Customizing Sanitization Protocols for Food-Borne Pathogens Based on Biofilm Formation, Surfaces and Disinfectants—Their Two- and Three-Way Interactions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors applied microbiology
Article
Customizing sanitization protocols for food-borne pathogens based on biofilm formation, surfaces and disinfectants – and their two- and three-way interactions

Food-borne pathogens are a serious challenge in food handling, processing and packaging systems.
Microorganisms within biofilms are difficult to eradicate with chemical disinfectants with an increased likelihood of survival, and subsequent contamination of food.

Authors have evaluated the responses of 3 bacterial biofilms formed by three food-borne bacterial pathogens on eight hard surfaces to seven chemical disinfectants.
Results are indicative of that fine-tuning of sanitization strategies along the food production, processing and packaging chain can
be achieved in specific scenarios by accounting for two- and three-way interactions between bacteria, surface and disinfectant.

The title is ok, and informative.
Figures were adequate, tables are ok, has this work been attempted previously?
The literature review was done adequately, and English can be improved to a large extent.

Statistical analyses were performed using Minitab Statistical Software  
Despite the challenges of investigating biofilms' two- and three-way interactions with surfaces and disinfectants, the BEST™ Assay provided an effective platform for evaluating these influences and interactions.
Additionally, interactions between pathogen, surface and biocide were evaluated.

Accept it for publication with minor mandatory changes.  

With Regards,

Comments on the Quality of English Language applied microbiology
Article
Customizing sanitization protocols for food-borne pathogens based on biofilm formation, surfaces and disinfectants – and their two- and three-way interactions

Food-borne pathogens are a serious challenge in food handling, processing and packaging systems.
Microorganisms within biofilms are difficult to eradicate with chemical disinfectants with an increased likelihood of survival, and subsequent contamination of food.

Authors have evaluated the responses of 3 bacterial biofilms formed by three food-borne bacterial pathogens on eight hard surfaces to seven chemical disinfectants.
Results are indicative of that fine-tuning of sanitization strategies along the food production, processing and packaging chain can
be achieved in specific scenarios by accounting for two- and three-way interactions between bacteria, surface and disinfectant.

The title is ok, and informative.
Figures were adequate, tables are ok, has this work been attempted previously?
The literature review was done adequately, and English can be improved to a large extent.

Statistical analyses were performed using Minitab Statistical Software  
Despite the challenges of investigating biofilms' two- and three-way interactions with surfaces and disinfectants, the BEST™ Assay provided an effective platform for evaluating these influences and interactions.
Additionally, interactions between pathogen, surface and biocide were evaluated.

Accept it for publication with minor mandatory changes.  

With Regards,

Author Response

Reviewer #1: 

Thank you for your kind review.

In response to your question yes, variations on this work have been done previously in support of internal and commercial R&D efforts.  This is the first time this work has been published in a peer-reviewed format for academic purposes however.

The manuscript has been revised with minor edits to improve the English and grammar throughout.

Reviewer 2 Report

Comments and Suggestions for Authors

The paper describes the biofilm formation of three food borne pathogens on eight different hard surfaces and the bacterial biofilm reduction to seven different chemical disinfectants. Results demonstrate the ability of forming biofilms by these microorganisms and the differential efficiency of the disinfectants on the various surfaces.

The introduction is detailed, but points out the problems of biofilm examinations well. The method section is described in detail so that the test series can be easily reproduced and the results section reflect all the data collected. In the discussion, the data obtained are compared very well with results from the current references. The aim of the study was to show a method for biofilm formation on various surfaces following the reduction of cell numbers after disinfection.

The paper is high interesting; however, it needs some revisions and amendments.

The initial solution was 1.5 x 8 log10, the resuspension (population densities) was e.g. 4.85 for E. coli (Table 2). Are the missing numbers cells that were in the planktonic state? Or was there a loss of cell numbers due to the biofilm removal method? Please explain in more detail. I think it is easier to follow when you add the initial cell number in each experimental set up.

It is written in the legend texts of the figures and in few tables that error bars represent the standard error of the mean. How many assays were calculated in the means and why have you calculated the errors and not the standard deviation?

The initial biofilm cell count should be specified in order to be able to clearly track the reduction. The log reduction on stainless steel (Table 3) has always the identical values independent on the biocide used. Is this the limit of detection?

Figure 3 and others: the population density after 48h is much higher than after 24h. Starting on the initial cell count of 8 log10, the density after 24 h is about 3-4 log10 and after 48h 5 to 6 log10. If the cell count in the biofilm increased so significantly, then the cell count of the planktonic cells should decrease accordingly, or is the increase due to bacterial growth? Please explain.

