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ABSTRACTDuring the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent—something that is unknown at the time of sequencing—and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

Abstract Streptococcus pneumoniae colonizes the human nasopharynx, a multi-species microbial niche. Pneumococcal Ami-AliA/AliB oligopeptide permease is an ABC transporter involved in environmental sensing with peptides AKTIKITQTR, FNEMQPIVDRQ, and AIQSEKARKHN identified as ligands of its substrate binding proteins AmiA, AliA, and AliB, respectively. These sequences match ribosomal proteins of multiple bacterial species, including Klebsiella pneumoniae. By mass spectrometry, we identified such peptides in the Klebsiella pneumoniae secretome. AmiA and AliA peptide ligands suppressed pneumococcal growth, but the effect was dependent on peptide length. Growth was suppressed for diverse pneumococci, including antibiotic-resistant strains, but not other bacterial species tested, with the exception of Streptococcus pseudopneumoniae, whose growth was suppressed by the AmiA peptide ligand. By multiple sequence alignments and protein and peptide binding site predictions, for AmiA we have identified the location of an amino acid in the putative binding site whose mutation appears to result in loss of response to the peptide. Our results indicate that pneumococci sense the presence of Klebsiella pneumoniae peptides in the environment.

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach called NASCarD (Nanopore Adaptive Sampling with Carrier DNA), which allows a low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using COVID-19 positive samples available during the omicron wave, we demonstrate how the method may lead to >99% genome completeness of the SARS-CoV-2 genome sequences within 7 h of sequencing at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples.

Poliomyelitis-like acute flaccid myelitis associated with enterovirus D68 (EV-D68) has emerged globally during the past decade. Here we describe the first documented case reported from Switzerland, and a second, suspected case occurring in temporal association. AFM occurs primarily in children, is usually heralded by a febrile, respiratory prodrome followed by acute-onset, usually asymmetrical, limb weakness with some predilection for the upper extremities, and respiratory muscle compromise in one third of reported cases. There is no specific therapy and the majority of cases result in permanent neurological sequelae. A comprehensive diagnostic workup and timely reporting to the health authorities are essential. Surveillance of respiratory and stool samples for EV-D68 and other neurotropic enteroviruses is in place in several European countries and warrants consideration in Switzerland. This could entail the extension of the poliomyelitis surveillance program of the Federal Office of Public Health by monitoring and enteroviral typing of respiratory samples from patients with acute flaccid paralysis.

Abstract Human adenoviruses (HAdVs) are highly contagious pathogens of clinical importance, especially among the pediatric population. Studies on comparative viral genomic analysis of cases associated with severe and mild infections due to HAdV are limited. Using whole-genome sequencing (WGS), we investigated whether there were any differences between circulating HAdV strains associated with severe infections (meningitis, sepsis, convulsion, sudden infant death syndrome, death, and hospitalization) and mild clinical presentations in pediatric patients hospitalized between the years 1998 and 2017 in a tertiary care hospital group in Bern, Switzerland covering a population base of approx. 2 million inhabitants. The HAdV species implicated in causing severe infections in this study included HAdV species C genotypes (HAdV1, HAdV2, and HAdV5). Clustering of the HAdV whole-genome sequences of the severe and mild cases did not show any differences except for one sample (isolated from a patient presenting with sepsis, meningitis, and hospitalization) that formed its own cluster with HAdV species C genotypes. This isolate showed intertypic recombination events involving four genotypes, had the highest homology to HAdV89 at complete genome level, but possessed the fiber gene of HAdV1, thereby representing a novel genotype of HAdV species C. The incidence of potential recombination events was higher in severe cases than in mild cases. Our findings confirm that recombination among HAdVs is important for molecular evolution and emergence of new strains. Therefore, further research on HAdVs, particularly among susceptible groups, is needed and continuous surveillance is required for public health preparedness including outbreak investigations.

