Vet Dermatol 2014; 25: 35–e12
DOI: 10.1111/vde.12091
Canine antimicrobial peptides are effective against resistant bacteria and yeasts Domenico Santoro* and Carol W. Maddox† *Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Drive, Urbana, IL 61802, USA †Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USA Correspondence: Domenico Santoro, Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, 2015 SW 16th Avenue, Gainesville, FL 32608, USA. E-mail: [email protected]
Background – Antimicrobial peptides (AMPs) are small proteins present in most living species and they are involved in the defence against pathogenic organisms. b-Defensins and cathelicidin are the most frequently studied AMPs in both people and dogs. Hypothesis/Objectives – Our objectives were to determine the minimal inhibitory concentration and minimal bactericidal concentration of two canine b-defensins and a cathelicidin against antibiotic-sensitive and antibioticresistant bacteria and yeasts using a broth microdilution method. Methods – The micro-organisms tested were ATCC strains of meticillin-sensitive and meticillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively), meticillin-sensitive Staphylococcus pseudintermedius (MSSP), Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Malassezia pachydermatis. Field strains of meticillin-resistant S, pseudintermedius (MRSP, n = 1) and MSSP (n = 11) were also tested. Mann–Whitney U-test and Friedman test were used for statistical analysis. Results – The MSSP and MRSP were more susceptible to canine AMPs than MSSA and MRSA. Malassezia pachydermatis was more sensitive than C. albicans to canine AMPs. The b-defensin cBD103 was most effective against Staphylococci and P. aeruginosa, while the cathelicidin cCath was the most effective AMP against E. coli. Additionally, cBD103 was the most effective AMP for both yeasts studied, with M. pachydermatis being more susceptible than C. albicans. All AMPs tested exhibited killing within 2 h of exposure. Conclusions and clinical importance – We demonstrated that natural canine AMPs are effective against canine-specific pathogens, are equally effective against meticillin-resistant or -susceptible strains, and are more effective against M. pachydermatis than against C. albicans organisms.
Introduction Over the past decade, an increase in antibacterial resistance has been reported in dogs and human patients with atopic dermatitis.1,2 The frequent use of antibiotics may have contributed to an increase in bacterial resistance worldwide.1,2 Antimicrobial peptides (AMPs) are small, predominantly cationic proteins; they play a fundamental role in the defence against micro-organisms.3 They are produced by epithelial and immune cells.4 Antimicrobial peptides such as b-defensins (BDs) and cathelicidin (Cath) have been widely studied. They attach to micro-organisms based upon a high affinity for anionic molecules on the microbes’ surface and lead to membrane damage, disruption of the ion gradient and subsequent death of the micro-organism.5 Due to their mechanism of action,
Accepted 13 September 2013 Sources of Funding: This study was funded by the American College of Veterinary Dermatology. Conflict of Interest: No conflicts of interest have been declared. © 2013 ESVD and ACVD, Veterinary Dermatology, 25, 35–e12.
AMPs have shown very low bacterial, fungal and viral resistance.3 Four BDs and one Cath have been isolated in human skin. They have antimicrobial properties against bacteria, including meticillin-resistant Staphylococcus aureus (MRSA), yeasts and viruses. Six BDs (cBD1-like, cBD2like/122, cBD3-like, cBD102, cBD103 and cBD127) and one cathelicidin (cCath) have been identified in canine epithelia.6–11 The conserved sequences of four cBDs (cBD1like, cBD2-like/122, cBD3-like and cBD102),8 cBD1039,10 and cCath11 have antimicrobial activity against meticillinsensitive S. aureus (MSSA), meticillin-sensitive and meticillin-resistant Staphylococcus pseudintermedius (MSSP and MRSP, respectively), Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. To date, no reports have quantified the effects [minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)] of cAMPs against antibiotic-sensitive or -resistant bacteria (including MRSA and MRSP), nor against Malassezia pachydermatis. The objective of this study, therefore, was to determine the MIC and MBC of cAMPs for common pathogenic micro-organisms (see Table 1). 35
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Table 1. Bacteria and yeasts tested for susceptibility to canine antimicrobial peptides
All the micro-organisms were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Field strains of MSSP were isolated by the Veterinary Diagnostic Laboratory staff from dogs with pyoderma.
The bacteria were grown overnight on Columbia blood agar (Remel, Lenexa, KS, USA) at 37°C in air supplemented with 5% CO2. Yeasts were grown for 48 h on Sabouraud dextrose agar plates (Remel) at 35°C in an air-forced incubator. Isolated colonies were suspended in sterile double distilled water to achieve an optical density equal to a McFarland 0.5 standard (Sensititre Inc., Westlake, OH, USA). The bacterial/yeast suspension was then diluted ~1:500 to obtain a final concentration of 1 x 106 colony-forming units (CFU)/ mL. Twofold serial dilutions of test peptide in SPB (final concentrations of 200–3.125 lg/mL) were added (100 lL) to each well containing 100 lL of inoculum. Negative control wells contained 100 lL of SPB without bacteria, while positive control wells contained 50 lL of SPB instead of the test peptides. Initial inoculum density was confirmed by plate count. The number of bacteria after 2 h of incubation at 35°C in SPB was counted to assess antimicrobial effects of SPB. For each well containing cAMPs, 100 lL was plated on Columbia blood agar to determine the viability after the 2 h exposure. The MIC was reported as the lowest concentration of cAMPs without visible €ller-Hinton broth (100 lL) with TES (MHB; Sensititre Inc.) growth. Mu was added to each well, and the plates were incubated at 35°C for another 18–20 h. The same methodology was used to determine the MIC and MBC for C. albicans and M. pachydermatis with the exception of the medium used, Yeast-One (Trek Diagnostic Systems, Cleveland, OH, USA) instead of MHB. Given that M. pachydermatis growth was difficult to detect after overnight incubation, the inoculum/peptide and the control wells were plated on Columbia blood agar at 35°C for 24–48 h.
