Cytotoxic Activity of Coagulase-Negative Staphylococci in Bovine Mastitis

Abstract

Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34-to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially in Staphylococcus chromogenes strains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1,10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34-to 36-kDa protease, which may provide an indication of strain virulence.Coagulase-negative staphylococcus (CoNS) has been considered a minor pathogen of bovine mastitis; however, many studies recently have shown the importance of CoNS infection in the bovine mammary gland. Several studies indicated that CoNS is the most frequently recovered isolate from mastitis samples, especially in first lactation and unbred heifers (16,18,26,30). CoNS also caused mastitis problems in other species such as milking goats (11). CoNS infections usually are more mild than Staphylococcus aureus (one coagulase-positive Staphylococcus sp.) which is a major and contagious pathogen of bovine mastitis. The somatic cell counts of CoNS-infected cows are generally two-to threefold higher than that of uninfected cows (18, 30). CoNS-infected mammary tissues exhibited greater leukocyte infiltration and increased connective tissue stroma over an uninfected control (37). One study showed that CoNS infections caused an 8.7% loss in milk production from a 305-day milk yield total (36).Even though more researchers have realized the importance of CoNS intramammary infections, the virulence factors of CoNS remain poorly understood. The virulence factors of staphylococci have been studied most extensively in the species of S. aureus. Secreted toxins play very important roles in the pathogenesis of S. aureus (9, 10, 41). S. aureus strains isolated from bovine mastitis have been shown to express alpha, beta, gamma, and delta toxins, leukocidins, enterotoxin, and coagulase (8, 25). CoNS strains also produce several toxins and enzymes that could contribute to virulence, such as hemolysin, leucocidin, lipase, proteases, and DNase (17, 34, 39). Many CoNS strains isolated from mastitis samples had higher protease, DNase, and lecitinase activity than that of CoNS from normal cows (19). However, the roles of these enzymes on the pathogenesis of CoNS are unclear. A delta-like toxin from CoNS strains causes membrane ble...

Oral Administration of Lactobacillus Strains Isolated from Breast Milk as an Alternative for the Treatment of Infectious Mastitis during Lactation

“…At the species level, S. aureus has been traditionally considered the most common agent; however, recent studies have shown the increasing importance of S. epidermidis in bovine mastitis and have revealed that its incidence could be even higher than that of S. aureus (3,25,29). In fact, in the present study, 40% of the women carried S. aureus in their milk, while 60% of them harbored S. epidermidis isolates.…”

“…Traditionally, Staphylococcus aureus has been considered the main etiological agent, although Staphylococcus epidermidis is emerging as the leading cause in both human and veterinary medicine (3,25,29). Multidrug resistance to antibiotics and/or formation of biofilms is very common among clinical isolates of these two staphylococcal species; therefore, it is not strange that ca.…”

Virulence factors and ability of staphylococci from bovine milk and the cowshed environment to biofilm formation

“…Stepanović et al (2001) also showed strong proteolytic and DNase activities in 121 human and animal S. sciuri isolates. Zhang and Maddox (2000) stated that cytotoxic activity and casein hydrolysis activity of CNS from bovine mastitis was associated with production of 34-to 36-kDa protein. In this study, we also investigated esterase activity and we found the highest percentage of strains with this activity among S. epidermidis.…”

“…The pathogenicity of staphylococci is related to the production of several virulence factors responsible for the development of disease process ( factors were found in S. aureus strains isolated from bovine mastitis (Silva et al 2005, Hata et al 2006, Peles et al 2007, Melchior et al 2009, Capurro et al 2010, but only few studies involved search of virulence factors of CNS isolated from bovine mastitis (Zhang andMaddox 2000, Bochniarz andWawron 2012). Virulence factors of staphylococci have been investigated by measuring phenotypic expression of products associated with virulence (Taponen and Pyörälä 2009).…”

“…CNS strains isolated from milk of cows with mastitis produced proteases and haemolysins (Zhang andMaddox 2000, Bochniarz andWawron 2012). Moreover, biofilm formation has been proposed as an important virulence factor of CNS, by which mastitis-causing staphylococci avoid the immune response of the cow and cause persistent intramammary infections (Simojoki et al 2012).…”