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      Antimicrobial Protein and Peptide Concentrations and Activity in Human Breast Milk Consumed by Preterm Infants at Risk of Late-Onset Neonatal Sepsis

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          Abstract

          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.

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          Most cited references39

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          Neonatal infectious diseases: evaluation of neonatal sepsis.

          Neonatal sepsis remains a feared cause of morbidity and mortality in the neonatal period. Maternal, neonatal, and environmental factors are associated with risk of infection, and a combination of prevention strategies, judicious neonatal evaluation, and early initiation of therapy are required to prevent adverse outcomes. This article reviews recent trends in epidemiology and provides an update on risk factors, diagnostic methods, and management of neonatal sepsis. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Impact of Early Human Milk on Sepsis and Health Care Costs in Very Low Birth Weight Infants

            Objective To study the incidence of sepsis and neonatal intensive care unit (NICU) costs as a function of the human milk (HM) dose received during the first 28 days post-birth for very low birth weight (VLBW) infants. Study Design Prospective cohort study of 175 VLBW infants. Average daily dose of HM (ADDHM) was calculated from daily nutritional data for the first 28 days post-birth (ADDHM-Days1-28). Other covariates associated with sepsis were used to create a propensity score, combining multiple risk factors into a single metric. Result The mean gestational age and birth weight were 28.1 ± 2.4 wk and 1087 ± 252 g, respectively. The mean ADDHM-Days1-28 was 54 ± 39 mL/kg/d (range 0-135). Binary logistic regression analysis controlling for propensity score revealed that increasing ADDHM-Days1-28 was associated with lower odds of sepsis (OR .981, 95%CI .967-.995, p=.008). Increasing ADDHM-Days1-28 was associated with significantly lower NICU costs. Conclusion A dose-response relationship was demonstrated between ADDHM-Days1-28 and a reduction in the odds of sepsis and associated NICU costs after controlling for propensity score. For every HM dose increase of 10 mL/kg/d, the odds of sepsis decreased by 19%. NICU costs were lowest in the VLBW infants who received the highest ADDHM-Days1-28.
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              Antibacterial activity and specificity of the six human {alpha}-defensins.

              We developed a kinetic, 96-well turbidimetric procedure that is capable of testing the antimicrobial properties of six human alpha-defensins concurrently on a single microplate. The defensins were prepared by solid-phase peptide synthesis and tested against gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Enterobacter aerogenes and Escherichia coli). Analysis of the growth curves provided virtual lethal doses (vLDs) equivalent to conventional 50% lethal doses (LD(50)s), LD(90)s, LD(99)s, and LD(99.9)s obtained from colony counts. On the basis of their respective vLD(90)s and vLD(99)s, the relative potencies of human myeloid alpha-defensins against S. aureus were HNP2 > HNP1 > HNP3 > HNP4. In contrast, their relative potencies against E. coli and E. aerogenes were HNP4 > HNP2 > HNP1 = HNP3. HD5 was as effective as HNP2 against S. aureus and as effective as HNP4 against the gram-negative bacteria in our panel. HD6 showed little or no activity against any of the bacteria in our panel, including B. cereus, which was highly susceptible to the other five alpha-defensins. The assay described provides a quantitative, precise, and economical way to study the antimicrobial activities of host-defense peptides. Its use has clarified the relative potencies of human alpha-defensins and raised intriguing questions about the in vivo function(s) of HD6.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2015
                2 February 2015
                : 10
                : 2
                : e0117038
                Affiliations
                [1 ]Centre for Neonatal Research and Education, University of Western Australia, Perth, Western Australia, Australia
                [2 ]School of Paediatrics and Child Health, University of Western Australia, Perth, Western Australia, Australia
                [3 ]Neonatal Clinical Care Unit, King Edward Memorial Hospital for Women, Perth, Western Australia, Australia
                [4 ]School of Public Health, Curtin University, Perth, Australia
                [5 ]School of Women’s and Infants’ Health, University of Western Australia, Perth, Australia
                [6 ]Murdoch Childrens Research Institute, Parkville, Victoria, Australia
                [7 ]University of Melbourne, Melbourne, Victoria, Australia
                [8 ]The University of Edinburgh/MRC Centre for Inflammation Research, Queen’s Medical Research Institute, Edinburgh, United Kingdom
                [9 ]School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia
                University Hospital Schleswig-Holstein, Campus Kiel, GERMANY
                Author notes

                Competing Interests: Dr. Tobias Strunk and Prof. David Burgner are currently listed as members of the editorial board. This does not alter the authors’ adherence to PLOS ONE editorial policies and criteria.

                Conceived and designed the experiments: ST AJC TS DJD PR GZ KS DAD. Performed the experiments: ST. Analyzed the data: ST. Wrote the paper: ST. Acquisition of data: ST JH CHK TS. Critical revision and content of manuscript: TS JH CHK GZ DAD PR DB KS DJD AJC.

                Article
                PONE-D-14-35297
                10.1371/journal.pone.0117038
                4314069
                25643281
                b0ca15c7-3ce8-4383-980b-360105d61ce2
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 6 August 2014
                : 17 December 2014
                Page count
                Figures: 6, Tables: 3, Pages: 20
                Funding
                Funding for this study was awarded to AC, TS, KS, PR, DAD and DB by the National Health and Medical Research Council (572548; www.nhmrc.gov.au), to AC, TS, KS and DB from the Princess Margaret Hospital Foundation ( www.pmhfoundation.com), to AC, DJD, TS, and DB from the Telethon New Childrens Hospital Research Fund ( http://www.health.wa.gov.au), and to TS from the Clive and Vera Ramaciotti Foundation ( www.perpetual.com.au/ramaciotti). DJD is supported by a Medical Research Council Senior Non-clinical Fellowship (G1002046; http://www.mrc.ac.uk) and a Distinguished Collaborator Award from Murdoch University ( www.murdoch.edu.au). DB is supported by a National Health and Medical Research Council Senior Research Fellowship (APP1064629; www.nhmrc.gov.au) and an Honorary National Heart Foundation Australia Future Leader Fellowship (100026; www.heartfoundation.org.au). Research at the Murdoch Childrens Research Institute is supported by the Victorian Government’s Operational Infrastructure Support Program ( www.vic.gov.au). GZ is supported by a Brightspark Foundation Fellowship ( www.brightsparkfoundation.com.au). ST is supported by an Australian postgraduate award ( www.education.gov.au), and postgraduate scholarships from the Women and Infants Research Foundation ( www.wirf.com.au) and the Princess Margaret Hospital Foundation ( www.pmhfoundation.com). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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