Genomic and phenotypic diversity of Clostridium difficile during long-term sequential recurrences of infection.

Infection with the emerging pathogen Clostridioides (Clostridium) difficile might lead to colonization of the gastrointestinal tract of humans and mammals eventually resulting in antibiotic-associated diarrhea, which can be mild to possibly life-threatening. Recurrences after antibiotic treatment have been described in 15-30% of the cases and are either caused by the original (relapse) or by new strains (reinfection). In this study, we describe a patient with ongoing recurrent C. difficile infections over 13 months. During this time, ten C. difficile strains of six different ribotypes could be isolated that were further characterized by phenotypic and genomic analyses including motility and sporulation assays, growth fitness and antibiotic susceptibility as well as whole-genome sequencing. PCR ribotyping of the isolates confirmed that the recurrences were a mixture of relapses and reinfections. One recurrence was due to a mixed infection with three different strains of two different ribotypes. Furthermore, genomes were sequenced and multi-locus sequence typing (MLST) was carried out, which identified the strains as members of sequence types (STs) 10, 11, 14 and 76. Comparison of the genomes of isolates of the same ST originating from recurrent CDI (relapses) indicated little within-patient microevolution and some concurrent within-patient diversity of closely related strains. Isolates of ribotype 126 that are binary toxin positive differed from other ribotypes in various phenotypic aspects including motility, sporulation behavior and cell morphology. Ribotype 126 is genetically related to ribotype 078 that has been associated with increased virulence. Isolates of the ribotype 126 exhibited elongated cells and a chaining phenotype, which was confirmed by membrane staining and scanning electron microscopy. Furthermore, this strain exhibits a sinking behavior in liquid medium in stationary growth phase. Taken together, our observation has proven multiple CDI recurrences that were based on a mixture of relapses and reinfections.

Management of Helicobacter pylori infection-the Maastricht V/Florence Consensus Report.

Important progress has been made in the management of Helicobacter pylori infection and in this fifth edition of the Maastricht Consensus Report, key aspects related to the clinical role of H. pylori were re-evaluated in 2015. In the Maastricht V/Florence Consensus Conference, 43 experts from 24 countries examined new data related to H. pylori in five subdivided workshops: (1) Indications/Associations, (2) Diagnosis, (3) Treatment, (4) Prevention/Public Health, (5) H. pylori and the Gastric Microbiota. The results of the individual workshops were presented to a final consensus voting that included all participants. Recommendations are provided on the basis of the best available evidence and relevance to the management of H. pylori infection in the various clinical scenarios.

Fungal epidemiology and diversity in cystic fibrosis patients over a 5-year period in a national reference center.

The knowledge on prevalence rates of yeasts and moulds in patients with cystic fibrosis (CF) in Germany is scarce. The aim of this report is to give an overview of the diversity and epidemiology of fungal species in CF patients. Over a 5-year period, all fungal isolates cultured from microbiological specimen from CF patients were recorded. Beside standard bacteriological culture media two fungal media were used for cultivation. Species were identified by microscopy, biochemical profiling, MALDI-TOF analysis or DNA sequencing methods. In sum, 25,975 clinical samples from CF patients were analyzed. About 75% of CF patients were colonized by yeasts, mainly Candida albicans (38%) and Candida dubliniensis (12%). In 35% of the patients Aspergillus spp. (Aspergillus fumigatus: 29%) were detected, followed by Exophiala dermatitidis and Scedosporium/Lomentospora complex isolates (4% each). Data for other fungal species are shown. Over a 5-year period, the epidemiology of fungal species detected in CF patients was relatively constant. Clinical microbiology laboratories should carefully monitor samples from CF patients for newly occurring fungal pathogens.

[S2k-guideline Helicobacter pylori and gastroduodenal ulcer disease]. / S2k-Leitlinie Helicobacter pylori und gastroduodenale Ulkuskrankheit.

In the line "bismuth-containing quadruple therapy" of Table 7 (p 342), in the column "dosage" incorrectly at the three antibiotics respectively 1-1-1-1. The correct is: 3-3-3-3.

[Frequency of polyphasic health-care-associated outbreaks is highest in surgical departments - results of a systematic literature search]. / Chirurgen sind am häufigsten von mehrphasischen nosokomialen Ausbrüchen betroffen - Ergebnisse einer systematischen Literaturrecherche.

