In vitro antimicrobial activity of miconazole and polymyxin B against canine meticillin-resistant Staphylococcus aureus and meticillin-resistant Staphylococcus pseudintermedius isolates

Background – Meticillin‐resistant Staphylococcus aureus (MRSA) and meticillin‐resistant Staphylococcus pseudintermedius (MRSP) infections are increasingly reported in dogs, and these bacteria may be isolated from ear infections. Hypothesis/Objectives – The main aim of the present study was to investigate the in vitro antimicrobial activity of miconazole, polymyxin B and a combination of both against 24 canine MRSA and 50 canine MRSP isolates. The minimal inhibitory concentration (MIC) values of 12 other antimicrobial agents were also determined. Methods – All MIC values were determined according to a broth microdilution assay. Results – Acquired resistance was found to all tested agents, except for linezolid, miconazole and polymyxin B. The MIC values for miconazole and polymyxin B against MRSA were in the range of 4–8 and 8–64 μg/mL, respectively, while the MIC values for miconazole and polymyxin B against MRSP were in the range of 1–2 and 0.25–4 μg/mL, respectively. Using a combination of miconazole and polymyxin B, there was no evidence for enhanced in vitro activity of the combination (i.e. synergy) of both products. Nevertheless, MIC₉₀ values of the combination of these antimicrobial agents and of a commercial product containing both agents were at least 1000 times lower than the concentration present in the commercial product. Conclusions and clinical importance – These results indicate that the topical use of a combination of miconazole and polymyxin B in a 43.5:1 ratio may have potential for the treatment of MRSA‐mediated and MRSP‐associated otitis externa in dogs.     

In vitro evaluation of topical biocide and antimicrobial susceptibility of Staphylococcus pseudintermedius from dogs

Background – Staphylococcus pseudintermedius is an important canine pathogen, and the emergence and widespread dissemination of meticillin‐resistant strains (MRSP) is of significant concern. Multidrug‐resistant infections may require alternative approaches, such as the use of topical therapy. There is minimal information about the in vitro susceptibility of meticillin‐susceptible S. pseudintermedius (MSSP) and MRSP to biocides and topical antimicrobials. Hypothesis/Objectives – The hypothesis was that clinical isolates of MSSP and MRSP would not have universal susceptibility to topical biocides and antimicrobials. The goal of this study was to assess the susceptibility of a collection of S. pseudintermedius isolates to selected antimicrobials and biocides. Animals – The study was performed on clinical isolates of MSSP and MRSP from dogs with skin and soft tissue infections collected throughout North America between 2006 and 2008. Methods – The minimal inhibitory concentrations (MICs) of chlorhexidine digluconate, benzalkonium chloride, triclosan, accelerated hydrogen peroxide, geranium oil, tea tree oil and grapefruit seed extract were tested for 25 MRSP and 25 MSSP isolates from dogs using the agar dilution method. The MICs of fusidic acid, bacitracin and mupirocin were determined using Etests. Results – Triclosan demonstrated excellent activity against all bacterial isolates, with no growth at the lowest concentration evaluated (MIC ≤ 0.5 μg/mL). Conversely, grapefruit seed extract did not inhibit growth at the highest concentration tested (MIC > 3.84 μg/mL). All isolates were susceptible to mupirocin, fusidic acid and bacitracin. There were no significant differences noted in the range, MIC₅₀ or MIC₉₀ between MSSP and MRSP isolates. Conclusions and clinical importance – While isolates were susceptible to most of the tested compounds, universal susceptibility to all compounds with potential antimicrobial activity cannot be assumed, and specific testing is required.     

