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An experiment was conducted in the Sheep Farming Laboratory at the Federal University of Santa Maria to examine the effect of different levels of replacement of sorghum silage with soybean pre-cleaning by-product (SPCB) on nutrient intake, performance and feeding behavior; as well as to undertake an economic analysis of feeding finishing lambs in a feedlot system. The study involved 32 uncastrated Texel × Ile de France crossbred male lambs weaned at 60 days of age. Treatments consisted of diets in which the roughage source (sorghum silage) was replaced with SPCB at the levels of 0, 33.5, 66.5 or 100% (DM basis). A 45:55 roughage:concentrate ratio was used (DM basis). The diets were composed of a roughage feed based on sorghum silage and/or SPCB, and a concentrate consisting of crushed maize, soybean meal and limestone. In addition, mineral salt was freely available to the animals. The lambs were slaughtered upon reaching the pre-established live weight of 35 kg. Intake on a DM basis showed a quadratic response (P ≤ 0.05) in all forms it was evaluated and expressed, except for the NDF fraction, which increased linearly (P ≤ 0.05) with SPCB inclusion. Average daily weight gain was not significantly influenced (P > 0.05) by the different SPCB levels, averaging 292 g/day. As regards the feeding behavior characteristics evaluated, the number of daily feeding bouts increased linearly (P ≤ 0.01) with the SPCB content of the diets. Total feed cost decreased linearly (P ≤ 0.01), whereas the profit per kilogram of gain and profit obtained per day in the feedlot period increased linearly (P ≤ 0.01) with SPCB inclusion. The use of soybean pre-cleaning by-product as the roughage source in the finishing of feedlot lambs proved to be a viable alternative in both the productive and economic terms.

Zusammenfassung Hintergrund Fur den Erfolg der Bemuhungen zur Eindammung einer Epidemie sind die grosflachige Umsetzung der Masnahmen und ihre Akzeptanz in der Bevolkerung wichtig. Diese Studie untersucht daher den Wissensstand und die Risikowahrnehmung in der fruhen Phase der COVID-19-Pandemie und analysiert, wie empfohlene und vorgeschriebene Masnahmen umgesetzt bzw. eingehalten wurden. Methode Im Marz 2020 wurden die im HeReCa-Online-Panel registrierten Teilnehmer/-innen aus Sachsen-Anhalt, Berlin und Schleswig-Holstein eingeladen, einen Fragebogen mit 65 Fragen zu beantworten. Ergebnisse 1 048 Teilnehmer/-innen beantworteten den Fragebogen. Dies entspricht einer Rucklaufquote von 3,5%. 83% der Befragten fuhlten sich gut oder sehr gut uber COVID-19 und das ursachliche Virus informiert. Die meisten Befragten gaben an, vor allem in Bezug auf die Gesundheit von Familienangehorigen (60%) und der Bevolkerung als Ganzes (45%) Angst vor der Pandemie zu haben und furchteten negative Folgen fur die Wirtschaft (79%). 79% der Befragten haben in Reaktion auf die Pandemie individuelle Masnahmen ergriffen, z. B. das Einhalten von Mindestabstanden zu anderen im offentlichen Raum und Kontaktreduktion. Die staatlich verordneten Masnahmen wurden uberwiegend als sinnvoll und der Situation angemessen wahrgenommen. Schlussfolgerung In der Fruhphase der Pandemie hat sich ein Grosteil der Bevolkerung gezielt mit COVID-19 beschaftigt und setzte individuelle Masnahmen zur Risikoreduktion um. Die Akzeptanz fur die staatlichen Masnahmen zur Eindammung der Pandemie war hoch.

Background: An EU directive holds the EU member states responsible for implementing the provision of health care for asylum seekers. However, current literature indicates insufficient care for asylum seekers in the German health system. This article aims to characterize the situation of the client population on the waiting list of a psychosocial center (PSZ). Methods: We conducted a retrospective observational study based on client files in Halle (Saale), Germany. We included 437 adults who were on the PSZ waiting list between 2016 and 2019. Questionnaires that collected information on the clientele at two different times were analyzed. Results: The average waiting time for psychotherapy was 50 weeks. In total, 85.6% of the 188 respondents reported sleep disorders (n = 161), 65.4% of clients reported pain (n = 123) and 54.8% suicide attempts/suicidal thoughts (n = 54). In the 16-week waiting period in which the clients waited for an initial appointment with a psychologist, the residence status deteriorated in 21.3% (n = 40). Conclusion: Improving asylum seekers’ access to the German health system is urgently needed in order to prevent unnecessary suffering in the future and to comply with EU law.

