<table><caption>TABLE 7 | Distribution of enterotoxin genes in all species.</caption><thead><tr><th rowspan="2">Virulence factors</th><th rowspan="2">Related gene</th><th rowspan="2">SAU<sup>1</sup><br>(45)</th><th rowspan="2">SAU<sup>1</sup><br>%</th><th rowspan="2">NAS<br>(48)</th><th rowspan="2">NAS<br>%</th><th colspan="2">Clade A</th><th colspan="2">Clade B</th><th colspan="2">Clade C</th><th colspan="4">Clade D</th><th colspan="4">Clade E</th></tr><tr><th>SVI<sup>1</sup><br>(1)</th><th>SSC<sup>1</sup><br>(5)</th><th>SHY<sup>1</sup><br>(1)</th><th>SCH<sup>1</sup><br>(6)</th><th>SSI<sup>1</sup><br>(2)</th><th>SHO<sup>1</sup><br>(4)</th><th>SDE<sup>1</sup><br>(2)</th><th>SHA<sup>1</sup><br>(9)</th><th>SWA<sup>1</sup><br>(2)</th><th>SEP<sup>1</sup><br>(7)</th><th>SAC<sup>1</sup><br>(1)</th><th>SAR<sup>1</sup><br>(1)</th><th>SCO<sup>1</sup><br>(2)</th><th>SEQ<sup>1</sup><br>(2)</th><th>SSA<sup>1</sup><br>(1)</th><th>SXY<sup>1</sup><br>(2)</th></tr></thead><tbody><tr><td>Enterotoxin A</td><td>sea</td><td>28</td><td>62%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin B</td><td>seb</td><td>21</td><td>46%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin C</td><td>sec</td><td>20</td><td>44%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin D</td><td>sed</td><td>3</td><td>7%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin E</td><td>see</td><td>16</td><td>35%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin G</td><td>seg</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin H</td><td>she</td><td>15</td><td>33%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin I</td><td>sei</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin J</td><td>sej</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like K</td><td>selk</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like L</td><td>sell</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like M</td><td>selm</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like N</td><td>seln</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like O</td><td>selo</td><td>23</td><td>51%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like P</td><td>selp</td><td>23</td><td>51%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like Q</td><td>selq</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like R</td><td>selr</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like U</td><td>selu</td><td>21</td><td>46%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin-like V</td><td>selv</td><td>18</td><td>40%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin Yent1</td><td>yent1</td><td>14</td><td>31%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Enterotoxin Yent2</td><td>yent2</td><td>45</td><td>100%</td><td>1</td><td>2%</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

NAS are shown according to clade. Green color indicates no virulence genes, light orange indicates the presence of virulence genes. For columns with S. aureus and total NAS dark orange indicates virulence gene present in over 50% of isolates.

withdraws free iron from body fluids to suppress pathogens (Haley and Skaar, 2012; Sheldon and Heinrichs, 2015). Mechanisms to acquire iron in a situation where the supply is scarce are well studied in S. aureus, who can take up iron directly from molecules using *isd* genes and produce siderophores along with surface transporters (Sheldon and Heinrichs, 2015). We did indeed detect all iron uptake and metabolism genes, except *isdA*, in all S. aureus isolates. In the NAS species, however, ABC transporter and staphyloferrin A genes were more frequently detected, compared to *isd* genes and staphyloferrin B genes. This is in accordance with the study by Naushad et al. (2019), and supports their hypothesis that staphyloferrin A production is the principal mechanism for iron acquisition in NAS.

The production of toxins is another important determinant of virulence in staphylococci, especially in S. aureus. These toxins, such as cytotoxins (hemolysins, leukotoxins, and leukocidins) and superantigens [enterotoxins, exfoliative toxins and toxic shock syndrome toxins (TSST)], promotes inflammation and leukocyte cell death (Tam and Torres, 2019). Of the cytotoxins, we detected *hlb*, encoding beta hemolysin, in all S. epidermidis isolates (clade D), similar to Naushad et al. (2019), who found *hlb* to be the most frequent hemolysin. Surprisingly, we did not detect *hlb* in any of the S. aureus isolates, whereas *hla* was present in all isolates. This contrasts the findings from a study on bovine

and humans S. aureus isolates where *hlb* appeared more common in bovine S. aureus isolates, while *hla* was more prevalent in the human isolates (Aarestrup et al., 1999). Similar to the study by Naushad et al. (2019), we did not detect any leukocidin genes or leukotoxin genes in the NAS isolates. Åvall-Jääskeläinen et al. (2018) found *lukD* in one S. simulans isolate, however, none of the other leukocidin or leukotoxin genes were detected in NAS in their study. All the S. aureus isolates in our study contained all genes for leukocidins and leukotoxins, including *lukS-PV* and *lukF-PV*. This resembles findings from another study on bovine S. aureus isolates that found leukocidin and leukotoxin genes in most isolates. However, that study did not detect any isolates carrying *lukS-PV* (Yamada et al., 2005). The Panton Valentine Leukocidin (PVL) genes are believed to be restricted to human strains of S. aureus (Vrieling et al., 2016) and it was surprising to find these genes in all our bovine S. aureus isolates. It is possible that this result appeared due to the method of similarity search, as there is a possibility for detecting genes that are similar to the gene in question, although not being the same gene. This could explain our results, as many of the *lukS-PV* and *lukF-PV* genes in our study had the same percentage identity in several S. aureus isolates, possibly indicating that there are sequence similarities between the genome and the genes, although the genes in question are not actually present. However, this warrants

