Aureimonas altamirensis: First Isolation from a Chicken Slaughterhouse in Italy Followed by Genotype and Phenotype Evaluations

Round 1

Reviewer 1 Report

In the present manuscript the authors perform Whole Genome Sequencing fo a Aureimonas altamirensis strain isolated from a chicken gut. This bacteria is a environmental contaminant. In a few cases this bacteria was present in human infections. This bacteria is poorly studied and the number of strain sequenced are just a few. However, the data present in this study is not enought for justifiyng the publication of the manuscript. The authors stress the importance of monitoring this bacterium because it may be a foodborne pathogen. However, this species is not currently associated with foodborne infections in humans. Perhaps if the study had focused on studying the prevalence of this bacterium in a larger number of samples and more isolated were sequenced it would have more relevance.  That said, I recognize the interest of having the sequenced genome available in the NCBI database.

Author Response

Dear reviewer 1,

Thank you for your multiple-choice form assessment and for the revisions.

The study of A. altamirensis isolated from the first time from a meat-related environment underlines the necessity of strain characterization. Even if was not possible the isolation of additional A. altamirensis strains our work allows to expose new data related to this species determining its resistance to gentamicin (remembering that resistance to antibiotics is rare in this species) and its pathogenic potential in vitro. Moreover, several genomic data are shown and discussed in the text. Unfortunately, the genomes lack didn’t allow a pangenome study, this aspect has been discussed in the conclusion section. Moreover, the bacteria first isolation from new sources is often related to a single or few strains (e.g. https://doi.org/10.3389/fmars.2022.824071; 10.1186/s13071-017-2104-x; https://doi.org/10.3390/plants11243487; https://doi.org/10.3390/microorganisms9091875). Even if A. altamirensis is not currently considered a foodborne pathogen the isolation of a potential pathogen from a slaughterhouse demonstrates a possible role as food contaminant and in cross-contamination as stated in the introduction. Our aim, exposed in the text, focuses  the attention on A. altamirensis presence in the slaughterhouse that considering the ways of cross-contamination of meat within the slaughterhouse (including the intestinal tract of animals) can lead to contamination of the final product. As stated in the text, future studies are needed to better describe the possible role as foodborne pathogen of A. altamirensis but initially some punctual evaluations must be carried out and reported (e.g. virulence potential, AR, genome characteristics). The study was focused on 49 flocks, however as stated in the text the absence of a specific isolation protocol allows the isolation of a single strain.

Reviewer 2 Report

The submitted manuscript presents results of the isolation and phenotypic and genotypic characteristics of an isolate of Aureimonas altamirensis that was isolated from a chicken slaughterhouse in Italy.

The manuscript is well-written and presents all the methodological steps, which followed all the necessary bioinformatics techniques.

Some notes are in the attached file, which I believe could be improved for the final version of the manuscript.

Comments for author File: Comments.pdf

Author Response

Dear reviewer 2,

Thank you for your revisions,

We modified the text considering the pdf file as follows:

• Gram Negative modified in Gram-negative
• The MIC to gentamicin was added
• AR studies have been added in the introduction
• Qbit modified to qubit
• Maldi modified to MALDI
• Results and discussion format adjusted.
• Added a sentence about possible mechanism linked to gentamicin resistance (L307-314).
• Sentence about one health inserted in conclusion section (L383-386).

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Reviewer 3 Report

The manuscript entitled “Aureimonas altamirensis: first isolation from a chicken’s slaughterhouse in Italy followed by genotype and phenotype evaluation” by Buzzanca et al. mentioned the analysis of A. altamirensis isolated from chicken guts using WGS. Although the authors tried to fulfill the work via both bioinformatic analysis and cell culture experiment, the current form of the manuscript could not be acceptable since no results of the cell culture test and WGS analysis were shown. Moreover, grammatical errors and writing errors were found throughout the manuscript. Therefore, the authors should go through to fix these issues first before submitting the manuscript.   

Grammatical errors and writing errors were found throughout the manuscripts.

