Accurate and comprehensive identification of enteropathogens, causing infectious gastroenteritis, is essential for optimal patient treatment and effective isolation processes in health care systems. Traditional diagnostic techniques are well established and optimised in low-cost formats. However, thorough testing for a wider range of causal agents is time consuming and remains limited to a subset of pathogenic organisms. Metagenomic next-generation sequencing (mNGS) allows the identification of all pathogens in a sample in a single test, without a reliance on culture or introduction of target selection bias. This study aims to determine the ability to routinely apply mNGS testing, in comparison to traditional culture or polymerase chain reaction (PCR) based tests, for the identification of causal pathogens for gastrointestinal infections. The performance of mNGS, PCR and microscopy, culture and sensitivity (MCS) assays was established using 2,619 prospectively collected faecal samples from patients with symptomology indicative of infectious gastroenteritiss. Commonly experienced pathogens including Aeromonas spp, Campylobacter spp, Salmonella spp and Giardia spp, in single and co-infected patients, were used to establish test outcomes. When testing for these organisms, using the combined result from either or both PCR and MCS testing as the comparator, the mNGS assay had clinically acceptable sensitivity (89.2-100%). Further, the mNGS assay detected 14 additional enteropathogens, that were either not detected or not tested, by initial PCR/MCS testing. The advantage of mNGS compared to other syndromic testing systems is the broad range of detectable targets and the ability to interrogate samples without clinician informed or assay specific bias. With the development of newer sequencing assays, it is now feasible to test for a wide range of target organisms in a sample using a single mNGS test. Overall, the mNGS based approach enabled pathogen detection that was comparable to conventional diagnostics and was shown to have the potential to be extended for the detection of many pathogens and genes of clinical interest. In conclusion, the mNGS assay offers an easy, sample to answer workflow with rapid detection of enteropathogens and has the potential to improve diagnosis, therapy and infection control precautions.
Abstract Escherichia coli ST131 is the most frequently isolated fluoroquinolone resistant (FQR) E. coli clone worldwide and a major cause of urinary tract and bloodstream infections. Although originally identified through its association with the CTX-M-15 extended-spectrum β-lactamase resistance gene, global genomic epidemiology studies have failed to resolve the geographical and temporal origin of the ST131 ancestor. Here, we developed a framework for the reanalysis of publicly available genomes from different sources and used this dataset to reconstruct the evolutionary steps that led to the emergence of FQR ST131. Using Bayesian estimation, we show that point mutations in chromosomal genes that confer FQR coincide with the first clinical use of fluoroquinolone in 1986, and illustrate the impact of this pivotal event in the rapid population expansion of ST131 worldwide from an apparent origin in North America. Furthermore, we identify key virulence factor acquisition events that predate the development of FQR, suggesting that the gain of virulence-associated genes followed by the tandem development of antibiotic resistance primed the successful global dissemination of ST131.
Fish mortality caused by Streptococcus iniae is a major economic problem in aquaculture in warm and temperate regions globally. There is also risk of zoonotic infection by S. iniae through handling of contaminated fish. In this study, we present the complete genome sequence of S. iniae strain QMA0248, isolated from farmed barramundi in South Australia. The 2.12 Mb genome of S. iniae QMA0248 carries a 32 kb prophage, a 12 kb genomic island and 92 discrete insertion sequence (IS) elements. These include nine novel IS types that belong mostly to the IS 3 family. Comparative and phylogenetic analysis between S. iniae QMA0248 and publicly available complete S. iniae genomes revealed discrepancies that are probably due to misassembly in the genomes of isolates ISET0901 and ISNO. Long-range PCR confirmed five rRNA loci in the PacBio assembly of QMA0248, and, unlike S. iniae 89353, no tandemly repeated rRNA loci in the consensus genome. However, we found sequence read evidence that the tandem rRNA repeat existed within a subpopulation of the original QMA0248 culture. Subsequent nanopore sequencing revealed that the tandem rRNA repeat was the most prevalent genotype, suggesting that there is selective pressure to maintain fewer rRNA copies under uncertain laboratory conditions. Our study not only highlights assembly problems in existing genomes, but provides a high-quality reference genome for S. iniae QMA0248, including manually curated mobile genetic elements, that will assist future S. iniae comparative genomic and evolutionary studies.
