Effects of a live versus heat-inactivated probiotic Bifidobacterium spp in preterm infants: a randomised clinical trial.

BACKGROUND: Heat-inactivated probiotics (HPs) may provide an effective alternative to live probiotics (P) by avoiding their risks (eg, probiotic sepsis) while retaining the benefits. We assessed the safety and efficacy of a HP in very preterm (VP: gestation <32 weeks) infants. METHODS: VP infants were randomly allocated to receive a HP or P mixture (Bifidobacterium breve M-16V, Bifidobacterium longum subsp. infantis M-63, Bifidobacterium longum subsp. longum BB536, total 3×109 CFU/day) assuring blinding. Primary outcome was faecal calprotectin (FCP) levels were compared after 3 weeks of supplementation. Secondary outcomes included faecal microbiota and short chain fatty acid (SCFA) levels. RESULTS: 86 VP infants were randomised to HP or P group (n=43 each). Total FCP and SCFA were comparable between HP and P groups within 7 days (T1) and between day 21 and 28 (T2) after supplementation. At T2, median (range) FCP was 75 (8-563) in the HP group and 80 (21-277) in the P group (p=0.71). Propionate was significantly raised in both groups, while butyrate was significantly raised in the HP group (all p<0.01). Bacterial richness and diversity increased but was comparable between HP and P (p>0.05). Beta diversity showed similar community structures in both groups (all p>0.05). Changes in faecal Actinobacteria, Bacteroidetes and Bifidobacteriacae levels were comparable in both groups at T1 and T2. There was no probiotic sepsis. CONCLUSIONS: HP was safe and showed no significant difference in FCP as compared with a live probiotic. Adequately powered trials are needed to assess the effects of HP on clinically significant outcomes in preterm infants. TRIAL REGISTRATION NUMBER: ACTRN12618000489291.

Probiotic supplementation for neonates with congenital gastrointestinal surgical conditions: guidelines for future research.

Our pilot RCT found that probiotic supplementation with the three-strain bifidobacterial product (B. breve M-16V, B. longum subsp. infantis M-63 and B. longum subsp. longum BB536) attenuates gut dysbiosis, increases stool short-chain fatty acid (SCFA) levels and improves the growth of head circumference in neonates with congenital gastrointestinal surgical conditions (CGISC). In this article, we have provided guidelines for designing future multicentre RCTs based on the experience gained from our pilot RCT. The recommendations include advice about sample size, potential confounders, outcomes of interest, probiotic strain selection, storage, dose, duration and microbial quality assurance, collection of stool samples, storage and analysis and reporting. Following these guidelines will increase the validity of future RCTs in this area and hence confidence in their results. IMPACT: Probiotic supplementation attenuates gut dysbiosis, increases stool short-chain fatty acid (SCFA) levels and improves the growth of head circumference in neonates with congenital gastrointestinal surgical conditions. The current review provides evidence-based guidelines to conduct adequately powered RCTs in this field.

Effect of single versus multistrain probiotic in extremely preterm infants: a randomised trial.

OBJECTIVE: Evidence indicates that multistrain probiotics benefit preterm infants more than single-strain (SS) probiotics. We assessed the effects of SS versus triple-strain (TS) probiotic supplementation (PS) in extremely preterm (EP) infants. DESIGN: EP infants (gestational age (GA) <28 weeks) were randomly allocated to TS or SS probiotic, assuring blinding. Reference (REF) group was EP infants in the placebo arm of our previous probiotic trial. PS was commenced with feeds and continued until 37 weeks' corrected GA. Primary outcome was time to full feed (TFF: 150 mL/kg/day). Secondary outcomes included short-chain fatty acids and faecal microbiota collected at T1 (first week) and T2 (after 3 weeks of PS) using 16S ribosomal RNA gene sequencing. RESULTS: 173 EP (SS: 86, TS: 87) neonates with similar GA and birth weight (BW) were randomised. Median TFF was comparable (11 (IQR 8-16) vs 10 (IQR 8-16) days, p=0.92). Faecal propionate (SS, p<0.001, and TS, p=0.0009) and butyrate levels (TS, p=0.029) were significantly raised in T2 versus T1 samples. Secondary clinical outcomes were comparable. At T2, alpha diversity was comparable (p>0.05) between groups, whereas beta-diversity analysis revealed significant differences between PS and REF groups (both p=0.001). Actinobacteria were higher (both p<0.01), and Proteobacteria, Firmicutes and Bacteroidetes were lower in PS versus REF. Gammaproteobacteria, Clostridia and Negativicutes were lower in both PS versus REF. CONCLUSION: TFF in EP infants was similar between SS and TS probiotics. Both probiotics were effective in reducing dysbiosis (higher bifidobacteria and lower Gammaproteobacteria). Long-term significance of increased propionate and butyrate needs further studies. TRIAL REGISTRATION NUMBER: ACTRN 12615000940572.

