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Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis
Research in Veterinary Science 2000, 68, 279–283 doi:10.1053/rvsc.2000.0378, available online at http://www.idealibrary.com on
Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis N. A. MCEWAN University of Glasgow Veterinary School, Department of Veterinary Clinical Studies, Bearsden, Glasgow, G61 1QH, UK SUMMARY 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.
CANINE atopic dermatitis is a common pruritic skin disorder characterised by a type I hypersensitivity to environmental allergens such as house dust mites, storage mites, animal epithelia, plant pollens and moulds. Although the pathogenesis of atopic dermatitis is not fully understood, clinical and immunological studies have shown similarities between the human and canine condition (Parish 1981, Scott 1981, Willemse et al 1985, Helton Rhodes et al 1987, Rhodes et al 1987, Nimmo Wilkie et al 1991, Zachary et al 1985, Olivry et al 1996, 1997). In atopic dermatitis of man and dogs, bacterial skin disease is a very common and important complication. The major microbial pathogens involved are the pathogenic staphylococci; Staphylococcus aureus in man and S. intermedius in the dog. The reason why individuals who suffer from atopic dermatitis show increased susceptibility to staphylococcal pyoderma is poorly understood and is probably multifactorial. Adherence by microbes to cell surfaces is recognised to be an important virulence factor and is appreciated to be an essential initial step in the colonisation and subsequent invasion of organic tissue. In human atopic dermatitis patients, increased adherence by Staphylococcus aureus to keratinocytes has been demonstrated and it has been postulated that this is one possible explanation for the high incidence of staphylococcal pyoderma in atopic patients (Cole and Silverberg 1986). The present study was conducted to test the hypothesis that S. intermedius shows increased adherence to keratinocytes in dogs suffering from atopic dermatitis compared with keratinocytes from normal dogs and dogs suffering from non-atopic skin disease. MATERIALS AND METHODS Populations studied Normal dogs This group consisted of 16 dogs of nine different breeds comprising 13 females and three males, with a mean age of 0034-5288/00/030279 + 05 $35.00/0
5·28 ± 2·58 years and a range of 1 to 10 years which were either kept at or belonged to staff of the University of Glasgow Veterinary School (UGVS) or were dogs referred with minor surgical conditions. All dogs in this group were given a full clinical examination, were free from skin disease and in good general health. Dogs suffering from atopic dermatitis Eighteen dogs comprising 13 different breeds of which 11 were females and seven males with a mean age of 3·78 ± 1·88 years and a range of 1 to 8 years were used in the study. All dogs were referred to the UGVS and fulfilled the criteria set out by Willemse (1986) to establish a diagnosis of atopic dermatitis and showed at least one positive intradermal skin test reaction to a panel of common environmental allergens. None of the dogs showed any gross clinical signs of pyoderma at the time of keratinocyte collection. Dogs suffering from pyoderma Five other dogs comprising two females and three males with a mean age of 3·24 ± 1·56 years and a range of 1 to 5 years, all referred cases at UGVS, were included in this group. Two dogs with deep generalised pyoderma had demodectic mange, two dogs had superficial pyoderma that was completely responsive to antibiotic therapy and one dog had a superficial and deep (interdigital) pyoderma which was also entirely antibiotic responsive. Dogs suffering from primary seborrhoea Eight other dogs referred to UGVS, comprising three females and five males with a mean age of 4·72 ± 1·83 years and a range of 4 to 9 years, were included in this group. All of these dogs were spaniels (five Cocker spaniels and three English Springer spaniels). In addition to standard dermatological investigations, hypothyroidism was excluded by demonstrating normal blood cholesterol and resting thyroxine values. Food intolerance as a cause of skin disease was excluded by failure of the skin condition to resolve after feeding a modified diet designed to identify the presence of this condition. © 2000 Harcourt Publishers Ltd
280
N. A. McEwan
Collection of keratinocytes The subject was placed on a clean table with a smooth surface and the skin vigorously rubbed using the fingertips. Using this technique, keratinocytes were dislodged from the skin over the dorsum, lateral thorax, flanks and thighs. The keratinocytes so dislodged were collected and any gross debris and hair manually removed. The keratinocytes were then placed in a centrifuge tube with 10 ml of sterile phosphate buffered saline (PBS 0·01 M, pH 7·4) and mixed vigorously in a vortex mixer (Autovortex Mixer SA2, Stuart Scientific, UK) for 5 minutes to break up any sheets of keratinocytes and so produce single cell suspensions. The keratinocytes were then washed by centrifugation in three changes of PBS. The resultant suspension of keratinocytes was adjusted to approximately 1 × 104 keratinocytes per ml of PBS, ascertained by counting in a haemocytometer.
