Knife Decontamination by Cold Water Treatment Supplemented with InspexxTM 210—A Validation Study in an Abattoir
Round 1
Reviewer 1 Report
In the manuscript titled “Knife decontamination by cold water treatment supplemented with InspexxTM 210 – a validation study in an abattoir” a new methodology of knifes’ disinfection is evaluated, specifically the use of Inspexx 210 (Ecolab) for disinfecting knives in abattoirs. The authors researched the possibility of exchanging standard protocol with hot water for a more efficient one in time and energy consumption.
In general, the research issue is important and necessary to study and find the best solution. It is particularly valuable to perform tests at the place of production where the method can be used. The authors put a lot of effort into preparing, conducting, and describing the experiment. However, they did not avoid a few mistakes that need to be clarified or corrected.
Firstly, in the introduction, the description of the veterinary inspection as a "tool" is incorrect. The inspection has the tools to carry out its tasks and these are listed in lines 35-37. Moreover, the paragraph (lines 66-71) should be clarified. Because Inspexx 210 is a commercial system, there is proof of its effectiveness but unpublished. The authors have to explain what is beneficial/the purpose of testing the Inspexx 210 that was tested and approved (in another case Ecolab would not sell it).
Secondly, in the material and methods, used in the challenge test only one strain is a serious drawback and does not allow for evaluating the efficiency of the method. It is generally known, that susceptibility to environmental factors is strain dependent. Regarding “field study” it is a next serious mistake that the time of exposure was not standardised. Moreover, there is no information about microbiological media used for the test and group of tested microorganisms.
In the results’ description, you do not have to repeat so many times the number of samples i.e. (n=…). It is enough to write it in the table of figure description.
Regarding the discussion, only 5 research are mentioned. In my opinion, there is an opportunity (numerous research) to deepen issues related to the topic, e.g. strain dependence, and biofilm formation, which are important for the development of an effective method of disinfection.
Finally, the results obtained for one strain of E. coli and one strain of S. aureus are not sufficient to conclude the effectiveness of the system. Especially when the field study has such a serious drawback as the lack of a specific duration of action.
Detailed comments:
Lines 37-39 It would be useful to give the source of such an approach to food safety.
Lines 39-41 What do you mean by “in recent years”? Food science and microbiology know this for many, many years.
Line 40: What do you mean by “healthy food animals”?
Lines 46-53: This paragraph needs information confirmation in the literature.
Line 100: Is this mean that only one side of the knife was swabbed? Or one before intervention and the other after?
Lines 108-109: Please, add the reason for using 5 or 10 ml of 0.85% saline solution depending on the test. And why were used different temperatures for incubation?
Author Response
In general, the research issue is important and necessary to study and find the best solution. It is particularly valuable to perform tests at the place of production where the method can be used. The authors put a lot of effort into preparing, conducting, and describing the experiment. However, they did not avoid a few mistakes that need to be clarified or corrected.
Answer: thank you for this valuable feedback.
Firstly, in the introduction, the description of the veterinary inspection as a "tool" is incorrect.
Answer: changed to “….the veterinary inspection is the main intervention step in abattoirs to protect consumer health…”
Moreover, the paragraph (lines 66-71) should be clarified. Because Inspexx 210 is a commercial system, there is proof of its effectiveness but unpublished. The authors have to explain what is beneficial/the purpose of testing the Inspexx 210 that was tested and approved (in another case Ecolab would not sell it).
Answer: The reviewer is right, that there has to be an effect of Inspexx 210, otherwise the company would not sell this system. However, as mentioned, so far, there are no published independent studies on the decontamination effect of the procedure. We have now also added: Moreover, based on a quality assurance system as e.g. ISO:9001 or GFSI standards (IFS Food), companies must validate processes when introducing them.
Secondly, in the material and methods, used in the challenge test only one strain is a serious drawback and does not allow for evaluating the efficiency of the method. It is generally known, that susceptibility to environmental factors is strain dependent. Regarding “field study” it is a next serious mistake that the time of exposure was not standardised. Moreover, there is no information about microbiological media used for the test and group of tested microorganisms.
Answer: We are convinced that the chosen approach in the challenge test to use an S. aureus strain as an example for the Gram-positive and an E. coli strain for the Gram-negative bacteria is technically adequate. As written in the title, it is about the validation of an existing system. In addition, disinfectant reports (e.g. according to DVG (Deutsche Veterinärmedizinische Gesellschaft) guideline lines) are also based on one strain in each case. In order to be as close as possible to reality, tests were then also carried out with knives from the slaughter process, in order to generate field data using the example of a real microbiological contamination in this environment.
