Journal of Applied Bacteriology 1992, 73, 421425
Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats L.A. Devriese, J.I. Cruz Colque’, P. De Herdt and F. Haesebrouck
’
Faculty of Veterinary Medicine, University of Gent, Belgium and Facultad de Medicina Veterinaria, Universidad Nacional del Altiplano , Puno, Peru 4140/02/92: accepted 9 May 1992
L . A . D E V R I E S E , J . I . C R U Z COLQUE, P. DE H E R D T A N D F. HAESEBROUCK. 1992. Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent. faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium-like strains were seen in dogs, while the related species Ent. rafJinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum, shared characteristics with Ent. columbae. T h e most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. T h e canine Strep. suis differed from the common porcine strains in fermenting mannitol. Forty-seven of the 288 isolates examined could not be identified or related to known species. T h e characteristics of two groups of these bacteria, provisionally called ‘Ton 3 1 group’ and ‘07group’ are described.
INTRODUCTION
Little information is available about the enterococci and streptococci that are found among the mouth and faecal flora of dogs and cats. Saphir & Carter (1976) examined the gingival flora of dogs with special reference to bacteria associated with bites but they did not identify the mainly alpha-haemolytic streptococci isolated. Bailie et al. (1978) found group D and G and few other streptococci in oral fluids from dogs. The flora of different compartments of the intestinal tract of dogs and cats was investigated by Smith (1965). The streptococci, which were not identified, were found in higher numbers than in the other animals examined, averaging 109/g in faeces of dogs and 108/g in faeces of cats. Weisser (1981) identified streptococci and enterococci from the faeces of dogs and cats as belonging to the Enterococcus faecium-Ent. durans group, Streptococcus bovis and Ent. faecalis. Few strains were identified as Streptococcus MG group, Lancefield G group or Strep. lactis, and other species were very rare. Devriese et al. (1987) found Ent. faecalis, Ent. faecium and Ent. hirae among a small collection of enterococci isolated from the intestine of dogs. Correspondence to :Dr L.A. Devriese, Faculty of Veterinary Medicine, Universit,y of Gent, Casinoplein 24, B 9000 Gmt, Belgium.
In the present investigation the classification, nomenclature and identification systems which have been in use in recent years, are applied to identify a larger and more representative collection of strains from tonsils and anal swabs of dogs and cats. MATERIALS AND METHODS
Animals that attended the Small Animal Clinic of the Veterinary Faculty in Gent and which belonged to different owners, were sampled. Anal swabs were taken from dogs and cats not suffering from enteric disease and which were not treated with antibiotics. Tonsils were collected at necropsy from animals which died or were euthanized. They were seared with a hot spatula and a loopful of fluid, taken from an incision, was inoculated on CNA Columbia Agar (Gibco) containing bovine blood and the selective agents colistin and nalidixic acid. Anal swabs were inoculated directly on the same medium. The cultures were incubated at 37°C for 2 d in 3-5% CO,. Representative colonies were purified and identified as described by Devriese et al. (1992). This involved growth in 6.5% NaCI, on Slanetz & Bartley (Oxoid), Rogosa (Oxoid) and kanamycin aesculin azide (Oxoid) selective agars, colony characteristics, haemolysis, motility, carbohydrate fermentation tests, amylase,
422 L . A . DEVRIESE E T A L .
arginine dehydrolase, pyrrolidonylarylamidase and coagglutination grouping tests. Cultures with similar phenotypical traits and which could not be identified with known species, were tested for growth at 10°C and their carbohydrate dissimilation patterns were determined in the API 50CH system.
