Glasgow Theses Service http://theses.gla.ac.uk/ theses@gla.ac.uk Beattie, Lynne Mary (2014) Gut bacterial activity in a cohort of preterm infants in health and disease. MD thesis. http://theses.gla.ac.uk/5312/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. 1 Gut bacterial activity in a cohort of preterm infants in health and disease Dr Lynne Mary Beattie, MRCPCH MBChB PGCertMedEd Submitted in fulfilment of the requirements for the degree of Doctorate of Medicine School of Medicine University of Glasgow February 2014 2 Summary Introduction Randomised controlled trials administering probiotic supplements to preterm infants to prevent sepsis and necrotising enterocolitis are already underway, despite the lack of a robust evidence base of normative values for gut microbiota, bacterial metabolites, and markers of inflammation and immunity. There are increasing calls for observational studies to establish baseline data in these infants. Most of these studies to date have involved the measurement of these analytes individually. In the studies presented in this thesis, we measured a range of stool markers collectively in a cohort of preterm infants in health and disease. Design 56 infants at <32 week gestation and less than 1500g birth weight were sequentially recruited from all three Glasgow Neonatal Units within week one of life after commencement of enteral feeds. Anthropometric, dietary and treatment data were collected. Stool samples were taken once weekly for the first four weeks, testing: short chain fatty acids; calprotectin, secretory immunoglobulin A; and microbial diversity by temporal temperature gel electrophoresis. Results Out of 61 live births meeting the study criteria, 56 infants were enrolled in the study, 62.5% of whom were female. 19.6% were between 24-26 weeks gestation, 28% were 26- 28 weeks, 30% were 28-30 weeks, and 21% were 30-32 weeks. 5.3% were between 490- 600g in birth weight, 17.8% were 600-800g, 21.4% were 801-1000g, 39.2% 1001-1250g, and 16% were between 1251-1500g. Feed regimen was heterogeneous, comprising 5 combinations of maternal, donor and formula milks. The highest social deprivation level as measured by the Carlisle ‘Depcat’ scoring system of level 7 was significantly higher in the study group than Glasgow or Scotland-wide averages. Sepsis rates were low, with a group median of only 1 per infant. Overall mortality: 7%. 32 with any NEC (56%), 20 with Bells’ ≥2a NEC. 8 (14%) with surgically treated NEC, 5 (8%) underwent ileostomy. SCFAs: (n=56) there were no correlations between gestation, weekly totals, feed type, or NEC and SCFA concentration. Acetate and lactate dominated each sample. Few significant changes were noted with respect to NEC, and these were in the less dominant SCFAs: stage 2a NEC showed higher concentrations of propionate in week 4 than week 3, and lower valerate in week 4 than 2. Stage 3b levels of isobutyrate and heptanoate were significantly 3 lower in week 4 than 3. FC: (n=56) there were no significant differences in FC levels between each week in infants with or without NEC, although the former illustrated a trend to lower levels by week 4. There were no significant differences in NEC before and after clinical signs were apparent, or in those before NEC and after stoma formation for stage 3b NEC. However, significantly lower FC levels were noted in stage 3b NEC requiring ileostomy compared to the immediate pre-operative sample. SIgA: (n=34) Levels rose significantly week on week, and were considerably higher in weeks three and four than week one. There were no significant differences in stool SIgA concentration between infants with and without NEC. A significant increase in mean stool SIgA concentration appeared from week 2 to week 3 in NEC infants, and from week 1 to week 2 for those without. For all breastfed preterm neonates (n=6), the level of milk SIgA was significant higher on week 1 (colostrum) than week 2 and week 3. TTGE: (n=22) There was large variability between number (1-17) and species diversity (25-36 different species). Bacterial composition varied largely between the 2 sample points. No difference in species richness or similarity within the 2 feeding groups was observed. 