Automated detection and classification of polioviruses from nanopore sequencing reads using piranha.

Widespread surveillance, rapid detection, and appropriate intervention will be critical for successful eradication of poliovirus. Using deployable next-generation sequencing (NGS) approaches, such as Oxford Nanopore Technologies' MinION, the time from sample to result can be significantly reduced compared to cell culture and Sanger sequencing. We developed piranha (poliovirus investigation resource automating nanopore haplotype analysis), a 'sequencing reads-to-report' solution to aid routine poliovirus testing of both stool and environmental samples and alleviate the bioinformatic bottleneck that often exists for laboratories adopting novel NGS approaches. Piranha can be used for efficient intratypic differentiation of poliovirus serotypes, for classification of Sabin-like polioviruses, and for detection of wild-type and vaccine-derived polioviruses. It produces interactive, distributable reports, as well as summary comma-separated values files and consensus poliovirus FASTA sequences. Piranha optionally provides phylogenetic analysis, with the ability to incorporate a local database, processing from raw sequencing reads to an interactive, annotated phylogeny in a single step. The reports describe each nanopore sequencing run with interpretable plots, enabling researchers to easily detect the presence of poliovirus in samples and quickly disseminate their results. Poliovirus eradication efforts are hindered by the lack of real-time detection and reporting, and piranha can be used to complement direct detection sequencing approaches.

Wastewater Surveillance in Europe for Non-Polio Enteroviruses and Beyond.

Wastewater surveillance (WWS) was developed in the early 1960s for the detection of poliovirus (PV) circulation in the population. It has been used to monitor several pathogens, including non-polio enteroviruses (NPEVs), which are increasingly recognised as causes of morbidity in children. However, when applying WWS to a new pathogen, it is important to consider the purpose of such a study as well as the suitability of the chosen methodology. With this purpose, the European Non-Polio Enterovirus Network (ENPEN) organised an expert webinar to discuss its history, methods, and applications; its evolution from a culture-based method to molecular detection; and future implementation of next generation sequencing (NGS). The first simulation experiments with PV calculated that a 400 mL sewage sample is sufficient for the detection of viral particles if 1:10,000 people excrete poliovirus in a population of 700,000 people. If the method is applied correctly, several NPEV types are detected. Despite culture-based methods remaining the gold standard for WWS, direct methods followed by molecular-based and sequence-based assays have been developed, not only for enterovirus but for several pathogens. Along with case-based sentinel and/or syndromic surveillance, WWS for NPEV and other pathogens represents an inexpensive, flexible, anonymised, reliable, population-based tool for monitoring outbreaks and the (re)emergence of these virus types/strains within the general population.

Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study.

Timely detection of outbreaks is needed for poliovirus eradication, but gold standard detection in the Democratic Republic of the Congo takes 30 days (median). Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method. Here we report prospective testing of stool samples from suspected polio cases, and their contacts, in the Democratic Republic of the Congo between 10 August 2021 and 4 February 2022. DDNS detected polioviruses in 62/2,339 (2.7%) of samples, while gold standard combination of cell culture, quantitative PCR and Sanger sequencing detected polioviruses in 51/2,339 (2.2%) of the same samples. DDNS provided case confirmation in 7 days (median) in routine surveillance conditions. DDNS enabled confirmation of three serotype 2 circulating vaccine-derived poliovirus outbreaks 23 days (mean) earlier (range 6-30 days) than the gold standard method. The mean sequence similarity between sequences obtained by the two methods was 99.98%. Our data confirm the feasibility of implementing DDNS in a national poliovirus laboratory.

Noninvasive Fecal Cytokine and Microbiota Profiles Predict Commencement of Necrotizing Enterocolitis in a Proof-of-Concept Study.

Background and Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell's staging is routinely used to guide diagnosis, but early diagnostic signs are nonspecific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated fecal cytokine levels, coupled with gut microbiota profiles, as a noninvasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary's or Queen Charlotte's & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologists grouped individuals by modified Bell's staging (healthy, NEC1, NEC2/3) and fecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota composition were observed for Bacillota (adj. P = .0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of fecal cytokine levels revealed significantly higher concentrations of IL-1α (P = .045), IL-5 (P = .0074), and IL-10 (P = .032) in Bell stage 1 NEC compared to healthy individuals. Conclusion: Differences in certain fecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains.

