Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. METHODS: Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. RESULTS: Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. CONCLUSIONS: Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Genomic Surveillance of Vancomycin-Resistant Enterococcus faecium Reveals Spread of a Linear Plasmid Conferring a Nutrient Utilization Advantage.

Healthcare-associated outbreaks of vancomycin-resistant Enterococcus faecium (VREfm) are a worldwide problem with increasing prevalence. The genomic plasticity of this hospital-adapted pathogen contributes to its efficient spread despite infection control measures. Here, we aimed to identify the genomic and phenotypic determinants of health care-associated transmission of VREfm. We assessed the VREfm transmission networks at the tertiary-care University Hospital of Zurich (USZ) between October 2014 and February 2018 and investigated microevolutionary dynamics of this pathogen. We performed whole-genome sequencing for the 69 VREfm isolates collected during this time frame and assessed the population structure and variability of the vancomycin resistance transposon. Phylogenomic analysis allowed us to reconstruct transmission networks and to unveil external or wider transmission networks undetectable by routine surveillance. Notably, it unveiled a persistent clone, sampled 31 times over a 29-month period. Exploring the evolutionary dynamics of this clone and characterizing the phenotypic consequences revealed the spread of a variant with decreased daptomycin susceptibility and the acquired ability to utilize N-acetyl-galactosamine (GalNAc), one of the primary constituents of the human gut mucins. This nutrient utilization advantage was conferred by a novel plasmid, termed pELF_USZ, which exhibited a linear topology. This plasmid, which was harbored by two distinct clones, was transferable by conjugation. Overall, this work highlights the potential of combining epidemiological, functional genomic, and evolutionary perspectives to unveil adaptation strategies of VREfm. IMPORTANCE Sequencing microbial pathogens causing outbreaks has become a common practice to characterize transmission networks. In addition to the signal provided by vertical evolution, bacterial genomes harbor mobile genetic elements shared horizontally between clones. While macroevolutionary studies have revealed an important role of plasmids and genes encoding carbohydrate utilization systems in the adaptation of Enterococcus faecium to the hospital environment, mechanisms of dissemination and the specific function of many of these genetic determinants remain to be elucidated. Here, we characterize a plasmid providing a nutrient utilization advantage and show evidence for its clonal and horizontal spread at a local scale. Further studies integrating epidemiological, functional genomics, and evolutionary perspectives will be critical to identify changes shaping the success of this pathogen.

Bacterial but no SARS-CoV-2 contamination after terminal disinfection of tertiary care intensive care units treating COVID-19 patients.

BACKGROUND: In intensive care units (ICUs) treating patients with Coronavirus disease 2019 (COVID-19) invasive ventilation poses a high risk for aerosol and droplet formation. Surface contamination of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) or bacteria can result in nosocomial transmission. METHODS: Two tertiary care COVID-19 intensive care units treating 53 patients for 870 patient days were sampled after terminal cleaning and preparation for regular use to treat non-COVID-19 patients. RESULTS: A total of 176 swabs were sampled of defined locations covering both ICUs. No SARS-CoV-2 ribonucleic acid (RNA) was detected. Gram-negative bacterial contamination was mainly linked to sinks and siphons. Skin flora was isolated from most swabbed areas and Enterococcus faecium was detected on two keyboards. CONCLUSIONS: After basic cleaning with standard disinfection measures no remaining SARS-CoV-2 RNA was detected. Bacterial contamination was low and mainly localised in sinks and siphons.

Protein SIC Secreted from Streptococcus pyogenes Forms Complexes with Extracellular Histones That Boost Cytokine Production.

Innate immunity relies on an effective recognition of the pathogenic microorganism as well as on endogenous danger signals. While bacteria in concert with their secreted virulence factors can cause a number of inflammatory reactions, danger signals released at the site of infection may in addition determine the amplitude of such responses and influence the outcome of the disease. Here, we report that protein SIC, Streptococcal Inhibitor of Complement, an abundant secreted protein from Streptococcus pyogenes, binds to extracellular histones, a group of danger signals released during necrotizing tissue damage. This interaction leads to the formation of large aggregates in vitro. Extracellular histones and SIC are abundantly expressed and seen colocalized in biopsies from patients with necrotizing soft-tissue infections caused by S. pyogenes. In addition, binding of SIC to histones neutralized their antimicrobial activity. Likewise, the ability of histones to induce hemolysis was inhibited in the presence of SIC. However, when added to whole blood, SIC was not able to block the pro-inflammatory effect of histones. Instead SIC boosted the histone-triggered release of a broad range of cytokines and chemokines, including IL-6, TNF-α, IL-8, IL-1ß, IL-1ra, G-CSF, and IFN-γ. These results demonstrate that the interaction between SIC and histones has multiple effects on the host response to S. pyogenes infection.

