrence, Bacillus species are often recovered from clinical specimens submitted to hospital bacteriology laboratories, and they usually tend to be dismissed as contaminants. Two recent experiences in this institution in which a blood culture, obtained while the patient was still alive, was reported to contain a Bacillus species that was not thought germane until after the patient died with evidence of disseminated Bacillus infection, led to a review of all possibly significant cultural isolates of Bacillus species in Memorial Hospital over a 6 year period. MATERIALS
AND
METHODS
Hospital records from the years 1966 to 1971 were reviewed in all cases in which a bacteriologic specimen revealed large gram-positive or gram-variable sporebearing rods on gram stain and in which a Bacillus species grew on culture. In two instances, speciation was confirmed at the Center for Disease Control, Atlanta, Georgia. The charts of 12 of 13 such patients were available for review. The patient population in this hospital consists mostly of persons with malignant neoplastic disease. Antibiotic sensitivity, when determined, was performed by standard disc sensitivity methods. CASE
1. A 63 year old white man was transferred to Memorial Hospital on November 24, 1971, with a diagnosis of acute myelogenous leukemia. He had presented with fatigue, spontaneous ecchymoses and
Case
Figure 2.
Case 1. Pathology slide of material from brain abscess obtained postmortem shows gram-positive spore-bearing rods.
840
December
1973
The American Journal of Medicine
REPORTS
Volume 55
INFECTION
Figure
3. Case (right).
e&m
fever
responsive
admission. 17,400 mic
2. Chest
to
The
/mm3
94
with
showing
cephalothin;
peripheral
with
therapy
films
per
he
pulmonary
was
afebrile
leukocyte cent
cytosine
blast
count
forms.
arabinoside
Infiltrate
on was
Antileuke-
and
6-thiogua-
nine produced a fall in the peripheral leukocyte count to 400/mm” within 4 days. At that point the patient had a shaking Rales
chill, were
a dry cough heard
and a temperature
in the
left
axilla,
and
of 105°F.
a chest
roent-
genogram revealed a wedge-shaped infiltrate in upper lobe of the left lung, which had been clear admission gentamicin
(Figure therapy
persisted
and,
pectorate
after
copious
following added
1). Intravenous was begun. Fever
day, to the
2 days, amounts
carbenicillin antibiotic
on the
regimen.
spore-bearing tilts on culture. the
patient
following
Postmortem of
the
patient
began
to ex-
sputum.
amphotericin Blood
taken
day
contained
comatose.
examination bone
marrow.
lung was firm in consistency neous appearance. Gross
revealed The
upper
for
cul-
right
noted.
of
Car-
Similar
organisms
tained
postmortem.
Case
2.
An
Memorial fever,
in
sev-
which
blood
old
patient
a
white
acute
leukocyte
mm3.
On the
ductive infiltrate were 103°F
7th
persistent or greater.
seen
on
(Figure
aerobic,
spore-
hospital
was 1970, cough.
was
admitted
to
because
of
She
had
leukemia
administered,
the
daily elevations Kanamycin was distress,
ob-
was and
1,700imm3
and
treatment count fell
day,
tissue
her temperature was negative,
with and
the
vincristine
of her leukebelow 1 ,OOO/
patient
a chest roentgenogram lower lobe of the right
Examination
lung
girl
Prednisone
begun for leukocyte
30, respiratory noted.
were
afebrile.
cough; in the
also
lymphoblastic
count
blast forms. and ampicillin
administration was mia. The peripheral
pleural
abscess
17,
nonproductive
with
became
from
on December
and
were
grew
cultured
since May 1970. On admission, 101.8”F. A chest roentgenogram peripheral
developing
the cerebral
were
relapse
ARMSTRONG
rods thought most similar to B. the Center for Disease Control.
11 year
fatigue
been
um were
revealed
rods,
heart’s
Hospital
cember
left
and had a gray homogesections of the brain re-
of the left lung
of the
SPECIES-IHDE.
rapidly
from
gram-positive on study at
infiltra-
vealed an area of soft, brownish necrosis, replacing the right cingulate gyrus and rupturing into the right lateral ventricle. Gram stain of sections
Culture
bearing cereus
the
leukemic lobe
gram-positive
72 per cent Oxacillin
dilated
were
2).
