Janet E. Berrington

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection

Aim:  To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS).

Development of the Preterm Gut Microbiome in Twins at Risk of Necrotising Enterocolitis and Sepsis

The preterm gut microbiome is a complex dynamic community influenced by genetic and environmental factors and is implicated in the pathogenesis of necrotising enterocolitis (NEC) and sepsis. We aimed to explore the longitudinal development of the gut microbiome in preterm twins to determine how shared environmental and genetic factors may influence temporal changes and compared this to the expressed breast milk (EBM) microbiome. Stool samples (n = 173) from 27 infants (12 twin pairs and 1 triplet set) and EBM (n = 18) from 4 mothers were collected longitudinally. All samples underwent PCR-DGGE (denaturing gradient gel electrophoresis) analysis and a selected subset underwent 454 pyrosequencing. Stool and EBM shared a core microbiome dominated by Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae. The gut microbiome showed greater similarity between siblings compared to unrelated individuals. Pyrosequencing revealed a reduction in diversity and increasing dominance of Escherichia sp. preceding NEC that was not observed in the healthy twin. Antibiotic treatment had a substantial effect on the gut microbiome, reducing Escherichia sp. and increasing other Enterobacteriaceae. This study demonstrates related preterm twins share similar gut microbiome development, even within the complex environment of neonatal intensive care. This is likely a result of shared genetic and immunomodulatory factors as well as exposure to the same maternal microbiome during birth, skin contact and exposure to EBM. Environmental factors including antibiotic exposure and feeding are additional significant determinants of community structure, regardless of host genetics.

Deaths in Preterm Infants: Changing Pathology Over 2 Decades

OBJECTIVE To establish how cause of death for live-born preterm infants (24-31 weeks gestation) has changed in a single large UK population over 2 decades. STUDY DESIGN This was an interrogation of a population-based survey of >680, 000 live births (between 1988 and 2008) for deaths in the first postnatal year. We collected cause of death grouped into major etiologies: respiratory, infection, malformation, necrotizing enterocolitis (NEC), and other. Data were analyzed in three 7-year epochs and 2 gestational groups (<27 and 28-31 weeks). Numbers, rates per 1000 live births, and proportional contributions to each epoch were analyzed. RESULTS A total of 1504 deaths occurred. The infants who died had a median gestational age of 26 weeks (IQR, 25-28 weeks) and a median birth weight of 880 g (IQR, 700-1170 g). The number of deaths decreased with each later epoch (from 671 to 473 and then to 360), as did the proportion of deaths from respiratory causes (64% to 62% and then to 49%). The proportion of deaths occurring after 40 weeks postmenstrual age remained stable across the 3 epochs (8.8%, 8%, and 8%). Deaths from infection and NEC increased with time (from 11% to 13% and then to 21%), as did median time to death (from 2.7 to 3.8 days). CONCLUSION Infection and NEC are increasingly prevalent causes of death in preterm infants.

Lymphocyte subsets in term and significantly preterm UK infants in the first year of life analysed by single platform flow cytometry

This observational study describes the ranges observed for lymphocyte subsets for significantly preterm infants (<32 weeks) in the first year of life, measured by single platform flow cytometry and compared to identically determined subsets in term infants. After ethical approval 39 term and 28 preterm infants had lymphocyte subset analysis before and after their primary immunization series. Median values with 5th and 95th percentiles of absolute counts and percentages are presented for total lymphocytes, T cells, NK cells, B cells, cytotoxic T cells, helper T cells, dual positive T cells, activated T cells, activated T helper cells (including T regulatory cells), pan memory T cells, pan naïve T cells, memory helper T cells, naïve helper T cells and the T helper/suppressor ratio. The lymphocyte profile of the preterm infants differed from that of the term infants.

