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Group A streptococcal (GAS) meningitis is a fulminant disease associated with high morbidity and mortality. To elucidate the mechanisms underlying the invasiveness of GAS in meningitis, we compared GAS isolates derived from five cases of meningitis, to otitis and colonizing isolates. We did not observe differences in adherence to and invasion of human brain microvascular endothelial cells, virulence factors activity or barrier disruption. Whole genome sequencing did not reveal particular invasiveness traits. Most patients previously suffered from otitis media suggesting that meningitis likely resulted from a continuous spread of the infection rather than being attributable to changes in pathogen’s virulence.

ObjectivesGroup AStreptococcus(GAS) necrotizing fasciitis (NF) is a difficult-to-treat bacterial infection associated with high morbidity and mortality despite extensive surgery and targeted antibiotic treatment. Bacteria surviving prolonged antibiotic exposure without displaying genetic resistance, so-called persisters, are associated with difficult-to-treat infections, such as GAS-NF. In the present study, we investigated the presence of persistent GAS in tissue freshly debrided from three NF patients and examined more in depth the persisters phenomenon in GAS-NF clinical isolates.MethodsTime-lapse imaging of freshly isolated GAS-NF clinical isolates, the image analysis software ColTapp and antibiotic challenge-based persisters assays were used to assess the presence of persisters.ResultsWe show for the first time that GAS recovered directly from freshly debrided NF patient’s tissue are characterized by increased colony appearance time heterogeneity, indicating the presence of persisters. Acidic pH or nutrient stress exposure, mimicking the NF-like environment in vitro, similarly leads to phenotypic heterogeneity resulting in enhanced antibiotic survival and confirming the presence of GAS persisters.ConclusionsGAS persisters are present in the tissue freshly debrided from GAS-NF patients and might be one explanation for antibiotic treatment failure and surgery requirement in GAS-NF. Tailored treatment options, including the use of persisters-targeting drugs, need to be developed to increase GAS-NF therapy success.

Prosthetic vascular graft infections of the thoracic aorta are rare but can be fatal. Our comparison of collagen- and gelatin-coated grafts showed that collagen-coated grafts were associated with increased biofilm formation and bacterial adherence in vitro and with higher rates of perioperative vascular graft infections in vivo.

Staphylococcus aureus carriage is a risk factor for invasive infections. Unique genetic elements favoring the transition from colonizing to invasive phenotype have not yet been identified and phenotypic traits are understudied. We therefore assessed pheno- and genotypic profiles of eleven S. aureus isolate pairs sampled from colonized patients simultaneously suffering from invasive S. aureus infections. Ten out of 11 isolate pairs presented the same spa and MLST suggesting colonization as origin for the invasive infection. Systematic analysis of colonizing and invasive isolate pairs showed similar adherence, hemolysis and reproductive fitness properties, minimal genetic differences, identical antibiotic tolerance and bacterial virulence in Galleria mellonella. Our results provide insights into the similar phenotype associated with limited adaptive genetic evolution between the colonizing and invasive isolates. Disruption of the physical barriers mucosa or skin were identified in the majority of patients further emphasizing colonization as a major risk factor for invasive disease.ImportanceS. aureus is a major pathogen of humans causing a wide range of disease. The failure to develop a vaccine and antibiotic warrant the exploration of novel treatment strategies. Asymptomatic colonization of the human nasal passages is a major risk factor for invasive disease and decolonization procedures have shown efficacy in preventing invasive infections. However, the transition of S. aureus from a benign colonizer of the nasal passages to a major pathogen is not well understood and host as well as bacterial properties have been discussed as being relevant for this behavioral change. We conducted a thorough investigation of patient-derived strain pairs reflecting colonizing and invasive isolates in a given patient. Although we identified limited genetic adaptation in certain strains as well as slight differences in adherence capacity of colonizing and invasive isolates, our work suggests that barrier breaches are a key event in the disease continuum of S. aureus.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite Ags presented by MHC class Ib–related protein (MR1) and play important roles in immune control of microbes that synthesize riboflavin. This includes the pathobiont Staphylococcus aureus, which can also express a range of virulence factors, including the secreted toxin leukocidin ED (LukED). In this study, we found that human MAIT cells are hypersensitive to LukED-mediated lysis and lost on exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid and occurred at toxin concentrations lower than those required for toxicity against conventional T cells. Furthermore, this coincided with high MAIT cell expression of CCR5, and loss of these cells was efficiently inhibited by the CCR5 inhibitor maraviroc. Interestingly, exposure and preactivation of MAIT cells with IL-12 and IL-18, or activation via TCR triggering, partially protected from LukED toxicity. Furthermore, analysis of NK cells indicated that LukED targeted the mature cytotoxic CD57(+) NK cell subset in a CCR5-independent manner. Overall, these results indicate that LukED efficiently eliminates immune cells that can respond rapidly to S. aureus in an innate fashion without the need for clonal expansion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED secretion may allow S. aureus to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

