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Evaluation of Methicillin Resistance in Field Isolates of Staphylococcus aureus: An Emerging Issue of Indigenous Bovine Breeds

PJZ_55_2_831-842

Evaluation of Methicillin Resistance in Field Isolates of Staphylococcus aureus: An Emerging Issue of Indigenous Bovine Breeds

Nauman Zaheer Ghumman, Muhammad Ijaz*, Arslan Ahmed, Muhammad Umar Javed, Iqra Muzammil and Ahmed Raza

Department of Veterinary Medicine, University of Veterinary and Animal Sciences, Lahore

ABSTRACT

The emergence of resistant strains of Staphylococcus aureus particularly methicillin-resistant S. aureus (MRSA) confers overwhelming economic losses to the global dairy industry. The current study was planned to investigate the S. aureus and MRSA associated subclinical mastitis in 345 milk samples (Cattle n=173, Buffalo n=172) collected from indigenous bovines of district Rawalpindi, Pakistan. The milk samples were screened for S. aureus and the confirmed isolates were subjected to disc diffusion test, PCR, and SDS-PAGE analysis for the confirmation of methicillin resistance. The farm-associated and individual animal-associated risk factors were analyzed to check the association with S. aureus and MRSA-associated subclinical mastitis. The results revealed an overall molecular prevalence of 28.70% for S. aureus among which MRSA-associated mastitis was found 47.62% prevalent. The SDS-PAGE analysis depicted the presence of a 78KDa protein band specific for PBP2a protein in MRSA. The comparative risk factor analysis showed significant variation among risk factors associated with S. aureus and MRSA-induced mastitis. The phylogenetic analysis of MRSA mecA gene showed a high resemblance of the study isolates with MRSA isolates of the USA, Turkey, India, Africa, and Brazil. This is the first study regarding molecular characterization and phylogenetic analysis of MRSA isolates from the study area.


Article Information

Received 16 March 2022

Revised 25 April 2022

Accepted 18 May 2022

Available online 21 June 2022

(early access)

Published 02 January 2023

Authors’ Contribution

NZG and MI conceptualization, methodology, data curation, investigation, resources, project administration, funding acquisition, writing review, and editing. MUJ and AA methodology, data curation, investigation, software, visualization, validation. IM writing original draft. AR writing review, and editing.

Key words

MRSA, Indigenous bovine, phylogenetic analysis, comparative risk factor analysis, SDS-PAGE

DOI: https://dx.doi.org/10.17582/journal.pjz/20220316080346

* Corresponding author: mijaz@uvas.edu.pk

0030-9923/2023/0002-831 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).



INTRODUCTION

Worldwide milk production is mainly dominated by dairy cows but water buffaloes also account for 13.2% of non-cattle milk according to Food and Agriculture Organization (Giovanni et al., 2020). In Pakistan, the major indigenous dairy breeds include Sahiwal cattle and Nili-Ravi buffalo (Afzal et al., 2007) among which the Nili-Ravi breed of buffalo is producing about 75% of all the milk produced in the country (Sharif et al., 2009). Among the major economically devastating health hazard of dairy animals, mastitis comes on top of the list (Ji et al., 2020). Staphylococcus aureus, due to its low cure rate, transmission between animals during milking, ability to reside in the mammary gland leading to subclinical infection, and theemergence of antibiotics resistant strains, is considered a major pathogen deteriorating both milk quality and production (El-Ashker et al., 2020; Monistero et al., 2018). The pathogenicity of S. aureus comprises a combination of its invasive property, pathogenicity-mediated genes, immune evasion, and antibiotic resistance which negates the therapeutic approaches (Chua et al., 2014).

The diverse genetic abilities of S. aureus have led to the emergence of strains that resist the β-lactam antibiotics, of which methicillin-resistant S. aureus (MRSA) is of paramount importance (Abdeen et al., 2021; Zaatout and Hezil, 2021). The methicillin resistance in S. aureus is primarily governed by the acquisition of the mecA gene which encodes penicillin-binding protein 2a (PBP2a) having a lower affinity for methicillin and all other β-lactam antibiotics (Zhao et al., 2021; Javed et al., 2021). MRSA is an emerging zoonotic bacteria of veterinary and public health importance (Algammal et al., 2020b) and has been listed by the World Health Organization as a pathogen of utmost priority for further research and treatment (Shrestha et al., 2021). The prevalence of MRSA in livestock is higher in Asia as compared to other continents (Zaatout and Hezil, 2021) which might be due to inadequate accomplishment of standard farm and milking hygiene practices with indiscriminate and undue usage of antibiotics prescribed without veterinary consultation in underdeveloped and developing countries (Kayitsinga et al., 2017).

