- Research article
- Open Access
Prevalence of virulence factors in Staphylococcus intermediusisolates from dogs and pigeons
© Futagawa-Saito et al; licensee BioMed Central Ltd. 2006
Received: 08 August 2005
Accepted: 26 January 2006
Published: 26 January 2006
Staphylococcus intermedius has been isolated from healthy dogs and pigeons as well as diseased dogs. Similar to Staphylococcus aureus, S. intermedius is known to carry many virulence factors but most of these factors remain to be studied. In this study, we examined 106 S. intermedius isolates (44 dog isolates and 62 pigeon isolates) for their hemolytic activity, biofilm formation, protease activity, and clumping factor and protein A production.
Forty-three dog isolates (97.7%) and all pigeon isolates were hemolytic on sheep RBCs with a mean hemolytic titer of 336.7 and 47.32, respectively, whereas 43 dog isolates (97.7%) and 11 pigeon isolates (17.7%) exhibited a significant difference in their hemolytic activity on rabbit RBCs with a mean hemolytic titer of 11.04 and 3.76, respectively (p < 0.0005). The mean biofilm formation activity for dog isolates was 0.49, which was significantly higher than that (0.33) for pigeon isolates (p < 0.0005). Twenty-four dog isolates (54.5%) and 11 pigeon isolates (17.7%) were protease positive. Twenty-four dog isolates (54.5%) were clumping factor- and protein A- positive.
S. intermedius strains carrying the virulence factors examined in this study were more prevalent in dogs than pigeons.
Staphylococcus intermedius is found in a wide range of animal species including pigeons and dogs. It is recognized as normal flora of dogs and pigeons  and a principal causative agent of skin infections, mainly pyoderma, in dogs. It has been reported that the isolation frequency of S. intermedius from pigeons was two times higher than that from dogs  and that there is a genotypic diversity between S. intermedius isolates from dogs and pigeons [2–4].
Similar to Staphylococcus aureus, S. intermedius produces many virulence factors such as protease, coagulase, clumping factor, enterotoxins, exfoliative toxin, leukotoxin, and alpha and beta hemolysins [1, 2, 5–8]. It has been reported that the enterotoxin- and leukotoxin-producing S. intermedius strains are more prevalent in dogs than pigeons [1, 2].
Biofilm formation by S. aureus strains isolated from bovine mastitis has been reported . Biofilm formation is considered to be one of the virulence factors in Staphylococci, which helps Staphylococci adhere to its target tissues, mainly implants and other foreign body materials, through adhesive mechanisms [10, 11]. Microcolonies encased in extracellular polysaccharide of biofilm are protected from antimicrobial agents . The biofilm formation in S. intermedius has not yet been investigated.
In this study, we have examined S. intermedius isolates from dogs and pigeons with regards to their hemolytic activity, biofilm formation, protease activity, and clumping factor and protein A production.
Clumping factor and protein A production
Twenty-four dog isolates (24/44, 54.5%) and none of the pigeon isolates were positive for clumping factor and protein A.
Protease production was significantly higher in dog isolates (24/44, 54.5%) than pigeon isolates (11/62, 17.7%) (p < 0.0005, Fisher's exact test).
Hemolytic activity and biofilm formation activity of Staphylococcus intermedius isolates from dogs and pigeons
Animal (no. of isolates)
Hemolytic titer on sheep RBC (1/dilution)
Hemolytic titer on rabbit RBC (1/dilution)
Biofilm formation activity (absorbance value at 490 nm)
The range of biofilm formation activity for all S. intermedius isolates was 0.02 to 1.00. The mean of biofilm formation activity for dog isolates was 0.49, which was significantly higher than that (0.33) for pigeon isolates (Table 1, p < 0.0005, t test).