Gut microbiota in preterm infants: assessment and relevance to health and disease

In adults the microbial community of the gut (microbiota) influences a diverse range of health outcomes from obesity, diabetes, asthma and allergy to seemingly ‘remote’ diseases like Parkinsons disease.1 In preterm infants, establishment of the gut microbiota is also of importance for key morbidities like late onset sepsis (LOS) and necrotising enterocolitis (NEC), both significant causes of mortality.2 Many episodes of LOS are with gut derived organisms3 and changes in the intestinal barrier contribute to both LOS and NEC. The gut microbiota are key to developing barrier function, integrity, and mucosal and systemic immune function. They also ‘educate’ the gut associated lymphoid tissue, allowing the establishment of a ‘tolerant’ state between microbiota and the immune system, affecting intestinal function including tight junction structure and immune function.4–6 Patterns of initial colonisation affect host metabolic function: fat deposition, circulating leptin levels, and insulin resistance.6 In the preterm gut structural and immunological immaturity contribute to inflammatory necrosis and abnormal bacterial colonisation (dysbioses). This may result in decreased microbial diversity and an increased inflammatory response exacerbated by an immature innate immune response that increases the risk of diseases like NEC or LOS. An improved understanding of the microbiota of infants cared for in neonatal intensive care, and how this is affected by current practices may allow clinicians to promote more ‘healthy’ gut microbiota patterns, and may be associated with reductions in mortality and improvements in long term outcomes.7 ### What factors associated with preterm birth affect the gut microbiota? Early differences in delivery mode and care alter the development of the gut microbiota. Vaginally delivered term infants acquire their gut microbiota from maternal exposures (genital, stool, skin and breast milk flora) and the wider environment. In contrast, preterm infants are more commonly delivered by caesarean section, and have additional ‘risks and exposures’: antenatal and postnatal antibiotics, infection control measures …

Unsuspected Pneumocystis carinii pneumonia at presentation of severe primary immunodeficiency.

BACKGROUND Pneumocystis carinii is an important pathogen in immunodeficiency but may be an unrecognised cause of respiratory compromise. OBJECTIVES To ascertain the incidence of P cariniipneumonia (PCP) at presentation of severe combined immunodeficiency (SCID), whether it had been diagnosed, and the effect of treatment on outcome. SETTING The supraregional paediatric bone marrow transplant unit for primary immunodeficiencies at Newcastle General Hospital. METHODS Retrospective case note review of infants referred with a diagnosis of SCID from 1992 to 1998. RESULTS Ten of 50 infants had PCP at presentation; only one was diagnosed before transfer. Eight were diagnosed by bronchoalveolar lavage and two by lung biopsy. In only one was P cariniiidentified in nasopharyngeal secretions. Five required ventilation for respiratory failure but all were successfully treated with co-trimoxazole and methylprednisolone with or without nebulised budesonide. Nine survived to bone marrow transplantation and four are long term survivors after bone marrow transplantation; no deaths were related to PCP. CONCLUSIONS PCP is a common presenting feature of SCID but is rarely recognised. Bronchoalveolar lavage or lung biopsy are needed for diagnosis. Treatment with co-trimoxazole is highly successful.

Bacterial and fungal viability in the preterm gut: NEC and sepsis

Background and aims Evidence suggests that microbial communities in the preterm gut may influence the development of necrotising enterocolitis (NEC) and sepsis. Existing data often neglect fungi and whether bacteria were metabolically active or not. We sought to characterise the bacterial and fungal stool flora of preterm neonates and organism viability and evaluate any associations with NEC and sepsis. Patients 136 stools from 32 patients (<32 weeks gestation) were collected between birth and day 95. Seven infants developed NEC and 13 sepsis. Methods Stools were analysed by PCR-DGGE for assessment of the total bacterial and fungal communities by analysis of 16S rRNA and 28S rRNA, respectively. In 65 samples (25 infants), the viable (RNA) bacterial and fungal communities were analysed. Analyses were performed to examine the possible effects of demographic or treatment related factors and the development of NEC or sepsis. Results 80 (66 viable) bacterial species were identified overall and 12 fungal (none viable). Total bacterial communities significantly differed between healthy infants and those with NEC or sepsis, with Sphingomonas spp. significantly associated with NEC. Significant drivers of community structure differed based on either total or viable analysis. Antifungal prophylaxis was associated with altered bacterial community and a reduction in bacterial richness was observed in week 4, correlating with high antibiotic exposure. Conclusions Total and viable communities differ in preterm infants, and non-viable fungal species are present in infants on fungal prophylaxis. Exploration of viability and non-bacterial contributors to the total community may increase understanding of NEC and sepsis.

