{"paper_id":"3fdfbb51-9ea3-4161-a478-258c46393a78","body_text":"Comparison of bacterial profiles in human milk from mothers of term and preterm infants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of bacterial profiles in human milk from mothers of term and preterm infants Kumiko Miura, Miori Tanaka, Midori Date, Mizuho Ito, Noriko Mizuno, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2449355/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jun, 2023 Read the published version in International Breastfeeding Journal → Version 1 posted 7 You are reading this latest preprint version Abstract Background Bacteria in human milk (HM) can be endogenous or exogenous, and the latter can carry the risk of various infections in very low-birth weight infants because of the possibility of contamination with pathogenic bacteria. The mother's lifestyle and environment have a major influence on such bacterial contamination, and it is thought that there are differences in the number and types of bacteria cultured from HM between term mothers whose infants are at home and mothers of preterm infants in neonatal intensive care units (NICUs). This research aimed to compare the bacterial profiles of HM among mothers of term and preterm infants. Methods The data comprised 214 milk samples (term: 75, preterm: 139) donated by 47 registered donors (term: 31, preterm: 16) from January to November 2021. Bacterial culture results were compared between term and preterm HM samples. Differences in the mean total bacterial count and bacterial species count per batch were analyzed using Welch’s t-test and Student's t-test, respectively. The bacterial contamination rate was analyzed using Chi-square test or Fisher's exact test. Results Coagulase-negative Staphylococci , Staphylococcus aureus , and Pseudomonas fluorescens were frequently found in both term and preterm HM. Serratia liquefaciens (p < 0.001) and two other bacteria contaminated term HM, while five types of bacteria, including Enterococcus faecalis and Enterobacter aerogenes (p < 0.001) contaminated preterm HM. The mean (SD) total bacterial count was 351,141 (1,060,949) CFU/100 µL for term HM and 872,272 (2,324,477) CFU/100 µL for preterm HM (p = 0.026). Similarly, the number of bacterial species in HM was more diverse in preterm donors (p < 0.001). Conclusions This study revealed that HM from preterm donors has a higher total bacterial count and greater diversity and characterization of bacterial types compared with HM from term donors. These results also suggested there was a trend toward greater contamination with nosocomial-infection-causing bacteria in the NICU. Enhanced hygiene instructions for preterm donors may reduce the need to dispose of valuable donated HM as well as the risk of BM pathogen transmission to infants in the NICU. Neonatal Intensive Care Unit human milk donor milk milk bank bacterial profiles bacteriological test Figures Figure 1 Background Human milk (HM) is recognized as the most ideal nutrient source for infants, and in particular, for preterm infants. It not only reduces the risk of developing necrotizing enterocolitis [ 1 – 3 ], sepsis, and other diseases but also contributes to better long-term outcomes and the neurodevelopment of the infant [ 4 , 5 ]. HM contains various kinds of bacteria, such as Bifidobacteria spp., Lactobacillus spp., coagulase-negative Staphylococci (CoNS), diphtheroid, Acinetobacter spp., oral Streptococcus (viridans group streptococci), Staphylococcus aureus , Group B Streptococcus , Escherichia coli , Pseudomonas spp., Klebsiella spp., Enterococcus spp., Enterobacter spp., Bacillus spp., and Moraxella spp. [ 6 – 9 ], in addition to nutrients and micronutrients. Bacteria in HM are known to play important roles in infant health that are both positive and negative. The bacteria can be classified into two main categories: endogenous and exogenous (contamination) [ 10 ]. The former consists of part of the maternal gastrointestinal microbiota that transitions through the mammary glands via an entero-mammary pathway and is highly beneficial to an infant’s intestinal tract development and immune function [ 11 ]. The latter, however, comprises contaminants from the external environment, such as normal bacteria on the mother's and infant's skin, flora in the infant's oral cavity, and bacteria in the breast pump and milk bottle. These bacteria are sometimes pathogenic and may pose a risk of infections in preterm infants. Contamination with HM is not a major problem for term infants, but this is not the case for preterm infants. There are several pathways resulting in HM contamination, such as breast milk expression, freezing HM at home, shipping, processing in human milk banks (HMBs), and handling in the NICU. Among these, the likelihood of contamination during expression and storage at home are considered to be affected by the donor's living environment conditions and hygiene. Based on the hypothesis that the type of bacteria in HM is related to the donor's living environment, this study focused on the differences in bacteria in HM between term donors whose infants were at home and preterm donors whose infants were hospitalized in an NICU. The research aim was to compare the bacterial profile of HM from term and preterm mothers. Methods Design This was a retrospective analysis of data from Japan Human Milk Bank Association (JHMBA) from January to November 2021. The study compared bacterial culture results for HM between term maternal donors (37 to 41 weeks) and preterm maternal donors (<37 weeks). This study was approved by Showa University Research Ethics Review Board (Permit Number: 2714). Setting and Participants Forty-seven donors who registered at JHMBA from January to November 2021 were included in the study. The donors’ health conditions were confirmed based on a health checklist. The donors were screened based on their detailed medical history, physical examinations, and laboratory data according to the Guidelines for the Establishment and Operation of a Donor Human Milk Bank 2018. Donors who did not fulfill the eligibility criteria were excluded from donating milk. In Japan, term donors register themselves via the HMB websites, while preterm donors are generally registered through a referral from the medical staff in NICU. The expression method was either by hand or with an electric or manual pump. From the time of milk expression at the donor’s home, the milk was kept frozen until sent to JHMBA. We pasteurized the donated milk using the Holder pasteurization (HP) method (62.5°C for 30 min), and pre- and post-pasteurization samples were tested for bacterial culture. The acceptance criteria for donated milk were total bacterial count of ≤10⁵ CFU/ml, Enterobacteriaceae count of ≤10⁴ CFU/ml, Staphylococcus aureus count of ≤10⁴ CFU/ml, and the absence of spore-forming bacteria. The donated HM was accepted as pasteurized donor HM if no bacteria were detected after HP. In addition to the strict safety standards, hygienic instructions for adequate expression, freezing, and shipping were provided to all donors by JHMBA staff at registration. Measurement For the culture test, we send the samples to the clinical laboratory test company (BML Co.Ltd, Tokyo, Japan). 