Treatment outcome of dogs with meticillin-resistant and meticillin-susceptible Staphylococcus pseudintermedius pyoderma

Background – The prevalence of meticillin‐ and multidrug‐resistant Staphylococcus pseudintermedius (MRSP) in canine pyoderma has been increasing in recent years; thus, treatment of these cases has become challenging. Hypothesis/Objectives – To compare treatment outcome (clinical resolution and treatment duration), adverse effects of medication, and concurrent diseases and medications in dogs with meticillin‐susceptible S. pseudintermedius (MSSP) and MRSP pyoderma. Animals/Methods – Medical records were reviewed retrospectively, and 123 MSSP and 93 MRSP clinical cases between January 2008 and April 2010 were included. Results – In MSSP infections, cefalexin and cefpodoxime were the most commonly prescribed antimicrobials, accounting for 43.2 and 34.4% of cases, respectively. In MRSP infections, chloramphenicol and doxycycline were most commonly prescribed, accounting for 52.6 and 14.4% of cases, respectively. Adverse effects were reported in seven MSSP and 31 MRSP cases. The most commonly reported adverse effects were gastrointestinal, prompting antibiotic discontinuation in three MSSP and 20 MRSP cases. Chloramphenicol was associated with the highest incidence of adverse reactions (27 of 51 cases). Of 164 cases with follow up, 43 of 88 MSSP infections and 29 of 76 MRSP infections achieved complete clinical resolution at the first recheck examination. Three MSSP and seven MRSP cases failed to improve or resolve at subsequent visits assessed at 3–4 week intervals. Conclusions and clinical importance – Results from this study showed that the majority of pyodermas resolved regardless of meticillin susceptibility. Although some cases of MRSP pyoderma took longer to treat, this is likely to be because of chronicity and not the organism. In addition, adverse effects were frequently associated with chloramphenicol administration.     

Prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) from skin and carriage sites of dogs after treatment of their meticillin-resistant or meticillin-sensitive staphylococcal pyoderma

Background – Meticillin‐resistant staphylococci are significant pathogens in veterinary dermatology, yet longitudinal studies of the impact of routine antimicrobial therapy on emergence or resolution of resistance are lacking. Objectives – To determine the prevalence of meticillin‐resistant staphylococci on skin and carriage sites in dogs with bacterial pyoderma and evaluate the prevalence of meticillin‐resistant Staphylococcus pseudintermedius (MRSP) colonization after successful treatment of pyoderma. Animals – One hundred and seventy‐three dogs that presented to a dermatology referral service with pyoderma and 41 healthy control dogs. Methods – Skin, nasal and rectal swabs for bacterial culture were collected at the time of referral and after clinical resolution of the pyoderma. Meticillin resistance was confirmed by demonstration of penicillin binding protein 2a antigen. Results – Initially, skin cultures yielded MRSP in 70 (40.5%) dogs, meticillin‐resistant Staphylococcus aureus (MRSA) in three (1.7%) and meticillin‐resistant Staphylococcus schleiferi ssp. coagulans (MRSScoag) in five (2.9%). Samples collected from the nose and rectum (carriage sites) yielded MRSP in 59 (34.1%) dogs, MRSA in 11 (6.4%) and MRSScoag in seven (4.0%). One hundred and two dogs were available for follow‐up cultures after clinical cure. Of 42 dogs initially diagnosed with MRSP pyoderma, MRSP was isolated at follow‐up from skin in 19 (45.2%) and carriage sites in 20 (47.6%). Of 60 dogs that did not have MRSP pyoderma initially, MRSP was isolated post‐treatment from the skin in 17 (28.3%), and MRSP from carriage sites increased from 7.8% (initially) to 26.7% (P = 0.0022). Conclusions and clinical importance – Colonization by MRSP often persists after resolution of MRSP pyoderma. Acquisition of MRSP during treatment appears to be common.     

