Identification by High-Throughput Real-Time PCR of 30 Major Circulating Listeria monocytogenes Clonal Complexes in Europe

ABSTRACT Listeria monocytogenes is a ubiquitous bacterium that causes a foodborne illness, listeriosis. Most strains can be classified into major clonal complexes (CCs) that account for the majority of outbreaks and sporadic cases in Europe. In addition to the 20 CCs known to account for the majority of human and animal clinical cases, 10 CCs are frequently reported in food production, thereby posing a serious challenge for the agrifood industry. Therefore, there is a need for a rapid and reliable method to identify these 30 major CCs. The high-throughput real-time PCR assay presented here provides accurate identification of these 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations, along with the molecular serogroup of a strain. Based on the BioMark high-throughput real-time PCR system, our assay analyzes 46 strains against 40 real-time PCR arrays in a single experiment. This European study (i) designed the assay from a broad panel of 3,342 L. monocytogenes genomes, (ii) tested its sensitivity and specificity on 597 sequenced strains collected from 24 European countries, and (iii) evaluated its performance in the typing of 526 strains collected during surveillance activities. The assay was then optimized for conventional multiplex real-time PCR for easy implementation in food laboratories. It has already been used for outbreak investigations. It represents a key tool for assisting food laboratories to establish strain relatedness with human clinical strains during outbreak investigations and for helping food business operators by improving their microbiological management plans. IMPORTANCE Multilocus sequence typing (MLST) is the reference method for Listeria monocytogenes typing but is expensive and takes time to perform, from 3 to 5 days for laboratories that outsource sequencing. Thirty major MLST clonal complexes (CCs) are circulating in the food chain and are currently identifiable only by sequencing. Therefore, there is a need for a rapid and reliable method to identify these CCs. The method presented here enables the rapid identification, by real-time PCR, of 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations. The assay was then optimized on different conventional multiplex real-time PCR systems for easy implementation in food laboratories. The two assays will be used for frontline identification of L. monocytogenes isolates prior to whole-genome sequencing. Such assays are of great interest for all food industry stakeholders and public agencies for tracking L. monocytogenes food contamination.

Editor Comments: In the article, "Identification by high-throughput real-time PCR of 30 major circulating Listeria monocytogenes clonal complexes in Europe," the authors describe the development of a high-throughput real-time PCR assay for the identification of 30 Listeria monocytogenes clonal complexes. They further adapt the assay to conventional PCR systems used in diagnostic labs. Overall, the significance and need for the study is well presented. Nevertheless, as this is an assay development study, additional explanations and details of the methods are required. The assays were designed from and tested against several "panels" of Listeria strains and genomes, however, these different groups quickly became confusing and hard to distinguish (strains vs. genomes, # included, sources, used in which part of the study). Perhaps a table or labeling of the panels A, B, C, etc. with descriptions would aid the reader throughout the paper. Three different DNA extraction methods are described but the only result given was a statement in L337 that it did not affect the assay's performance. However, no information is given on the details of how this was determined. What was the starting amount of cells? Probes were labeled with either FAM or HEX but no description is given to which dye was used for which probe or was one chosen over the other. Table 1 should show dye with probe sequence. Likewise, the conventional PCR assays were based on duplex and triplex reactions (L243) yet the groups of primers/probes used together in reactions are not described. It doesn't appear the same set of strains or panel was analyzed by both the high-throughput assay as well as the 2 conventional assays. How then can the assays be compared between the 3 platforms? Minor comments: L32 -how can nomenclature provide info on persistence in food chain? Overall sentence structure is awkward L28-57 All section headers need to be in same font L96-97 rapid and reliable duplicated L 100 remove "of" L 108-116 no mention of Lm even though it's implied L 144-145 incomplete sentence L169 awkward "placed on the alleles" L245 Cyanine was added to some probes. Again, which ones, how were they grouped? L251 an should be "and" Table 1: need dye used on probe sequence Table 2: typo in header, "corss" I didn't see Table S1 and S2. Fig 1: I don't understand what this is supposed to show. The title says it's an interpretation of a multiplex PCR assay but the chart contents seem higher level with WGS and verification of concordance shown.
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Reviewer comments:
Reviewer #1 (Comments for the Author): Please see below the suggestions / comments. Line-49: please put the hyphen between "real time" to make it uniform with other similar entries -> Done Lines-50-82-83: please put "s" to CC Line-369: please insert the space between "the" and "77" -> Done Line-435: please put the hyphen between "false negatives" to make it uniform with other similar entries -> Done Line-441: please change 1000 to 1,000 -> Done Line-465: please put "s" to CC -> Done Line-572 (not line 576) : please put "s" to strain -> For "strain DNA" strain should not be plural Line-604: please consider updating the reference (8) with the correct version referring to 2020 version -> Date was updated in the text to 2019 Line-754: please consider updating the reference (51) with the correct version, Part 1 is refferred only to Vocabulaire; moreover the link doesn't work -> The references were changed for English version of the standards ISO16140 and ISO17025. Access to standard is charged and requires specific subscription. Therefore we preferred to removed the link to the French accreditation body website.
Line-896: please standardize the title character of Table 1 -> Done Conclusions: please consider to add the limitation of the study or of the assays performed.

