Growth of Escherichia coli in Human Milk and Powdered Infant Formula under Various Treatments and Feeding Conditions in Neonatal Units

Round 1

Reviewer 1 Report

The manuscript summarizes the main findings related to the growth of Escherichia coli (E. coli) in human milk (HM) and powdered infant formula (PIF) under different conditions. A few general suggestions and requests for clarity are specified below.

While the introduction covers the importance of breastfeeding and the use of HM banks in neonatal units, it would be beneficial to explicitly state the need for this study. Consider emphasising the knowledge gap or research question that prompted the investigation. For example, you could mention that the growth of Escherichia coli in HM and its handling practices have implications for neonatal health and the management of neonatal units. Other pathogen might be mentioned (importance of C.sakazakii in PIF) and/or as the study focuses on the growth of Escherichia coli, it would be helpful to briefly explain why E. coli was chosen as the microorganism of interest. Discuss its potential pathogenicity and its significance in the neonatal context. This will provide readers with a better understanding of the specific risks associated with E. coli growth in neonatal units. The introduction mentions the recommended methods for thawing and milk administration, consider discussing the limitations or challenges associated with these methods. Addressing these limitations will highlight the need for further research and support the importance of the study. Mention also a clearer roadmap for the study, and explicitly state the objectives at the end of the introduction. For example, you can mention that the study aims to evaluate the growth of Escherichia coli in HM and PIF under various treatments and conditions, as well as determine the influence of different thawing methods on microbial growth in HM. It is mentioned that HM samples were donated voluntarily, it would be helpful to provide additional details on the collection process. Include information on how the milk was taken, the frequency, and any specific instructions provided to the donors and mention the brand and model of the electric or manual breast pump used for standardised extraction.

When describing the selection of PIF, provide additional details on the criteria used for choosing the specific brands. Include factors such as nutritional composition, popularity, or other relevant considerations. Clarify whether the four thawing methods were performed in separate experiments or sequentially on the same samples and the duration of the thawing methods for each treatment. 

Compare the findings of the present study with the research cited and discuss how the current study adds to the existing knowledge and whether the observed results support or challenge previous findings and discuss the implications of the study's findings for milk handling and administration practices in neonatal intensive care units.  Also, as mentioned above, propose investigating the behaviour of other pathogenic microorganisms of interest in HM/PIF with varying levels of contamination.

Author Response

human milk (HM) and powdered infant formula (PIF) under different conditions. A few general suggestions and requests for clarity are specified below.

While the introduction covers the importance of breastfeeding and the use of HM banks in neonatal units, it would be beneficial to explicitly state the need for this study. Consider emphasising the knowledge gap or research question that prompted the investigation. For example, you could mention that the growth of Escherichia coli in HM and its handling practices have implications for neonatal health and the management of neonatal units.

ANSWER:

The recommended aspects have been included in the introduction. The need for the study has been emphasized since the recommendations for maximum holding time of milk at room temperature in nurseries are varied and sometimes contradictory. The recommended methods of thawing are also varied. There are not enough studies to support these recommendations and the results depend on the previous treatments that the milk has undergone, as they affect its antimicrobial capacity. It has also been mentioned that the growth of Escherichia coli in HM and its handling practices have implications for neonatal health and the management of neonatal units.

Other pathogen might be mentioned (importance of C.sakazakii in PIF) and/or as the study focuses on the growth of Escherichia coli, it would be helpful to briefly explain why E. coli was chosen as the microorganism of interest. Discuss its potential pathogenicity and its significance in the neonatal context. This will provide readers with a better understanding of the specific risks associated with E. coli growth in neonatal units.

ANSWER:

The reason for choosing Escherichia coli as the microorganism for the study is given in lines 65-71. This microorganism is one of the most common pathogens causing neonatal infections (Flannery et al. 2021; Xiao et al, 2023), and is the cause of outbreaks due to consumption of unpasteurised human milk in NICUs (Chishiki et al, 2023; Nakamura et al. 2016)

In the introduction, the importance of other pathogens such as Cronobacter sakazakii has not been included, because we wanted to highlight the interest of investigating E. coli growth, which is the subject of this study, but it has been included in the discussion section.

