Review Reports - Domestic Food Safety Risks: A Two-Year Assessment of Refrigerator Hygiene and Egg Contamination

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper reported on a study that attempted to correlate refrigerator temperature and sanitation practices to pathogen and indicator counts from cloth samples, along with collecting shelled eggs from participants' fridges. The concept of the study is interesting, although there are concerns about the experimental approach, interpretation of data and structure of the script. Specifically, the egg testing and refrigeration sampling appear to be two separate studies. The eggs were screened for Salmonella and Campylobacter which would be more likely to be derived during production/grading stages. Moreover, there were participants who didn’t even store eggs in the refrigerator.

Sampling the refrigerator interior could be of interest, but it is unclear how consistent the sampling was across sites. It would have been more informative to include additional information on the types of foods stored, the length of time, and whether sanitation decisions were based on visible cleanliness. The efficacy of sanitation using the different methods should have been evaluated.

Title: Assessment of what? The authors should use a more descriptive title to reflect the conclusion of the study.

Line 18: Humidity and temperature fluctuations are also of concern in relation to egg storage.

Line 20: Indicators and Listeria?

Line 27: 72 or 50?

Line 30: What is considered a high TVC?

Line 35: What do the authors mean by changes in temperature?

Line 38: What is considered moderate temperature fluctuations?

Line 39: Wouldent the results suggest that there are no significant food safety issues relating to refrigerator stored eggs?

Line 52: Statements should be supported by citing references.

Line 57: What would be considered acceptable microbial counts in fridges?

Line 63: Microaerophilic than thermophilic.

Line 94: The authors need to provide a hypothesis. It is unclear if the researchers are assessing if contamination on the egg influence the counts (surface & internal) of the shell or if contamination is introduced by refrigerated storage. This should be clarified in the revised script.

Line 101: Was there any selection criteria or just the first 50 households? It would have been informative to have a more diverse range of fridge types with respect to temperature.

Line 124: Why didn’t the authors look to sample the RTE foods given the increased risk of contamination?

Line 137: Which part of the fridge were participants asked to collect samples from?

Line 157: What confirmation tests were performed with pathogen analysis?

Line 159: Were the eggs washed or unwashed?

Line 197: Unclear what point the authors are trying to make. What is ensuring proper preservation?

Line 201: Would this include eggs?

Line 204: Bleach and acid would release chlorine gas.

Line 220 and 255: Would cost also be a factor with the older generation?

Line 259: The concentration of sanitizer would also be a factor to consider in relation to efficacy.

Line 270: Why were eggs selected if a proportion were stored at room temperature?

Line 282: Would the counts depend if the condenser tray was sampled?

Line 291: What are considered high counts?

Line 320: Foods stored and humidity would have affected counts.

Line 332: What was the Total Aerobic Count associated with eggs.

Line 339: The authors should discuss the results of the study. This part would be better placed in the introduction.

Line 348: Would a 0-6C range be significant given most enteric pathogens grow at >10C?

Line 359: Would the cleaning frequency depend on the visible cleanliness of the refrigerator?

Line 381: By above 6C would this also mean >10C which is a more critical temperature.

Line 303: Was the low TAC more related using a 30C incubation temp which would not support the growth of psychrophiles.

Line 435: The prevalence of Salmonella and Campylobacter would be carried on the eggs rather than introduced within the refrigerator.

Line 449: Is this relevant to refrigerator sanitation?

Line 473: The study microbial count data suggests there are no food safety risks.

Author Response

Thank you for the valuable revision and comments on our paper. The updated version was reformulated accordingly The lines number are related to the new file.

Comment: The paper reported on a study that attempted to correlate refrigerator temperature and sanitation practices to pathogen and indicator counts from cloth samples, along with collecting shelled eggs from participants' fridges. The concept of the study is interesting, although there are concerns about the experimental approach, interpretation of data and structure of the script. Specifically, the egg testing and refrigeration sampling appear to be two separate studies. The eggs were screened for Salmonella and Campylobacter which would be more likely to be derived during production/grading stages. Moreover, there were participants who didn’t even store eggs in the refrigerator.

Answer: The study was designed from the outset as a two-component consumer-based assessment, in which egg sampling and refrigerator sampling were conducted simultaneously within the same households. Although the two components address different aspects of domestic food safety, they were intentionally integrated: the refrigerator sampling documented storage conditions and hygiene practices, while the egg sampling assessed the microbiological status of eggs as purchased and handled by consumers.

We have now clarified this point in the Introduction to avoid misinterpretation. “The present study addresses this gap through a two-year longitudinal investigation of domestic refrigerators and table eggs in Porto, Portugal. The research combined temperature monitoring, microbiological analysis of key pathogens and hygiene indicators, and a structured evaluation of household food-safety practices. Conducting the study over two consecutive years enabled the assessment of temporal consistency. The study comprised two complementary components implemented within the same households: (i) sampling of domestic refrigerators to characterise temperature performance and surface microbial contamination, and (ii) microbiological screening of eggs as purchased and stored by consumers. These components were designed to capture different but interrelated aspects of domestic food safety behaviours. Importantly, the egg analysis aimed to characterise the microbiological status of eggs at the consumer level and was not intended to infer causal relationships with refrigerator conditions. By integrating both storage environments and food matrices, the study provides a comprehensive overview of potential microbiological hazards and opportunities for risk reduction in domestic settings.” Lines 91-101.

