In vitro activity of selected antimicrobials against methicillin-resistant Staphylococcus pseudintermedius of canine origin in Poland.

BACKGROUND: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important veterinary pathogen. In general, only a few antimicrobials show in vitro activity against MRSP isolates. OBJECTIVES: The objective of this study was to determine the in vitro activity of selected antimicrobials, including last-choice drugs, against clinical MRSP isolates of canine origin. The activity of 10 selected agents was evaluated against 41 clinical MRSP isolates. METHODS: The disk diffusion method and minimal inhibitory concentration values were used for antimicrobial susceptibility testing (AST). The guidelines for staphylococci of canine or human origin were employed for the interpretation of the results. RESULTS: Among the examined MRSP isolates, resistance to enrofloxacin and clindamycin was the most prevalent (n = 40; 97.6%). Resistance to doxycycline and gentamicin was observed in 83.0% (n = 34) and 68.3% (n = 28) of the isolates, respectively. Single isolates were resistant to chloramphenicol (n = 5; 12.2%) and rifampicin (n = 3; 7.3%), whereas all showed susceptibility to amikacin, vancomycin, mupirocin and linezolid. Predominantly, the results of AST obtained by both methods were consistent. Some discrepancies were observed for gentamicin; however, clinical breakpoints for staphylococci of human origin were used. CONCLUSIONS: Amikacin and chloramphenicol constitute potential treatment options in infections caused by MRSP and may be included in extended susceptibility testing in our geographical region. The determination of clinical breakpoints for some antimicrobials not incorporated in the recommendations should be a high priority in the veterinary diagnostics.

Multidrug-Resistant Commensal and Infection-Causing Staphylococcus spp. Isolated from Companion Animals in the Valencia Region.

The emergence of antimicrobial resistance (AMR) and multidrug resistance (MDR) among microorganisms to commonly used antibiotics is a growing concern in both human and veterinary medicine. Companion animals play a significant role in the epidemiology of AMR, as their population is continuously increasing, posing a risk of disseminating AMR, particularly to strains of public health importance, such as methicillin-resistant Staphylococcus strains. Thus, this study aimed to investigate the prevalence of AMR and MDR in commensal and infection-causing Staphylococcus spp. in dogs and cats in Valencia region. For this purpose, 271 samples were taken from veterinary centers to assess antimicrobial susceptibility against 20 antibiotics, including some of the most important antibiotics for the treatment of Staphylococcus infections, including the five last resort antibiotics in this list. Of all the samples, 187 Staphylococcus spp. strains were recovered from asymptomatic and skin-diseased dogs and cats, of which S. pseudintermedius (≈60%) was more prevalent in dogs, while S. felis (≈50%) was more prevalent in cats. In the overall analysis of the isolates, AMR was observed for all antibiotics tested, including those crucial in human medicine. Furthermore, over 70% and 30% of the strains in dogs and cats, respectively, exhibited MDR. This study highlights the significance of monitoring the trends in AMR and MDR among companion animals. The potential contribution of these animals to the dissemination of AMR and its resistance genes to humans, other animals, and their shared environment underscores the necessity for adopting a One Health approach.

Antibacterial activity of silver nanoparticles against Escherichia coli and methicillin-resistant Staphylococcus pseudintermedius is affected by incorporation into carriers for sustained release.

