FAIR+E pathogen data for surveillance and research: lessons from COVID-19.

The COVID-19 pandemic has exemplified the importance of interoperable and equitable data sharing for global surveillance and to support research. While many challenges could be overcome, at least in some countries, many hurdles within the organizational, scientific, technical and cultural realms still remain to be tackled to be prepared for future threats. We propose to (i) continue supporting global efforts that have proven to be efficient and trustworthy toward addressing challenges in pathogen molecular data sharing; (ii) establish a distributed network of Pathogen Data Platforms to (a) ensure high quality data, metadata standardization and data analysis, (b) perform data brokering on behalf of data providers both for research and surveillance, (c) foster capacity building and continuous improvements, also for pandemic preparedness; (iii) establish an International One Health Pathogens Portal, connecting pathogen data isolated from various sources (human, animal, food, environment), in a truly One Health approach and following FAIR principles. To address these challenging endeavors, we have started an ELIXIR Focus Group where we invite all interested experts to join in a concerted, expert-driven effort toward sustaining and ensuring high-quality data for global surveillance and research.

Development of early life gut resistome and mobilome across gestational ages and microbiota-modifying treatments.

BACKGROUND: Gestational age (GA) and associated level of gastrointestinal tract maturation are major factors driving the initial gut microbiota composition in preterm infants. Besides, compared to term infants, premature infants often receive antibiotics to treat infections and probiotics to restore optimal gut microbiota. How GA, antibiotics, and probiotics modulate the microbiota's core characteristics, gut resistome and mobilome, remains nascent. METHODS: We analysed metagenomic data from a longitudinal observational study in six Norwegian neonatal intensive care units to describe the bacterial microbiota of infants of varying GA and receiving different treatments. The cohort consisted of probiotic-supplemented and antibiotic-exposed extremely preterm infants (n = 29), antibiotic-exposed very preterm (n = 25), antibiotic-unexposed very preterm (n = 8), and antibiotic-unexposed full-term (n = 10) infants. The stool samples were collected on days of life 7, 28, 120, and 365, and DNA extraction was followed by shotgun metagenome sequencing and bioinformatical analysis. FINDINGS: The top predictors of microbiota maturation were hospitalisation length and GA. Probiotic administration rendered the gut microbiota and resistome of extremely preterm infants more alike to term infants on day 7 and ameliorated GA-driven loss of microbiota interconnectivity and stability. GA, hospitalisation, and both microbiota-modifying treatments (antibiotics and probiotics) contributed to an elevated carriage of mobile genetic elements in preterm infants compared to term controls. Finally, Escherichia coli was associated with the highest number of antibiotic-resistance genes, followed by Klebsiella pneumoniae and Klebsiella aerogenes. INTERPRETATION: Prolonged hospitalisation, antibiotics, and probiotic intervention contribute to dynamic alterations in resistome and mobilome, gut microbiota characteristics relevant to infection risk. FUNDING: Odd-Berg Group, Northern Norway Regional Health Authority.

Comparative analysis of HiSeq3000 and BGISEQ-500 sequencing platform over whole genome sequencing metagenomics data.

Recent advances in sequencing technologies and platforms have enabled to generate metagenomics sequences using different sequencing platforms. In this study, we analyzed and compared shotgun metagenomic sequences generated by HiSeq3000 and BGISEQ-500 platforms from 12 sediment samples collected across the Norwegian coast. Metagenomics DNA sequences were normalized to an equal number of bases for both platforms and further evaluated by using different taxonomic classifiers, reference databases, and assemblers. Normalized BGISEQ-500 sequences retained more reads and base counts after preprocessing, while a slightly higher fraction of HiSeq3000 sequences were taxonomically classified. Kaiju classified a higher percentage of reads relative to Kraken2 for both platforms, and comparison of reference database for taxonomic classification showed that MAR database outperformed RefSeq. Assembly using MEGAHIT produced longer assemblies and higher total contigs count in majority of HiSeq3000 samples than using metaSPAdes, but the assembly statistics notably improved with unprocessed or normalized reads. Our results indicate that both platforms perform comparably in terms of the percentage of taxonomically classified reads and assembled contig statistics for metagenomics samples. This study provides valuable insights for researchers in selecting an appropriate sequencing platform and bioinformatics pipeline for their metagenomics studies.

