Genomic characterization of Staphylococcus aureus from Canastra Minas Artisanal Cheeses.

Canastra Minas Artisanal Cheese is produced in the Brazilian State of Minas Gerais using raw milk, rennet, and pingo, a natural endogenous starter culture (fermented whey) collected from the previous day's production. Due to the use of raw milk, the product can carry microorganisms that may cause foodborne diseases (FBD), including Staphylococcus aureus. Genomic characterization of S. aureus is an important tool to assess diversity, virulence, antimicrobial resistance, and the potential for causing food poisoning due to enterotoxin production. This study is aimed at exploring the genomic features of S. aureus strains isolated from Canastra Minas Artisanal Cheeses. Multilocus sequence typing (MLST) classified these strains as ST1, ST5, and a new profile ST7849 (assigned to the clonal complex CC97). These strains belonged to four spa types: t008, t127, t359, and t992. We identified antimicrobial resistance genes with phenotypic correlation against methicillin (MRSA) and tetracycline. Virulome analysis revealed genes associated with iron uptake, immune evasion, and potential capacity for adherence and biofilm formation. The toxigenic potential included cyto- and exotoxins genes, and all strains presented the genes that encode for Panton-Valentine toxin and hemolysin, and two strains encoded 4 and 8 Staphylococcal enterotoxin (SE) genes. The results revealed the pathogenic potential of the evaluated S. aureus strains circulating in the Canastra region, representing a potential risk to public health. This study also provides useful information to monitor and guide the application of control measures to the artisanal dairy food production chain.

Food Safety Issues Related to Eating In and Eating Out.

Because of growing urbanization and lack of time to prepare meals at home, eating out or getting food delivered have become common trends for many people. The consumption of food from unknown sources may impose an increased chance of contamination with microbiological hazards, especially if sanitary conditions are not met. We evaluated data from health surveillance agencies and scientific articles on foodborne diseases (FBD) reported internationally according to the exposure sites. We observed that the data are influenced by cultural, political, and socioeconomic differences. For instance, in New Zealand, Australia, United States, Denmark and India, the occurrence of FBD outbreaks was greater from foods prepared in commercial establishments and street vendors than from households. Conversely, in China, countries of the European Union and Brazil, the results are the opposite. Additionally, the pandemic imposed new eating behavior patterns, increasing delivery services and foods prepared in so-called "Dark Kitchens". The underreporting and heterogeneity of data among countries prevented a precise conclusion to the question of whether homemade foods are inherently safer than foods prepared out. Nevertheless, a lower level of development in a country influences its sanitation conditions, as well as the number of street food vendors, the search for cheaper foods, and insufficient knowledge of the population on good hygiene practices, which can all increase the chances of FBD cases.

Persistent symptoms and decreased health-related quality of life after symptomatic pediatric COVID-19: A prospective study in a Latin American tertiary hospital.

OBJECTIVES: To prospectively evaluate demographic, anthropometric and health-related quality of life (HRQoL) in pediatric patients with laboratory-confirmed coronavirus disease 2019 (COVID-19). METHODS: This was a longitudinal observational study of surviving pediatric post-COVID-19 patients (n=53) and pediatric subjects without laboratory-confirmed COVID-19 included as controls (n=52) was performed. RESULTS: The median duration between COVID-19 diagnosis (n=53) and follow-up was 4.4 months (0.8-10.7). Twenty-three of 53 (43%) patients reported at least one persistent symptom at the longitudinal follow-up visit and 12/53 (23%) had long COVID-19, with at least one symptom lasting for >12 weeks. The most frequently reported symptoms at the longitudinal follow-up visit were headache (19%), severe recurrent headache (9%), tiredness (9%), dyspnea (8%), and concentration difficulty (4%). At the longitudinal follow-up visit, the frequencies of anemia (11% versus 0%, p=0.030), lymphopenia (42% versus 18%, p=0.020), C-reactive protein level of >30 mg/L (35% versus 0%, p=0.0001), and D-dimer level of >1000 ng/mL (43% versus 6%, p=0.0004) significantly reduced compared with baseline values. Chest X-ray abnormalities (11% versus 2%, p=0.178) and cardiac alterations on echocardiogram (33% versus 22%, p=0.462) were similar at both visits. Comparison of characteristic data between patients with COVID-19 at the longitudinal follow-up visit and controls showed similar age (p=0.962), proportion of male sex (p=0.907), ethnicity (p=0.566), family minimum monthly wage (p=0.664), body mass index (p=0.601), and pediatric pre-existing chronic conditions (p=1.000). The Pediatric Quality of Live Inventory 4.0 scores, median physical score (69 [0-100] versus 81 [34-100], p=0.012), and school score (60 [15-100] versus 70 [15-95], p=0.028) were significantly lower in pediatric patients with COVID-19 at the longitudinal follow-up visit than in controls. CONCLUSIONS: Pediatric patients with COVID-19 showed a longitudinal impact on HRQoL parameters, particularly in physical/school domains, reinforcing the need for a prospective multidisciplinary approach for these patients. These data highlight the importance of closer monitoring of children and adolescents by the clinical team after COVID-19.

