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Background: An altered immune response against Staphylococcus aureus might contribute to inflammation and barrier damage in atopic dermatitis (AD)., Objectives: To profile IgG antibodies against 55 S. aureus antigens in sera of children with mild-to-severe AD and to evaluate the association between IgG levels and disease severity., Methods: In this cross-sectional study, we included children with AD from two interventional study cohorts, the Shared Medical Appointment (SMA) cohort (n = 131) and the older DAVOS cohort (n = 76). AD severity was assessed using the Self-Administered Eczema Area and Severity Index (SA-EASI) and levels of thymus and activation-regulated chemokine (TARC) in serum. IgG antibody levels against 55 S. aureus antigens were quantified simultaneously using a Luminex assay. Pair-wise correlations were calculated between the 55 IgG levels using the Spearman rank correlation test. Linear regression analysis was performed to test for associations between 55 IgG levels and SA-EASI and TARC, adjusting for age, sex and S. aureus colonization., Results: In the SMA cohort, 16 antigens were associated with SA-EASI and 12 with TARC (10 overlapping antigens; P-values 0·001-0·044). The associated IgG antibodies targeted mainly secreted proteins with immunomodulatory functions. In the DAVOS study, IgG levels against only four and one S. aureus antigen(s) were associated with SA-EASI and TARC, respectively (no overlap)., Conclusions: In young children, severity of AD is associated with an IgG response directed against S. aureus antigens with mainly immunomodulatory functions. These findings encourage further evaluation of the role of S. aureus in the pathogenesis of AD., (© 2017 British Association of Dermatologists.)

Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine-aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets.

Multiplex bead-based flow cytometry is an attractive way for simultaneous, rapid and cost-effective analysis of multiple analytes in a single sample. Previously, we developed various bead-based assays using non-magnetic beads coated with Staphylococcus aureus and Streptococcus pneumoniae antigens for the detection of antibodies. Here, we compared the performance of the assay using non-magnetic beads with one based on the newly developed magnetic beads. We optimized the magnetic beads' coupling procedure and antibody detection assays for S. aureus and S. pneumoniae antigens and we measured IgG in human pooled serum against a series of S. aureus and S. pneumoniae-derived antigens in a singleplex and in a multiplex assay, respectively. For the multiplex assay, the comparison between magnetic and non-magnetic beads showed: i) in the majority of the cases (13 of the 17 tested S. pneumoniae antigens) significantly higher Median Fluorescence Intensity (MFI) values, ii) lower detection limits, iii) lower coefficient of variation (CV: 12% vs. 7% for non-magnetic vs. magnetic beads), so lower inter-assay variation and hence higher reproducibility. Magnetic bead coupling is cost effective, as we used 25% of the normal amount of antigen and only 50% of the beads in comparison to the non-magnetic beads. This optimized magnetic-based assay, which combines ease of use with an improved assay performance, allows detection of antibodies with a low titer that are potentially missed with the non-magnetic-based assay., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to the d-Ala-d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variant d-Ala-d-Lac, which is produced by VanA. Here we show that exogenous d-Ala competes with d-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of the d-Ala-d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations of d-Ala led to the production of a significant amount of wild-type cell wall precursors, while vanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organism Streptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates of Enterococcus faecium (VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity of S. coelicolor cells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.

Pneumococcal surface proteins are potential candidates for the development of protein-based vaccines and serological assays. The objective of the study was to develop a multiple bead-based immunoassay using Luminex xMAP® technology for the quantitation of natural antibodies against Streptococcus pneumoniae proteins and the characterization of the acute serum response following pneumococcal pneumonia in children. Sixty-four recombinantly produced pneumococcal proteins, which were selected based on their proteomic experimental identification by "shaving" live cells with trypsin followed by LC/MS/MS analysis, were coupled to fluorescent SeroMAP® beads and anti-pneumococcal specific IgG levels were determined in sera. Multiplex assay was validated through comparison of IgG levels to 14 randomly chosen pneumococcal antigens by using multiplex and singleplex assays. Acute serum IgG levels against RrgB were significantly lower in children ≤ 4 years old with pneumococcal pneumonia than those in controls. In addition, there was a small trend toward slightly lower antibody levels for PrsA, RrgC and RrgB in pneumonia patients of the all age group., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Fifteen percent of all methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) human carriers detected in The Netherlands had not been in direct contact with pigs or veal calves. To ensure low MRSA prevalence, it is important to investigate the likely origin of this MRSA of unknown origin (MUO). Recently, it was shown that CC398 strains originating from humans and animals differ in the presence of specific mobile genetic elements (MGEs). We hypothesized that determining these specific MGEs in MUO isolates and comparing them with a set of CC398 isolates of various known origin might provide clues to their origin. MUO CC398 isolates were compared to MRSA CC398 isolates obtained from humans with known risk factors, a MRSA CC398 outbreak isolate, livestock associated (LA) MRSA CC398 isolates from pigs, horses, chickens, and veal calves, and five methicillin-susceptible Staphylococcus aureus (MSSA) CC398 isolates of known human origin. All strains were spa typed, and the presence or absence of, scn, chp, φ3 int, φ6 int, φ7 int, rep7, rep27, and cadDX was determined by PCRs. The MRSA CC398 in humans, MUO, or MRSA of known origin (MKO) resembled MRSA CC398 as found in pigs and not MSSA CC398 as found in humans. The distinct human MSSA CC398 spa type, t571, was not present among our MRSA CC398 strains; MRSA CC398 was tetracycline resistant and carried no φ3 bacteriophage with scn and chp. We showed by simple PCR means that human MUO CC398 carriers carried MRSA from livestock origin, suggestive of indirect transmission. Although the exact transmission route remains unknown, direct human-to-human transmission remains a possibility as well., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Chronic nasal carriage of the bacterium Staphylococcus aureus in patients with the autoimmune disease granulomatosis with polyangiitis (GPA) is a risk factor for disease relapse. To date, it was neither known whether GPA patients show similar humoral immune responses to S. aureus as healthy carriers, nor whether specific S. aureus types are associated with GPA. Therefore, this study was aimed at assessing humoral immune responses of GPA patients against S. aureus antigens in relation to the genetic diversity of their nasal S. aureus isolates. A retrospective cohort study was conducted, including 85 GPA patients and 18 healthy controls (HC). Humoral immune responses against S. aureus were investigated by determining serum IgG levels against 59 S. aureus antigens. Unexpectedly, patient sera contained lower anti-staphylococcal IgG levels than sera from HC, regardless of the patients' treatment, while total IgG levels were similar or higher. Furthermore, 210 S. aureus isolates obtained from GPA patients were characterized by different typing approaches. This showed that the S. aureus population of GPA patients is highly diverse and mirrors the general S. aureus population. Our combined findings imply that GPA patients are less capable of mounting a potentially protective antibody response to S. aureus than healthy individuals.