The inclusion of households that stored eggs at room temperature reflects real consumer behaviour in Portugal, where egg refrigeration is not universal. These households were intentionally retained, as excluding them would reduce ecological validity and introduce bias into the consumer-level analysis. We have added a short explanation to the Methods to clarify that egg storage practices. “Eggs were sampled as they were found in the household, whether stored in the refrigerator or at room temperature, to reflect actual consumer practices.” Lines 169 -170. We have refined the manuscript to clarify the integrated nature of the study and to ensure that the objectives and limitations of the egg and refrigerator components are presented transparently.

Comment: Sampling the refrigerator interior could be of interest, but it is unclear how consistent the sampling was across sites. It would have been more informative to include additional information on the types of foods stored, the length of time, and whether sanitation decisions were based on visible cleanliness. The efficacy of sanitation using the different methods should have been evaluated.

Answer: We would like to clarify that the study was intentionally designed as a consumer-based survey, in which the sampling procedures had to remain simple, standardised and practical for participants to perform at home. For this reason, the protocol did not include in-person observations, detailed food inventories, records of storage duration or assessments of whether cleaning decisions were based on visual cues. Collecting such data would have required on-site inspections or participant diaries, which were beyond the scope and feasibility of this study. Although, we agree that collecting more information on refrigeration habits would improve the manuscript we also wonder how many refrigerators should be sampled to find a suitable and statically valid conclusion. Sampling consistency was maintained by providing all participants with identical sampling kits and standardised written instructions following ISO 18593:2018, specifying the sampling location and procedure. We have now clarified this more explicitly in the Methods with the introduction of a new sentence “Surface sampling using the sterile cloths provided to participants followed the procedures described in ISO 18593:2018 [21].” Lines 145-146. We hope that these clarifications help demonstrate that the study adhered to its intended design, focusing on real-world consumer practices while maintaining feasibility and participant compliance.

Comment: Title: Assessment of what? The authors should use a more descriptive title to reflect the conclusion of the study.

Answer: We thank the reviewer for this suggestion. We agree that the original title could be made more descriptive to better reflect the scope and conclusions of the study. We have therefore revised the title to specify the key components assessed—refrigerator temperature performance, surface hygiene indicators and the microbiological status of household eggs. The revised title reads: “Domestic Food Safety Risks: A Two-Year Assessment of Refrigerator Hygiene and Egg Contamination”

Comment: Line 18: Humidity and temperature fluctuations are also of concern in relation to egg storage.

Answer: We thank the reviewer for this observation. The sentence referred specifically to refrigerator conditions in general and not to egg storage in particular. We agree that humidity and temperature fluctuations are relevant factors for egg integrity and quality; however, this paragraph aimed only to contextualise the role of temperature control and hygiene in the growth and survival of foodborne pathogens on refrigerator surfaces.

Comment: Line 20: Indicators and Listeria?

Answer: We are not sure if we understand your comment. The microbiological targets differed between the two components of the study. For eggs (both shell surface and internal contents), only Salmonella spp. and Campylobacter spp. were analysed, as these are the primary pathogens of epidemiological relevance in eggs and egg-related outbreaks. Microbiological indicators were not assessed in the egg component.

In contrast, the refrigerator sampling included both hygiene indicators (TVC, Enterobacteriaceae, E. coli) and screening for Campylobacter spp., L. monocytogenes and Salmonella spp.

Comment: Line 27: 72 or 50?

Answer: We are not sure if we understand your comment. The manuscript does not report a value of 72; the number presented is 76, which corresponds to the total number of refrigerator-stored egg samples across the two sampling years. Specifically, 37 households stored eggs in the refrigerator in 2024 and 39 households stored eggs in the refrigerator in 2025, resulting in a combined total of 76 samples.

Comment: Line 30: What is considered a high TVC?

Answer: The sentence referred only to the year-to-year increase in TVC and Enterobacteriaceae, not to the absolute magnitude of these values.