Lactoferrin: Antimicrobial activity and therapeutic potential

Lactoferrin is a highly conserved protein from an evolutionary perspective, with a wide range of roles related to protection from infection and promotion of nutritional status. Infection, malnutrition and intestinal pathologies are key inter-related problems, represent important threats to survival and are associated with adverse long-term health outcomes after preterm birth. Lactoferrin is available as a commercial extract from bovine milk and offers potential as a therapeutic intervention for preterm infants modulating infections and intestinal pathologies. In this review we explore the structure, direct antimicrobial effects, modification of host immune function and gastrointestinal effects of lactoferrin. Current trial data are reviewed, and research priorities and challenges identified and discussed.

The neonatal bowel microbiome in health and infection

Purpose of review In newborns, interactions between the host and the microbiome operate synergistically, modulating host immune function and shaping the microbiome. Next generation molecular sequencing methodologies in tandem with modeling complex communities allow insights into the role of the microbiome in health and disease states. Infection-related disease states in which dysbiosis is integral include late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), which still cause deaths and morbidity. Understanding microbiomic interactions may lead to alternative prevention, monitoring or treatment strategies, and modulation of long-term health outcomes especially in the preterm population. Recent studies have advanced understanding of the microbiome in NEC and LOS. Recent findings Mechanisms of host–microbiome interaction have been demonstrated. Patterns of microbiomic change in association with NEC and LOS have been observed, with community changes dominated by Proteobacteria and Firmicutes appearing to precede NEC, and very early microbiomic signatures influencing LOS. Data on viral and fungal elements are emerging. Summary Greater understanding of the neonatal bowel microbiome may allow tailored clinical practice and therapeutic intervention. Data handling and interpretation is challenging. Mechanistic studies of clinical interventions that affect the gut microbiome are important next steps.

Haemophilus influenzae type b immunization in infants in the United Kingdom: Effects of diphtheria/tetanus/acellular pertussis/Hib combination vaccine, significant prematurity, and a fourth dose

OBJECTIVE. To measure anti-polyribosylribitolphosphate (PRP) antibody and anti–tetanus toxoid (TT) antibody responses in UK infants to explore the effects of (1) immunization with an acellular diphtheria/tetanus/pertussis/Haemophilus influenzae type b (DTPHib) combination vaccine, (2) significant preterm delivery, and (3) a fourth dose of conjugated Hib vaccine (PRP-T) in those with a low anti-PRP antibody (<1.0 μg/mL) after primary immunization. METHODS. A prospective study was conducted in 4 tertiary neonatal units at a time when 2 types of DTPHib vaccines were used interchangeably in the United Kingdom for primary immunization: acellular (DTPaHib) and whole cell. Timing and type of all vaccine doses were as per standard UK practice. Blood was taken before and after immunization. A total of 166 preterm and 45 term infants completed the study; 97 (15 term) infants who had anti-PRP antibody <1.0 μg/mL were offered a fourth dose of PRP-T; 61 (55 preterm) then had repeat antibody measurements. Anti-PRP and anti-TT antibody after primary immunization relative to gestation and number of whole cell vaccine doses received was measured, as well as anti-PRP antibody after a fourth dose of PRP-T. RESULTS. A total of 49% of preterm and 33% of term infants had anti-PRP antibody <1.0 μg/mL after full primary immunization. Receipt of 1 or more acellular vaccine doses was associated with lower anti-PRP antibody, a dose response effect being observed. Preterm infants were less likely to have anti-PRP antibody >1.0 μg/mL compared with term infants. A total of 93% of infants who were given a fourth dose had anti-PRP antibody >1.0 μg/mL. Anti-TT antibody responses were satisfactory for all infants but also reduced by each DTPaHib dose received. CONCLUSION. Infants who receive DTPaHib, are significantly preterm, or who do not receive a fourth dose of conjugated Hib vaccine may be at increased risk for Hib disease.