4 bands were identified in >50% of infants. Intra-individual similarity varied greatly and ranged from a similarity index (Cs) of 0% to 66.8%. There was no statistical difference between the similarity indices of the feeding groups or between those with and without NEC. There were no significant correlations between any of the analytes. Conclusions Only extreme prematurity and extremely low birth weight were associated with NEC, which was at a strikingly high incidence. A limitation was therefore the unexpected onset of severe NEC resulting in prolonged paralytic ileus with low stool production. No correlations were found between analytes, indicating that each set of stool investigations may signify independent physiological, biochemical and immunological gut processes. Despite the severity of NEC, the levels of each analyte were remarkably consistent. High levels of deprivation within the study population may provide the constellation for an as of yet undefined genetic and epigenetic predisposition to NEC in this cohort, similar to that of other illnesses endemic to different geographical areas – notably Multiple Sclerosis in the North East of Scotland – and both follow up of these infants into childhood as well as further analysis of future inborn infants with NEC is planned. 4 Contents Chapter 1: BACKGROUND Page 1.1 Introduction 29 1.2 Definition and Evolution of Gut Microbiota 30 1.2.1 Definition 30 1.2.2 Functions 31 i) Fermentation, energy absorption and micronutrient 33 production a) Carbohydrates 33 b) Protein 35 c) Lipids 36 d) Micronutrients 37 ii) Trophic factors 37 iii) Immunological, antibiotics and anti-inflammatory properties 38 iv) Anti-carcinogenic properties 39 v) Reduction of serum cholesterol and morbid obesity 40 vi) Hormonal interactions 41 vii) Modulation of neurological development 43 1.3 Microbiota, Metabolism and Markers of Gut Inflammation: Short Chain Fatty Acids 45 1.3.1 Definition and relevance 45 1.3.2 Branched Chain Fatty Acids and products of protein degradation 46 1.3.3 General Functions of Short Chain Fatty Acids 48 1.4 Evolution and Identification of Gut Microflora 51 1.4.1 Introduction 51 1.4.2 Methods of identification 52 i) Culture 53 ii) Culture-independent methods 54 1.5 Acquisition of Gut Microbiota 54 1.5.1 Influencing the infant microbiota perinatally 54 i) In utero effects of maternal dietary pre and probiotic supplementation 54 ii) Establishment of the microbiota at birth 56 iii) Ex utero influences: Nutrition and Environment 56 5 a) Nutrition 56 b) Antibiotics 60 c) Environment 61 1.6 Gut Microbiota and the Preterm Infant 64 1.6.1 Demography and Definitions 64 1.6.2 Effects of Prematurity on the Development and Composition of the Gut Microbiota 65 i) Gestation 65 ii) Preterm versus Small for Gestational Age 65 iii) Effect of Method of Delivery and Incubation 66 iv) Maternal Environment 66 v) Antibiotics 67 vi) Nutrition 68 a) Donor EBM 68 b) Maternal Postnatal Probiotic Supplementation 70 1.6.3 Evidence of Gut Microbiota Species Diversity and Abundance in Preterm Infants 70 1.6.4 Evidence for Normative Data in Stool Metabolites, Inflammation and Immunological Markers of Gut Health in Preterm Infants 75 i) Variation in Stool Bacterial Metabolites in Healthy Preterm Infants 75 ii) Inflammation and Immunoprotection: Calprotectin and Secretory IgA 79 a) Calprotectin 79 b) Secretory IgA 85 1.6.5 Necrotising Enterocolitis 88 i) Definition and incidence 88 ii) Associations with Morbidity and Mortality 88 iii) Aetiology of NEC 89 iv) Diagnosis and Management of NEC 90 1.6.6 Trends in Microbiological Stool Studies of Preterm Infants with NEC 91 1.6.7 Potential Biomarkers of NEC 95 i) Bacterial Metabolites: Toxic Products or Innocent Bystanders? 