ABSTRACT: Objectives: Patients hospitalised abroad can become colonised with multidrug-resistant bacteria and import them to their home countries. In this study, we characterised an OXA-484 carbapenemase-producing Escherichia coli strain from a Swiss patient infected by SARS-CoV-2 and repatriated from India. Methods: At admission to Switzerland (April 2021), the patient undertook a nasopharyngeal swab to search for SARS-CoV-2 and a rectal swab to detect multidrug-resistant bacteria. Both SARS-CoV-2 and E. coli isolates were whole-genome sequenced and analysed for phylogenetic relatedness. Results: The patient was infected with the SARS-CoV-2 B.1.617.2 lineage (VOC Delta), a lineage that began to be reported across Switzerland at that time. He was also colonised with a sequence type 410 (ST410) E. coli strain (L3452210II) producing OXA-484, a single amino acid variant of OXA-181. The blaOXA-484 gene was carried by a 51.5 kb IncX3 plasmid identical to those described in blaOXA-181-harbouring ST410 E. coli strains. Core genome analysis showed that L3452210II was identical (ΔSNV ≤23) to two ST410 OXA-484 producers recently reported in Qatar and Germany, but differed from other ST410 OXA-181 producers reported worldwide. Conclusion: The patient was infected by an emerging SARS-CoV-2 variant and also imported an E. coli producing OXA-484, an OXA-48-like carbapenemase not yet reported in Switzerland. The genetic background of L3452210II indicated that blaOXA-484 shared the same plasmid as blaOXA-181, but its bacterial host differed from most of the pandemic OXA-181-producing ST410 strains reported previously. This case description underlines that the COVID-19 crisis can contribute to the worldwide spread of emerging carbapenemase producers.

Abstract Streptococcus pneumoniae causes life-threatening meningitis. Its capsular polysaccharide determines the serotype and influences disease severity but the mechanism is largely unknown. Due to evidence of elevated cytokines levels in the meningeal inflammatory response, we measured 41 cytokines/chemokines and growth factors in cerebrospinal fluid (CSF) samples from 57 South African meningitis patients (collected in the period 2018–2019), with confirmed S. pneumoniae serotypes, using a multiplexed bead-based immunoassay. Based on multivariable Bayesian regression, using serotype 10A as a reference and after adjusting for HIV and age, we found IL-6 concentrations significantly lower in patients infected with serotypes 6D (undetectable) and 23A (1601 pg/ml), IL-8 concentrations significantly higher in those infected with 22A (40,459 pg/ml), 7F (32,400 pg/ml) and 15B/C (6845 pg/ml), and TNFα concentration significantly higher in those infected with serotype 18A (33,097 pg/ml). Although a relatively small number of clinical samples were available for this study and 28% of samples could not be assigned to a definitive serotype, our data suggests 15B/C worthy of monitoring during surveillance as it is associated with in-hospital case fatality and not included in the 13-valent polysaccharide conjugate vaccine, PCV13. Our data provides average CSF concentrations of a range of cytokines and growth factors for 18 different serotypes (14, 19F, 3, 6A, 7F, 19A, 8, 9N, 10A, 12F, 15B/C, 22F, 16F, 23A, 31, 18A, 6D, 22A) to serve as a basis for future studies investigating host–pathogen interaction during pneumococcal meningitis. We note that differences in induction of IL-8 between serotypes may be particularly worthy of future study.

The SARS-CoV-2 Delta variant, corresponding to the Pangolin lineage B.1.617.2, was first detected in India in July 2020 and rapidly became dominant worldwide. The ARTIC v3 protocol for SARS-CoV-2 whole-genome sequencing, which relies on a large number of PCR primers, was among the first available early in the pandemic, but may be prone to coverage dropouts that result in incomplete genome sequences. A new set of primers (v4) was designed to circumvent this issue in June 2021. In this study, we investigated whether the sequencing community adopted the new sets of primers, especially in the context of the spread of the Delta lineage, in July 2021. Because information about protocols from individual laboratories is generally difficult to obtain, the aims of the study were to identify whether large under-sequenced regions were present in deposited Delta variant genome sequences (from April to August 2021), to investigate the extent of the coverage dropout among all the currently available Delta sequences in six countries, and to propose simple PCR primer modifications to sequence the missing region, especially for the first circulating Delta variants observed in 2021 in Switzerland. Candidate primers were tested on few clinical samples, highlighting the need to further pursue primer optimization and validation on a larger and diverse set of samples.

