Eike Steinmann

Adaptation of Stenotrophomonas maltophilia in cystic fibrosis: molecular diversity, mutation frequency and antibiotic resistance.

Due to the continuous exposure to a challenging environment and repeated antibiotic treatment courses, bacterial populations in cystic fibrosis (CF) patients experience selective pressure causing the emergence of mutator phenotypes. In this study we investigated the genotypic diversity, mutation frequency and antibiotic resistance of S. maltophilia isolates chronically colonizing CF patients. S. maltophilia was isolated from a total of 90 sputum samples, collected sequentially from 19 CF patients admitted between January 2008 and March 2012 at the University Hospital Essen, Germany. DNA fingerprinting by repetitive-sequence-based PCR revealed that 68.4% (n=13) of CF patients harbored different S. maltophilia genotypes during the 4-year study course. Out of 90 S. maltophilia isolates obtained from chronically colonized CF patients, 17.8% (n=16) were hypomutators, 27.7% (n=25), normomutators, 23.3% (n=21), weak hypermutators and 31.2% (n=28) strong hypermutators. We also found that mutation rates of the most clonally related genotypes varied over time with the tendency to become less mutable. Mutator isolates were found to have no significant increase in resistance against eight different antibiotics versus nonmutators. Sequencing of the mismatch repair genes mutL, mutS and uvrD revealed alterations that resulted in amino acid changes in their corresponding proteins. Here, we could demonstrate that several different S. maltophilia genotypes are present in CF patients and as a sign of adaption their mutation status switches over time to a less mutator phenotype without increasing resistance. These results suggest that S. maltophilia attempts to sustain its biological fitness as mechanism for long-term persistence in the CF lung.

Development of a quantitative immunofluorescence assay for detection of Stenotrophomonas maltophilia antibodies in patients with cystic fibrosis.

BACKGROUNDnTo describe a simple quantitative immunofluorescence assay (IFA) for the detection of specific Stenotrophomonas maltophilia antibodies in serum of CF patients.nnnMETHODSnA total of 100 sera (64 CF patients and 36 healthy subjects) were collected over a period of 2 years at the University Hospital Essen, Germany. Sputum culture status classified CF patients into groups. Serologic response was determined after Pseudomonas aeruginosa absorption by indirect IFA to Sm whole cell.nnnRESULTSnCF patients with chronic S. maltophilia showed significantly higher S. maltophilia antibody levels compared with healthy individuals (P<0.0001) and CF patients with intermittent (P=0.0315) or never S. maltophilia/P. aeruginosa (P=0.0002). A discriminant cut-off value of >1:120 titre was established to differentiate CF chronic S. maltophilia from the other groups. For CF chronic S. maltophilia, the IFA showed sensitivity and specificity values of 70.7% and 84.7%, respectively.nnnCONCLUSIONnOur data demonstrated that quantitative IFA is a simple serological assay for the detection of specific S. maltophilia antibodies, which could be useful as a diagnostic tool for monitoring immune response of CF patients to S. maltophilia.

Emergence of linezolid- and vancomycin-resistant Enterococcus faecium in a department for hematologic stem cell transplantation.

BackgroundPrevalence of vancomycin-resistant enterococci has increased in Germany. Here, we report the cluster of linezolid- and vancomycin-resistant Enterococcus faecium (LVRE) in a German department for hematologic stem cell transplantation (HSCT).MethodsIn this retrospective analysis we included all patients with LVRE in a university-based department for HSCT in 2014 and 2015. Patients chart reviews were used to investigate the epidemiology and clinical outcome. Available LVRE isolates underwent detailed microbiological characterization and genotyping by pulsed-field gel electrophoresis (PFGE).ResultsIn total, 20 patients with LVRE were identified within the observed time period. All except two patients underwent allogeneic HSCT. Surveillance culture results from incoming patients and chart review revealed that 10 of 20 patients were colonized at hospital admission. Eight of 10 patients with in-hospital acquired LVRE had previous linezolid treatment. Analysis of spatio-temporal patterns showed no evidence for LVRE patient-to-patient or environment-to-patient transmission within the HSCT department. In five cases (25xa0%) LVRE bloodstream infection occurred. Nine LVRE isolates could be saved for characterization. Eight isolates carried vanA, one isolate vanB. PFGE analysis showed that four different LVRE clones were responsible for the cluster. One single genotype was present in six LVRE isolates whereupon the corresponding patients were all referred from the same hospital to the HSCT department.ConclusionsThis is the first report demonstrating the emergence of LVRE in a German HSCT department. (L)VRE screening at patients’ admission and appropriate infection control strategies were sufficient to prevent any transmission. Further studies in this predisposed patient collective are warranted.

Leitlinie der Deutschen Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) e. V. und des Robert Koch-Instituts (RKI) zur Prüfung von chemischen Desinfektionsmitteln auf Wirksamkeit gegen Viren in der Humanmedizin

In dieser Leitlinie wird die Durchfühxad rung von Suspensionsversuchen zum Nachweis der Wirksamkeit von Desinxad fektionsmitteln gegen Viren beschrieben. Diese Versuche sind sowohl ohne als auch mit zusätzlicher Belastung durch foetales Kälberserum (FKS) im Testansatz durchxad zuführen. Eine Titerreduktion von minxad destens 4 logarthmischen Stufen (4 log10) lässt den Schluss zu, dass das Mittel unter den geprüften Bedingungen viruzide Eigenschaften besitzt. Verschiedene Parameter des in-vitro Tests können die Ergebnisse beeinxad flussen (u. a. Virus, Zellen, Passagenzahl, Zytotoxizität). Weiterhin bestimmen die Titrationsbedingungen zur Bestimmung der Viruskonzentration (u. a. Probenverxad dünnungsfaktor und Anzahl der getestexad ten Replikate pro Verdünnung) die Gexad nauigkeit der Prüfung und haben damit ebenfalls Einfluss auf die Aussage über die viruzide Wirksamkeit des zu testenxad den Desinfektionsmittels. In der vorliexad genden Leitlinie finden daher biometrixad sche Aspekte besondere Aufmerksamkeit. H.F. Rabenau · I. Schwebke · J. Blümel · M. Eggers · D. Glebe · I. Rapp · A. Sauerbrei · E. Steinmann · J. Steinmann · H. Willkommen · P. Wutzler