Previous research on Staphylococcus aureus in pigs focused on livestock-associated methicillin-resistant S. aureus (MRSA) and had a qualitative cross-sectional design. This study aimed to elucidate the frequency, load, and stability of S. aureus nasal carriage in pigs over time and investigated possible associations between carriage and immune response. Nasal swabs were collected three times weekly from 480 tagged adult pigs in 20 Danish production farms. S. aureus and MRSA were quantified on selective media by the most-probable-number method. The levels of IgG against 10 S. aureus antigens in serum were quantified in selected pigs by a Luminex assay. All the farms were positive for S. aureus and 15 for MRSA, leading to overall prevalences of persistent and intermittent carriers and noncarriers of 24, 52, and 23%, respectively. Carriage frequency and nasal loads were significantly higher on MRSA-positive farms. Logistic-regression modeling revealed the presence of individual pigs characterized by high nasal loads (>10,000 CFU per swab) and stable carriage regardless of farm- and pen-associated factors. On the other hand, the humoral response was strongly influenced by these environmental factors. The existence of a minority of shedders contributing to maintenance of S. aureus within farms opens up new perspectives on the control of MRSA in pig farming.

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

The majority of Staphylococcus aureus virulence- and colonization-associated surface proteins contain a pentapeptide recognition motif (LPXTG). This motif can be recognized and cleaved by sortase A (SrtA) which is a membrane-bound transpeptidase. After cleavage these proteins are covalently incorporated into the peptidoglycan. Therefore, SrtA plays a key role in S. aureus virulence. We aimed to generate a substrate mimicking this SrtA recognition motif for several purposes: to incorporate this substrate into the S. aureus cell-wall in a SrtA-dependent manner, to characterize this incorporation and to determine the effect of substrate incorporation on the incorporation of native SrtA-dependent cell-surface-associated proteins. We synthesized substrate containing the specific LPXTG motif, LPETG. As a negative control we used a scrambled version of this substrate, EGTLP and a S. aureus srtA knockout strain. Both substrates contained a fluorescence label for detection by FACScan and fluorescence microscope. A spreading assay and a competitive Luminex assay were used to determine the effect of substrate treatment on native LPXTG containing proteins deposition in the bacterial cell-wall. We demonstrate a SrtA-dependent covalent incorporation of the LPETG-containing substrate in wild type S. aureus strains and several other Gram-positive bacterial species. LPETG-containing substrate incorporation in S. aureus was growth phase-dependent and peaked at the stationary phase. This incorporation negatively correlated with srtA mRNA expression. Exogenous addition of the artificial substrate did not result in a decreased expression of native SrtA substrates (e.g. clumping factor A/B and protein A) nor induced a srtA knockout phenotype.

The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In this assay, various S. aureus bacteria or culture supernatant dilutions are exposed to human pooled serum. S. aureus specific IgGs from the serum are captured to the IgG-accessible staphylococcal antigens present on the bacterial surface, or in supernatant, in a dose dependent manner. The remaining non-captured IgGs are then quantified by incubation with Luminex beads to which the individual recombinant S. aureus surface proteins and carbohydrates are covalently linked. The signals obtained are inversely proportional to the antigen concentration measured, and binding patterns were dependent on the particular S. aureus isolate used, growth conditions and growth phase. For example, the detection of cell wall teichoic acid (WTA) and peptidoglycan (PG) carbohydrate structures increased significantly when bacteria entered the stationary growth phase, whereas the amounts of clumping factors (ClfA and ClfB) decreased significantly. Using this technique, the appearance of several S. aureus gene knockout strains were studied. We demonstrated that in an S. aureus srtA gene knockout strain, srtA-dependent cell-surface-associated proteins were detectable yet not covalently linked to the PG layer. In conclusion, we show that this multiplex competitive Luminex assay is a suitable method for the rapid and simultaneous determination of S. aureus IgG-accessible cell-surface-associated proteins and carbohydrates in various growth phases and growth conditions in different S. aureus strains., (© 2013. Published by Elsevier B.V. All rights reserved.)