RESULTS
Multiple isolates from the same sample which belonged to the same species and which showed the same characteristics in the tests used, were considered to be representatives of the same strain and counted only once. In all 85 and 86 strains were isolated from anal swabs of 60 dogs and 40 cats respectively and 57 and 60 strains from the tonsils of 25 dogs and 26 cats respectively. Percentages of strains identified to different species and percentages of animals positive, are listed in Tables 1 and 2. T h e characteristics of Ent. faecalis, Ent. avium and the rare species Enterococcus durans and Enterococcus gallinarum were similar to those of other strains from animals (Devriese et al. 1987) and typical strains from humans (Facklam & Collins 1989). Also, Enterococcus hirae reacted as described by Farrow & Collins (1985) but many of the strains belonging to other known enterococcal species, had unusual characteristics as is shown in Table 3. A homogenous group of six canine anal strains showed growth characteristics and biochemical reactions similar to those described for Ent. avium and Ent. pseudoavium. They differed from Ent. avium, however, in failing to produce acid from sorbitol, sorbose, adonitol, melezitose, L-arabitol, wtagatose and rhamnose. They also differed from Ent. pseudoavium in their positive L-arabinose and glycerol reactions as well as in their negative sorbitol, sorbose and adonitol reactions. Two dog strains from tonsils reacted similarly to this group, except for showing a negative Larabinose and a positive rhamnose reaction. The Streptococcus canis reacted as described earlier (Devriese et al. 1986). Also none of the other streptococci isolated showed any unusual characteristics, except for the five canine strains of Strep. suis and also one of the seven feline Strep. suis strains (Table 3). Forty-seven strains remained unidentified. Two homogenous groups of strains were phenotypically characterized as shown in Table 4. The first group consisting of four strains, was provisionally called ‘07’ group. T h e second, represented by 12 strains, received the provisional designation ‘Ton 31’ group. These names are derived from the designations of the first strains isolated. Colonies of the ‘Ton 3 1’ group were relatively large and somewhat opaque, resembling those of enterococci belonging to the Ent. faecium species group. They continued to grow at room
Table 1 Percentages of canine strains identified to different species and percentages of animals positive*
Species Enterococcus faecalis hirae faecium avium avium-like raginosus durans cecorum
Anus
Tonsils
(45) (37) (12) (12) (13) (2) (2) (2)
21 (48) 4 (8) 9 (20)
Streptococcus canis bovis suis alactolyticur dysgalactiae
8 (12) 6 (10) 0 1 (2) 0
15 (32) 4 (8) 9 (20) 4 (8) 4 (8)
Unidentified 0 7 group Ton 31 group Other
0 0 8
27 22 8 8
10 1 1 1
0 6 (12) 0 0 4 (8)
4 (8) 6 (12)
13
* In brackets. temperature after being removed from the incubator. They were not haemolytic on bovine blood plates after incubation for 24 h but weak haemolysis was seen after 2-3 d. Colonies of the ‘07’ group were alpha-haemolytic, smaller and
Table 2 Percentages of feline strains identified to different species and percentages of animals positive*
Species
Anus
Tonsils
Enterococcus faecalis hirae faecium raginosus gallinarum cecorum
28 7 7 0 1 8
17 2 8 12 2 8
Streptococcus canis bovis suis alactolyticus
14 15 2 2
Unidentified 0 7 group Ton 31 group Other
2 (5) 3 (7) 12
* In brackets.
(60)
(15) (15) (2)
(14) (30) (32) (5) (5)
(40) (4) (19) (27)
(4) (12)
8 (19) 6 (12) 8 (12) 0 0 10 (23) 22
ENTEROCOCCI AND STREPTOCOCCI FROM DOGS AND CATS 423
Table 3 Unusual characteristics of feline and canine enterococci and streptococci
Species
Source
Enterococcus faecium faecium faecium raffinosus cecorum cecorum cecorum cecorum cecorum cecorum cecorum Streptococcus suis suis suis suis suis
Table 4 Variable characteristics and characteristics differentiating
between strains belonging to the groups ‘Ton 31’ and ‘07’ isolated from dogs and cats Characteristic
‘Ton 31’
‘07’
Growth at 10°C Growth in 6.5% NaCl broth Growth on Rogosa medium
12/12* 8wt/12 0112
014 014 414 414 014 014 414 014 014
Arginine hydrolysis
Hippurate hydrolysis Pyrrolidonylary lamidase Beta-galactosidase Alkaline phosphatase Group D antigen Acid from Ribose L-Arabinose
12/12 7w/12
10112 0112 3w/12 9/12 12/12 2/12
Mannitol Sorbitol Lactose
8/12
Trehalose
12/12 12/12
Amidon Saccharose Galactose D-Xylose Am ygdalin Aesculin Arbutin
Salicin Cellobiose Beta-gentiobiose D-Tagatose Gluconate
* Number of positives/number tested. Weak or delayed reactions.
