Shevchenko M. Staphylococcus pseudintermedius and Staphylococcus aureus: prevalence in animals, microbiological and molecular genetic characterization and antibiotic resistance

The dissertation work of M. Shevchenko is devoted to the study of the prevalence and characterization of microorganisms Staphylococcus pseudintermedius and Staphylococcus aureus among dogs, cats and cows in Ukraine. The study included the determination of antibiotic resistance of these bacteria, the ability to form biofilms, and the presence of genes associated with pathogenicity factors, including toxin production and adhesion. The results showed that Staph. pseudintermedius actively colonizes the nasal cavities (24.1%) and ear canals (7.6%) of clinically healthy dogs. This species was the dominant pathogen of dermatologic and wound infections, it was detected in 23.4% of sick dogs and 12.5% of cats. Among all the detected staphylococci from dogs, Staph. pseudintermedius accounted for the vast majority (58%), while in cats its share reached 30.8%. Staph. aureus was much less common – only 5.2% of dogs and 6.3% of cats. In cattle, Staph. aureus was one of the main causative agents of intrauterine infections, it was detected in 33.3% of mastitis cases. At the same time, it was Staph. aureus that caused the clinical form of the disease more often than other types of staphylococci (16.6%). For a complete characterization of field isolates, their morphological and cultural properties were studied. Species identification and differentiation of Staph. pseudintermedius and Staph. aureus were performed using a set of microbiological tests, PCR with species-specific and genus-specific primers, sequencing of a fragment of the thermonuclease gene and high-resolution MALDI-TOF mass spectrometry. An important aspect of the study was the study of antibiotic resistance of the isolates. A meta–analysis of the literature on Staph. pseudintermedius and Staph. aureus resistance in dogs and cats in Europe in 2016 – 2020 revealed significant heterogeneity of results between different research groups. Overall, a high level of resistance was observed in Staph. pseudintermedius isolates to erythromycin (52.9%), tetracycline (43.8%), and penicillin (34.8%). Staph. aureus was most often resistant to penicillin (66.9%) and tetracycline (55%). Among the field isolates of Staph. pseudintermedius isolated from dogs and cats with infections in Ukraine, the highest resistance was found to trimethoprim with sulfamethoxazole (36.8%), penicillin (18.2%) and erythromycin (18.2%). Staph. aureus isolates from sick animals were most often resistant to penicillin (50%). Particular attention is drawn to the detection of methicillin–resistant strains – one isolate of Staphylococcus pseudintermedius from a clinically healthy dog and 23.3% of methicillin-resistant Staphylococcus aureus among isolates obtained from cows with mastitis. In parallel with the phenotypic determination of methicillin resistance, a molecular screening was performed to identify mecA genes, the presence of which determines the resistance of these strains to methicillin. The majority of Staph. pseudintermedius (77.3%) and Staph. aureus (83.4%) isolates showed different abilities to form biofilms on abiotic surfaces. PCR analysis of these bacteria revealed the icaA and icaD genes, which play a key role in the process of intercellular adhesion and biofilm formation. Additionally, the presence of genes encoding toxins and colonization factors was determined. All analyzed strains of Staph. pseudintermedius contained the siet gene, which is responsible for the production of a potent exfoliative toxin. In addition, 90% of the isolates contained the lukS gene and 100% contained the lukF gene, which encode two components of the highly active staphylococcal leukotoxin. These data indicate a high pathogenic potential of Staph. pseudintermedius as a pathogen of infections in animals. For the final verification of the species affiliation of the isolates to Staphylococcus pseudintermedius, the PCR-amplified fragment of the nuc gene, which encodes a thermostable nuclease, was sequenced. Comparison of the obtained nucleotide sequences with the reference genomic sequences available in the GenBank database showed their high identity (99.3% and 99.2%, respectively) to the sequences of Staph. pseudintermedius. At the same time, phylogenetic analysis showed that the obtained sequences differ from closely related species of the SIG group, such as Staph. delphini and Staph. intermedius. The obtained nucleotide sequences were registered in the international database under the numbers OR555770 and OR555771. The results of the dissertation research are important for assessing the current state of distribution of Staphylococcus pseudintermedius and Staphylococcus aureus among different animal species in Ukraine, their antibiotic resistance profiles and the potential danger posed by resistant and pathogenic strains of these bacteria. The proposed diagnostic approaches can be implemented and used in the work of veterinary and medical diagnostic laboratories.