I SO L A T IO N A N D C H A R A C T E R I S A T I O N O F BACTERIA
F R O M ABSCESSES I N T H E SUBCUTIS O F CATS
DARIA
N. LOVE*,R. F. JONES*,MARILYN
BAILEY*
AND R. s. JOHNSON
*Department of Veterinary Pathology, University of Sydney, NS W 2006, and
Blacktown Veterinary Hospital, Blacktown, NS W 2148; Australia
A L T H o u G H subcutaneous abscesses in cats, resulting from bites sustained in
fights, constitute one of the conditions most frequently encountered in smallanimal veterinary practice, there is a need for much more information on the
causative prganisms. Most of the existing research on the bacteriology of
subcutaneous abscesses in cats was carried out more than 15 years ago when
identification methods for anaerobes were less definitive than they are now.
PasteureZZa muZtocida was cultured from the nasopharynx of 94 % of normal
cats by Smith (1964), who considered that it was invariably present in abscesses
that occurred as a result of fighting; he noted that beta-haemolytic streptococci
and anaerobic fusiform bacilli were often present with Past. multocida. Earlier
work in this laboratory (Love et aZ., 1978) revealed the importance of anaerobic
bacteria in subcutaneous abscesses in cats. It showed however that opened
abscess cavities yielded few or no anaerobic species; on occasion it was possible
to see organisms resembling anaerobes in stained smears of pus although they
could not be cultured.
This paper presents the results of a study of selected clinical cases in which
modern methods for the characterisation of anaerobic isolates were used.
MATERIALS
AND METHODS
Specimens and transport. Specimens received for culture consisted of fluid pus from subcutaneous abscesses in cats. Each specimen was collected from an unopened lesion after the
skin had been clipped, scrubbed, and prepared as for aseptic surgery. A needle attached to a
syringe was introduced and pus aspirated into the syringe. The needle was then covered
with a sterile plastic cap and any air was expelled from the syringe. A portion of the specimen
was placed in an Anaerobic Specimen Collector (Becton-Dickinson, Rutherford, New Jersey,
USA) and, with the portion remaining in the syringe, was submitted to the laboratory as
quickly as possible.
Culture media. None of the media used was sterilised under anaerobic conditions.
Blood agar (Oxoid Blood Agar Base No. 2 plus 5 % defibrinated sheep blood) and brain-heart
infusion agar (Holdeman, Cat0 and Moore, 1977)plates were poured on the day of use. Pure
cultures of isolates were grown anaerobically in a medium (CMC) that consisted of BVF
broth (Turner, Campbell and Dick, 1935) containing cooked meat particles and supplemented
with glucose 0-4%, cellobiose 0.1 %, maltose 0.1 %, and starch 0.1 %. The basal medium for
fermentation studies and the biochemical tests for each genus were as described by Holdeman
Received 4 Oct. 1978; accepted 19 Oct. 1978.
J. MED. MICROBI0L.-VOL.
12 (1979)
207
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208
D A N A LOVE, R. JONES, MARILYN BAILEY AND R. JOHNSON
et al. (1977). Some of the bacterial species isolated required supplementation of the basal
medium with sterile horse serum 5 %.
Culture procedures. Each specimen was processed immediately on receipt at the laboratory. A direct smear was made and stained with Gram's stain. The relative numbers of the
various types of bacteria that could be distinguished on the basis of morphology and gramstaining reaction were then recorded.
A loopful of pus from the Anaerobic Specimen Collector and a drop from the syringe
were streaked on agar plates in duplicate to allow for aerobic and anaerobic incubation.
Anaerobic plates were transferred immediately to a GasPak anaerobic system in which the
catalyst had just been rejuvenated by heating (Holdeman et al., 1977). Pus in which organisms
resembling Actinomyces spp. were seen was in addition plated and incubated in a candle jar
containing CO2 10% in air. Aerobic plates were examined after incubation for 24 h and
colonies were picked off for identification. Primary microaerophilic and anaerobic plates
were dealt with in a similar fashion after incubation for 48 h. Primary plates were reincubated for another 5 days and any further colonies were removed for identification.
Identi3cation of aerobes and facultative anaerobes was by the methods outlined by Cowan
(1974).
Identification of anaerobes. Colonies of bacteria from anaerobic and microaerophilic
plates were tested for their atmospheric requirements as follows. Those from anaerobic
plates were transferred to blood agar and incubated aerobically, while those from microaerophilic plates were transferred to duplicate blood-agar plates to allow for aerobic and
anaerobic incubation. To confirm its anaerobic nature, each isolate was subjected, on
several occasions during the course of its identification, to aerobic subculture. Organisms
growing only on anaerobic plates were identified by the techniques described below.
