Microbiological Risk Assessment Research Articles

Integrating quantitative microbiological risk assessment and disability-adjusted life years to evaluate the effects of urbanization on health risks for river recreationists

Urban rivers provide an excellent opportunity for water recreation. This study probabilistically assessed health risks associated with water recreation in urban rivers in the Bitan Scenic Area, Taiwan, by employing quantitative microbial risk assessment and disability-adjusted life years (DALYs). Moreover, the effects of urbanization on the health risks of river recreation induced by waterborne pathogenic Escherichia coli (E. coli) were investigated. First, data on river E. coli levels were collected in both the Bitan Scenic Area and the upstream river section, and model parameters were obtained through a questionnaire administered to river recreationists. Monte Carlo simulation was then employed to address parameter uncertainty. Finally, DALYs were calculated to quantify the cumulative effects in terms of potential life lost and years lived with disability. The results indicated that the 90 % confidence intervals for the disease burden (DB) were 0.2–74.1 × 10−6, 0.01–94.0 × 10−6, and 0.3–128.9 × 10−6 DALY per person per year (pppy) for canoeing, swimming, and fishing, respectively, in the Bitan Scenic Area. Furthermore, urbanization near the Bitan Scenic Area approximately doubled the DB risks to river recreationists in upstream rural areas. At the 95th percentile, the DB risks exceeded the tolerances recommended by the World Health Organization (1 × 10−6) or U.S. Environmental Protection Agency (1 × 10−4). The findings suggest that the simultaneous implementation of effluent sewer systems and best management practices can reduce health risks to river recreationists by at least half, reducing the DALY levels below 1 × 10−4 or even 1 × 10−5 pppy.

A secondary model for the effect of pH on the variability in growth fitness of Listeria innocua strains

Variability in microbial growth is a keystone of modern Quantitative Microbiological Risk Assessment (QMRA). However, there are still significant knowledge gaps on how to model variability, with the most common assumption being that variability is constant. This is implemented by an error term (with constant variance) added on top of the secondary growth model (for the square root of the growth rate). However, this may go against microbial ecology principles, where differences in growth fitness among bacterial strains would be more prominent in the vicinity of the growth limits than at optimal growth conditions.This study coins the term “secondary models for variability”, evaluating whether they should be considered in QMRA instead of the constant strain variability hypothesis. For this, 21 strains of Listeria innocua were used as case study, estimating their growth rate by the two-fold dilution method at pH between 5 and 10. Estimates of between-strain variability and experimental uncertainty were obtained for each pH using mixed-effects models, showing the lowest variability at optimal growth conditions, increasing towards the growth limits. Nonetheless, the experimental uncertainty also increased towards the extremes, evidencing the need to analyze both sources of variance independently. A secondary model was thus proposed, relating strain variability and pH conditions. Although the modelling approach certainly has some limitations that would need further experimental validation, it is an important step towards improving the description of variability in QMRA, being the first model of this type in the field.

Estimation and evaluation of the risks of protozoa infections associated to the water from a treatment plant in southern Brazil using the Quantitative Microbiological Risk Assessment Methodology (QMRA).

In this study, the Quantitative Microbial Risk Assessment (QMRA) methodology was applied to estimate the annual risk of Giardia and Cryptosporidium infection associated with a water treatment plant in southern Brazil. The efficiency of the treatment plant in removing protozoa and the effectiveness of the Brazilian legislation on microbiological protection were evaluated, emphasizing the relevance of implementing the QMRA in this context. Two distinct approaches were employed to estimate the mechanical removal of protozoa:The definitions provided by the United States Environmental Protection Agency (USEPA), and the model proposed by Neminski and Ongerth.Although the raw water collected had a higher concentration of Giardia cysts than Cryptosporidium oocysts, the estimated values for the annual risk of infection were significantly higher for Cryptosporidium than for Giardia. From a general perspective, the risk values of protozoa infection were either below or very near the limit set by the World Health Organization (WHO). In contrast, all the risk values of Cryptosporidium infection exceeded the threshold established by theUSEPA. Ultimately, it was concluded that the implementation of the QMRA methodology should be considered by the Brazilian authorities, as the requirements and guidelines provided by the Brazilian legislation proved to be insufficient to guarantee the microbiological safety of drinking water. In this context, the QMRA application can effectively contribute to the prevention and investigation of outbreaks of waterborne disease.

