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Article

Characterization of Staphylococcus aureus CC1 and CC1660 of Human and Equine Origin

by
Johanna Jahnen
1,2,
Christiane Cuny
3,
Wolfgang Witte
3,
Ralf Ehricht
4,5,6,
Stefan Monecke
4,5,
Dennis Hanke
1,2,
Tanja Ahrens
1,2,
Marta Leal
7,
Sofia S. Costa
7,
Isabel Couto
7,
Stefan Schwarz
1,2,* and
Andrea T. Feßler
1,2,*
1
Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
2
Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
3
Robert Koch Institute, 38855 Wernigerode, Germany
4
Leibniz Institute of Photonic Technology, Member of the Research Alliance “Leibniz Health Technologies” and the Leibniz Centre for Photonics in Infection Research (LPI), 07745 Jena, Germany
5
InfectoGnostics Research Campus Jena, Center for Applied Research, 07743 Jena, Germany
6
Institute of Physical Chemistry, Friedrich Schiller University Jena, 07743 Jena, Germany
7
Global Health and Tropical Medicine (GHTM), LA-REAL, Instituto de Higiene e Medicina Tropical (IHMT), Universidade NOVA de Lisboa, 1349-008 Lisbon, Portugal
*
Authors to whom correspondence should be addressed.
Antibiotics 2025, 14(11), 1082; https://doi.org/10.3390/antibiotics14111082
Submission received: 1 September 2025 / Revised: 21 October 2025 / Accepted: 22 October 2025 / Published: 27 October 2025
(This article belongs to the Special Issue Antibiotic Resistance in Bacterial Isolates of Animal Origin)
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Abstract

Background/Objectives: Staphylococcus aureus isolates from humans and horses of the equine-associated clonal complexes (CCs) CC1 and CC1660 were comparatively investigated for their genomic relationships. Methods: A total of 91 S. aureus isolates (64 human, 27 equine) were subjected to whole-genome sequencing (WGS), sequence analysis, and antimicrobial susceptibility testing. Results: WGS confirmed 75 CC1 and 16 CC1660 isolates, comprising nine sequence types (STs) in CC1 and four STs in CC1660. Ten spa types were present in CC1 and five in CC1660. In the arcC gene of three CC1 isolates, a 285 bp deletion was detected, and a nucleotide deletion causing a premature stop codon was found in one CC1660 isolate. Core genome (cg) MLST revealed a minimum difference of 1398/1492 alleles between the two CCs. All CC1 isolates harbored agr group III and capsule type 8 alleles, whereas all CC1660 isolates had agr group II and capsule type 5 alleles. Antimicrobial susceptibility testing revealed 18 phenotypic and 19 genotypic resistance patterns. All isolates were susceptible to vancomycin, linezolid and quinupristin–dalfopristin. Several virulence genes were detected in different combinations. The equine leukocidin genes lukP/lukQ were found in 22 isolates from horses and 38 isolates from humans, of which 35 had confirmed contact with horses. No Panton–Valentine leukocidin genes were found. Three human CC1660 isolates carried the toxic shock syndrome toxin-1 gene tst-1. Conclusions: The analysis of the 91 isolates might suggest intra- and interspecies transmission among and between humans and horses, which should be monitored in the future.

1. Introduction

Staphylococcus aureus (S. aureus) is mainly a harmless colonizer of humans and animals, but it can also cause severe infections in cases of predisposition. It is involved in several infections, including wound infections, skin and soft tissue infections, dermatitis, infective endocarditis, pneumonia, bacteremia, sepsis, osteomyelitis and septic arthritis in humans [1]. This pathogen is also capable of inducing similar infections in animals with notable examples being bovine mastitis [2] and wound infections affecting various animal species [3], including horses [4].
To facilitate the phylogenetic classification of S. aureus, Multilocus Sequence Typing (MLST) was established based on sequence variability within seven housekeeping genes [5]. MLST assigns isolates to sequence types (STs), which are further grouped into clonal complexes (CCs) [6,7,8]. Interestingly, these CCs usually share common characteristics, which may include partial host specificity. For example, CC5, CC15, CC30, CC45, and CC121 are mainly human-associated [9,10,11], while mainly animal-associated CCs include CC49 (rodents) [12], CC692 (birds) [13,14], CC425 (lagomorphs, badgers and ruminants) [14,15,16,17], CC133 and CC522 (goats and sheep) [18,19], and CC479 and CC705 (cattle) [20]. However, host range is not strictly defined by the MLST genes themselves, but rather by broader features of the genome, including mobile genetic elements [21]. In comparison, other CCs, such as CC398, display a broad host spectrum and can be found in humans [22], pigs [22], cattle [23], horses [24] and poultry [25], among others.
Several S. aureus clonal complexes have been described among horses, including for example CC1, CC8, CC22, CC130, CC398 and CC1660 [26]. They were associated with various infections, such as soft tissue and joint infections, pneumonia, sinusitis, metritis, omphalophlebitis or wound infections [26]. Horses can be infected by human S. aureus strains and some of these strains spread nosocomially in horse clinics. Examples are a CC8 methicillin-resistant S. aureus (MRSA) strain that was brought from Southern Africa into Australia [27] and the “Hannover Epidemic Strain”, another nosocomial CC8 MRSA that was common in Germany during the 1990s in humans and during the 2000s in horses [26]. Among horses, other ST8 isolates have also been found, including, e.g., USA500 and/or the Canadian epidemic MRSA lineage CMRSA-5 with spa type t064 and SCCmecIV elements, which was not present in our collection [28]. This might also be true for CC22 as CC22-MRSA-IV occurs in horses and, commonly, in humans [28,29]. Horses might acquire livestock-associated MRSA, which, at least in Western Europe, is usually represented by CC398. CC130 might also be livestock-associated, as it is common among small ruminants [30,31,32]. For some lineages, such as CC816 and CC8115, not much is known; they might have been transmitted from other animals to horses, or vice versa [33].
Finally, there are lineages that might be considered naturally adapted to horses. These include CC1 and CC1660 (also known as CC350) which have already been observed in Switzerland, Germany, Austria and other countries [34,35,36]. This raises the question of which virulence factors or other genetic determinants are associated with the ability to infect or to colonize horses. These lineages also might be observed in humans and there are several ubiquitous, near-pandemic CC1 strains, methicillin-susceptible S. aureus (MSSA), and MRSA in humans [37,38,39] as well as in livestock [40]. Therefore, it would be interesting to study differences between human and equine CC1 strains to check whether strains from humans can colonize and/or infect horses or vice versa, and to see which genes might pre-determine host specificity.
S. aureus isolates of CC1 and CC1660 have been detected in the Robert Koch Institute’s isolate collection obtained from human and equine samples, respectively. The detection of these equine-associated CCs in human samples is notable and warrants monitoring since a zoonotic spillover of such CCs might be a remarkable aspect for public health in the One Health context. Therefore, the aim of this study was to investigate the genetic relationships of these isolates using whole-genome sequencing (WGS) and bioinformatic analyses, as well as their virulence and resistance properties.

