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Article

Prevalence and Diversity of Staphylococcus aureus in Bulk Tank Milk from Community-Based Alpine Dairy Pastures in Tyrol, Austria

by
Nasrin Ramezanigardaloud
1,
Igor Loncaric
1,
Patrick Mikuni-Mester
2,
Masoumeh Alinaghi
1,
Monika Ehling-Schulz
1,
Johannes Lorenz Khol
3 and
Tom Grunert
1,*
1
Centre of Pathobiology, Department of Biological Sciences and Pathobiology, University of Veterinary Medicine, 1210 Vienna, Austria
2
Centre for Food Science and Veterinary Public Health, Clinical Department for Farm Animals and Food System Science, University of Veterinary Medicine, 1210 Vienna, Austria
3
VetmedRegio Tyrol, Clinical Centre for Ruminant and Camelid Medicine, University of Veterinary Medicine, 1210 Vienna, Austria
*
Author to whom correspondence should be addressed.
Animals 2025, 15(14), 2153; https://doi.org/10.3390/ani15142153
Submission received: 22 June 2025 / Revised: 11 July 2025 / Accepted: 15 July 2025 / Published: 21 July 2025
(This article belongs to the Section Animal Products)
Browse Figures
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Simple Summary

Healthy dairy cows are crucial for producing high-quality milk, ensuring food safety, and promoting sustainable milk production. However, udder infections (bovine mastitis), which are frequently caused by the Gram-positive bacterial pathogen Staphylococcus (S.) aureus, are among the most common diseases in dairy cows. These infections are costly for farmers, reduce milk production, and raise concerns for cow health and milk safety. Because S. aureus spreads from cow to cow, it poses a particular challenge to community-based Alpine dairy pastures. Sharing grazing areas, stables, and milking infrastructure among animals from different farms raises the risk of cross-infection. Here, we collected bulk tank milk samples from 156 community-based Alpine dairy pastures in Tyrol, Austria, throughout the 2023 Alpine season to investigate the prevalence and genetic diversity of S. aureus circulating in these farms. We found at least one instance of S. aureus in 60% of these pastures. Furthermore, genotyping revealed that a specific subtype of S. aureus was widespread, detected in approximately 35% of the pastures, highlighting its significant presence in the European Alpine region. These findings emphasise the need for effective control measures to prevent bovine mastitis caused by S. aureus, which is vital for ensuring animal health, sustainable dairy farming, and producing safe food.

Abstract

Staphylococcus aureus frequently causes intramammary infections in dairy cows (bovine mastitis), which impair animal welfare, milk yield, and food safety. This study determined the prevalence and genetic diversity of S. aureus in bulk tank milk (BTM) samples from community-based Alpine dairy pastures in Tyrol, a major milk-producing region in Austria. Throughout the 2023 Alpine season (May–September), 60.3% (94/156) of BTM samples tested positive for S. aureus at least once over the course of up to four samplings. A total of 140 isolates collected from the 94 S. aureus-positive community-based Alpine dairy pastures revealed 33 distinct spa types, with t2953 (n = 33), t529 (n = 12), t267 (n = 11), and t024 (n = 10) being the most common. Selected isolates representing the different spa types were characterised by DNA microarray-based genotyping, multi-locus sequence typing (MLST), and antimicrobial susceptibility testing. Isolates with spa types associated with bovine-adapted CC8 (CC8bov/GTB) were identified as the most common subtype, being detected in BTM samples from 35.3% (55/156) of the pastures. This emphasises the high prevalence of this subtype in dairy herds across European Alpine countries. Other common bovine-associated subtypes were also detected, including CC97, CC151, and CC479. While antimicrobial resistance was rare, enterotoxin-producing genes were detected in all CC8bov-associated spa types. Overall, these findings underscore the importance of rigorous hygiene practices in dairy farming, particularly in community-based Alpine dairy pastures, where the risk of transmission is particularly high. It also emphasises the need for continued surveillance and subtyping to improve animal health, ensure food safety, and promote sustainable milk production.