However, the authors acknowledge that the original manuscript did not specify the criteria used to classify TVC levels, and we agree that this information was missing. To address this, we have now added a detailed explanation in the Materials and Methods section where we justify the choice of thresholds and describe how reference limits for food-contact surfaces were adapted to CFU/cloth values. Specifically, we now state: “To evaluate surface contamination, we applied the criteria defined by Düven et al. (2021) [29], with <5 CFU/cm² (≈0.7 Log₁₀) considered acceptable, 5–25 CFU/cm² (0.7–1.4 Log₁₀) borderline, and >25 CFU/cm² (>1.4 Log₁₀) unsatisfactory. To enable direct com-parison with our results, which are expressed as CFU per cloth, the area-based limits were converted to CFU/cloth by approximating the sampled surface area. Based on meas-urements from a representative sample of domestic refrigerators in Portugal, a standard shelf area of approximately 1500 cm² was assumed. Multiplying each value by this surface area resulted in the following operational limits for this study: <7.5 × 10³ CFU/cloth (<3.88 Log₁₀) classified as acceptable, 7.5 × 10³–3.75 × 10⁴ CFU/cloth (3.88–4.57 Log₁₀) as borderline, and >3.75 × 10⁴ CFU/cloth (>4.57 Log₁₀) as unsatisfactory.” (lines 207-216)

In addition, we incorporated the classification results into the Results section (lines 362-367) to clearly show how refrigerators were categorized in each year: “Based on the TVC results, in 2024, 26% (n = 13) of the refrigerators were classified within the acceptable limit, 18% (n = 9) within the borderline range, and 56% (n = 28) as unsatisfactory. In 2025, the distribution shifted considerably: only 20% (n = 10) of refrigerators fell within the acceptable limit, 14% (n = 7) were borderline, and 66% (n = 33) were classified as unsatisfactory. This increase in the proportion of unsatisfactory units from 2024 to 2025 indicates a marked deterioration in overall hygiene status.”

These additions clarify the criteria used to define high TVC values and strengthen the interpretation of our findings.

Comment: Line 35: What do the authors mean by changes in temperature?

Answer: In this context, “changes in temperature (ΔT)” refers to the difference in mean refrigerator temperature between the two sampling years for each household (i.e., 2025 mean temperature minus 2024 mean temperature). This year-to-year temperature difference was used to assess whether variations in temperature were associated with variations in microbial indicators. A new paragraph was written to clarify “Data analysis focused on assessing the year-to-year correlations between temperature variation and microbial load. For each refrigerator, the change in mean temperature between 2024 and 2025 (ΔT)—defined as the difference in mean refrigerator temperature between the two sampling years (i.e., 2025 mean temperature minus 2024 mean temperature)—was calculated along with the corresponding change in surface counts (Δ Log₁₀ CFU/cloth). These values were computed separately for total viable counts (TVC), Enterobacteriaceae, and β-glucuronidase–positive E. coli, allowing the evaluation of whether temperature fluctuations were associated with variations in microbial indicators.” Line 217-224

Comment: Line 38: What is considered moderate temperature fluctuations?

Answer: In this context, “modest temperature fluctuations” was not intended as a technical classification but simply as a qualitative description indicating that the year-to-year changes in mean refrigerator temperature (ΔT) were generally small. To avoid misinterpretation, we revised the sentence to explicitly state that the observed temperature differences were limited in magnitude and therefore unlikely to explain the variation in microbial indicators. New paragraph was rewritten “The absence of correlation between ΔT and Δ microbial counts suggests that behaviour-driven hygiene factors (e.g., cleaning, segregation, spill management, door-opening patterns), rather than the relatively small year-to-year temperature differences observed, are more influential in determining household bioburden.” Lines 36-39

Comment: Line 39: Wouldn’t the results suggest that there are no significant food safety issues relating to refrigerator stored eggs?

Answer: We agree that the findings indicate no food-safety concerns for the eggs analysed, as neither Salmonella nor Campylobacter were detected. However, the sentence in question refers to general food-safety guidance for domestic refrigerators and not specifically to eggs. Maintaining temperatures ≤ 6 °C is a standard recommendation intended to reduce risks for a wide range of perishable foods stored in the refrigerator, including ready-to-eat products and leftovers.

Comment: Line 52: Statements should be supported by citing references.

Answer: We agree that the statement should be supported, and an appropriate reference has now been added to substantiate the point regarding the impact of inadequate temperature control, poor cleaning habits and improper food storage on food-safety risks. The new added reference is: [5] Farber, J.M.; Peterkin, P.I. Listeria monocytogenes, a Food-Borne Pathogen. Microbiol Rev 1991, 55, 476–511. Doi:10.1128/mr.55.3.476-511.1991.

Comment: Line 57: What would be considered acceptable microbial counts in fridges?

Answer: The sentence in line 57 “Such conditions favour the survival, and in some cases even the proliferation, of foodborne pathogens, thereby amplifying potential risks within households”, did not refer to numerical values but to the general principle that inadequate temperature control and hygiene can support the survival or growth of foodborne pathogens.

Comment: Line 63: Microaerophilic than thermophilic.

Answer: Our intention was to refer specifically to the temperature-related behaviour of Campylobacter spp. rather than its oxygen requirements. To ensure microbiological accuracy, we have replaced the term “thermophilic” with “thermotolerant”, which describes more appropriately its growth characteristics in the context of temperature. The sentence has been revised accordingly. Lines 64-66

Comment: Line 94: The authors need to provide a hypothesis. It is unclear if the researchers are assessing if contamination on the egg influence the counts (surface & internal) of the shell or if contamination is introduced by refrigerated storage. This should be clarified in the revised script.

