## 1 Introduction  
Human milk (HM) is the preferred food for infants, at least for the first six months of life, since it is one of the most effective ways to ensure children's health and survival [1]. In addition to its optimal nutrient supply, HM transfers immune factors to protect newborns during their first years of life. For this reason, breastfeeding is the preferred method for feeding infants, including those who are preterm and hospitalized.  
In the case of preterm infants admitted to neonatal intensive care units (NICUs), it is often not possible to directly breastfeed the infant; therefore, bottle feeding or feeding through an enteral feeding tube are used instead [2]. If the mother's milk is insufficient or unavailable, donated HM can be used; thus, many hospitals have incorporated human milk banks (HMBs) in their facilities. In these facilities, milk usually undergoes different processes that can vary between the different HMBs. In general, some steps are common to all HMBs: freezing, thawing, and Holder pasteurization [3; 4; 5]. Lastly, before administration, milk is fortified with proteins, fats, minerals, and vitamins that are necessary in the case of preterm infants [6]. With regard to thawing, the recommended methods from different organizations include carrying thawing out overnight under refrigeration, at room temperature, or in a thermal bath [3; 4]. In some of these methods, the milk remains at a temperature that could allow microbial growth for a considerable period of time; therefore, it is necessary to evaluate the different methods in relation to this issue.  
In addition, it must be considered that treatments such as freezing and pasteurization can affect the antimicrobial capacity of milk [7; 8; 9]. Preterm infants are an extremely vulnerable population; therefore, it is essential to control hygiene during all treatments and handling of HM, as well as powdered infant formula (PIF), which is used as an alternative in some cases [8].  
Finally, during the milk administration process (both HM and PIF) through an enteral feeding tube in the NICU, milk is kept at room temperature for some time. Guidelines from various countries [10; 11] and several authors [5; 12; 13] recommend that milk delivered through enteral feeding tubes should be kept for a maximum of 4 h and containers must also be changed every 4 h. However, the United States Centers for Diseases Control and Prevention [14] recommend keeping thawed HM at room temperature for a maximum of 1-2 h at 25 \({}^{\circ}\)C. These differences in recommendations justify the need to obtain experimental information about the microbial growth in HM and PIF over time, under the conditions of administration in NICUs, to rationalize these recommendations. The holding time of milk at room temperatures is key, because if it exceeds the duration of the lag phase of a microorganism, it can lead to undesirable microbial growth. Among the different microorganisms that can contaminate milk, _Escherichia coli_ is one of the most common pathogens that causes neonatal infections [15; 16], and is a cause of outbreaks due to the consumption of unpasteurized human milk in NICUs [17; 18]. The growth of _E. coli_ in HM and PIF and its handling practices have implications for neonatal health and the management of neonatal units. Therefore, it is of interest to study the maximum time that milk can be kept at room temperature during its administration in NICUs without posing a risk of possible microbial growth.  
Furthermore, evidence-based standards for recommending the optimal HM thawing method and feeding temperature for infants are limited [19].  
This study aims to evaluate the growth of _E. coli_ in HM and PIF under various treatments and conditions, as well as to determine the influence of different thawing methods on microbial growth in HM.