OBJECTIVE: To establish the lowest effective dose of commercially available nanoparticulate silver (AgNP) for antibacterial activity against Escherichia coli (E coli) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), in vitro, and to establish the effect of incorporating AgNP into carriers for sustained release on this antibacterial activity. SAMPLES: Silver nanoparticle dispersion (0.02 mg/mL) composed of citrate-stabilized, spherical, 10 nm diameter nanoparticles in aqueous buffer. PROCEDURES: E coli and MRSP were treated with 0.01 mg/mL AgNP. The highest concentration of bacteria where growth was inhibited by AgNP was selected for treatment with 0.01 mg/mL AgNP incorporated 3 carriers for sustained release: calcium sulfate hemihydrate (CSH) beads, poloxamer 407 gel, and gelatin sponge, respectively. The antibacterial activity of AgNP and AgNP incorporated into carriers for sustained release was compared with a mixed linear effects model. RESULTS: AgNP inhibited bacterial growth at a concentration of 101 for MRSP and 103 for E coli. For MRSP, the treatment group was associated with bacterial growth (P < .001) while the concentration of bacteria and time were not (P = .292 and P = .289, respectively). For E coli, the treatment group and concentration of bacteria were associated with bacterial growth (P < .001 and = .029, respectively) while time was not (P = .095). Poloxamer 407 gel exerted standalone antibacterial activity against both species of bacteria; sponge and CSH beads did not. CLINICAL RELEVANCE: AgNP has antibacterial activity against E coli and MRSP, which can be reduced when incorporated into carriers for sustained release. Poloxamer 407 gel alone and combined with AgNP exerts antibacterial activity against E coli and MRSP.

Characterization and antimicrobial susceptibility of coagulase-positive Staphylococcus isolated in a veterinary teaching hospital in Chile / Caracterización y sensibilidad antimicrobiana de Staphylococcus coagulasa-positivos aislados en un hospital clínico veterinario en Chile

Abstract There is limited information about the prevalence and antimicrobial susceptibility of coagulase-positive Staphylococcus (CoPS) strains in veterinary settings in Chile. The aim of this observational study was to identify and characterize CoPS strains from dogs, owners, veterinary professionals and surfaces in a veterinary teaching hospital at Universidad de Chile to determine the presence of methicillin-resistant strains and evaluate the genetic relationship among the strains. Veterinarians (n = 24), surfaces (n = 10), and healthy dogs (n = 40) and their respective owners (n = 40) were sampled for CoPS. Isolates were identified by PCRand antimicrobial susceptibility was assessed by the disk diffusion method and MIC. The presence of the mecA gene was evaluated by PCR, and the genetic relationship among the strains was established by PFGE. A total of 45 CoPS strains were obtained, eight from veterinary professionals, three from hospital surfaces, eight from owners and 26 from dogs. Nine of the strains were resistant to methicillin (20%), and all of them carried the mecA gene. A high percentage of the strains was resistant to clindamycin (33.3%). Additionally, the isolated CoPS showed high genetic diversity. This study suggests that veterinarians are in high risk of harboring methicillin-resistant CoPS (25% versus 2.5% from owners) and our results provide evidence that clindamycin could not be an empiric alternative for CoPS in the analyzed hospital. This is the first report of methicillin-resistant CoPS in veterinary settings in Chile, considering humans, pets and surfaces.

Resumen Existe información limitada sobre prevalencia y sensibilidad antimicrobiana de cepas de Staphylococcus coagulasa-positivas (CoPS) en entornos veterinarios en Chile. El objetivo de este estudio observacional fue identificar y caracterizar cepas CoPS de perros, duenos, veterinarios y superficies de un hospital veterinario de la Universidad de Chile, determinar la presencia de cepas meticilino-resistentes y evaluar la relación genética entre las cepas. Se colectaron muestras de veterinarios (n = 24), de superficies hospitalarias (n = 10) y de perros sanos (n =40) y sus respectivos duenos (n = 40). Los aislamientos se identificaron mediante PCR y la sensibilidad antimicrobiana se evaluó por difusión en discos y CIM. También se empleó PCR para detectar la presencia del gen mecA; la relación genética entre las cepas se estableció mediante electroforesis de campos pulsantes (PFGE). Se obtuvo un total de 45 cepas de CoPS, 8 de veterinarios, 3 de superficies hospitalarias, 8 de duenos y 26 de perros. Nueve cepas fueron meticilino-resistentes (20%), todas portadoras del gen mecA. Un porcentaje importante de cepas fue resistente a clindamicina (33,3%). Además, las cepas aisladas mostraron una alta diversidad genética. Este estudio sugiere que los veterinarios tienen alto riesgo de portar CoPS resistentes a meticilina (25% versus 2,5% propietarios). Asimismo, nuestros resultados proporcionan evidencia de que la clindamicina podría no ser una alternativa empírica para CoPS en el hospital analizado. Este es el primer estudio de CoPS meticilino-resistentes en entornos veterinarios en Chile que considera humanos, mascotas y superficies.