SummaryBackgroundDifficult-to-treat infections caused by antibiotic susceptible strains have been linked with the occurrence of persisters. Persisters are a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. They can be identified phenotypically upon plating on nutrient agar because of their altered growth dynamics, resulting in colony size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remain unknown.MethodsWe plated bacteria derived from 132 patient-samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time-lapse imaging. Of these, we retained 36 Staphylococcus aureus mono-cultures for further analysis. We investigated clinical factors potentially associated with increased colony growth-delay with regression analyses. Additionally, we corroborated the clinical findings using in vitro grown static biofilms, exposed to distinct antibiotics.ResultsThe extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients. Increased heterogeneity coincided with increased median growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay. S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with either clindamycin or levofloxacin exhibited prolonged growth-delay, correlating with a strain-dependent increase in antibiotic tolerance.ConclusionsUpon direct cultivation on nutrient-rich agar, S. aureus from difficult-to-treat infections commonly exhibited colony size heterogeneity. This was due to heterogeneous delays in growth resumption, with delays larger than two days in the most extreme cases. Since bacteria in a dormant state are tolerant to antibiotics, the observation of large growth-delays might have direct clinical implications. Future studies are needed to assess the potential of bacterial phenotypic heterogeneity quantification for staphylococcal infections prognosis.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite antigens presented by MR1 and play an important role in antimicrobial immune defense. Staphylococcus aureus is a pathobiont expressing a range of virulence factors including the secreted toxin Leukocidin ED (LukED), which binds to certain chemokine receptors and causes cell death by osmolysis. Here, we investigated the effect of LukED on subsets of human T cells and NK cells that are involved in the early innate response to infection. MAIT cells were strikingly hypersensitive to LukED-mediated lysis and rapidly lost from the peripheral blood T cell pool upon exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid, occurred at lower LukED concentration compared to effects on the overall T cell pool, and coincided with extraordinarily high and uniform expression of CCR5. Furthermore, loss of MAIT cells was efficiently inhibited by the CCR5 inhibitor Maraviroc. Interestingly, pre-activation of MAIT cells with IL-12 and IL-18 also partially rescued these cells from LukED toxicity. Among NK cells, LukED targeted the more mature and cytotoxic CD57+ NK cell subset in a CXCR1-dependent manner. Overall, these results indicate that LukED efficiently eliminates cells of the human immune system that have the capacity to respond rapidly to S. aureus in an innate fashion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED functions as a S. aureus immune evasion mechanism to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1β/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies.

Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required.We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics.One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation.Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates.In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Rapid detection of antibiotic tolerance offers a new diagnostic readout that might allow more tailored treatments in the future.

The impact of secondary bacterial infections (superinfections) in COVID-19 is not well understood. In this prospective, monocentric cohort study we aim to investigate the impact of superinfections in COVID-19 patients with acute respiratory distress syndrome. Patients are assessed for concomitant microbial infections by longitudinal analysis of tracheobronchial secretions, bronchoalveolar lavages and blood cultures. In 45 critically ill patients, we identify 19 patients with superinfections (42.2%). Superinfections are detected on day 10 after intensive care admission. The proportion of participants alive and off invasive mechanical ventilation at study day 28 (ventilator-free days (VFDs) at 28 days) is substantially lower in patients with superinfection (subhazard ratio 0.37, 95%-CI 0.15-0.90, p=0.028). Patients with pulmonary superinfections have a higher incidence of bacteraemia, virus reactivations, yeast colonization, and required intensive care treatment for a longer time. Superinfections are frequent and associated with reduced VFDs at 28 days despite a high rate of empirical antibiotic therapy., Graphical Abstract, Buehler et al. show that detection of bacterial pulmonary superinfection is associated with a more severe disease course in COVID-19 patients, especially a lower likelihood of being alive and off invasive mechanical ventilation at study day 28 (ventilator-free days at 28 days).

