Whole-genome analysis of vancomycin-resistant Enterococcus faecium causing nosocomial outbreaks suggests the occurrence of few endemic clonal lineages in Bavaria, Germany.

OBJECTIVES: Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) represent a major public health concern due to limited treatment options. Among invasive isolates of VREfm, ST117, ST80 and ST78 represent the most frequently detected STs by MLST in Germany. In this study, we investigated the genetic diversity of isolates of VREfm recovered from different nosocomial outbreaks in Bavaria, Germany, by WGS. METHODS: Between January 2018 and April 2019, 99 non-replicate isolates of VREfm originating from nosocomial outbreaks at eight different hospitals in Bavaria were investigated for genetic diversity by WGS. In detail, complex types (CTs) were identified by core-genome MLST. Furthermore, an SNP analysis was performed for all VREfm strains. RESULTS: Most of the isolates of this study (76%) belonged to three major clonal groups, which occurred in at least three hospitals: ST80/CT1065 vanB (n = 45; six hospitals), ST117/CT71 vanB (n = 11; four hospitals) and ST78/CT894like vanA (n = 19; three hospitals). Moreover, isolates of the predominant lineage ST80/CT1065 vanB showed a maximum difference of 36 SNPs as revealed by SNP analysis. CONCLUSIONS: Whole-genome analysis of VREfm causing nosocomial outbreaks suggests the occurrence of few endemic clonal lineages in Bavarian hospital settings, namely ST80/CT1065 vanB, ST117/CT71 vanB and ST78/CT894like vanA. Further studies are needed for a better understanding of the factors affecting the successful spread of the above-mentioned lineages.

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.

Microbiological Surveillance of Peritoneal Dialysis Associated Peritonitis: Antimicrobial Susceptibility Profiles of a Referral Center in GERMANY over 32 Years.

OBJECTIVES: Peritonitis is one of the most important causes of treatment failure in peritoneal dialysis (PD) patients. This study describes changes in characteristics of causative organisms in PD-related peritonitis and antimicrobial susceptibility. METHODS: In this single center study we analyzed retrospective 487 susceptibility profiles of the peritoneal fluid cultures of 351 adult patients with peritonitis from 1979 to 2014 (divided into three time periods, P1-P3). RESULTS: Staphylococcus aureus decreased from P1 compared to P2 and P3 (P<0.05 and P<0.01, respectively). Methicillin-resistant S. aureus (MRSA) occurred only in P3. Methicillin-resistant Staphylococcus epidermidis (MRSE) increased in P3 over P1 and P2 (P <0.0001, respectively). In P2 and P3, vancomycin resistant enterococci were detected. The percentage of gram-negative organisms remained unchanged. Third generation cephalosporin resistant gram-negative rods (3GCR-GN) were found exclusively in P3. Cefazolin-susceptible gram-positive organisms decreased over the three decades (93% in P1, 75% in P2 and 58% in P3, P<0.01, P<0.05 and P<0.0001, respectively). Vancomycin susceptibility decreased and gentamicin susceptibility in gram-negatives was 94% in P1, 82% in P2 and 90% in P3. Ceftazidim susceptibility was 84% in P2 and 93% in P3. CONCLUSIONS: Peritonitis caused by MSSA decreased, but peritonitis caused by MRSE increased. MRSA peritonitis is still rare. Peritonitis caused by 3GCR-GN is increasing. An initial antibiotic treatment protocol should be adopted for PD patients to provide continuous surveillance.

Clinical course and long-term outcome of hantavirus-associated nephropathia epidemica, Germany.

Human infection with Puumala virus (PUUV), the most common hantavirus in Central Europe, causes nephropathia epidemica (NE), a disease characterized by acute kidney injury and thrombocytopenia. To determine the clinical phenotype of hantavirus-infected patients and their long-term outcome and humoral immunity to PUUV, we conducted a cross-sectional prospective survey of 456 patients in Germany with clinically and serologically confirmed hantavirus-associated NE during 2001-2012. Prominent clinical findings during acute NE were fever and back/limb pain, and 88% of the patients had acute kidney injury. At follow-up (7-35 mo), all patients had detectable hantavirus-specific IgG; 8.5% had persistent IgM; 25% had hematuria; 23% had hypertension (new diagnosis for 67%); and 7% had proteinuria. NE-associated hypertension and proteinuria do not appear to have long-term consequences, but NE-associated hematuria may. All patients in this study had hantavirus-specific IgG up to years after the infection.

Acute kidney injury and tools for risk-stratification in 456 patients with hantavirus-induced nephropathia epidemica.