more adherent cocci was recorded. The adherence index, i.e., the number of keratinocytes with 20 or more adherent cocci out of the 100 keratinocytes examined, was then determined (Cole and Silverberg 1986). Statistical analysis Statistical analysis was conducted using a computerised statistical package (SigmaStat for Windows version 2·03, SPSS Inc.). One way analysis of variance was used to compare groups with specific differences identified using the all pairwise multiple comparison procedure (Tukey test). As the data for the control groups was not normally distributed, Kruskal-Wallis one way analysis of variance on ranks was used to compare these groups. A value of P < 0·05 was considered to be significant.
Staphylococcus
Adherence assay One millilitre of the keratinocyte suspension was incubated with 1 ml of the S. intermedius suspension. A negative control consisting of 1 ml of keratinocytes with 1 ml of PBS was included. The suspensions were placed in a shaking water bath (Grant Instruments (Cambridge) Ltd., Cambridge, UK) and incubated at a temperature of 38°C for 45 minutes. At the end of this time, the keratinocytes were collected on polycarbamate filters (Nucleopore® Corporation Filtration Products, Nucleopore Canada, Inc., Toronto) with a pore size of 12 µm and washed with 300 ml of sterile PBS. The filters were then placed on clean glass microscope slides and allowed to air dry before staining by Gram’s method. After air drying, the stained filter was mounted in DPX under a number one glass cover slip. Each filter was examined under oil immersion microscopy (× 1000 magnification) and 100 keratinocytes were examined. The number of keratinocytes with 20 or
RESULTS The adherence indices for the four groups studied are detailed in Table 1. When keratinocytes were incubated with S. intermedius, the adherence index for the four groups was normal 17·19 ± 14·06; atopy 35·28 ± 15·95; pyoderma 34·00 ± 8·83 and seborrhoea 18·25 ± 12·00. The groups are illustrated in Fig 1, where box and whisker plots depict the median for each group and the boxes define the 25th and 75th percentiles, whiskers indicate the 10th and 90th percentiles. Outlying data points are included. There were no significant differences between the groups when the controls were compared. Where keratinocytes were incubated with S. intermedius, the atopic group was significantly different from the normal group (P = 0·003) and the seborrhoea group (P = 0·035).
DISCUSSION The results of this study are very similar to those obtained by Cole and Silverberg (1986) who studied the adherence by S. aureus to human keratinocytes. Both studies demonstrated that pathogenic staphylococci adhere to a greater extent to keratinocytes from patients with atopic dermatitis when compared with normal keratinocytes and those col70 Adherence Index (percentage)
An isolate of S. intermedius, identified by a microtray method (API Staph system, bioMérieux, Lyon, France) was obtained from a clinical case of canine pyoderma, cultured on sheep blood agar, subcultured into liquid medium (Oxoid Nutrient Broth. No. 2. Unipath Ltd., Basingstoke, Hampshire, England) and frozen at –70°C in 1 ml aliquots. Suspensions of S. intermedius in sterile PBS were made at optical densities (OD) ranging from 0·2 to 0·8 at 570 nanometers (nm) on a spectrophotometer (Cecil instruments, Cambridge, England) and plated on sheep blood agar at 10-fold dilution to determine the number of colony forming units (CFU) per ml. An OD of 0·5 was found to give approximately 1 × 106 CFU per ml and this dilution was used for the assay. When required for an adhesion assay, a frozen aliquot of S. intermedius was thawed and plated on sheep blood agar incubated at 38°C for 48 hours. Colonies were harvested and placed in a centrifuge tube with 10 ml of sterile PBS. The cocci were washed in three changes of PBS by centrifugation and the resulting suspension adjusted to an OD of 0·5 at 570 nm.
60 50 40 30 Pyoderma
20 10 0
Atopic dermatitis
Seborrhoea
Normal
FIG 1: Box plot of the adherence indices shown by S. intermedius to canine keratinocytes.
Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis TABLE 1:
Adherence indices for staphylococcal adherence to canine keratinocytes Normal n = 16
Max Min
Atopy n = 18 Control Staph
Pyoderma n=5 Control Staph
Seborrhoea n=8 Control Staph
Control
Staph
0 3 0 1 1 1 2 1 1 1 1 4 1 2 3 7
20 15 8 13 23 27 52 5 31 2 1 38 12 7 7 14
3 1 0 2 1 0 5 8 1 0 1 1 0 2 2 1 0 7
24 30 58 48 38 31 32 56 29 54 4 55 43 30 17 14 50 22
25 7 2 1 1
43 27 24 43 33
2 2 1 0 2 0 0 0
23 10 36 13 6 19 34 5
1·81 1·76 4·00 0·00
17·19 14·06 52·00 1·00
1·94 2·39 8·00 0·00
35·28 15·95 58·00 4·00
7·20 10·26 25·00 1·00
34·00 8·83 43·00 24·00
0·88 0·99 2·00 0·00
18·25 12·00 36·00 5·00
Mean SD
281
lected from patients with non-atopic skin diseases. It could be speculated that this finding is due to the presence of larger numbers of receptor sites exposed or available on keratinocytes from human and canine atopic dermatitis patients. Cole and Silverberg (1986) also demonstrated that the increased adherence exhibited by S. aureus was not seen when other Gram-positive cocci (S. epidermidis and Bacillus species) were assayed with atopic keratinocytes, suggesting that these bacteria do not possess adhesins which bind to atopic keratinocytes. An interesting feature of this study was the variable numbers of cocci adhering to individual keratinocytes in the same sample. Some keratinocytes attracted hundreds of adherent cocci while others very few (Fig 2). This was a finding for all the groups studied and suggests that there is variability in the expression of receptors on keratinocytes for the adherence of S. intermedius. Bibel et al (1982) demonstrated that S. aureus adhered to a greater extent to fully keratinised (older) nasal epithelial cells when compared with younger, less keratinised, nasal epithelial cells. A key feature determining adherence by S. aureus to keratinocytes may therefore be the degree of keratinisation of these cells. It is interesting to note that in this study, the adherence by S. intermedius to keratinocytes from primary seborrhoea patients was not significantly different from normal dogs. Dogs that suffer from primary seborrhoea are known to have larger numbers of pathogenic staphylococci on their skin (Ihrke et al 1978) and may show increased susceptibility to develop secondary skin infections including staphylococcal pyoderma (Scott et al 1995), but from this study it would appear that increased adherence by S. intermedius is not involved. Canine primary seborrhoea is a condition characterised by increased epidermal turnover and histologically this appears as epidermal hyperplasia with both orthokeratotic and parakeratotic hyperkeratosis (Gross et al 1992, Yager and Wilcox 1994). Epidermal turnover in Cocker spaniels suffering from primary seborrhoea has been shown
to be reduced from a normal of 21 days to as little as 8 days (Kwochka and Radmakers 1989). Thus it can be hypothesised that the keratinocytes collected from the skin surface of patients suffering from primary seborrhoea are the product of altered or abnormal keratinisation which results in smaller numbers of receptor sites for adherence by pathogenic staphylococci, so explaining the low adherence index found in this group. Statistically significant increased adherence was shown by S. intermedius to keratinocytes from the pyoderma group when compared with both the normal and the seborrhoea group. Although the mean adherence index for the pyoderma control group was higher than the controls for the other groups, this did not alter the statistical findings when the four groups were compared after subtracting the control adherence indices from the staphylococcal incubated keratinocytes in each group. A high number of adherent cocci on the keratinocytes from the pyoderma group present prior to the adherence assay would therefore not appear to be the cause of the high adherence index in this group. Within the pyoderma group, two cases had generalised demodectic mange which was liable to predispose these dogs to pyoderma. In the remaining three cases, no underlying skin disease was detected to explain the development of pyoderma and these cases were classed as idiopathic. It is, however, possible that these cases may have suffered from mild or early atopic dermatitis. The low numbers in the pyoderma group dictate that the findings for this group should be interpreted with care and further investigation is warranted. Staphylococci are capable of invasion of various body tissues so it seems likely that they have multiple mechanisms for adhesion. Various studies have demonstrated that lipoteichoic acid, wall teichoic acid and protein A may all act as adhesins for S. aureus which bind to a variety of extracellular matrix protein receptors present around epithelial cells. Fibronectin, a glycoprotein present in the extracellular matrix of animal tissues as well as a soluble plasma
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A
B
FIG 2: A. Atopic keratinocyte with few adherent Gram-positive cocci. (1000 × magnification). B. Atopic keratinocyte with numerous adherent Gram-positive cocci. (1000 × magnification)
compound, is known to bind S. aureus (Kuusela 1978) and has been shown to play a role in the binding of S. aureus to the skin (Kanzaki et al 1996). S. intermedius has also been shown to bind to extracellular matrix proteins (Cree and Noble 1995). It can be hypothesised that S. intermedius ini-
tially colonises the skin of canine atopic dermatitis patients by adherence to keratinocytes that express large numbers of receptors for S. intermedius. After this initial colonisation, toxins released by S. intermedius result in inflammation and capillary leakage, exposing fibronectin, which further
Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis
enhances adherence by S. intermedius so creating a vicious cycle ultimately resulting in tissue invasion of S. intermedius. Although the study presented here tested only a single strain of S. intermedius, the findings would support the conclusion that S. intermedius adheres to canine keratinocytes and adheres preferentially to atopic keratinocytes when compared with adherence to normal keratinocytes and those from some other dermatoses. A major benefit of the elucidation of the mechanisms of bacterial adherence to atopic keratinocytes would be to open the potential to development of novel therapies which prevent or interfere with the adherence process.