Whole genome sequencing (WGS) enables high resolution typing of bacteria up to the single nucleotide polymorphism (SNP) level. WGS is used in clinical microbiology laboratories for infection control, molecular surveillance and outbreak analyses. Given the large palette of WGS reagents and bioinformatics tools, the Swiss clinical bacteriology community decided to conduct a ring trial (RT) to foster harmonization of NGS-based bacterial typing. The RT aimed at assessing methicillin-susceptible Staphylococcus aureus strain relatedness from WGS and epidemiological data. The RT was designed to disentangle the variability arising from differences in sample preparation, SNP calling and phylogenetic methods. Nine laboratories participated. The resulting phylogenetic tree and cluster identification were highly reproducible across the laboratories. Cluster interpretation was, however, more laboratory dependent, suggesting that an increased sharing of expertise across laboratories would contribute to further harmonization of practices. More detailed bioinformatic analyses unveiled that while similar clusters were found across laboratories, these were actually based on different sets of SNPs, differentially retained after sample preparation and SNP calling procedures. Despite this, the observed number of SNP differences between pairs of strains, an important criterion to determine strain relatedness given epidemiological information, was similar across pipelines for closely related strains when restricting SNP calls to a common core genome defined by S. aureus cgMLST schema. The lessons learned from this pilot study will serve the implementation of larger-scale RT, as a mean to have regular external quality assessments for laboratories performing WGS analyses in a clinical setting.

Polymicrobial infections of the respiratory tract due to antibiotic resistant bacteria are a great concern in patients with cystic fibrosis (CF). We therefore aimed at establishing a functional metagenomic method to analyze the nasal resistome in infants with CF within the first year of life. We included samples from patients before antibiotic treatment, which allowed obtaining information regarding natural status of the resistome. In total, we analyzed 130 nasal swabs from 26 infants with CF and screened for β-lactams (ampicillin, amoxicillin-clavulanic acid, and cefuroxime) and other classes of antibiotic resistances (tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole). For 69 swabs (53% of total), we found at least one non-susceptible phenotype. Analyses of the inserts recovered from non-susceptible clones by nanopore MinION sequencing revealed a large reservoir of resistance genes including mobile elements within the antibiotic naïve samples. Comparing the data of the resistome with the microbiota composition showed that the bacterial phyla and operational taxonomic units (OTUs) of the microbiota rather than the antibiotic treatment were associated with the majority of non-susceptible phenotypes in the resistome. Future studies will reveal if characterization of the resistome can help in clinical decision-making in patients with CF.

Facing multidrug resistant (MDR) bacterial pathogens is one of the most important challenges for our society. The spread of highly virulent and resistant pathogens can be described using molecular typing technologies; in particular, whole genome sequencing (WGS) data can be used for molecular typing purposes with high resolution. WGS data analysis can explain the spatiotemporal patterns of pathogen transmission. However, the transmission between compartments (human, animal, food, environment) is very complex. Interoperable and curated metadata are a key requirement for fully understanding this complexity. In addition, high quality sequence data are a key element between centres using WGS data for diagnostic and epidemiological applications. We aim to describe steps to improve WGS data analysis and to implement a molecular surveillance platform allowing integration of high resolution WGS typing data and epidemiological data.

The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological and microbiological definitions. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 13,387 VoCs have been identified since the detection of the first Swiss case in October 2020, with 4194 being B.1.1.7, 172 B.1.351, and 7 P.1. The remaining 9014 cases of VoCs have been described without further lineage specification. Overall, all diagnostic centers reported a rapid increase of the percentage of detected VOCs, with a range of 6 to 46% between 25 to 31 of January 2021 increasing towards 41 to 82% between 22 to 28 of February. A total of 739 N501Y positive genomes were analysed and show a broad range of introduction events to Switzerland. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs.

