FOCUS ON PYODERMA Ross Bond, Department of Clinical Sciences and Services Royal Veterinary College

FOCUS ON PYODERMA Ross Bond, Department of Clinical Sciences and Services Royal Veterinary College Brought to you by Bayer, makers of Veraflox EDUCATION NOW ANTIM IC R O BIALS

CONTENTS Page 4: Introduction Page 5: History taking in canine pyoderma Page 6 7: Clinical examination Page 8 9: Diagnostic testing in canine pyoderma Page 10 12: Antimicrobial therapy Page 13: Summary Page 14: References Ross Bond BVMS PhD DVD MRCVS DipECVD European and RCVS Recognised Specialist in Veterinary Dermatology Professor of Veterinary Dermatology, Royal Veterinary College Ross graduated from Glasgow Veterinary School in 1985, spent 5 years in farm and small animal practice, and then joined the Royal Veterinary College in 1990, where he is currently Professor in Veterinary Dermatology and Head of the Dermatology Service. His research work into yeast infections in dogs led to an award of a PhD in 1996. Ross holds RCVS and European College Diplomas in Veterinary Dermatology and is recognised as a specialist in veterinary dermatology by the RCVS. His research interests relate primarily to microbial skin infections in dogs and cats. 2 3

INTRODUCTION HISTORY TAKING IN CANINE PYODERMA Pyoderma is best defined as a cutaneous pyogenic infection. The dog seems particularly susceptible to bacterial skin disease amongst the veterinary species, and pyoderma is a common diagnosis in canine practice. Canine pyoderma commonly poses diagnostic challenges, due to its varied clinical presentation and tendency to be super-imposed on other skin diseases. Therapy is also potentially challenging, especially in relapsing cases, cases where concurrent diseases are not corrected, and / or where infection is associated with multidrug-resistant bacteria. In one survey of 428 dogs with skin disease seen in first opinion practice in the UK, pyoderma (62 cases, 14.5%) was second only to otitis (104, 24.3%) in cases with a specific dermatological diagnosis (Hill et al., 2006). In another review of 54,600 dogs presented to 73 UK veterinary practices in 2010, a diagnosis of pyoderma was retrieved from electronic patient records in 683 cases (1.3%) (Summers et al., 2014). The goals in history-taking in cases of pyoderma are to establish the course and duration of disease (differentiating first episodes from recurrent or persistent cases), to ascertain outcomes of any previous diagnostic testing and treatments, and to evaluate the case for evidence of concurrent diseases that might increase likelihood of infection or interfere with the diagnostic or therapeutic plan. The majority of canine cases are associated with Staphylococcus pseudintermedius (formerly S. intermedius). This is a commensal organism on healthy canine mucosa / skin and perturbation of normal skin defence in the presence of strains of appropriate virulence is necessary for infection to develop. It therefore follows that dogs presenting with pyoderma should be carefully evaluated for diseases that impair immunity or skin barrier function because incomplete clinical response and or relapsing infection can be anticipated if these are not diagnosed and treated. Other bacteria, including other staphylococci, streptococci and Gram-negative rods, are less frequently isolated. It is traditional to classify pyoderma according to depth of infection into surface, superficial and deep forms of the disease. Whilst this is technically a histological classification, it is often possible to determine which form is most likely from the clinical signs (Table 1). This scheme has practical implications for therapy since response to topical treatments can be anticipated with surface and sometimes superficial infection (assuming correct application and compliance), whereas deep pyoderma cases most often require a systemic route of administration (Fig. 1). It is important to differentiate cases of persistent infection (suggesting poor compliance, wrong drug / dose / duration, resistance, or wrong diagnosis) from truly recurrent cases (as seen in cases where pyoderma has fully resolved but ongoing underlying disease or immune failure allows re-infection). Persistence of pruritus following successful treatment of pyoderma with antibiotics alone is a typical observation in dogs with underlying allergic diseases, and or concurrent Malassezia dermatitis; dorso-lumbar pruritus is most common in flea allergy whereas a facial, aural, ventral and/or pedal distribution is reported routinely in cases of (food and nonfood-induced) atopic dermatitis. A combination of owner report and record scrutiny should establish the specific details of prescribed treatments and results of any sampling procedures. An assessment of general health is critical for the detection of systemic and metabolic diseases that might favour pyoderma. Review of ecto-parasite control measures and the likelihood of their reliable