Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis

The adherence of Staphylococcus intermedius to canine keratinocytes in normal dogs was compared to that in dogs suffering from atopic dermatitis, primary seborrhoea and bacterial pyoderma. Statistically significant greater adherence by S. intermedius to keratinocytes occurred in atopic dogs and dogs suffering from pyoderma when compared with the normal group (P < 0.01) and dogs suffering from primary seborrhoea (P < 0.05). This is similar to the results of a study of human atopic dermatitis by Cole and Silverberg (1986) who demonstrated increased adherence by S. aureus to keratinocytes from atopic dermatitis patients when compared with adherence to keratinocytes in a variety of non-atopic dermatoses. This increased adherence by pathogenic staphylococci to keratinocytes may in part explain the high incidence of staphylococcal pyoderma seen in both canine and human patients suffering from atopic dermatitis.     

Adherence by Staphylococcus intermedius to canine corneocytes: a preliminary study comparing noninflamed and inflamed atopic canine skin

The adherence by three strains of Staphylococcus intermedius to corneocytes collected from healthy dogs was compared to the adherence to corneocytes collected from the inflamed (erythematous) and noninflamed (normal appearing) skin of dogs suffering from atopic dermatitis. All three strains of S. intermedius adhered in greater numbers to corneocytes from both inflamed and noninflamed atopic skin than to corneocytes from healthy dogs. Adherence was greatest to corneocytes from inflamed atopic skin but one strain showed no statistical difference for adherence to inflamed and noninflamed atopic skin. These findings suggest that S. intermedius adheres extensively to both inflamed and noninflamed canine atopic skin. This may be important in the colonization of atopic skin by this microorganism. Strain variation in the ability of S. intermedius to adhere to canine atopic corneocytes is probable.     

Adherence of Staphylococcus intermedius to canine corneocytes in vitro

This study investigated the in vitro adherence of Staphylococcus intermedius to canine corneocytes, collected from a healthy dog using double-sided adhesive tape. Adherence was shown to depend on duration (P < 0.001) and temperature of incubation (P < 0.001) and the concentration of bacteria (P < 0.001). Isolates of S. intermedius from lesions of pyoderma were not generally more adherent to healthy canine skin than were isolates from healthy dogs. Significant differences in adherence were demonstrated between individual isolates within both groups (P < 0.001). The study suggests that among S. intermedius there is no correlation between virulence and adherence to canine corneocytes in vitro. The finding may be important for the potential use of avirulent variants of S. intermedius as antagonistic strains against canine pyoderma. However, more studies are needed to compare the adherence of the isolates to skin cells obtained from dogs with diseases predisposed to pyoderma.     

Reduced in vitro adherence of Staphylococcus species to feline corneocytes compared to canine and human corneocytes

It is apparent that in-contact humans and animals exchange commensal staphylococci. Previous in vitro studies, however, indicate that staphylococci preferentially adhere to corneocytes from host species. This study compared adherence of meticillin-sensitive and -resistant Staphylococcus aureus (MSSA/MRSA), S. intermedius, S. felis and S. hominis to feline, canine and human corneocytes acquired from 10 healthy subjects using adhesive tape discs. Adherent bacteria were counted using an image processing and analysis programme. Mean adherence of MSSA (P = 0.0009), MRSA (P = 0.0162) and S. intermedius (P = 0.0117), but not S. felis or S. hominis, to feline corneocytes was significantly lower than that to canine and human corneocytes. All the isolates had similar adherence to both human and canine corneocytes. S. felis was the most adherent species to feline corneocytes followed by S. intermedius, and then MSSA, MRSA and S. hominis. For dogs and humans, S. intermedius and S. felis were the most adherent, followed by MRSA and MSSA, and then S. hominis. These results do not reveal any preferential adherence of staphylococci to canine or human corneocytes. Poor adherence to feline corneocytes could suggest that cats are relatively resistant to pyoderma and cross-species transmission of staphylococci.     