Figure 5 represents the log reduction for each pathogen as means from seven disinfectants. I take a critical view of this statement, because there is a cell-specific (inactivation-) behavior on every surface. The comparability and the surface-specific behavior is not expressed in a differentiated way. Summarizing the data from all the different surfaces does not reflect the reality on each individual surfaces.

Figure 8: The standard errors of wood for all three pathogens are very high. I know, wood has no smooth surface, but this need a sentence of explanation.

I can´t find Figure 11. Was this forgotten?

Lines 211/212 and 414: the functional disinfection is defined at 3 log10 reduction and ….is widely described… please add references for this statement and please add a statement explaining the difference of “functional disinfection” and “disinfection” that should be higher in log reduction.

Author Response

Reviewer #2:

Thank you for your kind review.  Please find suggested revisions included in the attached revised manuscript and answered here for convenience:

The initial solution was 1.5 x 8 log10, the resuspension (population densities) was e.g. 4.85 for E. coli (Table 2). Are the missing numbers cells that were in the planktonic state? Or was there a loss of cell numbers due to the biofilm removal method? Please explain in more detail. I think it is easier to follow when you add the initial cell number in each experimental set up.  Thank you for this excellent comment.  The population densities reported in table #2 are recovered biofilm numbers from the surface.  The recovery method described in the methods sections describes the recovery of biofilm organisms from the surface by first removing the planktonic challenge, rinsing the coupons several times to remove planktonic organisms and finally recovering the organisms off the surface through sonication.  The reported numbers in table 2 describes the character of the biofilm formed on the surface of the coupon by relating back to the number of viable cells recovered in 4.5 mL of recovery fluid after sonication.  The reduction in numbers from the initial challenge inoculum reflects the reduced numbers of recovered cells due to multiple reasons which include reduced adherence to coupon, reduced recovery from surface, change in metabolic state.  The methods section has been updated to better describe this.  Thank you for your input.

It is written in the legend texts of the figures and in few tables that error bars represent the standard error of the mean. How many assays were calculated in the means and why have you calculated the errors and not the standard deviation?  Thank you for this.  Each disinfectant challenge was compared to a control and done in triplicate and each experiment repeated once, for this reason we elected to present the SEM rather than the SD to provide the reader with insight into the confidence interval.  This is now defined in the methods section, thank you.

The initial biofilm cell count should be specified in order to be able to clearly track the reduction. The log reduction on stainless steel (Table 3) has always the identical values independent on the biocide used. Is this the limit of detection?  Excellent point.  A Control Recovery row has been added to table 2 to calibrate the reader.  The repeated numbers indicate maximum kill (i.e. no organisms were recovered after treatment and the control average log recovery number remains i.e. Control recovery = 4.47 – test recovery 0.00 CFU, Log Reduction = 4.47-0.00).

Figure 3 and others: the population density after 48h is much higher than after 24h. Starting on the initial cell count of 8 log10, the density after 24 h is about 3-4 log10 and after 48h 5 to 6 log10. If the cell count in the biofilm increased so significantly, then the cell count of the planktonic cells should decrease accordingly, or is the increase due to bacterial growth? Please explain.  Thank you for this interesting point.  Similar to the answer above, the recovery on the coupons is not strictly related to the planktonic population recovery (which was not evaluated) but rather speaks to the maturity of the biofilm over the 48h time frame, increased adherence, changes in metabolic status in the matrix etc. at the time of recovery.   It would be interesting to look at planktonic changes over time in detail in future work (this was out of scope for this particular research effort).

Figure 5 represents the log reduction for each pathogen as means from seven disinfectants. I take a critical view of this statement, because there is a cell-specific (inactivation-) behavior on every surface. The comparability and the surface-specific behavior is not expressed in a differentiated way. Summarizing the data from all the different surfaces does not reflect the reality on each individual surfaces.  Thank you for this, we agree with your critical review.  We acknowledge the significant contributions of the various modes of action of the disinfectants evaluated and the equally important contributions of the surfaces.  This is precisely why evaluated all of these individually and presented the various data in separate analyses.  Figure 5 is an attempt by the authors to generally compare the log reductions compared across the species for quick reference of the reader.

Figure 8: The standard errors of wood for all three pathogens are very high. I know, wood has no smooth surface, but this need a sentence of explanation.  Agreed, a valuable input.  This has been added to the discussion in 3.4.

I can´t find Figure 11. Was this forgotten?  No.  This is meant to refer to figure 10.  Thank you for catching the typo.

Lines 211/212 and 414: the functional disinfection is defined at 3 log10 reduction and ….is widely described… please add references for this statement and please add a statement explaining the difference of “functional disinfection” and “disinfection” that should be higher in log reduction.  Good point.  Three references from CDC, EPA and USDA have now been added.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewer's comments were well implemented, supplemented and explained. From my point of view, this manuscript can be accepted in this form.