Isolation and Characterization of Staphylococcus aureus in Bovine Milk from Rewa, India

Abstract

Mastitis in dairy animals affect milk quality and quantity, animal health and welfare, antimicrobial use and economics of dairy farm, and antimicrobial residues in milk. Staphylococcus aureus is most common mastitis pathogen with ability to cause infections which are difficult to treat. The present study aimed to characterize the S. aureus strains associated with dairy animal with reference to its virulence, biofilm formation, antimicrobial resistance including methicillin and penicillin G resistance. A total of 100 bovine milk samples were screened by bacterial culture method, out of which 18 S. aureus and 6 methicillin-resistant S. aureus (MRSA) isolates were identified and characterized for virulence determinants. The strains were uniformly positive for the virulence determinants. However, the hemolysis in blood agar was found to be specific but not a sensitive criterion for virulence. The biofilm formation ability of the isolates showed 61.66% of S. aureus and 83.33% of MRSA strains were positive by Microtiter plate method. The biofilm formation genes (icaA and icaD) were detected in all the strains. The multi-drug resistance profile of the strains was studied by disk diffusion assay where over 70% of S. aureus strains were sensitive to all the anti-microbial agents (except penicillin) and only 33.33% of the strains had the MAR index above 0.2. All the MRSA strains (100%) had a MAR index of ≥ 0.2. All the strains showed resistance to penicillin which is considered as a prognostic marker for mastitis. The presence of penicillin and/or methicillin resistant, biofilm forming S. aureus mastitis strains can severely affect the treatment outcomes and economics of small dairy farmers of the region. Further studies to understand the population structure of the strains, by whole genome or traditional sequence-based methods and MIC values of antibiotics are required.

Introduction

Despite considerable research on mastitis of dairy animals, the disease still remains a major problem to the dairy farmer and dairy industry. There is a demand for wholesome, nutritious and safe milk which can originate only from healthy animal. Further the need to control mastitis is driven by multiple considerations including milk quality and quantity, producer economic viability, reductions in antimicrobial use and animal welfare [1].

Staphylococcus aureus is the most important and prevalent bacteria which cause mastitis in dairy cattle and buffalo. Because of its contagiousness and capacity to induce long-lasting chronic infections, S. aureus is among the few major pathogens associated with endemic mastitis all over the world. As the herd is the epidemiological unit for mastitis, the farm specific control measures for mastitis are required. Owing to the phenotypic and genomic plasticity of S. aureus the outcome of mastitis is highly variable and can range from subclinical to acute gangrenous mastitis or persistent, chronic mastitis. The ability to invade mammary epithelial cells (MEC) and exist as Small Colony Variants (SCV), form a biofilm and polysaccharide capsules seems to be most common attributes to S. aureus persistence in the host. This complicates the identification of affected animal even by bacteriological culture [2].

A significant proportion of mastitis strains (40–60%, depending on the reports) are able to form biofilms in vitro [3]. This may be associated with reduced antimicrobial susceptibility, resulting in disappointing cure rates, especially for infections of long duration [4]. The primary reason for antibiotic use on the dairy farm is mastitis. The mastitis causing S. aureus strains remain susceptible to common antibiotics used in the treatment. Penicillin resistance is probably the most well-known antibiotic resistance of S. aureus. Antimicrobial treatment with penicillin-resistant S. aureus strains results in a lower cure rate for treatment with either β-lactam or non-β-lactam antibiotics [5, 6].

Staphylococcus aureus is a multi-faceted pathogen that can exist as contagious and environment pathogen, extracellular and intracellular, form biofilm, a clonal bacteria but its prevalence varies with geographical locations. The emergence of MRSA as a cause of mastitis may also impact human health due to its potential zoonotic transmission. This makes necessary to monitor the prevalence and characteristics of this important pathogen. The present study aimed to characterize the S. aureus strains associated with dairy animal with reference to its biofilm formation, antimicrobial resistance including methicillin and penicillin G resistance. The latter is also an important prognostic marker.