BACKGROUND: Puumala virus (PUUV) is the most common species of hantavirus in Central Europe. Nephropathia epidemica (NE), caused by PUUV, is characterized by acute kidney injury (AKI) and thrombocytopenia. The major goals of this study were to provide a clear clinical phenotyping of AKI in patients with NE and to develop an easy prediction rule to identify patients, who are at lower risk to develop severe AKI. METHODS: A cross-sectional prospective survey of 456 adult patients with serologically confirmed NE was performed. Data were collected from medical records and prospectively at follow-up visit. Severe AKI was defined by standard criteria according to the RIFLE (Risk, Injury, Failure, Loss, End-stage kidney disease) classification. Fuller statistical models were developed and validated to estimate the probability for severe AKI. RESULTS: During acute NE, 88% of the patients had AKI according to the RILFE criteria during acute NE. A risk index score for severe AKI was derived by using three independent risk factors in patients with normal kidney function at time of diagnosis: thrombocytopenia [two points; odds ratios (OR): 3.77; 95% confidence intervals (CI): 1.82, 8.03], elevated C-reactive protein levels (one point; OR: 3.02; 95% CI: 1.42, 6.58) and proteinuria (one point; OR: 3.92; 95% CI: 1.33, 13.35). On the basis of a point score of one or two, the probability of severe AKI was 0.18 and 0.28 with an area under the curve of 0.71. CONCLUSION: This clinical prediction rule provides a novel and diagnostically accurate strategy for the potential prevention and improved management of kidney complications in patients with NE and, ultimately, for a possible decrease in unnecessary hospitalization in a high number of patients.

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.

A single standardized practical training for surgical scrubbing according to EN1500: effect quantification, value of the standardized method and comparison with clinical reference groups.

UNLABELLED: The standardized training of practical competences in skills labs is relatively new among German Medical Faculties. The broad acceptance and outstanding evaluation results do not provide objective data on the efficiency and cost-efficiency of these trainings. This study aims on the quantification of the teaching effect of the surgical scrubbing technique EN1500 and its comparison with clinical references of OR personnel. METHODS: 161 4(th) year medical students were randomized into intervention and control group. The intervention group received a 45 minute standardized peer-teaching training of practical competences necessary in the OR including the scrubbing according to EN1500. Fluorescence dye was mixed in the disinfectant solution. After hand disinfection, standardized fotographs and semi-automated digital processing resulted in quantification of the insufficiently covered hand area. These results were compared with the control group that received the training after the test. In order to provide information on the achieved clinical competence level, the results were compared with the two clinical reference groups. RESULTS: The intervention group remained with 4,99% (SD 2,34) insufficiently covered hand area after the training compared to the control group 7,33% (SD 3,91), p<0,01. There was no significant difference between control group and reference groups: surgeons 9,32% (SD 4,97), scrub nurses 8,46% (SD 4,66). The student intervention group showed results that were significantly better than the clinical references. The methodic mistake remained negligible. In the sub-group analysis, the students with low or medium experience in surgical scrubbing and hand disinfection derived highest benefit from the training, whereas students with no or high experience did benefit less. All participants showed better results on hand palms compared to back of hand areas. DISCUSSION: A single standardized peer-teaching of surgical scrubbing and hand disinfection according to EN1500 is sufficient to improve the measurable coverage of hand area and reduce the disinfection gap by 1/3. In absolute measures, the competence level of experienced surgeons and scrub nurses is achieved or even exceeded.

Improving health from the inside: Use of engineered intestinal microorganisms as in situ cytokine delivery system.

The anti-inflammatory cytokine interleukin-10 and its viral homologs were chosen as model proteins for the development of drug delivery systems based on probiotic carriers like E. coli Nissle 1917, E. coli G3/10, and Saccharomyces boulardii. Exterior cytokine secretion was achieved by a modified E. coli hemolysin transporter. Release of interleukin-10 transported to the periplasm via the OmpF signal peptide was enabled by a T4 phage lysis system under control of the araC PBAD activator-promoter. The yield of interleukin-10 delivered by the phage lysis system was too low for functional analysis whereas the fusion protein secreted by the hemolysin transporter proved to be biologically inactive. Moreover, partial processing of the fusion protein by the E. coli membrane protease OmpT had no effect on the protein's functionality. Using the α-mating factor signal sequence, the yeast S. boulardii proved to be suitable for secretory expression of biologically active viral interleukin-10.

Quantitative phenotyping of inflammatory bowel disease in the IL-10-deficient mouse by use of noninvasive magnetic resonance imaging.