1/12
8/12 4/12 819
019 919 919 919 919 919 919
319 319
314 314 114 014 014 114 014 214 114 314 214 314 314 314 214 014 014 314
Characteristic
No. positive/no. tested
Sorbitol+ Sorbitol + Rafinose + L-Arabinose B-Glucuronidase 8-GlucuronidaseAlkaline phosphatase Pyrrolidonylarylamidase + Mannitol +, sorbitol + Mannitol +, sorbitol + Mannitol +
7/13 115 211 3 215 213 611 1 311 1
/3-Glucuronidase B-Glucuronidase Mannitol + , sorbitol+ Mannitol + , sorbitol+ Mannitol +
515
1111
213 511 1 211 1
117 215 117 315
Streptococcus-like. Other variable characters or characters which differed between the two groups, are listed in Table 4. All strains were non-motile, catalase-negative, Grampositive, coccal or slightly elongated. They gave positive reactions in tests for leucine arylaminidase and acetoin and acid production from glucose, fructose, mannose, maltose and N-acetylglucosamine. All strains of both groups gave negative reactions to the following: alpha-galactosidase, beta-glucuronidase, amylase, acid production from glycerol, glycogen, inulin, raffinose, erythritol, D-arabinose, L-xylose, adonitol, beta-methylxyloside, melezitose, xylitol, melibiose, L-sorbose, L-rhamnose, dulcitol, inositol, alphamethyl-D-mannoside, alpha-methyl-D-glucoside, 2- and 5-ketogluconate, D and L-arabitol, D and L-fucose, D turanose and L-lyxose. DISCUSSION
During this work there were identification problems which were difficult to solve. Acid production from sorbitol and raffinose, which is often used to assist in the identification of Ent. faecium, was atypical in some of our strains: rare sorbitol-positive strains have been described by Facklam & Collins (1989) among those from humans. Raffinosepositive Ent. faecium strains have been found only in poultry (Devriese et al. 1987, 1991a). T h e atypical canine and feline strains were identified as Ent. faecium because they were typical in all other reactions investigated, They differed in four or more test results from known sorbitol or raffinose fermenting species. Identification problems with atypical Enterococcus aviumlike and Ent. cecorum-like strains presented more difficulties. T h e canine Ent. avium-like strains listed in Table 1,
424 L . A . DEVRIESE ET A L .
differed in no fewer than seven carbohydrate reactions from typical Ent. avium, which were also found in dogs. Diagnostically important reactions such as acid production from L-arabinose and sorbitol differed also from those described with Ent. pseudoavium (Collins et al. 1989). Enterococcus raflnosus was differentiated from Ent. avium and Ent. avium-like strains by its positive raffinose and melibiose reactions. Two cat strains were atypical, however. They resembled Ent. malodoratus in being L-arabinose-negative, but they differed from this species in their negative betagalactosidase. All strains belonging to the Ent. avium-Ent. pseudoavium-Ent. raflnosus-Ent malodoratus species group (Williams et al. 1991), typical as well as atypical, produced broad, sharply demarcated zones of greening haemolysis with an inner dark zone surrounding the colonies and a narrow clear margin. This trait which became more evident when incubation was prolonged or when the plates were left at room temperature, was most useful in their identification. Some characters of the atypical Ent. recorum strains, notably the positive mannitol and sorbitol and negative beta-glucuronidase reactions have been described earlier (Devriese et al. 1991b) but pyrrolidonylarylamidasepositive or alkaline phosphatase-negative Ent. cecorum were not known to exist. Enterococcus cecorum is very similar in its growth and biochemical characteristics to Ent. columbae, from which it can be differentiated usually by its negative L-arabinose- and positive beta-glucuronidase reactions (Devriese et al. 1990, 1991b). All our canine and feline strains failed to produce acid from L-arabinose but many were beta-glucuronidase-negative. However, following the original species description, a minority of Ent. columbae from pigeons has been found L-arabinose-negative (Devriese, unpublished observations). Therefore, our decision to identify the beta-glucuronidase-negative strains as Ent. cecorum rather than Ent. columbae should be considered with caution. Definite confirmation of the identifications of these strains must await results of further genomic and phenotypic characterization. Streptococcus canis was easily identified and differentiated, as described earlier (Devriese et al. 1986), from the human beta-haemolytic Streptococcus group G which belongs to the serologically and biochemically heterogenic species Strep. dysgalactiae (Farrow & Collins 1984). Betahaemolytic Strep. dysgalactzae of the Lancefield group C also occur in dogs (Biberstein et al. 1980). The three strains considered here belonged to the ‘equisimilis’ biotype and they reacted similarly to the porcine ecovar of this biotype in the tests described by Devriese (1991). All Strep. bovis strains produced a strong amylase reaction on Columbia Agar Base plates enriched with starch. This test, originally developed for Strep. suis (Devriese et al. 1991c), was most useful in the identification of both species. All Strep. bovis