Identification of cost-effective biosecurity measures to reduce Salmonella along the pork production chain.

The continued occurrence of salmonellosis cases in Europe attributed to the consumption of pork products highlights the importance of identifying cost-effective interventions. Certain biosecurity measures (BSMs) may be effective in reducing the prevalence of specific pathogens along the pork production chain and their presence in food products. The objective of this study was to identify pathogen-specific, cost-effective BSMs to reduce Salmonella at different stages of the pork production chain in two European countries - Austria (AT) and the United Kingdom (UK). For this purpose, a cost-benefit analysis was conducted based on the epidemiological output of an established quantitative microbiological risk assessment that simulated the implementation effect of the BSMs based on their risk ratios. For each of the BSMs, the associated costs and benefits were assessed individually and country-specifically. For both AT and UK, nine different BSMs were evaluated assuming a countrywide implementation rate of 100%. The results showed that four BSMs were cost-effective (benefit-cost ratio > 1) for AT and five for the UK. The uncertainty regarding the cost-effectiveness of the BSMs resulted from the variability of individual risk ratios, and the variability of benefits associated with the implementation of the BSMs. The low number of cost-effective BSMs highlights the need for holistic risk-based models and economic assessments. To increase the willingness to implement BSMs and maximize the benefits for stakeholders, who carry the majority of the implementation costs, epidemiological assessments of BSM effectiveness should consider the impact on several relevant pathogens simultaneously.

Addition of selected cereal grains as non-dairy ingredients to dairy products: A microbiological risk assessment approach

The addition of cereal grains to dairy products in the dairy industry has the potential to contaminate final products with pathogenic and spoilage microorganisms. In this study, the microbial risks involved in the addition of cereal grains to dairy products with low, intermediate, and high-water activity/moisture content were assessed using a semi-quantitative risk assessment method.The results showed that the most critical microbiological hazard in the selected cereal grains is Bacillus cereus (B. cereus) due to its ability to form spores and persist in cereal grains. The addition of cereal grains to dairy products with high water activity/moisture content such as liquid breakfast products were found to pose the highest theoretical risk, and processing mitigations, such as UHT, would need to be implemented. The results of this study have identified some knowledge gaps in conducting risk assessments and have also provided background information about the microbial risks involved in the addition of cereal grains to dairy products.

Microbiological risk assessment and resistome analysis from shotgun metagenomics of bovine colostrum microbiome

Colostrum is known for its nutraceutical qualities, probiotic attributes, and health benefits. The aim of this study was to profile colostrum microbiome from bovine in rural sites of a developing country. The focus was on microbiological safety assessments and antimicrobial resistance, taking into account the risks linked with the consumption of raw colostrum. Shotgun sequencing was employed to analyze microbiome in raw buffalo and cow colostrum. Alpha and beta diversity analyses revealed increased inter and intra-variability within colostrum samples' microbiome from both livestock species. The colostrum microbiome was mainly comprised of bacteria, with over 90% abundance, whereas fungi and viruses were found in minor abundance. Known probiotic species, such as Leuconostoc mesenteroides, Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus paracasei, were found in the colostrum samples. A relatively higher number of pathogenic and opportunistic pathogenic bacteria were identified in colostrum from both animals, including clinically significant bacteria like Clostridium botulinum, Pseudomonas aeruginosa, Escherichia coli, and Listeria monocytogenes. Binning retrieved 11 high-quality metagenome-assembled genomes (MAGs), with three MAGs potentially representing novel species from the genera Psychrobacter and Pantoea. Notably, 175 antimicrobial resistance genes (ARGs) and variants were detected, with 55 of them common to both buffalo and cow colostrum metagenomes. These ARGs confer resistance against aminoglycoside, fluoroquinolone, tetracycline, sulfonamide, and peptide antibiotics. In conclusion, this study describes a thorough overview of microbial communities in buffalo and cow colostrum samples. It emphasizes the importance of hygienic processing and pasteurization in minimizing the potential transmission of harmful microorganisms linked to the consumption of colostrum.

Benzalkonium chloride disinfectant residues stimulate biofilm formation and increase survival of Vibrio bacterial pathogens.

