Analysis of the Virulence and Inflammatory Markers Elicited by Enteroaggregative Escherichia coli Isolated from Clinical and Non-Clinical Sources in an Experimental Infection Model, India

Round 1

Reviewer 1 Report

The author present a study where they compare the virulence of EAEC bacteria from human, animal and environmental source in a mouse model of infection. While the presented data are interesting many changes have to be done.

Lane 72 space missing

Lane 101 space missing

Lane 82 please rephrase “Since EAEC virulence is highly heterogeneous, and none of the virulence factors are observed in all EAEC isolates”

the authors want to say that no isolate present all the EAEC associated virulence factors

lane 103 please rephrase “ten EAEC strains from each animal and environmental sources were used for animal study”

I do not understand the “from each”

Section 3.1.1 where are the data about the Bodyweight measurement ?

Lane 206 “)”

Figure 1 and 2 are very poor in definition and should be merge into only one figure to be able to compare lactoferrin release mediated by human vs non-human EAEC isolates

Figure 1 and 2 Y axis rename lectoferrin to lactoferrin

Same comment for figure 3 and 4 than for figure 1&2

Please define the attA gene, it is present in all the EAEC ?

Lane 266, “of life » is confusing, please remove or add “after challenge”

Figure S2 should be as primary figure not supplemental

Lane 267 “Taken together 267

these results suggest that colonization by EAEC strains from different sources (human, 268

animal, and environmental) occurs up to 10-15 days of life » this is wrong as no attA gene was detected after 15 days from environmental EAEC only up to 10 days

lane 275 there is no figure S7

lane 277 “Figure 5 (data C and A).” should be Figure 5 panel A and C

lane 282 “)” missing

part 3.6 to be able to compare the data presented in the paper a histopathological analysis of the 3 different sources of EAEC should be done 1 day and 15 days after challenge

lane 357, the author conclude that “the non clinical isolates may present less virulence genes that would affect their virulence in the animal model”. They also conclude lane 375, that “EAEC, even from non-diarrhoeal stools and non-clinical sources, had the potential to cause prolonged colonization, weight loss, and inflammation”.

 However what about the animal strain M51 and M82 that present elevated lactoferrin and calprotectin releases

 what are the gene contents of these strains and the associated phylogroups ?

more generally can you define for the animal and environmental strains that present a virulence phenotype (elevated lactoferrin and calprotectin releases) theirs gene content and phylogroups.

More generally bacteria names have to be in italic

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Vinay et al. investigated the pathogenic potential of EAEC from various sources by using mice model, and claimed that both the clinical and non-clinical EAEC have the pathogenic potential. This research revealed the importance in the understanding of EAEC pathogensis. However, more data should be supplimented to support the issue. 

Methods- PCR detection of virulence factors:  M-PCR must be testified to avoid dimers.

Results-3.1: Data must be provided to show the changes in the body weight and bacterial load.

Results-3.3&3.4: There were 2 strains and 4 strains that didn't cause elevations of lactoferrin and calprotectin, respectively. How about the virulence genes of these strains as compare with other strains? the related correlation analysis should be performed to interpret the data one step further. 

Restuls 3.5: there were EAEC strains occuring up to 10-15 days from human and animal sources. Which strain specificly? What's the relation between their colonization and the virulence gene, and between their colonization and the inflammation? Further analysis should be conducted.  

Results 3.6: at day 15, there is little histology changes. More histology observation should be added to observe the time-course change (eg. day 1, 2, 5, 10, 15), and to compare different strains, in order ot correspond with other results (eg, inflammation marker and virulence gene expression). 

Disscussion: 

line 306-307. the level of lactoferrin and calprotectin is only compared with the PBS/blank control group. This conclusion cannot be made unless the author conducted the multiple comparision within the group3.

line 311&321. Such conclusion cannot be made. The author did not compare the level of lactoferrin and calprotectin between the EAEC of diarrheal  and the non-diarrheal strain, or clinical and non-clinical source. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

accept