except the two canine tonsillar strains examined, were mannitol-negative. T h e canine Strep. suis clearly differed from the commonly occurring porcine strains in their positive mannitol and negative beta-glucuronidase reactions and in the case of some strains also in their positive sorbitol reaction (Table 3). They appear to belong to a separate host-associated ecovar, as has been described also in other animal species (Devriese et al. 1991~). T h e ‘Ton 3 1 group’ presented several characteristics which are more often found in the genus Enterococcus than in the genus Streptococcus; their colonies were relatively large and not transparent and they grew at 10°C. However, only a proportion of the strains was resistant to 6.5% NaCl or possessed the group D antigen. They did not grow on Slanetz & Bartley selective medium and grew only poorly on kanamycin aesculin azide agar. They share these features, however, with several other enterococci such as Ent. avium and Ent. cecorum. T h e ‘07 group’ was unusual in growing on Rogosa selective medium, a characteristic found only in Strep. bovis among streptococcal species ; ‘07’ strains can be differentiated from Strep. bovis by their positive arginine and ribose reactions and also by their failure to grow on the commonly used Slanetz & Bartley and kanamycin aesculin azide agar. Enterococcus faecalis was the most frequently isolated species in all types of samples examined. Enterococcus hirae ranked next in anal swabs from dogs but was isolated relatively infrequently from other samples. This species is potentially enteropathogenic in puppies : an entero-adhesive strain which was originally described as Ent. durans (Collins et al. 1988), was found to be an Ent. hirae (Devriese & Haesebrouck 1991). Typical Ent. avium and Ent. avium-like strains were seen in approximately 10% of anal and tonsillar samples of dogs, while Ent. raflnosus was the only member of the Ent. avium-Ent. pseudoavium-Ent. malodoratus-Ent. rafinosus species group found in cats. Feline anal samples did not contain any members of this group. Also Ent. cecorum was found more often in cats than in dogs. Streptococcus canis appears to be associated as often with cats as with dogs. This corresponds with reports of severe infections, including lymphadenitis, pharyngitis, pneumonia, septicaemia, abscesses and arthritis (Iglauer et al. 1991), which are more frequent in cats than in dogs. T h e feline Strep. canis did not differ from the canine strains in the biochemical tests used. Streptococcus suis infections have been described recently in cats (Devriese & Haesebrouck 1992). Similar infections have not been reported in dogs. In our sampling series 20% of dog tonsils were found to harbour Strep. suis. Possibly the biotype which appears to be associated with dogs is of low virulence. Differences in virulence have been documented in porcine strains (Vecht et al. 1989).
ENTEROCOCCI AND STREPTOCOCCI F R O M DOGS AND CATS
ACKNOWLEDGEMENTS This work was made possible by the generous help of staff and personnel of the Small Animal Clinic and the Department of Pathology of the Faculty of Veterinary Medicine, University of Gent, Belgium.
REFERENCES B A I L I E ,W.E., STOWE,E . C . & S C H M I T T A,. M . (1978) Aerobic bacterial flora of oral and nasal fluids of canines with reference to bacteria associated with bites. Journal of Clinical Microbiology 7, 223-23 1. BIBERSTEIN E ,. L . , B R O W N C , . & S M I T H ,T . (1980) Serogroups and biotypes among beta-hemolytic streptococci of canine origin. Journal of Clinical Microbiology 11, 558-561. C O L L I N SJ,. E . , B E R G E L A N M D ,. E . , L I N D E M A NC.J. , & D U I M S T R AJ ,. R. (1988) Enterococcus (Streptococcus) durans adherence in the small intestine of a diarrheic pup. Veterinary Pathology 25, 396398. C O L L I N S ,M . D . , F A C K L A M R.R., , FARROW J.A.E. , & W I L L I A M S O NR,. (1989) Enterococcus raflnosus sp. nov., Enterococcus pseudoavium sp. nov. and Enterococcus solitarius sp. nov. F E M S Microbiology Letters 57, 283-288. D E V R I E S EL.A. , (1991) Streptococcal ecovars associated with different animal species : epidemiological significance of serogroups and biotypes. Journal of Applied Bacteriology 71, 478483. D E V R I E S EL, . A . & H A E S E B R O U CF. K , (1991) Enterococcus hirae in different animal species. The Veterinary Record 129, 391-392. D E V R I E S EL,. A . & H A E S E B R O U CF.K ,(1992) Streptococcus suis infections in horses and in cats. The Veterinary Record 130, 380. DEVRIESE, L . A . , H O M M E ZJ, . , KILPPER-BALz, R . & S C H L E I F E RK, . H . (1986) Streptococcus canis sp. nov.: a species of group G streptococci from animals. International Journal of Systematic Bacteriology 36, 422425. DEVRIESE, L.A., VAN DE K E R C K H O V E , A., KILPPER-B'ALz,R. & S C H L E I F E RK, . H . (1987) Characterization and identification of Enterococcus species isolated from animals. International Journai of Systematic Bacteriology 37,257-259. D E V R I E S EL,. A . , CEYSSENS,K . , R O D R I G U E SU, . M . & C O L L I N SM , .D . (1990) Enterococcus columbae, a species from pigeon intestines. F E M S Microbiology Letters 71, 247-252. D E V R I E S EL, . A . , H O M M E ZJ,. , WYFFELS,R . & HAESEB R O U C KF, . (1991a) Composition of the enterococcal and streptococcal intestinal flora of poultry. Journal of Applied Bacteriology 71, 46-50.