Molecular-based detection of bacterial pathogens directly from clinical specimens permits rapid initiation of effective antimicrobial treatment and adequate patient management. Broad-range polymerase chain reaction (PCR) amplification of the 16S rRNA gene (16S rDNA qPCR) is used in many diagnostic laboratories as a complement to cultural identification of bacterial pathogens. However, efforts for automation of 16S rDNA PCR workflows are needed in order to reduce turnaround times and to enhance reproducibility and standardization of the technique. In this retrospective method evaluation study, clinical specimens (N = 499) from patients with suspected bacterial infections were used to evaluate 2 diagnostic semiautomated workflows for rapid bacterial pathogen detection. The workflows included automated DNA extraction (QIASymphony), 16S rDNA qPCR, fragment or melting curve analysis, and amplicon sequencing. Our results support the use of the 16S rDNA qPCR and fragment analysis workflow as it enabled rapid and accurate identification of bacterial pathogens in clinical specimens.

Zusammenfassung Hintergrund Für den Erfolg der Bemühungen zur Eindämmung einer Epidemie sind die großflächige Umsetzung der Maßnahmen und ihre Akzeptanz in der Bevölkerung wichtig. Diese Studie untersucht daher den Wissensstand und die Risikowahrnehmung in der frühen Phase der COVID-19-Pandemie und analysiert, wie empfohlene und vorgeschriebene Maßnahmen umgesetzt bzw. eingehalten wurden. Methode Im März 2020 wurden die im HeReCa-Online-Panel registrierten Teilnehmer/-innen aus Sachsen-Anhalt, Berlin und Schleswig-Holstein eingeladen, einen Fragebogen mit 65 Fragen zu beantworten. Ergebnisse 1 048 Teilnehmer/-innen beantworteten den Fragebogen. Dies entspricht einer Rücklaufquote von 3,5%. 83% der Befragten fühlten sich gut oder sehr gut über COVID-19 und das ursächliche Virus informiert. Die meisten Befragten gaben an, vor allem in Bezug auf die Gesundheit von Familienangehörigen (60%) und der Bevölkerung als Ganzes (45%) Angst vor der Pandemie zu haben und fürchteten negative Folgen für die Wirtschaft (79%). 79% der Befragten haben in Reaktion auf die Pandemie individuelle Maßnahmen ergriffen, z. B. das Einhalten von Mindestabständen zu anderen im öffentlichen Raum und Kontaktreduktion. Die staatlich verordneten Maßnahmen wurden überwiegend als sinnvoll und der Situation angemessen wahrgenommen. Schlussfolgerung In der Frühphase der Pandemie hat sich ein Großteil der Bevölkerung gezielt mit COVID-19 beschäftigt und setzte individuelle Maßnahmen zur Risikoreduktion um. Die Akzeptanz für die staatlichen Maßnahmen zur Eindämmung der Pandemie war hoch.

Phototrophic biofilms are ubiquitous in freshwater and marine environments where they are critical for biogeochemical cycling, food webs and in industrial applications. In streams, phototrophic biofilms dominate benthic microbial life and harbour an immense prokaryotic and eukaryotic microbial biodiversity with biotic interactions across domains and trophic levels. Here, we examine how community structure and function of these biofilms respond to varying light availability, as the crucial energy source for phototrophic biofilms. Using metatranscriptomics, we found that under light limitation‐dominant phototrophs, including diatoms and cyanobacteria, displayed a remarkable plasticity in their photosynthetic machinery manifested as higher abundance of messenger RNAs (mRNAs) involved in photosynthesis and chloroplast ribosomal RNA. Under higher light availability, bacterial mRNAs involved in phosphorus metabolism, mainly from Betaproteobacteria and Cyanobacteria, increased, likely compensating for nutrient depletion in thick biofilms with high biomass. Consumers, including diverse ciliates, displayed community shifts indicating preferential grazing on algae instead of bacteria under higher light. For the first time, we show that the functional integrity of stream biofilms under variable light availability is maintained by structure–function adaptations on several trophic levels. Our findings shed new light on complex biofilms, or “microbial jungles”, where in analogy to forests, diverse and multitrophic level communities lend stability to ecosystem functioning. This multitrophic level perspective, coupling metatranscriptomics to process measurements, could advance understanding of microbial‐driven ecosystems beyond biofilms, including planktonic and soil environments., https://doi.org/10.1111/mec.14733