In the field study, the exposure time was deliberately not standardized, in contrast to the challenge test, as this does not correspond to the reality in a slaughterhouse. The employee will never comply with a standardized time. Our goal was to reflect the real situation in the working environment.
In addition, we point out possible limitations in the discussion.
It is mentioned in the “microbiological analysis text section” that plate count agar (PC agar) was used. This is the method recommended in ISO 4833-2 for counting TVC.
In the results’ description, you do not have to repeat so many times the number of samples i.e. (n=…). It is enough to write it in the table of figure description.
Answer: the number of samples were deleted here as requested.
Regarding the discussion, only 5 research are mentioned. In my opinion, there is an opportunity (numerous research) to deepen issues related to the topic, e.g. strain dependence, and biofilm formation, which are important for the development of an effective method of disinfection.
Answer: the paper is in the format of a communication (short paper). The aspect of biofilm is mentioned in the discussion part, the aspect of possible strain/species dependence in the part limitations of the study.
Finally, the results obtained for one strain of E. coli and one strain of S. aureus are not sufficient to conclude the effectiveness of the system. Especially when the field study has such a serious drawback as the lack of a specific duration of action.
Answer: We are convinced that the chosen approach in the challenge test to use an S. aureus strain as an example for the Gram-positive and an E. coli strain for the Gram-negative bacteria is technically adequate. As written in the title, it is about the validation of an existing system. In addition, disinfectant reports (e.g. according to DVG (Deutsche Veterinärmedizinische Gesellschaft) guideline lines) are also based on one strain in each case (S. aureus, E. coli). In order to be as close as possible to reality, tests were then also carried out with knives from the slaughter process, in order to generate field data using the example of a real microbiological contamination in this environment.
Detailed comments:
Lines 37-39 It would be useful to give the source of such an approach to food safety.
Answer: the example bovine tuberculosis was added.
Lines 39-41 What do you mean by “in recent years”? Food science and microbiology know this for many, many years.
Answer: in recent years was deleted. The sentence is now: However, also healthy food animals were recognized as carriers of pathogen responsible for human illness.
Line 40: What do you mean by “healthy food animals”?
Answer: these are e.g. fattening pigs without clinical signs (healthy animals), but carriers e.g of Salmonella or cattle which are often carriers of Shigatoxin-producing E. coli
Lines 46-53: This paragraph needs information confirmation in the literature.
Answer: we have now added in view of risk factors for carcass contamination a reference
J.C. Galland. Risks and prevention of contamination of beef carcasses during the slaughter process in the United States of America. Revue Scientifique et Technique, 16(2), (1997), pp. 395-404. doi: 10.20506/rst.16.2.1023.
Line 100: Is this mean that only one side of the knife was swabbed? Or one before intervention and the other after?
Answer: For the challenge test we have used the same knife but two different sampling areas, see line 81: two areas of 5 cm2 were inoculated with several colonies of the respective bacterium. For the field experiments, different sides of the knives before and after the intervention were sampled.
Lines 108-109: Please, add the reason for using 5 or 10 ml of 0.85% saline solution depending on the test. And why were used different temperatures for incubation?
Answer: The final results are in the format of cfu/cm2. For the challenge test we sampled 5 cm2, for the field study approx. 68 cm2. For the surface plating technique 0.1ml of the dilution steps are used. We varied the volume of 0.85% saline solution which was added the swab samples to have an easy formula for calculation of grown colonies on the plates to the format of cfu/cm2.
For the challenge test: incubation 24h at 37°C was used since S. aureus and E. coli are growing very well at 37°C for 24h on plate count agar; for the field study (where we have not known which bacteria were present on the knives) we used the incubation conditions of ISO 4833-2 (30°C for 3d) to get the TVC.
Reviewer 2 Report
Manuscript ID: hygiene-2366158-peer-review
Knife decontamination by cold water treatment supplemented with InspexxTM 210 – a validation study in an abattoir
The authors present an article focusing on the potential use of disinfectant to contain the microbial contamination of knives used in an abattoir and, therefore, cross-contamination during ordinary procedures. As reported by the authors in the introduction, European regulation provides that knives must be decontaminated with water at a minimum of 82°C or with an alternative system with an equal efficacy. However, this common practice negatively affects the costs and causes accidents in slaughterhouses. For these reasons, the authors investigated the effect of InspexxTM 210 as promising alternative that could replace the use of hot water.
