STUDIES ON MALLEOMYCES
PSEUDOMALLEI
ISOLATED FROM
MELIOIDOSIS ORIGINATING IN T H E WESTERN HEMISPHERE
P A R K E R R. B E A M E R , M . D . , P H I L I P L. V A R N E Y , P H . D . , WILSON G.
BROWN, M . D . , AND F R A N K M c D O W E L L , M . D .
Indiana University Medical Center, Indianapolis,
Indiana, Washington
University
School of Medicine, St. Louis, Missouri, and Baylor University College of
Medicine, Houston, Texas
Melioidosis is a specific glanders-like infection of man caused by MaUeomyces
pseudomallei. The disease is endemic in the Eastern Hemisphere in such regions
as Burma, Thailand, Indo-China, Malay States, Ceylon and the East Indies.
Although human-to-human transmission has been suspected, the authors could
find no reports wherein this was proved. Rats seem to be the primary reservoir
of the organism, but the exact route of transmission is not known. Some investigators believe that contamination of food by rat excreta may be important,
but attempts to isolate M. 'pseudomallei from rats are frequently unsuccessful,
even in the immediate area where human cases are found. The disease can be
transmitted experimentally by the rat flea (Xenopsylla cheopsis)3 and by Aedes
aegypti}
Whitmore' 3 was the first to isolate the causal agent, a small, pleomorphic,
motile, g+am-negative, rod-shaped bacterium. Although it resembles the glanders
bacillus (M. mallei), it is distinctly different serologically and in some of its
growth characteristics. The organism is now classified as M. pseudomallei,2 but
it is known under several names, i.e., Bacillus pseudomallei, B. whitmori, Flavobacterium pseudomallei, Pfeifferella whitmori, Actinobacillus pseudomallei and
Loefflerella whitmori.
Ordinarily, the disease in man develops as an acute pulmonary infection,
followed by hematogenous dissemination to several viscera, miliary abscesses,
septicemia and death within a few days. The vast majority of the scores of
cases reported in the literature followed this type of course. Relatively few
instances of chronic melioidosis are reported, and most of these occurred in
persons who survived an illness regarded as the acute phase of melioidosis. Some
of the cases of chronic disease occurred in persons who were not natives of the
endemic regions, but were believed to have contracted the disease in those
areas. 6 " 7,9 Only 2 instances of chronic melioidosis have been discovered in
persons who were known to have never been outside the Western Hemisphere. 1 ' 8
One of these patients had been in the Panama Canal Zone several years prior
to the onset of his illness,8 and the other had never left the United States of
Received for publication August 23, 1954.
D r . Beamer is Professor of Pathology, Indiana University School of Medicine; D r .
Varney is Assistant Professor of Microbiology, and D r . McDowell is Assistant Professor
of Clinical Surgery, Washington University School of Medicine; D r . Brown is Associate
Professor of Pathology, Baylor University College of Medicine, and Director of Laboratories, Hermann Hospital.
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America.1 The sources of infection and modes of transmission of the disease in
these patients were not determined and are still unknown.
Clinical pathologists may be encountering a case of melioidosis from time to
time. When first observed in cultures, this organism may be overlooked and not
identified because of its rare and unexpected occurrence, and because of certain
similarities to other, more common bacteria, some of which are regarded as
nonpathogenic forms. I t is of value to review briefly the 2 cases cited above and
to describe the morphologic, biochemical and serologic properties of the 2 strains
of M. pseudomallei isolated from the lesions.