Peptide preparation
Minimal bactericidal concentration assays
Based on previous studies,8,9,11 the following three peptides were synthesized (Peptide Protein Research Ltd, Fareham, UK) and tested: the 34-amino-acid peptide shared by four cBDs (cBD); a 45-aminoacid peptide synthesized from the C-terminus of cBD103; and a 38amino-acid peptide synthesized from the C-terminus of cCath. One milligram of each lyophilized peptide was diluted in 10 mM 0.01% acetic acid to generate a stock concentration of 4 mg/mL. The 0.01% acetic acid solution activates the bonds between the cysteine residues without adverse effects on fungi or other bacteria.8,11–13 Each peptide was further diluted (1:10) with 10 mM sodium phosphate buffer at pH 7.4 (SPB).12
All assays were performed in duplicate wells. After 18–20 h of incubation, 100 lL of inoculum/peptide solution at the MIC and the concentrations above and below the MIC were spirally plated on Columbia blood agar and incubated at 35°C for 24 h. The MBC was defined as the lowest dilution at which micro-organisms were no longer viable on subculture.
Micro-organism
Isolates
MSSA
ATCC-29213 + 1 UIUC-VDL clinical isolate ATCC-43300 ATCC-49444 + 11 UIUC-VDL clinical isolates UIUC-VDL clinical isolate ATCC-25922 ATCC-27853 ATCC-16231 ATCC-14522
MRSA MSSP MRSP Escherichia coli Pseudomonas aeruginosa Candida albicans Malassezia pachydermatis
Abbreviations: MRSA, meticillin-resistant Staphylococcus aureus; MSSA, meticillin-sensitive S. aureus; MRSP, meticillin-resistant Staphylococcus pseudintermedius; MSSP, meticillin-sensitive S. pseudintermedius; and UIUC-VDL, University of Illinois - Veterinary Diagnostic Laboratory.
Materials and methods Micro-organism isolates
Minimal inhibitory concentration assays All assays were performed in duplicate wells in sterile 96-well polystyrene round-bottomed plates (Costarâ; Corning Inc., Corning, NY, USA) using a broth microdilution method adapted from Clinical and Laboratory Standards Institute (CLSI) methods.14,15 All assays were independently repeated three times, and the MIC was calculated as the median of the triplicates. The MIC was defined as the lowest antimicrobial concentration inhibiting growth of the organism in the well. The experimental error was accepted if falling within the doubling dilution.
Statistical analysis The Mann–Whitney U-test was used to compare MICs and MBCs of ATCC isolates versus field isolates of MSSP. The Friedman test was used to compare MICs and MBCs of ATCC strains for the different antimicrobials. P-Values of 200 >200 50 100 >200
100 100 25 50 50
100 100 25 50 25
>200 >200 50 100 >200
100 100 25 50 50
100 >200 25 50 25
50
12.5
25
25
25
25
50
25
>200
100
50
>200
25
12.5
25
100
50
50
difference was noted for MICs of cBDs and cCath for MSSP isolates. cBD was less effective than cBD103 and cCath against both field (MIC, P < 0.001; MBC, P = 0.049) and ATCC isolates of MSSP (MIC, P = 0.004; MBC, P = 0.004).
Discussion
Abbreviations: cBDs, conserved sequence of three canine b-defensins; cBD103, canine b-defensin 103; cCath, canine cathelicidin; MBC, minimal bactericidal concentration; MIC, minimal inhibitory concentration; other abbreviations are as for Table 1. *Average of three repeats of duplicate wells observed visually and reported as no growth. †Average of three repeats of duplicate wells plated to detect viable growth.
except E. coli (Table 3). cCath was the most effective cAMP against E. coli. Most of the MBCs were within the doubling dilution compared with the MICs of each cAMP. At the MIC values, no growth was observed after 2 h of pre-incubation, suggesting that cAMPs are effective within 2 h of contact time. However, even at the lowest peptide concentration (3.125 lg/mL), growth of bacteria was dramatically inhibited after 2 h (range, 99.4–100%). No effect of the SPB was seen on the growth of bacteria (Table 2). Eleven field isolates of MSSP were screened for susceptibility to the three cAMPs. A significant difference between the MIC of cBD and cBD103 and cCath was observed (P = 0.049). The MIC for cBD was ≥200 lg/mL, while the MIC for both cBD103 and cCath was 50 lg/mL (Table 4). A statistically significant decrease in susceptibility (higher MICs) of field isolates to cBD103 and cCath was seen when compared with the MICs of ATCC isolates of MSSP (P = 0.013 and P = 0.048). Likewise, a higher MBC for cBD103 was present between the field isolates and the ATCC isolates of MSSP (P = 0.044). No
We demonstrated that cBD103 and cCath are highly effective against Gram-positive bacteria, Gram-negative bacteria and yeasts. Different peptides have a different spectrum of activity, with cBD103 being overall the most effective against the micro-organisms tested. Few studies have been published on the antimicrobial effects of cAMPs.8–11 Due to differences in methods and/or in peptides used, a comparison between them and the present study is impossible. In agreement with previous results,11 cCath was more effective against Gramnegative than against Gram-positive bacteria. Malassezia pachydermatis was highly sensitive to cCath, whereas C. albicans was resistant at the highest concentration tested (200 lg/mL). Results for cBD and cBD103 were different when compared with previous studies.8,10 cBD was reported to be highly effective against E. coli, but less effective against C. albicans and MSSA.8 In our study, cBD was highly effective against both yeasts tested, P. aeruginosa and the ATCC isolates but not the field isolates of S. pseudintermedius (MSSP > MRSP), but it was not effective against E. coli or MSSA/MRSA. Similar discordant results exist between this and a previous study for cBD103.10 Leonard et al.10 reported that cBD103 was effective against E. coli, MRSP and MSSP. In the present study, cBD103 was the most effective cAMP for all the micro-organisms tested, except E. coli. Differences between this and previously published studies on cBD8 and cBD10310 may be due to different ATCC strains, to the different methodology used and field isolates used, which may have a different pattern of resistance to cAMPs from the ATCC strains. Field strains may have been more in contact with AMPs than ATCC strains, changing their sensitivity to cAMPs. One limitation of this study is the number of field isolates tested. However, it is possible that field isolates of other bacteria have similar results that MSSP had
Table 4. Summary of the field strains of Staphylococcus spp. tested and their susceptibility to canine antimicrobial peptides MIC (lg/mL)
MBC (lg/mL)
Staphylococcus spp. strains
UIUC–VDL accession no.
