Molecular Detection and Antibiogram of Bacteria and Fungi in Table Eggs Under Different Storage Durations with Organoleptic Properties
, Saiduzzaman 1, Marzia Rahman 1 and K. H. M. Nazmul Hussain Nazir 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis is regarding a manuscript entitled Molecular Detection and Antibiogram of Bacteria and Fungi in Table Eggs at Different Storage Durations with Organoleptic Properties
The content of the article is interesting.
The introduction can be improved
Author Response
Reviewer: The introduction can be improved.
Answer: We added a few more passages of information and organized the whole introduction again to make a good presentation of the study.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors describe a topic which is relevant for their country for food and nutrition supply, and also for other countries. Studies of prevalience and growth of microbes in egg shells and egg interior during storage is relevant for food safety risk assessment in general. Eggs as reservoir for microbes harbouring antimicrobial resistance genes is also highly relevant.
The study contains a lot of data. However, the text has incomplete sentences and some sentences appear to contradict each other. This is a major drawback, and makes it difficult both to judge and understand the content, particularly in the results and discussion sessions.
Some of the materials and methods are only partly explained. For instance, the storage conditions in terms of temperature, hygienic status of the surroundings of the eggs during storage are not described. Are the eggs washed or dusted before placed in a box, or are they stored in soil or grass? I suppose this is obvious for the authors, but not for the readers. Increased concentrations of microbes in the egg shell will depend on whether there is cross contamination from the surroundings or growth of microbes already in the egg shell pores.
The organoleptical evaluation is only partly explained, as the scoring system is not described. Also, are the judgement done as a group or individually? Please explain.
Results: the concentrations are given as 1013 cfu/lot. This is unusual and looks unlikely. The saturation level of microbes is normally around 109 cfu/g. How large is a lot? Please consider the calculations and explain the use of lots.
Figure 1: The symbols for shell and interior should be more different to improve clarity. The number of storage days could be given on the x-axis.
All in all, there is a lot of potentially relevant information in the manuscript, but the text needs to be clarified before further assessment can be done.
Comments on the Quality of English LanguageParts of the text is clear, while other parts hva incomplete sentences.
Author Response
Comment 1. The study contains a lot of data. However, the text has incomplete sentences and some sentences appear to contradict each other. This is a major drawback, and makes it difficult both to judge and understand the content, particularly in the results and discussion sessions.
Response 1. We appreciate your feedback. To address this issue, we organize the introduction and discussion part once again and add necessary information where it is needed (track changes). For the result section, we added more information in some steps, for example, sample collection, TVC count, and organoleptic evaluation, for better understanding.
Comment 2. Some of the materials and methods are only partly explained. For instance, the storage conditions in terms of temperature, hygienic status of the surroundings of the eggs during storage are not described. Are the eggs washed or dusted before placed in a box, or are they stored in soil or grass? I suppose this is obvious for the authors, but not for the readers. Increased concentrations of microbes in the egg shell will depend on whether there is cross-contamination from the surroundings or growth of microbes already in the egg shell pores.
Response 2. Thanks for pointing that out. Explanation is added in the Collection of Egg Samples passage.
Comment 3. The organoleptical evaluation is only partly explained, as the scoring system is not described. Also, are the judgement done as a group or individually? Please explain.
Response 3. Nice observation. Additional information is added about organoleptical evaluation in the evaluation method section. Also we added a citation of the method that we used. The judgment was carried out individually (10 people total). And a citation is added as a reference to the scoring system.
Comment 4. the concentrations are given as 10^13 cfu/lot. This is unusual and looks unlikely. The saturation level of microbes is normally around 10^9 cfu/g. How large is a lot? Please consider the calculations and explain the use of lots.
Response 4. We appreciate your insightful feedback. We calculated the TVC for each egg. As it would be a 5 big table with all the TVC counts, we merged them into a single table where we described them as per lot. In this context, the lot refers to the sample that has been testified for each respective storage day. We are providing a supplementary file for the reader. Additionally, yes, it is quite a high count of TVC compared to the other study. The reason is that in Bangladesh, small farmers don’t do any treatments before selling them in the market. Also, the hygienic condition of the frames is not satisfactory. Which is addressing high number of TVC counts. We also added a passage in the discussion part describing this issue (Line 430-438).
Comment 5. The symbols for shell and interior should be more different to improve clarity. The number of storage days could be given on the x-axis.