with
of material
the
gram-positive
A fixed globe
large
sections
On the
rods on smear and colonies of B. subOn the morning of the 6th day of fever,
became
eral
associated
B were
a Bacillus species later reto B. cereus, and sputum
pupil and proptosis of the right dioresprratory arrest followed. tion
oxacillin and and leukopenia
of bloody and
ture at this trme contained ported to be most similar obtained
the
the on
(leff)
DUE TO BACILLUS
had
a pro-
disclosed an lung; and there
in temperature to then given. By De-
hemoptysis
of the
sputum
and deliriand
a tra-
cheal aspirate revealed gram-variable rods on smear and B. subtilis on culture. Blood cultures on the following day contained B. subtilis. Chest films revealed an increase in the pulmonary infiltrate, with a rapidly progressive pleural effusion (Figure 3). The administration
December 1973
of kanamycin
was
discontinued,
The American Journal of Medicine
and carbenicil-
Volume 55
841
INFECTION
DUE TO BACILLUS
SPECIES-IHDE.
ARMSTRONG
lin and gentamicin therapy was begun, but on the 16th hospital day respiratory distress and delirium worsened, and the patient died. At autopsy, leukemic infiltrates were found in the bone marrow, lymph nodes, liver, kidneys and meninges. The right pleural cavity contained 1,000 cc of cloudy yellow fluid, and areas of consolidation with abscess formation were present in the right lung. Numerous pleomorphic spore-bearing gram-positive rods were seen in the periphery of the abscesses. Multiple, necrotic, green abscess cavities were present in the cerebral cortex and mid-brain. One collection in the right posterior temporal lobe had ruptured into the occipital pole of the lateral ventricle. Clusters of gramvariable rods similar to those in the lung were seen on gram stain. B. subtillis grew from a culture of the heart’s blood, but rio material for culture was obtained from thelung or brain.
Case 3.
A 70 year old white woman underwent right hepatic lobectomy at Memorial Hospital on July 20, 1971, with resection of a hepatocellular carcinoma. A tube was inserted into the right side of her chest at operation. On the 1st postoperative day, bloody drainage was noted from the chest tube and the abdominal drains, and the patient experienced her first elevation in temperature to 101” to 102°F which continued for 5 weeks; cephalothin was administered. The chest tube was removed on the 5th postoperative day, and a right pleural effusion was noted on roentgenograms. Culture of the serosanguineous drainage from the abdominal wound on July 27 revealed moderate numbers of B. subtilis, Esch. coli and enterococci. Several gram-positive spore-bearing rods were seen on smear of the material. On the following day, a right thoracentesis was performed, and moderate numbers of B. subtilis and Esch. coli were cultured from the bloody fluid. Cephalothin therapy was discontinued, and ampicillin and then chloramphenicol therapy was instituted. Culture of fluid from a second thoracentesis on the 9th postoperative day grew B. subtilis, Esch. coli and enterococci, as did drainage from the abdominal wound which still remained serosanguineous, on the 20th postoperative day. Five days later, the abdominal drainage, which had become bile-stained, grew only Esch. coli and enterococci. The patient was discharged with a low grade fever at the end of August, but she returned a few days later with a high fever, persistent drainage from the abdominal wound and a right pleural effusion. A subphrenic pus collection was drained surgically, and revealed Esch. coli and enterococci on culture.