Answer: The study was designed to test a causal relationship between egg contamination and refrigerator contamination. The study followed an observational, consumer-based approach with a descriptive hypothesis: that domestic refrigerators and household eggs, when sampled as found, would reflect real-world variability in hygiene practices, temperature control and microbiological status. The two components of the study—refrigerator sampling and egg microbiological analysis—were intentionally conducted in parallel to characterise different, complementary aspects of domestic food safety. We have now clarified this explicitly in the Introduction: “The study comprised two complementary components implemented within the same households: (i) sampling of domestic refrigerators to characterise temperature performance and surface hygiene, and (ii) microbiological screening of eggs and egg-holders and stored by consumers. These components were designed to capture different but interrelated aspects of domestic food safety behaviours. By integrating both storage environments and food matrices, the study provides a comprehensive overview of potential microbiological hazards and opportunities for risk reduction in domestic settings.” Lines 94-101

Comment: Line 101: Was there any selection criteria or just the first 50 households? It would have been informative to have a more diverse range of fridge types with respect to temperature.

Answer: Participants were recruited on a voluntary basis, and no specific selection criteria was applied beyond residence in the study area. The sample was therefore not restricted to the “first 50” households but reflected voluntary enrolment. This approach was chosen to maximise feasibility and participant compliance within a community-based study design. We agree that including a wider range of refrigerator types could provide additional insight into performance variability. However, this was beyond the scope of the present consumer-based survey, which did not involve household visits or technical characterisation of appliances. A new phrase was added to clarify: “Recruitment followed an open voluntary approach; households were not selected based on refrigerator model or characteristics, and no prioritisation or ordering was applied.” Lines 107-109.

Comment: Line 124: Why didn’t the authors look to sample the RTE foods given the increased risk of contamination?

Answer: The study was designed as a consumer-led, in-home sampling approach, in which participants collected surface samples and provided eggs as purchased and stored. For feasibility, safety, and compliance reasons, the protocol did not include collecting ready-to-eat (RTE) foods, as this would have required either researcher-led household visits or cold-chain controlled transport of perishable foods, which were beyond the scope of the study design. The objective of the questionnaire was therefore to document behaviours relevant to safe storage of RTE foods—such as organisation, segregation, packaging removal, and cleaning practices—rather than to analyse the foods themselves.

Comment: Line 137: Which part of the fridge were participants asked to collect samples from?

Answer: As described in Section 2.1 (Sampling), all participants were instructed to collect a surface sample from the bottom refrigerator shelf using the sterile cloth provided, and to use a sterile swab pre-moistened with Neutralizing Rinse Solution to sample the egg-holder area.

Comment: Line 157: What confirmation tests were performed with pathogen analysis?

Answer: Confirmatory testing only can be done when presumptive colonies are detected. In the present study, no presumptive colonies of either pathogen were obtained, and therefore no confirmation tests were required. However, the confirmatory tests are described in the standards used, lines 169-172.

Comment: Line 159: Were the eggs washed or unwashed?

Answer: The authors appreciate the opportunity to clarify this matter. A new paragraph was written: “Eggs were sampled as they were found in each household—whether stored in the refrigerator or at room temperature—to accurately reflect consumer practices. In Portugal, eggs are sold unwashed and their shells may naturally carry microorganisms. Although participants were instructed to collect the eggs as they stored them, we had no information on whether any of them washed the eggs before refrigeration. Domestic egg washing is considered a poor food-safety practice, as it can disperse microorganisms through splashing, remove the protective cuticle, and facilitate microbial penetration, thereby increasing the risk of cross-contamination to the egg-holder or other refrigerator surfaces. This uncertainty in handling practices may therefore introduce a potential source of pathogenic contamination.” Lines 175-184

Comment: Line 197: Unclear what point the authors are trying to make. What is ensuring proper preservation?

Answer: By “proper preservation,” we refer to storing foods in the appropriate temperature zones of the refrigerator to ensure adequate conservation and reduce the risk of microbial growth or cross-contamination. Refrigerators are designed with zones that differ in temperature, and placing each type of food in its recommended area helps maintain food safety.

Comment: Line 201: Would this include eggs?

Answer: Yes, when they store the eggs in the egg’s holder

Comment: Line 204: Bleach and acid would release chlorine gas.

Answer: We thank the reviewer for this important observation. We fully agree that mixing hypochlorite-based products with acids can release chlorine gas and is unsafe. A clarification has now been added to the Discussion to explicitly acknowledge this and to emphasise that such combinations should not be used in domestic cleaning. A new sentence was added “It should be noted that mixing hypochlorite-based bleach with acidic cleaning products is hazardous because it can release chlorine gas. Such combinations should never be used without a thorough rinsing step between applications; nevertheless, this unsafe practice was reported by a small number of participants.” Lines 250-254

Comment: Line 220 and 255: Would cost also be a factor with the older generation?

