The role of bacterial colonisation in severity, symptoms and aetiology of hand eczema: The importance of Staphylococcus aureus and presence of commensal skin flora.

BACKGROUND: The role and causality of the microbial ecosystem on the skin in relation to the development of hand eczema (HE) is still unknown. OBJECTIVES: To investigate the prevalence of different bacterial colonisations in HE patients and their association with the severity, symptoms and aetiology of the disease. METHODS: In a retrospective cohort study of 167 HE patients, bacterial swabs from lesional skin were collected for culturing. Patients were categorised according to bacterial colonisation, HE severity, HE symptoms and HE aetiology. RESULTS: The majority of the patients were tested positive for Staphylococcus aureus (S. aureus) (n = 131, 78.4%) and/or commensal skin flora (CSF; n = 130, 77.8%), while other bacteria species were found only sporadically. Severe HE was significantly more prevalent in skin with S. aureus (odds ratio [OR]: 5.13, 95% confidence interval [CI]: 2.21-11.94) and less common in skin with CSF (OR: 0.20, 95% CI: 0.05-0.88). S. aureus colonisation was also associated with atopic HE aetiology (p < 0.001) and acute HE symptoms such as blisters, erosions and crusts (p = 0.003). CONCLUSIONS: The main colonisation of HE patients is with S. aureus and is associated with disease severity, acute HE symptoms and atopic HE aetiology. CSF is associated with mild HE, which could result in new therapeutic approaches.

Bruton's tyrosine kinase inhibition attenuates disease progression by reducing renal immune cell invasion in mice with hemolytic-uremic syndrome.

Hemolytic-uremic syndrome (HUS) can occur as a complication of an infection with Shiga-toxin (Stx)-producing Escherichia coli. Patients typically present with acute kidney injury, microangiopathic hemolytic anemia and thrombocytopenia. There is evidence that Stx-induced renal damage propagates a pro-inflammatory response. To date, therapy is limited to organ-supportive strategies. Bruton's tyrosine kinase (BTK) plays a pivotal role in recruitment and function of immune cells and its inhibition was recently shown to improve renal function in experimental sepsis and lupus nephritis. We hypothesized that attenuating the evoked immune response by BTK-inhibitors (BTKi) ameliorates outcome in HUS. We investigated the effect of daily oral administration of the BTKi ibrutinib (30 mg/kg) and acalabrutinib (3 mg/kg) in mice with Stx-induced HUS at day 7. After BTKi administration, we observed attenuated disease progression in mice with HUS. These findings were associated with less BTK and downstream phospholipase-C-gamma-2 activation in the spleen and, subsequently, a reduced renal invasion of BTK-positive cells including neutrophils. Only ibrutinib treatment diminished renal invasion of macrophages, improved acute kidney injury and dysfunction (plasma levels of NGAL and urea) and reduced hemolysis (plasma levels of bilirubin and LDH activity). In conclusion, we report here for the first time that BTK inhibition attenuates the course of disease in murine HUS. We suggest that the observed reduction of renal immune cell invasion contributes - at least in part - to this effect. Further translational studies are needed to evaluate BTK as a potential target for HUS therapy to overcome currently limited treatment options.

Targeting the innate repair receptor axis via erythropoietin or pyroglutamate helix B surface peptide attenuates hemolytic-uremic syndrome in mice.

Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli and is characterized by microangiopathic hemolytic anemia and acute kidney injury. Hitherto, therapy has been limited to organ-supportive strategies. Erythropoietin (EPO) stimulates erythropoiesis and is approved for the treatment of certain forms of anemia, but not for HUS-associated hemolytic anemia. EPO and its non-hematopoietic analog pyroglutamate helix B surface peptide (pHBSP) have been shown to mediate tissue protection via an innate repair receptor (IRR) that is pharmacologically distinct from the erythropoiesis-mediating receptor (EPO-R). Here, we investigated the changes in endogenous EPO levels in patients with HUS and in piglets and mice subjected to preclinical HUS models. We found that endogenous EPO was elevated in plasma of humans, piglets, and mice with HUS, regardless of species and degree of anemia, suggesting that EPO signaling plays a role in HUS pathology. Therefore, we aimed to examine the therapeutic potential of EPO and pHBSP in mice with Stx-induced HUS. Administration of EPO or pHBSP improved 7-day survival and attenuated renal oxidative stress but did not significantly reduce renal dysfunction and injury in the employed model. pHBSP, but not EPO, attenuated renal nitrosative stress and reduced tubular dedifferentiation. In conclusion, targeting the EPO-R/IRR axis reduced mortality and renal oxidative stress in murine HUS without occurrence of thromboembolic complications or other adverse side effects. We therefore suggest that repurposing EPO for the treatment of patients with hemolytic anemia in HUS should be systematically investigated in future clinical trials.