100 µL sample from each batch (before pasteurization) were cultured on blood agar to allow the growth of aerobic organisms. Bacterial counts are expressed as colony-forming units (CFU) in 100 μL. Statistical analyses The normality and variability of all parameters were evaluated with the Kolmogorov–Smirnov test and F-test, respectively. The total bacterial count and the bacterial species count per batch are presented as the mean (SD) and compared between term and preterm HM using Welch's t-test and Student's t-test, respectively. As for bacterial contamination rates, the number of batches in which a particular bacterial species was detected among all batches of HM is presented as a percentage, and two groups were compared using the Chi-square test or Fisher's exact test. The rate of milk sample rejection was also analyzed using the Chi-square test. Donor age, gestational age, birth weight, start date of HM donation (calculated using the expression and delivery dates of the donated milk), and the number of times milk samples were donated per mother are presented as the mean (SD) and median (range) values and compared between two groups using the Mann–Whitney test. All statistical analyses were performed using StatMate V (ATMS Co., Ltd., Tokyo, Japan) Results Characteristics of study participants The characteristics of all donors are shown in Table 1. Among the 47 donors that registered, 31 were term donors, and the other 16 were preterm donors. The total amount of donated milk batches was 214, including 75 batches from term donors and 139 batches from preterm donors. In some cases, a single donor provided milk multiple times over the study period. There was no significant difference in the mean maternal ages between the groups (term: 32.3, preterm: 32.6; p = 0.75), but there were significant differences in the gestational ages and birth weights of the infants (38.8 weeks vs. 27.6 weeks; 3,080 g vs. 1,040 g; p < 0.001). In addition, mothers who gave birth to preterm infants tended to begin donating breast milk earlier (18.9 weeks vs. 9.8 weeks; p<0.001). Total bacterial count and bacterial species count The mean total bacterial count was 351,141 CFU/100 μL in the term group and 872,272 CFU/100 μL in the preterm group (Table 2). There was a significant difference in the mean total bacterial count between the two groups (p = 0.026). A total of 29 bacterial species were detected in the batches from both groups. The mean bacterial species count was more diverse in the preterm group than the term group (p < 0.001). Bacterial contamination rate The bacterial contamination rate of HM is shown in Table 3. Staphylococcus epidermidis was the most prevalent bacteria (term: 65.6% of batches, preterm: 85.3% of batches), followed by Staphylococcus lugdunensis (14.4%, 32.9%), Staphylococcus aureus (20.0%, 21.7%), and Pseudomonas fluorescens (12.2%, 27.3%), which were detected at high frequencies in both groups (Figure 1). Four bacterial species were more prevalent in term HM, Pseudomonas putida (p = 0.008), Serratia liquefaciens (p < 0.001), Pantoea agglomerans (p = 0.014), and Bacillus cereus (p = 0.042); while five species were more prevalent in preterm HM, Pseudomonas fluorescens (p = 0.027) , Enterococcus faecalis and Enterobacter aerogenes (p < 0.001) , Staphylococcus lugdunensis (p = 0.01) , and Stenotrophomonas maltophilia (p = 0.003) (Table 3). Discussion After bacterial culture studies were conducted on term and preterm HM, the results revealed three major findings. First, high-ranked bacteria were common to both groups. Staphylococcus epidermidis , a commensal skin bacterium, was the most frequent bacterial pathogen in both groups, being detected in approximately 80% of the batches. CoNS, Staphylococcus aureus , and Pseudomonas fluorescens were contaminants found at high rates. These results are in line with previous studies [ 6 – 9 ]. These bacteria were reaffirmed to be common commensal bacteria with a high risk of causing contamination, regardless of the donor's living environment. Second, the total bacterial count was significantly higher in preterm than term HM, and the bacterial species count was greater in preterm HM. Third, there were differences in bacterial profiles between term and preterm HM. The characteristics of four bacteria species in the term HM are as follows. Serratia liquefaciens and Pseudomonas putida are often isolated from water and soil environments [ 12 , 13 ]; Pseudomonas putida , especially, is rarely isolated from clinical specimens [ 13 ]. Pantoea agglomerans is a Gram-negative bacterium commonly present in fecal material and soil, but it is an uncommon cause of infection in children[ 14 ]. The spore-forming bacterium Bacillus cereus was detected in three batches. Preterm HM had more Enterococcus faecalis and Enterobacter aerogenes contamination. These bacteria are classified as enterococci that reside in the human gastrointestinal tract, and they are frequently reported to cause nosocomial infections [ 15 , 16 ]. There have been cases of outbreaks in the NICU due to contamination of tap water [ 17 ]. Pseudomonas fluorescens is widely found in water supplies and can also be isolated from medical devices [ 18 ]. Staphylococcus lugdunensis , the CoNS species, is sometimes clinically treated the same as Staphylococcus aureus [ 19 ]. It is a commensal skin bacterium [ 20 ] and is reportedly a common cause of community-acquired and nosocomial infections [ 19 , 21 ]. Stenotrophomonas maltophilia is commonly isolated from water, soil, and fecal material and detected in hospitals, especially in the water supply [ 17 , 22 ]. According to a report by Urrea et al. [ 23 , 24 ], Enterococcus species, Staphylococcus aureus , and CoNS such as Staphylococcus epidermidis are the leading Gram-positive bacteria, while Escherichia coli , Enterobacter species, Pseudomonas species, and Klebsiella species have been reported to be the organisms most frequently responsible for nosocomial infections in NICU. Of the five species present at significantly different rates in the preterm HM, four species were relevant. The result suggests that preterm HM tends to be contaminated with bacteria that can cause nosocomial infections in NICUs. We made three main conclusions based on these results. The first is that preterm donors visit the NICU to meet their infants, and bacteria prevalent in the NICU environment may adhere to their clothing, resulting in them bringing the bacteria home. Therefore, there is a contamination risk by such bacteria, not only when HM is expressed in the NICU but also when expressed at home. Although data related to the expression environment (location, methods) were not collected in this study, it is likely most donors expressed at home because the study period overlapped with the coronavirus outbreak, and many NICUs had restricted visiting times. Second, several preterm samples included HM that had been expressed before donor registration. First of all, as a basic premise, HMBs always provide hygiene instructions, such as pre-breast expression wiping and disinfection of the breast pump, at donor registration, regardless