<footnote n="1">¹Species abbreviations: SAU, S. aureus; SVI, S. vitulinus; SSC, S. sciuri; SHY, S. hyicus; SCH, S. chromogenes; SSI, S. simulans; SHO, S. hominis; SDE, S. devriesei; SHA, S. haemolyticus; SWA, S. warneri; SEP, S. epidermidis; SAC, S. auricularis; SAR, S. arlettae; SCO, S. cohnii; SEQ, S. equorum; SSA, S. saprophyticus; SXY, S. xylosus.</footnote>
further investigation. The PVL genes have been reported in a few
NAS isolates of bovine origin in India (Mahato et al., 2017). Of the
exfoliative toxin genes, we detected etc in all NAS isolates and etb
in all S. sciuri isolates. This contrasts the findings of Naushad et al.
(2019) who found eta in all isolates of three NAS species and etb in
a few isolates of S. agnetis and Ävall-Jääskeläinen et al. (2018) who
also found etb in S. agnetis. However, the latter did not test for eta,
etc, and etd. We did not have any S. agnetis in our collection, and
it is unknown whether there are geographical differences between
the distribution of the different exfoliative toxin genes or if there
are other factors affecting our results. Of the exfoliative toxin
genes, only the etc gene was detected in S. aureus isolates. The lack
of eta and etb has been shown previously by Haveri et al. (2007),
however, these were the only two exfoliative toxin genes included
in this study. The eta gene has been detected in a few S. aureus
isolates from bovines (Hayakawa et al., 2001).

Phenol soluble modulins (PSM) are also involved in the killing
of leukocytes and can act synergistically with leukocidins (Hongo
et al., 2009), contributing to the leukotoxicity of S. aureus, as
well as being involved in biofilm formation (Otto, 2014; Vrieling
et al., 2016). The PSMs are considered major determinants of the
virulence of S. aureus and α-type PSMs are thought to be more
aggressive than β-type PSMs (Otto, 2014; Naushad et al., 2019).
Being encoded on the core genome, the PSMs are present in
virtually all staphylococci (Otto, 2014). We detected β-type PSMs
genes, encoding the least aggressive PSMs, in most NAS species,
but not in species of clade A and the S. arlettae isolate (clade E).
This concurs with the results reported by Naushad et al. (2019).
However, in the species of clade A (S. sciuri and S. vitulinus) we
detected the PSMmec gene. This is the only exception to the core
genome-encoded PSMs, as the PSMmec is often found on the
SCCmec cassette carrying the mecA genes conferring methicillin
resistance (Qin et al., 2016). The isolates in our study in which
the PSMmec was found did indeed harbor mec genes, as the
S. sciuri isolates harbored the mecA1 and the S. vitulinus isolate
harbored the mecA2 gene. The PSMmec has been identified in
S. vitulinus carrying SCCmec previously (Monecke et al., 2012).
In S. aureus PSMα1, as well as PSMβ1-6 were detected in all
isolates. The finding of α-type PSM in S. aureus and not in NAS
can be due to the aggressive potential of S. aureus, as the α-type
PSM are considered more aggressive and this type of PSMs is
mostly associated with S. aureus (Wang et al., 2007). The fact
that we only detected PSMα1 and not the other PSMα can be
due to limitations in the method used (similarity search) and the
short sizes of PSMα, as this often does not give meaningful results
(Cheung et al., 2014; Otto, 2014).

Superantigens are responsible for much of the toxicity in
staphylococci. They are robust toxins, resilient to heat, proteolysis
and desiccation (Spaulding et al., 2013; Tam and Torres, 2019).
Toxic shock syndrome toxin gene (tsst) was not detected in the
NAS isolates in our study. This concurs with several previous
studies (Xu et al., 2015; Mello et al., 2016; Naushad et al., 2019).
The tsst gene is mostly associated with S. aureus and has been
detected in bovine S. aureus previously (Artursson et al., 2016;
Vaughn et al., 2020). We only detected staphylococcal exotoxins
in species of clade B (S. hyicus and S. chromogenes), however,
several exotoxins were present in all S. aureus isolates. We only

detected enterotoxin genes in one S. hyicus isolates (clade B).
Naushad et al. (2019) also detected enterotoxin genes only in
species from clade B. However, unlike Naushad et al. (2019)
we did not identify any enterotoxin genes in S. chromogenes.
We also detected several enterotoxin genes in S. aureus isolates.
The S. aureus enterotoxins can cause acute and severe food
poisoning, making it important to avoid contamination of
enterotoxin-producing S. aureus isolates throughout the food
production chain (Nia et al., 2016). Raw milk and cheese made
of unpasteurized milk are well-known food sources of food
poisoning caused by S. aureus.

Regarding the total virulence gene content, S. aureus stands
out from NAS with, on average, five times as many virulence
genes as NAS, highlighting the large virulence potential of
S. aureus and the limited virulence potential of NAS. When
looking at the total number of virulence genes in NAS,
S. chromogenes had a higher virulence potential than most other
species, mostly due to the presence of exotoxins. The single
S. hyicus isolate stood apart with the highest virulence potential,
due to host immune evasion, exotoxin and enterotoxin genes.
This concurs with the findings of Naushad et al. (2019) who also
found the highest virulence potential in species from clade B,
including S. chromogenes and S. hyicus. Virulence is dependent
on context and is often characterized by an intricate interplay
between the microorganism and the host, making it difficult to
predict virulence based on virulence gene content alone (Balloux
et al., 2018). Nor is it a given that the virulence genes are
expressed in the microorganism, even if they are present (Chaves-
Moreno et al., 2016), which further complicates the matter.
Considering the apparent complexity of virulence and the limited
knowledge on the subject in bovine NAS, further studies on the
association between virulence and clinical impact of these species
is important. As several studies indicates that S. chromogenes
could have a greater impact on udder health (Supré et al.,
2011; Fry et al., 2014; Valckenier et al., 2019) and both this
study and Naushad et al. (2019) found S. chromogenes among
those with the highest virulence potential, further studies of the
virulence and pathogenesis of this species should be emphasized
(De Buck et al., 2021).

