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.

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.

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.

Pan genome and CRISPR analyses of the bacterial fish pathogen Moritella viscosa.

BACKGROUND: Winter-ulcer Moritella viscosa infections continue to be a significant burden in Atlantic salmon (Salmo salar L.) farming. M. viscosa comprises two main clusters that differ in genetic variation and phenotypes including virulence. Horizontal gene transfer through acquisition and loss of mobile genetic elements (MGEs) is a major driving force of bacterial diversification. To gain insight into genomic traits that could affect sublineage evolution within this bacterium we examined the genome sequences of twelve M. viscosa strains. Matches between M. viscosa clustered, regularly interspaced, short palindromic, repeats and associated cas genes (CRISPR-Cas) were analysed to correlate CRISPR-Cas with adaptive immunity against MGEs. RESULTS: The comparative genomic analysis of M. viscosa isolates from across the North Atlantic region and from different fish species support delineation of M. viscosa into four phylogenetic lineages. The results showed that M. viscosa carries two distinct variants of the CRISPR-Cas subtype I-F systems and that CRISPR features follow the phylogenetic lineages. A subset of the spacer content match prophage and plasmid genes dispersed among the M. viscosa strains. Further analysis revealed that prophage and plasmid-like element distribution were reflected in the content of the CRISPR-spacer profiles. CONCLUSIONS: Our data suggests that CRISPR-Cas mediated interactions with MGEs impact genome properties among M. viscosa, and that patterns in spacer and MGE distributions are linked to strain relationships.

Analysis of the distribution and evolution of the ATP-dependent DNA ligases of bacteria delineates a distinct phylogenetic group 'Lig E'.

Prior to the discovery of a minimal ATP-dependent DNA ligase in Haemophilus influenzae, bacteria were thought to only possess a NAD-dependent ligase, which was involved in sealing of Okazaki fragments. We now know that a diverse range of bacterial species possess up to six of these accessory bacterial ATP-dependent DNA ligases (b-ADLs), which vary in size and enzymatic domain associations. Here we compare the domain structure of different types of b-ADLs and investigate their distribution among the bacterial domain to describe possible evolutionary trajectories that gave rise to the sequence and structural diversity of these enzymes. Previous biochemical and genetic analyses have delineated three main classes of these enzymes: Lig B, Lig C and Lig D, which appear to have descended from a common ancestor within the bacterial domain. In the present study, we delineate a fourth group of b-ADLs, Lig E, which possesses a number of unique features at the primary and tertiary structural levels. The biochemical characteristics, domain structure and inferred extracellular location sets this group apart from the other b-ADLs. The results presented here indicate that the Lig E type ligases were horizontally transferred into bacteria in a separate event from other b-ADLs possibly from a bacteriophage.

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.

Bifidobacterium Bacteremia: Clinical Characteristics and a Genomic Approach To Assess Pathogenicity.

Bifidobacteria are commensals that colonize the orogastrointestinal tract and rarely cause invasive human infections. However, an increasing number of bifidobacterial blood culture isolates has lately been observed in Norway. In order to investigate the pathogenicity of the Bifidobacterium species responsible for bacteremia, we studied Bifidobacterium isolates from 15 patients for whom cultures of blood obtained from 2013 to 2015 were positive. We collected clinical data and analyzed phenotypic and genotypic antibiotic susceptibility. All isolates (11 Bifidobacterium longum, 2 B. breve, and 2 B. animalis isolates) were subjected to whole-genome sequencing. The 15 patients were predominantly in the extreme lower or upper age spectrum, many were severely immunocompromised, and 11 of 15 had gastrointestinal tract-related conditions. In two elderly patients, the Bifidobacterium bacteremia caused a sepsis-like picture, interpreted as the cause of death. Most bifidobacterial isolates had low MICs (≤0.5 mg/liter) to beta-lactam antibiotics, vancomycin, and clindamycin and relatively high MICs to ciprofloxacin and metronidazole. We performed a pangenomic comparison of invasive and noninvasive B. longum isolates based on 65 sequences available from GenBank and the sequences of 11 blood culture isolates from this study. Functional annotation identified unique genes among both invasive and noninvasive isolates of Bifidobacterium Phylogenetic clusters of invasive isolates were identified for a subset of the B. longum subsp. longum isolates. However, there was no difference in the number of putative virulence genes between invasive and noninvasive isolates. In conclusion, Bifidobacterium has an invasive potential in the immunocompromised host and may cause a sepsis-like picture. Using comparative genomics, we could not delineate specific pathogenicity traits characterizing invasive isolates.

Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis.

BACKGROUND: Aliivibrio wodanis and Moritella viscosa have often been isolated concurrently from fish with winter-ulcer disease. Little is known about the interaction between the two bacterial species and how the presence of one bacterial species affects the behaviour of the other. RESULTS: The impact on bacterial growth in co-culture was investigated in vitro, and the presence of A. wodanis has an inhibitorial effect on M. viscosa. Further, we have sequenced the complete genomes of these two marine Gram-negative species, and have performed transcriptome analysis of the bacterial gene expression levels from in vivo samples. Using bacterial implants in the fish abdomen, we demonstrate that the presence of A. wodanis is altering the gene expression levels of M. viscosa compared to when the bacteria are implanted separately. CONCLUSIONS: From expression profiling of the transcriptomes, it is evident that the presence of A. wodanis is altering the global gene expression of M. viscosa. Co-cultivation studies showed that A. wodanis is impeding the growth of M. viscosa, and that the inhibitorial effect is not contact-dependent.

Whole-genome sequencing reveals clonal expansion of multiresistant Staphylococcus haemolyticus in European hospitals.

OBJECTIVES: Staphylococcus haemolyticus is an emerging cause of nosocomial infections, primarily affecting immunocompromised patients. A comparative genomic analysis was performed on clinical S. haemolyticus isolates to investigate their genetic relationship and explore the coding sequences with respect to antimicrobial resistance determinants and putative hospital adaptation. METHODS: Whole-genome sequencing was performed on 134 isolates of S. haemolyticus from geographically diverse origins (Belgium, 2; Germany, 10; Japan, 13; Norway, 54; Spain, 2; Switzerland, 43; UK, 9; USA, 1). Each genome was individually assembled. Protein coding sequences (CDSs) were predicted and homologous genes were categorized into three types: Type I, core genes, homologues present in all strains; Type II, unique core genes, homologues shared by only a subgroup of strains; and Type III, unique genes, strain-specific CDSs. The phylogenetic relationship between the isolates was built from variable sites in the form of single nucleotide polymorphisms (SNPs) in the core genome and used to construct a maximum likelihood phylogeny. RESULTS: SNPs in the genome core regions divided the isolates into one major group of 126 isolates and one minor group of isolates with highly diverse genomes. The major group was further subdivided into seven clades (A-G), of which four (A-D) encompassed isolates only from Europe. Antimicrobial multiresistance was observed in 77.7% of the collection. High levels of homologous recombination were detected in genes involved in adherence, staphylococcal host adaptation and bacterial cell communication. CONCLUSIONS: The presence of several successful and highly resistant clones underlines the adaptive potential of this opportunistic pathogen.

Exploring the Frozen Armory: Antiphage Defense Systems in Cold-Adapted Bacteria with a Focus on CRISPR-Cas Systems.

Our understanding of the antiphage defense system arsenal in bacteria is rapidly expanding, but little is known about its occurrence in cold-adapted bacteria. In this study, we aim to shed light on the prevalence and distribution of antiphage defense systems in cold-adapted bacteria, with a focus on CRISPR-Cas systems. Using bioinformatics tools, Prokaryotic Antiviral Defense LOCator (PADLOC) and CRISPRCasTyper, we mapped the presence and diversity of antiphage defense systems in 938 available genomes of cold-adapted bacteria from diverse habitats. We confirmed that CRISPR-Cas systems are less frequent in cold-adapted bacteria, compared to mesophilic and thermophilic species. In contrast, several antiphage defense systems, such as dXTPases and DRTs, appear to be more frequently compared to temperate bacteria. Additionally, our study provides Cas endonuclease candidates with a potential for further development into cold-active CRISPR-Cas genome editing tools. These candidates could have broad applications in research on cold-adapted organisms. Our study provides a first-time map of antiphage defense systems in cold-adapted bacteria and a detailed overview of CRISPR-Cas diversity.