of whether the donor gave birth prematurely or not. Term mothers may voluntarily register as a donor if they delivered a term baby, are currently breastfeeding, and have excessive breast milk supply. After registration, they donate milk to the HMB. However, preterm mothers may provide HMB with milk, which is kept in stock for infants admitted to the NICU. Therefore, there is a higher risk that preterm HM is contaminated with bacteria. Our third point is that the unique circumstances of preterm donors should also be considered. Several factors contribute to a stressful expression environment for preterm donors, including physical separation from their infants, the provision of a structured feeding schedule, the lack of privacy (when expressing in a hospital), the exhaustion and anxiety associated with an infant’s hospitalization, and long expression periods. These factors can also affect HM production [ 25 – 28 ]. Considering the situation, it is understandably more difficult to pay attention to hygiene precautions compared with when expressing at home, and HM may be contaminated by more bacteria. These results tell us that hygiene education is more important for preterm donors. However, their physical and psychological circumstances need to be taken into consideration. HMBs need to provide less burdensome and more hygienic expression instructions for these mothers. It may also be necessary to communicate more frequently with preterm donors and to follow up the instructions by observing how they are expressing and storing milk at home. In addition, it may be necessary to survey the NICU situation at each institution and discuss hygiene instructions for preterm donors with the NICU staff. Although the results of this study did not show a significant difference in the pass/fail score according to the bacterial culture test criteria established by the HMB (Table 1), in the future, better hygiene instructions will not only reduce the breast milk transmission risk but also contribute to reducing the wastage of valuable donated HM, a pertinent issue for HMBs. Limitations We did not investigate the location or method of expression, so the influence of these factors on the culture results is unknown. Additional environmental factors, such as living arrangements and sibling status, may also affect the bacterial profile of HM and will be investigated in the future. Conclusions This study revealed that preterm HM has a higher total bacterial count and greater diversity of bacterial species. In addition, it tends to be more highly contaminated by nosocomial-infection-causing bacteria in the NICU. Taken together, these findings suggest the need for more focused hygiene education for preterm donors. Abbreviations HM human milk NICU Neonatal Intensive Care Unit CoNS coagulase-negative Staphylococci HMB human milk bank Declarations Ethics approval and consent to participate Ethical Approval was obtained from Showa University Research Ethics Review Board (Permit Number: 2714). Additionally, written informed consent was obtained from all donors for using their milk for clinical and research purposes. Consent for publication Not applicable. Availability of data and materials The datasets used and / or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments We gratefully thank the donors for their willing participation in this study. Competing interests The authors declare that they have no competing interests. Author contribution All authors have read and approved the final manuscript to be published and agree to be accountable for all aspects of the work. K.M., K.M conceived and designed the study; K.M, M.T., M.D., M.I, and N.M contributed to the recruitment and data collection; K.M was responsible for data analysis and writing the manuscript; K.M, M.T. and K.M contributed to reviewing the manuscript critically. Authors' information Corresponding author Correspondence to Katsumi Mizuno. Funding Not applicable. References Herrmann K, Carroll K. An Exclusively Human Milk Diet Reduces Necrotizing Enterocolitis. Breastfeeding Medicine. 2014;9:184–90. Silano M, Milani GP, Fattore G, Agostoni C. Donor human milk and risk of surgical necrotizing enterocolitis: A meta-analysis. Clinical Nutrition. 2019;38:1061–6. Altobelli E, Angeletti PM, Verrotti A, Petrocelli R. The Impact of Human Milk on Necrotizing Enterocolitis: A Systematic Review and Meta-Analysis. Nutrients. 2020;12:1322. Llanos M A, Mena N P, Uauy D R. Nutritional tendencies in the premature infant. Rev Chil Pediatr. 2004;75. Embleton N, Cleminson J. Randomized trial of exclusive human milk versus preterm formula diets in extremely premature infants. Acta Paediatr. 2017;106:1538–1538. Soto A, Martín V, Jiménez E, Mader I, Rodríguez JM, Fernández L. Lactobacilli and Bifidobacteria in Human Breast Milk. J Pediatr Gastroenterol Nutr. 2014;59:78–88. Lindemann PC. Characteristics of breast milk and serology of women donating breast milk to a milk bank. Arch Dis Child Fetal Neonatal Ed. 2004;89:F440–1. Almutawif Y, Hartmann B, Lloyd M, Erber W, Geddes D. A retrospective audit of bacterial culture results of donated human milk in Perth, Western Australia. Early Hum Dev. Elsevier Ireland Ltd; 2017;105:1–6. Landers S, Updegrove K. Bacteriological Screening of Donor Human Milk Before and After Holder Pasteurization. Breastfeeding Medicine. 2010;5:117–21. Lyons KE, Ryan CA, Dempsey EM, Ross RP, Stanton C. Breast Milk, a Source of Beneficial Microbes and Associated Benefits for Infant Health. Nutrients. 2020;12:1039. Rodríguez JM. The Origin of Human Milk Bacteria: Is There a Bacterial Entero-Mammary Pathway during Late Pregnancy and Lactation? Advances in Nutrition. 2014;5:779–84. Mahlen SD. Serratia infections: From military experiments to current practice. Clin Microbiol Rev. 2011. Boullègue O, Mzoughi R, Weill FX, Mahdhaoui N, ben Salem Y, Sboui H, et al. Outbreak of Pseudomonas putida bacteraemia in neonatal intensive care unit. Journal of Hospital Infection. 2004;57. Cruz AT, Cazacu AC, Allen CH. Pantoea agglomerans, a plant pathogen causing human disease. J Clin Microbiol. 2007;45. Furtado I, Xavier PCN, Tavares LVM, Alves F, Martins SF, Martins A de S, et al. Enterococcus faecium AND Enterococcus faecalis IN BLOOD OF NEWBORNS WITH SUSPECTED NOSOCOMIAL INFECTION. Rev Inst Med Trop Sao Paulo. 2014;56. Loiwal V, Kumar A, Gupta P, Gomber S, Ramachandran VG. Enterobacter aerogenes outbreak in a neonatal intensive care unit. Pediatrics International. 1999;41. Verweij PE, Meis JFGM, Christmann V, van der Bor M, Melchers WJG, Hilderink BGM, et al. Nosocomial outbreak of colonization and infection with Stenotrophomonas maltophilia in preterm infants associated with contaminated tap water. Epidemiol Infect. 1998;120. Aa L, Kharat VM. Biochemical and physiological characterizations of Pseudomonas fluorescens. ~ 1785 ~ International Journal of Chemical Studies. 2019;7:1785–8. Rupp ME, Fey PD. Staphylococcus epidermidis and Other Coagulase-Negative Staphylococci. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. Elsevier Inc.; 2014. p. 2272-2282.e5. Akiyama H, Kanzaki H, Tada J, Arata J. Coagulase-negative staphylococci isolated from various skin lesions. Journal of Dermatology. 