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D E V R I E S EL.A., , CEYSSENS,K . & H A E S E B R O U C F.K , (1991b) Characteristics of Enterococcus cecorum strains from the intestines of different animal species. Letters in Applied Microbiology 12, 137-139. D E V R I E S E , L.A., CEYSSENS, K . , H O M M E Z , J . , KILPPER-BALz,R . & S C H L E I F EKR. H, . (1991~)Characteristics of different Streptococcus suis ecovars and unusual strains isolated from farm animals. Veterinary Microbiology 26, 141-150. D E V R I E S EL.A., , L A U R I E RL, . , D E H E R D T ,P. & H A E S E B R O U C K , F. (1992) Enterococcal and streptococcal species isolated from faeces of calves, young cattle and dairy cows. Journal of Applied Bacteriology 72, 29-3 1. F A C K L ARM . R, . & C O L L I N S ,M . D . (1989) Identification of Enterococcus species isolated from human infections by a conventional test scheme. Journal of Clinical Microbiology 27, 731734. FARROW J .,A . E . & C O L L I N S , M . D . (1984) Taxonomic studies on streptococci of serological groups C, G and L and possibly related taxa. Systematic and Applied Microbiology 5 , 483-493. F A R R O WJ.A.E. , & C O L L I N S M . D . (1985) Enterococcus hirae, a new species that includes amino acid assay strain NCDO 1258 and strains causing growth depression in young chickens. International Journal of Systematic Bacteriology 35, 73-75. I G L A U E RF., , K U N S T Y RI., , MORSTEDT,R., F A R O U Q , H., WULLENWEBER, W. & DAMSCH, S. (1991) Streptococcus canis in a cat breeding colony. Journal of Experimental Animal Science 34, 5%65. S M I T HH.W. , (1965) Observations on the flora of the alimentary tract of animals and factors affecting its composition. Journal of Pathology and Bacteriology 89, 95-122. S A P H I RD,. A . & C A R T E RG, . R . (1976) Gingival flora of the dog with special reference to bacteria associated with bites. Journal of Clinical Microbiology 3, 344-349. VECHT, U., ARENDS, J . P . , V A N D E R M O L E N ,E . J . & VANL E E N G O E DL,. A . M . G . (1989) Differences in virulence between two strains of Streptococcus suis type I1 after experimentally induced infection of newborn germ-free pigs. American Journal of Veterinary Research 50, 1037-1043. W E I S S E RW , . (1981) Beobachtungen uber die fakale streptokokkenflora bei fleischfressenden Haustieren. Zentralblatt f u r Bakteriologie A248, 455-462. W I L L I A M SA.M., , R O D R I G U E SU, . M . & L O L L I N SM, . U . (1991) Intrageneric relationship of enterococci as determined by transscriptase sequencing of small-subunit rRNA. Research in Microbiology 142, 67-74.
Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats L.A. Devriese, J.I. Cruz Colque’, P. De Herdt and F. Haesebrouck
’
Faculty of Veterinary Medicine, University of Gent, Belgium and Facultad de Medicina Veterinaria, Universidad Nacional del Altiplano , Puno, Peru 4140/02/92: accepted 9 May 1992
L . A . D E V R I E S E , J . I . C R U Z COLQUE, P. DE H E R D T A N D F. HAESEBROUCK. 1992. Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent. faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium-like strains were seen in dogs, while the related species Ent. rafJinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum, shared characteristics with Ent. columbae. T h e most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. T h e canine Strep. suis differed from the common porcine strains in fermenting mannitol. Forty-seven of the 288 isolates examined could not be identified or related to known species. T h e characteristics of two groups of these bacteria, provisionally called ‘Ton 3 1 group’ and ‘07group’ are described.
INTRODUCTION
Little information is available about the enterococci and streptococci that are found among the mouth and faecal flora of dogs and cats. Saphir & Carter (1976) examined the gingival flora of dogs with special reference to bacteria associated with bites but they did not identify the mainly alpha-haemolytic streptococci isolated. Bailie et al. (1978) found group D and G and few other streptococci in oral fluids from dogs. The flora of different compartments of the intestinal tract of dogs and cats was investigated by Smith (1965). The streptococci, which were not identified, were found in higher numbers than in the other animals examined, averaging 109/g in faeces of dogs and 108/g in faeces of cats. Weisser (1981) identified streptococci and enterococci from the faeces of dogs and cats as belonging to the Enterococcus faecium-Ent. durans group, Streptococcus bovis and Ent. faecalis. Few strains were identified as Streptococcus MG group, Lancefield G group or Strep. lactis, and other species were very rare. Devriese et al. (1987) found Ent. faecalis, Ent. faecium and Ent. hirae among a small collection of enterococci isolated from the intestine of dogs. Correspondence to :Dr L.A. Devriese, Faculty of Veterinary Medicine, Universit,y of Gent, Casinoplein 24, B 9000 Gmt, Belgium.
