Staphylococcus pseudintermedius-associated canine skin disorders: isolation, identification, antibiotic resistance patterns, sequence typing and alternative therapeutic strategies.

Sthaphylococcus pseudintermedius (S. pseudintermedius) is the main inhabitant of skin and mucosa of dogs, where it represents the major bacterial pathogen causing skin diseases. Firstly described in 2005, S. pseudintermedius is a member of the Staphylococcus intermedius Group (SIG), together with S. intermedius and S. delphini. The recent emergence and rapid dissemination of multidrug-resistant S. pseudintermedius strains, particulary methicillin-resistant S. pseudintermedius (MRSP), showing more and more often resistance to the antibiotic agents licensed for use in small animal practice, represent a relevant threat to small animal and public health. In the last decade, the antibiotic resistance issue has become increasingly relevant, highlighting the need to control and limit it by the means of a continuous surveillance of both methicillin-resistant and methicillin-susceptible S. pseudintermedius isolates as well as the development of new and alternative therapeutic options. This study aims to describe the phenotypic and genotypic features, the antibiotic resistance patterns, the prevalence of virulence genes of both MRSP and methicillin-susceptible S. pseudintermedius (MSSP) strains, isolated from dogs suffering from otitis externa and pyoderma. MRSP sequence type clones circulating in Southern Italy were also investigated. Moreover, the antimicrobial activity of hydroxypiridone-based iron chelating co-polymer DIBI and abietic acid was analyzed against S. pseudintermedius isolates, as alternative treatment options. From a total of 259 staphylococcal cultures, 126 S. pseudintermedius strains were selected during the years 2015-2017. S. pseudintermedius strains (49%) were identified by proteomic (MALDI-TOF MS) and molecular profiling (nuc and hlb genes). S. pseudintermedius strains were recovered from dogs suffering from otitis externa (84%) and pyoderma (16%), originating from Naples, Campania Region (52%) and Latina, Lazio Region (48%). Amongst the 126 S. pseudintermedius strains, 18% were MRSP, carrying mecA gene. The remaining 82% were classified as MSSP. None of the isolates was positive to mecC gene. Furthermore, all the MRSP showed a complete resistance to amoxicillin-clavulanate and ampicillin (100%), while the highest resistance rates to selected non-β-lactam antibiotics were registered for erythromycin (91%); tetracycline (87%); sulfamethoxazole/trimethoprim (78%); kanamycin (78%); streptomycin (78%); clindamycin (65%) and enrofloxacin (61%). However, there was no resistance to vancomycin and linezolid. It is worth noting that 91% MRSP strains were found to be multidrug-resistant strains. The 82% MSSP isolates, showed interesting antibiotic resistant profiles, but their resistance rates were of about 50% lower than MRSP strains. The phenotypic tetracycline- and erythromycin-resistant MRSP and MSSP strains harbored tetK and tetM genes, alone or in association, and ermB gene. Referring to MRSP isolates, they mainly belonged to multidrug-resistant sequence type ST71 (26%) European clone. In this study, 9 new clones of MRSP were identified and described in Italy and worldwide for the first time. The newly described sequence types (STs) were named from ST1053 up to ST1061. In addition, DIBI and abietic acid resulted to be effective against MRSP and MSSP strains, inhibiting their growth. Thus, they may be considered as valid non-antibiotic alternative treatment approaches for S. pseudintermedius infections

Antimicrobial Resistant Staphylococcus Species Colonization in Dogs, Their Owners, and Veterinary Staff of the Veterinary Teaching Hospital of Naples, Italy