Changes in the riparian vegetation along stream channels, diurnal light availability, and longitudinal fluctuations in the local light regime in streams influence primary production and carbon (C) cycling in benthic stream biofilms. To investigate the influence of light availability on the uptake dynamics of autochthonous and allochthonous dissolved organic carbon (DOC) in benthic biofilms, we experimentally added 13C-labeled allochthonous DOC to biofilms grown under light intensities ranging from 5 to 152 μmol photons m- 2 s- 1. We calculated the net C flux, which showed that benthic biofilms released autochthonous DOC across the entire light gradient. Light availability and diurnal light patterns influenced C uptake by benthic biofilms. More allochthonous DOC was respired under low light availability and at night, whereas under high light availability and during the day mainly autochthonous C was respired by the benthic biofilm community.

The thioredoxin system protects against oxidative stress through the reversible oxidation of the thioredoxin active center dithiol to a disulphide. The genome of Oenococcus oeni PSU-1 contains three thioredoxin genes (trxA1, trxA2, trxA3), one thioredoxin reductase (trxB) and one ferredoxin reductase (fdr) which, until recently, was annotated as a second thioredoxin reductase. For the first time, the entire thioredoxin system in several O. oeni strains isolated from wine has been analysed. Comparisons at the DNA and protein levels have been undertaken between sequences from O. oeni and other genera and species, and the genera Leuconostoc and Lactobacillus were found to present the highest similarities. The gene most frequently absent from a collection of 34 strains and the sequences annotated in the NCBI database was trxA1. Moreover, phylogenetic analysis suggested that this gene was horizontally transferred from Lactobacillus to O. oeni. Strain-dependent expression profiles were determined in rich and in wine-like media. General over-expression was detected after inoculation into wine-like medium, with trxA3 being the most highly expressed gene. The increased transcriptional levels of the thioredoxin genes are indicative of the crucial role of this system in the O. oeni response to wine harsh conditions.

Line probe assays enable rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens. This study shows that defining threshold values in microscopic examination or PCR detection of M. tuberculosis optimizes line probe assay efficiency, thereby avoiding costs and loss of time due to unnecessary molecular testing.

Helicobacter pylori (H. pylori) infection is highly prevalent in the human population and may lead to severe gastrointestinal pathology including gastric and duodenal ulcers, mucosa associated tissue lymphoma and gastric adenocarcinoma. In recent years, an alarming increase in antimicrobial resistance and subsequently failing empiric H. pylori eradication therapies have been noted worldwide, also in many European countries. Therefore, rapid and accurate determination of H. pylori's antibiotic susceptibility prior to the administration of eradication regimens becomes ever more important. Traditionally, detection of H.pylori and its antimicrobial resistance is done by culture and phenotypic drug susceptibility testing that are cumbersome with a long turn-around-time. Recent advances in diagnostics provide new tools, like real-time polymerase chain reaction (PCR) and line probe assays, to diagnose H. pylori infection and antimicrobial resistance to certain antibiotics, directly from clinical specimens. Moreover, high-throughput whole genome sequencing technologies allow the rapid analysis of the pathogen's genome, thereby allowing identification of resistance mutations and associated antibiotic resistance. In the first part of this review, we will give an overview on currently available diagnostic methods for detection of H. pylori and its drug resistance and their implementation in H. pylori management. The second part of the review focusses on the use of next generation sequencing technology in H. pylori research. To this end, we conducted a literature search for original research articles in English using the terms "Helicobacter", "transcriptomic", "transcriptome", "next generation sequencing" and "whole genome sequencing". This review is aimed to bridge the gap between current diagnostic practice (histology, rapid urease test, H. pylori culture, PCR and line probe assays) and new sequencing technologies and their potential implementation in diagnostic laboratory settings in order to complement the currently recommended H. pylori management guidelines and subsequently improve public health.