1998;25. Frank KL, del Pozo JL, Patel R. From clinical microbiology to infection pathogenesis: How daring to be different works for Staphylococcus lugdunensis. Clin Microbiol Rev. 2008. Cervia JS, Ortolano GA, Canonica FP. Hospital tap water as a source of Stenotrophomonas maltophilia infection. Clinical Infectious Diseases. 2008. p. 1485–6. Urrea M, Iriondo M, Thio M, Krauel X, Serra M, LaTorre C, et al. A prospective incidence study of nosocomial infections in a neonatal care unit. Am J Infect Control. 2003;31. Couto RC, Carvalho EAA, Pedrosa TMG, Pedroso ÊR, Neto MC, Biscione FM. A 10-year prospective surveillance of nosocomial infections in neonatal intensive care units. Am J Infect Control. 2007;35. Alves E, Rodrigues C, Fraga S, Barros H, Silva S. Parents’ views on factors that help or hinder breast milk supply in neonatal care units: Systematic review. Arch Dis Child Fetal Neonatal Ed. 2013;98. Boucher CA, Brazal PM, Graham-Certosini C, Carnaghan-Sherrard K, Feeley N. Mothers’ breastfeeding experiences in the NICU. Neonatal Network. 2011;30. Alves E, Magano R, Amorim M, Nogueira C, Silva S. Factors Influencing Parent Reports of Facilitators and Barriers to Human Milk Supply in Neonatal Intensive Care Units. Journal of Human Lactation. 2016;32. Dewey KG. Maternal and fetal stress are associated with impaired lactogenesis in humans. J Nutr. 2001. Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Table2TotalbacterialcountandBacterialspeciescountperbatch.docx Table3.xlsx Cite Share Download PDF Status: Published Journal Publication published 08 Jun, 2023 Read the published version in International Breastfeeding Journal → Version 1 posted Editorial decision: Major revision 04 Feb, 2023 Reviews received at journal 14 Jan, 2023 Reviewers agreed at journal 06 Jan, 2023 Reviewers invited by journal 06 Jan, 2023 Editor assigned by journal 06 Jan, 2023 Submission checks completed at journal 06 Jan, 2023 First submitted to journal 06 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-2449355\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":165639432,\"identity\":\"677213cb-2ea1-4519-80ab-28ee633f02b2\",\"order_by\":0,\"name\":\"Kumiko Miura\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The Nippon Foundation Human Milk Bank\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kumiko\",\"middleName\":\"\",\"lastName\":\"Miura\",\"suffix\":\"\"},{\"id\":165639433,\"identity\":\"82a51978-72d6-4f04-b7e6-bba1815fd33b\",\"order_by\":1,\"name\":\"Miori 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rate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe graphs show the bacteria with the highest contamination rates in each of the term and preterm HM.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2449355/v1/28f304dbedfb8e00e5a443ef.png\"},{\"id\":44730679,\"identity\":\"7858544c-5119-42a4-afd2-870f8069e16e\",\"added_by\":\"auto\",\"created_at\":\"2023-10-16 21:33:15\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":330376,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2449355/v1/4c87b7f7-8c6d-4528-8664-b2990c1b31d1.pdf\"},{\"id\":31373704,\"identity\":\"1177cfbd-4b85-497c-86b0-e65ee97a871a\",\"added_by\":\"auto\",\"created_at\":\"2023-01-10 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16:01:41\",\"extension\":\"xlsx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":14123,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Table3.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2449355/v1/1abce977211557e1d7e73ddf.xlsx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Comparison of bacterial profiles in human milk from mothers of term and preterm infants\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eHuman milk (HM) is recognized as the most ideal nutrient source for infants, and in particular, for preterm infants. It not only reduces the risk of developing necrotizing enterocolitis [\\u003cspan additionalcitationids=\\\"CR2\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e], sepsis, and other diseases but also contributes to better long-term outcomes and the neurodevelopment of the infant [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. HM contains various kinds of bacteria, such as \\u003cem\\u003eBifidobacteria\\u003c/em\\u003e spp., \\u003cem\\u003eLactobacillus\\u003c/em\\u003e spp., coagulase-negative \\u003cem\\u003eStaphylococci\\u003c/em\\u003e (CoNS), diphtheroid, \\u003cem\\u003eAcinetobacter\\u003c/em\\u003e spp., oral \\u003cem\\u003eStreptococcus\\u003c/em\\u003e (viridans group streptococci), \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e, Group B \\u003cem\\u003eStreptococcus\\u003c/em\\u003e, \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e, \\u003cem\\u003ePseudomonas\\u003c/em\\u003e spp., \\u003cem\\u003eKlebsiella\\u003c/em\\u003e spp., \\u003cem\\u003eEnterococcus\\u003c/em\\u003e spp., \\u003cem\\u003eEnterobacter\\u003c/em\\u003e spp., \\u003cem\\u003eBacillus\\u003c/em\\u003e spp., and \\u003cem\\u003eMoraxella\\u003c/em\\u003e spp. [\\u003cspan additionalcitationids=\\\"CR7 CR8\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], in addition to nutrients and micronutrients.\\u003c/p\\u003e \\u003cp\\u003eBacteria in HM are known to play important roles in infant health that are both positive and negative. The bacteria can be classified into two main categories: endogenous and exogenous (contamination) [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. The former consists of part of the maternal gastrointestinal microbiota that transitions through the mammary glands via an entero-mammary pathway and is highly beneficial to an infant\\u0026rsquo;s intestinal tract development and immune function [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. The latter, however, comprises contaminants from the external environment, such as normal bacteria on the mother's and infant's skin, flora in the infant's oral cavity, and bacteria in the breast pump and milk bottle. These bacteria are sometimes pathogenic and may pose a risk of infections in preterm infants.\\u003c/p\\u003e \\u003cp\\u003eContamination with HM is not a major problem for term infants, but this is not the case for preterm infants. There are several pathways resulting in HM contamination, such as breast milk expression, freezing HM at home, shipping, processing in human milk banks (HMBs), and handling in the NICU. Among these, the likelihood of contamination during expression and storage at home are considered to be affected by the donor's living environment conditions and hygiene.\\u003c/p\\u003e \\u003cp\\u003eBased on the hypothesis that the type of bacteria in HM is related to the donor's living environment, this study focused on the differences in bacteria in HM between term donors whose infants were at home and preterm donors whose infants were hospitalized in an NICU. The research aim was to compare the bacterial profile of HM from term and preterm mothers.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eDesign\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis was a retrospective analysis of data from Japan Human Milk Bank Association (JHMBA) from January to November 2021. The study compared bacterial culture results for HM between term maternal donors (37 to 41 weeks) and preterm maternal donors (\\u0026lt;37 weeks). This study was approved by Showa University Research Ethics Review Board (Permit Number: 2714).