Consistent biofilm formation by Streptococcus pyogenes emm 1 isolated from patients with necrotizing soft tissue infections

Objectives: Biofilm formation has been demonstrated in muscle and soft tissue samples from patients with necrotizing soft tissue infection (NSTI) caused by Streptococcus pyogenes, but the clinical importance of this observation is not clear. Although M-protein has been shown to be important for in vitro biofilm formation in S. pyogenes, the evidence for an association between emm type and biofilm forming capacity is conflicting. Here we characterize the biofilm forming capacity in a collection of S. pyogenes isolates causing NSTI, and relate this to emm type of the isolates and clinical characteristics of the patients. Methods: Bacterial isolates and clinical data were obtained from NSTI patients enrolled in a multicenter prospective observational study. Biofilm forming capacity was determined using a microtiter plate assay. Results: Among 57 cases, the three most frequently encountered emm types were emm1 (n = 22), emm3 (n = 13), and emm28 (n = 7). The distribution of biofilm forming capacity in emm1 was qualitatively (narrow-ranged normal distribution) and quantitatively (21/22 isolates in the intermediate range) different from other emm types (wide ranged, multimodal distribution with 5/35 isolates in the same range as emm1). There were no significant associations between biofilm forming capacity and clinical characteristics of the patients. Conclusions: The biofilm forming capacity of emm1 isolates was uniform and differed significantly from other emm types. The impact of biofilm formation in NSTI caused by S. pyogenes on clinical outcomes remains uncertain.publishedVersio

Genomic epidemiology of Streptococcus dysgalactiae subsp. equisimilis strains causing invasive disease in Norway during 2018

Background: Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen, yet the epidemiology and population genetics of SDSE species have not been extensively characterized. Methods: We carried out whole genome sequencing to characterize 274 SDSE isolates causing bloodstream infections obtained through national surveillance program in 2018. We conducted multilocus sequence typing (MLST), emm-typing, core genome phylogeny, as well as investigated key features associated with virulence. Moreover, comparison to SDSE from other geographic regions were performed in order to gain more insight in the evolutionary dynamics in SDSE. Results: The phylogenetic analysis indicated a substantial diversity of emm-types and sequence types (STs). Briefly, 17 emm-types and 58 STs were identified that formed 10 clonal complexes (CCs). The predominant ST-types were ST20 (20%), ST17 (17%), and ST29 (11%). While CC17 and CC29 clades showed a substantial heterogeneity with well-separated emm-associated subclades, the CC20 clade harboring the stG62647 emm-type was more homogenous and the most prevalent in the present study. Moreover, we observed notable differences in the distribution of clades within Norway, as well as several disseminated CCs and also distinct geographic variations when compared to data from other countries. We also revealed extensive intra-species recombination events involving surface exposed virulence factors, including the emm gene important for phylogenetic profiling. Conclusion: Recombination events involving the emm as well as other virulence genes in SDSE, are important mechanisms in shaping the genetic variability in the SDSE population, potentially offering selective advantages to certain lineages. The enhanced phylogenetic resolution offered by whole genome sequencing is necessary to identify and delimitate outbreaks, monitor and properly characterize emerging strains, as well as elucidate bacterial population dynamics.publishedVersio

Skåringsverktøy for tidlig oppdagelse av sepsis på sengepost

BAKGRUNN Sepsis er en av de vanligste årsakene til død i norske sykehus. Tilstanden er ofte oversett og behandlingen forsinket. Skåringsverktøy for å oppdage pasienter med sepsis er hyppigst undersøkt i akuttmottak, sjeldnere på sengepost. Målet med studien var å undersøke evnen ulike kliniske skåringsverktøy har til å identifisere sepsis blant pasienter på sengepost. MATERIALE OG METODE Skåringsverktøyene SIRS (Systemic Inflammatory Response Syndrome), qSOFA (quick Sequential Organ Failure Assessment) og NEWS2 (National Early Warning Score 2) sin evne til å identifisere pasienter med sepsis ble sammenlignet i en studie av voksne pasienter med klinisk mistenkt alvorlig infeksjon på sengeposter ved Haukeland universitetssjukehus i perioden mars–desember 2019. Diagnosen sepsis ble stilt på bakgrunn av en økning i SOFA-skår (Sequential Organ Failure Assessment) på 2 eller mer innen de første 24 timene etter inklusjon. RESULTATER 89 pasienter med mistenkt nyoppstått alvorlig infeksjon ble identifisert. Av disse fikk 55 stilt diagnosen sepsis. SIRS hadde en sensitivitet på 82 % og en spesifisitet på 6 % for diagnostisering av sepsis, qSOFA sensitivitet 20 % og spesifisitet 97 %, mens NEWS2 med grense på ≥ 5 totalt og/eller 3 på en enkelt parameter hadde sensitivitet på 96 % og spesifisitet på 59 %. Oksygenmetning med grense på 95 % var det mest sensitive målet for sepsis. FORTOLKNING NEWS2 var bedre egnet enn qSOFA og SIRS til å tidlig oppdage sepsis på sengepost.publishedVersio