Material and Methods

Collection of Milk Samples

A total of 100 samples of milk were collected aseptically from quarter/composite milk from bovines with mastitis (clinical or subclinical) from participating dairies and pooled milk from participating and non-participating dairies (Table 1). The clinical mastitis (CM) was identified as abnormal milk, abnormal udder and/or abnormal cow. The subclinical mastitis (SCM) was identified by California mastitis test (CMT) with a score of above 1 [7].

Table 1.

Details of the Total milk samples collected for the study

S. no	Source of sample	No. of sample
1	Mastitis milk from farm*	26
2	Teaching Veterinary Clinical Complex (TVCC)	36
3	Pooled milk	38
Total		100

*A total of 116 bovines (cow = 103 and Buffalo = 13) from dairy farms were screened for mastitis by CMT

Screening of Milk Samples for Staphylococcus aureus

Isolation of Staphylococcus aureus from milk was done as per the method of [8] with slight modification. Briefly, the milk sample was inoculated in Mannitol Salt Agar (MSA) and thereafter in Baird-Parker Agar (BPA) with egg yolk tellurite after pre-enrichment with 6.5% NaCl and incubated overnight at 37 °C. The typical single pure colony on Tryptone Soy Agar (TSA) were phenotypically and genotypically characterized for identification and virulence determinants. The phenotypic characterization was done by Gram’s staining, catalase, pigmentation, Tube coagulase (TC), Voges-Proskauer (VP), hemolysis in Sheep Blood Agar (SBA), beta-galactosidase tests, Novobiocin (5 μg) resistance (> 16 mm) and Polymyxin B (300 U) susceptibility (≤ 10 mm) [9, 10]. When the incomplete (α) haemolysis turned complete on storage of the SBA plates at 4 °C, it was regarded as hot–cold lysis. The presence of thermostable nuclease (nuc), staphylococcal protein A (spa) and Panton–Valentine leucocidin (pvl) gene were confirmed by PCR as detailed below (Table 2).

Table 2.

Sequences of primer sets with their corresponding PCR protocols and product

PCR	Gene	Primer sequence	size (bp)	Reference
1	spa	spa- F: TAA AGA CGA TCC TTC GGT GAG C spa- R: CAG CAG TAG TGC CGT TTG CTT	180–600 bp	[12]
1	mecA	mecA-F: TCC AGA TTA CAA CTT CAC CAG G mecA-R: CCA CTT CAT ATC TTG TAA CG	162 bp
1	pvl	pvl -F: GCT GGA CAA AAC TTC TTG GAA TAT pvl- R: GAT AGG ACA CCA ATA AAT TCT GGA TTG	87 bp
2	nuc	nuc-F: GCG ATT GAT GGT GAT ACG GTT nuc-R: AGC CAA GCC TTG ACG AAC TAA AGC	279 bp	[13]
3	blaZ	blaZ-F: AAG AGA TTT GCC TAT GCT TC blaZ-R: GCT TGA CCA CTT TTA TCA GC	518 bp	[14]
4	icaA	icaA-F: CCTAAC TAA CGA AAG GTA G icaA-R: AAG ATATAG CGA TAA GTG C	1315 bp	[15]
5	icaD	icaD-F: AAA CGT AAG AGA GGT GG icaD-R: GGC AAT ATG ATC AAG ATA C	318 bp	[15]

¹94 °C for 5 min, 30 ×  (94ºC for 30 s, 59 °C for 1 min, 72 °C for 1 min), 72 °C for 10 min, and 4 °C for ∞

²94 °C for 5 min, 37 ×  (94 °C for 1 min, 55 °C for 30 s, 72 °C for 1.5 min), 72 °C for 3.5 min, and 4 °C for ∞

³94 °C for 5 min, 35 × (94 °C for 5 min, 55 °C for 30 s, 72 °C for 30 s), 72 °C for 10 min, and 4 °C for ∞

^4,530 × (92 °C for 45 s, 49 °C for 45 s, 72 °C for 1 min), 72 °C for 7 min, and 4 °C for ∞

Screening of Milk Samples for Methicillin-Resistant Staphylococcus aureus (MRSA)

Isolation and identification of MRSA from the milk was done as above except that cefoxitin (3.5 mg/L) and Aztreonam (75 mg/L) were used for selective enrichment in all the steps [8].