The rapid determination of S. aureus isolates, either to be methicillin-resistant or not, is critically important for both treatment and control measures. For the identification of bacteria various phenotypic and genotypic methods have been used (Elhaig and Selim, 2015). Phenotypic methods like E-tests, micro-dilution, and oxacillin disc diffusion tests have been used for MRSA detection but some of these techniques are not specific and may overestimate the MRSA prevalence leading to false-positive results (Olowe et al., 2013). In addition, these methods are also not reliable to distinguish between MRSA and methicillin-sensitive S. aureus (MSSA). However, the differentiation of MRSA from MSSA can be done based on the mecA gene, an important marker for the indication of methicillin-resistance in S. aureus, which is present in resistant isolates and absent in susceptible ones (Baddour et al., 2007). The mecA gene confirmation can be done using molecular methods like PCR which is considered reliable and the ‘Gold Standard’ test for MRSA detection. Moreover, to evaluate the protein expression of MRSA and MSSA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis can be performed. The SDS PAGE of whole-cell MRSA revealed the separation of mutant PBP2a protein at 78kDa, which is exclusively present in MRSA isolates (Doan et al., 2013). PBP2a protein is encoded by the mecA gene in the chromosomal cassette of the foreign DNA region that is integrated into the MRSA chromosome (Senna et al., 2003).

The current study was designed to estimate the phenotypic and molecular prevalence of S. aureus and MRSA associated subclinical mastitis along with a comparative analysis of associated risk factors in indigenous breeds of Pakistan. In addition, molecular characterization of local MRSA isolates was performed and their genetic relatedness with the reported MRSA isolates was also checked. Moreover, protein expression of MRSA and MSSA was also evaluated using SDS-PAGE.

MATERIALS AND METHODS

Sampling design

The study was set to investigate the S. aureus and MRSA-associated subclinical mastitis in indigenous dairy cattle and buffalo located in three tehsils (Rawalpindi, Gujar Khan, and Taxila) of district Rawalpindi, Pakistan (Fig. 1), between the period of December 2020 to July 2021. A total of 345 bovine milk samples (n=173 cattle; n=172 buffalo from each tehsil) were collected aseptically as per guidelines of the National Mastitis Council, USA (Reyher and Dohoo, 2011). The milk samples were examined for subclinical mastitis by California mastitis test (CMT) and positive samples were immediately dispatched to Medicine Research Laboratory, University of Veterinary and Animal Sciences, Lahore, maintaining the cold chain at 4°C.

 

The information regarding animal-based and herd-based management practices was collected on a predesigned data capture form for the comparative analysis of assumed risk factors associated with S. aureus and MRSA-associated subclinical mastitis in indigenous dairy animals. The purpose was to pinpoint the potential risk factors more significantly associated with MRSA-associated mastitis compared to S. aureus-associated mastitis.

Isolation and identification of S. aureus

The bacterial growth was isolated from milk samples by primarily streaking on 5% sheep blood agar followed by an incubation of 24-48 hours at 37°C. The bacterial colonies were subjected to Gram staining and identified by standard biochemical tests (catalase and coagulase) as per recommendations of Bergey’s Manual of Determinative Bacteriology (Holt, 1977). Presumptive S. aureus colonies were further streaked on mannitol salt agar (MSA) for the confirmation of S. aureus (Altaf et al., 2019). Molecular confirmation of S. aureus was done by amplification of nuc gene using primers as (F= GCG ATT GAT GGT GAT ACG GTT, R= AGC CAA GCC TTG ACG AAC TAA AGC) and conditions as initial denaturation 94°C for 5 min, followed by 35 cycles and final denaturation at 94°C for 30s, annealing 55°C for 30s, with initial extension at 72ºC for 1 min and final extension for 10-min at 72°C as reported by Louie et al. (2002). The phenotypic and genotypic confirmed S. aureus isolates were further analyzed for the evaluation of methicillin resistance.