Molecular mimicry between Campylobacter jejuni sialylated lipooligosaccharides (LOS) and human nerve gangliosides can trigger the production of cross-reactive antibodies which induce Guillain-Barré syndrome (GBS). To better understand the immune events leading to GBS, it is essential to know how sialylated LOS are recognized by the immune system. Here, we show that GBS-associated C. jejuni strains bind to human sialoadhesin (hSn), a conserved, mainly macrophage-restricted I-type lectin. Using hSn-transduced THP-1 cells, we observed that C. jejuni strains with α(2,3)-sialylated LOS, including strains expressing GM1a- and GD1a-like epitopes, bind to hSn. This observation is of importance, as these epitopes are frequently the targets of the cross-reactive antibodies detected in GBS patients. Interestingly, the Sn binding domains were not constitutively exposed on the surface of C. jejuni. Heat inactivation and the environmental conditions which food-borne C. jejuni encounters during its passage through the intestinal tract, such as low pH and contact with bile constituents, exposed LOS and facilitated Sn binding. Sn binding enhanced bacterial uptake and increased the production of interleukin-6 (IL-6) by primary human Sn-expressing monocyte-derived macrophages compared to control conditions, where Sn was blocked using neutralizing antibodies or when nonsialylated C. jejuni was used. Sn-mediated uptake has been reported to enhance humoral immune responses. As C. jejuni strains expressing ganglioside mimics GD1a and GM1a are closely associated with GBS, Sn binding may be a determining event in the production of cross-reactive antibodies and the development of GBS.

Due to molecular mimicry, Campylobacter jejuni lipo-oligosaccharides can induce a cross-reactive antibody response to nerve gangliosides, which leads to Guillain-Barré syndrome (GBS). Cross-reactive antibodies to ganglioside GQ1b are strongly associated with oculomotor weakness in GBS and its variant, Miller Fisher syndrome (MFS). Antigen recognition is a crucial first step in the induction of a cross-reactive antibody response, and it has been shown that GQ1b-like epitopes expressed on the surface of C. jejuni are recognized by sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7). We aimed to determine the epitope specificity of C. jejuni binding to Siglec-7, and correlate the outcome to disease symptoms in GBS and MFS patients. Using a well-defined GBS/MFS-associated C. jejuni strain collection, which included three sialic acid knockout strains, we found that Siglec-7 exclusively binds to C. jejuni strains that express terminal disialylated ganglioside mimics. When serological and diagnostic patient records were correlated with the Siglec-7-binding properties, we observed an association between Siglec-7 binding and the presence of anti-GQ1b antibodies in patient serum. In addition, Siglec-7 binding was associated with oculomotor weakness in GBS and MFS patients. Lipo-oligosaccharide-specific binding of C. jejuni to Siglec-7 may be an initiating event in immune recognition and presentation, and lead to anti-GQ1b antibody production and the development of ocular weakness in GBS or MFS., (© 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

At present, the immune response of pigs in relation to Staphylococcus aureus carriage is poorly understood. This study was aimed at investigating the dynamics of the anti-staphylococcal humoral immune response in methicillin-susceptible S. aureus (MSSA)-positive piglets and at assessing the effect of the experimental introduction of a methicillin-resistant S. aureus (MRSA) Sequence Type (ST) 398 strain. Therefore, serum samples were collected at different times from 31 weaned piglets originating from four different sows. Twenty-four out of the 31 piglets were challenged with MRSA ST398. The serum samples were analyzed for IgG antibodies to 39 S. aureus antigens, using a multiplex bead-based assay (xMAP technology, Luminex Corporation). Though antibody responses showed broad inter-individual variability, serological results appeared to be clustered by litter of origin. For most antigens, an age-related response was observed with an apparent increase in antibody titers directed against staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMM), which have been shown to play a role in S. aureus colonization. In most animals, antibody titers directed against staphylococcal toxins or immune-modulating proteins decreased with age, possibly reflecting the absence of bacterial invasion. The introduction of MRSA ST398 did not elicit a significant humoral immune reaction.This study describes, for the first time, the humoral immune response in weaned pigs colonized with S. aureus.

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BµG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688.

Translocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether sialylation of C. jejuni lipooligosaccharide (LOS) structures, generating human nerve ganglioside mimics, is important for intestinal epithelial translocation. We here show that C. jejuni isolates expressing ganglioside-like LOS bound in larger numbers to the Caco-2 intestinal epithelial cells than C. jejuni isolates lacking such structures. Next, we found that ganglioside-like LOS facilitated endocytosis of bacteria into Caco-2 cells, as visualized by quantitative microscopy using the early and late endosomal markers early endosome-associated protein 1 (EEA1), Rab5, and lysosome-associated membrane protein 1 (LAMP-1). This increased endocytosis was associated with larger numbers of surviving and translocating bacteria. Next, we found that two different intestinal epithelial cell lines (Caco-2 and T84) responded with an elevated secretion of the T-cell attractant CXCL10 to infection by ganglioside-like LOS-expressing C. jejuni isolates. We conclude that C. jejuni translocation across Caco-2 cells is facilitated by ganglioside-like LOS, which is of clinical relevance since C. jejuni ganglioside-like LOS-expressing isolates are linked with severe gastroenteritis and bloody stools in C. jejuni-infected patients.