Comparative in vitro efficacy of antimicrobial shampoos: a pilot study

This study compared the antimicrobial efficacy of shampoos against meticillin‐sensitive Staphylococcus pseudintermedius (MSSP), meticillin‐resistant S. pseudintermedius (MRSP), antibiotic‐sensitive Pseudomonas aeruginosa (PA), multidrug‐resistant P. aeruginosa (MDR‐PA) and Malassezia pachydermatis. Three isolates were incubated for 10, 30 and 60 min with each shampoo diluted in phosphate‐buffered saline. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud’s dextrose agar (Malassezia). The minimal bactericidal concentrations (MBCs) for chlorhexidine products (Malaseb^®, Pyoderm^®/Microbex^® and Hibiscrub^®) were 1:1,024–1:2,048 for MSSP and MRSP, 1:512–1:1,024 for PA and MDR‐PA, and 1:2,048–1:5,096 for Malassezia at all time points. The MBCs for benzoyl peroxide (Paxcutol^®) for MSSP and MRSP were 1:2–1:8 at 10 min, and 1:256 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and 1:512–1:1,024 dilutions were effective against Malassezia at all time points. The MBCs for ethyl lactate (Etiderm^®) for MSSP and MRSP were 1:2 at 10 min, and 1:2–1:16 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and a 1:512 dilution was effective against Malassezia at all time points. Chloroxylenol (Coatex^®) and acetic acid–boric acid (Malacetic^®) were not effective against MSSP, MRSP or Pseudomonas. Both were effective against Malassezia at 1:8–1:16 dilution at 10 min, and at 1:8–1:32 dilution after 30 and 60 min. In conclusion, chlorhexidine appeared to be the most effective topical biocide, and MRSP and MDR‐PA were no less susceptible than antibiotic‐sensitive organisms. These results should, however, be confirmed with larger numbers of isolates.     

In vitro antiseptic susceptibilities for Staphylococcus pseudintermedius isolated from canine superficial pyoderma in Japan

Background –  Topical therapy, particularly with chlorhexidine, is becoming increasingly common as a treatment option for canine pyoderma; however, there are limited studies on the susceptibility of Staphylococcus pseudintermedius to chlorhexidine compounds. Objectives –  To determine the in vitro susceptibility of both meticillin‐resistant and meticillin‐susceptible S. pseudintermedius isolates to chlorhexidine and other antiseptic agents and the presence of multidrug efflux pump genes. Samples –  One hundred S. pseudintermedius isolates from 23 initial and 77 recurrent cases of canine pyoderma. Methods –  After bacterial identification and mecA testing, minimal inhibitory concentrations (MICs) of antiseptic agents were determined. Multidrug efflux pump genes, including qacA, qacB and smr, were identified. Results –  Of the 100 isolates, 57 were identified as meticillin‐resistant S. pseudintermedius. The MIC₉₀ of chlorhexidine acetate, chlorhexidine gluconate, acriflavine, ethidium bromide and benzalkonium chloride were 1, 1, 2, 0.5 and 2 μg/mL, respectively. Multidrug efflux pump genes qacA, qacB and smr were not detected in any of the isolates. Conclusions and clinical importance –  The MICs for chlorhexidine and other antiseptics remain low, and multidrug efflux pump genes were not found in the tested isolates.     

In vitro assessment of chloramphenicol and florfenicol as second‐line antimicrobial agents in dogs

The aim of this study was to evaluate the potential of chloramphenicol and florfenicol as second‐line antimicrobial agents for treatment of infections caused by methicillin‐resistant Staphyococcus pseudintermedius (MRSP) and extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli in dogs, through a systematic in vitro assessment of the pharmacodynamic properties of the two drugs. Minimum inhibitory concentrations (MIC) and phenicol resistance genes were determined for 169 S. pseudintermedius and 167 E. coli isolates. Minimum bactericidal concentrations (MBC), time‐killing kinetics, and postantibiotic effect (PAE) of both agents against wild‐type isolates of each species were assessed. For S. pseudintermedius, the chloramphenicol MIC₉₀ was 32 μg/mL. No florfenicol resistance was detected in this species (MIC_90 = 4 μg/mL). The MIC₉₀ of both agents against E. coli was 8 μg/mL. Resistance genes found were cat_pC221 in S. pseudintermedius and catA1 and/or floR in E. coli. The phenicols displayed a time‐dependent, mainly, bacteriostatic effect on both species. Prolonged PAEs were observed for S. pseudintermedius, and no PAEs were detected for E. coli. More research into determination of PK/PD targets of efficacy is needed to further assess the clinical use of chloramphenicol and florfenicol as second‐line agents in dogs, optimize dosage regimens, and set up species‐specific clinical break points.     