The article cited as reference 17 (N. Stoesser et al.) was originally available as a preprint in BioRxiv ( ) on 6 November 2015. It was subsequently published in mBio while our article was in proof
AbstractBackground Accurate and comprehensive identification of enteropathogens, causing infectious gastroenteritis, is essential for optimal patient treatment and effective isolation processes in health care systems. Traditional diagnostic techniques are well established and optimised in low-cost formats. However, thorough testing for a wider range of causal agents is time consuming and remains limited to a subset of pathogenic organisms. Metagenomic next-generation sequencing (mNGS) allows the identification of all pathogens in a sample in a single test, without a reliance on culture or introduction of target selection bias. This study aims to determine the ability to routinely apply mNGS testing, in comparison to traditional culture or polymerase chain reaction (PCR) based tests, for the identification of causal pathogens for gastrointestinal infections. Results The performance of mNGS, PCR and microscopy, culture and sensitivity (MCS) assays was established using 2,619 prospectively collected faecal samples from patients with symptomology indicative of infectious gastroenteritis. Commonly experienced pathogens including Aeromonasspp, Campylobacter spp, Salmonella spp and Giardia spp, in single and co-infected patients, were used to establish test outcomes. When testing for these organisms, using the combined result from both PCR and MCS testing as the comparator, the mNGS assay had clinically acceptable sensitivity (89.2-100%). Further, the mNGS assay detected 14 additional enteropathogens, that were either not detected or not tested, by initial PCR/MCS testing. Conclusions The advantage of mNGS compared to other syndromic testing systems is the broad range of detectable targets and the ability to interrogate samples without clinician informed or assay specific bias. With the development of newer sequencing assays, it is now feasible to test for a wide range of target organisms in a sample using a single mNGS test. Overall, the mNGS based approach enabled pathogen detection that was comparable to conventional diagnostics and was shown to have the potential to be extended for the detection of many pathogens and genes of clinical interest. In conclusion, the mNGS assay offers an easy, sample to answer workflow with rapid detection of enteropathogens and has the potential to improve diagnosis, therapy and infection control precautions.
ABSTRACT Endozoicomonas bacteria were found highly associated with the coral Stylophora pistillata , and these bacteria are also ubiquitously associated with diverse corals worldwide. Novel Endozoicomonas -specific probes revealed that Endozoicomonas bacteria were abundant in the endodermal tissues of S. pistillata and appear to have an intimate relationship with the coral.
Abstract Fish mortality caused by Streptococcus iniae is a major economic problem in fish aquaculture in warm and temperate regions globally. There is also risk of zoonotic infection by S. iniae through handling of contaminated fish. In this study, we present the complete genome sequence of S. iniae strain QMA0248, isolated from farmed barramundi in South Australia. The 2.12 Mb genome of S. iniae QMA0248 carries a 32 Kb prophage, a 12 Kb genomic island, and 92 discrete insertion sequence (IS) elements. These include 9 novel IS types that belong mostly to the IS 3 family. Comparative and phylogenetic analysis between S. iniae QMA0248 and publicly available complete S. iniae genomes revealed discrepancies that are likely due to misassembly in the genomes of isolates ISET0901 and ISNO. We also determined by long-range PCR that a tandem duplication of an rRNA region in the PacBio assembly of QMA0248 was an assembly error. A similar rRNA duplication in the PacBio genome of S. iniae 89353 may also be a misassembly. Our study not only highlights assembly problems in existing genomes, but provides a high quality reference genome for S. iniae QMA0248, including manually curated mobile genetic elements, that will assist future S. iniae comparative genomic and evolutionary studies.
