Quantitative microbial risk assessment of Salmonella in fresh chicken patties.

Quantitative microbial risk assessment (QMRA) has witnessed rapid development within the context of food safety in recent years. As a means of contributing to these advancements, a QMRA for Salmonella spp. in fresh chicken patties for the general European Union (EU) population was developed. A two-dimensional (Second Order) Monte-Carlo simulation method was used for separating variability and uncertainty of model's parameters. The stages of industrial processing, retail storage, domestic storage, and cooking in the domestic environment were considered in the exposure assessment. For hazard characterization, a dose-response model was developed by combining 8 published dose-response models using a Pert distribution for describing uncertainty. The QMRA model predicted a mean probability of illness of 1.19*10-4 (5.28*10-5 - 3.57*10-4 95 % C.I.), and a mean annual number of illnesses per 100,000 people of 2.13 (0.96 - 6.59 95 % C.I.). Moreover, sensitivity analysis was performed, and variability in cooking preferences was found to be the most influential model parameter (r = -0.39), followed by dose-response related variability (r = 0.22), and variability in the concentration of Salmonella spp. at the time of introduction at the processing facility (r = 0.11). Various mitigation strategy scenarios were tested, from which, "increasing the internal temperature of cooking" and "decreasing shelf life" were estimated to be the most effective in reducing the predicted risk of illness. Salmonella-related illnesses exhibit particularly high severity, making them some of the most prominent zoonotic diseases in the EU. Regular monitoring of this hazard in order to further highlight its related parameters and causes is a necessary procedure. This study not only provides an updated assessment of Salmonella spp. risk associated with chicken patties, but also facilitates the identification of crucial targets for scientific investigation and implementation of real-world intervention strategies.

Analysis of the assessment of antimicrobial susceptibility. Non-typhoid Salmonella in meat and meat products as model (systematic review).

BACKGROUND: The scientific publications of antimicrobial susceptibilities and resistance must be precise, with interpretations adjusted to the standard. In this frame, knowledge of antimicrobial resistance is fundamental in pathogenic microorganisms such as Salmonella spp., known for many annual deaths worldwide. The objective of this work was to compare the interpretation of standards, the concentrations, and the breakpoints, to study antimicrobial resistance in Non-Typhoidal Salmonella (NTS) isolated from beef, pork, and chicken meat, meat products, and propose additional considerations that improve the use and usefulness of published results. RESULTS: After refining the search based on meeting the inclusion and exclusion criteria, 48 papers were selected. In 33 (68.8%) of them, the disc diffusion method was used, in 11 (22.9%) the MIC determination method, and in 4 (8.33%) were used both. In 24 (50%) of the articles, the selection of a different (correct) standard could have had an impact on the interpretation of antimicrobial susceptibility, which observed when considering three scenarios, i) comparison between the year of the isolation versus the implemented standard, ii) comparison between the year of submission versus implemented standard and iii) comparison between the year of publication versus implemented standard. CONCLUSIONS: The most frequent scenario was the inadequate selection of standards, indicating that some studies had not ensured that applied standards kept in line with the date of isolation, date of publication and interpretation of susceptibilities. We proposed 2 years for standards use for resistance and multi-resistance interpretations. On the other hand, we invite researchers to publish their results in the shortest possible time, and editors and reviewers of scientific journals to prioritise these types of studies and verify the correspondence between the standard cited and the one used and the one to be taken into account.

Antibiotic resistance and biofilm formation ability of Salmonella serotypes isolated from beef, mutton, and meat contact surfaces at retail.

