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Quantitative real-time polymerase chain reactions (qPCRs) of the most prevalent bacteria causing foodborne diseases worldwide, such as Salmonella spp., Escherichia coli, and Staphylococcus aureus, can be an important tool for quantitative microbial risk assessment, which requires numerical data to determine the level of contamination at a specific stage of food production. However, most of qPCR assays described in the literature for these pathogens are qualitative; their objective is pathogen detection and not pathogen quantification. Thus, the aim of our work was to develop a qPCR for the simultaneous quantification of Salmonella spp., E. coli, and S. aureus and to propose its use in the analysis of foods, as a tool for microbiological quality monitoring. For this, a multiplex qPCR was standardized for the simultaneous quantification of specific fragments of target genes (ssf, phoA, and nuc) corresponding to each one of the mentioned bacteria. The limit of detection of the technique was 13, 10, and 12 gene copies for ssf, phoA, and nuc, respectively; standard curves showed R2 > 0.99, with efficiencies ranging from 99 to 110%, and inter- and intraexperiment reproducibility presented a low coefficient of variation in all trials. This methodology was applied in different food matrices (milk, ground beef, and oyster meat), and the results were compared with official microbiological culture methodology and with ready-to-use test. Advantages and disadvantages of each methodology used in this study are pointed out. We suggest that this multiplex qPCR can be used as a rapid screening technique for the analysis of food microbiological quality.

The objective of this study was to standardize the high-resolution melting method for identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by amplification of 18S ribosomal DNA (rDNA) using a single primer pair. The analyses were performed on individual reactions (containing DNA from a single species of a protozoan), on duplex reactions (containing DNA from two species of protozoa in each reaction), and on a multiplex reaction (containing DNA of four parasites in a single reaction). The proposed method allowed us to identify and discriminate the four species by analyzing the derivative, normalized, and difference melting curves, with high reproducibility among and within the experiments, as demonstrated by low coefficients of variation (less than 2.2% and 2.0%, respectively). This is the first study where this method is used for discrimination of these four species of protozoa in a single reaction.

One of the greatest challenges in poultry production is maintaining intestinal mucosal barrier integrity and gut microbiota balance. Safe alternative antimicrobials that can regulate the microbial community through animal feed have been the subject of research in poultry production. This study evaluated the effect of Mentha piperita and Melaleuca alternifolia essential oils (EOs) on the gut microbiome and morphometry of broiler quails under normal feeding conditions. The gut microbiome was studied using a completely randomized design consisting of 4 treatments, namely control, bacitracin zinc, and the Eos M. piperita and M. alternifolia, with 8 repetitions and 7 quails per treatment, totaling 224 quails from 1 to 42 days old. The intestinal contents of the slaughtered quails were collected to evaluate the gut microbiome profile of their digestive tract. Gut morphometry was analyzed using a completely randomized factorial design, with four experimental rations for three intestinal sections (4x3) and five replications. The variables studied were villus surface area and height, crypt depth, villus height to cryptdepth ratio (VH:CD), villus-crypt ratio (V:C), villus width to height ratio (VW:H), and height of the intestinal epithelium and musculature. M. alternifolia (50 mg/kg of feed) in the diet of broiler quails improved gut morphometry, similar to the results obtained with bacitracin zinc. This EO also altered the gut microbiome of quails and reduced pathogenic bacterial diversity. RESUMO: Um dos maiores desafios na produção de aves é manter a integridade da barreira da mucosa intestinal e o equilíbrio da microbiota intestinal. Antimicrobianos alternativos seguros, que podem regular a comunidade microbiana através da alimentação animal, têm sido objeto de pesquisa na produção avícola. O objetivo deste estudo foi avaliar o efeito dos óleos essenciais (OEs) Mentha piperita e Melaleuca alternifolia no microbioma intestinal e na morfometria de codornas de corte em condições normais de alimentação. O microbioma intestinal foi estudado em delineamento inteiramente casualizado com 4 tratamentos: controle, bacitracina zinco e os OEs M. piperita e M. alternifolia, com 8 repetições, por tratamento,com sete codornas totalizando 224 aves no período de um a 42 dias. O conteúdo intestinal das codornas abatidas foi coletado para avaliar o perfil do microbioma intestinal de seu trato digestivo. A morfometria intestinal foi estudada usando um delineamento fatorial inteiramente casualizado, consistindo de quatro rações experimentais para três secções intestinais (4x3), com cinco repetições. As variáveis estudadas foram área das vilosidades intestinais, altura das vilosidades intestinais, profundidade da cripta intestinal, relação entre a altura das vilosidades e a profundidade das criptas, relação cripta e vilosidades intestinais, relação largura e altura das vilosidades intestinais, altura do epitélio intestinal e altura da musculatura intestinal. A utilização do OE M. alternifolia (50 mg/kg de ração) na dieta de codornas de corte resultou em melhora da morfometria intestinal, semelhante aos resultados obtidos com bacitracina zinco. Além disso, alterou o microbioma intestinal de codornas e reduziu a diversidade bacteriana patogênica.