implementation, history of potential transmission of skin disease between other pet animals, wildlife and owners, along with reports of observed parasites, should help quantify the risk of a parasitic cause. Table 1. Classification of canine pyoderma and associated histological and clinical features Type of pyoderma Anatomical location Histological inflammatory pattern Typical clinical signs* Route of treatment administration Surface Inter-follicular epidermis Erosive neutrophilic epidermitis# Moist exudation and erosion, (e.g. hot spots, fold dermatitis) Primarily topical Superficial Inter-follicular epidermis + within intact follicle Neutrophilic luminal folliculitis Pustule, preceded by papule, evolving to collarette/crust Topical and/or systemic Deep Extension into dermis from ruptured follicle Neutrophilic luminal folliculitis / furunculosis Nodules, furuncles, sinus tracts Primarily systemic *Not comprehensive; # rarely biopsied Surface Superficial Deep Fig. 1. Schematic representation of depth of infection in canine pyoderma. 4 5

CLINICAL EXAMINATION CLINICAL EXAMINATION The clinical assessment should include a general physical examination of other organ systems (in case of systemic disease favouring infection) as well as a methodical examination of the entire integument so that all lesions can be identified and considered in the diagnostic process. Surface pyoderma Fold dermatitis (intertrigo) is clinically distinctive wherein inflammatory skin lesions are confined to facial, lip, neck, vulval (Fig. 2) or body folds. Accordingly specific breeds are most likely to be affected (for example, facial fold dermatitis in brachycephalic dogs (Fig. 3). Lesions comprise erythema and varying degrees of exudation and erosion. Differential diagnoses are normally limited to Malassezia dermatitis (which may mimic or co-exist with bacterial infection), mucocutaneous pyoderma (usually involving the lips and face) and rare cases of immunemediated erosive diseases, most often associated with the vulvar folds. When suspected, these disorders cannot be excluded on clinical grounds alone and further tests are necessary to confirm the diagnosis. There is controversy whether pyotraumatic dermatitis (acute moist dermatitis, hot spot ) represents a surface pyoderma (an infection by definition) or mere bacterial colonisation of an area of self-trauma (Holm et al., 2004, Cobb et al., 2005). These lesions typically develop rapidly in dogs that traumatise a single area, usually on the rump or neck, with intense pruritus, erosion and purulent exudation that mats overlying hair. These lesions must be differentiated from the less common pyotraumatic folliculitis, a deep necrotising folliculitis and furunculosis; satellite papules and pustules around the erosive lesion are suggestive of this deep pyoderma (Fig. 4). Fig. 2. Vulvar fold pyoderma: purulent exudation in the skin folds of a dog with a recessed vulva. Superficial pyoderma (bacterial folliculitis) The typical primary lesion of this most common form of canine pyoderma is a pustule that may occur in the interfollicular epidermis or centred on a hair follicle (hair emerges from lesion). These transient lesions are preceded by papules and evolve to crusts or epidermal collarettes; intact pustules are usually less frequently seen than the other lesions (Fig. 5). The groin and medial thighs are important sites although lesions commonly affect the dorsal and lateral trunk in some patients. Sterile pustular diseases are rare but important differential diagnoses, especially in cases where lesions occur on the face or pinnae, where bacteria are absent on cytology/ culture, and when rational antibiotic therapy is ineffective. Fig. 5. The primary lesion of superficial pyoderma is a follicular pustule (arrow), in this case with an intensely erythematous base, that rapidly evolves into epidermal collarettes and focal crusts. Deep pyoderma Deep pyoderma can present in a variety of different ways. Examples of localised disease include callus pyoderma (secondary infection of elbow and hock pressure calluses), pyotraumatic folliculitis (Fig. 4), and focal digital (Fig. 6) or interdigital lesions (most commonly a complication of conformational pododermatitis). More generalised disease can be seen in dogs with concurrent superficial lesions; important target sites include the trunk, although the feet are commonly infected in cases of pododemodicosis. Typically purulent exudation can be expressed from inflamed, swollen or thickened skin. Some cases should be differentiated from infection caused by fungi or atypical bacteria, or immune-mediated processes such as perianal fistulae or panniculitis. All deep pyoderma cases in dogs must be systematically and thoroughly evaluated for underlying demodicosis. Superficial pyoderma may commonly present as moth-eaten alopecia in shortcoated breeds; primary lesions of papules and pustules are often hard to locate and the