Methicillin-resistant Staphylococcus Pseudintermedius Research Articles

In vitro antimicrobial activity of a black currant oil based shampoo versus a chlorhexidine 4% shampoo on bacteria strains isolated from canine pyoderma: A comparative study

Over the last few years, antimicrobial shampoo therapy has been increasingly used to treat skin infections in order to reduce systemic use of antibiotics. This study was aimed to compare the In vitro bactericidal effect of a black currant oil based shampoo (S1) to a chlorhexidine 4% shampoo (S2) against methicillin-sensitive Staphylococcus pseudintermedius (MSSP), methicillin-resistant Staphylococcus pseudintermedius (MRSP), Staphylococcus aureus (SA), Escherichia coli (EC) and Pseudomonas aeruginosa (PA) isolates. A collection of 50 bacterial strains from skin swabs of dogs with superficial recurrent pyoderma was selected: 10 MSSP, 10 MRSP, 10 SA, 10 EC and 10 PA. The two shampoos were blindly tested in duplicate with a microdilution plate method, with scalar concentrations from 1:2 to 1: 256. The MBC was performed for each dilution. A linear regression was used to detect a statistically significance between the two shampoos. All isolates were completely killed at 1:2 up to 1:16 dilution of the two antiseptic products. At the 1:32 dilution the first bacterial growths were observed, in particular for 2 and 4 strains of MRSP by S1 and S2 respectively. The first lethal dilution for SA was at 1:64 for S1/S2 and only for S2 against SP. No significant difference was observed between the two shampoos according to the results of linear regression significant for: i) MRSP, PA and EC (p < 0.05); ii) MSSP and SA (p < 0.1). This study showed that both black currant oil based shampoo and chlorhexidine 4% shampoo have a similar In vitro bactericidal activity.

Methicillin-resistant Staphylococcus pseudintermedius: an underestimated risk at pet clinic

The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) as a cause of infectious disease in companion animals remains unknown. The emergence of MRSP is a challenge in veterinary medicine as multidrug-resistant strains began to emerge, resulting in treatment failures. This study provides an overview of the characterization of S. pseudintermedius strains from clinical pet samples and the prevalence of MRSP strains. A total of 123 S. pseudintermedius strains were characterized by phenotypic testing and the MALDI-TOF technique and evaluated for susceptibility to methicillin and the presence of the mecA gene. Of these, 49 (39.8%) were identified as MRSP. The results confirm the importance of monitoring resistant pathogens and the need for further studies to determine the prevalence of MRSP in companion animals. The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) as a cause of infectious disease in companion animals remains unknown. The emergence of MRSP is a challenge in veterinary medicine as multidrug-resistant strains began to emerge, resulting in treatment failures. This study provides an overview of the characterization of S. pseudintermedius strains from clinical pet samples and the prevalence of MRSP strains. A total of 123 S. pseudintermedius strains were characterized by phenotypic testing and the MALDI-TOF technique and evaluated for susceptibility to methicillin and the presence of the mecA gene. Of these, 49 (39.8%) were identified as MRSP. The results confirm the importance of monitoring resistant pathogens and the need for further studies to determine the prevalence of MRSP in companion animals.

METHICILLIN-RESISTANT STAPHYLOCOCCUS PSEUDINTERMEDIUS, A NEW THREAT IN HUMAN AND VETERINARY MEDICINE?

In this paper we briefl y described the worldwide distribution of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs and cats. Th e most common sequence type of MRSP strains in dogs is ST71 as it was detected in isolates on four continents (Europe, Asia, North and South America). However, several diff erent MRSP sequence types are detected in small animals, and the presence of new genetic variants is continually reported. Sometimes isolates belonging to the same sequence type (ST) are detected in dogs, cats, their owners, and veterinarians. MRSP is oft en multidrug-resistant and its resistance patterns are usually linked to certain sequence types. Th e resistance to non-beta-lactam antibiotics such as erythromycin, clindamycin, tetracycline, gentamicin, enrofl oxacin and sulfamethoxazole/trimethoprim is also recorded. Taking into account that MRSP tends to confer multidrug-resistant phenotype, it is quite challenging for veterinarians to give adequate therapy in clinically ill animals. It would seem as if the signifi cance of MRSP in the clinical epidemiology of humans is not fi rmly established. However, the importance of MRSP in human medicine should not be underestimated given the fact that all methicillinresistant Staphylococcus spp. carry resistance and virulence genes and have the potential to share their genetic elements with other bacteria.