• Flannery, D. D.; Akinboyo, I. C.; Mukhopadhyay, S.; Tribble, A. C.; Song, L.; Chen, F.; Li, Y.; Gerber, J. S.; Puopolo, K. M. Antibiotic susceptibility of Escherichia coli among infants admitted to neonatal intensive care units across the US from 2009 to 2017. JAMA Pediatr. 2021, 175, 168–175. https://doi.org/10.1001/jamapediatrics.2020.4719
• Xiao, R.; Li, Y.; Liu, X.; Ding, Y.; Lai, J.; Li, Y.; Kang, W.; Zou, P.; Wang, J.; Du, Y.; et al. Antibiotic susceptibility of Escherichia coliisolated from neonates admitted to neonatal intensive care units across China from 2015 to 2020.  Cell. Infect. Microbiol. 2023, 13, 1183736. https://doi.org/10.3389/fcimb.2023.1183736
• Chishiki, M.; Nishiyama, K.; Suzutani, T.; Hiruta, S.; Ichikawa, H.; Haneda, K.; Maeda, H.; Shimizu, H.; Kanai, Y.; Ogasawara, K.; et al. Sterilization efficacy of a new water-free breast milk pasteurizer. Int., 2023, 65, e15574. https://doi.org/10.1111/ped.15574
• Nakamura, K.; Kaneko, M.; Abe, Y.; Yamamoto, N.; Mori, H.; Yoshida, A.; Ohashi, K., Miura, S.; Yang, T. T.; Momoi, N; et al. Outbreak of extended-spectrum β-lactamase-producing Escherichia coli transmitted through breast milk sharing in a neonatal intensive care unit. Hosp. Infect., 2016, 92, 42–46. https://doi.org/10.1016/j.jhin.2015.05.002

 

 

The introduction mentions the recommended methods for thawing and milk administration, consider discussing the limitations or challenges associated with these methods. Addressing these limitations will highlight the need for further research and support the importance of the study. Mention also a clearer roadmap for the study, and explicitly state the objectives at the end of the introduction. For example, you can mention that the study aims to evaluate the growth of Escherichia coli in HM and PIF under various treatments and conditions, as well as determine the influence of different thawing methods on microbial growth in HM.

ANSWER:

In lines 44-48, the need to assess the best thawing treatment from the point of view of microbial growth has been highlighted, since in this case the recommended methods are also varied and some of them could favour microbial growth.

Lines 60-64 indicate the need to carry out studies to assess the maximum time that milk could be kept under certain conditions of administration of NICUs, without posing a risk of possible microbial growth.

At the end of the introduction (lines 74-76) the objectives have been changed to those proposed.

 It is mentioned that HM samples were donated voluntarily, it would be helpful to provide additional details on the collection process. Include information on how the milk was taken, the frequency, and any specific instructions provided to the donors and mention the brand and model of the electric or manual breast pump used for standardised extraction.

ANSWER:

On lines 99-104, additional information on the sample collection process has been added, including information on how it was taken, the frequency and other instructions. The model and brand of the electric or manual breast pump has been indicated.

When describing the selection of PIF, provide additional details on the criteria used for choosing the specific brands. Include factors such as nutritional composition, popularity, or other relevant considerations.

ANSWER:

The criteria used for the selection of specific brands, including popularity and nutritional criteria, have been explained in lines 111-113.

 

Clarify whether the four thawing methods were performed in separate experiments or sequentially on the same samples and the duration of the thawing methods for each treatment. 

ANSWER:

It is specified in lines 158-159 that the four thawing methods were applied at the same time on different aliquots of the same sample.  The durations of each treatment are specified in lines 154-156.

Compare the findings of the present study with the research cited and discuss how the current study adds to the existing knowledge and whether the observed results support or challenge previous findings and discuss the implications of the study's findings for milk handling and administration practices in neonatal intensive care units.  Also, as mentioned above, propose investigating the behaviour of other pathogenic microorganisms of interest in HM/PIF with varying levels of contamination.

ANSWER:

The discussion has been modified by comparing the results obtained in the present study with those cited indicating whether the observed results support or challenge previous findings and we are discussing the study's implications in relation to milk handling and administration practices in neonatal intensive care units.  At the end of the discussion, lines 396-399, future studies on the behaviour of other pathogens, such as Cronobacter sakazakii, and with other levels of contamination are proposed.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript "Growth of Escherichia coli under neonatal feeding conditions in neonatal units" has an interesting outcome. However, for publication in Microorganisms, the manuscript needs to be improved.
The discussion needs to review and compare the data with the literature. Many grammatically problematic sentences were found throughout the manuscript, which must be checked and corrected precisely by English editing services.

 L73: put references

L85: Did the authors consider the collection of milk only from one breast? If yes, why? 

