Staphylococcus aureus is a Gram-positive coccoid bacterium and a facultative pathogen, usually colonizing the skin and mucosal surfaces of healthy humans and animals. S. aureus infections range from mild skin diseases to surgical site infections, life threatening pneumonia or sepsis. S. aureus harbors a great zoonotic potential and humans can get infected via close contact to humans, animals or contaminated animal products or the consumption of contaminated food. Subject of this thesis were 19 S. aureus isolates from 17 horses admitted to a veterinary clinic between 2015 and 2017. All but one isolate, which was sampled from an injury, originated from surgical site infections. These isolates were further investigated since they showed unusual results in antimicrobial susceptibility testing (AST). They were characterized via multi locus sequence typing (MLST), spa typing and phenotypic and genotypic resistance profiles against antimicrobial agents and biocides. Whole genome sequencing (WGS) allowed the in-depth investigation of resistance determinants and virulence factors. The isolates belonged to two sequence types (STs), ST1 (n = 3) and ST1660 (n = 16), which are both common colonizers and pathogens of animals (especially horses) and humans (especially veterinary personnel). The respective spa types were t127 for the ST1 isolates, and t3043 (n = 14); t2484 (n = 1) and t549 (n = 1) for the ST1660 isolates. All isolates were classified as multiresistant, since all were resistant to penicillins (blaZ), aminoglycosides (aacA-aphD) and trimethoprim (dfrG or dfrS1). The ST1 isolates harbored an additional aminoglycoside resistance gene (aadD) and were also resistant to tetracyclines (tet(L)). Within the STs the isolates seemed to be closely related, but there was no relatedness between the ST1 isolates and the ST1660 isolates. None of the isolates harbored the toxic shock syndrome toxin 1 gene tst or the Panton-Valentin leukocidin (PVL) genes lukF-PV and lukS-PV. All isolates were positive for staphylococcal enterotoxins or enterotoxin like proteins associated with the enterotoxin gene cluster egc and the leukocidin LukD/E. Saeq1, a bacteriophage harboring the leukocidin LukP/Q and the equine staphylococcal complement inhibitor eqScin, was present in all isolates. Biocide susceptibility testing (BST) via broth macrodilution and broth microdilution (BMD) showed that both methods generated comparable and reproducible results and are suitable for future investigations of biocide susceptibility. The minimal inhibitory concentrations (MICs) for benzalkonium chloride (BAC), chlorhexidine and glutardialdehyde did not differ vastly from those of the reference strains and were below the used concentrations of the respective agents, except for a few isolates for BAC. Since BAC is rarely used as a single agent and the MIC was merely at the lowest used concentration of the agent, this does not necessarily result in BAC resistance. The isolates presented reduced susceptibility to oxacillin, while lacking the mec genes, commonly responsible for oxacillin resistance, thus being classified as borderline oxacillin-resistant S. aureus (BORSA). Using AST, WGS and a nitrocefin test, inducible hyperproduction of the β-lactamase BlaZ was identified as the reason for the reduced oxacillin susceptibility. Interestingly, all but one of the ST1660 isolates produced remarkably higher amounts of BlaZ than the ST1 isolates and the ST1660 isolate IMT37083. IMT37083 and the ST1 isolates shared 19 amino acid (aa) exchanges in the β-lactam sensor protein BlaR1, which could explain the lower inducibility of these isolates. When using different test methods and set-ups to assess susceptibility to the combination sulfamethoxazole/trimethoprim (SXT), and the single compounds – a sulfonamide (SUL, here: sulfisoxazole) and trimethoprim (TMP) – varying results occurred. Again, a genetic approach was chosen to elucidate the true resistance properties. The ST1 isolates were the only ones consistently classified as resistant to SXT. Resistance to SULs (BMD) and TMP (BMD and agar disk diffusion (DD)) was also detected. These isolates harbored the TMP resistance gene dfrG and a mutation within their dihydropteroate synthase gene folP, resulting in the aa exchange F17L, which confers SUL resistance. Therefore, these isolates are truly resistant to SXT, TMP and SULs. The ST1660 isolates harbored the TMP resistance gene dfrS1 and were consistently classified as TMP-resistant. BMD and DD identified these isolates as SUL-susceptible. Regarding SXT, the isolates were inconsistently classified as resistant, susceptible, and intermediate. There was no relevant aa alteration in their FolP proteins. This highlights the necessity on further research in this field, since the mechanisms and tangible SUL resistance determinants are barely unveiled. This thesis also confirmed the need for further development and establishment of diagnostic procedures to identify unusual antimicrobial resistance properties and their mechanisms. AST protocols used in veterinary medicine also lack animal specific clinical breakpoints which impedes the transferability of AST results to expectable clinical outcomes and, by extension antimicrobial stewardship.