Case 4. A 60 bear old white man had a wide excision of a large malignant melanoma of the right flank on November 30, 1966. Primary wound closure could not be achieved, and pedicle flaps and split thickness skin grafts were placed. Penicillin was given during the first 7 postoperative days; the patient was febrile for the first 4 of these days. On the 2nd postoperative day, musty serosanguineous drainage was noted from the site of the skin flaps. Spore-bearing gram-positive rods later identified as B. macerans were seen on .gram
842
December 1973
The American Journal of Medicine
stain and in cultures of the drainage. They were resistant to penicillin and sensitive to tetracycline. On 3 subsequent days B. macerans was obtained from the drainage, and Staph. aureus began to grow on culture as well. On December 5, tetracycline was added to the drug regimen, and by the following day, the wound drainage was no longer serosanguineous. Many Staph. aureus and few B. macerans grew from the fluid on December 7, but four subsequent cultures revealed only Staph. aureus. The wound began to granulate and eventually healed well. RESULTS
Clinical Features and Microbiology. Bacillus species were responsible for a life-threatening infection in only two patients (Cases 1 and 2). The organism was recovered from surgical wound drainage in 8 of the remaining 10 patients. In one of them (Case 3), B. subtilis was also isolated from a pleural effusion contiguous to a subphrenic abscess. A breast prosthesis, removed because of pain and serous drainage after repositioning, and a necrotic, draining axillary tumor mass in a patient with carcinoma of the breast were the sources of the other two cultures. Characteristics of the cultured material, the age and sex of the patients, and the presence or absence of fever at the time of the first positive culture for a Bacillus species are recorded in Table I. In two cases, organisms were recovered in multiple specimens and typed by the Center for Disease Control B. cereus and
as being
most
closely
related
to
B. macerans. All other isolates were classed as B. subtilis. The organisms were found in four, five or seven specimens in the cases reported in detail, but in the other cases only one or two specimens contained the organism. Discounting blood cultures, other bacteria were recovered from the same specimens which yielded the Bacillus species in all but three cases. These organisms were considered significant only when they were also seen on gram-stained smear. Esch. coli and entercocci in Case 3 and Staph. aureus in Case 4 were present in multiple specimens, and persisted in subsequent cultures after the Bacillus could no longer be isolated. Nine of 12 patients were febrile at the time the Bacillus was cultured. In most instances, the surgical wound or tumor drainage from which Bacillus species were isolated was purulent, bloody or serosanguineous. Other pathogenic bacteria were sometimes present in such culture material as well. In two cases physicians caring for the patients commented on the character of the drainage which changed from bloody to serous; thereafter the Bacillus was no longer recovered on culture. Antibiotic sensitivities on the isolated Bacillus
Volume 55
INFECTION
TABLE I
Characteristics
of Patients
* $ 0 t
with Bacillus
Source of Bacillus Species Isolate*
AgeW) Case No.
Infected
and Sex
DUE
TO
No. Positive Cultures
+
Serous Serosanguineous
1 2
Blood and purulent Purulent
2
None Gram-positive micrococci Staph. albus
+
1
None
+
Serous
1
None
-
Bile stained
2
+
Serosanguineous
2
Yellow and gelatinous, malodorous
1
Gram-positive micrococcl Gram-positive micrococci Staph. al bus, gram-positive diphtheroides
Hemoptysis, grayish consolidation in lung
4
2
11.
F
Hemoptysis
4
3
70, F
Bloody, serosanguineus
5
4
60,
M
Serosanguineus
5 6
31, 22,
F M
7
11, F
a
50, F
9
43, M
10
52, F
11
59, M
12
39, F
Sputum, tracheal aspirate, blood, postmortem heart’s blood Abdominal wound drainage, pleural fluid Drainage under skin flaps over flank wound3 Breast prosthesis Abdominal wound drainage Abdominal wound drainage Abdominal wound drainage Abdominal wound drainage Abdominal wound drainage Axillary wound drainage Necrotic axillary tumor
a 9 10 11 12 Totals
Recent Surgery
+ + + + 9112
Underlying
Fever
Staph.
Sputum, blood, postmortem lung sections, heart’s blood$
Case No.
Other Bacterlat
7
M
with Infection
ARMSTRONG
+
64,
Factors Associated
IHDE.
None in blood, Pr. mirabilis in sputum, enterococci in lung sections None in blood or sputum, alphastreptococcus in tracheal aspirate Esch. coli, enterococci in both
1
TABLE II
SPECIES
Species Character of Drainage
B. subtilis unless otherwise indicated. B. cereus, see case report. B. macerans see case report. Cultured from same specimen(s) as Bacillus
BACILLUS
+
+
aureus
+
+
and seen on smear.
with Bacillus Species*
Neoplasm
Acute leukemia Acute leukemia Hepatoma Melanoma None Hepatoma Malignant neurilemoma Cancer of breast None Cancer of breast Melanoma Cancer of breast 10/12
Antibiotics
Leukopenia
Recent Radiotherapy
+ -
+ + + +
+
-
+ +
+
+ +
3/12
5112
3112
+ + -
+ + + -
3112
* Measurements of total globulins or total gamma globulins, as determined by total protein electrophoresis or serum immunoelectrophoresis, were normal in all 11 patients
December 1973
Chemotherapy
+ -
+ + + + -
+ + + IO/12
Steroids
serum protein tested.
minus
The American Journal of Medicine
albumin,
Volume 55
serum
843
INFECTION
DUE TO BACILLUS
SPECIES-IHDE.