1. Introduction

Staphylococcus (S.) aureus presents a significant threat to human and animal health. In dairy cows, S. aureus is a primary cause of intramammary infections (IMIs) globally, negatively impacting animal welfare, food safety, and dairy production. Bovine IMI caused by S. aureus is predominantly characterised by subclinical, chronic persistent infections that are difficult to treat. S. aureus strains can be grouped into clonal complexes (CCs) based on their phylogenetic relationship, which may differ in their ability to adapt to the host. While some CCs demonstrate strict host specificity, others display remarkable adaptability across species barriers [1]. Among bovine-associated lineages, CC151 and CC97 are the most widely distributed globally [2]. CC151 is an archetypal bovine-specific lineage, whereas CC97 exhibits a broader host range, primarily being isolated from bovine but also from human hosts. In the European Alpine region, the bovine-adapted CC8 variant (CC8bov), also referred to as Genotype B (GTB), has been frequently isolated from dairy cows with IMI [3]. CC8 exemplifies interspecies transmission, as evidenced by a recent human-to-bovine host jump event [4,5]. Both lineages, CC97 and CC8bov, highlight the significance of S. aureus as a zoonotic pathogen.
Phylogenetic grouping into CCs has been correlated with key phenotypic traits, including biofilm formation capacity, cellular internalisation capabilities, transmission dynamics, and antimicrobial resistance profiles. To improve the prevention, detection, and treatment of S. aureus IMI, subtyping techniques such as genotyping could potentially help farmers and veterinarians implement targeted control and treatment strategies.
In Austria, while the bovine-associated lineages CC151 and CC97 have been detected throughout the country, previous studies suggest that CC8bov is primarily present in the western Alpine region [6,7]. In Vorarlberg, Austria’s westernmost province, an analysis of bulk tank milk (BTM) samples revealed that 10 out of 18 dairy farms were positive for S. aureus, with CC8bov being present in half of the positive cases [6]. Studies in Swiss dairy farms also reported a high prevalence of CC8bov, with cow prevalence reaching up to 87% (median 47%) and herd prevalence at 37% [8,9,10]. CC8bov is considered highly contagious, with its transmission largely being attributed to cow movements between herds and the sharing of milking equipment within the same herds [11,12]. This poses a particular challenge for Alpine farming systems, where animals from different farms are commingled on pastures, sharing grazing areas, stables, and milking infrastructure, which increases the risk of transmission. Moreover, CC8bov can enter the dairy production chain, especially raw milk products, posing a potential health risk to consumers due to its secretion of heat-stable enterotoxins [6,13].
In this study, we examined the prevalence and diversity of S. aureus in BTM samples from community-based pastures located in the western Austrian Alpine region, in the federal province of Tyrol. Tyrol accounts for the largest proportion of milking pastures (53.7%, 2018) and Alpine dairy cows (62%, 2018) in Austria [14]. Investigating the prevalence and diversity of S. aureus, particularly the CC8bov variant, is essential for improving animal welfare and ensuring consumer safety in the context of dairy production.

2. Materials and Methods

2.1. Sampling

During the summer season in the Alpine region of western Austria, including Tyrol, dairy farming practices incorporate a communal grazing system where dairy cows from several herds are brought together on high-altitude common pastures. These community-based pastures provide shared access to grazing areas, stables, and milking infrastructure. As part of the routine monitoring programme of the Tyrol Milch dairy, all shared pastures that host dairy cows from at least two participating farms and deliver milk to the facility in Wörgl were examined (n = 163). The selection of these pastures was not influenced by any prior information, such as clinical history and herd status. Traditionally, the summer grazing period extends from May/June to late August to mid-September, depending on weather conditions. While the initial plan outlined three sampling time points (beginning, middle, and end of the season), unfavourable weather conditions in 2023 delayed the transfer of livestock to the high-altitude communal pastures. This delay necessitated the addition of an extra sampling point between the start and midpoint of the season. In total, we collected 465 bulk tank milk (BTM) samples from 163 pastures across four time points between late May and early September 2023, as follows: first sampling—30–31 May; second sampling—4–5 July; third sampling—9–10 August; and fourth sampling—6–7 September. Most BTM samples were collected from pastures in replicates with varying sampling frequencies, as follows: 26.9% (n = 42) were sampled four times from June to September 2023, 48.1% (n = 75) were samples three times, 21.2% (n = 33) were samples twice, and 3.9% (n = 6) were sampled once.

2.2. Bacterial Isolation and Identification

Samples were transported to the laboratory in sterile tubes maintained at 4 °C. Because many samples were collected on the respective sampling dates, they were stored at −20 °C pending analysis. Bacterial enrichment was conducted using Matrix-Lysis, a sample preparation method recognised for its simplicity, reliability, and cost-effectiveness in detecting pathogenic bacteria in food matrices [15]. Briefly, 20 mL of milk was combined with lysis buffer (comprising 1 M Tricin pH 7.4 and 1 M MgCl2) to a final volume of 40 mL. The mixture was homogenised by vigorous shaking and incubated at room temperature for 15 min. Subsequently, samples were centrifuged at 32,200 × g for 30 min, and the remaining pellet was resuspended in 1000 µL PBS. A 100 µL aliquot was used to culture viable bacteria on Columbia Nalidixic Acid agar (BD Columbia CNA Agar with 5% Sheep Blood, Improved II; C = colistin, n = nalidixic acid, A = aztreonam) at 37 °C for 18–24 h. Up to four presumptive, morphologically distinct staphylococci colonies were subcultured onto mannitol salt agar, identified using matrix-assisted laser desorption ionisation–time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen, Germany), and S. aureus was confirmed via the detection of the nuc gene [16].