Regarding the S. aureus isolates, the results from
VirulenceFinder supported the results obtained using the
database by Naushad et al. (2019). However, some genes were
not detected as frequently with the VirulenceFinder, possibly due
to stricter thresholds for identity in VirulenceFinder. In addition,
the VirulenceFinder returned less virulence genes compared to
the database from Naushad et al. (2019). This could be because
VirulenceFinder contains less virulence genes compared to the
database by Naushad et al. (2019), as the database was expanded
by doing blast similarity search to identify genes. Although the
similarity search method does identify true genes, there is a
possibility for misinterpretations due to detection of similar, but
not identical genes.

In conclusion, our data support the opinion that there
are more antimicrobial resistance genes in NAS compared to
S. aureus. Regarding virulence, our S. aureus isolates had a
higher virulence potential compared to NAS, but there are also
differences in virulence gene content within the NAS group,
further investigation. The PVL genes have been reported in a few
NAS isolates of bovine origin in India (Mahato et al., 2017). Of the
exfoliative toxin genes, we detected etc in all NAS isolates and etb
in all S. sciuri isolates. This contrasts the findings of Naushad et al.
(2019) who found eta in all isolates of three NAS species and etb in
a few isolates of S. agnetis and Ävall-Jääskeläinen et al. (2018) who
also found etb in S. agnetis. However, the latter did not test for eta,
etc, and etd. We did not have any S. agnetis in our collection, and
it is unknown whether there are geographical differences between
the distribution of the different exfoliative toxin genes or if there
are other factors affecting our results. Of the exfoliative toxin
genes, only the etc gene was detected in S. aureus isolates. The lack
of eta and etb has been shown previously by Haveri et al. (2007),
however, these were the only two exfoliative toxin genes included
in this study. The eta gene has been detected in a few S. aureus
isolates from bovines (Hayakawa et al., 2001).

Phenol soluble modulins (PSM) are also involved in the killing
of leukocytes and can act synergistically with leukocidins (Hongo
et al., 2009), contributing to the leukotoxicity of S. aureus, as
well as being involved in biofilm formation (Otto, 2014; Vrieling
et al., 2016). The PSMs are considered major determinants of the
virulence of S. aureus and α-type PSMs are thought to be more
aggressive than β-type PSMs (Otto, 2014; Naushad et al., 2019).
Being encoded on the core genome, the PSMs are present in
virtually all staphylococci (Otto, 2014). We detected β-type PSMs
genes, encoding the least aggressive PSMs, in most NAS species,
but not in species of clade A and the S. arlettae isolate (clade E).
This concurs with the results reported by Naushad et al. (2019).
However, in the species of clade A (S. sciuri and S. vitulinus) we
detected the PSMmec gene. This is the only exception to the core
genome-encoded PSMs, as the PSMmec is often found on the
SCCmec cassette carrying the mecA genes conferring methicillin
resistance (Qin et al., 2016). The isolates in our study in which
the PSMmec was found did indeed harbor mec genes, as the
S. sciuri isolates harbored the mecA1 and the S. vitulinus isolate
harbored the mecA2 gene. The PSMmec has been identified in
S. vitulinus carrying SCCmec previously (Monecke et al., 2012).
In S. aureus PSMα1, as well as PSMβ1-6 were detected in all
isolates. The finding of α-type PSM in S. aureus and not in NAS
can be due to the aggressive potential of S. aureus, as the α-type
PSM are considered more aggressive and this type of PSMs is
mostly associated with S. aureus (Wang et al., 2007). The fact
that we only detected PSMα1 and not the other PSMα can be
due to limitations in the method used (similarity search) and the
short sizes of PSMα, as this often does not give meaningful results
(Cheung et al., 2014; Otto, 2014).

Superantigens are responsible for much of the toxicity in
staphylococci. They are robust toxins, resilient to heat, proteolysis
and desiccation (Spaulding et al., 2013; Tam and Torres, 2019).
Toxic shock syndrome toxin gene (tsst) was not detected in the
NAS isolates in our study. This concurs with several previous
studies (Xu et al., 2015; Mello et al., 2016; Naushad et al., 2019).
The tsst gene is mostly associated with S. aureus and has been
detected in bovine S. aureus previously (Artursson et al., 2016;
Vaughn et al., 2020). We only detected staphylococcal exotoxins
in species of clade B (S. hyicus and S. chromogenes), however,
several exotoxins were present in all S. aureus isolates. We only

detected enterotoxin genes in one S. hyicus isolates (clade B).
Naushad et al. (2019) also detected enterotoxin genes only in
species from clade B. However, unlike Naushad et al. (2019)
we did not identify any enterotoxin genes in S. chromogenes.
We also detected several enterotoxin genes in S. aureus isolates.
The S. aureus enterotoxins can cause acute and severe food
poisoning, making it important to avoid contamination of
enterotoxin-producing S. aureus isolates throughout the food
production chain (Nia et al., 2016). Raw milk and cheese made
of unpasteurized milk are well-known food sources of food
poisoning caused by S. aureus.