bacterial aetiology of these lesions is sometimes over-looked and confused with dermatophytosis and demodicosis. Bacterial overgrowth presents with inflamed, lichenified, often pigmented malodourous skin with a greasy surface, usually involving the ventral trunk. Pus is not a clinical feature (although neutrophilic inflammation is seen histologically) and Malassezia dermatitis is the principle differential diagnosis. Fig. 3. Erythema and scaling in a brachycephalic dog with surface (facial fold) pyoderma. Fig. 4. Pyotraumatic furunculosis (illustrated) is a form of deep pyoderma that must be differentiated from the more common surface pyoderma, pyotraumatic dermatitis. The deep form is more likely to occur on the face or neck, be palpably thicker, and have satellite papules or pustules. Mucocutaneous pyoderma is an ill-defined disease of the muco-cutaneous junctions that is diagnosed in dogs with erosive lesions that are otherwise suggestive of immune-mediated diseases; response to systemic antibiotics is arguably the most compelling diagnostic procedure in these cases. Fig. 6. Deep pyoderma: multiple erythematous and alopecic papules have coalesced to form a large plaque with small sinus tracts. 6 7

CANINE PYODERMA DIAGNOSTIC TESTING CANINE PYODERMA DIAGNOSTIC TESTING Canine pyoderma commonly poses diagnostic challenges, due to its varied clinical presentation and tendency to be superimposed on other skin diseases. The goals of diagnostic testing are: to confirm the presence (thus supporting a clinical diagnosis) or absence of bacteria by cytological evaluation, to accurately identify the nature of the potential pathogen(s) by culture and likely effective therapy by sensitivity testing, to identify or exclude concurrent diseases likely to mimic or favour skin infection. A key clinical step is the recognition of the lesion types displayed by the patient, as this then dictates what investigations are most appropriate. Interpretation of test results from skin samples is complicated by the presence of a commensal microbiota which includes common pathogens such as Staphylococcus pseudintermedius and Malassezia spp. yeasts. The nature and extent of the investigations are commonly modified according to duration and severity of clinical disease, as well as the wishes, expectations and resources of the owner. Skin biopsy Skin biopsy specimens might show inflammatory patterns that support a diagnosis of pyoderma. However, histopathology is arguably most useful for differentiating pyoderma from other inflammatory diseases when the latter are suspected, and in the search for underlying diseases once pyoderma has been resolved. Additional tests A variety of additional tests should be routinely considered in cases of superficial and deep pyoderma, in case of alternative diagnoses or concurrent diseases (see flowchart below). In cases where dermatophytosis is possible, Wood s lamp examination, direct microscopy and fungal culture should be considered. Evaluation for ectoparasitic infestation should be routinely considered, for example for fleas in superficial pyoderma, and for demodicosis in superficial and especially deep pyoderma. Coat brushings, skin scrapings and hair plucks are most useful in these circumstances. Surveys indicate that cytological testing is infrequently done in dogs with skin disease in general veterinary practice 7 whereas it is routine in referral practice. The cytological techniques are rapid and inexpensive to perform, and require only basic laboratory facilities. Tape-stripping Tape-stripping is a versatile method for skin sampling and is especially indicated for the sampling of skin folds (surface pyoderma) and lichenified plaques (bacterial overgrowth). In either scenario, bacteria and or Malassezia spp. may be observed. In skin fold lesions, mixed infections with rods and cocci are often seen, whereas in bacterial overgrowth a monomorphous population of (staphylo)cocci can be expected. Clear adhesive tape is applied to the lesion of interest, removed, stained with Diff-Quik, and examined using the x100 oil-immersion objective. Tape-stripping provides a semiquantitative assessment of microbial populations: occasional bacteria might represent commensal bacteria whereas very large numbers can be expected in cases of infection. This method is also useful in monitoring the response to therapy. Bacterial culture Bacterial culture is normally performed using swabs from intact pustules (or epidermal collarettes) in superficial pyoderma, or discharging lesions in deep pyoderma. Culture and sensitivity testing is less often indicated in surface pyoderma where topical therapy with biocides is popular, and since in vitro culture results are not optimised for the high concentrations of drug achieved by the topical use of antibiotics. International concern over the emergence of multi-drug resistant bacteria has led to an increased awareness for responsible antibiotic use, which