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Journal of Applied Microbiology, 2023, 134,1–11
https://doi.org/10.1093/jambio/lxad057
Advance access publication date: 17 March 2023
Research Article
Virulence and antimicrobial resistance genes proles of
spa type t605 methicillin-susceptible Staphylococcus
aureus isolated from subclinical bovine mastitis
Karen Vanessa Munive Nuñez 1, Anderson Clayton da Silva Abreu1, Juliano Leonel Gonçalves2,
Marcos Veiga dos Santos3, Liliana de Oliveira Rocha1, Nathália Cristina Cirone Silva1,*
1Department of Food Science and Nutrition, School of Food Engineering, University of Campinas (UNICAMP), Campinas, São Paulo
13083-862, Brazil
2Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA
3Department of Animal Nutrition and Production, School of Veterinary Medicine and Animal Sciences, University of São Paulo (USP),
Pirassununga, São Paulo 13635-900, Brazil
∗Corresponding author. Department of Food Science and Nutrition, School of Food Engineering, University of Campinas, Campinas, São Paulo 13083-862, Brazil.
E-mail: ncirone@unicamp.br
Abstract
Aims: Staphylococcus aureus is one of the most common pathogens associated with mastitis in dairy herds worldwide. This study evaluated the
prole of virulence and antimicrobial resistance genes of spa type t605 methicillin-susceptible Staphylococcus aureus isolated from subclinical
bovine mastitis in São Paulo, Brazil.
Methods and results: A total of 57 S. aureus strains were screened by conventional PCR (Polymerase Chain Reaction) for 49 virulence genes.
The most prevalent virulence genes detected were icaD (94.7%), b (93%), fnbA (82.5%), clfA (80.7%), bap (78.9%), clfB (73.7%), icaA (66.7%),
see (64.9%), and sed (61.4%). The blaZ (94.7%), aac6’aph2’ (15.8%), and ant4 (12.3%) genes were the most common antimicrobial resistance
genes; however, mecAandmecC genes were not found. All methicillin-susceptible S. aureus (MSSA) strains were characterized through spa
and agr typing. The spa type t605 was found in all isolates. By agr typing, the most prevalent were type II (56.1%). Antimicrobial resistance was
determined by the disk diffusion method, and 93% showed resistance to at least one antibiotic. Penicillin resistance was the most prevalent
(87.7%), followed by tetracycline (12.3%), oxacillin (10.5%), and gentamicin (10.5%) resistance.
Conclusion: Our study conrmed the spa type t605 as endemic, carrying a wide variety of virulence factors and high-level penicillin resistance.
The prole seems to be associated with the colonization of MSSA and its persistence in subclinical mastitis.
Signicance and impact of the study
The methicillin-susceptible Staphylococcus aureus characteristics found here evidence its potential risk to human and bovine health and em-
phasize the need for continuous surveillance to control its presence. These ndings improve the comprehension of its pathogenesis, genomic
variations, and antibiotic resistance, which can help to enhance therapeutic recommendations and optimize bovine mastitis treatment.
Keywords: MSSA, MSCRAMMs, antibiotic resistance, spa typing, mastitis
Introduction
Subclinical mastitis (SM) represents the most prevalent type
of bovine mastitis, and Staphylococcus aureus is one of the
main etiologic agents causing contagious intramammary in-
fections (Ruegg 2017, Côté-Gravel and Malouin 2019,Dyson
et al. 2022). The risk of S. aureus infection is increased when
unhygienic practices are performed during milking; these in-
clude contaminated equipment, unsanitary environments, and
the lack of trained personnel in dairy operations (Zaatout et
al. 2019). Cows with SM caused by S. aureus present high
SCC (>200 ×103cells mL−1), but no visual changes in milk
are observed (Gonçalves et al. 2018). However, it can cause
human intoxication since bacteria can be eliminated through
a thermal process, but enterotoxins may remain, increasing
the possibility of foodborne poisoning (Grispoldi et al. 2021,
Campos et al. 2022). In this regard, S. aureus has been associ-
ated with 9.4% and 7.8% of food-poisoning cases concerning
milk and dairy products, respectively, with 120 584 cases re-
ported by the Brazilian Ministry of Health between 2009 and
2018 (Brasil 2019).
Molecular studies are important to evaluate the genetic di-
versity of pathogens and contribute to understanding sources,
routes of transmission, prognosis, and adaptation mechanisms
(McMillan et al. 2016). Staphylococcus aureus represents a
signicant issue for bovine udder health. Its adaptation to the
tissue has been associated with persistence and specic genetic
and phenotypic traits (Schmidt et al. 2017, Santos et al. 2020).
In addition, this pathogen has various virulence factors and
survival strategies to persist and defeat host defences, such
as adhesion capacity and biolm formation (Bhunia 2008,
Zhu et al. 2018). The expression of these virulence factors
is rigorously controlled by a series of regulatory genes inu-
enced by the accessory gene regulator (agr). Their presence
or absence plays an important role in the persistence of the
pathogen causing mastitis (Rossi et al. 2021). Overall, mas-
titis can be reduced in the dairy industry by identifying the
Received: June 10, 2022. Revised: January 17, 2023. Accepted: March 16, 2023
C
The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International. All rights reserved. For permissions, please
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2Munive Nuñez et al.
pathogenesis and virulence factors present in the pathogens
(Khan et al. 2013).
Among the virulence factors, various studies have revealed
that staphylococcal enterotoxins (SEs) play an important role
in the pathogenesis of bovine mastitis since most S. aureus
strains isolated from this disease harbour one or more entero-
toxin genes (Rall et al. 2014, Bonsaglia et al. 2018, Ren et
al. 2020). Additionally, it is important to highlight that these
toxins are a major cause of food poisoning since foods con-
taining sufcient amounts of preformed SEs can cause human
intoxication (Grispoldi et al. 2021).
The onset of the intramammary infection caused by S. au-
reus is inuenced by diverse virulence factors, such as biolm
formation and microbial surface components recognizing ad-
hesive matrix molecules (MSCRAMMs) genes (Zuniga et al.
