Characterization of an International High-Risk Escherichia coli ST410 Clone Coproducing NDM-5 and OXA-181 in a Food Market in China

ABSTRACT During a 2020 routine epidemiological investigation of carbapenem-resistant Enterobacterales at a local food market in Guangzhou, China, two Escherichia coli ST410 isolates coproducing NDM-5 and OXA-181 were obtained from environmental samples. Antimicrobial susceptibility testing, whole-genome sequencing, and conjugation assays were applied to identify their resistance phenotypes, phylogenetic relatedness, and genetic characteristics. Phylogenetic analysis showed that the two isolates were clonally related with only one core-genome single-nucleotide polymorphism (SNP) difference and clustered into a branch with 87 E. coli ST410 isolates deposited in GenBank. These 89 ST410 isolates were closely related (≤51 SNPs), and most were from humans in Southeast Asian countries (n = 47). A Vietnamese clinical isolate collected in 2017 showed the strongest epidemiological link (seven SNPs) to the two ST410 isolates detected in this study. Complete-genome analysis revealed that the carbapenem resistance determinants blaNDM-5 and blaOXA-181 were located on an IncF1:A1:B49-IncQ1 plasmid and IncX3 plasmid, respectively. Conjugation experiments confirmed that the IncX3 plasmid was self-transmissible while the IncF1:A1:B49-IncQ1 plasmid was nonconjugative. BLASTn analysis indicated that the two plasmids showed high similarity to other blaNDM-5-bearing IncF1:A1:B49-IncQ1 and blaOXA-181-bearing IncX3 plasmids from other countries. Altogether, the high similarity of the core genomes and plasmids between the ST410 isolates found in this study and those human source isolates from foreign countries suggested the clonal spread of E. coli ST410 strains and horizontal transmission of blaOXA-181-bearing IncX3 plasmids across Southeast Asian countries. Stringent sanitary management of food markets is important to prevent the dissemination of high-risk clones to the public. IMPORTANCE This is the first report of an Escherichia coli ST410 clone that coproduces NDM-5 and OXA-181 in China. The high similarity of the core genomes and plasmids between the ST410 isolates characterized in this study and human source isolates from foreign countries strongly suggests that this ST410 lineage is an international high-risk clone, highlighting the need for continuous global surveillance of ST410 clones.