Single colonies from pure cultures were placed in CMC medium and incubated anaerobically. After 48 h, samples were removed and ether and chloroform extracts were prepared
as described by Holdeman et al. (1977). Analysis of acid metabolic products was carried out
by gas-liquid chromatography on a Hewlett-Packard Gas Chromatograph 5830A. A glass
column (length 160 cm, internal diameter 2 mm) packed with 10 % AT1200 plus 1 % H3P04 on
Chromosorb W-AW, 80/100 mesh (AT1200) was used. The carrier gas was nitrogen at a
flow of 30 ml/minute, the oven temperature was 115"C, and the flame ioniser detector was
run at 225°C. From the gram-staining reaction, morphology and metabolic profile of an
organism it was possible to determine its genus (Buchanan and Gibbons, 1974; Holdeman
et al., 1977). Biochemical and other tests listed by Holdeman et al. (1977) were then carried
out.
Identification to the level of species was based on the descriptions given by Buchanan
and Gibbons (1974) and Holdeman et al. (1977).
RESULTS
Samples from 36 abscesses were examined in this study. All had a foul
odour and all but four contained anaerobes (1-8 species) as shown in table I.
Six abscesses contained anaerobes only. From 26 of the samples, a mixed
growth of facultative and strict anaerobes was obtained. Table I presents
details of the numbers of facultative and strict anaerobes isolated from each
specimen. Of a total of 168 isolates identified, 121 (72%) were strict anaerobes
and 47 (28%) were facultative. Tables I1 and I11 show the genera and species
of the strictly and facultatively anaerobic isolates respectively. In all, 66-1%
of the anaerobic isolates were gram-negative bacilli belonging to the genera
Bacteroides and Fusobacterium, 14-9% were anaerobic cocci and the remainder
(19.0%) were anaerobic gram-positive bacilli. As shown in tables I1 and 111,
Bacteroides was the genus encountered most frequently (28.6 % of all isolates),
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SUBCUTANEOUS ABSCESSES IN CATS
209
TABLE
I
Distribution of numbers of facultatively anaerobic and strictly anaerobic isolates in 36
subcutaneous abscesses
Number of abscesses
(of a total of 36)
Number of
facultatively
anaerobic
species present
6
17
9
4
0
1
2
3
Totals
Number of abscesses containing the
stated number of anaerobic species
A
r
\
0
1
2
3
4
5
6
7
8
0
3
1
0
0
0
2
1
2
3
1
1
1
4
1
1
0
2
1
1
0
3
0
0
2
1
2
0
1
1
0
0
0
0
1
0
4
3
7
7
4
3
5
2
1
TABLE
I1
Strictly anaerobic isolates
Percentage of
Organisms
Number of isolates
Y
anaerobic
all
isolates
isolates
Bacteroides
B. fragilis
B. asaccharolyticus
B. rnelaninogenicus
Other species
48
12
11
9
16
39.7
28.6
Fusobacteria
F. nucleaturn
F. necrophoruni
F. russii
Other species
32
11
8
7
6
26.4
19.0
Peptostreptococci
Pept. anaerobius
Pept. interrnedius
18
16
2
14.9
10.7
Clostridia
11
9.1
6.5
Propionibacteria
Prop. acnes
Prop. freudenreichii
7
6
1
5.8
4.2
Bifidobacter ia
2
1.7
1.2
Lactobacilli
2
1.7
1.2
Eubacteria
1
0.8
0.6
The total for each group or genus is given in bold type.
0
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D A N A LOVE, R . JONES, MARILYN BAILEY AND R. JOHNSON
--
TABLEIII
Facultatively anaerobic isolates
Percentage of
Organisms
Number of isolates
facultatively
anaerobic
isolates
all
isolates
Pasteurella multocida
22
46.8
13.1
Actinomyces
A. viscosus
A . odontolyticus
13
8
27.7
7.7
Streptococci
8
17.0
48
Lactobacilli
3
6.4
1.8
Escherichia coli
1
2.1
0.6
5
The total for each group or species given in bold type.
followed by Fusobacterium (19*0%)and Past. multocida (13.1 %). Thus 61.3 %
of all bacteria isolated were gram-negative bacilli. Peptostreptococcus
anaerobius was the anaerobic species isolated most frequently.