ACKNOWLEDGEMENTS The author is grateful for the help and support of Professor Max Murray and the University of Glasgow, Department of Veterinary Clinical Studies. Professor Andrew Nash, Dr Joanna Dukes McEwan and Mr Peter Forsythe provided advise and helpful criticism in the preparation of this paper. This work was supported by funds from the Wellcome Trust. REFERENCES BIBEL, D.J., ALY, R., SHINFIELD, H.R., MAIBACH, H.I. & STAUSS, W.G. (1982) Importance of the keratinized epithelial cell in bacterial adherence. The Journal of Investigative Dermatology 79, 250–253 COLE, G.W. & SILVERBERG, N.L. (1986) The adherence of Staphylococcus aureus to human corneocytes. Archives of Dermatology 122, 166–169 CREE, R.G.A. & NOBLE, W.C. (1995) In vitro indices of tissue adherence in Staphylococcus intermedius. Letters in Applied Microbiology 20, 168–170 GROSS, T.L., IHRKE, P.J. & WALDER, E.J. (1992) Veterinary Dermatohistopathology. A Macroscopic and Microscopic Evaluation of Canine and Feline Skin Diseases. Mosby Year Book. St Louis. PP. 88–90 HELTON RHODES, K., KERDEL, F. & SOTER, N.A. (1987) Investigation into the immunopathogenesis of canine atopy. Seminars in Veterinary Medicine and Surgery (Small Animal) 2, 199–201
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IHRKE, P.J., SCHWARTZMAN, R.M., MCGINLEY, K., HOROWITZ, L. & MARPLES, R.R. (1978) Microbiology of normal and seborrhoeic canine skin. American Journal of Veterinary Research 39, 1487–1489 KANZAKI, H., MORISHITA, Y., AKIYAMA, H. & ARATA, J. (1996) Adhesion of Staphylococcus aureus to horny layer: role of fibrinogen. Journal of Dermatological Science 12, 132–139 KUUSELA, P. (1978) Fibronectin binds to Staphylococcus aureus. Nature 276, 718–720 KWOCHKA, K.W. & RADMAKERS, A.M. (1989) Cell proliferation kinetics of epidermis, hair follicles, and sebaceous glands of Cocker spaniels with primary seborrhea. American Journal of Veterinary Research 50, 1918–1922 NIMMO WILKIE, J.S., YAGER, J.A., WILKIE, B.N. & PARKER, W.M. (1991) Abnormal cutaneous response to mitogens and a contact allergen in dogs with atopic dermatitis. Veterinary Immunology and Immunopathology 28, 97–106 OLIVRY, T., MOORE, P.F., AFFOLTER, V.K. & NAYDAN, D.K. (1996) Langerhans cell hyperplasia and IgE expression in canine atopic dermatitis. Archives of Dermatological Research 288, 579–585 OLIVRY, T., NAYDAN, D.K. & MOORE, P.F. (1997) Characterisation of the cutaneous inflammatory infiltrate in canine atopic dermatitis. American Journal of Dermatopathology 19, 477–486 PARISH, W.E. (1981) The clinical relevance of heat-stable, short-term sensitizing anaphylactic IgG antibodies (IgG S-TS) and of related activities of IgG4 and IgG2. British Journal of Dermatology 105, 223–231 RHODES, K.H., KERDEL, F., SOTER, N.A. & CHINNICI, R. (1987) Comparative aspects of canine and human atopic dermatitis. Seminars in Veterinary Medicines and Surgery (Small Animal) 2, 168–172 SCOTT, D.W. (1981) Observations on canine atopy. Journal of the American Animal Hospital Association 17, 91–100 SCOTT, D.W., MILLER, W.H. Jr. & GRIFFIN, C.E. (1995) Muller and Kirk’s Small Animal Dermatology. 5th edition. W.B. Saunders Company, Philadelphia. P. 738 WILLEMSE, A., NOORDZIJ, A., VAN DEN BROM, W.E. & RUTTEN, V.P. (1985) Allergen specific IgGd in dogs with atopic dermatitis as determined by enzyme linked immunosorbent assay (ELISA). Clinical and Experimental Immunology 59, 359–363 WILLEMSE, T. (1986) Atopic skin disease: a review and a reconsideration of diagnostic criteria. Journal of Small Animal Practice 27, 771–778 YAGER, J.A. & WILCOCK, B.P. (1994) Color Atlas and Text of Surgical Pathology of the Dog and Cat. Dermatopathology and Skin Tumors. Wolfe Publishing. PP. 66–67 ZACHARY, C.B., ALLEN, M.H. & MCDONALD, D.M. (1985) In situ quantification of T-lymphocyte subsets and Langerhans’ cells in the inflammatory infiltrate of atopic eczema. British Journal of Dermatology 112, 149–156