cBDs
cBD103
cCath
cBDs
cBD103
cCath
(1) MSSP (2) MSSP (3) MSSP (4) MSSP (5) MRSP (6) MSSP (7) MSSP (8) MSSP (9) MSSP (10) MSSP (11) MSSP (12) MSSP
12-46786 12-46873 13-30073 11-43740 12-49755 12-41388 12-37915 12-37957 12-32217 12-312512-30314 11-43451
200 >200 >200 >200 >200 >200 >200 >200 200 200 200 200
50 50 50 50 50 50 50 50 50 50 50 50
50 50 50 50 25 50 50 50 100 50 50 100
>200 >200 >200 >200 >200 >200 >200 >200 200 200 200 200
50 50 50 50 50 50 50 50 100 50 50 100
100 100 100 50 25 200 100 50 100 50 50 100
Abbreviations are as for Table 3.
© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 35–e12.
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compared to ATCC strains. The authors recognize that the antimicrobial peptide concentrations tested exceed those reasonable for systemic therapy. However, these concentrations of cAMPs still have potential for topical applications. Further investigations to establish the MICs for field isolates of bacteria and yeasts commonly involved in dermatitis are necessary and warranted.
Acknowledgements The authors are grateful to Mariangela Segre for the suggestions and critical comments made during the entire study.
References 1. DeBoer DJ, Marsella R. The ACVD task force on canine atopic dermatitis (XII): the relationship of cutaneous infections to the pathogenesis and clinical course of canine atopic dermatitis. Vet Immunol Immunopathol 2001; 81: 239–249. ska-Mrowiecka M, Czubak-Macugowska M 2. Kedzierska A, Kapin et al. Susceptibility testing and resistance phenotype detection in Staphylococcus aureus strains isolated from patients with atopic dermatitis, with apparent and recurrent skin colonization. Br J Dermatol 2008; 159: 1290–1299. 3. Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clin Microbiol Rev 2006; 19: 491–511. 4. Braff MH, Bardan A, Nizet V et al. Cutaneous defense mechanisms by antimicrobial peptides. J Invest Dermatol 2005; 125: 9–13. 5. Boman HG, Agerberth B, Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect Immun 1993; 61: 2978–2984.
6. Leonard BC, Affolter VK, Bevins CL. Antimicrobial peptides: agents of border protection for companion animals. Vet Dermatol 2012; 23: 177–e36. 7. Santoro D, Bunick D, Graves TK et al. Expression and distribution of antimicrobial peptides in the skin of healthy beagles. Vet Dermatol 2011; 22: 61–67. 8. Sang Y, Ortega T, Blecha F et al. Molecular cloning and characterization of three b-defensins from canine testes. Infect Immun 2005; 73: 2611–2620. 9. Erles K, Brownlie J. Expression of b-defensins in the canine respiratory tract and antimicrobial activity against Bordetella bronchiseptica. Vet Immunol Immunopathol 2010; 135: 12–19. 10. Leonard BC, Marks SL, Outerbridge CA et al. Activity, expression and genetic variation of canine b-defensin 103: a multifunctional antimicrobial peptide in the skin of domestic dogs. J Innate Immun 2012; 4: 248–259. 11. Sang Y, Teresa Ortega M, Rune K et al. Canine cathelicidin (K9CATH): gene cloning, expression, and biochemical activity of a novel pro-myeloid antimicrobial peptide. Dev Comp Immunol 2007; 31: 1278–1296. 12. Fazakerley J, Crossley J, McEwan N et al. In vitro antimicrobial efficacy of b-defensin 3 against Staphylococcus pseudintermedius isolates from healthy and atopic canine skin. Vet Dermatol 2010; 21: 463–468. 13. Fritz P, Beck-Jendroschek V, Brasch J. Inhibition of dermatophytes by the antimicrobial peptides human b-defensin-2, ribonuclease 7 and psoriasin. Med Mycol 2012; 50: 579–584. 14. Watts JL, Shryock TR, Apley M et al. Performance standards for antimicrobial disk and dilution susceptibility tests of bacteria isolated from animals; approved standard – third edition. CLSI Bulletin 2008; 28: 23–37. 15. Rex JH, Alexander BD, Andes D et al. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard – third edition. CLSI Bulletin 2008; 28: 6–11.