Staphylococcus aureus is a Gram-positive coccoid bacterium and a facultative pathogen, usually colonizing the skin and mucosal surfaces of healthy humans and animals. S. aureus infections range from mild skin diseases to surgical site infections, life threatening pneumonia or sepsis. S. aureus harbors a great zoonotic potential and humans can get infected via close contact to humans, animals or contaminated animal products or the consumption of contaminated food. Subject of this thesis were 19 S. aureus isolates from 17 horses admitted to a veterinary clinic between 2015 and 2017. All but one isolate, which was sampled from an injury, originated from surgical site infections. These isolates were further investigated since they showed unusual results in antimicrobial susceptibility testing (AST). They were characterized via multi locus sequence typing (MLST), spa typing and phenotypic and genotypic resistance profiles against antimicrobial agents and biocides. Whole genome sequencing (WGS) allowed the in-depth investigation of resistance determinants and virulence factors. The isolates belonged to two sequence types (STs), ST1 (n = 3) and ST1660 (n = 16), which are both common colonizers and pathogens of animals (especially horses) and humans (especially veterinary personnel). The respective spa types were t127 for the ST1 isolates, and t3043 (n = 14); t2484 (n = 1) and t549 (n = 1) for the ST1660 isolates. All isolates were classified as multiresistant, since all were resistant to penicillins (blaZ), aminoglycosides (aacA-aphD) and trimethoprim (dfrG or dfrS1). The ST1 isolates harbored an additional aminoglycoside resistance gene (aadD) and were also resistant to tetracyclines (tet(L)). Within the STs the isolates seemed to be closely related, but there was no relatedness between the ST1 isolates and the ST1660 isolates. None of the isolates harbored the toxic shock syndrome toxin 1 gene tst or the Panton-Valentin leukocidin (PVL) genes lukF-PV and lukS-PV. All isolates were positive for staphylococcal enterotoxins or enterotoxin like proteins associated with the enterotoxin gene cluster egc and the leukocidin LukD/E. Saeq1, a bacteriophage harboring the leukocidin LukP/Q and the equine staphylococcal complement inhibitor eqScin, was present in all isolates. Biocide susceptibility testing (BST) via broth macrodilution and broth microdilution (BMD) showed that both methods generated comparable and reproducible results and are suitable for future investigations of biocide susceptibility. The minimal inhibitory concentrations (MICs) for benzalkonium chloride (BAC), chlorhexidine and glutardialdehyde did not differ vastly from those of the reference strains and were below the used concentrations of the respective agents, except for a few isolates for BAC. Since BAC is rarely used as a single agent and the MIC was merely at the lowest used concentration of the agent, this does not necessarily result in BAC resistance. The isolates presented reduced susceptibility to oxacillin, while lacking the mec genes, commonly responsible for oxacillin resistance, thus being classified as borderline oxacillin-resistant S. aureus (BORSA). Using AST, WGS and a nitrocefin test, inducible hyperproduction of the β-lactamase BlaZ was identified as the reason for the reduced oxacillin susceptibility. Interestingly, all but one of the ST1660 isolates produced remarkably higher amounts of BlaZ than the ST1 isolates and the ST1660 isolate IMT37083. IMT37083 and the ST1 isolates shared 19 amino acid (aa) exchanges in the β-lactam sensor protein BlaR1, which could explain the lower inducibility of these isolates. When using different test methods and set-ups to assess susceptibility to the combination sulfamethoxazole/trimethoprim (SXT), and the single compounds – a sulfonamide (SUL, here: sulfisoxazole) and trimethoprim (TMP) – varying results occurred. Again, a genetic approach was chosen to elucidate the true resistance properties. The ST1 isolates were the only ones consistently classified as resistant to SXT. Resistance to SULs (BMD) and TMP (BMD and agar disk diffusion (DD)) was also detected. These isolates harbored the TMP resistance gene dfrG and a mutation within their dihydropteroate synthase gene folP, resulting in the aa exchange F17L, which confers SUL resistance. Therefore, these isolates are truly resistant to SXT, TMP and SULs. The ST1660 isolates harbored the TMP resistance gene dfrS1 and were consistently classified as TMP-resistant. BMD and DD identified these isolates as SUL-susceptible. Regarding SXT, the isolates were inconsistently classified as resistant, susceptible, and intermediate. There was no relevant aa alteration in their FolP proteins. This highlights the necessity on further research in this field, since the mechanisms and tangible SUL resistance determinants are barely unveiled. This thesis also confirmed the need for further development and establishment of diagnostic procedures to identify unusual antimicrobial resistance properties and their mechanisms. AST protocols used in veterinary medicine also lack animal specific clinical breakpoints which impedes the transferability of AST results to expectable clinical outcomes and, by extension antimicrobial stewardship.
