ISOLATION AND CHARACTERISATION OF BACTERIA FROM ABSCESSES IN THE SUBCUTIS OF CATS

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1 ISOLATION AND CHARACTERISATION OF BACTERIA FROM ABSCESSES IN THE SUBCUTIS OF 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 J. MED. MICROBI0L.-VOL. 12 (1979) 207

2 208 DANA 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 (1 974). 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),

3 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 r Number of abscesses containing the stated number of anaerobic species A \ Totals TABLE I1 Strictly anaerobic isolates Organisms Bacteroides B. fragilis B. asaccharolyticus B. rnelaninogenicus Other species Fusobacteria F. nucleaturn F. necrophoruni F. russii Other species Peptostreptococci Pept. anaerobius Pept. interrnedius Clostridia Propionibacteria Prop. acnes Prop. freudenreichii Bifidobacter ia Lactobacilli Eubacteria Percentage of Number of isolates anaerobic all isolates isolates Y The total for each group or genus is given in bold type. 0

4 210 DANA LOVE, R. JONES, MARILYN BAILEY AND R. JOHNSON Organisms Pasteurella multocida Actinomyces A. viscosus A. odontolyticus Streptococci Lactobacilli Escherichia coli TABLE III -- Facultatively anaerobic isolates Percentage of Number of isolates facultatively all anaerobic isolates isolates 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

5 SUBCUTANEOUS ABSCESSES IN CATS 21 1 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 infections that 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 faculta- tive 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*

6 212 DARIA LOVE, R. JONES, MARILYN BAILEY AND R. JOHNSON REFERENCES BUCHANAN, R. E. AND GIBBONS, N. E Bergey s Manual of determinative bacteriology, 8th ed., Baltimore. COWAN, S. T Cowan and Steel s Manual for the identification of medical bacteria, 2nd ed., Cambridge. FINEGOLD, S. M Anaerobic infections of the lung. In Anaerobic infections, edited by J. 0. Godden and I. B. R. Duncan. The Upjohn Symposium, Toronto, p. 50. GORBACH, S. L Anaerobic gynecological infections. In Anaerobic infections, edited by J. 0. Godden and I. B. R. Duncan. The Upjohn Symposium, Toronto, p. 77, HOLDEMAN, L. V., CATO, E. P. AND MOORE, W. E. C Anaerobic laboratory manual, 4t h ed., Blacksburg, Virginia. LOVE, D. N., JONES, R. F., BAILEY, M., CANFIELD, R. AND MCNAUGHT, H Bacteria isolated from subcutaneous abscesses in cats. Aust. vet. Pract., 8, 87. SMITH, J. E Symposium on diseases of cats Some pathogenic bacteria of cats with special reference to their public health significance. J. small Anim. Pract., 5, 517. SUTTER, V. L Collection and transport of specimens for anaerobic culturing. In Anaerobic infections, edited by J. 0. Godden and I. B. R. Duncan. The Upjohn Symposium, Toronto, p. 20. TINDALL, J. P. AND HARRISON, C. M Pasteurella multocida infections following animal injuries, especially cat bites. Arch. Derm. Syph., 105, 412. TURNER, A. W., CAMPBELL, A. D. AND DICK, A. T Recent work on pleuropneumonia contagiosa bovum in North Queensland. Aust. vet. J., 11,63. WREN, M. W. D., BALDWIN, A. W. F., ELDON, C. P. AND SANDERSON, P. J The anaerobic culture of clinical specimens. A 14-month study. J. med. Microbiol., 10,49.