This study aimed to identify Staphylococcus species isolated from nasal swabs of both healthy and diseased dogs, and those of human origin, obtained from nasal swabs of both owners and veterinary staff. Firstly, pet owners were requested to complete a questionnaire relating to the care and relationship with their pets, whose results mainly showed a statistically significant higher frequency of hand washing in diseased dogs’ owners than in healthy dogs’ owners. Canine nasal swabs were obtained from 43 diseased dogs and 28 healthy dogs, while human nasal swabs were collected from the respective dogs’ owners (71 samples) and veterinary staff (34 samples). The isolation and identification of Staphylococcus spp. were followed by disk diffusion method to define the antimicrobial resistance profiles against 18 different molecules. Staphylococcus pseudintermedius was the most frequent isolated strain in both diseased (33.3%) and healthy (46.1%) dogs. Staphylococcus epidermidis was the most frequent isolated bacterium in diseased dogs’ owners (66.6%), while in nasal samples of healthy dogs’ owners, the same frequency of isolation (38.4%) was observed for both Staphylococcus epidermidis and Staphylococcus aureus. All the isolated strains showed good susceptibility levels to the tested antimicrobials; however, the carriage of oxacillin-resistant strains was significantly higher in diseased dogs than in healthy ones (71% and 7.7%, respectively). Only in three cases the presence of the same bacterial species with similar antimicrobial resistance profiles in dogs and their owners was detected, suggesting the potential bacterial transmission. In conclusion, this study suggests potential transmission risk of staphylococci from dogs to humans or vice versa, and highlights that the clinical relevance of Staphylococcus pseudintermedius transmission from dog to human should not be underestimated, as well as the role of Staphylococcus aureus from human to dog transmission

A Preliminary Study on Antimicrobial Susceptibility of Staphylococcus spp. and Enterococcus spp. Grown on Mannitol Salt Agar in European Wild Boar ( Sus scrofa) Hunted in Campania Region-Italy

The importance of wild boar lies in its role as a bioindicator for the control of numerous zoonotic and non-zoonotic diseases, including antibiotic resistance. Mannitol Salt Agar (MSA) is a selective medium used for isolation, enumeration, and differentiation of pathogenic staphylococci. Other genera such as Enterococcus spp. are also salt tolerant and able to grow on MSA. The present study focused on the identification, by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), of bacteria grown on MSA isolated from the nasal cavities of 50 healthy wild boars hunted in Campania Region (southern Italy) in the year 2019. In addition, the antimicrobial resistance phenotype of the isolated strains was determined by disk diffusion method. Among genus Staphylococcus, coagulase-negative Staphylococcus (CoNS) were the most common isolated species, with Staphylococcus xylosus as the most prevalent species (33.3%). Furthermore, Enterococcus spp. strains were isolated, and Enterococcus faecalis was the species showing the highest frequency of isolation (93.8%). For staphylococci, high levels of resistance to oxacillin (93.3%) were recorded. Differently, they exhibited low frequencies of resistance to tested non-β-lactams antibiotics. Among enterococci, the highest resistances were observed for penicillin (93.7%), followed by ampicillin (75%), and ciprofloxacin (68.7%). Interestingly, 43.7% of the isolated strains were vancomycin-resistant. In conclusion, this study reports the phenotypic antibiotic resistance profiles of Staphylococcus spp. and Enterococcus spp. strains isolated from nasal cavities of wild boars hunted in Campania Region, highlighting that these wild animals are carriers of antibiotic resistant bacteria

a successful vancomycin treatment of multidrug resistant mrsa associated canine pyoderma

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Detection of a novel clone of Acinetobacter baumannii isolated from a dog with otitis externa

In this study, the isolation ofAcinetobacter baumanniiin a dog with clinical bilateral otitis externa is described.Moreover, to investigate the zoonotic potential of the isolate, microbiological examinations on the familymembers were performed. AnA. baumanniistrain was isolated from nasal swab in one of the dog owners. Theidentity of bacterial strains, either from dog and owner, was confirmed by phenotypic and molecular typing(wgMLST). Furthermore, to assess the pathogenic potential of the isolates a deep characterization of virulenceand antibiotic resistance genes was done by Whole Genome Sequencing (WGS). Finally, the susceptibility to-wards a wide panel of antimicrobials was investigated. In our knowledge, this is thefirst recorded case ofA.baumanniiisolation from canine auricular swabs in Italy. And interestingly, this study underlines the possiblespread of this microorganism from human to anima

Endometritis associated with Enterococcus casseliflavus in a mare: A case report

Abstract Infectious endometritis is one of the main causes of subfertility/infertility in the mare. In this report, we present the first case of endometritis in mare associated with a strain of Enterococcus casseliflavus, an unusual gram-positive bacterium which can also be a zoonotic agent. Furthermore, the isolated strain showed a worrying multidrug-resistant profile. The accurate finding of a successful antimicrobial treatment and consequently, the pregnancy diagnosis indicate the importance to isolate, identify and define the antibiotic resistance profile of bacteria associated with endometritis

Genotypic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of mares with fertility problems

