The molecular epidemiology and antimicrobial resistance of Staphylococcus pseudintermedius canine clinical isolates submitted to a veterinary diagnostic laboratory in South Africa

Abstract

Staphylococcus pseudintermedius is an important cause of clinical infections in small-animal-veterinary medicine. Evolutionary changes of strains using multilocus sequence typing (MLST) have been observed among S. pseudintermedius in European countries and the United States. However, there are limited or no studies on the detection of methicillin resistant Staphylococcus pseudintermedius (MRSP) and predominating MLST strains in South Africa. Therefore, this study aimed to determine the molecular epidemiology of S. pseudintermedius in South Africa. Twenty-six, non-duplicate, clinical isolates from dogs were obtained as convenience samples from four provinces in South Africa. The Kirby Bauer disk diffusion test was used to determine antimicrobial susceptibility. We used Resfinder and the Comprehensive Antibiotic Resistance Database (CARD) to detect antimicrobial resistance genes. Virulence genes were identified using the virulence factor database and Basic Local Alignment Search Tool (BLASTN) on Geneious prime. geoBURST analysis was used to study relationships between MLST. Finally, the maximum likelihood phylogeny was determined using Randomized Axelerated Maximum Likelihood (RAxML). Twenty-three isolates were confirmed as S. pseudintermedius of which 14 were MRSP. In addition to β-lactam antimicrobials, MRSP isolates were resistant to tetracycline (85.7%), doxycycline (92.8%), kanamycin (92.8%), and gentamicin (85.7%). The isolates harbored antimicrobial resistance genes (tetM, ermB, drfG, cat, aac(6')-Ie-aph(2")-Ia, ant(6)-Ia, and aph(3')-III) and virulence genes (AdsA, geh, icaA, and lip). MLST analysis showed that ST2228, ST2229, ST2230, ST2231, ST2232, ST2318, ST2326 and ST2327 are unique sequence types in South Africa. Whereas, previously reported major STs including ST45, ST71, ST181, ST551 and ST496 were also detected. The geoBURST and phylogenetic analysis suggests that the isolates in South Africa are likely genetically related to isolates identified in other countries. Highly resistant MRSP strains (ST496, ST71, and ST45) were reported that could present challenges in the treatment of canine infections in South Africa. Hence, we have gained a better understanding of the epidemiology of MRSP in the African continent, the genes involved in resistance and virulence factors associated with these organisms.

Fig 1. Virulence genes detected among S. pseudintermedius isolates submitted to a veterinary diagnostic laboratory in South Africa, 2021.

S. pseudintermedius cell wall anchored proteins genes (spsM, spsL, spsD, spsO, spsI, spsK, spsF), fibrinogen binding protein gene (fnbB), Intercellular adhesion (icaA, icaB), leukotoxin (lukF-I, lukS-I), ser-asp rich fibrinogen binding protein (sdrE), immune evasion (adsA), exfoliative toxin (se-int, SpeX, speta, siet) and exoenzyme (coagulase, nanB).

Fig 2. Clonal relationship of MLST using isolates identified in South Africa and major STs identified worldwide.

The geoBURST full MST algorithm showed links connected by SLV, DLV, TLV and 4 link variants. Each node represents a ST. The size of the node denotes the sample size of each ST. The distance between each node is represented by 1 = SLV, 2 = DLV, 3 = TLV and 4 = four locus variants. The predicted founder clonal complexes are represented as green.

Fig 3. Phylogenetic tree based on the time-reversible nucleotide substitution model and four gamma categories for rate heterogeneity of S. pseudintermedius isolates.

The phylogenetic tree includes isolates of the present study and selection of previously sequenced genomes representing phylogenetic diversity found across the species.