sume Re ines pre sentes chez la plupart des Contexte – Les peptides antimicrobiens (AMPs) sont de petites prote ces vivantes et ils sont implique s dans la de fense contre les organismes pathoge nes. Les b-de fenespe licidine sont les AMPs les plus fre quemment e tudie s sines et la cathe a la fois chez l’homme et chez le chien. ses/Objectifs – Nos objectifs e taient de de terminer la concentration minimale inhibitrice et la Hypothe ricide pour deux b-de fensines et une cathe licidine de chien contre des bacte concentration minimale bacte sistantes aux antibiotiques thode de microdilution en ries et des levures sensibles et re a l’aide d’une me milieu liquide. thodes – Les micro-organismes teste s e taient des souches ATCC de Staphylococcus aureus sensibles Me a la me ticilline et re sistantes a la me ticilline (respectivement MSSA et MRSA), Staphylococcus pseudinterticilline (MSSP), Escherichia coli, Pseudomonas aeruginosa, Candida albicans et medius sensibles a la me sistantes ticilline (MRSP, n = 1) Malassezia pachydermatis. Les souches de S. pseudintermedius re a la me galement e te teste es. Des statistiques ont e te re alise es par les tests Mann–Whitet MSSP (n = 11) ont e ney U-test et Friedman. sultats – Les MSSP er MRSP e taient plus sensibles aux AMPs canins que MSSA et MRSA. Malassezia Re tait plus sensible que C. albicans aux AMP canins. La b-de fensine cBD103 e tait plus efficpachydermatis e licidine cCath e tait l’AMP la plus efficace conace contre Staphylococci et P. aeruginosa, alors que la cathe tait l’AMP le plus efficace pour les deux levures, avec M. pachydermatis tre E. coli. En outre, cBD103 e s ont e te efficaces dans les 2 h d’exposition. plus sensible que C. albicans. Tous les AMPs teste montrons que les AMPs naturels canins sont efficaces Conclusions et importance clinique – Nous de nes spe cifiques du chien, sont tout autant efficaces contre les souches re sistantes ou contre les pathoge ticilline et sont plus efficaces contre M. pachydermatis que contre C. albicans. sensibles a la me Resumen n – los pe ptidos antimicrobianos (AMPs) son pequen ~as proteınas presentes en la mayorıa de Introduccio genos. Las ß-defensinas y la catlos seres vivos y estan implicadas en la defensa frente a organismos pato elicidina son las AMPs mas frecuentemente estudiadas tanto en humanos como en perros.
38
© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 35–e12.
Canine antimicrobial peptides
tesis/Objetivos – nuestros objetivos fueron determinar la concentracio n inhibitoria mınima y la conHipo n bactericida mınima de dos ß-defensinas caninas y de la catecilidina frente a bacterias sensibles centracio ticos y levaduras utilizando un me todo de microdilucio n en caldo de cultivo. y resistentes a antibio todos – los microorganismos probados fueron cepas ATCC de Staphylococcus aureus sensibles y reMe sistentes a meticilina (MSSA y MRSA, respectivamente), Staphylococcus pseudintermedius sensible a meticilina (MSSP), Escherichia coli, Pseudomonas aeruginosa, Candida albicans y Malassezia pachydermatis. Cepas de campo resistentes a meticilina de S. pseudintermedius (MRSP, n = 1) y MSSP (n = 11) tamn fueron probadas. En el analisis estadıstico se utilizaron la prueba U de Mann-Whitney y la prueba de bie Friedman. Resultados – las cepas MSSP y MRSP fueron mas susceptibles a las AMPs caninas que MSSA y MRSA. Malassezia pachydermatis fue mas sensible que C. albicans a las AMPs caninas. La defensina ß cBD103 fue la AMP mas eficaz frente a E. coli. Adem as, cBD103 fue la AMP mas eficaz frente a ambas especies de levadura, siendo M. pachydermatis mas susceptible que C. albicans. Todas las AMPs probadas den inicial. struyeron los microorganismos en menos de 2 h tras la exposicio Conclusiones e importancia clınica – hemos demostrado que las AMPs caninas son efectivas frente a genos caninos, son igual de eficaces frente a cepas sensibles y resistentes a antibio ticos, y son mas pato efectivas frente a M. pachydermatis que C. albicans. Zusammenfassung Hintergrund – Antimikrobielle Peptide (AMPs) sind kleine Proteine, die in den meisten lebenden Spezies vorkommen und bei der Abwehr pathogener Organismen involviert sind. b-Defensine und Cathelicidin sind beim Menschen wie auch beim Hund die am h€ aufigsten untersuchten AMPs. Hypothese/Ziele – Unsere Ziele waren es, die minimale inhibitorische Konzentration und die minimale €r Antibiobakterizide Konzentration zweier b-Defensine des Hundes festzustellen, sowie ein Cathelicidin fu tika-sensitive und Antibiotika-resistente Bakterien und Hefepilze mittels Bouillon-Mikrodilutionsmethode zu ermitteln. Methoden – Die untersuchten Mikroorganismen waren ATCC St€ amme Methicillin-sensitiver und Methicillin-resistenter Staphylococcus aureus (MSSA bzw. MRSA), Methicillin-sensitiver Staphylococcus pseudintermedius (MSSP), Escherichia coli, Pseudomonas aeruginosa, Canida albicans und Malassezia pachydermatis. Es wurden ebenfalls Feldst€ amme Methicillin-resistenter S. pseudintermedius (MRSP, n = 1) und MSSP (n = 11) getestet. Zur statistischen Analyse wurden Mann-Whitney U-Test und Friedman €hrt. Test durchgefu €r die AMPs des Hundes als MSSA und MRSA. Ergebnisse – Die MSSP und MRSP waren empf€ anglicher fu €r die AMPs des Hundes als Canida albicans. Die b-Defensine Malassezia pachydermatis war sensibler fu €ber Staphylokokken und P. aeruginosa, w€ cBD103 waren effektiver gegenu ahrend die Cathelicidine cCATH €r bedie effektivsten AMP gegen E. coli darstellten. Zus€ atzlich waren die cBD103 die effektivsten AMP fu ide untersuchten Hefepilze, wobei M. Pachydermatis sensibler war als C. albicans. Alle getesteten AMPs €teten Keime innerhalb von 2 h nach Verabreichung ab. to €rlichen AMPs des Hundes geSchlussfolgerungen und klinische Bedeutung – Wir zeigten, dass die natu €ber Hunde-spezifischen Pathogenen wirksam sind, dass sie gleichermaßen wirksam sind gegenu €ber genu €ber M. pachyMethicillin-resistenten oder –empf€anglichen St€ ammen, und dass sie wirksamer sind gegenu €ber C. albicans Organismen. dermatis als gegenu
© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 35–e12.
e11
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© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 35–e12.