Genotypic Characterization of the Isolates

The isolation of genomic DNA was done using Instagene™ matrix (Bio-Rad) as per the manufacturer’s instruction [11].

Multiplex PCR for the Detection of spa, mecA and pvl

The detection of polymorphic Xr portion of spa gene for identification of S. aureus isolates, mecA gene for confirmation of methicillin resistance and lukF-PV or pvl gene for detection of Panton–Valentine leucocidin was done in a 25 μL reaction using DreamTaq™ Green PCR Master Mix (2X) with 2 μL of DNA purified as above and 2 μL each of Forward and Reverse Primer mix. The 1000 μL of primer mix was made using 25 μL of 100 μM of each primer. The primer sequence and cycling conditions is mentioned in Table 2.

PCR for the Detection of nuc, icaA/icaD and blaZ gene

The detection of nuc, ica (intercellular adhesion) A and icaD and blaZ gene for the presence of thermostable nuclease, biofilm production and penicillinase resistance, respectively was done in a 25 μL reaction using DreamTaq™ Green PCR Master Mix (2×) with 2 μL of DNA purified as above and 2 μL each of Forward and Reverse Primer (10 μM). The primer sequence and cycling conditions is mentioned in Table 2.

Biofilm Formation Ability of the Isolates

The biofilm forming ability of the isolates was studied using following three methods:

Congo Red Agar (CRA) method:An overnight culture of the isolates was adjusted to 0.5 McFarland and 4 µL of it was spot inoculated in the CRA (Brain Heart Infusion Agar with 0.08% Congo red and 5% sucrose) [16]. The plates were incubated 35 °C for 18–24 h. A positive result (biofilm formation) was indicated by the presence of black or pink colonies with a dry crystalline surface (rough colony phenotype) while negative result (non-biofilm formation) was indicated by the presence of smooth, pink colonies.

Microtitre plate (Mtp) assay for biofilm formation: The isolates were grown overnight in Tryptone Soy Broth (TSB) supplemented with 0.25% glucose (TSB-glucose) which was diluted 1:40 in TSB-glucose, and 200 µL of this cell suspension was used per well to inoculate sterile, 96-well tissue culture plates (Tarson). Each isolate was run in triplicate and negative control wells contained broth only. The plate was incubated at 37 °C for 24 h. The wells were gently washed three times with 200 µL of sterile phosphate-buffered saline, air dried in an inverted position, and stained with 0.1% safranin for 10 min. The biofilms were washed once with distilled water and then dried at 37 °C for 15 min. The stain was then released with 200 μL of the destaining solution (50% (v/v) ethanol, 50% (v/v) glacial acetic acid), and quantified by measuring the absorbance at 490 nm (A490) with a microplate reader [17, 18]. The isolates were classified as non-biofilm, weak, moderate and strong biofilm producer [19].

The Tube Method for testing biofilm formation: Determination of biofilm formation on a glass surface was carried out essentially in the same way as of Microtiter plate method, except that glass tubes were used instead of microtiter plates and 5 ml of TSB + 0.25% glucose was inoculated instead of 200 μL of TSB + 0.25% glucose.

Antibiotic Sensitivity Profile of the Isolates

The Antibiotic sensitivity test of S. aureus isolates against 11 antimicrobial agents [20] were done by disk diffusion assay (Table 3) as per the method of CLSI [21].

Table 3.