Vibrio spp. are opportunistic human and animal pathogens found ubiquitously in marine environments. Globally, there is a predicted rise in the prevalence of Vibrio spp. due to increasing ocean temperatures, which carries significant implications for public health and the seafood industry. Consequently, there is an urgent need for enhanced strategies to control Vibrio spp. and prevent contamination, particularly in aquaculture and seafood processing facilities. Presently, these industries employ various disinfectants, including benzalkonium chloride (BAC), as part of their management strategies. While higher concentrations of BAC may be effective against these pathogens, inadequate rinsing post-disinfection could result in residual concentrations of BAC in the surrounding environment. This study aimed to investigate the adaptation and survival of Vibrio spp. exposed to varying concentrations of BAC residues. Results revealed that Vibrio bacteria, when exposed, exhibited a phenotypic adaptation characterized by an increase in biofilm biomass. Importantly, this effect was found to be strain-specific rather than species-specific. Exposure to BAC residues induced physiological changes in Vibrio biofilms, leading to an increase in the number of injured and alive cells within the biofilm. The exact nature of the "injured" bacteria remains unclear, but it is postulated that BAC might heighten the risk of viable but non-culturable (VBNC) bacteria development. These VBNC bacteria pose a significant threat, especially since they cannot be detected using the standard culture-based methods commonly employed for microbiological risk assessment in aquaculture and seafood industries. The undetected presence of VBNC bacteria could result in recurrent contamination events and subsequent disease outbreaks. This study provides evidence regarding the role of c-di-GMP signaling pathways in Vibrio adaptation mechanisms and suggests that c-di-GMP mediated repression is a potential avenue for further research. The findings underscore that the misuse and overuse of BAC may increase the risk of biofilm development and bacterial survival within the seafood processing chain.

Spice-Induced Metal Contamination and Microbiological Risk Assessment of Instant Noodles Prepared for Human Consumption.

With the rising worries of potential metal contamination in cooked noodles, this study aimed at unravelling, for the first time, the possible source of metal contamination in cooked noodles. Noodles cooked with full spices (CWFS), cooked with half spices (CWHS), cooked with quarter spices (CWQS), and cooked with no spices (CWNS: control) and their spices were analysed for metals using inductively coupled plasma-atomic emission spectrometry. The microbiological quality of the noodles was also evaluated. Metal concentrations in cooked noodles varied with spice quantity. Noodles CWFS had the highest significant (p < 0.05) concentration of Pb (0.36 ± 0.12mg/kg), Ni (1.05 ± 0.01mg/kg), Cd (0.07 ± 0.04mg/kg), Co (0.02 ± 0.002mg/kg), and Na (9.45 ± 0.04mg/kg), compared to the control (CWNS). The mean Pb and Ni of spice and cooked noodles were above the WHO acceptable limits for food and could be harmful to consumers. Pearson's correlation and PCA showed that packed noodle spices introduced metals into the cooked noodles. Although the hazard indices (adults and children) in all noodle's groups were less than 1, children still had the potential to contract cancer from Ni exposure because the carcinogenic risk values of CWQS (2.87 × 10-4), CWHS (3.03 × 10-4), and CWFS (3.21 × 10-4) were greater than 10-4. Microbiological analysis revealed the presence of potential pathogens that showed multidrug resistance and the ability to elaborate protease and amylase enzymes. Given the impending chronic health risks inherent in processed noodles, consistent consumption should be avoided.

A review on the application of UV‐C treatment on food and food surfaces: association with food microbiology, predictive microbiology and quantitative microbial risk assessment

SummaryThe so‐called emerging technologies have gained prominence in the field of food processing, compared to conventional heat treatments, due to technological, sensorial and microbiological safety benefits. Ultraviolet radiation (UV), more specifically UV‐C radiation, is inexpensive and has been one of the most studied technologies. This work seeks to summarise the application of UV‐C treatment, contextualising its efficiency in the inactivation of microorganisms in different food matrices and surfaces associated with food handling and allowing the visualisation of trends in the combination of UV‐C radiation with other technologies. Still, the technology is related to quantitative tools associated with food microbiology. Factors related to processing parameters, characteristics of the microbial population and food properties have a direct influence on the effectiveness of UV‐C technology. The UV‐C treatment parameters must be rigorously defined, considering the particularities of each food matrix, so that undesirable identity or sensory changes do not occur. The application of mathematical tools, such as predictive microbiology and quantitative microbial risk assessment, is fundamental in the planning and definition of processing parameters for UV‐C radiation technology.