Detection of Klebsiella pneumoniae human gut carriage: a comparison of culture, qPCR, and whole metagenomic sequencing methods.

Klebsiella pneumoniae is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with K. pneumoniae is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of K. pneumoniae at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting K. pneumoniae in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting K. pneumoniae in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified K. pneumoniae to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman's rho = 0.91). WMS also supported accurate intra-sample K. pneumoniae sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of K. pneumoniae from fecal samples with potential to support infection control and enhance insights in K. pneumoniae gastrointestinal ecology.

What is the context?Klebsiella pneumoniae is a major cause of healthcare-associated infections and a key contributor to the spread of resistance to last-line antimicrobials.Gastrointestinal colonization by K. pneumoniae is a risk factor for developing infection and can facilitate the spread of antimicrobial resistance.Culture-based detection may lack sensitivity and is ill-suited to detecting within-sample K. pneumoniae abundance and diversity.Developing molecular methods to improve K. pneumoniae abundance and strain diversity detection are essential in understanding human gut colonization and ecology.What is new? We compared culture-based detection of K. pneumoniae within human fecal samples to two molecular-based techniques: 1) qPCR, which amplifies K. pneumoniae species complex-specific DNA targets with high sensitivity, and 2) whole metagenomic sequencing (WMS), which sequences the entire DNA content of complex microbial communities.Our findings show:qPCR had the highest sensitivity, detecting K. pneumoniae in all (52/52) culture-positive samples and 11/51 and 23/47 culture-negative samples, using a direct-fecal and culture-sweep method, respectively. qPCR could accurately quantify K. pneumoniae to 16 genome copies/reaction.WMS had lower sensitivity, positive in 37/52 culture-positive samples, and demonstrated false positives arising from closely related bacterial species. Relative abundance estimates could be performed to 0.01%.WMS performed accurate strain-level detection of K. pneumoniae to 0.1% relative abundance and could detect within-sample strain diversity.What is the impact?qPCR and WMS are valid methods for the detection and characterization of colonizing K. pneumoniae with potential to enhance our understanding of the gastrointestinal ecology of this important pathogen.

Quorum Sensing Controls the CRISPR and Type VI Secretion Systems in Aliivibrio wodanis 06/09/139.

For bacteria to thrive in an environment with competitors, phages and environmental cues, they use different strategies, including Type VI Secretion Systems (T6SSs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to compete for space. Bacteria often use quorum sensing (QS), to coordinate their behavior as the cell density increases. Like other aliivibrios, Aliivibrio wodanis 06/09/139 harbors two QS systems, the main LuxS/LuxPQ system and an N-acyl homoserine lactone (AHL)-mediated AinS/AinR system and a master QS regulator, LitR. To explore the QS and survival strategies, we performed genome analysis and gene expression profiling on A. wodanis and two QS mutants (ΔainS and ΔlitR) at two cell densities (OD600 2.0 and 6.0) and temperatures (6 and 12°C). Genome analysis of A. wodanis revealed two CRISPR systems, one without a cas loci (CRISPR system 1) and a type I-F CRISPR system (CRISPR system 2). Our analysis also identified three main T6SS clusters (T6SS1, T6SS2, and T6SS3) and four auxiliary clusters, as well about 80 potential Type VI secretion effectors (T6SEs). When comparing the wildtype transcriptome data at different cell densities and temperatures, 13-18% of the genes were differentially expressed. The CRISPR system 2 was cell density and temperature-independent, whereas the CRISPR system 1 was temperature-dependent and cell density-independent. The primary and auxiliary clusters of T6SSs were both cell density and temperature-dependent. In the ΔlitR and ΔainS mutants, several CRISPR and T6SS related genes were differentially expressed. Deletion of litR resulted in decreased expression of CRISPR system 1 and increased expression of CRISPR system 2. The T6SS1 and T6SS2 gene clusters were less expressed while the T6SS3 cluster was highly expressed in ΔlitR. Moreover, in ΔlitR, the hcp1 gene was strongly activated at 6°C compared to 12°C. AinS positively affected the csy genes in the CRISPR system 2 but did not affect the CRISPR arrays. Although AinS did not significantly affect the expression of T6SSs, the hallmark genes of T6SS (hcp and vgrG) were AinS-dependent. The work demonstrates that T6SSs and CRISPR systems in A. wodanis are QS dependent and may play an essential role in survival in its natural environment.