Regarding the total virulence gene content, S. aureus stands
out from NAS with, on average, five times as many virulence
genes as NAS, highlighting the large virulence potential of
S. aureus and the limited virulence potential of NAS. When
looking at the total number of virulence genes in NAS,
S. chromogenes had a higher virulence potential than most other
species, mostly due to the presence of exotoxins. The single
S. hyicus isolate stood apart with the highest virulence potential,
due to host immune evasion, exotoxin and enterotoxin genes.
This concurs with the findings of Naushad et al. (2019) who also
found the highest virulence potential in species from clade B,
including S. chromogenes and S. hyicus. Virulence is dependent
on context and is often characterized by an intricate interplay
between the microorganism and the host, making it difficult to
predict virulence based on virulence gene content alone (Balloux
et al., 2018). Nor is it a given that the virulence genes are
expressed in the microorganism, even if they are present (Chaves-
Moreno et al., 2016), which further complicates the matter.
Considering the apparent complexity of virulence and the limited
knowledge on the subject in bovine NAS, further studies on the
association between virulence and clinical impact of these species
is important. As several studies indicates that S. chromogenes
could have a greater impact on udder health (Supré et al.,
2011; Fry et al., 2014; Valckenier et al., 2019) and both this
study and Naushad et al. (2019) found S. chromogenes among
those with the highest virulence potential, further studies of the
virulence and pathogenesis of this species should be emphasized
(De Buck et al., 2021).

Regarding the S. aureus isolates, the results from
VirulenceFinder supported the results obtained using the
database by Naushad et al. (2019). However, some genes were
not detected as frequently with the VirulenceFinder, possibly due
to stricter thresholds for identity in VirulenceFinder. In addition,
the VirulenceFinder returned less virulence genes compared to
the database from Naushad et al. (2019). This could be because
VirulenceFinder contains less virulence genes compared to the
database by Naushad et al. (2019), as the database was expanded
by doing blast similarity search to identify genes. Although the
similarity search method does identify true genes, there is a
possibility for misinterpretations due to detection of similar, but
not identical genes.

In conclusion, our data support the opinion that there
are more antimicrobial resistance genes in NAS compared to
S. aureus. Regarding virulence, our S. aureus isolates had a
higher virulence potential compared to NAS, but there are also
differences in virulence gene content within the NAS group,
supporting the view that NAS should not be considered as one
uniform group of bacteria.

DATA AVAILABILITY STATEMENT

The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and
accession number(s) can be found below: https://www.ncbi.nlm.
nih.gov/, PRJNA609060.

AUTHOR CONTRIBUTIONS

FT was in charge of the methodology, performed the methods
used, and wrote sections of the manuscript. MF organized the
databases, analyzed the results, and wrote the first draft of the
manuscript. SD, AD, and YW contributed to the analysis of the
results. All authors contributed to the conception and design of

the study, the manuscript revision, and read and approved the
submitted version.

FUNDING

This project was part of the ERA-Net Animal Health and Welfare (ANIHWA), MRSA_bacteriophages project. The project was funded by the Research Council of Norway (Project No. 258877), the Research Foundation - Flanders (FWO), and Service Public Fédéral - Santé publique, Sécurité de la Chaîne alimentaire et Environnement (Wallonia). Salary was provided by each home institution.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.715851/full#supplementary-material

REFERENCES

Aarestrup, F. M., Larsen, H. D., Eriksen, N. H., Elsberg, C. S., and Jensen, N. E. (1999). Frequency of alpha- and beta-haemolysin in *Staphylococcus aureus* of bovine and human origin. A comparison between pheno- and genotype and variation in phenotypic expression. *Apmis* 107, 425–430.

Andreis, S. N., Perreten, V., and Schwendener, S. (2017). Novel β-lactamase bla(ARL) in *Staphylococcus arlettae*. *mSphere* 2:e00117-17. doi: 10.1128/mSphere.00117-17

Anthonisen, I. L., Sunde, M., Steinum, T. M., Sidhu, M. S., and Sorum, H. (2002). Organization of the antiseptic resistance gene qaC and Tn552-related beta-lactamase genes in multidrug- resistant *Staphylococcus haemolyticus* strains of animal and human origins. *Antimicrob. Agents Chemother.* 46, 3606–3612. doi: 10.1128/aac.46.11.3606-3612.2002

Antignac, A., and Tomasz, A. (2009). Reconstruction of the phenotypes of methicillin-resistant Staphylococcus aureus by replacement of the staphylococcal cassette chromosome mec with a plasmid-borne copy of *Staphylococcus sciuri* pbpD gene. *Antimicrob. Agents Chemother.* 53, 435–441. doi: 10.1128/aac.01099-08

Artursson, K., Söderlund, R., Liu, L., Monecke, S., and Schelin, J. (2016). Genotyping of *Staphylococcus aureus* in bovine mastitis and correlation to phenotypic characteristics. *Vet. Microbiol.* 193, 156–161. doi: 10.1016/j.vetmic. 2016.08.012

Åvall-Jääskeläinen, S., Taponen, S., Kant, R., Paulin, L., Blom, J., Palva, A., et al. (2018). Comparative genome analysis of 24 bovine-associated *Staphylococcus* isolates with special focus on the putative virulence genes. *PeerJ* 6:e4560. doi: 10.7717/peerj.4560

Balloux, F., Brønstad Brynildsrud, O., van Dorp, L., Shaw, L. P., Chen, H., Harris, K. A., et al. (2018). From theory to practice: translating whole-genome sequencing (WGS) into the clinic. *Trends Microbiol.* 26, 1035–1048. doi: 10.1016/j.tim.2018.08.004

