Markers of epidemiological success of methicillin-resistant Staphylococcus aureus isolates in European populations.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) infections impose a considerable burden on health systems, yet there is remarkable variation in the global incidence and epidemiology of MRSA. The MACOTRA consortium aimed to identify bacterial markers of epidemic success of MRSA isolates in Europe using a representative MRSA collection originating from France, the Netherlands and the United Kingdom. METHODS: Operational definitions of success were defined in consortium meetings to compose a balanced strain collection of successful and sporadic MRSA isolates. Isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing; genes were identified and phylogenetic trees constructed. Markers of epidemiological success were identified using genome-based time-scaled haplotypic density analysis and linear regression. Antimicrobial usage data from ESAC-Net was compared with national MRSA incidence data. RESULTS: Heterogeneity of MRSA isolate collections across countries hampered the use of a unified operational definition of success; therefore, country-specific approaches were used to establish the MACOTRA strain collection. Phenotypic antimicrobial resistance varied within related MRSA populations and across countries. In time-scaled haplotypic density analysis, fluoroquinolone, macrolide and mupirocin resistance were associated with MRSA success, whereas gentamicin, rifampicin and trimethoprim resistance were associated with sporadicity. Usage of antimicrobials across 29 European countries varied substantially, and ß-lactam, fluoroquinolone, macrolide and aminoglycoside use correlated with MRSA incidence. DISCUSSION: Our results are the strongest yet to associate MRSA antibiotic resistance profiles and antibiotic usage with the incidence of infection and successful clonal spread, which varied by country. Harmonized isolate collection, typing, resistance profiling and alignment with antimicrobial usage over time will aid comparisons and further support country-specific interventions to reduce MRSA burden.

The survival of epidemic and sporadic MRSA on human skin mimics is determined by both host and bacterial factors.

Bacterial survival on, and interactions with, human skin may explain the epidemiological success of MRSA strains. We evaluated the bacterial counts for 27 epidemic and 31 sporadic MRSA strains on 3D epidermal models based on N/TERT cells (NEMs) after 1, 2 and 8 days. In addition, the expression of antimicrobial peptides (hBD-2, RNase 7), inflammatory cytokines (IL-1ß, IL-6) and chemokine IL-8 by NEMs was assessed using immunoassays and the expression of 43 S. aureus virulence factors was determined by a multiplex competitive Luminex assay. To explore donor variation, bacterial counts for five epidemic and seven sporadic MRSA strains were determined on 3D primary keratinocyte models (LEMs) from three human donors. Bacterial survival was comparable on NEMs between the two groups, but on LEMs, sporadic strains showed significantly lower survival numbers compared to epidemic strains. Both groups triggered the expression of immune factors. Upon interaction with NEMs, only the epidemic MRSA strains expressed pore-forming toxins, including alpha-hemolysin (Hla), gamma-hemolysin (HlgB), Panton-Valentine leucocidin (LukS) and LukED. Together, these data indicate that the outcome of the interaction between MRSA and human skin mimics, depends on the unique combination of bacterial strain and host factors.

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.

Dispersing and Sonoporating Biofilm-Associated Bacteria with Sonobactericide.

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 µg/mL). Biofilms were exposed to ultrasound (2 MHz, 100-400 kPa, 100-1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy.

Real time monitoring of Staphylococcus aureus biofilm sensitivity towards antibiotics with isothermal microcalorimetry.

Biofilm-associated infections with Staphylococcus aureus are difficult to treat even after administration of antibiotics that according to the standard susceptibility assays are effective. Currently, the assays used in the clinical laboratories to determine the sensitivity of S. aureus towards antibiotics are not representing the behaviour of biofilm-associated S. aureus, since these assays are performed on planktonic bacteria. In research settings, microcalorimetry has been used for antibiotic susceptibility studies. Therefore, in this study we investigated if we can use isothermal microcalorimetry to monitor the response of biofilm towards antibiotic treatment in real-time. We developed a reproducible method to generate biofilm in an isothermal microcalorimeter setup. Using this system, the sensitivity of 5 methicillin-sensitive S. aureus (MSSA) and 5 methicillin-resistant S. aureus (MRSA) strains from different genetic lineages were determined towards: flucloxacillin, cefuroxime, cefotaxime, gentamicin, rifampicin, vancomycin, levofloxacin, clindamycin, erythromycin, linezolid, fusidic acid, co-trimoxazole, and doxycycline. In contrast to conventional assays, our calorimetry-based biofilm susceptibility assay showed that S. aureus biofilms, regardless MSSA or MRSA, can survive the exposure to the maximum serum concentration of all tested antibiotics. The only treatment with a single antibiotic showing a significant reduction in biofilm survival was rifampicin, yet in 20% of the strains, emerging antibiotic resistance was observed. Furthermore, the combination of rifampicin with flucloxacillin, vancomycin or levofloxacin was able to prevent S. aureus biofilm from becoming resistant to rifampicin. Isothermal microcalorimetry allows real-time monitoring of the sensitivity of S. aureus biofilms towards antibiotics in a fast and reliable way.