Response 5. Thanks. Color contrast is changed for the legend, and the number of storage days is added to the X axis.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript is interesting but some aspect should be improved:
- Admission should be enriched with information referring to the title. There is little information in the introduction regarding the impact of organoleptic features on consumer choices
- Is the physicochemical features of the evaluated eggs in the work? It is difficult to interpret the data from organoleptic assessment when they cannot be referred to the value of physico-chemical parameters.
- Discussion and description of results should be extended.
- There is no statistical assessment of the results obtained - it must be added
the language is quite ok. The manuscript can be chceked by native speaker.
Author Response
Comment 1. Admission should be enriched with information referring to the title. There is little information in the introduction regarding the impact of organoleptic features on consumer choices
Response 1. Thanks for the comment. Yes, we just added a passage at that point.
Comment 2. Is the physicochemical features of the evaluated eggs in the work? It is difficult to interpret the data from organoleptic assessment when they cannot be referred to the value of physico-chemical parameters.
Response 2. Thanks for the observation. Yes, it would be best if we could add the physico-chemical analysis. But due to funding unavailability, we could not do the physicochemical analysis. It’s a solo work, and no funding was provided. The statement was also added in the discussion part.
Comment 3. Discussion and description of results should be extended.
Response 3. Thanks for the comment. Discussion is elaborated where it is necessary. Although we believe the description of the result is appropriate, we have made some minor changes where necessary.
Comment 4. There is no statistical assessment of the results obtained - it must be added.
Response 4. Thanks for the observation. A statistical analysis, including the respective p-values, is presented in Table 3. Other than antibiotics resistance table are in prevalence statistics. TVC table is in CFU/ml condition, which is a universal method now added as a supplementary file for the reader. And the organoleptic analysis is done according to the previous researcher (citation is added).
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript has been much improved.
Still, the high concentrations observed needs more clarification, as they appear to be above the theoretical maximum limit.
Abstract: The term antibiogram needs to be explained.
Material and methods
Line 89:
It looks like the eggs are from one, small producer, only. What is then random? Were all collected at the same time or at different days, different times of the year etc? Such information is useful to assess the variability of the samples.
Line 94: “without any delay” could be better explained by for instance “within xx-yy hours after the egg was laid”.
Line 94: The additional 32 eggs from the same batch (new text) indicates that the first 32 eggs were applied for microbial analyses and treated differently from the first 32 eggs. What does it mean that the additional eggs were transported to the lab without any treatment? Were the first 32 eggs treated in any way, or were the addiotional eggs not controlled for cracks, double yolk or similar? Please explain, as treatment may influence cross contamination and removal of microbes.
2.3 organoleptical evaluation
This part is well described in this version of the manuscript.
Results
Line 164: The concentrations 1013 on eggshell need further consideration. What does the number refer to? Per egg, per ml solution, per batch or something else? If the microbiom include alive cells, it means that the sample needs to be more than 1 kg to give place to so many microbes. The high numbers can be explained to some extent if there are spores included in the microbiome, as a spore has a lower weight and volume than an alive microbe. Please explain, and check if the high numbers could be due to a dilution or calculation mistake, non-sterile dilution solution or cross contamination during the analyses.
Figure 1 indicates the unit “bacteria per lot”. Please explain what a lot is, and how large the lot is. Preferably, use a unit which is more intuitive, like per egg, per square cm of egg shell, per ml of washin solution, or similar.
Figure 1: quantitative results are often given in logarithmic scale. With that approach, the observed contents of microbes are more or less stable during storage. Please add the variability, for instance by including data for single analyses. If the samples have different amounts, it indicates natural variability, which is useful information.
Table 2: The high content of specific microbes at day 15 and onwards indicates growth on the egg shell and maybe in the interior, or a contamination during storage. Comments about possible reasons would be useful. If it comes in the discussion, please tell so.
Line 198 – Tabel 3
Please explain what the purpose of calculation of P values for each bacterium in this context. I understand from the presentation of data that all samples are included, i.e that the samples analysed are not a selection of samples intended to be representative for a larger group of samples. If that is correct, the positive samples give the information about presence or not presence.
Discussion
Line 286: There is hardly any decrease of TVC counts after day 20 if a log scale is used to assess the data. The measurement uncertainty for microbial analyses is normally 0.3-0.5 log units, and if that is the case also for these analyses, the observed decrease is likely to be insignificant.
Author Response
Comment: Abstract: The term antibiogram needs to be explained.
Response: Thanks for the comment. The word changed to antibiotic resistance profile.