Author Response

Dear reviewer 3,

Thank you for your considerations,

This study has been performed on a A. altamirensis strain isolated from a chicken slaughterhouse. Although its isolation was not the initial focus of the work, its isolation by our point of view turns out to be of significant importance and have to  be reported considering its pathogenic potential (even if not considered a foodborne pathogen yet). For this reason, we performed several tests to study A. altamirensis characteristics, this to allow other authors to be easily aware of the characteristics of this bacterium and its pathogenic potential in vitro.

The results of WGS analysis are shown in the text (now with additional discussion). Unfortunately, also considering the high number of samples, it was not possible the isolation of additional strains and for this reason the WGS data are related only to AAI strain. The results of gene enrichment (COGs classes) are shown in the polar plot (We apologize for the Figure 3, now figure 4, missed in first version due formatting error) while even if it was not possible produce analysis on multiple strains (e.g. to obtain SNPs analysis) the WGS data of AAI are reported in the text and discussed.

The data about strain colonization are reported in the text indicating average delta Log and st. err and statistic. In the new version we added the figure 3 to better present the results.

The English was revised in the new version.

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Reviewer 4 Report

The study by Buzzanca and colleagues reports the isolation of Aureimonas altamirensis in chicken slaughterhouse, and the phenotypic and molecular characterization of the isolate.

In general the manuscript is well written. However, it needs a review of the English language, as some sentences are not written clearly. The manuscript is technically sound, and all phenotypic tests have been validated using appropriate controls.

I have minor comments:

Lines 28 and 42: AR – antibiotic resistant. It is more suitable.

Line 42-43: “The presence of antibiotic resistant (AR) strains increases the level of risks related to their presence on meat.” Please, clarify.

Line 45-46: “Pathogenic AR contaminants are considered a possible negative risk for human health” Please clarify.

Line 102: “and” can be removed

Line 145: “Antibiotics resistance evaluation”. Why were MIC, 2xMIC and 3xMIC concentration values ​​chosen? Rather than determining the MIC?

Line 256: It is important to make it clear that the bacterium was not able to form a biofilm on the polystyrene surface, as genes possibly related to biofilms were found in its genome.

In general the manuscript is well written. However, it needs a review of the English language, as some sentences are not written clearly. 

Author Response

Authors reply.

 

Dear reviewer 4,

Thank you for your revisions, you will find our answers below.

Lines 28 and 42: AR – antibiotic resistant. It is more suitable.

Author reply.

Modified with antibiotic resistant (AR) (L30).

Line 42-43: “The presence of antibiotic resistant (AR) strains increases the level of risks related to their presence on meat.” Please, clarify.

Author reply.

Thank for your comment, the following sentence has been added/modified in introduction (L49-50):

Line 45-46: “Pathogenic AR contaminants are considered a possible negative risk for human health” Please clarify.

Author reply.

modified (L50-53).

 

Line 102: “and” can be removed

Author reply.

Thank you for your revision,

“and” removed.

Line 145: “Antibiotics resistance evaluation”. Why were MIC, 2xMIC and 3xMIC concentration values ​​chosen? Rather than determining the MIC?

Author reply.

Following your revision we modify the section related to AR evaluation in m e m (section 2.5)

The MIC considered were collected from EUCAST breakpoint table. The antibiotics concentrations to which a strain is considered resistant is indicated in EUCAST breakpoint table t (indicated as MIC value). In our case, not having official guidelines for A. altamirensis, we took these data into consideration defining the strain resistant in case of growth at 1X MIC. The aim was to evaluate and define the strain AR and not the specific MIC evaluating the AAI resistance also to high antibiotic concentrations (e.g. MIX 3X).

Line 256: It is important to make it clear that the bacterium was not able to form a biofilm on the polystyrene surface, as genes possibly related to biofilms were found in its genome.

Author reply.

Thank you for this consideration, we improve the sentence about BFI (L301-303).

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Round 2

Reviewer 1 Report

Based in the response of the authors and the comments of the other reviewers the manuscript can be accepted. 

Reviewer 3 Report

All queries have been sufficiently addressed. This form of manuscript is now suitable for publication.

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