Becker, K., Heilmann, C., and Peters, G. (2014). Coagulase-negative staphylococci. *Clin. Microbiol. Rev.* 27, 870–926. doi: 10.1128/cmr.00109-13

Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for illumina sequence data. *Bioinformatics* 30, 2114–2120. doi: 10.1093/ bioinformatics/btu170

Bortolaia, V., Kaas, R. S., Ruppe, E., Roberts, M. C., Schwarz, S., Cattoir, V., et al. (2020). ResFinder 4.0 for predictions of phenotypes from genotypes. *J. Antimicrob. Chemother.* 75, 3491–3500. doi: 10.1093/jac/dka a345

Cameron, M., Barkema, H. W., De Buck, J., De Vliegher, S., Chaffer, M., Lewis, J., et al. (2017). Identification of bovine-associated coagulase-negative

staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol. *J. Dairy Sci.* 100, 2137–2147. doi: 10.3168/jds.2016-12020

Cameron, M., Perry, J., Middleton, J. R., Chaffer, M., Lewis, J., and Keefe, G. P. (2018). Short communication: evaluation of MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci. *J. Dairy Sci.* 101, 590–595. doi: 10.3168/jds.2017-13226

Chaves-Moreno, D., Wos-Oxley, M. L., Jáuregui, R., Medina, E., Oxley, A. P., and Pieper, D. H. (2016). Exploring the transcriptome of *Staphylococcus aureus* in its natural niche. *Sci. Rep.* 6:33174. doi: 10.1038/srep33174

Cheung, G. Y., Joo, H. S., Chatterjee, S. S., and Otto, M. (2014). Phenol-soluble modulins—critical determinants of staphylococcal virulence. *FEMS Microbiol. Rev.* 38, 698–719. doi: 10.1111/1574-6976.12057

Condas, L. A. Z., De Buck, J., Nobrega, D. B., Carson, D. A., Naushad, S., De Vliegher, S., et al. (2017). Prevalence of non-aureus staphylococci species causing intramammary infections in Canadian dairy herds. *J. Dairy Sci.* 100, 5592–5612. doi: 10.3168/jds.2016-12478

Costa, S. S., Sobkowiak, B., Parreira, R., Edgeworth, J. D., Viveiros, M., Clark, T. G., et al. (2018). Genetic diversity of norA, coding for a main efflux pump of *Staphylococcus aureus*. *Front. Genet.* 9:710. doi: 10.3389/fgene.2018.0 0710

Couto, I., de Lencastre, H., Severina, E., Kloos, W., Webster, J. A., Hubner, R. J., et al. (1996). Ubiquitous presence of a mecA homologue in natural isolates of *Staphylococcus sciuri*. *Microb. Drug Resist.* 2, 377–391. doi: 10.1089/mdr.1996. 2.377

De Buck, J., Ha, V., Naushad, S., Nobrega, D. B., Luby, C., Middleton, J. R., et al. (2021). Non-aureus staphylococci and bovine udder health: current understanding and knowledge gaps. *Front. Vet. Sci.* 8:658031. doi: 10.3389/fvets. 2021.658031

De Visscher, A., Supré, K., Haesebrouck, F., Zadoks, R. N., Piessens, V., Van Coillie, E., et al. (2014). Further evidence for the existence of environmental and host-associated species of coagulase-negative staphylococci in dairy cattle. *Vet. Microbiol.* 172, 466–474. doi: 10.1016/j.vetmic.2014.06.011

De Vliegher, S., Fox, L. K., Piepers, S., McDougall, S., and Barkema, H. W. (2012). Invited review: mastitis in dairy heifers: nature of the disease, potential impact, prevention, and control. *J. Dairy Sci.* 95, 1025–1040. doi: 10.3168/jds.2010- 4074

DeMarco, C. E., Cushing, L. A., Frempong-Manso, E., Seo, S. M., Jaravaza, T. A., and Kaatz, G. W. (2007). Efflux-related resistance to norfloxacin, dyes, and biocides in bloodstream isolates of *Staphylococcus aureus*. *Antimicrob. Agents Chemother.* 51, 3235–3239. doi: 10.1128/aac.00430-07
supporting the view that NAS should not be considered as one
uniform group of bacteria.

DATA AVAILABILITY STATEMENT

The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and
accession number(s) can be found below: https://www.ncbi.nlm.
nih.gov/, PRJNA609060.

AUTHOR CONTRIBUTIONS

FT was in charge of the methodology, performed the methods
used, and wrote sections of the manuscript. MF organized the
databases, analyzed the results, and wrote the first draft of the
manuscript. SD, AD, and YW contributed to the analysis of the
results. All authors contributed to the conception and design of

the study, the manuscript revision, and read and approved the
submitted version.

FUNDING

This project was part of the ERA-Net Animal Health and Welfare (ANIHWA), MRSA_bacteriophages project. The project was funded by the Research Council of Norway (Project No. 258877), the Research Foundation - Flanders (FWO), and Service Public Fédéral - Santé publique, Sécurité de la Chaîne alimentaire et Environnement (Wallonia). Salary was provided by each home institution.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.715851/full#supplementary-material

REFERENCES

Aarestrup, F. M., Larsen, H. D., Eriksen, N. H., Elsberg, C. S., and Jensen, N. E. (1999). Frequency of alpha- and beta-haemolysin in *Staphylococcus aureus* of bovine and human origin. A comparison between pheno- and genotype and variation in phenotypic expression. *Apmis* 107, 425–430.

