High Production of LukMF’ in Staphylococcus aureus Field Strains Is Associated with Clinical Bovine Mastitis

Abstract

Staphylococcus aureus, a major cause of bovine mastitis, produces a wide range of immune-evasion molecules. The bi-component leukocidin LukMF’ is a potent killer of bovine neutrophils in vitro. Since the role of LukMF’ in development of bovine mastitis has not been studied in natural infections, we aimed to clarify whether presence of the lukM-lukF’ genes and production levels of LukMF’ are associated with clinical severity of the disease. Staphylococcus aureus was isolated from mastitis milk samples (38 clinical and 17 subclinical cases) from 33 different farms. The lukM-lukF’ genes were present in 96% of the isolates. Remarkably, 22% of the lukM-lukF’-positive S. aureus isolates displayed a 10-fold higher in vitro LukMF’ production than the average of the lower-producing ones. These high producing isolates were cultured significantly more frequently from clinical than subclinical mastitis cases. Also, the detection of LukM protein in milk samples was significantly associated with clinical mastitis and high production in vitro. The high producing LukMF’ strains all belonged to the same genetic lineage, spa-type t543. Analysis of their global toxin gene regulators revealed a point mutation in the Repressor of toxins (rot) gene which results in a non-functional start codon, preventing translation of rot. This mutation was only identified in high LukMF’ producing isolates and not in low LukMF’ producing isolates. Since rot suppresses the expression of various toxins including leukocidins, this mutation is a possible explanation for increased LukMF’ production. Identification of high LukMF’ producing strains is of clinical relevance and can potentially be used as a prognostic marker for severity of mastitis.

Symposium review: Intramammary infections—Major pathogens and strain-associated complexity

“…Intramammary challenge of cows with S. aureus strain S1444, which produces high levels of LukMF ′, resulted in more severe clinical signs than challenge with strains S1449 and S1463, which produce intermediate levels of LukMF ′, although SCC was similar between the groups (Vrieling et al, 2016). However, S1444 belongs to CC479 (Hoekstra et al, 2018), whereas the lineage to which S1449 and S1463 belong was not reported. Therefore, the strains may vary in more than just production of LukMF ′ and challenge of cows with strain S1444 lacking lukMF ′ would confirm the role of this toxin in virulence of bovine-adapted S. aureus.…”

Activation of a Bovine Mammary Epithelial Cell Line by Ruminant-Associated Staphylococcus aureus is Lineage Dependent

“…In addition, these regulatory systems also control expression and release of PAMPs, such as PSMs [38], and immune evasion factors that inhibit TLR-2 activation [12]. Since the CCs tested in this study belong to different agr types (CC133 to agr type I and CC151, CC479, CC425 to type II) [16,39] and CC479 S. aureus have been reported to carry a non-functional copy of the rot gene [23], differences in expression of these agr/rot controlled genes are likely and could be a possible explanation for the differential bMEC response towards ruminant-associated CCs. In addition, a previous study associated reduced bMEC activation by S. aureus with a negative Staphaurex latex agglutination test (SLAT) phenotype [29].…”

“…Although the level of bMEC activation by S. aureus likely contributes to the severity of an IMI, it is important to emphasize that the clinical severity of IMI depends on several factors, such host conditions and production of bacterial virulence factors [44]. Recently, we observed that S. aureus belonging to CC479 are associated with clinical rather than subclinical mastitis cases in cattle [23]. In the current study, CC479 isolates induced a stronger reaction from bMEC than the other bovine-associated lineage CC151, which could potentially contribute to the severity of mastitis.…”

“…Small ruminant associated S. aureus have been shown to occasionally infect cattle (e.g., [16]), but it is unknown to what extent the immune response to these lineages differs from infections with bovine associated S. aureus lineages. Clonal complex 479 has been associated with severe bovine mastitis cases [16,23], hence, the effects of CC479 strains on bMEC, which have not been studied thus far, may yield valuable insights into the pathogenesis of this lineage. Since the strength of proinflammatory responsiveness of bMEC towards invading pathogens is expected to influence the course and outcome of IMI, increased understanding of the interaction between S. aureus belonging to different CC and bMEC during IMI can give insight in the variable pathogenicity of S. aureus lineages in bovine mastitis.…”

Leukotoxins

“…Jeannoel et al, [3] studied the synergistic effects of influenza virus and S. aureus in a monocytic cell-line model, and report that influenza virus potentiates the pro-inflammatory action of heat-killed S. aureus and contributes to the cytotoxicity of alpha-hemolysin on monocytes, but only few synergistic interactions were relevant in the ex vivo model. As regards the S. aureus infection in animals, Hoekstra et al, [4] report that production of the bi-component leucocidin LukMF' at higher levels is associated with clinical mastitis in cattle.…”