Answer: Cost-related considerations and their potential influence on cleaning behaviours, particularly among older participants are included in the manuscript. Lines 303-306

Comment: Line 259: The concentration of sanitizer would also be a factor to consider in relation to efficacy.

Answer: The authors agree that the concentration of sanitizing agents is an important determinant of cleaning efficacy. However, the aim of the present study was to document self-reported household cleaning practices rather than to evaluate the microbiological performance of specific products. For this reason, participants were not asked to report sanitizer concentrations, and the study did not include any assessment of product efficacy. Furthermore, we note that the study did not evaluate whether cleaning agents were used correctly (e.g., appropriate dilution, application time, or contact conditions), which is an additional limitation. A new sentence was added to the manuscript. “The study did not evaluate the correct use of cleaning products, such as appropriate dilution or application practices.” Lines 306-307

Comment: Line 270: Why were eggs selected if a proportion were stored at room temperature?

Answer: In Portugal, as in other European countries, eggs are sold unwashed, and the “best before” date printed on the shell corresponds to ambient storage. For this reason, all eggs were sampled “as found”, whether kept at room temperature or in the refrigerator. This approach is consistent with the observational nature of the study and allowed us to explore whether contamination levels differed between eggs stored under different conditions.

Comment: Line 282: Would the counts depend if the condenser tray was sampled?

Answer: The condenser tray is not readily accessible to participants, and the study did not include sampling sites that do not contact foods items.

Comment: Line 291: What are considered high counts?

Answer: The term “high” is not used in the manuscript to classify the absolute level of microbial counts. In the sentence highlighted by the reviewer, “higher counts” refers only to an increase from 2024 to 2025 within the same households, not to counts being “high” in an absolute sense. We have clarified this distinction in the revised text. “Thirty-three (66%) refrigerators showed an increase in Enterobacteriaceae counts in 2025 compared with 2024 (Table 2).” Lines 342-343

Comment: Line 320: Foods stored and humidity would have affected counts.

Answer: We agree that factors such as the types of foods stored, humidity, and general household handling practices can influence microbial loads on refrigerator surfaces. These variables were not measured in the present study and fall outside the scope of the statistical comparison performed. Nevertheless, the statement in the manuscript refers specifically to the finding that temperature differences (ΔT) explained little of the year-to-year variation in microbial indicators.

Comment: Line 332: What was the Total Aerobic Count associated with eggs.

Answer: we are not sure if we understood your comment. Total Aerobic Counts were not assessed in eggs in this study. The egg component focused exclusively on the detection of Salmonella spp. and Campylobacter spp., in accordance with the study objectives.

Comment: Line 339: The authors should discuss the results of the study. This part would be better placed in the introduction.

Answer: The reference to Masson et al. (2017) is included in the Discussion not as background information, but to contextualise and contrast our findings with previous research on consumer refrigerator-storage behaviours. This sentence serves to introduce a direct comparison with our results rather than to provide general introductory material. To ensure this intention is clear, we have slightly adjusted the surrounding text to strengthen the connection between their observations and the findings of the present study. “Masson et al. (2017) [33] conducted an observational study in France to assess how consumers organised food items within domestic refrigerators under controlled laboratory conditions. Their findings are directly comparable to those of the present study: they reported that only 30% of participants removed external packaging before storage, particularly for products such as yogurts and fresh vegetables. In our study, similar behaviours were observed, with most participants also retaining external packaging for several food categories, suggesting that this practice is common across different European populations.” Lines 392 -398

Comment: Line 348: Would a 0-6C range be significant given most enteric pathogens grow at >10C?

Answer: We agree that most enteric pathogens, excluding L. monocytogenes do not grow well below approximately 10 °C. However, the 0–6 °C range referred to in the manuscript does not imply microbial growth at these temperatures; rather, it reflects widely accepted food-safety recommendations for domestic refrigeration. Maintaining temperatures ≤ 6 °C is intended to slow microbial multiplication, limit spoilage, reduce the risk of pathogen survival and proliferation and maximise shelf life should temperature abuse occur. Our statement therefore relates to preventive storage guidance rather than to the growth characteristics of enteric pathogens.

Comment: Line 359: Would the cleaning frequency depend on the visible cleanliness of the refrigerator?

Answer: Based on participants’ questionnaire responses, a proportion of households reported cleaning the refrigerator “when necessary”, which likely reflects a visually driven assessment of cleanliness. However, many other participants indicated fixed cleaning intervals (e.g., weekly, monthly), suggesting that their behaviour was not dependent on the immediate visual condition of the appliance. Because the study relied on self-reported practices and did not evaluate perceived cleanliness directly, we cannot quantify the extent to which visual assessment influenced cleaning frequency, but the questionnaire responses indicate variability between households.

Comment: Line 381: By above 6C would this also mean >10C which is a more critical temperature.