Phenotypic identification of MRSA and MSSA isolates

Methicillin susceptibility of confirmed S. aureus isolates was assessed by performing a disc diffusion assay according to the guidelines of the Clinical Laboratory Standards Institute (CLSI, 2019). The (0.5 McFarland) adjusted bacterial suspension of S. aureus was streaked on Muller Hinton agar (MHA). The oxacillin (1µg) and cefoxitin (30 µg) antibiotics discs (Bioanalyse ® Turkey) were aseptically applied using a sterile disc dispenser and subjected to incubation at 37°C for 20-24 h. The zones of inhibition were compared with standards, provided by CLSI (2019). The isolates exhibiting inhibition zone of size ≤17mm and ≤21 mm respectively for each disc were considered MRSA while isolates having greater zones were considered as MSSA.

Molecular confirmation of mecA gene

To evaluate the molecular confirmation of methicillin resistance, the genomic DNA was extracted using a bacterial DNA extraction kit (Vivantis Technologies Sdn. Bhd, Malaysia) following the manufacturer’s instructions. All phenotypically confirmed MRSA isolates were subjected to conventional PCR (Scilogex TC1000-G) for the amplification of 310bp fragment of mecA gene using primers (P1: 5′-TGGCATTCGTGTCACAATCG-3′ and P2: 5′- CTGGAACTTGTTGAGCAGAG-3′) reported by Galdiero et al. (2003) using initial denaturation at 95ºC for 5 min followed by 35 cycle with final denaturation of 95ºC for 30s with annealing at 58ºC for 30s and extension at 72ºC for 30s and 72ºC for 10 min, respectively. The PCR products obtained after amplification were subjected to gel electrophoresis at 120 volts using 200 mAmp and run electrophoresis for 30 min using a gel electrophoresis apparatus (Cleaver Scientific Ltd). The resultant bands were visualized on a UV trans-illuminator along with standard 100bp DNA ladder (Bioshop® Canada Inc.) as a molecular weight marker. The isolates showing bands on 310bp were considered MRSA (mecA positive) and those found negative for bands were assumed MSSA (mecA negative) isolates.

Sequencing and phylogenetic analysis

The mecA positive bands, after the gel purification, were shipped to a renowned sequencing lab for sequencing. The resulted obtained sequences of study isolates were evaluated using the basic local alignment search tool (BLAST) of NCBI (National Centre for Biotechnology Information) to check the similarity of the study isolate’s nucleotide sequence with other S. aureus isolates showing methicillin-resistance reported in various countries of the world. The multiple sequence alignment was done using the Clustal W method by using BioEdit software (Version 7.2.5). A phylogenetic tree was then constructed based on sequence distance using neighborhood joining methods on MEGA-X software.

Protein expression by SDS PAGE

The whole-cell protein profile of mecA positive and mecA negative isolates was analyzed for general protein expression by SDS PAGE analysis using (omni PAGE mini vertical Protein electerophoresis by Cleaver Scientific Ltd) apparatus. The difference between proteins expression of both isolates was noted specifically for the expression of PBP2a protein at 78kDa present in MRSA and absent in MSSA isolates (Hartman and Tomasz, 1986). The SDS PAGE analysis was performed according to procedures followed by (Berber et al., 2003). The protein bands were visualized with fluorescent light and relative flow (Rf) values of each sample were measured. Molecular weights of all isolates were determined through a standard curve made between Rf values and logarithmic molecular weights of known protein marker bands (Fig. 4). The presence of protein bands was assessed and compared with each other for differentiation between MRSA and MSSA isolates.

Data analysis

The phenotypic and genotypic prevalence of S. aureus, MRSA, and MSSA isolated from bovine milk samples was estimated using pre-described formula reported by (Thrushfield, 2013). The risk factors related to S. aureus and MRSA-associated mastitis were initially analyzed and compared using the chi-square method and multivariable logistic regression technique at 5% probability. All the statistical analysis was performed using SPSS statistical computer program.

RESULTS

Prevalence of S. aureus and MRSA associated subclinical mastitis

The current study revealed that an overall prevalence of subclinical mastitis (SCM) in district Rawalpindi was 52.75% based on CMT. While the S. aureus-associated SCM were found 28.70% prevalent. The phenotypic evaluation of methicillin resistance by oxacillin/cefoxitin disc diffusion test revealed that 42.42% S. aureus isolates were found resistant (MRSA) while 57.58% isolates were sensitive (MSSA) to antibiotic discs. Molecular analysis of phenotypically positive MRSA revealed that 47.62% of isolates were found positive for the mecA gene (MRSA) while the rest of the isolates were found negative for the mecA gene (MSSA) as shown in (Table I). The genotypic results of the current study showed that among indigenous bovines, the cattle were more prone to S. aureus and MRSA-associated mastitis compared to buffalo (Table I). The prevalence of MRSA associated SCM was found more in tehsil Gujar khan (58.82%) followed by tehsil Rawalpindi (42.86%) and tehsil Taxila (36.36%) as shown in (Table II). The results depicted that phenotypic methods like bacterial culture and disc diffusion test can overestimate the infection load of S. aureus and MRSA in bovine mastitis and molecular methods like PCR and sequencing are more specific and reliable methods for methicillin resistance screening.