Helicobacter pylori is a major human pathogen that causes a wide range of gastrointestinal pathology. Progression of H. pylori induced gastritis to more severe disease has been found to highly correlate with the array of virulence factors expressed by the pathogen. The objective of this study was twofold: first, to characterize the genetic diversity of H. pylori strains isolated from 41 non-atrophic gastritis patients in Switzerland, an issue that has not been investigated to date. And second, to assess the prevalence and sequence variation of H. pylori virulence factors (cagA, vacA, iceA and dupA) and genes encoding outer membrane proteins (OMPs, babA, babB, sabA, sabB, hopZ, hopQ and oipA) by whole genome sequencing (WGS) using an Illumina MiSeq platform. WGS identified high genetic diversity in the analyzed H. pylori strains. Most H. pylori isolates were assigned to hpEurope (95.0%, 39/41), and the remaining ones (5.0%, 2/41) to hpEastAsia, subpopulation hspEAsia. Analysis of virulence factors revealed that 43.9% of the strains were cagA-positive, and the vacA s1 allele was detected in 56.0% of the isolates. The presence of cagA was found to be significantly associated (P &lt, 0.001) with the presence of vacA s1, babA2 and hopQ allele 1 as well as expression of oipA. Moreover, we found an association between the grade of gastritis and H. pylori abundance in the gastric mucosa, respectively and the presence of cagA, vacA s1 and hopQ allele 1. Among our 41 gastritis patients, we identified seven patients infected with H. pylori strains that carried a specific combination of virulence factors (i.e cagA, vacA s1 allele and babA2 allele), recently implicated in the development of more severe gastrointestinal pathology, like peptic ulcer disease and even gastric cancer. To this end, WGS can be employed for rapid and detailed characterization of virulence determinants in H. pylori, providing valuable insights into the pathogenic capacity of the bacterium. This could ultimately lead to a higher level of personalized treatment and management of patients suffering from H. pylori associated infections.

Background Interpretative reading of antimicrobial susceptibility test (AST) results allows inferring biochemical resistance mechanisms from resistance phenotypes. For aminoglycosides, however, correlations between resistance pathways inferred on the basis of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints and expert rules versus genotypes are generally poor. This study aimed at developing and validating a decision tree based on resistance phenotypes determined by disc diffusion and based on epidemiological cut-offs (ECOFFs) to infer the corresponding resistance mechanisms in Escherichia coli. Methods Phenotypic antibiotic susceptibility of thirty wild-type and 458 aminoglycoside-resistant E. coli clinical isolates was determined by disc diffusion and the genomes were sequenced. Based on well-defined cut-offs, we developed a phenotype-based algorithm (Aminoglycoside Resistance Mechanism Inference Algorithm - ARMIA) to infer the biochemical mechanisms responsible for the corresponding aminoglycoside resistance phenotypes. The mechanisms inferred from susceptibility to kanamycin, tobramycin and gentamicin were analysed using ARMIA- or EUCAST-based AST interpretation and validated by whole genome sequencing (WGS) of the host bacteria. Findings ARMIA-based inference of resistance mechanisms and WGS data were congruent in 441/458 isolates (96·3%). In contrast, there was a poor correlation between resistance mechanisms inferred using EUCAST CBPs/expert rules and WGS data (418/488, 85·6%). Based on the assumption that resistance mechanisms can result in therapeutic failure, EUCAST produced 63 (12·9%) very major errors (vME), compared to only 2 (0·4%) vME with ARMIA. When used for detection and identification of resistance mechanisms, ARMIA resolved >95% vMEs generated by EUCAST-based AST interpretation. Interpretation This study demonstrates that ECOFF-based analysis of AST data of only four aminoglycosides provides accurate information on the resistance mechanisms in E. coli. Since aminoglycoside resistance mechanisms, despite having in certain cases a minimal effect on the minimal inhibitory concentration, may compromise the bactericidal activity of aminoglycosides, prompt detection of resistance mechanisms is crucial for therapy. Using ARMIA as an interpretative rule set for editing AST results allows for better predictions of in vivo activity of this drug class., eBioMedicine, 46, ISSN:2352-3964