In this study, Salmonella isolates recovered from meat (beef and mutton) and meat contact surfaces at retail were investigated to determine their serotype, antibiotic resistance, and biofilm formation ability. Salmonella was found in 29 (24.17%) samples out of 120 samples including 14/50 (28%) of beef, 10/40 (25%) of mutton, and 5/30 (16.67%) of meat contact surfaces. Seven isolates were identified as S. Enteritidis, three as S. Typhimurium, and two as S. Typhi, while the rest of the isolates were considered as other Salmonella spp. All of the isolates were resistant to at least one antimicrobial agent and 48.27% of them were identified as multidrug-resistant (MDR) Salmonella. All (100%) of meat contact surfaces isolates, 42.8% of beef isolates, and 30% of mutton isolates were found to be MDR Salmonella. Resistance to nalidixic acid (100%), tetracycline (79.3%), and sulphamethoxazole/trimethoprim (44.8%) were observed. The gyrA gene was detected in 19 of 29 isolates, but tetA was found in one isolate. All of the serotypes were able to form biofilm (75.86 % moderate and 24.14 % strong) and S. Enteritidis was the strongest biofilm producer. The findings indicated that the majority of Salmonella isolates in this study were MDR and biofilm producer. Then, safety measures such as cleaning and disinfection must be taken to control Salmonella and promote public health. PRACTICAL APPLICATION: The present study provides useful information on the prevalence of Salmonella serotypes in meat and meat contact surfaces and their antibiotic resistance patterns as well as biofilm formation capacities. Improving hygiene practices in livestock, slaughterhouses, and at retails may reduce the risk of meat contamination to Salmonella. Meanwhile, high levels of antibiotic resistance in Salmonella isolates emphasized on the improper use of antibiotics.

Assessment of Pediococcus acidilactici ATCC 8042 as potential Salmonella surrogate for thermal treatments of toasted oats cereal and peanut butter.

The control of Salmonella in low water activity foods poses a challenge for the food industry because of its thermal resistance. The use of surrogate bacteria in a food plant is considered a critical component to validate processing steps. The objective of this study was to evaluate the use of Pediococcus acidilactici ATCC 8042, a generally recognized as safe bacterium (GRAS), as potential surrogate for Salmonella in commercial toasted oats cereal (TOC) and peanut butter. P. acidilactici was compared to a five-serovar cocktail of Salmonella and Enterococcus faecium NRRL-B2354, separately. Cultures were inoculated into TOC and thermal kinetic parameters (δ, ß) were determined at 80, 85, 90, and 95 °C using the Weibull model. In peanut butter, δ and ß parameters were obtained at 63, 68, 73, and 77 °C. In TOC, the δ values (initial decimal reduction time) of P. acidilactici were 63 and 7 min at 80 and 95 °C, respectively, and at all four temperatures they were not significantly different from δ values of E. faecium. The δ value of Salmonella at 80 °C (139 min) was two-fold greater than the other two bacteria's values (p < 0.05). In peanut butter, δ values of P. acidilactici ranged from 31 min at 63 °C to 2.6 min at 77 °C, and at all temperatures they were not significantly different from E. faecium's δ values. In peanut butter, all Salmonella cocktail's δ values were significantly smaller than P. acidilactici's with values of 2 min at 63 °C and 0.4 min at 77 °C. These results indicated that P. acidilactici was as heat tolerant as E. faecium in these food matrices. However, the thermal inactivation kinetic parameters suggested that P. acidilactici can only be considered a Salmonella surrogate in TOC at temperatures above 85 °C. Because of its greater thermal tolerance in peanut butter, P. acidilactici may be used as Salmonella surrogate if an additional safety factor is recommended.

Spotlight on avian pathology: red mite, a serious emergent problem in layer hens.

Dermanyssus gallinae, the poultry red mite, is currently the most important ectoparasite of the egg laying industry worldwide with an expanding global prevalence. As a blood-feeder, it causes anaemia and severe welfare issues to the hens and it is a major cause of economic losses. It is also a vector for Salmonella species, avian influenza and potentially for other vector-borne pathogens. Paradoxically, there is a notable lack of funding for research into poultry red mite and an urgent need for effective and safe control strategies, sustainable therapies, prophylactics and integrated pest management.