Microcalorimetry: A Novel Application to Measure In Vitro Phage Susceptibility of Staphylococcus aureus in Human Serum.

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

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms.

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0-12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.

Direct detection of SARS-CoV-2 antisense and sense genomic RNA in human saliva by semi-autonomous fluorescence in situ hybridization: A proxy for contagiousness?

Saliva is a matrix which may act as a vector for pathogen transmission and may serve as a possible proxy for SARS-CoV-2 contagiousness. Therefore, the possibility of detection of intracellular SARS-CoV-2 in saliva by means of fluorescence in situ hybridization is tested, utilizing probes targeting the antisense or sense genomic RNA of SARS-CoV-2. This method was applied in a pilot study with saliva samples collected from healthy persons and those presenting with mild or moderate COVID-19 symptoms. In all participants, saliva appeared a suitable matrix for the detection of SARS-CoV-2. Among the healthy, mild COVID-19-symptomatic and moderate COVID-19-symptomatic persons, 0%, 90% and 100% tested positive for SARS-CoV-2, respectively. Moreover, the procedure allows for simultaneous measurement of viral load ('presence', sense genomic SARS-CoV-2 RNA) and viral replication ('activity', antisense genomic SARS-CoV-2 RNA) and may yield qualitative results. In addition, the visualization of DNA in the cells in saliva provides an additional cytological context to the validity and interpretability of the test results. The method described in this pilot study may be a valuable diagnostic tool for detection of SARS-CoV-2, distinguishing between 'presence' (viral load) and 'activity' (viral replication) of the virus. Moreover, the method potentially gives more information about possible contagiousness.

Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains.

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

Exploring Virulence Factors and Alternative Therapies against Staphylococcus aureus Pneumonia.

Pneumonia is an acute pulmonary infection associated with high mortality and an immense financial burden on healthcare systems. Staphylococcus aureus is an opportunistic pathogen capable of inducing S. aureus pneumonia (SAP), with some lineages also showing multidrug resistance. Given the high level of antibiotic resistance, much research has been focused on targeting S. aureus virulence factors, including toxins and biofilm-associated proteins, in an attempt to develop effective SAP therapeutics. Despite several promising leads, many hurdles still remain for S. aureus vaccine research. Here, we review the state-of-the-art SAP therapeutics, highlight their pitfalls, and discuss alternative approaches of potential significance and future perspectives.

Novel use of culturomics to identify the microbiota in hospital sink drains with and without persistent VIM-positive Pseudomonas aeruginosa.

In hospitals, Verona Integron-encoded Metallo-beta-lactamase (VIM)-positive Pseudomonas aeruginosa may colonize sink drains, and from there, be transmitted to patients. These hidden reservoirs are difficult to eradicate since P. aeruginosa forms biofilms that resist disinfection. However, little is known on the composition of these biofilms. Therefore, culturomics was used for the first time to investigate the viable microbiota in four hospital sink drain samples with longstanding VIM-positive P. aeruginosa drain reservoirs (inhabited by high-risk clone, sequence type ST111), and four drain samples where VIM-positive P. aeruginosa was not present. Microbial load and composition varied between samples, yielding between 471-18,904 distinct colonies and 8-20 genera. In two VIM-positive drain samples, P. aeruginosa was the most abundantly-isolated microorganism, and found in combination with other Gram-negative bacteria, Citrobacter, Enterobacter, or Stenotrophomonas. P. aeruginosa was in low abundance in the other two VIM-positive samples, and found with Gram-positive cocci (Enterococcus and Staphylococcus) or Sphingomonas. In VIM-negative drain samples, high abundances of Gram-negative non-fermenting bacteria, including Acinetobacter, non-aeruginosa Pseudomonas spp., Acidovorax, Chryseobacterium, Flavobacterium, and Sphingobium, as well as Candida, were cultured. Although additional experiments are needed to draw more firm conclusions on which microorganisms enable or inhibit VIM-positive P. aeruginosa persistence, our data provide unique insights into the microbial compositions of sink drain inlets.

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, which we have termed sonobactericide. This review summarizes investigations that have provided evidence for ultrasound-activated microbubble or droplet treatment of bacteria and biofilm. In particular, we review the types of bacteria and therapeutics used for treatment and the in vitro and pre-clinical experimental setups employed in sonobactericide research. Mechanisms for ultrasound enhancement of sonobactericide, with a special emphasis on acoustic cavitation and radiation force, are reviewed, and the potential for clinical translation is discussed.

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.