Andreis, S. N., Perreten, V., and Schwendener, S. (2017). Novel β-lactamase bla(ARL) in *Staphylococcus arlettae*. *mSphere* 2:e00117-17. doi: 10.1128/mSphere.00117-17

Anthonisen, I. L., Sunde, M., Steinum, T. M., Sidhu, M. S., and Sorum, H. (2002). Organization of the antiseptic resistance gene qaC and Tn552-related beta-lactamase genes in multidrug- resistant *Staphylococcus haemolyticus* strains of animal and human origins. *Antimicrob. Agents Chemother.* 46, 3606–3612. doi: 10.1128/aac.46.11.3606-3612.2002

Antignac, A., and Tomasz, A. (2009). Reconstruction of the phenotypes of methicillin-resistant Staphylococcus aureus by replacement of the staphylococcal cassette chromosome mec with a plasmid-borne copy of *Staphylococcus sciuri* pbpD gene. *Antimicrob. Agents Chemother.* 53, 435–441. doi: 10.1128/aac.01099-08

Artursson, K., Söderlund, R., Liu, L., Monecke, S., and Schelin, J. (2016). Genotyping of *Staphylococcus aureus* in bovine mastitis and correlation to phenotypic characteristics. *Vet. Microbiol.* 193, 156–161. doi: 10.1016/j.vetmic. 2016.08.012

Åvall-Jääskeläinen, S., Taponen, S., Kant, R., Paulin, L., Blom, J., Palva, A., et al. (2018). Comparative genome analysis of 24 bovine-associated *Staphylococcus* isolates with special focus on the putative virulence genes. *PeerJ* 6:e4560. doi: 10.7717/peerj.4560

Balloux, F., Brønstad Brynildsrud, O., van Dorp, L., Shaw, L. P., Chen, H., Harris, K. A., et al. (2018). From theory to practice: translating whole-genome sequencing (WGS) into the clinic. *Trends Microbiol.* 26, 1035–1048. doi: 10.1016/j.tim.2018.08.004

Becker, K., Heilmann, C., and Peters, G. (2014). Coagulase-negative staphylococci. *Clin. Microbiol. Rev.* 27, 870–926. doi: 10.1128/cmr.00109-13

Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for illumina sequence data. *Bioinformatics* 30, 2114–2120. doi: 10.1093/ bioinformatics/btu170

Bortolaia, V., Kaas, R. S., Ruppe, E., Roberts, M. C., Schwarz, S., Cattoir, V., et al. (2020). ResFinder 4.0 for predictions of phenotypes from genotypes. *J. Antimicrob. Chemother.* 75, 3491–3500. doi: 10.1093/jac/dka a345

Cameron, M., Barkema, H. W., De Buck, J., De Vliegher, S., Chaffer, M., Lewis, J., et al. (2017). Identification of bovine-associated coagulase-negative

staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol. *J. Dairy Sci.* 100, 2137–2147. doi: 10.3168/jds.2016-12020

Cameron, M., Perry, J., Middleton, J. R., Chaffer, M., Lewis, J., and Keefe, G. P. (2018). Short communication: evaluation of MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci. *J. Dairy Sci.* 101, 590–595. doi: 10.3168/jds.2017-13226

Chaves-Moreno, D., Wos-Oxley, M. L., Jáuregui, R., Medina, E., Oxley, A. P., and Pieper, D. H. (2016). Exploring the transcriptome of *Staphylococcus aureus* in its natural niche. *Sci. Rep.* 6:33174. doi: 10.1038/srep33174

Cheung, G. Y., Joo, H. S., Chatterjee, S. S., and Otto, M. (2014). Phenol-soluble modulins—critical determinants of staphylococcal virulence. *FEMS Microbiol. Rev.* 38, 698–719. doi: 10.1111/1574-6976.12057

Condas, L. A. Z., De Buck, J., Nobrega, D. B., Carson, D. A., Naushad, S., De Vliegher, S., et al. (2017). Prevalence of non-aureus staphylococci species causing intramammary infections in Canadian dairy herds. *J. Dairy Sci.* 100, 5592–5612. doi: 10.3168/jds.2016-12478

Costa, S. S., Sobkowiak, B., Parreira, R., Edgeworth, J. D., Viveiros, M., Clark, T. G., et al. (2018). Genetic diversity of norA, coding for a main efflux pump of *Staphylococcus aureus*. *Front. Genet.* 9:710. doi: 10.3389/fgene.2018.0 0710

Couto, I., de Lencastre, H., Severina, E., Kloos, W., Webster, J. A., Hubner, R. J., et al. (1996). Ubiquitous presence of a mecA homologue in natural isolates of *Staphylococcus sciuri*. *Microb. Drug Resist.* 2, 377–391. doi: 10.1089/mdr.1996. 2.377

De Buck, J., Ha, V., Naushad, S., Nobrega, D. B., Luby, C., Middleton, J. R., et al. (2021). Non-aureus staphylococci and bovine udder health: current understanding and knowledge gaps. *Front. Vet. Sci.* 8:658031. doi: 10.3389/fvets. 2021.658031

De Visscher, A., Supré, K., Haesebrouck, F., Zadoks, R. N., Piessens, V., Van Coillie, E., et al. (2014). Further evidence for the existence of environmental and host-associated species of coagulase-negative staphylococci in dairy cattle. *Vet. Microbiol.* 172, 466–474. doi: 10.1016/j.vetmic.2014.06.011

De Vliegher, S., Fox, L. K., Piepers, S., McDougall, S., and Barkema, H. W. (2012). Invited review: mastitis in dairy heifers: nature of the disease, potential impact, prevention, and control. *J. Dairy Sci.* 95, 1025–1040. doi: 10.3168/jds.2010- 4074