2015). Of these genes, bap (encoding biolm-associated pro-
tein), ica (encoding intercellular adhesion gene cluster), clfA
and clfB (encoding clumping factors A and B), fnbAand
fnbB (encoding bronectin-binding protein A and B), ebps
(encoding elastin binding protein), eno (encoding laminin-
binding protein), cna (encoding collagen adhesin), b (en-
coding brinogen-binding protein), and bbp (encoding bone
sialoprotein-binding protein) play an important role in the ini-
tial attachment, recruitment, and colonization of mammary
glands; consequently, it is associated with the persistence of
bovine mastitis infection (Zuniga et al. 2015,Aslanta¸sand
Demir 2016, Zaatout et al. 2020).
The large quantity of antimicrobials used in dairy cattle is
a potential source for the emergence and dissemination of re-
sistant bacteria, which is the main reason for the restrictions
on using critically important antimicrobials (CIAs) in veteri-
nary medicine (Zaatout et al. 2020). They are called CIAs be-
cause they can treat potentially serious bacterial infections in
humans. Therefore, the use of ‘highest priority’ CIAs in vet-
erinary medicine (e.g. a broad-spectrum β-lactam antibiotic,
third generation cephalosporin as ceftiofur) has already been
restricted by legislation in their use (Koops et al. 2018). Peni-
cillins and other β-lactam antibiotics (e.g. intramammary cef-
tiofur that represented 52.8% of rst cases of treated clini-
cal mastitis) remain the drugs of choice, most frequently used
for treating mastitis (Gonçalves et al. 2022).Although methi-
cillin is not used to treat cattle, methicillin-resistant S. aureus
(MRSA) is regarded as a superbug that poses a concern to
human health, and the emergence of methicillin resistance is
a marker for the emergence of universal resistance to other
β-lactam medicines (e.g. as ceftiofur also used to treat hu-
man infections). Unfortunately, acquired resistance remains
a notable concern; in this regard, evaluating the antimicro-
bial resistance proles becomes essential to guide manage-
ment decisions and implement prudent and effective antimi-
crobial usage in dairy cattle (Ruegg 2017, Zaatout et al. 2020).
The pathogen adapts to different stress conditions due to its
genotypic and phenotypic plasticity, which explains the varia-
tions in its susceptibility to antimicrobial compounds (Erick-
son et al. 2017). Genes that confer less susceptibility to phe-
nolic and quaternary ammonium compounds (such as ben-
zalkonium chloride and cetrimide) are called qacs. Quater-
nary ammonium compound (QAC) sanitizers can break the
microbial cell membrane and are considered efcient antimi-
crobial agents (Wassenaar et al. 2015, Zhang et al. 2018).
Methicillin-susceptible S. aureus (MSSA) constantly evolves
through horizontal gene transfer and mutations. When the
mecAormecC gene carried on the staphylococcal cassette
chromosome mec (SCCmec) is acquired by MSSA, new MRSA
lineages may emerge, and these could establish infections, af-
fecting the health of animals, including cattle (Silva et al. 2013,
Bal et al. 2016, Moore-Lotridge et al. 2022). Furthermore, as-
sessing the risk of MSSA exposure for those involved in milk-
ing processes and for consumers of raw dairy products is cru-
cial due to the potential exchange between human and bovine
bacterial reservoirs (Sheet et al. 2019).
Analyses concerning the evolution and genomic variation
of S. aureus from SM could provide information to formulate
surveillance measures that decrease or eliminate this pathogen
and reduce the potential risks to public health. From this per-
spective, the present study aimed to screen the virulence gene
proles and determine the antibiotic resistance of spa type
t605 MSSA strains isolated from cows with chronic SM in
the State of São Paulo, Brazil.
Material and methods
Bacterial strains
A total of 57 viable S. aureus strains were recovered from a
bacterium library in the Milk Quality Research Laboratory
of the University of São Paulo that had been cryopreserved at
−80◦C from two companion studies of SM cases (Gonçalves
et al. 2018,2020) without taking into account the study de-
sign or exclusion criteria prementioned in both studies (e.g.
contralateral quarters).
Briey, six commercial dairy herds in the Midwest area
of São Paulo State, Brazil, were enrolled in the companion
studies during a 9-month sampling period (Feb–Oct 2014)
(Gonçalves et al. 2018,2020). Cow selection (stage 1, three
samplings; timeframe, 6 weeks) was designed to identify cows
with evidence of chronic SM and to disregard cows with pre-
vious episodes of clinical mastitis. All lactating Holstein cows
(n=647) with four functional quarters and milk yield ≥10 kg
day−1per cow were selected. Dairy cows with two out of three
biweekly consecutive SCC testing >200 000 cells mL−1and
culture-positive results were selected for quarter milk sam-
pling. Mammary quarter milk samples (stage 2, three sam-
plings; timeframe, 6 weeks) were collected during the three
subsequent consecutive sampling occasions from all selected
cows (n=117) at intervals of 15 days. A total of 22 cows were
excluded from the study because they lacked the third sam-
pling, had clinical mastitis (n=11), were dried off (n=7), or
were culled from the herd (n=4). A total of 57 S. aureus were
isolated from three biweekly aseptic mammary quarter milk
samples (n=1140 from 95 cows) using bacteriological culture
analyses (Oliver et al. 2004). Of 57 isolates, 26 S. aureus were
isolated in herd 1, 20 were isolated in herd 2, 2 were isolated
in herd 3, and 9 were isolated in herd 4. In two herds,no S. au-
reus was detected. Milk samples with more than one pathogen
detected were not included. Quarters were considered infected
with S. aureus when at least one colony of S. aureus was iso-
lated from 10 μL of milk plated (≥100 CFU mL−1).