Answer: The temperatures reported in this sentence reflect the findings of the cited studies, which used 6 °C as the benchmark because it corresponds to the recommended maximum temperature for domestic refrigeration, not to a microbiological growth threshold. Although temperatures >10 °C are indeed more critical from a food-safety perspective, the cited publications did not provide detailed stratification above this level. Our sentence therefore reports the values as presented in those studies and does not imply that >6 °C represents equivalent risk to >10 °C; rather, it reflects how domestic refrigerator performance has been described in the literature.

Comment: Line 303: Was the low TAC more related using a 30C incubation temp which would not support the growth of psychrophiles.

Answer: We agree that this method does not detect psychrophiles microorganisms. We used aerobic count at 30 °C as indicator of general hygiene on food and food-contact surfaces which can be comparable with other studies.

Comment: Line 435: The prevalence of Salmonella and Campylobacter would be carried on the eggs rather than introduced within the refrigerator.

Answer: We agree that any Salmonella or Campylobacter detected at the consumer level would originate from contamination on the eggs themselves, rather than being introduced within the refrigerator, as in the Serbian study that suggests eggs carrying Salmonella (line 484)

Comment: Line 449: Is this relevant to refrigerator sanitation?

Answer: The sentence in question refers specifically to upstream control programmes implemented in egg production (e.g., flock vaccination, biosecurity, feed control), not to refrigerator sanitation. Its purpose is to contextualise the observed absence of Salmonella and Campylobacter in consumer eggs by highlighting the effectiveness of primary-production control measures.

Comment: Line 473: The study microbial count data suggests there are no food safety risks.

Answer: We are not sure if understand your comment. We think that controlling and maintaining refrigeration temperatures under 5ºC as well cleaning and disinfection practices potential reduce domestic microbial food safety risks. Lines 540-544

Reviewer 2 Report

Comments and Suggestions for Authors

The authors conducted a survey using "A Longitudinal Assessment of Refrigerators and Eggs in Domestic Environments."

The manuscript is well-structured and the survey instruments are clear. However, in my opinion, the use of sampling kits administered to consumers may affect the reliability of the results. However, the authors chose this method and cannot modify it for this manuscript. For this reason, they should reiterate in the conclusions that the sampling was conducted by the survey participants.

Line 71: Enterobacteriaceae - not italicized throughout the text

Line 133: Was the data logger in the sampling kit?

Author Response

Thank you for the valuable revision and comments on our paper. The updated version was reformulated accordingly The lines number are related to the new file.

Comment: The authors conducted a survey using "A Longitudinal Assessment of Refrigerators and Eggs in Domestic Environments."

Answer: The authors agree that the use of sampling kits administered by participants may affect the reliability of the results. As noted, this limitation is inherent to the study design. Following the reviewer’s suggestion, we have now explicitly reiterated in the Discussion section that the samples were collected by the survey participants, emphasizing this limitation more clearly. To this end, a new paragraph has been added: “It is important to note that all samples were collected by the participants themselves using the sampling kits provided. While this approach was necessary for the logistics of the longitudinal study conducted in domestic environments, it may introduce variability due to differences in how individuals performed the sampling. Therefore, the results should be interpreted with this limitation in mind.” Lines 466-470

Comment: Line 71: Enterobacteriaceae - not italicized throughout the text

Answer: We have corrected the formatting of the term Enterobacteriaceae throughout the manuscript.

Comment: Line 133: Was the data logger in the sampling kit?

Answer: We have clarified in the corresponding section that the data logger was included in the sampling kit. New paragraph: “Temperature measurements were conducted in situ over an 8-hour overnight period. An Elitech RC-5 data logger, included in the sampling kit and programmed to record at 15-minute intervals, was placed on the bottom shelf of the refrigerator to capture the lowest expected temperatures.” Lines139- 142

Reviewer 3 Report

Comments and Suggestions for Authors

The study deals with an interesting research field that fits with the scope of Hygiene. The paper is well organized with a suitable length. I have hardly detected typographical errors and also adequate is the use of English language.

Nonetheless, I believe that there are some areas for improvement.

Introduction

Lines 77-79. The sentence “Evidence from domestic settings shows that Salmonella can be recovered from refrigerator surfaces, including in case-control work from the UK, and was detected in 14% of egg-storage compartments across 100 households in Belgrade” should be divided into two sentences with its corresponded cited each.

Material and methods

A cloth has been given to the participating household to sample the bottom shelf surface for the microbial analysis. However, the reference to the adequation to this methodology for this purpose is not included in ISO 4833-1:2013 but in ISO 18593:2018.

Line 169: First time that EQF is named the meaning of the acronym should be specified.

Line 183. The sign should not be < ?

Results

The authors place great emphasis on analyzing the types of cleaning used by the study participants, but the correlation between the type of cleaning procedure used and the microorganism counts obtained on the surfaces of the refrigerators analyzed has not been discussed or studied. I believe that the effectiveness of the different cleaning methods, assessed by microbiological analysis of refrigerator surfaces, should be analyzed.