Comparative risk factor analysis

The relationship of various animal-based and herd-based assumed risk factors with the occurrence of S. aureus and MRSA-associated mastitis was analyzed and compared using statistical tools. A significant variation between an association of risk factors with S. aureus and MRSA-induced mastitis was observed.

The results revealed that among animal-based risk factors, physiological status of animals, mastitis history, and teat lesion were found significantly (p<0.05) associated with both S. aureus and MRSA induced SCM (Table III). The parity (p=0.01) was found significantly associated with S. aureus but not with MRSA-associated mastitis (Table III). It was also noted that animals having teat lesions were 2.215 times more prone to S. aureus-associated mastitis (Table VI). The results further showed that usage of antibiotics (p=0.02) was significantly associated with MRSA-associated mastitis but was found non-significant risk factor for S. aureus-based mastitis. Animals, in which beta-lactam antibiotics usage was common, were 3.054 times more prone to MRSA-associated mastitis compared to S. aureus-associated mastitis (Table V).

The data analysis of herd-base risk factors (Table IV) showed that herd size, hygiene during milking, milking person hygiene, and milking methods were found to be significantly (p<0.05) associated with both S. aureus and MRSA associated mastitis while the presence of vectors and other livestock species were found to be non-significant (p>0.05) risk factors for S. aureus-associated mastitis and MRSA associated mastitis as shown in (Table IV). The final logistic regression model showed that animals with hand milking method and poor milking person hygiene were 3.134 and 2.825 times more prone to MRSA infection (Table V).

 

Table I. Prevalence of subclinical mastitis (SCM), methicillin resistant (MRSA) and methicillin sensitive (MSSA) Staphylococcus aureus from indigenous breeds of cattle and buffalo.

Animal

No. of animals

SCM (%)

S. aureus

Phenotypic (%)

Genotypic (%)

MRSA

MSSA

MRSA

MSSA

Cattle

173

97/173 (56.07)

57/173 (32.95)

23/57 (40.35)

34/57 (59.65)

12/23 (52.17)

11/23 (47.83)

Buffalo

172

85/172 (49.42)

42/172 (24.42)

19/42 (45.24)

23/42 (54.76)

8/19 (42.11)

11/19 (57.89)

Total

345

182/345 (52.75)

99/345 (28.70)

42/99 (42.42)

57/99 (57.58)

20/42 (47.62)

22/42 (52.38)

 

MRSA, methicillin resistant Staphylococcus aureus; MSSA, Methicillin Sensitive Staphylococcus aureus; SCM, Sub clinical mastitis.

 

Table II. Prevalence of SCM, MRSA and MSSA in indigenous cattle and buffalo breeds of tehsil Rawalpindi, Gujar khan and Taxila.

Area

Animal Spp.

No. of animals

SCM (%)

S. aureus

Phenotypic (%)

Genotypic (%)

MRSA

MSSA

MRSA

MSSA

Rawalpindi

Cattle

58

32(55.17)

19(32.76)

08(42.11)

11(57.89)

04(50.00)

04(50.00)

Buffalo

57

28(49.12)

12(21.05)

06(50.00)

06(50.00)

02(33.33)

04(66.67)

Total

115

60(52.17)

31(26.96)

14(45.16)

17(54.84)

06(42.86)

08(57.14)

Gujar Khan

Cattle

58

36(62.07)

20(34.48)

09(45.00)

11(55.00)

06(66.67)

03(33.33)

Buffalo

57

33(57.89)

14(24.56)

08(57.14)

06(42.86)

04(50.00)

04(50.00)

Total

115

69(60.00)

34(29.57)

17(50.00)

17(50.00)

10(58.82)

07(41.18)

Taxila

Cattle

57

29(50.88)

18(31.58)

06(33.33)

12(66.66)

02(33.33)

04(66.66)

Buffalo

58

24(43.38)

16(27.59)

05(31.25)

11(68.75)

02(40.00)

03(60.00)

Total

115

53 (46.09)

34(29. 57)

11(32.35)

23(67.65)

04(36.36)

07(63.64)

Total animals

345

182(52.75)

99(28.70)

42(42.42)

57(57.58)

20(47.62)

22(52.38)

 

For abbreviation see Table I.