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.

COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections

Background SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. Methods Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. Findings COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. Interpretation ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. Funding No external funding was necessary for this study.

Background: While superinfections are associated with unfavourable disease course, their impact on clinical outcomes in critically ill COVID-19 patients remains largely unknown. We aimed to investigate the impact of superinfections in critically ill patients with COVID-19. Methods: In this prospective single centre cohort study in an intensive care unit (ICU) setting patients aged ≥ 18 years with COVID-19 Acute Respiratory Distress Syndrome (CARDS) and confirmed SARS-CoV-2 infection were assessed for concomitant microbial infections by longitudinal analysis of tracheobronchial secretions (TBS), bronchoalveolar lavages (BAL) and blood. Our primary outcome was ventilator-free survival on day 28 in patients with and without clinically relevant superinfection. Further outcomes included the association of superinfection with ICU length of stay, incidence of bacteremia, viral reactivations, and fungal colonization. Findings: In a total of 45 critically ill COVID-19 patients, we identified 19 patients with superinfections (42·2%) by longitudinal analysis of 433 TBS, 35 BAL and 455 blood samples, respectively. On average, superinfections were detected on day 10 after ICU admission despite the majority of patients already being on empiric antibiotic therapy. The most frequently isolated clinically relevant bacteria were Enterobacter/Citrobacter , Klebsiella spp., Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa. Ventilator-free survival (VFS) at 28 days was substantially lower in patients with superinfection (subhazard ratio 0·37, 95%-CI 0·15-0·90, p=0·028). Patients with pulmonary superinfections more often had bacteraemia, virus reactivations, yeast colonization, and needed ICU treatment for a significantly longer time. Interpretation: The detection of superinfections was frequent and associated with reduced ventilator-free survival. A more severe disease course in critically ill COVID-19 patients with superinfections thus seems likely, also reflected by a substantially increased ICU length of stay despite empirical broad-spectrum antimicrobial therapy. Longitudinal microbiologic sampling in COVID-19 patients could allow targeted antimicrobial therapy, and therefore minimize the use of broad-spectrum and reserve antibiotics. Funding: Promedica-Foundation, unrestricted grant. Declaration of Interests: All other authors have no conflict of interest. Ethics Approval Statement: The study was approved by the local ethics committee of the Canton of Zurich, Switzerland (Kantonale Ethikkommission Zurich BASEC ID 2020 - 00646).

The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections., Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus.

Staphylococcus aureus causes chronic and relapsing infections, which may be difficult to treat. So-called small colony variants (SCVs) have been associated with chronic infections and their occurrence has been shown to increase under antibiotic pressure, low pH and intracellular localization. In clinics, S. aureus isolated from invasive infections often show a dysfunction in the accessory gene regulator (agr), a major virulence regulatory system in S. aureus. To assess whether intracellular environment and agr function influence SCV formation, an infection model was established using lung epithelial cells and skin fibroblasts. This allowed analyzing intracellular survival and localization of a panel of S. aureus wild type strains and their isogenic agr knock out mutants as well as a natural dysfunctional agr strain by confocal laser scanning microscopy (CLSM). Furthermore, bacterial colonies were quantified after 1, 3, and 5 days of intracellular survival by time-lapse analysis to determine kinetics of colony appearance and SCV formation. Here, we show that S. aureus strains with an agr knock out predominantly resided in a neutral environment, whereas wild type strains and an agr complemented strain resided in an acidic environment. S. aureus agr mutants derived from an intracellular environment showed a higher percentage of SCVs as compared to their corresponding wild type strains. Neutralizing acidic phagolysosomes with chloroquine resulted in a significant reduction of SCVs in S. aureus wild type strain 6850, but not in its agr mutant indicating a pH dependent formation of SCVs in the wild type strain. The in-depth understanding of the interplay between intracellular persistence, agr function and pH should help to identify new therapeutic options facilitating the treatment of chronic S. aureus infections in the future.