Carprofen-induced depletion of proton motive force reverses TetK-mediated doxycycline resistance in methicillin-resistant Staphylococcus pseudintermedius

We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the proton gradient. We showed that CPF is a proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising proton-motive-force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.

Identification and molecular epidemiology of methicillin resistant Staphylococcus pseudintermedius strains isolated from canine clinical samples in Argentina

BackgroundStaphylococcus pseudintermedius is the leading cause of pyoderma in dogs and the frequent use of antimicrobial treatment is associated to the development of resistance to nearly all classes of antibiotics. Despite S. pseudintermedius significance, our understanding of the molecular mechanism of β-lactam resistance and its genetic diversity remains limited. We aimed to: i) determine the phenotypic resistance profile of methicillin resistant Staphylococcus pseudintermedius (MRSP) isolated from infected dogs in three different veterinary hospitals in Buenos Aires, Argentina; ii) identify the SCCmec elements and resistance genes; and iii) analyze the clonal relationship between isolates and in regard of dominant lineages found in the world.ResultsIn addition to the differential levels of β-lactam resistance, MRSP isolates (n = 10) showed resistance to 5–6 families of antibiotics, and were therefore categorized as multidrug-resistant. All the isolates were variant of SCCmec V homologous to S. aureus; additional SCCmecFinder analysis classified five of the genomes as SCCmec type V (5C2&5) with mecA (encodes for PBP2a), mecRI and mecI and all the genes closely related to the reference SCCmec type V S. aureus TSGH17 strain. In the remaining five strains, mecA was present, although other genes associated with SCCmec V including mecR1 and mecI were missing. PBP2a was inducible in low level resistance strains (MRSP 8151), and constitutively expressed in MRSP 8150, suggesting different mecA regulatory mechanisms. MRSP isolates showed significant genetic diversity: eight PFGE clonal types and six multilocus-sequence typing (MLST) sequence types (STs) (339, 649, 919, 920, 921 and 922), including four new STs genetically distinct from STs reported in other geographic areas. Comparative genomics and phylogenetic analyses of the MRSP showed a correlation between the genetic content and the phenotypes, and established the genetic relationship between the isolates.ConclusionsMRSP could be a threat to animal health due to it concerning level of antimicrobial resistance. Our study highlights genetic and epidemiological aspects of multidrug-resistant MRSP strains from Argentina showing high degree of correlation between the resistance genes and the phenotype of the isolates and, furthermore, they appeared evolutionary closer to major worldwide reported ST68 and ST71.

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.

Analysis host-recognition mechanism of staphylococcal kayvirus ɸSA039 reveals a novel strategy that protects Staphylococcus aureus against infection by Staphylococcus pseudintermedius Siphoviridae phages.

Following the emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), phage therapy has attracted significant attention as an alternative to antibiotic treatment. Bacteriophages belonging to kayvirus (previously known as Twort-like phages) have broad host range and are strictly lytic in Staphylococcus spp. Previous work revealed that kayvirus ɸSA039 has a host-recognition mechanism distinct from those of other known kayviruses: most of kayviruses use the backbone of wall teichoic acid (WTA) as their receptor; by contrast, ɸSA039 uses the β-N-acetylglucosamine (β-GlcNAc) residue in WTA. In this study, we found that ɸSA039 could switch its receptor to be able to infect S. aureus lacking the β-GlcNAc residue by acquiring a spontaneous mutation in open reading frame (ORF) 100 and ORF102. Moreover, ɸSA039 could infect S. pseudintermedius, which has a different WTA structure than S. aureus. By comparison, with newly isolated S. pseudintermedius-specific phage (SP phages), we determined that glycosylation in WTA of S. pseudintermedius is essential for adsorption of SP phages, but not ɸSA039. Finally, we describe a novel strategy of S. aureus which protects the bacteria from infection of SP phages. Notably, glycosylation of ribitol phosphate (RboP) WTA by TarM or/and TarS prevents infection of S. aureus by SP phages. These findings could help to establish a new strategy for the treatment of S. aureus and S. pseudintermedius infection, as well as provide valuable insights into the biology of phage-host interactions.