Objectives Rapid detection of macrolide resistance–associated mutations in Mycoplasma pneumoniae is crucial for effective antimicrobial treatment. We evaluated the Lightmix Mycoplasma macrolide assay for the detection of point mutations at nucleotide positions 2063 and 2064 in the 23S ribosomal RNA (rRNA) gene of M. pneumoniae that confer macrolide resistance. Methods Samples from 3438 patients with a respiratory tract infection were analysed by M. pneumoniae real-time PCR, and 208 (6%) of them were tested positive. In this retrospective study, 163 M. pneumoniae real-time PCR–positive samples were analysed by the Lightmix assay, and results were compared to targeted 23S rRNA sequencing. Results Macrolide-resistant M. pneumoniae were found in 15 (9%) of 163 retrospectively analysed samples. The Lightmix assay showed a sensitivity of 100% (95% confidence interval, 78.2–100) and a specificity of 100% (95% confidence interval, 97.5–100) as the detected M. pneumoniae genotype (148 wild type and 15 non–wild type) was confirmed by 23S rRNA sequencing in all samples. Conclusions The Lightmix assay is an easy-to-use and accurate molecular test that allows rapid determination of macrolide resistance in M. pneumoniae.

Objectives In this study, the commercially available AID AmpC line probe assay (LPA) was evaluated for detection of plasmid-mediatedblaAmpC β-lactamase genes in Enterobacterales as well as chromosomal mutations in the blaAmpC promoter/attenuator regions in Escherichia coli. Methods Accuracy of the AID AmpC probes was assessed using Enterobacterales clinical isolates harbouring diverse plasmid-mediated AmpC enzymes (ACC, ACT, DHA, FOX, CMY and MOX) and E. coli clinical isolates with mutations in the chromosomal blaAmpC promoter/attenuator regions. The diagnostic performance of the AID AmpC LPA for blaAmpC detection directly from clinical specimens was determined using 99 clinical urine specimens with bacterial cell counts >105 CFU/mL and the results were compared with culture-based phenotypic drug susceptibility testing (DST). Results Detection of blaAmpC genes in Enterobacterales clinical isolates showed 100% congruence with phenotypic DST results. The AID AmpC LPA showed 100% specificity [95% confidence interval (CI) 96–100%] and 100% sensitivity (95% CI 75–100%) for detection of plasmid-meditated blaAmpC and E. coli genomic blaAmpC promoter/attenuator mutations directly from clinical urine specimens. The AID AmpC LPA detected three AmpC-producers in urine specimens with bacterial cell counts >105 CFU/mL that were missed by culture-based phenotypic DST, thereby displaying higher diagnostic sensitivity. Conclusion The AID AmpC LPA is an accurate, sensitive and easy-to-use test that can be readily implemented in any diagnostic laboratory for molecular detection of blaAmpC genes in Enterobacterales.

Helicobacter pylori is a major human pathogen. Diagnosis of H. pylori infection and determination of its antibiotic susceptibility still mainly rely on culture and phenotypic drug susceptibility testing (DST) that is time-consuming and laborious. Whole genome sequencing (WGS) has recently emerged in medical microbiology as a diagnostic tool for reliable drug resistance prediction in bacterial pathogens. The aim of this study was to compare phenotypic DST results with the predictions based on the presence of genetic determinants identified in the H. pylori genome using WGS. Phenotypic resistance to clarithromycin, metronidazole, tetracycline, levofloxacin, and rifampicin was determined in 140 clinical H. pylori isolates by E-Test&reg, and the occurrence of certain single nucleotide polymorphisms (SNPs) in target genes was determined by WGS. Overall, there was a high congruence of &gt, 99% between phenotypic DST results for clarithromycin, levofloxacin, and rifampicin and SNPs identified in the 23S rRNA, gyrA, and rpoB gene. However, it was not possible to infer a resistance phenotype for metronidazole based on the occurrence of distinct SNPs in frxA and rdxA. All 140 H. pylori isolates analysed in this study were susceptible to tetracycline, which was in accordance with the absence of double or triple nucleotide substitutions in the 16S rRNA gene.