2. Results and Discussion

2.1. Genetic Relationships

In total, 75 isolates were confirmed as CC1, including five cases with two different phenotypes from the same sample (Figure S1), and 16 isolates as CC1660, based on the seven MLST alleles. Among the CC1 isolates, nine STs were detected, 61 isolates belonged to ST1, four to ST81, two to ST9569 and single isolates belonged to ST9300, ST9570, ST9571, ST9572 and ST9573, with the latter four being new STs identified during this study. Moreover, three isolates (18-02051-36, 18-02052-37, 18-02054-39), also assigned to CC1 as single-locus variants, were identified, in which a deletion of 285 bp within the arcC gene had occurred. Because the 285 bp deletion includes the region used to define the arcC allele in MLST, no ST could be assigned for these isolates. Among the CC1660 isolates, four STs were detected, namely ST1660 present in 13 isolates, as well as ST9574, ST9575 and one with a different arcC deletion in single isolates. It should be noted that the novel ST also has a single bp deletion in the homopolymeric region between nucleotides 594 and 605 within the arcC gene. This deletion resulted in a frame shift which produced an early stop codon at amino acid position 211 (see also Section 2.2).
In comparison to the conventional MLST with only seven target genes (https://pubmlst.org/; accessed on 22 October 2025), cgMLST targets a fixed set of genome-wide distributed core genes, including about 400 to over 4000 alleles (https://www.cgmlst.org/ncs; accessed on 22 October 2025), for different bacterial species, including 1861 alleles for S. aureus (https://www.ridom.de/seqsphere/cgmlst/; accessed on 22 October 2025) [41]. The cgMLST revealed a minimum difference between the two CCs of 1398/1492 alleles and clearly separated the two CCs (Table S2). The difference between the isolates ranged from 0 to 288 alleles in CC1 and from 0 to 249 alleles in CC1660 (Figure 1, Table S2).
Because multiple samples were collected from nine equine clinics (ECs), we compared the cgMLST results, sample metadata, and geographic origin (Table S1, Figures S2 and S3) to assess isolate relatedness within and between clinics. Related isolates could be detected in six of the nine equine clinics. The three equine isolates of each of the clinics EC6 and EC7 (including two isolates from the same sample) were classified as related, using the classification proposed by Cunningham et al. [42]. Fourteen isolates from clinic EC1 were investigated, including 12 CC1 isolates (nine human, with two samples containing paired isolates, and three equine). All CC1 isolates were classified as possibly related, with some subsets (nine and three isolates) showing close relatedness. Within the ten CC1 isolates (one human, nine equine) from EC2 as well as the six human CC1 isolates, including two isolates from the same sample, from EC4, all but one of the isolates per equine clinic were closely related. Two of four human isolates in EC5 were also related. In contrast, the seven isolates from EC3 as well as the two isolates from EC8 and EC9 were not related. The detection of equine-related S. aureus isolates of these CCs among humans in equine clinics could be explained as simple contamination due to close contact with horses. Since no repeated testing was performed as in previous studies, e.g., on CC398 [43], a differentiation between contamination, transient or permanent colonization of the CCs among humans cannot be made. Therefore, further investigation of the presence and carriage of S. aureus CC1 and CC1660 among horses would be interesting to monitor a possible spillover of these equine-related CCs to humans and elucidate a potential zoonotic risk of these isolates.
The spa typing revealed ten spa types among the CC1 isolates comprising t114 (n = 1), t127 (n = 62), t273 (n = 1), t559 (n = 2), t922 (n = 1), t1383 (n = 2), t1491 (n = 1), t1508 (n = 3), t1405 (n = 1), and t18599 (n = 1), with t127 being the most prevalent, which is in accordance with the data from the ridom database https://spa.ridom.de/spa-t127.shtml (accessed on 22 October 2025). All spa types were related, but differed in the number of repeats, ranging from four repeats (t18599) to eleven repeats (t1491), which can mainly be explained by one or two deletion or insertion events in the variable region of the spa gene. Moreover, single point mutations in three different positions, compared to the most common alleles, were observed (Figure S4a).
Among the CC1660 isolates, five spa types were detected, comprising four isolates with t549, seven isolates with t3043, three isolates with t11926, as well as single isolates with t12047 and t15977. The five spa types detected among the CC1660 isolates comprised five to twelve repeats. All of the detected spa types were related and differed in the number of repeats and or exchanges of repeats, which can be explained as point mutations in two different positions (Figure S4b).

2.2. Deletions Within the arcC Gene

A 285 bp deletion (corresponding to 95 amino acids, namely the amino acids at positions 117–211) was identified in the arcC gene (Figure 2), which—if complete—encodes a 313-amino acids carbamate kinase, during whole-genome sequencing of three isolates (18-02051-36, 18-02052-37, 18-02054-39). The deletion of amino acids 117–211 is located between the primer binding sites (Figure 2) and results in a change in the PCR product size from 570 bp to 285 bp (Figure 2 and Figure S5). This finding was confirmed via PCR amplification and sequencing of the PCR products (Figure S5). Sequence analysis suggested that the deletion likely resulted from an illegitimate recombination event (Figure 2).
As shown by Ramón-Maiques et al. [44], the positions N51, K128, K209, and D210 play a role in the ternary complex active-site architecture and the catalytic mechanism proposed. These positions correspond to the positions N51, K128, K213, and D214 in the S. aureus ArcC carbamate kinase (GenBank: BAB58794.1; ABD21772.1). Ramón-Maiques et al. [44] observed an extreme negative effect of a mutation in position 128 (K128A) pointing towards a key role of the protruding subdomain, hanging over the carbamate kinase active center, as an exclusive and characteristic carbamate kinase feature for substrate binding and catalysis. A mechanism connecting carbamoyl phosphate site occupation with the protruding subdomain approach, involving V206-I207 (positions V210-I211 in S. aureus) in the carbamoyl phosphate site and P162-S163 (positions A164-S165 in S. aureus) in the protruding subdomain stem, was also identified [44]. From the important amino acid positions described by Ramón-Maiques et al. [44], the respective correlating positions K128, V210-I211 and P162-S163 are part of the deleted region in the three S. aureus isolates of the present study. Since the positions K213 and D214 are involved in the putative recombination event, it cannot be clarified whether this position is part of the deletion, and if so, whether the homologous part can take over its function (Figure 2). Therefore, it can be assumed that the deletion detected within the arcC gene results in the loss of function of the ArcC protein, inhibiting the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin [44].
The delayed and incomplete reaction of the anaerobic arginine dehydrolase (Figure S6) might be explained by the presence of an additional carbamate kinase gene, which showed 99.9% identity at the nucleotide sequence level and 100% at the amino acid level [GenBank: AFD28597.1] to an arcC gene as indicated in locus 2 [GenBank: JQ031649.1] by Zhang et al. [45]. This gene showed only 47.3% homology at the amino acid level compared to the arcC gene used in the S. aureus MLST scheme indicated as locus 1 (GenBank: NC_007793.1 [2,779,133–2,778,192]) by Zhang et al. [45]. This additional carbamate kinase can possibly cover, at least partly, for the function of the truncated carbamate kinase ArcC as supported by the results from the anaerobic arginine dehydrolase testing. It should be noted that the arginine catabolic mobile element (ACME), described to harbor a third carbamate kinase [45], is not present in the investigated S. aureus collection.
The human CC1660 isolate 13-03859-1660.7 also carried an adenine nucleotide deletion in the arcC gene within the homopolymeric region (594–605). This results in changes in the protein (difference of 10 amino acids, positions 201 to 210) as well as an earlier stop codon at position 211. The newly described deleted arcC is designated as “allele 613” in the locus SAUR2853 (=arcC) at pubmlst.org (accessed on 22 October 2025). This deletion resulted in a delayed reaction during anaerobic arginine dehydrolase testing, which, however, did not seem as impacted as the one observed for the isolates with the 95 amino acids deletion (Figure S6).