Panel of Antimicrobial agents for Antibiotic Sensitivity profile of the isolates

S. No	Antimicrobial agent	Disc (µg)	Group	Remark
1	Cefoxitin	30	Cephalosporin	Methicillin-Resistance
2	Chloramphenicol	30	Chloramphenicol
3	Ciprofloxacin	5	Fluoroquinolone
4	Clindamycin	2	Lincosamide
5	Co-trimoxazole	25	Sulfonamide
6	Erythromycin	15	Macrolide
7	Gentamicin	10	Aminoglycoside
8	Linezolid	30	Oxazolidinones
9	Mupirocin	200	Monocarboxylic acid
10	Penicillin	10	Penicillin	Penicillinase
11	Tetracycline	30	Tetracycline

Multiple Antibiotic Resistance (MAR) Index

The MAR index is defined as a/b, where a represents the number of antibiotics to which the isolate was resistant, and b represents the number of antibiotics to which the isolate was exposed.

Result and Discussion

For the treatment, prevention and control of mastitis, the laboratory diagnosis by culture method is still the most reliable method. It is of particular importance for S. aureus as it can be a commensal or mastitis pathogen. Moreover, S. aureus cause mastitis which can extend over the multiple lactation periods of the dairy cow and therefore, is the important reason for dry cow therapy.

In this study, the California Mastitis Test (CMT) was used to screen the herd for subclinical mastitis and the prevalence in dairy cattle was found to be 25.24% (26/103) while no cases (0/13) were found in buffaloes (Table 4). In another similar study in Rewa district, the overall prevalence of subclinical mastitis in dairy cows was found to be 31.40% on animal wise, 7.85% on quarter wise and 2.48% on blind teat wise [22]. Several studies show that there is spatio-temporal distribution of mastitis pathogens including S. aureus which in turn help to inform programs for the successful control and management of mastitis [23, 24].

Table 4.

Prevalence of Mastitis in bovines in the studied population using CMT

S. No	Particular	Cattle	Buffalo
1	Animals screened for mastitis	103	13
2	Subclinical mastitis (SCM)	26	0
3	Prevalence of subclinical mastitis (SCM)	25.24	0

Characterization of Staphylococcus aureus in the Milk

The milk samples (n = 100) were screened for S. aureus using the criteria-salt tolerance at 6.5% NaCl, growth in selective agars (Mannitol salt agar and Baird–Parker agar with egg yolk-tellurite supplement) and standard biochemical tests as shown in Table 5. Unlike the strains of human origin which produce typical yellow colour colonies, the colony of all the isolates were white or off-white in colour which is very typical of isolates of ruminant origin [25]. To ensure the isolates had pathogenicity, the virulence determinants-coagulase, staphylococcal protein, thermostable nuclease, Panton–Valentine leucocidin, biofilm, penicillinase production, mannitol fermentation and haemolysis in sheep blood agar of each isolate was studied by phenotypic and/or genotypic method. These milk samples were also screened for MRSA using cefoxitin and aztreonam as selective enrichment method. This method of selective enrichment is effective for isolation of MRSA.

Table 5.

Phenotypic characterization of Staphylococcus aureus and MRSA in the milk

S.No	Isolates	Growth in MSA	Growth in BPA	G + ve cocci	NV 5 μg	PB 300 U	TC	Haemolysis
								α	α, β	γ
1	Staphylococcus aureus	48	39	18	> 16 mm	≤ 10 mm	18	7	4	7
2	MRSA	35	20	10	> 16 mm	≤ 10 mm	6	3	0	3

Haemolysis in blood agar represents an important criterion for rapid presumptive identification of S. aureus isolates of bovine origin [25, 26]. The variation in haemolysis by the isolates are shown in Table 5. The phenotypic alpha (α) and beta (β) haemolysis shows geographical variation [27]. Our findings are not consistent with previous findings where around 80% of bovine S. aureus isolates showed α and/or β haemolysis [28]. In another study all the S. aureus isolates from clinical mastitis showed α haemolysis as determined by hot–cold lysis [22]. The α haemolysis is very characteristic of isolates from ruminant origin. The hemolysis pattern in MRSA strains seen in our study is in agreement with the study in which 62.7% showed non-haemolytic, 23.2% showed α and β haemolysis, 12.5% showed β-haemolysis and only 1.6% showed α-haemolysis [29]. Our study is in agreement with the studies which suggests that haemolysis is not a very sensitive criteria of the isolates from ruminant origin so the use of SBA for the isolation of pathogenic S. aureus using haemolysis as criterion should be done in conjunction with coagulase test [26, 30].