\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eSetting and Participants\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eForty-seven donors who registered at JHMBA from January to November 2021 were included in the study. The donors\\u0026rsquo; health conditions were confirmed based on a health checklist. The donors were screened based on their detailed medical history, physical examinations, and laboratory data according to the Guidelines for the Establishment and Operation of a Donor Human Milk Bank 2018. Donors who did not fulfill the eligibility criteria were excluded from donating milk.\\u003c/p\\u003e\\n\\n\\u003cp\\u003eIn Japan, term donors register themselves via the HMB websites, while preterm donors are generally registered through a referral from the medical staff in NICU.\\u003c/p\\u003e\\n\\n\\u003cp\\u003eThe expression method was either by hand or with an electric or manual pump. From the time of milk expression at the donor\\u0026rsquo;s home, the milk was kept frozen until sent to JHMBA. We pasteurized the donated milk using the Holder pasteurization (HP) method (62.5\\u0026deg;C for 30 min), and pre- and post-pasteurization samples were tested for bacterial culture.\\u003c/p\\u003e\\n\\n\\u003cp\\u003eThe acceptance criteria for donated milk were total bacterial count of \\u0026le;10⁵ CFU/ml, Enterobacteriaceae count of \\u0026le;10⁴ CFU/ml, \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e count of \\u0026le;10⁴ CFU/ml, and the absence of spore-forming bacteria. The donated HM was accepted as pasteurized donor HM if no bacteria were detected after HP. In addition to the strict safety standards, hygienic instructions for adequate expression, freezing, and shipping were provided to all donors by JHMBA staff at registration. \\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasurement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor the culture test, we send the samples to the clinical laboratory test company (BML Co.Ltd, Tokyo, Japan). 100 \\u0026micro;L sample from each batch (before pasteurization) were cultured on blood agar to allow the growth of aerobic organisms. Bacterial counts are expressed as colony-forming units (CFU) in 100 \\u0026mu;L.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eStatistical analyses\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe normality and variability of all parameters were evaluated with the Kolmogorov\\u0026ndash;Smirnov test and F-test, respectively. The total bacterial count and the bacterial species count per batch are presented as the mean (SD) and compared between term and preterm HM using Welch\\u0026apos;s t-test and Student\\u0026apos;s t-test, respectively. As for bacterial contamination rates, the number of batches in which a particular bacterial species was detected among all batches of HM is presented as a percentage, and two groups were compared using the Chi-square test or Fisher\\u0026apos;s exact test. The rate of milk sample rejection was also analyzed using the Chi-square test.\\u003c/p\\u003e\\n\\n\\u003cp\\u003eDonor age, gestational age, birth weight, start date of HM donation (calculated using the expression and delivery dates of the donated milk), and the number of times milk samples were donated per mother are presented as the mean (SD) and median (range) values and compared between two groups using the Mann\\u0026ndash;Whitney test. All statistical analyses were performed using StatMate V (ATMS Co., Ltd., Tokyo, Japan)\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacteristics of study participants\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe characteristics of all donors are shown in Table 1. Among the 47 donors that registered, 31 were term donors, and the other 16 were preterm donors. The total amount of donated milk batches was 214, including 75 batches from term donors and 139 batches from preterm donors. In some cases, a single donor provided milk multiple times over the study period. There was no significant difference in the mean maternal ages between the groups (term: 32.3, preterm: 32.6; p = 0.75), but there were significant differences in the gestational ages and birth weights of the infants (38.8 weeks vs. 27.6 weeks; 3,080 g vs. 1,040 g; p \\u0026lt; 0.001). In addition, mothers who gave birth to preterm infants tended to begin donating breast milk earlier (18.9 weeks vs. 9.8 weeks; p\\u0026lt;0.001).\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eTotal bacterial count and bacterial species count\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe mean total bacterial count was 351,141 CFU/100 \\u0026mu;L in the term group and 872,272 CFU/100 \\u0026mu;L in the preterm group (Table 2). There was a significant difference in the mean total bacterial count between the two groups (p = 0.026). A total of 29 bacterial species were detected in the batches from both groups. The mean bacterial species count was more diverse in the preterm group than the term group (p \\u0026lt; 0.001).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBacterial contamination rate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe bacterial contamination rate of HM is shown in Table 3. \\u003cem\\u003eStaphylococcus epidermidis\\u003c/em\\u003e was the most prevalent bacteria (term: 65.6% of batches, preterm: 85.3% of batches), followed by \\u003cem\\u003eStaphylococcus lugdunensis\\u003c/em\\u003e (14.4%, 32.9%), \\u003cem\\u003eStaphylococcus\\u003c/em\\u003e\\u003cem\\u003e aureus\\u003c/em\\u003e (20.0%, 21.7%), and \\u003cem\\u003ePseudomonas fluorescens\\u003c/em\\u003e (12.2%, 27.3%), which were detected at high frequencies in both groups (Figure 1). \\u003c/p\\u003e\\n\\u003cp\\u003eFour bacterial species were more prevalent in term HM, \\u003cem\\u003ePseudomonas putida \\u003c/em\\u003e(p = 0.008), \\u003cem\\u003eSerratia liquefaciens \\u003c/em\\u003e(p \\u0026lt; 0.001), \\u003cem\\u003ePantoea agglomerans \\u003c/em\\u003e(p = 0.014), and \\u003cem\\u003eBacillus cereus \\u003c/em\\u003e(p = 0.042); while five species were more prevalent in preterm HM,\\u003cem\\u003e \\u003c/em\\u003e\\u003cem\\u003ePseudomonas fluorescens \\u003c/em\\u003e(p = 0.027)\\u003cem\\u003e, Enterococcus faecalis\\u003c/em\\u003e and\\u003cem\\u003e Enterobacter aerogenes \\u003c/em\\u003e(p \\u0026lt; 0.001)\\u003cem\\u003e, Staphylococcus lugdunensis \\u003c/em\\u003e(p = 0.01)\\u003cem\\u003e, \\u003c/em\\u003eand\\u003cem\\u003e Stenotrophomonas maltophilia \\u003c/em\\u003e(p = 0.003) (Table 3).