95 ii) SCFA: Friend or Foe? 97 6 iii) Calprotectin in NEC 99 iv) Secretory IgA in NEC 102 1.6.8 Management of NEC 103 1.6.9 Animal Models: Relevance to Research into NEC 104 1.6.10 Therapeutics: 107 i) Prebiotics 107 ii) Probiotics 108 iii) Synbiotics 110 1.6.11 Therapeutic Alteration in the Gut Microbiota of Preterm Infants 110 i) Prebiotics 110 ii) Probiotics 111 a) Probiotic safety 112 b) Current Randomised Controlled Trials 113 1.7 Introduction to Study Hypothesis 116 Chapter 2: METHODOLOGY 2.1 Introduction 117 2.1.1 Hypotheses 117 i) Primary 117 ii) Secondary 117 2.2 Study Design and Methodology 118 2.2.1 Study Design 118 i) Recruitment 118 ii) Sample collection 119 2.2.2 Analyses 120 2.2.3 NEC 120 2.2.4 Demographical and Clinical Data 121 2.3 Methodology 122 2.3.1 Stool samples 122 2.3.2 Breast milk samples 123 2.3.3 SCFA: GCMS 124 i) Measurement of SCFAs 124 ii) Lactate analysis by GC: trial protocols 125 iii) tBDMS: final protocol 126 iv) Method development: derivatisation and GCMS 127 7 2.3.4 Calprotectin by ELISA 128 2.3.5 Secretory IgA by ELISA 130 2.3.6 Molecular techniques: TTGE 132 i) DNA extraction 132 ii) PCR amplification and protocol optimisation 133 iii) Optimised PCR protocol 134 iv) TTGE 136 v) Data analysis 136 2.3.7 General statistical analysis and data interpretation 138 Chapter 3: Clinical and Demographical Results 3.1 Study population 140 3.1.1 Gender by gestation and birth weight 142 3.1.2 CRIB in preceding 12 hours prior to recruitment 143 3.1.3 Method of delivery 143 3.1.4 Multiparity and chorionicity 144 3.1.5 Depcat scores 145 3.1.6 Apgars 146 3.1.7 PPROM 147 3.1.8 PIH contributing to delivery 147 3.1.9 Presence of umbilical lines, by gestation 148 3.1.10 IUGR and AEDF 148 3.1.11 Duration of incubation 149 3.1.12 Duration of invasive and non-invasive ventilation 150 3.1.13 IVH 150 3.1.14 PDA and ROP 151 3.1.15 Mortality 152 3.1.16 Feed types 153 i) Feed regimen by volume 155 ii) Demography by feed regimen 156 3.1.17 Birth weight and weight gain 158 i) By gestation and feed type 159 ii) Comparison with national z scores 161 3.1.18 Sepsis 162 i) By gestation and feed type 162 3.1.19 Demography by Unit 164 8 3.1.20 NEC: demographical and clinical associations 166 i) All-stage NEC associations 167 ii) Surgical management 169 iii) ≥Stage 2a NEC associations 172 iv) All-stage NEC: significant correlations 173 v) Demographical associations 175 3.1.21 Discussion 178 i) Demography 178 ii) Clinical features 179 iii) Unit differences 180 iv) Feeds 182 v) Growth 182 vi) Sepsis 182 vii) NEC 183 Chapter 4: Bacteria and Bacterial Metabolites 188 4.1 Metabolites: SCFAs and BCFAs 188 4.1.1 Total SCFA concentrations 188 4.1.2 By gestation 190 i) Week by week analysis 191 ii) Week on week comparisons by gestation 196 iii) Ratiometric data 198 4.1.3 By Feed type 199 i) EEBM by week 199 ii) EEBM vs mixed SCFAs, by week 200 iii) Ratiometric data 201 4.1.4 NEC: ≥ Stage 2a 201 i) Total SCFA: ≥ stage 2a NEC versus those without 201 ii) Weekly comparisons 202 iii) Ratiometric data 203 iv) Stage-by-stage comparisons: 2a and 2b, 3a and 3b 205 v) Before and after NEC diagnosis 211 4.1.5 Correlations between analytes 212 4.1.6 Discussion 213 i) Individual and total SCFAs: gestation and feed influences 213 9 ii) Comparison of infants with and without NEC 214 iii) Ratiometric data 215 iv) Comparison with evidence base in healthy preterm infants 216 v) Comparison with the evidence base in NEC 220 4.1.7 Conclusions 221 4.2 TTGE 222 4.2.1 Introduction 222 4.2.2 Clinical and demographical results 222 4.2.3 Outcomes of TTGE analysis 225 i) Number of species present 228 ii) Change in microbiota over time 229 iii) Interindividual similarity 231 iv) Relative abundance of species 232 v) Correlations between analytes 233 4.2.4 Discussion 235 i) Introduction 235 ii) DNA yield 235 iii) Similarities 236 iv) Feed type 238 v) Band numbers 238 vi) Correlations with metabolites 239 vii) Study limitations 239 4.2.5 Conclusions 241 Chapter 5: Gut Inflammation and Immunological Markers 242 5.1 Calprotectin 242 5.1.1 Totals over study period 242 5.1.2 Totals by gestation 242 5.1.3 Week on week totals, by gestation 243 5.1.4 Totals by feed type 244 5.1.5 Totals by ≥ 2a NEC 247 i) By stages of NEC 248 5.1.6 Correlations 249 5.1.7 Discussion 251 [...]... 