Preterm Infant Feeding: A Mechanistic Comparison between a Vacuum Triggered Novel Teat and Breastfeeding.

The goal for preterm infants is to achieve full oral feeds quickly and ultimately progress to full breastfeeding. Supplementary oral feeds are often given when the mother is not available to breastfeed. Bottles typically deliver milk in a different fashion compared to breastfeeding, which is thought to hamper transition to full breastfeeding. The aim of this study was to compare the sucking dynamics of preterm infants fed at the breast to feeding with an experimental novel teat (NT) designed to release milk only upon the application of vacuum. Simultaneous ultrasound imaging of the infant oral cavity and measurement of intra-oral vacuum was performed during a breastfeed and a feed with the NT. Test weighs were used to measure milk intake. Linear mixed effects models were performed to investigate differences by feed type, and simultaneous linear regression was performed to investigate individual patterns. Tongue movement was not different between breastfeeding and the NT. Intra-oral vacuums (median (interquartile range: IQR)) were significantly lower with the NT (Baseline vacuum: -5.8 mmHg (-11.0, 0.1); Peak: 40.0 mmHg (-54.6, -27.1)) compared to breastfeeding (Baseline: -31.1 mmHg (-60.0, -12.7); Peak: -106.2 mmHg (-153.0, -65.5)). Milk intake was significantly higher with the NT (33 mL (22.5, 42.5)) compared to the breastfeed (12 mL (3, 15.5)). The novel teat encouraged a similar tongue action to breastfeeding, and infants transferred a greater volume of milk with the novel teat. Intra-oral vacuums were lower in strength with the novel teat compared to the breast. Use of the novel teat for the training of sucking dynamics in preterm infants has the potential to improve breastfeeding success and requires further investigation.

Levels of innate immune factors in preterm and term mothers' breast milk during the 1st month postpartum.

There is a paucity of data on the effect of preterm birth on the immunological composition of breast milk throughout the different stages of lactation. We aimed to characterise the effects of preterm birth on the levels of immune factors in milk during the 1st month postpartum, to determine whether preterm milk is deficient in antimicrobial factors. Colostrum (days 2-5 postpartum), transitional milk (days 8-12) and mature milk (days 26-30) were collected from mothers of extremely preterm (<28 weeks of gestation, n 15), very preterm (28-<32 weeks of gestation, n 15), moderately preterm (32-<37 weeks of gestation, n 15) and term infants (37-41 weeks of gestation, n 15). Total protein, lactoferrin, secretory IgA, soluble CD14 receptor (sCD14), transforming growth factor-ß2 (TGF-ß2), α defensin 5 (HD5), ß defensins 1 (HBD1) and 2, IL-6, IL-10, IL-13, interferon-γ, TNF-α and lysozyme (LZ) were quantified in milk. We examined the effects of lactation stage, gestational age, volume of milk expressed, mode of delivery, parity and maternal infection on milk immune factor concentrations using repeated-measures regression analysis. The concentrations of all factors except LZ and HD5 decreased over the 1st month postpartum. Extremely preterm mothers had significantly higher concentrations of HBD1 and TGF-ß2 in colostrum than term mothers did. After controlling for other variables in regression analyses, preterm birth was associated with higher concentrations of HBD1, LZ and sCD14 in milk samples. In conclusion, preterm breast milk contains significantly higher concentrations of some immune proteins than term breast milk.

Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry.

BACKGROUND: Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood. METHODS: Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28-31 wk), and moderately preterm (32-36 wk), as well as term (37-41 wk) infants were recruited. Colostrum (d2-5), transitional (d8-12) and mature milk (d26-30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry. RESULTS: The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed. CONCLUSIONS: Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk.

