This manuscript studied the microbial environment of neonatal intensive care units by sampling various room surfaces and air. ddPCR allowed the quantification of the biomass and amplicon-based sequencing permitted diversity analyses. Premature infants present a unique framework as their microbiome differs from the ones of traditional births. The authors tried to answer the question of how the microbiome of infants and caregivers shape the microbiome of intensive care units. Exciting findings can come from this field of research bringing new information about acquired microbiomes.

Nonetheless, significant flaws were noted while reading this manuscript. The authors refer to infant gut microbiome shaping the microbiome of neonatal intensive care unit rooms while gut microbiome represented a small fraction of their work (90% of the paper is about room surfaces and air). This creates a significant disconnection between the title, the aim and the rest of the manuscript. Also, no statistical approach was used or described which make the reliability of the findings questionable.

Specific comments are discussed in the following revision:

The objective of the study is clearly stated in the abstract. However, some information about the methodology used is confusing at this stage of reading. Without additional clarification, it is difficult to understand why only very low birthweight (< 1,500g) were considered for this study. I would suggest erasing this information from the abstract. Also, the authors talk about essential taxa being identified without giving an example of the most dominant ones. Additionally, with the methodology described in the abstract (ddPCR and 16S gene survey), it is not clear how the authors determined that bacterial cells were actively dividing. Finally, the authors did not mention a sustainable significance of their results and how it may affect this field of science.

The title of the manuscript is misleading as the authors mention only premature infant gut microbiome as the significant factor shaping the microbiome of the care unit rooms, while it is not the primary focus of their work.

References seem to be recent, relevant and correctly cited but, I would’ve expected more references in the field. Also, the link in reference one does not work, and reference 17 is wrongly mentioned in the text (line 159)

Although the research question is clearly outlined, the authors failed to showcase two primary objectives of an introduction: the importance and the purpose of their work. Multiple reads were needed to identify the logical thinking structure that leads to the research question.

Given what is already known about the topic, implications and contribution to the literature are hardly defined. How will the findings of this study improve the question of hospital-acquired infections?

The process of subject selection is clearly described and the methodology applied is valid and reliable to measure the variables of the research question. However, not enough details are provided to replicate the study. For example, the authors did not give information about the 22 locations of surface sampling in the room. Also, there is no information about the exact location of air sampling in the room. A diagram describing the sample collection section would be handy to make the methodology of the paper clearer.

Even though a reference is given, ddPCR protocol and how its outcome was analyzed should be provided and described in this manuscript. Thus, the reader can have a general idea about how the results are obtained and have a thorough critical reading.

It is impossible to reproduce the same analyses with the bioinformatics information provided in this manuscript. Crucial information about the workflow for sequence processing (amplicon-based and metagenomics) is missing. The reader should be able to understand what happened to sequences from raw reads to the step before diversity analyses to make a clear judgment about the methodology used.

The major flaw of the results is the absence of appropriate statistical tests. Thus, it is not possible to determine the statistical significance of the results and to stipulate if the results are practically meaningful.

In Fig.1A, the color legend is not clear and not described in the figure caption. In Fig.2, the difference between cyclone and cyclone hall samples is not defined. This definition should be present in the methodology section at least. The title of Table 1 does not describe the result presented in the table. What does the % of abundance represent? The total is 55.3%. What does this percentage represent compared to the total relative abundance?

The source tracker results (additional file 4) are impressive but unfortunately were not explained further. I am not familiar with this software and, I would’ve appreciated a more detailed explanation of the results it provides.

Fig.5, additional file 7 and 8 are impossible to observe. The result should be presented differently if the authors want the reader to understand the message in them. This lack of clarity makes the text associated with these figures hard to understand. What may seem obvious to the authors is not necessarily apparent to the rest of the scientific readers.

The authors used the discussion section to re-state the results and cite the same figures. This section should be kept for data interpretation (line 352 to 432)

The authors refer to the dominant taxa detected in most environments as 5-10 OTUs. In this case, taxonomy level identification is mandatory as it makes a big difference if the authors are talking about 5-10 genera of bacteria or 5-10 classes of bacteria (line 376 to 380)

More references should support conclusions about possible contamination from sample processing in regards to the skin associated taxa (line 395 to 397). There is plenty of literature available about this bias (Mbareche et al., 2017: Bioaerosol sampling and detection methods based on molecular approaches: No pain no gain)

Limitations of the study are not mentioned in the manuscript, and no opportunities to inform future research in the field are discussed.

Conclusions are disconnected from the aim of the study mentioned in the introduction.