2.3. Antimicrobial Resistance

2.3.1. Antimicrobial Susceptibility Testing and Interpretation of the Results

Antimicrobial susceptibility testing was performed for 31 antimicrobial agents, and the respective MIC distributions of all isolates are displayed in Table 1. For the interpretation of the susceptibility testing results for penicillin, different breakpoints for human and equine isolates were available and classified 16 human isolates (10 CC1, 6 CC1660) with MICs of ≤0.12 µg/mL and three equine isolates (all CC1660) with MICs of ≤1 µg/mL as susceptible. The remaining 48 human (45 CC1, 3 CC1660) and 24 equine isolates (20 CC1, 4 CC1660) were classified as resistant. For horses, clinical breakpoints for ampicillin are also available, classifying all but three isolates (all CC1660) as resistant, which is in accordance with the results from the penicillin testing. For oxacillin, the human-specific clinical breakpoints were used for all isolates, since no veterinary-specific clinical breakpoints are available. Three human and five equine CC1 as well as two equine CC1660 are classified as oxacillin-resistant, which are, therefore, considered as MRSA (see also Section 2.3.2).
Tetracycline resistance was detected among 18 human and ten equine CC1 isolates as well as single human and equine CC1660 isolates. For doxycycline, all but one of the human CC1 isolates were classified as doxycycline-susceptible with the remaining one being classified as intermediate. Among the equine isolates, 12 (eight CC1, four CC1660) were classified as susceptible, four (two of each CC) as intermediate, and ten CC1 isolates as well as the remaining CC1660 isolate as resistant to doxycycline based on the horse-specific clinical breakpoints. It should be noted that there was a good correlation between tetracycline and doxycycline resistance among the equine isolates. However, this correlation could not be observed for the human isolates when using only the breakpoint-based classification. Still having a closer look at the MIC distributions, it can be seen that all but one of the human tetracycline-resistant isolates had elevated doxycycline MICs of 2 µg/mL to 4 µg/mL and were classified as doxycycline-susceptible, whereas the remaining isolate (MIC: 8 µg/mL) was classified as intermediate to tetracycline.
Aminoglycoside resistance was also commonly seen in this collection, with 31 isolates, including 18 human (17 CC1, one CC1660) and 13 equine (ten CC1, three CC1660) isolates being gentamicin-resistant. A single equine CC1660 isolate was classified as intermediate. Moreover, elevated MICs ≥ 4 µg/mL for neomycin and ≥128 µg/mL for streptomycin were detected among 30 and 11 isolates, respectively.
Erythromycin resistance was detected only in nine CC1 isolates (five human, four equine). The observation that all isolates had comparably low clindamycin MICs of ≤2 µg/mL might point towards inducible macrolide resistance [46] (see Section 2.3.3). Resistance to the combination sulfamethoxazole/trimethoprim was detected in 19 isolates based on MICs of ≥4/76 µg/mL, comprising 16 human CC1 and three equine CC1 isolates.
All human isolates were classified as ciprofloxacin-susceptible with MICs of ≤1 µg/mL. In comparison, all but three isolates of the equine collection were classified as susceptible based on enrofloxacin MICs of ≤0.12 µg/mL. The remaining three CC1 isolates were classified as resistant by their MIC of 0.5 µg/mL.
A single human CC1 isolate (10-00442-18) had an elevated florfenicol MIC of 64 µg/mL. All isolates were susceptible to the oxazolidinone linezolid, the glycopeptide vancomycin, the streptogramin combination quinupristin/dalfopristin and the pleuromutilin tiamulin. This is a rather favorable situation, but it should be noted that the Antimicrobial Advice ad hoc Expert Group (AMEG) classified glycopeptides, oxazolidinones and streptogramins in Category A (“Avoid”) of the European Medicines Agency (EMA), which are, therefore, not authorized in veterinary medicine and restricted to use in human medicine in the EU [47].

2.3.2. Correlation of Phenotypic and Genotypic Resistance Properties

Antimicrobial susceptibility testing revealed 18 distinct phenotypic resistance patterns among the isolates, whereas whole-genome sequence analysis identified 19 genotypic resistance patterns (Table 2). Among the 91 S. aureus isolates, 18 were susceptible to all antimicrobial agents tested and 31 isolates were only resistant to penicillin. One penicillin-resistant isolate showed additional phenotypic resistance to oxacillin only. Nine additional isolates were phenotypically methicillin-resistant. Methicillin resistance is commonly mediated by the genes mecA and mecC [48,49]. This includes all β-lactams licensed for veterinary use. The β-lactams, which are effective against MRSA, such as the cephalosporin ceftobiprole [50], are restricted for use in humans by the AMEG category A [47]. In the present collection, only seven methicillin-resistant isolates carried the mecA gene (see also Section 2.3.3). In total, three equine isolates were phenotypically oxacillin-resistant, with a low oxacillin MIC of 4 µg/mL (12-00973-22 [CC1]; 12-00626-1660.2 and 12-00971-1660.3 [CC1660]), but negative for the respective resistance genes mecA and mecC. The finding of phenotypic methicillin resistance with low MICs without a known methicillin resistance gene is described as borderline oxacillin-resistant S. aureus (BORSA). A possible reason for the observation of phenotypic methicillin resistance without the presence of a mec gene or mutations in the genes for penicillin-binding proteins could be due to an overexpression of the blaZ gene as described previously [51]. However, the comparison of the blaZ operons (blaZ, blaI, blaR) in the present study revealed that the same nucleotide sequence variants were not only present in the methicillin-resistant but also in methicillin-susceptible isolates.
Tetracycline resistance was detected in 30 isolates, among which 22 harbored the gene tet(L), whereas the remaining eight carried a tet(K) gene. This finding also corresponds well with the study by Scholtzek et al. with all three tetracycline-resistant isolates being positive for tet(L) [51], which is also the most common tetracycline resistance gene in our collection. In comparison to other studies on S. aureus that describe the presence of two or more tetracycline resistance genes in the same isolate [52,53], only one tetracycline resistance gene was detected per isolate.
Resistance to gentamicin was present in 31 isolates and it was in all isolates mediated by the aacA-aphD gene (also known as aac(6′)-aph(2″)). The single gentamicin-intermediate isolate was also positive for aacA-aphD. This is in accordance with previous studies with BORSA isolates of CC1 and CC1660, where all of these isolates showed aacA-aphD-mediated gentamicin resistance [34,51]. Gentamicin resistance is also common among MRSA ST8 isolates isolated from horses in the USA [28]. Neomycin resistance was also very common in this collection, being present in 30 isolates, 22 of which carried the aadD gene (also known as ant(4′)-Ia), whereas the remaining eight carried aphA3. The eight aad(E) (also known as ant(6)-Ia)-positive and the three str-positive isolates had streptomycin MICs of ≥128 µg/mL.
In total, 19 isolates proved to be phenotypically resistant to the combination trimethoprim/sulfamethoxazole, which was in agreement with the presence of dfrS1 and/or dfrG (mediating trimethoprim resistance) as well as the mutations F17L and A184V in the folP gene (mediating sulfonamide resistance). However, an additional 15 isolates harbored a dfr gene (dfrS1, also known as dfrA [n = 10], dfrK [n = 4], dfrG [n = 1]) and two had the F17L mutation in the folP gene, but were classified as trimethoprim/sulfamethoxazole-susceptible by broth microdilution. This finding is in accordance with previous studies showing that resistance mechanisms for both components, trimethoprim (dfr genes) and sulfonamides (folP gene mutations), are needed to confer a trimethoprim/sulfamethoxazole resistance phenotype [54]. The finding that dfrS1 is the most common trimethoprim resistance gene is in accordance with the findings of Sieber et al. [34] who detected this gene in all 18 CC1 and CC1660 BORSA isolates tested. Among the resistant isolates, at least one dfr gene is present in combination with two mutations (F17L and A184V) within the folP gene (Table 2).
Nine isolates were resistant to erythromycin mediated by the erm(C) gene (conferring resistance to macrolide, lincosamide, and streptogramin B antibiotics), but showed unimodal distributions for the other macrolides and lincosamides tested (Table 1). This points towards inducible macrolide resistance [46]. As described previously [46], the regulatory region upstream of the erm(C) gene was complete and showed no mutations. This finding is important for the choice of the antimicrobial agents, since erm genes are only induced by 14- and 15-membered macrolides, such as erythromycin and azithromycin, but not by 16-membered macrolides (e.g., tylosin), lincosamides (e.g., clindamycin) and streptogramin B antibiotics [46]. In the presence of inducibly expressed erm genes, the aforementioned non-inducers should not be used for therapeutic purposes, since mutations in the regulator region can occur rapidly and result in constitutive erm(C) expression [46].
Fluoroquinolone resistance is known to be mediated by mutations in the quinolone resistance-determining regions (QRDRs) of the genes for DNA topoisomerase IV (grlA and grlB) and DNA gyrase (gyrA, gyrB). In three equine and 12 human CC1 isolates, the GrlA amino acid replacement S80Y has been detected. This corresponds well with the classification of equine isolates as resistant. In contrast, the human isolates with the same GrlA amino acid replacement were classified as susceptible. Having a closer look at the MIC values, it can be noted that these human isolates also had elevated MIC values, corresponding well with the MIC values of the equine isolates. Therefore, the different clinical breakpoints used for the human and equine isolates resulted in a different categorization despite similar MIC values and the presence of a mutation in the QRDR (see also Section 2.3.1; Table 2).
A single isolate was classified as phenicol-resistant and carried the fexA gene mediating resistance to non-fluorinated and fluorinated phenicols [55].

