Pseudomonas putida: Sensitivity to Various Antibiotics, Genetic Diversity, Virulence, and Role of Formic Acid to Modulate the Immune-Antioxidant Status of the Challenged Nile tilapia Compared to Carvacrol Oil
, Hend S. Nada 3, Shaimaa A. A. Ahmed 4, Asmaa W. Zaglool 5, Sherif M. Shawky 6, Mohamed Alkafafy 7 and Heba H. Mahboub 3,*
Round 1
Reviewer 1 Report
In the title of their manuscript, the authors very ambitiously announce the characterization of the bacterial pathogen Pseudomonas putida, with a view to determining its susceptibility to various antibiotics, genetic diversity, virulence, and the role of formic acid in modulating the immune-antioxidant status of challenged Nile tilapia compared to carvacrol oil. It is a very welcome work, it would only be desirable if the authors would improve it.
The improvements relate primarily to the spelling of the nomenclature of bacteria according to the rules for writing bacterial genera and species, such as in lines 54 and 84, etc.
Also, it is not necessary to write the full name of the bacterium and then its abbreviation in parentheses, as in line 66.
Also, citing literature in the text must follow the instructions for authors.
in line 108, please check the use of parentheses.
When you write abbreviations in the text, sometimes their first mention is followed by the full name, but somewhere it is not, so it must be consistent. Check the writing in lines 144 and 148.
The determination of the virulence gene is accompanied by two lines and three figures in the text and has been ignored in the discussion. In any case, genetic diversity and virulence need to be presented in more detail in an article that has this in the title.
Author Response
Reviewer 1
In the title of their manuscript, the authors very ambitiously announce the characterization of the bacterial pathogen Pseudomonas putida, with a view to determining its susceptibility to various antibiotics, genetic diversity, virulence, and the role of formic acid in modulating the immune-antioxidant status of challenged Nile tilapia compared to carvacrol oil. It is a very welcome work, it would only be desirable if the authors would improve it.
Response: Thank you so much for your valuable comment.
The improvements relate primarily to the spelling of the nomenclature of bacteria according to the rules for writing bacterial genera and species, such as in lines 54 and 84, etc.
Response: Corrected.
Also, it is not necessary to write the full name of the bacterium and then its abbreviation in parentheses, as in line 66.
Response: Corrected.
Also, citing literature in the text must follow the instructions for authors.
Response: Corrected.
in line 108, please check the use of parentheses.
Response: Corrected.
When you write abbreviations in the text, sometimes their first mention is followed by the full name, but somewhere it is not, so it must be consistent. Check the writing in lines 144 and 148.
Response: Corrected.
The determination of the virulence gene is accompanied by two lines and three figures in the text and has been ignored in the discussion. In any case, genetic diversity and virulence need to be presented in more detail in an article that has this in the title.
Response: Thank you so much for your valuable comment. For clarity, more details were added in the text and now reads:
2.1.4. Molecular characterization
- 16S rDNA amplification
The suspected pseudomonus species were molecularly confirmed using PCR with specific 16S rDNA primers.Then, according to the manufacturer’s guidelines, bacterial DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany). Strain was cultured in brain heart infusion broth (Oxid,Baasigngstoke, United Kingdom) and incubated under aerobic condition, then, 200 μl of AL buffer was added to 200 μl of the sample and 20 μl QIAGEN protease into the bottom of a 1.5 ml microcentrifuge tube. The tube was incubated at 56ËšC for 10 min. 200 μl ethanol (96%) were added to the sample, and mixed again by pulse vortexing for 15 seconds ,the sample was then washed and centrifuged according to the manufacturer s recommendation. nucleic acid was eluted with 100ul of elution buffer provided in the kit to measure the quantity and purity .