Association between hemolytic activity and biofilm formation activity
Association between hemolytic activity on rabbit RBC and biofilm formation activity in Staphylococcus intermedius isolates from dogs and pigeons
Hemolytic activity on Rabbit RBC
Animal (no. of isolates)
Biofilm formation activityc (absorbance value at 490 nm)
S. intermedius isolates from dogs and pigeons have been reported to be genotypically distinguishable [2–4]. Here, we observed a difference in their virulence traits such as hemolytic activity, biofilm formation, protease activity, and clumping factor and protein A production.
Hemolytic activity of S. intermedius isolates from healthy and infected dogs and pigeons has been described previously [5, 7, 13]. The number of hemolysis-positive isolates in this study (99.1% on sheep RBC and 50.9% on rabbit RBC) is higher than that (88.2% on sheep RBC and 1.5% on rabbit RBC) in a previous study in which blood agar plate was used for assay . This observed difference could be due to the fact that a sensitive microplate technique was used in our study to determine the hemolytic activity. On the other hand, the mean hemolytic titers on rabbit and sheep RBC for dog isolates were significantly higher than that for pigeon isolates. Since dog isolates also exhibited a very high leukotoxic activity in a previous study , cytotoxin-producing S. intermedius strains seem to be prevalent among dogs.
Biofilm-forming S. epidermidis and S. aureus isolates have been recovered from hospitalized patients and non-hospitalized people , and instruments of dialysis , and bovine mastitis , and food and food processing environments , respectively. We tested a large number of S. intermedius isolates for their biofilm formability. Biofilm formation was significantly higher in the isolates from dogs than pigeons. Bacteria in biofilms are generally resistant to environmental stress , antibiotics , and phagocytosis by macrophage . Therefore biofilm-forming S. intermedius isolates from dogs may have the potential to cause opportunistic and biomaterial-related infections.
Alpha-hemolysin, which is hemolytic on rabbit RBC , has been shown to be required for cell-to-cell interactions during biofilm formation in S. aureus . Likewise, the association between biofilm formation and alpha-hemolysin production in S. intermedius was also observed in this study as the number of hemolysis-positive isolates was significantly higher than that of the hemolysis-negative isolates among the biofilm-forming S. intermedius isolates.
The accessory gene regulator (agr) of a two-component regulatory system in S. aureus is implicated in biofilm formation and alpha-hemolysin production . In S. intermedius, an agr-like locus has also been identified by PCR , but the alpha hemolysin (hla) gene has not been reported. Therefore, it is of interest to further examine the production and regulation of virulence factors in S. intermedius strains.
S. aureus clinical isolates produce a variety of extracellular proteases . Several in-vitro studies have suggested that extracellular protease is an important virulence factor in S. aureus [24, 25]. Clumping factor promotes binding of fibrinogen and fibrin to the bacterial cell surface , and is shown to act as a virulence factor in experimental septic arthritis in S. aureus . S. aureus isolates from patients with Kawasaki disease produce high levels of protein A , which is reportedly associated with inflammation of lungs . More than half of S. intermedius isolates from dogs in this study produced protease and they were clumping factor and protein A positive. It is interesting to note that the clumping factor- and protein A- positive S. intermedius were isolated only from dogs. It is not known if protease, clumping factor, and protein A are associated with pathogenesis of S. intermedius infections in animals, but the carriage of these virulence factors indicate the pathogenic potential of the isolates. Besides, the production of many virulence traits tested in this study are susceptible or dependent on in-vitro conditions and it should cautious in interpretation of the virulence properties of S. intermedius isolates.
This study demonstrated that S. intermedius strains carrying tested virulence factors are more prevalent in dogs than pigeons.
S. intermedius isolates (n = 106), including 44 isolates from dogs and 62 isolates from pigeons, were used in this study. Isolation and identification of S. intermedius isolates were done as described previously . S. aureus RN4220  and S. hyicus JCM2423T  were used respectively as a positive control and a negative control in the hemolytic activity assay. S. epidermidis ATCC35984 was used as a positive control in the quantitative assay of biofilm formation.