2.3.3. SCC Elements

Seven CC1 isolates were identified as MRSA. All harbored SCCmec IVa (2B) elements. For the seven mecA-positive isolates, dru typing—a recognized MRSA subtyping method targeting a variable-number tandem repeat region located within the SCCmec element between mecA and IS431—was performed [56]. Four isolates harbored the dru type dt9y (5a-2d-4a-0-2d-5b-3a-3b-4e), whereas the remaining ones represented dru type dt10a (5a-2d-4a-0-2d-5b-3a-2g-3b-4e). Actually, the detected dru types are related and dt9y lacks repeat 2g, which is the eighth repeat in dt10a.
Of note, in all seven CC1-MRSA-IVa sequences, genes ydil2-SCC and C5QAP8 (locus tags KOJLHLHF_00002 and _00004 in GenBank MT380478.1) were identified as well as npd = pnoA-SCC (KOJLHLHF_00006) in five out of the seven isolates. The presence of these genes corresponds to a large insertion directly downstream of orfX previously described to cause false-negative results in some commercial assays for molecular MRSA screening [57].
In addition, two CC1 isolates carried an SCC element without mecA or mecC genes, but comprising of the fusidic acid resistance gene fusC, the tirS gene for a staphylococcal Toll-like/interleukin-1 receptor and recombinase genes ccrA1/B1. The element comprising these genes is already known to occur in CC1 strains, corresponding, for instance, to locus tags SAS0043, SAS0038, SAS0033 and SAS0032, respectively, in MSSA476 (GenBank BX571857.1).

2.4. Clonal Complex-Specific Virulence Factors

All CC1 isolates (n = 75) harbored agr group III and capsule type 8 alleles. The lukF/lukS/hemolysin gamma locus (corresponding to locus tags MW2342 to MW2344 in the CC1 reference sequence of MW2, GenBank BA000033.2) and the leukocidin D/E genes (MW1767/1768) were always present. Another leukocidin locus, lukA/B (MW1941/1942), was detected in 73 out of 75 (97.3%) isolates. The enterotoxin H gene (seh; MW0051), located on a genomic island downstream of orfX and the SCCmec integration site, was identified in 74 (98.7%) isolates. Two genes encoding adhesion factors known to correlate with CC affiliations were also examined. These were the sasG gene (MW2416), encoding the S. aureus surface protein G, and cna (MW2612), encoding a collagen adhesion protein, which were present in 74 (98.7%) and 70 (93.3%) isolates, respectively.
The combination of agr group III, capsule type 8, seh, sasG and cna is characteristic for CC1. Agr group III and capsule type 8 can also be found in isolates of CC93, CC30 (which also carried the cna gene), CC80 and CC88 (which also carried the sasG gene), CC509/ST 207 as well as ST154 (which also carried the cna and sasG genes) isolates. The seh gene is rarely—if at all—found outside of CC1 isolates [37], whereas lukA/B genes can be found in virtually all S. aureus isolates [33]. All CC1660 isolates (n = 16) carried agr group II alleles and capsule type 5 genes. Genes of the lukF/lukS/hemolysin gamma and the lukA/B and lukD/E loci were universally present. Isolates of this clonal complex lacked the seh gene, but they always carried the enterotoxin gene cluster (egc, comprising selg, seli, selm, sen, selo, selu), although the selg gene remained undetected as well as seli and selm in one isolate. The absence of selg appears to be a general property of CC1660 as shown by array experiments with additional, epidemiologically unrelated isolates (Monecke, personal communication). All isolates harbored an additional enterotoxin-like gene (“selu2”, locus tag UC18_08930 from CP010526.1). The cna gene was present whereas sasG was absent in all CC1660 sequences.
The combination of agr group II and capsule type 5 alleles was seen in all isolates of CC5 (which also carried the egc and sasG genes, but lacked cna), CC9 (which also harbored only egc), as well as ST573 and ST1772 (which also carried the egc, sasG and cna genes) [37].