B.Virulotyping
Specific primers targeting the entertoxin gene (tox A , exo S, and nan1), genes were screened in the confirmed P. putida isolates. Positive controls for P. putida were run alongside the tested isolates and were generously supplied by the Biotechnology Unit, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute (Dokki, Giza, Egypt). The oligonucleotide primers are listed in Table 2 , the cycling conditions which used in this study are listed in Table 1. All PCR reaction were done on a total volume of 50 μl reaction containing 13 μl of DNA template , 25 μl of Emerald Amp GT PCR mastermix, 1 μl of each primer (20 pmol concentration) and 6 μl PCR grade water. PCR amplification was carried out in T3 Thermal cycler (Biometra). The amplified product was electrophoresed on 1.5% agarose gel in 100 ml TBE buffer at room temperature, stained with 0.5μg/ml ethedium bromide. A 100bp plus DNA ladder (QIAGEN (USA) was used to determine the fragment size (Table 2). The gel was photographed by aGelDoc UVgel documentation system.
Table (1): Cycling conditions of the different primers during cPCR
|
No. |
Pseudomonas Genes |
Primary denaturation |
Secondary denaturation |
Annealing |
Extension |
No. of cycles |
Final extension |
|
1 |
exoS |
94ËšC 5 min. |
94ËšC 30 sec. |
55ËšC 30 sec. |
72ËšC 30 sec. |
35 |
72ËšC 7 min. |
|
2 |
nan1 |
95ËšC 10 min. |
94ËšC 1 min. |
58°C 1 m |
72ËšC 1 min. |
30 |
72ËšC 10 min. |
|
3 |
toxA |
95ËšC 10 min. |
94ËšC 1 min. |
58ËšC 30 sec. |
72ËšC 1 min. |
35 |
72ËšC 10 min. |
Table (2): Oligonucleotide primers sequences
|
Pseudomonas Genes |
Primer Sequence 5'-3' |
Amplified product |
Reference |
|
exoS |
CTT GAA GGG ACT CGA CAA GG |
504 bp |
Khattab et al., 2015 |
|
’ TTC AGG TCC GCG TAG TGA AT |
|||
|
nan1 |
AGG ATG AAT ACT TAT TTT GAT |
1316 bp |
|
|
TCA CTA AAT CCA TCT CTG ACC CGA TA |
|||
|
toxA |
GGT AAC CAG CTC AGC CAC AT |
352 bp |
|
|
TGA TGT CCA GGT CAT GCT TC |
Reviewer 2 Report
The manuscript "Pseudomonas putida: Sensitivity to various antibiotics, genetic diversity, virulence, and role of formic acid to modulate the immune-antioxidant status of the challenged Nile tilapia compared to carvacrol oil" (manuscript ID: fishes-2065685) collects data from various experiments conducted using a P. putida strain, including an experimental infection, a characterization of the strain and the use of antimicrobial substances in place of antibiotics.
Although the data may be of interest from a scientific point of view, their presentation does not meet the minimum quality required for a publication. The manuscript, as it is currently structured, must be completely revised. The data are reported in an excessively fragmentary and confusing way, which makes it difficult to understand whether they are actually interesting or not. For this reason, I decided to give the manuscript a major revision: if the changes made make the data usable, the manuscript will be revised again. Conversely, I will consider the manuscript as non-editable upon publication.
I list below the changes I think are necessary:
- The manuscript aims to analyze many aspects related to the isolated P. putida strain: could the hypothesis of publishing only part of the reported data be considered?
- From a stylistic point of view, it is necessary to check the whole manuscript to eliminate errors, repetitions, correctly report the scientific names (in italics) and citations according to the MDPI guidelines.
- The abstract must be completely rewritten.
- The introduction, as well as the whole article, should be reviewed following a logical and precise sense. It is advisable to review the manuscript following a precise scheme of topics, not jumping from one topic to another randomly. Regarding the introduction specifically, I recommend expanding the part relating to the breeding of the nile tilapia in Egypt and globally, to emphasize the importance of this species.
- The materials and methods are written in a confusing way. I suggest a succession based on isolation of the strain, biochemical and molecular characterization and antibiotic resistance tests; once these aspects have been described, the authors can decide how to report the experimental study or the data relating to the use of compounds with antimicrobial action. All the procedures used must be described in detail (PCR conditions, information regarding positive/negative controls).
