Cultures of Holder-Pasteurized Donor Human Milk After Use in a Neonatal Intensive Care Unit

Introduction

Human milk is the recommended form of enteral nutrition for infants, including those born high-risk and prematurely.^1,2 When maternal milk production is inadequate to support the high-risk or premature infant while in the newborn intensive care unit (NICU), it may be necessary to supplement the milk supply. Many NICUs in the United States choose to use donor human milk (DHM) provided by a milk bank. Current recommendations are that frozen human milk, once thawed, may be kept refrigerated for up to 24 hours.^3,4 These recommendations were for unpasteurized maternal milk initially but have been extrapolated to DHM. Recent data indicate that the bactericidal effect of human milk is decreased by pasteurization, with higher temperature and longer time exposure causing greater degradation.^5,6 Milk banks operating under guidelines ⁷ of the Human Milk Banking Association of North America (HMBANA) distribute DHM that has been screened for bacterial contamination and undergone Holder pasteurization. Studies have described the initial efficacy of Holder pasteurization of DHM. ⁸ The present study was designed to assess the results of bacterial cultures of pasteurized DHM after thawing, refrigeration, and use under clinical conditions.

Materials and Methods

This study was done using DHM from the Mothers' Milk Bank (MMB) of San Jose, CA, which distributes over 250,000 ounces of milk annually. Following HMBANA guidelines,^4,7 pumped milk from screened donors is brought to the MMB frozen. There, each individual donor's milk bottles are thawed, combined, and cultured, and samples with spore-forming bacteria at this stage are discarded. Remaining bottles are then pooled with milk from two to five other donors in sterile glass bottles and capped. Bottles are then Holder-pasteurized as 90–100-mL aliquots in glass bottles for 30 minutes at 62.5°C. The pooled and pasteurized DHM is cultured for bacterial growth and then stored frozen at −20°C in 3-ounce glass bottles. Milk with a positive culture at this step in the process is not distributed.

Bottles of pasteurized DHM were delivered in the usual fashion to the freezers of a local Regional Level 3 NICU. Bottles were kept frozen at −20°C until needed clinically. They were then defrosted by the NICU nursing staff and handled in the usual fashion for feeding NICU patients. Bottles were not preselected, and the NICU staff did not know in advance which bottles might be collected for study. As was the preexisting protocol in this NICU, bottles were marked with the date and time they were thawed so that the staff could discard them within 24 hours thereafter. After DHM was thawed, the protocol indicated that the nursing staff withdrew aliquots of milk into feeding bottles in a sterile fashion. The glass bottles were then recapped and returned to the refrigerator. Once thawed for 24 hours, bottles were deemed “expired” and were not used further by the NICU staff. During the study, the NICU staff was instructed to mark “expired” bottles and leave them in the refrigerator so MMB staff could collect them. Bottles that were “expired” or otherwise no longer needed clinically by the NICU were collected by the MMB staff and returned to the MMB. Decisions as to which baby was fed DHM and how much, and whether or not to use human milk fortifier (HMF) (Mead-Johnson), were left to the clinical staff who were managing the patients and were unaware of the study.

Bottles returned to the MMB on days when one of the investigators (C.-F.R.H.) was available were studied. The samples were cultured using the same standard plate count method used to determine bacterial growth in the pasteurized DHM prior to distribution. Under sterile conditions in the MMB, 1-mL samples were collected from the bottles, diluted with 99 mL of phosphate buffer, and pipetted into sterile Petri dishes with plate count agar, according to standard practice. ⁹ These were then incubated for at least 48 hours at 32°C. Bottles that had HMF added were excluded from this analysis because the addition of a powdered additive might be considered a risk factor for contamination.^10–12

Results

In total, 30 bottles were collected for the study, including six to which HMF had been added by the NICU nursing staff in the usual fashion. Two additional bottles were excluded because they were found unrefrigerated.

The remaining 22 bottles (Table 1) had been thawed for 7–122 hours (mean, 46.7±28.4 hours). None of the samples had any bacterial growth, including 18 bottles thawed and refrigerated appropriately for more than 24 hours.

Discussion

DHM is considered the best alternative to formula for feeding high-risk premature infants for whom adequate maternal milk may not be available.^1–3 Several studies have reported a decreased incidence of necrotizing enterocolitis in premature infants fed human milk, including DHM.^13–15 Nevertheless, for whatever reasons, many NICUs do not use DHM. We believe that the cost-benefit analysis is in favor of using DHM,^16,17 although it is expensive. Further reduction in the cost of using DHM would be beneficial financially and might encourage wider usage. If milk could be used longer after thawing, there might be a decrease in wastage and thus significant cost savings, further tipping the financial balance in favor of using DHM. Furthermore, unnecessary wastage of a commodity as valuable as human milk should be avoided whenever it can be done safely.

There were too few bottles with HMF added to provide significant data; however, two of the six bottles were culture positive. The process of adding powders to the milk may introduce contaminants.^10–12 Although some have reported that HMF may decrease the antibacterial properties of DHM, ¹⁸ there also are reports that HMF per se may not increase bacterial growth.^19–21 This needs to be studied further.

Dilution techniques are used commonly for testing milk.^9,21–24 This technique facilitated our ability to culture the small residual volumes left in many of the samples, as 13 of the 22 samples had 3 mL or less residual volume. It is unlikely that this decreased the sensitivity of our cultures, although it may have decreased the colony count. Thus, we considered any growth to be significant.

These data confirm that unfortified Holder-pasteurized DHM handled appropriately and refrigerated for 24 hours after thawing remained culture negative. Additionally, they suggest that DHM processed according to HMBANA guidelines may be safe for consumption for longer than 24 hours after thawing, if adequately refrigerated and if nothing has been added to the bottle. If DHM could be used for up to 48 hours after thawing, for example, the decreased wastage would result in improved availability of a valuable and limited resource. Additionally, the more cost-efficient usage might contribute to wider use and potentially better NICU outcomes without increased healthcare expense. The addition of powdered fortifier remains a concern for possible bacterial contamination. Further studies are warranted to confirm this, and individual NICUs should consider studying their own DHM, as handling may vary from unit to unit.