 

Table III. Comparative analysis of animal-based risk factors associated with Staph and MRSA-associated subclinical mastitis.

Variable

Variable

level

S. aureus associated mastitis

MRSA associated mastitis

Total (n=345)

Positive (%)

p value

Total (n=345)

Positive (%)

p value

Body condition

Healthy

166

43 (25.90)

0.27

122

05 (04.10)

0.20

Thin

88

24 (27.27)

127

06 (04.72)

Emaciated

91

32 (35.16)

96

09 (09.37)

Parity

First

30

09 (30.00)

0.01*

32

02 (06.25)

0.77

Second

101

19 (18.81)

102

06 (05.88)

Third

123

39 (31.71)

128

09 (07.03)

> Third

84

32 (38.10)

83

03 (03.61)

Physiological status

Lactating

280

94 (33.57)

0.00*

184

15 (08.15)

0.04*

Dry

65

05 (07.69)

161

05 (03.10)

Mastitis history

Present

211

72 (34.12)

0.00*

198

16 (08.08)

0.03*

Absent

134

27 (20.15)

147

04 (02.72)

Use of β-lactam antibiotics

Frequent use

203

60 (29.56)

0.67

177

15 (08.47)

0.02*

Seldom use

142

39 (27.46)

168

05 (02.98)

Treatment person

Veterinarian

141

34 (24.11)

0.11

199

09 (04.52)

0.23

Self

204

65 (31.86)

146

11 (07.53)

Teat lesion

Present

227

77 (33.92)

0.00*

176

15 (08.52)

0.02*

Absent

118

22 (18.64)

169

05 (02.96)

 

*Indicate significantly associated risk factors.

 

Table IV. Comparative analysis of herd-based risk factors associated with Staph and MRSA-associated subclinical mastitis.

Variable

Variable level

S. aureus associated mastitis

MRSA associated mastitis

Total (n=345)

Positive (%)

p value

Total (n=345)

Positive (%)

p value

Milking person hygiene

Yes

159

36 (22.64)

0.02*

164

05 (03.05)

0.03*

No

186

63 (33.87)

181

15 (8.29)

Presence of vectors

Present

135

43 (31.85)

0.29

207

13 (6.28)

0.63

Absent

210

56 (26.67)

138

07 (5.07)

Hygiene during milking

Good

142

26 (18.31)

0.00*

146

04 (2.74)

0.03*

Poor

203

73 (35.96)

199

16 (8.04)

Herd size

Small

115

47 (40.87)

0.00*

94

11 (11.70)

0.006*

medium

129

33 (25.58)

120

07 (05.83)

large

101

19 (18.81)

131

02 (01.53)

Milking methods

Hand milking

169

58 (34.32)

0.02*

148

13 (8.78)

0.04*

Machine method

176

41 (23.29)

197

07 (3.55)

Other livestock species

Present

199

60 (30.15)

0.48

180

11 (06.11)

0.79

Absent

146

39 (26.71)

165

09 (05.45)

 

*indicate significantly associated risk factors.

 

Characterization of MRSA mecA gene

After sequencing, one representative isolate was selected each for cattle and buffalo population of all three tehsils, and the BLAST and CLUSTAL W alignment tool was used to align and compare these local isolates nucleotide sequences with already reported mecA gene sequences. The local isolates revealed up to 99% homology with MRSA isolates reported from the USA, Turkey, India, and Brazil. After alignment, the similarity and significant variations among already reported sequences and current study sequences were explained by constructing a phylogenetic tree.

 

Table V. Risk factors included in final logistic regression model for MRSA-associated mastitis.

Variable

Response

OR

95% C.I

S.E

p value

Mastitis history

Present

2.92

1.056-8.035

0.518

0.039

Absent

1

Use of β-lactam antibiotics

Frequently

3.054

1.015-9.184

0.562

0.047

Seldom

1

Milking person hygiene

Poor

2.825

1.132-7.054

0.467

0.026

Good

1

Milking method

Hand

3.134

1.042-9.425

0.562

0.042

Machine

1

 

Table VI. Risk factors included in final logistic regression model for S. aureus-associated mastitis.