2.3. Molecular Subtyping and Microarray

All S. aureus isolates were genotyped by spa typing [17]. Sample preparation and spa sequence typing were performed as previously described [18]. For spa typing, the polymorphic X-region of the protein A (spa) was amplified and sequenced according to the Ridom Spa Server protocol (https://spa.ridom.de/, last accessed on 25.01.2025). Spa types were determined using Ridom SeqSphere + Software v8.4 (Ridom, Münster, Germany) and the Ridom SpaServer website (http://www.spaserver.ridom.de, last accessed on 25.04.2024). One isolate per detected spa type (n = 33) was chosen randomly and subjected to DNA microarray-based technology (INTER-ARRAY Genotyping Kit S. aureus, Bad Langensalza, Germany) to detect antimicrobial resistance and virulence genes [19], as well as being subjected to multi-locus sequence typing (MLST) [20] to identify the respective ST type and clonal complex (CC). The visualisation of the DNA microarray data was performed using a SplitsTree4 phylogenetic network tool [21]. In addition, to gain an overview of the distribution of CCs across community-based Alpine dairy pastures, we inferred all isolates of a specific spa type as belonging to the same CC, based on the strong, established correlations between known spa types and specific MLST-defined CCs within the S. aureus population structure [19,22,23].

2.4. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing (AST) was conducted using the agar disc diffusion test according to the Clinical and Laboratory Standards Institute (CLSI M100, 2025), testing for the following antimicrobial agents: penicillin (PEN 10 units), cefoxitin (FOX 30 μg), ciprofloxacin (CIP 5 μg), gentamicin (GEN 10 μg), tetracycline (TET 30 μg), erythromycin (ERY 15 μg), clindamycin (CLI 2 μg), chloramphenicol (CHL 30 μg), trimethoprim-sulfamethoxazole (SXT 1.25/23.75 μg), nitrofurantoin (NIT 300 μg), rifampicin (RIF 5 μg), and linezolid (LZD 30 μg) (Beckton Dickinson (BD); Heidelberg, Germany). S. aureus ATCC® 25,923 was used as a quality control strain.

2.5. Moran’s I Spatial Autocorrelation

Spatial analysis was performed on S. aureus prevalence data to assess the spatial autocorrelation across the study area. A data point was classified as positive if S. aureus was detected in BTM at least once during the season. The geographic location of each pasture was based on latitude and longitude coordinates. Global and Local Moran’s I statistics were calculated in Python (v3.11) using the GeoPandas v1.0.1, PySAL’s ESDA v2.7.0, libpysal v4.12.1, and Matplotlib libraries v3.8.4. The k-nearest neighbours (k = 4) approach was used to define the spatial weights matrix with row standardisation. The statistical significance was assessed by Monte Carlo permutation with 999 simulations. Global Moran’s I provided a single measure of spatial dependence across the study area, while the use of the Local Moran’s I is important to identify patterns of spatial association. A Moran’s I cluster map highlights areas of high or low spatial autocorrelation based on different categories (e.g., clusters of high values, clusters of low values, etc.) [24]. The Local Moran’s I cluster map categorises observations into four types, as follows: High–High (HH)—areas with high presence surrounded by other high presence (hot spot); Low–Low (LL)—areas with low presence surrounded by other low presence (cold spot); High–Low (HL)—areas with high presence surrounded by low presence (spatial outliers, hot spot in a cold region); Low–High (LH)—areas with low presence surrounded by high presence (spatial outliers, cold spot in a hot region).

