Comparative study of recurrent mastitis caused by methicillin-resistant Staphylococcus aureus and non-MRSA S. aureus in Holstein-Fresian cows

Abstract

Mastitis is one of the most common diseases in dairy cows, causing significant economic losses due to reduced productivity and the need for treatment. In recent years, MRSA (methicillin-resistant Staphylococcus aureus) has emerged as an important cause of chronic and recurrent mastitis, posing additional challenges for herd health management. The aim of this study was to conduct a comparative analysis of recurrent mastitis in Holstein-Friesian cows caused by MRSA and non-MRSA S. aureus, with a particular focus on the number of recurrences, somatic cell count (SCC), milk production, and risk factors. The study included 120 cows, divided into two groups of 60, raised under identical conditions on a single farm. Milk samples were collected under sterile conditions, cultured on Blood agar and Mannitol Salt agar, and analyzed for the presence of MRSA using PCR for the mecA gene. Cows were monitored for the number of recurrences, SCC, daily milk yield, and response to therapy. Statistical analyses included t-tests, ANOVA, correlation analysis, and logistic regression. The results showed that MRSA-infected cows had significantly higher recurrence rates, along with elevated SCC and reduced milk yield compared to the non-MRSA group. MRSA was identified as the strongest independent risk factor for mastitis recurrence. These findings highlight the importance of regular bacteriological monitoring, appropriate therapy, and preventive measures to reduce economic losses and protect herd health.

INTRODUCTION

Mastitis is one of the most common and significant diseases in dairy cows, causing serious economic losses due to reduced milk production, increased SCC, and higher treatment costs [2, 3, 17]. Several studies indicate that Staphylococcus aureus is one of the main etiological agents of clinical and subclinical mastitis in cows, and its ability to cause chronic and recurrent infections makes it particularly problematic for farms [2, 5, 26]. In recent years, the emergence of methicillin-resistant S. aureus (MRSA) has raised concern among researchers and practitioners, as these strains are resistant to standard antibiotic therapies and can cause persistent infections that are difficult to control [23, 28]. Recurrent mastitis, especially that caused by MRSA, presents a major challenge to herd health and productivity. High SCC, reduced milk yield, and frequent recurrences create a significant economic burden and complicate therapy selection [20, 21, 25]. MRSA infections are not just a local problem; research shows that these resistant strains can circulate within farms, spread between animals, and even pose a risk to the health of humans working with cows [1, 9, 13, 25]. Several studies have reported that MRSA-associated mastitis is characterized by elevated somatic cell counts, more severe mammary gland tissue damage, reduced milk yield, and increased recurrence rates [6, 21, 22], further supporting its role as a major pathogen contributing to chronic and difficult-to-control infections in dairy cows. Additionally, the type of dairy management, parity, and previous infection history have been identified as risk factors for recurrent mastitis [11, 12, 14, 16].

However, direct comparative evidence on recurrent mastitis caused by MRSA and non-MRSA S. aureus under identical herd conditions, particularly regarding recurrence dynamics, SCC, milk yield, and associated risk factors, remains limited.

Although previous studies have highlighted the potential role of virulence factors in S. aureus strains in contributing to persistence and antimicrobial resistance, the present study did not include molecular characterization of virulence genes or genotyping of isolates, which may be considered a limitation for deeper epidemiological interpretation.

We hypothesize that MRSA-related recurrent mastitis exhibits a distinct and more severe clinical and epidemiological profile than non-MRSA infections, with higher recurrence risk, increased SCC, reduced milk yield, and stronger association with host and management risk factors. The aim of this study is to investigate the differences in prevalence and clinical outcomes of recurrent mastitis caused by MRSA and non-MRSA S. aureus in Holstein-Friesian cows, to determine the impact of etiology on SCC, milk production, and the number of recurrences, and to identify risk factors for repeat infections. The results of this study may help develop improved strategies for mastitis control, optimize treatment, and reduce economic losses on dairy farms.