Antimicrobial resistance in Staphylococcus pseudintermedius on the environmental surfaces of a recently constructed veterinary hospital in Southern Thailand.

Background and Aim: Staphylococcus pseudintermedius is a zoonotic bacterium commonly found in animals, especially dogs. These bacteria can survive on environmental surfaces for several months. The infection of S. pseudintermedius from the environment is possible, but properly cleaning surface objects can prevent it. This study aimed to investigate the prevalence of methicillin-resistant S. pseudintermedius (MRSP) in the environment of a recently constructed veterinary hospital in Southern Thailand, where we hypothesized that the prevalence of MRSP might be very low. Materials and Methods: At three different time points, 150 samples were collected from different environmental surfaces and wastewater across the veterinary hospital. The collection was done after the hospital's cleaning. Bacteria were purified in the culture before being identified as species by biochemical tests and polymerase chain reaction (PCR). Next, the antimicrobial-resistant profile was performed using an automated system (Vitek 2). Finally, the antimicrobial resistance genes were identified using PCR. Results: Fifteen colonies of S. pseudintermedius were isolated from the surfaces of eight floors, four tables, two chairs, and one rebreathing tube. Fourteen colonies (93.3%) were multidrug-resistant (MDR) and carried the blaZ gene (93.3%). The majority of colonies were resistant to benzylpenicillin (93.3%), cefovecin (93.3%), ceftiofur (93.3%), kanamycin (93.3%), and neomycin (93.3%). Notably, only four colonies (26.7%) were methicillin-susceptible S. pseudintermedius, whereas 11 colonies (73.3%) were MRSP and carried both the mecA and blaZ genes. Five MRSP (45.5%) were resistant to at least 14 antimicrobial drugs, represented as extensively drug-resistant (XDR) bacteria. Ten of eleven MRSP (90.9%) were Staphylococcal chromosomal mec type V, while another displayed untypeable. Despite the routine and extensive cleaning with detergent and disinfectant, MRSP isolates were still detectable. Conclusion: Many isolates of MRSP were found in this veterinary hospital. Almost all of them were MDR, and nearly half were XDR, posing a threat to animals and humans. In addition, the current hospital cleaning procedure proved ineffective. Future research should be conducted to determine the bacterial biofilm properties and bacterial sensitivity to certain detergents and disinfectants.

Characterization and antimicrobial susceptibility of coagulase-positive Staphylococcus isolated in a veterinary teaching hospital in Chile.

There is limited information about the prevalence and antimicrobial susceptibility of coagulase-positive Staphylococcus (CoPS) strains in veterinary settings in Chile. The aim of this observational study was to identify and characterize CoPS strains from dogs, owners, veterinary professionals and surfaces in a veterinary teaching hospital at Universidad de Chile to determine the presence of methicillin-resistant strains and evaluate the genetic relationship among the strains. Veterinarians (n=24), surfaces (n=10), and healthy dogs (n=40) and their respective owners (n=40) were sampled for CoPS. Isolates were identified by PCR and antimicrobial susceptibility was assessed by the disk diffusion method and MIC. The presence of the mecA gene was evaluated by PCR, and the genetic relationship among the strains was established by PFGE. A total of 45 CoPS strains were obtained, eight from veterinary professionals, three from hospital surfaces, eight from owners and 26 from dogs. Nine of the strains were resistant to methicillin (20%), and all of them carried the mecA gene. A high percentage of the strains was resistant to clindamycin (33.3%). Additionally, the isolated CoPS showed high genetic diversity. This study suggests that veterinarians are in high risk of harboring methicillin-resistant CoPS (25% versus 2.5% from owners) and our results provide evidence that clindamycin could not be an empiric alternative for CoPS in the analyzed hospital. This is the first report of methicillin-resistant CoPS in veterinary settings in Chile, considering humans, pets and surfaces.

Duration of carriage of multidrug-resistant bacteria in dogs and cats in veterinary care and co-carriage with their owners.