Clostridium perfringens is an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link between C. perfringens and the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundant C. perfringens termed C. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272 C. perfringens isolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O, pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typical pfoA-encoding virulent lineages. We determined that infant-associated pfoA+ strains caused significantly more cellular damage than pfoA- strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance of pfoA+ C. perfringens as a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies.

Comparison of Eleven RNA Extraction Methods for Poliovirus Direct Molecular Detection in Stool Samples.

Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods (7 manual and 4 semiautomated) for poliovirus molecular detection using stool samples (n = 59) that had been previously identified as poliovirus positive by cell culture. To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant (P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time. IMPORTANCE Poliovirus, the causative agent of poliomyelitis, is a target of global eradication led by the World Health Organization since 1988. Direct molecular detection and genomic sequencing without virus propagation in cell culture is arguably a critical tool in the final stages of polio eradication. Efficient recovery of good-quality viral RNA from stool samples is a prerequisite for direct detection by nucleic acid amplification. We tested 11 nucleic acid extraction methods to identify those facilitating sensitive, fast, simple, and cost-effective extraction, with flexibility for manual and automated protocols considered. Several different PCR assays were used to compare the recovered viral RNA to test suitability for poliovirus direct molecular detection. Our findings highlight the importance of choosing a suitable RNA extraction protocol and provide useful information to diagnostic laboratories and researchers facing the choice of RNA extraction method for direct molecular virus detection from stool.

Development of the gut microbiota during early life in premature and term infants.

BACKGROUND: The gastrointestinal (GI) microbiota has been linked to health consequences throughout life, from early life illnesses (e.g. sepsis and necrotising enterocolitis) to lifelong chronic conditions such as obesity and inflammatory bowel disease. It has also been observed that events in early life can lead to shifts in the microbiota, with some of these changes having been documented to persist into adulthood. A particularly extreme example of a divergent early GI microbiota occurs in premature neonates, who display a very different GI community to term infants. Certain characteristic patterns have been associated with negative health outcomes during the neonatal period, and these patterns may prove to have continual damaging effects if not resolved. RESULTS: In this study we compared a set of premature infants with a paired set of term infants (n = 37 pairs) at 6 weeks of age and at 2 years of age. In the samples taken at 6 weeks of age we found microbial communities differing in both diversity and specific bacterial groups between the two infant cohorts. We identified clinical factors associated with over-abundance of potentially pathogenic organisms (e.g. Enterobacteriaceae) and reduced abundances of some beneficial organisms (e.g. Bifidobacterium). We contrasted these findings with samples taken at 2 years of age, which indicated that despite a very different initial gut microbiota, the two infant groups converged to a similar, more adult-like state. We identified clinical factors, including both prematurity and delivery method, which remain associated with components of the gut microbiota. Both clinical factors and microbial characteristics are compared to the occurrence of childhood wheeze and eczema, revealing associations between components of the GI microbiota and the development of these allergic conditions. CONCLUSIONS: The faecal microbiota differs greatly between infants born at term and those born prematurely during early life, yet it converges over time. Despite this, early clinical factors remain significantly associated with the abundance of some bacterial groups at 2 years of age. Given the associations made between health conditions and the microbiota, factors that alter the makeup of the gut microbiota, and potentially its trajectory through life, could have important lifelong consequences.

Gastrointestinal, vaginal, nasopharyngeal, and breast milk microbiota profiles and breast milk metabolomic changes in Gambian infants over the first two months of lactation: A prospective cohort study.