in turn makes culture more desirable ( the right drug for the right bug ). Culture is especially indicated in deep pyoderma (often severe lesions, mixed infections), in superficial cases that relapse or are refractory to therapy, and when rods are identified in cytological specimens (Fig.8), as these bacteria tend to have unpredictable sensitivity patterns. Care should be taken to sample representative lesions, and to rapidly deliver a swab in transport medium to the laboratory with sufficient clinical history to enable the laboratory to select appropriate cultural conditions. In cases of pyoderma, routine procedures are likely to be sufficient but observation of unusual/rare organisms such as actinomycetes on cytology should be reported. Direct impression Direct impression with a glass slide is more appropriate for lesions of superficial pyoderma characterised by pustules and epidermal collarettes. Intact pustules are ideal lesions to sample: a fine needle is used to open the head and then the slide is applied to the bead of pus, air-dried and stained. In cases of superficial pyoderma, large numbers of degenerate neutrophils with intra- and extracellular bacteria (usually cocci) are readily demonstrated (Fig. 7). When pustules cannot be identified, an impression of inflamed skin exposed by peeling back the scale at the margins of epidermal collarettes is a useful alternative. In deep pyoderma, impressions from furuncles and sinus tracts are likely to yield degenerate neutrophils and varying numbers of macrophages whereas bacteria can be expected to be much less abundant when compared with superficial lesions. Fig. 7. Impression smear from superficial pyoderma with typical coccoid bacteria amongst abundant neutrophils. Fig. 8. The presence of numerous rod-shaped bacteria amongst neutrophils in impression smears from lesions of pyoderma should prompt consideration of bacterial culture and sensitivity testing. Possibility of dermatophytosis? Wood's lamp examination Microscopy of hair plucks Fungal culture (Skin biopsy) Laboratory investigation of suspect pyoderma lesions Cytological methods to establish presence of microbes Malassezia spp. detected Topical (or systemic) antifungal therapy Consider tests for underlying cause Empirical topical (antiseptic or antibiotic) and/or systemic antibiotics depending on clinical aspects Bacteria detected (cocci,rods) Fig. 9. Approach to diagnosis in dogs with suspect pyoderma lesions utilising routine laboratory tests. Culture and sensitivity testing to identify possible pathogen(s) and guide specific therapy Concurrent check for ectoparasites (by microscopy of coat brushings, scrapings and/or hair plucks) routinely No bacteria detected Re-consider diagnosis Consider biopsy in case of sterile pustular / other inflammatory diseases 8 9

ANTIMICROBIAL THERAPY ANTIMICROBIAL THERAPY a Overview of pathogens Coagulase-positive staphylococci are the dominant pathogens in canine pyoderma; S. pseudintermedius accounts for the majority of cases (Fig. 10) although S. aureus and S. schlieferi subsp. coagulans are also of importance (Hillier et al., 2014). Historically, coagulasenegative staphylococci have tended to be regarded as non-pathogenic commensals (Bloom, 2014), but there is increasing evidence of their importance as pathogens (Cain et al., 2011). The coagulase negative S. schlieferi subsp. schlieferi is challenging because this zoonotic pathogen of canine skin and ears may be overlooked since many diagnostic laboratories do not currently speciate coagulase-negative staphylococci, contrary to current guidelines (Hillier et al., 2014, Cain et al., 2011). Pseudomonas aeruginosa is infrequent in pyoderma and is more often associated with deep infection, usually but not always in combination with staphylococci (Hillier et al., 2006). Recently, deep pyoderma caused by Burkholderia cepacia was reported in six dogs receiving oral ciclosporin (Banovic et al., 2015). b Are systemic antibacterial drugs necessary? The traditional therapeutic approach to superficial and deep pyoderma was to provide oral antibiotics; in an extensive survey of primary-care prescribing practices conducted in the UK in 2010, 91.9% of 659 dogs received at least one systemic antimicrobial (Summers et al., 2014). The need to limit the emergence of antimicrobial resistance prompted a FECAVA working group on antimicrobial use to present guidelines for a more critical appraisal of their need (http://www.fecava.org/content/guidelinespolicies); they suggest the use of antiseptics as a preferred alternative for conditions such as surface and superficial pyoderma, and when a delay to the use of a systemic antimicrobial will not negatively impact on the animal s wellbeing. Whilst the primary-care survey mentioned above showed that concurrent topical therapy was prescribed in 182 of 659 (27.7%) cases of pyoderma, topical therapy alone was used in only 31 dogs (4.7%) (Summers et al., 