The Netherlands is known for its low methicillin-resistant Staphylococcus aureus (MRSA) prevalence. Yet MRSA with no link to established Dutch risk factors for acquisition, MRSA of unknown origin (MUO), has now emerged and hampers early detection and control by active screening upon hospital admittance. We assessed the magnitude of the problem and determined the differences between MUO and MRSA of known origin (MKO) for CC398 and non-CC398. National MRSA Surveillance data (2008-2009) were analysed for epidemiological determinants and genotypic characteristics (Panton-Valentine leukocidin, spa). A quarter (24%) of the 5545 MRSA isolates registered were MUO, i.e. not from defined risk groups. There are two genotypic MUO groups: CC398 MUO (352; 26%) and non-CC398 MUO (998; 74%). CC398 MUO needs further investigation because it could suggest spread, not by direct contact with livestock (pigs, veal calves), but through the community. Non-CC398 MUO is less likely to be from a nursing home than non-CC398 MKO (relative risk 0.55; 95% CI 0.42-0.72) and Panton-Valentine leukocidin positivity was more frequent in non-CC398 MUO than MKO (relative risk 1.19; 95% CI 1.11-1.29). Exact transmission routes and risk factors for non-CC398 as CC398 MUO remain undefined., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Knowledge of the immunological correlates of Staphylococcus aureus and Streptococcus pneumoniae colonization is required for the search for future protein vaccines. We evaluated natural antibody levels against pneumococcal and staphylococcal proteins in relation to previous bacterial colonization with both pathogens. In a randomized controlled trial, nasopharyngeal samples were obtained from children at 1.5, 6, 12, 18, and 24 months and cultured for S. aureus and S. pneumoniae. Approximately 50% of the children were PCV7 vaccinated. Serum IgG against 18 pneumococcal and 40 staphylococcal proteins was semiquantified by Luminex technology from 111 12 month olds and 158 24 month olds. Previous culture-proven S. aureus colonization was associated with higher IgG levels against 6/40 staphylococcal proteins (ClfB, ClfA, Efb, CHIPS, LukD, and LukF [P ≤ 0.001]) compared to noncarriers. Previous pneumococcal colonization was associated with increased IgG levels against 12/18 pneumococcal proteins compared to noncarriers (P ≤ 0.003). Increasing age was associated with higher levels of antibodies to most pneumococcal proteins and lower levels of antibodies to over half the staphylococcal proteins, reflecting natural colonization dynamics. Anti-S. pneumoniae and anti-S. aureus protein antibodies at the age of 12 months were not negatively correlated with subsequent colonization with the homologous species in the following year and did not differ between PCV7-vaccinated and nonvaccinated children. Colonization with S. aureus and S. pneumoniae induces serum IgG against many proteins, predominantly proteins with immune-modulating functions, irrespective of PCV7 vaccination. None of them appeared to be protective against new acquisition with both pathogens, possibly due to the polymorphic nature of those proteins in the circulating bacterial population.

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.

There is evidence that MRSA ST398 of animal origin is only capable of temporarily occupying the human nose, and it is therefore, often considered a poor human colonizer.We inoculated 16 healthy human volunteers with a mixture of the human MSSA strain 1036 (ST931, CC8) and the bovine MSSA strain 5062 (ST398, CC398), 7 weeks after a treatment with mupirocin and chlorhexidine-containing soap. Bacterial survival was studied by follow-up cultures over 21 days. The human strain 1036 was eliminated faster (median 14 days; range 2-21 days) than the bovine strain 5062 (median 21 days; range 7-21 days) but this difference was not significant (p = 0.065). The bacterial loads were significantly higher for the bovine strain on day 7 and day 21. 4/14 volunteers (28.6%) showed elimination of both strains within 21 days. Of the 10 remaining volunteers, 5 showed no differences in bacterial counts between both strains, and in the other 5 the ST398 strain far outnumbered the human S. aureus strain. Within the 21 days of follow-up, neither human strain 1036 nor bovine strain 5062 appeared to acquire or lose any mobile genetic elements. In conclusion, S. aureus ST398 strain 5062 is capable of adequately competing for a niche with a human strain and survives in the human nose for at least 21 days.

Staphylococcus aureus (S. aureus) colonizes the anterior nares in part of the population and the persistent carrier state is associated with increased infection risk. Knowledge concerning the determinants of S. aureus nasal carriage is limited. Previously, we found that glucocorticoid receptor polymorphisms influence carrier risk, suggesting involvement of glucocorticoids. Our aim was to study long-term cortisol levels in non-carriers, intermittent, and persistent carriers of S. aureus. We hypothesized that cortisol levels are higher in carriers, since cortisol-induced immune suppression would enhance S. aureus colonization. We determined nasal carrier state and long-term hair cortisol levels in 72 healthy subjects. Nasal swabs were collected twice with an interval of 2 weeks. Cortisol levels were determined in hair segments of 3 cm, which corresponds to a period of roughly 3 months. Of all 72 participants, 38 were non-carriers, 10 were intermittent carriers, and 24 were persistent carriers of S. aureus. Cortisol levels did not differ between these carrier groups (p=0.638). Long-term cortisol levels are not associated with S. aureus nasal carriage.

It remains largely unknown which factors determine the clinical outcome of human metapneumovirus (HMPV) infections. The aim of the present study was to analyse whether exposure to bacterial pathogens can influence HMPV infections. From 57 children, serum samples and colonization data for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pneumoniae were collected at 1.5, 6, 14 and 24 months of age. Seroconversion rates to HMPV were determined and related to bacterial carriage. Frequent nasopharyngeal carriage (≥2 times in the first 2 years of life) of S. pneumoniae, but not of the other three pathogens, was associated with increased seroconversion rates of infants to HMPV at the age of 2 years (frequently vs. less exposed, 93% vs. 59%; p <0.05). Subsequently, the susceptibility of well-differentiated normal human bronchial epithelial cells (wd-NHBE) pre-incubated with bacterial pathogens to in vitro HMPV infection was evaluated. Pre-incubation of wd-NHBE with S. pneumoniae resulted in increased susceptibility to infection with HMPV-enhanced green fluorescent protein (EGFP), as determined by enumeration of EGFP-positive cells. This was not the case for cells pre-incubated with H. influenzae, M. catarrhalis on S. aureus. We conclude that exposure to S. pneumoniae can modulate HMPV infection., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Staphylococcus aureus is a major cause of life-threatening infections such as bacteremia and endocarditis. Unfortunately, many strains of this bacterial species have become resistant to certain antibiotics, including methicillin and amoxicillin. These strains are known as methicillin-resistant S. aureus (MRSA). Therefore, the prophylactic and therapeutic potential of antistaphylococcal vaccines is currently being explored with priority. In animal models, (passive) immunization with (antibodies directed against) certain S. aureus surface components, staphylococcal toxins and capsular polysaccharides protects against S. aureus colonization or infection. However, immunization studies performed in humans show less promising results. So far, not a single antistaphylococcal vaccine successfully passed clinical trials. This article focuses on the results that were obtained with immunotherapeutic approaches directed against S. aureus in animal and human studies. In addition, it is discussed whether effective immunization approaches against S. aureus are feasible in humans.

Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP(®) Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner.

The currently available pneumococcal vaccines do not protect against all serotypes of Streptococcus pneumoniae. A shift toward nonvaccine serotypes causing colonization and invasive disease has occurred, and studies on protein-based vaccines have been undertaken. We assessed the association between specific antibodies against pneumococcal virulence proteins and colonization and respiratory tract infections (RTIs). Additionally, we assessed the extent to which colonization induces a humoral immune response. Nasopharyngeal swabs collected from children at 1.5, 6, 14, and 24 months of age were cultured for pneumococcus. Serum samples were obtained at birth and at 6, 14, and 24 months (n = 57 children providing 177 serum samples). Data were collected prior to the pneumococcal vaccine era. IgG, IgA, and IgM levels against 17 pneumococcal protein vaccine candidates were measured using a bead-based flow cytometry technique (xMAP; Luminex Corporation). Information regarding RTIs was questionnaire derived. Levels of IgG against all proteins were high in cord blood, decreased in the first 6 months and increased again thereafter, in contrast to the course of IgA and IgM levels. Specific antibodies were induced upon colonization. Increased levels of IgG against BVH-3, NanA, and SP1003 at 6 months, NanA, PpmA, PsaA, SlrA, SP0189, and SP1003 at 14 months, and SlrA at 24 months were associated with a decreased number of RTIs in the third year of life but not with colonization. Maternal antipneumococcal antibodies did not protect against pneumococcal colonization and infection. Certain antibodies against pneumococcal virulence proteins, some of which are induced by colonization, are associated with a decreased number of RTIs in children. This should be taken into account in future pneumococcal vaccine studies.

The Staphylococcus aureus immune evasion cluster (IEC), located on β-haemolysin-converting bacteriophages (βC-Φs), encodes the immune-modulating proteins chemotaxis inhibitory protein, staphylococcal complement inhibitor (SCIN), staphylococcal enterotoxin A and staphylokinase. Its precise role in S. aureus colonization is unclear. We studied the presence of the IEC-carrying bacteriophages in human and animal S. aureus isolates, using PCR for the gene encoding SCIN (scn). Human isolates were obtained by collecting serial nasal swabs from 21 persistent carriers. S. aureus strains from 19 (90%) persistent carriers contained an IEC that was present and indistinguishable in 95% of cases at all five sampling moments over a 3-month period. Of the 77 infectious animal strains included in the study, only 26 strains (34%) were IEC-positive. Integration of these IEC-positive strains into an amplified fragment length polymorphism genotype database showed that 24 of 53 (45%) strains were human-associated and only two of 24 (8%) were 'true' animal isolates (p < 0.001). The high prevalence and stability of IEC-carrying βC-Φs in human strains suggested a role for these βC-Φs in human nasal colonization. To test this hypothesis, 23 volunteers were colonized artificially with S. aureus strain NCTC 8325-4 with or without the IEC type B-carrying βC-Φ13. Intranasal survival was monitored for 28 days after inoculation. The strain harbouring βC-Φ13 was eliminated significantly faster (median 4 days; range 1-14 days) than the strain without βC-Φ13 (median 14 days; range 2-28 days; p 0.011). In conclusion, although IEC-carrying βC-Φs are highly prevalent among human colonizing S. aureus strains, they are not essential in the first stages of S. aureus nasal colonization., (© 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.)

Staphylococcus aureus causes a variety of infections. Knowledge about the physiological role of most S. aureus antigens in colonization and infection is only limited. This can be studied by measuring antigen-specific antibody responses. In this study, we optimized the multiplex microsphere bead-based flow cytometry technique for mouse serum samples. We analysed immunoglobulin G (IgG) levels directed against 26 S. aureus proteins in a single small-volume mouse serum sample. We assessed possible cross reactivity. Furthermore, we analysed serum samples from mice with different types of S. aureus infections caused by different S. aureus strains. The results show that cross reactivity between proteins on microspheres and serum antibodies towards other proteins was limited. We found that lung-infected mice had a higher and broader IgG response than skin-infected mice. Clearly, the site of infection influences the IgG profile. Next, we compared sera from mice with intravenously-induced bacteraemia caused by different S. aureus strains. We showed different IgG responses depending on the causing S. aureus strain. It is concluded that the bead-based multiplex S. aureus antibody assay can be successfully applied to determine the immunogenicity of different S. aureus proteins in relation to the site of infection and the S. aureus strain causing the infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Colonization rates of Streptococcus pneumoniae and Staphylococcus aureus are inversely correlated in infants. Several studies have searched for determinants of this negative association. We studied the association between antipneumococcal antibodies with Staphylococcus aureus colonization and the association between antistaphylococcal antibodies with pneumococcal colonization in healthy children in the pneumococcal vaccine era. In the first year of life, no association between maternal IgG levels and colonization was seen. In addition, no association between the IgG and IgA levels in the child versus colonization status was seen.

Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa repeat sequencing and multi-locus sequence typing (MLST), and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs). Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention.