Clumping factor and protein A assay
Simultaneous detection of clumping factor and protein A was performed as described previously by Essers et al. . S. intermedius isolates were cultured on brain heart infusion agar plate (Becton, Dickinson and Company, MD, USA) for 18 h at 37°C. A mixture of one drop each of culture (approximately 108 cfu) and saline was mixed with PS latex (Eiken, Tokyo Japan). Agglutination that occurred within one minute while stirring was considered a positive reaction.
Protease activity was determined on casein agar plates following the procedure described by Bjorklind et al. . The production of protease was recognized as a clear zone or a broad zone of precipitation around the bacterial streak [32, 33].
Assay for hemolytic activity
Hemolytic assay was performed by the microplate method  using sheep and rabbit erythrocytes (RBCs). A culture supernatant of overnight-grown bacteria at 37°C in brain heart infusion broth (Becton, Dickinson and Company, MD, USA) was used. Two-fold dilutions of the culture supernatant in PBS (pH 7.0) containing 0.1% bovine serum albumin (BSA) (50 μl each) were mixed with 50 μl of 1% RBC in PBS in a 96-well microtiter plate. The microtiter plate was incubated at 37°C for 1 h with gentle shaking and, for sheep RBC, further incubated at 4°C for 1 h without shaking. The microtiter plate was centrifuged at 600 × g for 5 min. The hemolytic activity titer was defined as the inverse of the last dilution that caused complete hemolysis. The isolates with hemolytic titer ≥2 were considered positive for hemolytic activity.
Quantitative assay for biofilm formation
The assay was performed as previously described [9, 34] with some modifications. Bacteria were cultivated overnight in trypticase soy broth, TSB, (Becton, Dickinson and Company, MD, USA) containing 0.25% glucose. Each culture was diluted 1:200 in the same broth. The cell suspension (200 μl) was inoculated into each well of sterile 96-well polystyrene tissue culture plates (Becton Dickinson Labware, NJ, USA) and incubated at 37°C for 16 h. The wells were washed twice with 200 μl of PBS (pH 7.4) and stained with 100 μl of 0.1% safranin-O solution per well for 30 s. After removal of the staining solution, the wells were washed once again with PBS. Then, 100 μl of a 97% ethanol-3% ether solution was added to each well and mixed. The absorbance of the adherent biofilm was measured at 490 nm in a microplate reader (Model 680, BioRad, CA, USA) and the absorbance value was expressed as the biofilm formation activity. The results were reported after subtracting the reading for a blank (TSB plus 0.25% glucose, without bacterial cells) from the experimental readings. Each assay was performed in triplicate.
We would like to thank Drs. M. Fukuyama, K. Furuhata, and K. Oonaka of the College of Environmental Health, Azabu University, Kanagawa, Japan, for their kind assistance in collecting the samples.