In the present investigation the classification, nomenclature and identification systems which have been in use in recent years, are applied to identify a larger and more representative collection of strains from tonsils and anal swabs of dogs and cats. MATERIALS AND METHODS
Animals that attended the Small Animal Clinic of the Veterinary Faculty in Gent and which belonged to different owners, were sampled. Anal swabs were taken from dogs and cats not suffering from enteric disease and which were not treated with antibiotics. Tonsils were collected at necropsy from animals which died or were euthanized. They were seared with a hot spatula and a loopful of fluid, taken from an incision, was inoculated on CNA Columbia Agar (Gibco) containing bovine blood and the selective agents colistin and nalidixic acid. Anal swabs were inoculated directly on the same medium. The cultures were incubated at 37°C for 2 d in 3-5% CO,. Representative colonies were purified and identified as described by Devriese et al. (1992). This involved growth in 6.5% NaCI, on Slanetz & Bartley (Oxoid), Rogosa (Oxoid) and kanamycin aesculin azide (Oxoid) selective agars, colony characteristics, haemolysis, motility, carbohydrate fermentation tests, amylase,
422 L . A . DEVRIESE E T A L .
arginine dehydrolase, pyrrolidonylarylamidase and coagglutination grouping tests. Cultures with similar phenotypical traits and which could not be identified with known species, were tested for growth at 10°C and their carbohydrate dissimilation patterns were determined in the API 50CH system.
RESULTS
Multiple isolates from the same sample which belonged to the same species and which showed the same characteristics in the tests used, were considered to be representatives of the same strain and counted only once. In all 85 and 86 strains were isolated from anal swabs of 60 dogs and 40 cats respectively and 57 and 60 strains from the tonsils of 25 dogs and 26 cats respectively. Percentages of strains identified to different species and percentages of animals positive, are listed in Tables 1 and 2. T h e characteristics of Ent. faecalis, Ent. avium and the rare species Enterococcus durans and Enterococcus gallinarum were similar to those of other strains from animals (Devriese et al. 1987) and typical strains from humans (Facklam & Collins 1989). Also, Enterococcus hirae reacted as described by Farrow & Collins (1985) but many of the strains belonging to other known enterococcal species, had unusual characteristics as is shown in Table 3. A homogenous group of six canine anal strains showed growth characteristics and biochemical reactions similar to those described for Ent. avium and Ent. pseudoavium. They differed from Ent. avium, however, in failing to produce acid from sorbitol, sorbose, adonitol, melezitose, L-arabitol, wtagatose and rhamnose. They also differed from Ent. pseudoavium in their positive L-arabinose and glycerol reactions as well as in their negative sorbitol, sorbose and adonitol reactions. Two dog strains from tonsils reacted similarly to this group, except for showing a negative Larabinose and a positive rhamnose reaction. The Streptococcus canis reacted as described earlier (Devriese et al. 1986). Also none of the other streptococci isolated showed any unusual characteristics, except for the five canine strains of Strep. suis and also one of the seven feline Strep. suis strains (Table 3). Forty-seven strains remained unidentified. Two homogenous groups of strains were phenotypically characterized as shown in Table 4. The first group consisting of four strains, was provisionally called ‘07’ group. T h e second, represented by 12 strains, received the provisional designation ‘Ton 31’ group. These names are derived from the designations of the first strains isolated. Colonies of the ‘Ton 3 1’ group were relatively large and somewhat opaque, resembling those of enterococci belonging to the Ent. faecium species group. They continued to grow at room
Table 1 Percentages of canine strains identified to different species and percentages of animals positive*
Species Enterococcus faecalis hirae faecium avium avium-like raginosus durans cecorum
Anus
Tonsils
(45) (37) (12) (12) (13) (2) (2) (2)
21 (48) 4 (8) 9 (20)
Streptococcus canis bovis suis alactolyticur dysgalactiae
8 (12) 6 (10) 0 1 (2) 0
15 (32) 4 (8) 9 (20) 4 (8) 4 (8)
Unidentified 0 7 group Ton 31 group Other
0 0 8
27 22 8 8
10 1 1 1
0 6 (12) 0 0 4 (8)
4 (8) 6 (12)
13
* In brackets. temperature after being removed from the incubator. They were not haemolytic on bovine blood plates after incubation for 24 h but weak haemolysis was seen after 2-3 d. Colonies of the ‘07’ group were alpha-haemolytic, smaller and
Table 2 Percentages of feline strains identified to different species and percentages of animals positive*
Species
Anus
Tonsils
Enterococcus faecalis hirae faecium raginosus gallinarum cecorum
28 7 7 0 1 8
17 2 8 12 2 8
Streptococcus canis bovis suis alactolyticus
14 15 2 2
Unidentified 0 7 group Ton 31 group Other
2 (5) 3 (7) 12
* In brackets.