DeMarco, C. E., Cushing, L. A., Frempong-Manso, E., Seo, S. M., Jaravaza, T. A., and Kaatz, G. W. (2007). Efflux-related resistance to norfloxacin, dyes, and biocides in bloodstream isolates of *Staphylococcus aureus*. *Antimicrob. Agents Chemother.* 51, 3235–3239. doi: 10.1128/aac.00430-07
Dos Santos, D. C., Lange, C. C., Avellar-Costa, P., Dos Santos, K. R., Brito, M. A., and Giambiagi-deMarval, M. (2016). *Staphylococcus chromogenes*, a coagulase-negative *Staphylococcus* Species that can clot plasma. *J. Clin. Microbiol.* **54**, 1372–1375. doi: 10.1128/jcm.03139-15

Fergestad, M. E., De Visscher, A., L'Abee-Lund, T., Tchamba, C. N., Mainil, J. G., Thiry, D., et al. (2021). Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples. *J. Dairy Sci.* **104**, 10250–10267. doi: 10.3168/jds.2020-19988

Foster, T. J., Geoghegan, J. A., Ganesh, V. K., and Höök, M. (2014). Adhesion, invasion and evasion: the many functions of the surface proteins of *Staphylococcus aureus*. *Nat. Rev. Microbiol.* **12**, 49–62. doi: 10.1038/nrmicro3161

Fry, P. R., Middleton, J. R., Dufour, S., Perry, J., Scholl, D., and Dohoo, I. (2014). Association of coagulase-negative staphylococcal species, mammary quarter milk somatic cell count, and persistence of intramammary infection in dairy cattle. *J. Dairy Sci.* **97**, 4876–4885. doi: 10.3168/jds.2013-7657

Geoghegan, J. A., and Foster, T. J. (2017). Cell wall-anchored surface proteins of *Staphylococcus aureus*: many proteins, multiple functions. *Curr. Top. Microbiol. Immunol.* **409**, 95–120. doi: 10.1007/82_2015_5002

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Fergestad, M. E., De Visscher, A., L'Abee-Lund, T., Tchamba, C. N., Mainil, J. G., Thiry, D., et al. (2021). Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples. *J. Dairy Sci.* 104, 10250-10267. doi: 10.3168/jds.2020-19988

Foster, T. J., Geoghegan, J. A., Ganesh, V. K., and Höök, M. (2014). Adhesion, invasion and evasion: the many functions of the surface proteins of *Staphylococcus aureus*. *Nat. Rev. Microbiol.* 12, 49-62. doi: 10.1038/nrmicro3161

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Nobrega, D. B., Naushad, S., Naqvi, S. A., Condas, L. A. Z., Saini, V., Kastelic, J. P., et al. (2018). Prevalence and genetic basis of antimicrobial resistance in non-aureus staphylococci isolated from Canadian dairy herds. *Front. Microbiol.* 9:256. doi: 10.3389/fmicb.2018.00256

Olde Riekerink, R. G., Barkema, H. W., Kelton, D. F., and Scholl, D. T. (2008). Incidence rate of clinical mastitis on Canadian dairy farms. *J. Dairy Sci.* 91, 1366-1377. doi: 10.3168/jds.2007-0757

Olsen, J. E., Christensen, H., and Aarestrup, F. M. (2006). Diversity and evolution of blaZ from *Staphylococcus aureus* and coagulase-negative staphylococci. *J. Antimicrob. Chemother.* 57, 450-460. doi: 10.1093/jac/dki492

Østerås, O., Sølverød, L., and Reksen, O. (2006). Milk culture results in a large Norwegian survey-effects of season, parity, days in milk, resistance, and clustering. *J. Dairy Sci.* 89, 1010-1023. doi: 10.3168/jds.S0022-0302(06)72167-1

Otto, M. (2013). Coagulase-negative staphylococci as reservoirs of genes facilitating MRSA infection: staphylococcal commensal species such as *Staphylococcus epidermidis* are being recognized as important sources of genes promoting MRSA colonization and virulence. *Bioessays.* 35, 4-11. doi: 10.1002/bies. 201200112

Otto, M. (2014). Phenol-soluble modulins. *Int. J. Med. Microbiol.* 304, 164-169. doi: 10.1016/j.ijmm.2013.11.019

Piddock, L. J. (2006). Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. *Clin. Microbiol. Rev.* 19, 382-402. doi: 10.1128/cmr.19.2.382-402.2006

Piessens, V., De Vliegher, S., Verbist, B., Braem, G., Van Nuffel, A., De Vuyst, L., et al. (2012). Characterization of coagulase-negative *Staphylococcus* species from cows' milk and environment based on bap, icaA, and mecA genes and phenotypic susceptibility to antimicrobials and teat dips. *J. Dairy Sci.* 95, 7027-7038. doi: 10.3168/jds.2012-5400

Piessens, V., Van Coillie, E., Verbist, B., Supré, K., Braem, G., Van Nuffel, A., et al. (2011). Distribution of coagulase-negative *Staphylococcus* species from milk and environment of dairy cows differs between herds. *J. Dairy Sci.* 94, 2933-2944. doi: 10.3168/jds.2010-3956

Pitkälä, A., Haveri, M., Pyörälä, S., Myllys, V., and Honkanen-Buzalski, T. (2004). Bovine mastitis in Finland 2001-prevalence, distribution of bacteria, and antimicrobial resistance. *J. Dairy Sci.* 87, 2433-2441. doi: 10.3168/jds.S0022-0302(04)73366-4

Poole, K. (2007). Efflux pumps as antimicrobial resistance mechanisms. *Ann. Med.* 39, 162-176. doi: 10.1080/0785389071195262