ICESluvan, a 94-kilobase mosaic integrative conjugative element conferring interspecies transfer of VanB-type glycopeptide resistance, a novel bacitracin resistance locus, and a toxin-antitoxin stabilization system.

A 94-kb integrative conjugative element (ICESluvan) transferable to Enterococcus faecium and Enterococcus faecalis from an animal isolate of Streptococcus lutetiensis consists of a mosaic of genetic fragments from different Gram-positive bacteria. A variant of ICESluvan was confirmed in S. lutetiensis from a patient. A complete Tn5382/Tn1549 with a vanB2 operon is integrated into a streptococcal ICESde3396-like region harboring a putative bacteriophage exclusion system, a putative agglutinin receptor precursor, and key components of a type IV secretion system. Moreover, ICESluvan encodes a putative MobC family mobilization protein and a relaxase and, thus, in total has all genetic components essential for conjugative transfer. A 9-kb element within Tn5382/Tn1549 encodes, among others, putative proteins similar to the TnpX site-specific recombinase in Faecalibacterium and VanZ in Paenibacillus, which may contribute to the detected low-level teicoplanin resistance. Furthermore, ICESluvan encodes a novel bacitracin resistance locus that is associated with reduced susceptibility to bacitracin when transferred to E. faecium. The expression of a streptococcal pezAT toxin-antitoxin-encoding operon of ICESluvan in S. lutetiensis, E. faecium, and E. faecalis was confirmed by reverse transcription (RT)-PCR, indicating an active toxin-antitoxin system which may contribute to stabilizing ICESluvan within new hosts. Junction PCR and DNA sequencing confirmed that ICESluvan excised to form a circular intermediate in S. lutetiensis, E. faecalis, and E. faecium. Transfer between E. faecalis cells was observed in the presence of helper plasmid pIP964. Sequence analysis of the original S. lutetiensis donor and enterococcal transconjugants showed that ICESluvan integrates in a site-specific manner into the C-terminal end of the chromosomal tRNA methyltransferase gene rumA.

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.

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.

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.

Unique core genomes of the bacterial family vibrionaceae: insights into niche adaptation and speciation.

BACKGROUND: The criteria for defining bacterial species and even the concept of bacterial species itself are under debate, and the discussion is apparently intensifying as more genome sequence data is becoming available. However, it is still unclear how the new advances in genomics should be used most efficiently to address this question. In this study we identify genes that are common to any group of genomes in our dataset, to determine whether genes specific to a particular taxon exist and to investigate their potential role in adaptation of bacteria to their specific niche. These genes were named unique core genes. Additionally, we investigate the existence and importance of unique core genes that are found in isolates of phylogenetically non-coherent groups. These groups of isolates, that share a genetic feature without sharing a closest common ancestor, are termed genophyletic groups. RESULTS: The bacterial family Vibrionaceae was used as the model, and we compiled and compared genome sequences of 64 different isolates. Using the software orthoMCL we determined clusters of homologous genes among the investigated genome sequences. We used multilocus sequence analysis to build a host phylogeny and mapped the numbers of unique core genes of all distinct groups of isolates onto the tree. The results show that unique core genes are more likely to be found in monophyletic groups of isolates. Genophyletic groups of isolates, in contrast, are less common especially for large groups of isolate. The subsequent annotation of unique core genes that are present in genophyletic groups indicate a high degree of horizontally transferred genes. Finally, the annotation of the unique core genes of Vibrio cholerae revealed genes involved in aerotaxis and biosynthesis of the iron-chelator vibriobactin. CONCLUSION: The presented work indicates that genes specific for any taxon inside the bacterial family Vibrionaceae exist. These unique core genes encode conserved metabolic functions that can shed light on the adaptation of a species to its ecological niche. Additionally, our study suggests that unique core genes can be used to aid classification of bacteria and contribute to a bacterial species definition on a genomic level. Furthermore, these genes may be of importance in clinical diagnostics and drug development.

Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida.

BACKGROUND: Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. RESULTS: We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. CONCLUSIONS: We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin.

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.

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.

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ(54) and the σ(54)-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.