Comment: Line 89, It looks like the eggs are from one, small producer, only. What is then random? Were all collected at the same time or at different days, different times of the year etc? Such information is useful to assess the variability of the samples.
Response: Although the farm is small, they produce between 500-800 eggs per day. So from there we collected randomly 32 (for microbe isolation) and 32 (for organoleptic profile) egg samples. Yes it was collected at the same time. Line 98.
Comment: Line 94, “without any delay” could be better explained by for instance “within xx-yy hours after the egg was laid”.
Response: Thanks for the suggestion. Time duration is added in line 96.
Comment: Line 94, The additional 32 eggs from the same batch (new text) indicates that the first 32 eggs were applied for microbial analyses and treated differently from the first 32 eggs. What does it mean that the additional eggs were transported to the lab without any treatment? Were the first 32 eggs treated in any way, or were the addiotional eggs not controlled for cracks, double yolk or similar? Please explain, as treatment may influence cross contamination and removal of microbes.
Response: Thanks for the suggestion. Yes, we edited the whole passage once again for better clarification. No, eggs were not treated in any way. They are all collected the same way, on the same day.
Comment: organoleptical evaluation, This part is well described in this version of the manuscript.
Response: Thanks
Comment: Line 164, The concentrations 10^13 on eggshell need further consideration. What does the number refer to? Per egg, per ml solution, per batch or something else? If the microbiom include alive cells, it means that the sample needs to be more than 1 kg to give place to so many microbes. The high numbers can be explained to some extent if there are spores included in the microbiome, as a spore has a lower weight and volume than an alive microbe. Please explain, and check if the high numbers could be due to a dilution or calculation mistake, non-sterile dilution solution or cross contamination during the analyses.
Response: Thanks once again for raising the question. All the data is given as a supplementary file. We suggest that the reviewer consult the supplementary file if there is any confusion. 10^13 means per lot. And what it means per lot is explained in the data set. We also give an explanation in our previous reviewer response.
Comment: Figure 1 indicates the unit “bacteria per lot”. Please explain what a lot is, and how large the lot is. Preferably, use a unit which is more intuitive, like per egg, per square cm of egg shell, per ml of washin solution, or similar.
Response: 4 eggs per lot for 5- and 10-day samples. 8 eggs per lot for 15, 20 and 25 days samples. The calculation for each egg can be found in the supplementary file.
Comment: Figure 1 quantitative results are often given in logarithmic scale. With that approach, the observed contents of microbes are more or less stable during storage. Please add the variability, for instance by including data for single analyses. If the samples have different amounts, it indicates natural variability, which is useful information.
Response: Thanks. We have provided a supplementary file where data is included in single analysis.
Comment: Table 2: The high content of specific microbes at day 15 and onwards indicates growth on the egg shell and maybe in the interior, or a contamination during storage. Comments about possible reasons would be useful. If it comes in the discussion, please tell so.
Response: Yes, it is explained in the discussion line 316-323.
Comment: Line 198 – Tabel 3. Please explain what the purpose of calculation of P values for each bacterium in this context. I understand from the presentation of data that all samples are included, i.e that the samples analysed are not a selection of samples intended to be representative for a larger group of samples. If that is correct, the positive samples give the information about presence or not presence.
Response: Thanks. Since our table includes both eggshells and egg contents, and we are comparing the presence of microbes in each, it makes sense to include the p-value. It strengthens our findings by showing whether differences are statistically meaningful.
Comment: Line 286, There is hardly any decrease of TVC counts after day 20 if a log scale is used to assess the data. The measurement uncertainty for microbial analyses is normally 0.3-0.5 log units, and if that is the case also for these analyses, the observed decrease is likely to be insignificant.
Response: Thank you for your insightful comment regarding the interpretation of TVC data beyond day 20. We appreciate your observation and have re-evaluated the TVC trend using a logarithmic scale to assess biological significance in light of typical measurement uncertainty (0.3–0.5 log units).
While our raw data indicated a numerical decrease in the mean Total Viable Count (TVC) on the eggshells from 3.25×10¹³ CFU (day 20) to 2.34×10¹³ CFU (day 25), the change corresponds to approximately a 0.14 log₁₀ reduction (from log₁₀ 13.51 to 13.37), which falls well within the standard range of analytical variability. Therefore, we agree that this decrease is likely not statistically or biologically significant. We have revised the manuscript accordingly to reflect this more accurate interpretation of the data (line 319-322).
