TllK AMKH1CAN Joi.'KNAli OF CuNlCAl, PATHOLOOY
V.il. 41. No. 2,, pp. 150-1 S3
Frbniiirv. 1061
Copyri«M iO 1UU4 by Tin- yYilli:mis \- Wilkins Co.
J'ritttrd in U.S.A.
PATHOGENICITY OK PSEUDOMONAS
FLUORESCENS
AND
PSEUDOMONADS TO WARM-BLOODED ANIMALS
RELATED
PINGHUI V. LIU, M.D.
Department of Microbiology, University of Louisville School of Medicine, Louisville, Kentucky
Pscudomonas fluorescens is generally regarded as being nonpathogenic to animals,
especially the warm-blooded types. In
addition to their failure to produce
pyocyanine, the chief difference between
this group of organisms and P. aeruginosa
seems to be the inability of the former to
grow readily in a range of temperature
(37 C. to 42 C.) in which the latter are
capable of growth. The nonpathogenic!ty of
P. fluorescens to warm-blooded animals,
therefore, may be a reflection of their
inability to grow at the body temperature of
these animals. The pathogenicity of P.
fluorescens for cold-blooded animals has
already been described (Bullock, 19612).
I t has been suggested from this laboratory
(Esselmann and Liu, 19G15) that some
phenazine pigment-producing members of
the genus Pscudomonas, such as P. aureofaciens and P. chlororaphis, are merely
variants of P. fluorescens and not distinct
species, because the enzymes of these
organisms were serologically indistinguishable. Most of these organisms produce high
titers of extracellular enzymes, such as
lecithinase and protease, which were quite
toxic when injected into animals (Liu,
19G19). There are some parts of the bodies of
warm-blooded animals, such as tips of
extremities and tails, in which the temperature is less than 37 C ; such sites are
within the range in which P. fluorescens can
grow readily. The possibility of infecting
such parts of the bodies of warm-blooded
animals with P. fluorescens and related
pseudomonads was studied.
MATERIALS AND
METHODS
Strains of pseudomonads used. Strains 8/3
(P. fluorescens), B-1543P (P. aureofaciens),
Received, June 15, 19G3; revision received,
September 7; accepted for publication November
5.
Dr. Liu is Associate Professyr of Microbiology.
and B-5G0 (P. chlororaphis) were used in
this study, as well as a strain of P. aeruginosa
(P-A-7) for comparison. All of these strains
were used in a previous study of the. production of hemolysin by pseudomonads, as
described in a previous paper (Liu, 1(J57S).
Pathogenicity test. Each of a group of
mice was lightly anesthetized with ether and
a superficial burned lesion, approximately 1
cm. in length and 3 mm. in width, was made
with a hot platinum loop near the base of
the mouse's tail; a loopful of 18-hr.-old
broth culture of each organism was then
smeared on the lesion. The animal was
placed in a cage with wood shavings that
were autoclaved previously, along with the
cage, in order to minimize the effects of
other bacterial flora on the development of
the lesion. Ten mice were inoculated with
each of the strains of pseudomonads. Two
mice from each group were sacrificed every
day, and the heart's blood, lung, spleen,
liver, kidney, and a piece of the lesion on the
tail were cultured on a blood agar plate
incubated at 24 C.
RESULTS
The burned lesion per se healed, with
formation of a scar, within 4 to 5 days, when
it was not inoculated with the organisms.
When the organisms were inoculated, the
lesion manifested redness and edema within
24 hr., and, at the end of 3 days, extensive
necrosis was observed in all cases, as
illustrated in Figure 1. The lesion produced
by B-1")43P was even more extensive than
that produced by P-A-7. The tails of mice
inoculated with the former frequently fell
off after 4 days. In Table 1, the positive
cultures obtained from various organs of the
mice are listed. As may be seen in this table,
positive cultures from heart's blood and
lung could be obtained with animals
inoculated with B-1">43P and B-">('>0 only
the day after the inoculation. The animals
150
Feb. 1964
PATHOGENICITY OF P.
151
FLUOHESCEXS
3
&*
t
Hi 1
iku %\mmmkii*
FIG. 1. Pathogenicity of Pscudomonas fluorescens and related pseudomonads compared with that
of P. aeruginosa. The photograph was made 3 days after the inoculation of 1 loopful of broth culture
on a superficial burned lesion of the tail. Observing from left to right, the mice were treated, respectively, with P. aeruginosa, P. aureojaciens, P. chlororaphis, and P. fluorescens. Note the extensive
necrosis of the tail of the mouse inoculated with P. aureojaciens (B-1543P).
apparently were able to eliminate the
organisms from the blood sti'eam readily,
and prevent the occurrence of sepsis. In
the case of P. aeruginosa, the infection of
the lung persisted much longer, and some
of the mice died with hemorrhagic pneumonia.
DISCUSSION
The findings described in the preceding
paragraphs confirm the fact that the inability of P. fluorescens and related organisms
to produce a generalized infection in warmblooded animals results from their failure
to grow at the body temperature of the
animals. In the parts of the bodies that have
a temperature less than 37 C , the Pseudomonas organisms were able to produce
advanced lesions. Invasion of the blood
stream from the lesions does occur, as
demonstrated by positive cultures from
heart's blood and lung, but the organisms
were apparently unable to establish an
infection in the parts of the body that have
higher temperatures. As part of the test for
pathogenicity of the organisms, they were
injected through subcutaneous, intramuscular, intravenous, and intraperitoneal routes
into mice and rabbits, but no significant
pathologic change developed in such animals.