L88: Did the authors check the microbial flora in collected milk prior to the experiment? Add this information.

L107: make a correction "62°C"

L111: What was the basis for using 104 CFU/ml of E. coli in your experiment? 

1. The introduction and results sections are poor. Need to maintain a logical flow of the writing. 

2. The discussion is feeble. A sound discussion includes principal, relationships, and generalizations supported by the results.

 

Many grammatically problematic sentences were found throughout the manuscript, which must be checked and corrected precisely by English editing services.

Author Response

The manuscript "Growth of Escherichia coli under neonatal feeding conditions in neonatal units" has an interesting outcome. However, for publication in Microorganisms, the manuscript needs to be improved.

The discussion needs to review and compare the data with the literature. Many grammatically problematic sentences were found throughout the manuscript, which must be checked and corrected precisely by English editing services.

ANSWER:

The discussion has been modified by comparing the results obtained in the present study with those cited indicating whether the observed results support or challenge previous findings and we are discussing the study's implications in relation to milk handling and administration practices. The manuscript has been reviewed using the English editing services of MPDI (certificate attached)

 L73: put references

ANSWER:

The reference has been indicated in line 73 (line 84 of the current document).

L85: Did the authors consider the collection of milk only from one breast? If yes, why? 

ANSWER:

As indicated on lines 102-104, samples were taken only from one breast. The reason was to take advantage of all the properties of the milk, as its composition varies throughout the expression and we were interested in having a representative sample of all the components in the milk. In this way, the milk from the other breast is also available for the infant. This justification has been included in the manuscript.

L88: Did the authors check the microbial flora in collected milk prior to the experiment? Add this information.

ANSWER:

The initial count before inoculation was not carried out on the samples analysed, but after inoculation, an initial count of the total Escherichia coli present in the milk samples was made, which of course included the one that had been inoculated plus the one that may have been present initially in milk. Subsequently, the study of the growth of the microorganism at different times or after different thawing methods is compared with this initial count data.

L107: make a correction "62°C"

ANSWER:

The proposed correction has been made.

L111: What was the basis for using 104 CFU/ml of E. coli in your experiment? 

ANSWER:

The study aims to assess what could happen in the event of possible contamination. These inoculation levels of E. coli have been used because with high initial concentrations we put in the most risky situation where the bacteria will grow faster. Other authors also use these concentrations, for example Gao et al (2020) use similar loads of 10⁴ CFU/mL of Enterococcus faecalis. Moreover, several studies have detected gram-negative colony counts of around 10⁴ in fresh refrigerated human milk (Arhabi et al, 2016; Handa et al. 2024; Slutzah et al 2010).

• Ahrabi, A. F., Handa, D., Codipilly, C. N., Shah, S., Williams, J. E., McGuire, M. A., Potak, D., Aharon, G. G., & Schanler, R. J. (2016). Effects of Extended Freezer Storage on the Integrity of Human Milk. The Journal of pediatrics, 177, 140–143. https://doi.org/10.1016/j.jpeds.2016.06.024
• Handa, D.; Ahrabi, A. F.; Codipilly, C. N.; Shah, S.; Ruff, S.; Potak, D.; Williams, J. E.; McGuire, M. A.; Schanler, R. J. Do thawing and warming affect the integrity of human milk?. Perinatol. 2014, 34, 863–866. https://doi.org/10.1038/jp.2014.113
• Slutzah, M., Codipilly, C. N., Potak, D., Clark, R. M., & Schanler, R. J. (2010). Refrigerator storage of expressed human milk in the neonatal intensive care unit. The Journal of pediatrics, 156(1), 26–28. https://doi.org/10.1016/j.jpeds.2009.07.023

 

The introduction and results sections are poor. Need to maintain a logical flow of the writing. 

ANSWER:

The introduction and the results have been modified. The introduction has been reorganised to include what we consider to be a logical flow: first, the importance of human milk for infants has been included. Then the feeding system for preterm infants admitted to neonatal intensive care units has been explained, using donated milk, including the treatments that this milk undergoes before being administered. In relation to these two aspects (milk treatments and administration in neonatal nursery conditions), the different recommendations made by guidelines and organisations have been indicated, highlighting the differences, and the effect of the treatments on the antimicrobial capacity of the milk. Based on these aspects, emphasis has been placed on the research question that motivated the research.

 

2. The discussion is feeble. A sound discussion includes principal, relationships, and generalizations supported by the results.

