Antimicrobial Susceptibility Profiles and Resistance Genes in Genus Aeromonas spp. Isolated from the Environment and Rainbow Trout of Two Fish Farms in France

Round 1

Reviewer 1 Report

The theme of this study is greatly relevant in present times where the concern over antimicrobial resistance among pathogenic bacteria is rising.

It is very surprising that there are no line numbers in the manuscript, which makes extremely hard to put the comments on the manuscript.

Comments:

1. What was the basis for the statistical tests used Kruskal-Wallis and logistic regression?
2. What is the meaning of healthy Aeromonas strains?
3. Please add water temperature of fish farms.
4. What was the size and weight of sampled fish?
5. Table 1: add the size of each amplified primer set.
6. The quality of Figure 1, 2 and 3 is very poor. Please provide high quality of Figures.
7. What are the three distinct populations for quinolones which the current study claims to find? From section 4.Discussion MIC distributions and the MIC50 and MIC90 values calculated showed a few differences compared to a previous study [31], with our study finding three distinct populations for quinolones and much higher values for quinolones and OXY.

8. Language of text may be simplified to provide better clarity for the readers. As an example, in section 2.6 Biofilms samples “One biofilm surface was taken for the bacteriological analysis of cumulative previous months, and another biofilm surface was also collected from a previous month and then replaced with the biofilm surface of the following month. Each biofilm plate was placed in a sterile bottle filled with the corresponding pond water “.
9. Few grammatical errors were noticed. As an example In lines from section 4.Discussion “Although 105 out 211 Aeromonas strains (49%) were consider as NWT for the three quinolones (FLUQ, OA and ENRO)”.

Author Response

Dear reviewer,

Thank you for your letter and the opportunity to revise our paper. The suggestions offered have been immensely helpful, and we also appreciate your insightful comments on revising the manuscript and other aspects of the paper.

I have included your comments immediately after this letter and responded to them individually indicating exactly how we addressed each concern or problem and describing the changes we have made. The changes are inserted and also marked in “Track Changes” in the revised paper. The revised manuscript is also submitted online by using the Editorial Manager system.

Comments:

• It is very surprising that there are no line numbers in the manuscript, which makes extremely hard to put the comments on the manuscript:

Response: We are very sorry for this inconvenience but we have uploaded the manuscript (PDF) with line numbers.

1. What was the basis for the statistical tests used Kruskal-Wallis and logistic regression?

Response:

Kruskal-Wallis: Nonparametric _ANOVA 

logistic regression: Univariate analysis and binary logistic regression

2. What is the meaning of healthy Aeromonas strains?

Response: the sentences are modified and added.

In “Aeromonas spp. isolation and identification” part:

These isolates were classified as healthy isolates when no episode of furunculosis or no antibiotic treatment were observed; furunculosis isolates when furunculosis occurred; treated isolates when followed by an antibiotic treatment.

In “Antimicrobial susceptibility” part:

Among these strains, 153 environmental and fish isolates were considered as healthy Aeromonas strains including 58 and 98 strains isolated from farms A and B respectively, when no episode of furunculosis and no antibiotic treatment have been observed.

3. Please add water temperature of fish farms:

Response:  The average water temperature on both fish farms is recorded at 10± 0.5 °C and 14± 0.5 °C in winter and summer season respectively.

1. What was the size and weight of sampled fish?

 Response: These fish farms are composed of upstream ponds for juvenile trout and downstream ponds for larger trout until they reach the commercial weight. In this study, the large trout from 40± 5 cm/ 800± 200 g to 55± 5 cm/ 2000± 200 g were considered at the beginning and end of the study respectively.