3. Results

3.1. Prevalence of S. aureus in BTM Samples of Pastures

To assess the presence of S. aureus at the farm level, we analysed 156 community-based Alpine dairy pastures from June to September 2023. The median herd size was 34 dairy cows per pasture, with a range of 9 to 133 cows (Figure 1a), totalling around 5500 dairy cows across all participating pastures. Each community-based Alpine dairy pasture received dairy cows from a median of 3 farms (ranging from 2 to 20, Figure 1b).
Throughout the 2023 season, 60.3% of the pastures (94 out of 156) had at least one BTM sample test positive for S. aureus. The first sampling detected 34.9% of Alpine dairy pastures to be positive (30 out of 86 samples), the second sampling detected 31.7% (46 out of 145 samples), the third sampling detected 33.8% (42 out of 124 samples), and the fourth sampling detected 17.3% (19 out of 110 samples). Notably, a lower percentage of S. aureus-positive BTM samples was detected on the last sampling date, despite a similar total sample count.
Next, we used Moran’s I analysis to statistically assess if the spatial distribution of pastures with positive S. aureus BTM samples was random or showed clustering. The global Moran’s I indicated a weak but statistically significant spatial autocorrelation in the data (I = 0.0912, p-value = 0.034). The Moran’s I cluster map identified geographic areas where the spatial clustering of pastures with positive S. aureus BTM samples exists (Figure 1c).

3.2. Diversity of S. aureus Isolates in BTM Samples of Pastures

We used spa typing to subtype all 140 isolates collected from the 94 S. aureus-positive community-based Alpine dairy pastures. Thereby, 33 distinct spa types were identified, comprising eight previously unreported spa types: t21587, t21588, t21610, and t21743-47. The most frequently detected spa types included t2953 (n = 33), t529 (n = 12), t267 (n = 11), and t024 (n = 10) (Figure 1d). A single spa type was identified in most pastures (79%; 78/94). Two distinct spa types were present in 19% (18/94), and three spa types were found in 2% (2/94) of the pastures, respectively.

3.3. DNA Microarray-Based Genotyping, MLST, and Antimicrobial Susceptibility Testing of Selected Isolates

One isolate per spa type (n = 33) was subjected to DNA microarray-based genotyping, multi-locus sequence typing (MLST), and antimicrobial susceptibility testing. SplitsTree analysis used the microarray data of more than 330 genetic markers, revealing several distinct clonal groups, which were categorised according to the similarity of DNA microarray profiles (Figure 2a).
Using MLST, all isolates of a distinct clonal group could be aligned to a specific CC. Notably, 14 different spa types were assigned to CC8bov, revealing high spa gene diversity for this CC. Spa type t2953 represented the most dominant (n = 33), followed by t024 (n = 10), while the remaining twelve spa types assigned to CC8bov were detected sporadically (n = 1 or 2). These include seven new spa-types (t21587, t21588, t21610, t21743, t21745, t21746, and t21747). During the season, S. aureus isolates with spa types associated with CC8bov were detected at least once in BTM samples from 35.3% (55/156) of all pastures, making it the most prevalent clonal complex. CC97 and CC151 were detected in 16.7% (26/156) and 8.3% (13/156) of pastures, respectively, followed by CC479, at 3.2% (5/156) (Figure 2b). Notably, while the prevalence of CC97 and CC151 in BTM samples decreased at the last sampling point, the prevalence of CC8bov remained relatively stable across all four samplings (Figure 2c).
Among the genetic markers tested by DNA microarray (n = 33 isolates; 1 isolate per spa type), we focused on the enterotoxin and leukocidin genes due to their potential relevance to S. aureus food intoxication and virulence, respectively (Table 1).
All examined spa types of CC8bov (n = 13) contained enterotoxin genes in one of the following three variants: either the sea gene alone; sed, sej, and ser; or in combination with the sea gene. In CC97, one spa type (t359) was found to carry the sea gene. The enterotoxin gene cluster egc, comprising the genes seg, sei, sem, sen, seo, and seu, was associated with CC97, CC151, and CC479. A single isolate (t223, CC22) carried the human variant of the toxic shock syndrome toxin 1 gene (tst-1). None of the isolates exhibited the sec gene.
We found the following five leukocidin gene profiles: lukMF’-lukED-lukFS; lukED-lukFS; lukD-lukFS; lukED-lukS; and lukFS. LukMF’, also referred to as bovine LukM/LukF-P83, explicitly targeting bovine leukocytes, was detected in the combination lukMF’-lukED-lukFS associated with CC49, CC97, CC151, and CC479. In contrast, lukFS-lukED (without lukMF’) was associated with the lineages CC1, CC5, CC8bov, CC15, and CC97. All CC8bov harboured the leukocidin genes lukFS-lukED.
All examined BTM isolates (one isolate for each spa type, n = 33) were susceptible to cefoxitin, and the mecA or mecC genes were not detected (Table 1). In total, 29 out of 33 isolates were susceptible to all tested antimicrobial agents—penicillin, cefoxitin, ciprofloxacin, amikacin, gentamicin, tetracycline, erythromycin, clindamycin, chloramphenicol, trimethoprim-sulfamethoxazole, nitrofurantoin, rifampicin, and linezolid. Four isolates were resistant to penicillin mediated by the bla operon genes (blaZ, blaI, and blaR), associated with CC5, CC15, CC45, and CC398. One isolate (CC398) was resistant to tetracycline mediated by the tet(K) gene. One isolate (CC5) was resistant to erythromycin (erm(A) gene-positive) with a positive D-zone test (inducible clindamycin resistance). These four isolates, showing phenotypic resistance, were associated with lineages that were non-typical for bovine-associated CCs (CC5, CC15, CC45, and CC398). Interestingly, 12 out of 14 isolates of CC8bov were positive for all bla genes but exhibited no phenotypic resistance to penicillin—a discrepancy just recently reported for this CC [25].