In my opinion, the manuscript is focused on a very interesting topic, however, some integrations and revisions are needed.
Some revisions are suggested and listed below.
Overall, the text needs a moderate English check. some grammatical and typo errors are present.
Some doubts and missing information are related the section “Materials and Methods”:
- Line 81: What inoculum concentrations were used for the challenge test? How were the inocula prepared?
- Line 89: It is not clear whether 0.16% is the concentration of PAA and POA in disinfectant or the % of InspexxTM210 in water. Please, specify and indicate the percentage of InspexxTM 210 in water.
- Line 89: Please, indicate the temperature of the water used.
- As far for the methods, was the same knife used for sampling before and after treatment for the test? In this case, after having sampled the entire area of the knife, the first sampling by mechanical action of the swab may have already reduced (albeit slightly) the initial contamination. Did the authors consider that the first sampling (operating before the treatment) could reduce the microbial concentration?
- Lines 90-92: Please, indicate the surface size sampled.
- Lines 103-104: Even if not standardized, please indicate an interval of exposure time to the disinfectant.
- Line 108: Please, specify the microbiological methods used (respectively ISO, medium used, etc.)
Regards the results, please specify that the reduction is calculate on the means.
- Figure 1 and 2:
· Please, check the statistic (for example the Fig.1 f);
· Please, replace “Colony forming units (CFU)” with “Logarithm (CFU/cm2)”;
· Why weren't the knives pre-treated with protein and then rinsed under a tap for 15 seconds for the E. coli test?
· Is it possible to put the error bar before the dots, so as to make it more visible?
· Line 155: please, rewrite the point (a).
I would like to suggest to:
- Insert more references and discussions in “discussions” paragraph;
- No longer repeat the results in “conclusion”, already indicated in paragraphs of results and discussions.
Comments for author File: 
Comments.docx
Overall, the text needs a moderate English check. some grammatical and typo errors are present.
Author Response
Overall, the text needs a moderate English check. some grammatical and typo errors are present.
Answer: done by a native English-speaking scientist
Materials and Methods
Line 81: What inoculum concentrations were used for the challenge test? How were the inocula prepared?
Answer: We have first evaluated different procedure. With most of them it was not possible to bring enough bacteria concentration on the surface of a knife. We wanted to make sure, that after the intervention a log 5 reduction could be measured. The best procedure, which is described in the Material and Method part was the following: Several colonies of S. aureus or E. coli were taken from sheep blood plates (incubated at 37°C for 24 h) using sterile cotton swabs by smearing them on the defined surface. Using this procedure we could guarantee a more or less standardized initial colony count per cm2 on the knives (see results in Figure 1, ante interventionem).
Line 89: It is not clear whether 0.16% is the concentration of PAA and POA in disinfectant or the % of InspexxTM 210 in water. Please, specify and indicate the percentage of InspexxTM 210 in water.
Answer: 0.16% is the concentration of InspexxTM 210 in water (now clarified in the text). Inspexx 210 contains 122.0g peracetic acid / kg liquid and 14.0g peroctanoic acid / kg liquid.
Line 89: Please, indicate the temperature of the water used.
Answer: done
As far for the methods, was the same knife used for sampling before and after treatment for the test? In this case, after having sampled the entire area of the knife, the first sampling by mechanical action of the swab may have already reduced (albeit slightly) the initial contamination. Did the authors consider that the first sampling (operating before the treatment) could reduce the microbial concentration?
Answer: For the challenge test we have used the same knife but two different sampling areas, see line 81: two areas of 5 cm2 were inoculated with several colonies of the respective bacterium. For the field experiments, different sides of the knives before and after the intervention were sampled.
Lines 90-92: Please, indicate the surface size sampled.
Answer: this is described in line 81: two areas of 5 cm2 were inoculated with several colonies of the respective bacterium.
Lines 103-104: Even if not standardized, please indicate an interval of exposure time to the disinfectant.
Answer: This is not possible, since we wanted to show the real situation in such an abattoir. The time differs depending on the employee and the position in the slaughter.
Line 108: Please, specify the microbiological methods used (respectively ISO, medium used, etc.)
Answer: this is mentioned lines 111ff: The surface plating method on plate count agar (PC agar, Oxoid AG, Pratteln, Switzerland) was used. For the challenge test: incubation 24h at 37°C was used since S. aureus and E. coli are growing very well on plate count agar at 37°C for 24h; for the field study (where we have not known which bacteria were present on the knives) we used the incubation conditions of ISO 4833-2 (30°C for 3d) to get the TVC.
Regards the results, please specify that the reduction is calculate on the means.