The matrix-assisted laser desorption ionization time-of-
ight mass spectrometry (MALDI-TOF MS) method was used
for the microbial identication of S. aureus isolates (Barce-
los et al. 2019). The strains were subjected to DNA extrac-
tion using the ‘InstaGene Matrix’ kit (BIO-RAD) according
to the manufacturer’s instructions. The nuclease gene (nucA)
was amplied to reconrm S. aureus species using the method
described by Sasaki et al. (2010).
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Virulence prole of MSSA 3
Phenotypic characterization of antibiotic resistance
The isolates were subjected to the disk diffusion test us-
ing Mueller–Hinton agar (MHA) plates (OXOID, USA)
with disks impregnated with nine antimicrobial drugs: peni-
cillin (PEN, 10 μg), oxacillin (OXA, 1 μg), cefoxitin (CEF,
30 μg), gentamicin (GEN, 10 μg), tobramycin (TOB, 10 μg),
erythromycin (ERY, 15 μg), tetracycline (TET, 30 μg), clin-
damycin (CLI, 2 μg), and chloramphenicol (CHL, 30 μg; all
from Laborclin, Paraná, Brazil). These antibiotics were se-
lected based on their human and veterinary use. The zones of
inhibition (halos) were measured according to the guidelines
provided by the Clinical and Laboratory Standards Institute
(CLSI 2021).
PCR assays for the detection of
virulence-associated genes
The PCR assays were performed according to the reference
protocols cited in Supplementary Table S1, with modica-
tions. All the 49 virulence genes investigated were those re-
sponsible for enterotoxin production (sea,seb,sed,see,seg,
seh,sei,andsej); resistance to penicillin (blaZ), oxacillin
and cefoxitin (mecAandmecC), gentamicin (aac6’aph2’), to-
bramycin (ant4), erythromycin and clindamycin (ermA, ermB,
ermC, ermF, ermT, msrA, cfr,vgaC, lnuA/A’, lnuB, lsaB, and
lsaE), and tetracycline (tetK, tetL, and tetM); antiseptic (QAC)
resistance (qacA/B and qacC); biolm formation (icaA, icaD,
and bap); and encoding MSCRAMMs (fnbA, fnbB, b,clfA,
clfB, cna,eno,bbp,andebps).
Each PCR was carried out with a nal volume of 25 μL
containing 1X PCR Buffer (Promega Corp., Madison, USA),
0.2 mM of each deoxynucleotide (dNTP, Invitrogen, Carlsbad,
CA, USA), 1.5 to 4 mM MgCl2(25 mM; Promega Corp.) de-
pending on the gene, 0.4 μM of the respective primers (In-
vitrogen, or Sigma, St. Louis, MO, USA; Exxtend, São Paulo,
Brazil), 2.5 U of GoTaq Hot Start Polymerase (Promega Corp.,
Madison, USA), 50 ng of template DNA, and sterilized ultra-
pure Milli-Q water (Merck KGaA, Darmstadt, Germany).
PCR for all reactions was performed in a thermocycler
(Thermo Fisher Scientic, Waltham, MA, USA). The condi-
tions included initial denaturation at 94◦C–95◦C for 2–5 min
for all primers, followed by different amplication cycles (25–
40) of denaturation (94◦C–95◦C for 30 s to 1 min), annealing
(Supplementary Table S1), and extension at 72◦Cfor1min.
The nal extension step was performed at 72◦C for 10 min.
Positive controls from the collection of the Laboratory of
Microbial Toxins at the Department of Food Science and Nu-
trition at UNICAMP (Campinas, Brazil) were included in each
PCR reaction. We used Ultrapure water (Merck KGaA, Darm-
stadt, Germany) as the negative control. PCR products were
analysed by electrophoresis in a 1.5% (w/v) agarose gel with
0.5X TBE buffer (Promega Corp., Madison, WI, USA) and
stained with SYBR® Safe (Invitrogen, Carlsbad, CA, USA).
A 100-bp molecular weight DNA ladder (New England Bi-
olabs, Ipswich, MA, USA) was used to validate the length of
the products and then visualized using a UV Gel Documenta-
tion system (Uvitec, Cambridge, UK).
Molecular typing
The agr system groups were classied based on the hyper-
variable domain of the agr locus (I–IV) through amplica-
tion using the oligonucleotide primers described by Shopsin
et al. (2003). For spa typing, DNA amplication was per-
formed according to the protocol from the ofcial Ridom
website, www.ridom.com. The samples were sequenced using
the Sanger method, and DNA sequencing data were analysed
using the spa typing database to determine the repeat prole
and spa type of each isolate (http://spa.ridom.de/repeats.shtml
and http://spa.ridom.de/spatypes.shtml). Description of PCR
primers used for agr and spa typing are shown in Supplemen-
tary Table S1.
Statistical analyses
Statistical analysis of the genes was performed using
Cochran’s Q test, after multiple comparisons using the Mc-
Nemar (Bonferroni) procedure, using the XLSTAT 2020 soft-
ware for Microsoft Excel® (Microsoft®, WA, USA). Because
of the dichotomous variables, a gene’s presence was equal to
one (1), and its absence was zero. A signicance level of 5%
was considered; therefore, all results with a P-value <0.05
were considered statistically signicant.
Results
All S. aureus isolates were identied at the species level with
ascore>2.0 by MALDI-TOF MS. Regarding the phenotypic
antibiotic resistance, 7% showed no resistance to any antibi-
otics and 93% showed resistance to at least one antibiotic (Ta-
ble 1). Penicillin resistance was the most prevalent (87.7%),
followed by tetracycline (12.3%), oxacillin (10.5%), gentam-
icin (10.5%), and erythromycin (3.5%) resistance. In addi-
tion, 5.3% exhibited intermediate resistance to tobramycin
and 1.8% to erythromycin. Resistance to clindamycin was ob-
served in one isolate (1.7%). Eight antibiotic resistance pro-
les were detected; penicillin (68.4%) was the most abundant,
followed by penicillin and oxacillin (7%).
