Identification of influenza urban transmission patterns by geographical, epidemiological and whole genome sequencing data: protocol for an observational study.

INTRODUCTION: Urban transmission patterns of influenza viruses are complex and poorly understood, and multiple factors may play a critical role in modifying transmission. Whole genome sequencing (WGS) allows the description of patient-to-patient transmissions at highest resolution. The aim of this study is to explore urban transmission patterns of influenza viruses in high detail by combining geographical, epidemiological and immunological data with WGS data. METHODS AND ANALYSIS: The study is performed at the University Hospital Basel, University Children's Hospital Basel and a network of paediatricians and family doctors in the Canton of Basel-City, Switzerland. The retrospective study part includes an analysis of PCR-confirmed influenza cases from 2013 to 2018. The prospective study parts include (1) a household survey regarding influenza-like illness (ILI) and vaccination against influenza during the 2015/2016 season; (2) an analysis of influenza viruses collected during the 2016/2017 season using WGS-viral genomic sequences are compared with determine genetic relatedness and transmissions; and (3) measurement of influenza-specific antibody titres against all vaccinated and circulated strains during the 2016/2017 season from healthy individuals, allowing to monitor herd immunity across urban quarters. Survey data and PCR-confirmed cases are linked to data from the Statistics Office of the Canton Basel-City and visualised using geo-information system mapping. WGS data will be analysed in the context of patient epidemiological data using phylodynamic analyses, and the obtained herd immunity for each quarter. Profound knowledge on the key geographical, epidemiological and immunological factors influencing urban influenza transmission will help to develop effective counter measurements. ETHICS AND DISSEMINATION: The study is registered and approved by the regional ethics committee as an observational study (EKNZ project ID 2015-363 and 2016-01735). It is planned to present the results at conferences and publish the data in scientific journals. TRIAL REGISTRATION NUMBER: NCT03010007.

Phenotypic and Genomic Analyses of Burkholderia stabilis Clinical Contamination, Switzerland.

A recent hospital outbreak related to premoistened gloves used to wash patients exposed the difficulties of defining Burkholderia species in clinical settings. The outbreak strain displayed key B. stabilis phenotypes, including the inability to grow at 42°C; we used whole-genome sequencing to confirm the pathogen was B. stabilis. The outbreak strain genome comprises 3 chromosomes and a plasmid, sharing an average nucleotide identity of 98.4% with B. stabilis ATCC27515 BAA-67, but with 13% novel coding sequences. The genome lacks identifiable virulence factors and has no apparent increase in encoded antimicrobial drug resistance, few insertion sequences, and few pseudogenes, suggesting this outbreak was an opportunistic infection by an environmental strain not adapted to human pathogenicity. The diversity among outbreak isolates (22 from patients and 16 from washing gloves) is only 6 single-nucleotide polymorphisms, although the genome remains plastic, with large elements stochastically lost from outbreak isolates.

Quantitative contribution of efflux to multi-drug resistance of clinical Escherichia coli and Pseudomonas aeruginosa strains.

BACKGROUND: Efflux pumps mediate antimicrobial resistance in several WHO critical priority bacterial pathogens. However, most available data come from laboratory strains. The quantitative relevance of efflux in more relevant clinical isolates remains largely unknown. METHODS: We developed a versatile method for genetic engineering in multi-drug resistant (MDR) bacteria, and used this method to delete tolC and specific antibiotic-resistance genes in 18 representative MDR clinical E. coli isolates. We determined efflux activity and minimal inhibitory concentrations for a diverse set of clinically relevant antibiotics in these mutants. We also deleted oprM in MDR P. aeruginosa strains and determined the impact on antibiotic susceptibility. FINDINGS: tolC deletion abolished detectable efflux activity in 15 out of 18 tested E. coli strains, and modulated antibiotic susceptibility in many strains. However, all mutant strains retained MDR status, primarily because of other, antibiotic-specific resistance genes. Deletion of oprM altered antibiotic susceptibility in a fraction of clinical P. aeruginosa isolates. INTERPRETATION: Efflux modulates antibiotic resistance in clinical MDR isolates of E. coli and P. aeruginosa. However, when other antimicrobial-resistance mechanisms are present, inhibition of MDR efflux pumps alone is often not sufficient to restore full susceptibility even for antibiotics with a dramatic impact of efflux in laboratory strains. We propose that development of novel antibiotics should include target validation in clinical MDR isolates. FUND: Innovative Medicines Initiative of European Union and EFPIA, Schweizerischer Nationalfonds, Swiss National Research Program 72, EU Marie Sklodowska-Curie program. The funders played no role in design, data collection, data analysis, interpretation, writing of the report, and in the decision to submit the paper for publication.

