Research Article: Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model

Date Published: January 27, 2017

Publisher: Public Library of Science

Author(s): Jully Gogoi-Tiwari, Vincent Williams, Charlene Babra Waryah, Paul Costantino, Hani Al-Salami, Sangeetha Mathavan, Kelsi Wells, Harish Kumar Tiwari, Nagendra Hegde, Shrikrishna Isloor, Hesham Al-Sallami, Trilochan Mukkur, Suzan HM Rooijakkers.


Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model.

Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection.

Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (p<0.05) in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain. This finding suggests an important role of TNF-α in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

Partial Text

Despite advances in diagnosis and management practices aimed at reducing the incidence of ruminant mastitis associated with the contagious pathogens, Staphylococcus aureus (S. aureus) is one of the major contagious causative agents of clinical and subclinical bovine mastitis worldwide causing significant economic loss to the dairy industry [1–3]. Reduced milk production due to mastitis is associated with irreversible mammary tissue damage in majority of the cases [4]. A wide array of virulence factors including surface-associated polysaccharide antigens including capsular polysaccharides (CP), teichoic acid (TA) and Poly-β-1, 6-N-acetylglucosamine (PNAG), MSCRAMM (microbial surface components recognizing adhesive matrix molecules), cytotoxins and enterotoxins are produced by S. aureus, which compromise host’s innate and adaptive immune systems [5, 6], resulting in the establishment of infection. Staphylococcus aureus enters the mammary gland, most likely via the orifice of chapped or injured teats [7]. Once the organism enters into the mammary gland, it adheres to epithelial cell receptors for bacterial adhesins [8] resulting in the production of virulence factors mentioned above and intracellular uptake of the small colony variants of S. aureus. Once the intra-mammary infection is established, damage to the mammary gland epithelial lining is initiated by ulceration and occlusion of lactiferous ducts, alveoli and infiltration of inflammatory cells in the parenchyma [4]. Mammary tissue damage is further compounded by various toxins and extracellular enzymes produced by S. aureus, including α, β, γ, and δ toxins, toxic shock syndrome toxin (TSST-1), enterotoxins, nuclease, coagulase, catalase, hyaluronidase, phosphatase, lipase, staphylokinase and proteases [9]. Some toxins act as super-antigens promoting the release of inflammatory cytokines, while others are leukocidins and cytolysins including haemolysins [10, 11]. Inflow of somatic cells, predominantly of polymorphonuclear (PMN) leukocytes into mammary gland is possibly due to breach of blood-milk barrier, worsens the damage to mammary epithelial cells owing to release of proteolytic enzymes and superoxide resulting in mammary tissue damage [12].

Biofilm formation by S. aureus has been studied extensively with respect to methods of detection, potential role in chronicity of infection and resistance to antibiotics and innate immune defences for both human and animal infectious diseases [13–15, 38, 41, 58]. Demonstrated ability of S. aureus biofilm to resist innate immune defences against intra-mammary infections (IMI) in the last few years [31, 37, 59, 60] and the difficulty encountered in treating such infections with antibiotics [9] has highlighted the urgency of developing effective vaccines against bovine mastitis. This also highlights the urgency of discovering ways to disperse the biofilms for improvement in the efficacy of the available antibiotics in the treatment of intra-mammary infections. Potential reasons underpinning mammary tissue damage due to S. aureus infection have been reported previously [4, 61]. However, limited studies have been reported on the potential impact of biofilm formation by S. aureus on the integrity of the mammary tissue post-infection with the pathogen [26, 62]. Most studies have been confined to characterization of the inflammatory immune responses to porcine dermal explants [63], murine tibial implants [64], nasal explant model [65] and prosthetic implant infections [64, 66]. Baselga and co-workers (1993) reported slime producing (SP) S. aureus isolates from ruminant mastitis cases to exhibit higher colonisation capacity than the non-slime producing (NSP) variant of the same strain obtained by repeated subculturing on Congo red agar at a medium frequency [26]. The NSP variants were shown to have higher virulence than the SP S. aureus revertants as measured by moderate to severe clinical signs and inflammatory lesions in mammary glands of lactating ewes at 48 h post-infection. It was hypothesized that the SP isolates of S. aureus were responsible for attachment to mammary epithelia and colony formation, whereas the NSP S. aureus variant led to the development of severe mastitis. Since some NSP strains can produce slime in vivo as demonstrated by immunoperoxidase staining of mammary tissue [26], it is important to obtain information on the strength of the biofilm formation of SP versus NSP strains cultured in vitro as well as for isolates cultured from infected mammary tissues for confirmation of the stated hypotheses.