CHRONIC M E L I O I D O S I S O R I G I N A T I N G I N T H E W E S T E R N
HEMISPHERE
8
McDowell and Vaniey reported the first case of melioidosis which seems to have originated in the Western Hemisphere. T h e patient was a white man, 31 years old, who was
employed in the Panama Canal Zone in 1927-28. Except for these 2 years, he worked as a
machinist in Oklahoma, Missouri and Illinois. There was no history of any bites from
animals or unusual exposure to insects, and the patient knew of no contacts with a similar
disease. In 1945 he was admitted to the hospital for diagnosis and treatment of ulcers and
sinuses of the right buttock and thigh. T h e patient said t h a t these developed following a
fall with injury to the coccygeal region in 1937. F o r 8 years he was treated many times,
having been subjected to almost a hundred operations for drainage of abscesses of the
right buttock and thigh, down to the knee, posteriorly. M. •pseudomallei was recovered
several times from blood agar cultures of purulent exudate from the lesions.* Numerous
colonies of Proteus ammoniac and moderate numbers of Micrococcus pyogenes var. aureus
and Pseudomonas aeruginosa were present, also. T h e patient was treated with sulfonamide derivatives, antibiotics and several topically-applied drugs, but there was little improvement until the lesions were widely excised by cautery and t h e skin grafted over the
area. H e developed no new lesions the last few months, and felt so much better t h a t he
could not be persuaded to stay in the hospital. After a continuous hospitalization of about
.1.0 months he left, although observers thought t h a t he was not free of the disease.
T h e second case of melioidosis believed to have originated in the Western Hemisphere
was reported by Beamer and his associates. 1 T h e patient was a white woman, 25 years
old, who had never been outside the United States. Approximately 11 months previously,
she was treated with x-rays for a small mass in the left axilla. No tissue was removed for
biopsy, and the exact nature of this lesion is not known. T h e mass receded almost immediately, and, so far as is known, caused no further trouble. Six weeks prior to admission, the patient noted a swelling and soreness in the right lower portion of t h e abdomen.
Subsequently, this area desquamated, exposing deeper layers of the skin, from which thick
purulent fluid oozed. Several enlarged, tender lymph nodes developed in both inguinal
regions. Adjacent areas of previously uninvolved skin developed vesicles and bullae with
turbid fluid, and then desquamation occurred. Several cultures of exudate from the bullae
and denuded areas .yielded growth of Micrococcus pyogenes var. aureus, Escherichia coil
and diphtheroids. Despite intensive therapy with penicillin, streptomycin, sulfadiazine
and various supportive measures, the skin lesion spread slowly to the left inguinal area
and over both thighs. During the course of the extensive skin lesion, the patient's general
condition grew worse steadily. I n t e r m i t t e n t bouts of "septic, spiking fever" became cons t a n t and persisted for the last several weeks. Finally, during the last few days of life, the
patient suffered clonic convulsions and died after approximately 9 months of hospitalization.
Postmortem examination revealed a huge multiloculated region of liquefactive necrosis, underlying the skin lesion, involving the retroperitoneal connective tissues and mus* This strain is designated as " A " in the bacteriologic studies.
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MALLEOMYCES
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cles, anteriorly, laterally and posteriorly on the right side. I t extended upward to the
perirenal tissues where it was directly contiguous with a similar lesion of the left side of
the body, which extended downward into the muscles and subcutaneous tissues of the left
thigh. T h e large, irregular region described above was rilled with viscid, purulent material.
Several sections taken from the irregular, shaggy wall of the large abscess revealed essentially similar findings. T h e outer portion of the wall was composed of slighth' to moderately dense, cellular fibrous tissue with numerous thick-walled blood vessels and
inflammatory cells, most of which were macrophages and lymphocytes, with relatively
few polymorphonuclear leukocytes and plasma cells. T h e inner portion of the wall was
composed principally of irregular deposits of fibrin, some of which were partially organized. Numerous polymorphonuclear leukocytes were enmeshed within the fibrin network.
Sections of the liver revealed an advanced degree of hepatocellular disarray, with necrosis of hepatic cells, singly or in small groups. There were several, irregularly scattered,
minute foci of nonspecific granulomatous inflammation, consisting chiefly of macrophages
with a few lymphocytes, and cellular debris. Similar small granulomas were found in the
kidneys, predominantly in the region of the distal convoluted tubules. Examination of
the other organs revealed no significant anatomic alterations t h a t were regarded as a part
of the primary disease. Actually, one could not be certain as to the exact nature of the
granulomatous lesions in the liver and kidneys, b u t it was thought t h a t the histologic
findings were not inconsistent with the possibility t h a t they represented focal lesions of
chronic melioidosis.