Molecular characterization of community‐associated methicillin‐resistant Staphylococcus aureus from pet dogs

Community‐associated methicillin‐resistant Staphylococcus aureus (MRSA) is a serious public health concern and in Australia, one that disproportionately affects Aboriginal people. Paralleling MRSA in human medicine, methicillin‐resistant S. pseudintermedius (MRSP) is an increasingly prevalent pathogen in veterinary medicine. We aimed to characterize the carriage of MRSA and MRSP in dogs and cats from predominantly Aboriginal communities in a very remote region of New South Wales (NSW), Australia. Pets (303 dogs and 80 cats) were recruited from six communities in western NSW. Three swabs were collected from each animal (anterior nares, oropharynx and perineum) and from skin lesions or wounds (if present) and cultured on selective media for methicillin‐resistant staphylococci. Human host‐adapted community‐associated MRSA representing four multilocus sequence types (ST1‐IV, ST5‐IV, ST72‐IV, ST93‐IV) were isolated from eight dogs (prevalence 2.6%, 95% confidence interval 1.3%–5.1%). Two ST5‐IV isolates from a single dog were phenotypically trimethoprim‐resistant, harbouring trimethoprim‐resistant gene dfrG within the SCCmec type IVo mobile genetic element. MRSA was not isolated from any cats and MRSP was not isolated from any dogs or cats. This study estimated a high prevalence of human host‐adapted community‐associated MRSA carriage in dogs despite an absence of MRSP. This suggests MRSA carried by dogs in remote NSW originate from human hosts. The cycle of transmission between people, dogs and common environmental sources warrants further investigation. To our knowledge, this is the first report of trimethoprim‐resistant ST5‐IV in eastern Australia and the first report of trimethoprim‐resistant ST5‐IV from a dog.     

The prevalence of carriage of meticillin‐resistant staphylococci by veterinary dermatology practice staff and their respective pets

It has been shown that people and pets can harbour identical strains of meticillin‐resistant (MR) staphylococci when they share an environment. Veterinary dermatology practitioners are a professional group with a high incidence of exposure to animals infected by Staphylococcus spp. The objective of this study was to assess the prevalence of carriage of MR Staphylococcus aureus (MRSA), MR S. pseudintermedius (MRSP) and MR S. schleiferi (MRSS) by veterinary dermatology practice staff and their personal pets. A swab technique and selective media were used to screen 171 veterinary dermatology practice staff and their respective pets (258 dogs and 160 cats). Samples were shipped by over‐night carrier. Human subjects completed a 22‐question survey of demographic and epidemiologic data relevant to staphylococcal transmission. The 171 human‐source samples yielded six MRSA (3.5%), nine MRSP (5.3%) and four MRSS (2.3%) isolates, while 418 animal‐source samples yielded eight MRSA (1.9%) 21 MRSP (5%), and two MRSS (0.5%) isolates. Concordant strains (genetically identical by pulsed‐field gel electrophoresis) were isolated from human subjects and their respective pets in four of 171 (2.9%) households: MRSA from one person/two pets and MRSP from three people/three pets. In seven additional households (4.1%), concordant strains were isolated from only the pets: MRSA in two households and MRSP in five households. There were no demographic or epidemiologic factors statistically associated with either human or animal carriage of MR staphylococci, or with concordant carriage by person–pet or pet–pet pairs. Lack of statistical associations may reflect an underpowered study.     

Low Rate of Methicillin‐resistant Coagulase‐positive Staphylococcal Colonization of Veterinary Personnel in Hong Kong

Elevated rates of methicillin‐resistant Staphylococcus aureus (MRSA) carriage have been reported in veterinary personnel, suggesting an occupational colonization risk. Hong Kong veterinary personnel (n = 150) were sampled for coagulase‐positive staphylococci (CPS) nasal colonization. Risk factors for colonization were assessed by questionnaire. Isolates were identified and antibiotic susceptibility determined. All CPS isolates were investigated for mecA carriage, SCCmec type and PVL genes. Two subjects were colonized with methicillin‐resistant CPS: one with MRSA (spa type t002 (CC5), SCCmec type II) and one with methicillin‐resistant Staphylococcus pseudintermedius (MRSP) (MLST type ST71, SCCmec type II‐III). MLST type ST71 S. pseudintermedius strain is the predominant MRSP clone circulating in dogs in Europe and in Hong Kong. The low MR‐CPS colonization rate may be associated with low levels of large animal exposure or low rates of MRSA colonization of companion animals in Hong Kong. Colonization with non‐aureus CPS, which may cause human infection, must also be considered in veterinary personnel.     