Microbiota composition in breast milk affects intestinal and respiratory microbiota colonization and the mucosal immune system's development in infants. The metabolomic content of breast milk is thought to interact with the microbiota and may influence developing infant immunity. One hundred seven Gambian mothers and their healthy, vaginally delivered, exclusively breastfed infants were included in our study. We analyzed 32 breast milk samples, 51 maternal rectovaginal swabs and 30 infants' rectal swabs at birth. We also analyzed 9 breast milk samples and 18 infants' nasopharyngeal swabs 60 days post-delivery. We used 16S rRNA gene sequencing to determine the microbiota composition. Metabolomic profiling analysis was performed on colostrum and mature breast milk samples using a multiplatform approach combining 1-H Nuclear Magnetic Resonance Spectroscopy and Gas Chromatography-Mass Spectrometry. Bacterial communities were distinct in composition and diversity across different sample types. Breast milk composition changed over the first 60 days of lactation. α-1,4- and α-1,3-fucosylated human milk oligosaccharides, and other 33 key metabolites in breast milk (monosaccharides, sugar alcohols and fatty acids) increased between birth and day 60 of life. This study's results indicate that infant gut and respiratory microbiota are unique bacterial communities, distinct from maternal gut and breast milk, respectively. Breast milk microbiota composition and metabolomic profile change throughout lactation. These changes may contribute to the infant's immunological, metabolic, and neurological development and could consist the basis for future interventions to correct disrupted early life microbial colonization.

Sustained detection of type 2 poliovirus in London sewage between February and July, 2022, by enhanced environmental surveillance.

BACKGROUND: The international spread of poliovirus exposes all countries to the risk of outbreaks and is designated a Public Health Emergency of International Concern by WHO. This risk can be exacerbated in countries using inactivated polio vaccine, which offers excellent protection against paralysis but is less effective than oral vaccine against poliovirus shedding, potentially allowing circulation without detection of paralytic cases for long periods of time. Our study investigated the molecular properties of type 2 poliovirus isolates found in sewage with an aim to detect virus transmission in the community. METHODS: We performed environmental surveillance in London, UK, testing sewage samples using WHO recommended methods that include concentration, virus isolation in cell culture, and molecular characterisation. We additionally implemented direct molecular detection and determined whole-genome sequences of every isolate using novel nanopore protocols. FINDINGS: 118 genetically linked poliovirus isolates related to the serotype 2 Sabin vaccine strain were detected in 21 of 52 sequential sewage samples collected in London between Feb 8 and July 4, 2022. Expansion of environmental surveillance sites in London helped localise transmission to several boroughs in north and east London. All isolates have lost two key attenuating mutations, are recombinants with a species C enterovirus, and an increasing proportion (20 of 118) meet the criterion for a vaccine-derived poliovirus, having six to ten nucleotide changes in the gene coding for VP1 capsid protein. INTERPRETATION: Environmental surveillance allowed early detection of poliovirus importation and circulation in London, permitting a rapid public health response, including enhanced surveillance and an inactivated polio vaccine campaign among children aged 1-9 years. Whole-genome sequences generated through nanopore sequencing established linkage of isolates and confirmed transmission of a unique recombinant poliovirus lineage that has now been detected in Israel and the USA. FUNDING: Medicines and Healthcare products Regulatory Agency, UK Health Security Agency, Bill & Melinda Gates Foundation, and National Institute for Health Research Medical Research Council.

Time Taken to Detect and Respond to Polio Outbreaks in Africa and the Potential Impact of Direct Molecular Detection and Nanopore Sequencing.

BACKGROUND: Detection of poliovirus outbreaks relies on a complex laboratory algorithm of cell-culture, polymerase chain reaction (PCR), and sequencing to distinguish wild-type and vaccine-derived polioviruses (VDPV) from Sabin-like strains. We investigated the potential for direct molecular detection and nanopore sequencing (DDNS) to accelerate poliovirus detection. METHODS: We analyzed laboratory data for time required to analyze and sequence serotype-2 VDPV (VDPV2) in stool collected from children with acute flaccid paralysis in Africa (May 2016-February 2020). Impact of delayed detection on VDPV2 outbreak size was assessed through negative binomial regression. RESULTS: VDPV2 confirmation in 525 stools required a median of 49 days from paralysis onset (10th-90th percentile, 29-74), comprising collection and transport (median, 16 days), cell-culture (7 days), intratypic differentiation quantitative reverse transcription PCR (3 days), and sequencing, including shipping if required (15 days). New VDPV2 outbreaks were confirmed a median of 35 days (27-60) after paralysis onset, which we estimate could be reduced to 16 days by DDNS (9-37). Because longer delays in confirmation and response were positively associated with more cases (P < .001), we estimate that DDNS could reduce the number of VDPV2 cases before a response by 28% (95% credible interval, 12%-42%). CONCLUSIONS: DDNS could accelerate poliovirus outbreak response, reducing their size and the cost of eradication.