The results in Table 1show the total genotypic and phe-
notypic data of the 57 strains of S. aureus isolated from all
chronic SM cases. All these data were subjected to statistical
analysis by Cochran’s Q test. Based on the PCR assay, the rel-
ative frequencies of virulence-encoding genes for S. aureus are
shown in Table 2. Our results showed that 98.2% of isolates
contained at least one biolm-forming gene. There was no sig-
nicant difference (P>0.05) in the occurrence of icaA, icaD,
and bap. The most frequent biolm formation gene detected
was icaD, with 94.7% (n=54), followed by bap with 78.9%
(n=45), and icaA with 66.7% (n=38). The most frequent
MSCRAMMS genes detected were b (93%), fnbA (82.5%),
clfA (80.7%), clfB (73.7%), and eno (63.2%). On the other
hand, the less prevalent were cna (7%) and ebps (7%), while
the bbp and fnbB genes were not detected. For enterotoxin
genes, the frequencies were see (64.9%), sed (61.4%), and seh
(3.5%). A total of 47 isolates (82.5%) presented at least one
enterotoxin-encoding gene. Only 10 isolates (17.5%) did not
have any enterotoxin genes. Enterotoxin genes sea,seb,sec,
sec,sei,andsej were undetected.
The highest detection frequency for antibiotic resistance
genes was blaZ (94.7%), followed by aac6’aph2’ (15.8%),
and ant4 (12.3%). The lowest frequency was observed for
lnuA/A’ (1.8%). All isolates resistant to tetracycline had tetL
(10.5%), while the tetKandtetM genes were undetected. No
mecAandmecC (MRSA) positive strains were identied.Only
three isolates (5.26%) had no antibiotic-resistance genes. The
antibiotic resistance genes ermA, ermB, ermC, ermF, ermT,
msrA, cfr,vgaC, lsaB, lsaE, and lnuB were also not detected.
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4Munive Nuñez et al.
Ta b l e 1 . Genotypic and phenotypic prolesaof the 57 strains of S. aureus isolates from bovine SM.
Genotypic data Phenotypic data
Strain
Enterotoxin
genes
Biolm
formation
genes MSCRAMMs genes
Antibiotic
resistance genes agr type spa type
Antibiotic
resistance
262 seh icaDclfA, clfB, cna,fnbA,
eno,b,ebps
blaZagrIII t605 –
296 – icaDclfA, clfB, cna,fnbA,
eno,b,ebps
blaZ, aac6’aph2’agrII t605 PEN, OXA, TET
300 –icaD, bap clfA, clfB, fnbA, eno,
b
blaZ,
aac6’aph2’, tetL
agrII t605 PEN, ERY, CLI
313 –icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
315 –icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
316 see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZ – t605 PEN
345 – icaDclfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
354 – icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
356 –icaA, icaD, bap b blaZagrII t605 PEN
361 sed icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
375 – icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
376 – icaA, icaD, bap – – – t605 –
506 sed icaDclfA, clfB, cna,fnbA,
b,ebps
–agrIII t605 –
613 sed icaDclfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
614 sed icaDclfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN, OXA
615 sed icaA, icaDclfA, fnbA, eno,b blaZagrII t605 PEN
616 sed icaA, icaD, bap –blaZ – t605 PEN
661 – icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
663 sed icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN, OXA
664 sed icaA, icaD, bap clfA, fnbA, eno,b blaZ – t605 PEN
680 see icaA, icaD, bap clfA, fnbA, eno blaZ – t605 PEN
693 sed,see icaA, icaD, bap b blaZ – t605 PEN
697 sed,see icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
715 sed,see icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN, OXA
716 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
1009 sed,see –b blaZ – t605 PEN
1010 sed,see icaD, bap clfA, clfB, fnbA, eno,
b
blaZ – t605 PEN
1011 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
1012 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZ – t605 PEN
1041 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
1045 sed,see icaDclfA, fnbA, eno,b blaZagrII t605 PEN
1061 see icaD, bap clfA, clfB, fnbA, eno,
b
blaZ – t605 PEN
1063 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
1064 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZ – t605 PEN
1074 sed,see icaA, icaDfnbAblaZ, aac6’aph2’ – t605 PEN
1077 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
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Virulence prole of MSSA 5
Ta b l e 1 . Continued
Genotypic data Phenotypic data
Strain
Enterotoxin
genes
Biolm
formation
genes MSCRAMMs genes
Antibiotic
resistance genes agr type spa type
Antibiotic
resistance
1097 sed,see icaA, icaD, bap clfA, clfB, fnbA, b blaZagrII t605 PEN
1100 sed,see icaA, icaD, bap clfA, clfB, fnbA, b blaZ, lnuA/A’ – t605 PEN, ERY∗
1101 sed,see icaA, icaD, bap clfA, clfB, fnbA, b blaZ – t605 PEN
1111 sed,see icaA, icaD, bap clfA, clfB, fnbA, b blaZ – t605 PEN
1177 sed,see icaA, icaD, bap clfA, clfB, fnbA, b blaZ – t605 PEN
1185 sed,see icaD, bap clfA, clfB, fnbA, b blaZ – t605 PEN
1191 sed,see icaA, icaD, bap clfA, clfB, fnbA, b blaZagrII t605 PEN, OXA
1199 sed,see icaA, bap b blaZ – t605 PEN
1219 sed,see icaA, icaD, bap b blaZ, ant4 – t605 PEN
1261 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZ,
aac6’aph2’, tetL
agrII t605 GEN, TET
1269 sed,see icaA, icaD, bap clfA, clfB, fnbA, eno,
b
blaZ – t605 PEN
1286 sed,see icaA, icaD, bap b blaZ, ant4 – t605 GEN, TET
1288 see icaD, bap clfA, clfB, fnbA, b blaZ,
aac6’aph2’, ant4,
tetL
agrII t605 GEN, TET
1349 sed,see icaA, bap b blaZ,
aac6’aph2’, tetL
agrII t605 PEN, TOB∗,
GEN, TET
1365 see icaA, icaD, bap clfA, clfB, fnbA, b blaZ, ant4 – t605 PEN, OXA, ERY
1388 see icaA, icaD, bap b blaZ,
aac6’aph2’, ant4,
tetL
agrII t605 PEN, TOB∗,
GEN, TET
1393 see icaD, bap clfA, clfB, fnbA, eno,
b
blaZ,
aac6’aph2’, ant4,
tetL
agrII t605 PEN, TOB∗,
GEN, TET
1395 see icaD, bap clfA, clfB, fnbA, eno,
b
blaZ,
aac6’aph2’, ant4
agrII t605 PEN
1423 seh icaDclfA, clfB, cna,fnbA,
eno,b,ebps
–agrIII t605 –
1425 see icaA, bap clfA, clfB, fnbA, eno,
b
blaZagrII t605 PEN
1427 see icaD, bap clfA, clfB, fnbA, eno,
b
blaZ, ant4agrII t605 PEN
aCEF =cefoxitin, CLI =clindamycin, ERY =erythromycin, GEN =gentamicin, PEN =penicillin, OXA =oxacillin, TET =tetracycline, TOB =tobramycin;
–=gene not detected.