Comparative Study of Different Diagnostic Routine Methods for the Identification of Acinetobacter radioresistens.

Recent publications indicate that A. radioresistens can cause infections in humans, even though it is rarely reported in routine diagnostics. However, the fact that it is infrequently detected may be explained by the misidentification of the species by conventional methods. It is also likely that A. radioresistens is not considered clinically relevant and therefore not consistently included in diagnostic results. To elucidate the medical significance of this probably clinically underestimated bacterial species, we created a well-documented reference strain collection of 21 strains collected in routine diagnostics. For further analysis of A. radioresistens, it is essential to know which methods can be used to achieve a trustworthy identification. We, therefore, compared three methods widely used in routine diagnostics (MALDI-TOF MS, VITEK 2, and sequencing of housekeeping genes) in terms of secure and reliable identification of A. radioresistens. As reference methods, whole genome-based approaches were applied. VITEK 2 led to misidentification for four strains. However, MALDI-TOF MS and sequencing of housekeeping genes led to reliable and robust identifications.

Comparative Genomic Analysis of the Human Pathogen Wohlfahrtiimonas Chitiniclastica Provides Insight Into the Identification of Antimicrobial Resistance Genotypes and Potential Virulence Traits.

Recent studies suggest that Wohlfahrtiimonas chitiniclastica may be the cause of several diseases in humans including sepsis and bacteremia making the bacterium as a previously underappreciated human pathogen. However, very little is known about the pathogenicity and genetic potential of W. chitiniclastica; therefore, it is necessary to conduct systematic studies to gain a deeper understanding of its virulence characteristics and treatment options. In this study, the entire genetic repertoire of all publicly available W. chitiniclastica genomes was examined including in silico characterization of bacteriophage content, antibiotic resistome, and putative virulence profile. The pan-genome of W. chitiniclastica comprises 3819 genes with 1622 core genes (43%) indicating a putative metabolic conserved species. Furthermore, in silico analysis indicated presumed resistome expansion as defined by the presence of genome-encoded transposons and bacteriophages. While macrolide resistance genes macA and macB are located within the core genome, additional antimicrobial resistance genotypes for tetracycline (tetH, tetB, and tetD), aminoglycosides (ant(2'')-Ia, aac(6')-Ia,aph(3'')-Ib, aph(3')-Ia, and aph(6)-Id)), sulfonamide (sul2), streptomycin (strA), chloramphenicol (cat3), and beta-lactamase (blaVEB) are distributed among the accessory genome. Notably, our data indicate that the type strain DSM 18708T does not encode any additional clinically relevant antibiotic resistance genes, whereas drug resistance is increasing within the W. chitiniclastica clade. This trend should be monitored with caution. To the best of our knowledge, this is the first comprehensive genome analysis of this species, providing new insights into the genome of this opportunistic human pathogen.

Divergent roles of haptoglobin and hemopexin deficiency for disease progression of Shiga-toxin-induced hemolytic-uremic syndrome in mice.

Thrombotic microangiopathy, hemolysis and acute kidney injury are typical clinical characteristics of hemolytic-uremic syndrome (HUS), which is predominantly caused by Shiga-toxin-producing Escherichia coli. Free heme aggravates organ damage in life-threatening infections, even with a low degree of systemic hemolysis. Therefore, we hypothesized that the presence of the hemoglobin- and the heme-scavenging proteins, haptoglobin and hemopexin, respectively impacts outcome and kidney pathology in HUS. Here, we investigated the effect of haptoglobin and hemopexin deficiency (haptoglobin-/-, hemopexin-/-) and haptoglobin treatment in a murine model of HUS-like disease. Seven-day survival was decreased in haptoglobin-/- (25%) compared to wild type mice (71.4%), whereas all hemopexin-/- mice survived. Shiga-toxin-challenged hemopexin-/- mice showed decreased kidney inflammation and attenuated thrombotic microangiopathy, indicated by reduced neutrophil recruitment and platelet deposition. These observations were associated with supranormal haptoglobin plasma levels in hemopexin-/- mice. Low dose haptoglobin administration to Shiga-toxin-challenged wild type mice attenuated kidney platelet deposition and neutrophil recruitment, suggesting that haptoglobin at least partially contributes to the beneficial effects. Surrogate parameters of hemolysis were elevated in Shiga-toxin-challenged wild type and haptoglobin-/- mice, while signs for hepatic hemoglobin degradation like heme oxygenase-1, ferritin and CD163 expression were only increased in Shiga-toxin-challenged wild type mice. In line with this observation, haptoglobin-/- mice displayed tubular iron deposition as an indicator for kidney hemoglobin degradation. Thus, haptoglobin and hemopexin deficiency plays divergent roles in Shiga-toxin-mediated HUS, suggesting haptoglobin is involved and hemopexin is redundant for the resolution of HUS pathology.