Premature neonatal gut microbial community patterns supporting an epithelial TLR-mediated pathway for necrotizing enterocolitis.

BACKGROUND: Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data. RESULTS: We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation. CONCLUSIONS: The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

The role of genetic sequencing and analysis in the polio eradication programme.

Genetic sequencing of polioviruses detected through clinical and environmental surveillance is used to confirm detection, identify their likely origin, track geographic patterns of spread, and determine the appropriate vaccination response. The critical importance of genetic sequencing and analysis to the Global Polio Eradication Initiative has grown with the increasing incidence of vaccine-derived poliovirus (VDPV) infections in Africa specifically (470 reported cases in 2019), and globally, alongside persistent transmission of serotype 1 wild-type poliovirus in Pakistan and Afghanistan (197 reported cases in 2019). Adapting what has been learned about the virus genetics and evolution to address these threats has been a major focus of recent work. Here, we review how phylogenetic and phylogeographic methods have been used to trace the spread of wild-type polioviruses and identify the likely origins of VDPVs. We highlight the analysis methods and sequencing technology currently used and the potential for new technologies to speed up poliovirus detection and the interpretation of genetic data. At a pivotal point in the eradication campaign with the threat of anti-vaccine sentiment and donor and public fatigue, innovation is critical to maintain drive and overcome the last remaining circulating virus.

Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing.

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

Dynamics of toxigenic Clostridium perfringens colonisation in a cohort of prematurely born neonatal infants.

BACKGROUND: Clostridium perfringens forms part of the human gut microbiota and has been associated with life-threatening necrotising enterocolitis (NEC) in premature infants. Whether specific toxigenic strains are responsible is unknown, as is the extent of diversity of strains in healthy premature babies. We investigated the C. perfringens carrier status of premature infants in the neonatal intensive care unit, factors influence this status, and the toxic potential of the strains. METHODS: C. perfringens was isolated by culture from faecal samples from 333 infants and their toxin gene profiles analysed by PCR. A survival analysis was used to identify factors affecting probability of carriage. Competitive growth experiments were used to explore the results of the survival analysis. RESULTS: 29.4% of infants were colonized with C. perfringens before they left hospital. Three factors were inversely associated with probability of carriage: increased duration of maternal milk feeds, CPAP oxygen treatment and antibiotic treatment. C. perfringens grew poorly in breast milk and was significantly outperformed by Bifidobacterium infantis, whether grown together or separately. Toxin gene screening revealed that infants carried isolates positive for collagenase, perfringolysin O, beta 2, beta, becA/B, netB and enterotoxin toxin genes, yet none were observed to be associated with the development of NEC. CONCLUSIONS: Approximately a third of preterm infants are colonised 3 weeks after birth with toxin gene-carrying C. perfringens. We speculate that increased maternal breast milk, oxygen and antibiotic treatment creates an environment in the gut hostile to growth of C. perfringens. Whilst potentially toxigenic C. perfringens isolates were frequent, no toxin type was associated with NEC. TRIAL REGISTRATION: clinicaltrials.gov NCT01102738, registered 13th April 2010.

Antibiotic resistance potential of the healthy preterm infant gut microbiome.

BACKGROUND: Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. RESULTS: Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. CONCLUSIONS: We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.

Assessing the Colonic Microbiota in Children: Effects of Sample Site and Bowel Preparation.

OBJECTIVES: Inflammatory bowel disease states are associated with gastrointestinal dysbiosis. Mucosal biopsy sampling, retrieving the bacterial community that most directly interacts with the host, is an invasive procedure that, we hypothesis, may be sufficiently approximated by other sampling methods. We investigate the relatedness of samples obtained by different methods and the effects of bowel preparation on the gastrointestinal community in a paediatric population. METHODS: We recruited a cohort of patients undergoing colonoscopy, collecting serial samples via differing methods (rectal swabs, biopsies, and faecal matter/luminal contents) prebowel preparation, during colonoscopy and after colonoscopy. Next-generation sequencing was used to determine the structure of the microbial community. RESULTS: The microbial community in luminal contents collected during colonoscopy was found to be more similar to that of mucosal biopsies than rectal swabs. Community traits of the mucosal biopsies could be used to segregate patients with inflammatory bowel disease from other patients, and the similarity of the communities in the luminal contents was sufficient for the segregation to be reproduced. Microbial communities sampled by rectal swabs and prebowel preparation faeces were less similar to mucosal biopsies. Bowel preparation was found to have no significant long-term effects on the microbial community, despite the transient effects evident during colonoscopy. CONCLUSIONS: A clinically relevant description of the mucosal microbial community can be obtained via the noninvasive collection of luminal contents after bowel cleansing. Bowel preparation in a paediatric population results in no consistent sustained alterations to the gastrointestinal microbiota.