In order to develop novel antistaphylococcal strategies, understanding the determinants of carriage and how humans respond to Staphylococcus aureus exposure is essential. Here, the primary S. aureus-specific humoral immune response and its association with nasal colonization was studied in young children. Sera from 57 colonized or non-colonized children, serially collected at birth and at 6, 14 and 24 months, were analysed for IgG, IgA and IgM binding to 19 staphylococcal proteins, using flow cytometry-based technology. The antibody responses showed extensive inter-individual variability. On average, the levels of antistaphylococcal IgA and IgM increased from birth until the age of 2 years (p <0.05), whereas the levels of IgG decreased (p <0.001). Placentally transferred maternal IgG did not protect against colonization. In colonized children, IgG and IgA levels for a number of proteins were higher than in non-colonized children. At both 14 and 24 months, the levels of IgG against chemotaxis inhibitory protein of S. aureus (at 24 months; median fluorescence intensity, 4928 vs. 24, p <0.05), extracellular fibrinogen-binding protein (987 vs. 604, p <0.05), and iron-responsive surface determinant H (62 vs. 5, p <0.05) were significantly higher in colonized children. The levels of IgA against CHIPS, IsdH and IsdA were higher (p <0.05). Therefore, CHIPS, Efb, IsdA and IsdH seem to play a role in nasal colonization of young children.

In Campylobacter jejuni-induced Guillain-Barré syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for alpha(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.

Expanding knowledge on the humoral immune response in Staphylococcus aureus-infected patients is a mandatory step in the development of vaccines and immunotherapies. Here, we present novel insights into the antibody responses following S. aureus bacteremia. Fifteen bacteremic patients were followed extensively from diagnosis onwards (median 29 days, range 9-74). S. aureus strains (median 3, range 1-6) and serial serum samples (median 16, range 6-27) were collected. Strains were genotyped by pulsed-field gel electrophoresis (PFGE) and genes encoding 19 staphylococcal proteins were detected by polymerase chain reaction (PCR). The levels of IgG, IgA, and IgM directed to these proteins were determined using bead-based flow cytometry. All strains isolated from individual patients were PFGE-identical. The genes encoding clumping factor (Clf) A, ClfB, and iron-responsive surface-determinant (Isd) A were detected in all isolates. Antigen-specific IgG levels increased more frequently than IgA or IgM levels. In individual patients, different proteins induced an immune response and the dynamics clearly differed. Anti-ClfB, anti-IsdH, and anti-fibronectin-binding protein A IgG levels increased in 7 of 13 adult patients (p < 0.05). The anti-IsdA IgG level increased in 12 patients (initial to peak level: 1.13-10.72 fold; p < 0.01). Peak level was reached 7-37 days after diagnosis. In a bacteremic 5-day-old newborn, antistaphylococcal IgG levels declined from diagnosis onwards. In conclusion, each bacteremic patient develops a unique immune response directed to different staphylococcal proteins. Therefore, vaccines should be based on multiple components. IsdA is immunogenic and, therefore, produced in nearly all bacteremic patients. This suggests that IsdA might be a useful component of a multivalent staphylococcal vaccine.

Determinants of bacterial colonization in children have been described. In the Generation R Study, a population-based cohort study, we determined whether the colonization statuses of mothers and children are correlated. Such a correlation was observed for Staphylococcus aureus and Haemophilus influenzae. Direct transmission, genetic susceptibility and/or unidentified environmental factors may play a role here.

At the Veterinary Microbiological Diagnostic Center, the Netherlands, the percentage of methicillin-resistant Staphylococcus aureus (MRSA) isolates found in equine clinical samples increased from 0% in 2002 to 37% in 2008. MRSA of spa-type t064, belonging to MLST ST8 and spa-types t011 and t2123, both belonging to the livestock-associated MLST ST398, predominated. During an outbreak of post-surgical MRSA infections in horses at a veterinary teaching hospital in 2006/2007, MRSA isolates of spa-type t2123 were cultured from 7 horses and 4/61 personnel which indicated zoonotic transmission. After intervention the outbreak stopped. However, another outbreak occurred in 2008, where 17 equine MRSA isolates of spa-type t011 (n=12), t2123 (n=4), and t064 (n=1) were found. This time, 16/170 personnel were positive for MRSA with spa-type t011 (n=11) and t2123 (n=5). Personnel in close contact with horses were more often MRSA-positive (15/106) than those without (1/64). Screening of horses upon admission showed that 9.3% were MRSA-positive predominantly with spa-type t011. Weekly cross-sectional sampling of all hospitalized horses for 5 weeks showed that 42% of the horses were MRSA-positive at least once, again predominantly with spa-type t011, which suggests that nosocomial transmission took place. Fifty-three percent of the environmental samples were MRSA-positive, including samples from students' and staff members' rooms, and all were spa-type t011. This indicates that humans contribute to spreading the organism. Culturing of samples employing high-salt pre-enrichment performed better than a comparable method without pre-enrichment. Our results show that nosocomial transmission occurs in equine clinics and suggests that personnel play a role in the transmission., (Copyright 2009 Elsevier B.V. All rights reserved.)

We compared genotype and virulence gene profiles for strains from carriers with autologous invasive infection (n = 56), nasal isolates from matched carriers (n = 108), and invasive strains from noncarriers (n = 34). Superantigen gene profiles and presence of exfoliative toxin genes A and D were associated with clonal complex rather than with invasive disease., (2010 Elsevier Inc. All rights reserved.)