Identification and Antibiotic Profiling of Wohlfahrtiimonas chitiniclastica, an Underestimated Human Pathogen.

In the past 12 years, several case reports have clearly demonstrated that Wohlfahrtiimonas chitiniclastica is capable of causing sepsis and bacteremia in humans. However, since most clinicians are not familiar with this species, little is known about its pathogenicity and treatment options while it is as rare but underestimated human pathogen. Therefore, a larger strain collection is required so that methods can be identified that are most suitable to obtain rapid and reliable identification. Moreover, the antimicrobial resistance profile needs to be elucidated in order to explore possible treatment options. Over a period of 6 years, we therefore have collected a total of 14 W. chitiniclastica isolates in routine diagnostics, which now served as the basis for a comprehensive characterization with respect to identification and antibiotic profiling. We compared the accuracy and convenience of several identification techniques in which MALDI-TOF MS and sequencing of the 16S rRNA gene have proven to be suitable for identification of W. chitiniclastica. In addition, whole genome sequencing (WGS)-based digital DNA-DNA hybridization (dDDH) was used as a reference method for strain identification, and surprised with the detection of a novel W. chitiniclastica subspecies. A combination of in silico and in vitro analyses revealed a first insight into the antimicrobial resistance profile and the molecular basis of antimicrobial resistance. Based on our findings, trimethoprim/sulfamethoxazole, levofloxacin, and cephalosporins (e.g., ceftazidime) may be the best antibiotics to use in order to treat infections caused by W. chitiniclastica, while resistance to fosfomycin, amikacin and tobramycin is observed.

Clostridium Difficile infections in patients with AML or MDS undergoing allogeneic hematopoietic stem cell transplantation identify high risk for adverse outcome.

Clostridium difficile (CD) infection is the main cause of nosocomial enterocolitis in western countries and in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHCT). Recipients of alloHCT are at high risk for CD infection but large studies in this population are rare and conflicting results have been reported. We analyzed 727 patients with AML or MDS undergoing alloHCT in our center from 2004 to 2015. Ninety-six patients (13%) had CD infection and 103 patients (14%) were identified as asymptomatic carriers by screening at admission and once a week during aplasia. Patients with CD infection had a shorter median overall survival of 8 months (95% CI, 6-36 months) compared with 25 months (95% CI, 17-35 months) for patients without CD infection, (HR 1.4, p = 0.04). CD positive patients were less likely to develop acute graft-versus-host disease (aGvHD; HR 0.6, p = 0.004) compared with CD-negative patients, but did not show differences in gastrointestinal aGvHD (HR 0.9, p = 0.5). Symptomatic patients developed gastrointestinal aGvHD (HR 2.5, p = 0.02) more often compared with asymptomatic CD positive patients. This analysis demonstrates a high prevalence for CD infection in patients undergoing alloHCT. A significant lower overall survival for patients with CD infection could be demonstrated.

Comparison of VITEK 2, MALDI-TOF MS, 16S rRNA gene sequencing, and whole-genome sequencing for identification of Roseomonas mucosa.