BACKGROUND: The emergence and spread of multidrug-resistant organisms (MDROs) represent a threat to human and animal health. OBJECTIVES: To assess duration of carriage of MDROs in dogs and cats presented to veterinary clinics/hospitals in Switzerland. To estimate prevalence, duration of and risk factors for MDRO carriage in their owners and the occurrence of co-carriage in owner-pet pairs. METHODS: Prospective, longitudinal, observational study. Nasal swabs and fecal samples were collected from 50 owners of dogs and cats presented to 3 large veterinary hospitals, 1 medium-sized clinic and 1 practice. If pet or owner tested positive for a MDRO, follow-up samples were collected for up to 8 months. Methicillin-resistant (MR) Staphylococcus aureus, MR S. pseudintermedius, MR coagulase-negative staphylococci (MRCoNS), MR Macrococcus spp., cephalosporinase- and carbapenemase-producing (CP) Enterobacterales were isolated and further characterized by MALDI-TOF MS, microdilution, ß-lactam resistance gene detection, REP/ERIC-PCR, multilocus sequence typing or whole-genome sequencing. Risk factors for MDRO carriage in owners were explored based on questionnaire-derived data. RESULTS: Five out of 50 owners carried 3rd generation cephalosporin-resistant Enterobacterales (3GC-R-Ent.), and 5/50 MRCoNS. In 3 dogs and 4 cats carriage of 3GC-R-Ent. persisted for up to 136 days after discharge (median 99 days, IQR 83 days, range 36-136 days), in two cats isolates were carbapenem-resistant. Owner-pet co-carriage was not observed. No specific risk factors for MDRO carriage in owners were identified. CONCLUSIONS: After discharge from veterinary care, dogs and cats may carry 3GC-R-Ent. for prolonged time periods. Carriage of MDROs was common in owners, but pet-owner co-carriage of the same MDRO was not observed.

Molecular Epidemiology of Clinical and Colonizing Methicillin-Resistant Staphylococcus Isolates in Companion Animals.

In this study, we aimed to investigate the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) clinical and colonizing isolates of dogs and cats to profile contributing factors associated with their isolation. Nasal and rectal samples were collected from dogs and cats between 2015 and 2017 to identify colonizing isolates. Clinical isolates collected between 2003 and 2016 were retrieved from a Queensland university veterinary diagnostic laboratory. All isolates were identified using standard microbiological and molecular methods and were characterized by whole genome sequencing. Phylogenetic relationships and differences in epidemiological factors were investigated. Seventy-two MRSP isolates out of 1,460 colonizing samples and nine MRSP clinical isolates were identified. No MRSA was isolated. ST496 and ST749 were the most commonly isolated sequence types with different SCCmec types. ST496 clones spread both along the coast and more inland where ST749 was more centered in Brisbane. The resistance and virulence factors differed significantly between the two sequence types. ST496 colonizing and clinical isolates were similarly multidrug resistant. The virulence genes of ST749 colonizing and clinical isolates were similar as both contained the gene nanB for sialidase. There were no differences in the individual and clinical factors between predominant sequence types. High levels of antimicrobial resistance occurred in the majority of isolates, which is of potential concern to human and veterinary health. The phylogenetic clustering of isolates from this study and others previously identified in countries, particularly New Zealand, with which Australia has high volume of pet movements could suggest the importation of clones, which needs further investigation.

Genomic insights into the emergence and spread of methicillin-resistant Staphylococcus pseudintermedius in veterinary clinics.