Adherence of Staphylococcus intermedius to corneocytes of healthy and atopic dogs: effect of pyoderma, pruritus score, treatment and gender

Staphylococcal pyoderma occurs commonly in atopic dogs. Some studies have suggested that adherence of staphylococci to corneocytes of atopic dogs and humans is higher than to corneocytes of healthy individuals. This hypothesis and possible differences resulting from the presence or absence of pyoderma, the severity of pruritus or the effect of treatment or gender, were studied. Adherent bacteria (Staphylococcus intermedius) were quantified by computerized image analysis on corneocytes collected from healthy or atopic dogs using double-sided adhesive tape. The adherence of S. intermedius to the corneocytes of atopic dogs was significantly greater than to those of healthy dogs (P=0.005). Furthermore, adherence was significantly greater in dogs with high levels of pruritus compared to those with low scores. No significant differences were found between atopic dogs with no history of pyoderma, atopic dogs with a history of pyoderma and atopic dogs with pyoderma at the time of sampling (P=0.068), suggesting that factors other than adherence are necessary for clinical pyoderma to develop. Treatment did not generally influence the adherence of S. intermedius to corneocytes of atopic dogs and there was no gender difference in adherence in either healthy or atopic dogs.     

Enhanced adherence of methicillin-resistant Staphylococcus pseudintermedius sequence type 71 to canine and human corneocytes

The recent worldwide spread of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs is a reason for concern due to the typical multidrug resistance patterns displayed by some MRSP lineages such as sequence type (ST) 71. The objective of this study was to compare the in vitro adherence properties between MRSP and methicillin-susceptible (MSSP) strains. Four MRSP, including a human and a canine strain belonging to ST71 and two canine non-ST71 strains, and three genetically unrelated MSSP were tested on corneocytes collected from five dogs and six humans. All strains were fully characterized with respect to genetic background and cell wall-anchored protein (CWAP) gene content. Seventy-seven strain-corneocyte combinations were tested using both exponential- and stationary-phase cultures. Negative binomial regression analysis of counts of bacterial cells adhering to corneocytes revealed that adherence was significantly influenced by host and strain genotype regardless of bacterial growth phase. The two MRSP ST71 strains showed greater adherence than MRSP non-ST71 (p < 0.0001) and MSSP (p < 0.0001). This phenotypic trait was not associated to any specific CWAP gene. In general, S. pseudintermedius adherence to canine corneocytes was significantly higher compared to human corneocytes (p < 0.0001), but the MRSP ST71 strain of human origin adhered equally well to canine and human corneocytes, suggesting that MRSP ST71 may be able to adapt to human skin. The genetic basis of the enhanced in vitro adherence of ST71 needs to be elucidated as this phenotypic trait may be associated to the epidemiological success and zoonotic potential of this epidemic MRSP clone.     

Monosaccharide inhibition of adherence by Pseudomonas aeruginosa to canine corneocytes

The effect of d -galactose, d -mannose, l -rhamnose and dextrose on the adhesion to canine corneocytes by three strains of Pseudomonas aeruginosa was studied in six healthy dogs. Canine corneocytes were collected from the inner aspect of the pinna using adhesive discs (D-Squame®). Half millimetre of bacterial suspension in phosphate-buffered saline (PBS) with or without the addition of a monosaccharide was placed over the corneocyte layer and incubated in moist chambers. Image analysis was used to quantify bacterial adherence to corneocytes. The three strains of Pseudomonas adhered well to canine corneocytes. All monosaccharides tested inhibited the adherence of Pseudomonas to canine corneocytes. The mean reduction in adhesion for individual sugars at a concentration of 0.1% was 40.2% (dextrose), 30.8% ( l -rhamnose), 25.6% ( d -galactose) and 19.4% ( d -mannose). When d -galactose, d -mannose and l -rhamnose were used in combination at 0.1% concentration, the mean reduction in adherence was 52.9%. The monosaccharides studied may have a potential role in the management of Pseudomonas infections in dogs.     