BACKGROUND: Although magnetic resonance imaging (MRI) is an increasingly used diagnostic tool in the assessment of inflammatory bowel disease (IBD) in humans, diagnosis and quantitation of intestinal inflammation in animal models of IBD still depends on ex vivo techniques. The aim of this study was to evaluate whether high-field MRI is suitable for the quantitative phenotyping of gut inflammation in a dextran sulfate sodium (DSS)-triggered interleukin (IL)10-deficient (IL-10(-/-)) mouse model of IBD, especially in longitudinal studies. METHODS: Using colitis-susceptible and -resistant backgrounds, MRI and ex vivo analyses were applied to characterize this specific model, differentiating disease severity and time-dependent alterations. Colon wall thickness, cecum wall tissue intensity, spleen, and mesenteric lymph node (MLN) volumes were evaluated 1, 2, 4, and 12 weeks after disease onset by T2-weighted MRI. Ex vivo parameters included histology, spleen, and MLN weight and analysis of cytokine expression. RESULTS: MRI and ex vivo determined parameters correlated well, revealing a mouse strain-specific colitis development over time with characteristics typical for the DSS model in the initial and for the IL-10(-/-) model in the chronic phase. To evaluate the use of high-field MRI for monitoring therapeutic studies, mice with a profound colitis were treated with IL-10-producing Saccharomyces boulardii and monitored by MRI. CONCLUSIONS: MRI can be utilized to quantify colitis development in the IL-10(-/-) model of IBD. Therefore, this noninvasive technique might be highly advantageous for an individual follow-up of colitis development in chronic models of IBD, facilitating the reduction of animal numbers in this kind of research.

Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10.

Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.

Periplasmic delivery of biologically active human interleukin-10 in Escherichia coli via a sec-dependent signal peptide.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10.

Blastocystis sp. subtype 2 detection during recurrence of gastrointestinal and urticarial symptoms.

Blastocystis is a common unicellular intestinal parasite in humans. Its clinical relevance is still subject to discussion with numerous conflicting reports on its ability to cause disease. A remarkable genetic heterogeneity among isolates suggests an association between distinct subtypes (STs) and pathogenicity, although a clear correlation between symptoms and subtype is lacking. Here, we report on a clinical case which possibly links Blastocystis sp. ST2 infection with the simultaneous occurrence of gastrointestinal illness and generalized chronic urticaria. Despite repeated chemotherapy with different antimicrobial drugs, both the gastrointestinal and cutaneous disorders reoccurred after short symptom-free intervals. Eradication of the parasite and permanent resolution of the patient's medical condition was finally achieved with the combined application of metronidazole and paromomycin.

Cidofovir and foscarnet for treatment of human herpesvirus 6 encephalitis in a neutropenic stem cell transplant recipient.

We report a stem cell transplant recipient who developed human herpesvirus 6 encephalitis. Human herpesvirus 6 load indicated massive intrathecal viral replication. Administration of cidofovir followed by foscarnet was associated with total clearance of human herpesvirus 6 infection. Cidofovir and foscarnet combination therapy may be beneficial for reducing mortality of (neutropenic) stem cell transplant recipients with human herpesvirus 6 encephalitis.

Chlorpromazine combined with cidofovir for treatment of a patient suffering from progressive multifocal leukoencephalopathy.

We report on a stem cell-transplanted patient with B cell chronic lymphatic leukemia who presented with a subacute onset of focal neurological deficits, gait abnormalities, emotional lability and dementia. Progressive multifocal leukoencephalopathy was diagnosed by magnetic resonance imaging (MRI) of the brain and detection of JC virus genome in the cerebrospinal fluid. Cidofovir and the 5HT2A receptor antagonist chlorpromazine were subsequently administered. A follow-up MRI of the brain 2 weeks after initiation of the antiviral therapy displayed progress of the demyelination, and the patient died 3 months after onset of the neurological symptoms. This report highlights the need for the development of novel and potent strategies for treatment of progressive multifocal leukoencephalopathy.

Sustained CD8+ T-cell responses induced after acute parvovirus B19 infection in humans.

Murine models have suggested that CD8+ T-cell responses peak early in acute viral infections and are not sustained, but no evidence for humans has been available. To address this, we longitudinally analyzed the CD8+ T-cell response to human parvovirus B19 in acutely infected individuals. We observed striking CD8+ T-cell responses, which were sustained or even increased over many months after the resolution of acute disease, indicating that CD8+ T cells may play a prominent role in the control of parvovirus B19 and other acute viral infections of humans, including potentially those generated by live vaccines.