2.5. Virulence Factors on Mobile Genetic Elements

Virulence factors lukP/lukQ (=lukPQ leukocidin, horse-specific), sak (staphylokinase), sak_phi-42e (putative horse-associated kinase), scn (staphylococcal complement inhibitor), scn2 (complement inhibitor paralog from a staphylococcal pathogenicity island (SaPI), locus tag SAPIG0482 from AM990992.1), scn-eq (putative horse-associated complement inhibitor), enterotoxin genes sea, seb, sec, sek, sel, seq, the toxic shock syndrome toxin gene tst-1 and vwb3 (a SaPI-borne gene encoding a “van Willebrand factor”-binding protein, locus tag SAPIG0483 from AM990992.1) were present in various combinations. The distribution and co-occurrence of the aforementioned virulence genes in CC1 and CC1660 isolates from humans and horses are summarized in Table 3.
Horse-specific leukocidin genes lukP/lukQ as well as scn-eq genes are located on prophages that integrate into the chromosomal lip2 (=geh) gene [33]. The respective prophages differ with respect to their structures and gene contents into three groups, of which one was seen in CC1 and CC97, another one was detected mostly in CC350, but also in CC1, CC97, CC133 and CC398, whereas a third one was solely found in CC816 and CC8115 [33]. The enterotoxin gene sea has been identified on β-hemolysin-converting phages in isolates of CC5 and CC8, but occasionally also in CC1, CC6, CC15, CC30, CC59, CC398, ST239, ST426, and ST772. The enterotoxin B gene seb has so far also been detected in CC361 and together with the genes sek and seq in isolates of CC8 and CC59; sek and seq have also been seen in CC45, CC398, ST239 and ST426 [37]. The gene tst-1 for toxic shock syndrome toxin 1 also occurs in isolates of CC5, CC8, CC22, CC30, CC45, CC361, ST426, CC705 and ST834 [37].
Phage-borne leukocidin genes lukF-PV/lukS-PV (Panton–Valentine leukocidin), plasmid-borne enterotoxins (sed, sej, ser), the gene encoding a chemotaxis-inhibiting protein (chp), exfoliative toxins (etA, etB, etD, etE) and epidermal cell differentiation inhibitor genes (edinA, edinB, edinD) were not detected.

3. Materials and Methods

3.1. Isolate Collection

The study is based on a set of 91 S. aureus isolates belonging to CC1 (n = 75) and CC1660 (n = 16) of human and equine origin obtained within the period from 2006 to 2022 by the Robert-Koch-Institute, Wernigerode, Saxony-Anhalt, Germany, during previous studies, and some of the isolates have already been published previously [26,58]. The collection consists of isolates from humans (employees) and horses obtained from a study in equine clinics and from humans and horses in the community in Germany. It includes 75 CC1 and 16 CC1660 isolates. Among the 75 CC1 isolates, 55 were of human origin and 20 of equine origin. From five human samples, two phenotypically distinct isolates each were analyzed (Figure S1). Among the 16 CC1660 isolates, nine were of human and seven of equine origin. The geographic origin is only given on a federal state level for data protection reasons (Table S1, Figure S2). In eight cases, more than one sample of human and/or equine origin from the same equine clinics was investigated. In one case, three samples from the same horse (12-00973-22, 12-00974-23, 12-00975-24) were provided from a veterinary diagnostic laboratory and included.

3.2. Whole-Genome Sequencing (WGS) and Sequence Analysis

3.2.1. Whole-Genome Sequencing

All isolates were subjected to WGS. Whole-cell DNA extraction was performed using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany) with some adaptations for staphylococci as described previously [51]. Initially, staphylococcal cells were mixed with 25 µL lysostaphin solution (0.1 mg/mL) followed by incubation at 37 °C for 25 min. Subsequently, 25 µL proteinase K (0.1 mg/L) and 75 µL TE buffer were added and the mixture was incubated at 37 °C for another 25 min. Finally, 2 µL RNAse A (2 µg/µL) and 75 µL PBS were added and mixed. Thereafter, the protocol for the kit was followed starting with the addition of AL buffer [51]. The libraries were prepared using the Nextera XT library preparation kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. The 2 × 300 bp paired-end sequencing in 40-fold multiplexes was performed on the Illumina MiSeq platform. Trim Galore v0.6.10 (RRID: SCR_011847) and FastQC v0.12.1 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/; accessed on 22 October 2025) were used for adapter trimming and quality checking. The genome sequences were de novo assembled using Unicycler v0.4.9 [59] and SPAdes v3.15.5 [60].

3.2.2. Molecular Typing and Phylogenetic Analysis

Conventional MLST for S. aureus is based on seven housekeeping genes (arcC [carbamate kinase], aroE [shikimate dehydrogenase], glpF [glycerol kinase], gmk [guanylate kinase], pta [phosphate acetyltransferase], tpi [triosephosphate isomerase], and yqiL [acetyl coenzyme A acetyltransferase]) for S. aureus [5]. The respective alleles were deduced from the whole-genome sequences using the software program mlst from the PubMLST database [61]. For the sake of convenience, STs were then clustered in CCs. There are slightly different definitions of a CC, e.g., isolates with five of the seven alleles in common [6,7] or isolates that “match the central genotype (ST) at four or more loci unless they more closely match another central genotype” (https://pubmlst.org/organisms/staphylococcus-aureus/clonal-complexes; accessed on 22 October 2025). In our case, all isolates were single-locus variants of the central genotype, which is in accordance with both definitions. It should be noted that the CC assignment commonly concurs with the agr group, the capsule type affiliations and the carriage of markers on major genomic islands (seh, ORF CM14, egc locus, ssl locus) [37].
In addition, spa typing and SCCmec typing were extracted from the WGS sequences using the software programs spaTyper [62] and SCCmecFinder v1.2 (https://cge.food.dtu.dk/services/SCCmecFinder/; accessed on 22 October 2025), respectively. The dru types were identified according to the drutyping.org database [56] using the basic local alignment search tool (BLAST) function in Geneious v11.1.5 (Biomatters, Ltd., Auckland, New Zealand).
The 91 genomes were also subjected to phylogenetic analysis with chewBBACA v3.3.10 [63] using the S. aureus core genome MLST (cgMLST) approach [41]. A minimum spanning tree was built based on a distance matrix of the core genome allelic profiles to illustrate the clonal relationships between the isolates. Overall, 1492 of 1861 potential target genes were included in the analysis by removing 369 columns that were missing in at least one sample. GrapeTree [64] was used to visualize the minimum spanning tree.
PCR amplification of the arcC gene was performed for three isolates with a 285 bp deletion in the arcC gene, an isolate with a single bp deletion as well as an isolate with a complete arcC gene, using the primers and the protocol described previously for MLST by Enright et al. [5] (Table S3) and subsequent agarose gel electrophoresis before Sanger sequencing at LGC Genomics GmbH (Berlin, Germany) for confirmatory reasons.

3.3. Anaerobic Arginine Dehydrogenase Testing

The testing of the anaerobic use of arginine was performed for selected isolates using DIATABS™ ARGININE DIHYDROLASE (ADH) (Rosco Diagnostica A/S, Albertslund, Denmark) according to the manufacturer’s instructions (https://www.keyscientific.com/files/Other%20Manufacturers/Rosco/DiaTabs/Rosco%20DiaTabs.pdf; accessed on 22 October 2025). Bacterial suspensions of McFarland 4 were prepared in saline. One ADH Diagnostic tablet was added to 0.25 mL bacterial suspension and anaerobic conditions were generated by adding sterile paraffin oil. The results were read after 4 h and 24 h of incubation at 37 °C.