In this study, all the 18 S. aureus and 6 MRSA strains were positive for nuc and spa gene (Figs. 1 and 2). Two major roles have been proposed for the nuclease during infection, the disruption of neutrophil extracellular traps (NETs) and modulating biofilm development. It has been shown that the expression of nuclease results in reduced biofilm formation in vitro, while a nuc mutant displays enhanced biofilm formation [31]. The second role proposed for nuclease during infection is the evasion of NETs. NETs are a newly discovered killing mechanism utilized by neutrophils against bacterial infections. Activated neutrophils secrete nuclear DNA at the site of infection to entrap bacteria and enhance bacterial killing. Nuclease is able to degrade NETs and promote resistance against killing by neutrophils [32].

Fig. 1.

Agarose gel electrophoresis analysis showing nuc gene PCR amplification products (279 base pair) of Staphylococcus aureus. M = 100 base pair (bp) ladder, Lane1–6 nuc positive Staphylococcus aureus isolates

Fig. 2.

Agarose gel electrophoresis analysis showing multiplex PCR amplification products for the detection of the spa, mecA, pvl gene of Staphylococcus aureus. The amplification of spa gene show bands ranging from 300 to 400 bp. M = 100 base pair (bp) ladder, Lane 1, 3 spa and mecA positive MRSA isolates, Lane 2—negative control, Lane 4–6 spa positive Staphylococcus aureus

In this study, the presence of Panton–Valentine Leukocidin (PVL) was not detected in any of the strains by PCR. The presence of PVL is considered a rare finding in bovine isolates but is an important virulence determinant for isolates of human origin [33–35]. Although one study identified pvl gene in more than 50% of isolates from bovine mastitis [36] while another study detected pvl toxin gene in 41.5% of the isolates collected from three Chinese Holstein herds [37]. In India, the pvl gene was detected in 41.6–82.05% of the S. aureus isolates from bovine milk [38–40].

The methicillin resistance is due to the production of PBP2a or penicillin-binding protein 2a (target modification), which is encoded by the mecA gene located on the mobile element of the staphylococcal chromosome cassette mec (SCCmec). The presence of these genes in MRSA makes them resistant to multiple antibiotics, particularly all ß-lactam antibiotics. It has been suggested that cattle may serve as a source of MDR and new MRSA strains in humans [41, 42]. In this study, 6% prevalence of MRSA was reported in the bovine milk after genotypic confirmation by mecA PCR (Fig. 2) (Table 6). In India, the prevalence of MRSA in bovine milk has been reported to vary widely between 13.1 and 48.84% [35, 39, 43, 44].

Table 6.

Genotypic characterization of Positive Isolates

S.No	Sample	nuc	spa	mecA	pvl	blaZ
1	Staphylococcu aureus	18	18	0	0	18
2	MRSA	6	6	6	0	6

Biofilm Forming Ability of Staphylococcus aureus Strains

The formation of biofilm is a complex and dynamic three step process-initial attachment, biofilm formation and maturation, biofilm dispersal. The biofilm formation ability of all the S. aureus strains (n = 18) and MRSA strains (n = 6) were studied by three methods-Congo Red Agar (CRA), Microtiter plate (using Safranin) and tube method [17, 18]. The results are depicted in Table 7. In this study, a total of 33.33% S. aureus and 66.66% MRSA strains were slime producers by CRA method which is a not a quantitative assay because it is based on a subjective chromatic evaluation. Based on Microtiter plate method, all the S. aureus and MRSA strains were classified as weak or moderate biofilm producers. Multiple studies found that majority of S. aureus isolates from bovine mastitis cases can form biofilm in vitro by this assay [45–47]. In another study, half of S. aureus isolates from dairy cows with subclinical mastitis were able to produce biofilm by the CRA method [48, 49].