Shotgun-metagenomics based prediction of antibiotic resistance and virulence determinants in Staphylococcus aureus from periprosthetic tissue on blood culture bottles.

Shotgun-metagenomics may give valuable clinical information beyond the detection of potential pathogen(s). Identification of antimicrobial resistance (AMR), virulence genes and typing directly from clinical samples has been limited due to challenges arising from incomplete genome coverage. We assessed the performance of shotgun-metagenomics on positive blood culture bottles (n = 19) with periprosthetic tissue for typing and prediction of AMR and virulence profiles in Staphylococcus aureus. We used different approaches to determine if sequence data from reads provides more information than from assembled contigs. Only 0.18% of total reads was derived from human DNA. Shotgun-metagenomics results and conventional method results were consistent in detecting S. aureus in all samples. AMR and known periprosthetic joint infection virulence genes were predicted from S. aureus. Mean coverage depth, when predicting AMR genes was 209 ×. Resistance phenotypes could be explained by genes predicted in the sample in most of the cases. The choice of bioinformatic data analysis approach clearly influenced the results, i.e. read-based analysis was more accurate for pathogen identification, while contigs seemed better for AMR profiling. Our study demonstrates high genome coverage and potential for typing and prediction of AMR and virulence profiles in S. aureus from shotgun-metagenomics data.

Prevalence, diversity and transferability of the Tn916-Tn1545 family ICE in oral streptococci.

Background: The Tn916-Tn1545 family of Integrative Conjugative Elements (ICE) are mobile genetic elements (MGEs) that play a role in the spread of antibiotic resistance genes. The Tn916 harbors the tetracycline resistance gene tet(M) and it has been reported in various bacterial species. The increase in the levels of tetracycline resistance among oral streptococci is of great concern primarily due to the abundance of these species in the oral cavity and their ability to act as reservoirs for antibiotic resistance genes.Methods: In the current study, we screened 100 Norwegian clinical oral streptococcal isolates for the presence and diversity of the Tn916-Tn1545 family. In addition, we investigated the transferability the elements, and the associated transfer frequencies.Results: We observed that 21 isolates harboured the Tn916-Tn1545 family and that two of these elements were the novel Tn6815 and Tn6816. The most prevalent member of the Tn916 -Tn1545 family observed in the Norwegian clinical oral streptococcal isolates was the wild type Tn916.Conclusion: The detection of other members of this family of ICE and varying transfer frequencies suggests high versatility of the Tn916 element in oral streptococci in Norway.

Effects of fecal microbiota transplantation in subjects with irritable bowel syndrome are mirrored by changes in gut microbiome.

Irritable bowel syndrome (IBS) is a common disorder of the lower gastrointestinal tract. The pathophysiology is far from settled, but a gut microbial dysbiosis is hypothesized to be a contributing factor. We earlier published a randomized double-blind placebo-controlled clinical trial on fecal microbiota transplantation (FMT) for IBS - the REFIT trial. The present data set describes the engraftment and includes participants from the study who received active FMT; 14 participants with effect of FMT (Effect) and 8 without (No effect). Samples were collected at baseline, after 6 and 12 months. Samples from the transplants (Donor) served as a comparator. In total 66 recipient samples and 17 donor samples were subjected to deep metagenomic sequencing, and taxonomic and functional analyses were performed. Alpha diversity measures showed a significantly increased diversity and evenness in the IBS groups compared to the donors. Taxonomic profiles showed higher relative abundance of phylum Firmicutes, and lower relative abundance of phylum Bacteroidetes, compared to donors at baseline. This profile was shifted toward the donor profile following FMT. Imputed growth rates showed that the resulting growth pattern was a conglomerate of donor and recipient activity. Thirty-four functional subclasses showed distinct differences between baseline samples and donors, most of which were shifted toward a donor-like profile after FMT. All of these changes were less pronounced in the No effect group. We conclude that FMT induces long-term changes in gut microbiota, and these changes mirror the clinical effect of the treatment. The study was registered in ClinicalTrials.gov (NCT02154867).