The bacterial species Roseomonas mucosa is pathogenic in humans, and although it is rarely detected during routine diagnostics, it is becoming increasingly important clinically. For a long time, R. mucosa was regarded as a classic environmental bacterium. Recent studies, however, revealed that it is part of the physiological human skin flora and mainly affects immunocompromised patients. Furthermore, the use of catheter systems may increase the risk of contracting R. mucosa infections. The bacterium has been linked to severe infections, such as bacteraemia, osteomyelitis and cellulitis. Therefore, it is important to discern the best method of identifying R. mucosa in routine laboratory testing. To facilitate this testing, we compared three suitable methods for routine bacterial identification in the laboratory: VITEK 2, MALDI-TOF MS and 16S rRNA gene sequencing. Additionally, we conducted whole-genome sequencing (WGS) and calculated the average nucleotide identity (ANI). ANI is seen as the gold standard of strain identification; therefore, we decided to use it as a reference method. Both MALDI-TOF MS and 16S rRNA gene sequencing confidently identified the species. However, when using the VITEK 2 technique, isolates were misidentified as Roseomonas gilardii, Rhizobium radiobacter, or Sphingomonas paucimobilis. When conducting WGS and determining the ANI, it became obvious that one isolate belonged to the species R. gilardii rather than R. mucosa. Therefore (although not yet applicable in routine diagnostics), we suggest that WGS is presently the most appropriate technique to reliably identify Roseomonas mucosa. However, after expanding the Biotyper database, MALDI-TOF MS could also be an applicable method.

Detection of Robinsoniella peoriensis in multiple bone samples of a trauma patient.

We report a case of a 58-year-old male patient who underwent several surgeries following an accident. The bacterium Robinsoniella peoriensis was detected independently in multiple samples from both the right talus and tibia. The bacterium could only be identified using 16S rRNA gene sequencing.

Metabolization of the Advanced Glycation End Product N-ε-Carboxymethyllysine (CML) by Different Probiotic E. coli Strains.

N-ε-Carboxymethyllysine (CML) is formed during glycation reactions (synonym, Maillard reaction). CML is degraded by the human colonic microbiota, but nothing is known about the formation of particular metabolites. In the present study, six probiotic E. coli strains were incubated with CML in the presence or absence of oxygen in either minimal or nutrient-rich medium. CML was degraded by all strains only in the presence of oxygen. HPLC-MS/MS was applied for identification of metabolites of CML. For the first time, three bacterial metabolites of CML have been identified, namely N-carboxymethylcadaverine (CM-CAD), N-carboxymethylaminopentanoic acid (CM-APA), and the N-carboxymethyl-Δ1-piperideinium ion. During 48 h of incubation of CML with five different E. coli strains in minimal medium in the presence of oxygen, 37-66% of CML was degraded, while CM-CAD (1.5-8.4% of the initial CML dose) and CM-APA (0.04-0.11% of the initial CML dose) were formed linearly. Formation of the metabolites is enhanced when dipeptide-bound CML is applied, indicating that transport phenomena may play an important role in the "handling" of the compound by microorganisms.

Unimpaired Responses to Vaccination With Protein Antigen Plus Adjuvant in Mice With Kit-Independent Mast Cell Deficiency.

Innate inflammatory responses are crucial for induction and regulation of T cell and antibody responses. Mast cell (MC)-deficient Kit mutant mice showed impaired adaptive immunity, suggesting that MCs provide essential adjuvant activities, and pharmacological MC activation was proposed as a new adjuvant principle. However, the Kit mutations result in complex alterations of the immune system in addition to MC deficiency. We revisited the role of MCs in vaccination responses using Mcpt5-Cre R26DTA/DTA and Cpa3Cre/+ mice that lack connective tissue MCs or all MCs, respectively, but feature an otherwise normal immune system. These animals showed no impairment of T and B cell responses to intradermal vaccination with protein antigen plus complete Freund's adjuvant. Moreover, we demonstrate that the adjuvant effects of the MC secretagogue c48/80 in intradermal or mucosal immunization are independent of the presence of MCs. We hence find no evidence for a regulation by MCs of adaptive immune responses to protein antigens. The finding that immunological MC functions differ from those suggested by experiments in Kit mutants, emphasizes the importance of rigorous tests in Kit-independent MC-deficiency models.

Modeling Hemolytic-Uremic Syndrome: In-Depth Characterization of Distinct Murine Models Reflecting Different Features of Human Disease.