2014). These data indicate that a significant change in veterinary prescribing practice (and likely owner expectation and education) is needed if these guidelines are to be adopted. Should I perform a bacterial culture and susceptibility test in superficial and deep pyoderma? Historically it was unusual for an empiricallyselected anti-staphylococcal antibiotic to be ineffective in canine pyoderma but with the emergence of multidrug-resistant bacteria this is no longer the case. It has been usefully stated that a culture is never contra-indicated, and according to ISCAID guidelines (Hillier et al., 2014), culture is mandated in specific clinical circumstances in superficial pyoderma (Table 2.) Table 2. Factors to consider when deciding whether bacterial culture is indicated in canine pyoderma adapted from (Beco et al., 2013a) and (Hillier et al., 2014). Culture mandated should any of the following apply: <50% improvement within 2 weeks of starting treatment New lesions developing 2 weeks after starting treatment Ongoing lesions with cocci in cytological specimens after 6 weeks of treatment (intracellular) rods in cytological specimens History of multidrug resistant organism in patient or in-contact animal Empirical treatment without culture appropriate if: First episode of a superficial infection Following ineffective antiseptic therapy Only coccoid bacteria observed on cytology (not rods) No reasons to suspect antibiotic resistance Fig. 10. a) Staphylococcus pseudintermedius, the usual bacterial pathogen of dog skin, forms haemolytic flat, white, glistening, entire colonies on bovine and ovine sheep blood agar; b) that consist of Gram-positive cocci. Topical products of potential value include chlorhexidine alone or in combination with miconazole or EDTA, benzoyl peroxide, oxychlorine products (sodium hypochlorite and hypochlorous acid), and fusidic acid (Jeffers, 2013, Loeffler et al., 2012, Clark et al., 2015). In a recent evidence-based review (Mueller et al., 2012), good evidence of efficacy in canine pyoderma was identified for chlorhexidine, and to a lesser extent, benzoyl peroxide. Empirical antibiotic choices Various organisations have made recommendations on which antibiotics are appropriate for empirical use in canine pyoderma. FECAVA guidelines approve trimethoprim sulphonamide, clindamycin and cephalexin for superficial pyoderma, and cephalexin pending culture and sensitivity testing for deep pyoderma. ISCAID guidelines (which apply only to superficial pyoderma) refer to antibiotics suitable for empirical use as first-tier drugs ; these include potentiated sulphonamides, clindamycin, first generation cephalosporins (includes cephalexin), and co-amoxyclav (Hillier et al., 2014). Anecdotal reports suggest that cephalexin is strongly favoured by many dermatology specialists, although this approach is actively discouraged as a first-line approach in some countries due to the risk of selecting for methicillin-resistant staphylococci. The ISCAID working group was unable to reach consensus on whether cefovecin should be considered a first-tier or second-tier drug, in part due to concerns over potential for selection of extended-spectrum ß-lactamase-producing Escherichia coli (Hillier et al., 2014). Although evidence-based protocols are not currently available, it is generally accepted that treatment of superficial pyoderma should be extended for at least 7 days past clinical cure in order to prevent rapid recurrence, and for at least 14 days in cases of deep pyoderma (Hillier et al., 2014, Beco et al., 2013b). Duration of treatment The majority of cases of superficial pyoderma show a rapid response to treatment within 1-2 weeks and are resolved within 3 weeks, although individual cases may take up to 6 weeks to recover (Hillier et al., 2014). Cases of deep pyoderma normally respond more slowly and may require 6-12 weeks of treatment. Regular re-examination by the veterinary surgeon is necessary to establish when infection has resolved as this is beyond the skills of most pet owners, especially when signs of concurrent disease may need to be differentiated from those of the pyoderma. Unfortunately, a recent review of prescribing practices showed that dogs with pyoderma were commonly under-dosed or given courses that were inappropriately short (Summers et al., 2014). 10 11

