Human Adaptive Immunity Rescues an Inborn Error of Innate Immunity.

The molecular basis of the incomplete penetrance of monogenic disorders is unclear. We describe here eight related individuals with autosomal recessive TIRAP deficiency. Life-threatening staphylococcal disease occurred during childhood in the proband, but not in the other seven homozygotes. Responses to all Toll-like receptor 1/2 (TLR1/2), TLR2/6, and TLR4 agonists were impaired in the fibroblasts and leukocytes of all TIRAP-deficient individuals. However, the whole-blood response to the TLR2/6 agonist staphylococcal lipoteichoic acid (LTA) was abolished only in the index case individual, the only family member lacking LTA-specific antibodies (Abs). This defective response was reversed in the patient, but not in interleukin-1 receptor-associated kinase 4 (IRAK-4)-deficient individuals, by anti-LTA monoclonal antibody (mAb). Anti-LTA mAb also rescued the macrophage response in mice lacking TIRAP, but not TLR2 or MyD88. Thus, acquired anti-LTA Abs rescue TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP deficiency, accounting for incomplete penetrance. Combined TIRAP and anti-LTA Ab deficiencies underlie staphylococcal disease in this patient.

Staphylococcus aureus-specific skin resident memory T cells protect against bacteria colonization but exacerbate atopic dermatitis-like flares in mice.

BACKGROUND: The contribution of Staphylococcus aureus to the exacerbation of atopic dermatitis (AD) is widely documented, but its role as a primary trigger of AD skin symptoms remains poorly explored. OBJECTIVES: This study sought to reappraise the main bacterial factors and underlying immune mechanisms by which S aureus triggers AD-like inflammation. METHODS: This study capitalized on a preclinical model, in which different clinical isolates were applied in the absence of any prior experimental skin injury. RESULTS: The development of S aureus-induced dermatitis depended on the nature of the S aureus strain, its viability, the concentration of the applied bacterial suspension, the production of secreted and nonsecreted factors, as well as the activation of accessory gene regulatory quorum sensing system. In addition, the rising dermatitis, which exhibited the well-documented AD cytokine signature, was significantly inhibited in inflammasome adaptor apoptosis-associated speck-like protein containing a CARD domain- and monocyte/macrophage-deficient animals, but not in T- and B-cell-deficient mice, suggesting a major role for the innate response in the induction of skin inflammation. However, bacterial exposure generated a robust adaptive immune response against S aureus, and an accumulation of S aureus-specific γδ and CD4+ tissue resident memory T cells at the site of previous dermatitis. The latter both contributed to worsen the flares of AD-like dermatitis on new bacteria exposures, but also, protected the mice from persistent bacterial colonization. CONCLUSIONS: These data highlight the induction of unique AD-like inflammation, with the generation of proinflammatory but protective tissue resident memory T cells in a context of natural exposure to pathogenic S aureus strains.

Bacteriophage therapy reduces Staphylococcus aureus in a porcine and human ex vivo burn wound infection model.

Burn wounds are a major burden, with high mortality rates due to infections. Staphylococcus aureus is a major causative agent of burn wound infections, which can be difficult to treat because of antibiotic resistance and biofilm formation. An alternative to antibiotics is the use of bacteriophages, viruses that infect and kill bacteria. We investigated the efficacy of bacteriophage therapy for burn wound infections, in both a porcine and a newly developed human ex vivo skin model. In both models, the efficacy of a reference antibiotic treatment (fusidic acid) and bacteriophage treatment was determined for a single treatment, successive treatment, and prophylaxis. Both models showed a reduction in bacterial load after a single bacteriophage treatment. Increasing the frequency of bacteriophage treatments increased bacteriophage efficacy in the human ex vivo skin model, but not in the porcine model. In both models, prophylaxis with bacteriophages increased treatment efficacy. In all cases, bacteriophage treatment outperformed fusidic acid treatment. Both models allowed investigation of bacteriophage-bacteria dynamics in burn wounds. Overall, bacteriophage treatment outperformed antibiotic control underlining the potential of bacteriophage therapy for the treatment of burn wound infections, especially when used prophylactically.

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.

Effective antimicrobial combination in vivo treatment predicted with microcalorimetry screening.

OBJECTIVES: The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model. METHODS: The synergy of meropenem combined with colistin, rifampicin or amikacin was tested on 12 isolates (1 Escherichia coli, 5 Klebsiella pneumoniae, 3 Pseudomonas aeruginosa and 3 Acinetobacter baumannii) in an isothermal microcalorimeter measuring metabolic activity. One A. baumannii strain was tested with two individual pairings of antibiotic combinations. The microcalorimetric data were used to predict in vivo efficacy in a murine peritonitis/sepsis model. NMRI mice were inoculated intraperitoneally and after 1 h treated with saline, drug X, drug Y or X+Y. Bacterial load was determined by cfu in peritoneal fluid and blood after 4 h. RESULTS: In vitro, of the 13 combinations tested on the 12 strains, 3 of them exhibited a synergistic reduction in MIC (23% n = 3/13), 5 showed an additive effect (38.5% n = 5/13) and 5 had indifferent or antagonistic effects (38.5% n = 5/13). There was a significant correlation (P = 0.024) between microcalorimetry-screening FIC index values and the log reduction in peritoneal fluid from mice that underwent combination treatment compared with the most effective mono treatment. No such correlation could be found between chequerboard and in vivo results (P = 0.16). CONCLUSIONS: These data support microcalorimetic metabolic readout to predict additive or synergistic effects of combination treatment of MDR infections within hours.

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.

Staphylococcus aureus infections, some second thoughts.

PURPOSE OF REVIEW: Staphylococcus aureus (S. aureus) is well known for its ability to cause life-threatening infections. On the other hand, this bacterium can thrive as a commensal on and in human tissues without causing much problems. How big a threat is S. aureus actually? Furthermore, commensalism is associated with biofilms, where can we find them, and which natural and artificial components activate biofilm formation? RECENT FINDINGS: Recent findings on S. aureus carriage on skin, mucosa, and in wounds indicate the presence of large numbers of S. aureus, yet its abundance can be without major implications for the host. S. aureus is often present in biofilms, together with other microorganisms, which can stimulate biofilm formation of S. aureus, in addition medicine including antibiotics can do the same. SUMMARY: S. aureus can cause devastating infections, but when we take into consideration the ubiquitous presence of S. aureus, the risk seems to be relatively low. S. aureus forms biofilms in response to the 'hazards' on the human body, and signal to do so can come from various sources. All this has to be taken into consideration when we treat a patient as this might have enormous impact on the outcome.

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.

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.