Temperature status of domestic refrigerators and its effect on the risk of listeriosis from ready-to-eat (RTE) cooked meat products

Inadequate domestic refrigeration is frequently cited as a factor that contributes to foodborne poisoning and infection, and consumer behaviour in this regard can vary largely. This study provides insight into the temperature profiles of domestic refrigerators in the Netherlands and the impact on the number of listeriosis cases related to ready-to-eat (RTE) cooked meat products. A survey was conducted among Dutch consumers (n = 1020) to assess their knowledge and behaviour related to refrigerators. Out of these participants, 534 measured their refrigerator's temperature, revealing an average temperature of 5.7 °C (standard deviation (SD) of 2.2 °C) with a maximum of 17 °C. Elderly people (65 years and older) had refrigerators with temperatures that were on average 0.6 °C higher than those of younger people (35 years or younger). The 24-hour temperature profiles of an additional set of actively surveyed refrigerators (n = 50) showed that the temperature measured on the upper shelf was significantly higher (mean 7.7 °C, SD 2.7 °C) than the temperature measured on the bottom shelf (5.7 °C, SD 2.1 °C). Quantitative Microbiological Risk Assessment (QMRA) predicted that the primary factors contributing to the risk of listeriosis were the initial concentration and the time and temperature during household storage. Scenario analysis revealed that storing opened RTE cooked meat products at home for either <7 days or at temperatures <7 °C resulted in a significant reduction of over 80 % in predicted illness cases. Among all illness cases, the elderly represented nearly 90 %. When assessing the impact of the disease in terms of Years of Life Lost (YLL), the contribution of the elderly was 59 %. Targeted communication, particularly directed towards the elderly, on the importance of storing RTE cooked meat products at the recommended temperature on the bottom or middle shelf as well as consuming within two to three days after opening, holds the potential to significantly reduce the number of cases.

The Characteristics of Bacillus cereus Group Strains Isolated from Permafrost in Yakutia for Assessment of Microbiological Risks during Climate Change

The Characteristics of Bacillus cereus Group Strains Isolated from Permafrost in Yakutia for Assessment of Microbiological Risks during Climate Change

Development of a quantitative microbiological spoilage risk assessment (QMSRA) model for cooked ham sliced at retail

A quantitative microbiological spoilage risk assessment model (QMSRA) for cooked ham sliced at retail was developed based on a stochastic growth model for lactic acid bacteria (LAB), which are considered as the specific spoilage organisms (SSO), and a “spoilage-response” relationship characterizing the variability in consumer's perception of spoilage. In a simulation involving 10,000 cooked ham purchases, the QMSRA model predicted a median of zero spoilage events for up to 4.5 days of storage. After storage times of 5 and 6 days, the model predicted 1,790 and 8,570 spoilage events, respectively. A sensitivity analysis showed that domestic storage temperature was the most significant factor affecting LAB concentration in cooked ham, followed by the LAB contamination level at slicing. A scenario analysis was performed testing better temperature control of consumer's refrigerators, better hygiene conditions during slicing and a combination of the two strategies. Among the tested scenarios, a 2 log reduction in the LAB contamination at slicing combined with a 2 °C decrease in domestic storage temperature resulted in zero risk of spoilage for up to 12 days of storage. The QMSRA model developed in the present study can be a useful tool for quality management decisions.

Characterization of the Virulency of Vibrio spp. in Giant Tiger Shrimp (Penaeus monodon) from Peninsular Malaysia

Nowadays, bacterial diseases caused by Vibrio spp. pose a significant obstacle to aquaculture growth, affecting socioeconomic development. The presence of pathogenic Vibrio spp. are gaining attention due to widespread mortality in fish and shellfish worldwide. This study investigates the distribution of virulence genes and their pathogenicity by experimentally infecting a healthy group of cultured shrimp, specifically Penaeus monodon. A total of 225 Vibrio spp. isolated from 210 cultured shrimp from various geographical regions in Peninsular Malaysia were examined, resulting in 13 Vibrio spp. being identified and characterized. According to virulence gene assessment, only eight of the 11 virulence genes were detected in the isolates, including chiA (89%), flaC (74%), tlh (72%), toxR (70%), luxR (40%), pirA (11%), pirB (11%), and hlyA (0.4%). Other virulence genes, such as tdh, trh, and ctxA, were not found in any of the isolates (0%). Then, three Vibrio isolates, including V. parahaemolyticus S12-3, V. campbellii S10-4, and V. rotiferianus S24-4, were selected for pathogenicity test based on highest number of virulence genes in each species. These species were subjected to immersion challenge test against postlarvae (PL) P. monodon approximately PL-20 with three different concentrations of 1 × 106, 1 × 107, and 1 × 108 CFU/mL. The V. parahaemolyticus S12-3 was determined to have lethal concentration 50 (LC50) 1 × 105 CFU/mL after 168 hr of exposure. The immersion challenge test resulted in a pale hepatopancreas and empty guts in P. monodon. Histopathological examination showed sloughing of epithelial cells in the hepatopancreatic tubule, hemocytic infiltration, massive vacuolation, and loss of hepatopancreatic tubule structure. Overall, the study demonstrated the virulence and pathogenicity of V. parahaemolyticus S12-3 toward P. monodon, which could be associated with its virulence properties. Furthermore, the current findings will be useful in developing microbiological risk assessments for shrimp farming and aquaculture management.