ARMSTRONG
were determined in nine cases; unfortunately, Cases 1 and 2 were not included. The strains isolated were uniformly resistant to penicillin, ampicillin, oxacillin, methicillin and colistin, whereas they were uniformly sensitive to tetracyclin, chloramphenicol and kanamycin (and to gentamicin in two instances when tested), and variably sensitive to cephalothin and erythromycin. Only once (Case 4) was a clinical decision to alter antibiotic coverage made on the basis of these sensitivity tests, and, at least coincident with the new antibiotic and the disappearance of the Bacillus from cultures, the character of the drainage changed and wound healing accelerated. Predisposing Factors. Factors possibly predisposing to infection in these cases are presented in Table II. Two patients (Cases 1 and 2) were severely leukopenic and were being treated intensively with antileukemic agents and several antibiotics at the time Bacillus species were first isolated. Nine of the remaining 10 patients had undergone recent surgery which was followed by wound drainage; in 8 of these, the operations were for malignant neoplastic disease. Aside from the postoperative administration of antibiotics, factors often associated with increased susceptibility to infection in our patients [ll], such as leukopenia, diminished levels of gamma globulins, and recent treatment with adrenal corticosteroids, chemotherapeutic agents or radiation, were not often present in these latter 10 cases. Significance of Isolations. It was often difficult to evaluate the contribution of the isolated Bacillus species to the subsequent clinical course of the patient. Two patients (Cases 1 and 2), who had leukemia, bacteremia, pneumonia and brain abscess, obviously died of their infections. In one patient (Case 4), in whom a different antibiotic was administered because of the organism’s drug sensit$!ities, the presence of B. macerans was very possibly exacerbating a wound infection. In the remaining patients, however, no specific therapy other than drainage of an infected space was instituted because of the recovery of the Bacillus organism, although one patient (Case 3) was given chloramphenicol therapy for reasons unrelated to the Bacillus isolation. Shortly thereafter cultures from her pleural and abdominal spaces grew only Esch. coli and enterococci and not B. subtilis. On review of the other cases we found no convincing evidence that the presence of Bacillus species altered the patient’s course for good or ill, except that their presence seemed to indicate infection of the operative wound or infection of a tumor mass. Blood cultures were frequently obtained in febrile or deteriorating patients, but with two exceptions (Cases 1 and 2), no Bacillus bac044
December
1973
The American
Journal
of Medicine
teremia was found in association infection or colonization elsewhere
with a Bacillus in the body.
COMMENTS Farrar [2] divided human infections caused by “nonpathogenic” Bacillus species into three groups: (1) local infections of a closed space, such as the eye, that is usually violated by trauma or manipulation; (2) mixed infections in which the Bacillus is found in company with another organism of recognized pathogenicity; and (3), disseminated infections in which the organism is recovered from multiple sites, usually including the blood, in a seriously ill patient. Examples of the second and third groups, and possibly of the first, are included in this series. The B. subtilis recovered from the breast prosthesis in a setting of pain and drainage shortly after surgical repositioning could be considered to have arisen in a closed space subjected to manipulation, and several of the present isolates from surgical wound drainage were recovered in the company of an organism that is a recognized pathogen. The twelve disseminated infections discussed by Farrar [2] included eight cases of meningitis, often accompanied by bacteremia. Meningitis occurred in three patients following spinal anesthesia, in one patient with a subdural hematoma, in one alcoholic with rheumatic heart disease, in one patient with chronic mastoiditis, in one patient with urinary tract instrumentation and obstruction, and in one infant. The remaining four infections occurred in a patient with nephrotic syndrome and in three patients with bacteremia (one with leukemia, one with hydrocephalis and a ventriculoatrial shunt, and one a newborn infant). In the patient of Allen and Wilkinson [4], an apparently previously healthy man, meningitis, bacteremia and possible Schwartzman reaction developed, and the patient described by Coonrod and colleagues [3], who died of B. cereus pneumonia and bacteremia, had “subacute” lymphatic leukemia. Two of our patients (Cases 1 and 2) had leukemia and profound treatment-induced leukopenia. The tendency of disseminated Bacillus infections to occur in debilitated hosts or in those with compromised resistance to infection has been commented upon [l]. Nevertheless, it should be noted that in at least 7 of the 14 patients recently discussed in the English literature, the infection disseminated, albeit sometimes after instrumentation, in apparently immunologically normal subjects [2-41. Antibiotic sensitivities of these organisms are of interest in that they could be variable in different institutions or could be undergoing a change. Of the six patients discussed by Farrar [2] who reVolume