Background: Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated., Methods: Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects were analyzed for immunoglobulin (Ig) G binding to 20 toxins, using flow-cytometry based technology. Antibody levels were associated with polymerase chain reaction-defined presence of toxin genes in homologous S. aureus isolates., Results: IgG levels directed to exfoliative toxin (ET) A, ETB, gamma hemolysin B (HlgB), leukocidin (Luk) D, LukE, LukS, staphylococcal enterotoxin (SE) A, SEE, SEH, SEI, and SElM were higher in S. aureus-infected patients than in control subjects (P < .05). Furthermore, in the S. aureus-infected patient group, IgG levels were higher if genes encoding ETA, ETB, SEA, SEC, SEH, SElQ, toxic shock syndrome toxin-1 (TSST-1), or Panton-Valentine leukocidin (PVL) were present in the infectious isolate (P< .05). Levels of anti-SEA IgG increased during infections with sea-positive (median fluorescence intensity from 11,555 to 12,388; P<.05) but not sea-negative strains. In addition, anti-LukS IgG levels increased during skin and soft-tissue infections with luk-PV-positive (median fluorescence intensity from 15,231 to 15,911; P<.05) but not luk-PV-negative strains. Bacteremia was associated with sea (odds ratio, 3.4; 95% confidence interval, 1.2-10.0) and tst (odds ratio, 5.7; 95% confidence interval, 1.6-20.8). Skin and soft-tissue infections and bone and joint infections were associated with luk-PV (odds ratio, 2.5; 95% confidence interval, 1.2-5.2)., Conclusions: Many toxins are expressed in vivo and recognized by the immune system during staphylococcal infections, suggesting their involvement in S. aureus pathogenesis.

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.

Hospital-acquired Enterococcus faecium isolates responsible for nosocomial outbreaks and invasive infections are enriched in the orf2351 and orf2430 genes, encoding the SgrA and EcbA LPXTG-like cell wall-anchored proteins, respectively. These two surface proteins were characterized to gain insight into their function, since they may have favored the rapid emergence of this nosocomial pathogen. We are the first to identify a surface adhesin among bacteria (SgrA) that binds to the extracellular matrix molecules nidogen 1 and nidogen 2, which are constituents of the basal lamina. EcbA is a novel E. faecium MSCRAMM (microbial surface component recognizing adhesive matrix molecules) that binds to collagen type V. In addition, both SgrA and EcbA bound to fibrinogen; however, SgrA targeted the alpha and beta chains, whereas EcbA bound to the gamma chain of fibrinogen. An E. faecium sgrA insertion mutant displayed reduced binding to both nidogens and fibrinogen. SgrA did not mediate binding of E. faecium cells to biotic materials, such as human intestinal epithelial cells, human bladder cells, and kidney cells, while this LPXTG surface adhesin is implicated in E. faecium biofilm formation. The acm and scm genes, encoding two other E. faecium MSCRAMMs, were expressed at the mRNA level together with sgrA during all phases of growth, whereas ecbA was expressed only in exponential and late exponential phase, suggesting orchestrated expression of these adhesins. Expression of these surface proteins, which bind to extracellular matrix proteins and are involved in biofilm formation (SgrA), may contribute to the pathogenesis of hospital-acquired E. faecium infections.

Lactating cows were immunized with inactivated Staphylococcus aureus strains and concentrated culture supernatants. Application of a repeated mucosal immunization scheme resulted in significant levels of S. aureus-specific IgA in milk of dairy cows. Average IgA titers against whole cell S. aureus increased during the first 10 weeks of immunization after which a plateau level was reached and maintained during lactation. Immune whey agglutinated both bovine and human S. aureus strains including methicillin-resistant S. aureus (MRSA) strains and recognized extracted S. aureus proteins on Western blot. ELISAs to quantify milk IgA reactive with a number of S. aureus virulence proteins (e.g. enterotoxins, microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) and immune modulating proteins) and cell wall components, demonstrated the polyclonality of the IgA. Correlations observed between agglutination and specific IgA titers for whey and for purified IgA suggested functionality of the induced antibodies. Milk from immunized cows may provide a way of producing potentially therapeutic polyclonal antibodies against S. aureus colonization and infection.

Background: Persistent carriers have a higher risk of Staphylococcus aureus infections than noncarriers but a lower risk of bacteremia-related death. Here, the role played by anti-staphylococcal antibodies was studied., Methods: Serum samples from 15 persistent carriers and 19 noncarriers were analyzed for immunoglobulin (Ig) G, IgA, and IgM binding to 19 S. aureus antigens, by means of Luminex technology. Nasal secretions and serum samples obtained after 6 months were also analyzed., Results: Median serum IgG levels were significantly higher in persistent carriers than in noncarriers for toxic shock syndrome toxin (TSST)-1 (median fluorescence intensity [MFI] value, 11,554 vs. 4291; P < .001) and staphylococcal enterotoxin (SE) A (742 vs. 218; P < .05); median IgA levels were higher for TSST-1 (P < .01), SEA, and clumping factor (Clf) A and B (P < .05). The in vitro neutralizing capacity of anti-TSST-1 antibodies was correlated with the MFI value (R(2) = 0.93) and was higher in persistent carriers (90.6% vs. 70.6%; P < .05). Antibody levels were stable over time and correlated with levels in nasal secretions (for IgG, R(2) = 0.87; for IgA, R(2) = 0.77)., Conclusions: Antibodies to TSST-1 have a neutralizing capacity, and median levels of antibodies to TSST-1, SEA, ClfA, and ClfB are higher in persistent carriers than in noncarriers. These antibodies might be associated with the differences in the risk and outcome of S. aureus infections between nasal carriers and noncarriers.

In a household setting within a residential care facility for visually and intellectually disabled people, a resident (index case) was diagnosed with dermal abscesses caused by a methicillin-resistant Staphylococcus aureus (MRSA) which was non-typeable by standard pulsed-field gel electrophoresis. All residents and staff in contact with the index case (a total of 200 people) were screened for MRSA.

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.