Variable

Response

OR

95% C.I

S.E

p value

Mastitis history

Present

1.952

1.191 - 3.198

0.252

0.008

Absent

1

Teat lesion

Present

2.215

1.312 - 3.739

0.267

0.003

Absent

1

 

The phylogenetic tree, constructed by using the neighbor-joining method, showed that the study isolates clustered together exhibiting high similarity among themselves. All isolates showed similarity and making in group with all other countries isolates except Brazil (Accession no: KF058902) which is out grouped and showing significant variations in nucleotide pattern to local study isolates (Fig. 2). Furthermore, the phylogenetic tree also reported current study isolates showed more evolutionary relationships among themselves as compared to other reported mecA gene sequences from NCBI.

SDS PAGE analysis

The protein profile of both mecA positive and mecA negative isolates was compared by SDS PAGE using 12.5% polyacrylamide resolving gel. The molecular weight of protein bands produced by MRSA isolates ranges from 26.90KDa to 122.38KDa along with a characteristic band of 78KDa indicative of PBP2a which is present in MRSA and not seen in MSSA. Moreover, some other proteins bands were found in MRSA and absent in MSSA isolates. The comparative protein profile reveals a significant difference between MRSA and MSSA isolates as shown in Figure 3.

 

 

 

DISCUSSION

Bovine mastitis caused by S. aureus is a major cause of milk production losses in the Asian buffalo and cattle populations (Badua et al., 2020). S. aureus is a leading pathogen in the causation of clinical, sub-clinical, and chronic mastitis in bovines (Aqib et al., 2017). The current study revealed that S. aureus associated subclinical mastitis was prevalent in 32.95% of cattle and 24.42% of buffaloes with an overall prevalence of 28.70%. The pathogen is involved in one-third of subclinical and clinical mastitis cases in the cattle population (Li et al., 2017). The current results were supported by the findings of various other researchers who reported an overall prevalence of 28%-33% in various countries (Abo-Shama, 2014; Pamuk et al., 2012; Saka et al., 2018; Sharma et al., 2011). However, contrary to the findings of the current study, some reports depicting the higher prevalence rates of S. aureus have also been documented (Abera et al., 2013; Ganai et al., 2016). The wide-ranging dissemination of this pathogen on the udder and teats surface along with survival inside the mammary gland is responsible for the higher prevalence of S. aureus induced subclinical mastitis in bovines (Ganai et al., 2016).

In the last few decades, MRSA has become a major threat having zoonotic potential, and is of central importance for public health (Vandenesch et al., 2003). Various reports have been published on the confirmation of MRSA in dairy animals and related products in various countries. The MRSA prevalence of 47.62% found in the current study was close to the findings reported in China (47.6%), Ethiopia (42.9%), and Egypt (35.7%), However, a relatively closer prevalence has also been reported in Pakistan (34%) (Algammal et al., 2020a; Aqib et al., 2017; Girmay et al., 2020; Yang et al., 2020). Lack of hygienic measures and standard pre and post-milking practices, environmental contamination, undue usage of antibiotics without consulting the veterinarian, and use of hand milking method instead of machine milking in indigenous cattle and buffalo are the prime reasons which may be responsible for the higher prevalence of MRSA in the current study. While the lower prevalence from the Philippines (23.08%), Iran (20%), China (19%), Germany (16.7%), and Turkey (15.89%) has also been reported (Badua et al., 2020; Buyukcangaz et al., 2014; Havaei et al., 2015; Spohr et al., 2011; Vandenesch et al., 2003; Zhang et al., 2016). Contrary to the findings of the current study, a very low prevalence of 0.46% in China (Li et al., 2017), 9% and 13.1% in India (Kumar et al., 2010; Venugopal et al., 2019), 11.57% in Iran (Khazaie and Ahmadi, 2021), and 6.9% in Nepal (Shrestha et al., 2021) has also been documented from bovine mastitis. The higher prevalence (42.10%) of MRSA in indigenous dairy buffaloes might be due to hand milking practices and the lack of hygienic measures adopted in backyard buffalo farming (Badua et al., 2020). The discrepancies in overall MRSA prevalence in various reports might be due to variations in sampling strategies, sample size, geographical location of the study area, seasons, and farming system (backyard farming or commercial farming) (Girmay et al., 2020; Klibi et al., 2018).