Adherence to food hygiene and personal protection recommendations for prevention of COVID-19.

BACKGROUND: Several actions in the realm of public policies and programs have been implemented worldwide to prevent and control the spread of COVID-19. As Brazil and many parts of the world are marked by regional, cultural, and social inequalities, risk communication and assimilation of protection recommendations by the public may be erratic, hampering the proper measurement of their effectiveness. SCOPE AND APPROACH: Even though transmission of SARS-CoV-2 by foods is unlikely, this work aimed to evaluate the population's adherence to the recommended food hygiene and personal protection measures to prevent COVID-19. An online survey containing questions on food hygiene and personal protection was publicly disseminated via internet and social networks. Data from 3000 respondents were collected and discussed herein.Key findings and conclusions: Most respondents adopted the recommended hygienic and personal protection measures to avoid contamination with SARS-CoV-2. However, some important flaws were detected, such as lack of use of face masks when in public places (6%), improper hand washing and sanitizing (10-12%) and use of incorrect products for food or environment cleaning and sanitization (28%), indicating that adequate risk communication and preventive recommendations must continue in order to avoid contamination with the coronavirus. The heighten awareness with food hygiene during the pandemic may have an enduring positive effect on food safety.

Behavior of the Biological Control Agent Bacillus thuringiensis subsp. aizawai ABTS-1857 and Salmonella enterica on Spinach Plants and Cut Leaves.

Fresh produce has been identified as an important vehicle for the transmission of foodborne pathogens. This study evaluated the behavior of vegetative cells and spores of Bacillus thuringiensis, one of the main biological control agents (BCAs) used in the world, and Salmonella enterica on spinach plants (pre-harvest) and spinach cut leaves (post-harvest) at 12°C, experimentally inoculated as single or co-cultures. The results evidenced that spray-inoculated commercial BCA containing Bacillus thuringiensis subsp. aizawai ABTS-1857 (BTa ABTS-1857) spores persisted well on spinach leaves in both pre- and post-harvest simulations. However, when BTa ABTS-1857 vegetative cells were spray-inoculated, more than 2 log reductions in the counts of B. thuringiensis were observed during 20 days pre- and 5 days post-harvest simulations, respectively. The counts of S. Montevideo on the spinach cut leaves during post-harvest storage at 12°C for 5 days remained unchanged, whereas 1 log reduction was noted during pre-harvest. Moreover, during pre-harvest simulation, when co-inoculated with BTa ABTS-1857 vegetative cells or spores, additional 0.5 or 1.0 log reductions were detected on the counts of S. Montevideo in the spinach leaves on the 10th day. These results were obtained under laboratory conditions, and further findings in longitudinal studies from farm (in the agricultural field) to retail (end of shelf life) will contribute to understanding of the role of B. thuringiensis as a BCA on growth/survival of Salmonella spp. in fresh produce.

Persistent symptoms and decreased health-related quality of life after symptomatic pediatric COVID-19: A prospective study in a Latin American tertiary hospital