This study aimed to identify Leishmania species in small non-flying mammals captured in semi-deciduous forest fragments of the Atlantic Forest and pastures in the Southwest region of Bahia state, Northeast Brazil. A total of 445 animals belonging to 11 different species were captured, the majority being rodents (75.7%; 337), followed by marsupials (24.2%; 108), and the most prevalent species were Cerradomys vivoi, Calomys expulsus, Necromys Lasiurus, and Marmosops incanus. Liver, spleen, kidney, heart, and lung fragments were collected for subsequent molecular diagnosis. Leishmania spp. kDNA amplification in positive samples was performed using real-time polymerase chain reaction (qPCR). Species identification of Leishmania was conducted through nested PCR, followed by sequencing. Leishmania spp. infection was detected in 2.92% (13/445) of the animals. Sequencing revealed that L. infantum infected three animals, while the species of the agent in the other animals could not be determined. The results indicate the presence of Leishmania spp. in the studied region, primarily affecting the local wildlife. These findings not only highlight the risk of transmission to domestic animals and humans in close contact with forest remnants, but also underscore the critical role of these fragments in supporting native fauna. However, it is worth noting that the continuous deforestation of these forest remnants could lead to increased contact between wildlife, domestic animals, and humans, thereby elevating the risk of transmission.

Wild animals have an ecological function and can serve as sentinels to identify infectious agents and as indicators of environmental health. Among the zoonotic pathogens, Salmonella spp. deserve special attention due to their high worldwide prevalence and their ubiquity of hosts. With the aim of investigating the presence of Salmonella spp. in wild birds from the Atlantic Forest in southern Bahia, Brazil, we collected 114 fecal samples of wild birds (14 families) between 2016 and 2017. Fecal samples were collected by means of cloacal swab and subjected to microbiological culture to isolate and serotype Salmonella spp. specifically. Antibiotic susceptibility was determined using the disk diffusion test protocol. Only one bird, Ceratopipra rubrocapilla, tested positive for Salmonella enterica subsp. enterica serotype Agona, which is the first record for this bird species. This isolate exhibited intermediate sensitivity to amikacin and gentamicin and sensitivity to the other 13 antibiotics tested. Results may indicate environmental preservation since the studied areas had minimal human activity and good sanitary quality. Despite the low prevalence, it is necessary to monitor wildlife and establish disease control and surveillance systems, especially for zoonotic diseases.

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), challenged public health systems worldwide. Individuals in low-income countries/regions are still at individual and community risk concerning inequality, sanitation, and economic conditions. Besides, during the pandemic, the transmission in municipalities and communities in the countryside and less developed regions kept viral spread and required structured and strengthened clinical and laboratory surveillance. Here, we present an observational, analytic, cross-sectional study conducted using secondary data from the Laboratório de Farmacogenômica e Epidemiologia Molecular (LAFEM)-Universidade Estadual de Santa Cruz (UESC), to evaluate individual and community factors associated to SARS-CoV-2 infection in outpatients from different cities from Southern Region of Bahia State, in Brazil. The data were collected between June 2021 and May 2022. The SARS-CoV-2 positivity by RT-qPCR was correlated with low socio-economic indicators, including the Human development index (HDIc) and Average worker salary (AWSc). Besides, in general, females were less likely to test positive for SARS-CoV-2 (OR = 0.752; CI 95% 0.663–0.853; p < 0.0001), while brown individuals had more positivity for infection (p < 0.0001). In addition, those who had clinical symptoms were more likely to test positive for SARS-CoV-2 (OR = 6.000; CI 95% 4.932–7.299; p < 0.0001). Although dry cough, headache, and fever were the most frequent, loss of taste (OR = 5.574; CI 95% 4.334–7.186) and loss of smell (OR = 6.327; CI 95% 4.899–8.144) presented higher odds ratio to be positive to SARS-CoV-2 by RT-qPCR. Nonetheless, the distribution of these characteristics was not homogenous among the different cities, especially for age and gender. The dynamic of SARS-CoV-2 positivity differed between cities and the total population and reinforces the hypothesis that control strategies for prevention needed to be developed based on both individual and community risk levels to mitigate harm to individuals and the health system.