MATERIALS AND METHODS

Herd under study

The study was conducted on a commercial Holstein–Friesian dairy farm operating under an intensive production system. The total herd size consisted of 411 cows. A total of 120 cows with confirmed

S. aureus mastitis were included in the study, representing 29.2% of the herd population. These animals were divided into two groups of 60 cows each: the MRSA group (methicillin-resistant S. aureus) and the non-MRSA group (methicillin-susceptible S. aureus).The study was conducted from May 2024 to April 2025, thereby covering all four seasons. This allowed the inclusion of potential seasonal variation in mastitis occurrence within the analysis.The cows were distributed across different lactation groups (I–IV), with ages ranging from 3.5 to 7 years, and were maintained under identical housing, feeding, and hygiene conditions throughout the study period. Housing consisted of enclosed barns with straw-bedded resting areas, with continuous access to fresh water. The diet included hay, silage, and concentrated feed supplemented with minerals and vitamins to support optimal milk production and udder health. Routine preventive veterinary management, including vaccination and ectoparasite control, was regularly implemented.

Disease diagnosis and definition of recurrent mastitis

Mastitis was diagnosed through a combination of clinical examination and laboratory confirmation. Clinical mastitis was defined as the presence of visible inflammatory changes in the udder and/or abnormal milk secretion, including swelling, heat, pain, and the presence of clots, flakes, or watery milk. Subclinical mastitis was defined by an elevated (SCC) exceeding 200 × 10³ cells/mL, without visible clinical signs.

All suspected cases were confirmed by bacteriological examination of milk samples, with the isolation of S. aureus serving as the definitive etiological diagnosis.

Recurrent mastitis was defined as the reappearance of clinical signs and/or re-isolation of S. aureus from milk samples after complete clinical recovery and successful antimicrobial therapy. A minimum interval of 21 days following the end of treatment was required to distinguish recurrence from treatment failure or persistent infection. This criterion ensured differentiation between relapse of the same infection and new infection events, allowing consistent classification of cases included in the study.

Sample collection

Milk samples were collected from all cows according to International Dairy Federation (IDF) guidelines for aseptic milk sampling procedures. Prior to sampling, udders and teats were thoroughly cleaned and disinfected with 70% ethanol, and the first streams of milk were discarded to minimize contamination. Samples were collected in sterile tubes and labeled with unique identification codes for each cow.

Sample transport and storage

Samples were transported under cooled conditions (4°C) to the laboratory within 2–4 hr of collection. Upon arrival, samples were processed immediately or stored at 4°C for a maximum of 24 hr to preserve bacteriological integrity. For molecular analysis, aliquots of milk samples were stored at −20°C until DNA extraction and subsequent PCR analysis.

Microbiological analysis

Milk samples were cultured on Blood agar (HiMedia Laboratories, Mumbai, India) and Mannitol Salt agar (HiMedia Laboratories, Mumbai, India) for the preliminary isolation of S. aureus. Presumptive colonies were identified based on colony morphology, hemolytic pattern, and mannitol fermentation.

Confirmation of MRSA was performed using phenotypic and molecular methods. Methicillin resistance was initially assessed by the disk diffusion method according to CLSI guidelines. For molecular confirmation, only colonies phenotypically identified as S. aureus were used. Genomic DNA was extracted from isolated bacterial colonies using a standard boiling method in which bacterial cells were lysed by thermal treatment [17].

PCR amplification of the mecA gene was performed using the following primers:

Forward primer: 5′-AAA ATC GAT GGT AAA GGT TGG C-3′

Reverse primer: 5′-AGT TCT GCA GTA CCG GAT TTG C-3′

The expected amplicon size was approximately 310 bp. The PCR reaction was carried out in a final volume of 25 µL under the following cycling conditions: initial denaturation at 94°C for 5 min, followed by 30–35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min, with a final extension step at 72°C for 5 min. Antimicrobial susceptibility profiles of the isolated S. aureus strains were determined using the disk diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) M100, Performance Standards for Antimicrobial Susceptibility Testing, 36th edition, to evaluate resistance patterns against commonly used veterinary antimicrobials.

Clinical and productive assessment

The following parameters were recorded for each cow:

Number of mastitis recurrences, recorded per animal during the study period.

SCC, defined as the number of somatic cells per milliliter of milk and measured using a DeLaval Cell Counter as an indicator of intramammary inflammatory response. SCC and daily milk yield were recorded at the time of mastitis diagnosis and monitored throughout the clinical course of the disease, with mean values used for statistical analysis.

Daily milk yield (L), recorded using automatic milking systems.

Percentage of milk loss, calculated based on the difference in milk yield before and after infection.

