Plasmid copy number underlies adaptive mutability in bacteria.

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F'lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F'lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F'lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F'lac copies divide very little under selection but have enough energy to replicate their F'lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced

Antigenspezifische und neutralisierende Antikörper bei Patienten mit Clostridioides difficile-Infektion

Das Bakterium Clostridioides difficile hat als weltweit häufigster Erreger einer nosokomialen infektiösen Diarrhoe eine besondere humanmedizinische Bedeutung. Die Inzidenz der Infektion stieg in den letzten zwei Jahrzehnten an und stellt wegen einer erhöhten Morbidität und hoher Behandlungskosten eine Belastung für das Gesundheitswesen dar. Das klinische Bild einer C. difficile-Infektion (CDI) reicht von leichten bis schweren Verläufen, die bis zur Entwicklung eines Megakolons führen können. Dabei liegt das Risiko für ein Rezidiv bei 30% und steigt mit jeder weiteren Krankheitsepisode nochmals an. Die orale Antibiotikatherapie gilt derzeit als Therapie der ersten Wahl, doch birgt gerade auch sie das Risiko einer Rekurrenz, sodass das Bestreben nach neuen präventiven Therapiestrategien wächst. In der Pathogenese der CDI kommt der Bildung von erregerspezifischen Toxinen eine große Bedeutung zu. In früheren Studien korrelierten nachgewiesene Toxin-Antikörper bei Patienten mit einem geringeren Rezidivrisiko. Daraus haben sich vielversprechende Ansätze für neue präventive Therapieoptionen (z.B. Anti-Toxin-Antikörper Bezlotoxumab oder Impfstoffe) entwickelt. Allerdings ist die natürliche Immunantwort bei einer CDI noch nicht vollumfänglich verstanden. Ziel der vorliegenden Arbeit war es daher, die natürliche Antikörperantwort während einer CDI zu untersuchen und mit dem klinischen Verlauf zu korrelieren. Hierfür wurden in der ersten klinischen Studienphase 46 Patienten des Universitätsklinikums des Saarlandes mit diagnostisch gesicherter CDI rekrutiert. Daten zu Anamnese und Klinik wurden mittels Fragebogen und Gesprächen mit Patienten und behandelndem Stationspersonal prospektiv für die Akutphase der Erkrankung ermittelt. An Tag 1, 3 und 6 (±1 d) nach der CDI-Diagnose erfolgten die Blutabnahmen, um in einer zweiten experimentellen Studienphase die Plasmaproben auf antigenspezifische Antikörper gegen Toxin A (Anti-TcdA), Toxin B (Anti-TcdB), Glutamatdehydrogenase (Anti-GDH) und Cwp84 (Anti-Cwp84) sowie auf neutralisierende Antikörper zu untersuchen. Für den Nachweis der Neutralisation wurde ein zellkulturbasierter Toxin-Neutralisationstest etabliert. Die Toxine wurden aus konditionierten Medien von klinischen Isolaten der C. difficile-Ribotypen (RTs) RT014 und RT027 gewonnen. Zuvor wurde die Wachstums- und Toxinkinetik dieser entsprechenden RTs bewertet, um konditionierte Medien mit hohen Toxingehalten zu gewinnen. Bei den meisten Patienten waren Antikörper gegen GDH (85%) und Cwp84 (61%) nachweisbar, während Anti-TcdA (11%) und Anti-TcdB (28%) nur bei einer Minderheit zu finden waren. In insgesamt nur geringen Titerstufen (≤ 1:16) wiesen 26% der Erkrankten neutralisierende Antikörper gegen die Toxine von RT027 und eine Untergruppe (11%) zusätzlich gegen die von RT014 auf. Neutralisierende Antikörper einzig gegen RT014-Toxine fanden sich nicht. Im Verlauf des einwöchigen Untersuchungszeitraumes wurde unabhängig vom Schweregrad der Erkrankung keine Serokonversion und kein Antikörperanstieg beobachtet, sodass ein Booster-Effekt ausgeschlossen werden konnte. Es wurde kein Zusammenhang zwischen dem Vorhandensein von antigenspezifischen oder neutralisierenden Antikörpern und dem klinischen Verlauf der Krankheit festgestellt. Anti-TcdB-, nicht aber Anti-TcdA-Antikörper, korrelierten mit dem Auftreten neutralisierender Antikörper. Insgesamt waren die Antikörperprävalenzen und Titer neutralisierender und Toxin-abhängiger Antikörper niedrig; ihr Vorhandensein oder Fehlen stand zudem nicht in Zusammenhang mit dem Krankheitsverlauf. Es wurden sowohl neutralisierende Antikörper mit breitem Spektrum als auch mit stammbeschränkter Neutralisierungskapazität nachgewiesen, was auf unterschiedliche neutralisierende Epitope zurückzuführen sein könnte. Als weiteres Kernergebnis