DOI: 10.1111/vde.12091
Canine antimicrobial peptides are effective against resistant bacteria and yeasts Domenico Santoro* and Carol W. Maddox† *Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Drive, Urbana, IL 61802, USA †Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USA Correspondence: Domenico Santoro, Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, 2015 SW 16th Avenue, Gainesville, FL 32608, USA. E-mail: [email protected]
Background – Antimicrobial peptides (AMPs) are small proteins present in most living species and they are involved in the defence against pathogenic organisms. b-Defensins and cathelicidin are the most frequently studied AMPs in both people and dogs. Hypothesis/Objectives – Our objectives were to determine the minimal inhibitory concentration and minimal bactericidal concentration of two canine b-defensins and a cathelicidin against antibiotic-sensitive and antibioticresistant bacteria and yeasts using a broth microdilution method. Methods – The micro-organisms tested were ATCC strains of meticillin-sensitive and meticillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively), meticillin-sensitive Staphylococcus pseudintermedius (MSSP), Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Malassezia pachydermatis. Field strains of meticillin-resistant S, pseudintermedius (MRSP, n = 1) and MSSP (n = 11) were also tested. Mann–Whitney U-test and Friedman test were used for statistical analysis. Results – The MSSP and MRSP were more susceptible to canine AMPs than MSSA and MRSA. Malassezia pachydermatis was more sensitive than C. albicans to canine AMPs. The b-defensin cBD103 was most effective against Staphylococci and P. aeruginosa, while the cathelicidin cCath was the most effective AMP against E. coli. Additionally, cBD103 was the most effective AMP for both yeasts studied, with M. pachydermatis being more susceptible than C. albicans. All AMPs tested exhibited killing within 2 h of exposure. Conclusions and clinical importance – We demonstrated that natural canine AMPs are effective against canine-specific pathogens, are equally effective against meticillin-resistant or -susceptible strains, and are more effective against M. pachydermatis than against C. albicans organisms.
Introduction Over the past decade, an increase in antibacterial resistance has been reported in dogs and human patients with atopic dermatitis.1,2 The frequent use of antibiotics may have contributed to an increase in bacterial resistance worldwide.1,2 Antimicrobial peptides (AMPs) are small, predominantly cationic proteins; they play a fundamental role in the defence against micro-organisms.3 They are produced by epithelial and immune cells.4 Antimicrobial peptides such as b-defensins (BDs) and cathelicidin (Cath) have been widely studied. They attach to micro-organisms based upon a high affinity for anionic molecules on the microbes’ surface and lead to membrane damage, disruption of the ion gradient and subsequent death of the micro-organism.5 Due to their mechanism of action,
Accepted 13 September 2013 Sources of Funding: This study was funded by the American College of Veterinary Dermatology. Conflict of Interest: No conflicts of interest have been declared. © 2013 ESVD and ACVD, Veterinary Dermatology, 25, 35–e12.
AMPs have shown very low bacterial, fungal and viral resistance.3 Four BDs and one Cath have been isolated in human skin. They have antimicrobial properties against bacteria, including meticillin-resistant Staphylococcus aureus (MRSA), yeasts and viruses. Six BDs (cBD1-like, cBD2like/122, cBD3-like, cBD102, cBD103 and cBD127) and one cathelicidin (cCath) have been identified in canine epithelia.6–11 The conserved sequences of four cBDs (cBD1like, cBD2-like/122, cBD3-like and cBD102),8 cBD1039,10 and cCath11 have antimicrobial activity against meticillinsensitive S. aureus (MSSA), meticillin-sensitive and meticillin-resistant Staphylococcus pseudintermedius (MSSP and MRSP, respectively), Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. To date, no reports have quantified the effects [minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)] of cAMPs against antibiotic-sensitive or -resistant bacteria (including MRSA and MRSP), nor against Malassezia pachydermatis. The objective of this study, therefore, was to determine the MIC and MBC of cAMPs for common pathogenic micro-organisms (see Table 1). 35
Santoro and Maddox
Table 1. Bacteria and yeasts tested for susceptibility to canine antimicrobial peptides
All the micro-organisms were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Field strains of MSSP were isolated by the Veterinary Diagnostic Laboratory staff from dogs with pyoderma.
The bacteria were grown overnight on Columbia blood agar (Remel, Lenexa, KS, USA) at 37°C in air supplemented with 5% CO2. Yeasts were grown for 48 h on Sabouraud dextrose agar plates (Remel) at 35°C in an air-forced incubator. Isolated colonies were suspended in sterile double distilled water to achieve an optical density equal to a McFarland 0.5 standard (Sensititre Inc., Westlake, OH, USA). The bacterial/yeast suspension was then diluted ~1:500 to obtain a final concentration of 1 x 106 colony-forming units (CFU)/ mL. Twofold serial dilutions of test peptide in SPB (final concentrations of 200–3.125 lg/mL) were added (100 lL) to each well containing 100 lL of inoculum. Negative control wells contained 100 lL of SPB without bacteria, while positive control wells contained 50 lL of SPB instead of the test peptides. Initial inoculum density was confirmed by plate count. The number of bacteria after 2 h of incubation at 35°C in SPB was counted to assess antimicrobial effects of SPB. For each well containing cAMPs, 100 lL was plated on Columbia blood agar to determine the viability after the 2 h exposure. The MIC was reported as the lowest concentration of cAMPs without visible €ller-Hinton broth (100 lL) with TES (MHB; Sensititre Inc.) growth. Mu was added to each well, and the plates were incubated at 35°C for another 18–20 h. The same methodology was used to determine the MIC and MBC for C. albicans and M. pachydermatis with the exception of the medium used, Yeast-One (Trek Diagnostic Systems, Cleveland, OH, USA) instead of MHB. Given that M. pachydermatis growth was difficult to detect after overnight incubation, the inoculum/peptide and the control wells were plated on Columbia blood agar at 35°C for 24–48 h.