Therapeutic outcome, classified as successful or unsuccessful based on combined clinical improvement and bacteriological cure. Treatment of infected cows was carried out according to antimicrobial susceptibility testing (antibiogram) to ensure targeted therapy and to minimize the risk of antimicrobial resistance development. Differences in therapeutic protocols between MRSA and non-MRSA groups were applied according to the identified resistance profiles.

MRSA mastitis cases were treated with a combination of local intramammary therapy and systemic antimicrobial therapy, depending on infection severity and culture results. Local treatment included intramammary preparations containing gentamicin, tetracycline, or combinations with newer lincosamides, selected according to the susceptibility profile of the isolated MRSA strains. Systemic therapy consisted of lincosamides (e.g., clindamycin or linezolid) or gentamicin, applied in combination with local therapy in accordance with CLSI guidelines and current veterinary therapeutic protocols. In contrast, non-MRSA S. aureus infections were treated with beta-lactam antibiotics, such as oxacillin or amoxicillin-clavulanate, based on susceptibility results.

Dosages and treatment durations were strictly controlled: local intramammary treatments were administered for 3–5 days, while systemic therapy lasted 5–7 days, with continuous monitoring of clinical signs and milk parameters. Each cow was regularly observed for reduction of inflammation symptoms, milk changes, and SCC values.

To identify recurrent infections, cows were monitored for 60–90 days post-therapy, with repeated bacteriological milk analyses and SCC measurements. This allowed assessment of therapy efficacy and the potential for chronic mastitis development, particularly in MRSA-infected cows, which tend to show persistence and higher risk of reinfection. Treatment failure was defined as the persistence or recurrence of clinical signs and/or bacteriological positivity after completion of the full antimicrobial treatment course.

Ethical approval

All procedures were conducted in accordance with the Institutional Animal Care and Use Committee guidelines, which authorized animal handling, sample collection, and continuous monitoring (Ethics approval number: 001328298 2024 14841 002 001 323 022).

Statistical analysis

Data were analyzed using SPSS 27.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism (GraphPad Software, Boston, MA, USA). Normality of continuous variables was assessed prior to analysis. Since somatic cell count (SCC) showed a non-normal distribution, appropriate statistical approaches were applied. Differences between MRSA and non-MRSA groups were evaluated using t-test or Z-test for normally distributed variables. One-way ANOVA was used to assess the effect of etiology on SCC and daily milk yield, while two-way ANOVA (multifactorial design) was applied to evaluate the interaction between etiology and lactation number on recurrence rates. Pearson correlation analysis was performed to assess relationships between SCC, number of recurrences, and milk production. To identify independent risk factors for recurrent mastitis, multivariate logistic regression analysis was conducted, and results were expressed as odds ratios (OR) with 95% confidence intervals (95% CI).

Statistical significance was set at P<0.05, and data are presented as mean ± standard deviation.

RESULTS

The results presented in Table 1 show that recurrent mastitis was more frequent in cows infected with MRSA compared to those infected with non-MRSA strains. A higher proportion of cows in the MRSA group experienced recurrence, whereas the non-MRSA group showed a lower recurrence rate. Statistical analysis (χ² test) revealed a significant difference between the two groups, indicating that recurrence is associated with the etiological factor.

Table 1. Etiological distribution and recurrence of mastitis.

Etiology	Total cows	Clinical mastitis n (%)	Recurrent mastitis n (%)	No recurrence n (%)
MRSA	60	48 (80.0)	42 (70.0)	18 (30.0)
Staphylococcus aureus (non-MRSA)	60	39 (65.0)	27 (45.0)	33 (55.0)
Total	120	87 (72.5)	69 (57.5)	51 (42.5)

Values are shown as number of cows (n) and percentages (%). Clinical mastitis: cows with observable signs; Recurrent mastitis: cows with ≥1 additional episode; No recurrence: cows without further episodes. MRSA: methicillin-resistant S. aureus; non-MRSA: methicillin-sensitive S. aureus.

The results presented in Table 2 show that cows infected with MRSA had a significantly higher mean number of recurrence episodes per cow compared to the non-MRSA group. In addition, the interval to recurrence was significantly shorter in MRSA-infected cows. Furthermore, the duration of active infection was significantly longer in the MRSA group. All observed differences were statistically significant (P<0.001), confirming distinct recurrence dynamics depending on the etiological agent.