Bacterial membrane vesicle research has so far focused mainly on Gram-negative bacteria. Only recently have Gram-positive bacteria been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MV formation, the underlying mechanisms are poorly understood. In this study, we show that antibiotics can induce MVs through different routes in the important human pathogen Staphylococcus aureus DNA-damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of S. aureus but not in their phage-devoid counterparts. The β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is greater than that for MVs formed by β-lactam antibiotic-induced blebbing. The purified MVs derived from S. aureus protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both in vitro and ex vivo in whole blood. In addition, the MVs protected S. aureus from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.

Novel approaches for adjunctive therapy are urgently needed for complicated infections and patients with compromised immunity. Necrotizing fasciitis (NF) is a destructive skin and soft tissue infection. Despite treatment with systemic antibiotics and radical debridement of necrotic tissue, lethality remains high. The key iron regulatory hormone hepcidin was originally identified as a cationic antimicrobial peptide (AMP), but its putative expression and role in the skin, a major site of AMP production, have never been investigated. We report here that hepcidin production is induced in the skin of patients with group A Streptococcus (GAS) NF. In a GAS-induced NF model, mice lacking hepcidin in keratinocytes failed to restrict systemic spread of infection from an initial tissue focus. Unexpectedly, this effect was due to its ability to promote production of the CXCL1 chemokine by keratinocytes, resulting in neutrophil recruitment. Unlike CXCL1, hepcidin is resistant to degradation by major GAS proteases and could therefore serve as a reservoir to maintain steady-state levels of CXCL1 in infected tissue. Finally, injection of synthetic hepcidin at the site of infection can limit or completely prevent systemic spread of GAS infection, suggesting that hepcidin agonists could have a therapeutic role in NF.

Necrotizing soft tissue infections (NSTIs) are critical clinical conditions characterized by extensive necrosis of any layer of the soft tissue and systemic toxicity. Group A streptococci (GAS) and Staphylococcus aureus are two major pathogens associated with monomicrobial NSTIs. In the tissue environment, both Gram-positive bacteria secrete a variety of molecules, including pore-forming exotoxins, superantigens, and proteases with cytolytic and immunomodulatory functions. The present review summarizes the current knowledge about streptococcal and staphylococcal toxins in NSTIs with a special focus on their contribution to disease progression, tissue pathology, and immune evasion strategies.

Bacterial membrane vesicle research has so far focused mainly on Gram-negative bacteria. Only recently have Gram-positive bacteria been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MV formation, the underlying mechanisms are poorly understood. In this study, we show that antibiotics can induce MVs through different routes in the important human pathogen Staphylococcus aureus. DNA-damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of S. aureus but not in their phage-devoid counterparts. The β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is greater than that for MVs formed by β-lactam antibiotic-induced blebbing. The purified MVs derived from S. aureus protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both in vitro and ex vivo in whole blood. In addition, the MVs protected S. aureus from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.

Group A Streptococcus causes severe invasive infections, including necrotizing fasciitis. The expression of an array of virulence factors targeting specific host immune functions impedes successful bacterial clearance. The virulence factor streptococcal DNase Sda1 was previously shown to interfere with the entrapment of bacteria through neutrophil extracellular traps and TLR9 signaling. In this study, we showed that plasmacytoid dendritic cells are recruited to the infected tissue during group A streptococcal necrotizing fasciitis. We found that the streptococcal DNase Sda1 impairs plasmacytoid dendritic cell recruitment by reducing IFN-1 levels at the site of infection. We found that streptococcal DNase Sda1 interferes with stabilization of the DNA by the host molecule HMGB1 protein, which may account for decreased IFN-1 levels at the site of infection.

