Biofilm Formation by Multidrug-Resistant Serotypes of Salmonella Isolated from Fresh Products: Effects of Nutritional and Environmental Conditions

Round 1

Reviewer 1 Report

1. The title does not reflect the content of the manuscript.
2. No 'genetic'/'molecular' analyses were done so there should be no reporting on genetic characteristics.
3. Aims of research written in introduction are not same as in abstract.
4. It is not clear how isolates were obtained and identified. Throughout the manuscript it is necessary to unify the writing of the names of bacteria.
5. Methodology on evaluation of biofilm formation (2.2.2., 2.2.3.) is not clear - preparation of inocula, repetitions, controls, method and enumeration. It is also not described how TSBS and TSBA were prepared. It is also not described what designates strong/weak biofilm producers.
6. There is no need to double quote numbers - percentages and ratio.
7. For many reported p-values it is not clear what was compared.
8. If 91 strains were tested all results should be shown and discussed.

L90 If CRA is abbreviation for Congo red agar it should not be used in this line where Congo red is added.

L91-94: What this designations mean? How this is seen?

Fig. 1 is not clear.

L151-153: Where this comparison is done/reported in the manuscript?

Tables 2 and 3 should be improved in terms of providing information.

Author Response

The title does not reflect the content of the manuscript.

New submission: The title has been re-written.

 

No 'genetic'/'molecular' analyses were done so there should be no reporting on genetic characteristics.

New submission: Changes are made in the context of the sentence.

 

Aims of research written in introduction are not same as in abstract.

New submission: Similar aims are incorporated in the introduction and abstract.

 

It is not clear how isolates were obtained and identified. Throughout the manuscript it is necessary to unify the writing of the names of bacteria.

New submission: The methodology has been re-written and unify the writing of the names of bacteria.

 

• Gómez Olmos I. 2020. Evaluación de la efectividad del ácido peracético y ácido láctico para la remoción de biopelículas de Salmonella spp provenientes de productos hortofructícolas. Thesis. Centro Universitario de la Ciénega (CuCiénega), Universidad de Guadalajara, Ocotlán, Jalisco, México

 

Methodology on evaluation of biofilm formation (2.2.2., 2.2.3.) is not clear - preparation of inocula, repetitions, controls, method and enumeration. It is also not described how TSBS and TSBA were prepared. It is also not described what designates strong/weak biofilm producers.

New submission: The methodology has been re-written. Based on the optical density (OD) of bacterial films, strains were classified into the following categories:

 

- non-biofilm producer = O.D. < O.D.c

- weak biofilm producer = O.D. c < O.D.c < (2 x O.D.c)

- moderate biofilm producer = (2 x O.D.c) < O.D. < (4 x O.D.c)

- strong biofilm producer = (4 x O.D.c) < O.D.

 

The cut-off O.D. (O.D.c) was defined as three standard deviations above the mean O.D. of the negative control.

 

• Stepanović, S.; Ćirković, I.; Ranin, L.; Švabić-Vlahović, M. Biofilm formation by Salmonella and Listeria monocytogenes on plastic surface. Lett. Appl. Microbiol. 2004, 38, 428–432. https://doi.org/10.1111/j.1472-765X.2004.01513.x

 

There is no need to double quote numbers - percentages and ratio.

New submission: The requested changes are made.

 

For many reported p-values it is not clear what was compared.

New submission: The requested changes are made.

 

If 91 strains were tested all results should be shown and discussed.

New submission: The discussion is carried out considering the 91 strains of Salmonella.

 

L90 If CRA is abbreviation for Congo red agar it should not be used in this line where Congo red is added.

New submission: The requested changes are made.

L91-94: What this designations mean? How this is seen? Fig. 1 is not clear.

New submission: The requested changes are made.

L151-153: Where this comparison is done/reported in the manuscript?Tables 2 and 3 should be improved in terms of providing information.

New submission: The requested changes are made.

Thank you very much for your attention.

The authors.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study investigated the attachment and biofilm formation by multidrug-resistant serotypes of Salmonella Isolated from fresh products: Influence of organic matter and food contact surface.

In my opinion, the content of this manuscript is not related to the title and mislead. Also, the formation of biofilm on the polystyrene plate is not an innovative idea and it was reported in several studies as part of the work. Hence, I could not recommend it for publication at present form.

