Dendritic cells internalize staphylococcus aureus more efficiently than staphylococcus epidermidis, but do not differ in induction of antigen-specific t cell proliferation

Staphylococcus aureus and Staphylococcus epidermidis are related species which can cause predominantly acute and subacute infections, respectively. Differences in human adaptive immune responses to these two species are not well understood. Dendritic cells (DCs) have an important role in the control and regulation of anti-staphylococcal T cell responses. Therefore, we aimed to compare the ability of S. aureus and S. epidermidis to influence the essential steps in human DC activation and subsequent antigen-specific CD4+ T cell proliferation, and to investigate the underlying mechanisms. Using multiple strains of both species, we observed that S. aureus was internalized more effectively than S. epidermidis by DCs but that both species were equally potent in activating these host cells, as evidenced by similar induction of DC maturation marker expression and antigen loading onto MHC-II molecules. The DCs stimulated by S. aureus strains not harboring superantigen (SAg) genes or by any of the S. epidermidis strains, induced low, likely physiological levels of T cell proliferation. Only DCs stimulated with S. aureus strains harboring SAg genes induced high levels of T cell proliferation. Taken together, S. aureus and S. epidermidis do not differently affect DC activation and ensuing antigen-specific T cell proliferation, unless a strain has the capacity to produce SAgs

Synthetic LPETG-containing peptide incorporation in the Staphylococcus aureus cell-wall in a sortase a- and growth phase-dependent manner

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

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

An in vitro proof-of-principle study of sonobactericide

Infective endocarditis (IE) is associated with high morbidity and mortality rates. The predominant bacteria causing IE is Staphylococcus aureus (S. aureus), which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro flow study, we evaluated sonobactericide as a novel strategy to treat IE, using ultrasound and an ultrasound contrast agent with or without other therapeutics. We developed a model of IE biofilm using human whole-blood clots infected with patient-derived S. aureus (infected clots). Histology and live-cell imaging revealed a biofilm layer of fibrin-embedded living Staphylococci around a dense erythrocyte core. Infected clots were treated under flow for 30 minutes and degradation was assessed by time-lapse microscopy imaging. Treatments consisted of either continuous plasma flow alone or with different combinations of therapeutics: oxacillin (antibiotic), recombinant tissue plasminogen activator (rt-PA; thrombolytic), intermittent continuous-wave low-frequency ultrasound (120-kHz, 0.44 MPa peak-to-peak pressure), and an ultrasound contrast agent (Definity). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% loss, which was greater than the other treatment arms. Effluent size measurements suggested low likelihood of emboli formation. These results support the continued investigation of sonobactericide as a therapeutic strategy for IE

Sonobactericide: An Emerging Treatment Strategy for Bacterial Infections

Ultrasound has been developed as both a diagnostic tool and a potent promoter of beneficial bio-effects for the treatment of chronic bacterial infections. Bacterial infections, especially those involving biofilm on implants, indwelling catheters and heart valves, affect millions of people each year, and many deaths occur as a consequence. Exposure of microbubbles or droplets to ultrasound can directly affect bacteria and enhance the efficacy of antibiotics or other therapeutics

In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review

__Background__ Multiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing. __Methods__ In vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging. __Results__ PMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis. __Conclusion__ Our systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events

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

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

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