Salmonella and Campylobacter biofilm formation: a comparative assessment from farm to fork.

It takes several steps to bring food from the farm to the fork (dining table), and contamination with food-borne pathogens can occur at any point in the process. Campylobacter spp. and Salmonella spp. are the main microorganisms responsible for foodborne disease in the EU. These two pathogens are able to persist throughout the food supply chain thanks to their ability to form biofilms. Owing to the high prevalence of Salmonella and especially of Campylobacter in the food supply chain and the huge efforts of food authorities to reduce these levels, it is of great importance to fully understand their mechanisms of persistence. Diverse studies have evaluated the biofilm-forming capacity of foodborne pathogens isolated at different steps of food production. Nonetheless, the principal obstacle of these studies is to reproduce the real conditions that microorganisms encounter in the food supply chain. While there are a wide number of Salmonella biofilm studies, information on Campylobacter biofilms is still limited. A comparison between the two microorganisms could help to develop new research in the field of Campylobacter biofilms. Therefore, this review evaluates relevant work in the field of Salmonella and Campylobacter biofilms and the applicability of the data obtained from these studies to real working conditions. © 2018 Society of Chemical Industry.

Modeling the survival kinetics of Salmonella in tree nuts for use in risk assessment.

Salmonella has been shown to survive in tree nuts over long periods of time. This survival capacity and its variability are key elements for risk assessment of Salmonella in tree nuts. The aim of this study was to develop a mathematical model to predict survival of Salmonella in tree nuts at ambient storage temperatures that considers variability and uncertainty separately and can easily be incorporated into a risk assessment model. Data on Salmonella survival on raw almonds, pecans, pistachios and walnuts were collected from the peer reviewed literature. The Weibull model was chosen as the baseline model and various fixed effect and mixed effect models were fit to the data. The best model identified through statistical analysis testing was then used to develop a hierarchical Bayesian model. Salmonella in tree nuts showed slow declines at temperatures ranging from 21°C to 24°C. A high degree of variability in survival was observed across tree nut studies reported in the literature. Statistical analysis results indicated that the best applicable model was a mixed effect model that included a fixed and random variation of δ per tree nut (which is the time it takes for the first log10 reduction) and a fixed variation of ρ per tree nut (parameter which defines the shape of the curve). Higher estimated survival rates (δ) were obtained for Salmonella on pistachios, followed in decreasing order by pecans, almonds and walnuts. The posterior distributions obtained from Bayesian inference were used to estimate the variability in the log10 decrease levels in survival for each tree nut, and the uncertainty of these estimates. These modeled uncertainty and variability distributions of the estimates can be used to obtain a complete exposure assessment of Salmonella in tree nuts when including time-temperature parameters for storage and consumption data. The statistical approach presented in this study may be applied to any studies that aim to develop predictive models to be implemented in a probabilistic exposure assessment or a quantitative microbial risk assessment.

Assessment of potentially probiotic properties of Lactobacillus strains isolated from chickens.

This study was performed in order to isolate lactobacilli from chicken droppings and to select strains with the most promising probiotic properties. Lactobacillus strains were isolated from a flock of healthy laying hens. The first selection criterion was the ability to inhibit the growth of Salmonella Enteritidis. Then the tolerance to low pH and bile salt, the ability to coaggregate with pathogenic bacteria and hydrogen peroxide production were evaluated. Four isolates showing the best antagonistic activity against Salmonella Enetritidis were selected for further research. All isolates tested tolerated low pH and bile salt, likewise all produced hydrogen peroxide. They efficiently coaggregated with C. perfringens and relatively less with E. coli. Isolate 03'04 displayed above-average results in all criteria, thus it is considered as a potential probiotic for chickens, and will be further evaluated for health promoting effect in animals. The results presented in this study confirm the strain specific probiotic properties and prove the probiotic potential of isolate 03'04. Strong antagonistic properties against C. perfringens exhibited by certain Lactobacillus strains indicate the possibility to use them as a component of probiotic supplement in necrotic enteritis of poultry.