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eAfter bacterial culture studies were conducted on term and preterm HM, the results revealed three major findings. First, high-ranked bacteria were common to both groups. \\u003cem\\u003eStaphylococcus epidermidis\\u003c/em\\u003e, a commensal skin bacterium, was the most frequent bacterial pathogen in both groups, being detected in approximately 80% of the batches. CoNS, \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e, and \\u003cem\\u003ePseudomonas fluorescens\\u003c/em\\u003e were contaminants found at high rates. These results are in line with previous studies [\\u003cspan additionalcitationids=\\\"CR7 CR8\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. These bacteria were reaffirmed to be common commensal bacteria with a high risk of causing contamination, regardless of the donor's living environment.\\u003c/p\\u003e \\u003cp\\u003eSecond, the total bacterial count was significantly higher in preterm than term HM, and the bacterial species count was greater in preterm HM.\\u003c/p\\u003e \\u003cp\\u003eThird, there were differences in bacterial profiles between term and preterm HM. The characteristics of four bacteria species in the term HM are as follows. \\u003cem\\u003eSerratia liquefaciens\\u003c/em\\u003e and \\u003cem\\u003ePseudomonas putida\\u003c/em\\u003e are often isolated from water and soil environments [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]; \\u003cem\\u003ePseudomonas putida\\u003c/em\\u003e, especially, is rarely isolated from clinical specimens [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. \\u003cem\\u003ePantoea agglomerans\\u003c/em\\u003e is a Gram-negative bacterium commonly present in fecal material and soil, but it is an uncommon cause of infection in children[\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. The spore-forming bacterium \\u003cem\\u003eBacillus cereus\\u003c/em\\u003e was detected in three batches. Preterm HM had more \\u003cem\\u003eEnterococcus faecalis\\u003c/em\\u003e and \\u003cem\\u003eEnterobacter aerogenes\\u003c/em\\u003e contamination. These bacteria are classified as enterococci that reside in the human gastrointestinal tract, and they are frequently reported to cause nosocomial infections [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. There have been cases of outbreaks in the NICU due to contamination of tap water [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. \\u003cem\\u003ePseudomonas fluorescens\\u003c/em\\u003e is widely found in water supplies and can also be isolated from medical devices [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. \\u003cem\\u003eStaphylococcus lugdunensis\\u003c/em\\u003e, the CoNS species, is sometimes clinically treated the same as \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. It is a commensal skin bacterium [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e] and is reportedly a common cause of community-acquired and nosocomial infections [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. \\u003cem\\u003eStenotrophomonas maltophilia\\u003c/em\\u003e is commonly isolated from water, soil, and fecal material and detected in hospitals, especially in the water supply [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAccording to a report by Urrea \\u003cem\\u003eet al.\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e], \\u003cem\\u003eEnterococcus\\u003c/em\\u003e species, \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e, and CoNS such as \\u003cem\\u003eStaphylococcus epidermidis\\u003c/em\\u003e are the leading Gram-positive bacteria, while \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e, \\u003cem\\u003eEnterobacter\\u003c/em\\u003e species, \\u003cem\\u003ePseudomonas\\u003c/em\\u003e species, and \\u003cem\\u003eKlebsiella\\u003c/em\\u003e species have been reported to be the organisms most frequently responsible for nosocomial infections in NICU. Of the five species present at significantly different rates in the preterm HM, four species were relevant. The result suggests that preterm HM tends to be contaminated with bacteria that can cause nosocomial infections in NICUs.\\u003c/p\\u003e \\u003cp\\u003eWe made three main conclusions based on these results. The first is that preterm donors visit the NICU to meet their infants, and bacteria prevalent in the NICU environment may adhere to their clothing, resulting in them bringing the bacteria home. Therefore, there is a contamination risk by such bacteria, not only when HM is expressed in the NICU but also when expressed at home. Although data related to the expression environment (location, methods) were not collected in this study, it is likely most donors expressed at home because the study period overlapped with the coronavirus outbreak, and many NICUs had restricted visiting times.\\u003c/p\\u003e \\u003cp\\u003eSecond, several preterm samples included HM that had been expressed before donor registration. First of all, as a basic premise, HMBs always provide hygiene instructions, such as pre-breast expression wiping and disinfection of the breast pump, at donor registration, regardless of whether the donor gave birth prematurely or not. Term mothers may voluntarily register as a donor if they delivered a term baby, are currently breastfeeding, and have excessive breast milk supply. After registration, they donate milk to the HMB. However, preterm mothers may provide HMB with milk, which is kept in stock for infants admitted to the NICU. Therefore, there is a higher risk that preterm HM is contaminated with bacteria.\\u003c/p\\u003e \\u003cp\\u003eOur third point is that the unique circumstances of preterm donors should also be considered. Several factors contribute to a stressful expression environment for preterm donors, including physical separation from their infants, the provision of a structured feeding schedule, the lack of privacy (when expressing in a hospital), the exhaustion and anxiety associated with an infant\\u0026rsquo;s hospitalization, and long expression periods. These factors can also affect HM production [\\u003cspan additionalcitationids=\\\"CR26 CR27\\\" citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Considering the situation, it is understandably more difficult to pay attention to hygiene precautions compared with when expressing at home, and HM may be contaminated by more bacteria.\\u003c/p\\u003e \\u003cp\\u003eThese results tell us that hygiene education is more important for preterm donors. However, their physical and psychological circumstances need to be taken into consideration. HMBs need to provide less burdensome and more hygienic expression instructions for these mothers. It may also be necessary to communicate more frequently with preterm donors and to follow up the instructions by observing how they are expressing and storing milk at home. In addition, it may be necessary to survey the NICU situation at each institution and discuss hygiene instructions for preterm donors with the NICU staff.\\u003c/p\\u003e \\u003cp\\u003eAlthough the results of this study did not show a significant difference in the pass/fail score according to the bacterial culture test criteria established by the HMB (Table\\u0026nbsp;1), in the future, better hygiene instructions will not only reduce the breast milk transmission risk but also contribute to reducing the wastage of valuable donated HM, a pertinent issue for HMBs.