The International Virus Bioinformatics Meeting 2020 was originally planned to take place in Bern, Switzerland, in March 2020. However, the COVID-19 pandemic put a spoke in the wheel of almost all conferences to be held in 2020. After moving the conference to 8–9 October 2020, we got hit by the second wave and finally decided at short notice to go fully online. On the other hand, the pandemic has made us even more aware of the importance of accelerating research in viral bioinformatics. Advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks. The International Virus Bioinformatics Meeting 2020 has attracted approximately 120 experts in virology and bioinformatics from all over the world to join the two-day virtual meeting. Despite concerns being raised that virtual meetings lack possibilities for face-to-face discussion, the participants from this small community created a highly interactive scientific environment, engaging in lively and inspiring discussions and suggesting new research directions and questions. The meeting featured five invited and twelve contributed talks, on the four main topics: (1) proteome and RNAome of RNA viruses, (2) viral metagenomics and ecology, (3) virus evolution and classification and (4) viral infections and immunology. Further, the meeting featured 20 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting.

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

BACKGROUND:Cough in children is a common reason for medical consultations and affects quality of life. There are little population-based data on the epidemiology of recurrent cough in children and how this varies by age and sex, or between children with and without wheeze. We determined the prevalence of cough throughout childhood, comparing several standardised cough questions. We did this for the entire population and separately for girls and boys, and for children with and without wheeze. METHODS:In a population-based prospective cohort from Leicestershire, UK, we assessed prevalence of cough with repeated questionnaires from early childhood to adolescence. We asked whether the child usually coughed more than other children, with or without colds, had night-time cough or cough triggered by various factors (triggers, related to increased breathing effort, allergic or food triggers). We calculated prevalence from age 1 to 18 years using generalised estimating equations for all children, and for children with and without wheeze. RESULTS:Of 7670 children, 10% (95% CI 10-11%) coughed more than other children, 69% (69-70%) coughed usually with a cold, 34% to 55% age-dependently coughed without colds, and 25% (25-26%) had night-time cough. Prevalence of coughing more than peers, with colds, at night, and triggered by laughter varied little throughout childhood, while cough without colds and cough triggered by exercise, house dust or pollen became more frequent with age. Cough was more common in boys than in girls in the first decade of life, differences got smaller in early teens and reversed after the age of 14 years. All symptoms were more frequent in children with wheeze. CONCLUSIONS:Prevalence of cough in children varies with age, sex and with the questions used to assess it, suggesting that comparisons between studies are only valid for similar questions and age groups.

Breastfeeding is said to protect children from eczema (atopic dermatitis), but the available evidence is conflicting and subject to the influences of parental atopy and reverse causation (when mothers extended duration of breastfeeding because their children had eczema).In the prospective, population-based Leicester Respiratory Cohort study, we assessed duration of breastfeeding in children aged 1-4 years. Prevalence of eczema was determined by questionnaire surveys that were repeated until the children were 17 years old. We investigated the association between having been breastfed and current eczema using generalized estimating equations, adjusting for potential confounders, and tested for effect modification by parental atopy. We also assessed the association between having been breastfed and incident eczema at ages 2, 4, and 6 years using multivariable logistic regression.Among the 5,676 children in the study, 2,284 (40%) had never been breastfed, while 1,610 (28%), 705 (12%), and 1,077 (19%) had been breastfed for 0-3, 4-6, and >6 months, respectively. Prevalence of current eczema decreased from 36% in 1-year-olds to 18% in children aged 10-17 years. Breastfeeding was not associated with current eczema. Compared with children who had never been breastfed, the adjusted odds ratios for current eczema at any age were 1.02 (95% confidence interval 0.90-1.15) for children who had been breastfed for 0-3 months, 0.97 (0.82-1.13) for children breastfed for 4-6 months, and 0.98 (0.85-1.14) for children breastfed for >6 months. There was no strong evidence for an effect modification by parental atopy (p-value for interaction term was 0.061) and no association between having been breastfed and incident eczema later in childhood.This population-based cohort study found no evidence for protection of breastfeeding against childhood eczema at any age, from infancy through adolescence.