- Futagawa-Saito K, Suzuki M, Ohsawa M, Ohshima S, Sakurai N, Ba-Thein W, Fukuyasu T: Identification and prevalence of an enterotoxin-related gene, se-int, in Staphylococcus intermedius isolates from dogs and pigeons. J Appl Microbiol. 2004, 96: 1361-1366. 10.1111/j.1365-2672.2004.02264.x.View ArticlePubMedGoogle Scholar
- Futagawa-Saito K, Sugiyama T, Karube S, Sakurai N, Ba-Thein W, Fukuyasu T: Prevalence and characterization of leukotoxin-producing Staphylococcus intermedius isolates from dogs and pigeons. J Clin Microbiol. 2004, 42: 5324-5326. 10.1128/JCM.42.11.5324-5326.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Bes M, Saidi Slim L, Becharnia F, Meugnier H, Vandenesch F, Etienne J, Freney J: Population diversity of Staphylococcus intermedius isolates from various host species: typing by 16S-23S intergenic ribosomal DNA spacer polymorphism analysis. J Clin Microbiol. 2002, 40: 2275-2277. 10.1128/JCM.40.6.2275-2277.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Wakita Y, Shimizu A, Hajek V, Kawano J, Yamashita K: Characterization of Staphylococcus intermedius from pigeons, dogs, foxes, mink, and horses by pulsed-field gel electrophoresis. J Vet Med Sci. 2002, 64: 237-243. 10.1292/jvms.64.237.View ArticlePubMedGoogle Scholar
- Hajek V: Staphylococcus intermedius, a new species isolated from animals. Int J Syst Bacteriol. 1976, 26: 401-408.View ArticleGoogle Scholar
- Raus J, Love DN: Characterization of coagulase-positive Staphylococcus intermedius and Staphylococcus aureus isolated from veterinary clinical specimens. J Clin Microbiol. 1983, 18: 789-792.PubMed CentralPubMedGoogle Scholar
- Shimizu A, Kawano J, Kimura S: Biotyping of coagulase-positive Staphylococcus aureus and Staphylococcus intermedius strains isolated from various animals in Japan. Nippon Juigaku Zasshi. 1986, 48: 1227-1235.View ArticlePubMedGoogle Scholar
- Terauchi R, Sato H, Hasegawa T, Yamaguchi T, Aizawa C, Maehara N: Isolation of exfoliative toxin from Staphylococcus intermedius and its local toxicity in dogs. Vet Microbiol. 2003, 94: 19-29. 10.1016/S0378-1135(03)00048-8.View ArticlePubMedGoogle Scholar
- Vasudevan P, Nair MK, Annamalai T, Venkitanarayanan KS: Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation. Vet Microbiol. 2003, 92: 179-185. 10.1016/S0378-1135(02)00360-7.View ArticlePubMedGoogle Scholar
- Rupp ME, Ulphani JS, Fey PD, Bartscht K, Mack D, Lee J: Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model. Infect Immun. 1999, 67: 2627-2632.PubMed CentralPubMedGoogle Scholar
- Gotz F: Staphylococcus and biofilms. Mol Microbiol. 2002, 43: 1367-1378. 10.1046/j.1365-2958.2002.02827.x.View ArticlePubMedGoogle Scholar
- Olson ME, Ceri H, Morck DW, Buret AG, Read RR: Biofilm bacteria: formation and comparative susceptibility to antibiotics. Can J Vet Res. 2002, 66: 86-92.PubMed CentralPubMedGoogle Scholar
- Talan DA, Staatz D, Staatz A, Goldstein EJ, Singer K, Overturf GD: Staphylococcus intermedius in canine gingiva and canine-inflicted human wound infections: laboratory characterization of a newly recognized zoonotic pathogen. J Clin Microbiol. 1989, 27: 78-81.PubMed CentralPubMedGoogle Scholar
- Krepsky N, Rocha Ferreira RB, Ferreira Nunes AP, Casado Lins UG, Costa e Silva Filho F, de Mattos-Guaraldi AL, Netto-dosSantos KR: Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates. Curr Microbiol. 2003, 46: 280-286. 10.1007/s00284-002-3868-5.View ArticlePubMedGoogle Scholar
- Chaieb K, Mahdouani K, Bakhrouf A: Detection of icaA and icaD loci by polymerase chain reaction and biofilm formation by Staphylococcus epidermidis isolated from dialysate and needles in a dialysis unit. J Hosp Infect. 2005, 61: 225-230. 10.1016/j.jhin.2005.05.014.View ArticlePubMedGoogle Scholar
