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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
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.
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.
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).
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Abstract 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.
Abstract 4866
The first pandemic of the 21st century was caused by the novel influenza A (H1N1) virus, now known as pandemic H1N1-09 virus. Its first wave reached Germany in autumn 2009. Though for ...the German public health sector this pandemic was a challenge, a state of national emergency could not be recognized. A popular current believe is that the threat by this pandemic had been overestimated by national and international health care experts. Here, we report our experience with the pandemic H1N1-09 virus in hematologic patients when the first wave hit Germany between December 2009 and February 2010.
Viral diagnostic for all patients was performed in a central virologic laboratory. All patients with at least one sample tested positive by PCR for H1N1-09 between October 2009 and March 2010 were included into the analysis. Samples were obtained by nasal wash, nasopharyngeal swab or broncho-alveolary lavage. The medical charts of all patients with H1N1-09 infection were reviewed systematically.
15 patients with underlying hematologic diseases (10 male and 5 female) with a median age of 52 years (range 18–70 years) were tested positive for H1N1-09 virus by PCR in our department. Notably, in 12 Patients H1N1-09 was nosocomially acquired after a median of 16 days hospitalization (range 6 – 42 days). 13 patients (87%) got infected between December 2 and December 29. At the time of infection all patients were immunocompromised with 11 patients being cytopenic after chemotherapy and four patients after allogeneic hematopoietic cell transplantation (HCT) among the latter two patients were in aplasia after allogeneic HCT (day +5 and day +11 after HCT). CT scan was performed in 8 patients at the time of diagnosis. Seven patients presented with signs of atypical pneumonia on CT scan. Ground glass opacity, consolidation, airway wall thickening, airway dilatation, pleural effusion and lymphadenopathy were common findings.
In 10 patients viral clearance was monitored by RT-PCR. The median duration of viral shedding was 10 days (range 4 – 41 days). Prolonged viral persistence was associated with severe lung injury. All patients received Oseltamivir as first-line therapy, except three patients, who died prior to the confirmation of the diagnosis by RT-PCR. Simultaneously broad spectrum antibiotics and antimycotics were administered.
5 patients (33%) with respiratory failure needed invasive mechanical ventilation (MV) at the time of the H1N1-09 infection. Three out of these patients died.
Within a follow up of 6 months eight patients have died. Six patients (40%) have died from the infection. Among these three patients died from fulminant pulmonary failure whereas three patients died several weeks after H1N1-09 infection from subsequent respiratory or multiorgan failure. The impact on indirect mortality cannot yet be fully assessed, since in some patients the infection caused significant delay of anti-leukemic therapy and acquired comorbidities resulted in dose-reductions of chemotherapy.
In contrast to largely mild infections in the healthy German population pandemic H1N1-09 pneumonia represented a life-threatening infection for hematologic patients associated with a high mortality due to acute respiratory failure, late pulmonary complications and delay of antitumor treatment.
One alarming finding was the frequency of nosocomial infections. This observation points to the possibility of transmission of the virus from patient to patient, visitors to patient or even from medical staff to patient. With this observation in our institution patients admitted to hospital were put under quarantine before they were allowed to be accommodated in a double room in the hematologic unit. Transmission of the virus from asymptomatic staff or visitors to patient is another major concern. The suspected vaccination rate of medical staff in Germany was less than 20%. Especially, when asymptomatic or mild H1N1 infections occur - as it was the case in Germany – the medical staff and visitors could become important vectors of infection. The most effective measure against this threat is active immunization of visitors prior to patient contact and medical staff in hematologic units.
No relevant conflicts of interest to declare.
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. 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 + or - 24.9 ng/ml for HCMV IL-10 and 1.5 mug/ml + or - 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 alpha (TNF-alpha) 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. 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-alpha 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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