Escherichia coli is the bacterial pathogen most frequently associated with mare infertility. Here, we characterized 24 E. coli strains isolated from mares which presented signs of endometritis and infertility from a genotypic and phenotypic point of view. The majority of the isolates belonged to phylogenetic group B1 (9/24, 37.5%). Regarding antibiotic resistance profiles, 10 out of 24 (41.7%) were multidrug-resistant (MDR). Moreover, 17 out of 24 (70.8%) were strong or moderate biofilm producers, and of these eight were MDR strains. Interestingly, 21 out of 24 (87.5%) E. coli strains were phenotypically resistant to ampicillin and 10 of them were also resistant to amoxicillin with clavulanic acid. Regarding the presence of selected virulence factors, 50% of the examined strains carried at least three of them, with fimH detected in all strains, and followed by kpsMTII (11/24, 45.9%). No strain was able to invade HeLa cell monolayers. No relevant differences for all the investigated characteristics were shown by strains that grew directly on plates versus strains requiring the broth-enrichment step before growing on solid media. In conclusion, this work provides new insight into E. coli strains associated with mares' infertility. These results broaden the knowledge of E. coli and, consequently, add useful information to improve prevention strategies and therapeutic treatments contributing to a significant increase in the pregnancy rate in mares

An update on microbiological causes of canine otitis externa in Campania Region – Italy.

Objective: To update the recent knowledge of the microbiological causes of canine otitis externa in Campania Region (Italy) and the antibiotic susceptibility patterns of the isolated strains. Methods: A total of 122 dogs were examined by otoscopy, and auricular swab samples were collected from both ears in 74 dogs presenting clinical bilateral otitis and from single ears in 48 dogs displaying clinical unilateral otitis. Cytological examination, bacteriological analysis and antimicrobial susceptibility tests were performed. Results: Thirty-one out of 122 dogs were positive for yeast species (25.4%, 95% confidence interval (CI): 18.2%–34.2%) with a higher prevalence of Malassezia pachydermatis (21/31 isolates, 67.7%, CI: 48.5%–82.7%), and a total of 91 out of 122 dogs were positive for bacterial species (74.6%; CI: 65.8%–81.8%) with a higher prevalence of Staphylococcus pseudintermedius (45/143 isolates, 31.5%, CI: 24.1%–39.8%). These results are the first description of Streptococcus agalactiae-associated otitis. The yeasts isolated showed high levels of susceptibility to all antifungal agents tested; on the contrary all the isolated bacterial strains were highly resistant to at least four out of ten antimicrobial classes. Both Gram-positive and Gram-negative bacteria showed high resistance to amoxicillin/clavulanate and kanamycin hence they are not recommended as initial empirical therapy for the otitis treatment. Conclusions: This update illustrates an increase in antibiotic resistances providing an insight into the current knowledge of the therapeutic procedures followed on canine otitis externa in Italy. It also emphasizes the importance of considering the results of the microbiological and sensitivity tests to decide on an appropriate antibiotic therapy

Use of cytobrush for bacteriological and cytological diagnosis of endometritis in mares

Background and Aim: A combined microbial and cytological examination of uterine samples is the main diagnostic method for endometritis in mares. This study aimed to describe a procedure for using the same uterine cytobrush (CB) for both bacteriological and cytological evaluation. Materials and Methods: The procedure consists of rolling the CB onto a sterilized glass slide immediately after collection and before the transfer into a sterile saline solution. In Experiment 1, a comparison between bacteriological results of the cotton swab (CS) and CB or pellet was made in 10 mares; in Experiment 2, bacteriological and cytological results were compared between different processing methods of CB in 28 mares; in other 6 mares, a CB was processed for cytology only, to investigate the reasons for the low cellularity of the pellet. Results: The agreement between culture results from the CB and CS was evaluated, and a comparison between the cytological data obtained by different processing methods of CB was performed. The perfect agreement between the CB and CS microbiological results was found. The described procedure enables useful diagnostic smears for cytology. Moreover, the seeding of both the tip of CB and the saline solution used for the transport produced accurate bacteriological results. Conclusion: The protocol described in this study for the use of CB for both cytological and bacteriological analysis could be used for the diagnosis of endometritis. To maximize diagnostic sample quality, cytology slides must be prepared with meticulous care in the field to preserve cellular integrity and minimize artifacts