Shotgun-Metagenomics on Positive Blood Culture Bottles Inoculated With Prosthetic Joint Tissue: A Proof of Concept Study.

Clinical metagenomics is actively moving from research to clinical laboratories. It has the potential to change the microbial diagnosis of infectious diseases, especially when detection and identification of pathogens can be challenging, such as in prosthetic joint infection (PJI). The application of metagenomic sequencing to periprosthetic joint tissue (PJT) specimens is often challenged by low bacterial load in addition to high level of inhibitor and contaminant host DNA, limiting pathogen recovery. Shotgun-metagenomics (SMg) performed directly on positive blood culture bottles (BCBs) inoculated with PJT may be a convenient approach to overcome these obstacles. The aim was to test if it is possible to perform SMg on PJT inoculated into BCBs for pathogen identification in PJI diagnosis. Our study was conducted as a laboratory method development. For this purpose, spiked samples (positive controls), negative control and clinical tissue samples (positive BCBs) were included to get a comprehensive overview. We developed a method for preparation of bacterial DNA directly from PJT inoculated in BCBs. Samples were processed using MolYsis5 kit for removal of human DNA and DNA extracted with BiOstic kit. High DNA quantity/quality was obtained, and no inhibition was observed during the library preparation, allowing further sequencing process. DNA sequencing reads obtained from the BCBs, presented a low proportion of human reads (<1%) improving the sensitivity of bacterial detection. We detected a 19-fold increase in the number of reads mapping to human in a sample untreated with MolYsis5. Taxonomic classification of clinical samples identified a median of 96.08% (IQR, 93.85-97.07%; range 85.7-98.6%) bacterial reads. Shotgun-metagenomics results were consistent with the results from a conventional BCB culture method, validating our approach. Overall, we demonstrated a proof of concept that it is possible to perform SMg directly on BCBs inoculated with PJT, with potential of pathogen identification in PJI diagnosis. We consider this a first step in research efforts needed to face the challenges presented in PJI diagnoses.

Composition of Gut Microbiota of Children and Adolescents With Perinatal Human Immunodeficiency Virus Infection Taking Antiretroviral Therapy in Zimbabwe.

BACKGROUND: Human immunodeficiency virus (HIV) infection causes impairment of the gastrointestinal barrier, with substantial depletion of CD4+ T cells in the gut. Antiretroviral therapy (ART) restores CD4+ counts and may have beneficial effects on gut microbiota in adults. Little is known about effect of long-term ART on gut microbiome in HIV-infected children. We investigated composition of gut microbiota in HIV-infected and -uninfected children and assessed associations between gut microbiota and patient characteristics. METHODS: In a cross-sectional study, rectal swabs were collected from 177 HIV-infected and 103 HIV-uninfected controls. Gut microbial composition was explored using 16S ribosomal ribonucleic acid sequencing. RESULTS: Human immunodeficiency virus-infected children had significantly lower alpha-diversity and higher beta-diversity compared to HIV-uninfected. No association was observed between microbiome diversity and CD4+ T-cell count, HIV viral load, or HIV-associated chronic lung disease. We found enriched levels of Corynebacterium (P < .01), Finegoldia (P < .01), and Anaerococcus (P < .01) in HIV-infected participants and enrichment of Enterobacteriaceae (P = .02) in participants with low CD4+ counts (<400 cells/mm3). Prolonged ART-treatment (≥10 years) was significantly associated with a richer gut microbiota by alpha diversity. CONCLUSIONS: Human immunodeficiency virus-infected children have altered gut microbiota. Prolonged ART may restore the richness of the microbiota closer to that of HIV-uninfected children.

Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes.

BACKGROUND: Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. RESULTS: We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5'-fusion to stimulate protein production and solubility. Finally we tested production of a set of "difficult-to-produce" enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5'-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5'-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. CONCLUSIONS: Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.

Comparative Genomic Analysis of Staphylococcus haemolyticus Reveals Key to Hospital Adaptation and Pathogenicity.