Peptide preparation
Minimal bactericidal concentration assays
Based on previous studies,8,9,11 the following three peptides were synthesized (Peptide Protein Research Ltd, Fareham, UK) and tested: the 34-amino-acid peptide shared by four cBDs (cBD); a 45-aminoacid peptide synthesized from the C-terminus of cBD103; and a 38amino-acid peptide synthesized from the C-terminus of cCath. One milligram of each lyophilized peptide was diluted in 10 mM 0.01% acetic acid to generate a stock concentration of 4 mg/mL. The 0.01% acetic acid solution activates the bonds between the cysteine residues without adverse effects on fungi or other bacteria.8,11–13 Each peptide was further diluted (1:10) with 10 mM sodium phosphate buffer at pH 7.4 (SPB).12
All assays were performed in duplicate wells. After 18–20 h of incubation, 100 lL of inoculum/peptide solution at the MIC and the concentrations above and below the MIC were spirally plated on Columbia blood agar and incubated at 35°C for 24 h. The MBC was defined as the lowest dilution at which micro-organisms were no longer viable on subculture.
Micro-organism
Isolates
MSSA
ATCC-29213 + 1 UIUC-VDL clinical isolate ATCC-43300 ATCC-49444 + 11 UIUC-VDL clinical isolates UIUC-VDL clinical isolate ATCC-25922 ATCC-27853 ATCC-16231 ATCC-14522
MRSA MSSP MRSP Escherichia coli Pseudomonas aeruginosa Candida albicans Malassezia pachydermatis
Abbreviations: MRSA, meticillin-resistant Staphylococcus aureus; MSSA, meticillin-sensitive S. aureus; MRSP, meticillin-resistant Staphylococcus pseudintermedius; MSSP, meticillin-sensitive S. pseudintermedius; and UIUC-VDL, University of Illinois - Veterinary Diagnostic Laboratory.
Materials and methods Micro-organism isolates
Minimal inhibitory concentration assays All assays were performed in duplicate wells in sterile 96-well polystyrene round-bottomed plates (Costarâ; Corning Inc., Corning, NY, USA) using a broth microdilution method adapted from Clinical and Laboratory Standards Institute (CLSI) methods.14,15 All assays were independently repeated three times, and the MIC was calculated as the median of the triplicates. The MIC was defined as the lowest antimicrobial concentration inhibiting growth of the organism in the well. The experimental error was accepted if falling within the doubling dilution.
Statistical analysis The Mann–Whitney U-test was used to compare MICs and MBCs of ATCC isolates versus field isolates of MSSP. The Friedman test was used to compare MICs and MBCs of ATCC strains for the different antimicrobials. P-Values of 200 >200 50 100 >200
100 100 25 50 50
100 100 25 50 25
>200 >200 50 100 >200
100 100 25 50 50
100 >200 25 50 25
50
12.5
25
25
25
25
50
25
>200
100
50
>200
25
12.5
25
100
50
50
difference was noted for MICs of cBDs and cCath for MSSP isolates. cBD was less effective than cBD103 and cCath against both field (MIC, P < 0.001; MBC, P = 0.049) and ATCC isolates of MSSP (MIC, P = 0.004; MBC, P = 0.004).
Discussion
Abbreviations: cBDs, conserved sequence of three canine b-defensins; cBD103, canine b-defensin 103; cCath, canine cathelicidin; MBC, minimal bactericidal concentration; MIC, minimal inhibitory concentration; other abbreviations are as for Table 1. *Average of three repeats of duplicate wells observed visually and reported as no growth. †Average of three repeats of duplicate wells plated to detect viable growth.
except E. coli (Table 3). cCath was the most effective cAMP against E. coli. Most of the MBCs were within the doubling dilution compared with the MICs of each cAMP. At the MIC values, no growth was observed after 2 h of pre-incubation, suggesting that cAMPs are effective within 2 h of contact time. However, even at the lowest peptide concentration (3.125 lg/mL), growth of bacteria was dramatically inhibited after 2 h (range, 99.4–100%). No effect of the SPB was seen on the growth of bacteria (Table 2). Eleven field isolates of MSSP were screened for susceptibility to the three cAMPs. A significant difference between the MIC of cBD and cBD103 and cCath was observed (P = 0.049). The MIC for cBD was ≥200 lg/mL, while the MIC for both cBD103 and cCath was 50 lg/mL (Table 4). A statistically significant decrease in susceptibility (higher MICs) of field isolates to cBD103 and cCath was seen when compared with the MICs of ATCC isolates of MSSP (P = 0.013 and P = 0.048). Likewise, a higher MBC for cBD103 was present between the field isolates and the ATCC isolates of MSSP (P = 0.044). No
We demonstrated that cBD103 and cCath are highly effective against Gram-positive bacteria, Gram-negative bacteria and yeasts. Different peptides have a different spectrum of activity, with cBD103 being overall the most effective against the micro-organisms tested. Few studies have been published on the antimicrobial effects of cAMPs.8–11 Due to differences in methods and/or in peptides used, a comparison between them and the present study is impossible. In agreement with previous results,11 cCath was more effective against Gramnegative than against Gram-positive bacteria. Malassezia pachydermatis was highly sensitive to cCath, whereas C. albicans was resistant at the highest concentration tested (200 lg/mL). Results for cBD and cBD103 were different when compared with previous studies.8,10 cBD was reported to be highly effective against E. coli, but less effective against C. albicans and MSSA.8 In our study, cBD was highly effective against both yeasts tested, P. aeruginosa and the ATCC isolates but not the field isolates of S. pseudintermedius (MSSP > MRSP), but it was not effective against E. coli or MSSA/MRSA. Similar discordant results exist between this and a previous study for cBD103.10 Leonard et al.10 reported that cBD103 was effective against E. coli, MRSP and MSSP. In the present study, cBD103 was the most effective cAMP for all the micro-organisms tested, except E. coli. Differences between this and previously published studies on cBD8 and cBD10310 may be due to different ATCC strains, to the different methodology used and field isolates used, which may have a different pattern of resistance to cAMPs from the ATCC strains. Field strains may have been more in contact with AMPs than ATCC strains, changing their sensitivity to cAMPs. One limitation of this study is the number of field isolates tested. However, it is possible that field isolates of other bacteria have similar results that MSSP had
Table 4. Summary of the field strains of Staphylococcus spp. tested and their susceptibility to canine antimicrobial peptides MIC (lg/mL)
MBC (lg/mL)
Staphylococcus spp. strains
UIUC–VDL accession no.