Practical methodologies that include food safety and hygiene education in pedagogical activities are strategies to prevent foodborne diseases (FBDs). Thus, the aim of this study was to investigate the knowledge of 7th‐grade middle school students regarding food microbiology and food safety, and to apply workshop‐based educational strategies that focus on scientific literacy. The students (144) were initially evaluated using a Likert‐scale questionnaire (pre‐intervention, Q0) with ten objective questions on microbiology and food safety. Once the questionnaire was evaluated, interventions were conducted through five science workshops of 50 min, over a period of 5 months. The workshops included educational games, laboratory practices, videos, and lectures that addressed microorganisms that are known to cause the most common FBDs in Brazil. After each workshop, students were asked to express their opinions and understanding of the content through semi‐structured interviews. Six months after the end of the practical interventions, the students completed a second identical Likert‐scale questionnaire (post‐intervention, Q1), and the answers to both questionnaires (Q0 and Q1) were analyzed by calculating the middle rank. The middle rank of Q1 (mean = 0.65 ± 0.13) was 21% greater than the middle rank of Q0 (mean = 0.44 ± 0.16), and statistical significance was observed ( p  = .0135). This demonstrates that new information acquired during the workshops positively influenced learning. We believe that when practical approaches to food safety are included in school education as a priority, the prevalence of FBD will decrease.

Microbial bioprospecting in contaminated environments is a promising strategy for identifying biosurfactant-producing bacteria; however, translating environmentally adapted strains into predictable cultivation processes remains challenging. In this study, a microbial consortium subjected to long-term evolutionary laboratory adaptation in oily sludge was investigated to evaluate strain-specific phenotypic responses related to biosurfactant production. Phylogenetic analysis based on 16S rDNA sequencing identified three taxonomically distant isolates: Faucicola sp. strain BS5C, Pseudomonas sp. strain BS16B, and Enterobacter sp. BS14MR. Biosurfactant production was evaluated using a sequential Design of Experiments (DOE) approach, including fractional factorial and central composite rotatable designs, with the emulsification index (E24) used as a semi-quantitative response variable. Initial screening revealed a statistically significant negative effect (p < 0.10) of high dextrose concentrations for all isolates. Strain-specific differences in model adequacy were observed, with a statistically adequate quadratic model obtained for Pseudomonas sp. BS16B (R2 = 0.8658, p = 0.0225), whereas the other isolates showed significant lack of fit (p < 0.05). ATR-FTIR analysis revealed spectral profiles consistent with lipopeptide-like compounds. Overall, these results indicate that isolates derived from the same long-term adapted system may differ substantially in process predictability, suggesting that productivity-based screening alone may be insufficient for selecting robust strains.

In December 2019, a novel coronavirus was detected in Wuhan, China, and rapidly spread worldwide. In Brazil, to date, there have been more than 20,000,000 confirmed cases of COVID-19 and more than 550,000 deaths. The purpose of the current study was to determine the clinical and epidemiological profile of the population affected by COVID-19 that have attended referral hospitals in Southern region of Bahia State, to better understand the disease and its risk factors in order to enable more appropriate conduct for patients. An observational, descriptive, cross-sectional, exploratory study was conducted using secondary data collected from the Laboratório de Farmacogenômica e Epidemiologia Molecular, Universidade Estadual de Santa Cruz (LAFEM/UESC). Chi-squared and Fisher’s exact tests were applied to determine the association between clinical symptoms and laboratory results, and to identify risk factors associated with SARS-CoV-2 infection. A total of 3135 individuals with suspected severe respiratory illness were analyzed and 41.4% of them tested positive for SARS-CoV-2 infection. Male individuals and having comorbidities were risk factors significantly associated with SARS-CoV-2 infection (OR = 1.17 and OR = 1.37, respectively). Interestingly, being a healthcare professional was a significantly protective factor (OR = 0.81, p < 0.001). Our findings highlight the importance of routinely testing the population for early identification of infected individuals, and also provide important information to health authorities and police makers to improve control measures, management, and screening protocols.

Strains of diarrheagenic Escherichia coli (DEC) are involved in foodborne disease outbreaks worldwide, especially the enterohemorrhagic E. coli O157:H7. This study describes two multiplex quantitative real time PCR (qPCR) assays for simultaneous identification and quantification of genes related to virulence of DEC; a triplex reaction for detection and quantification of stxA1, stxA2, and eaeA genes, and a duplex reaction for detection and quantification of eaeA and virA genes. The technique was applied in raw oyster samples for direct quantification of DEC, thereby evaluating the applicability of this methodology for microbiological quality assessment of food. Using custom designed primers and specific MGB probes, a triplex qPCR assay was performed to quantify stxA1, stxA2, and eaeA, and a duplex reaction was performed to quantify virA and eaeA genes. The assays showed high sensitivity, with the detection limit varying between 5 and 17 copies of the genes. The coefficient of determination (R2) of the standard curves was 0.99. The coefficient of variation was < 1% indicated high intra- and inter-assay reproducibilities. The application of this methodology in oyster samples from tropical environment provided direct quantitative data that determined the presence of the genes stxA1 (32.1%), eaeA (28.6%), stxA2 (3.6%), and virA (3.6%). This would prove critical for immediate intervention of control strategies, particularly in oysters that are often ingested as raw food.