“Innovative high pressure/high temperature, multi-sensing bioreactors system for microbial risk assessment in underground hydrogen storage”

This study addresses the microbial risks associated with Underground Hydrogen Storage (UHS), a critical component in the transition towards renewable energy systems, by employing an innovative multi-reactor system (Bio-xplorer) to simulate UHS conditions in two Italian reservoirs. The microbiological risk assessment (MRA) of Reservoir A and B was evaluated by subjecting them to gas mixtures of 10 % H2 and 90 % CH4, and 99 % H2 and 1 % CO2, respectively.In Reservoir A, the stability of pressure and temperature, the negligible optical density, and lack of microbial metabolites suggested a low risk of microbial activation. Molecular analyses confirmed the absence of sulphate-reducing bacteria (SRB) and limited growth of hydrogenotrophic methanogens (HM). Similarly, in Reservoir B, the absence of SRB and limited occurrence of HM indicated a low microbiological risk. Overall, the present work supports the safe and efficient implementation of UHS, a promising mitigation technique for climate change, using an innovative tool for MRA.

Identifying ARG-carrying bacteriophages in a lake replenished by reclaimed water using deep learning techniques

As important mobile genetic elements, phages support the spread of antibiotic resistance genes (ARGs). Previous analyses of metaviromes or metagenome-assembled genomes (MAGs) failed to assess the extent of ARGs transferred by phages, particularly in the generation of antibiotic pathogens. Therefore, we have developed a bioinformatic pipeline that utilizes deep learning techniques to identify ARG-carrying phages and predict their hosts, with a special focus on pathogens. Using this method, we discovered that the predominant types of ARGs carried by temperate phages in a typical landscape lake, which is fully replenished by reclaimed water, were related to multidrug resistance and β-lactam antibiotics. MAGs containing virulent factors (VFs) were predicted to serve as hosts for these ARG-carrying phages, which suggests that the phages may have the potential to transfer ARGs. In silico analysis showed a significant positive correlation between temperate phages and host pathogens (R = 0.503, p < 0.001), which was later confirmed by qPCR. Interestingly, these MAGs were found to be more abundant than those containing both ARGs and VFs, especially in December and March. Seasonal variations were observed in the abundance of phages harboring ARGs (from 5.62 % to 21.02 %) and chromosomes harboring ARGs (from 18.01 % to 30.94 %). In contrast, the abundance of plasmids harboring ARGs remained unchanged. In summary, this study leverages deep learning to analyze phage-transferred ARGs and demonstrates an alternative method to track the production of potential antibiotic-resistant pathogens by metagenomics that can be extended to microbiological risk assessment.

Training in modern statistical methodologies and software tools for the definition and analysis of (stochastic) quantitative microbial risk assessment models with a comparison between the Hungarian and Spanish food supply chains.