In vitro adherence of Staphylococcus pseudintermedius to canine corneocytes is influenced by colonization status of corneocyte donors

The current knowledge of in vitro adherence of Staphylococcus pseudintermedius to canine corneocytes is limited to comparative analyses between strains, staphylococcal species or corneocytes collected from different breeds, body sites and hosts. However, the role played by colonization status of corneocyte donors remains unknown. The aim of this study was to evaluate the adherence properties of commensal S. pseudintermedius strains to corneocytes collected from dogs with different colonization status. For this purpose, corneocytes were collected from five dogs that were classified as persistently colonized (D1 and D2), intermittently colonized (D3 and D4) or non-colonized (D5) on the basis of the results of a previous longitudinal study. Adherence to corneocytes originating from each of the five dogs was assessed by an in vitro adhesion assay using four genetically unrelated strains isolated from the colonized dogs (S1 to S4). Irrespective of their host of origin, all strains adhered significantly better to corneocytes from D1 and D2 than to corneocytes from D3, D4 and D5 (P < 0.0001). The mean count of cells adhering to corneocytes from persistently colonized dogs was on average three times higher than the mean count using corneocytes from the other dogs. A significant difference between strains was only observed for one strain-corneocyte combination (S2-D4), indicating that S. pseudintermedius adherence to corneocytes is driven by host factors and only marginally influenced by strain factors. This finding has important implications for understanding and preventing S. pseudintermedius skin colonization and infection.     

Antimicrobial activity of enrofloxacin against Staphylococcus intermedius strains isolated from canine pyodermas

This study examined and compared the minimal inhibition concentrations (MICs) of enrofloxacin against 393 Staphylococcus intermedius strains isolated in France from canine pyodermas during three different years, 1995 (174 isolates), 1997 (101 isolates) and 1999 (118 isolates). The MICs of enrofloxacin against these strains ranged from 0.063 to 64 mg L^?1, with MIC₅₀ and MIC₉₀ equal to 0.125 and 0.25 mg L^?1, respectively. Two resistant strains were found, but only among isolates collected in 1999. The data show that resistance to enrofloxacin among S. intermedius strains is still rare in dogs, but the selection in vitro of variants in which the MICs were increased 4–16‐fold after 10 serial passages in subinhibitory concentrations of enrofloxacin suggests that inappropriate use might favour the development of resistant strains in vivo.     

Molecular characterization of Staphylococcus intermedius carriage by healthy dogs and comparison of antimicrobial susceptibility patterns to isolates from dogs with pyoderma

We conducted an epidemiological study of Staphylococcus intermedius using arbitrarily primed PCR (AP-PCR) and antibiograms. One hundred and twenty-five S. intermedius isolates were recovered from the oral cavity and/or cranial hair coat of healthy dogs enrolled in a pet therapy program. Commensal S. intermedius was cultured from 32% of the oral cavity cultures and 13% of the cranial hair coat cultures. We characterized the colonization of the dogs as transient, intermittent, or persistent. For dogs characterized as persistently colonized, 73% of the isolates came from the oral cavity. These isolates were also genotyped by AP-PCR. A single major AP-PCR type was observed in 91% of the dogs (n=22); minor variations were frequently observed in these major types. Antibiograms of these commensal isolates were compared to antibiograms from 97 historical clinical isolates (1988-1992) obtained from cases of canine pyoderma. Resistance was most often observed to penicillin (64% and 55%) and tetracycline (38% and 38%) among the commensal and clinical isolates, respectively. The commensal isolates were significantly less resistant to erythromycin, clarithromycin, clindamycin, and trimethoprim/sulfamethoxazole. Our data suggests that differences in both genotype and antimicrobial susceptibility phenotypes exist among S. intermedius strains isolated from different anatomic sites from the same dog and supports the opportunistic nature of S. intermedius in canine infections.     

Protein A in Staphylococcus intermedius isolates from dogs and cats

The presence and quantity of extracellular and cell-bound protein A of Staphylococcus intermedius isolates from dogs and cats were determined, using an enzyme-linked immunoglobulin-binding assay. Horseradish peroxidase-conjugated rabbit anti-bovine immunoglobulin G purified by affinity chromatography was reacted with whole cell and supernatant fractions of S intermedius (n = 139), a protein A-producing strain of S aureus, and a protein A-deficient strain of S epidermidis. Extracellular protein A was found in 118 (84.9%) of 139 isolates of S intermedius. Most (69/118; 58.5%) of these isolates produced greater than 0.2 micrograms of extracellular protein A/ml. Cell-bound protein A was found in 6 (4.3%) of 139 isolates. Only 1 of these isolates contained cell-bound protein A exclusively. The other 5 isolates produced significantly greater amounts of extracellular protein A than cell-bound protein A. Additionally, greater than 96% of extracellular protein A could be removed from supernatants by adsorption with agarose gel containing immunoglobulin G.     