Staphylococcus haemolyticus is a skin commensal gaining increased attention as an emerging pathogen of nosocomial infections. However, knowledge about the transition from a commensal to an invasive lifestyle remains sparse and there is a paucity of studies comparing pathogenicity traits between commensal and clinical isolates. In this study, we used a pan-genomic approach to identify factors important for infection and hospital adaptation by exploring the genomic variability of 123 clinical isolates and 46 commensal S. haemolyticus isolates. Phylogenetic reconstruction grouped the 169 isolates into six clades with a distinct distribution of clinical and commensal isolates in the different clades. Phenotypically, multi-drug antibiotic resistance was detected in 108/123 (88%) of the clinical isolates and 5/46 (11%) of the commensal isolates (p < 0.05). In the clinical isolates, we commonly identified a homolog of the serine-rich repeat glycoproteins sraP. Additionally, three novel capsular polysaccharide operons were detected, with a potential role in S. haemolyticus virulence. Clinical S. haemolyticus isolates showed specific signatures associated with successful hospital adaption. Biofilm forming S. haemolyticus isolates that are resistant to oxacillin (mecA) and aminoglycosides (aacA-aphD) are most likely invasive isolates whereas absence of these traits strongly indicates a commensal isolate. We conclude that our data show a clear segregation of isolates of commensal origin, and specific genetic signatures distinguishing the clinical isolates from the commensal isolates. The widespread use of antimicrobial agents has probably promoted the development of successful hospital adapted clones of S. haemolyticus clones through acquisition of mobile genetic elements or beneficial point mutations and rearrangements in surface associated genes.

Exploring the transcriptome of luxI- and ΔainS mutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation in Aliivibrio salmonicida.

BACKGROUND: Bacterial communication through quorum sensing (QS) systems has been reported to be important in coordinating several traits such as biofilm formation. In Aliivibrio salmonicida two QS systems the LuxI/R and AinS/R, have been shown to be responsible for the production of eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. We have previously demonstrated that inactivation of LitR, the master regulator of the QS system resulted in biofilm formation, similar to the biofilm formed by the AHL deficient mutant ΔainSluxI- . In this study, we aimed to investigate the global gene expression patterns of luxI and ainS autoinducer synthases mutants using transcriptomic profiling. In addition, we examined the influence of the different AHLs on biofilm formation. RESULTS: The transcriptome profiling of ΔainS and luxI- mutants allowed us to identify genes and gene clusters regulated by QS in A. salmonicida. Relative to the wild type, the ΔainS and luxI- mutants revealed 29 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation of luxI, but not ainS genes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI- ) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when the ΔainSluxI- mutant was supplemented with N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) or N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and for repression of EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor. CONCLUSION: These findings imply that the LuxI and AinS autoinducer synthases play a critical role in the regulation of biofilm formation, EPS production, and motility.

Closely-related Photobacterium strains comprise the majority of bacteria in the gut of migrating Atlantic cod (Gadus morhua).

BACKGROUND: The population of Atlantic cod (Gadus morhua), also known as Northeast Arctic cod, migrating Atlantic cod, or simply "skrei," lives mainly in the Barents Sea and Svalbard waters and migrates in annual cycles to the Norwegian coast in order to spawn eggs during late winter. It is the world's largest population of Atlantic cod, and the population is distinct from the Norwegian coastal cod (or "fjord" cod). Despite the biological, economic, and cultural importance of migrating Atlantic cod, current knowledge on the associated microbiota is very limited. Using shotgun metagenomics and metaproteomics approaches, we present here the gut microbiota, metagenome-assembled genomes (MAGs) of the most abundant bacterial species, DNA-based functional profile, and the metaproteome of Atlantic cod specimens caught at a spawning area in an open ocean outside of Tromsø, Norway. RESULTS: Our analyses identified 268 bacterial families in DNA isolated from feces of 6 individual migrating Atlantic cod. The most abundant family was Vibrionaceae (52%; 83% if unclassified reads are excluded), with Photobacterium (genus) representing the vast majority. The recovery of metagenome-assembled genomes provided further details and suggests that several closely related Photobacterium strains from the Photobacterium phosphoreum clade are the most abundant. A genomic-based functional profiling showed that the most abundant functional subsystems are "Carbohydrates"; "Amino Acids and Derivatives"; "Protein Metabolism"; "Cofactors, Vitamins, Prosthetic, Groups, and Pigments"; and "DNA Metabolism," which is in agreement with other studies of gut microbiomes of marine organisms. Finally, the MS-based metaproteomic dataset revealed that the functional category "Protein Metabolism" is highly overrepresented (3×) when compared to the genome-based functional profile, which shows that ribosomal proteins are rich in the bacterial cytosol. CONCLUSION: We present here the first study of bacterial diversity of the gut of migrating Atlantic cod using shotgun sequencing and metagenome-assembled genomes (MAGs). The most abundant bacteria belong to the Photobacterium genus (Vibrionaceae family). We also constructed functional profiles of the gut microbiome. These may be used in future studies as a platform for mining of commercially interesting cold-active enzymes.