The deep ocean floor covers more than 60% of the Earth's surface, and hosts diverse bacterial communities with important functions in carbon and nutrient cycles. The identification of key bacterial members remains a challenge and their patterns of distribution in seafloor sediment yet remain poorly described. Previous studies were either regionally restricted or included few deep-sea sediments, and did not specifically test biogeographic patterns across the vast oligotrophic bathyal and abyssal seafloor. Here we define the composition of this deep seafloor microbiome by describing those bacterial operational taxonomic units (OTU) that are specifically associated with deep-sea surface sediments at water depths ranging from 1000-5300 m. We show that the microbiome of the surface seafloor is distinct from the subsurface seafloor. The cosmopolitan bacterial OTU were affiliated with the clades JTB255 (class Gammaproteobacteria, order Xanthomonadales) and OM1 (Actinobacteria, order Acidimicrobiales), comprising 21% and 7% of their respective clades, and about 1% of all sequences in the study. Overall, few sequence-abundant bacterial types were globally dispersed and displayed positive range-abundance relationships. Most bacterial populations were rare and exhibited a high degree of endemism, explaining the substantial differences in community composition observed over large spatial scales. Despite the relative physicochemical uniformity of deep-sea sediments, we identified indicators of productivity regimes, especially sediment organic matter content, as factors significantly associated with changes in bacterial community structure across the globe.

Hydrogen cyanide (HCN) is a broad-spectrum antimicrobial compound involved in biological control of root diseases by many plant-associated fluorescent pseudomonads. The HCN synthase is encoded by three biosynthetic genes (hcnA,hcnB, and hcnC), but little is known about the diversity of these genes in fluorescent Pseudomonas spp. and in other bacteria. Here, the partial hcnBC sequence was determined for a worldwide collection of biocontrol fluorescent Pseudomonas spp. Phylogenies based on hcnBC and deduced protein sequences revealed four main bacterial groups, but topological incongruences were found between hcnBC and rrs-based phylogenies, suggesting past lateral transfer of hcnBC among saprophytic root-colonizing pseudomonads. Three of the four groups included isolates from different countries and host plants. Yet, these groups corresponded to distinct, ecologically-adapted populations of HCN-producing biocontrol fluorescent pseudomonads, as indicated by high hcnBC distinctness ratio values and the differences in production levels of HCN in vitro found between groups. This is in accordance with previous results on catabolic properties and biocontrol abilities of these strains. HCN synthase gene diversity may thus reflect the adaptive radiation of HCN+ biocontrol fluorescent pseudomonads. Positive correlations were found between HCN production in vitro and plant protection in the cucumber/Pythium ultimum and tomato/Fusarium oxysporum f. sp. radicis-lycopersici pathosystems.

Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro.

The methane-emitting cold seeps of Hikurangi margin (New Zealand) are among the few deep-sea chemosynthetic ecosystems of the Southern Hemisphere known to date. Here we compared the biogeochemistry and microbial communities of a variety of Hikurangi cold seep ecosystems. These included highly reduced seep habitats dominated by bacterial mats, partially oxidized habitats populated by heterotrophic ampharetid polychaetes and deeply oxidized habitats dominated by chemosynthetic frenulate tubeworms. The ampharetid habitats were characterized by a thick oxic sediment layer that hosted a diverse and biomass-rich community of aerobic methanotrophic Gammaproteobacteria. These bacteria consumed up to 25% of the emanating methane and clustered within three deep-branching groups named Marine Methylotrophic Group (MMG) 1-3. MMG1 and MMG2 methylotrophs belong to the order Methylococcales, whereas MMG3 methylotrophs are related to the Methylophaga. Organisms of the groups MMG1 and MMG3 are close relatives of chemosynthetic endosymbionts of marine invertebrates. The anoxic sediment layers of all investigated seeps were dominated by anaerobic methanotrophic archaea (ANME) of the ANME-2 clade and sulfate-reducing Deltaproteobacteria. Microbial community analysis using Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the different seep habitats hosted distinct microbial communities, which were strongly influenced by the seep-associated fauna and the geographic location. Despite outstanding features of Hikurangi seep communities, the organisms responsible for key ecosystem functions were similar to those found at seeps worldwide. This suggests that similar types of biogeochemical settings select for similar community composition regardless of geographic distance. Because ampharetid polychaetes are widespread at cold seeps the role of aerobic methanotrophy may have been underestimated in seafloor methane budgets.