Diarrhea-positive hemolytic-uremic syndrome (HUS) is a renal disorder that results from infections with Shiga-toxin (Stx)-producing Escherichia coli. The aim of this study was to establish well-defined refined murine models of HUS that can serve as preclinical tools to elucidate molecular mechanisms of disease development. C57BL/6J mice were subjected to different doses of Stx2 purified from an E. coli O157:H7 patient isolate. Animals received 300 ng/kg Stx2 and were sacrificed on day 3 to establish an acute model with fast disease progression. Alternatively, mice received 25 ng/kg Stx2 on days 0, 3, and 6, and were sacrificed on day 7 to establish a subacute model with moderate disease progression. Indicated by a rise in hematocrit, we observed dehydration despite volume substitution in both models, which was less pronounced in mice that underwent the 7-day regime. Compared with sham-treated animals, mice subjected to Stx2 developed profound weight loss, kidney dysfunction (elevation of plasma urea, creatinine, and neutrophil gelatinase-associated lipocalin), kidney injury (tubular injury and loss of endothelial cells), thrombotic microangiopathy (arteriolar microthrombi), and hemolysis (elevation of plasma bilirubin, lactate dehydrogenase, and free hemoglobin). The degree of complement activation (C3c deposition), immune cell invasion (macrophages and T lymphocytes), apoptosis, and proliferation were significantly increased in kidneys of mice subjected to the 7-day but not in kidneys of mice subjected to the 3-day regime. However, glomerular and kidney volume remained mainly unchanged, as assessed by 3D analysis of whole mount kidneys using CD31 staining with light sheet fluorescence microscopy. Gene expression analysis of kidneys revealed a total of only 91 overlapping genes altered in both Stx2 models. In conclusion, we have developed two refined mouse models with different disease progression, both leading to hemolysis, thrombotic microangiopathy, and acute kidney dysfunction and damage as key clinical features of human HUS. While intrarenal changes (apoptosis, proliferation, complement deposition, and immune cell invasion) mainly contribute to the pathophysiology of the subacute model, prerenal pathomechanisms (hypovolemia) play a predominant role in the acute model. Both models allow the further study of the pathomechanisms of most aspects of human HUS and the testing of distinct novel treatment strategies.

Whole-genome sequencing of a large collection of Myroides odoratimimus and Myroides odoratus isolates and antimicrobial susceptibility studies.

The genus Myroides comprises several species of Gram-negative, non-motile, and non-fermenting bacteria, which have been regarded as non-pathogenic for decades. Multiple recent reports, however, underscore the pathogenic potential that Myroides sp. possesses for humans. These bacteria seem to be resistant to a wide range of antibiotics (including ß-lactams and aminoglycosides). Therefore, treatment options are limited. Knowledge of antimicrobial resistance, however, is based on only one meaningful comprehensive study and on data published from case reports. This lack of data motivated us to test 59 strains from our Myroides collection (43 M. odoratimimus and 16 M. odoratus) for resistance against 20 commonly used antibiotics. We also performed molecular analyses to reveal whether our bacteria harbor the genus-specific M. odoratimimus metallo-ß-lactamase (MUS-1) or the M. odoratus metallo ß-lactamase (TUS-1), and other ß-lactamases, which may provide an explanation for the extended antimicrobial resistance.

Comparison of clinical and immunological findings in gnotobiotic piglets infected with Escherichia coli O104:H4 outbreak strain and EHEC O157:H7.

BACKGROUND: Shiga toxin (Stx) producing Escherichia coli (E. coli) (STEC) is the most frequent cause of diarrhoea-positive haemolytic uraemic syndrome (D + HUS) in humans. In 2011, a huge outbreak with an STEC O104:H4 strain in Germany highlighted the limited possibilities for causative treatment of this syndrome. The responsible STEC strain was found to combine Stx production with adherence mechanisms normally found in enteroaggregative E. coli (EAEC). Pathotypes of E. coli evolve and can exhibit different adhesion mechanisms. It has been shown previously that neonatal gnotobiotic piglets are susceptible for infection with STEC, such as STEC O157:H7 as well as for EAEC, which are considered to be the phylogenetic origin of E. coli O104:H4. This study was designed to characterise the host response to infection with the STEC O104:H4 outbreak strain in comparison to an STEC O157:H7 isolate by evaluating clinical parameters (scoring) and markers of organ dysfunction (biochemistry), as well as immunological (flow cytometry, assessment of cytokines/chemokines and acute phase proteins) and histological alterations (light- and electron microscopy) in a gnotobiotic piglet model of haemolytic uraemic syndrome. RESULTS: We observed severe clinical symptoms, such as diarrhoea, dehydration and neurological disorders as well as attaching-and-effacing lesions (A/E) in the colon in STEC O157:H7 infected piglets. In contrast, STEC O104:H4 challenged animals exhibited only mild clinical symptoms including diarrhoea and dehydration and HUS-specific/severe histopathological, haematological and biochemical alterations were only inconsistently presented by individual piglets. A specific adherence phenotype of STEC O104:H4 could not be observed. Flow cytometric analyses of lymphocytes derived from infected animals revealed an increase of natural killer cells (NK cells) during the course of infection revealing a potential role of this subset in the anti-bacterial activity in STEC disease. CONCLUSIONS: Unexpectedly, E. coli O104:H4 infection caused only mild symptoms and minor changes in histology and blood parameters in piglets. Outcome of the infection trial does not reflect E. coli O104:H4 associated human disease as observed during the outbreak in 2011. The potential role of cells of the innate immune system for STEC related disease pathogenesis should be further elucidated.

