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. 2022 Apr 18;10(4):833.
doi: 10.3390/microorganisms10040833.

Antibiotic Resistance and Molecular Profiling of the Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis from India

Umarani Brahma  1 , Akash Suresh  1   2 , Shweta Murthy  1 , Vasundhra Bhandari  1   3 , Paresh Sharma  1
Affiliations

Antibiotic Resistance and Molecular Profiling of the Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis from India

Umarani Brahma et al. Microorganisms. .

Abstract

Staphylococcus aureus is an opportunistic bacterium known to cause severe infections in humans and animals. It is one of the major bacteria causing subclinical and clinical mastitis, leading to significant economic losses in livestock industry. In this study, we have isolated and characterized 80 S. aureus clinical isolates from mastitis-infected animals. The analysis of antimicrobial susceptibility, molecular typing, biofilm production and genetic determinants was performed to understand molecular and phenotypic features of the prevalent pathogen. Our antibiotic susceptibility assays showed the majority (57.5%) of isolates to be multidrug-resistant (MDR), 38.75% resistant and 3.75% sensitive. We found 25% isolates to be methicillin-resistant S. aureus (MRSA) based on oxacillin susceptibility assays. In the MRSA group, maximum isolates (95%) were MDR compared to 45% in MSSA. Multilocus sequence typing (MLST) revealed 15 different STs; ST-97 was the most common ST, followed by ST-2459, ST-1, ST-9 and ST-72. The agr typing showed agr-I as the most common type, followed by type II and III. Most isolates developed biofilms, which ranged in intensity from strong to weak. The presence or absence of lukS, a virulence-related gene, was found to have a substantial relationship with the biofilm phenotype. However, no significant association was found between biofilm formation and antimicrobial resistance or other virulence genes. We also found four MRSA isolates that were mecA negative based on molecular assays. Our findings reveal the prevalence of multidrug-resistant S. aureus clinical isolates in India that are biofilm positive and have critical genetic factors for disease pathogenesis causing bovine mastitis. This study emphasizes the need for the comprehensive surveillance of S. aureus and other mastitis-causing pathogens to control the disease effectively.

Keywords: MRSA; antimicrobial susceptibility; biofilm; bovine mastitis; multidrug-resistance; virulence.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Distribution of sequence types (ST) of 80 clinical isolates of S. aureus and prevalence of MRSA among three different states: Telangana, Andhra Pradesh and Tamil Nadu. ST was determined using the multilocus sequence typing (MLST) method.
Figure 2
Figure 2
Molecular characterization of the S. aureus clinical isolates using accessory gene regulator locus (agr) typing: agr Type-I was the most common among the isolates followed by type III. Type-II was not found in the MRSA isolates but there were 2 and 1 NTs in MRSA and MSSA, respectively.
Figure 3
Figure 3
Classification of the biofilm-forming intensity of S. aureus isolates: The biofilm formation ability was determined using the crystal violet (CV) method and the biofilm formed was differentiated into strong (red), moderate (green) and weak (blue) biofilms.
Figure 4
Figure 4
Genetic determinants profiling of markers: PCR-based detection of genes associated with biofilm formation, adhesion, virulence, toxin production and pathogenesis across S. aureus isolates (n = 80). (A) Biofilm and adhesion genes (icaA, icaD, clfA, fnbpA, fnbpB and bap), virulence and toxin genes (aHLS, bHLS, cHLS, dHLS, lukF, pvl, lukMF and lukS), pathogenesis genes (can, adsA, sbi and scn). (B) Comparison of antibiotic resistance in each isolate with respect to their biofilm formation. (C) Comparison of virulence genes in each isolate with respect to their biofilm formation.
Figure 5
Figure 5
Significance of virulence genes and antimicrobial resistance across S. aureus isolates (n = 80). (A) eBURST analysis showing the random distribution of antimicrobial resistance profile across different MLST. (B) eBURST analysis showing the random distribution of virulence genes across different MLST. (C) Chi-square analysis of antimicrobial resistance and susceptibility with respect to biofilm formation. (D) Chi-square analysis of virulence genes with respect to biofilm formation. The red-dotted line marks the cutoff value of 0.05, the values falling below the cutoff range are considered statistically significant and the red arrows mark the genes with a significant p-value (<0.05).
Figure 5
Figure 5
Significance of virulence genes and antimicrobial resistance across S. aureus isolates (n = 80). (A) eBURST analysis showing the random distribution of antimicrobial resistance profile across different MLST. (B) eBURST analysis showing the random distribution of virulence genes across different MLST. (C) Chi-square analysis of antimicrobial resistance and susceptibility with respect to biofilm formation. (D) Chi-square analysis of virulence genes with respect to biofilm formation. The red-dotted line marks the cutoff value of 0.05, the values falling below the cutoff range are considered statistically significant and the red arrows mark the genes with a significant p-value (<0.05).
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