Knowledge on spatial scales of the distribution of deep-sea life is still sparse, but highly relevant to the understanding of dispersal, habitat ranges and ecological processes. We examined regional spatial distribution patterns of the benthic bacterial community and covarying environmental parameters such as water depth, biomass and energy availability at the Arctic Long-Term Ecological Research (LTER) site HAUSGARTEN (Eastern Fram Strait). Samples from 13 stations were retrieved from a bathymetric (1,284-3,535 m water depth, 54 km in length) and a latitudinal transect (∼ 2,500 m water depth; 123 km in length). 454 massively parallel tag sequencing (MPTS) and automated ribosomal intergenic spacer analysis (ARISA) were combined to describe both abundant and rare types shaping the bacterial community. This spatial sampling scheme allowed detection of up to 99% of the estimated richness on phylum and class levels. At the resolution of operational taxonomic units (97% sequence identity; OTU3%) only 36% of the Chao1 estimated richness was recovered, indicating a high diversity, mostly due to rare types (62% of all OTU3%). Accordingly, a high turnover of the bacterial community was also observed between any two sampling stations (average replacement of 79% of OTU3%), yet no direct correlation with spatial distance was observed within the region. Bacterial community composition and structure differed significantly with increasing water depth along the bathymetric transect. The relative sequence abundance of Verrucomicrobia and Planctomycetes decreased significantly with water depth, and that of Deferribacteres increased. Energy availability, estimated from phytodetrital pigment concentrations in the sediments, partly explained the variation in community structure. Overall, this study indicates a high proportion of unique bacterial types on relatively small spatial scales (tens of kilometers), and supports the sampling design of the LTER site HAUSGARTEN to study bacterial community shifts in this rapidly changing area of the world's oceans.

Cold-water coral reefs are known to locally enhance the diversity of deep-sea fauna as well as of microbes. Sponges are among the most diverse faunal groups in these ecosystems, and many of them host large abundances of microbes in their tissues. In this study, twelve sponge species from three cold-water coral reefs off Norway were investigated for the relationship between sponge phylogenetic classification (species and family level), as well as sponge type (high versus low microbial abundance), and the diversity of sponge-associated bacterial communities, taking also geographic location and water depth into account. Community analysis by Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that as many as 345 (79%) of the 437 different bacterial operational taxonomic units (OTUs) detected in the dataset were shared between sponges and sediments, while only 70 (16%) appeared purely sponge-associated. Furthermore, changes in bacterial community structure were significantly related to sponge species (63% of explained community variation), sponge family (52%) or sponge type (30%), whereas mesoscale geographic distances and water depth showed comparatively small effects (

Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water's chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea-Dead Sea water conduit.

Ectosymbioses between invertebrates and sulfur-oxidizing bacteria are widespread in sulfidic marine environments and have evolved independently in several invertebrate phyla. The first example from a freshwater habitat, involving Niphargus ictus amphipods and filamentous Thiothrix ectosymbionts, was recently reported from the sulfide-rich Frasassi caves in Italy. Subsequently, two new Niphargus species, N. frasassianus and N. montanarius, were discovered within Frasassi and found to co-occur with N. ictus. Using a variety of microscopic and molecular techniques, we found that all three Frasassi-dwelling Niphargus species harbor Thiothrix ectosymbionts, which belong to three distinct phylogenetic clades (named T1, T2, and T3). T1 and T3 Thiothrix dominate the N. frasassianus ectosymbiont community, whereas T2 and T3 are prevalent on N. ictus and N. montanarius. Relative distribution patterns of the three ectosymbionts are host species-specific and consistent over different sampling locations and collection years. Free-living counterparts of T1-T3 are rare or absent in Frasassi cave microbial mats, suggesting that ectosymbiont transmission among Niphargus occurs primarily through inter- or intraspecific inoculations. Phylogenetic analyses indicate that the Niphargus-Thiothrix association has evolved independently at least two times. While ectosymbioses with T1 and T2 may have been established within Frasassi, T3 ectosymbionts seem to have been introduced to the cave system by Niphargus.