Staphylococcus pseudintermedius is a common cause of skin and soft tissue infections in dogs but can also cause infections in cats and humans. The frequency of methicillin-resistant S. pseudintermedius (MRSP) strains is increasing worldwide. Here, we obtained 43 MRSP isolates from dogs (n = 41), one cat (n = 1) and the small animal clinic environment (n = 1) in Slovenia from the period 2008-2018, which underwent whole-genome sequencing (WGS) and antimicrobial susceptibility testing. Five sequence types (STs) were identified, with ST71 (32/43) and ST551 (8/43) being the predominant. In Slovenia, ST551 was first detected in 2016, whereas a decrease in the frequency of ST71 was observed after 2015. All isolates were multidrug-resistant and most antimicrobial-resistant phenotypes could be linked to acquisition of the corresponding resistance genes or gene mutations. Core-genome multilocus sequence typing (cgMLST) revealed several potential MRSP transmission routes: (i) between two veterinary clinics by a single MRSP-positive dog, (ii) between the environment of a veterinary clinic and a dog, and (iii) between a canine and a feline patient through the contaminated environment of a veterinary clinic. Of the six dogs that were additionally sampled from 14 days to five months after the initial sampling, each harbored the same MRSP strain, suggesting a limited within-host diversity of MRSP in symptomatic dogs. The present results highlight the importance of MRSP-positive dogs in the spread of veterinary care-associated MRSP infections and call for the implementation of strict control measures to reduce MRSP contamination in veterinary clinic environments originating from animal-contact surfaces.

Two Novel Bacteriophages Control Multidrug- and Methicillin-Resistant Staphylococcus pseudintermedius Biofilm.

As a primary bacterial pathogen in companion animals, Staphylococcus pseudintermedius has zoonotic potential. This pathogen exhibits multidrug resistance, including resistance to methicillin, and biofilm-forming ability, making it hard to eradicate with antimicrobial agents. One potential alternative is bacteriophage therapy. In this study, we first characterized the antimicrobial resistance profile of S. pseudintermedius from canine samples and isolated two novel bacteriophages, pSp-J and pSp-S, from canine pet parks in South Korea to potentially control S. pseudintermedius. The biological characteristics of phages were assessed, and the phages could infect most of the methicillin-resistant S. pseudintermedius strains. We found that these phages were stable under the typical environment of the body (~37°C, pH 7). We also assessed bacterial lysis kinetics using the two phages and their cocktail, and found that the phages could prevent biofilm formation at low doses and could degrade biofilm at high doses. Taken together, this study demonstrates that bacteriophages pSp-J and pSp-S isolated in this study can be used to potentially treat methicillin-resistant S. pseudintermedius.

Epidemiology of methicillin resistant Staphylococcus species carriage in companion animals in the Greater Brisbane Area, Australia.

To detect methicillin resistant Staphylococcus aureus (MRSA) and S. pseudintermedius (MRSP) swab samples were collected from dogs, cats and horses from South East Queensland (SE QLD). MRSP carriage in dogs was 8.7% and no MRSP was isolated from cats and horses; no MRSA was isolated. Risk factors for carriage included previous hospitalisation, previous bacterial infection, consultation type, average precipitation, and human population density. The probability of MRSP carriage was highest in Brisbane city, Sunshine Coast and Gympie. This suggests that MRSP carriage in dog populations from SE QLD is geographically clustered and associated with clinical and environmental factors.

Synergistic Effect of Abietic Acid with Oxacillin against Methicillin-Resistant Staphylococcus pseudintermedius.

Resin acids are valued in traditional medicine for their antiseptic properties. Among these, abietic acid has been reported to be active against methicillin-resistant Staphylococcus aureus (MRSA) strains. In veterinary healthcare, the methicillin-resistant Staphylococcus pseudintermedius (MRSP) strain is an important reservoir of antibiotic resistance genes including mecA. The incidence of MRSP has been increasing, and treatment options in veterinary medicine are partial. Here, we investigated the antimicrobial and antibiofilm properties of abietic acid against three MRSP and two methicillin-susceptible Staphylococcus pseudintermedius (MSSP) strains, isolated from diseased pet animals and human wound samples. Abietic acid showed a significant minimal inhibitory concentration (MIC) value ranging from 32 to 64 µg/mL (MRSPs) and 8 µg/mL (MSSP). By checkerboard method we demonstrated that abietic acid increased oxacillin susceptibility of MRSP strains, thus showing a synergistic interaction with oxacillin. Abietic acid was also able to contrast the vitality of treated MSSP and MRSP1 biofilms at 20 µg/mL and 40 µg/mL, respectively. Finally, the compound moderately reduced mecA, mecR1 and mec1 gene expression. In conclusion, the results here reported demonstrate the antimicrobial activity of abietic acid against MRSP and support the use of this compound as a potential therapeutic agent to be used in combinatorial antibiotic therapy.