Differential expression profiling of ΔlitR and ΔrpoQ mutants reveals insight into QS regulation of motility, adhesion and biofilm formation in Aliivibrio salmonicida.

BACKGROUND: The coordination of group behaviors in bacteria is achieved by a cell-cell signaling process called quorum sensing (QS). QS is an intercellular communication system, which synchronously controls expression of a vast range of genes in response to changes in cell density and is mediated by autoinducers that act as extracellular signals. Aliivibrio salmonicida, the causative agent of cold-water vibrosis in marine aquacultures, uses QS to regulate several activities such as motility, biofilm formation, adhesion and rugose colony morphology. However, little is known about either genes or detailed mechanisms involved in the regulation of these phenotypes. RESULTS: Differential expression profiling allowed us to define the genes involved in controlling phenotypes related to QS in A. salmonicida LFI1238. RNA sequencing data revealed that the number of expressed genes in A. salmonicida, ΔlitR and ΔrpoQ mutants were significantly altered due to changes in cell density. These included genes that were distributed among the 21 functional groups, mainly presented in cell envelope, cell processes, extrachromosomal/foreign DNA and transport-binding proteins functional groups. The comparative transcriptome of A. salmonicida wild-type at high cell density relative to low cell density revealed 1013 genes to be either up- or downregulated. Thirty-six downregulated genes were gene clusters encoding biosynthesis of the flagellar and chemotaxis genes. Additionally we identified significant expression for genes involved in acyl homoserine lactone (AHL) synthesis, adhesion and early colonization. The transcriptome profile of ΔrpoQ compared to the wild-type revealed 384 differensially expressed genes (DEGs) that allowed us to assign genes involved in regulating motility, adhesion and colony rugosity. Indicating the importance of RpoQ in controlling several QS related activities. Furthermore, the comparison of the transcriptome profiles of ΔlitR and ΔrpoQ mutants, exposed numerous overlapping DEGs that were essential for motility, exopolysaccharide production via syp operon and genes associated with tad operon. CONCLUSION: Our findings indicate previously unexplained functional roles for LitR and RpoQ in regulation of different phenotypes related to QS. Our transcriptome data provide a better understanding of the regulation cascade of motility, wrinkling colony morphology and biofilm formation and will offer a major source for further research and analysis on this important field.

Effects of Probiotic Supplementation on the Gut Microbiota and Antibiotic Resistome Development in Preterm Infants.

Objectives: In 2014 probiotic supplementation (Lactobacillus acidophilus and Bifidobacterium longum subspecies infantis; InfloranⓇ) was introduced as standard of care to prevent necrotizing enterocolitis (NEC) in extremely preterm infants in Norway. We aimed to evaluate the influence of probiotics and antibiotic therapy on the developing gut microbiota and antibiotic resistome in extremely preterm infants, and to compare with very preterm infants and term infants not given probiotics. Study design: A prospective, observational multicenter study in six tertiary-care neonatal units. We enrolled 76 infants; 31 probiotic-supplemented extremely preterm infants <28 weeks gestation, 35 very preterm infants 28-31 weeks gestation not given probiotics and 10 healthy full-term control infants. Taxonomic composition and collection of antibiotic resistance genes (resistome) in fecal samples, collected at 7 and 28 days and 4 months age, were analyzed using shotgun-metagenome sequencing. Results: Median (IQR) birth weight was 835 (680-945) g and 1,290 (1,150-1,445) g in preterm infants exposed and not exposed to probiotics, respectively. Two extremely preterm infants receiving probiotic developed NEC requiring surgery. At 7 days of age we found higher median relative abundance of Bifidobacterium in probiotic supplemented infants (64.7%) compared to non-supplemented preterm infants (0.0%) and term control infants (43.9%). Lactobacillus was only detected in small amounts in all groups, but the relative abundance increased up to 4 months. Extremely preterm infants receiving probiotics had also much higher antibiotic exposure, still overall microbial diversity and resistome was not different than in more mature infants at 4 weeks and 4 months. Conclusion: Probiotic supplementation may induce colonization resistance and alleviate harmful effects of antibiotics on the gut microbiota and antibiotic resistome. Clinical Trial Registration: Clinicaltrials.gov: NCT02197468. https://clinicaltrials.gov/ct2/show/NCT02197468.