- Figures 1-4 are superfluous: I would recommend removing them.
- The results must be completely reviewed, following the scheme used in the materials and methods. The division into mirror paragraphs for materials and methods and results could help the authors in this.
- Discussions and conclusions are difficult to understand in relation to the above. A clearer description of the procedures used and the results obtained will certainly help to better understand what the authors want to report in this manuscript.
Author Response
Reviewer 2
The manuscript "Pseudomonas putida: Sensitivity to various antibiotics, genetic diversity, virulence, and role of formic acid to modulate the immune-antioxidant status of the challenged Nile tilapia compared to carvacrol oil" (manuscript ID: fishes-2065685) collects data from various experiments conducted using a P. putida strain, including an experimental infection, a characterization of the strain and the use of antimicrobial substances in place of antibiotics.
Although the data may be of interest from a scientific point of view, their presentation does not meet the minimum quality required for a publication. The manuscript, as it is currently structured, must be completely revised. The data are reported in an excessively fragmentary and confusing way, which makes it difficult to understand whether they are actually interesting or not. For this reason, I decided to give the manuscript a major revision: if the changes made make the data usable, the manuscript will be revised again. Conversely, I will consider the manuscript as non-editable upon publication.
Response: Thank you for your valuable comment, basically, many parts in the manuscript were amended and more detailed information about the genetic identification of the bacterium is included.
I list below the changes I think are necessary:
- The manuscript aims to analyze many aspects related to the isolated P. putida strain: could the hypothesis of publishing only part of the reported data be considered?
Response: We tried to focus on this bacterial species as it is recently detected to cause mortalities in fish and we assessed many points related to P. putida strain including antimicrobial susceptibility, molecular characterization to identify the virulence genes, and trial for treatment this bacterial infection after testing the sensitivity of P. putida to carvacrol oil (c) and formic acid (f) using Disc diffusion method via bacterial challenge.
- From a stylistic point of view, it is necessary to check the whole manuscript to eliminate errors, repetitions, correctly report the scientific names (in italics) and citations according to the MDPI guidelines.
Response: We apologize for the mistyping; all these points were considered and corrected.
- The abstract must be completely rewritten.
Response: Corrected.
- The introduction, as well as the whole article, should be reviewed following a logical and precise sense. It is advisable to review the manuscript following a precise scheme of topics, not jumping from one topic to another randomly. Regarding the introduction specifically, I recommend expanding the part relating to the breeding of the nile tilapia in Egypt and globally, to emphasize the importance of this species.
Response: Thank you for your suggestion, the introduction was expanded showing more details about the breeding of the Nile tilapia and more details about the bacterium.
- The materials and methods are written in a confusing way. I suggest a succession based on isolation of the strain, biochemical and molecular characterization and antibiotic resistance tests; once these aspects have been described, the authors can decide how to report the experimental study or the data relating to the use of compounds with antimicrobial action. All the procedures used must be described in detail (PCR conditions, information regarding positive/negative controls).
Response: Thank you for your suggestion. Corrected following your suggestion.
- Figures 1-4 are superfluous: I would recommend removing them.
Response: The first figure was deleted, while the remaining three figures modified and included in one plate as they represent important results about the virulence genes.
- The results must be completely reviewed, following the scheme used in the materials and methods. The division into mirror paragraphs for materials and methods and results could help the authors in this.
Response: Corrected.
- Discussions and conclusions are difficult to understand in relation to the above. A clearer description of the procedures used and the results obtained will certainly help to better understand what the authors want to report in this manuscript.
Response: Corrected.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
The manuscript entitled “Pseudomonas putida: Sensitivity to various antibiotics, genetic diversity, virulence, and role of formic acid to modulate the immune-antioxidant status of the challenged Nile tilapia compared to carvacrol oil” by Alzahrani et al. The manuscript is well written however the objectives of the study were not clear in some section.
These comments are presented below:
1. Will Pseudomonas putida have a serious impact on tilapia farming?
2. In line 147, how ammonia and nitrite are determined？
3. Is there a clearer photo of Figure 1？
4. In line 191-193, the description of this part is too brief, in addition, why choose the three genes toxA, nan1 and exoS ?