First report on sepsis caused by Porphyromonas pogonae.

We report on a 62 year old patient who developed sepsis due to an infection caused by Porphyromonas pogonae, a recently described species of the bacterial genus Porphyromonas. This is the first case of an invasive infection with this pathogen.

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli.

Wild-type human interleukin-10 (hIL-10) is a non-covalent homodimer with a short half-life, thus limiting its therapeutic applications in vivo. To avoid loss of function due to dimer dissociation, we designed a synthetic hIL-10 analog by bridging both monomers via a 15 amino acid-long peptide spacer in a C-terminal to N-terminal fashion. For secretory expression in Escherichia coli, a 1156 bp fragment was generated from template vector pAZ1 by fusion PCR encoding a T7 promoter region and the signal sequence of the E. coli outer membrane protein F fused in frame to two tandem E. coli codon-optimized mature hIL-10 genes connected via a 45 nucleotide linker sequence. The construct was cloned into pUC19 for high-level expression in E. coli BL21 (DE3). The mean concentrations of hIL-10 fusion protein in the periplasm and supernatant of E. coli at 37 °C growth temperature were 130 ± 40 and 2 ± 1 ng/ml, respectively. The molecular mass of the recombinant protein was assessed via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, indicating correct processing of the signaling sequence in E. coli. In vitro biological activity was shown by phosphorylation of signal transducer and activator of transcription protein 3 and suppression of tumor necrosis factor α secretion in lipopolysaccharide-stimulated macrophages.

Therapy of solid tumors using probiotic Symbioflor-2: restraints and potential.

To date, virulent bacteria remain the basis of most bacteria mediated cancer therapies. For clinical application attenuation is required. However, this might result in a drastically lowered therapeutic capacity. Herein we argue that the E. coli probiotic Symbioflor-2, with a history of safe application may constitute a viable tumor therapeutic candidate. We demonstrate that Symbioflor-2 displays a highly specific tumor targeting ability as determined in murine CT26 and RenCa tumor models. The excellent specificity was ascribed to reduced levels of adverse colonization. A high safety standard was demonstrated in WT and Rag1-/- mice. Thus, Symbioflor-2 may represent an ideal tumor targeting delivery system for therapeutic molecules. Moreover, Symbioflor-2 was capable of inducing CT26 tumor clearance as result of an adjuvant effect on tumor specific CD8+ T cells analogous to the Salmonella variant SL7207. However, lower therapeutic efficacy against RenCa tumors suggested a generally reduced therapeutic potency for probiotics. Interestingly, concurrent depletion of Gr-1+ or Ly6G+ cells installed therapeutic efficacy equal to SL7207, thus highlighting the role of innate effector cells in restraining the anti-tumor effects of Symbioflor-2. Collectively, our findings argue for a strategy of safe strain application and a more sustainable use of bacteria as a delivery system for therapeutic molecules.

The prediction of virulence based on presence of virulence genes in E. coli may not always be accurate.

Now that microbial whole genome sequencing is in reach of many researchers, it is common to infer virulent properties of a given bacterial isolate based on the presence of virulence genes. However, this may lead to inaccurate presumptions of virulence. Using the findings of a recent publication (Da Silva Santos et al. Gut Pathog 7:2, 2015) where virulence was inferred from a genome sequence and subsequently confirmed by in vitro analysis, we present an alternative view on the case described in that publication. Our alternative view point, which is further substantiated by whole genome sequencing of probiotic E. coli strains, may contribute to a more balanced vision on the interactions between pathogens and host.