Identification of protein A from Staphylococcus intermedius isolated from canine skin

Protein A was identified in cell wall-bound and secreted forms from Staphylococcus intermedius isolated from canine skin. A direct binding radioimmunoassay for the detection of bacterial surface Fc receptors identified 48 of 50 S intermedius isolates that contained cell wall-bound protein A. Using a competitive binding radioimmunoassay for the detection of Fc-reactive proteins in bacterial culture supernatants, we identified 9 of 50 clinical isolates of S intermedius that secreted measurable quantities of an Fc receptor into the culture medium. Concentrated culture supernatants from these isolates were analyzed by western blotting techniques and probed with either a radiolabeled human IgG Fc-specific probe or a radiolabeled affinity-purified chicken antibody against protein A. The studies reported here confirmed that Fc receptors are secreted by S intermedius isolates from dogs and are antigenically and functionally similar or are identical to staphylococcal protein A. Analysis of Fc receptor secretion by S intermedius strains, isolated from dogs with a variety of dermatologic conditions, suggested a trend between severity of skin disease and the extent of Fc receptor secretion.     

Determination of antigenic proteins of housedust mites in 90 dogs suffering from atopic dermatitis

Housedust mites, Dermatophagoides pteronyssinus (D. pteronyssinus) and Dermatophagoides farinae (D. farinae), are the important causative agents of allergic diseases in human and animals. By using 165 dogs suffering from atopic dermatitis (AD), serum levels of immunogloblin E (IgE) antibody against 25 kinds of allergen including housedust mites were determined. Housedust mites were the most frequent allergen against which 90 of the 165 allergic dogs (54.5%) by IMMUNODOT assay. With the further analysis of immunoblotting assay in the 90 dogs sensitized with housedust mites, antigenic proteins of housedust mites recognized by IgE antibodies were with the apparent molecular masses of 15, 76, 90, 98, and 170-kD. Among them, the 15-kD protein that might be identical to Group 2 antigens (Der f2, Der p2) was prominently observed (52/90). This study indicates that about a half of dogs with AD were sensitized to housedust mites, suggesting that Group 2 antigens of housedust mites may be a major allergen in canine AD.     

Pilot investigation of a model for canine atopic dermatitis: environmental house dust mite challenge of high-IgE-producing beagles, mite hypersensitive dogs with atopic dermatitis and normal dogs

Although canine atopic dermatitis (cAD) is common, few models are available. The aim of this study was to evaluate high-IgE beagles epicutaneously sensitized to house dust mite (HDM) as a possible model for cAD. Six high-IgE beagles were environmentally challenged with HDM using various doses and protocols. Similar challenge protocols were used in positive and negative control dogs: three dogs with naturally occurring cAD and positive intradermal skin test (IDT) to HDM and three normal dogs without history of skin disease and negative IDT to HDM. All high-IgE beagles and all atopic dogs developed severe cutaneous lesions and pruritus after challenge. Lesions were erythematous papules and macules in contact areas such as face, ears, ventral abdomen, groin, axillae and feet. They were first visible after 6 h and increased in severity over time. No normal dog developed pruritus or lesions. Biopsies of representative lesions in the high-IgE beagles were taken for histopathology and immunohistochemistry. There was superficial perivascular dermatitis with mononuclear infiltrates and spongiosis. Lymphocytes and eosinophils accumulated in small epidermal micro-abscesses with hyperplasia of epidermal IgE-bearing dendritic cells. These findings suggest that this colony of high-IgE beagles develops a dermatitis that clinically, histopathologically and immunologically resembles the naturally occurring canine disease. It is also concluded that this modality of challenge is not irritating to normal dogs but induces flare-ups in hypersensitive atopic dogs.     