BACKGROUND: So-called polyphasic nosocomial outbreaks describe a situation in which additional infections occur after a certain case-free interval - despite the detection of the outbreak's source. This article summarises the results of a systematic search of the medical literature on polyphasic outbreaks. MATERIALS AND METHODS: For this purpose, the Outbreak Worldwide-Database, PubMed and reference lists of relevant articles were screened. RESULTS: A total of 124 polyphasic outbreaks (median duration of 50 weeks) was included in the analysis and then compared to 2089 monophasic nosocomial outbreaks. Surgical departments were significantly more often involved in polyphasic outbreaks than they were in monophasic events (33.9 % vs. 24.5 %; p < 0.05). Hepatitis B virus outbreaks were significantly more often seen as poly-phasic events. Either there had been more than one source initially, or a new source developed during the first phase of the outbreak and led to additional cases thereafter. CONCLUSIONS: Up to now, only little is known about polyphasic nosocomial outbreaks. Thus, there is a further need to close this gap of information in the future. Personnel on the ward as well as -infection control staff should always consider the possibility of the existence of more than one -source when investigating a nosocomial outbreak.

Surveillance of extended-spectrum beta-lactamase-producing bacteria and routine use of contact isolation: experience from a three-year period.

The usefulness and applicability of isolation precautions were questioned for extended-spectrum beta-lactamase (ESBL)-producing strains of Enterobacteriaceae in the endemic setting. We performed a surveillance programme for ESBL-positive organisms and the infection control management of patients colonized or infected with these organisms. Between 1 January 2002 and 31 December 2004, a total of 147 cases of ESBL-producing strains of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis from 123 patients were noted. The overall incidence of ESBL-producing-strain-positive cases was 0.12/1000 patient-days. The proportion of referred cases was 35% (N=51); 65% of cases (N=96) were acquired in our institution. Infections developed in 57 cases (38.8%), of which 36 (63.3%) were nosocomial. Contact isolation precautions were carried out for 79.6% of the cases, with a median duration of contact isolation precautions for 14 days (range: 0-144). The contact isolation precautions resulted in 2985 isolation days in total, i.e. 995 isolation days per year. Typing by pulsed-field gel electrophoresis showed clonal diversity in 94.2% of the isolates from patients. Seven patient-to-patient transmissions were noted. Only in 10 cases (6.8%) was colonization with ESBL-producing strains cleared. Considering the large number of immunocompromised patients treated in our institution (>1500 bone marrow or solid organ transplantations performed during 2002-2004), we will continue to isolate patients who are colonized or infected with ESBL-producing organisms.

Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation.

Foxp3 (forkhead box P3 transcription factor)-expressing regulatory T cells (Tregs) are essential for immunological tolerance, best illustrated by uncontrolled effector T-cell responses and autoimmunity upon loss of Foxp3 expression. Tregs can adopt specific effector phenotypes upon activation, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3(+)CD4(+) T cells coexpressing retinoic acid-related orphan receptor-γt (RORγt), the master transcription factor for T helper type 17 (Th17) cells, represent a stable effector Treg lineage. Transcriptomic and epigenetic profiling revealed that Foxp3(+)RORγt(+) T cells display signatures of both Tregs and Th17 cells, although the degree of similarity was higher to Foxp3(+)RORγt(-) Tregs than to Foxp3(-)RORγt(+) T cells. Importantly, Foxp3(+)RORγt(+) T cells were significantly demethylated at Treg-specific epigenetic signature genes such as Foxp3, Ctla-4, Gitr, Eos, and Helios, suggesting that these cells have a stable regulatory rather than inflammatory function. Indeed, adoptive transfer of Foxp3(+)RORγt(+) T cells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and revealed an enhanced suppressive capacity as compared with Foxp3(+)RORγt(-) Tregs. Thus, our data suggest that RORγt expression in Tregs contributes to an optimal suppressive capacity during gut-specific immune responses, rendering Foxp3(+)RORγt(+) T cells as an important effector Treg subset in the intestinal system.

Prevalence of Scedosporium species and Lomentospora prolificans in patients with cystic fibrosis in a multicenter trial by use of a selective medium.

OBJECTIVE: Detection of hyphomycetes of the Scedosporium apiospermum complex and Lomentospora prolificans (Sac-Lp) is not yet standardized. Prevalence rates in patients with cystic fibrosis (CF) and the resistance pattern of these pathogens in Germany are unknown. METHODS: In a one-year prospective study 11 laboratories used a selective medium for isolation of Sac-Lp, examining >11,600 respiratory samples from 2346 patients with CF. Isolates were identified by molecular methods and tested for susceptibility to antifungal drugs. RESULTS: The prevalence of Sac-Lp in patients with CF in Germany varied from 0.0 to 10.5% (mean: 3.1%) among the clinical centres. The benefit of the selective medium SceSel(+) compared to standard media for fungi was documented for >5000 samples. High antifungal resistance was detected in the S. apiospermum complex, and the multiresistance of L. prolificans was confirmed. CONCLUSION: Microbiology laboratories should be aware of these resistant species in patients with CF and consider using a selective medium.

Topoisomerase I of Helicobacter pylori: juxtaposition with a flagellin gene (flaB) and functional requirement of a fourth zinc finger motif.