Author Response

In my opinion, the content of this manuscript is not related to the title and mislead. Also, the formation of biofilm on the polystyrene plate is not an innovative idea and it was reported in several studies as part of the work

 

The title is modified according to the content of the investigation. Regarding his opinion, it is not only to determine the formation of biofilm on the polystyrene plates. The context of the article should be reviewed, where the formation capacity of some serotypes is raised, not only in polystyrene microplates, but also that environmental conditions (temperatura) affect the expression of Salmonella morphotypes and multi-resistance is associated with the ability to form biofilms. In turn, these are determining inclusion criteria to continue with the next stages; generating Salmonella serotype biofilms on type B polypropylene surfaces, which is a useful material within the fresh produce industry with a determining factor in the medium of culture of development that is the incorporation or organic residues of products fresh that can favor some stage of biofilm formation. Finally, this shows that the deficiency in the cleaning and disinfection of food contact surfaces favors the development of biofilms by the presence of organic residues.

 

Finally, a document of the English revision is attached.

 

Thank you very much for your attention.

The authors.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

1. The title is still misleading - no nutritional data are given for broth media used and only different plastic surfaces are used (but are not even compared). Biofilm is evaluated only at 35 °C.
2. Why two different plastic surfaces were used? Why two different methods were used? this further makes it impossible to compare the results obtained.
3. the aims still do not coincide with the further records in the mansucript
4. Morphotype is not evaluation of biofilm.
5. Methods 2.2.4 and 2.2.5 should be shortly described.
6. Salmonella should be written in italic.
7. All results for 91 strains should be presented. Such presentation of results would add value to the manuscript.
8. Results presented in figures 2 and 3 should be described in more detail.
9. The discussion writes about things that were not included in the actual work presented in the manuscript. The comments in the previous points should also be taken into account in the text in the discussion.

L24: for what comparison does this p stand for?

L25-26: this was the highest? why reporting range if this are results for one strain? why not reported as average and standard deviation for each broth?  

L65: polyethylene? 

L71: only reference was added but no other information - this should be added, also accession numbers if available; it is still not clear what was source of isolation (what kind of fruit, from where, etc.). Also conditions of storage, revitalization and preparation of working suspension are not described.

L89-92: how much agar was added.

L93: what are these macroscopic characteristics

L98: Cut-off values for 2.2.2 should be included. As described, only three technical replications were done. Why diluted TSB was used.

L119: why only 'food' broths are used with this method? in such way you can not evaluate how this media influence formation of biofilm. This should be added. Also add information on selected strains used for this part.

L143: does this refer to reference 21?

L160: it is still not clear what was compared and what does this p stand for.

L161: additionally comment, describe results from Table 1.

L177: what does this p stand for?

L188: when written like 'all the tested microorganisms' it looks like from 91 tested strains... 

L189: higher cell density than what? Also in L192 - what does p stand for?

L188-198: why results are reported in ranges and not average values and sd as only 5 strains were tested here?

L233-235: not all of these data are even presented in the manuscript, neither biofilm formation data are analysed for their correlation/comparison based on data of antimicrobial resistance or morphotype

L278-279: only TSBS and TSBA were used and not compared to TSB (based on described method) so it can not really be evaluated how these changes influence biofilm formation.

L280-281: missing reference and why talk only about fats? this is not the only nutrient found in avocado and strawberries. Also missing discussion on this two media used.

L289-290: biofilm was not compared at 22 °C and 35 °C - based on methodology tests were done only at 35 °C

L293-294: this is based only on one strain - why not all results are presented to really show influence of serotype on biofilm formation?

Figure 1 in not clear. As only frequency is presented and there are no sd values, different type of graph should be used to present these data and make it more clear.

L304: AO/acridine orange

L307: how living and dead cells were distinguished with SEM?

Table 2: why this is presented if this data is already given in text. it is still not clear why these strains were selected.

Table3: the strain names are repeated twice in the table which makes it confusing; also no statistical data are given despite comparisons.

Author Response

We send the paper titled "Biofilm Formation by Multidrug-Resistant Serotypes of Salmonella Isolated from Fresh Products: Effects of Nutritional and Environmental Conditions", which was reviewed in the journal that you lead. The reviewers suggested some comments, which ones were fully realized; then you can find enclosed how was explained the information in the previous submission how you will find it in the new submission in attention to reviewers’ comments, which are highlighted in yellow.

1. The title is still misleading - no nutritional data are given for broth media used and only different plastic surfaces are used (but are not even compared). Biofilm is evaluated only at 35 °C.

 New submission: The requested changes are made. The title is justified because the development of research incorporates a culture (1/20 diluted TSB), temperature (35 ºC) and type of surface (polystyrene microtiter plates). The temperature of 22 ºC and 35 ºC is implemented for the determination of Salmonella morphotypes in order to see if it influences the expression of morphological characteristics in CRA. In addition, to this it must be considered that the methodology (2.2), (2.3) and (2.4.1) allow to establish inclusion criteria for the proposed methodology of (2.4.2) and select of (n=91strains of Salmonella (five serotypes), four Salmonella isolates from different serotypes for the development of in vitro models in culture media with organic residues (TSBA  and TSBS) to evaluate the biofilm formation capacity according to a type of surface  (Polypropylene Type B) used in the fresh produce industry.