Portions of the wall of the abscess, the liver, the spleen and the kidneys were ground
with sterile sand in a mortar and pestle prior to culturing on blood agar, blood tryptosc
broth and several other mediums. Inasmuch as the authors had no idea of what causal
agent was present, cultures were prepared in triplicate, so t h a t they could be incubated
aerobically, anaerobically and under increased concentration of carbon dioxide (10 per
cent). Almost pure cultures of M. pseudomallei* were obtained from several of the specimens t h a t came from the deeper portions of the wall of the abscess. Only a few colonies
were found in cultures derived from the more superficial portions of the abscess, and, a t
first, these were overlooked, owing to the more impressive growth of Micrococcus pyogenes
var. aureus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and diphtheroids.
Cultures of the liver, spleen, kidneys and blood from the heart yielded no growth.
BACTERIOLOGIC
STUDIES
Both strains of M. pseudomallei used in these studies were isolated on blood
tryptose agar (approximately 5 per cent of defibrinated rabbit blood), some of
which continued 0.3 per cent hog bile to inhibit overgrowth by spreaders. Paraaminobenzoic acid was added to counteract the effect of sulfonamide drugs
which both patients were receiving. Several rough (R) and smooth (S) colonies,
slightly different from all the others, were detected among the relatively numerous
colonies of more common bacteria (listed in the above summaries). At first the
R and S colonies were regarded as different organisms, but further study revealed that they were variant forms of M. pseudomallei. The S colonies were
dark tan, whereas the R colonies were smaller and pale yellow or yellow-gray.
Closer examination revealed that the R colonies were slightly folded or corrugated, and that transmitted light produced a scintillating surface. Both types
of colonies were slightly to moderately mucoid in consistency, and were surrounded by narrow hemolytic zones in the blood agar. Inasmuch as the authors
* This strain is designated " B " in the bacteriologic studies.
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recognized that these were either not present or not identified in previous
cultures from the patients, representative colonies were subcultured on blood
tryptose agar and several other mediums for the observation of biochemical
characteristics. A review of appropriate sections of Bergey's Manual 2 suggested
the possibility that the organisms might be M. pseudomallei. Subcultures were
made on tryptose agar with 3 to 5 per cent glycerol, and the characteristic
corrugated colonies developed, as described by Whitmore13 and Stanton and his
associates.10-'2 With this tentative identification as a basis, further studies were
performed. Stock cultures of the strains were maintained by frequent transfers
on blood tryptose agar, inasmuch as the authors found that longevity of lyophilized preparations was not dependable. In general, strains A and B of these
studies were identical in their characteristics, but a few exceptions are noted
in the detailed descriptions. Slight variations in the cultural characteristics of
M. •pseudomallei may be noted in several published descriptions of different
strains. Some of the variations were probably the result of growing the organisms
on different mediums, but others seem to represent slight differences among
the strains, even when observed under similar conditions of growth.
Morphologic characteristics. The organisms from both strains were poorly
staining, pleomorphic, bipolar, non-acid-fast, gram-negative rods (Fig. 1).
Unstained or faintly stained, indefinite "halos" around many of the organisms
suggested that they were encapsulated, but no capsules were demonstrable in
special preparations. Motility was variable, even in young cultures, apparently
owing to viscous material adherent to the bacteria; when washed in physiologic
salt solution, motility was enhanced, rapid and serpentine in character.
Smooth (S) colonies yielded relatively long, slender organisms, averaging 5-6
by 0.6 ix, with rounded or slightly pointed ends. In each field a few short, plump
rods were noted, also. In general, the S forms were in parallel arrangement
within irregular groups or clumps (Fig. 2), or they formed an irregular network
somewhat reminiscent of the alveolar walls of a lung in microscopic section
(Fig. 3). Such arrangements seemed to be the result of viscous material that
stained palely in the background (Figs. 2 and 3). This was not observed in
younger cultures, and the peculiar arrangements were not noted in older cultures
after the bacteria were washed in physiologic salt solution.
Organisms from rough (11) colonies were relatively short and plump, averaging
2 by 0.6 ii, with rounded ends. The predominant forms were single, but end-toend pairs were noted occasionally. There was little tendency to parallel or
"palisade" grouping as noted in S forms.