Pneumonia caused by the bacterium Staphylococcus aureus can vary in severity, from milder to necrotizing cases. In Staphylococcal pneumonia the cooperative actions of pathogen- and host-derived factors that contribute to disease progression and severity are relatively unknown. The objective of this project is to substantially deepening the understanding of the pathophysiology of Staphylococcal pneumonia, by pin-pointing key host and bacterial factors that contribute to the inflammatory reactions and that are targetable in new therapeutic strategies. The hypothesis is that S. aureus toxins, in addition to have cytotoxic properties, also trigger pulmonary epithelial cells and fibroblasts in ways leading to aggravation of inflammation and pathology. In this study, a complex three-dimensional model of airway epithelium combined with live cell imaging was used to delineate tissue responses to specific staphylococcal toxins. Tissue models were exposed to exotoxins produced by clinical isolates of S. aureus . At sublytic concentrations of toxins, inflammatory responses were induced and chemotactic gradients defined by the redistribution of CXCL8 and CX3CL1 were evident. This coincided with enhanced migration of phagocyte cells towards the apical side of the pulmonary epithelium. Using this innovative model of bacterial infection of the airway epithelium, has the potential to pinpoint novel disease traits and identify new potential targets for treatment aimed at managing detrimental inflammatory reactions.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of skin and soft tissue infections. One of the highly successful and rapidly disseminating clones is MRSA ST22 commonly associated with skin tropism. Here we show that a naturally occurring single amino acid substitution (tyrosine to cysteine) at position 223 of AgrC determines starkly different ST22 S. aureus virulence phenotypes, e.g. cytotoxic or colonizing, as evident in both in vitro and in vivo skin infections. Y223C amino acid substitution destabilizes AgrC-AgrA interaction leading to a colonizing phenotype characterized by upregulation of bacterial surface proteins. The colonizing phenotype strains cause less severe skin tissue damage, show decreased susceptibility towards the antimicrobial LL-37 and induce autophagy. In contrast, cytotoxic strains with tyrosine at position 223 of AgrC cause infections characterized by inflammasome activation and severe skin tissue pathology. Taken together, the study demonstrates how a single amino acid substitution in the histidine kinase receptor AgrC of ST22 strains determines virulence properties and infection outcome.

Necrotizing fasciitis caused by group A streptococcus (GAS) is a life-threatening, rapidly progressing infection. At present, biofilm is not recognized as a potential problem in GAS necrotizing soft tissue infections (NSTI), as it is typically linked to chronic infections or associated with foreign devices. Here, we present a case of a previously healthy male presenting with NSTI caused by GAS. The infection persisted over 24 days, and the surgeon documented the presence of a "thick layer biofilm" in the fascia. Subsequent analysis of NSTI patient tissue biopsies prospectively included in a multicenter study revealed multiple areas of biofilm in 32% of the patients studied. Biopsies associated with biofilm formation were characterized by massive bacterial load, a pronounced inflammatory response, and clinical signs of more severe tissue involvement. In vitro infections of a human skin tissue model with GAS NSTI isolates also revealed multilayered fibrous biofilm structures, which were found to be under the control of the global Nra gene regulator. The finding of GAS biofilm formation in NSTIs emphasizes the urgent need for biofilm to be considered as a potential complicating microbiological feature of GAS NSTI and, consequently, emphasizes reconsideration of antibiotic treatment protocols.

S. aureusis a major cause of hospital and community acquired pneumonia. Several virulence factors such as α-toxin, PVL and cytolytic peptides are implicated as major mediators of tissue pathology. Here we use a novel organotypic model of human lung to address the role of exotoxins produced by clinical S. aureus strains causing varying severity of lung infection i.e necrotizing pneumonia(NP) and lung empyema(LE). Exposure of lung tissue models to bacterial supernatants revealed that the NP strains were highly cytotoxic, demonstrating accentuated inflammation, necrosis and loss of Ecadherin, whereas the LE strain induced limited tissue damage. This corresponded to higher levels of α-toxin and PVL being produced by the NP strains, as compared to the LE strain that produced moderate levels of PVL but no α-toxin. A direct role of α-toxin in epithelial disruption was verified by toxin deficient mutants and pure α-toxin. PVL contributed to epithelial injury indirectly through lysis of neutrophils. Notably combined effects of α-toxin and PVL further augmented the tissue pathology. The study identifies a novel immunomodulatory effect of these toxins resulting in strong upregulation and polarization of CXCL8 and CX3CL1 chemokines towards the apical side of the epithelium translating into increased chemotaxis. Association between toxin levels, cytotoxicity and clinical outcome was confirmed in a larger cohort of pneumonia isolates. This study provides novel insight into toxin-mediated roles in lung tissue pathology implicating both α-toxin and PVL in chemotactic and cytolytic events contributing to severe tissue injury linked to NP.

Affiliations ECOFECT; International audience; 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.

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772 suggests that widespread mobility of the sea enterotoxin might be a selective force behind its ‘transfer’ to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside the genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.