Prevalence of virulence factors in Staphylococcus intermedius isolates from dogs and pigeons

Background 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. Results 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. Conclusion S. intermedius strains carrying the virulence factors examined in this study were more prevalent in dogs than pigeons.


Background
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 [1] 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 [1] and that there is a genotypic diversity between S. intermedius isolates from dogs and pigeons [2][3][4].
Biofilm formation by S. aureus strains isolated from bovine mastitis has been reported [9]. 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 [12]. 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.

Biofilm formation
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

Association between hemolytic activity and biofilm formation activity
The association between hemolytic activity on rabbit RBC and biofilm formation activity is shown in Table 2.
Regardless of the origin, the isolates with positive hemolytic activity had the mean biofilm formation activity of 0.49, whereas the isolates with negative hemolytic activity had significantly less biofilm formation activity with the mean activity of 0.29 (p < 0.0005). In addition, there was a significant difference in biofilm formation activity between hemolysis-positive and -negative pigeon isolates (0.52 vs. 0.28, p < 0.0005). However, there was no significant difference in biofilm formation activity between dog and pigeon isolates that had positive hemolytic activity on rabbit RBC (p = 0.49).

Discussion
S. intermedius isolates from dogs and pigeons have been reported to be genotypically distinguishable [2][3][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 [7]. 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 [2], 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 [14], and instruments of dialysis [15], and bovine mastitis [9], and food and food processing environments [16], 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 [17], antibiotics [12], and phagocytosis by macrophage [18]. Therefore biofilmforming S. intermedius isolates from dogs may have the potential to cause opportunistic and biomaterial-related infections.
Alpha-hemolysin, which is hemolytic on rabbit RBC [19], has been shown to be required for cell-to-cell interactions during biofilm formation in S. aureus [20]. 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 [21]. In S. intermedius, an agr-like locus has also been identified by PCR [22], 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 [23]. 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 [26], and is shown to act as a virulence factor in experimental septic arthritis in S. aureus [27]. S. aureus isolates from patients with Kawasaki disease produce high levels of protein A [28], which is reportedly associated with inflammation of lungs [29]. 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.

Conclusion
This study demonstrated that S. intermedius strains carrying tested virulence factors are more prevalent in dogs than pigeons.

Bacterial strains
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 [1]. S. aureus RN4220 [30] and S. hyicus JCM2423 T [5] 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. [31]. 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 10 8 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
Protease activity was determined on casein agar plates following the procedure described by Bjorklind et al. [32]. 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 [19] 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 over-