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.

Whole-genome sequencing analysis and CRISPR genotyping of rare antibiotic-resistant Salmonella enterica serovars isolated from food and related sources.

For decades, Salmonella Typhimurium and Salmonella Enteritidis have prevailed in several countries as agents of salmonellosis outbreaks. In Brazil, the largest exporter of poultry meat, relatively little attention has been paid to infrequent serovars. Here, we report the emergence and characterization of rare serovars isolated from food and related sources collected between 2014 and 2016 in Brazil. Twenty-two Salmonella enterica isolates were analyzed through the use of whole-genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats (CRISPR) genotyping. These isolates were classified into 10 infrequent serovars, including S. Abony, S. Isangi, S. Rochdale, S. Saphra, S. Orion, S. Ouakam, S. Grumpensis, S. Carrau, S. Abaetetuba, and S. Idikan. The presence of six antimicrobial resistance (AMR) genes, qnrB19, blaCMY-2, tetA, aac(6')-Iaa, sul2 and fosA7, which encode resistance to quinolones, third-generation cephalosporin, tetracycline, aminoglycoside, sulfonamide and fosfomycin, respectively, were confirmed by WGS. All S. Isangi harbored qnrB19 with conserved genomic context across strains, while S. Abony harbored blaCMY-2. Twelve (54.5%) strains displayed chromosomal mutations in parC (Thr57→Ser). Most serovars were classified as independent lineages, except S. Abony and S. Abaetetuba, which phylogenetically nested with Salmonella strains from different countries. CRISPR analysis revealed that the spacer content was strongly correlated with serovar and multi-locus sequence type for all strains, independently confirming the observed phylogenetic patterns, and highlighting the value of CRISPR-based genotyping for Salmonella. These findings add valuable information to the epidemiology of S. enterica in Brazil, where the emergency of antibiotic-resistant Salmonella continues to evolve.

Genomic insights of Klebsiella pneumoniae isolated from a native Amazonian fish reveal wide resistome against heavy metals, disinfectants, and clinically relevant antibiotics.

A multidrug-resistant CTX-M-15-producing Klebsiella pneumoniae (KpP1 strain) was isolated from a native Amazonian fish (Brachyplatystoma filamentosum) at the Brazilian Amazon. The strain was identified by MALDI-TOF. The genome was extracted, purified and a Nextera DNA Flex library was prepared and sequenced by Illumina platform. The sequenced genome was de novo assembled using Unicycler and in silico prediction accomplished by curated bioinformatics tools. The size of the genome is 5.6 Mb with 5715 genes. Whole-genome sequencing analysis revealed the presence of wide resistome, with genes conferring resistance to clinically relevant antibiotics, heavy metals and disinfectants. The KpP1 strain was assigned to the sequence type ST3827, KL111 (wzi113) and O3b locus. Native freshwater fish sold in wet markets of the Amazonian region could be an important vehicle for transmission of multidrug-resistant bacteria to humans. This study may give genomic insights on the spread of critical-priority WHO pathogens in a One Health context.

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.

Silent dissemination of colistin-resistant Escherichia coli in South America could contribute to the global spread of the mcr-1 gene.

During a Brazilian multicentric antimicrobial resistance surveillance study, colistin resistance was investigated in 4,620 Enterobacteriaceae isolated from human, animal, food and environmental samples collected from 2000 to 2016. We present evidence that mcr-1-positive Escherichia coli has been emerging in South America since at least 2012, supporting a previous report on the possible acquisition of mcr-1-harbouring E. coli by European travellers visiting Latin American countries.

High pressure spray with water shows similar efficiency to trimming in controlling microorganisms on poultry carcasses.