 

2. Why two different plastic surfaces were used? Why two different methods were used? this further makes it impossible to compare the results obtained.

 New submission: The requested changes are made. Due to the characteristics of the surfaces and the uses, polypropylene type B surfaces are more frequent due to activities within the industry compared to polystyrene. In addition, the two materials are not compared in the investigation, it is not the purpose, the development of the biofilm is evaluated independently on each type of surface.

Reference:

• Shen, ; Luo, Y.; Nou, X.; Bauchan, G.; Zhou, B.; Wang, Q.; Millner, P. Enhanced inactivation of salmonella and pseudomonas biofilms on stainless steel by Use of T-128, a fresh-produce washing aid, in chlorinated wash solutions. Appl. Environ. Microbiol. 2012, 78, 6789–6798. https://doi.org/10.1128/AEM.01094-12
• Singla, R.; Goel, H.; Ganguli, A. Novel synergistic approach to exploit the bactericidal efficacy of commercial disinfectants on the biofilms of Salmonella enterica serovar Typhimurium. Biosci. Bioeng. 2014, 118, 34–40. https://doi.org/10.1016/j.jbiosc.2013.12.025
• De Oliveira, D.C.V.; Fernandes Júnior, A.; Kaneno, R.; Silva, M.G.; Araújo Júnior, J.P.; Silva, N.C.C.; Rall, V.L.M. Ability of Salmonella spp. to produce biofilm is dependent on temperature and surface material. Foodborne Pathog. Dis. 2014, 11, 478–483. https://doi.org/10.1089/fpd.2013.1710
• Cook, K.L.; Givan, E.C.; Mayton, H.M.; Parekh, R.R.; Taylor, R.; Walker, S.L. Using the agricultural environment to select better surrogates for foodborne pathogens associated with fresh produce. J. Food Microbiol. 2017, 262, 80–88. https://doi.org/10.1016/j.ijfoodmicro.2017.09.017

 

 

3. the aims still do not coincide with the further records in the manuscript

New submission: The requested changes are made.

 

4. Morphotype is not evaluation of biofilm.

              New submission: The requested changes are made.

 

5. Methods 2.2.4 and 2.2.5 should be shortly described.

New submission: The requested changes are made.

 

6. Salmonella should be written in italic.

       New submission: The requested changes are made.

 

7. All results for 91 strains should be presented. Such presentation of results would add value to the manuscript.

      New submission: The requested changes are made.

 

8. Results presented in figures 2 and 3 should be described in more detail.

New submission: The requested changes are made.

 

9. The discussion writes about things that were not included in the actual work presented in the manuscript. The comments in the previous points should also be taken into account in the text in the discussion.

      New submission: The requested changes are made.

 

L24: for what comparison does this p stand for?

The cell density of the Salmonella serotypes was compared when using the TSBS medium and TSBA on polypropylene type B, deducing that there is a higher cell density in the biofilms developed in the TSBS medium (p < 0.05).

L25-26: this was the highest? why reporting range if this are results for one strain? why not reported as average and standard deviation for each broth?  

New submission: The requested changes are made.

L65: polyethylene? 

New submission: The requested changes are made.

L71: only reference was added but no other information - this should be added, also accession numbers if available; it is still not clear what was source of isolation (what kind of fruit, from where, etc.). Also conditions of storage, revitalization and preparation of working suspension are not described.

New submission: The requested changes are made. However, it should be noted that the antecedents of the samples are still found in data not published by the original author.

L89-92: how much agar was added.

New submission: The requested changes are made.

L93: what are these macroscopic characteristics

New submission: The requested changes are made.

L98: Cut-off values for 2.2.2 should be included. As described, only three technical replications were done. Why diluted TSB was used.

New submission: The requested changes are made. There is research that supports the biofilm formation capacity in diluted TSB.

References:

1. Stepanović, S.; Ćirković, I.; Ranin, L.; Švabić-Vlahović, M. Biofilm formation by Salmonella and Listeria monocytogenes on plastic surface. Lett. Appl. Microbiol. 2004, 38, 428–432. https://doi.org/10.1111/j.1472-765X.2004.01513.x
2. Singla, R.; Goel, H.; Ganguli, A. Novel synergistic approach to exploit the bactericidal efficacy of commercial disinfectants on the biofilms of Salmonella enterica serovar Typhimurium. Biosci. Bioeng. 2014, 118, 34–40. https://doi.org/10.1016/j.jbiosc.2013.12.025
3. Cook, K.L.; Givan, E.C.; Mayton, H.M.; Parekh, R.R.; Taylor, R.; Walker, S.L. Using the agricultural environment to select better surrogates for foodborne pathogens associated with fresh produce. J. Food Microbiol. 2017, 262, 80–88. https://doi.org/10.1016/j.ijfoodmicro.2017.09.017

L119: why only 'food' broths are used with this method? in such way you can not evaluate how this media influence formation of biofilm. This should be added. Also add information on selected strains used for this part.