During the Early Stages of Staphylococcus aureus Biofilm Formation, Induced Neutrophil Extracellular Traps Are Degraded by Autologous Thermonuclease.

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

Production of Staphylococcal Complement Inhibitor (SCIN) and Other Immune Modulators during the Early Stages of Staphylococcus aureus Biofilm Formation in a Mammalian Cell Culture Medium.

Immune modulators are known to be produced by matured biofilms and during different stages of planktonic growth of Staphylococcus aureus Little is known about immune modulator production during the early stages of biofilm formation, thus raising the following question: how does S. aureus protect itself from the innate immune responses at these stages? Therefore, we determined the production of the following immune modulators: chemotaxis inhibitory protein of staphylococci (CHIPS); staphylococcal complement inhibitor (SCIN); formyl peptide receptor-like 1 inhibitor; gamma-hemolysin component B; leukocidins D, E, and S; staphylococcal superantigen-like proteins 1, 3, 5, and 9; and staphylococcal enterotoxin A. Production was determined during in vitro biofilm formation in Iscove's modified Dulbecco's medium at different time points using a competitive Luminex assay and mass spectrometry. Both methods demonstrated the production of the immune modulators SCIN and CHIPS during the early stages of biofilm formation. The green fluorescence protein promoter fusion technology confirmed scn (SCIN) and, to a lesser extent, chp (CHIPS) transcription during the early stages of biofilm formation. Furthermore, we found that SCIN could inhibit human complement activation induced by early biofilms, indicating that S. aureus is able to modulate the innate immune system already during the early stages of biofilm formation in vitro These results form a stepping stone toward elucidating the role of immune modulators in the establishment of biofilms in vivo and present opportunities to develop preventive strategies.

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 follow-up 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.

Human Immunoglobulin G Cannot Inhibit Fibrinogen Binding by the Genetically Diverse A Domain of Staphylococcus aureus Fibronectin-Binding Protein A.

The fibronectin-binding protein A (FnBPA) is a cell surface-associated protein of Staphylococcus aureus which mediates adherence to the host extracellular matrix and is important for bacterial virulence. Previously, substantial sequence diversity was found among strains in the fibrinogen-binding A domain of this protein, and 7 different isotypes were described. The effect of this sequence diversity on the human antibody response, in terms of both antibody production and antibody function, remains unclear. In this study, we identify five different FnBPA A domain isotypes based on the sequence results of 22 clinical S. aureus isolates, obtained from the same number of patients suffering from bacteremia. Using a bead-based Luminex technique, we measure the patients' total immunoglobulin G (IgG) against the 7 FnBPA isotypes at the onset and during the time course of bacteremia (median of 10 serum samples per patient over a median of 35 days). A significant increase in IgG against the FnBPA A domain, including the isotype carried by the infecting strain, is observed in only three out of 22 patients (14%) after the onset of bacteremia. Using a Luminex-based FnBPA-fibrinogen-binding assay, we find that preincubation of recombinant FnBPA isotypes with IgG from diverse patients does not interfere with binding to fibrinogen. This observation is confirmed using an alternative Luminex-based assay and enzyme-linked immunosorbent assay (ELISA). IMPORTANCE Despite the many in vitro and murine in vivo studies involving FnBPA, the actual presence of this virulence factor during human infection is less well established. Furthermore, it is currently unknown to what extent sequence variation in such a virulence factor affects the human antibody response and the ability of antibodies to interfere with FnBPA function. This study sheds new light on these issues. First, the uniform presence of a patient's IgG against FnBPA indicates the presence and importance of this virulence factor during S. aureus pathogenesis. Second, the absence of an increase in antibody production in most patients following bacteremia indicates the complexity of S. aureus-host interactions, possibly involving immune evasion or lack of expression of FnBPA during invasive infection. Finally, we provide new insights into the inability of human antibodies to interfere with FnBPA-fibrinogen binding. These observations should be taken into account during the development of novel vaccination approaches.

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.

Staphylococcal Protein A Is a Key Factor in Neutrophil Extracellular Traps Formation.

Staphylococcus aureus are strong inducers of neutrophil extracellular traps (NETs), a defense mechanism of neutrophils against pathogens. Our aim was to explore the role of Protein A in S. aureus-induced NETosis. We determined the Protein A production of four different S. aureus strains and found a direct relationship between the degree of NETosis induction and Protein A production: strains producing higher concentrations of Protein A evoke significantly more NETs. A S. aureus strain in which Protein A as well as a second binding protein for immunoglobulins (Sbi) have been knocked-out (ΔSpA ΔSbi) induced significantly less NETosis than the wild-type strain. NETosis induction by this knockout strain can be rescued by the addition of purified Protein A. Dead S. aureus did not induce NETosis. In conclusion, Protein A is a determinant for NETosis induction by S. aureus.

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.