-> Done
Reviewer #2 (Comments for the Author): This study describes two novel real-time PCR methods (high-throughput and multiplex PCRs), which can rapidly discriminate 30 clonal complexes that are frequently encountered in the food chain. The methods developed by the authors will definitely attract a great interest from the public health sector and food industry by allowing to identify the CC of a strain without relying on the whole genome sequencing. Also, primers and probes were systematically designed using a large panel of genomes and the novel methods were thoroughly validated in various aspects. However, the current manuscript could be further improved by addressing weaknesses shown below.
1. The conventional multiplex real-time PCR was not sufficiently described, at least not to the degree that a reader can replicate this method after reading this manuscript. For instance, "3 duplex and 10 triplex PCR reactions" in lines 243 and 244 were not specified in the text or tables and "several steps" comprising this real-time PCR (lines 249 and 250) were not adequately delineated. Also, Figure 1, which was cited in lines 249 and 250, did not clearly convey the process and thus needs to be revised. Given that this conventional real-time PCR method was developed so that diagnostic laboratories can perform this assay routinely, this could be a major pitfall that could reduce the usefulness of this study. In the Materials and Methods section in general, more details are needed. See specific comments.
-> The multiplex scheme was further detailed in table 1 with a multiplex code and dye and quencher indications. Figure 1 was simplified and made more explanatory for routine performance of the assay.
2. If the authors have included the features/functions of the inserted or deleted genes when discussing false-negative strains, it could have pleased many readers, including this reviewer, who are interested in the genetic diversity of L. monocytogenes population.
-> We have pushed the identification of integrative elements responsible of false negative identification for the PCR targeting CC9, CC193 and CC204. Unfortunately we did not find further annotation on them. When possible we identified the gene where the insertion or the deletion happened.
3. Show the GitHub site for the in-house bioinformatics pipeline, if available.
-> We provided the scientific article for BIOHIT tool, which is the core of our in-house pipeline. The rest of the analysis was performed in BioNumerics.
4. Many statements, in particular in the Results and Discussion sections, should be worked on to improve clarity. See specific comments.
-> Comment were followed to improve clarity. 5. References are often missing. See specific comments.
6. Cite tables and figures more frequently in Results.
-> Done Abstract Line 35: Change "serogroup of the strain" to "serogroup of a strain".
-> Done Line 39: Change "its sensitivity and its specificity" to "its sensitivity and specificity".
-> Done Importance Line 51: Change "four CC, splitting each CC in" to "four CCs, splitting each CC into".
-> Reference were added Lines 82 and 83: Provide references.
-> Reference were added Line 93: Change "the cost and the time" to "the cost and time".
-> Done Line 115: Do "both assays" mean the high-throughput and conventional real-time PCR assays? Clarify this.