Using the mini-VIDAS(®) Easy Salmonella protocol to assess contamination in transitional and coastal waters.

Classical methodologies for Salmonella detection may be too long in time to assure public safety. Presently, one of the fastest assays for Salmonella detection using the mini-VIDAS(®) system is the Easy Salmonella protocol. This assay, developed for food matrixes analysis, was here assessed for the applicability on the detection of these bacteria in transitional and saltwaters. The presence of Salmonella was detected in 4.2 % of the samples studied. In these transitional waters, the proposed protocol presented an efficiency of 79.1 %, due to a high false positive rate (20.8 %), and a false negative rate of 0 %-implying reducing analysis time, the use of enrichment broths, and making it more cost effective. Despite the multitude of samples nature, the method here described revealed to be an efficient and promising tool for transitional waters analysis.

Assessment of cecal microbiota, integron occurrence, fermentation responses, and Salmonella frequency in conventionally raised broilers fed a commercial yeast-based prebiotic compound.

Prebiotics are defined as nondigestible food ingredients that can stimulate the growth of one or more beneficial bacteria in the gastrointestinal tract. The Biolex(®) MB40 is a commercial prebiotic that contains mannanoligosaccharides. The aims of this study were to evaluate the effects of prebiotic Biolex(®) MB40 on cecal microbiota of conventionally raised chickens using PCR-based denaturing gradient gel electrophoresis (DGGE) and assessing Salmonella prevalence. Chickens were randomly selected and distributed into three groups; a negative control (NC) and two treatment groups (T1 and T2). The NC group was fed a non-medicated feed, while the treatment groups were fed either T1 or T2, 0.05% antibiotic (BMD50) or 0.2% Biolex(®) MB40 respectively. During the study, cecal contents and bird feed were plated on selective media for Salmonella, yeast and mold prevalence analysis. Ten chickens from each group were randomly selected at 1, 2, 4 and 6 wk and ceca were extracted for DNA isolation for PCR-based DGGE. Also, short-chain fatty acids (SCFAs) were analyzed from collected cecal material by gas chromatography. Only 4.2% of the samples were Salmonella positive. Presence of class 1 integron from cecal material were analyzed by PCR and 97.5% of the cecal samples were positive for integron presence, but no class I integrons were detected in the Salmonella isolates. According to the PCR-based DGGE analysis, the T2 group exhibited a cecal microbial population pattern that was similar to the T1 group prior to wk 4 and the T2 group appeared to be almost identical with the NC group after wk 4 but T2 exhibited less Bacteroides rodentium prior to wk 4. Overall results showed that the commercial prebiotic, MB40 did not lead to a detectable reduction of Salmonella but the general frequency of Salmonella was minimal in all treatments. However, feeding an MB40 supplement did result in similar DGGE band patterns as the T1 group indicating that cecal microbiotia were potentially similar in these 2 groups. Overall, it appears that MB40 (T2) exhibited similar DGGE-cecal population patterns as BMD50 (T1) which suggests that these treatments may have influenced the populations in a comparable fashion.

Salmonellosis Hospitalizations in the United States: Associated Chronic Conditions, Costs, and Hospital Outcomes, 2011, Trends 2000-2011.

