Methicillin resistance genes and in vitro biofilm formation among Staphylococcus aureus isolates from bovine mastitis in India

Highlights

• The present work highlights a positive correlation between presence of methicillin resistant genes (mecA) and SCC mec I-V among Staphylococcus aureus strains and in vitro biofilm forming ability and in vitro antibiotic sensitivity assay.

• The study observes a higher occurrence of biofilm forming ability among methicillin resistance strains of S. aureus which augments their pathogenicity.

• MRSA have emerged as serious threat to animal and human welfare worldwide owing to their association with serious infections and the biofilms formation.

Abstract

Introduction

Biofilms, an assemblage of microbial cells irreversibly associated with a surface and enclosed in a matrix of polysaccharide material pose serious health challenges, resulting in high economic losses. The emergence of methicillin-resistant S. aureus (MRSA) infections and ability to form biofilms in dairy animals is of emerging concern for livestock and public health owing to their association with serious infections. The present study was undertaken to examine the presence of methicillin resistance genes among the biofilm forming Staphylococcus aureus strains isolated from cases of acute and subacute bovine mastitis. A total of 150 mastitic milk samples referred to Veterinary Clinical Complex, Shuhama (Aulesteng) SKUAST-K were screened in present study. The methicillin resistant Staphylococcus aureus isolates were also screened for in vitro biofilm forming ability.

Results

A total of 80 (53.33%) S. aureus isolates were recovered from cases of bovine mastitis of which 20 (25%) were methicillin (mecA) gene positive. Of the 20 mecA positive isolates, 20% were positive for SCCmec I, 35% for SCCmec IV and 45% for SCCmec V subtypes. In vitro antibiotic sensitivity testing of MRSA revealed complete resistance towards methicillin and other pencillin group of antibiotics.

Conclusion

A significant correlation was observed between in vitro biofilm formation and presence of methicillin resistance gene in S aureus isolates recovered from acute and subacute mastitis. The Staphylococcus aureus isolates positive for methicillin resistance gene (mecA) were either strong or moderate biofilm formers.

Introduction

Mastitis, a major disease for dairy cattle is characterized by decreased milk quality, production, reduced profitability due to cost of treatment and milk discard [1]. Many Gram positive and negative pathogens are implicated in etiology of bovine mastitis. In most cases Staphylococcus aureus has been associated with subclinical and persistent intra-mammary infection which responds poorly to antibiotic treatment [2]. Biofilms are microbially derived sessile communities, characterized by cells attached to a substratum interface, to each other, embedded in a matrix of extracellular polymeric substance [3]. The production of biofilms by S aureus is under the control of intercellular adhesion operon (ica operon) that codes for polysaccharide intercellular adhesion (PIA). PIA independent factors like biofilm associated protein (bap), clumping factor A and B (Clf A and B) and fibronectin binding proteins A and B (FNBpA and FNBpB) can also promote biofilm production [4]. Biofilm production in S. aureus mastitis can be associated with antimicrobial resistance [5]. The resistance is attributed to physical and chemical diffusion barrier formed by exopolysaccharide matrix, hindering antimicrobial penetration, the existence of microenvironments that antagonize the antibiotic action, the activation of stress responses that cause changes in bacterial physiology, the slow and stable growth of these microorganisms due to nutrient limitation and the absence of antimicrobial targets [6]. Biofilms resist antibiotic concentration 10-10,000 folds higher than those required to inhibit the growth of free floating bacteria [7].

β-lactam antibiotics are preferred for treatment of Staphylococcal infections. The production of β-lactamase enzymes and low-affinity penicillin binding protein (PBP2a), has led to increase antimicrobial resistance globally [8]. Methicillin resistant S. aureus (MRSA) infections are life-threatening due to multidrug resistance of such strains and ability to form strong biofilms [7]. Presence of glycocalyx in biofilm protects the enclosed bacteria from host defenses and impedes delivery of antibiotics. The resistance to methicillin in S. aureus is mediated by mecA gene, carried on the mobile genetic element Staphylococcal cassette chromosome mec (SCCmec). Based on the structural organization and diversity these elements are classified as SCCmec type I- SCCmec XI. Methicillin susceptible strains (MSSA) show increased biofilm production by expressing PIA whereas MRSA form biofilm in an ica-independent manner by secreting surface proteins or release of extracellular DNA [9]. Studies have revealed that presence or absence of mecA gene influences the expression of biofilm phenotype. SCCmec typing (I–V) of MRSA has revealed expression of an altered penicillin binding protein, PBP2a, with decreased affinity for β-lactam antibiotics, thus allowing continuous cell-wall assembly.

There is a need to study the presence of methicillin resistance in biofilm producing S aureus strains. Identification and efficient control protocol against biofilm forming MRSA can be one of the essential steps towards the prevention of the most serious MRSA infections [10]. The present study was undertaken to screen the Staphylococcus aureus isolates recovered from cases of bovine mastitic (acute or subacute) for in vitro biofilm production and presence of methicillin resistance gene (mecA) and SCCmec I-V subtypes