-> Done
Materials and Methods Line 123: Change "fluorimeter, DNA high" to "fluorimeter and a DNA high".
-> To clarify the sentence "diluted if necessary" was removed as dilution rules are specified in sections Results, 1.2 Limit of detection (LoD) and 2.1 Limit of detection.
-> All units were modified accordingly Line 132: Show the manufacturer of the lysozyme and its location.

-> Done
Lines 148-152: Which program was used to download genomes from these databases? Remove "(EMBL-EBI)" in line 151, which is redundant with the acronym in line 152.
-> The SRA were downloaded via the BioNumerics Calculation engine. Precision was added.
-> Done Lines 157 and 158: Re-write this sentence, which does not make sense. Why is the quality of genomes discussed with genome sizes? -> Lm genomes size is between 2.8 and 3.1 Mb, assembly providing a genome out from this range must be related to a technical issue. The sentence was rephrased.
Line 158 and 159: What criteria were applied to select these representative genomes?
-> Selection criteria were added to the text.
Lines 160 and 161: Re-write this statement to enhance clarity.
-> The paragraph was modified to make it clearer.

-> Done
Line 164: I would recommend changing "retained" to "selected". Show criteria to select a single kmer for each CC.
-> The selection of k-mer was done without pre-selection criteria. The k-mer selection was performed at step 2.1.3 using BLAST screening. The selection criteria were further specified in this section.
Lines 168 and 169: Remove the comma after "(the 4807 gene scheme)". I recommend incorporating the statement in lines 170 and 171 into here.
-> The parapgraph was modified as well as section 2.1.3 Lines 169 and 170: Re-write this statement for clarity. Also, which program or assay was used to select the alleles with the best real-time PCR discrimination? -> Specification were provided in particular software functionality used.
Lines 174 and 175: I expected four numbers due to "between oligos, for hairpins, self-dimers and multiplex dimers" but only three were presented. Clarify this.
-> Energy tolerance between oligos was not a nominal parameter. The sentence was modified for clarity.
-> The statement was revised.
Lines 182 and 183: Which program was used to download fasta files of these genomes? -> Fasta files were were directly uploaded from NCBI RefSeq assembly database, on the 28th of June 2019 Lines 184 and 185: Change "6-Carboxyfluorescein" and "Hexachlorofluorescein" to "6carboxyfluorescein" and "hexachlorofluorescein". Show what "ex." and "em." represent. b -> Excitation ("ex.") was replaced by absorbance. Modification was changed in the text.
Line 201: "manually defined thresholds" sounds vague and I recommend showing specific thresholds and how they were determined.
-> The manual threshold set up was specified in the text.

-> Done
Lines 207 and 208: It is hard to understand what "The plasmids ... recombinant plasmids" means. I suggest revising it to improve clarity.
-> The sentence was simplified, pBluescriptIISK is the vector plasmid used to carry the PCR targeted sequenced.
Line 211-213: Revise this statement for clarity. How was genomic DNA concentration equivalence determined?
-> The information was added. The same methodology was applied for plasmid copy number estimation.
Lines 213-215: Revise this statement for clarity. Also, is "106 bp" a typo for "10^6 bp"? -> The calculation was false and replaced by the right formula.
-> Done Line 217: Include "ISO 16140, part 4" to references and cite it here.
-> Done Line 218: Change "for the testing and validating the assay." to "to test and validate the assay".
-> The statement was modified to make it clearer.
Line 233: I recommend merging the statement "They covered 43 STs (Table S1)" with the prior one.
-> Done Line 235: Provide references for "known to be frequent food-chain contaminants".
-> Reference were added Line 243: Change "was based on" to "consisted of".
-> Done Line 248: What does "similar" mean here? Any difference from the high-throughput real-time PCR assay regarding controls? -> They are the ones used for HT real time PCR. The text was modified accordingly.