The current study revealed that S. aureus-associated mastitis prevalence was significantly linked to the parity of the cow. Present study findings were in line with studies reported by Abrahmsén et al. (2014) and Tesfaye et al. (2019) and in contrast to the findings of Muzammil et al. (2021) that the risk of S. aureus associated mastitis significantly increased with the parity. The increased incidence of S. aureus-associated mastitis with parity could be explained in part by the probability of infection increases with age and parity. Furthermore, the greater the parity, or the number of times the cow has calved, the greater the chances of the pendulous udder and a mastitis history (Katsande et al., 2013). It has also been reported that cows with the most pendulous quarters tend to be the most susceptible to mammary infections, since the pendulous udder expose the teat and udder to damage, allowing bacteria to readily attach to the teat and get access to gland tissue (Abebe et al., 2016). In the current study, the mastitis history of the animal was highly significant towards the spread of S. aureus and MRSA. Similar results were also observed by Kemal et al. (2017) who concluded that previous mastitis record was a significant factor for having an antimicrobial-resistant S. aureus infection in cows. The use of antibiotics was also a significant factor in the spread of MRSA in animals. MRSA is resistant to β-lactam antibiotics, which are commonly used to treat mastitis. Therefore, treatment of mastitis caused by β-lactam resistant S. aureus, such as MRSA usually leads to a lower cure rate (Taponen et al., 2003). In addition, MRSA intra-mammary infection can be chronic as S. aureus can form microabscesses and invade host phagocytes in the mammary glands. As a result, multiple infections can be seen in animals with mastitis caused by MRSA (Cortimiglia et al., 2016).

The current study also found that large herds were at a higher risk of MRSA infection than smaller herds. This finding was correlated with the observations made by Mekonnen et al. (2017) that S. aureus was isolated more frequently from large dairy herds. Similarly, Tenhagen et al. (2018) and Cortimiglia et al. (2016) also reported that a higher MRSA prevalence was observed in the large herds of cows examined. The high risk of MRSA infection in large dairy herds may be due to a high number of mastitis cases, which can lead to an increased frequency of antibiotic treatments. In addition, larger herds tend to be more likely to import new animals on the farm, which increases the risk of introducing MRSA-infected animals into the herd (Tenhagen et al., 2018).

Protein profiles of MRSA and MSSA were compared using SDS-PAGE along with known protein markers to be used for the determination of polypeptides separated by resolving gel. The difference of protein profile among MRSA and MSSA isolates reported in the current study is supported by the findings of Tesařová et al. (2016) who reported significant differences among the band’s pattern of both isolates. Similar findings were also documented by Sacilik et al. (2000). This could be attributed due to the difference in protein profiles among both sensitive and resistant isolates. Moreover, SDS PAGE of MRSA isolates revealed the protein band of 78.2KDa which is designated for PBP2a altered protein particular for MRSA and absent in MSSA, and it is supported by Doan et al. (2013) and Hartman and Tomasz (1986). The presence of the PBP2a band explains the resistance mechanism of MRSA as this protein is encoded by the mecA gene which is exclusively related to the occurrence of resistance (Arsic et al., 2012; Wendlandt et al., 2013). However, the SDS-PAGE technique has proved to be a conventional technique to differentiate between both isolates based on their protein expression (Tesařová et al., 2016).

CONCLUSION

The study concluded a significant prevalence of MRSA from indigenous bovine breeds from district Rawalpindi. Molecular characterization revealed considerable variation among local field isolates of MRSA and also with already reported isolates from other countries. The comparative risk factor analysis showed that milking person hygiene, milking method, and usage of antibiotics were significantly associated with MRSA. However, mastitis history, physiological status, and teat lesion were found potential risk factors for both S. aureus and MRSA-associated mastitis. The current study proved the first molecular characterization report addressing resistance patterns in field isolates of MRSA which can be effective in devising control strategies.

ACKNOWLEDGMENT

The current study was conducted at Medicine Research Laboratory, Department of Veterinary Medicine, University of Veterinary and Animal Sciences (UVAS) and part of the research project number ALP AS-042 entitled as “Mechanistic study of MRSA from bovine and occupational workers, and its application in field conditions” funded by the Pakistan Agriculture Research Council (PARC). The authors would like to thank the funding agency.

Statement of conflict of interest

The authors have declared no conflict of interest.

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