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.

Isolation and characterization of bacteriophages active against methicillin-resistant Staphylococcus pseudintermedius

We aimed to isolate and characterize bacteriophages (phages) with preferential activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP), a multidrug-resistant canine pathogen. Four phages were isolated from canine faeces using two MRSP strains as initial hosts. Phage host range was evaluated by the spot test on 17 MRSP, 43 methicillin-susceptible S. pseudintermedius (MSSP), and six other staphylococci isolated from dogs. Transmission electron microscopy was used for presumptive identification followed by whole genome sequencing (WGS). All phages lysed all MRSP isolates whereas only 16–28% of MSSP were lysed. Their lytic activity was limited to S. pseudintermedius and S. schleiferi. All phages had similar morphology and belonged to the Siphoviridae family. WGS indicated that the phages were 93.8–99.7% identical to each other, and exhibited the highest similarity (87%) to the temperate S. aureus phage 187. Confirmatory lytic activity tests showed that phages were able to produce clear plaques on lysogens, which was enabled by recombination of the lysogeny modules as shown by WGS of the phages after propagation and plaque formation. This study provides insight into the genetic diversity and biology of S. pseudintermedius temperate phages, which could be further developed for topical therapy of MRSP skin and wound infections.

Occurrence and characterization of methicillin-resistant Staphylococcus pseudintermedius in successive parturitions of bitches and their puppies in two kennels in Italy

BackgroundMulti-drug methicillin-resistant Staphylococcus pseudintermedius (MRSP) detection is rapidly increasing in microbial specimens from pets across Europe. MRSP has also been isolated from bitches and newborns in dog breeding kennels. This study assessed whether MRSP lineage differs between breeding kennels and is maintained over time. Post-partum bitches (at day 3 vaginal and day 3, 9 and 35 milk samples) and their litters (at day 3, 9 and 35 oral and abdominal skin samples) from two Italian breeding kennels (A and B) were sampled and MRSP was subsequently characterized via whole-genome sequencing and antibiotic susceptibility testing. The study was carried out from October 2014 to March 2016 and included successive parturitions from the same animals.ResultsThe analysis revealed different situations in both investigated kennels. In kennel A, circulating strains were from 7-locus sequence types ST688, ST258 and closely related isolates of ST71, which included most isolates. In kennel B, only a new isolate, ST772, was detected. In addition, most isolates from both kennels had multi-resistant antibiotic profiles. MRSP was only isolated from litters of MRSP-positive bitches, thus suggesting that bitch-litter transmission is likely.ConclusionsOur data show that MRSP circulation can differ in different settings, that several clonal lineages can circulate together, and that vertical transmission appears common. MRSP colonization did not affect the health conditions of the bitches or of their litters.

Genetic characterisation of methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in pets and veterinary personnel in Iran: new insights into emerging methicillin-resistant S. pseudintermedius (MRSP).

Methicillin-resistant staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), pose a threat to animal and human health worldwide. Veterinary staff and pets may play a role in the spread of resistant clones. A total of 125 samples from veterinary staff (n=50), dogs (n=49) and cats (n=26) were investigated. Obtained isolates were tested for the methicillin resistance gene mecA and were subjected to multiplex PCR to differentiate coagulase-positive species. Following SCCmec and spa typing, isolates were tested for the presence of various toxin and virulence genes and phenotypic resistance to common antimicrobials. Overall, 4 MRSA were isolated from two veterinarians and two dogs and 19 MRSP were found in eleven dogs (12 isolates) and five cats (7 isolates). The MRSA isolates possessed sea (2) and eta (3) virulence genes and the MRSP isolates possessed sea (6), expA (15), expB (1) and siet (19) genes. SCCmec type II and three spa types (t186, t1816 and t10897) were identified in the MRSA isolates. Most of the MRSP isolates belonged to SCCmec types II (2 isolates) and V (10 isolates); however, the remaining 7 isolates were untypeable and contained class C1 mec. The majority of isolates were multidrug-resistant (MDR). These findings show that pets and veterinarians could be potential sources of MDR-MRSA and MDR-MRSP in Iran. Taken together, these findings warrant future investigations on the epidemiology and public-health significance of MDR-MRSA and MDR-MRSP both in veterinarians and companion animals in Iran.