-> Done
Line 255: Include the manufacturer of master mix. Change "until" to "up to".

-> Done
Line 258: It would be informative for readers if the authors mentioned how this threshold was determined.
Line 267: Change "Performance of the assay was performed" to "Performance of the assay was measured".
-> The sentence was clarified. The TaqMan 7500 fast system global runtime was adjusted from 30 min to 55 min. 30 min was reported by mistake.
Lines 281 and 282: What do all these acronyms (EURL, IT NRL and NL NRL) mean? Re-write this to improve clarity.
-> The acronyms were developed.

Results
Lines 290 and 291: Was this in silico analysis described in the Materials and Methods section? Which program was used.
-> The in silico analysis strategy was further detail in Material and Methods section, cross detection identification was performed by BLAST as detailed in section 2.1.3.
Line 293: I would recommend changing "to be between" to ", ranging between".
-> Done Lines 295 and 296: What does this mean? Clarify and re-write this statement.
Line 304 and 320: What do "CC9 locus" and "CC203 locus" mean? Regions targeted in the real-time PCR to identify CC9 and Cc203? Also, it would be informative if the authors briefly described genes located in these loci.
-> The statements have been changed.
-> Done Line 313: Change "GuaA" to "guaA" and italicize it. Change other instances as well throughout the manuscript.

-> Done
Lines 313 and 314: Why were these two strains mentioned here, not in the beginning of the paragraph along with strain 01CEB168LM? -> Both ST662 strains were cited apart from 01CEB168LM because we only have the assemblies for these strains and we never tested them by PCR. The statement was modified to make it clearer.
Lines 314-316: Do "strains reported from Canada and France from fish products" mean two ST662 strains mentioned in lines 313 and 314? Revise this statement for clarity.
-> The statement was revised to clarify.
Line 316: Which strain was "strain genome" obtained from? -> The statement was revised to clarify.
Line 319: I recommend moving " (Table S1)" to the end of the statement (line 320).
-> Done Line 321: Clarify "deletion of the CC204 locus". Was "CC204 locus" replaced with the 43 kb genomic insertion? Or is the 43 kb genomic insertion located in the middle of the CC204 locus? -> The genetic location of CC204 locus was clarified in the text.
-> This statement was removed and replaced by a clearer description.
Line 324: What strains are referred to in "strain genomes"? -> Precision was provided.

-> Done
Lines 334 and 335: This statement is vague and I recommend re-writing it.
-> The statement has been changed.
Line 341: Clarify "remaining strains". Are they those whose CCs could not be determined by the highthroughput real-time PCR? Or are they those that were not isolated from food products, FPEs, or ruminants?
-> Precision was provided Lines 344-346: Cite tables or figures. Change "same plant" to "the same plant".

-> Done
Lines 360-362: What does this statement mean? Re-write it to improve clarity.
-> Done Lines 371-374: This part could be merged with lines 365-370. Change "all primer and probes sets" to "all primer and probe sets".
-> Both sections were merged Line 377: Provide references.
-> A reference was provided Line 386: Change "less than 3 AD" to "strains with less than 3 AD".
-> Done Lines 388 and 389: This explanation sounds strange since the analysis output indicates that these processing plants were colonized by different strains of ST398.
-> The argue is not supported as you said. The statement was removed.
-> There is no publicly available reference for this outbreak investigation Line 393: I believe that "audits" instead of "controls" might sound more appropriate here.