- Moretro T, Hermansen L, Holck AL, Sidhu MS, Rudi K, Langsrud S: Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments. Appl Environ Microbiol. 2003, 69: 5648-5655. 10.1128/AEM.69.9.5648-5655.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science. 1999, 284: 1318-1322. 10.1126/science.284.5418.1318.View ArticlePubMedGoogle Scholar
- Shiau AL, Wu CL: The inhibitory effect of Staphylococcus epidermidis slime on the phagocytosis of murine peritoneal macrophages is interferon-independent. Microbiol Immunol. 1998, 42: 33-40.View ArticlePubMedGoogle Scholar
- Bhakdi S, Muhly M, Fussle R: Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal alpha-toxin. Infect Immun. 1984, 46: 318-323.PubMed CentralPubMedGoogle Scholar
- Caiazza NC, O'Toole GA: Alpha-toxin is required for biofilm formation by Staphylococcus aureus. J Bacteriol. 2003, 185: 3214-3217. 10.1128/JB.185.10.3214-3217.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Vuong C, Saenz HL, Gotz F, Otto M: Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J Infect Dis. 2000, 182: 1688-1693. 10.1086/317606.View ArticlePubMedGoogle Scholar
- Dufour P, Jarraud S, Vandenesch F, Greenland T, Novick RP, Bes M, Etienne J, Lina G: High genetic variability of the agr locus in Staphylococcus species. J Bacteriol. 2002, 184: 1180-1186. 10.1128/jb.184.4.1180-1186.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Karlsson A, Arvidson S: Variation in extracellular protease production among clinical isolates of Staphylococcus aureus due to different levels of expression of the protease repressor sarA. Infect Immun. 2002, 70: 4239-4246. 10.1128/IAI.70.8.4239-4246.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Potempa J, Watorek W, Travis J: The inactivation of human plasma alpha 1-proteinase inhibitor by proteinases from Staphylococcus aureus. J Biol Chem. 1986, 261: 14330-14334.PubMedGoogle Scholar
- Prokesova L, Potuznikova B, Potempa J, Zikan J, Radl J, Hachova L, Baran K, Porwit-Bobr Z, John C: Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus. Immunol Lett. 1992, 31: 259-265. 10.1016/0165-2478(92)90124-7.View ArticlePubMedGoogle Scholar
- McDevitt D, Francois P, Vaudaux P, Foster TJ: Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol Microbiol. 1994, 11: 237-248.View ArticlePubMedGoogle Scholar
- Palmqvist N, Josefsson E, Tarkowski A: Clumping factor A-mediated virulence during Staphylococcus aureus infection is retained despite fibrinogen depletion. Microbes Infect. 2004, 6: 196-201. 10.1016/j.micinf.2003.10.014.View ArticlePubMedGoogle Scholar
- Wann ER, Fehringer AP, Ezepchuk YV, Schlievert PM, Bina P, Reiser RF, Hook MM, Leung DY: Staphylococcus aureus isolates from patients with Kawasaki disease express high levels of protein A. Infect Immun. 1999, 67: 4737-4743.PubMed CentralPubMedGoogle Scholar
- Normark BH, Normark S, Norrby-Teglund A: Staphylococcal protein A inflames the lungs. Nat Med. 2004, 10: 780-781. 10.1038/nm0804-780.View ArticlePubMedGoogle Scholar
- Kreiswirth BN, Lofdahl S, Betley MJ, O'Reilly M, Schlievert PM, Bergdoll MS, Novick RP: The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature. 1983, 305: 709-712. 10.1038/305709a0.View ArticlePubMedGoogle Scholar
- Essers L, Radebold K: Rapid and reliable identification of Staphylococcus aureus by a latex agglutination test. J Clin Microbiol. 1980, 12: 641-643.PubMed CentralPubMedGoogle Scholar
- Bjorklind A, Arvidson S: Occurrence of an extracellular serineproteinase among Staphylococcus aureus strains. Acta Pathol Microbiol Scand [B]. 1977, 85: 277-280.Google Scholar
- Arvidson S: Hydrolysis of casein by three extracellular proteolytic enzymes from Staphylococcus aureus, strain V8. Acta Pathol Microbiol Scand [B]. 1973, 81: 538-544.Google Scholar
- Heilmann C, Gerke C, Perdreau-Remington F, Gotz F: Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect Immun. 1996, 64: 277-282.PubMed CentralPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.