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods.

OBJECTIVES: Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations. METHODS: We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq. RESULTS: We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient. CONCLUSION: Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

Burkholderia stabilis outbreak associated with contaminated commercially-available washing gloves, Switzerland, May 2015 to August 2016.

We describe an outbreak of Burkholderia stabilis associated with contaminated washing gloves, a commercially available Class I medical device. Triggered by an increase in Burkholderia cepacia complex (BCC) bacteremias and the detection of BCC in unopened packages of washing gloves, an ad hoc national outbreak committee comprising representatives of a public health organisation, a regulatory agency, and an expert association convened and commissioned an outbreak investigation. The investigation included retrospective case finding across Switzerland and whole genome sequencing (WGS) of isolates from cases and gloves. The investigation revealed that BCC were detected in clinical samples of 46 cases aged 17 to 91 years (33% females) from nine institutions between May 2015 and August 2016. Twenty-two isolates from case patients and 16 from washing gloves underwent WGS. All available outbreak isolates clustered within a span of < 19 differing alleles, while 13 unrelated clinical isolates differed by > 1,500 alleles. This BCC outbreak was rapidly identified, communicated, investigated and halted by an ad hoc collaboration of multiple stakeholders. WGS served as useful tool for confirming the source of the outbreak. This outbreak also highlights current regulatory limitations regarding Class I medical devices and the usefulness of a nationally coordinated outbreak response.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.

Correction: A Cross-Sectional Study of Colonization Rates with Methicillin-Resistant Staphylococcus aureus (MRSA) and Extended-Spectrum Beta-Lactamase (ESBL) and Carbapenemase-Producing Enterobacteriaceae in Four Swiss Refugee Centres.

[This corrects the article DOI: 10.1371/journal.pone.0170251.].

Corynebacterium diphtheriae in a free-roaming red fox: case report and historical review on diphtheria in animals.

OBJECTIVES: Corynebacterium diphtheriae, the classical causative agent of diphtheria, is considered to be nearly restricted to humans. Here we report the first finding of a non-toxigenic C. diphtheriae biovar belfanti strain in a free-roaming wild animal. METHODS: The strain obtained from the subcutis and mammary gland of a dead red fox (Vulpes vulpes) was characterized by biochemical and molecular methods including MALDI-TOF and Multi Locus Sequence Typing. Since C. diphtheriae infections of animals, usually with close contact to humans, are reported only very rarely, an intense review comprising also scientific literature from the beginning of the 20th century was performed. RESULTS: Besides the present case, only 11 previously reported C. diphtheriae animal infections could be verified using current scientific criteria. CONCLUSIONS: Our report is the first on the isolation of C. diphtheriae from a wildlife animal without any previous human contact. In contrast, the very few unambiguous publications on C. diphtheriae in animals referred to livestock or pet animals with close human contact. C. diphtheriae carriage in animals has to be considered as an exceptionally rare event.

The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription.

RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters.

Co-transcriptional mRNP formation is coordinated within a molecular mRNP packaging station in S. cerevisiae.

In eukaryotes, the messenger RNA (mRNA), the blueprint of a protein-coding gene, is processed and packaged into a messenger ribonucleoprotein particle (mRNP) by mRNA-binding proteins in the nucleus. The steps of mRNP formation - transcription, processing, packaging, and the orchestrated release of the export-competent mRNP from the site of transcription for nuclear mRNA export - are tightly coupled to ensure a highly efficient and regulated process. The importance of highly accurate nuclear mRNP formation is illustrated by the fact that mutations in components of this pathway lead to cellular inviability or to severe diseases in metazoans. We hypothesize that efficient mRNP formation is realized by a molecular mRNP packaging station, which is built by several recruitment platforms and coordinates the individual steps of mRNP formation.

Zoonotic transmission of toxigenic Corynebacterium ulcerans strain, Germany, 2012.

Severe necrotizing fasciitis was diagnosed in a 53-year-old man in Germany in 2012. Toxigenic Corynebacterium ulcerans was grown from a wound swab sample. One of the patient's 2 dogs was found to harbor a toxigenic C. ulcerans strain. Results of next generation sequencing of both isolates supported recent zoonotic transmission of this bacterial pathogen.

Multilocus sequence typing of Corynebacterium ulcerans provides evidence for zoonotic transmission and for increased prevalence of certain sequence types among toxigenic strains.