\\u003c/p\\u003e\\n\\u003ch3\\u003eLimitations\\u003c/h3\\u003e\\n\\u003cp\\u003eWe did not investigate the location or method of expression, so the influence of these factors on the culture results is unknown. Additional environmental factors, such as living arrangements and sibling status, may also affect the bacterial profile of HM and will be investigated in the future.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eThis study revealed that preterm HM has a higher total bacterial count and greater diversity of bacterial species. In addition, it tends to be more highly contaminated by nosocomial-infection-causing bacteria in the NICU. Taken together, these findings suggest the need for more focused hygiene education for preterm donors.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cspan type=\\\"BoldItalic\\\" class=\\\"BoldItalic\\\" name=\\\"Emphasis\\\"\\u003eHM\\u003c/span\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ehuman milk\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cspan type=\\\"BoldItalic\\\" class=\\\"BoldItalic\\\" name=\\\"Emphasis\\\"\\u003eNICU\\u003c/span\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eNeonatal Intensive Care Unit\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cspan type=\\\"BoldItalic\\\" class=\\\"BoldItalic\\\" name=\\\"Emphasis\\\"\\u003eCoNS\\u003c/span\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ecoagulase-negative \\u003cem\\u003eStaphylococci\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cspan type=\\\"BoldItalic\\\" class=\\\"BoldItalic\\\" name=\\\"Emphasis\\\"\\u003eHMB\\u003c/span\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ehuman milk bank\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEthical Approval was obtained from Showa University Research Ethics Review Board (Permit Number: 2714). Additionally, written informed consent was obtained from all donors for using their milk for clinical and research purposes.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and / or analyzed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe gratefully thank the donors for their willing participation in this study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests. \\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors have read and approved the final manuscript to be published and agree to be accountable for all aspects of the work. K.M., K.M conceived and designed the study; K.M, M.T., M.D., M.I, and N.M contributed to the recruitment and data collection; K.M was responsible for data analysis and writing the manuscript; K.M, M.T. and K.M contributed to reviewing the manuscript critically.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCorresponding author\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCorrespondence to Katsumi Mizuno.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eHerrmann K, Carroll K. An Exclusively Human Milk Diet Reduces Necrotizing Enterocolitis. Breastfeeding Medicine. 2014;9:184\\u0026ndash;90. \\u003c/li\\u003e\\n\\u003cli\\u003eSilano M, Milani GP, Fattore G, Agostoni C. Donor human milk and risk of surgical necrotizing enterocolitis: A meta-analysis. Clinical Nutrition. 2019;38:1061\\u0026ndash;6. \\u003c/li\\u003e\\n\\u003cli\\u003eAltobelli E, Angeletti PM, Verrotti A, Petrocelli R. The Impact of Human Milk on Necrotizing Enterocolitis: A Systematic Review and Meta-Analysis. Nutrients. 2020;12:1322. \\u003c/li\\u003e\\n\\u003cli\\u003eLlanos M A, Mena N P, Uauy D R. Nutritional tendencies in the premature infant. Rev Chil Pediatr. 2004;75. \\u003c/li\\u003e\\n\\u003cli\\u003eEmbleton N, Cleminson J. Randomized trial of exclusive human milk versus preterm formula diets in extremely premature infants. Acta Paediatr. 2017;106:1538\\u0026ndash;1538. \\u003c/li\\u003e\\n\\u003cli\\u003eSoto A, Mart\\u0026iacute;n V, Jim\\u0026eacute;nez E, Mader I, Rodr\\u0026iacute;guez JM, Fern\\u0026aacute;ndez L. Lactobacilli and Bifidobacteria in Human Breast Milk. J Pediatr Gastroenterol Nutr. 2014;59:78\\u0026ndash;88. \\u003c/li\\u003e\\n\\u003cli\\u003eLindemann PC. Characteristics of breast milk and serology of women donating breast milk to a milk bank. Arch Dis Child Fetal Neonatal Ed. 2004;89:F440\\u0026ndash;1. \\u003c/li\\u003e\\n\\u003cli\\u003eAlmutawif Y, Hartmann B, Lloyd M, Erber W, Geddes D. A retrospective audit of bacterial culture results of donated human milk in Perth, Western Australia. Early Hum Dev. Elsevier Ireland Ltd; 2017;105:1\\u0026ndash;6. \\u003c/li\\u003e\\n\\u003cli\\u003eLanders S, Updegrove K. Bacteriological Screening of Donor Human Milk Before and After Holder Pasteurization. Breastfeeding Medicine. 2010;5:117\\u0026ndash;21. \\u003c/li\\u003e\\n\\u003cli\\u003eLyons KE, Ryan CA, Dempsey EM, Ross RP, Stanton C. Breast Milk, a Source of Beneficial Microbes and Associated Benefits for Infant Health. Nutrients. 2020;12:1039.\\u003c/li\\u003e\\n\\u003cli\\u003eRodr\\u0026iacute;guez JM. The Origin of Human Milk Bacteria: Is There a Bacterial Entero-Mammary Pathway during Late Pregnancy and Lactation? Advances in Nutrition. 2014;5:779\\u0026ndash;84. \\u003c/li\\u003e\\n\\u003cli\\u003eMahlen SD. Serratia infections: From military experiments to current practice. Clin Microbiol Rev. 2011. \\u003c/li\\u003e\\n\\u003cli\\u003eBoull\\u0026egrave;gue O, Mzoughi R, Weill FX, Mahdhaoui N, ben Salem Y, Sboui H, et al. Outbreak of Pseudomonas putida bacteraemia in neonatal intensive care unit. Journal of Hospital Infection. 2004;57. \\u003c/li\\u003e\\n\\u003cli\\u003eCruz AT, Cazacu AC, Allen CH. Pantoea agglomerans, a plant pathogen causing human disease. J Clin Microbiol. 2007;45. \\u003c/li\\u003e\\n\\u003cli\\u003eFurtado I, Xavier PCN, Tavares LVM, Alves F, Martins SF, Martins A de S, et al. Enterococcus faecium AND Enterococcus faecalis IN BLOOD OF NEWBORNS WITH SUSPECTED NOSOCOMIAL INFECTION. Rev Inst Med Trop Sao Paulo. 2014;56. \\u003c/li\\u003e\\n\\u003cli\\u003eLoiwal V, Kumar A, Gupta P, Gomber S, Ramachandran VG. Enterobacter aerogenes outbreak in a neonatal intensive care unit. Pediatrics International. 1999;41. \\u003c/li\\u003e\\n\\u003cli\\u003eVerweij PE, Meis JFGM, Christmann V, van der Bor M, Melchers WJG, Hilderink BGM, et al. Nosocomial outbreak of colonization and infection with Stenotrophomonas maltophilia in preterm infants associated with contaminated tap water. Epidemiol Infect. 1998;120. \\u003c/li\\u003e\\n\\u003cli\\u003eAa L, Kharat VM. Biochemical and physiological characterizations of Pseudomonas fluorescens. ~ 1785 ~ International Journal of Chemical Studies. 2019;7:1785\\u0026ndash;8. \\u003c/li\\u003e\\n\\u003cli\\u003eRupp ME, Fey PD. Staphylococcus epidermidis and Other Coagulase-Negative Staphylococci. Mandell, Douglas, and Bennett\\u0026rsquo;s Principles and Practice of Infectious Diseases. Elsevier Inc.; 2014. p. 2272-2282.e5. \\u003c/li\\u003e\\n\\u003cli\\u003eAkiyama H, Kanzaki H, Tada J, Arata J. Coagulase-negative staphylococci isolated from various skin lesions. Journal of Dermatology. 1998;25. \\u003c/li\\u003e\\n\\u003cli\\u003eFrank KL, del Pozo JL, Patel R. From clinical microbiology to infection pathogenesis: How daring to be different works for Staphylococcus lugdunensis. Clin Microbiol Rev. 2008. \\u003c/li\\u003e\\n\\u003cli\\u003eCervia JS, Ortolano GA, Canonica FP. Hospital tap water as a source of Stenotrophomonas maltophilia infection. Clinical Infectious Diseases. 