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

A New Human Challenge Model for Testing Heat-Stable Toxin-Based Vaccine Candidates for Enterotoxigenic Escherichia Coli Diarrhea - Dose Optimization, Clinical Outcomes, and CD4+ T Cell Responses

Enterotoxigenic Escherichia coli (ETEC) are a common cause of diarrheal illness in young children and travelers. There is yet no licensed broadly protective vaccine against ETEC. One promising vaccine development strategy is to target strains expressing the heat-stable toxin (ST), particularly the human ST (STh), since infections with these strains are among the leading causes of diarrhea in children in low-and-middle income countries. A human challenge model based on an STh-only ETEC strain will be useful to evaluate the protective efficacy of new ST-based vaccine candidates. To develop this model, we experimentally infected 21 healthy adult volunteers with the epidemiologically relevant STh-only ETEC strain TW10722, identified a suitable dose, assessed safety, and characterized clinical outcomes and immune responses caused by the infection. Doses of 1×1010 colony-forming units (CFU) of TW10722 gave a suitable attack risk of 67% for moderate or severe diarrhea and an overall diarrhea attack risk of 78%. Non-diarrheal symptoms were mostly mild or moderate, and there were no serious adverse events. During the first month after ingesting the challenge strain, we measured significant increases in both activated CD4+ T cells and levels of serum IgG and IgA antibodies targeting coli surface antigen 5 (CS5) and 6 (CS6), as well as the E. coli mucinase YghJ. The CS5-specific CD4+ T cell and antibody responses were still significantly elevated one year after experimental infection. In conclusion, we have developed a safe STh-only ETEC-based human challenge model which can be efficiently used in Phase 2B trials to evaluate the protective efficacy of new ST-based vaccine candidates.publishedVersio

Analysis of host-pathogen gene association networks reveals patient-specific response to streptococcal and polymicrobial necrotising soft tissue infections

Background: Necrotising soft tissue infections (NSTIs) are rapidly progressing bacterial infections usually caused by either several pathogens in unison (polymicrobial infections) or Streptococcus pyogenes (mono-microbial infection). These infections are rare and are associated with high mortality rates. However, the underlying pathogenic mechanisms in this heterogeneous group remain elusive. Methods: In this study, we built interactomes at both the population and individual levels consisting of host-pathogen interactions inferred from dual RNA-Seq gene transcriptomic profiles of the biopsies from NSTI patients. Results: NSTI type-specific responses in the host were uncovered. The S. pyogenes mono-microbial subnetwork was enriched with host genes annotated with involved in cytokine production and regulation of response to stress. The polymicrobial network consisted of several significant associations between different species (S. pyogenes, Porphyromonas asaccharolytica and Escherichia coli) and host genes. The host genes associated with S. pyogenes in this subnetwork were characterised by cellular response to cytokines. We further found several virulence factors including hyaluronan synthase, Sic1, Isp, SagF, SagG, ScfAB-operon, Fba and genes upstream and downstream of EndoS along with bacterial housekeeping genes interacting with the human stress and immune response in various subnetworks between host and pathogen. Conclusions: At the population level, we found aetiology-dependent responses showing the potential modes of entry and immune evasion strategies employed by S. pyogenes, congruent with general cellular processes such as differentiation and proliferation. After stratifying the patients based on the subject-specific networks to study the patient-specific response, we observed different patient groups with different collagens, cytoskeleton and actin monomers in association with virulence factors, immunogenic proteins and housekeeping genes which we utilised to postulate differing modes of entry and immune evasion for different bacteria in relationship to the patients’ phenotype.publishedVersio

Prothrombotic and Proinflammatory Activities of the β-Hemolytic Group B Streptococcal Pigment

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1β, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system