Biofilm-forming ability and virulence factors of methicillin-resistant Staphylococcus pseudintermedius from canine pyoderma

Staphylococcus pseudintermedius (SP) has been associated with high antibiotic-resistance rates (e.g. methicillin) in European countries [1,3]. This condition could be also related to the ability to produce biofilm [2]. The aim of this study was to investigate the presence of methicillin-resistant SP strains and determine their ability to produce biofilm and some crucial virulence factors.Forty-two SP strains, previously determined as multi drug resistant (MDR) by the disk diffusion method using a panel of 17 antimicrobial agents, were selected from our collection and tested phenotypically for the minimum inhibitory concentration (MIC) of methicillin and genotypically for the presence of mecA and blaZ genes. The ability to produce biofilm was assessed phenotypically by two different assays: the Congo Red Agar plates (CRA) and the Microtitre Plate test (MtP) and genetically by the amplification of icaA and icaD genes. Three virulence factors genes coding for bicomponent leukocidin and enterotoxins (luk-I, seC, se-int) were searched.Twenty-three strains revealed a value of MIC for the methicillin greater than 128 µg/mL. The 83% were mecA-positive and 86% resulted blaZ-positive; all the strains positive for mecA were also positive for blaZ. All SP strains resulted biofilm-producers by MtP assay and classified as weakly producers (4.7%), moderate producers (47.6 %) and strongly producers (47.6 %). In contrast, only 35.7% of all strains were considered biofilm-producers by CRA method. The amplification of icaA and icaD gene occurred respectively in 66.6% and 97.6%; only one strain was negative for both genes. Almost all strains were positive for luk-I (95%), seC (74%) and se-int (84%).Our data reveal the pathogenicity potential of SP strains from dogs, suggesting that they could be considered zoonotic potential agents and confirming other previous studies [3-5]. Moreover could be observed a clear linkage between antibiotic-resistance and ability to produce biofilm.

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees and in the environment of a small animal hospital

The aim of the study was to investigate methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees of a small animal hospital and the hospital environment. In total, 96 swabs from employees and 73 swabs from the clinic environment were investigated. Cation-adjusted-Mueller-Hinton broth (CAMHB) + 6.5% NaCl was used for enrichment before plating on Mueller-Hinton (MH) agar with 2% NaCl and 0.25 mg/L oxacillin. The staphylococcal species was determined using MALDI-TOF MS. The isolates were subjected to mecA-PCR, macrorestriction analysis, and antimicrobial susceptibility testing. MRSA were present in five nasal swabs of the 55 employees tested and in six environmental samples, MRSP in two employees (nasal and hand swabs, each) and in three environmental samples. All isolates harboured mecA. Susceptibility testing revealed that all but one of the isolates were multiresistant. All isolates were resistant to β-lactams and fluoroquinolones. All but one of the isolates were resistant to macrolides and lincosamides. A single MRSA was resistant to gentamicin. All MRSP were resistant to trimethoprim/sulfamethoxazole and non-susceptible to gentamicin. One isolate was also resistant to tetracycline. Macrorestriction analysis revealed three main SmaI patterns for MRSA and two main SmaI patterns for MRSP. All environmental isolates were found in areas of high people and animal traffic, such as dog ward areas, waiting and triage rooms. The finding of indistinguishable MRSA or MRSP among employees and in the environment of the small animal hospital suggests the possibility of transfer of these bacteria between humans, animals, and the hospital environment.

Enterotoxin gene profile of methicillin-resistant Staphylococcus pseudintermedius isolates from dogs, humans and the environment.