∗Indicates intermediate resistance.
Ta b l e 2 . Frequency of S. aureus genes detected from bovine SM using the Cochran’s Q test.
Genes No. of positive isolates Frequency, %
Enterotoxins seh 23.5
a
sed 35 61.4ab, b
see 37 64.9ab, b
Biolm formation icaA 38 66.7ab, b
bap 45 78.9ab, b
icaD 54 94.7b
MSCRAMMs cna 47
a
ebps 47
a
eno 36 63.2ab, b
clfB 42 73.7ab, b
clfA 46 80.7ab, b
fnbA 47 82.5ab, b
b 53 93b
Antibiotic resistance lnuA/A’ 1 1.8a
tetL 6 10.5a
ant4 7 12.3a
aac6’aph2’ 9 15.8a
blaZ 54 94.7b
a,bStatistically signicant difference (P<0.05) indicated by different letters in the same column.
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6Munive Nuñez et al.
Ta b l e 3 . Different gene combinations of S. aureus isolates from bovine SM.
Enterotoxin genes MSCRAMMs genes Antibiotic resistance genes Biolm formation genes
Gene Patterns
N◦of isolates
(%) Gene Patterns
N◦of isolates
(%) Gene Patterns
N◦of isolates
(%) Gene Patterns
N◦of isolates
(%)
sed,see 27 (47.4) clfA, clfB, fnbA, eno,
b
29 (50.9) blaZ 40 (70.2) icaA, icaD, bap 33 (57.9)
clfA, clfB, fnbA, b 9 (15.8)
see 10 (17.5) b 8 (14.0) blaZ, aac6’aph2’ 2 (3.5) icaD, bap 10 (17.5)
clfA,clfB, cna, fnbA,
eno, b, ebps
3 (5.3) blaZ, aac6’aph2’,
tetL
3 (5.3)
sed 8 (14.0) clfA, fnbA, eno,b 3 (5.3) blaZ, ant4 4 (7.0) icaD 8 (14.0)
clfA, clfB, cna,fnbA,
b,ebps
1 (1.8) blaZ, aac6’aph2’,
ant4, tetL
3 (5.3)
seh 2 (3.5) clfA,fnbA,eno 1 (1.8) blaZ, lnuA/A’ 1 (1.8) icaA, bap 3 (5.3)
Gene not
detected
10 (17.5) fnba 1 (1.8) blaZ, aac6’aph2’,
ant4
1 (1.8) icaA, icaD 2 (3.5)
Gene not detected 2 (3.5) Gene not detected 3 (5.3) Gene not detected 1 (1.8)
Total 100 100 100 100
Regarding the prevalence of antiseptic (QAC) resistance genes,
it is indicated that no S. aureus carried the QAC resistance
genes (qacA/B and qacC).
Concerning the gene patterns with different gene combi-
nations, there was only one type of combination for entero-
toxin genes (sed and see), representing 47.4% of the isolates.
MSCRAMMs patterns showed that the most prevalent com-
bination for these genes was clfA, clfB, fnbA, eno,b (29
isolates, 50.9%), followed by the combination of clfA, clfB,
fnbA, b (9 isolates, 15.8%). Thus, we identied that 3.5%
of the isolates did not present MSCRAMMs genes. Further-
more, high genetic diversity was detected in three isolates
(5.3%), which showed seven out of nine genes (clfA, clfB, cna,
fnbA, fnbB, eno,b, and ebps). For biolm formation patterns,
the most prevalent combination was icaA,icaD, bap,andb
(33 isolates, 57.9%), followed by icaDandbap (10 isolates,
17.5%). Table 3shows the gene combinations of the S. aureus
isolates.
All isolates belonged to the spa type t605 (100%). Through
agr typing, we determined that agr type II was the dominant
type (n=32, 56.14%) and that 5.26% (n=3) were agr type
III. The agr type I and IV were undetected, and 22 isolates
(38.6%) were non-typeable.
Discussion
Molecular studies are fundamental because they pro-
vide epidemiological information and knowledge regarding
pathogenicity mechanisms, making it possible to understand
the association of these mechanisms with the microorganism
virulence prole (Veh et al. 2015, Ren et al. 2020). Our study
demonstrated different virulence proles for S. aureus strains
involved in chronic SM. Knowledge regarding the genetic di-
versity and antimicrobial resistance of these S. aureus strains
isolated from chronic subclinically infected cows can help im-
plement control measures for this disease in dairy herds. The
results conrmed that an endemic type is found in Brazilian
dairy herds and carries virulence genes, which may explain
the difculty in eradicating and controlling chronic SM. Our
ndings improved the comprehension of the pathogenesis of
chronic S. aureus by tracking important virulence genes in-
volved in mastitis and determining genotypic variations of the
strains.