4. Discussion

The results of our in-depth monitoring of S. aureus in Alpine dairy pastures in Tyrol underline the importance of S. aureus surveillance, especially in community-based Alpine dairy pastures. Consistent with reports from other European Alpine regions [6,10,26], we found a high prevalence of S. aureus (60% in BTM samples) in community-based Alpine dairy pastures in Tyrol. Generally, the reported prevalence of S. aureus in BTM samples from dairy herds exhibits significant regional variations, ranging from 18% (Lower Saxony, Germany) [27] to 84% (Minnesota, U.S.) [28]. Apart from the region, the prevalence of the contagious mastitis-causing pathogen S. aureus can be influenced by various factors, such as milking procedures, housing systems, herd size, and season [27,29,30]. Moreover, it is important to note that methodological differences, including sampling intensity and (pre-) analytics, can significantly influence the reported prevalence of S. aureus in BTM samples. In our study, samples were collected up to four times throughout the season, which likely contributes to a higher detection sensitivity compared to studies employing less-frequent or single-farm samplings, which should be considered when relating our findings with reported prevalence rates from other studies.
The high prevalence of S. aureus in community-based Alpine dairy pastures presents a significant challenge in preventing the transmission of S. aureus during the Alpine grazing season. The higher presence of S. aureus observed in BTM samples in close geographic proximity indicates that factors such as the movement of animals, personnel, or shared equipment between neighbouring farms may influence transmission. As only BTM samples from community-based pastures were included in the study, the local clustering is not surprising but rather highlights the increased risk of transmission when cows from different farms share communal pastures. Rigorous hygiene practices, such as intermediate disinfection and the correct maintenance of milking equipment, are important measures to prevent the spread of S. aureus among farms sharing community-based Alpine dairy pastures.
Overall, our study confirms the high prevalence of CC8bov in dairy farming within the European Alpine region. CC8bov was detected in 35.3% of BTM samples during the season, which is comparable to the 36.9% reported in dairy herds in Ticino, Switzerland [10], and 29.3% in Lombardy, northern Italy [31]. The predominant lineages detected were CC8bov, followed by CC97, CC151, and CC479; collectively, these represented nearly 90% of S. aureus lineages in BTM samples. These lineages are also among the six most frequently associated with bovine IMI in Europe [3]. CC8bov, CC97, and CC151 were also most common in BTM samples in Lombardy, Italy [31]. CC479 has been detected in cows with IMI in Germany, Belgium, the Netherlands, Portugal, and Italy [3]. Notably, the fourth sampling at the end of the season showed a much lower prevalence of S. aureus (17.3%) compared to the first three (average: 33.5%). This decline appears to be associated with a shift in the relative composition of the S. aureus lineages in the BTM samples. Specifically, the prevalence of lineages CC97 and CC151 decreased compared to previous samplings, whereas that of CC8bov remained stable. The continuous detection of CC8bov throughout the season demonstrates its critical role in the herd management of Alpine dairy pastures, including efforts for eradication [10]. However, it remains elusive whether there is any causal relationship between the decline in S. aureus prevalence in the last sampling and the distribution of the lineages. Several factors may influence this observed correlation. For example, consultations with regional farmers and veterinarians confirmed a common practice, whereby cows exhibiting clinical signs or other health issues with adverse effects on milk production are preferentially selected for dry-off and receive intramammary antibiotic treatment towards the end of the grazing season as the calving season approaches. It is tempting to speculate that management decisions may be biassed by the lineage; however, this hypothesis needs to be investigated further. Isolates from BTM samples may also originate from extramammary sites, such as teat skin, the environment, or personnel operating on the farm [31,32]. Consistent with this, the remaining rarely detected lineages (including CC5, CC22, CC398, and CC45) are associated with colonisation and the infection of humans, as well as pig farming (CC398) [33].
Beyond lineage classification, the isolates also exhibited distinct profiles of virulence factors and antibiotic resistance. Among the leukocidin genes, eight isolates of different CCs harboured the lukMF’ operon, which is particularly associated with bovine isolates. LukMF’ can lyse bovine neutrophils and monocytes, and it is a significant virulence factor associated with the severity of S. aureus IMI [34]. Leukocidin genes encoding Panton-Valentine leukocidin (PVL, lukF-PV and lukS-PV) were not detected, which is unsurprising as they are rarely found in bovine isolates and are primarily linked to human S. aureus infections [35].
Among the enterotoxin genes, sea and sed are most frequently associated with human foodborne outbreaks, including those involving dairy products. These most relevant enterotoxins were detected in all 13 spa types of CC8bov examined. As we showed previously, CC8bov can enter BTM from cows with subclinical, mild IMI and persist through dairy processing and cheese production, remaining viable in cheese for up to 14 days of ripening [6]. This suggests that CC8bov appears to be particularly well adapted to the cheese production environment. Due to their production of enterotoxins, these S. aureus strains pose a potential health risk to consumers. For instance, an S. aureus strain with CC8bov characteristics, isolated from soft cheese, was linked to a foodborne outbreak at a Swiss boarding school [36]. In Austria, a CC8bov strain was linked to an outbreak among elementary school children in Lower Austria related to milk products [37]. Thus, reducing S. aureus contamination at the source enhances food safety by preventing potential toxin-producing subtypes from entering the food chain, lowering the risk of foodborne illnesses in consumers.
In our study, no methicillin-resistant S. aureus (MRSA) isolates were detected in BTM samples. In general, the prevalence of MRSA in BTM samples from dairy herds is considered low, averaging around 3% [38]. Phenotypic resistance to penicillin, tetracycline, erythromycin, and clindamycin was shared by a small number of isolates (n = 5/33) that are associated with the lineages CC5, CC15, CC45, and CC398, which are not typically of bovine origin. All other isolates, including the typical bovine-associated lineages, were susceptible to all antimicrobials tested. Interestingly, most analysed CC8bov isolates were positive for all bla genes, although they were susceptible to penicillin. The discrepant result was recently described as being exclusively associated with S. aureus CC8bov isolates, in which the promoter of the bla operon is inactivated by a 31 bp deletion [39]. Overall, our findings support recent observations that antibiotic resistance is becoming less prevalent in S. aureus mastitis isolates from Europe and that bovine mastitis-associated lineages are not a significant source of antimicrobial resistance [25,40].