Contamination of the urban environment with excrements of companion animals as an underestimated source of Staphylococcus species posing a threat to public health.

The aim of the study was to assess the incidence, resistance, virulence, and genotypic characteristics of Staphylococcus spp. residing in the gastrointestinal tract of dogs and cats, as a group of animals causing potential contamination of the urban space. A high percentage of strains resistant to penicillin (58%), oxacillin (9%) and tetracycline (60%) were found. All isolates resistant to penicillin, kanamycin, or chloramphenicol carried genes responsible for individual resistance (blaZ, aph(3')-IIIa, and cat (pC194)/cat (pC223), respectively. The mecA gene was detected in 45% of the oxacillin-resistant Staphylococcus pseudintermedius strains. The amplification of DNA fragments surrounding rare restriction sites analysis demonstrated high heterogeneity of genotypic profiles correlating with phenotypic resistance profiles. Multilocus sequence typing analysis classified the methicillin-resistant S. pseudintermedius strains as ST71, ST890, and the totally new ST1047. The presence of a high level of resistance among Staphylococcus strains may suggest a potential risk of transfer of these bacteria between companion animals and humans.

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.

Prevalence and characterization of methicillin-resistant Staphylococcus pseudintermedius from symptomatic companion animals in Northern Italy: Clonal diversity and novel sequence types.

The aim of this study was to assess the prevalence, the genotypic diversity, the antimicrobial resistance traits of canine and feline clinical methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates in a diagnostic laboratory in Italy during 2015-2016. All isolates were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome (SCC)-mec typing and staphylococcal protein A (spa)-typing. The resistance profiles were assessed by antimicrobial susceptibility testing and confirmed genotypically by the detection of mecA gene and by microarray analyses. The prevalence of MRSP isolates was high (31.6%). All the strains were multidrug resistant and the most frequent clone was ST71-SCCmec type II-III. These results confirm a high prevalence of MRSP amongst clinical samples from pets in Italy. These isolates show multidrug resistance features that are of concern both in veterinary and human medicine for clinical and epidemiological reasons.

Structure⁻Activity Study, Characterization, and Mechanism of Action of an Antimicrobial Peptoid D2 and Its d- and l-Peptide Analogues.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure⁻activity relationship study of D2, which included 19 peptide/peptoid analogs. Our best compound D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2⁻4 µg/mL) and P. aeruginosa (4 µg/mL) isolates as well as other selected dog pathogens (2⁻16 µg/mL). Time⁻kill assays demonstrated that D2D was able to inhibit MRSP in 30 min at 1× MIC, significantly faster than D2. Our results suggest that at high concentrations D2D is rapidly lysing the bacterial membrane while D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of D2, D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane.

Beta-defensin derived cationic antimicrobial peptides with potent killing activity against gram negative and gram positive bacteria.

BACKGROUND: Avian ß-defensins (AvBD) are cationic antimicrobial peptides (CAMP) with broad-spectrum antimicrobial activity, chemotactic property, and low host cytotoxicity. However, their bactericidal activity is greatly compromised under physiological salt concentrations which limits the use of these peptides as therapeutic agents. The length and the complex structure involving three conserved disulfide bridges are additional drawbacks associated with high production cost. In the present study, short linear CAMPs (11 to 25 a.a. residues) were developed based on the key functional components of AvBDs with additional modifications. Their biological functions were characterized. RESULTS: CAMP-t1 contained the CCR2 binding domain (N-terminal loop and adjacent α-helix) of AvBD-12 whereas CAMP-t2 comprised the key a.a. residues responsible for the concentrated positive surface charge and hydrophobicity of AvBD-6. Both CAMP-t1 and CAMP-t2 demonstrated strong antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus pseudintermedius. However, CAMP-t1 failed to show chemotactic activity and CAMP-t2, although superior in killing Staphylococcus spp., remained sensitive to salts. Using an integrated design approach, CAMP-t2 was further modified to yield CAMP-A and CAMP-B which possessed the following characteristics: α-helical structure with positively and negatively charged residues aligned on the opposite side of the helix, lack of protease cutting sites, C-terminal poly-Trp tail, N-terminal acetylation, and C-terminal amidation. Both CAMP-A and CAMP-B demonstrated strong antimicrobial activity against multidrug-resistant P. aeruginosa and methicillin-resistant S. pseudintermedius (MRSP) strains. These peptides were resistant to major proteases and fully active at physiological concentrations of NaCl and CaCl2. The peptides were minimally cytotoxic to avian and murine cells and their therapeutic index was moderate (≥ 4.5). CONCLUSIONS: An integrated design approach can be used to develop short and potent antimicrobial peptides, such as CAMP-A and CAMP-B. The advantageous characteristics, including structural simplicity, resistance to salts and proteases, potent antimicrobial activity, rapid membrane attacking mode, and moderate therapeutic index, suggest that CAMP-A and CAMP-B are excellent candidates for development as therapeutic agents against multidrug-resistant P. aeruginosa and methicillin-resistant staphylococci.

