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. Keywords: Enterococcus faecium; N-acetyl-galactosamine; horizontal gene transfer; linear plasmid; transmission network

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.publishedVersio

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

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

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

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-1β, 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-1β: 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 β-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-1β (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-1β had the strongest association with 30-day mortality

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.ISSN:2150-7511ISSN:2161-212

An intensive care unit outbreak with multidrug-resistant Pseudomonas aeruginosa - spot on sinks.

BACKGROUND Pseudomonas aeruginosa and other Gram-negative bacteria have the ability to persist in moist environments in healthcare settings, but their spread from these areas can result in outbreaks of healthcare-associated infections. METHODS We report the investigation and containment of a multidrug-resistant P. aeruginosa outbreak in 3 intensive care units of a Swiss university hospital. A total of 255 patients and 276 environmental samples were screened for the multidrug-resistant P. aeruginosa outbreak strain. We describe the environmental sampling and molecular characterization of patient and environmental strains, control strategies implemented, including waterless patient care. RESULTS Between March and November 2019, the outbreak affected 29 patients. Environmental sampling detected the outbreak strain in nine samples of sink siphons of three different intensive care units sharing an identical water sewage system and on one gastroscope. Three weeks after sink siphon replacement, the outbreak strain grew again in siphon-derived samples and newly-affected patients were identified. The outbreak ceased after removal of all sinks in the proximity of patients and in medication preparation areas and minimization of tap water use. Multilocus sequence typing indicated clonality (sequence type 316) in 28/29 patient isolates and all 10 environmental samples. CONCLUSIONS Sink removal combined with the introduction of waterless patient care terminated the multidrug-resistant P. aeruginosa outbreak. Sinks in intensive care units might pose a risk for point source outbreaks with P. aeruginosa and other bacteria persisting in moist environments

Biofilm in group A streptococcal necrotizing soft tissue infections

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Histone H4 binds to TLR4/MD-2 on THP-1 cells.

<p>(A) Overlapping peptides from histone H4 (100 μM) were incubated with heparinized blood for 12h at 37°C and CXCL10 release was measured by ELISA. (B) IRR20 was immobilized on a CM5 sensorchip and serial dilutions (200 nM – 12.5 nM) of the TLR4/MD-2 complex were applied in a flow. Steady-state affinity analysis was used to calculate the KD value (BIAevaluation software, GE Healthcare, Uppsala, Sweden). (C) The predicted structure of histone H4 was modeled using the UCSF Chimera software (<a href="http://www.cgl.ucsf.edu/chimera" target="_blank">http://www.cgl.ucsf.edu/chimera</a>) with a hairpin loop corresponding to the TLR4-binding 20-mer peptide IRR20 marked in red.</p