5. Conclusions

This study provides an in-depth insight into the S. aureus subtypes circulating in community-based Alpine dairy pastures in Tyrol, a region highly relevant for bovine milk production in Austria. Our findings indicate that S. aureus, particularly the CC8bov lineage, is highly prevalent in dairy herds throughout this region, consistent with findings in other European Alpine countries. Additionally, we identified the lineages CC97, CC151, and CC479, all of which are frequently associated with bovine IMI in Europe. Understanding the variability among these lineages could help inform targeted interventions and improve strategies for controlling infections caused by this zoonotic pathogen. Overall, our findings underscore the importance of continuous surveillance and subtyping, revealing the risks posed by antibiotic-resistant and toxin-producing bacteria, thereby enhancing animal health, food safety, and sustainable milk production.

Author Contributions

Conceptualisation: T.G. and J.L.K.; methodology: N.R., T.G., P.M.-M., and I.L.; validation: N.R. and T.G.; formal analysis: T.G., N.R. and M.A.; investigation: N.R., I.L., P.M.-M., T.G. and J.L.K.; resources: T.G., J.L.K. and M.E.-S.; data curation: N.R. and T.G.; writing—original draft preparation: N.R. and T.G.; writing—review and editing: T.G., N.R., M.E.-S., I.L., P.M.-M., M.A. and J.L.K.; visualisation: N.R., T.G. and I.L.; supervision: T.G.; project administration: T.G.; funding acquisition: T.G. All authors have read and agreed to the published version of the manuscript.

Funding

This project is funded by the Austrian Federal Ministry of Agriculture, Forestry, Regions and Water Management via dafne.at (funding number: 101722). The BML supports applied, problem-oriented research in its area of expertise.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data generated and/or analysed during the current study are available from the corresponding author upon reasonable request.