A study was conducted to evaluate a high pressure spray (HPS) with water as an alternative to trimming to remove gastrointestinal contamination on poultry carcasses and improve microbiological quality. The study was conducted under commercial conditions in 5 slaughter plants with one plant presenting approximately 5% of carcasses with visible gastrointestinal contamination (VGC), and the others showing approximately 12% of VGC. In all 5 plants, carcasses were sampled from the slaughter line and separated into 6 groups corresponding to 3 different treatments: A) carcasses with VGC before and after trimming; B) carcasses with VGC before and after HPS; and C) carcasses with no VGC before and after HPS. At the end of Trial A and prior to Trials B and C, an HPS equipment was installed before the end of the slaughter line. The HPS equipment was operated with 10 kgf/cm² of pressure and 1.5 L of potable water per carcass. Carcasses were analyzed using a rinsing procedure, and the following microbiological parameters were evaluated: the prevalence of Salmonella and Campylobacter, the abundance of Escherichia coli (EC), Enterobacteriaceae (EB), and the Total Viable Count (TVC). Salmonella was found in all plants at a prevalence ranging from 0.8% (plant 1) to 17.3% (plant 5), and the difference between plants was significant (P < 0.05%). The prevalence of Campylobacter ranged from 2.1 (plant 1) to 18.6% (plant 4) (P < 0.05%). The prevalence of Campylobacter was similar in plants 2, 3, and 5, and a significant difference (P < 0.05%) was observed compared to plants 1 and 4. In all plants, the EC, EB, and TVC counts did not show a significant difference (P > 0.05%) in any treatments. These results demonstrate that HPS with water is an alternative method for removing VGC and improving or maintaining the microbiological quality and safety of broiler carcasses.

Genome-based diagnostic of MDR Escherichia coli ONT:H19 ST10955 causing human gastrointestinal infection.

This study focuses on the genomic characterization of a multidrug-resistant Escherichia coli strain responsible for a severe gastrointestinal infection in a 33-year-old male. The patient initially received sulfamethoxazole/trimethoprim treatment, which proved ineffective. Fecal culture confirmed the presence of E. coli displaying a MDR profile to ampicillin, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, and tetracycline. Serotyping identified the strain as ONT:H19. Virulence analysis indicated a highly virulent profile with numerous virulence markers. Plasmid analysis uncovered various plasmids carrying both antimicrobial resistance and virulence genes. MLST assigned the strain to ST10955. Phylogenomic analysis revealed similarity to an older Brazilian isolate, suggesting the persistence of a common lineage with evolving antimicrobial resistance. This report highlights the first identification of a multidrug-resistant ST10955 E. coli strain with a wide resistome and virulence potential, emphasizing the importance of ongoing surveillance of E. coli strains due to their potential for severe infections, resistance development, and virulence.

Virulent shiga toxin-producing Escherichia coli (STEC) O157:H7 ST11 isolated from ground beef in Brazil.

In this study, a total of 248 ground beef samples were analyzed for the presence of Shiga toxin-producing Escherichia coli (STEC). Out of these samples, only one (0.4%) tested positive for STEC. Further analysis using PCR confirmed the presence of all tested genes associated with STEC, including stx1, stx2, eae, ehx, uid, rfbO157, and fliCH7 in this isolate. Interestingly, no STEC strains were detected in the remaining 100 beef cut samples or the 100 chicken cut samples, indicating the absence of detectable STEC contamination in those specific samples. The isolated strain exhibited significant cytotoxic activity in Vero cells, indicating its ability to produce cytotoxic Shiga toxins. To further investigate the strain, whole-genome sequencing (WGS) analyses were performed. The resistome analysis revealed the absence of acquired antimicrobial resistance genes, indicating a pan-susceptible phenotype. However, this strain presented chromosomal mutations in gyrA, gyrB, parC, parE, pmrA, pmrB, and folP. Plasmid analysis identified the presence of two plasmids, namely IncFIB(AP001918) and IncFII. The multi-locus sequence typing (MLST) identified the strain as belonging to sequence type (ST) 11, which is associated with E. coli O157:H7 strains. The virulome analysis confirmed the presence of several canonical virulence markers, including stx1, stx2, eae-g01-gamma, ehxA, stx1a-O157, and stx2a-O157. Overall, this study identified for the first time a rare occurrence of STEC contamination in ground beef, with the isolated strain belonging to the highly virulent O157:H7 serotype. These findings contribute to our understanding of STEC prevalence and characteristics in food samples, highlighting the importance of effective food safety measures to prevent potential health risks associated with STEC contamination.