Summary Menstrual toxic shock syndrome (mTSS) is a life-threatening disease caused by superantigen-producing Staphylococcus aureus. Incidence ranges from 0·03 to 0·50 cases per 100 000 people, with overall mortality around 8%. In this Grand Round, we present the case of a previously healthy 23-year-old menstruating woman who was diagnosed with mTSS after she presented at our hospital with a septic condition for the second time. The diagnosis was confirmed by fulfilment of the clinical criteria outlined by the US Centers for Disease Control and Prevention (CDC; fever, rash, desquamation, hypotension, and multi-system involvement) as well as a nasal swab positive for the S aureus strain and presence of the gene encoding for toxic shock syndrome toxin 1 (TSST-1). In the early 1980s, when mTSS was first described, use of tampons was considered the main risk factor. Today, the complex interplay between pathogenic factors of S aureus, immunological mechanisms of the host, and changes in the vaginal ecosystem during menstruation has broadened current understanding of the disease, and the CDC criteria have appreciable limitations in everyday clinical practice.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Acute bacterial meningitis is a medical emergency, and delays in initiating effective antimicrobial therapy result in increased morbidity and mortality. Culture-based methods, thus far considered the “gold standard” for identifying bacterial microorganisms, require 24 to 48 h to provide a diagnosis. In addition, antimicrobial therapy is often started prior to clinical sample collection, thereby decreasing the probability of confirming the bacterial pathogen by culture-based methods. To enable a fast and accurate detection of the most important bacterial pathogens causing meningitis, namely, Streptococcus pneumoniae , Haemophilus influenzae , Neisseria meningitidis , Streptococcus agalactiae , and Listeria monocytogenes , we evaluated a commercially available multiplex LightMix real-time PCR (RT-PCR) in 220 cerebrospinal fluid (CSF) specimens. The majority of CSF samples were collected by lumbar puncture, but we also included some CSF samples from patients with symptoms of meningitis from the neurology department that were recovered from shunts. CSF samples were analyzed by multiplex RT-PCR enabling a first diagnosis within a few hours after sample arrival at our institute. In contrast, bacterial identification took between 24 and 48 h by culture. Overall, a high agreement of bacterial identification between culture and multiplex RT-PCR was observed (99%). Moreover, multiplex RT-PCR enabled the detection of pathogens, S. pneumoniae ( n = 2), S. agalactiae ( n = 1), and N. meningitidis ( n = 1), in four culture-negative samples. As a complement to classical bacteriological CSF culture, the LightMix RT-PCR assay proved to be valuable by improving the rapidity and accuracy of the diagnosis of bacterial meningitis.

Headwater streams are tightly connected with the terrestrial milieu from which they receive deliveries of organic matter, often through the hyporheic zone, the transition between groundwater and streamwater. Dissolved organic matter (DOM) from terrestrial sources (that is, allochthonous) enters the hyporheic zone, where it may mix with DOM from in situ production (that is, autochthonous) and where most of the microbial activity takes place. Allochthonous DOM is typically considered resistant to microbial metabolism compared to autochthonous DOM. The composition and functioning of microbial biofilm communities in the hyporheic zone may therefore be controlled by the relative availability of allochthonous and autochthonous DOM, which can have implications for organic matter processing in stream ecosystems. Experimenting with hyporheic biofilms exposed to model allochthonous and autochthonous DOM and using 454 pyrosequencing of the 16S rRNA (targeting the “active” community composition) and of the 16S rRNA gene (targeting the “bulk” community composition), we found that allochthonous DOM may drive shifts in community composition whereas autochthonous DOM seems to affect community composition only transiently. Our results suggest that priority effects based on resource-driven stochasticity shape the community composition in the hyporheic zone. Furthermore, measurements of extracellular enzymatic activities suggest that the additions of allochthonous and autochthonous DOM had no clear effect on the function of the hyporheic biofilms, indicative of functional redundancy. Our findings unravel possible microbial mechanisms that underlie the buffering capacity of the hyporheic zone and that may confer stability to stream ecosystems.

Summary Changes in riparian vegetation or water turbidity and browning in streams alter the local light regime with potential implications for stream biofilms and ecosystem functioning. We experimented with biofilms in microcosms grown under a gradient of light intensities (range: 5–152 μmole photons s−1 m−2) and combined 454‐pyrosequencing and enzymatic activity assays to evaluate the effects of light on biofilm structure and function. We observed a shift in bacterial community composition along the light gradient, whereas there was no apparent change in alpha diversity. Multifunctionality, based on extracellular enzymes, was highest under high light conditions and decoupled from bacterial diversity. Phenol oxidase activity, involved in the degradation of polyphenolic compounds, was twice as high on average under the lowest compared with the highest light condition. This suggests a shift in reliance of microbial heterotrophs on biofilm phototroph‐derived organic matter under high light availability to more complex organic matter under low light. Furthermore, extracellular enzyme activities correlated with nutrient cycling and community respiration, supporting the link between biofilm structure–function and biogeochemical fluxes in streams. Our findings demonstrate that changes in light availability are likely to have significant impacts on biofilm structure and function, potentially affecting stream ecosystem processes.