Accepted January 19, 2000
Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis N. A. MCEWAN University of Glasgow Veterinary School, Department of Veterinary Clinical Studies, Bearsden, Glasgow, G61 1QH, UK SUMMARY 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.
CANINE atopic dermatitis is a common pruritic skin disorder characterised by a type I hypersensitivity to environmental allergens such as house dust mites, storage mites, animal epithelia, plant pollens and moulds. Although the pathogenesis of atopic dermatitis is not fully understood, clinical and immunological studies have shown similarities between the human and canine condition (Parish 1981, Scott 1981, Willemse et al 1985, Helton Rhodes et al 1987, Rhodes et al 1987, Nimmo Wilkie et al 1991, Zachary et al 1985, Olivry et al 1996, 1997). In atopic dermatitis of man and dogs, bacterial skin disease is a very common and important complication. The major microbial pathogens involved are the pathogenic staphylococci; Staphylococcus aureus in man and S. intermedius in the dog. The reason why individuals who suffer from atopic dermatitis show increased susceptibility to staphylococcal pyoderma is poorly understood and is probably multifactorial. Adherence by microbes to cell surfaces is recognised to be an important virulence factor and is appreciated to be an essential initial step in the colonisation and subsequent invasion of organic tissue. In human atopic dermatitis patients, increased adherence by Staphylococcus aureus to keratinocytes has been demonstrated and it has been postulated that this is one possible explanation for the high incidence of staphylococcal pyoderma in atopic patients (Cole and Silverberg 1986). The present study was conducted to test the hypothesis that S. intermedius shows increased adherence to keratinocytes in dogs suffering from atopic dermatitis compared with keratinocytes from normal dogs and dogs suffering from non-atopic skin disease. MATERIALS AND METHODS Populations studied Normal dogs This group consisted of 16 dogs of nine different breeds comprising 13 females and three males, with a mean age of 0034-5288/00/030279 + 05 $35.00/0
5·28 ± 2·58 years and a range of 1 to 10 years which were either kept at or belonged to staff of the University of Glasgow Veterinary School (UGVS) or were dogs referred with minor surgical conditions. All dogs in this group were given a full clinical examination, were free from skin disease and in good general health. Dogs suffering from atopic dermatitis Eighteen dogs comprising 13 different breeds of which 11 were females and seven males with a mean age of 3·78 ± 1·88 years and a range of 1 to 8 years were used in the study. All dogs were referred to the UGVS and fulfilled the criteria set out by Willemse (1986) to establish a diagnosis of atopic dermatitis and showed at least one positive intradermal skin test reaction to a panel of common environmental allergens. None of the dogs showed any gross clinical signs of pyoderma at the time of keratinocyte collection. Dogs suffering from pyoderma Five other dogs comprising two females and three males with a mean age of 3·24 ± 1·56 years and a range of 1 to 5 years, all referred cases at UGVS, were included in this group. Two dogs with deep generalised pyoderma had demodectic mange, two dogs had superficial pyoderma that was completely responsive to antibiotic therapy and one dog had a superficial and deep (interdigital) pyoderma which was also entirely antibiotic responsive. Dogs suffering from primary seborrhoea Eight other dogs referred to UGVS, comprising three females and five males with a mean age of 4·72 ± 1·83 years and a range of 4 to 9 years, were included in this group. All of these dogs were spaniels (five Cocker spaniels and three English Springer spaniels). In addition to standard dermatological investigations, hypothyroidism was excluded by demonstrating normal blood cholesterol and resting thyroxine values. Food intolerance as a cause of skin disease was excluded by failure of the skin condition to resolve after feeding a modified diet designed to identify the presence of this condition. © 2000 Harcourt Publishers Ltd