Hospitalized salmonellosis patients with concurrent chronic conditions may be at increased risk for adverse outcomes, increasing the costs associated with hospitalization. Identifying important modifiable risk factors for this predominantly foodborne illness may assist hospitals, physicians, and public health authorities to improve management of these patients. The objectives of this study were to (1) quantify the burden of salmonellosis hospitalizations in the United States, (2) describe hospitalization characteristics among salmonellosis patients with concurrent chronic conditions, and (3) examine the relationships between salmonellosis and comorbidities by four hospital-related outcomes. A retrospective analysis of salmonellosis discharges was conducted using the Agency for Healthcare Research and Quality's Nationwide Inpatient Sample for 2011. A supplemental trend analysis was performed for the period 2000-2011. Hospitalization characteristics were examined using multivariable regression modeling, with a focus on four outcome measures: in-hospital death, total amount billed by hospitals for services, length of stay, and disease severity. In 2011, there were 11,032 total salmonellosis diagnoses; 7496 were listed as the primary diagnosis, with 86 deaths (case-fatality rate = 1.2%). Multivariable regression analyses revealed a greater number of chronic conditions (≥4) among salmonellosis patients was associated with higher mean total amount billed by hospitals for services, longer length of stay, and greater disease severity (p ≤ 0.05). From 2000 to 2011, hospital discharges for salmonellosis increased by 27.2%, and the mean total amount billed by hospitals increased nearly threefold: $9,777 (2000) to $29,690 (2011). Observed increases in hospitalizations indicate the burden of salmonellosis remains substantial in the United States. The positive association between increased number of chronic conditions and the four hospital-related outcomes affirms the need for continual healthcare and public health investments to prevent and control this disease in vulnerable groups.

Epidemiology, Clinical Presentation, Laboratory Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of Invasive Salmonella Infections.

Salmonella enterica infections are common causes of bloodstream infection in low-resource areas, where they may be difficult to distinguish from other febrile illnesses and may be associated with a high case fatality ratio. Microbiologic culture of blood or bone marrow remains the mainstay of laboratory diagnosis. Antimicrobial resistance has emerged in Salmonella enterica, initially to the traditional first-line drugs chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole. Decreased fluoroquinolone susceptibility and then fluoroquinolone resistance have developed in association with chromosomal mutations in the quinolone resistance-determining region of genes encoding DNA gyrase and topoisomerase IV and also by plasmid-mediated resistance mechanisms. Resistance to extended-spectrum cephalosporins has occurred more often in nontyphoidal than in typhoidal Salmonella strains. Azithromycin is effective for the management of uncomplicated typhoid fever and may serve as an alternative oral drug in areas where fluoroquinolone resistance is common. In 2013, CLSI lowered the ciprofloxacin susceptibility breakpoints to account for accumulating clinical, microbiologic, and pharmacokinetic-pharmacodynamic data suggesting that revision was needed for contemporary invasive Salmonella infections. Newly established CLSI guidelines for azithromycin and Salmonella enterica serovar Typhi were published in CLSI document M100 in 2015.

Developing a two-step heat treatment for inactivating desiccation-adapted Salmonella spp. in aged chicken litter.

The effectiveness of a two-step heat treatment for eliminating desiccation-adapted Salmonella spp. in aged chicken litter was evaluated. The aged chicken litter with 20, 30, 40, and 50% moisture contents was inoculated with a mixture of four Salmonella serotypes for a 24-h adaptation. Afterwards, the inoculated chicken litter was added into the chicken litter with the adjusted moisture content for a 1-h moist-heat treatment at 65 °C and 100% relative humidity inside a water bath, followed by a dry-heat treatment in a convection oven at 85 °C for 1 h to the desired moisture level (<10-12%). After moist-heat treatment, the populations of Salmonella in aged chicken litter at 20 and 30% moisture contents declined from ≈6.70 log colony-forming units (CFU)/g to 3.31 and 3.00 log CFU/g, respectively. After subsequent 1-h dry-heat treatment, the populations further decreased to 2.97 and 2.57 log CFU/g, respectively. Salmonella cells in chicken litter with 40% and 50% moisture contents were only detectable by enrichment after 40 and 20 min of moist-heat treatment, respectively. Moisture contents in all samples were reduced to <10% after a 1-h dry-heat process. Our results demonstrated that the two-step heat treatment was effective in reducing >5.5 logs of desiccation-adapted Salmonella in aged chicken litter with moisture content at or above 40%. Clearly, the findings from this study may provide the chicken litter processing industry with an effective heat treatment method for producing Salmonella-free chicken litter.