(60)
(15) (15) (2)
(14) (30) (32) (5) (5)
(40) (4) (19) (27)
(4) (12)
8 (19) 6 (12) 8 (12) 0 0 10 (23) 22
ENTEROCOCCI AND STREPTOCOCCI FROM DOGS AND CATS 423
Table 3 Unusual characteristics of feline and canine enterococci and streptococci
Species
Source
Enterococcus faecium faecium faecium raffinosus cecorum cecorum cecorum cecorum cecorum cecorum cecorum Streptococcus suis suis suis suis suis
Table 4 Variable characteristics and characteristics differentiating
between strains belonging to the groups ‘Ton 31’ and ‘07’ isolated from dogs and cats Characteristic
‘Ton 31’
‘07’
Growth at 10°C Growth in 6.5% NaCl broth Growth on Rogosa medium
12/12* 8wt/12 0112
014 014 414 414 014 014 414 014 014
Arginine hydrolysis
Hippurate hydrolysis Pyrrolidonylary lamidase Beta-galactosidase Alkaline phosphatase Group D antigen Acid from Ribose L-Arabinose
12/12 7w/12
10112 0112 3w/12 9/12 12/12 2/12
Mannitol Sorbitol Lactose
8/12
Trehalose
12/12 12/12
Amidon Saccharose Galactose D-Xylose Am ygdalin Aesculin Arbutin
Salicin Cellobiose Beta-gentiobiose D-Tagatose Gluconate
* Number of positives/number tested. Weak or delayed reactions.
1/12
8/12 4/12 819
019 919 919 919 919 919 919
319 319
314 314 114 014 014 114 014 214 114 314 214 314 314 314 214 014 014 314
Characteristic
No. positive/no. tested
Sorbitol+ Sorbitol + Rafinose + L-Arabinose B-Glucuronidase 8-GlucuronidaseAlkaline phosphatase Pyrrolidonylarylamidase + Mannitol +, sorbitol + Mannitol +, sorbitol + Mannitol +
7/13 115 211 3 215 213 611 1 311 1
/3-Glucuronidase B-Glucuronidase Mannitol + , sorbitol+ Mannitol + , sorbitol+ Mannitol +
515
1111
213 511 1 211 1
117 215 117 315
Streptococcus-like. Other variable characters or characters which differed between the two groups, are listed in Table 4. All strains were non-motile, catalase-negative, Grampositive, coccal or slightly elongated. They gave positive reactions in tests for leucine arylaminidase and acetoin and acid production from glucose, fructose, mannose, maltose and N-acetylglucosamine. All strains of both groups gave negative reactions to the following: alpha-galactosidase, beta-glucuronidase, amylase, acid production from glycerol, glycogen, inulin, raffinose, erythritol, D-arabinose, L-xylose, adonitol, beta-methylxyloside, melezitose, xylitol, melibiose, L-sorbose, L-rhamnose, dulcitol, inositol, alphamethyl-D-mannoside, alpha-methyl-D-glucoside, 2- and 5-ketogluconate, D and L-arabitol, D and L-fucose, D turanose and L-lyxose. DISCUSSION
During this work there were identification problems which were difficult to solve. Acid production from sorbitol and raffinose, which is often used to assist in the identification of Ent. faecium, was atypical in some of our strains: rare sorbitol-positive strains have been described by Facklam & Collins (1989) among those from humans. Raffinosepositive Ent. faecium strains have been found only in poultry (Devriese et al. 1987, 1991a). T h e atypical canine and feline strains were identified as Ent. faecium because they were typical in all other reactions investigated, They differed in four or more test results from known sorbitol or raffinose fermenting species. Identification problems with atypical Enterococcus aviumlike and Ent. cecorum-like strains presented more difficulties. T h e canine Ent. avium-like strains listed in Table 1,