Cultural characteristics. Both strains were strictly aerobic, and grew rapidly
on plain tryptose agar and other common mediums. The rate of growth seemed
to vary little, if any, at temperatures from 25 to 37 C. The S and R forms of
both strains were emulsifiable, even though the colonies were mucoid or butyroid
in consistency. A faint, pungent odor devoid of ammonia was noted, especially
on mediums with blood added. A characteristic metallic luster developed often,
but not invariably, on the surface of the colonies, particularly in regions where
they had grown together. Some describe this as the appearance of "hammered
aluminum," but the authors feel that it was more similar to the metallic luster of
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F I G . 1 (upper). M. pseudomallei (strain A or B) from a smooth (S) colony on blood
tryptose agar, 24 hours at 37 C. Note relatively poor staining of many cells, bipolarity
and pleomorphism. Most cells are relatively long and slender, but a few shorter, plump
rods are present. Approximately X 1200.
F I G . 2 (lower left). M. pseudomallei (strain B) from a smooth colony on blood tryptose
agar, 72 hours at 37 C. Note parallelism or " p a l i s a d e " grouping of long, slender bacterial cells. A few pleomorphic forms are present, b u t in smaller number t h a n in older
cultures. Approximately X 1000.
F I G . 3 (lower right) M. pseudomallei (strain A) from a smooth colony on blood t r y p tose agar, 72 hours at 37 C. Note irregular network, somewhat resembling alveolar
walls of a lung. A few pleomorphic forms are present. Approximately X 1000.
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For convenience, the more significant findings are summarized below (all tests
incubated at 37 C ) :
Coagulated serum—
Strain A: digestion approximately two-thirds complete in 48 to 120 hours,
with S forms showing more rapid activity.
Strain B : digestion only slight after 120 hours.
Litmus milk—
Strain A: R forms produced coagulation in 48 hours and one-third digestion
in 120 hours; S forms produced less coagulation, but two-thirds digestion
in 120 hours.
Strain B : no acid or coagulation produced; digestion one-third complete in
120 hours.
Gelatin—
.. Strain A: almost complete liquefaction in 96 to 120 hours, with zoning of
growth (8 or 9 stratums, more dense near the top, only slight at the
bottom).
Strain B : partial liquefaction in 120 hours, usually only the upper third,
with uniform growth.
Carbohydrates—
Strain A: acid only (no gas) in dextrose, galactose, lactose, maltose, sucrose
and mannitol; ability to form acid from dextrose was maintained indefinitely, but ability to attack the other 5 was lost after 4 to 8 transplants.
Strain B : acid only (no gas) in dextrose, and maltose, apparently indefinitely.
Neither strain attacked dextrin, dulcitol, glycerol, inositol, raffinose or
salicin.
Indol—not formed by either strain.
Hydrogen sulfide—not formed by either strain.
Serologic characteristics. Considerable difficulty was encountered in attempts
to read the results of agglutination tests using suspensions of the S variants.
The viscid material associated with the organism tended to sediment and mask
the presence of truly agglutinated clumps. Consequently, most of the tests were
performed with suspensions of the R variants of the 2 strains.
A known culture of M. pseudomallei and a specimen of positive antipseudomallei serum were obtained* in order to make comparative studies with strains
A and B and with the patients' serums. The results of these tests were as follows:
1. Strain A was agglutinated by known antiserum to a titer of 1:1280; strain B
was agglutinated to a titer of 1:640, with doubtful results at 1:1280;
2. Serum from patient A agglutinated strain A and the known organism
(strain 295 Calcutta) to a titer of 1:1280;
3. Serum from patient B agglutinated strain B and strain A to a titer of
* T h e authors deeply appreciate the courtesy of Col. Raymond Randall, Veterinary
Division, Army Medical D e p a r t m e n t Research and Graduate School, in supplying these
materials.
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MALLEOMYCES
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1:640 (the latter test was substituted for agglutination with the 295
Calcutta strain because the known antigen was not available at the time).