Risk assessment of Salmonella in Danish meatballs produced in the catering sector.

A modular process risk model approach was used to assess health risks associated with Salmonella spp. after consumption of the Danish meatball product (frikadeller) produced with fresh pork in a catering unit. Meatball production and consumption were described as a series of processes (modules), starting from 1.3kg meat pieces through conversion to 70g meatballs, followed by a dose response model to assess the risk of illness from consumption of these meatballs. Changes in bacterial prevalence, concentration, and unit size were modelled within each module. The risk assessment was built using observational data and models that were specific for Salmonella spp. in meatballs produced in the catering sector. Danish meatballs are often pan-fried followed by baking in an oven before consumption, in order to reach the core temperature of 75°C recommended by the Danish Food Safety Authority. However, in practice this terminal heat treatment in the oven may be accidentally omitted. Eleven production scenarios were evaluated with the model, to test the impact of heat treatments and cooling rates at different room temperatures. The risk estimates revealed that a process comprising heat treatment of meatballs to core temperatures higher than 70°C, and subsequent holding at room temperatures lower than 20°C, for no longer than 3.5h, were very effective in Salmonella control. The current Danish Food Safety Authority recommendation of cooking to an internal temperature of 75°C is conservative, at least with respect to Salmonella risk. Survival and growth of Salmonella during cooling of meatballs not heat treated in oven had a significant impact on the risk estimates, and therefore, cooling should be considered a critical step during meatball processing.

Assessment of producers' response to Salmonella biosecurity issues and uptake of advice on laying hen farms in England and Wales.

High standards of biosecurity are known to reduce the risk of disease outbreaks; however, uptake of advice and implementation of biosecurity measures are dependent on many factors. This study assessed the uptake of targeted biosecurity advice by 60 laying hen farms provided during biosecurity audit visits. Advice was provided as bullet point cards focusing on specific areas identified as benefitting from improvement. These covered site entrance, site tidiness, vaccination, boot hygiene, hand hygiene, house tidiness, rodent control, fly control, red mite control and cleaning and disinfection between flocks. Background knowledge of Salmonella and biosecurity and farmers' willingness and intent to implement additional measures were assessed. About 50% of the principal decision-makers had basic background knowledge of Salmonella, with 22% considered well informed; almost all agreed that biosecurity could impact on Salmonella control and many appeared willing to implement additional biosecurity measures. Sixty-three per cent of study farms were categorised using the Defra Farmer Segmentation Model as Modern Family Businesses (MFBs), with 7-11% of farms being categorised as Custodian, Lifestyle Choice, Pragmatist or Challenged Enterprise; however, categorisation, did not determine uptake of advice. The most frequently used advice cards were boot hygiene, red mite control, hand hygiene, site entrance and cleaning and disinfection; uptake of advice ranged from 54 to 80% depending on the advice card. Uptake of advice by the farmers was encouraging, especially considering it was being provided by people other than their usual source of biosecurity information. Those who did not implement the recommended measures cited cost, difficulty of enforcement and practicality as the main reasons. However, the positive uptake of advice and implementation of recommended measures by many farmers demonstrates that targeted advice, discussed face to face with farmers, on a small number of key areas, is a potentially effective method of providing biosecurity information to complement more lengthy formal advisory reports.

Association between component costs, study methodologies, and foodborne illness-related factors with the cost of nontyphoidal Salmonella illness.