Six Heterocyclic Metabolites from the Myxobacterium Labilithrix luteola

Two new secondary metabolites, labindole A [2-methyl-3-(2-nitroethyl)-3H-indole] (1) and labindole B [2-methyl-3-(2-nitrovinyl)-3H-indole] (2), were isolated from the myxobacterium Labilithrix luteola (DSM 27648T). Additionally, four metabolites 3, 4, 5 and 6 already known from other sources were obtained. Their structures were elucidated from high resolution electrospray ionisation mass spectrometry (HRESIMS) and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy data and their relative configuration was assigned based on nuclear Overhauser effect (NOE) and vicinal 1H-NMR coupling data. The compounds where tested for biological activities; labindoles A (1) and B (2) exhibited significant activity against Hepatitis C Virus, 9H-carbazole (3), 3-chloro-9H-carbazole (4) and 4-hydroxymethyl-quinoline (5) showed antifungal activities. Moreover, compound 3 had weak to moderate antibacterial activities, while labindoles A (1) and B (2) were devoid of significant antifungal and antibacterial effects.

Lanyamycin, a macrolide antibiotic from Sorangium cellulosum, strain Soce 481 (Myxobacteria)

Lanyamycin (1/2), a secondary metabolite occurring as two epimers, was isolated from the myxobacterium Sorangium cellulosum, strain Soce 481. The structures of both epimers were elucidated from HRESIMS and 1D and 2D NMR data and the relative configuration of their macrolactone ring was assigned based on NOE and vicinal 1H NMR coupling constants and by calculation of a 3D model. Lanyamycin inhibited HCV infection into mammalian liver cells with an IC50 value of 11.8 µM, and exhibited a moderate cytotoxic activity against the mouse fibroblast cell line L929 and the human nasopharyngeal cell line KB3 with IC50 values of 3.1 and 1.5 μM, respectively, and also suppressed the growth of the Gram-positive bacterium Micrococcus luteus.

First detection and frequent occurrence of Equine Hepacivirus in horses on the African continent

Since the discovery of equine hepacivirus (EqHV) in 2011, the virus has been detected in horse populations from more than twelve countries across five continents. EqHV seroprevalence has been reported to be as high as 61.8% and EqHV ribonucleic acid (RNA) prevalence to range between 0.9% and 34.1%. Molecular and serological indications of EqHV infection have never been reported in equids on the African continent. Therefore, investigation of EqHV prevalence in South African horses and subsequent viral genetic characterization contribute to a better understanding of the global epidemiology of this virus. In a cross-sectional study, serum samples from 454 Thoroughbred foals (aged 58-183 days) were analysed for anti-EqHV non-structural protein 3 (NS3)-specific antibodies (abs) with a luciferase immunoprecipitation system (LIPS) and for EqHV RNA by quantitative real-time polymerase chain reaction (qRT-PCR). Farms of origin (nu2009=u200926) were situated in South Africas Western Cape Province. The associations between EqHV infection state and farm of origin, foal gender and foal age were subsequently described. Furthermore, nested PCRs were performed on parts of the 5UTR, NS3 and NS5B genes of 17 samples. Samples were sequenced and phylogenetic analyses were conducted. The populations seroprevalence was 83.70% and RNA was detected in 7.93% of samples. Increasing foal age was associated with decreasing ab prevalence and increasing prevalence of EqHV RNA. Sequences from South African EqHV strains did not show in-depth clustering with published sequences of EqHV isolates from particular continents. In conclusion, EqHV is present in the South African Thoroughbred population and appears more prevalent than reported in other horse populations worldwide.

Methylene Blue Treatment of Grafts During Cold Ischemia Time Reduces the Risk of Hepatitis C Virus Transmission

BackgroundnAlthough organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time.nnnMethodsnStandard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV.nnnResultsnThree different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys.nnnConclusionsnThis proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.

High environmental stability of hepatitis B virus and inactivation requirements for chemical biocides

Abstract Hepatitis B virus (HBV) infection is considered a major public health problem worldwide, and a significant number of reports on nosocomial and occupational outbreaks have been reported. This systematic investigation of HBV stability and susceptibility to different antiseptics revealed that HBV infectivity was very stable, with a half-life of >22 days at 37°C. At 4°C, infectivity was barely reduced for up to 9 months. Different alcohols and commercially available hand antiseptics had a virucidal effect against HBV. We propose that very strict compliance with established hygienic guidelines should be mandatory to avoid and prevent HBV infections.

Susceptibility of Chikungunya Virus to Inactivation by Heat and Commercially and World Health Organization-Recommended Biocides

Abstract Despite increasing clinical relevance of Chikungunya virus (CHIKV) infection, caused by a rapidly emerging pathogen, recommended guidelines for its inactivation do not exist. In this study, we investigated the susceptibility of CHIKV to inactivation by heat and commercially available hand, surface, and World Health Organization-recommended disinfectants to define CHIKV prevention protocols for healthcare systems.