The priming effect refers to quantitative changes in microbial decomposition of recalcitrant organic matter upon addition of labile organic matter and is a phenomenon that mainly has been reported and debated in soil science. Recently, priming effects have been indicated in aquatic ecosystems and have received attention due to the potential significance for ecosystem carbon budgets. Headwater stream biofilms, which are important degraders of both allochthonous, presumably recalcitrant, organic matter and labile autochthonous organic matter, may be sites where priming effects are important in aquatic environments. We have experimentally tested for priming effects in stream biofilms within microcosms mimicking the stream hyporheic zone. A (13)C labeled model allochthonous carbon source was used in combination with different carbon sources simulating autochthonous inputs. We did not detect changes in respiration, removal or incorporation of allochthonous organic matter in response to autochthonous treatments, thus not supporting the occurrence of priming effects under the experimental conditions. This study is the first to address priming effects in the hyporheic zone, and one of very few studies quantitatively assessing aquatic priming effects. The results contrast with existing studies, which highlights the need for quantitative approaches to determine the importance of priming effects in aquatic environments.

BACKGROUND Resistance to clarithromycin in Helicobacter pylori (H pylori) is mediated by mutations in the domain V of the 23S rRNA gene (A2142G, A2143G, A2142C). Other polymorphisms in the 23S rRNA gene have been reported to cause low-level clarithromycin resistance but their importance is still under debate. In this study, we aimed to develop and evaluate the CRHP Finder webtool for detection of the most common mutations mediating clarithromycin resistance from whole-genome sequencing (WGS) data. Moreover, we included an analysis of 23 H pylori strains from Danish patients between January 2017 and September 2019 in Copenhagen, Denmark. MATERIALS AND METHODS The CRHP Finder detects the fraction of each of the four nucleotides in nucleotide positions 2142, 2143, 2182, 2244 and 2712 of the 23S rRNA gene in H pylori (E coli numbering) by aligning raw sequencing reads (fastq format) with k-mer alignment (KMA). The nucleotide distribution in each position is compared to previously described point mutations mediating clarithromycin resistance in H pylori, and a genotypic prediction of the clarithromycin resistance phenotype is presented as output. For validation of the CRHP webtool, 137 fastq paired-end sequencing datasets originating from a well-characterized strain collection of H pylori were analyzed. RESULTS The CRHP Finder correctly identified all resistance mutations reported in the sequencing data of 137 H pylori strains. In the 23 Danish H pylori strains, CRHP Finder detected A2143G (13%) in all resistant strains, and T2182C (13%) and C2244T (4,3%) nucleotide exchanges in only susceptible strains. CONCLUSION In this study, we present the validation of the first webtool for H pylori resistance prediction based on the detection of 23S rRNA mutations (A2142C, A2142G, A2143G, T2182C, C2244T, T2712C) from WGS data of H pylori.

Background Mycoplasma sp. are well recognized as etiological agents of respiratory and sexually transmitted disease. Mycoplasma penetrans, a species of Mycoplasma sp., has been frequently detected in HIV-positive patients and associated with the progression of HIV-associated disease. To date, there is only a single case report describing M. penetrans as the causative agent of a severe respiratory tract infection in a HIV-negative patient. Case presentation In this report, we describe the case of M. penetrans bacteremia in a HIV-negative, 38-year-old, female, immunocompromised, solid organ transplant patient (combined kidney and pancreas transplantation in 2016), who was admitted to our hospital with anemic uterine bleeding and fever of 38.3 °C. Several hours before her admission at our university hospital, a latex bladder catheter was inserted into her uterus and she complained about fatigue, dizziness and ongoing vaginal bleeding. Laboratory examination showed severe anemia, but microbiological examination was inconspicuous (culture negative vaginal and cervical smears, negative urine culture). Bacterial blood cultures showed a growth signal after 4 h, but microscopic examination with Gram staining and subcultures on different agar media did not identify bacterial pathogens. To identify the bacterial cause of malignancy in the patient, metagenomic sequencing of the blood culture was performed that identified M. penetrans. Conclusion Metagenomic sequencing identified M. penetrans in an immunosuppressed patient with culture-negative bacteremia. Clinicians should be aware of the opportunistic potential of M. penetrans that may cause severe infections in certain vulnerable patient populations and the limitations of culture and Gram staining for confirming the presence of fastidious bacterial pathogens like Mycoplasma spp.