cBDs
cBD103
cCath
cBDs
cBD103
cCath
(1) MSSP (2) MSSP (3) MSSP (4) MSSP (5) MRSP (6) MSSP (7) MSSP (8) MSSP (9) MSSP (10) MSSP (11) MSSP (12) MSSP
12-46786 12-46873 13-30073 11-43740 12-49755 12-41388 12-37915 12-37957 12-32217 12-312512-30314 11-43451
200 >200 >200 >200 >200 >200 >200 >200 200 200 200 200
50 50 50 50 50 50 50 50 50 50 50 50
50 50 50 50 25 50 50 50 100 50 50 100
>200 >200 >200 >200 >200 >200 >200 >200 200 200 200 200
50 50 50 50 50 50 50 50 100 50 50 100
100 100 100 50 25 200 100 50 100 50 50 100
Abbreviations are as for Table 3.
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compared to ATCC strains. The authors recognize that the antimicrobial peptide concentrations tested exceed those reasonable for systemic therapy. However, these concentrations of cAMPs still have potential for topical applications. Further investigations to establish the MICs for field isolates of bacteria and yeasts commonly involved in dermatitis are necessary and warranted.
Acknowledgements The authors are grateful to Mariangela Segre for the suggestions and critical comments made during the entire study.
References 1. DeBoer DJ, Marsella R. The ACVD task force on canine atopic dermatitis (XII): the relationship of cutaneous infections to the pathogenesis and clinical course of canine atopic dermatitis. Vet Immunol Immunopathol 2001; 81: 239–249. ska-Mrowiecka M, Czubak-Macugowska M 2. Kedzierska A, Kapin et al. Susceptibility testing and resistance phenotype detection in Staphylococcus aureus strains isolated from patients with atopic dermatitis, with apparent and recurrent skin colonization. Br J Dermatol 2008; 159: 1290–1299. 3. Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clin Microbiol Rev 2006; 19: 491–511. 4. Braff MH, Bardan A, Nizet V et al. Cutaneous defense mechanisms by antimicrobial peptides. J Invest Dermatol 2005; 125: 9–13. 5. Boman HG, Agerberth B, Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect Immun 1993; 61: 2978–2984.
6. Leonard BC, Affolter VK, Bevins CL. Antimicrobial peptides: agents of border protection for companion animals. Vet Dermatol 2012; 23: 177–e36. 7. Santoro D, Bunick D, Graves TK et al. Expression and distribution of antimicrobial peptides in the skin of healthy beagles. Vet Dermatol 2011; 22: 61–67. 8. Sang Y, Ortega T, Blecha F et al. Molecular cloning and characterization of three b-defensins from canine testes. Infect Immun 2005; 73: 2611–2620. 9. Erles K, Brownlie J. Expression of b-defensins in the canine respiratory tract and antimicrobial activity against Bordetella bronchiseptica. Vet Immunol Immunopathol 2010; 135: 12–19. 10. Leonard BC, Marks SL, Outerbridge CA et al. Activity, expression and genetic variation of canine b-defensin 103: a multifunctional antimicrobial peptide in the skin of domestic dogs. J Innate Immun 2012; 4: 248–259. 11. Sang Y, Teresa Ortega M, Rune K et al. Canine cathelicidin (K9CATH): gene cloning, expression, and biochemical activity of a novel pro-myeloid antimicrobial peptide. Dev Comp Immunol 2007; 31: 1278–1296. 12. Fazakerley J, Crossley J, McEwan N et al. In vitro antimicrobial efficacy of b-defensin 3 against Staphylococcus pseudintermedius isolates from healthy and atopic canine skin. Vet Dermatol 2010; 21: 463–468. 13. Fritz P, Beck-Jendroschek V, Brasch J. Inhibition of dermatophytes by the antimicrobial peptides human b-defensin-2, ribonuclease 7 and psoriasin. Med Mycol 2012; 50: 579–584. 14. Watts JL, Shryock TR, Apley M et al. Performance standards for antimicrobial disk and dilution susceptibility tests of bacteria isolated from animals; approved standard – third edition. CLSI Bulletin 2008; 28: 23–37. 15. Rex JH, Alexander BD, Andes D et al. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard – third edition. CLSI Bulletin 2008; 28: 6–11.