Line 292. It is said that it can be seen in Table 1 that “Thirty-three (66%) refrigerators showed higher Enterobacteriaceae counts in 2025 than 291 in 2024”. However this information is not in table 1.

Discusion

Lines 362-364, it is said that results indicate that infrequent cleaning is a common household practice across European populations. Why authors conclude that cleaning frequency is low if the counts are not high and no pathogenic bacteria have been detected in the refrigerator environment? Which is the recommended frequency for cleaning refrigerators?. In fact, it is not said which is the recommended frequency for cleaning the refrigerators. Is there any indication of this in any treaty or recommendation from hygiene or public health institutes?

In discussing the results, the authors make a comparison with other studies in which the analysis methodology differs greatly (results expressed as UFC/cm²). (lines 396 to 408). I believe that, rather than comparing these results directly with those obtained using other methods, the authors should refer to these methodological differences in addition to searching for references that include the assessment of contamination of the surfaces using methods similar to those used in this study.

Conclusions

Lines 456-457: It is concluded that there are significant increases in the TVC and Enterobacteriaceae between 2024 and 2025. However, it is unclear whether these figures can be considered high.

Author Response

Thank you for the valuable revision and comments on our paper. The updated version was reformulated accordingly The lines number are related to the new file.

Comment: The study deals with an interesting research field that fits with the scope of Hygiene. The paper is well organized with a suitable length. I have hardly detected typographical errors and also adequate is the use of English language.

Nonetheless, I believe that there are some areas for improvement.

Lines 77-79. The sentence “Evidence from domestic settings shows that Salmonella can be recovered from refrigerator surfaces, including in case-control work from the UK, and was detected in 14% of egg-storage compartments across 100 households in Belgrade” should be divided into two sentences with its corresponded cited each.

Answer: We agree and have now divided the statement into two separate sentences, each clearly associated with the appropriate reference. New sentences: “Evidence from domestic settings shows that Salmonella can be recovered from refrigerator surfaces, as demonstrated in case–control work from the UK [19]. Moreover, Salmonella was found in 14% of egg-storage compartments in a survey of 100 households in Belgrade [20].” Lines 78-81

Materials and Methods

Comment: A cloth has been given to the participating household to sample the bottom shelf surface for the microbial analysis. However, the reference to the adequation to this methodology for this purpose is not included in ISO 4833-1:2013 but in ISO 18593:2018.

Response: We agree that ISO 18593:2018 (“Microbiology of the food chain — Horizontal methods for surface sampling”) is the appropriate standard for surface sampling using swabs or cloths. We have therefore corrected the text and now cite ISO 18593:2018 as the reference for the sampling methodology (line 146). ISO 4833-1:2013 remains cited only for the enumeration procedures applied in the laboratory.

Comment: Line 169: first occurrence of EQF — acronym should be defined.

Response: We have now defined EQF at first mention. Line 195

Comment: Line 183: The sign should not be “<”.

Response: Thank you for pointing this out. We agree that the original wording, which referenced “>LOQ”, was inaccurate because no formal limit of quantification had been established for this study. To correct this and avoid the use of inappropriate notation, we have revised the sentence to clearly indicate how undetermined results were handled. The updated text now reads:

“For refrigerators in which counts could not be determined due to insufficient dilution, the results were assigned the highest quantifiable value established for this study (TVC: 6.36 log₁₀; Enterobacteriaceae: 6.18 log₁₀; E. coli: 4.18 log₁₀). In case of growth absence, a value of 1.00 Log₁₀ CFU/cloth was assigned to enable statistical analysis.” Lines 224-228

Results

Comment: The authors place great emphasis on analyzing the types of cleaning used by the study participants, but the correlation between the type of cleaning procedure used and the microorganism counts obtained on the surfaces of the refrigerators analyzed has not been discussed or studied. I believe that the effectiveness of the different cleaning methods, assessed by microbiological analysis of refrigerator surfaces, should be analyzed.

Response:

We appreciate this important observation. We have now performed an additional analysis assessing the relationship between the type of cleaning procedure and microbial counts. These findings have been incorporated in the Results section and discussed in the Discussion section.

“A comparison of microbial contamination between refrigerators cleaned with detergent-based products (n = 26) and those cleaned with non-detergent methods (n = 20) showed no statistically significant differences in microbial contamination. The median values were similar between the two groups for TVC (4.70 vs. 4.95 Log₁₀ CFU/cloth), and for Enterobacteriaceae (5.79 vs. 7.12 Log₁₀ CFU/cloth).” Lines 312 -316

Comment: Line 292. It is said that it can be seen in Table 1 that “Thirty-three (66%) refrigerators showed higher Enterobacteriaceae counts in 2025 than 291 in 2024”. However this information is not in table 1.

Response: Thank you for noting this inconsistency. We have corrected the text to accurately reflect the data in Table 2.