-> Done
Line 414: Only one in-house pipeline was mentioned in the Materials and Methods section. What are "two original bio-informatics tools"? Change "bio-informatics" to "bioinformatics".
-> the reference to both bioinformatics pipelines were added in the text.
Line 420: Clarify "following the molecular serotyping method developed by Doumith et al.". Do 28 primer and probe sets target genes utilized in the PCR serotyping assay devised by Doumith et al.? Also, remove "(2004)".
-> Compare the primer design approach of Doumith et al. with the approach applied in this study was here only to compare the approach, that in both study target unique sequences on Lm chromosome. The statement was modified to clarify it.
-> The statement was changed accordingly.
Lines 431 and 432: I would recommend combining this statement with the one in lines 430 and 431.
Line 439: What does "confirmed analytically" mean? Confirmed via the high-throughput real-time PCR? Change "a false-negative ST662" to "a false-negative ST662 strain".
Line 440: Does "observed in silico" mean that only in silico analysis was conducted or that it was found in silico but not confirmed with a wet lab assay? -> Yes it mean that in silico analysis conducted without wet lab confirmation. We changed the statement accordingly.
Line 441: "with more than 1000 AD with the CC193 targeted strains" is confusing. Does this mean that ST662 and ST796 do not belong to CC193? Clarify and re-write this part. Also, I recommend starting a new paragraph from "All false-negative results".
-> Precision was provided on the genetic link between ST662 and ST796 and the other CC193 strains. ST796 in silico analysis were mentioned in the Results section 1.3 line . Mention to this ST in the results section Line 442: Remove the comma after "CC204" and change "the insertion or to the deletion" to "the insertion or deletion".

-> Done
Lines 444 and 445: Revise this statement, which is fragmentary.

-> Done
Lines 447 and 448: Change "resulting from our method" to "observed in our method".
-> The statement was clarified accordingly.
Lines 455-458: I suggest merging this paragraph with the prior one.

-> Done
Lines 459-466: I recommend moving this paragraph to after line 418.
The paragraph was moved.
-> Done Line 461: Is "isolated" a typo for "isolates"? -> It is a typo, isolated was changed for isolates Line 468: I suggest changing "tracking" to "trackdown of".
-> Done Lines 486 and 487: Change "the high-throughput and the conventional real-time PCR assay" to "the high-throughput and conventional real-time PCR assays".

-> Done
Lines 491, 493, and 494: It will be helpful to readers if the authors add some explanations for the acronyms of the NRLs in different countries.
-> The name of the institutes hosting the NRLs were specified when mention for the first time and after cited as NRL + two letters country acronym.
Lines 494 and 495: Revise this statement, which seems to pertain to the purpose of this study.
-> The statement was removed.
Line 497: Revise "in a timeframe ... mobile laboratory" to improve clarity.

-> Done
Lines and 505: Clarify and revise "pure strain DNA extracts" and "complex sample DNA extracts".
-> The term were changed to define them more accurately.

-> Done
Line 522: Revise "food Lm strain population structure" to "Lm population structure in foods".
-> Done for all tables and figures Figure 1 Line 913: Change "flow chart" and "real time" to "flowchart" and "real-time", respectively.