Absence of Shiga toxin-producing Escherichia coli (STEC) in organic leafy greens from the metropolitan region of São Paulo, Brazil.

Shiga toxin-producing Escherichia coli (STEC) is an important pathogen with public health implications, including its potential association with vegetables. In this study, we investigated the presence of STEC in vegetables obtained from organic producers located in São Paulo city, Brazil. As part of a routine surveillance study conducted over (years of isolation), a total of 200 samples of organic vegetables were screened using biochemical and PCR methods. Among the vegetable samples tested, 30 (15%) were positive for non-Shiga toxin-producing E. coli. While no STEC was detected in the organic vegetables in this study, the presence of non-STEC in vegetables raises concerns about the lack of proper hygiene practices during vegetable handling. This contamination represents a public health risk, particularly considering that these isolates can still be pathogenic, and vegetables are often consumed raw. To address this important issue, continuous monitoring of these farms is recommended to ensure the quality and safety of organic vegetables produced for both domestic consumption and exportation.

High Level of Interaction between Phages and Bacteria in an Artisanal Raw Milk Cheese Microbial Community.

Microbial starter cultures are used in the production of many cheeses around the world, such as Parmigiano-Reggiano, in Italy, Époisses, in France, and Canastra, in Brazil, providing many of the unique features of these cheeses. Bacteriophages (phages) are ubiquitous and well known to modulate the structure of bacterial communities, and recent data indicate that cheeses contain a high abundance of naturally occurring phages. Here, we analyze the viral and bacterial metagenomes of Canastra cheese: a traditional artisanal Brazilian cheese produced using an endogenous starter culture and raw milk. Over 1,200 viral operational taxonomic units were recovered using both isolated viral-like particles and complete metagenomic DNA. Common viral families identified included Siphoviridae and Myoviridae, with 40% of putative phage genomes unidentified at the family level of classification. We observed very high phage diversity, which varied greatly across different cheese producers, with 28% of phage genomes detected in only one producer. Several metagenome-assembled genomes were recovered for lactic acid-producing bacteria, as well as nonstarter bacterial species, and we identified several phage-bacterium interactions, at the strain level of resolution, varying across distinct cheese producers. We postulate that at least one bacterial strain detected could be endogenous and unique to the Canastra cheese-producing region in Brazil and that its growth seems to be modulated by autochthonous phages present in this artisanal production system. This phage-host relationship is likely to influence the fermentation dynamics and ultimately the sensorial profile of these cheeses, with implications for other similar cheese production systems around the world. IMPORTANCE Our work demonstrated a dynamic yet stable microbial ecosystem during cheese production using an endogenous starter culture. This was observed across several distinct producers and was marked by genomic evidence of continued phage-bacterium interactions, such as the presence of bacterial defense mechanisms. Furthermore, we provide evidence of unique microbial signatures for each individual cheese producer studied in the region, a fact that may have profound consequences on product traceability. This was the first effort to describe and understand the bacteriophage composition and ecological dynamics within the Brazilian Canastra cheese production system. The study of this prototypical backslopping production system provides a solid background for further mechanistic studies of the production of many cheeses around the world.

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.

Clustered Regularly Interspaced Short Palindromic Repeats Genotyping of Multidrug-Resistant Salmonella Heidelberg Strains Isolated From the Poultry Production Chain Across Brazil.