3.4. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was performed by broth microdilution according to CLSI standards. Tests were usually performed once, but were repeated if quality control (QC) was out of range or if results were inconclusive. The McFarland 0.5 is adjusted using the direct colony suspension method: 60 µL of this bacterial suspension is added to 12 mL cation-adjusted Mueller–Hinton broth and incubated for 16–20 h or 24 h (for oxacillin and vancomycin only) in ambient air. Custom-made microtiter plates (MSC diagnostics, Swalmen, The Netherlands) containing 30 antimicrobial agents [48,49,65] were used, which are also used in the German National Resistance Monitoring program GERM-Vet [66]. The test panel included β-lactams (oxacillin, penicillin, ampicillin, amoxicillin/clavulanic acid, imipenem, ceftiofur, cefquinome, cefalothin, cefotaxime, cefoperazone), macrolides (erythromycin, tylosin tartrate, tulathromycin, tilmicosin), lincosamides (clindamycin, pirlimycin), quinolones (ciprofloxacin, enrofloxacin, marbofloxacin), aminoglycosides (gentamicin, streptomycin, neomycin), tetracyclines (tetracycline, doxycycline), a phenicol (florfenicol), a folate pathway inhibitor combination (sulfamethoxazole/trimethoprim), a pleuromutilin (tiamulin), a glycopeptide (vancomycin), a streptogramin (quinupristin/dalfopristin) and an oxazolidinone (linezolid). S. aureus ATCC® 29213 served as the QC strain. Since the collection contains human and equine isolates, species-specific clinical breakpoints were applied as far as possible using the clinical breakpoints from CLSI document M100 for the human isolates [49] and from the CLSI document VET01S for the equine isolates [48]. For our collection, human-specific clinical breakpoints for 12 antimicrobial agents were available, namely penicillin, oxacillin, erythromycin, clindamycin, ciprofloxacin, gentamicin, tetracycline, doxycycline, trimethoprim/sulfamethoxazole, linezolid, vancomycin and quinupristin/dalfopristin [49]. For the equine isolates, equine-specific clinical breakpoints for penicillin, ampicillin, enrofloxacin, and doxycycline as well as human-adopted clinical breakpoints for oxacillin, erythromycin, gentamicin, tetracycline, trimethoprim/sulfamethoxazole, linezolid and vancomycin were applied [48]. Thus, in some cases, the human clinical breakpoints were adopted for veterinary isolates; however, these results should be interpreted with caution due to species-specific differences in dosage and pharmacokinetics/pharmacodynamics. As a consequence of the different species-specific breakpoints for certain compounds, any discussion on resistance properties and phenotypic effects of resistance genes must focus on raw MIC values rather than on mere susceptible/intermediate/resistant classifications, especially if it deals with pathogens affecting multiple host species.

3.5. Detection of Resistance Genes and Resistance-Mediating Mutations

Known antimicrobial resistance genes were identified with ABRicate v1.0.1 (https://github.com/tseemann/abricate; accessed on 22 October 2025) using the NCBI AMRfinder [67] and the CGE ResFinder database [68]. The assembled WGS sequences were used as fasta files for analysis using the default settings. Chromosomal point mutations conferring AMR were detected by applying PointFinder [69] as part of the ResFinder tool [68] using default settings. All results were confirmed with Geneious v11.1.5 (Biomatters, Ltd., Auckland, New Zealand), a bioinformatic tool for sequence analysis, BLAST [70], a Basic Local Alignment Search Tool to find regions of similarity between biological sequences, and the UniProt Knowledgebase [71] to identify protein sequences and their function.

3.6. Determination of Virulence Properties

Virulence genes were identified with ABRicate v1.0.1 (https://github.com/tseemann/abricate; accessed on 22 October 2025) using the VFDB database [72]. As described for the determination of antimicrobial resistance genes, the assembled WGS sequences were used as fasta files for analysis using the default settings. All results were confirmed with Geneious v11.1.5 (Biomatters, Ltd., Auckland, New Zealand), BLAST + 2.17.0 [70], and the UniProt Knowledgebase [71]. In addition, sequences of the probes of previously published DNA microarrays [37,73,74] were mapped against the genome sequences of the isolates.

4. Conclusions

In summary, comprehensive genetic and phenotypic characterization of S. aureus isolates from humans and horses revealed a clear separation between the 75 CC1 and the 16 CC1660 strains. No human- or horse-associated subpopulations within the CCs could be observed. Notable alterations were found in the arcC gene of four isolates with potential functional consequences. Isolates within each clonal complex carried related spa types, supporting the established epidemiological linkage.
A total of 18 phenotypic and 19 genotypic antimicrobial resistance patterns were identified, including numerous isolates that were resistant to more than three classes of antimicrobial agents. The application of species-specific clinical breakpoints for different fluoroquinolones used in human or veterinary medicine showed that isolates with similar MICs and the same grlA mutation may be classified either as susceptible or resistant. This observation points towards a problem in resistance surveillance studies when using species-specific clinical breakpoints rather than epidemiological cut-off values or raw, uninterpreted MIC values. The detection of diverse mobile genetic elements, such as SCCmec cassettes and phages, highlights the dynamic potential for horizontal gene transfer of virulence and antimicrobial resistance determinants. We also observed that equine-adapted strains carried genes such as lukP/lukQ, sak_phi-42e or scn-eq [33] and that human as well as human-adapted strains, carrying prophages with immune-evasion cluster genes in various combinations (scn, sak, chp, sea) [75,76], were present in horses. Therefore, it would be interesting to investigate whether these strains adapted to a specific host can stably colonize other host species, and whether species-specific virulence factors, such as the human toxic shock syndrome toxin or the horse-specific leukocidin, are also functional in other hosts. For this, a detailed characterization of the respective strains in terms of surveillance of zoonotic transmission—as provided in this study—is indispensable. The findings of this study underscore the importance of ongoing surveillance and molecular monitoring of equine-associated S. aureus clonal complexes—not only in horses and humans, but also in animals with close contact to these hosts—to better understand and mitigate potential risks to animal and public health in a One Health context. The observations showed that human-adapted strains might spill over to horses and, vice versa, that equine strains might colonize humans. This is of particular relevance in horse clinics. Therefore, it can be suggested to perform pre-surgical screening for S. aureus in both humans with horse contact as well as horses. In this context it would be an interesting topic to investigate the effect of the equine leukocidin on human cells as well as the pathogenicity of equine-adapted strains on humans and their possible persistence in human hosts. Further studies should investigate not only whether colonizations of horses by human strains, or of humans by equine strains, were permanent or transient, but also whether these strains are able to adapt to and cause invasive diseases in the new hosts.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antibiotics14111082/s1. Figure S1: Phenotypically different isolates within the samples; Figure S2: Geographic origin of the samples included; Figure S3: Distribution of the isolates from the different equine clinics (EC); Figure S4: Genetic relationship of the spa types detected among the CC1 (a) and CC1660 (b) isolates; Figure S5: arcC-PCR; Figure S6: Testing of the anaerobic arginine dehydrolase activity; Table S1: Background information of the 91 S. aureus isolates; Table S2: Heatmap of the cgMLST; Table S3: Primers used for MLST of Staphylococcus aureus according to reference [5].

Author Contributions

Conceptualization, C.C., S.S., I.C. and A.T.F.; methodology, J.J., T.A., M.L., S.S.C. and I.C.; software, D.H. and S.M.; validation, S.M., R.E., D.H. and A.T.F.; formal analysis, J.J., D.H., S.M., M.L., S.S.C., I.C. and A.T.F.; investigation, D.H., S.M., M.L., S.S.C., I.C. and A.T.F.; resources, C.C., R.E., I.C. and S.S.; data curation, J.J., D.H., S.M. and A.T.F.; writing—original draft preparation, J.J., S.M., D.H. and A.T.F.; writing—review and editing, all authors; visualization, J.J., D.H. and A.T.F.; supervision, A.T.F.; project administration, C.C., W.W. and A.T.F.; funding acquisition, C.C., W.W. and S.S. All authors have read and agreed to the published version of the manuscript.