Table 2. Average number of recurrences and duration.

Parameter	MRSA (mean ± SD)	Non-MRSA (mean ± SD)	Test	P
Number of recurrences per cow	2.8 ± 0.9	1.6 ± 0.7	t	<0.001
Time to recurrence (days)	38 ± 12	74 ± 18	z	<0.001
Duration of infection (days)	21 ± 6	12 ± 4	t	<0.001

Values are presented as mean ± SD. Number of recurrences per cow: average number of mastitis episodes per cow; Time to recurrence: interval in days until the next episode; Duration of infection: length of each mastitis episode in days. MRSA: methicillin-resistant S. aureus; non-MRSA: methicillin-sensitive S. aureus. Statistical tests: t-test (t) and z-test (z); P<0.05 indicates significance.

The results presented in Table 3 show significant differences in SCC, daily milk yield, and milk loss depending on the etiology of mastitis. Cows with MRSA infection exhibited significantly higher SCC values and lower milk yield compared to the non-MRSA group. In addition, the percentage of milk loss was significantly higher in MRSA-infected cows. One-way ANOVA confirmed statistically significant differences between groups (P<0.001), indicating a strong effect of etiology on inflammatory and productive parameters.

Table 3. One-way analysis of variance: effect of etiology on somatic cell count and milk yield.

Parameter	MRSA	non-MRSA	F	P
SCC (×103/mL)	1,450 ± 320	980 ± 210	42.6	<0.001
Daily milk yield (L)	21.4 ± 3.2	25.8 ± 3.6	31.2	<0.001
Milk loss (%)	18.7 ± 4.1	10.3 ± 3.5	29.4	<0.001

Values are presented as mean ± SD. SCC: somatic cell count (×10³/mL); Daily milk yield: milk produced per cow per day (L); Milk loss: percentage reduction in milk yield compared to healthy cows. MRSA: methicillin-resistant S. aureus; non-MRSA: methicillin-sensitive S. aureus. Statistical analysis was performed using one-way ANOVA; P<0.05 indicates statistical significance.

The results presented in Table 4 indicate that etiology has a statistically significant effect on the number of mastitis recurrences. Lactation number also significantly influences the variation in recurrence numbers among the studied cows. Two-way ANOVA analysis shows a statistically significant interaction between etiology and lactation. This means that the effect of etiology on recurrence numbers changes depending on the lactation group. Different lactation groups exhibit varying recurrence values in relation to the etiological factor. These findings suggest a multifactorial influence on the occurrence of recurrent mastitis.

Table 4. Two-way analysis of variance: effect of etiology and lactation on recurrence of mastitis.

Factor	F	P
Etiology	36.4	<0.001
Lactation (I–IV)	8.9	0.002
Interaction	6.1	0.011

F-values and P-values are presented for each factor and their interaction. Etiology: type of S. aureus infection (MRSA or non-MRSA); Lactation (I–IV): number of lactation cycles; Interaction: combined effect of etiology and lactation. Statistical significance was considered at P<0.05.

The results of the correlation analysis presented in Table 5 show statistically significant relationships among the studied variables. A strong positive correlation was observed between SCC and the number of recurrences. At the same time, a strong negative correlation was found between the number of recurrences and milk yield, indicating that an increase in recurrences is associated with decreased productivity. Additionally, a moderate positive correlation was observed between time to recurrence and therapeutic success. All reported correlations were statistically significant according to the Pearson correlation coefficient.

Table 5. Correlation analysis (Pearson correlation coefficient).

Variables	r	P
SCC ↔ Number of recurrences	0.72	<0.001
Number of recurrences ↔ Milk yield	–0.65	<0.001
Time to recurrence ↔ Therapeutic success	0.58	0.002

Correlation coefficients (r) and P-values are presented. SCC: somatic cell count (×10³/mL); Number of recurrences: total mastitis episodes per cow; Milk yield: daily milk production (L); Time to recurrence: interval until next mastitis episode (days); Therapeutic success: effectiveness of treatment in resolving mastitis. Statistical significance was considered at P<0.05.