The pathogenesis of chronic SM caused by S. aureus begins
with adherence, subsequent biolm production, and bacterial
adhesion to the epithelium of mammary glands. It is inuenced
by the polysaccharide intercellular adhesion (PIA), encoded by
the icaADBC, an intercellular adhesion (ica) operon, which
contains icaAandicaB(N-acteylglucosamine transferases),
icaC (predicted exporter), and icaD (deacetylase) genes (Miao
et al. 2019, Marques et al. 2021, Silva et al. 2022). From these
in S. aureus,icaAandicaD are important factors in promot-
ing intercellular adhesion and forming multilayered biolms
associated with biolm formation, icaC seems to be involved
in the transport of icaAandicaD synthesized oligomers across
the cell membrane. Further, the absence of the icaBgenemay
reduce the efciency in binding to the bacterial cell surface,
leading to weak biolm production (Miao et al. 2019, Silva
et al. 2021). Studies assessing icaAandicaD on milk sam-
ples of clinical or SM cases reported that these genes were
present at high frequencies (each 86.6%) (Aslanta¸sandDemir
2016). Our results showed that 98.2% of strains present genes
involved in biolm formation, similar to Vasudevan et al.
(2003), who found icaAandicaD genes in 35 (100%) S. au-
reus isolates from bovine mastitis cases. Prior studies showed
that the ability to form a biolm in S. aureus strains depends
on the expression of icaD (Namvar et al. 2013). In our study,
this gene was detected in high frequency (94.7%), followed by
the icaA (66.7%).
The Bap protein plays a crucial role in the initial attach-
ment and recruitment of S. aureus. Many studies showed
that the bap gene was present in SM mastitis at a low fre-
quency (Zuniga et al. 2015,Aslanta¸sandDemir2016,Fe-
lipe et al. 2017) or was not present (Ren et al. 2020). For
both occurrences, biolm formation may be attributed to the
ica-dependent mechanism, which encodes the PIA production
(Marques et al. 2021). However, in this study, the bap gene
was detected at a high rate (78.9%), which is consistent with
the results reported by Salimena et al. (2016), who determined
that this gene was present in 95.6% of the isolates from the
milk of dairy herds in Brazil, which is the highest prevalence
rate reported to date.
There is an association between the bap,icaA, and icaD
genes and the genes encoding for MSCRAMMs. They provide
a great capacity for adhesion and biolm production, which
is an important characteristic of the persistence of S. aureus
(Castilho et al. 2017). MSCRAMMs are proteins found on the
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Virulence prole of MSSA 7
surface of the bacteria that allow for the initial addition and
colonization of S. aureus and the intracellular internalization
and initiation of biolm. These adhesins play a vital role in the
onset of S. aureus infection (Klein et al. 2012). Their presence
may explain the current difculties in controlling and erad-
icating chronic SM caused by S. aureus in dairy herds. Our
ndings show that biolm and MSCRAMMs genes are im-
portant virulence factors that can help with the colonization
and persistence of S. aureus.
Some adhesins are important virulence factors in binding
host cells, colonization, and invasion, such as bronectin-
binding proteins (fnbA and fnbB) and clumping factors (clfA
and clfB)(Aslanta¸sandDemir2016, Zhang et al. 2018).
Zhang et al. (2018) revealed that the most frequent adhesion
genes were clfA, clfB, fnbA, and fnbB, showing that >85% of
S. aureus isolated from bovine mastitis cases in China pre-
sented these genes. Similar to our ndings, Zaatout et al.
(2019) found a high diversity of MSCRAMMs genes (12 out
of 14 genes), such as clfA, clfB, ebpS,eno,fnbA, fnbB, and
bbp. This diversity in virulence factors, and their different
combinations, can cause changes in the level of pathogenic-
ity and the spread of infections among dairy cows inside or
across herds (Veh et al. 2015). Furthermore, Gogoi-Tiwari et
al. (2015) found a difference in the rates of fnbAandfnbB
genes. They reported that 54.5% of isolates present the fnbA
gene, while fnbB was found at a low frequency (1.3%) among
Australian bovine S. aureus isolates. Another study evaluated
the aforementioned genes of S. aureus isolated from SM cases
and showed a difference in rates with higher frequency in fnbA
(72.6%) compared to fnbB (5.7%) (Zuniga et al. 2015). In an-
other study with isolates from bovine mastitis in Argentinean
dairy farms, clfAandclfB genes were present at high rates,
comparable to our results (Felipe et al. 2017). Kumar et al.
(2011) and Zuniga et al. (2015) found the eno gene (gene en-
coding the laminin-binding protein) in 100% and 82.1% of
S. aureus isolated from clinical and SM, respectively. The eno
gene was also frequently detected in the isolates of our study
(63.2%). On the other hand, we detected the cna gene at a
low frequency (7%), and similar studies have demonstrated a
lower percentage of this gene in S. aureus isolates from SM
cases (Klein et al. 2012,Xuetal.2015).
The major enterotoxin gene detected in our investigation
was the see gene. Similarly, Fursova et al. (2018) reported that
this gene was present in S. aureus strains from cows with high
SM frequency (46.6%). Furthermore, another abundant en-
terotoxin gene was sed, detected in 35 of 57 (61.4%) isolates.
Previous studies reported the gene encoding for SED, which
is a common enterotoxin produced by S. aureus isolated from
dairy products (Vitale et al. 2015) and cows with clinical and
SM (Costa et al. 2018, Fursova et al. 2018, Grispoldi et al.
2019, Ren et al. 2020). Another study indicated variable rates
of enterotoxigenicity for subclinical mastitic S. aureus isolates
(Rall et al. 2014). Liu et al. (2014) showed that SEH toxin
plays an important role in the mastitis process because it can
induce apoptosis of the bovine mammary epithelial cells. Our
results showed that the seh gene was present but not domi-
nant; likewise, Cândido et al. (2020) found this gene in 13.1%
of isolates from dairy products. Another study found the seh
gene from SM cases in high frequency (41.5%) (Ren et al.