ANSWER:

The discussion has been modified. Findings of the present study have been compared with cited research and it has been discussed what the current study adds to the existing knowledge.  It has also been indicated whether the observed results support or challenge previous findings.  Finally, it has been discussed the implications of the study's findings for milk handling and administration practices in neonatal intensive care units.

Author Response File: Author Response.pdf

Reviewer 3 Report

Table 1 unreadable 

Table 2 illegible 

 Proposals for change

Summary: to change

Title to be changed

No DOI number

Table 1 unreadable 

Table 2 illegible 

 Proposals for change

Summary: to change

Title to be changed

No DOI number

Author Response

Table 1 unreadable 

Table 2 illegible 

ANSWER:

Certain modifications have been made to the tables 1 and 2 to clarify their interpretation and their format has also been modified to make it more readable. 

Proposals for change

Summary: to change

ANSWER:

Summary has been modified and some errors have been corrected, such as the types of HM used in the study (it was indicated that fresh HM had been studied, but this was not the case).

Title to be changed

ANSWER:

The title has been modified to make it more appropriate to the contents of the study.

No DOI number

ANSWER:

The doi number has been added to the reference of Lemons et al (1983), which was missing.

Author Response File: Author Response.pdf

Reviewer 4 Report

I do not recommend the publication of this work for several important reasons:

- It is not new to the scientific community.

- for the study of microbial growth it would be necessary to carry it out in many more microorganisms, not only E. coli. In particular some that are resistant to freezing for long periods such as Listeria monocytogenes as well as other common pathogens in hospital environments

Author Response

I do not recommend the publication of this work for several important reasons:

- It is not new to the scientific community.

ANSWER:

Currently the UK and USA Guidelines for the Preparation and Handling of Expressed and Donor Breast Milk [1,2] and studies by several authors [3-5], recommend keeping the milk for a maximum of 4 h at 22ºC during administration in neonatal intensive care units.  In contrast, the United States CDC recommendation [6], states not to keep thawed human milk for more than 1-2 h at a temperature of 25ºC. These recommendations are contradictory. Furthermore, it is important to bear in mind that human milk from Human Milk Banks administered to neonates has usually undergone different treatments that affect its antimicrobial capacity [7,8]. Few studies have been found that assess microbial growth in human milk, previously subjected to different treatments (freezing and pasteurisation), maintained at these neonatal nursery temperature conditions. The results of the study show that these recommendations need to be revised, since microbial growth is detected much earlier than the recommended 4 hours. The aim of this study is to demonstrate that this time is excessive and to urge that it be revised. Previous studies such as that of Handa et al [9] conclude that concerns about maintaining warmed milk at room temperature need to be explored.

As for thawing methods, several methods are also proposed by different organisations [10,11], some of which involve keeping the milk at temperatures above refrigeration for a certain period of time. This can obviously favour microbial growth. We also believe that scientific evidence is needed to justify a modification of the established recommendations.

References:

1. British Dietetic Association. Guidelines for the Preparation and Handling of Expressed and Donor Breast Milk and Specialist Feeds for Infants and Children in Neonatal and Paediatric Health Care Settings. Available online: https://www.bda.uk.com/uploads/assets/913a1f78-c805-42c1-8d85e37ca75e0fc0/2019sfuguidelines.pdf (accessed on 16 January 2023).
2. Strengthening Human Milk Banking: A Resource Toolkit for Establishing and Integrating Human Milk Bank Programs--A Global Implementation Framework. Version 2.0. PATH: Seattle, Washington, USA, 2019. https://www.aeblh.org/images/publicaciones/2019/20191009_path_hmb_toolkit_0.global_implementation_framework.pdf
3. Steele, C.; Collins, E. (Eds.). Infant and Pediatric Feedings: Guidelines for Preparation of Human Milk and Formula in Health Care Facilities, 3rd ed.; Academy of Nutrition and Dietetics: Chicago, USA, 2018: pp. 1–248.
4. Eglash, A.; Simon, L. ABM Clinical Protocol #8: Human milk storage information for home use for full-term infants, Revised 2017. Med. 2017, 12, 390-395. doi: 10.1089/bfm.2017.29047.aje. (Erratum published 2018, Breastfeed. Med 13[6], 459).
5. Boullata, J. I.; Carrera, A. L.; Harvey, L.; Escuro, A. A.; Hudson, L.; Mays, A.; McGinnis, C.; Wessel, J. J.; Bajpai, S.; Beebe, M. L.; et al. ASPEN safe practices for enteral nutrition therapy. Parenter. Enter. Nutr., 2017, 41, 15–103. https://doi.org/10.1177/0148607116673053
6. United States Centers for Diseases Control and Prevention. Proper Storage and Preparation of Breast Milk. Available online: https://www.cdc.gov/breastfeeding/recommendations/handling_breastmilk.htm (accessed on 1 March 2023).
7. Fernández-Pastor, S.; Silvestre, D.; López-Mendoza, M. C. Stability of the antimicrobial capacity of human milk against Cronobacter sakazakiiduring handling. Hum. Lact. 2021, 37, 139–146. https://doi.org/10.1177/0890334420932574
8. Van Gysel, M., Cossey, V., Fieuws, S., & Schuermans, A. (2012). Impact of pasteurization on the antibacterial properties of human milk.  J. Pediatr. 2012, 171(8), 1231–1237. https://doi.org/10.1007/s00431-012-1750-4
9. Handa, D.; Ahrabi, A. F.; Codipilly, C. N.; Shah, S.; Ruff, S.; Potak, D.; Williams, J. E.; McGuire, M. A.; Schanler, R. J. Do thawing and warming affect the integrity of human milk?. Perinatol. 2014, 34, 863–866. https://doi.org/10.1038/jp.2014.113
10. Calvo, J.; García, N. R.; Gormaz, M.; Peña, M.; Martínez, M. J.; Ortiz, P.; Brull, J. M.; Samaniego, C. M.; Gaya, A. Recomendaciones para la creación y el funcionamiento de los bancos de leche materna en España [Recommendations for the creation and operation of maternal milk banks in Spain]. Pediatr. 2018, 89, 65.e1–65.e6. https://doi.org/10.1016/j.anpedi.2018.01.010
11. Scott, H.; Sweet, L.; Strauch, L.; Muller, A. Expressed breastmilk handling and storage guidelines available to mothers in the community: A scoping review. Women birth 2020, 33(5), 426–432. https://doi.org/10.1016/j.wombi.2019.09.009

 

- for the study of microbial growth it would be necessary to carry it out in many more microorganisms, not only E. coli. In particular some that are resistant to freezing for long periods such as Listeria monocytogenes as well as other common pathogens in hospital environments

ANSWER:

Escherichia coli has been chosen because it is one of the most common pathogens causing neonatal infections [12,13], and is the cause of outbreaks due to consumption of unpasteurised human milk in NICUs [14,15]. It is also a microorganism that is frequently detected in human milk and can also reach the milk through contamination from handlers and equipment. We agree with the reviewer that other microorganisms need to be investigated in future studies. It is included in the manuscript, lines 396-399.

 

12. Flannery, D. D.; Akinboyo, I. C.; Mukhopadhyay, S.; Tribble, A. C.; Song, L.; Chen, F.; Li, Y.; Gerber, J. S.; Puopolo, K. M. Antibiotic susceptibility of Escherichia coli among infants admitted to neonatal intensive care units across the US from 2009 to 2017. JAMA Pediatr. 2021, 175, 168–175. https://doi.org/10.1001/jamapediatrics.2020.4719
13. Xiao, R.; Li, Y.; Liu, X.; Ding, Y.; Lai, J.; Li, Y.; Kang, W.; Zou, P.; Wang, J.; Du, Y.; et al. Antibiotic susceptibility of Escherichia coliisolated from neonates admitted to neonatal intensive care units across China from 2015 to 2020.  Cell. Infect. Microbiol. 2023, 13, 1183736. https://doi.org/10.3389/fcimb.2023.1183736
14. Chishiki, M.; Nishiyama, K.; Suzutani, T.; Hiruta, S.; Ichikawa, H.; Haneda, K.; Maeda, H.; Shimizu, H.; Kanai, Y.; Ogasawara, K.; et al. Sterilization efficacy of a new water-free breast milk pasteurizer. Int., 2023, 65, e15574. https://doi.org/10.1111/ped.15574
15. Nakamura, K.; Kaneko, M.; Abe, Y.; Yamamoto, N.; Mori, H.; Yoshida, A.; Ohashi, K., Miura, S.; Yang, T. T.; Momoi, N; et al. Outbreak of extended-spectrum β-lactamase-producing Escherichia coli transmitted through breast milk sharing in a neonatal intensive care unit. Hosp. Infect., 2016, 92, 42–46. https://doi.org/10.1016/j.jhin.2015.05.002

 

 

Author Response File: Author Response.pdf