280
N. A. McEwan
Collection of keratinocytes The subject was placed on a clean table with a smooth surface and the skin vigorously rubbed using the fingertips. Using this technique, keratinocytes were dislodged from the skin over the dorsum, lateral thorax, flanks and thighs. The keratinocytes so dislodged were collected and any gross debris and hair manually removed. The keratinocytes were then placed in a centrifuge tube with 10 ml of sterile phosphate buffered saline (PBS 0·01 M, pH 7·4) and mixed vigorously in a vortex mixer (Autovortex Mixer SA2, Stuart Scientific, UK) for 5 minutes to break up any sheets of keratinocytes and so produce single cell suspensions. The keratinocytes were then washed by centrifugation in three changes of PBS. The resultant suspension of keratinocytes was adjusted to approximately 1 × 104 keratinocytes per ml of PBS, ascertained by counting in a haemocytometer.
more adherent cocci was recorded. The adherence index, i.e., the number of keratinocytes with 20 or more adherent cocci out of the 100 keratinocytes examined, was then determined (Cole and Silverberg 1986). Statistical analysis Statistical analysis was conducted using a computerised statistical package (SigmaStat for Windows version 2·03, SPSS Inc.). One way analysis of variance was used to compare groups with specific differences identified using the all pairwise multiple comparison procedure (Tukey test). As the data for the control groups was not normally distributed, Kruskal-Wallis one way analysis of variance on ranks was used to compare these groups. A value of P < 0·05 was considered to be significant.
Staphylococcus
Adherence assay One millilitre of the keratinocyte suspension was incubated with 1 ml of the S. intermedius suspension. A negative control consisting of 1 ml of keratinocytes with 1 ml of PBS was included. The suspensions were placed in a shaking water bath (Grant Instruments (Cambridge) Ltd., Cambridge, UK) and incubated at a temperature of 38°C for 45 minutes. At the end of this time, the keratinocytes were collected on polycarbamate filters (Nucleopore® Corporation Filtration Products, Nucleopore Canada, Inc., Toronto) with a pore size of 12 µm and washed with 300 ml of sterile PBS. The filters were then placed on clean glass microscope slides and allowed to air dry before staining by Gram’s method. After air drying, the stained filter was mounted in DPX under a number one glass cover slip. Each filter was examined under oil immersion microscopy (× 1000 magnification) and 100 keratinocytes were examined. The number of keratinocytes with 20 or
RESULTS The adherence indices for the four groups studied are detailed in Table 1. When keratinocytes were incubated with S. intermedius, the adherence index for the four groups was normal 17·19 ± 14·06; atopy 35·28 ± 15·95; pyoderma 34·00 ± 8·83 and seborrhoea 18·25 ± 12·00. The groups are illustrated in Fig 1, where box and whisker plots depict the median for each group and the boxes define the 25th and 75th percentiles, whiskers indicate the 10th and 90th percentiles. Outlying data points are included. There were no significant differences between the groups when the controls were compared. Where keratinocytes were incubated with S. intermedius, the atopic group was significantly different from the normal group (P = 0·003) and the seborrhoea group (P = 0·035).