Salmonella enterica subsp. enterica serovar Heidelberg has been associated with a broad host range, such as poultry, dairy calves, swine, wild birds, environment, and humans. The continuous evolution of S. Heidelberg raises a public health concern since there is a global dispersal of lineages harboring a wide resistome and virulome on a global scale. Here, we characterized the resistome, phylogenetic structure and clustered regularly interspaced short palindromic repeats (CRISPR) array composition of 81 S. Heidelberg strains isolated from broiler farms (n = 16), transport and lairage (n = 5), slaughterhouse (n = 22), and retail market (n = 38) of the poultry production chain in Brazil, between 2015 and 2016 using high-resolution approaches including whole-genome sequencing (WGS) and WGS-derived CRISPR genotyping. More than 91% of the S. Heidelberg strains were multidrug-resistant. The total antimicrobial resistance (AMR) gene abundances did not vary significantly across regions and sources suggesting the widespread distribution of antibiotic-resistant strains from farm to market. The highest AMR gene abundance was observed for fosA7, aac(6')-Iaa, sul2, tet(A), gyrA, and parC for 100% of the isolates, followed by 88.8% for bla CMY-2. The ß-lactam resistance was essentially driven by the presence of the plasmid-mediated AmpC (pAmpC) bla CMY-2 gene, given the isolates which did not carry this gene were susceptible to cefoxitin (FOX). Most S. Heidelberg strains were classified within international lineages, which were phylogenetically nested with Salmonella strains from European countries; while CRISPR genotyping analysis revealed that the spacer content was overall highly conserved, but distributed into 13 distinct groups. In summary, our findings underscore the potential role of S. Heidelberg as a key pathogen disseminated from farm to fork in Brazil and reinforce the importance of CRISPR-based genotyping for salmonellae. Hence, we emphasized the need for continuous mitigation programs to monitor the dissemination of this high-priority pathogen.

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.

Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State, Brazil.

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide, and is often associated with gastroenteritis in humans following consumption of raw bivalve mollusks, especially raw oysters. The occurrence of total and pathogenic V. parahaemolyticus in 74 samples of raw oysters collected in restaurants, supermarkets, groceries and beach huts in Sao Paulo State, was monitored between February 2006 and January 2007. Enumeration of V. parahaemolyticus was performed according to the most probable number (MPN) procedure. Five to ten typical colonies were selected from thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates for confirmation by the presence of the species-specific gene tlh and the virulence genes tdh and trh by multiplex PCR. V. parahaemolyticus was detected in 100% of samples. The densities of total V. parahaemolyticus varied from 1.78 to 6.04 log(10) (MPN/g), with higher densities being detected in fall and summer, and lower densities in winter (P < 0.05). There was no statistical difference among densities of V parahaemolyticus regarding the site of collection. None of the 1943 V. parahaemolyticus isolates contained tdh and/or trh. These data provide information for the assessment of exposure to V. parahaemolyticus in oysters consumed in Sao Paulo, State, Brazil.

Prevalence and counts of Salmonella spp. in minimally processed vegetables in São Paulo, Brazil.

Minimally processed vegetables (MPV) may be important vehicles of Salmonella spp. and cause disease. This study aimed at detecting and enumerating Salmonella spp. in MPV marketed in the city of São Paulo, Brazil. A total of 512 samples of MPV packages collected in retail stores were tested for Salmonella spp. and total coliforms and Escherichia coli as indication of the hygienic status. Salmonella spp. was detected in four samples, two using the detection method and two using the counting method, where the results were 8.8 × 10(2) CFU/g and 2.4 × 10(2) CFU/g. The serovars were Salmonella Typhimurium (three samples) and Salmonella enterica subsp. enterica O:47:z4,z23:- (one sample). Fourteen samples (2.7%) presented counts of E. coli above the maximum limit established by the Brazilian regulation for MPV (10(2) CFU/g). Therefore, tightened surveillance and effective intervention strategies are necessary in order to address consumers and governments concerns on safety of MPV.