Discussion

Comment: Lines 362-364, it is said that results indicate that infrequent cleaning is a common household practice across European populations. Why authors conclude that cleaning frequency is low if the counts are not high and no pathogenic bacteria have been detected in the refrigerator environment? Which is the recommended frequency for cleaning refrigerators? In fact, it is not said which is the recommended frequency for cleaning the refrigerators. Is there any indication of this in any treaty or recommendation from hygiene or public health institutes?

Response: We agree that the manuscript did not explicitly present recommended cleaning frequencies for domestic refrigerators. Our intention was not to classify the reported cleaning habits as inadequate, but to describe the heterogeneity observed across households and to compare these findings with available consumer-behaviour surveys from Europe.

Regarding recommended frequency, there is no harmonised public health or hygiene guideline within the EU that specifies how often a domestic refrigerator should be cleaned. Existing regulations (e.g., Regulation (EC) No 852/2004 on the hygiene of foodstuffs) address general domestic hygiene but do not define cleaning intervals for home refrigeration.

Importantly, we acknowledge that our microbiological results showed nonpathogenic bacteria, which is consistent with the generally low risk associated with household refrigerators. For this reason, our intention was not to imply that participants' cleaning frequency was insufficient, but simply to note that cleaning practices vary widely and that no official standard exists against which these behaviours could be evaluated. We have revised the wording in the manuscript to avoid any unintended suggestion of inadequacy. A new sentence was added “There is currently no EU-level public health guideline defining how frequently domestic refrigerators should be cleaned, and therefore the practices reported by participants cannot be evaluated against a formal standard.” Lines 434-436

Comment: In discussing the results, the authors make a comparison with other studies in which the analysis methodology differs greatly (results expressed as UFC/cm2). (lines 396 to 408). I believe that, rather than comparing these results directly with those obtained using other methods, the authors should refer to these methodological differences in addition to searching for references that include the assessment of contamination of the surfaces using methods similar to those used in this study.

Response: We agree and have clarified this point in the Discussion and revised the text to emphasize that our comparisons are qualitative rather than quantitative. Given these differences, only general trends were considered and the discussion has been adjusted accordingly. New paragraph was added: “It should be noted that published studies vary greatly in sampling methodology and reporting units. Many investigations express results as CFU/cm² using swabs or contact plates, whereas the present study applied ISO 18593-based cloth sampling and reports results as CFU/cloth. These methodological differences influence microbial recovery efficiency and therefore limit direct numerical comparison. For this reason, comparisons with the literature are interpreted cautiously and focus on qualitative trends rather than absolute values.” Lines 453 – 459

Conclusions

Comment: Lines 456-457: It is concluded that there are significant increases in the TVC and Enterobacteriaceae between 2024 and 2025. However, it is unclear whether these figures can be considered high.

Response: The intention of this sentence was to indicate a year-to-year increase, not to imply that the absolute microbial levels were high.

However, the authors acknowledge that the original manuscript did not specify the criteria used to classify TVC levels, and we agree that this information was missing. To address this, we have now added a detailed explanation in the Materials and Methods section, where we justify the choice of thresholds and describe how reference limits for food-contact surfaces were adapted to CFU/cloth values. Specifically, we now state: “To evaluate surface contamination, we applied the criteria defined by Düven et al. (2021) [29], with <5 CFU/cm² (≈0.7 Log₁₀) considered acceptable, 5–25 CFU/cm² (0.7–1.4 Log₁₀) borderline, and >25 CFU/cm² (>1.4 Log₁₀) unsatisfactory. To enable direct com-parison with our results, which are expressed as CFU per cloth, the area-based limits were converted to CFU/cloth by approximating the sampled surface area. Based on measurements from a representative sample of domestic refrigerators in Portugal, a standard shelf area of approximately 1500 cm² was assumed. Multiplying each value by this surface area resulted in the following operational limits for this study: <7.5 × 10³ CFU/cloth (<3.88 Log₁₀) classified as acceptable, 7.5 × 10³–3.75 × 10⁴ CFU/cloth (3.88–4.57 Log₁₀) as borderline, and >3.75 × 10⁴ CFU/cloth (>4.57 Log₁₀) as unsatisfactory.” Lines 207-216

These additions clarify the criteria used to define high TVC values and strengthen the interpretation of our findings.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have attempted to address the issues raised in the previous review but in doing so revealed a further weakness. Specifically, the egg sampling was part of a separate study to determine the baseline prevalence of Salmonella and Campylobacter. This part of the study is out of place in the current study, which was focused on the temperature and sanitation status of domestic refrigerators. Apart from the temperature profiles, there is little that can be concluded from the microbial counts due to the wide range of confounding factors. Overall, it is unclear how the research contributes to the area.

Reviewer 3 Report

Comments and Suggestions for Authors

I appreciate for the amendments made to the article in response to the recommendations provided during the review process.
I believe that it now meets the requiredments for its publication in the journal Hygiene.
I encourage you to continue your research in this area and wish you the best of luck with your future investigations.