Kathleen Sim

Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing

The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, ‘bifidobacteria-optimised’ universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.

Dysbiosis Anticipating Necrotizing Enterocolitis in Very Premature Infants

Using 16S rRNA gene sequencing and targeted culture, we compared microbiota in fecal samples from infants with necrotizing enterocolitis (NEC) and controls. Two significant signatures were associated with NEC: 1 with dominant Clostridium perfringens and 1 with dominant Enterobacteriaceae.

Quantification of HTLV-1 Clonality and TCR Diversity.

Estimation of immunological and microbiological diversity is vital to our understanding of infection and the immune response. For instance, what is the diversity of the T cell repertoire? These questions are partially addressed by high-throughput sequencing techniques that enable identification of immunological and microbiological “species” in a sample. Estimators of the number of unseen species are needed to estimate population diversity from sample diversity. Here we test five widely used non-parametric estimators, and develop and validate a novel method, DivE, to estimate species richness and distribution. We used three independent datasets: (i) viral populations from subjects infected with human T-lymphotropic virus type 1; (ii) T cell antigen receptor clonotype repertoires; and (iii) microbial data from infant faecal samples. When applied to datasets with rarefaction curves that did not plateau, existing estimators systematically increased with sample size. In contrast, DivE consistently and accurately estimated diversity for all datasets. We identify conditions that limit the application of DivE. We also show that DivE can be used to accurately estimate the underlying population frequency distribution. We have developed a novel method that is significantly more accurate than commonly used biodiversity estimators in microbiological and immunological populations.

“Vaginal seeding” of infants born by caesarean section

How should health professionals engage with this increasingly popular but unproved practice?

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.

The neonatal gastrointestinal microbiota: the foundation of future health?

There is a developing appreciation of the abundance and diversity of the trillions of micro-organisms that live on and within us,1 ,2 and how they influence human health and disease.3 Outnumbering human cells in our bodies by 10:1, and their genes outnumbering ours by 100:1,4 bacteria in the gastrointestinal (GI) tract have the potential to significantly modulate human metabolism.5 Previous studies of the microbiota (all the microbes in a given environment) have been hampered by the difficulties of culturing complex samples containing fastidious or unculturable organisms, resulting in inaccurate depictions of microbial communities. Microbiota studies have now progressed to identifying organisms by their DNA sequence. A particular focus has been on the bacterial component, exploiting sequence variation in the ubiquitous 16S rRNA gene. By careful choice of primers hybridising to highly conserved domains within 16S rRNA ,6 a sufficiently large and variable region of this gene can be amplified and sequenced for organisms to be identified at least at genus level, without the bias arising from the need for culture. Utilising next-generation sequencing, which allows millions of sequencing reactions to be performed in parallel, we can determine in quantitative fashion, as never before, the composition of complex bacterial communities,7 such as exist in faecal samples, a pragmatic surrogate for the microbiota of the GI tract mucosal surface.8 Two large international initiatives, The Human Microbiome Project (HMP)9 and Metagenomics of the Human Intestinal Tract (MetaHIT),10 have used this approach to characterise the adult human microbiota at different body sites in health and disease. There is a burgeoning interest in the neonatal GI microbiota, and its association with diseases of prematurity,11 normal child development4 and future health.12 Our group is conducting a longitudinal prospective study assessing the importance of …

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.

Intestinal microbiota in infants at high risk for allergy: Effects of prebiotics and role in eczema development

Background: Development of the gut microbiota in infancy is important in maturation of the immune system. Deviations in colonization patterns have been associated with allergic manifestations such as eczema, but exact microbiome dysfunctions underlying allergies remain unclear. We studied the gut microbiota of 138 infants at increased risk of allergy, participating in a clinical trial investigating the effectiveness of a partially hydrolyzed protein formula supplemented with nondigestible oligosaccharides on the prevention of eczema. Objective: The effects of interventions and breast‐feeding on fecal microbiota were investigated. Additionally, we aimed to identify microbial patterns associated with the onset of eczema. Methods: Bacterial taxonomic compositions in the first 26 weeks of life were analyzed by using 16S rRNA gene sequencing. Additionally, fecal pH and microbial metabolite levels were measured. Results: Fecal microbial composition, metabolites, and pH of infants receiving partially hydrolyzed protein formula supplemented with nondigestible oligosaccharides was closer to that of breast‐fed infants than that of infants receiving standard cows milk formula. Infants with eczema by 18 months showed discordant development of bacterial genera of Enterobacteriaceae and Parabacteroides species in the first 26 weeks, as well as decreased acquisition of lactate‐utilizing bacteria producing butyrate, namely Eubacterium and Anaerostipes species, supported by increased lactate and decreased butyrate levels. Conclusions: We showed that a partially hydrolyzed protein infant formula with specific prebiotics modulated the gut microbiota closer to that of breast‐fed infants. Additionally, we identified a potential link between microbial activity and onset of eczema, which might reflect a suboptimal implementation of gut microbiota at specific developmental stages in infants at high risk for allergy. GRAPHICAL ABSTRACT Figure. No caption available.

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.

Determinants of Carboxyhemoglobin Levels and Relationship with Sepsis in a Retrospective Cohort of Preterm Neonates

Carboxyhemoglobin levels in blood reflect endogenous carbon monoxide production and are often measured during routine blood gas analysis. Endogenous carbon monoxide production has been reported to be increased during sepsis, but carboxyhemoglobin levels have not been thoroughly evaluated as a biomarker of sepsis. We sought to determine whether carboxyhemoglobin levels were elevated during sepsis in a high risk population of premature neonates. We conducted a retrospective cohort study of 30 infants in two neonatal intensive care units using electronic medical and laboratory records. The majority of infants were extremely premature and extremely low birth weight, and 25 had at least one episode of sepsis. We collected all carboxyhemoglobin measurements during their in-patient stay and examined the relationship between carboxyhemoglobin and a variety of clinical and laboratory parameters, in addition to the presence or absence of sepsis, using linear mixed-effect models. We found that postnatal age had the most significant effect on carboxyhemoglobin levels, and other significant associations were identified with gestational age, hemoglobin concentration, oxyhemoglobin saturation, and blood pH. Accounting for these covariates, there was no significant relationship between the onset of sepsis and carboxyhemoglobin levels. Our results show that carboxyhemoglobin is unlikely to be a clinically useful biomarker of sepsis in premature infants, and raise a note of caution about factors which may confound the use of carbon monoxide as a clinical biomarker for other disease processes such as hemolysis.