Human-to-human-transmitted Corynebacterium diphtheriae was historically the main pathogen causing diphtheria and has therefore been studied extensively in the past. More recently, diphtheria caused by toxigenic Corynebacterium ulcerans is an emerging disease in several industrial countries, including the United Kingdom, the United States, France, and Germany. However, toxigenic C. ulcerans has so far been almost neglected in the development of epidemiologic tools. One of the most important tools in modern epidemiology to understand transmission pathways is sequence typing of pathogens. Here, we provide a protocol for multilocus sequence typing (MLST) to type C. ulcerans strains rapidly and relatively cost-effectively. Applying MLST to C. ulcerans for the first time, we show that related sequence types (STs) might be associated with the presence of the diphtheria toxin gene, which encodes diphtheria toxin (DT), the most important diphtheria-causing virulence factor. Interestingly, we found only two very closely related STs in the isolates derived from six dogs. Additionally, our data show that all STs derived from animals which were at least twice present in our analysis were found in humans as well. This finding is congruent with zoonotic transmission of C. ulcerans.

Next generation sequencing analysis of nine Corynebacterium ulcerans isolates reveals zoonotic transmission and a novel putative diphtheria toxin-encoding pathogenicity island.

BACKGROUND: Toxigenic Corynebacterium ulcerans can cause a diphtheria-like illness in humans and have been found in domestic animals, which were suspected to serve as reservoirs for a zoonotic transmission. Additionally, toxigenic C. ulcerans were reported to take over the leading role in causing diphtheria in the last years in many industrialized countries. METHODS: To gain deeper insights into the tox gene locus and to understand the transmission pathway in detail, we analyzed nine isolates derived from human patients and their domestic animals applying next generation sequencing and comparative genomics. RESULTS: We provide molecular evidence for zoonotic transmission of C. ulcerans in four cases and demonstrate the superior resolution of next generation sequencing compared to multi-locus sequence typing for epidemiologic research. Additionally, we provide evidence that the virulence of C. ulcerans can change rapidly by acquisition of novel virulence genes. This mechanism is exemplified by an isolate which acquired a prophage not present in the corresponding isolate from the domestic animal. This prophage contains a putative novel virulence factor, which shares high identity with the RhuM virulence factor from Salmonella enterica but which is unknown in Corynebacteria so far. Furthermore, we identified a putative pathogenicity island for C. ulcerans bearing a diphtheria toxin gene. CONCLUSION: The novel putative diphtheria toxin pathogenicity island could provide a new and alternative pathway for Corynebacteria to acquire a functional diphtheria toxin-encoding gene by horizontal gene transfer, distinct from the previously well characterized phage infection model. The novel transmission pathway might explain the unexpectedly high number of toxigenic C. ulcerans.

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

Prp19C and TREX: interacting to promote transcription elongationand mRNA export.

During transcription of protein coding genes by RNA Polymerase II the mRNA is processed and packaged into an mRNP. Among the proteins binding cotranscriptionally to the mRNP are mRNA export factors. One of the protein complexes thus coupling transcription to mRNA export is the TREX complex. However, despite the fact that TREX was identified and characterized about a decade ago, it had remained enigmatic how TREX is recruited to genes. The conserved Prp19 complex (Prp19C) has long been known for its function in splicing. We recently identified Prp19C to be essential for a second step in gene expression namely TREX occupancy at transcribed genes, answering this long-standing question but also raising new ones.

La-motif-dependent mRNA association with Slf1 promotes copper detoxification in yeast.

The La-motif (LAM) is an ancient and ubiquitous RNA-binding domain defining a superfamily of proteins, which comprises the genuine La proteins and La-related proteins (LARPs). In contrast to La, which binds and stabilizes pre-tRNAs and other RNA polymerase III transcripts, data on function and RNA targets of the LARPs have remained scarce. We have undertaken a global approach to elucidate the previously suggested role of the yeast LARP Slf1p in copper homeostasis. By applying RNA-binding protein immunopurification-microarray (RIP-Chip) analysis, we show that Slf1p and its paralog Sro9p copurify with overlapping sets of hundreds of functionally related mRNAs, including many transcripts coding for ribosomal proteins and histones. Interestingly, among these potential RNA targets were also mRNAs coding for proteins critical for protection of cells against elevated copper concentrations. Mutations introduced in the conserved aromatic patch of the LAM in Slf1p drastically impaired both association with its targets and Slf1-mediated protection of cells against toxic copper concentrations. Furthermore, we show that Slf1p stabilizes copper-related mRNA targets in a LAM-dependent manner. These results provide the first evidence for post-transcriptional regulation of factors/pathways implicated in copper homeostasis by a cytoplasmic RBP.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.