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.

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.

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.

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.

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.

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.].

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.

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.

International lineages, such as Salmonella Typhimurium sequence type (ST) 19, are most often associated with foodborne diseases and deaths in humans. In this study, we compared the whole-genome sequences of five S. Typhimurium strains belonging to ST19 recovered from clinical human stool samples in North Carolina, United States. Overall, S. Typhimurium strains displayed multidrug-resistant profile, being resistance to critically and highly important antimicrobials including ampicillin, ticarcillin/clavulanic acid, streptomycin and sulfisoxazole, chloramphenicol, tetracycline, respectively. Interestingly, all S. Typhimurium strains carried class 1 integron (intl1) and we were able to describe two genomic regions surrounding blaCARB-2 gene, size 4,062 bp and 4,422 bp for S. Typhimurium strains (HS5344, HS5437, and HS5478) and (HS5302 and HS5368), respectively. Genomic analysis for antimicrobial resistome confirmed the presence of clinically important genes, including blaCARB-2, aac(6')-Iaa, aadA2b, sul1, tetG, floR, and biocide resistance genes (qacEΔ1). S. Typhimurium strains harbored IncFIB plasmid containing spvRABCD operon, as well as rck and pef virulence genes, which constitute an important apparatus for spreading the virulence plasmid. In addition, we identified several virulence genes, chromosomally located, while the phylogenetic analysis revealed clonal relatedness among these strains with S. enterica isolated from human and non-human sources obtained in European and Asian countries. Our results provide new insights into this unusual class 1 integron in virulent S. Typhimurium strains that harbors a pool of genes acting as potential hotspots for horizontal gene transfer providing readily adaptation to new surrounds, as well as being crucially required for virulence in vivo. Therefore, continuous genomic surveillance is an important tool for safeguarding human health.

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.

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.

Challenges of teaching food microbiology in Brazil.

Food Microbiology is included in majors such as Food Engineering, Food Science and Technology, Nutrition, Veterinary Medicine, Gastronomy, Pharmaceutical Sciences, among others. Food safety and hygiene are usually the focus, but the technological applications of microbes through fermentations are also covered. During an education symposium at the Brazilian Congress of Microbiology in 2017, a group of professors expressed their difficulties associated with teaching to new generations, the use of technology in the classroom, and the application of new learning tools. The objective of this study was to gather information about the educational practices among Brazilian professors who teach Food Microbiology throughout the country. The results indeed confirmed the diversity of careers in which food microbiology is taught. We verified that professors mixed traditional teaching strategies with modern active learning methods, even though some difficulties associated with lack of time, pedagogical training, and low adherence of students for adopting these modern methods were commonly highlighted. The preferred teaching approaches were dialogued or discussed lectures, seminars, homework, case studies, and field visits. It is noteworthy that most professors still use traditional teaching methods. It is crucial that awareness concerning the educational needs in different careers and the challenges and dilemmas facing education for the new generations should be dealt with by using effective teaching approaches in food microbiology education. We suggest that a more permanent discussion forum among faculty members dealing with food microbiology in the country should be launched and this work is a step towards this goal.

Genomic Features of High-Priority Salmonella enterica Serovars Circulating in the Food Production Chain, Brazil, 2000-2016.

Multidrug-resistant (MDR) Salmonella enterica has been deemed a high-priority pathogen by the World Health Organization. Two hundred and sixty-four Salmonella enterica isolates recovered over a 16-year period (2000 to 2016) from the poultry and swine production chains, in Brazil, were investigated by whole-genome sequencing (WGS). Most international lineages belonging to 28 serovars, including, S. enterica serovars S. Schwarzengrund ST96, S. Typhimurium ST19, S. Minnesota ST548, S. Infantis ST32, S. Heidelberg ST15, S. Newport ST45, S. Brandenburg ST65 and S. Kentucky ST198 displayed MDR and virulent genetic backgrounds. In this regard, resistome analysis revealed presence of qnrE1 (identified for the first time in S. Typhimurium from food chain), qnrB19, qnrS1, blaCTX-M-8, blaCTX-M-2 and blaCMY-2 genes, as well as gyrA mutations; whereas ColpVC, IncHI2A, IncHI2, IncFIA, Incl1, IncA/C2, IncR, IncX1 and po111 plasmids were detected. In addition, phylogenetic analysis revealed multiple independent lineages such as S. enterica serovars S. Infantis, S. Schwarzengrund, S. Minnesota, S. Kentucky and S. Brandenburg. In brief, ocurrence and persistence of international lineages of S. enterica serovars in food production chain is supported by conserved genomes and wide virulome and resistome.

Microbiological Testing for the Proper Assessment of the Hygiene Status of Beef Carcasses.

Microbiological testing is an important quality management tool in the food industry. In this study, the hygiene status of beef carcasses sampled in eight Brazilian slaughterhouses was assessed by enumeration of different hygiene indicator microorganisms, and a model to establish potential associations among these counts was proposed. The carcasses (n = 464) were surface sampled at four slaughtering steps (step 1: Hide after bleeding; step 2: Carcass after hide removal; step 3: Carcass after evisceration; step 4: Carcass after end washing) and subjected to a counting of mesophilic aerobes (MA), Enterobacteriaceae (EB), total coliforms (TC), and Escherichia coli (EC) using Petrifilm™ plates. Among the sampled beef carcasses (step 4), 32 (6.9%) and 71 (15.3%) presented counts above the microbiological criteria established by (EC) No. 1441/2007 for MA and EB, respectively. Thus, indicating that improvements in slaughter hygiene and a review of process controls are demanded in some of the studied slaughterhouses. The log count differences of EC, TC, and EB from MA were considered as response variables as a function of the slaughtering steps. Differential log counts changed consistently with the steps. The measurements, including the patterns in their inherently random variability, were fairly predictable from steps 1 and 4. The results indicated that differential log counts for TC and EC are not relevant, as their concentrations and random pattern can be inferred from counts of MA and EB. The proposed model can be used as a valuable tool for the design and adoption of feasible quality control programs in beef industries. The adoption of such a tool should have a positive contribution on consumers' health and enhance product quality.

Genome Sequencing of an Escherichia coli Sequence Type 617 Strain Isolated from Beach Ghost Shrimp (Callichirus major) from a Heavily Polluted Ecosystem Reveals a Wider Resistome against Heavy Metals and Antibiotics.

Here, we present the draft genome sequence of a multidrug-resistant (MDR) Escherichia coli strain belonging to sequence type 617 (ST617), isolated from beach ghost shrimp from polluted coastal waters in Brazil. These data provide valuable information for comparative genomic analysis, related to the dissemination of MDR E. coli in marine ecosystems.

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.

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.