Association between cytokine response, the LRINEC score and outcome in patients with necrotising soft tissue infection: a multicentre, prospective study.

Early assessment of necrotising soft tissue infection (NSTI) is challenging. Analysis of inflammatory markers could provide important information about disease severity and guide decision making. For this purpose, we investigated the association between cytokine levels and the Laboratory Risk Indicator for Necrotising Fasciitis (LRINEC)-score, disease severity and mortality in NSTI patients. In 159 patients, plasma was analysed for IL-1ß, IL-6, IL-10 and TNF-α upon admission. The severity of NSTI was assessed by SAPS, SOFA score, septic shock, microbial aetiology, renal replacement therapy and amputation. We found no significant difference in cytokine levels according to a LRINEC- score above or below 6 (IL-1ß: 3.0 vs. 1.3; IL-6: 607 vs. 289; IL-10: 38.4 vs. 38.8; TNF-α: 15.1 vs. 7.8 pg/mL, P > 0.05). Patients with ß-haemolytic streptococcal infection had higher level of particularly IL-6. There was no difference in mortality between patients with a LRINEC-score above or below 6. In the adjusted analysis assessing 30-day mortality, the association was strongest for IL-1ß (OR 3.86 [95% CI, 1.43-10.40], P = 0.008) and IL-10 (4.80 [1.67-13.78], P = 0.004). In conclusion, we found no significant association between the LRINEC-score and cytokine levels on admission. IL-6 was consistently associated with disease severity, whereas IL-1ß had the strongest association with 30-day mortality.

Biofilm in group A streptococcal necrotizing soft tissue infections.

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.

Local activation of coagulation factor XIII reduces systemic complications and improves the survival of mice after Streptococcus pyogenes M1 skin infection.

Coagulation is a mechanism for wound healing after injury. Several recent studies delineate an additional role of the intrinsic pathway of coagulation, also known as the contact system, in the early innate immune response against bacterial infections. In this study, we investigated the role of factor XIII (FXIII), which is activated upon coagulation induction, during Streptococcus pyogenes-mediated skin and soft tissue infections. FXIII has previously been shown to be responsible for the immobilization of bacteria within a fibrin network which may prevent systemic bacterial dissemination. In order to investigate if the FXIII-mediated entrapment of S. pyogenes also influences the disease outcome we used a murine S. pyogenes M1 skin and soft tissue infection model. Here, we demonstrate that a lack of FXIII leads to prolonged clotting times, increased signs of inflammation, and elevated bacterial dissemination. Moreover, FXIII-deficient mice show an impaired survival when compared with wildtype animals. Additionally, local reconstitution of FXIII-deficient mice with a human FXIII-concentrate (Fibrogammin®P) could reduce the systemic complications, suggesting a protective role for FXIII during early S. pyogenes skin infection. FXIII therefore might be a possible therapeutically application to support the early innate immune response during skin infections caused by S. pyogenes.

Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo.

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.

Increased cytotoxicity and streptolysin O activity in group G streptococcal strains causing invasive tissue infections.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) has emerged as an important cause of severe skin and soft tissue infections, but little is known of the pathogenic mechanisms underlying tissue pathology. Patient samples and a collection of invasive and non-invasive group G SDSE strains (n = 69) were analyzed with respect to virulence factor expression and cytotoxic or inflammatory effects on human cells and 3D skin tissue models. SDSE strains efficiently infected the 3D-skin model and severe tissue pathology, inflammatory responses and altered production of host structural framework proteins associated with epithelial barrier integrity were evident already at 8 hours post-infection. Invasive strains were significantly more cytotoxic towards keratinocytes and expressed higher Streptokinase and Streptolysin O (SLO) activities, as compared to non-invasive strains. The opposite was true for Streptolysin S (SLS). Fractionation and proteomic analysis of the cytotoxic fractions implicated SLO as a factor likely contributing to the keratinocyte cytotoxicity and tissue pathology. Analyses of patient tissue biopsies revealed massive bacterial load, high expression of slo, as well as immune cell infiltration and pro-inflammatory markers. Our findings suggest the contribution of SLO to epithelial cytotoxicity and tissue pathology in SDSE tissue infections.