1. Table 1: add the size of each amplified primer set: Response: Table1 is modified.

Primer Name			Amplicon Size (bp)
	F	R
	Sequence (5’ à 3’)
qnrA	AGGATTTCTCACGCCAGGATT	CCGCTTTCAATGAAACTGCAA	123
qnrB	GCGACGTTCAGTGGTTCAGA	GCTGCTCGCCAGTCGAA	61
aac(6')-Ib-01	GTTTGAGAGGCAAGGTACCGTAA	GAATGCCTGGCGTGTTTGA	72
aac(6')-Ib-02	CGTCGCCGAGCAACTTG	CGGTACCTTGCCTCTCAAACC	65
dfrA1-01	GGAATGGCCCTGATATTCCA	AGTCTTGCGTCCAACCAACAG	94
dfrA1-02	TTCAGGTGGTGGGGAGATATAC	TTAGAGGCGAAGTCTTGGGTAA	149
dfrA12	CCTCTACCGAACCGTCACACA	GCGACAGCGTTGAAACAACTAC	84
sul1	CAGCGCTATGCGCTCAAG	ATCCCGCTGCGCTGAGT	128
sul2	TCCGATGGAGGCCGGTATCTGG	CGGGAATGCCATCTGCCTTGAG	101
sul3	GCCGATGAGATCAGACGTATTG	CGCATAGCGCTGGGTTTC	189
strA	AATGAGTTTTGGAGTGTCTCAACGTA	AATCAAAACCCCTATTAAAGCCAAT	147
strB	GCTCGGTCGTGAGAACAATCT	CAATTTCGGTCGCCTGGTAGT	100
mcr-1	CGGTCAGTCCGTTTGTTC	CTTGGTCGGTCTGTAGGG	308
mcr-2	TGTTGCTTGTGCCGATTGGA	AGATGGTATTGTTGGTTGCTG	562
mcr-3	TTGGCACTGTATTTTGCATTT	TTAACGAAATTGGCTGGAACA	542
mcr-4	ATTGGGATAGTCGCCTTTTT	TTACAGCCAGAATCATTATCA	487
mcr-5	ATGCGGTTGTCTGCATTTATC	TCATTGTGGTTGTCCTTTTCTG	1644
tetA-01	GCTGTTTGTTCTGCCGGAAA	GGTTAAGTTCCTTGAACGCAAACT	62
tetA-02	CTCACCAGCCTGACCTCGAT	CACGTTGTTATAGAAGCCGCATAG	100
tetB-01	AGTGCGCTTTGGATGCTGTA	AGCCCCAGTAGCTCCTGTGA	62
tetB-02	GCCCAGTGCTGTTGTTGTCAT	TGAAAGCAAACGGCCTAAATACA	100
tetC-01	CATATCGCAATACATGCGAAAAA	AAAGCCGCGGTAAATAGCAA	77
tetC-02	ACTGGTAAGGTAAACGCCATTGTC	ATGCATAAACCAGCCATTGAGTAAG	104
tetD-01	TGCCGCGTTTGATTACACA	CACCAGTGATCCCGGAGATAA	85
tetD-02	TGTCATCGCGCTGGTGATT	CATCCGCTTCCGGGAGAT	100
tetE	TTGGCGCTGTATGCAATGAT	CGACGACCTATGCGATCTGA	73
tetG-01	TCAACCATTGCCGATTCGA	TGGCCCGGCAATCATG	92
tetG-02	CATCAGCGCCGGTCTTATG	CCCCATGTAGCCGAACCA	139
tetM-01	CATCATAGACACGCCAGGACATAT	CGCCATCTTTTGCAGAAATCA	100
tetM-02	TAATATTGGAGTTTTAGCTCATGTTGATG	CCTCTCTGACGTTCTAAAAGCGTATTAT	146
tetM-03	GCAATTCTACTGATTTCTGC	CTGTTTGATTACAATTTCCGC	185
floR-01	ATTGTCTTCACGGTGTCCGTTA	CCGCGATGTCGTCGAACT	60
catA1	GGGTGAGTTTCACCAGTTTTGATT	CACCTTGTCGCCTTGCGTATA	100
blaACC	CACACAGCTGATGGCTTATCTAAAA	AATAAACGCGATGGGTTCCA	67
blaCMY	CCGCGGCGAAATTAAGC	GCCACTGTTTGCCTGTCAGTT	107
blaCTX-M-01	GGAGGCGTGACGGCTTTT	TTCAGTGCGATCCAGACGAA	91
blaDHA	TGGCCGCAGCAGAAAGA	CCGTTTTATGCACCCAGGAA	120
blaIMP-01	AACACGGTTTGGTGGTTCTTGTA	GCGCTCCACAAACCAATTG	100
blaIMP-02	AAGGCAGCATTTCCTCTCATTTT	GGATAGATCGAGAATTAAGCCACTCT	232
blaIMP-03	GGAATAGAGTGGCTTAATTC	GGTTTAACAAAACAACCACC	71
blaKPC-02	CAGCTCATTCAAGGGCTTTC	GGCGGCGTTATCACTGTATT	195
blaKPC-03	GCCGCCGTGCAATACAGT	GCCGCCCAACTCCTTCA	59
blaSHV-01	TCCCATGATGAGCACCTTTAAA	TTCGTCACCGGCATCCA	69
mexF	CCGCGAGAAGGCCAAGA	TTGAGTTCGGCGGTGATGA	287
16S-01	GGGTTGCGCTCGTTGC	ATGGYTGTCGTCAGCTCGTG	60
16S-02	CCTACGGGAGGCAGCAG	ATTACCGCGGCTGCTGGC	195
rpoB	CGAACATCGGTCTGATCAACTC	GTTGCATGTTCGCACCCAT	359

 

 

 

6. The quality of Figure 1, 2 and 3 is very poor. Please provide high quality of Figures. Response: the figures are modified.

