Prevalence and Characteristics of Streptococcus canis Strains Isolated from Dogs and Cats

Lysková P., M. Vydržalová, D. Královcová, J. Mazurová: Prevalence and Characteristics of Streptococcus canis Strains Isolated from Dogs and Cats. Acta Vet. Brno 2007, 76: 619-625. To determine the prevalence of Streptococcus canis in dogs and cats, a total of 926 swabs were examined bacteriologically in the period from 2003 to 2005. Eighty-six isolates obtained from various anatomical locations were further characterized for their phenotypic properties. The most frequently isolated biotype produced phosphatase, leucine amidopeptidase, arginine dihydrolase, alpha-Dand beta-D-galactosidase and fermented lactose and ribose. Additional identification by species-specific amplification of the 16S-23S rRNA intergenic spacer region was consistent with S. canis. All isolates were susceptible to penicillin G and ampicillin. The least effective antimicrobial agent was found to be tetracycline (only 33.8% of susceptible strains). Prevalence, S. canis, biochemical properties, 16S-23S rRNA intergenic spacer region Streptococcus (S.) canis belongs to Lancefield serogroup G, which forms heterogeneous group of beta-hemolytic streptococci. Presently, three taxonomic types of this serological group can be distinguished, as follows: minute colony formers from humans (S. anginosus group), and large colony formers from humans (S. dysgalactiae ssp. equisimilis) and animals (S. canis). The species S. canis was officially described by Devriese et al. (1986) and is considered to be part of healthy microbiota of skin and mucosa of animals, especially dogs and cats, and the udders of cows, although it may be responsible for opportunistic infections. In dogs, S. canis is isolated from a variety of diseases including skin infections, infections of urogenital and respiratory tracts, otitis externa, septicaemia, necrotizing fasciitis and streptococcal toxic shock syndrome (Bornand 1992; Miller et al. 1996; DeWinter et al. 1999). S. canis also causes various infections in cats, including arthritis, wound infections, septicaemia and streptococcal toxic shock syndrome, and it can cause mastitis in cows (Iglauer et al. 1991; Hassan et al. 2005). Very few human infections with S. canis have been documented; however, human infections with this species may be underestimated because many clinical isolates are reported to only as “group G Streptococcus”. Syndromes that have been associated specifically with S. canis include septicaemia, meningitis and peritonitis (Bert and Lambert-Zechovsky 1997; Takeda et al. 2001; Whatmore et al. 2001). In the present study, we isolated S. canis from specimens collected from healthy household pets and from pets with various opportunistic infections. These strains were characterized for their phenotypic properties and examined for their susceptibility to selected antimicrobial agents. Identification of all S. canis strains was confirmed by species-specific polymerase chain reaction (PCR). Materials and Methods Bacterial strains Eighty six S. canis strains included in this study had been recovered from patients at the Veterinary Clinic of Pardubice during the period from 2003 to 2005. Samples were taken from 124 dogs and 21 cats with proceeding ACTA VET. BRNO 2007, 76: 619-625; doi:10.2754/avb200776040619 Address for correspondence: Mgr. Petra Lysková Department of Biology and Biochemistry Faculty of Chemical Technology University of Pardubice Štrossova 239, 530 03 Pardubice, Czech Republic Phone: + 420 466 037 704 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm infection. In addition, 199 dogs and 50 cats without clinical signs of infection were included in the study. S. canis ATCC 43496, kindly provided by Dr. J. Motlová (National Institute of Public Health, Prague, CZ) was used as a positive control for PCR reaction. Staphylococcus aureus ATCC 25923 and S. agalactiae CCM 6187, from the Department of Microbiology culture collection, were used to perform CAMP reaction. Isolation Samples were cultured by using blood agar base supplemented with 5% sheep blood, Edward’s modified medium supplemented with 7% sheep blood (both Oxoid, UK) and Slanetz and Bartley medium (HiMedia Laboratories, India). The plates were incubated at 37 °C for 24 h in normal atmosphere. Colonies resembling streptococci and surrounded by a zone of beta-haemolysis were selected for further investigation. Subsequently, all streptococcal strains were cultured by using Todd-Hewitt broth and blood agar base (both Oxoid, UK) supplemented with 5% sheep blood. The plates and tubes were incubated at 37 °C for 24 h in normal atmosphere. After the incubation period the strains were subjected to species identification and also evaluated for antimicrobial susceptibility. Phenotypic identification The biotype of each S. canis strain was determined by the STREPTOtest 16 identification system using the Identification program TNW lite 6.0 (PLIVA-Lachema a.s., CZ). Lancefield typing was performed by latex agglutination using ITEST STREPTO GROUP (ITEST plus s.r.o., CZ). For the CAMP test, each isolate was streaked on 5% sheep blood agar plates at right angles to beta-haemolysin producing strain of Staphylococcus aureus and incubated overnight at 37 °C. S. agalactiae was included as a positive control. After incubation, plates were examined for synergistic haemolysis between the two organisms (Lammler et al. 1987). Antimicrobial susceptibility testing Following antimicrobial agents were used for disk diffusion testing: penicillin G, ampicillin, chloramphenicol, vancomycin, tetracycline, erythromycin and clindamycin (Oxoid, UK). In preparation for testing, the strains were grown overnight on 5% sheep blood agar plates at 37 °C. A bacterial suspension equal to a McFarland standard of 0.5 prepared in 0.85% saline was used to inoculate plates containing Mueller-Hinton agar (Oxoid, UK) supplemented with 5% sheep blood. The inhibitory zone diameters obtained around the antibiotic disks were measured after incubation for 24 h at 37 °C. Susceptibility, intermediate susceptibility and resistance to antibiotics were interpreted according to the recommendations of National Committee for Clinical Laboratory Standards (2001). Species-specific PCR PCR was performed with primer c-I 5 ́-TAAACCGAAAACGCTGTAAGTATTA-3 ́ and primer c-II 5 ́ACCATTAGTTAGTGGGTTCCCCC-3 ́ as described previously by Hassan et al. (2003). The species-specific primers were targeted to 16S-23S rRNA intergenic spacer region and forming the amplicon with a size of 215 bp. The PCR reaction mixture (24 μl) contained 0.18 μl primer c-I (100 pmol·μl-1) and 0.18 μl primer c-II (100 pmol·μl-1), 2 μl dNTP (2.5 mM each, TAKARA BIO INC., Japan), 2.5 μl 10 × PCR buffer (TAKARA BIO INC.), 1.5 μl MgCl2 (25 mM, TAKARA BIO INC.), 0.13 μl Taq DNA polymerase (5 U·μl -1, TAKARA BIO INC.) and 17.52 μl distilled water. Finally one colony of tested strain was added to each reaction tube. The PCR assay was performed using Robocycler Gradient 96 (Stratagene, USA) as follows: initial denaturation at 94 °C for 4 min, 30 cycles of denaturation at 94 °C for 10 s, annealing at 61 °C for 30 s and extension at 72 °C for 10 s. Finally, a 10-min extension period at 72 °C was carried out. Positive and negative controls were included in each run of amplification. The presence of a specific PCR product was controlled by electrophoresis of 10 μl of the reaction product in a 1.5% agarose gel (SERVA Electrophoresis GmbH, Germany). The molecular weight of amplification product was determined using 100-bp DNA ladder (FERMENTAS INTERNATIONAL INC., Canada).