Southeast Asia, the study lacks evidence to suggest for that.
The study would benefit screening the individuals working in the same market (probably too late for that) to show possible spread to human beings. Is there any data on screening of individuals who worked with or stayed near the find? I.e. meat sellers or customers who have bought from there? Have the authors examine the farms where the animals come from and see if the same clone is found there? These attempts would make the study and the conclusions stronger.
It could be relevant to describe the screening process more in detail. Is it part of a general screening of food markets in China?
Reviewer #2 (Comments for the Author): Comments for the Author： He et al. analysed two E. coli ST410 isolates co-producing NDM-5 and OXA-181 from environmental samples in China. The genetic comparisons have been deposited and similarity have been identified with the ST410 strains mainly from human sources from other countries. These results are in my opinion interesting in order to obtain better insights in the spread of E. coli ST410 carrying antimicrobial resistance genes. Nevertheless, the manuscript needs some important revision to make it clearer and allow a complete comprehension. I also suggest that the authors have their manuscript checked by a native English speaker. Below are my remarks: Title: Considering only two ST410 (co-producing NDM-5 and OXA-181) isolates identified in the study, I do not think the title of the manuscript is proper, especially the word "Emergence". "Genetic comparison" or "Characterization" maybe better. Also on Line 68, it's better to revise the writing "the first emergence...".
Abstract: the abstract is difficult to follow and gives not much information that can be interesting for the reader to read this paper. I suggest to re-write the abstract giving more information so that it will be more attractive to read the whole paper.
About the Vietnamese clinical isolate, no description about its plasmids? Line 2, in the title "market" please revised as "markets"; Line 23, please revised the "a farmers' market"; and also double check the manuscript for the description about "food markets" or "a farmers' market"? Maybe "a local food market" is a better consistent description. Line 24, "The present study aimed to characterize the two isolates." I do not think this sentence is proper here. Line 26, "conjugation assay" please change as "conjugation assays" Line 70, "This has provoked our interest given this clone's global spread and potential to infect humans via the food chain." Please delete this sentence.
Line 108, "in the animal food sales environment"? It is a wrong description, please correct.
Line 74-93, four meropenem-non-susceptible isolates have been sequenced. Three are belonging to ST410. How about the fourth one? It's better to give a mention of its sequence type during the description. And also, only one isolate, GYX208DH4E-2, has been deposited for the WGS by long-read sequencing. "GYX208DH5-2 carried one (blaNDM-5), and GYX208DH6-1 carried two (blaNDM-5 and blaOXA-181)" only for short-read WGS, suggest to acquire complete genomes for these two as well. Since lacking of the complete genomes of the two strains, it's uneasy to follow the Figure 2. For example, I noticed in the Fig. 2a, "GYX208DH6-1 contigs" were deposited for comparison with the blaNDM-5-carrying plasmids. Couldn't tell where is the GAPs happened on the contigs? The same questions for Fig. 2c. It's better to acquire the WGS for the two strains, and then make the comparison.
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Response to Reviewers
Reviewer comments: Reviewer #1 (Comments for the Author): The present study He et al, describes the detection of ST410 E. coli carrying both blaNDM-5 and blaOXA-181 in the environment in China.
Detection of anti-microbial resistant microorganisms in the environment is interesting. However, strains with high similarity of the core genomes of ST410, IncF1:A1:B49-IncQ1 plasmid carrying blaNDM-5, and IncX3 plasmid carrying blaOXA-181 between the isolates in this study and clinical strains from other countries have already been described. Even though the authors claim that the ST410 isolates detected in pork sales environments may originate from human isolates from other countries, most likely from Southeast Asia, the study lacks evidence to suggest for that.
The study would benefit screening the individuals working in the same market (probably too late for that) to show possible spread to human beings. Is there any data on screening of individuals who worked with or stayed near the find? I.e. meat sellers or customers who have bought from there? Have the authors examine the farms where the animals come from and see if the same clone is found there? These attempts would make the study and the conclusions stronger. Answer: We agree with the reviewer. However, since meat sellers and customers were unwilling to cooperate with us, there was no data on them. And since we could not trace the origin of the meat-producing animals, we did not examine the farms where the animals come from.
It could be relevant to describe the screening process more in detail. Is it part of a general screening of food markets in China? Answer: Yes, it is part of our routine screening of food markets in Guangzhou, China. We have described this clearer in the manuscript (Lines 76-78).

Reviewer #2 (Comments for the Author):
Comments for the Author： He et al. analysed two E. coli ST410 isolates co-producing NDM-5 and OXA-181 from environmental samples in China. The genetic comparisons have been deposited and similarity have been identified with the ST410 strains mainly from human sources from other countries. These results are in my opinion interesting in order to obtain better insights in the spread of E. coli ST410 carrying antimicrobial resistance genes. Nevertheless, the manuscript needs some important revision to make it clearer and allow a complete comprehension. I also suggest that the authors have their manuscript checked by a native English speaker. Below are my remarks: Answer: The manuscript has been carefully revised to make it clearer and more comprehensible, and the manuscript has been checked by a native English speaker.
Title: Considering only two ST410 (co-producing NDM-5 and OXA-181) isolates identified in the study, I do not think the title of the manuscript is proper, especially the word "Emergence". "Genetic comparison" or "Characterization" maybe better. Also on Line 68, it's better to revise the writing "the first emergence...". Answer: We agree with the reviewer. We have revised "Emergence" as "Characterization" in the title and rewritten the sentence in Lines 69-71.
Abstract: the abstract is difficult to follow and gives not much information that can be interesting for the reader to read this paper. I suggest to re-write the abstract giving more information so that it will be more attractive to read the whole paper.