Cloning and nucleotide sequence analysis showed that in Helicobacter pylori the gene encoding topoisomerase I (topA) lies about 170 nucleotides upstream from flaB, a gene encoding one of the two flagellin proteins that is required for virulence. The topA and flaB genes are divergently transcribed. The orientation and spatial relationship between flaB and topA are remarkably conserved among strains of a bacterium in which genomic rearrangements are common. The deduced amino acid sequence of topoisomerase I revealed four zinc finger motifs, one more than has been reported previously for the Escherichia coli homologue. The additional motif, which is near the C-terminus of the protein, appears to be essential for function since mutations in that region are lethal. These data show that TopA proteins can be divided into several classes on the basis of zinc finger motifs and raise the interesting possibility that the H. pylori enzyme has local topological effects focussed on a flagellin gene.

Green fluorescent protein as a novel marker and reporter system in Helicobacter sp.

In order to be able to study gene regulation in single, live Helicobacter pylori bacteria in vitro or in contact with host cells, we established the green fluorescent protein gene gfp from Aequorea victoria as a reporter gene for use with Helicobacter species. We describe here the construction of genomic transcriptional fusions of the promoterless gfp gene with the flaA and flaB promoters of H. pylori. We have also constructed a Mini-Tn3-km-gfp transposon to be used for shuttle transposon mutagenesis in H. pylori and H. mustelae. A marker strain with wild-type phenotype, carrying multiple plasmid-borne copies of gfp under the control of the H. pylori flaB promoter, was constructed for studies of bacterial distribution and transmission in animal models.

Ultrastructure and biochemical studies of the flagellar sheath of Helicobacter pylori.

Helicobacter pylori flagellar sheaths were isolated by sucrose density-gradient centrifugation and analysed by electronmicroscopy, SDS-PAGE and gas-liquid chromatography. Electronmicroscopy of thin sections of flagella showed an internal electron-dense filament and a surrounding flagellar sheath with the typical bilayer structure of a membrane. The flagellar filaments could be disintegrated by acid treatment and the resulting isolated flagellar sheaths formed vesicles, sometimes with characteristic structures. Centrifugation of flagellar preparations after acid treatment resulted in the enrichment of flagellar sheaths in the pellet. SDS-PAGE analysis of the pellet showed a reduction of the flagellin band and a number of protein bands of 150, 76, 67, 65, 53, 51, 49, 29.5, 18, 17 and 16 kDa. However, there were no major protein bands characteristic for the sheath. Differences between the protein profiles of Sarkosyl-insoluble membranes and flagellar sheaths appeared in the lower M(r) range of 30-14 kDa. Major fatty acids of isolated flagellar sheaths were C 14:0, C 19:0 cyc, C 18:0, and the LPS-specific fatty acids 3-OH C 16:0 and 3-OH C 18:0. The results demonstrate that the flagellar sheaths of H. pylori are membranes and contain LPS and proteins.

Serum procalcitonin levels in the postmortem diagnosis of sepsis.

Procalcitonin is regarded as a valuable marker for sepsis in living persons and even in post-mortem investigations. At the Institute of Legal Medicine, 25 autopsy cases with suspected bacterial infectious diseases or sepsis were examined using the semi-quantitative PCT-Q(®)-test (B.R.A.H.M.S., Germany) in 2010 and 2011. As controls, 75 cadavers were used for which there was no suspicion of a bacterial infectious disease or sepsis. Femoral blood was cultured from the cases and from controls, and samples from the brain, heart, lungs, liver, spleen and kidneys were examined histologically for findings seen in sepsis. Twelve cases in the sepsis/infectious disease group (48%) were classifiable as sepsis following synopsis of PCT levels, autopsy results, and histopathological and microbiological findings. This study shows that the semi-quantitative PCT-Q(®)-test is a useful supplementary marker in routine autopsy investigations, capable of classifying death as due to sepsis.

[S3-guideline "Helicobacter pylori and gastroduodenal ulcer disease"]. / S3-Leitlinie "Helicobacter pylori und gastroduodenale Ulkuskrankheit" der Deutschen Gesellschaft für Verdauungs- und Stoffwechselkrankheiten (DGVS).

This guideline updates a prior concensus recommendation of the German Society for Digestive and Metabolic Diseases (DGVS) from 1996. It was developed by an interdisciplinary cooperation with representatives of the German Society for Microbiology, the Society for Pediatric Gastroenterology and Nutrition (GPGE) and the German Society for Rheumatology. The guideline is methodologically based on recommendations of the Association of the Scientific Medical Societies in Germany (AWMF) for providing a systematic evidence-based consensus guideline of S 3 level and has also implemented grading criteria according to GRADE (Grading of Recommendations Assessment, Development and Evaluation). Clinical applicability of study results as well as specifics for Germany in terms of epidemiology, antibiotic resistance status, diagnostics and therapy were taken into account.