sume Re ines pre sentes chez la plupart des Contexte – Les peptides antimicrobiens (AMPs) sont de petites prote ces vivantes et ils sont implique s dans la de fense contre les organismes pathoge nes. Les b-de fenespe licidine sont les AMPs les plus fre quemment e tudie s sines et la cathe a la fois chez l’homme et chez le chien. ses/Objectifs – Nos objectifs e taient de de terminer la concentration minimale inhibitrice et la Hypothe ricide pour deux b-de fensines et une cathe licidine de chien contre des bacte concentration minimale bacte sistantes aux antibiotiques thode de microdilution en ries et des levures sensibles et re a l’aide d’une me milieu liquide. thodes – Les micro-organismes teste s e taient des souches ATCC de Staphylococcus aureus sensibles Me a la me ticilline et re sistantes a la me ticilline (respectivement MSSA et MRSA), Staphylococcus pseudinterticilline (MSSP), Escherichia coli, Pseudomonas aeruginosa, Candida albicans et medius sensibles a la me sistantes ticilline (MRSP, n = 1) Malassezia pachydermatis. Les souches de S. pseudintermedius re a la me galement e te teste es. Des statistiques ont e te re alise es par les tests Mann–Whitet MSSP (n = 11) ont e ney U-test et Friedman. sultats – Les MSSP er MRSP e taient plus sensibles aux AMPs canins que MSSA et MRSA. Malassezia Re tait plus sensible que C. albicans aux AMP canins. La b-de fensine cBD103 e tait plus efficpachydermatis e licidine cCath e tait l’AMP la plus efficace conace contre Staphylococci et P. aeruginosa, alors que la cathe tait l’AMP le plus efficace pour les deux levures, avec M. pachydermatis tre E. coli. En outre, cBD103 e s ont e te efficaces dans les 2 h d’exposition. plus sensible que C. albicans. Tous les AMPs teste montrons que les AMPs naturels canins sont efficaces Conclusions et importance clinique – Nous de nes spe cifiques du chien, sont tout autant efficaces contre les souches re sistantes ou contre les pathoge ticilline et sont plus efficaces contre M. pachydermatis que contre C. albicans. sensibles a la me Resumen n – los pe ptidos antimicrobianos (AMPs) son pequen ~as proteınas presentes en la mayorıa de Introduccio genos. Las ß-defensinas y la catlos seres vivos y estan implicadas en la defensa frente a organismos pato elicidina son las AMPs mas frecuentemente estudiadas tanto en humanos como en perros.
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Canine antimicrobial peptides
tesis/Objetivos – nuestros objetivos fueron determinar la concentracio n inhibitoria mınima y la conHipo n bactericida mınima de dos ß-defensinas caninas y de la catecilidina frente a bacterias sensibles centracio ticos y levaduras utilizando un me todo de microdilucio n en caldo de cultivo. y resistentes a antibio todos – los microorganismos probados fueron cepas ATCC de Staphylococcus aureus sensibles y reMe sistentes a meticilina (MSSA y MRSA, respectivamente), Staphylococcus pseudintermedius sensible a meticilina (MSSP), Escherichia coli, Pseudomonas aeruginosa, Candida albicans y Malassezia pachydermatis. Cepas de campo resistentes a meticilina de S. pseudintermedius (MRSP, n = 1) y MSSP (n = 11) tamn fueron probadas. En el analisis estadıstico se utilizaron la prueba U de Mann-Whitney y la prueba de bie Friedman. Resultados – las cepas MSSP y MRSP fueron mas susceptibles a las AMPs caninas que MSSA y MRSA. Malassezia pachydermatis fue mas sensible que C. albicans a las AMPs caninas. La defensina ß cBD103 fue la AMP mas eficaz frente a E. coli. Adem as, cBD103 fue la AMP mas eficaz frente a ambas especies de levadura, siendo M. pachydermatis mas susceptible que C. albicans. Todas las AMPs probadas den inicial. struyeron los microorganismos en menos de 2 h tras la exposicio Conclusiones e importancia clınica – hemos demostrado que las AMPs caninas son efectivas frente a genos caninos, son igual de eficaces frente a cepas sensibles y resistentes a antibio ticos, y son mas pato efectivas frente a M. pachydermatis que C. albicans. Zusammenfassung Hintergrund – Antimikrobielle Peptide (AMPs) sind kleine Proteine, die in den meisten lebenden Spezies vorkommen und bei der Abwehr pathogener Organismen involviert sind. b-Defensine und Cathelicidin sind beim Menschen wie auch beim Hund die am h€ aufigsten untersuchten AMPs. Hypothese/Ziele – Unsere Ziele waren es, die minimale inhibitorische Konzentration und die minimale €r Antibiobakterizide Konzentration zweier b-Defensine des Hundes festzustellen, sowie ein Cathelicidin fu tika-sensitive und Antibiotika-resistente Bakterien und Hefepilze mittels Bouillon-Mikrodilutionsmethode zu ermitteln. Methoden – Die untersuchten Mikroorganismen waren ATCC St€ amme Methicillin-sensitiver und Methicillin-resistenter Staphylococcus aureus (MSSA bzw. MRSA), Methicillin-sensitiver Staphylococcus pseudintermedius (MSSP), Escherichia coli, Pseudomonas aeruginosa, Canida albicans und Malassezia pachydermatis. Es wurden ebenfalls Feldst€ amme Methicillin-resistenter S. pseudintermedius (MRSP, n = 1) und MSSP (n = 11) getestet. Zur statistischen Analyse wurden Mann-Whitney U-Test und Friedman €hrt. Test durchgefu €r die AMPs des Hundes als MSSA und MRSA. Ergebnisse – Die MSSP und MRSP waren empf€ anglicher fu €r die AMPs des Hundes als Canida albicans. Die b-Defensine Malassezia pachydermatis war sensibler fu €ber Staphylokokken und P. aeruginosa, w€ cBD103 waren effektiver gegenu ahrend die Cathelicidine cCATH €r bedie effektivsten AMP gegen E. coli darstellten. Zus€ atzlich waren die cBD103 die effektivsten AMP fu ide untersuchten Hefepilze, wobei M. Pachydermatis sensibler war als C. albicans. Alle getesteten AMPs €teten Keime innerhalb von 2 h nach Verabreichung ab. to €rlichen AMPs des Hundes geSchlussfolgerungen und klinische Bedeutung – Wir zeigten, dass die natu €ber Hunde-spezifischen Pathogenen wirksam sind, dass sie gleichermaßen wirksam sind gegenu €ber genu €ber M. pachyMethicillin-resistenten oder –empf€anglichen St€ ammen, und dass sie wirksamer sind gegenu €ber C. albicans Organismen. dermatis als gegenu
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© 2013 ESVD and ACVD, Veterinary Dermatology, 25, 35–e12.