Answer: This was added in the text section: microbiological analysis
Figure 1 and 2:
Please, check the statistic (for example the Fig.1 f);
Answer: The statistic analysis was redone, it is correct as shown
Please, replace “Colony forming units (CFU)” with “Logarithm (CFU/cm2)”;
Answer: was changed – thank you
Why weren't the knives pre-treated with protein and then rinsed under a tap for 15 seconds for the E. coli test?
Answer: Since the reduction of S. aureus was much more influence on the presence of protein compared to E. coli, this additional experiment was only performed with S. aureus (worst case scenario).
Is it possible to put the error bar before the dots, so as to make it more visible?
Answer: done as requested
Line 155: please, rewrite the point (a).
Answer: done
No longer repeat the results in “conclusion”, already indicated in paragraphs of results and discussions.
Answer: the results were deleted in the conclusion part.
Reviewer 3 Report
My compliments to the authors. Many years ago my study group conducted an experiment similar to the one presented by the authors, but it was not possible to publish the results due to property rights issues of the product studied (another than the one under study).
The survey was designed and conducted with skill, the results are interesting (in the context in which we are). I suggest improving the introduction by updating references.
I have inserted my annotations, the corrections of the typos and the suggestions directly in the pdf of the article that I enclose. Please, see and read it.
Comments for author File: 
Comments.pdf
My compliments to the authors. Many years ago my study group conducted an experiment similar to the one presented by the authors, but it was not possible to publish the results due to property rights issues of the product studied (another than the one under study).
The survey was designed and conducted with skill, the results are interesting (in the context in which we are). I suggest improving the introduction by updating references.
I have inserted my annotations, the corrections of the typos and the suggestions directly in the pdf of the article that I enclose.
I I believe that the article is of medium interest to readers, but still valid for publication in your journal.
Author Response
There is certainly more recent data on the epidemiology of foodborne diseases, in the US as well as in the European Union. I suggest to see for example the data from the EFSA reports which are updated to 2021 and the WHO data which is also more recent - I would propose to update the bibliographic references.
Answer: We have replaced based on the EFSA report 2021 the sentence. The new sentence is: In 2021, the European Food Safety Authority (EFSA) reported 4005 foodborne outbreaks, 32543 cases of illness, 2495 hospitalisations and 31 deaths in the European Union.
Clear space between E. coli and ,
Answer: corrected
In 2019, the ISO 20876-2 standard was published which provides guidelines for setting up challenge tests to validate "treatments" such as the one being studied could also be. Did the Authors deliberately choose NOT to consider the ISO standard mentioned above or did they simply not know it?
It would be appropriate to justify the reason for their choice (e.g. by declaring that according to them the ISO was not entirely suitable to satisfy the study needs and that they preferred to set up the test according to their criteria).
Answer: Thank you for this comment: This ISO standard is not suitable for this study aims. Therefore, we have set up the procedure as described. Moreover, the same set up was already successfully used to test UV decontamination of knives in abattoirs (Raschle et al. (2019) Italian Journal of Food Safety, 8(2).)
I imagine that we are talking about TVC here but it has not been specified in the figure.
Answer: the reviewer is right – thank you: corrected
Spelling error conlcusion
Answer: corrected
Round 2
Reviewer 1 Report
The authors made sufficient corrections to the text.
However, only 17 references look modest, taking into account the current access to literature data.
It will be to everyone's advantage if the authors compare their own hypotheses and results with more literature data.
Author Response
The authors made sufficient corrections to the text.
ANSWER: thank you.
However, only 17 references look modest, taking into account the current access to literature data. It will be to everyone's advantage if the authors compare their own hypotheses and results with more literature data.
ANSWER: We have once again done a literature search with the search term “Knife decontamination and slaughterhouse” on google and on pubmed. Please see:
https://duckduckgo.com/?t=ftsa&q=Knife+decontamination+and+slaughterhouse&ia=web
https://pubmed.ncbi.nlm.nih.gov/28290317/
There is not really very much literature on this topic. Most of it is already considered in our paper.
We have now additionally added:
C. Bauermann Brasil, J. S. Barin, E. Jacob-Lopes, C. R. Menezes,L. Q. Zepka, R. Wagner, P. C. Bastianello Campagnol, A. J. Cichoski. Single step non-thermal cleaning/sanitation of knives used in meat industry with ultrasound. Food Research International, 91, (2017), pp. 133–139.
Reviewer 2 Report
No comments to add. The communication could be accepted.
Author Response
Answer: Thank you.