Acknowledgments

The authors wish to acknowledge the support of Stefan Lindner, Anton Pollinger, Valentin Unterrainer, and Theresa Blasiker from Tyrol Milch eGen, Wörgl, Austria; Christian Mader from the Animal Health Service Tyrol (Tiroler Tiergesundheitsdienst, T-TGD); and the participating premises and their personnel for this study.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BTMbulk tank milk
CCclonal complex
CC8bovbovine-adapted CC8 variant
GTBgenotype B
IMIintramammary infection
MALDI-TOF-MSmatrix-assisted laser desorption ionisation–time-of-flight mass spectrometry
MLSTmulti-locus sequence typing

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Animals 15 02153 g001
Figure 1. (a,b) Dot plots illustrating the variation in the number of (a) dairy cows and (b) farms per community-based Alpine dairy pasture examined. Each dot represents an individual Alpine dairy pasture; the horizontal line indicates the median value. (c) Local Moran’s I cluster map showing the spatial autocorrelation of S. aureus in community-based Alpine dairy pastures in Tyrol. The colours denote four spatial clusters derived from Local Moran’s I analysis based on similarity or dissimilarity to surrounding points, as follows: High–High clusters (HH, red) indicate hotspots—positive points surrounded by positive points; Low–Low clusters (LL, dark blue) represent coldspots—negative points surrounded by negative points; Low–High (LH, light orange) and High–Low (HL, light blue) show spatial outliers—negative points surrounded by positive points or positive points surrounded by negative points, respectively. (d) Distribution of spa types across surveyed community-based Alpine dairy pastures. Number of pastures where a particular spa type was detected at least once during the season.
Figure 1. (a,b) Dot plots illustrating the variation in the number of (a) dairy cows and (b) farms per community-based Alpine dairy pasture examined. Each dot represents an individual Alpine dairy pasture; the horizontal line indicates the median value. (c) Local Moran’s I cluster map showing the spatial autocorrelation of S. aureus in community-based Alpine dairy pastures in Tyrol. The colours denote four spatial clusters derived from Local Moran’s I analysis based on similarity or dissimilarity to surrounding points, as follows: High–High clusters (HH, red) indicate hotspots—positive points surrounded by positive points; Low–Low clusters (LL, dark blue) represent coldspots—negative points surrounded by negative points; Low–High (LH, light orange) and High–Low (HL, light blue) show spatial outliers—negative points surrounded by positive points or positive points surrounded by negative points, respectively. (d) Distribution of spa types across surveyed community-based Alpine dairy pastures. Number of pastures where a particular spa type was detected at least once during the season.
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Figure 2. (a) SplitsTree network visualisation showing the genetic relationships among the detected spa types from S. aureus BTM isolates, as determined by DNA microarray hybridisation patterns. Isolates in closer proximity to each other are more closely phylogenetically related, with clustered strains belonging to the same CC. Bovine-associated CCs are highlighted in blue. (b,c) Distribution of CCs across surveyed community-based Alpine dairy pastures. (b) Number pastures where a particular CC was detected at least once during the season. (c) The relative proportion of CCs at four sampling time points between the end of May and the beginning of September 2023. N indicates the number of pastures sampled at a particular sampling time point.
Figure 2. (a) SplitsTree network visualisation showing the genetic relationships among the detected spa types from S. aureus BTM isolates, as determined by DNA microarray hybridisation patterns. Isolates in closer proximity to each other are more closely phylogenetically related, with clustered strains belonging to the same CC. Bovine-associated CCs are highlighted in blue. (b,c) Distribution of CCs across surveyed community-based Alpine dairy pastures. (b) Number pastures where a particular CC was detected at least once during the season. (c) The relative proportion of CCs at four sampling time points between the end of May and the beginning of September 2023. N indicates the number of pastures sampled at a particular sampling time point.
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Table 1. Summarised molecular characterisation and antimicrobial resistance profiles of one S. aureus isolate per detected spa type (n = 33).
Table 1. Summarised molecular characterisation and antimicrobial resistance profiles of one S. aureus isolate per detected spa type (n = 33).
Strain-ID (1)spaCCSTagr TypeAntimicrobial Resistance ProfileToxins/SuperantigensLeukocidins (4)
PhenotypeGenes Detected
502_1t2953CC8ST8agr INRblaZsed, sej, serlukF/S, lukD/E
69t529CC151ST504agr IINR egc cluster (3)lukMF’, lukF/S, lukD/E
229t267CC97ST352agr INR lukMF’, lukF/S, lukD/E
304t024CC8ST8agr INRblaZsea, sed, sej, serlukF/S, lukD/E
244t521CC97ST97agr INR lukMF’, lukF/S, lukD/E
41t524CC97ST71agr INR lukF/S, lukD/E
17t13487CC479ST1380agr IINR egc cluster (3)lukMF’, lukF/S, lukD/E
54t359CC97ST97agr INR sealukF/S, lukD/E
174t011CC398ST2199agr IPEN, TETblaZ, tet(K), tet(M) lukF/S
146t21588CC8ST8agr INRblaZsed, sej, serlukF/S, lukD/E
44t21610CC8ST6180agr INR sealukF/S, lukD/E
18t223CC22 ST5974agr INR tst-1 (human), egc cluster (3)lukF/S
291t3802CC8ST7509agr INR sealukF/S, lukD/E
274t5268CC8ST8agr INRblaZsea, sed, sej, serlukF/S, lukD/E
155t5270CC8ST8agr INRblaZsea, sed, sej, serlukF/S, lukD/E
16t002CC5ST5agr IIERY, CLI (2)erm(A)egc cluster (3)lukF/S, lukD/E
89t065CC45ST45agr IPENblaZegc cluster (3)lukF/S
68t084CC15ST15agr IIPENblaZ lukF/S, lukD/E
171t127CC1ST1agr IIINR sehlukF/S, lukD/E
502_2t1340CC5ST5agr IINR lukF/S, lukD/E
430t179CC5ST5agr IIPENblaZsed, sej, ser, egc cluster (3)lukF/S, lukD/E
491t18776CC151ST504agr IINR egc cluster (3)lukMF’, lukF/S, lukD/E
489t19341CC8ST8384agr INRblaZsea, sed, sej, serlukF/S, lukD/E
31t208CC49ST49agr IINR lukMF’, lukF/S, lukD/E
223t21587CC8ST6180agr INRblaZsea, sed, sej, serlukF/S, lukD/E
135t21743CC8ST8agr INRblaZsea, sed, sej, serlukF/S, lukD/E
147t21744CC479ST1380agr IINR egc cluster (3)lukMF’, lukF/S, lukD/E
25t21745CC8ST9273agr INRblaZsea, sed, sej, serlukS, lukD/E
446t21746CC8ST8384agr INRblaZsea, sed, sej, serlukF/S, lukD/E
95t21747CC8ST8agr INRblaZsed, sej, serlukF/S, lukD/E
150t527CC97ST352agr INR lukMF’, lukF/S, lukD/E
513t528CC97ST71agr INR egc cluster (3)lukF/S, lukD/E
125t843CC130ST2490agr IIINR lukF/S, lukD
(1) Selected for MLST and DNA microarray-based technology and antimicrobial susceptibility testing. (2) Erythromycin-induced resistance to clindamycin. (3) egc cluster comprising seg, sei, sem, sen, seo, seu. (4) lukMF’, also referred to as lukF-P83/lukM (bovine).
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Ramezanigardaloud, N.; Loncaric, I.; Mikuni-Mester, P.; Alinaghi, M.; Ehling-Schulz, M.; Khol, J.L.; Grunert, T. Prevalence and Diversity of Staphylococcus aureus in Bulk Tank Milk from Community-Based Alpine Dairy Pastures in Tyrol, Austria. Animals 2025, 15, 2153. https://doi.org/10.3390/ani15142153