konnte eine enge Korrelation zwischen dem Nachweis von Anti-TcdB und der Fähigkeit zur Neutralisation nachgewiesen werden, was die Annahme bestärkt, dass Anti-TcdB als funktioneller Antikörper für die CDI-Immunantwort sehr bedeutsam zu sein scheint. Schlussfolgernd sind alternative Therapiestrategien wie z.B. Impfungen nötig, um hohe Titer der nach natürlicher intestinaler Exposition mit C. difficile bereits vorhandenden niedrigtitrigen oder gar noch fehlenden neutralisierenden Antikörper zu erzielen.Clostridioides difficile is the most common agent of nosocomial infectious diarrhea, and has therefore considerable medical importance. On a global scale, incidence rates of C. difficile infection (CDI) have risen in the course of the last two decades. Thus, for the healthcare system, CDI has been recognized as a large burden leading to high mortality and morbidity. The clinical spectrum ranges from mild to severe courses (e.g. toxic megacolon). The risk of recurrence is 30% and increases with each additional infection episode. Oral antibiotic therapy is currently considered the treatment of choice. However, even this treatment poses the risk of recurrence; hence, efforts to find new preventive treatment strategies are needed. Previous studies described the correlation between antibodies and a lower risk of disease recurrence. This finding has led to new promising therapeutic approaches for new therapy concepts like passive immunization (e.g. with the antibody bezlotoxumab) and vaccination. However, the specific CDI immune response is not yet completely understood. In the present study, the prevalence and the course of antigen-specific and neutralizing C. difficile antibodies during CDI and the correlating clinical course were investigated. For this purpose, in a first study phase, 46 patients of Saarland University Hospital with diagnostically confirmed CDI were recruited. Data on medical history and acute symptoms were prospectively obtained by questionnaire and interviews with patients or attending ward staff. Blood samples were taken on days 1, 3, and 6 (±1 d) after diagnose. In the second experimental study phase, patients’ plasma samples were tested for the presence of antibodies against toxin A (anti-TcdA), toxin B (anti-TcdB), glutamate-dehydrogenase (anti-GDH) and Cwp84 (anti-Cwp84). Additionally neutralizating antibodies against toxigenic conditioned media generated by clinical isolates of the ribotypes (RT’s) RT027 and RT014 was invastigated. For this reason, a cell culture-based toxin neutralization assay was established. Previously, the growth and toxin kinetics of these RT's were evaluated to harvest conditioned media with high toxin levels. Most patients had antibodies against GDH (85%) and Cwp84 (61%), but antibodies against TcdA (11%) and TcdB (28%) were only detected in some patients. Neutralizing antibodies against C. difficile toxins generated by RT027 were found in 26% of samples and a subgroup of these samples (11%) could also neutralize toxins from RT014; however, no single sample neutralized RT014 alone. Overall, neutralizing antibody titers were low (≤ 1:16). In a one-week follow-up, neither a seroconversion nor a significant antibody increase was observed, irrespective of disease severity. Consequently, a C. difficile-associated booster effect could be ruled out. No correlation was found between the presence of antigen-specific or neutralizing antibodies and the clinical course of disease. Anti-TcdB, but not anti-TcdA antibodies correlated closely with the occurrence of neutralizing antibodies. In conclusion, the prevalence and titers of detected toxin-related and neutralizing antibodies were low. The presence or absence of specific antibodies was not related to the clinical course of infecion. Furthermore, both broad-range and strain-restricted neutralizing antibodies were found, which might be related to different neutralizing epitopes. As another main finding, a close correlation between the detection of anti-TcdB and the capacity for neutralization was demonstrated, strengthening the significance of anti-TcdB as a functional antibody for the immune response of CDI. Thus, alternative therapeutic strategies such as vaccination are needed to achieve protective antibody titers which are not sufficiently generated after natural intestinal exposure to C. difficile

Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1A

The kinesin-3 motor KIF1A is in charge of vesicular transport in neuronal axons. Its single-headed form is known to be very inefficient due to the presence of a diffusive state in the mechanochemical cycle. However, recent theoretical studies have suggested that these motors could largely enhance force generation by working in teams. Here we test this prediction by challenging single-headed KIF1A to extract membrane tubes from giant vesicles along microtubule filaments in a minimal in vitro system. Remarkably, not only KIF1A motors are able to extract tubes but they feature a novel phenomenon: tubes are wound around microtubules forming tubular helices. This finding reveals an unforeseen combination of cooperative force generation and self-organized manoeuvreing capability, suggesting that the diffusive state may be a key ingredient for collective motor performance under demanding traffic conditions. Hence, we conclude that KIF1A is a genuinely cooperative motor, possibly explaining its specificity to axonal trafficking.Peer ReviewedPostprint (published version

Late-onset native valve endocarditis caused by Corynebacterium kroppenstedtii

Corynebacterium kroppenstedtii is an emerging cause of granulomatous mastitis and recurrent breast abscesses in women, but data on its clinical relevance in nongynecological disease conditions are limited. Here, we report the first case of a late-onset endocarditis of a native aortic valve in a 73-year-old male patient who presented with symptomatic aortic insufficiency. Echocardiography and cardiac computed tomography revealed the perforation of the noncoronary cusp and a large perivalvular abscess cavity. Hence, the surgical replacement of the aortic valve and aortic root were performed. Intraoperatively obtained tissue specimens grew C. kroppenstedtii and the patient made a full recovery after a 6-week course of antibiotic treatment. We briefly review the literature pertaining to antimicrobial susceptibility patterns of C. kroppenstedtii and available treatment recommendations. Our report calls for further studies to assess the role of this bacterium as a causative agent of infections other than granulomatous mastitis

Tissue Sodium Content and Arterial Hypertension in Obese Adolescents

Early-onset obesity is known to culminate in type 2 diabetes, arterial hypertension and subsequent cardiovascular disease. The role of sodium (Na+) homeostasis in this process is incompletely understood, yet correlations between Na+ accumulation and hypertension have been observed in adults. We aimed to investigate these associations in adolescents. A cohort of 32 adolescents (13-17 years), comprising 20 obese patients, of whom 11 were hypertensive, as well as 12 age-matched controls, underwent 23Na-MRI of the left lower leg with a standard clinical 3T scanner. Median triceps surae muscle Na+ content in hypertensive obese (11.95 mmol/L [interquartile range 11.62-13.66]) was significantly lower than in normotensive obese (13.63 mmol/L [12.97-17.64]; p = 0.043) or controls (15.37 mmol/L [14.12-16.08]; p = 0.012). No significant differences were found between normotensive obese and controls. Skin Na+ content in hypertensive obese (13.33 mmol/L [11.53-14.22] did not differ to normotensive obese (14.12 mmol/L [13.15-15.83]) or controls (11.48 mmol/L [10.48-12.80]), whereas normotensive obese had higher values compared to controls (p = 0.004). Arterial hypertension in obese adolescents is associated with low muscle Na+ content. These findings suggest an early dysregulation of Na+ homeostasis in cardiometabolic disease. Further research is needed to determine whether this association is causal and how it evolves in the transition to adulthood

The anxiolytic effects of cognitive behavior therapy for insomnia: preliminary results from a web-delivered protocol