The failure of the organisms to produce
infections when injected into "closed tissue
space" may be a reflection of their requirements for oxygen, inasmuch as they are strict
aerobes. When they are smeared on an open
wound, the supply of oxygen is readily
available, thereby providing suitable conditions for the growth of the organisms.
Inasmuch as P. fluorescens is generally
present in soil and water, it is likely that
many open lesions of the extremities of
warm-blooded animals contain this organism. The pathogenicity of P. chlororaphis
in mice and guinea pigs, as well as some coldblooded animals, was described by Lasseur
(19137). In one of the previous studies of the
author (Esselmann and Liu, 19G15), it was
found that serum from some "normal"
rabbits contained antibodies that neutralized
the lecithinase of this species. Such findings
seem to indicate that infections of warmblooded animals with P. fluorescens and
related organisms are more frequent than is
usually realized. The paucity of literature on
the isolation of P. fluorescens from human
infections, even from gangrenous extremities, may be the result of the common
practice of workers in medical laboratories to
incubate all of the mediums at 37 C.
The findings described in this paper also
152
TABLE 1
Heait's
Blood
1 Days
POSITIVE CULTURES FROM VARIOUS ORC.ANS OF
MICE AFTER INOCULATION OF 4 STRAINS OF
PSEUDOMONADS ON THE LESIONS
OF THE TAILS*
Pseudomonas
aeruginosa (P-
Lung Spleen Liver
Kid- Tail
ney Lesion
1 1/2 2/2 0/2 1/2 0/2 2/2
2 1/2 2/2 0/2 0/2 0/2 2/2
3 0/2 1/2 0/2 0/2 0/2 2/2
A-7)
P. aureofaciens
1 1/2 2/2 0/2 0/2 0/2 2/2
2 0/2 0/2 0/2 0/2 0/2 2/2
(B-1543P) 3 0/2 0/2 0/2 0/2 0/2 2/2
P. chlororaphis
(B-560)
P. jlvorescens
(8/3)
Vol. J,l
LIU
1 0/2 1/2 0/2 0/2 0/2 2/2
2 0/2 0/2 0/2 0/2 0/2 2/2
3 0/2 0/2 0/2 0/2 0/2 2/2
1 0/2 0/2 0/2 0/2 0/2 2/2
2 0/2 0/2 0/2 0/2 0/2 2/2
3 0/2 0/2 0/2 0/2 0/2 2/2
* The denominators indicate the number of
mice sacrificed for examination, and the numerators indicate the number of positive cultures
from each organ.
indicate that the division of pseiidomonads
into animal pathogens and plant pathogens
is not a rational approach in the classification
of these organisms, inasmuch as P. aeruginosa, which is generally believed to be the
representative animal pathogen in this
genus, has been demonstrated to be also
phytopathogenic (Elrod and Braun, 1942 ;4
Liu and associates, 196110). Demonstration
of the pathogenicity of P. fluorescens in
animals provides additional evidence that
such a division is not practical, inasmuch as
that organism is generally regarded as a
plant pathogen. Furthermore, the findings
described in this paper also indicate that
zoopathogenicity is not acceptable as a
criterion for distinguishing apyocyanogenic
P. aeruginosa from other pseiidomonads, as
claimed by some workers (Hiihlmann and
co-workers, 19011)- It was demonstrated by
Charrin in 1889,3 Wassermann in 18%," and
Gaby and Logan in 1901'') that the virulence
of /'. aeruginosa varies greatly from strain
to strain, and some strains are entirely nonpathogenic.
The fact that P. aeruginosa and P.
Jluaresccns can be pathogenic for animals
and also plants does not indicate that the
2 groups of pseiidomonads are closely
related. In addition to the difference in
their optimal temperatures for growths, the
enzyme systems of these organisms are
serologically distinct, as reported from this
laboratory (Liu and associates in 1961,10
and Esselmann and Liu in 1901s). There is
no good reason, therefore, to confuse these
organisms in the future.
SUMMARY
Pseudomonas fluorescens and related
species of pseiidomonads that are generally
thought to be plant pathogens seemed to be
effective in producing lesions in warmblooded animals when the organisms were
placed in contact with open wounds of the
parts of the bodies that have temperatures
less than 37 C. Invasion of the blood stream
from such lesions occurred, but the oi-ganisms
seemed to be ineffective in causing infection
in the internal organs of the animals. The
classification of pseiidomonads into animal
pathogens and plant pathogens, therefore,
does not seem to be a sound practice,
because~P. aeruginosa, which is generally
thought to be an animal pathogen, has been
demonstrated to be also phytopathogenic.
SUMMARIO IX IXTEKLIXGUA
Pseudomonas jluorescente e altcre species »
de pscudomonades que es generahnente
reguardate como phytopathogenic pareva
esser capace a producer lesioncs in animates
de sanguine calide quando le organismos
esscva ponite in contacto con aperite
vulneres in partes del corpore que ha temperaturas de minus que 37 C. Invasion del
circulation sanguinee ab tal lesiones
occurreva, sed le organismos pareva esser
ineliicace in causar infectiones del organos
interne de tal animates. Le classification del
pseudomonades in zoopathogenes e phytopathogencs, per consequento, non pare
esser justificate, proque P. aeruginosa, que
es generahnente reguardate como zoopathogene, es dcmonstrateinentc ctiam
phytopathogene.
Acknowledgment. This work was supported by
a research career development award of the
Feb.
J9G4
PATHOGENICITY OF J'. FLUOKESCEXS
Public Health Service (CM-KIMS), D e p a r t m e n t
of H e a l t h , Education, and Welfare.
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153
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