Norwegian e-Infrastructure for Life Sciences (NeLS).

The Norwegian e-Infrastructure for Life Sciences (NeLS) has been developed by ELIXIR Norway to provide its users with a system enabling data storage, sharing, and analysis in a project-oriented fashion. The system is available through easy-to-use web interfaces, including the Galaxy workbench for data analysis and workflow execution. Users confident with a command-line interface and programming may also access it through Secure Shell (SSH) and application programming interfaces (APIs).  NeLS has been in production since 2015, with training and support provided by the help desk of ELIXIR Norway. Through collaboration with NorSeq, the national consortium for high-throughput sequencing, an integrated service is offered so that sequencing data generated in a research project is provided to the involved researchers through NeLS. Sensitive data, such as individual genomic sequencing data, are handled using the TSD (Services for Sensitive Data) platform provided by Sigma2 and the University of Oslo. NeLS integrates national e-infrastructure storage and computing resources, and is also integrated with the SEEK platform in order to store large data files produced by experiments described in SEEK.   In this article, we outline the architecture of NeLS and discuss possible directions for further development.

Ten steps to get started in Genome Assembly and Annotation.

As a part of the ELIXIR-EXCELERATE efforts in capacity building, we present here 10 steps to facilitate researchers getting started in genome assembly and genome annotation. The guidelines given are broadly applicable, intended to be stable over time, and cover all aspects from start to finish of a general assembly and annotation project. Intrinsic properties of genomes are discussed, as is the importance of using high quality DNA. Different sequencing technologies and generally applicable workflows for genome assembly are also detailed. We cover structural and functional annotation and encourage readers to also annotate transposable elements, something that is often omitted from annotation workflows. The importance of data management is stressed, and we give advice on where to submit data and how to make your results Findable, Accessible, Interoperable, and Reusable (FAIR).

Teicoplanin resistance in Staphylococcus haemolyticus is associated with mutations in histidine kinases VraS and WalK.

We investigated the genetic basis of glycopeptide resistance in laboratory-derived strains of S. haemolyticus with emphasis on differences between vancomycin and teicoplanin. The genomes of two stable teicoplanin-resistant laboratory mutants selected on vancomycin or teicoplanin were sequenced and compared to parental S. haemolyticus strain W2/124. Only the two non-synonymous mutations, VraS Q289K and WalK V550L were identified. No other mutations or genome rearrangements were detected. Increased cell wall thickness, resistance to lysostaphin-induced lysis and adaptation of cell growth rates specifically to teicoplanin were phenotypes observed in a sequenced strain with the VraS Q289K mutation. Neither of the VraS Q289K and WalK V550L mutations was present in the genomes of 121S. haemolyticus clinical isolates. However, all but two of the teicoplanin resistant strains carried non-synonymous SNPs in vraSRTU and walKR-YycHIJ operons pointing to their importance for the glycopeptide resistance.

Haem-iron plays a key role in the regulation of the Ess/type VII secretion system of Staphylococcus aureus RN6390.

The Staphylococcus aureus type VII protein secretion system (T7SS) plays important roles in virulence and intra-species competition. Here we show that the T7SS in strain RN6390 is activated by supplementing the growth medium with haemoglobin, and its cofactor haemin (haem B). Transcript analysis and secretion assays suggest that activation by haemin occurs at a transcriptional and a post-translational level. Loss of T7 secretion activity by deletion of essC results in upregulation of genes required for iron acquisition. Taken together these findings suggest that the T7SS plays a role in iron homeostasis in at least some S. aureus strains.