Pyörälä, S., and Taponen, S. (2009). Coagulase-negative staphylococci-emerging mastitis pathogens. *Vet. Microbiol.* 134, 3-8. doi: 10.1016/j.vetmic.2008.09.015

Qin, L., McCausland, J. W., Cheung, G. Y., and Otto, M. (2016). PSM-Mec-a virulence determinant that connects transcriptional regulation, virulence, and antibiotic resistance in staphylococci. *Front. Microbiol.* 7:1293. doi: 10.3389/fmicb.2016.01293

Reksen, O., Sølverød, L., Branscum, A. J., and Østerås, O. (2006). Relationships between milk culture results and treatment for clinical mastitis or culling in Norwegian dairy cattle. *J. Dairy Sci.* 89, 2928-2937. doi: 10.3168/jds.S0022-0302(06)72565-6

Salimena, A. P., Lange, C. C., Camussone, C., Signorini, M., Calvinho, L. F., Brito, M. A., et al. (2016). Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in Staphylococcus aureus isolated from bovine milk collected from Brazilian dairy farms. *Vet. Res. Commun.* 40, 97-106. doi: 10.1007/s11259-016-9658-5

Santos Costa, S., Viveiros, M., Rosato, A. E., Melo-Cristino, J., and Couto, I. (2015). Impact of efflux in the development of multidrug resistance phenotypes in *Staphylococcus aureus*. *BMC Microbiol.* 15:232. doi: 10.1186/s12866-015-072-8

Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. *Bioinformatics* 30, 2068-2069. doi: 10.1093/bioinformatics/btu153

Seemann, T., Edwards, R., da Silva, A. G., and Kiil, K. (2020). *Shovill*. Available online at: https://github.com/tseemann/shovill

Sheldon, J. R., and Heinrichs, D. E. (2015). Recent developments in understanding the iron acquisition strategies of gram positive pathogens. *FEMS Microbiol. Rev.* 39, 592-630. doi: 10.1093/femsre/fuv009

Spaulding, A. R., Salgado-Pabón, W., Kohler, P. L., Horswill, A. R., Leung, D. Y., and Schlievert, P. M. (2013). Staphylococcal and streptococcal superantigen exotoxins. *Clin. Microbiol. Rev.* 26, 422-447. doi: 10.1128/cmr.00104-12

Supré, K., Haesebrouck, F., Zadoks, R. N., Vaneechoutte, M., Piepers, S., and De Vliegher, S. (2011). Some coagulase-negative *Staphylococcus* species affect udder health more than others. *J. Dairy Sci.* 94, 2329-2340. doi: 10.3168/jds.2010-3741

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Publisher's Note:** All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Copyright © 2021 Fergestad, Touzain, De Vliegher, De Visscher, Thiry, Ngassam Tchamba, Mainil, L'Abee-Lund, Blanchard and Wasteson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Wassenaar, T. M., Ussery, D. W., and Ingmer, H. (2016). The qacC gene has recently spread between rolling circle plasmids of *Staphylococcus*, indicative of a novel gene transfer mechanism. *Front. Microbiol.* 7:1528. doi: 10.3389/fmicb.2016.01528

Wu, S., Piscitelli, C., de Lencastre, H., and Tomasz, A. (1996). Tracking the evolutionary origin of the methicillin resistance gene: cloning and sequencing of a homologue of mecA from a methicillin susceptible strain of *Staphylococcus sciuri*. *Microb. Drug Resist.* 2, 435–441. doi: 10.1089/mdr.1996.2.435

Wuytack, A., De Visscher, A., Piepers, S., Boyen, F., Haesebrouck, F., and De Vliegher, S. (2020). Distribution of non-aureus staphylococci from quarter milk, teat apices, and rectal feces of dairy cows, and their virulence potential. *J. Dairy Sci.* 103, 10658–10675. doi: 10.3168/jds.2020-18265

Xu, J., Tan, X., Zhang, X., Xia, X., and Sun, H. (2015). The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd. *Microb. Pathog.* 88, 29–38. doi: 10.1016/j.micpath.2015.08.004

Yamada, T., Tochimaru, N., Nakasuji, S., Hata, E., Kobayashi, H., Eguchi, M., et al. (2005). Leukotoxin family genes in *Staphylococcus aureus* isolated from domestic animals and prevalence of lukM-lukF-PV genes by bacteriophages in bovine isolates. *Vet. Microbiol.* 110, 97–103. doi: 10.1016/j.vetmic.2005.07.006

Zankari, E., Allesøe, R., Joensen, K. G., Cavaco, L. M., Lund, O., and Aarestrup, F. M. (2017). PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens. *J. Antimicrob. Chemother.* 72, 2764–2768. doi: 10.1093/jac/dkx217

Zhang, H. Z., Hackbarth, C. J., Chansky, K. M., and Chambers, H. F. (2001). A proteolytic transmembrane signaling pathway and resistance to beta-lactams in staphylococci. *Science* 291, 1962–1965. doi: 10.1126/science.1055144

Zhou, Y., Antignac, A., Wu, S. W., and Tomasz, A. (2008). Penicillin-binding proteins and cell wall composition in beta-lactam-sensitive and -resistant strains of *Staphylococcus sciuri*. *J. Bacteriol.* 190, 508–514. doi: 10.1128/jb.01549-07

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Publisher's Note:** All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Copyright © 2021 Fergestad, Touzain, De Vliegher, De Visscher, Thiry, Ngassam Tchamba, Mainil, L'Abee-Lund, Blanchard and Wasteson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.