DISCUSSION The results of this study are very similar to those obtained by Cole and Silverberg (1986) who studied the adherence by S. aureus to human keratinocytes. Both studies demonstrated that pathogenic staphylococci adhere to a greater extent to keratinocytes from patients with atopic dermatitis when compared with normal keratinocytes and those col70 Adherence Index (percentage)
An isolate of S. intermedius, identified by a microtray method (API Staph system, bioMérieux, Lyon, France) was obtained from a clinical case of canine pyoderma, cultured on sheep blood agar, subcultured into liquid medium (Oxoid Nutrient Broth. No. 2. Unipath Ltd., Basingstoke, Hampshire, England) and frozen at –70°C in 1 ml aliquots. Suspensions of S. intermedius in sterile PBS were made at optical densities (OD) ranging from 0·2 to 0·8 at 570 nanometers (nm) on a spectrophotometer (Cecil instruments, Cambridge, England) and plated on sheep blood agar at 10-fold dilution to determine the number of colony forming units (CFU) per ml. An OD of 0·5 was found to give approximately 1 × 106 CFU per ml and this dilution was used for the assay. When required for an adhesion assay, a frozen aliquot of S. intermedius was thawed and plated on sheep blood agar incubated at 38°C for 48 hours. Colonies were harvested and placed in a centrifuge tube with 10 ml of sterile PBS. The cocci were washed in three changes of PBS by centrifugation and the resulting suspension adjusted to an OD of 0·5 at 570 nm.
60 50 40 30 Pyoderma
20 10 0
Atopic dermatitis
Seborrhoea
Normal
FIG 1: Box plot of the adherence indices shown by S. intermedius to canine keratinocytes.
Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis TABLE 1:
Adherence indices for staphylococcal adherence to canine keratinocytes Normal n = 16
Max Min
Atopy n = 18 Control Staph
Pyoderma n=5 Control Staph
Seborrhoea n=8 Control Staph
Control
Staph
0 3 0 1 1 1 2 1 1 1 1 4 1 2 3 7
20 15 8 13 23 27 52 5 31 2 1 38 12 7 7 14
3 1 0 2 1 0 5 8 1 0 1 1 0 2 2 1 0 7
24 30 58 48 38 31 32 56 29 54 4 55 43 30 17 14 50 22
25 7 2 1 1
43 27 24 43 33
2 2 1 0 2 0 0 0
23 10 36 13 6 19 34 5
1·81 1·76 4·00 0·00
17·19 14·06 52·00 1·00
1·94 2·39 8·00 0·00
35·28 15·95 58·00 4·00
7·20 10·26 25·00 1·00
34·00 8·83 43·00 24·00
0·88 0·99 2·00 0·00
18·25 12·00 36·00 5·00
Mean SD
281
lected from patients with non-atopic skin diseases. It could be speculated that this finding is due to the presence of larger numbers of receptor sites exposed or available on keratinocytes from human and canine atopic dermatitis patients. Cole and Silverberg (1986) also demonstrated that the increased adherence exhibited by S. aureus was not seen when other Gram-positive cocci (S. epidermidis and Bacillus species) were assayed with atopic keratinocytes, suggesting that these bacteria do not possess adhesins which bind to atopic keratinocytes. An interesting feature of this study was the variable numbers of cocci adhering to individual keratinocytes in the same sample. Some keratinocytes attracted hundreds of adherent cocci while others very few (Fig 2). This was a finding for all the groups studied and suggests that there is variability in the expression of receptors on keratinocytes for the adherence of S. intermedius. Bibel et al (1982) demonstrated that S. aureus adhered to a greater extent to fully keratinised (older) nasal epithelial cells when compared with younger, less keratinised, nasal epithelial cells. A key feature determining adherence by S. aureus to keratinocytes may therefore be the degree of keratinisation of these cells. It is interesting to note that in this study, the adherence by S. intermedius to keratinocytes from primary seborrhoea patients was not significantly different from normal dogs. Dogs that suffer from primary seborrhoea are known to have larger numbers of pathogenic staphylococci on their skin (Ihrke et al 1978) and may show increased susceptibility to develop secondary skin infections including staphylococcal pyoderma (Scott et al 1995), but from this study it would appear that increased adherence by S. intermedius is not involved. Canine primary seborrhoea is a condition characterised by increased epidermal turnover and histologically this appears as epidermal hyperplasia with both orthokeratotic and parakeratotic hyperkeratosis (Gross et al 1992, Yager and Wilcox 1994). Epidermal turnover in Cocker spaniels suffering from primary seborrhoea has been shown