AMA Style

Ramezanigardaloud N, Loncaric I, Mikuni-Mester P, Alinaghi M, Ehling-Schulz M, Khol JL, Grunert T. Prevalence and Diversity of Staphylococcus aureus in Bulk Tank Milk from Community-Based Alpine Dairy Pastures in Tyrol, Austria. Animals. 2025; 15(14):2153. https://doi.org/10.3390/ani15142153

Chicago/Turabian Style

Ramezanigardaloud, Nasrin, Igor Loncaric, Patrick Mikuni-Mester, Masoumeh Alinaghi, Monika Ehling-Schulz, Johannes Lorenz Khol, and Tom Grunert. 2025. "Prevalence and Diversity of Staphylococcus aureus in Bulk Tank Milk from Community-Based Alpine Dairy Pastures in Tyrol, Austria" Animals 15, no. 14: 2153. https://doi.org/10.3390/ani15142153

APA Style

Ramezanigardaloud, N., Loncaric, I., Mikuni-Mester, P., Alinaghi, M., Ehling-Schulz, M., Khol, J. L., & Grunert, T. (2025). Prevalence and Diversity of Staphylococcus aureus in Bulk Tank Milk from Community-Based Alpine Dairy Pastures in Tyrol, Austria. Animals, 15(14), 2153. https://doi.org/10.3390/ani15142153

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