Latitude in sample handling and storage for infant faecal microbiota studies: the elephant in the room?

BACKGROUND: In this manuscript, we investigate the "stones best left unturned" of sample storage and preparation and their implications for the next-generation sequencing of infant faecal microbial communities by the 16S ribosomal ribonucleic acid (rRNA) gene. We present a number of experiments that investigate the potential effects of often overlooked methodology factors, establishing a "normal" degree of variation expected between replica sequenced samples. Sources of excess variation are then identified, as measured by observation of alpha diversity, taxonomic group counts and beta diversity magnitudes between microbial communities. RESULTS: Extraction of DNA from samples on different dates, by different people and even using varied sample weights results in little significant difference in downstream sequencing data. A key assumption in many studies is the stability of samples stored long term at -80 °C prior to extraction. After 2 years, we see relatively few changes: increased abundances of lactobacilli and bacilli and a reduction in the overall OTU count. Where samples cannot be frozen, we find that storing samples at room temperature does lead to significant changes in the microbial community after 2 days. Mailing of samples during this time period (a common form of sample collection from outpatients for example) does not lead to any additional variation. CONCLUSIONS: Important methodological standards can be drawn from these results; painstakingly created archives of infant faecal samples stored at -80 °C are still largely representative of the original community and varying factors in DNA extraction methodology have comparatively little effect on overall results. Samples taken should ideally be either frozen at -80 °C or extracted within 2 days if stored at room temperature, with mail samples being mailed on the day of collection.

Late-Onset Bloodstream Infection and Perturbed Maturation of the Gastrointestinal Microbiota in Premature Infants.

BACKGROUND: Late-onset bloodstream infection (LO-BSI) is a common complication of prematurity, and lack of timely diagnosis and treatment can have life-threatening consequences. We sought to identify clinical characteristics and microbial signatures in the gastrointestinal microbiota preceding diagnosis of LO-BSI in premature infants. METHOD: Daily faecal samples and clinical data were collected over two years from 369 premature neonates (<32 weeks gestation). We analysed samples from 22 neonates who developed LO-BSI and 44 matched control infants. Next-generation sequencing of 16S rRNA gene regions amplified by PCR from total faecal DNA was used to characterise the microbiota of faecal samples preceding diagnosis from infants with LO-BSI and controls. Culture of selected samples was undertaken, and bacterial isolates identified using MALDI-TOF. Antibiograms from bloodstream and faecal isolates were compared to explore strain similarity. RESULTS: From the week prior to diagnosis, infants with LO-BSI had higher proportions of faecal aerobes/facultative anaerobes compared to controls. Risk factors for LO-BSI were identified by multivariate analysis. Enterobacteriaceal sepsis was associated with antecedent multiple lines, low birth weight and a faecal microbiota with prominent Enterobacteriaceae. Staphylococcal sepsis was associated with Staphylococcus OTU faecal over-abundance, and the number of days prior to diagnosis of mechanical ventilation and of the presence of centrally-placed lines. In 12 cases, the antibiogram of the bloodstream isolate matched that of a component of the faecal microbiota in the sample collected closest to diagnosis. CONCLUSIONS: The gastrointestinal tract is an important reservoir for LO-BSI organisms, pathogens translocating across the epithelial barrier. LO-BSI is associated with an aberrant microbiota, with abundant staphylococci and Enterobacteriaceae and a failure to mature towards predominance of obligate anaerobes.

Dysbiosis anticipating necrotizing enterocolitis in very premature infants.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants. METHODS: Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization-time of flight. Clostridial isolates were typed and toxin genes detected. RESULTS: A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk. CONCLUSIONS: Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC. Clinical Trials Registration. NCT01102738.