Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis

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

Dehydration Tolerance in Epidemic versus Nonepidemic MRSA Demonstrated by Isothermal Microcalorimetry.

Methicillin-resistant Staphylococcus aureus (MRSA) clusters are considered epidemic or nonepidemic based on their ability to spread effectively. Successful transmission could be influenced by dehydration tolerance. Current methods for determination of dehydration tolerance lack accuracy. Here, a climate-controlled in vitro dehydration assay using isothermal microcalorimetry (IMC) was developed and linked with mathematical modeling to determine survival of 44 epidemic versus 54 nonepidemic MRSA strains from France, the United Kingdom, and the Netherlands after 1 week of dehydration. For each MRSA strain, the growth parameters time to end of first growth phase (tmax [h]) and maximal exponential growth rate (μm) were deduced from IMC data for 3 experimental replicates, 3 different starting inocula, and before and after dehydration. If the maximal exponential growth rate was within predefined margins (±36% of the mean), a linear relationship between tmax and starting inoculum could be utilized to predict log reduction after dehydration for individual strains. With these criteria, 1,330 of 1,764 heat flow curves (data sets) (75%) could be analyzed to calculate the post-dehydration inoculum size, and thus the log reduction due to dehydration, for 90 of 98 strains (92%). Overall reduction was ~1 log after 1 week. No difference in dehydration tolerance was found between the epidemic and nonepidemic strains. Log reduction was negatively correlated with starting inoculum, indicating better survival of higher inocula. This study presents a framework to quantify bacterial survival. MRSA strains showed great capacity to persist in the environment, irrespective of epidemiological success. This finding strengthens the need for effective surface cleaning to contain MRSA transmission. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of infections globally. While some MRSA clusters have spread worldwide, others are not able to disseminate successfully beyond certain regions despite frequent introduction. Dehydration tolerance facilitates transmission in hospital environments through enhanced survival on surfaces and fomites, potentially explaining differences in transmission success between MRSA clusters. Unfortunately, the currently available techniques to determine dehydration tolerance of cluster-forming bacteria like S. aureus are labor-intensive and unreliable due to their dependence on quantitative culturing. In this study, bacterial survival was assessed in a newly developed assay using isothermal microcalorimetry. With this technique, the effect of drying can be determined without the disadvantages of quantitative culturing. In combination with a newly developed mathematical algorithm, we determined dehydration tolerance of a large number of MRSA strains in a systematic, unbiased, and robust manner

An experimental Staphylococcus aureus carriage and decolonization model in rhesus macaques (Macaca mulatta)

Our human model of nasal colonization and eradication of S. aureus is limited by safety issues. As rhesus macaques are closely related to humans and natural hosts for S. aureus, we developed an experimental decolonization and inoculation protocol in these animals. Animals were screened for nasal carriage of S. aureus and 20 carriers were selected. Decolonization was attempted using nasal mupirocin (10 animals) or mupirocin plus trimethoprim/ sulfadiazine intramuscularly (10 animals) both once daily for 5 days, and checked by followup cultures for 10 weeks. Intranasal inoculation was performed with S. aureus strain 8325–4 in culture-negative animals. 11/20 animals, of which 5 received mupirocin and 6 the combination treatment, became culture-negative for S. aureus for 10 weeks and these 11 animals were subsequently inoculated. Swabs were taken once a week for 5 weeks to test for the presence of the inoculated strain. In 3 animals, strain 8325–4 was cultured from the nose 1 week after inoculation, indicating short-term survival of this strain only, a finding similar to that previously found in our human model. These data demonstrate that rhesus macaques may constitute a relevant animal model to perform S. aureus eradication and inoculation studies with relatively limited invasive handling of the animals

Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

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

Genomic evolution of staphylococcus aureus during artificial and natural colonization of the human nose

Staphylococcus aureus can colonize the human vestibulum nasi for many years. It is unknown whether and, how S. aureus adapts to this ecological niche during colonization. We determined the short (1 and 3 months) and mid-term (36 months) genomic evolution of S. aureus in natural carriers and artificially colonized volunteers. Eighty-five S. aureus strains were collected from 6 natural carriers during 3 years and 6 artificially colonized volunteers during 1 month. Multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis based on whole-genome sequencing (WGS) were carried out. Mutation frequencies within resident bacterial populations over time were quantified using core genome SNP counts (comparing groups of genomes) and pairwise SNP divergence assessment (comparing two genomes from strains originating from one host and sharing identical MLST). SNP counts (within 1-3 months) in all naturally colonizing strains varied from 0 to 757 (median 4). These strains showed random and independent patterns of pairwise SNP divergence (0 to 44 SNPs, median 7). When the different core genome SNP counts over a period of 3 years were considered, the median SNP count was 4 (range 0-26). Host-specific pairwise SNP divergence for the same period ranged from 9 to 57 SNPs (median 20). During short term artificial colonization the mutation frequency was even lower (0-7 SNPs, median 2) and the pairwise SNP distances were 0 to 5 SNPs (median 2). Quantifying mutation frequencies is important for the longitudinal follow-up of epidemics of infections and outbreak management. Random pattern of pairwise SNP divergence between the strains isolated from single carriers suggested that the WGS of multiple colonies is necessary in this context. Over periods up to 3 years, maximum median core genome SNP counts and SNP divergence for the strains studied were 4 and 20 SNPs or lower. During artificial colonization, where median core genome SNP and pairwise SNP distance scores were 2, there is no early stage selection of different genotypes. Therefore, we suggest an epidemiological cut off value of 20 SNPs as a marker of S. aureus strain identity during studies on nasal colonization and also outbreaks of infection

Genetic loci of Staphylococcus aureus associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides

The proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at comparing the genetic make-up of S. aureus isolates from PR3-ANCA-positive GPA patients with that of isolates from patients suffering from myeloperoxidase (MPO)-ANCA-positive AAV, and isolates from healthy controls. Based on a DNA microarray-based approach, we show that not only PR3-ANCA-positive GPA patients, but also MPO-ANCA-positive AAV patients mainly carried S. aureus types that are prevalent in the general population. Nonetheless, our data suggests that MPO-ANCA-associated S. aureus isolates may be distinct from healthy control- and PR3-ANCA-associated isolates. Furthermore, several genetic loci of S. aureus are associated with either PR3-ANCA- or MPO-ANCA-positive AAV, indicating a possible role for pore-forming toxins, such as leukocidins, in PR3-ANCA-positive GPA. Contrary to previous studies, no association between AAV and superantigens was detected. Our findings also show that a lowered humoral immune response to S. aureus is common for PR3-ANCA- and MPO-ANCA-positive AAV. Altogether, our observations imply that the presence or absence of particular virulence genes of S. aureus isolates from AAV patients contributes to disease progression and/or relapse

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection

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

Low anti-staphylococcal IgG responses in granulomatosis with polyangiitis patients despite long-term Staphylococcus aureus exposure

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

Reshuffling of Aspergillus fumigatus cell wall components chitin and β-glucan under the influence of caspofungin or nikkomycin Z alone or in combination

Chitin and β-glucan are major cell wall components of Aspergillus spp. We investigated the antifungal activity of chitin synthesis inhibitors nikkomycin Z, polyoxin D, flufenoxuron, lufenuron, and teflubenzuron, alone and combined with the β-glucan synthesis inhibitor caspofungin. Only nikkomycin Z and caspofungin were found to act synergistically. The nikkomycin Z-induced chitin decrease corresponded with a β-glucan increase, while with the caspofungin-induced β-glucan decrease, an increase in chitin was found. This could explain the synergistic activity of this combination of drugs. Copyrigh