ANTIMICROBIAL THERAPY Use of fluoroquinolones in canine pyoderma. Fluoroquinolones are considered by the World Health Organisation to be of critical importance in the treatment of certain life-threatening bacterial infections of humans (WHO, 2011). Whilst several drugs of this class are currently licensed for use in veterinary medicine, there is an ethical imperative that their use in animal health is strictly limited. The ISCAID working group consider these to be second tier treatment options to be considered only when topical therapy and first tier antimicrobials are not appropriate and when cultures indicate susceptibility (Hillier et al., 2014). In the author s clinic, they are most often used when deep pyoderma is complicated by Gram-negative pathogens such as Pseudomonas. LEARN MORE ABOUT THE MPC Simon Tappin Simon Tappin MA VetMB CertSAM DipECVIM-CA MRCVS European and RCVS Recognised Specialist in Veterinary Internal Medicine Hon. Assoc. Professor of Small Animal Medicine, University of Nottingham Simon graduated from the University of Cambridge and after two years in small animal practice undertook a residency at the University of Bristol in Small Animal Medicine and Intensive Care. He gained the European Diploma in Veterinary Internal Medicine in 2008. He is currently head of small animal medicine at Dick White Referrals, where he sees cases in all areas of internal medicine. Pradofloxacin Pradofloxacin is a third-generation veterinary fluoroquinolone with proven activity against S. pseudintermedius (Korber- Irrgang et al., 2012). Clinical trials have shown reliable efficacy in dogs with pyoderma. In one study of 107 dogs, clinical remission in pradofloxacin-treated dogs (86%) was higher than co-amoxyclav (73%); pradofloxacin was also more effective in preventing recurrence within 2 weeks of cessation (Mueller and Stephan, 2007). In pharmacokinetic-pharmacodynamic in vitro models, pradofloxacin showed faster and more sustained killing of 3 strains of S. intermedius than marbofloxacin. Pradofloxacin has also been shown to kill S. pseudintermedius more rapidly than cefazolin, cefovecin and doxycycline (Blondeau and Shebelski, 2016). The mutant prevention concentration (MPC) is a relatively recent concept in antimicrobial drug research; whilst the MIC predicts the likely activity of the drug, the MPC indicates the concentration needed to inhibit the development of drug resistant mutants (Fig 11). Studies indicate that pradofloxacin MPCs against coliforms and staphylococci are significantly lower than those of other veterinary fluoroquinolones (Wetzstein, 2005). This should theoretically translate into a reduced opportunity for therapeutic use of pradofloxacin to select for fluoroquinolone resistant bacteria; this may reflect in part dual activity and increased affinity of pradofloxacin against DNA gyrase and topo-isomerase enzymes. (Lees, 2013). Whilst there are arguments for limited use of third generation cephalosporins in animal health (Hillier et al., 2014), the use of this third-generation fluoro-quinolone may therefore be advantageous over second-generation products, provided use of a drug of this class is justified in the context of responsible antibiotic use (Lees, 2013). This hypothesis should be tested by large-scale prospective monitoring of antibacterial resistance in isolates from treated dogs. Bacterial genetic mutations resulting in antimicrobial resistance occur at a rate of around 1 in 10 million (10 7 ) bacteria. MIC testing utilises a standard inoculum of 100,000, or 10 5 bacterial colony forming units; this is a very useful test, but does not determine the concentration of antibiotic needed to kill spontaneously occurring mutant strains, as it isn t evaluating large enough bacterial populations. MPC testing uses larger inoculums of bacteria (10 9 CFU/ml) and as a result allows the identification of resistance arising from spontaneous mutations. This is an important concept as it allows the identification of the mutant selection window (MSW) which occurs at drug concentrations between the MIC and MPC. The mutant prevention concentration (MPC) offers a novel approach for the treatment of infectious diseases (Dong et al. 1999). Essentially, the MPC-approach favours a dose selection that not only aims at clinical cure but also at reducing selection of bacterial resistance. Bacterial genetic mutations resulting in antimicrobial resistance occur at a rate of around 1 in 10 million (10 7 ) bacteria. MIC testing utilises a standard inoculum of 100,000, or 10 5 bacterial colony forming units; this is a very useful test, but does not determine the concentration of antibiotic needed to kill spontaneously occurring mutant strains, as it isn t evaluating large enough bacterial populations. MPC testing uses larger inoculums of bacteria (10 9 CFU/ml) and as a result allows the identification of resistance arising from spontaneous mutations. This is an important concept as it allows the identification of the mutant selection window (MSW) which occurs at drug concentrations between the MIC and MPC. Summary The diagnosis and management of canine pyoderma is challenging due to the varied clinical presentation and the frequent association with concurrent skin diseases. Therapy is evolving with a greater appreciation of the role of topical therapy, which may replace or enhance the efficacy of traditional oral antibacterial treatment. Emergence of multidrug-resistant strains of Staphylococcus pseudintermedius represents a significant threat to the effectiveness of routine