Frequency of superantigen-producing Staphylococcus intermedius isolates from canine pyoderma and proliferation-inducing potential of superantigens in dogs

This preliminary study investigated the potential role of staphylococcal superantigens in the pathogenesis of canine pyoderma. The staphylococcal enterotoxins A (SEA), SEB, SEC and SED, and the toxic shock syndrome toxin-1 (TSST-1) were assayed in isolates from skins of dogs with pyoderma. Culture supernatants from 25 of 96 isolates were positive for multiple superantigens, with SEA and SEC being the most frequently detected. In in vitro stimulation of canine peripheral blood mononuclear cells and quantitative flow cytometry revealed that low concentrations of SEA and SEB were potent stimulators of blastogenesis of T cells.     

Use of multilocus enzyme electrophoresis to distinguish clinically important strains of Staphylococcus intermedius from the skin of dogs

AIMS: To use multilocus enzyme electrophoresis to determine the genetic structure of Staphylococcus intermedius from normal skin of dogs and those isolated from a variety of disease conditions and to distinguish clinically important strains in dogs. METHODOLOGY: The diversity amongst 129 isolates of S intermedius from the skin and mucosa of 32 healthy dogs and 120 isolates from diseased sites in 120 individual dogs was examined using multilocus enzyme electrophoresis. Associations among ETs were examined to determine the diversity of isolates. RESULTS: Twenty two ETs were distinguished comprising 21 containing isolates from diseased sites and 11 containing isolates from normal dogs. The majority of isolates (171 of 249; 69% were located in two ETs (ET1 and ET 4), that were not distinguishable phenotypically. ET 1 contained 94 isolates (54 isolates from healthy dogs and 40 isolates from diseased sites) and ET 4 contained 77 isolates (46 from healthy dogs and 31 isolates from diseased sites). Further, 77.5% of isolates from healthy dogs were present in ET 1 and ET 4 and 59% of isolates from diseased dogs belonged to the same two ETs. There was only a small difference in genetic diversity among isolates taken from healthy dogs (11 ETs; H = 0.182) and those isolates taken from clinical specimens from diseased dogs (21 ETs; H = 0.218). Of the 21 ETs from diseased sites, ET 16 contained all six isolates from Staphylococcal Scalded Skin Syndrome in racing Greyhounds. CONCLUSIONS: The small difference in genetic diversity between isolates from the skin and mucosa of healthy dogs and isolates from various diseases, as well as the presence of the majority of isolates in two ETs, is consistent with the role of S intermedius as an opportunistic pathogen. The confinement of all Staphylococcal Scalded Skin Syndrome isolates within one ET is confirmation of this entity as a distinct disease of dogs.     

Characteristics of Staphylococcus intermedius isolates from diseased and healthy dogs

Staphylococcus intermedius isolates from diseased and healthy dogs were examined for production of extracellular enzymes and toxins, and phage patterns. There were no significant differences between the two groups of isolates in the production rates of DNase, protease, lipase, gelatinase, hyaluronidase, hemolysins, protein A, and TSST-1, or in phage patterns. But the production rate of enterotoxins in isolates from diseased dogs was significantly higher than that in isolates from healthy dogs. PFGE analysis was performed with isolates from different body sites in individual dogs. In 3 of 6 healthy dogs, identical PFGE patterns were seen in isolates from the nares, external auditory meatus or skin. The remaining 3 dogs yielded isolates of different patterns. In 4 of 6 diseased dogs, identical patterns were seen in isolates from lesions as well as from the other normal sites.     

Occurrence of Staphylococcus intermedius on the hair and skin of normal dogs.

Aerobic bacterial populations were studied on the distal hair coat and at the skin surface of the shoulder, rump and abdomen of 10 healthy dogs. Coagulase negative staphylococci (CNS) were more frequently isolated from the hair than the skin at the shoulder and rump. There was no difference in the isolation rate of coagulase positive staphylococci (CPS) (Staphylococcus intermedius) between the hair and skin. Total skin counts were greatest on the abdomen whereas CNS counts from the hair were least at this site. There were no differences between CPS counts at the three sites on either hair or skin. The populations on the relatively unfavourable microenvironment of the distal hair may represent contamination rather than colonisation. The low populations of CPS at the skin surface also indicate contamination or transient colonisation rather than true resident status.