2020). None of the isolates harboured the sec gene. Based on
previous studies, this gene was rarely present in bovine mas-
titis, as reported by Cândido et al. (2020). We did not detect
sea genes comparable to previous studies of S. aureus from
dairy cows with clinical or SM (Costa et al. 2018, Zhang et
al. 2018). Therefore, our results suggest that S. aureus isolated
from milk samples of chronic subclinically infected cows has
the enterotoxigenic potential of food poisoning, which is wor-
risome since SM may not be detected visually but can be easily
distributed for human consumption (Hussein et al. 2018).
Due to the low recovery rates and the tendency to evolve
into chronic infection processes of longer duration, treatment
for cows subclinically infected with S. aureus is not commonly
adopted; however, antimicrobial treatment could result in a
higher bacteriological cure rate in young cows that had rst
contact with this pathogen (Mcdougall et al. 2022). Then
again, the nonassertive antibiotic might cause variability in
S. aureus resistance proles, as evidenced by the results ob-
tained in this study. Furthermore, there is a risk of exchang-
ing clones among humans and animals since they adapt to
the host and acquire virulence factors to colonize and gener-
ate new infections (Veh et al. 2015). In our study, S. aureus
isolates harboured the β-lactamase gene blaZ, indicative of
penicillin resistance, at an alarming frequency (94.7%). How-
ever, phenotypically we found that resistance to penicillin was
87.72%. These results are in line with previous studies from
different parts of the world. In China, Xu et al. (2015)and
Qu et al. (2019) detected a high prevalence of the blaZ gene,
95% and 82.1%, respectively, in S. aureus strains from dairy
cows with SM. Aslanta¸sandDemir(2016) reported a higher
rate (100%) of blaZ among S. aureus from mastitis cases in
Turkey. In addition, Zaatout et al. (2019) showed their pres-
ence in all nine S. aureus strains from dairies in Algeria. Some
studies corroborate the blaZ gene in dairies in Brazil, with a
prevalence of 25.3% and 40% (Soares et al. 2017, Abreu et al.
2021). Our results revealed a divergence between the pheno-
typic and genotypic prevalence of blaZ, which is in agreement
with earlier studies on bovine mastitis. Research conducted by
Martini et al. (2017) found 88% of blaZ-positive isolates and
83.3% expressed phenotypical resistance to penicillin; like-
wise, Neelam et al. (2022) showed that 92.73% harboured
the blaZ gene and 83.64% were resistant to penicillin. These
results demonstrate that gene detection does not necessarily
indicate that the gene has a detectable phenotype. Our nd-
ings are of great concern to bovine health, given the fact that
β-lactams are commonly used antibiotics to treat mastitis dis-
eases (Aslanta¸sandDemir2016). The extensive use of these
antibiotics results in a bacterial adaptation by developing re-
sistance to penicillin amongst S. aureus isolates (Abreu et al.
2021). Therefore, the alarming prevalence of the blaZgenein
the current study is probably an indicator of the intensive use
of penicillin to treat bovine mastitis in herds.
Remarkably, our study did not identify mecAandmecCre-
sistance genes among the isolates, which would help to classify
these strains as MSSA. Similar studies with no positive isolates
for the mecAandmecC genes in raw milk and dairy products
emphasized the need for signicant attention towards MSSA;
since they may be involved in the establishment and evolu-
tion of MRSA lineages, which may be derived through the
acquisition of mecAormecC genes (Silva et al. 2013, Bon-
saglia et al. 2018). In a previous study conducted in Michi-
gan, the USA, very few MRSA (0.6%) were isolated from the
milk of cows suspected of having an intramammary infec-
tion (Erskine et al. 2002). MRSA was found in <1% of bulk
tank milk samples collected from smaller organic and con-
ventional dairy herds (n=288 farms) in New York, Wiscon-
sin, and Oregon (Cicconi-Hogan et al. 2014) and 4% from
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8Munive Nuñez et al.
50 Minnesota farms (Haran et al. 2012). Other studies
showed that MRSA was detected in raw milk and dairy prod-
ucts (Aslanta¸sandDemir2016, Rodrigues et al. 2017a,b,Ren
et al. 2020, Abreu et al. 2021). Previous studies have demon-
strated that MRSA strains hold different gene proles, with
more enterotoxin genes (Papadopoulos et al. 2019), and are
more resistant to antimicrobials (Zayda et al. 2020)when
compared to MSSA strains. Therefore, to control the con-
tagious MSSA associated with mastitis in dairy herds, pro-
cedures must be implemented to prevent the spread of the
pathogen (i.e. considering all milk production stages), includ-
ing an optimal milking routine, teat disinfection, and segrega-
tion of infected cows.
In contrast, phenotypic analyses determined that six strains
were resistant to oxacillin. The absence of the mec gene and
phenotypic resistance to oxacillin may be associated with the
excessive production of the β-lactamase by the blaZgene
(Scholtzek et al. 2019). It should be noted that all strains that
expressed resistance to oxacillin carried the blaZ gene, which
may explain the phenotypic resistance to oxacillin. Another
study suggests that the appearance of this type of strain,
without the mec gene, is potentially attributed to previously
unknown selection pressure, such as the use of antiseptics
or disinfectants (Speck et al. 2020). Another cow-level study
found zero proportion of resistance to oxacillin of 357
bovine mastitis-causing S. aureus from 24 farms in North
Carolina and Virginia farms (Anderson et al. 2006). Further
investigations targeting phenotypic and genotypic methicillin
resistance mechanisms should be carried out to increase our
understanding of the genetic basis of antimicrobial resistance
in these strains.
We detected the aac6’aph2’ gene associated with gentam-
icin resistance in nine strains, but only ve were identied with
the resistance phenotype. Although one strain had phenotypic
resistance to gentamicin, it did not present the resistance geno-
type. These ndings are consistent with other studies from
dairies where the authors found strains with these character-
istics (Abreu et al. 2021, Szczuka et al. 2022). Concerning the
ant4 gene responsible for resistance to tobramycin, three of
the eight strains that possessed the gene showed intermedi-
ate phenotypic resistance. The other ve did not express the
phenotype. It is important to emphasize the alarming problem
that could cause the rapid worldwide spread of these antibi-
otic resistance genes amongst the huge diversity of S. aureus
isolates. It highlights the crucial point of using these antimi-
crobials that are commonly applied for mastitis treatment in
Brazilian dairy herds.