Funding

RKI: Sampling in equine clinics and basic diagnostics were supported by the German Ministry of Education and Research (project 01KI1727F) in the cluster of #1Health- Prevent. IHMT-NOVA: Fundacão para a Ciência e a Tecnologia (FCT, Portugal) through funds to UID/04413/2025 (GHTM), LA/P/0117/2020 (LA-REAL), 2023.02437.BD (ML), CEECINST/00042/2021/CP1773/CT0009 (SSC) and Bilateral PT- Germany (FCT/DAAD) mobility project (2022.15300.CBM). IPHT: Federal Ministry of Research, Technology and Space Project “ADA” (13GW0456), aiming to develop rapid assays for the detection and characterization of resistance genes and virulence factors in S. aureus (S.M. and R.E). The publication of this article was funded by Freie Universität Berlin.

Institutional Review Board Statement

For sampling of humans working in equine clinics, the study protocol including data was approved by the ethical committee of the Otto von Guericke University Magdeburg, affiliated with the faculty of medicine (file#47/09).

Informed Consent Statement

Not applicable.

Data Availability Statement

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under BioProject Accession number PRJNA1263585. The individual accession numbers (BioSample, Genome, SRA) are displayed in Table S1.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BORSAborderline oxacillin-resistant Staphylococcus aureus
bpbase pair
CCclonal complex
ECequine clinic
drudirect repeat unit
MICminimal inhibitory concentration
MLSTmultilocus sequence typing
MRSAmethicillin-resistant Staphylococcus aureus
MSSAmethicillin-susceptible Staphylococcus aureus
QRDRquinolone resistance-determining region
S.Staphylococcus
SaPIstaphylococcal pathogenicity island
SCCStaphylococcal Cassette Chromosome
SNPsingle nucleotide polymorphism
spastaphylococcal protein A
STsequence type
WGSwhole-genome sequencing