2008. p. 1485\\u0026ndash;6. \\u003c/li\\u003e\\n\\u003cli\\u003eUrrea M, Iriondo M, Thio M, Krauel X, Serra M, LaTorre C, et al. A prospective incidence study of nosocomial infections in a neonatal care unit. Am J Infect Control. 2003;31. \\u003c/li\\u003e\\n\\u003cli\\u003eCouto RC, Carvalho EAA, Pedrosa TMG, Pedroso \\u0026Ecirc;R, Neto MC, Biscione FM. A 10-year prospective surveillance of nosocomial infections in neonatal intensive care units. Am J Infect Control. 2007;35. \\u003c/li\\u003e\\n\\u003cli\\u003eAlves E, Rodrigues C, Fraga S, Barros H, Silva S. Parents\\u0026rsquo; views on factors that help or hinder breast milk supply in neonatal care units: Systematic review. Arch Dis Child Fetal Neonatal Ed. 2013;98. \\u003c/li\\u003e\\n\\u003cli\\u003eBoucher CA, Brazal PM, Graham-Certosini C, Carnaghan-Sherrard K, Feeley N. Mothers\\u0026rsquo; breastfeeding experiences in the NICU. Neonatal Network. 2011;30. \\u003c/li\\u003e\\n\\u003cli\\u003eAlves E, Magano R, Amorim M, Nogueira C, Silva S. Factors Influencing Parent Reports of Facilitators and Barriers to Human Milk Supply in Neonatal Intensive Care Units. Journal of Human Lactation. 2016;32. \\u003c/li\\u003e\\n\\u003cli\\u003eDewey KG. Maternal and fetal stress are associated with impaired lactogenesis in humans. J Nutr. 2001. \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTables 1 to 3 are available in the Supplementary Files section.\\u003c/p\\u003e\\n\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"international-breastfeeding-journal\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ibfj\",\"sideBox\":\"Learn more about [International Breastfeeding Journal](http://internationalbreastfeedingjournal.biomedcentral.com/)\",\"snPcode\":\"13006\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13006/3\",\"title\":\"International Breastfeeding Journal\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Neonatal Intensive Care Unit, human milk, donor milk, milk bank, bacterial profiles, bacteriological test\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2449355/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2449355/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eBacteria in human milk (HM) can be endogenous or exogenous, and the latter can carry the risk of various infections in very low-birth weight infants because of the possibility of contamination with pathogenic bacteria. The mother's lifestyle and environment have a major influence on such bacterial contamination, and it is thought that there are differences in the number and types of bacteria cultured from HM between term mothers whose infants are at home and mothers of preterm infants in neonatal intensive care units (NICUs). This research aimed to compare the bacterial profiles of HM among mothers of term and preterm infants.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e \\u003cp\\u003eThe data comprised 214 milk samples (term: 75, preterm: 139) donated by 47 registered donors (term: 31, preterm: 16) from January to November 2021. Bacterial culture results were compared between term and preterm HM samples. Differences in the mean total bacterial count and bacterial species count per batch were analyzed using Welch\\u0026rsquo;s t-test and Student's t-test, respectively. The bacterial contamination rate was analyzed using Chi-square test or Fisher's exact test.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eCoagulase-negative \\u003cem\\u003eStaphylococci\\u003c/em\\u003e, \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e, and \\u003cem\\u003ePseudomonas fluorescens\\u003c/em\\u003e were frequently found in both term and preterm HM. \\u003cem\\u003eSerratia liquefaciens\\u003c/em\\u003e (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) and two other bacteria contaminated term HM, while five types of bacteria, including \\u003cem\\u003eEnterococcus faecalis\\u003c/em\\u003e and \\u003cem\\u003eEnterobacter aerogenes\\u003c/em\\u003e (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) contaminated preterm HM. The mean (SD) total bacterial count was 351,141 (1,060,949) CFU/100 \\u0026micro;L for term HM and 872,272 (2,324,477) CFU/100 \\u0026micro;L for preterm HM (p\\u0026thinsp;=\\u0026thinsp;0.026). Similarly, the number of bacterial species in HM was more diverse in preterm donors (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001).\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eThis study revealed that HM from preterm donors has a higher total bacterial count and greater diversity and characterization of bacterial types compared with HM from term donors. These results also suggested there was a trend toward greater contamination with nosocomial-infection-causing bacteria in the NICU. Enhanced hygiene instructions for preterm donors may reduce the need to dispose of valuable donated HM as well as the risk of BM pathogen transmission to infants in the NICU.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Comparison of bacterial profiles in human milk from mothers of term and preterm infants\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-01-10 16:01:36\",\"doi\":\"10.21203/rs.3.rs-2449355/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2023-02-04T06:33:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2023-01-14T12:12:30+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"c4ab3907-263c-44ad-876b-5f8a7b760143\",\"date\":\"2023-01-07T02:42:03+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-01-07T00:45:54+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2023-01-06T15:17:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2023-01-06T15:17:11+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"International Breastfeeding Journal\",\"date\":\"2023-01-06T08:07:57+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"international-breastfeeding-journal\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ibfj\",\"sideBox\":\"Learn more about [International Breastfeeding Journal](http://internationalbreastfeedingjournal.biomedcentral.com/)\",\"snPcode\":\"13006\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13006/3\",\"title\":\"International Breastfeeding Journal\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f8a29a42-6add-40da-bb47-62a0a3d247e1\",\"owner\":[],\"postedDate\":\"January 10th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-10-16T21:17:55+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-2449355\",\"link\":\"https://doi.org/10.1186/s13006-023-00563-3\",\"journal\":{\"identity\":\"international-breastfeeding-journal\",\"isVorOnly\":false,\"title\":\"International Breastfeeding Journal\"},\"publishedOn\":\"2023-06-08 21:08:50\",\"publishedOnDateReadable\":\"June 8th, 2023\"},\"versionCreatedAt\":\"2023-01-10 16:01:36\",\"video\":\"\",\"vorDoi\":\"10.1186/s13006-023-00563-3\",\"vorDoiUrl\":\"https://doi.org/10.1186/s13006-023-00563-3\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2449355\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2449355\",\"identity\":\"rs-2449355\",\"version\":[\"v1\"]},\"buildId\":\"_2-kVJe1T_tPrBINL-cwx\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}