Antimicrobial resistance of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius, Staphylococcus intermedius and Staphylococcus delphini together comprise the S. intermedius group (SIG). Within the SIG, S. pseudintermedius represents the major pathogenic species and is involved in a wide variety of infections, mainly in dogs, but to a lesser degree also in other animal species and humans. Antimicrobial agents are commonly applied to control S. pseudintermedius infections; however, during recent years S. pseudintermedius isolates have been identified that are meticillin‐resistant and have also proved to be resistant to most of the antimicrobial agents approved for veterinary applications. This review deals with the genetic basis of antimicrobial resistance properties in S. pseudintermedius and other SIG members. A summary of the known resistance genes and their association with mobile genetic elements is given, as well as an update of the known resistance‐mediating mutations. These data show that, in contrast to other staphylococcal species, S. pseudintermedius seems to prefer transposon‐borne resistance genes, which are then incorporated into the chromosomal DNA, over plasmid‐located resistance genes.     

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

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

P‐17 In vitro activity of posaconazole and other antifungals against Malasseziapachydermatis isolated from dogs

The aim of this study was to determine the in vitro antifungal activity of several antifungal drugs (posaconazole, nystatin, miconazole and clotrimazole) against Malassezia pachydermatis with microdilution and agar dilution techniques. Malassezia pachydermatis isolates were obtained from the skin and ears of dogs. Tests on solid media were performed using 25‐well Petri dishes (2 mL/well containing Sabouraud's dextrose agar and diluted antifungal drug) inoculated with 5 μL suspensions of M. pachydermatis. Microtitre broth dilution used 96‐well microtitre plates containing Sabourauds dextrose broth and appropriate dilutions of antifungal drugs, inoculated with 10 μL standard suspensions of M. pachydermatis. Plates were inoculated in duplicate and incubated at 30°C for 5 days and growth assessed. The four antifungal drugs were tested in 10 dilutions (4.0‐0.007 μg/mL for posaconazole, and 32‐‐0.06 μg/mL for clotrimazole, miconazole and nystatin). Results obtained for 83 strains of M. pachydermatis and a control reference strain (CBS 1879) exhibited the same pattern. Results of the MIC between microtitre and agar methodologies showed no significant differences (≤ 2‐fold) across all drugs. For both solid and liquid methods, posaconazole was the most effective antifungal drug of the four tested with MIC₉₀ of 1–2 μg/mL for posaconazole, 16–32 μg/mL for clotrimazole, and ≥ 32 μg/mL for miconazole and nystatin. Funding: Schering‐Plough.     

Disposition of oral telithromycin in foals and in vitro activity of the drug against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates

Javsicas, LH., Giguère, S., Womble, AY. Disposition of oral telithromycin in foals and in vitro activity of the drug against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01151.x. The objectives of this study were to determine the serum and pulmonary disposition of telithromycin in foals and to determine the minimum inhibitory concentration (MIC) of telithromycin against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates. A single dose of telithromycin (15 mg/kg of body weight) was administered to six healthy 6–10‐week‐old foals by the intragastric route. Activity of telithromycin was measured in serum, pulmonary epithelial lining fluid (PELF), and bronchoalveolar lavage (BAL) cells using a microbiological assay. The broth macrodilution method was used to determine the MIC of telithromycin, azithromycin, clarithromycin and erythromycin against R. equi. Following intragastric administration, mean ± SD time to peak serum telithromycin activity (T_max) was 1.75 ± 0.76 h, maximum serum activity (C_max) was 1.43 ± 0.37 μg/mL, and terminal half‐life (t_½) was 3.81 ± 0.40 h. Telithromycin activity, 4 h postadministration was significantly higher in BAL cells (50.9 ± 14.5 μg/mL) than in PELF (5.07 ± 2.64 μg/mL), and plasma (0.84 ± 0.25 μg/mL). The MIC₉₀ of telithromycin for macrolide‐resistant R. equi isolates (8 μg/mL) was significantly higher than that of macrolide‐susceptible isolates (0.25 μg/mL). The MIC of telithromycin for macrolide‐resistant isolates (MIC₅₀ = 4.0 μg/mL) was significantly lower than that of clarithromycin (MIC₅₀ = 24.0 μg/mL), azithromycin (MIC₅₀ =256 μg/mL) and erythromycin (MIC₅₀ = 24 μg/mL).     