Characterization of class 1 integrons and antibiotic resistance genes in multidrug-resistant Salmonella enterica isolates from foodstuff and related sources.

In recent years, an increase in the occurrence of antimicrobial resistance among Salmonella enterica has been observed in several countries, which is worrisome because S. enterica is one of the most common causes of human gastroenteritis worldwide. The aim of this study was to characterize class 1 integrons and antibiotic resistance genotypes in Salmonella enterica isolates recovered from foodstuff and related sources. Nineteen multidrug-resistant (MDR) Salmonella enterica isolates were recovered. Higher resistance rates to tetracycline (90%), streptomycin (80%), sulfamethoxazole-trimethoprim (80%), ampicillin (60%) and nalidixic acid (70%) were related to the presence of the tetA, aadA, sul1/sul2, bla TEM-1 genes, and a codon mutation at position 83 of the gyrA gene, respectively. Class 1 integrons harboring aadA, bla TEM-1, sul1 or dhfr1 genes were detected in nine (45%) Salmonella enterica strains belonging to serotypes Brandenburg, Panama, Agona, Mbandaka and Alachua. Finally, clonal dissemination of S. Panama, S. Derby and S. Mbandaka was confirmed by PFGE. Detection of clonally related MDR Salmonella enterica suggests that endemic serotypes can be supported by class 1 integron-borne gene cassettes and/or mutations in drug targets. Emergence and dissemination of multidrug-resistant Salmonella enterica can have a major public health impact in an environment where large-scale suppliers ship their products.

Correction: Class 1 integron-borne cassettes harboring blaCARB-2 gene in multidrug-resistant and virulent Salmonella Typhimurium ST19 strains recovered from clinical human stool samples, United States.

[This corrects the article DOI: 10.1371/journal.pone.0240978.].

Correlation between environmental factors and prevalence of Vibrio parahaemolyticus in oysters harvested in the southern coastal area of Sao Paulo State, Brazil.

The presence of Vibrio parahaemolyticus in 123 oyster samples collected from an estuary on the southern coast of Sao Paulo state, Brazil, was investigated. Of the 123 samples, 99.2% were positive with densities ranging from < 3 to 10(5) most probable number (MPN)/g. Densities correlated significantly with water temperature (r = 0.48; P < 0.001) but not with salinity (r = -0.09; P = 0.34). The effect of harvest site on counts was not significant (P > 0.05). These data provide information for the assessment of exposure of V. parahaemolyticus in oysters at harvest.

Highly clonal relationship among Salmonella Enteritidis isolates in a commercial chicken production chain, Brazil.

In this study, we described the comparison among pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), ribotyping, and PCR-ribotyping methods for subtyping Salmonella Enteritidis isolated from an industrial chicken production chain. One hundred and eight S. Enteritidis were isolated at all stages of poultry meat processing plant. These isolates were pheno- and genotypically characterized by using antimicrobial susceptibility test, phage typing, RAPD, PFGE, ribotyping, and PCR-ribotyping. The highest antibiotic resistance rates were observed for enrofloxacin (18.5%) followed by furazolidone (15.7%), cefoxitin (1.8%), ciprofloxacin, and ampicillin with 0.9% each one, while seven isolates (6.4%) were pan-susceptible. Most strains belonged to the globally disseminated phage type PT4 (n = 74; 69.2%). Additionally, we identified strains belonging to phage types PT1 (n = 19; 17.8%) and PT7a (n = 14; 13.1%). Moreover, our results showed that these four molecular methods indicate similar results showing high similarity (≥ 90%) among S. Enteritidis strains, suggesting that these isolates appear to be from a common ancestor being spread at all stages of the poultry production chain. In summary, the combined molecular approaches of these methods remain a suitable alternative to efficiently subtyping S. Enteritidis in the absence of high-resolution genotyping methods and these results may serve as a baseline study for development of mitigation strategies.