This study aimed to detect and identify staphylococcal enterotoxin (SE) genes in methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains from different sources, and to investigate the relationship between their sequence types (STs) and SE gene patterns. The profiles of 17 SE genes in 93 MRSP strains isolated from dogs (n=43), humans (n=18) and the environment (n=32) were detected by PCR. Multilocus sequence typing (MLST), SCCmec typing and pulsed-field gel electrophoresis (PFGE) were used to analyse the clonal relatedness between the molecular type and SE gene profiles.Results/Key findings. The human MRSP strains harboured the greatest number of SE genes (12/17; sea, sec, seg, sei, sek, sel, sem, sen, seo, sep, seq and tst-1) compared to those from dogs (5/17; sec, sel, sem, seq and tst-1) and environmental sources (3/17; sec, seq and tst-1). Using MLST and PFGE, different SE gene profiles were found within the same clonal type. We show that isolates of MRSP vary in their virulence gene profiles, depending on the source from which they have been isolated. This insight should encourage the development of appropriate monitoring and mitigation strategies to reduce the transmission of MRSP in veterinary hospitals and households.

Draft Genome Sequences of 12 Clinical and Environmental Methicillin-Resistant Staphylococcus pseudintermedius Strains Isolated from a Veterinary Teaching Hospital in Washington State.

ABSTRACTMethicillin-resistant Staphylococcus pseudintermedius (MRSP) is a globally emergent multidrug-resistant pathogen of dogs associated with nosocomial transmission in dogs and with potential zoonotic impacts. Here, we report the draft whole-genome sequences of 12 hospital-associated MRSP strains and their resistance genotypes and phenotypes.

Ever-increasing emergence of multi-drug resistant Staphylococcus pseudintermedius in the dog and its zoonotic potentials

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged as an important pathogen in the dog over the past 10 years with an ever increasing incidence worldwide. This review focuses mainly on the epidemiology and detection of MRSP, emphasizing on the interpretation and pitfalls of screening laboratory tests and antimicrobial susceptibility tests. Risk factors for colonization and infection with MRSP and molecular analysis of the dominant clonal lineages are also described. The zoonotic potential and worldwide epidemiology of multidrug resistant Staphylococcus pseudintermedius (MDRSP) are presented. Finally, control options for the colonization of healthy dogs as well as infection by MRSP and MDRSP are described emphasizing on the indications for bacterial culture and susceptibility testing and the principles of topical therapy which may prove to be the sole effective treatment in several canine pyoderma cases.

An investigation of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in domestic and shelter dogs in Montenegro (RS-Brazil)

SUMMARY The interaction established between man and their pets has been increasing progressively. The diagnosis of animals carrying methicillin-resistant Staphylococcus pseudintermedius (MRSP) becomes important since positive results for this opportunistic bacteria has been found in humans. The aim of this study was to investigate methicillin-resistant Staphylococcus pseudintermedius (MRSP) in domestic dogs attended in a veterinary clinic and shelter dogs. Anal and nasal swabs from 53 domestic dogs and 46 shelter dogs were collected between the months of August and October of 2015, resulting in 198 samples, which were sent for routine laboratory analysis and subsequently used for this study. Phenotypic tests for bacteria identification and susceptibility to oxacillin were performed for the investigation of MRSP. A total of 23 samples (11.6%) from 21 dogs were positive for S. pseudintermedius, however none of the isolates were MRSP. Nasal and anal swabs had similar positivity and two dogs were carriers of the bacteria in both analyzed sites (anal and nasal). Our results showed a higher positivity for S. pseudintermedius in domestic dogs compared to shelter dogs, as well as indicating the importance of collecting more than one colonization site. This is the first study to perform a comparison among different animal creating sites in Brazil.

Methicillin-resistant Staphylococcus pseudintermedius among dogs in the description of novel SCCmec variants

The presence and genetic characteristics of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in Sri Lanka was investigated to add additional insight into global spread, emergence and evolution of MRSP. A total of 234 samples from dogs visiting veterinary clinics were cultured for staphylococci and the genomes of the MRSP isolates were sequenced, to identify resistance genes, the multilocus sequence types (MLST) and spa types. From a questionnaire the history of antimicrobial treatment and patient information was obtained.S. pseudintermedius was isolated from 116/229 samples, eight of these were MRSP. Six MRSP CC45 isolates contained a pseudo-SCC element ΨSCCmec57395. Two isolates belonging to ST429 (CC761) and ST121 (CC121) contained novel variants of the SCCmec Type V(T) element. The elements were designated SCCmecV(T)SL/066, that carried additional transposon-related genes, and SCCmecV(T)SL/154 that carried a type III restriction-modification system, a type 7 ccr gene complex, and a cadA coding sequence. Thirty-seven percent of the dogs received antimicrobial treatment at the time of sampling of which four dogs were MRSP-positive. The proportion of MRSP among S. pseudintermedius is low compared to other countries, despite the fact that in Sri Lanka antimicrobials for treatment of dogs are available over the counter. Important is the finding of novel type V(T) SCCmec elements, which further underlines the high recombination frequency of SCC elements. The ΨSCCmec57395 was found in isolates of CC45, which is the only sequence type of MRSP known to contain this pseudo-cassette.