Nontyphoidal Salmonella spp. are one of the most common causes of bacterial foodborne illness. Variability in cost inventories and study methodologies limits the possibility of meaningfully interpreting and comparing cost-of-illness (COI) estimates, reducing their usefulness. However, little is known about the relative effect these factors have on a cost-of-illness estimate. This is important for comparing existing estimates and when designing new cost-of-illness studies. Cost-of-illness estimates, identified through a scoping review, were used to investigate the association between descriptive, component cost, methodological, and foodborne illness-related factors such as chronic sequelae and under-reporting with the cost of nontyphoidal Salmonella spp. illness. The standardized cost of nontyphoidal Salmonella spp. illness from 30 estimates reported in 29 studies ranged from $0.01568 to $41.22 United States dollars (USD)/person/year (2012). The mean cost of nontyphoidal Salmonella spp. illness was $10.37 USD/person/year (2012). The following factors were found to be significant in multiple linear regression (p≤0.05): the number of direct component cost categories included in an estimate (0-4, particularly long-term care costs) and chronic sequelae costs (inclusion/exclusion), which had positive associations with the cost of nontyphoidal Salmonella spp. illness. Factors related to study methodology were not significant. Our findings indicated that study methodology may not be as influential as other factors, such as the number of direct component cost categories included in an estimate and costs incurred due to chronic sequelae. Therefore, these may be the most important factors to consider when designing, interpreting, and comparing cost of foodborne illness studies.

Microbiological quality and safety assessment of lettuce production in Brazil.

The microbiological quality and safety of lettuce during primary production in Brazil were determined by enumeration of hygiene indicators Escherichia coli, coliforms and enterococci and detection of enteric pathogens Salmonella and E. coli O157:H7 in organic fertilizers, soil, irrigation water, lettuce crops, harvest boxes and worker's hands taken from six different lettuce farms throughout the crop growth cycle. Generic E. coli was a suitable indicator for the presence of Salmonella and E. coli O157:H7, while coliforms and enterococci were not. Few pathogens were detected: 5 salmonellae and 2 E. coli O157:H7 from 260 samples, of which only one was lettuce and the others were manure, soil and water. Most (5/7) pathogens were isolated from the same farm and all were from organic production. Statistical analysis revealed the following environmental and agro-technical risk factors for increased microbial load and pathogen prevalence in lettuce production: high temperature, flooding of lettuce fields, application of contaminated organic fertilizer, irrigation with water of inferior quality and large distances between the field and toilets. Control of the composting process of organic fertilizers and the irrigation water quality appear most crucial to improve and/or maintain the microbiological quality and safety during the primary production of lettuce.

Ability of Salmonella spp. to produce biofilm is dependent on temperature and surface material.

Salmonella, one of the most important pathogens transmitted by food, especially poultry, has the ability to form biofilms on surfaces. Its adhesion can be influenced by different physicochemical properties of these surfaces, while Salmonella uses fimbriae and produces cellulose as the main matrix components of biofilms. Their synthesis is co-regulated by a LuxR-type regulator, the agfD (aggregative fimbriae, curli), and adrA genes, respectively. Thus, this study investigated the production of biofilm by Salmonella spp. isolated from raw poultry (breast fillet), purchased in Botucatu, Sao Paulo, Brazil, on glass, polyvinyl chloride, and stainless steel at different temperatures (16°, 20°, 28°, and 35°C). We analyzed the frequency of the agfD and adrA genes and the rdar morphotype at 28°C and 35°C in isolated strains. We found Salmonella in 112 of 240 poultry samples (46.7%), and 62 strains previously isolated from the same kind of food were included in the study on biofilm development, gene expression, and rdar morphotype. All of them were positive for both genes, and 98.3% were able to produce biofilm in at least one temperature. The rates of rdar morphotype at 28°C and at 35°C were 55.2% (96 strains) and 2.3% (4 strains), respectively. Glass was the best material to avoid biofilm production, while Salmonella grew even at 16°C on stainless steel. These results point out the need for more effective sanitizing processes in the slaughter plants in order to avoid the permanence of these bacteria in food and eventual human foodborne diseases.

Quantitative assessment of cytosolic Salmonella in epithelial cells.

Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.