Table 7.

Biofilm forming ability of Staphylococcus aureus strains

S.No	Detection of biofilm	Staphylococcus aureus	MRSA (%)
1	Congo Red Agar (CRA)	33.33%	66.66
2	Micro-titer plate Method (MTP)	61.66%	83.33
3	Tube Method (TM)	0	50
4	icaA gene	100%	100
5	icaD gene	100%	100

The most important genes involved at different stages of the biofilm are ica, bap and agr genes. In this study all the isolates were found to carry icaA and icaD genes (Table 7 and Fig. 3). The bap gene has been found exclusively in isolates from bovine mastitis [17]. The intercellular gene cluster adhesion operon (ica) has been found in 40% of S. aureus isolates from bovine mastitis by analyzing their biofilm forming abilities within the microtiter assay and then sequencing the isolates [50]. However, whether the isolates carrying the ica genes actually produce biofilm in vitro, depends on the biofilm assay. Some studies found that even if the isolates carried the ica genes, not all of the isolates produced biofilm in the microtiter plate [3] and that some isolates would form black colonies (indicating slime formation) when grown on CRA plates but not necessarily form biofilm in the microtiter assay [15]. A study found that over 90% of isolates carried icaADBC genes and of these 25% carried the bap genes. When the isolates were positive for both icaADBC and bap, they were strong biofilm producers in vitro, however, when only positive for icaADBC, they produced less biofilm. However, in other studies, the bap gene was not found at all in S. aureus isolates from bovine mastitis cases [3, 4].

Fig. 3.

Agarose gel (1.5%) electrophoresis analysis showing PCR amplification product of 1315 and 381 base pair (bp) for the detection of icaA and icaD gene of biofilm positive isolates. M = 100 base pair ladder, Lane 1—negative control, Lane 2–4 icaD and lane 5–7 icaA positive isolates

Penicillin Resistance

All the S. aureus and MRSA strains were resistant to penicillin both phenotypically by zone-edge test and genotypically by blaZ gene PCR (Fig. 4). This is in agreement with the other studies which found 85–100% presence of penicillin resistance isolates in bovine mastitis [39, 43, 51, 52]. The Penicillin resistance is an important virulence determinant in mastitis caused by S. aureus in bovines. The presence of β-lactamase (blaZ) gene is shown to influence the prognosis of bovine mastitis. Surprisingly, successful treatment rates are low when mastitis is caused by a penicillin-resistant strain, even when the treatment is not based on antibiotics of the β -lactam family. Since penicillin-resistance determinants are borne by pathogenicity islands, this phenotype may be accompanied by other virulence genes, which could explain this phenomenon [5, 6].

Fig. 4.

Agarose gel (1.5%) electrophoresis analysis showing PCR amplification product of 518 base pair (bp) for the detection of blaZ gene of Staphylococcus aureus. M = 100 base pair ladder, Lane 1–18 blaZ positive isolates

Antibiotic Sensitivity Profile of Staphylococcus aureus

A total of 18 S. aureus isolates were studied for antimicrobial susceptibility against the 11 different categories of antimicrobial agents by disk diffusion assay. Except for the penicillin resistance, at least 70% of S. aureus strains were sensitive to all the antimicrobial agents tested (Fig. 5). Only 33.33% of the strains had the MAR index above 0.2. The MAR index greater than 0.2 implies the origin of the isolate from a high-risk source of contamination where antibiotics are often used [53, 54].

Fig. 5.

Susceptibility pattern of the Staphylococcus aureus isolates to the antimicrobial agents in the disk diffusion assay

A third of the isolates were found to be pan-susceptible to all the antimicrobial agents except for penicillin used in this study. The pan-susceptible nature of the S. aureus from bovine mastitis milk has been widely reported [55–58]. It has been proposed that the dairy industry does not strongly promote the emergence of antibiotic-resistant S. aureus, despite the widespread use of antibiotics for treating bovine mastitis. It has been speculated that this reflects the ability of bovine strains to invade and survive within bovine mammary epithelial cells, a niche which may have a markedly reduced antibiotic selective pressure [59].