Though the efficacy of cognitive behavior therapy for insomnia (CBTI) is well-established, the paucity of credentialed providers hinders widespread access. Further, the impact of alternatives such as web-delivered CBTI has not been adequately tested on common insomnia comorbidities such as anxiety. Therefore, we assessed the impact of an empirically validated web-delivered CBTI intervention on insomnia and comorbid anxiety symptoms. A sample of 22 adults (49.8±13.5 yo; 62.5% female) with DSM-5 based insomnia were randomized to either an active CBTI treatment group (n = 13) or an information-control (IC) group (n = 9). Participants in the CBTI group underwent a standard CBTI program delivered online by a 'virtual' therapist, whereas the IC group received weekly 'sleep tips' and general sleep hygiene education via electronic mail. All participants self-reported sleep parameters, including sleep onset latency (SOL), insomnia symptoms per the Insomnia Severity Index (ISI), and anxiety symptoms per the Beck Anxiety Inventory (BAI) at both baseline as well as follow- up assessment one week post-treatment. There were no significant differences between the CBTI and IC groups on baseline measures. The CBTI group showed significantly larger reductions in BAI scores (t = 2.6; p < .05; Cohen's d = .8) and ISI scores (t = 2.1; p < .05; Cohen's d = .9) at follow-up than did the IC group. Further, changes in SOL from baseline (62.3±44.0 minutes) to follow-up (22.3±14.4 minutes) in the CBTI group were also significantly greater (t = 2.3; p < .05; Cohen's d = .9) than in the IC group (baseline: 55.0±44.2 minutes; follow-up: 50.±60.2 minutes).This study offers preliminary evidence that a web-delivered CBTI protocol with minimal patient contact can improve comorbid anxiety symptoms among individuals with insomnia

Assessing the macroeconomic impact of Brexit through trade and migration channels

Este trabajo conjunto de Bundesbank, Banque de France y Banco de España analiza en detalle algunos de los numerosos canales a través de los cuales el brexit afectará a la economía del Reino Unido y a la de sus socios comerciales. En particular, se centra en los canales comercial y migratorio, haciendo una evaluación más general de los costes de la salida de la UE utilizando un modelo de gravedad. El canal comercial por sí solo puede reducir el PIB del Reino Unido un 2 % a medio plazo si el Reino Unido vuelve a las reglas de la OMC, mientras que un modelo de gravedad más general apuntaría a que el PIB del Reino Unido se reduciría casi un 6 % en comparación con el escenario de no salida. Por lo tanto, de acuerdo con nuestro análisis, el «coste de estar fuera de Europa» (como se estableció originalmente en el trabajo seminal de Cecchini en 1988) se encuentra entre el 2 % y el 6 % en términos de pérdidas del PIB real para el Reino Unido. Este impacto es en gran medida asimétrico, ya que el PIB de la zona del euro no se ve prácticamente afectado por este evento, al situarse menos de un 1 % por debajo del escenario de no salida en 2023. El estudio también pone de manifiesto cómo los resultados son sensibles a la reacción de las políticas económicas. En general, las políticas monetarias y fiscales pueden actuar para amortiguar el shock del brexitsin embargo, su efectividad depende de la fuente subyacente de la perturbaciónThis joint work by the Bundesbank, the Banque de France and the Banco de España highlights some of the numerous channels through which Brexit will affect the UK economy and its economic partners. In particular, it focuses on trade and migration channels, adding a more general assessment of exiting the EU through the use of a gravity model. The trade channel alone may cut UK GDP by 2% over the medium term if the UK reverts to WTO rules, while a more general gravity model would point to UK GDP falling by almost 6% compared to baseline. According to our analysis, the ‘cost of non-Europe’ (such as originally stated by Cecchini’s seminal work in 1988) lies therefore between 2% and 6% in terms of real GDP losses for the UK. With the shock being largely asymmetric, the EA remains relatively unscathed by the UK’s exit, with GDP less than 1% lower than baseline by 2023. The study also shows that results are sensitive to the envisaged policy response. In general, monetary and fiscal policies may act to cushion a Brexit-related shockhowever, the potency of the policy response depends on the underlying source of the shoc

Antigen-Specific vs. Neutralizing Antibodies Against Conditioned Media of Patients With Clostridioides difficile Infection: A Prospective Exploratory Study

