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.

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.

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.

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.

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.

Development of a rapid microarray-based DNA subtyping assay for the alleles of Shiga toxins 1 and 2 of Escherichia coli.

In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise.

Pathogenic vibrios in environmental, seafood and clinical sources in Germany.

Bacteria of the family Vibrionaceae naturally occur in marine and estuarine environments. Only few species of Vibrionaceae are associated with human cases of gastroenteritis, ear and wound infections, caused by ingestion of seafood or contact with Vibrio containing water. Increasing consumption of seafood (fish, fishery products and shellfish) poses a possible source of Vibrio infections in Germany. Additionally, there is a growing concern that abundances of pathogenic vibrios may increase in German coastal waters as a result of e.g. climate change resulting in probably rising surface water temperatures. According to the One Health concept the VibrioNet consortium started in 2010 to investigate the occurrence and relevance of non-cholera vibrios of human concern in Germany. Vibrios from environmental, seafood and clinical sources were analyzed with the aim to find connections between different reservoirs or sources and to identify potential ways of transmission of these pathogens to assess the risk of infections associated with them. Potentially pathogenic strains mostly belong to the species Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae. Investigations on imported seafood and mussels from primary production areas confirmed the frequent occurrence of these species. Moreover, studies of German coastal waters and sediments showed the presence and seasonality of these marine bacteria. So far the incidence of clinical cases of vibriosis in Germany is low. Between 1994 and 2013 thirteen cases of Vibrio spp. associated wound infections and/or septicaemia have been reported. However, the high prevalence of vibrios in aquatic environments and aquatic organisms is of concern and demands continued control of food and surveillance for clinical infections with pathogenic vibrios.

Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct.

BACKGROUND: Interleukin-10 homologues encoded by Herpes viruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) hold interesting structural and biological characteristics compared to human interleukin-10 (hIL-10) that render these proteins promising candidates for therapeutic application in inflammatory bowel disease (IBD). Intestinal delivery of cytokines using bacterial carriers as chassis represents a novel approach for treatment of IBD patients. For proof of concept, a Sec-dependent transporter construct was designed for secretory expression of recombinant viral IL-10 proteins in the periplasm of Escherichia coli laboratory strain BL21 (DE3), which might serve as part of a prospective lysis based delivery and containment system. RESULTS: The signal peptide of E. coli outer membrane protein F fused to the mature form of the viral IL-10 proteins enabled successful transport into the periplasm, a compartment which seems crucial for proper assembly of the dimeric configuration of the cytokines. Cytokine concentrations in different bacterial compartments were determined by ELISA and achieved yields of 67.8 ng/ml ± 24.9 ng/ml for HCMV IL-10 and 1.5 µg/ml ± 841.4 ng/ml for EBV IL-10 in the periplasm. Immunoblot analysis was used to confirm the correct size of the E. coli-derived recombinant cytokines. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the signal transduction cascade after IL-10 receptor interaction, as well as suppression of tumor necrosis factor α (TNF-α) release of lipopolysaccharide-stimulated mouse macrophages were used as read-out assays for proving in vitro biological activity of the E. coli derived, recombinant viral IL-10 counterparts. CONCLUSIONS: In this study, proof of principle is provided that E. coli cells are a suitable chassis for secretory expression of viral IL-10 cytokines encoded by codon-optimized synthetic genes fused to the E. coli ompF signal sequence. In vitro biological activity evidenced by activation of transcription factor STAT3 and suppression of TNF-α in mammalian cell lines was shown to be strictly dependent on export of viral IL-10 proteins into the periplasmic compartment. E. coli might serve as carrier system for in situ delivery of therapeutic molecules in the gut, thus representing a further step in the development of novel approaches for treatment of IBD.

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.

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.

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.

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.

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.

High-throughput microarray technology in diagnostics of enterobacteria based on genome-wide probe selection and regression analysis.

BACKGROUND: The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. RESULTS: We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. CONCLUSIONS: Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.

Selective activation of human intestinal mast cells by Escherichia coli hemolysin.

Mast cells (MCs) are recognized to play an important role in bacterial host defense in the murine system. In this study, we studied the interaction of human MCs, isolated from the intestine and purified to homogeneity, with different Escherichia coli and Shigella flexneri strains. We show that alpha-hemolysin (Hly)-producing E. coli strains induce the release of histamine, leukotrienes, and proinflammatory cytokines in intestinal MCs. In contrast, MCs were virtually unresponsive to S. flexneri and several Hly-negative E. coli strains, including the isogenic Hly-deficient mutants of Hly(+) strains. Hly(+) E. coli but not Hly(-) E. coli caused an increase in intracellular Ca(2+) levels. Blocking of extracellular Ca(2+) and of the calmodulin/calcineurin pathway by cyclosporin A inhibited the response to Hly(+) E. coli. Furthermore, inhibition of MAPKs p38 and ERK reduces activation of MCs by Hly(+) E. coli. In addition, using an ex vivo system, we directly record the histamine release by MCs located in the lamina propria after infection with Hly(+) E. coli. Our data indicate that human intestinal mast cells interact with selected Gram-negative bacteria, establish E. coli Hly as a factor regulating MC effector functions, and argue further for a role of human MCs in innate immunity.

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.

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.

Sensitivity to Escherichia coli Nissle 1917 in mice is dependent on environment and genetic background.

Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic bacterium. Although genomic comparisons of EcN with the uropathogenic E. coli strain CFT073 revealed high degrees of similarity, EcN is generally considered a non-pathogenic organism. However, as recent evidence suggests that EcN is capable of inducing inflammatory responses in host intestinal epithelial cells, we aimed to investigate potential pathogenic properties of EcN in an in vivo model using various germ-free (GF) mouse strains. With the exception of C3H/HeJZtm mice, which carry a defective toll-like receptor (TLR)4-allele, no lesions were obvious in mice of different strains orally inoculated with EcN for 1 week, although organ cultures (blood, lung, mesenteric lymph node, pancreas, spleen, liver and kidney) tested positive to various degrees. C3H/HeJZtm mice inoculated with EcN became clinically ill and the majority died or had to be euthanized. Organs of all gnotobiotic C3H/HeJZtm mice were positive for EcN by culture; major histological findings were moderate to severe pyogranulomatous serositis, typhlitis and pancreatitis. Histological findings were corroborated by highly elevated tumour necrosis factor (TNF) serum levels. Lesions were not detected in specified pathogen free maintained C3H/HeJZtm mice, GF C3H/HeJ mice lacking the interleukin-10 gene, or GF C3H/HeJZtm mice that were inoculated with E. coli K12 strain MG1655 as a control. In addition, mild histological lesions were detected in Ztm:NMRI mice 3 months after oral inoculation with EcN. This study shows that EcN is capable of displaying a virulent phenotype in GF C3H/HeJZtm mice. Whether this phenotype is linked to the bacterium's probiotic nature should be the focus of further studies.

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.