424 L . A . DEVRIESE ET A L .
differed in no fewer than seven carbohydrate reactions from typical Ent. avium, which were also found in dogs. Diagnostically important reactions such as acid production from L-arabinose and sorbitol differed also from those described with Ent. pseudoavium (Collins et al. 1989). Enterococcus raflnosus was differentiated from Ent. avium and Ent. avium-like strains by its positive raffinose and melibiose reactions. Two cat strains were atypical, however. They resembled Ent. malodoratus in being L-arabinose-negative, but they differed from this species in their negative betagalactosidase. All strains belonging to the Ent. avium-Ent. pseudoavium-Ent. raflnosus-Ent malodoratus species group (Williams et al. 1991), typical as well as atypical, produced broad, sharply demarcated zones of greening haemolysis with an inner dark zone surrounding the colonies and a narrow clear margin. This trait which became more evident when incubation was prolonged or when the plates were left at room temperature, was most useful in their identification. Some characters of the atypical Ent. recorum strains, notably the positive mannitol and sorbitol and negative beta-glucuronidase reactions have been described earlier (Devriese et al. 1991b) but pyrrolidonylarylamidasepositive or alkaline phosphatase-negative Ent. cecorum were not known to exist. Enterococcus cecorum is very similar in its growth and biochemical characteristics to Ent. columbae, from which it can be differentiated usually by its negative L-arabinose- and positive beta-glucuronidase reactions (Devriese et al. 1990, 1991b). All our canine and feline strains failed to produce acid from L-arabinose but many were beta-glucuronidase-negative. However, following the original species description, a minority of Ent. columbae from pigeons has been found L-arabinose-negative (Devriese, unpublished observations). Therefore, our decision to identify the beta-glucuronidase-negative strains as Ent. cecorum rather than Ent. columbae should be considered with caution. Definite confirmation of the identifications of these strains must await results of further genomic and phenotypic characterization. Streptococcus canis was easily identified and differentiated, as described earlier (Devriese et al. 1986), from the human beta-haemolytic Streptococcus group G which belongs to the serologically and biochemically heterogenic species Strep. dysgalactiae (Farrow & Collins 1984). Betahaemolytic Strep. dysgalactzae of the Lancefield group C also occur in dogs (Biberstein et al. 1980). The three strains considered here belonged to the ‘equisimilis’ biotype and they reacted similarly to the porcine ecovar of this biotype in the tests described by Devriese (1991). All Strep. bovis strains produced a strong amylase reaction on Columbia Agar Base plates enriched with starch. This test, originally developed for Strep. suis (Devriese et al. 1991c), was most useful in the identification of both species. All Strep. bovis
except the two canine tonsillar strains examined, were mannitol-negative. T h e canine Strep. suis clearly differed from the commonly occurring porcine strains in their positive mannitol and negative beta-glucuronidase reactions and in the case of some strains also in their positive sorbitol reaction (Table 3). They appear to belong to a separate host-associated ecovar, as has been described also in other animal species (Devriese et al. 1991~). T h e ‘Ton 3 1 group’ presented several characteristics which are more often found in the genus Enterococcus than in the genus Streptococcus; their colonies were relatively large and not transparent and they grew at 10°C. However, only a proportion of the strains was resistant to 6.5% NaCl or possessed the group D antigen. They did not grow on Slanetz & Bartley selective medium and grew only poorly on kanamycin aesculin azide agar. They share these features, however, with several other enterococci such as Ent. avium and Ent. cecorum. T h e ‘07 group’ was unusual in growing on Rogosa selective medium, a characteristic found only in Strep. bovis among streptococcal species ; ‘07’ strains can be differentiated from Strep. bovis by their positive arginine and ribose reactions and also by their failure to grow on the commonly used Slanetz & Bartley and kanamycin aesculin azide agar. Enterococcus faecalis was the most frequently isolated species in all types of samples examined. Enterococcus hirae ranked next in anal swabs from dogs but was isolated relatively infrequently from other samples. This species is potentially enteropathogenic in puppies : an entero-adhesive strain which was originally described as Ent. durans (Collins et al. 1988), was found to be an Ent. hirae (Devriese & Haesebrouck 1991). Typical Ent. avium and Ent. avium-like strains were seen in approximately 10% of anal and tonsillar samples of dogs, while Ent. raflnosus was the only member of the Ent. avium-Ent. pseudoavium-Ent. malodoratus-Ent. rafinosus species group found in cats. Feline anal samples did not contain any members of this group. Also Ent. cecorum was found more often in cats than in dogs. Streptococcus canis appears to be associated as often with cats as with dogs. This corresponds with reports of severe infections, including lymphadenitis, pharyngitis, pneumonia, septicaemia, abscesses and arthritis (Iglauer et al. 1991), which are more frequent in cats than in dogs. T h e feline Strep. canis did not differ from the canine strains in the biochemical tests used. Streptococcus suis infections have been described recently in cats (Devriese & Haesebrouck 1992). Similar infections have not been reported in dogs. In our sampling series 20% of dog tonsils were found to harbour Strep. suis. Possibly the biotype which appears to be associated with dogs is of low virulence. Differences in virulence have been documented in porcine strains (Vecht et al. 1989).
ENTEROCOCCI AND STREPTOCOCCI F R O M DOGS AND CATS
ACKNOWLEDGEMENTS This work was made possible by the generous help of staff and personnel of the Small Animal Clinic and the Department of Pathology of the Faculty of Veterinary Medicine, University of Gent, Belgium.
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