The immunological response against Clostridioides difficile (C. difficile) is crucial for an improved understanding of disease mechanisms and the development of novel therapeutic strategies. From April 2014 to February 2015, adult patients with C. difficile infection (CDI) were recruited, and the clinical course and treatment response were carefully monitored. On day 1, 3, and 6 after diagnosis, patient plasma samples were screened for anti-GDH (glutamate dehydrogenase), anti-TcdA, anti-TcdB, and anti-CWP84 (cell-wall protein 84) antibodies by ELISA. Additionally, neutralization assays of toxins from conditioned media of clinical isolates (RT010, RT014, and RT027) were performed. Most patients with CDI (n=46) had antibodies against GDH (85%) and CWP84 (61%), but only few had antibodies against TcdA (11%) and TcdB (28%). We found patients with neutralizing antibodies against C. difficile toxins (conditioned media) produced by RT027 (26%). A subgroup of these samples could neutralize both toxins from RT027 and RT014 [11%, (5/46)]; however, no single sample neutralized only RT014. Overall, neutralizing antibody titers were low (≤1:16). In a one week follow-up of acute infection, we never observed an early booster effect with seroconversion or antibody increases, irrespective of disease severity. No correlation was found between the presence of antigen-specific (ELISA) or neutralizing antibodies and the clinical course of disease. Anti-TcdB but not anti-TcdA antibodies correlated with the occurrence of neutralizing antibodies. In conclusion, natural antibody titers against C. difficile toxins were absent or low and were not associated with disease severity. The correlation between the anti-TcdB with toxin neutralization confirms the importance of TcdB for virulence of CDI. Alternative sensitization strategies, e.g., through vaccine development, are required to overcome the regular low-titer antibody production following natural intestinal C. difficile exposure

Application and clinical impact of the RESIST-4 O.K.N.V. rapid diagnostic test for carbapenemase detection in blood cultures and clinical samples

Invasive infections caused by carbapenemase-producing bacteria are associated with excess mortality. We applied a rapid diagnostic test (RDT) on clinical samples with an elevated likelihood of carbapenemase-producing bacteria and documented its impact on antibiotic treatment decisions. Among 38 patients, twelve tested positive for infections caused by carbapenemase-producing bacteria (31.6%), mainly in blood cultures. KPC (n = 10) was more frequent than OXA-48 (n = 2). RDT-based carbapenemase detection led to a treatment modification to ceftazidime/avibactam-containing regimens in all patients before detailed antibiotic testing results became available. Eleven patients (92%) survived the acute infection, whereas one patient with a ceftazidime/avibactam- and colistin-resistant OXA-48-positive isolate died

Tocilizumab in early progressive rheumatoid arthritis: FUNCTION, a randomised controlled trial

OBJECTIVES: The efficacy of tocilizumab (TCZ), an anti-interleukin-6 receptor antibody, has not previously been evaluated in a population consisting exclusively of patients with early rheumatoid arthritis (RA). METHODS: In a double-blind randomised controlled trial (FUNCTION), 1162 methotrexate (MTX)-naive patients with early progressive RA were randomly assigned (1:1:1:1) to one of four treatment groups: 4 mg/kg TCZ+MTX, 8 mg/kg TCZ+MTX, 8 mg/kg TCZ+placebo and placebo+MTX (comparator group). The primary outcome was remission according to Disease Activity Score using 28 joints (DAS28-erythrocyte sedimentation rate (ESR) \u3c 2.6) at week 24. Radiographic and physical function outcomes were also evaluated. We report results through week 52. RESULTS: The intent-to-treat population included 1157 patients. Significantly more patients receiving 8 mg/kg TCZ+MTX and 8 mg/kg TCZ+placebo than receiving placebo+MTX achieved DAS28-ESR remission at week 24 (45% and 39% vs 15%; p \u3c 0.0001). The 8 mg/kg TCZ+MTX group also achieved significantly greater improvement in radiographic disease progression and physical function at week 52 than did patients treated with placebo+MTX (mean change from baseline in van der Heijde-modified total Sharp score, 0.08 vs 1.14 (p=0.0001); mean reduction in Health Assessment Disability Index, -0.81 vs -0.64 (p=0.0024)). In addition, the 8 mg/kg TCZ+placebo and 4 mg/kg TCZ+MTX groups demonstrated clinical efficacy that was at least as effective as MTX for these key secondary endpoints. Serious adverse events were similar among treatment groups. Adverse events resulting in premature withdrawal occurred in 20% of patients in the 8 mg/kg TCZ+MTX group. CONCLUSIONS: TCZ is effective in combination with MTX and as monotherapy for the treatment of patients with early RA. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, number NCT01007435