55
INFECTION
covered, four were treated with penicillin, alone or in combination, and the organism recovered by Allen and Wilkinson [4] was sensitive to penicillin and most other antibiotics tested. Sensitivities were not performed in our Cases 1 and 2, but the results in another nine of our cases of uniform resistance to penicillin and its congeners, and sensitivity to kanamycin, chloramphenicol and tetracycline, parallel those found by Coonrod and colleagues [3] in their patient with leukemia. However, in our Case 1 the patient received gentamitin and in Case 2, kanamycin followed by gentamicin, all without effect. Although patients with dissemination of Bacillus species are relatively infrequent, the pathogenicity of the organisms in these instances is easily recognized in retrospect. Infections of ocular structures by Bacillus species are well accepted [1,5]. More difficult to evaluate are the local and mixed infections. The usual event is recovery of the organism from the site of a wound (in conjunction with bacteria on smear of the cultured material that are morphologically identical with Bacillus species) followed by arousal of suspicion that the isolation may be significant. Forty-eight such isolates over a 2 year period in a general hospital were discussed by Pearson [12]. Various Bacillus species were found in skin ulcers, conjunctival ulcers, traumatic and surgical wounds, burns, bone fracture sites and sites of osteomyelitis, urogenital infection and infection of the biliary tree. In over half, or 25, of the cases, another bacterium was present in the isolate, although it is not stated
DUE TO BACILLUS
SPECIES----ibiDE.
ARMSTRONG
whether the second organism was seen on smear. Bacillus species were grown from more than one culture in six patients, and from more than one site in the same patient twice. The uncertainty of significant pathogenicity of the Bacillus species in most of these cases is similar to that in all but Cases 1 and 2 of the current series. Isolation of the bacteria from multiple sites and/or on multiple occasions in a single patient, as was observed in several instances by Pearson [12] and especially in Cases 3 and 4 by us, could tend to confirm that a significant Bacillus infection, or at least colonization, is present. This hypothesis may be further substantiated when the character of a wound drainage or other material is altered in association with treatment and eradication of the Bacillus from cultures. Bacillus species are known to produce hemolysins [l], and alteration in the sanguineous nature of drainage could be consistent with their eradication. Most recoveries of Bacillus species from clinical material are thought to be due to contamination. But the observation of gram-positive rods on smear of the material, especially in a seriously ill patient with compromised immune status, should raise the possibility of an invasive infection and lead to further cultures, including blood cultures. Much more common, however, is the culturing of a Bacillus from local drainage in the absence of bacteremia or a critical clinical situation. The significance of these isolates, even in large numbers and on repeated cultures, remains to be ascertained by further observations.
REFERENCES 1.
2.
3.
4.
5. 6.
Wilson GS. Miles AA: Topley and Wilson’s Principles of Bacteriology and Immunity, 5th ed, Baltimore, Williams 8 Wilkins Co., 1964. Farrar WE: Serious infections due to “non-pathogenic” organisms of the genus Bacillus. Am J Med 34: 134, 1963. Coonrod JE. Leadley PJ, Eickhoff TC: Bacillus cereus pneumonia and bacteremia. Am Rev Resp Dis 103: 711,197l. Allen BT, Wilkinson HA: A case of meningitis and generalized Schwartzman reaction caused by BacilIus sphaericus. John Hopkins Med J 125: 8.1969. Van Bijsterveld GP, Richards RD: Bacillus infections of the cornea. Arch Ophthal 74: 91, 1965. Curtis JR, Wing AJ, Coleman JC: Bacillus cereus bacteremia: a complication of intermittent haemodialysis. Lancet 1: 136. 1967.
7.
8.
9. 10.
11.
12.
December 1973
Flindt MLH: Pulmonary disease due to inhalation of derivatives of Bacillus subtilis containing proteolytic enzyme. Lancet 1: 1177, 1969. Greenberg M: Survey of workers exposed to dusts containing derivatives of Bacillus subtilis. Br Med J 1: 629, 1970. Dubos R: Toxic factors in enzymes used in laundry products. Science 173: 259, 1971. Spira WM, Goepfert JM: Bacillus cereus-induced fluid accumulation in rabbit ileal loops. Appl Microbial 24: 341,1972. Armstrong D, Young LS, Meyer RD, Blevins AH: Infectious complications of neoplastic disease. Med Clin North Am 55: 729, 1971. Pearson HE: Human infections caused by organisms of the Bacillus species. Am J Clin Pathol 53: 506, 1970.
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