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Antibiotics 14 01082 g001
Figure 1. Minimum spanning tree based on the cgMLST allelic profile of the 91 S. aureus isolates (1492 columns; 1861 columns for distance calculation, 369 columns with missing values with at least one sample removed). The distance between isolates is indicated by the number next to the line (length of line not proportionate). The correlations of numbers and isolate IDs are given in Table S1. The distance information of the single isolates is displayed as a heatmap in Table S2.
Figure 1. Minimum spanning tree based on the cgMLST allelic profile of the 91 S. aureus isolates (1492 columns; 1861 columns for distance calculation, 369 columns with missing values with at least one sample removed). The distance between isolates is indicated by the number next to the line (length of line not proportionate). The correlations of numbers and isolate IDs are given in Table S1. The distance information of the single isolates is displayed as a heatmap in Table S2.
Antibiotics 14 01082 g001
Antibiotics 14 01082 g002
Figure 2. Illegitimate recombination resulting in a 285 bp deletion within the arcC sequence. (a) Complete arcC sequence, (b) Putative recombination event and (c) Truncated arcC sequence. The recombination sites are underlined and the deleted area is indicated in yellow. The primer binding sites within the arcC are displayed in blue. |= identity of bases in the recombination site (b); – = deleted base (c).
Figure 2. Illegitimate recombination resulting in a 285 bp deletion within the arcC sequence. (a) Complete arcC sequence, (b) Putative recombination event and (c) Truncated arcC sequence. The recombination sites are underlined and the deleted area is indicated in yellow. The primer binding sites within the arcC are displayed in blue. |= identity of bases in the recombination site (b); – = deleted base (c).
Antibiotics 14 01082 g002
Table 1. MIC distributions of the 91 S. aureus isolates.
Table 1. MIC distributions of the 91 S. aureus isolates.
Antimicrobial AgentsMIC in (µg/mL)SIR
0.0080.0150.030.060.120.250.512481632641282565121024(µg/mL)
Oxacillin (total) ---214461723-7
  Equine ----110 + 34 + 111 + 2-4 ≤2-≥4
  Human ---1 + 112 + 129 + 49 + 31--3 ≤2-≥4
Penicillin (total) 3142---1101782484
  Equine 12-----23 + 13 + 14 + 153 + 1 ≤0.51≥2
  Human 1 + 17 + 52---1810 + 34193 ≤0.12-≥0.25
Ampicillin (total) -21611-1612228112-
  Equine -21---325 + 22 + 171 + 1- ≤0.250.5≥1
  Human --9 + 611-137 + 31554--
Amoxicillin/Clavulanic Acid (total) --217193511--61--
  Equine --12310 + 23 + 2--31
  Human --110 + 51622 + 14 + 2--3---
Imipenem (total) 3153----3-13---
  Equine 7 + 29 + 5------13---
  Human 17 + 535 + 4----3------
Ceftiofur (total) -----58251---34
  Equine -----14 + 42 + 3-----4
  Human -----33 + 718 + 21---3-
Cefquinome (total) ---1-34472-151-
  Equine ---1-8 + 37 + 31--31-
  Human -----19 + 432 + 51-12--
Cefalothin (total) 1218567---331--
  Equine 11113 + 32 + 2----31--
  Human -112 + 536 + 43---3----
Cefotaxime (total) -------33501--7
  Equine -------4 + 211 + 51 4
  Human -------23 + 429 + 5 --3
Cefoperazone (total) -----215211--7
  Equine -----1 + 312 + 13 + 3--4
  Human -----12 + 535 + 45--3
Erythromycin (total) ----9703-----9
  Equine ----5 + 311 + 4------4 ≤0.51–4≥8
  Human ----147 + 83-----5 ≤0.51–4≥8
Tylosin tartrate (total) ----56719------
  Equine ----3 + 215 + 52------
  Human -----38 + 917------
Tulathromycin (total) -----111561742
  Equine -----12 + 412 + 314-
  Human ------1 + 436 + 516-2
Tilmicosin (total) --154837-------
  Equine --13 + 27 + 59------
  Human ----28 + 827 + 1------
Clindamycin (total) -222661--------
  Equine -1 + 16 + 212 + 41--------
  Human --1441 + 9--------- ≤0.51–2≥4
Pirlimycin (total) ---222661------
  Equine ---29 + 39 + 4-------
  Human ----1045 + 81------
Tiamulin (total) --1113742------
  Equine --112 + 317 + 3-------
  Human ----845 + 92------
Ciprofloxacin (total)--133724818-----
  Equine--119 + 47 + 2-3-----
  Human---1 + 118 + 614 + 17 + 115----- ≤12≥4
Enrofloxacin (total)--34024717------
  Equine--1 + 19 + 67-3------ ≤0.120.25≥0.5
  Human--119 + 614 + 3714------
Marbofloxacin (total)----15481810-----
  Equine----5 + 18 + 652-----
  Human----8 + 127 + 712 + 18-----
Gentamicin (total) 1272551-1213142--
  Equine -4 + 24 + 111---2 + 362-- ≤48≥16
  Human 118 + 316 + 44--1287 + 1--- ≤48≥16
Streptomycin (total) ----1256111-110--
  Equine ----2 + 310 + 34--14--
  Human ----6 + 136 + 771--6--
Neomycin (total) -44012578672-
  Equine -18 + 51121 + 1241-
  Human -1 + 223 + 49 + 2455 + 1431-
Tetracycline (total) 24316----2217---
  Equine 16 + 53 + 1----28 + 1- --- ≤48≥16
  Human 126 + 610 + 2-----11 + 1 7--- ≤48≥16
Doxycycline (total) 333196-10191-----
  Equine 1 + 17 + 32 + 2--6 + 14------ ≤0.120.25≥0.5
  Human 119 + 413 + 25 + 1-314 + 11----- ≤48≥16
Sulfamethoxazole/trimethoprim (total) -348971316121--
  Equine -29 + 11 + 34-2 + 11-3--- ≤2/38-≥4/76
  Human -131 + 74 + 131--691-- ≤2/38-≥4/76
Florfenicol (total) ----26919--1---
  Equine ----1 + 117 + 62------
  Human -----39 + 715 + 2--1---
Linezolid (total) -----86221-----
  Equine -----2 + 317 + 41----- ≤4-≥8
  Human -----336 + 516 + 4----- ≤4-≥8
Vancomycin (total) -----6832-----
  Equine -----320 + 4------
  Human -----1 + 252 + 72----- ≤24–8≥16
Quinupristin/Dalfopristin (total) ----18703------
  Equine ----4 + 416 + 3-------
  Human ----6 + 446 + 53------ ≤12≥4
S = susceptible; I = intermediate; R = resistant. Dark gray shading indicates concentrations not included in the test panel. Isolates with growth throughout the panel have MIC values equal to or larger than the highest concentration tested and are, therefore, displayed in the next higher concentration with gray shading. Amoxicillin and trimethoprim MIC values were used for the combinations amoxicillin/clavulanic acid (2:1) and sulfamethoxazole/trimethoprim (19:1), respectively. Light gray shading indicates the adoption of human clinical breakpoints for veterinary use. For the distributions of the equine and human isolates, CC1 isolates are displayed in purple, and CC1660 isolates are displayed in blue. In cases with clinical breakpoints, the start of an intermediate category is indicated by yellow vertical bars and the start of a resistant category is indicated by red vertical bars.
Table 2. Phenotypic and genotypic resistance patterns of the 91 S. aureus isolates.
Table 2. Phenotypic and genotypic resistance patterns of the 91 S. aureus isolates.
Isolates Phenotype TotalIsolates Genotype TotalCC1 HorsesCC1 HumansCC1660 HorsesCC1660 HumansPENOXATETGENERYSXTFQNEOSTRFFN
1816 - 835
2 2 - - folP (F17L)
3132919 - 3blaZ
11 - - - blaZ
11 - 1 - - blaZ fexA
22 - 2 - - blaZ dfrG + folP (F17L and A184V)
22 - 2 - - blaZ erm(C) str
31 - 1 - - blaZ aacA-aphD dfrS1grlA (S80Y)
4 - 11 - blaZ aacA-aphD dfrS1
1 - - 1 - blaZ aacA-aphD dfrS1
1 - - 1 - blaZ aacA-aphD dfrS1
11 - 1 - - blaZ aacA-aphD dfrS1/dfrG + folP (F17L and A184V)
11 - 1 - - blaZ tet(K) aphA3aad(E)
11 - - - 1 tet(L)aacA-aphD dfrS1 aadD
433 - - - blaZ tet(L)aacA-aphD dfrK aadD
1 - 1 - - blaZ tet(L)aacA-aphD dfrG aadD
11 - - 1 - blaZ tet(L)aacA-aphD dfrK aadD str
33 - 3 - - blaZmecAtet(K) erm(C) aphA3aad(E)
132 - 2 - - blaZ tet(L)aacA-aphD dfrG + folP (F17L and A184V) aadD
1 - 1 - - blaZ tet(L)aacA-aphD dfrS1/dfrG + folP (F17L and A184V)grlA (S80Y)aadD
13-10 - - blaZ tet(L)aacA-aphD dfrG + folP (F17L and A184V)grlA (S80Y)aadD
33- - - blaZ tet(L)aacA-aphD dfrG + folP (F17L and A184V)grlA (S80Y)aadD
444 - - - blaZmecAtet(K)aacA-aphD erm(C)dfrS1 aphA3aad(E)
Abbreviations: PEN = penicillins; OXA = oxacillin; TET = tetracyclines; GEN = gentamicin; ERY = erythromycin; SXT = sulfamethoxazole/trimethoprim; FQ = fluoroquinolones; NEO = neomycin; STR = streptomycin; FFN = florfenicol; red shading = phenotypic resistance; genes/mutations = presence of genetic resistance determinants. Fluoroquinolone resistance was determined based on clinical breakpoints for ciprofloxacin (humans) and enrofloxacin (equine/veterinarian), respectively. For some antimicrobial agents without clinical breakpoints, “resistance” was classified based on the MICs as follows: neomycin ≥ 4 µg/mL, streptomycin ≥ 128 µg/mL, florfenicol ≥ 64 µg/mL.
Table 3. Distribution of virulence genes among the 75 CC1 and 16 CC1660 isolates.
Table 3. Distribution of virulence genes among the 75 CC1 and 16 CC1660 isolates.
Virulence GenesLocalizationIn CC1
Isolates
(n = 75)		In CC1660 Isolates
(n = 16)		In Human Isolates
(n = 67)		In Equine Isolates
(n = 24)
tst-1 + sec + selSaPI03 (18.75%)3 (4.48%)0
sebSaPI4 (5.33%)04 (5.97%)0
seb + sek + seqSaPI4 (5.33%)04 (5.97%)0
sek + seqSaPI1 (1.33%)01 (1.49%)0
scn2 + vwb3SaPI49 (65.33%)16 (100%)42 (62.69%) 23 (95.83%)
sea + sak + scnprophage7 (9.33%)07 (10.45%)0
sak + scnprophage8 (10.67%)04 (5.97%)4 (16.67%)
lukP + lukQ + scn-eqprophage46 (61.33%)14 (87.50%)38 (56.72%) 22 (91.67%)
sak_phi-42eprophage46 (61.33%)13 (81.25%)41 (61.19%) 18 (75%)
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Jahnen, J.; Cuny, C.; Witte, W.; Ehricht, R.; Monecke, S.; Hanke, D.; Ahrens, T.; Leal, M.; Costa, S.S.; Couto, I.; et al. Characterization of Staphylococcus aureus CC1 and CC1660 of Human and Equine Origin. Antibiotics 2025, 14, 1082. https://doi.org/10.3390/antibiotics14111082

AMA Style

Jahnen J, Cuny C, Witte W, Ehricht R, Monecke S, Hanke D, Ahrens T, Leal M, Costa SS, Couto I, et al. Characterization of Staphylococcus aureus CC1 and CC1660 of Human and Equine Origin. Antibiotics. 2025; 14(11):1082. https://doi.org/10.3390/antibiotics14111082

Chicago/Turabian Style

Jahnen, Johanna, Christiane Cuny, Wolfgang Witte, Ralf Ehricht, Stefan Monecke, Dennis Hanke, Tanja Ahrens, Marta Leal, Sofia S. Costa, Isabel Couto, and et al. 2025. "Characterization of Staphylococcus aureus CC1 and CC1660 of Human and Equine Origin" Antibiotics 14, no. 11: 1082. https://doi.org/10.3390/antibiotics14111082

APA Style

Jahnen, J., Cuny, C., Witte, W., Ehricht, R., Monecke, S., Hanke, D., Ahrens, T., Leal, M., Costa, S. S., Couto, I., Schwarz, S., & Feßler, A. T. (2025). Characterization of Staphylococcus aureus CC1 and CC1660 of Human and Equine Origin. Antibiotics, 14(11), 1082. https://doi.org/10.3390/antibiotics14111082

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