New submission: The requested changes are made.

L143: does this refer to reference 21?

New submission: The requested changes are made.

However, the methodology of Solis-Velazquez et al., 2020 for the development, quantification and visualization of biofilms is implemented and verified. Clearly the variable of in this investigation was the culture medium, temperature, time and strains.

L160: it is still not clear what was compared and what does this p stand for.

New submission: The requested changes are made.

Antimicrobial resistance of ampicillin is compared against resistance to amikacin, carbenicillin and ciprofloxacin.

L161: additionally comment, describe results from Table 1.

New submission: The requested changes are made.

L177: what does this p stand for?

New submission: The requested changes are made.

L188: when written like 'all the tested microorganisms' it looks like from 91 tested strains... 

New submission: The requested changes are made.

L189: higher cell density than what? Also in L192 - what does p stand for?

New submission: The requested changes are made.

L188-198: why results are reported in ranges and not average values and sd as only 5 strains were tested here?

New submission: The requested changes are made.

L233-235: not all of these data are even presented in the manuscript, neither biofilm formation data are analysed for their correlation/comparison based on data of antimicrobial resistance or morphotype

New submission: The requested changes are made.

L278-279: only TSBS and TSBA were used and not compared to TSB (based on described method) so it can not really be evaluated how these changes influence biofilm formation.

New submission: The requested changes are made. It is worth mentioning that the biofilm formation capacity is first evaluated using diluted TSB (TSB 1/20) determining that the 95.6 % of Salmonella serotypes were strong biofilm producers and 4.39% were weak biofilm producers in this study, Finally, inclusion criteria are established for the selection of four strains and continue with the evaluation in TSBA and TSBS.  

L280-281: missing reference and why talk only about fats? this is not the only nutrient found in avocado and strawberries. Also missing discussion on this two media used.

New submission: The requested changes are made.

L289-290: biofilm was not compared at 22 °C and 35 °C - based on methodology tests were done only at 35 °C

The temperature of 22ºC and 35ºC was used to determine the morphotypes of Salmonella strains (five serotypes) (n=91) (Methodology 2.3). In the detection of Salmonella serotype morphotypes was not observed significative influence of temperature on all tested microorganisms (p > 0.05). Subsequently, the temperature of 35ºC was implemented for the methodology of point (2.4.1) and (2.4.2).

L293-294: this is based only on one strain - why not all results are presented to really show influence of serotype on biofilm formation?

New submission: The requested changes are made.

Figure 1 in not clear. As only frequency is presented and there are no sd values, different type of graph should be used to present these data and make it more clear.

New submission: The requested changes are made.  The figure 1 illustrates the frequency (%) of the resistance, susceptible and intermediate of the 91 Salmonella strains (five serotypes) corresponding to each of the antimicrobial.

L304: AO/acridine orange

New submission: The requested changes are made.

L307: how living and dead cells were distinguished with SEM?

New submission: The requested changes are made.

Table 2: why this is presented if this data is already given in text. it is still not clear why these strains were selected.

New submission: The requested changes are made.

Table3: the strain names are repeated twice in the table which makes it confusing; also no statistical data are given despite comparisons.

New submission: The requested changes are made.

Finally, a document of the English revision is attached.

Thank you very much for your attention.

The authors.

Author Response File: Author Response.pdf

Reviewer 2 Report

I agree with the author's corrections. However, from the application point of view, the biofilm formation if tested in biological substance would be appreciatable. The name of the microorganisms should be written in italics.

Author Response

We send the paper titled "Biofilm Formation by Multidrug-Resistant Serotypes of Salmonella Isolated from Fresh Products: Effects of Nutritional and Environmental Conditions", which was reviewed in the journal that you lead. The reviewers suggested some comments, which ones were fully realized; then you can find enclosed how was explained the information in the previous submission how you will find it in the new submission in attention to reviewers’ comments, which are highlighted in yellow.

Reviewer #2:

I agree with the author's corrections. However, from the application point of view, the biofilm formation if tested in biological substance would be appreciatable. The name of the microorganisms should be written in italics.

New submission: The requested changes are made.

 

Finally, a document of the English revision is attached.

Thank you very much for your attention.

The authors.

Author Response File: Author Response.pdf