treatments. Recognition and correction where possible of underlying diseases is critical for successful control of relapsing cases, and is likely to limit the need for repeated courses of antibacterial treatments, and therefore selection pressure for resistance. Mutant Prevention Concentration (MPC) and Mutant Selection Window (MSW) (Blondeau et al. 2004) MSW Sub MIC neither susceptible nor first-step resistant mutants inhibited no selective amplification of resistant subpopulation Above MPC both susceptible and first-step resistant cells inhibited no selective amplification of resistant subpopulation MPC MSW - susceptible cells inhibited - first-step resistant cells not inhibited - selective amplification of resistant subpopulation MIC Time post-administration 12 13

LEARN MORE ABOUT THE MPC (CONT.) Simon Tappin REFERENCES: At drug concentrations above the MIC susceptible bacteria are killed but those that possess first-step mutations giving drug resistance are not, giving these isolates a selective advantage. Above the MPC, both susceptible bacteria and those with first-step mutations are killed, so there is no longer a selective advantage to the isolates with these first-step mutations. Fluoroquinolones and the MPC Fluoroquinolones are not considered to be a first line antimicrobial choice, their use should be reserved for those cases that haven't responded, or are unlikely to respond, to first line therapy, and should ideally be based on appropriate culture and sensitivity results. When the decision to use a fluoroquinolone has been made, it is worth knowing that there FLUOROQUINOLONE MPC PROFILE Pradofloxacin Enrofloxacin Marbofloxacin Orbifloxacin Difloxacin 0.6 3.5 3.5 0 2 4 6 8 10 12 14 16 18 9 MPC (μg/ml) 18 are differences between members of this class, in terms of how likely they are to select for fluoroquinolone resistance. Fluoroquinolones that are able to exceed MPC levels during therapeutic dosing, are less likely to fall into the MSW, and therefore less likely to leave spontaneous mutants behind in the population. At present MPC testing is technically challenging and is not commercially available, however theoretical knowledge still provides useful information for our patients. For example, the newer fluoroquinolone, pradofloxacin (Veraflox ), has the lowest available MPC values when compared to older generation fluoroquinolones such as marbofloxacin or enrofloxacin (Wetzstein 2005). Pradofloxacin (Veraflox ) is therefore able to exceed MPC levels during therapeutic dosing in these cases, making it less likely that early, first-stage mutant strains will be left behind(wetzstein 2005). This means that once a decision to use a fluoroquinolone has been made, pradofloxacin is the best choice in terms of limiting the development of resistance. Cmax: Peak level of antibiotic achieved in the serum of the animal with normal therapeutic dosing. The bars represent the Mutant Prevention Concentration(MPC) value for each antibiotic. This is the amount of antibiotic needed to kill both susceptible bacteria and bacteria that have spontaneously mutated. Where the bars turn dark purple beyond the Cmax, this indicates that the amount of antibiotic needed to reach MPC (and thus kill spontaneous mutants) is not achieved during normal dosing with the fluoroquinolone specified. Therapeutic Cmax Compared to other veterinary fluoroquinolones, Veraflox is the least likely to select for resistance By achieving drug concentrations that exceed the MPC, the potential for selection of resistant bacteria is reduced (Blondeau 2009) (Mateus et al.2011) With the lowest available MIC and MPC fluoroquinolone values overall, Veraflox fights both susceptible and first-step resistant bacteria at therapeutic concentrations Comparative MPC values of veterinary fluoroquinolones against Staphylococcus sp. in relation to serum drug levels reached in dogs (Wetzstein 2005) BANOVIC, F., KOCH, S., ROBSON, D., JACOB, M. & OLIVRY, T. 2015. Deep pyoderma caused by Burkholderia cepacia complex associated with ciclosporin administration in dogs: a case series. Vet Dermatol, 26, 287-e64. BECO, L., GUAGUERE, E., LORENTE MENDEZ, C., NOLI, C., NUTTALL, T. & VROOM, M. 2013a. Suggested guidelines for using systemic antimicrobials in bacterial skin infections (1): diagnosis based on clinical presentation, cytology and culture. Vet Rec, 172, 72-8. BECO, L., GUAGUERE, E., LORENTE MENDEZ, C., NOLI, C., NUTTALL, T. & VROOM, M. 2013b. Suggested guidelines for using systemic antimicrobials in bacterial skin infections: part 2-- antimicrobial choice, treatment regimens and compliance. Vet Rec, 172, 156-60. BLONDEAU, J. M. (2009). New concepts in antimicrobial susceptibility testing: the mutant prevention concentration and mutant selection window approach. Veterinary Dermatology 20: 383-396 BLONDEAU, J. M., HANSEN G. et al, (2004). The role of PK PD parameters to avoid selection and increase of resistance: mutant prevention