The results of the logistic regression analysis presented in Table 6 indicate a statistically significant effect of multiple factors on the occurrence of recurrent mastitis. MRSA infection shows the highest odds ratio, indicating an increased risk of recurrence compared to non-MRSA infections. An elevated SCC above 1,000 × 10³/mL is also significantly associated with recurrence. Lactation number equal to or greater than three demonstrates a statistically significant association with recurrent mastitis. Unsuccessful therapy is linked to the highest increased risk of reinfection. All these factors were identified as independent predictors in the logistic regression model.

Table 6. Logistic regression analysis of risk factors for recurrence.

Factor	OR	95% CI	P
MRSA infection	3.12	1.85–5.27	<0.001
SCC >1,000 × 103/mL	2.48	1.42–4.33	0.002
≥III lactation	1.96	1.11–3.44	0.019
Unsuccessful therapy	4.05	2.12–7.75	<0.001

Odds ratios (OR), 95% confidence intervals (CI), and P-values are presented for each factor. MRSA infection refers to infection with methicillin-resistant S. aureus. SCC >1,000 × 10³/mL indicates elevated somatic cell count. ≥III lactation refers to cows in their third or higher lactation. Unsuccessful therapy refers to ineffective treatment of the initial mastitis episode. Statistical significance was set at P<0.05.

DISCUSSION

The data obtained from this study clearly demonstrated that recurrent mastitis is more frequent in cows infected with MRSA than in those infected with non-MRSA S. aureus. Similar trends have been observed in other studies, reporting a significant presence of MRSA in the milk of cows with subclinical and clinical mastitis, indicating a persistent problem with methicillin-resistant bacterial infections on dairy farms [20, 21, 23]. Although global meta-analytical data show higher heterogeneity in MRSA prevalence, strains of this bacterium have been detected in dairy herds across different regions, highlighting its importance as an etiological factor in bovine mastitis [1, 4, 13, 15].

The relatively high proportion of MRSA isolates observed in the present study compared to some previously published reports may be associated with several factors. Since the investigation was conducted on a single intensive dairy farm, farm-specific epidemiological conditions, including antimicrobial usage patterns, herd management practices, biosecurity measures, and circulation of persistent resistant strains within the herd, may have contributed to the increased prevalence. In addition, differences in sampling strategies, diagnostic methodologies, geographical regions, and herd characteristics among studies may partially explain the variability in reported MRSA prevalence rates. Recent studies have also indicated that MRSA-associated mastitis is increasingly recognized in dairy farms worldwide, although prevalence remains highly heterogeneous depending on regional and management-related factors [1, 13].

Consistent with our findings, the higher SCC observed in MRSA-positive cows reflects a more pronounced immune response, often associated with persistent and chronic infections. Elevated SCC values are widely recognized as indicators of udder inflammation and show strong positive correlations with the presence of infectious agents, including S. aureus [2, 8, 10, 18]. Studies on mastitis and somatic cells have documented that increased SCC is frequently associated with subclinical infections such as those caused by S. aureus, which aligns with the higher SCC observed in MRSA-infected cows in this study [5,6,7]. Our data also indicate that MRSA mastitis is associated with a higher number of recurrences and a shorter interval to the next recurrence, suggesting a chronic disease course. These findings are consistent with studies linking low treatment efficacy to the presence of antimicrobial-resistant S. aureus strains, which hinder complete pathogen elimination and result in frequent recurrent infections [9, 20, 24].

The one-way ANOVA results demonstrated significant differences in productivity parameters, including daily milk yield and percentage milk loss, between MRSA and non-MRSA groups, confirming the negative impact of S. aureus mastitis on milk production [21, 27, 29]. The multifactorial ANOVA further indicated a significant interaction between etiology and lactation number, suggesting that recurrence risk increases with advancing age and higher parity [12, 14, 16]. Additionally, logistic regression identified MRSA infection as the strongest independent risk factor for recurrent mastitis compared to other evaluated variables [19, 22, 25]. These findings are consistent with previous studies reporting the detrimental effects of S. aureus infections, particularly antimicrobial-resistant strains, on dairy cow productivity and disease chronicity, although reported MRSA prevalence varies depending on regional and methodological differences [1, 13, 20, 28]. Overall, the results of this study are supported by relevant research emphasizing the importance of S. aureus, particularly MRSA, as causative agents of mastitis in cows, their impact on productivity, and the necessity of systematic monitoring and management strategies to control infections effectively.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.