Clonal diversity and geographic distribution of methicillin-resistant Staphylococcus pseudintermedius from Australian animals: Discovery of novel sequence types

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an increasingly prevalent pathogen in veterinary medicine. This study examined the molecular epidemiology of clinical MRSP isolated from Australian animals. Clinical staphylococci submitted to all Australian veterinary diagnostic laboratories were collected during 2013 and identified using traditional phenotypic tests and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Phenotypic antimicrobial resistance was determined using broth microdilution and disk diffusion. MRSP isolates were characterized by whole genome sequencing which included identification of the mecA gene. Phylogenetic relationships were inferred by comparison of single nucleotide polymorphisms. Of the 669 S. pseudintermedius isolates collected from dogs, cats and cattle, 77 (11.5%) were MRSP. Nineteen multilocus sequence types (STs) were identified, with most isolates belonging to one of five STs (ST71, ST497, ST316, ST496 and ST45). Phylogenetic analysis revealed that Australian ST71 appears closely related to ST71 from overseas. ST497 and ST496 represented novel sequence types, not previously reported outside Australia. Most other STs were novel and only distantly related to each other. Geographical clustering of STs was observed. All isolates belonging to the five main STs were multi- to extensively- drug resistant while isolates from singleton STs generally had lower levels of antimicrobial resistance. The frequency of ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin, chloramphenicol and tetracycline resistance varied significantly between STs (p<0.01). Australian MRSP isolates are phylogenetically diverse, with a mix of previously unreported and well known international MRSP clones that demonstrate geographic clustering and exhibit both multidrug-resistant and extensively drug-resistant phenotypes.

Frequency, antimicrobial susceptibility and clonal distribution of methicillin-resistant Staphylococcus pseudintermedius in canine clinical samples submitted to a veterinary diagnostic laboratory in Italy: A 3-year retrospective investigation

In the last decade there has been a rapid global spread of methicillin-resistant Staphylococcus pseudintermedius (MRSP) clones displaying multidrug resistance in dogs. We investigated prevalence, antimicrobial susceptibility and clonal distribution of MRSP isolated from clinical canine samples between during 2011–2014. Following species identification by nuc PCR, MRSP were confirmed by the presence of mecA and characterized by antimicrobial susceptibility testing, Pulsed Field Gel Electrophoresis (PFGE), SCCmec typing, and Multi-Locus Sequence Typing (MLST) of a few isolates having distinct PFGE profiles. Both the MRSP isolation frequency in the 175 samples tested (12%) and the prevalence of methicillin resistance amongst the 63S. pseudintermedius isolates (33%) were high compared to a previous study in Italy. Sequence type (ST)71 carrying SCCmec type II–III, described as the epidemic European MRSP clone, accounted for approximately half of the isolates. The remaining isolates belonged to ST410-SCCmec type II–III, ST258-SCCmec type IV and other three clones associated with SCCmec type IV (ST261, ST290 and ST477). MRSP were consistently resistant to potentiated sulfonamides, and more frequently to clindamycin, ciprofloxacin and doxycycline than methicillin-susceptible isolates. Gentamicin was the only antibiotic showing good in vitro activity on all MRSP with 20 of the 21 isolates being susceptible. Results confirm a high prevalence of MRSP amongst clinical samples in Italy, revealing the emergence of new clones other than ST71, such as ST258, ST410, ST261, ST290 and ST477, here describe for the first time. Implementation of antimicrobial stewardship and surveillance programmes are required to prevent the emergence of new MRSP clones and reducing transmission in small animal practice.