Little is known about QAC resistance genes in S. aureus
from SM cases. However, evaluating the potential resistance
of S. aureus strains to QAC helps to use and control the ef-
fectiveness of these disinfectants. Unlike other studies that re-
ported the presence of QAC-resistant genes in staphylococcal
strains from bovines (Ergun et al. 2017, Kotb and Gafer 2020)
and goat herds (Bjorland et al. 2005), we did not observe the
presence of these genes, which is supported by other ndings
similar to our study (Qu et al. 2019, Abreu et al. 2021). Zero
detection of the QAC-resistant genes affords us more possibil-
ities for controlling SM cases caused by S. aureus with disin-
fectants in Brazilian dairy cattle.
In our research, agr II was the most frequent type, followed
by agr III. None of the isolates tested was agr type I or IV,
and 22 were negative for all known types. These ndings are
important from an epidemiological point of view since other
studies conducted in São Paulo State, Brazil, reported simi-
larly the prevalence of agr type II. For instance, research per-
formed by Bonsaglia et al. (2018) regarding S. aureus isolates
from cows with SM conrmed a high prevalence of agr type II
(47.7%), followed by type III (20%); likewise, agr type IV was
not detected. Cândido et al. (2020) also reported a high preva-
lence of agr type II (29%) and III (44.7%) in S. aureus isolates
from organic and conventional dairies. Silva et al. (2013) iden-
tied agr types II (41.1%) and III (48.2%) in MSSA isolates
from dairy cows with mastitis. Rodrigues et al. (2017b) iden-
tied agr type II in 88.1% of isolates of Staphylococcus spp.
from mastitic milk. Other studies reported the presence of agr
type II in S. aureus isolates from dairies (Kumar et al. 2011,
Schmidt et al. 2017,Soaresetal.2017). Our ndings and pre-
vious studies infer that agr type II could be well adapted to
dairy environments in São Paulo State, Brazil, and may cause
chronic SM.
By molecular typing, we have shown that we only detected
the presence of spa type t605; this spa type consists of two
spa repeat successions (r07–r23). The Ridom spa server net-
work database shows that t605 has a relative global fre-
quency (0.09%) (http://spaserver.ridom.de—Data collected in
January 2021). A study performed in Minas Gerais, Brazil,
showed that the udder is the most important reservoir for S.
aureus t605 and emphasized that t605 is widespread in dairy
herds and can be responsible for the persistence of bovine
intramammary infections (Santos et al. 2020). Furthermore,
other studies conducted in São Paulo State, Brazil, such as the
study conducted by Bonsaglia et al. (2018), found a high fre-
quency of spa type t605 (70.5%) in S. aureus isolates from
cows with SM. Likewise, Silva et al. (2013) observed a pre-
dominance of spa type t605 (37.5%) in milk from cows sub-
clinically infected. This spa type was also discovered in high
frequency (83.3%) in Staphylococcus spp. from milk samples
with mastitis (Rodrigues et al. 2017a), but a low frequency
(32.9%) was reported in samples from dairies (Rodrigues et
al. 2017b). The high prevalence of spa type t605 suggests that
these strains are well adapted to bovine herds in Brazil, indi-
cating the presence of an endemic S. aureus widely dissemi-
nated, which can invade and remain in the bovine mammary
cells, causing chronic SM. These ndings corroborate what
other studies have reported by showing the persistence of spa
type t605 (Veh et al. 2015, Cândido et al. 2020).
Some limitations of the study should be noted, such as the
relatively small sample size that may not represent the entire
population of S. aureus involved in bovine chronic SM cases.
In addition, despite covering a broad spectrum of virulence
factors, research on virulence genes, such as icaBandicaCof
the icaADBC loci, were not included in this study. However,
the results found here follow the trends observed in other
studies.
We determined a great pathogenicity potential among
MSSA. Our results suggest that S. aureus isolated from milk
samples of chronic subclinically infected cows has the en-
terotoxigenic potential for food poisoning. The most pre-
dominant strains for enterotoxin genes were see and sed.A
unique endemic spa type (t605) was detected in the Brazil-
ian dairy herds evaluated. Our ndings showed that biolm
and MSCRAMMs genes are important virulence factors that
can help with the colonization and persistence of S. au-
reus. Clearly, agr type II is well adapted to dairy environ-
ments, increasing the risk of causing the disease. Although
no mecAandmecC (MRSA) positive strains were identied,
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Virulence prole of MSSA 9
we observed high phenotypic resistance levels to at least one
antibiotic class, mainly in β-lactams, which are widely used in
dairy herds. Further studies are needed to provide detailed in-
sights to guide clinical decisions around disease management
and develop effective preventive measures to control S. aureus
causing chronic SM.
Supplementary data
Supplementary data is available at JAMBIO Journal online.
Conict of interest
The authors declare no conict of interest.
Funding
This work was supported by a Ph.D. fellowship from the
Brazilian government through National Council for Scien-
tic and Technological Development (CNPq—grant numbers
141320/2018–4).
Author contributions
Karen Vanessa Munive Nuñez (Conceptualization, Formal
analysis, Funding acquisition, Investigation, Methodology, Vi-
sualization, Writing – original draft), Anderson Clayton da
Silva Abreu (Investigation, Methodology, Writing – review &
editing), Juliano Leonel Gonçalves (Investigation, Resources,
Writing – review & editing), Marcos Veiga dos Santos (Inves-
tigation, Resources, Writing – review & editing), Liliana de
Oliveira Rocha (Investigation, Writing – review & editing),
and Nathália Cristina Cirone Silva (Conceptualization, Fund-
ing acquisition, Resources, Supervision, Validation, Writing –
review & editing)
Data availability
The authors conrm the data supporting the ndings of this
study are available within the article.
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