Inoculation of experimental animals. The authors did not attempt exhaustive
studies on several species of animals but, for practicable reasons, selected guinea
pigs and Swiss mice. Several animals were used for each series of studies wherein
the intraperitoneal doses of organisms were varied. The results are summarized
below inasmuch as detailed observations would require too much space.
In general, both strains were virulent for guinea pigs when relatively large
doses (viz., 0.5 ml. of undiluted broth culture, 24 to 48 hours old) were given
intraperitoneally. The animals developed a fulminating illness with fever and
shock; most of them died in 4 or 5 days, but a few survived to the eighth or
ninth day. Postmortem examination revealed thick purulent exudate in the
peritoneal cavity, usually extending into the scrotum (Strauss reaction). The
organisms could be recovered from the exudate and from the blood. In the animals that survived a little longer, focal lesions containing thick exudate were
found in the liver, spleen, lungs and, sometimes, in the kidneys. Small doses
(viz., 0.5 ml. of a similar broth culture diluted 1:10) caused no detectable disease
in guinea pigs. The animals revealed restlessness after the injection and a few
developed a slight fever, but all of them recovered without signs of serious
illness.
Mice inoculated intraperitoneally with 0.1 ml. of undiluted broth culture,
24 to 48 hours old, derived from either strain, became prostrated within a few
hours after the injection. Within 24 hours flaccidity of muscles developed and
the eyelids were matted together with thick exudate. Most of the animals died
within 36 to 48 hours, only occasional ones surviving as long as 5 days. Postmortem findings were similar to those observed in guinea pigs. Mice receiving
varying degrees of smaller doses survived the illness for longer times, and the
viscera of these animals contained several focal yellow-white lesions. Some of
these were abscess-like with thick purulent exudate, whereas others were caseous
in character.
It is interesting to note that (1) cultures derived from the corrugated rough (R)
colonies of either strain were more virulent than those from the smooth variant,
and (2) strain B tended to be more virulent than strain A in both types of experimental animal. Variation in virulence for guinea pigs and mice was evident, also,
in the known strain (295 Calcutta). Even larger doses, such as those described
above, failed to cause serious illness, and most of the animals recovered.
Sensitivity to antibiotics. The tube dilution method was employed, using
tryptose broth as the basic medium (9.9 ml. per tube) and an inoculum of 0.1
ml. of a repeatedly transferred broth culture of the organism, 6 to 8 hours old
at the time of the test. The detailed results were too numerous to be given here,
but the essential findings are summarized:
Penicillin—growth of both strains occurred in concentrations as high as 250 jug.
per ml.
Streptomycin—growth of strain B occurred in concentrations as high as
250 Mg- per ml.; initially, 5 Mg- per ml. produced bactericidal action on
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strain A, but, after 3 passages in streptomycin broth, growth occurred in
250 jug- per ml.
Aureomycin—growth of both strains occurred in concentrations as high as
50 /ig- per ml.
Chloramphenicol—growth of both strains occurred in concentrations as high
as 100 Mg- per ml.
Tyrothricin—both strains were inhibited slightly, but bactericidal action
was not noted in concentrations up to the limit of solubility.
Streptothricin—growth of strain A in concentrations as high as 80 units per
ml.; strain B was not tested with this substance.
SUMMARY
1. A strain of Malleomyces pseiidomallei was isolated from each of 2 patients
with chronic melioidosis, both of whom were never outside the Western Hemisphere, and one of whom was never outside the United States.
2. The essential features of the clinical histories are reviewed, and the postmortem findings in 1 patient are summarized.
3. The morphologic, biochemical and serologic characteristics of the 2 strains
of M. pseiidomallei are described, together with a summary of results obtained
in experimental animals and sensitivity tests with antibiotics.
4. Certain strains of M. pseiidomallei may resemble other bacteria that occur
more commonly in bacteriologic cultures; therefore, cases of melioidosis may be
overlooked or diagnosed incorrectly.
Acknowledgment.
T h e authors deeply appreciate t h e assistance of M r . Cramer Lewis
and his staff, D e p a r t m e n t of Illustration, Washington University School of Medicine, in
preparation of t h e photographic material.
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