concentration. Journal of Chemotherapy 16: 1-19 BLONDEAU, J. M. & SHEBELSKI, S. D. 2016. Comparative in vitro killing of canine strains of Staphylococcus pseudintermedius and Escherichia coli by cefovecin, cefazolin, doxycycline and pradofloxacin. Vet Dermatol, 27, 267-e63. BLOOM, P. 2014. Canine superficial bacterial folliculitis: current understanding of its etiology, diagnosis and treatment. Vet J, 199, 217-22. CAIN, C. L., MORRIS, D. O. & RANKIN, S. C. 2011. Clinical characterization of Staphylococcus schleiferi infections and identification of risk factors for acquisition of oxacillin-resistant strains in dogs: 225 cases (2003-2009). J Am Vet Med Assoc, 239, 1566-73. CLARK, S. M., LOEFFLER, A. & BOND, R. 2015. Susceptibility in vitro of canine methicillin-resistant and -susceptible staphylococcal isolates to fusidic acid, chlorhexidine and miconazole: opportunities for topical therapy of canine superficial pyoderma. J Antimicrob Chemother, 70, 2048-52. COBB, M. A., EDWARDS, H. J., JAGGER, T. D., MARSHALL, J. & BOWKER, K. E. 2005. Topical fusidic acid/betamethasone-containing gel compared to systemic therapy in the treatment of canine acute moist dermatitis. Vet J, 169, 276-80. DONG, Y., ZHAO, X. et al, (1999). Effect of fluoroquinolone concentration on selection of resistant mutants of Mycobacterium bovis BCG and Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43 (7): 1756-1758 HILL, P. B., LO, A., EDEN, C. A., HUNTLEY, S., MOREY, V., RAMSEY, S., RICHARDSON, C., SMITH, D. J., SUTTON, C., TAYLOR, M. D., THORPE, E., TIDMARSH, R. & WILLIAMS, V. 2006. Survey of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice. Vet Rec, 158, 533-9. HILLIER, A., ALCORN, J. R., COLE, L. K. & KOWALSKI, J. J. 2006. Pyoderma caused by Pseudomonas aeruginosa infection in dogs: 20 cases. Vet Dermatol, 17, 432-9. HILLIER, A., LLOYD, D. H., WEESE, J. S., BLONDEAU, J. M., BOOTHE, D., BREITSCHWERDT, E., GUARDABASSI, L., PAPICH, M. G., RANKIN, S., TURNIDGE, J. D. & SYKES, J. E. 2014. Guidelines for the diagnosis and antimicrobial therapy of canine superficial bacterial folliculitis (Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases). Vet Dermatol, 25, 163-75, e42-3. HOLM, B. R., REST, J. R. & SEEWALD, W. 2004. A prospective study of the clinical findings, treatment and histopathology of 44 cases of pyotraumatic dermatitis. Vet Dermatol, 15, 369-76. JEFFERS, J. G. 2013. Topical therapy for drug-resistant pyoderma in small animals. Vet Clin North Am Small Anim Pract, 43, 41-50. KORBER-IRRGANG, B., WETZSTEIN, H. G., BAGEL-TRAH, S., HAFNER, D. & KRESKEN, M. 2012. Comparative activity of pradofloxacin and marbofloxacin against coagulase-positive staphylococci in a pharmacokinetic-pharmacodynamic model based on canine pharmacokinetics. J Vet Pharmacol Ther, 35, 571-9. LEES, P. 2013. Pharmacokinetics, pharmacodynamics and therapeutics of pradofloxacin in the dog and cat. J Vet Pharmacol Ther, 36, 209-21. LOEFFLER, A., COBB, M. A. & BOND, R. 2012. Comparison of a chlorhexidine and a benzoyl peroxide shampoo as sole treatment in canine superficial pyoderma. Vet Rec, 169, 249. MATEUS, A., BRODBELT, D. et al, (2011). Evidence-based use of antimicrobials in veterinary practice. In Practice 33: 194 202 MUELLER, R. S., BERGVALL, K., BENSIGNOR, E. & BOND, R. 2012. A review of topical therapy for skin infections with bacteria and yeast. Vet Dermatol, 23, 330-41, e62. MUELLER, R. S. & STEPHAN, B. 2007. Pradofloxacin in the treatment of canine deep pyoderma: a multicentred, blinded, randomized parallel trial. Vet Dermatol, 18, 144-51. SUMMERS, J. F., HENDRICKS, A. & BRODBELT, D. C. 2014. Prescribing practices of primary-care veterinary practitioners in dogs diagnosed with bacterial pyoderma. BMC Vet Res, 10, 240. WETZSTEIN, H. G. 2005. Comparative mutant prevention concentrations of pradofloxacin and other veterinary fluoroquinolones indicate differing potentials in preventing selection of resistance. Antimicrob Agents Chemother, 49, 4166-73. WHO. 2011. WHO AGISAR Critically important antimicrobials for human medicine. [Online]. Available: www.who.int/entity/foodborne_disease/resistance/cia_3.pdf. 14 15

Use Medicines Responsibly (www.noah.co.uk/responsible) Veraflox 15 mg tablets contain 15 mg pradofloxacin. Veraflox 60 mg tablets contain 60 mg pradofloxacin. Veraflox 120 mg tablets contain 120 mg pradofloxacin. Veraflox 25 mg/ml oral suspension for cats contains 25 mg/ml pradofloxacin. Further information is available from the datasheet at noahcompendium.co.uk or on request. Advice should be sought from the medicine prescriber. Registered Trade Mark of Bayer AG. Bayer plc, Animal Health Division, Bayer House, Strawberry Hill, Newbury, Berkshire RG14 1JA Tel: 01635 563000 POM-V Bayer Ltd, Animal Health Division, The Atrium, Blackthorn Road, Dublin 18, Ireland Tel: 01 299 9313 POM EU/2/10/107/003 EU/2/10/107/007 EU/2/10/107/009 EU/2/10/107/013. L.GB.MKT.01.2017.16672 16 RESPONSIBLE CHOICE FOR SEVERE INFECTIONS