Antimicrobial protein and Peptide concentrations and activity in human breast milk consumed by preterm infants at risk of late-onset neonatal sepsis.

OBJECTIVE: We investigated the levels and antimicrobial activity of antimicrobial proteins and peptides (AMPs) in breast milk consumed by preterm infants, and whether deficiencies of these factors were associated with late-onset neonatal sepsis (LOS), a bacterial infection that frequently occurs in preterm infants in the neonatal period. STUDY DESIGN: Breast milk from mothers of preterm infants (≤ 32 weeks gestation) was collected on days 7 (n = 88) and 21 (n = 77) postpartum. Concentrations of lactoferrin, LL-37, beta-defensins 1 and 2, and alpha-defensin 5 were measured by enzyme-linked immunosorbent assay. The antimicrobial activity of breast milk samples against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae was compared to the activity of infant formula, alone or supplemented with physiological levels of AMPs. Samples of breast milk fed to infants with and without subsequent LOS were compared for levels of AMPs and inhibition of bacterial growth. RESULTS: Levels of most AMPs and antibacterial activity in preterm breast milk were higher at day 7 than at day 21. Lactoferrin was the only AMP that limited pathogen growth >50% when added to formula at a concentration equivalent to that present in breast milk. Levels of AMPs were similar in the breast milk fed to infants with and without LOS, however, infants who developed LOS consumed significantly less breast milk and lower doses of milk AMPs than those who were free from LOS. CONCLUSIONS: The concentrations of lactoferrin and defensins in preterm breast milk have antimicrobial activity against common neonatal pathogens.

Phagocytosis of neonatal pathogens by peripheral blood neutrophils and monocytes from newborn preterm and term infants.

BACKGROUND: Deficiencies in phagocytosis may contribute to the increased susceptibility of infants to early life infections. Data on phagocytosis of the major neonatal pathogens Staphylococcus epidermidis (SE), Staphylococcus aureus (SA), and Escherichia coli (EC) by preterm infant leukocytes are inconsistent. METHODS: Cord and <24-h peripheral blood were collected from very preterm (<30.1 wks gestational age (GA)) and term (37-42 wks GA) infants. Monocyte and neutrophil phagocytosis of pHrodo-labeled SE, SA, and EC were analyzed using a small-volume flow cytometry assay, with simultaneous characterization of surface activation marker expression. RESULTS: Preterm infants had lower proportions of monocytes and neutrophils capable of phagocytosis than term infants, but preterm infant phagocytes had higher phagocytic capacity. Phagocytosis was strongly correlated between cord and <24-h peripheral blood. Supplementation with exogenous complement significantly increased phagocytosis of EC but not of SE or SA. Monocyte human leukocyte antigen (HLA)-DR expression was lower in preterm infants but did not correlate with phagocytosis. CONCLUSION: There is no defect in phagocytosis by monocytes and neutrophils from preterm compared with term infants, although preterm infants possess fewer phagocytes, possibly contributing to susceptibility to bacterial infection. Further investigation into the development of postnatal phagocytic competence is warranted.

A comparison of radiometric correction techniques in the evaluation of the relationship between LST and NDVI in Landsat imagery.

Atmospheric corrections for multi-temporal optical satellite images are necessary, especially in change detection analyses, such as normalized difference vegetation index (NDVI) rationing. Abrupt change detection analysis using remote-sensing techniques requires radiometric congruity and atmospheric correction to monitor terrestrial surfaces over time. Two atmospheric correction methods were used for this study: relative radiometric normalization and the simplified method for atmospheric correction (SMAC) in the solar spectrum. A multi-temporal data set consisting of two sets of Landsat images from the period between 1991 and 2002 of Penang Island, Malaysia, was used to compare NDVI maps, which were generated using the proposed atmospheric correction methods. Land surface temperature (LST) was retrieved using ATCOR3_T in PCI Geomatica 10.1 image processing software. Linear regression analysis was utilized to analyze the relationship between NDVI and LST. This study reveals that both of the proposed atmospheric correction methods yielded high accuracy through examination of the linear correlation coefficients. To check for the accuracy of the equation obtained through linear regression analysis for every single satellite image, 20 points were randomly chosen. The results showed that the SMAC method yielded a constant value (in terms of error) to predict the NDVI value from linear regression analysis-derived equation. The errors (average) from both proposed atmospheric correction methods were less than 10%.