19: Demographic and clinical features of those with all-stage NEC versus those without Table 20: Comparison of demographical and clinical features in infants with stage 2a, 2b, 3a and 3b NEC Table 21: Clinical and demographical features of those with >stage 2a NEC versus those without NEC Table 22: Table of clinical and demographical characteristics of patients included for TTGE analysis 12 Table 23:... material daily in a healthy adult Adults also both ferment and recycle the products of protein metabolism, including hydrolysis of urea, deamination of amino acids, and recycling of ammonia Nitrosation reactions of secondary amines from amino acid fermentation are associated with an increased risk of colo-rectal cancer (Hughes, Magee et al 2000, Kuhnle and Bingham 2007, Kuhnle, Story et al 2007, Lunn,... Department of Child Health, GU Mrs Karyn Cooper, for her incomparable organisational skills My parents Meg and Graham, my brother Paul, sister in law Yan, and nephew Noah Andy and my daughters Kate and Zed: for everything; for without whom, this is all meaningless Alicia, Study Baby 59, at age 2 – taken and included at parental suggestion ‘Keep calm and carry on’ - British World War II propaganda poster,... setting, and oligosaccharides are now added to sweeteners, baking products, yoghurts, and milkshakes (Sangwan, Tomar et al 2011) • Infants: Term and Preterm Infants delivered at term have higher concentrations of short chain fatty acids earlier in infancy than those born prematurely, owing to a faster rate of colonisation Marked differences are noted according to feed type – particularly between infants. .. chain fatty acids), bacteria (transient temperature gradient electrophoresis), an immunological marker (secretory immunoglobulin A) , and an inflammatory marker (calprotectin) are seen individually in observational studies to vary in preterm infants with and without NEC and sepsis As a panel however, they had not, at the inception of this project, been tested concurrently in a cohort of preterm infants. .. supplementation, given the well-established effect of exclusive maternal breast milk feeding in preventing NEC and sepsis in preterm infants Notably, none of the meta-analyses to date can extrapolate data according to feed type As such, this effect requires ascertainment with comparative analyses in ‘healthy’ preterm infants without probiotic supplementation The stool analyses of: metabolites (short and branched... et al 2013) Many studies, however, are still in animal models, although increasingly, paired data matching qualitative and quantitative molecular analyses with metabolites confirms the ability of prebiotics to promote growth of selective strains, and, in adults, producing beneficial butyrate and reducing parameters linked with protein fermentation (Vitali, Ndagijimana et al 2012, Walton, Lu et al 2012)... data Within the body of the background text, I performed all systematic reviews of the evidence as presented in table form and discussed thereafter, as well as creating all figures and tables Graphs and tables for the SCFA and FC results were created by me All others were created by me and collaborators KG, KB and MW 24 Common Abbreviations AEDF Absent End Diastolic Flow ANOVA Analysis of Variance APGAR... 81: a) Individual SCFA concentrations in infants with 3a+ b NEC, week 1; b) Individual SCFA concentrations in infants with 3a+ b NEC, week 2 Figure 82: a) Individual SCFA concentrations in infants with 3a+ b NEC, week 3; b) Individual SCFA concentrations in infants with 3a+ b NEC, week 4 Figure 83: a) : Concentrations of acetic acid in week 1 and week 4 in those with 3a + b NEC; b): Concentrations of acetic... Number of species present at the two sample points Table 24: Clinical and Demographical Features of infants included in SIgA analysis Table 25: T–test for equality of means of four weeks stool SIgA concentration (in log) between infants with and without NEC Table 26: Stool SIgA concentration (in log) in exclusively breast fed and mix breast milk and formula fed preterm neonates Table 27: Differences of . Metabolites, Inflammation and Immunological Markers of Gut Health in Preterm Infants 75 i) Variation in Stool Bacterial Metabolites in Healthy Preterm Infants 75 ii) Inflammation and Immunoprotection:. for gut microbiota, bacterial metabolites, and markers of inflammation and immunity. There are increasing calls for observational studies to establish baseline data in these infants. Most of. Glasgow Theses Service http://theses.gla.ac.uk/ theses@gla.ac.uk Beattie, Lynne Mary (2014) Gut bacterial activity in a cohort of preterm infants in health and disease. MD thesis.