Of the facultative anaerobes, Past. multocida was isolated most frequently
(46.8 %) followed by members of the genus Actinomyces (27.7 %) ; haemolytic
and non-haemolytic streptococci, Lactobacillus spp. and Escherichia coli
together accounted for the remainder (table 111). No abscess yielded a pure
culture of Past. multocida, but 22 contained the organism. However, three of
the four abscesses that contained facultative anaerobes only (table I), yielded a
pure culture of haemolytic streptococci.
It was possible to identify to the level of genus all the morphological types
of bacteria present in pus smears. If the specimens were delivered to the
laboratory within 24 h of collection, the syringe method of transport ensured
the survival of all the types of bacteria seen in smears. If specimens took longer
than 24 h to reach the laboratory, greater numbers of organisms, but not species,
survived in the anaerobic specimen collector than in the syringe.
It is admitted that the methods used may not have allowed the isolation
and identification of all anaerobes present in the lesions, and our laboratory is
now using the anaerobically sterilised and prereduced media described by
Holdeman et al. (1977) in an attempt to minimise losses as far as possible.
DISCUSSION
This report demonstrates the large number and variety of species of anaerobe
that can be recovered from cat " fight abscesses ". Although it is difficult to
distinguish between pathogenic and non-pathogenic isolates, the numbers of
organisms seen in direct smears suggested that all isolates had been actively
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21 1
SUBCUTANEOUS ABSCESSES IN CATS
multiplying in vivo. In this study, attempts were made to exclude the possibility
of exogenous contamination.
Studies on the normal flora of the hunian oral cavity (Sutter, 1974) have
revealed the following anaerobes, listed in order of prevalence: cocci (e.g.,
peptostreptococci), gram-negative bacilli (Bacteroides spp. and Fusobacterium
spp.), gram-positive non-sporing bacilli (propionibacteria, bifidobacteria), and
clostridia. The normal flora of the feline mouth has not been described to
our knowledge, but we suggest that it is likely to bear some resemblance to that
of the human mouth. The frequencies with which we isolated the various
gram-positive and gram-negative anaerobic genera were consistent with such a
flora.
Past. inultocida appears to be a common resident in the feline mouth and
pharynx (Smith, 1964) and there is no dispute over its role in cat-bite infections of man (Tindall and Harrison, 1972) from which the organism can be
isolated in pure culture. In the present study no abscess yielded Past. multocida
in pure culture although it was the most common facultative anaerobe isolated.
Neither Past. multocida nor any other organism isolated appeared to play a
dominant role in the production of the subcutaneous abscesses that we examined.
The many recent studies in which modern anaerobic-culture techniques were
used have revealed the diversity of human infectionsthat are caused by anaerobes
or mixtures of anaerobes (Finegold, 1974; Gorbach, 1974; Wren et al., 1977).
In such human infections, gram-negative anaerobic bacilli were the most
common isolates and B. fragilis was the species most commonly encountered.
In cat abscesses we found that the species isolated most frequently was Peptostreptococcus anaerobius. This may reflect the predominance of anaerobic
gram-positive cocci in the normal flora of the oral cavity.
From the work presented here, it is apparent that many abscesses in the
subcutis of cats, resulting from bite wounds, are caused by infections characterised by the proliferation of a number of facultative anaerobes and strict
anaerobes. The source of these organisms is presumably the mouth of the
feline assailant.
SUMMARY
Thirty-six closed abscesses in the subcutis of cats were examined. Of 168
bacterial strains isolated, 121 (72 %) were anaerobes and 47 (28 %) were facultative anaerobes. Twenty-six abscesses contained mixtures of facultative
anaerobes and anaerobes, six contained anaerobes only and four contained
facultative anaerobes only. Bacteroides was the genus most commonly isolated
(28.6 % of all isolates) followed by Fusobacterium (19.0 %) and Pasteurella
(multocida) (13.1 %). Peptostreptococcus anaerobius was the most commonly
isolated anaerobic species (13.2 % of anaerobic isolates and 9.5 % of all isolates)
and Past. multocida was the most commonly isolated facultative anaerobe
(46.8 %; 13.1 % of all isolates).
We wish to thank Mrs A. M. Whittington for diligent and skilful preparation of media,
R. Merriman and R. Whittington for clinical specimens, and the Sydney University Research
Grant for financial assistance.
O*
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DARIA LOVE, R. JONES, MARILYN BAILEY AND R. JOHNSON
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