5. What is cPCR technology, there is no description in the materials and methods.
6. Can Formic acid and carvacrol be applied in field culture?
As the experimental design has critical faults, the results are not reliable and the present work can not be accepted as a real experiment for publication in this journal. Based on these comments, my final decision is not to accept this manuscript. However, the final decision will be determined by the editor.
Author Response
Reviewer 3
The manuscript entitled “Pseudomonas putida: Sensitivity to various antibiotics, genetic diversity, virulence, and role of formic acid to modulate the immune-antioxidant status of the challenged Nile tilapia compared to carvacrol oil” by Alzahrani et al. The manuscript is well written however the objectives of the study were not clear in some section.
These comments are presented below:
- Will Pseudomonas putida have a serious impact on tilapia farming?
Response: Pseudomonas species exist throughout the aquatic environment and are associated with healthy or diseased fish. These bacteria can be opportunistic pathogens or produce damaging secondary infection (Daly, 1999) or cause high mortality among fish (Ahne et al., 1982; Fernandez et al., 1990). Pseudomonas septicaemia which caused by Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas aeruginosa is the most prevailing bacterial disease affecting tilapia (Abd El-Rahman et al., 2002). Pseudomonas putida is a fast growing bacterium found in most temperate soil and water habitats where oxygen is present (Krieg and Holt, 1984). It was isolated from haemorrhagic ascites of and yellowtail fish in Japan (Kusuda and Toyoshima, 1976; Wakabayashi et al., 1996) and from rainbow trout in Turkey (IIhan et al., 2006) and cause red disease in carp (Schäperclaus, 1992).
Salama and Gharib (2009), reported that P. putida was isolated from naturally infected cultured Oreochromis niloticus which suffered from exophthalmia, eye cloudiness, dark pigmentation, ascites, frayed fins and ulceration on the dorsal surface of the fish
Eissa et al. (2010) also isolated P. putida from Oreochromis niloticus reared in Qaroun and El Rayan lakes in Faiyum Governorate, Egypt. These isolates were approved to be pathogenic through inoculation in juvenile Oreochromis niloticus (70 ± 5 g). The challenged fish exhibited signs of dark pigmentation, easily detached scales, petechial hemorrhage on different parts of the body surface, ulceration, especially at dorsum part and at the base of fins with eroded and fins.
In line 147, how ammonia and nitrite are determined？
Response: Ammonia and nitrite were determined by using ammonia and nitrite assay kits (abcam company, USA).
- Is there a clearer photo of Figure 1？
Response: Unfortunately, we obtained only this one, and for clarity, the picture was deleted.
- In line 191-193, the description of this part is too brief, in addition, why choose the three genes toxA, nan1 and exoS?
Response: In order to find any relation between special virulence factors and special manifestation of S. putida.
- What is cPCR technology, there is no description in the materials and methods.
Response: More details are provided.
- Can Formic acid and carvacrol be applied in field culture?
According to our results obtained in this study; both formic acid and carvacrol are promising and potent alternatives which could be used to enhance fish immunity and resistance against bacterial infections. Furthermore, some recent researches supported our findings and recorded the positive impacts of formic acid and carvacrol after testing them as growth promoters, immune-stimulants and antioxidant feed additives (Reda et al., 2021 and Tartour and Mahboub 2020).
As the experimental design has critical faults, the results are not reliable and the present work can not be accepted as a real experiment for publication in this journal. Based on these comments, my final decision is not to accept this manuscript. However, the final decision will be determined by the editor.
Response: We apologize for the faults, all the required amendments were carefully considered and we hope these corrections will meet your expectations and improve the quality of the paper.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
the authors have made the suggested corrections in the manuscript and with an additional check of the English, I think they have made the requested corrections.
Reviewer 3 Report
Basically agree with the author's revised statement. Based on these statements, my final decision is accept this manuscript. However, the final decision will be determined by the editor.