-> Done
Editor Comments: In the article, "Identification by high-throughput real-time PCR of 30 major circulating Listeria monocytogenes clonal complexes in Europe," the authors describe the development of a highthroughput real-time PCR assay for the identification of 30 Listeria monocytogenes clonal complexes. They further adapt the assay to conventional PCR systems used in diagnostic labs. Overall, the significance and need for the study is well presented. Nevertheless, as this is an assay development study, additional explanations and details of the methods are required. The assays were designed from and tested against several "panels" of Listeria strains and genomes, however, these different groups quickly became confusing and hard to distinguish (strains vs. genomes, # included, sources, used in which part of the study). Perhaps a table or labeling of the panels A, B, C, etc. with descriptions would aid the reader throughout the paper. Three different DNA extraction methods are described but the only result given was a statement in L337 that it did not affect the assay's performance. However, no information is given on the details of how this was determined. What was the starting amount of cells? Probes were labeled with either FAM or HEX but no description is given to which dye was used for which probe or was one chosen over the other. Table 1 should show dye with probe sequence. Likewise, the conventional PCR assays were based on duplex and triplex reactions (L243) yet the groups of primers/probes used together in reactions are not described. It doesn't appear the same set of strains or panel was analyzed by both the high-throughput assay as well as the 2 conventional assays. How then can the assays be compared between the 3 platforms? -> Details on the biological and genomic resources were better described and referenced in the manuscript using two distinct denominations for genomic panel: GP-A and B and for strain panel: SP-C, D and E. Details were provided on extraction method assessment, the cell amount used to carry out these methods were specified, the 25 strains used for extraction method comparison were flagged in Table S1 and the results were further detailed. Probes were annoted with dyes and quencher and multiplex groups of primer and probe sets were provided in Table 1. Annotation of strain panel SP-C, D was reported in Table 2, Table 3 and Table S1 providing a link between the text and the strains used for the high-throughput assay as well as conventional assays validation.
Minor comments: L32 -how can nomenclature provide info on persistence in food chain? -> Recurrent isolation of strains assigned to the same CC in the same area provide an information on their presumable persistence. The presumable persistence nuances the statement.
Overall sentence structure is awkward L28-57 -> The section was re-written All section headers need to be in same font -> Done L245 Cyanine was added to some probes. Again, which ones, how were they grouped? -> All dyes and multiplex groups were specified in Table 1 and reference was made in the text.
L251 an should be "and" -> Done Table 1: need dye used on probe sequence -> Dyes were specified on the sequence Table 2: typo in header, "corss" -> Done I didn't see Table S1 and S2.
-> These tables are very large and were provided in PDF as supplementary material. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see from the reviewer's comments, there are still a lot of grammatical errors that needs to be fixed. I will suggest that the authors thoroughly look over the manuscript again before resubmitting.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Table 1 Lines 978 and 979: I believe that "WGS, whole-genome sequencing; CC, clonal complex" should be shown after the table as a legend. My understanding is that probes are marked with "_P" and primers are signified with "_F" or "_R". I recommend including this information to the legend. Show which dye was used for each Cy5-labeled probe in the high-throughput real-time PCR assay. Table 2 In the "Analytical sensitivity" column, is "0,992" a typo for "0.992"? If so, revise all the numbers in this column appropriately. In the "Cross-reaction with analytical confirmation" column, why is there a comma before brackets in several rows while there is not in other rows? I recommend changing "clonal complex" to "CC" and "strain panel C" to "SP-C". "(1) figures reported beside CC or ST report" could be re-written to "The number within parentheses beside CC or ST reports". Table 3 Line 989: I expected two panels since 526 and 77 strains were mentioned in line 988. I recommend changing "strain panel D" to "SP-D".

Figure 1
Lines 992 and 993: Change "follow" to "followed". Provide the reference for Vittulo et al.
Line 993: Start a new sentence from "CC id". Change "abbreviation and means" to "abbreviation for". In Figure 1, what do the numbers in rectangles under each molecular serotype mean? CCs with the numbers are sequentially identified according to the numbers? How were these numbers determined?
Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
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-Line 97 Line 106: Change "species identification or the five". to "the identification of species or the five".
-Line 111 Materials and methods Line 124: Remove the comma in "strains, from".
-Line 145 Line 157: Change "two bacterial genome public databases" to "two public bacterial genome databases".
-Line 159 Line 166 and 167: Change "Only genomes, consistent with Lm genomic size, between 2.8 and 3.1 Mb," to "Only genomes between 2.8 and 3.1 Mb, consistent with Lm genomic size,".
-Line 168 Line 182: Change "Primer and probes parameters" to "Primer and probe parameters".
-Lien 244 Line 259: Change "in France and in Hungary" to "in France and Hungary".

Results
Line 308: Change "designed in this study" to "designed for this study".
-Line 303 Line 311: Change "within the CC" to "within each CC".