Biofilms play an important role as a settlement cue for invertebrate larvae and significantly contribute to the nutrient turnover in aquatic ecosystems. Nevertheless, little is known about how biofilm community structure generally responds to environmental changes. This study aimed to identify patterns of bacterial dynamics in coral reef biofilms in response to associated macrofouling community structure, microhabitat (exposed vs. sheltered), seasonality, and eutrophication. Settlement tiles were deployed at four reefs along a cross-shelf eutrophication gradient and were exchanged every 4 months over 20 months. The fouling community composition on the tiles was recorded and the bacterial community structure was assessed with the community fingerprinting technique Automated Ribosomal Intergenic Spacer Analysis (ARISA). Bacterial operational taxonomic unit (OTU) number was higher on exposed tiles, where the fouling community was homogenous and algae-dominated, than in sheltered habitats, which were occupied by a variety of filter feeders. Furthermore, OTU number was also highest in eutrophied near-shore reefs, while seasonal variations in community structure were most pronounced in the oligotrophic mid-shelf reef. In contrast, the macrofouling community structure did not change significantly with seasons. Changes in bacterial community patterns were mostly affected by microhabitat, seasonal and anthropogenically derived changes in nutrient availability, and to a lesser extent by changes in the macrofouling community structure. Path analysis revealed a complex interplay of various environmental and biological factors explaining the spatial and temporal variations in bacterial biofilm communities under natural conditions.

BACKGROUND: Cold-water coral reef ecosystems are recognized as biodiversity hotspots in the deep sea, but insights into their associated bacterial communities are still limited. Deciphering principle patterns of bacterial community variation over multiple spatial scales may however prove critical for a better understanding of factors contributing to cold-water coral reef stability and functioning. METHODOLOGY/PRINCIPAL FINDINGS: Bacterial community structure, as determined by Automated Ribosomal Intergenic Spacer Analysis (ARISA), was investigated with respect to (i) microbial habitat type and (ii) coral species and color, as well as the three spatial components (iii) geomorphologic reef zoning, (iv) reef boundary, and (v) reef location. Communities revealed fundamental differences between coral-generated (branch surface, mucus) and ambient microbial habitats (seawater, sediments). This habitat specificity appeared pivotal for determining bacterial community shifts over all other study levels investigated. Coral-derived surfaces showed species-specific patterns, differing significantly between Lophelia pertusa and Madrepora oculata, but not between L. pertusa color types. Within the reef center, no community distinction corresponded to geomorphologic reef zoning for both coral-generated and ambient microbial habitats. Beyond the reef center, however, bacterial communities varied considerably from local to regional scales, with marked shifts toward the reef periphery as well as between different in- and offshore reef sites, suggesting significant biogeographic imprinting but weak microbe-host specificity. CONCLUSIONS/SIGNIFICANCE: This study presents the first multi-scale survey of bacterial diversity in cold-water coral reefs, spanning a total of five observational levels including three spatial scales. It demonstrates that bacterial communities in cold-water coral reefs are structured by multiple factors acting at different spatial scales, which has fundamental implications for the monitoring of microbial diversity and function in those ecosystems.

BackgroundMarine microbial communities have been essential contributors to global biomass, nutrient cycling, and biodiversity since the early history of Earth, but so far their community distribution patterns remain unknown in most marine ecosystems.Methodology/principal findingsThe synthesis of 9.6 million bacterial V6-rRNA amplicons for 509 samples that span the global ocean's surface to the deep-sea floor shows that pelagic and benthic communities greatly differ, at all taxonomic levels, and share Conclusions/significanceThis first synthesis of global bacterial distribution across different ecosystems of the World's oceans shows remarkable horizontal and vertical large-scale patterns in bacterial communities. This opens interesting perspectives for the definition of biogeographical biomes for bacteria of ocean waters and the seabed.