In vitro antimicrobial activity of a commercial ear antiseptic containing chlorhexidine and Tris–EDTA

Minimum bactericidal concentrations (MBCs) of a commercial ear antiseptic containing chlorhexidine 0.15% and Tris–EDTA (Otodine^®) were determined by broth microdilution for 150 isolates representing the most common pathogens associated with canine otitis. The microorganisms were classified into three groups according to their levels of susceptibility. The most susceptible group included Staphylococcus pseudintermedius, Malassezia pachydermatis, Streptococcus canis and Corynebacterium auriscanis, which were generally killed by 1 : 64 dilution of the antiseptic product (MBC = 23/0.8 μg/mL of chlorhexidine/Tris–EDTA). The most resistant organism was Proteus mirabilis, which survived up to 1 : 8 dilution of the product (MBC = 375/12 μg/mL). Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus displayed intermediate MBCs ranging between 188/6 and 47/1.5 μg/mL. Interestingly, S. pseudintermedius was more susceptible than S. aureus, and no significant difference was observed between meticillin‐resistant and meticillin‐susceptible isolates within each species, indicating that antiseptic use is unlikely to co‐select for meticillin resistance. Although the concentrations required for killing (MBCs) varied considerably with microorganism type, the combination of chlorhexidine 0.15% and Tris–EDTA was active against all the pathogens most commonly involved in canine otitis.     

In vitro antimicrobial efficacy of β‐defensin 3 against Staphylococcus pseudintermedius isolates from healthy and atopic canine skin

β‐Defensins (BDs) are highly conserved antimicrobial peptides important in innate defence against bacteria. β‐Defensin 3 has a specific role in protecting the skin. This study quantified the minimal inhibitory concentration (MIC) of human (h)BD3 against Staphylococcus pseudintermedius isolates from atopic and healthy dogs. Single colony isolates (1 × 10⁵ colony‐forming units/mL log phase) were cultured with doubling dilutions of hBD3 in sodium phosphate buffer from 0.8 to 50 μg/mL at 37 °C for 2 h, before adding 100 μL of tryptone soy broth and incubating for a further 20 h. Bacterial growth was assessed as the mean optical density at 540 nm corrected for background. The median MIC was 12.5 μg hBD3/mL (range 3.125–25 μg/mL; n = 22). Forty‐five percent of the isolates were inhibited at ≤6.25 μg hBD3/mL, and 90% were inhibited at ≤12.5 μg hBD3/mL. Bacterial growth was not inhibited at ≤1.6 μg hBD3/mL. There were no significant differences in the inhibition by hBD3 of isolates from atopic (median MIC 12.5 μg/mL, range 6.25–25 μg/mL, n = 14) and healthy dogs (median MIC 9.4 μg/mL, range 3.125–12.5 μg/mL, n = 8); from noninfected colonized sites (median MIC 12.5 μg/mL, range 3.125–25 μg/mL, n = 16) and infected lesions (median MIC 9.4 μg/mL, range 6.25–12.5 μg/mL, n = 6); or between sample sites (nose median MIC 12.5 μg/mL, range 6.25–25 μg/mL, n = 5; perineum median MIC 12.5 μg/mL, range 3.125–25 μg/mL, n = 7; ear median MIC 6.25 μg/mL, range 6.25–12.5 μg/mL, n = 4; lesions median MIC 9.4 μg/mL, range 6.25–12.5 μg/mL, n = 6). In conclusion, hBD3 inhibited the growth of canine S. pseudintermedius isolates in vitro irrespective of origin.     

