Micro-evolutionary analysis of 70 ST398 isolates by pulsed-field gel electrophoresis (PFGE) using Cfr9I revealed three sub-clones with abundant inter- and intra-sub-clone heterogeneity in spa- and SCCmec-types. In addition, we developed two specific PCRs for the detection of Staphylococcus aureus sequence type 398 (ST 398) isolates with 100% specificity and high sensitivity.

Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium. Using a fluorescence resonance energy transfer (FRET)-based assay, we detected thermonuclease activity of S. aureus biofilms grown in Iscove’s modified Dulbecco’s medium (IMDM) earlier than that of widely studied biofilms grown in tryptic soy broth (TSB). The thermonuclease found was Nuc1, confirmed by mass spectrometry and competitive Luminex assay. These results indicate that biofilm development in IMDM may not rely on eDNA for structural stability. A bacterial viability assay in combination with wheat germ agglutinin (WGA) staining confirmed the accumulation of dead cells and eDNA in biofilms grown in TSB. However, in biofilms grown in IMDM, minimal amounts of eDNA were found; instead, polysaccharide intercellular adhesin (PIA) was detected. To investigate if this early production of thermonuclease plays a role in immune modulation by biofilm, we studied the effect of thermonuclease on human neutrophil extracellular trap (NET) formation using a nuc knockout and complemented strain. We confirmed that thermonuclease produced by early-stage biofilms grown in IMDM degraded biofilm-induced NETs. Additionally, neither the presence of biofilms nor thermonuclease stimulated an increase in reactive oxygen species (ROS) production by neutrophils. Our findings indicated that S. aureus, during the early stages of biofilm formation, actively evades the host immune responses by producing thermonuclease.

Immune modulators are known to be produced by matured biofilms and during different stages of planktonic growth ofStaphylococcus aureus. Little is known about immune modulator production during the early stages of biofilm formation, thus raising the following question: how doesS. aureusprotect itself from the innate immune responses at these stages? Therefore, we determined the production of the following immune modulators: chemotaxis inhibitory protein of staphylococci (CHIPS); staphylococcal complement inhibitor (SCIN); formyl peptide receptor-like 1 inhibitor; gamma-hemolysin component B; leukocidins D, E, and S; staphylococcal superantigen-like proteins 1, 3, 5, and 9; and staphylococcal enterotoxin A.

Infections involving antibiotic resistant Staphylococcus aureus (S. aureus) represent a major challenge to successful treatment. Further, although bacteriophages (phages) could be an alternative to antibiotics, there exists a lack of correlation in phage susceptibility results between conventional in vitro and in vivo assays. This discrepancy may hinder the potential implementation of bacteriophage therapy. In this study, the susceptibility of twelve S. aureus strains to three commercial phage cocktails and two single phages was assessed. These S. aureus strains (including ten clinical isolates, five of which were methicillin-resistant) were compared using four assays: the spot test, efficiency of plating (EOP), the optical density assay (all in culture media) and microcalorimetry in human serum. In the spot test, EOP and optical density assay, all cocktails and single phages lysed both methicillin susceptible and methicillin resistant S. aureus strains. However, there was an absence of phage-mediated lysis in high concentrations of human serum as measured using microcalorimetry. As this microcalorimetry-based assay more closely resembles in vivo conditions, we propose that microcalorimetry could be included as a useful addition to conventional assays, thereby facilitating more accurate predictions of the in vivo susceptibility of S. aureus to phages during phage selection for therapeutic purposes.

IgG4 responses are considered indicative for long-term or repeated exposure to particular antigens. Therefore, studying IgG4-specific antibody responses against Staphylococcus aureus might generate new insights into the respective host-pathogen interactions and the microbial virulence factors involved. Using a bead-based flow-cytometry assay, we determined total IgG (IgGt), IgG1 and IgG4 antibody responses to 40 different S. aureus virulence factors in sera from healthy persistent (non) nasal carriers and patients with various staphylococcal infections from three distinct countries. IgGt responses were detected against all tested antigens. These were mostly IgG1 responses. In contrast, IgG4 antibodies were detected to alpha toxin, CHIPS, ETA and B, HlgB, IsdA, LukD, E, F, and S, SCIN, SEC, SSL1, 3, 5 and 9 and TSST-1 only. Large inter-patient variability was observed, and the type of infection or geographical location did not reveal conserved patterns of response. As persistent S. aureus carriers trended towards IgG4 responses to a larger number of antigens than persistent non-carriers, we also investigated sera from patients with epidermolysis bullosa (EB), a genetic blistering disease associated with high S. aureus carriage rates. EB patients responded immunologically to significantly more antigens than non-carriers and trended towards even more responses than carriers. Altogether, we conclude that the IgG4 responses against a restricted panel of staphylococcal antigens consisting primarily of immune modulators and particular toxins indicate important roles for these virulence factors in staphylococcal pathogen-host interactions, such as chronicity of colonization and/or (subclinical) infections.

During routinely screening (50.000 milk samples on an annual basis) 14 MRSA ST398 strains were identified in the period of January 2008 to September 2008 in 14 different dairy herds located in the provinces Overijssel and Gelderland, The Netherlands. Molecular analysis was performed by Cfr9I PFGE, ST398-specific diagnostic PCR, spa typing, SCCmec typing and Panton-Valentine Leukocidin (PVL) gene PCR. The molecular analyses of 14 MRSA (one MRSA strain per herd) strains revealed that all strains belong to ST398 with 3 closely related spa types (t011, t108 and t889, all commonly found in pigs) and carry 2 different SCCmec types, IVa and V. All MRSA strains were resistant to two or more classes of antibiotics and also PVL negative. The majority of farms (n = 9, 64%) harboured combined livestock with both cows and pigs present. Our study contributes to the growing evidence that MRSA ST398 is transmitted among various animal species and can be considered as an etiological agent of mastitis in dairy cows.