All the 6 MRSA isolates can be designated as multi-drug resistant (MDR) by virtue of being MRSA (Fig. 6) and had a MAR index of ≥ 0.2.

Fig. 6.

Susceptibility pattern of the MRSA isolates to the antimicrobial agents in the disk diffusion assay

In one Russian study the isolates (n = 21) were resistant to erythromycin (100%), gentamicin (75%), penicillin (62.5%), and ciprofloxacin (25.5%). However, all the isolates were sensitive to Oxacillin (1 μg) in disk diffusion assay [60]. Antibiotic sensitivity profiling revealed the majority of the strains (n = 24) to be multidrug resistant and eleven strains showed reduced susceptibility to vancomycin (MICs = 2 μg/ml). In another study from Southern India the isolates (n = 39) showed high resistance rates towards clindamycin (76.92%), erythromycin (64.10%), ampicillin (56.41%), tetracycline (41.02%) and ciprofloxacin (35.89%) whereas low resistance was seen against rifampicin (20.51%) and gentamicin (15.38%). All strains (n = 39) were oxacillin sensitive, but 19 strains were positive for the mecA gene, which revealed the occurrence of oxacillin susceptible mecA positive strains (OS-MRSA) for the first time from India [39]. While the antimicrobial susceptibility study in Bangladesh revealed that 79.3% S. aureus strains were resistant to at least one antimicrobial, 49.0% to two or more antimicrobials, and clinical isolates showed more resistance to all tested antibiotics. The highest resistance rate was found to oxytetracycline, and no resistance to ceftriaxone and azithromycin [61].

In recently published systematic review and meta-analysis of antimicrobial resistance of S. aureus isolated from bovine mastitis, the highest overall prevalence of resistant S. aureus was against penicillin, followed by clindamycin, erythromycin, and gentamicin. Ceftiofur and cephalotin presented the lowest overall prevalence of antimicrobial resistance. The AMR to almost all the antimicrobials evaluated presented an increasing pattern over time, more apparent from 2009 onwards. The antimicrobials with a higher increase in their AMR prevalence over time were clindamycin, gentamycin, and oxacillin. Africa, Asia and Latin America were the continents with higher AMR to most compounds included in this study. No differences in AMR were detected regarding the clinical origin of the isolates (subclinical vs clinical mastitis) for almost all antibiotics evaluated [62].

Conclusion

Mastitis is a costly disease not only to the dairy farmer but also to the dairy industry and consumer. Depending upon the strain, the mastitis caused by S. aureus can be peracute to chronic and therefore, may require treatment during lactation and dry period as well. The presence of methicillin and penicillin resistance in the biofilm forming S. aureus strains will complicate the treatment outcomes as the prevention and control of mastitis is mainly antibiotic based in the region. It could also have One Health implications. Further study of these S. aureus and MRSA strains using whole genome or by traditional method for spa typing, Multi-Locus Sequence Type (MLST) and SCCmec typing (for MRSA strains) will help to understand their population structure and the zoonotic potential. The MIC values of the antibiotics used in the study will provide the better understanding about the Epidemiological cut-off (ECOFF) values of the strains which would be more helpful in treatment decisions.

Authors Contributions

SR carried out sample collection, lab experiments, and writing of the manuscript. NS and AS gave design, supervision, and revision of the manuscript. SS and PKS supervised the research work and revised the article. AKN and RR contributed in lab experiments and manuscript writing. All authors have read and approved the final manuscript.

Funding

The authors thank the MP Council of Science and Technology, Bhopal, MP, India (F. No.: R and D(BS)/17–18/19) for their support in the form of research grant.

Data Availability

All datasets are presented in the main manuscript.

Declarations

Conflict of interest

Authors do not have any competing interest.