along withmicroorganisms including bacteria, archaea, fungi, and viruses.The significance of the bacterial assemblage to the coral animal isnot well understood, although coral bacteria have been character-ized as species specific (1) and may have roles in nitrogen fixation,carbon fixation, antibiotic production, and other features that en-able their health and functioning (1–5). A substantial componentof the coral bacterial community resides within the mucus layer(6, 7), and there is little understanding of which microbial part-ners are actually in residence within the coral tissues and poten-tially interacting with the coral. Here, we address this lack ofknowledge by examining if and how a dominant group of bacteriafrequently recovered in sequencing-based studies is located inter-nally within the coral tissues of the Red Sea coral
Advances in genomic sequencing technologies resulted in massive microbial diversity data (16S ribosomal gene sequences, rDNA) being generated in every possible environment. However, the majority of microorganisms have never been cultured, and therefore, nor cataloged. This poses a problem for molecular microbial ecologists because a large portion of the marker sequences can not be taxonomically resolved past the phylum taxon level. This tells very little about who or what these microorganisms are doing in relation to their environment. Our study describes an approach to assist in drawing ecological information from a sample when the taxon resolution is poor. We generated 16S rDNA libraries from a hypersaline marine sediment (coastal Sabkha) and saline mangrove soil in Abu Dhabi and then compared the compositional features to a database of 20,470 publicly available microbial community profiles (comprising the entire Earth Microbiome Project, EMP) that were annotated with terms from the Environmental Ontology (EnvO). An accurate taxonomic classification was not possible for 80% of the Sabkha operational taxonomic units (OTUs) beyond phylum level with widely used taxonomy classification tools, but habitat profiling performed on the community revealed strong links to bacterial assemblages of soil and marine origins. To capture the notion of generalist vs. specialist formally, we developed an algorithm to derive empirical probability distributions of OTUs over ecosystems from observed occurrences in the sample database, which then give rise to OTU-specific ecosystem entropies. We observed very low average ecosystem entropy of the Sabkha in contrast to other environmental samples. Based on this concept, the Sabkha community, while of midrange alpha diversity, presented largely specialist characteristics, with most OTUs identified to be unique to the Sabkha habitat. This finding is further corroborated by the observation that the Sabkha sample is unique with respect to the EMP-derived dataset (which contains 74 hypersaline and thousands of marine samples), as a comprehensive UniFrac similarity search did not yield any significant matches. Finally, we show that the ecosystem entropy formalism, which intrinsically accounts for the ability of OTUs to cross ecosystem borders according to a context database, is a novel, informative tool to describe and identify extreme environments in addition to conventional ecological diversity measures.
ABSTRACT Escherichia coli ST131 is the most frequently isolated fluoroquinolone-resistant (FQR) E. coli clone worldwide and a major cause of urinary tract and bloodstream infections. Although originally identified through its association with the CTX-M-15 extended-spectrum β-lactamase resistance gene, global genomic epidemiology studies have failed to resolve the geographical and temporal origin of the ST131 ancestor. Here, we developed a framework for the reanalysis of publically available genomes from different countries and used this data set to reconstruct the evolutionary steps that led to the emergence of FQR ST131. Using Bayesian estimation, we show that point mutations in chromosomal genes that confer FQR coincide with the first clinical use of fluoroquinolone in 1986 and illustrate the impact of this pivotal event on the rapid population expansion of ST131 worldwide from an apparent origin in North America. Furthermore, we identify virulence factor acquisition events that predate the development of FQR, suggesting that the gain of virulence-associated genes followed by the tandem development of antibiotic resistance primed the successful global dissemination of ST131. IMPORTANCE Escherichia coli sequence type 131 (ST131) is a recently emerged and globally disseminated multidrug-resistant clone frequently associated with human urinary tract and bloodstream infections. In this study, we have used two large publically available genomic data sets to define a number of critical steps in the evolution of this important pathogen. We show that resistance to fluoroquinolones, a class of broad-spectrum antibiotic used extensively in human medicine and veterinary practice, developed in ST131 soon after the introduction of these antibiotics in the United States, most likely in North America. We also mapped the acquisition of several fitness and virulence determinants by ST131 and demonstrate these events occurred prior to the development of fluoroquinolone resistance. Thus, ST131 has emerged by stealth, first acquiring genes associated with an increased capacity to cause human infection, and then gaining a resistance armory that has driven its massive population expansion across the globe.
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