OBJECTIVES: To prospectively evaluate demographic, anthropometric and health-related quality of life (HRQoL) in pediatric patients with laboratory-confirmed coronavirus disease 2019 (COVID-19) METHODS: This was a longitudinal observational study of surviving pediatric post-COVID-19 patients (n=53) and pediatric subjects without laboratory-confirmed COVID-19 included as controls (n=52) was performed. RESULTS: The median duration between COVID-19 diagnosis (n=53) and follow-up was 4.4 months (0.8-10.7). Twenty-three of 53 (43%) patients reported at least one persistent symptom at the longitudinal follow-up visit and 12/53 (23%) had long COVID-19, with at least one symptom lasting for >12 weeks. The most frequently reported symptoms at the longitudinal follow-up visit were headache (19%), severe recurrent headache (9%), tiredness (9%), dyspnea (8%), and concentration difficulty (4%). At the longitudinal follow-up visit, the frequencies of anemia (11% versus 0%, p=0.030), lymphopenia (42% versus 18%, p=0.020), C-reactive protein level of >30 mg/L (35% versus 0%, p=0.0001), and D-dimer level of >1000 ng/mL (43% versus 6%, p=0.0004) significantly reduced compared with baseline values. Chest X-ray abnormalities (11% versus 2%, p=0.178) and cardiac alterations on echocardiogram (33% versus 22%, p=0.462) were similar at both visits. Comparison of characteristic data between patients with COVID-19 at the longitudinal follow-up visit and controls showed similar age (p=0.962), proportion of male sex (p=0.907), ethnicity (p=0.566), family minimum monthly wage (p=0.664), body mass index (p=0.601), and pediatric pre-existing chronic conditions (p=1.000). The Pediatric Quality of Live Inventory 4.0 scores, median physical score (69 [0-100] versus 81 [34-100], p=0.012), and school score (60 [15-100] versus 70 [15-95], p=0.028) were significantly lower in pediatric patients with COVID-19 at the longitudinal follow-up visit than in controls. CONCLUSIONS: Pediatric patients with COVID-19 showed a longitudinal impact on HRQoL parameters, particularly in physical/school domains, reinforcing the need for a prospective multidisciplinary approach for these patients. These data highlight the importance of closer monitoring of children and adolescents by the clinical team after COVID-19.

Tilapia fish microbial spoilage monitored by a single optical gas sensor.

As consumption of fish and fish-based foods increases, non-destructive monitoring of fish freshness also becomes more prominent. Fish products are very perishable and prone to microbiological growth, not always easily detected by organoleptic evaluation. The analysis of the headspace of fish specimens through gas sensing is an interesting approach to monitor fish freshness. Here we report a gas sensing method for monitoring Tilapia fish spoilage based on the application of a single gas sensitive gel material coupled to an optical electronic nose. The optical signals of the sensor and the extent of bacterial growth were followed over time, and results indicated good correlation between the two determinations, which suggests the potential application of this simple and low cost system for Tilapia fish freshness monitoring.

Characterization of multiple antilisterial peptides produced by sakacin P-producing Lactobacillus sakei subsp. sakei 2a.

Antimicrobial compounds produced by lactic acid bacteria can be explored as natural food biopreservatives. In a previous report, the main antimicrobial compounds produced by the Brazilian meat isolate Lactobacillus sakei subsp. sakei 2a, i.e., bacteriocin sakacin P and two ribosomal peptides (P2 and P3) active against Listeria monocytogenes, were described. In this study, we report the spectrum of activity, molecular mass, structural identity and mechanism of action of additional six antilisterial peptides produced by Lb. sakei 2a, detected in a 24 h-culture in MRS broth submitted to acid treatment (pH 1.5) and proper fractionation and purification steps for obtention of free and cell-bound proteins. The six peptides presented similarity to different ribosomal proteins of Lb. sakei subsp sakei 23K and the molecular masses varied from 4.6 to 11.0 kDa. All peptides were capable to increase the efflux of ATP and decrease the membrane potential in Listeria monocytogenes. The activity of a pool of the obtained antilisterial compounds [enriched active fraction (EAF)] against Listeria monocytogenes in a food model (meat gravy) during refrigerated storage (4 °C) for 10 days was also tested and results indicated that the populations of L. monocytogenes in the food model containing the acid extract remained lower than those at time 0-day, evidencing that the acid extract of a culture of Lb. sakei 2a is a good technological alternative for the control of growth of L. monocytogenes in foods.

Draft Genome Sequences of Colistin-Resistant MCR-1-Producing Escherichia coli ST1850 and ST74 Strains Isolated from Commercial Chicken Meat.

We present here the draft genome sequences of two colistin-resistant mcr-1-carrying Escherichia coli strains belonging to sequence type 74 (ST74) and ST1850, isolated from commercial chicken meat in Brazil. Assembly of this draft genome resulted in 5,022,083 and 4,950,681 bp, respectively, revealing the presence of the IncX4 plasmid-mediated mcr-1 gene responsible for resistance to colistin.

Quantitative assessment of the impact of cross-contamination during the washing step of ready-to-eat leafy greens on the risk of illness caused by Salmonella.