Human pathogenic Salmonella enterica strains have been infecting people since historical times. The original human pathogens, typhoid Salmonella strains (e.g. S. Typhi) played a huge role in the previous centuries but nowadays in the developed world the number of cases or outbreaks caused by these serotypes deceased due to the development of personal and public hygiene. Nowadays in these regions the animal-borne zoonotic serotypes (e.g. S. Enteritidis) became more important because of their high prevalence in intensive animal husbandry. But these bacteria can also appear in fruits and vegetables. The fellow joined the scientific work of the Polytechnic University of Cartagena, Spain about the safety of plant-based products, where he could gain experience in microbiological laboratory exercises and theoretical calculations of statistics and modelling. The activities in the laboratory were part of the research lines already established at the host institution, being based on the protocols they have already implemented. Nonetheless, the fellow had the opportunity to design his own experiment, do the experimental work required and analysed the data within the context of a qualitative microbiological risk assessment. The main focus was on the heat resistance of two strains of zoonotic Salmonella spp. at different temperatures. Experiments were done using a reference strain and an extremely resistant variant to evaluate this rare phenotype. The experiments were executed using a Mastia thermoresistometer, a device patented by the host institution that provides more control when studying thermal treatments than traditional methods. The data was analysed using the principles of predictive microbiology, using the D-value as an estimate of heat resistance that provides insight into the bacterial behaviour. For this, the fellow used the bioinactivation software, developed within the host group. Through the work and results the fellow learned the principles of quantitative microbiological risk assessment (QMRA) and predictive microbiology, which was the aim for the EU-FORA programme.

Characteristics of &lt;i&gt;Bacillus cereus&lt;/i&gt; complex Group Strains Isolated from Permafrost in Yakutia for Assessment of Microbiological Risks During Climate Change

Characteristics of &lt;i&gt;Bacillus cereus&lt;/i&gt; complex Group Strains Isolated from Permafrost in Yakutia for Assessment of Microbiological Risks During Climate Change

Estimating the rejection risk of Indonesian shrimp exports from Salmonella spp. contamination using the food safety objective approach

Contamination by non-typhoidal Salmonella spp. in aquacultured shrimp poses a risk for human health. In Indonesia, the presence of the pathogen in shrimp raises a major concern for international trade because shrimp is one of the important exported fishery commodities. The aim of this study was to use food safety objective approach in combination with a quantitative microbiological risk assessment to determine the contamination of Salmonella spp. in raw materials, to estimate the risk of export rejection and to identify the contributing factors of export rejections. The prevalence and concentration of Salmonella spp. in shrimp based on real-time polymerase chain reaction of 7.02% and 0.72–1.46 log CFU/gr (mean: 1.00 ± 0.28 log CFU/g), respectively, were used to develop an exposure model. The exposure model was used to estimate the level of Salmonella spp. in frozen shrimp at the border of an export destination country, which was ca. −6.02 to −2.07 (mean −3.73 ± 0.73) log CFU/g. The value was compared to the food safety objective value determined from the required microbiological criterion of Salmonella spp. of n = 30, c = 0, and m = negative/25 g. As a result, the risk estimate for rejection was 27 rejections per 100,000 exports. The Salmonella spp. contamination level in raw materials and freezing technology were the primary factors contributing to the rejections. Moreover, maintaining the quality control of raw materials purchased or controlled quick freezing may be the mitigation formulae of export rejection of frozen shrimp due to Salmonella spp.

Annual report of the Scientific Network on Microbiological Risk Assessment 2023

Annual report of the Scientific Network on Microbiological Risk Assessment 2023

Unveiling Fresh-Cut Lettuce Processing in Argentine Industries: Evaluating Salmonella Levels Using Predictive Microbiology Models

A survey was performed to gather information on the processing steps, conditions, and practices employed by industries processing ready-to-eat (RTE) leafy vegetables in Argentina. A total of seven industries participated in the survey. A cluster analysis of the data obtained was performed to identify homogeneous groups among the participating industries. The data collected were used as inputs of two predictive microbiology models to estimate Salmonella concentrations after chlorine washing, during storage and distribution of final products, and to rank the different practices according to the final estimated Salmonella levels. Six different clusters were identified by evaluating the parameters, methods, and controls applied in each processing step, evidencing a great variability among industries. The disinfectant agent applied by all participating industries was sodium hypochlorite, though concentrations and application times differed among industries from 50 to 200 ppm for 30 to 110 s. Simulations using predictive models indicated that the reductions in Salmonella in RTE leafy vegetables would vary in the range of 1.70–2.95 log CFU/g during chlorine-washing depending on chlorine concentrations applied, washing times, and vegetable cutting size, which varied from 9 to 16 cm2 among industries. Moreover, Salmonella would be able to grow in RTE leafy vegetables during storage and distribution, achieving levels of up to 2 log CFU/g, considering the storage and transportation temperatures and times reported by the industries, which vary from 4 to 14 °C and from 18 to 30 h. These results could be used to prioritize risk-based sampling programs by Food Official Control or determine more adequate process parameters to mitigate Salmonella in RTE leafy vegetables. Additionally, the information gathered in this study is useful for microbiological risk assessments.