 

 

 

 

 

1. What are the three distinct populations for quinolones which the current study claims to find? From section 4. Discussion MIC distributions and the MIC50 and MIC90 values calculated showed a few differences compared to a previous study [31], with our study finding three distinct populations for quinolones and much higher values for quinolones and OXY.

Response: MIC distributions and the MIC50 and MIC90 values calculated showed a few differences compared to a previous study [31], with our study finding three distinct populations for quinolones (FLUQ: <0.125; 0.25- 2; > 4 µg/ml/ OA: <0.032; 0.064- 1; > 2 µg/ml/ ENRO: <0.032; 0.064- 0.25; > 0.5 µg/ml) and much higher values for quinolones and OXY.

1. Language of text may be simplified to provide better clarity for the readers. As an example, in section 2.6 Biofilms samples “One biofilm surface was taken for the bacteriological analysis of cumulative previous months, and another biofilm surface was also collected from a previous month and then replaced with the biofilm surface of the following month. Each biofilm plate was placed in a sterile

Response: the sentence is modified.

 One biofilm surface was taken for the bacteriological analysis of cumulative previous months. Another biofilm surface was also collected from a previous month and then replaced with the biofilm surface of the following month.  Each biofilm plate was placed in a sterile bottle filled with the corresponding pond water bottle filled with the corresponding pond water.

1. Few grammatical errors were noticed. As an example In lines from section 4. Discussion “Although 105 out 211 Aeromonas strains (49%) were consider as NWT for the three quinolones (FLUQ, OA and ENRO)”.

Response: the sentence is corrected.

Although 105 out 211 Aeromonas strains (49%) were considered as NWT for the three quinolones (FLUQ, OA and ENRO).

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Antibiotic resistance emergence is an actual and on-trend topic in both human and veterinary medicine. And in this sense, the manuscript gives interesting and valuable information on the establishing and development of antibiotic resistance in rainbow trout aquaculture, and draws opportune and helpful conclusions that, no doubt, will be of great help for the use of antibiotics in the very near future. However, there could be some confusion as in the tittle, and later in the text, authors only refer to Aeromonas in general, although they only tested strains of Aeromonas salmonicida subsp. salmonicida.  Therefore, it might be advisable that they change references to the genus in general, Aeromonas, to the real aeromonad representative they have tested (A. salmonicida subsp. salmonicida).

 

On the other hand, there are also a couple of other things that should be corrected, although these are much less important, and do not jeopardise the correct understanding of the text:

1. In results, at the end of the second paragraph of “3.2 Antimicrobial susceptibility”, authors say “…presented the most difference…”, and maybe it would be more correct “…presented the biggest (or highest) difference…”;
2. In discussion: all “et al.” should be in italics.

Author Response

 

Dear reviewer,

Thank you for your letter and the opportunity to revise our paper. The suggestions offered have been immensely helpful, and we also appreciate your insightful comments on revising the manuscript and other aspects of the paper.

I have included your comments immediately after this letter and responded to them individually indicating exactly how we addressed each concern or problem and describing the changes we have made. The changes are inserted and also marked in “Track Changes” in the revised paper. The revised manuscript is also submitted online by using the Editorial Manager system.

Comments:

• There could be some confusion as in the tittle, and later in the text, authors only refer to Aeromonas in general, although they only tested strains of Aeromonas salmonicida salmonicida. Therefore, it might be advisable that they change references to the genus in general, Aeromonas, to the real aeromonad representative they have tested (A. salmonicida subsp. salmonicida).

Response: This study did not characterize Aeromonas strains. Therefore, we have tested Aeromonas in general including A. salmonicida subsp. salmonicida. We are only sure of the presence of A. salmonicida subsp. salmonicida on fish farm regarding to clinical and laboratory analysis.

Title is modified to clarify the subject of study:

Antimicrobial susceptibility profiles and resistance genes in genus Aeromonas spp. isolated from environment and rainbow trout of two fish farms in France

In manuscript text: Material and methods:

Aeromonas spp. isolation and identification

 

 

 

 

1. In results, at the end of the second paragraph of “3.2 Antimicrobial susceptibility”, authors say “…presented the most difference…”, and maybe it would be more correct “…presented the biggest (or highest) difference…”;

Response: Oxolinic acid (OA) and flumequine (FLUQ) showed five and six dilutions, respectively, and trimethoprim-sulfamethoxazole (TMP) presented the highest difference with seven dilutions between MIC50 and MIC90 values. 

1. In discussion: all “et al.” should be in italics.

 

Response: corrected.

 

Author Response File: Author Response.pdf