The aim of this study was to develop a quantitative microbial risk assessment (QMRA) model to estimate the risk of illness caused by Salmonella in ready-to-eat (RTE) leafy greens, based on common practices in Brazilian processing plants. The risk assessment model considered five modules: in field, washing step, retail storage, home storage and dose-response. Fifty thousand iterations of a @Risk model built in Excel were run for each of sixty scenarios. These scenarios considered different initial pathogen concentrations, fractions of contaminated produce and chlorine concentrations. For chlorine, seven pre-set concentrations (0, 5, 10, 25, 50, 150 and 250mg/L) and three triangular distributions were considered [RiskTriang(0,5,10mg/L), RiskTriang(0,80,250mg/L) and RiskTriang(10,120,250mg/L)]. The outputs were risk of infection, estimated number of illnesses and estimated percent of illnesses arising from cross-contamination. The QMRA model indicated quantitatively that higher chlorine concentrations resulted in lower risk of illness. When simulation was done with <5mg/L of chlorine, most (>96%) of the illnesses arose from cross-contamination, but when a triangular distribution with 10, 120 and 250mg/L of chlorine was simulated, no illnesses arising from cross-contamination were predicted. Proper control of the sanitizer in the washing step is essential to reduce initial contamination and avoid cross-contamination.

Chicken Meat as a Reservoir of Colistin-Resistant Escherichia coli Strains Carrying mcr-1 Genes in South America.

The detection and rapid spread of colistin-resistant Enterobacteriaceae carrying the mcr-1 gene has created an urgent need to strengthen surveillance. In this study, eight clonally unrelated colistin-resistant Escherichia coli isolates carrying mcr-1 and blaCTX-M or blaCMY-2 genes were isolated from commercial chicken meat in Brazil. Most E. coli strains carried IncX4 plasmids, previously identified in human and animal isolates. These results highlight a new reservoir of mcr-1-harboring E. coli strains in South America.

Microbiology of organic and conventionally grown fresh produce

ABSTRACT Fresh produce is a generalized term for a group of farm-produced crops, including fruits and vegetables. Organic agriculture has been on the rise and attracting the attention of the food production sector, since it uses eco-agricultural principles that are ostensibly environmentally-friendly and provides products potentially free from the residues of agrochemicals. Organic farming practices such as the use of animal manure can however increase the risk of contamination by enteric pathogenic microorganisms and may consequently pose health risks. A number of scientific studies conducted in different countries have compared the microbiological quality of produce samples from organic and conventional production and results are contradictory. While some have reported greater microbial counts in fresh produce from organic production, other studies do not. This manuscript provides a brief review of the current knowledge and summarizes data on the occurrence of pathogenic microorganisms in vegetables from organic production.

Microbiology of organic and conventionally grown fresh produce.

Fresh produce is a generalized term for a group of farm-produced crops, including fruits and vegetables. Organic agriculture has been on the rise and attracting the attention of the food production sector, since it uses eco-agricultural principles that are ostensibly environmentally-friendly and provides products potentially free from the residues of agrochemicals. Organic farming practices such as the use of animal manure can however increase the risk of contamination by enteric pathogenic microorganisms and may consequently pose health risks. A number of scientific studies conducted in different countries have compared the microbiological quality of produce samples from organic and conventional production and results are contradictory. While some have reported greater microbial counts in fresh produce from organic production, other studies do not. This manuscript provides a brief review of the current knowledge and summarizes data on the occurrence of pathogenic microorganisms in vegetables from organic production.

Microbiology of organic and conventionally grown fresh produce

ABSTRACT Fresh produce is a generalized term for a group of farm-produced crops, including fruits and vegetables. Organic agriculture has been on the rise and attracting the attention of the food production sector, since it uses eco-agricultural principles that are ostensibly environmentally-friendly and provides products potentially free from the residues of agrochemicals. Organic farming practices such as the use of animal manure can however increase the risk of contamination by enteric pathogenic microorganisms and may consequently pose health risks. A number of scientific studies conducted in different countries have compared the microbiological quality of produce samples from organic and conventional production and results are contradictory. While some have reported greater microbial counts in fresh produce from organic production, other studies do not. This manuscript provides a brief review of the current knowledge and summarizes data on the occurrence of pathogenic microorganisms in vegetables from organic production.

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a.

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2(4) factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R (2) = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L(-1) and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL(-1)) occurred in the MRS broth supplemented with 5.5 g L(-1) glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL(-1).

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2⁴ factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R² = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L⁻¹ and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL⁻¹) occurred in the MRS broth supplemented with 5.5 g L⁻¹ glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL⁻¹.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese.

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain (Lc. lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: evaluation of the probiotic potential.

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.