Prevalence of and Resistance to Anti‐Microbial Drugs in Selected Microbial Species Isolated from Bulk Milk Samples

The prevalence of strains of Staphylococcus aureus, coagulase‐negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti‐microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co‐trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC₅₀, MIC₉₀, MIC range) were determined for each microbial species. Resistance against one or more anti‐microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E. faecalis, 55% of E. faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC₅₀ 4 μg/ml). A high percentage of staphylococci were resistant to β‐lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 μg/ml) and strains resistant to CMP (MIC₉₀ 16 μg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coli (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.     

Detection and characterization of methicillin-resistant Staphylococcus pseudintermedius in healthy dogs in La Rioja, Spain

The objective was to identify the methicillin-resistant coagulase-positive staphylococci (MRCoPS) nasal carriage rate of healthy dogs in La Rioja (Spain) and to characterize the recovered isolates by different molecular techniques. Nasal samples from 196 dogs were obtained (98 household-dogs, 98 pound-dogs). Isolates were identified and characterized by spa-, SCCmec- and MLST-typing, SmaI-PFGE, antimicrobial susceptibility, determination of antimicrobial resistance and toxin genes profiling. S. pseudintermedius was the only species recovered. Nine methicillin-resistant S. pseudintermedius (MRSP) were obtained from 9 of 196 sampled dogs (8% pound-dogs, 1% household-dogs). MRSP isolates were typed (MLST/PFGE/spa/SCCmec) as: ST71/A/t02/II–III (7 isolates), ST92/C/t06/V (1 isolate), and ST26/B/non-typable/non-typable (1 isolate). All MRSP were resistant to [resistance gene/number isolates]: β-lactams [mecA + blaZ/9], tetracycline [tet(K)/7, tet(M)/2], macrolides and lincosamides [erm(B)/9], aminoglycosides [aacA-aphD + aadE + aphA-3/9], and co-trimoxazol [dfr(G)/9]. Eight MRSP isolates showed also resistance to fluoroquinolones and amino acid changes in GyrA [Ser84Leu + Glu714Lys, 7 isolates; Ser84Leu, 1 isolate] and GrlA [Ser80Ile, 8 isolates] proteins were detected. The remaining isolate was chloramphenicol resistant and harboured cat_pC221 gene. All MRSP isolates harboured the aadE-sat4-aphA-3 multiresistance-gene-cluster linked to erm(B) gene as well as the siet, si-ent and lukS/F-I toxin genes. MRSP is a moderately common (4.6%) colonizer of healthy dogs in Spain. A major MRSP lineage (ST71) was detected and its future evolution should be tracked.     

Genetic relatedness of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from a dog and the dog owner

In the present study four methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains isolated from a dog (n = 3) and the anterior nares of the dog owner (n = 1) were investigated by conventional and molecular methods. The species identity of the four S. pseudintermedius strains was confirmed by conventional methods, by PCR mediated amplification of S. intermedius/S. pseudintermedius specific segments of thermonuclease encoding gene nuc and by restriction fragment length polymorphism analysis of phosphoacetyltransferase encoding gene pta. Investigation of the four S. pseudintermedius for toxinogenic potential revealed that all four strains were positive for the exfoliative toxin encoding gene siet and the leukotoxin encoding genes lukS, lukF. The oxacillin and penicillin resistance of the four S. pseudintermedius strains could be determined by cultivation of the strains on oxacillin resistant screening agar base, ChromID MRSA Agar and Brilliance MRSA Agar and by multiplex PCR detecting the resistance genes mecA and blaZ. The genetic relatedness of the strains was studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). According to PFGE all four S. pseudintermedius strains represent an identical bacterial clone indicating a cross transmission between the dog and the dog owner.