A genetic subpopulation of Enterococcus faecium, called clonal complex 17 (CC-17), is strongly associated with hospital outbreaks and invasive infections. Most CC-17 strains contain a putative pathogenicity island encoding the E. faecium variant of enterococcal surface protein (Esp). Western blotting, flow cytometric analyses, and electron microscopy showed that Esp is expressed and exposed on the surface of E. faecium, though Esp expression and surface exposure are highly varied among different strains. Furthermore, Esp expression depends on growth conditions like temperature and anaerobioses. When grown at 37 degrees C, five of six esp-positive E. faecium strains showed significantly increased levels of surface-exposed Esp compared to bacteria grown at 21 degrees C, which was confirmed at the transcriptional level by real-time PCR. In addition, a significant increase in surface-exposed Esp was found in half of these strains when grown at 37 degrees C under anaerobic conditions compared to the level in bacteria grown under aerobic conditions. Finally, amounts of surface-exposed Esp correlated with initial adherence to polystyrene (R(2) = 0.7146) and biofilm formation (R(2) = 0.7535). Polystyrene adherence was competitively inhibited by soluble recombinant N-terminal Esp. This study demonstrates that Esp expression on the surface of E. faecium (i) varies consistently between strains, (ii) is growth condition dependent, and (iii) is quantitatively correlated with initial adherence and biofilm formation. These data indicate that E. faecium senses and responds to changing environmental conditions, which might play a role in the early stages of infection when bacteria transit from oxygen-rich conditions at room temperature to anaerobic conditions at body temperature. In addition, variation of surface exposure may explain the contrasting findings reported on the role of Esp in biofilm formation.

Enterococcus faecium, an ubiquous colonizer of humans and animals, has evolved in the last 15 years from an avirulent commensal to the third most frequently isolated nosocomial pathogen among intensive care unit patients in the United States. E. faecium combines multidrug resistance with the potential of horizontal resistance gene transfer to even more pathogenic bacteria. Little is known about the evolution and virulence of E. faecium, and genomic studies are hampered by the absence of a completely annotated genome sequence. To further unravel its evolution, we used a mixed whole-genome microarray and hybridized 97 E. faecium isolates from different backgrounds (hospital outbreaks (n = 18), documented infections (n = 34) and asymptomatic carriage of hospitalized patients (n = 15), and healthy persons (n = 15) and animals (n = 21)). Supported by Bayesian posterior probabilities (PP = 1.0), a specific clade containing all outbreak-associated strains and 63% of clinical isolates was identified. Sequencing of 146 of 437 clade-specific inserts revealed mobile elements (n = 74), including insertion sequence (IS) elements (n = 42), phage genes (n = 6) and plasmid sequences (n = 26), hypothetical (n = 58) and membrane proteins (n = 10), and antibiotic resistance (n = 9) and regulatory genes (n = 11), mainly located on two contigs of the unfinished E. faecium DO genome. Split decomposition analysis, varying guanine cytosine content, and aberrant codon adaptation indices all supported acquisition of these genes through horizontal gene transfer with IS16 as the predicted most prominent insert (98% sensitive, 100% specific). These findings suggest that acquisition of IS elements has facilitated niche adaptation of a distinct E. faecium subpopulation by increasing its genome plasticity. Increased genome plasticity was supported by higher diversity indices (ratio of average genetic similarities of pulsed-field gel electrophoresis and multi locus sequence typing) for clade-specific isolates. Interestingly, the previously described multi locus sequence typing-based clonal complex 17 largely overlapped with this clade. The present data imply that the global emergence of E. faecium, as observed since 1990, represents the evolution of a subspecies with a presumably better adaptation than other E. faecium isolates to the constraints of a hospital environment.

Chemotaxis inhibitory protein of staphylococci (CHIPS) and Staphylococcal complement inhibitor (SCIN) are small, excreted molecules that play a crucial role in the staphylococcal defence against the human innate immune system. Here we show that they both counteract crucial acute responses of our immune system such as complement activation, neutrophil chemotaxis and neutrophil activation. By studying gene expression via promoter-green fluorescent protein fusions, Northern blots and protein expression analyses, we show that SCIN and CHIPS are produced during the early (exponential) growth stages. Although the SCIN and CHIPS genes are expressed simultaneously, they are differently regulated by various Staphylococcus aureus regulatory loci. However, the sae locus is crucial for upregulation of both SCIN and CHIPS. This is the first study that presents the expression of two extracellular S. aureus proteins early during growth. Because SCIN and CHIPS are both efficient modulators of neutrophil chemotaxis, phagocytosis and killing, their early expression is necessary for efficient modulation of the early immune response.

Staphylococcus aureus staphylococcal cassette chromosome mec type IV (SSCmec IV) is associated with virulent community-acquired methicillin-resistant Staphylococcus aureus (MRSA) and frequent horizontal transfer among staphylococci. To gain insight into the mechanism of transfer, we studied the ccrA/B type 2 recombinase-mediated excision of SCCmec IV (n = 5 strains) and SCCmec II (n = 2). In SCCmec IV- but not SCCmec II-containing strains, spontaneous excision of the cassette was observed. Introduction of ccrA/B type 2 recombinase genes under control of an S. aureus bacterial phage promoter in the different strains yielded excision of SCCmec II and multiple excision variants of SCCmec IV. Sequencing of the alternatively excised products in SCCmec IV strains identified a 100-bp shortened SCCmec' variant and a 5,877-bp, conserved SCC-like element that lacks mecA and ccrA/B recombinases. Excision of the SCC-like element in wild-type S. aureus was dependent on the presence of SCCmec. The element could be excised separately or as part of a novel composite cassette together with SCCmec. The relative abundance of and variety in SCCmec IV excisions may contribute to the frequency of horizontal transfer and genetic plasticity in SCCmec IV MRSA strains.