Search Engine for Antimicrobial Resistance: A Cloud Compatible Pipeline and Web Interface for Rapidly Detecting Antimicrobial Resistance Genes Directly from Sequence Data.

BACKGROUND: Antimicrobial resistance remains a growing and significant concern in human and veterinary medicine. Current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria are limited in their effectiveness and scope. With the rapidly developing field of whole genome sequencing beginning to be utilised in clinical practice, the ability to interrogate sequencing data quickly and easily for the presence of antimicrobial resistance genes will become increasingly important and useful for informing clinical decisions. Additionally, use of such tools will provide insight into the dynamics of antimicrobial resistance genes in metagenomic samples such as those used in environmental monitoring. RESULTS: Here we present the Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally acquired antimicrobial resistance genes in raw sequencing data. The pipeline provides gene information, abundance estimation and the reconstructed sequence of antimicrobial resistance genes; it also provides web links to additional information on each gene. The pipeline utilises clustering and read mapping to annotate full-length genes relative to a user-defined database. It also uses local alignment of annotated genes to a range of online databases to provide additional information. We demonstrate SEAR's application in the detection and abundance estimation of antimicrobial resistance genes in two novel environmental metagenomes, 32 human faecal microbiome datasets and 126 clinical isolates of Shigella sonnei. CONCLUSIONS: We have developed a pipeline that contributes to the improved capacity for antimicrobial resistance detection afforded by next generation sequencing technologies, allowing for rapid detection of antimicrobial resistance genes directly from sequencing data. SEAR uses raw sequencing data via an intuitive interface so can be run rapidly without requiring advanced bioinformatic skills or resources. Finally, we show that SEAR is effective in detecting antimicrobial resistance genes in metagenomic and isolate sequencing data from both environmental metagenomes and sequencing data from clinical isolates.This research was funded by GlaxoSmithKline, the Centre for Environment, Fisheries and Aquaculture Science and the Biotechnology and Biological Sciences Research Council under an industrial CASE studentship. The funder Centre for Environment, Fisheries and Aquaculture Science provided support in the form of salaries, research materials and facilities for authors DVJ and CBA, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funder GlaxoSmithKline provided support in the form of salaries for author JR, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version. It was first published by PLOS at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133492

Optimising Antibiotic Usage to Treat Bacterial Infections

The increase in antibiotic resistant bacteria poses a threat to the continued use of antibiotics to treat bacterial infections. The overuse and misuse of antibiotics has been identified as a significant driver in the emergence of resistance. Finding optimal treatment regimens is therefore critical in ensuring the prolonged effectiveness of these antibiotics. This study uses mathematical modelling to analyse the effect traditional treatment regimens have on the dynamics of a bacterial infection. Using a novel approach, a genetic algorithm, the study then identifies improved treatment regimens. Using a single antibiotic the genetic algorithm identifies regimens which minimise the amount of antibiotic used while maximising bacterial eradication. Although exact treatments are highly dependent on parameter values and initial bacterial load, a significant common trend is identified throughout the results. A treatment regimen consisting of a high initial dose followed by an extended tapering of doses is found to optimise the use of antibiotics. This consistently improves the success of eradicating infections, uses less antibiotic than traditional regimens and reduces the time to eradication. The use of genetic algorithms to optimise treatment regimens enables an extensive search of possible regimens, with previous regimens directing the search into regions of better performance

A Longitudinal Study of Bacteriophages as Indicators of Norovirus Contamination of Mussels (Mytilus edulis) and Their Overlying Waters

Sewage pollution leads to the contamination of bivalve shellfish by pathogenic microorganisms. Bacterial indicators support the management of risks associated with the consumption of shellfish; however, they often fail to indicate adequately the potential hazard to human health posed by certain human enteric viruses. Bacteriophages have been proposed as alternative indicators that may more effectively predict the presence of enteric viral pathogens. This study explored the relationships between bacterial indicators (Escherichia coli (E. coli), faecal coliforms (FC) and intestinal enterococci (IE)), phages (somatic (SOMPH), F-specific RNA (F + PH) and human-specific Bacteroides GB-124 phages (GB124PH)) and Norovirus (NoV) (GI/GII) in mussels (Mytilus edulis) and their overlying waters. The bioaccumulation of these indicators and Norovirus in shellfish matrices (e.g., flesh, digestive gland) was investigated bimonthly over a 12-month period in an English estuary. The findings revealed a marked seasonality in the distribution of all organisms, with the highest levels occurring during the autumn/winter months. The levels of all phages in shellfish and their overlying waters correlated better with the levels of Norovirus than with those of bacterial indicators. Somatic coliphages were the indicator that exhibited the strongest correlations with NoV (rho = 0.929). This study suggests that relatively low-cost culture-based phage enumeration appears to offer a more accurate indication of the likely presence of Norovirus in mussels than traditional bacterial indicators

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay for the detection of pathogenic V. vulnificus strains based on a polymorphism in pilF, a gene previously indicated to be associated with human pathogenicity. Compared to human serum reactivity, the real-time PCR assay successfully detected pathogenic strains in 46 out of 47 analysed V. vulnificus isolates (97.9%). The method is also rapid, sensitive, and more importantly can be reliably utilised on biotype 2 and 3 strains, unlike other current methods for V. vulnificus virulence differentiation

Impact of analytic provenance in genome analysis

Many computational methods are available for assembly and annotation of newly sequenced microbial genomes. However, when new genomes are reported in the literature, there is frequently very little critical analysis of choices made during the sequence assembly and gene annotation stages. These choices have a direct impact on the biologically relevant products of a genomic analysis - for instance identification of common and differentiating regions among genomes in a comparison, or identification of enriched gene functional categories in a specific strain. Here, we examine the outcomes of different assembly and analysis steps in typical workflows in a comparison among strains of Vibrio vulnificus

New invasive Nemertean species (Cephalothrix Simula) in England with high levels of tetrodotoxin and a microbiome linked to toxin metabolism

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety

High Prevalence of Multidrug-Tolerant Bacteria and Associated Antimicrobial Resistance Genes Isolated from Ornamental Fish and Their Carriage Water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes

Comparative metagenomics reveals a diverse range of antimicrobial resistance genes in effluents entering a river catchment.

The aquatic environment has been implicated as a reservoir for antimicrobial resistance genes (ARGs). In order to identify sources that are contributing to these gene reservoirs, it is crucial to assess effluents that are entering the aquatic environment. Here we describe a metagenomic assessment for two types of effluent entering a river catchment. We investigated the diversity and abundance of resistance genes, mobile genetic elements (MGEs) and pathogenic bacteria. Findings were normalised to a background sample of river source water. Our results show that effluent contributed an array of genes to the river catchment, the most abundant being tetracycline resistance genes tetC and tetW from farm effluents and the sulfonamide resistance gene sul2 from wastewater treatment plant (WWTP) effluents. In nine separate samples taken across 3 years, we found 53 different genes conferring resistance to seven classes of antimicrobial. Compared to the background sample taken up river from effluent entry, the average abundance of genes was three times greater in the farm effluent and two times greater in the WWTP effluent. We conclude that effluents disperse ARGs, MGEs and pathogenic bacteria within a river catchment, thereby contributing to environmental reservoirs of ARGs.This research was funded by the Centre for Environment, Fisheries and Aquaculture Science, GlaxoSmithKline and the Biotechnology and Biological Sciences Research Council. Metagenomic sequencing was performed by the Sequencing Service and Wellcome Trust Biomedical Informatics Hub, Exeter (UK).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by IWA Publishing