Taurolidine susceptibility of methicillin-resistant Staphylococcus pseudintermedius isolates.

Infections caused by methicillin-resistant Staphylococcus pseudintermedius (MRSP) can be challenging to treat because they are often resistant to many other antimicrobial agents. We assessed the susceptibility of 29 MRSP isolates from dogs to taurolidine in vitro. There was no growth at 0.12% taurolidine and light growth at 0.06% for all isolates. Taurolidine was reliable at inhibiting growth of MRSP at a concentration of 1,200 µg/mL.

Molecular characteristics of clinical methicillin-resistant Staphylococcus pseudintermedius harboring arginine catabolic mobile element (ACME) from dogs and cats.

This study aimed to investigate the presence of arginine catabolic mobile element (ACME) and its associated molecular characteristics in methicillin-resistant Staphylococcus pseudintermedius (MRSP). Among the 72 S. pseudintermedius recovered from various infection sites of dogs and cats, 52 (72.2%) were MRSP. ACME-arcA was detected commonly (69.2%) in these MRSP isolates, and was more frequently detected in those from the skin than from other body sites (P=0.047). There was a wide genetic diversity among the ACME-arcA-positive MRSP isolates, which comprised three SCCmec types (II-III, III and V) and 15 dru types with two predominant clusters (9a and 11a). Most MRSP isolates were multidrug-resistant. Since S. pseudintermedius could serve as a reservoir of ACME, further research on this putative virulence factor is recommended.

Resistance of canine methicillin-resistant Staphylococcus pseudintermedius strains to pradofloxacin.

We investigated in vitro activity of a novel veterinary fluoroquinolone, pradofloxacin, against methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates and compared with other fluoroquinolones. A total of 38 MRSP isolates were subjected to agar disk diffusion tests for sensitivity to pradofloxacin, orbifloxacin, marbofloxacin, enrofloxacin, and ciprofloxacin. The minimal inhibitory concentration (MIC) values of pradofloxacin, ciprofloxacin, and enrofloxacin were determined. Mutations in the genes encoding DNA gyrase subunit A (GyrA) and topoisomerase IV (GrlA) proteins associated with fluoroquinolone resistance were studied by an analysis of partial sequences of the genes encoding these proteins. Two MRSP isolates were susceptible in disk diffusion and microdilution test to all fluoroquinolones tested, including pradofloxacin. Based on the results of the disk diffusion testing, 33 of 38 isolates showed resistance to pradofloxacin and 3 were intermediate, whereas, by pradofloxacin MIC testing, 35 isolates were classified as resistant and 1 as intermediate. Single alterations in GyrA and GrlA proteins were observed in the 35 resistant isolates and the 1 intermediate isolate (MIC results). These same 36 isolates were also resistant to the other tested fluoroquinolones. The results of the current study showed that MRSP isolates are usually resistant to all fluoroquinolones, including pradofloxacin. Therefore, in routine susceptibility testing to pradofloxacin by disk diffusion, the results should be carefully interpreted for MRSP isolates, especially those resistant to other fluoroquinolones and, in questionable cases, the pradofloxacin MIC should be determined to confirm the susceptibility testing results.