to be reduced from a normal of 21 days to as little as 8 days (Kwochka and Radmakers 1989). Thus it can be hypothesised that the keratinocytes collected from the skin surface of patients suffering from primary seborrhoea are the product of altered or abnormal keratinisation which results in smaller numbers of receptor sites for adherence by pathogenic staphylococci, so explaining the low adherence index found in this group. Statistically significant increased adherence was shown by S. intermedius to keratinocytes from the pyoderma group when compared with both the normal and the seborrhoea group. Although the mean adherence index for the pyoderma control group was higher than the controls for the other groups, this did not alter the statistical findings when the four groups were compared after subtracting the control adherence indices from the staphylococcal incubated keratinocytes in each group. A high number of adherent cocci on the keratinocytes from the pyoderma group present prior to the adherence assay would therefore not appear to be the cause of the high adherence index in this group. Within the pyoderma group, two cases had generalised demodectic mange which was liable to predispose these dogs to pyoderma. In the remaining three cases, no underlying skin disease was detected to explain the development of pyoderma and these cases were classed as idiopathic. It is, however, possible that these cases may have suffered from mild or early atopic dermatitis. The low numbers in the pyoderma group dictate that the findings for this group should be interpreted with care and further investigation is warranted. Staphylococci are capable of invasion of various body tissues so it seems likely that they have multiple mechanisms for adhesion. Various studies have demonstrated that lipoteichoic acid, wall teichoic acid and protein A may all act as adhesins for S. aureus which bind to a variety of extracellular matrix protein receptors present around epithelial cells. Fibronectin, a glycoprotein present in the extracellular matrix of animal tissues as well as a soluble plasma
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A
B
FIG 2: A. Atopic keratinocyte with few adherent Gram-positive cocci. (1000 × magnification). B. Atopic keratinocyte with numerous adherent Gram-positive cocci. (1000 × magnification)
compound, is known to bind S. aureus (Kuusela 1978) and has been shown to play a role in the binding of S. aureus to the skin (Kanzaki et al 1996). S. intermedius has also been shown to bind to extracellular matrix proteins (Cree and Noble 1995). It can be hypothesised that S. intermedius ini-
tially colonises the skin of canine atopic dermatitis patients by adherence to keratinocytes that express large numbers of receptors for S. intermedius. After this initial colonisation, toxins released by S. intermedius result in inflammation and capillary leakage, exposing fibronectin, which further
Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis
enhances adherence by S. intermedius so creating a vicious cycle ultimately resulting in tissue invasion of S. intermedius. Although the study presented here tested only a single strain of S. intermedius, the findings would support the conclusion that S. intermedius adheres to canine keratinocytes and adheres preferentially to atopic keratinocytes when compared with adherence to normal keratinocytes and those from some other dermatoses. A major benefit of the elucidation of the mechanisms of bacterial adherence to atopic keratinocytes would be to open the potential to development of novel therapies which prevent or interfere with the adherence process.
ACKNOWLEDGEMENTS The author is grateful for the help and support of Professor Max Murray and the University of Glasgow, Department of Veterinary Clinical Studies. Professor Andrew Nash, Dr Joanna Dukes McEwan and Mr Peter Forsythe provided advise and helpful criticism in the preparation of this paper. This work was supported by funds from the Wellcome Trust. REFERENCES BIBEL, D.J., ALY, R., SHINFIELD, H.R., MAIBACH, H.I. & STAUSS, W.G. (1982) Importance of the keratinized epithelial cell in bacterial adherence. The Journal of Investigative Dermatology 79, 250–253 COLE, G.W. & SILVERBERG, N.L. (1986) The adherence of Staphylococcus aureus to human corneocytes. Archives of Dermatology 122, 166–169 CREE, R.G.A. & NOBLE, W.C. (1995) In vitro indices of tissue adherence in Staphylococcus intermedius. Letters in Applied Microbiology 20, 168–170 GROSS, T.L., IHRKE, P.J. & WALDER, E.J. (1992) Veterinary Dermatohistopathology. A Macroscopic and Microscopic Evaluation of Canine and Feline Skin Diseases. Mosby Year Book. St Louis. PP. 88–90 HELTON RHODES, K., KERDEL, F. & SOTER, N.A. (1987) Investigation into the immunopathogenesis of canine atopy. Seminars in Veterinary Medicine and Surgery (Small Animal) 2, 199–201
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Accepted January 19, 2000