-Line 338
Lines 359 and 360: Merge this statement with the prior paragraph. Change "SP-C closely related strains" to "closely related strains in SP-C".
-Line 358 Line 392: Change "until a concentration" to "up to a concentration".
-Line 419 Discussion Line 434: Change "developed in the study proved able" to "developed for this study proved to be able".
-Line 429 Line 445: Change "from meat products The 30 CCs set covers" to "from meat products. However, the 30 CC set covers".
-Line 436 Line 467: Change "primers and probes set" to "primer and probe set".
-Line 457 Line 468: Change "as characteristics of" to "as a characteristic of".
-Line 458 Lines 465 and 474: Does "five false-negative results" in line 465 mean "five false-negative strains"? Also, show the number of false-negative CC193 strains in line 474.
Line 480: Change "and was not" to "but not".
-Line 470 Line 482: Change "insertion or to the deletion" to "insertion or deletion".
-Line 473 Line 491: "conventional serotyping methods" could sound confusing since some readers could think that it refers to the traditional serological serotyping method. I recommend revising this to avoid confusion.
-Line 482 conventional was changed to molecular.
Line 494: I recommend starting a new statement from "27 of them".
-Line 486 Line 499: Change "track down the sources" to "trackdown of the sources".
-Line 490 Line 503: Change "encountered by the food" to "encountered in the food".
-Line 494 Lines 504-508: This statement summarizes the accomplishments achieved by this paper and I cannot understand why a paper on Biomark is cited here. Unless there is a due reason, I would recommend not citing any paper for this statement.
-The reference was removed Lines 513-516: Combine this paragraph to the prior one.
-Line 505-506 Line 521: Change "the high-throughput and the conventional" to "the high-throughput and conventional".
-Line 526 Lines 541 and 542: Change "strain DNA already isolated and purified" to "genomic DNA purified from an isolated strain".
-Two references were provided Line 549: Merge "Analyses are currently underway" with the previous sentence.
-Line 539 Line 549: Change "may be able to investigate" to "may be able to be used to investigate" -Line 540

Conclusions
Line 558: Change "The method do not cover the described the" to "The methods do not cover the described".
-Line 559 Acknowledgements Line 570: Change "The authors thanks" to "The authors thank".
-Line 596 to 601 Author Contribution Line 624: Change "contributed to the project design, contributed to the writing" to "contributed to the project design and the writing".
-Line 615 Table 1 Lines 978 and 979: I believe that "WGS, whole-genome sequencing; CC, clonal complex" should be shown after the table as a legend.
CC was added in column header, WGS was removed as not cited in the table 1.
My understanding is that probes are marked with "_P" and primers are signified with "_F" or "_R". I recommend including this information to the legend.
-Done, in foot note "c" Show which dye was used for each Cy5-labeled probe in the high-throughput real-time PCR assay.
-Alternative dyes used for the high throughput real-time PCR assay were reported under bracket in Table 1 following footnote "b". Table 2 In the "Analytical sensitivity" column, is "0,992" a typo for "0.992"? If so, revise all the numbers in this column appropriately. -Done In the "Cross-reaction with analytical confirmation" column, why is there a comma before brackets in several rows while there is not in other rows?
-It was a typo, comma were removed I recommend changing "clonal complex" to "CC" and "strain panel C" to "SP-C".
-Done "(1) figures reported beside CC or ST report" could be re-written to "The number within parentheses beside CC or ST reports".
-Done Table 3 Line 989: I expected two panels since 526 and 77 strains were mentioned in line 988. I recommend changing "strain panel D" to "SP-D".
-Line 250, 281, 359, 393 and 981-982: the legend was modified by defining two SP-D subpanels, SP-D.1 including 526 strains and SP-D.2 including 77 strains. Both were used for performance assessment of the high throughput and the conventional multiplex real-time PCR assay, respectively.

Figure 1
Lines 992 and 993: Change "follow" to "followed". Provide the reference for Vittulo et al.
-Line 986, the reference was provided Line 993: Start a new sentence from "CC id". Change "abbreviation and means" to "abbreviation for".
-Line 987 In Figure 1, what do the numbers in rectangles under each molecular serotype mean? CCs with the numbers are sequentially identified according to the numbers? How were these numbers determined?
-The number in each square left corner refers to the multiplex number reported in Table 1. This is a linear numbering. The legend was completed accordingly.