quantification-violacein-chromobacterium-violaceum-its-inhibition

Quantification of Violacein in <em>Chromobacterium violaceum</em> and Its Inhibition by Bioactive Compounds

Bacteria communicate through a system known as quorum sensing (QS), which allows them to coordinate their behavior in response to changes in cell density. ...

University of São Paulo (USP)

We describe the characterization of the centesimal composition, mineral and phenolic content of Eugenia uniflora fruit and the determination of the antioxidant, antimicrobial and quorum quenching activities of the pulp phenolic extract. Centesimal composition was determined according to standard methods; trace elements were measured by total reflection X-ray fluorescence spectroscopy. The phenolic compounds were extracted by solid-phase chromatography and quantified by spectrophotometry. Antioxidant activity was determined by using 3 different methods. Antimicrobial activity was evaluated against a panel of foodborne microorganisms and antiquorum sensing activity in Chromobacterium violaceum was performed by measuring inhibition of quorum sensing dependent violacein production. The centesimal composition (per 100 g of pulp) was as follows: protein 3.68 ± 0.21 g, lipids 0.02 ± 0.03 g, carbohydrates 10.31 g and fiber 2.06 g. Trace elements (mg/g of pulp) were determined as: K 0.90, Ca 3.36, Fe 0.60, Zn 0.17, Cl 0.56, Cr 0.06, Ni 0.04, and Cu 0.07. The pulp is a source of phenolic compounds and presents antioxidant activity similar to other berries. The fruit phenolic extract inhibited all tested bacteria. We also found that the fruit phenolic extract at low subinhibitory concentrations inhibited up to 96% of violacein production in C. violaceum, likely due to the fruit's phenolic content. This study shows the contribution of E. uniflora phenolic compounds to the antioxidant, antimicrobial and the newly discovered quorum quenching activity, all of which could be used by the food and pharmaceutical industries to develop new functional products.

Quorum Quenching and Microbial Control through Phenolic Extract of Eugenia Uniflora Fruits.

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.

Dual-species biofilm of Listeria monocytogenes and Escherichia coli on stainless steel surface.

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.

Challenges of teaching food microbiology in Brazil.

This study aimed to assess the foodborne diseases (FBD) outbreaks reported in Brazil between 2000 and 2018, based on data from the Brazilian Ministry of Health (official data) and from scientific literature. According to official data, 13,163 FBD outbreaks were reported in the country during this period, involving 247,570 cases and 195 deaths. The largest prevalence of FBD outbreaks was observed in the Southeast region of Brazil (45.6%). In most outbreaks it was not possible to determine the food implicated (45.9%) but among those identified, water was the most frequently associated (12.0%). The etiological agent was not identified in most outbreaks (38.0%), while  (14.4%) was the most frequently reported among those identified. Homes were the main site of FBD occurrence (12.5%). Regarding data obtained from the scientific literature, 57 articles dealing with FBD in the country throughout the same period were selected and analyzed. Based on these articles, mixed foods were the most prevalent in the outbreaks (31.6%),  spp. was the pathogen most frequently reported (22.8%) and homes were also the main site of FBD occurrence (45.6%). Despite under-notification, the records of FBD outbreaks that have occurred in Brazil in the past recent years show alarming data, requiring attention from health authorities. The notification of outbreaks is essential to facilitate public health actions.

Overview of Foodborne Disease Outbreaks in Brazil from 2000 to 2018.

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.

Genomic characterization of Staphylococcus aureus from Canastra Minas Artisanal Cheeses.

Minimally processed vegetables (MPVs) are marketed as convenient and healthy choices for consumers. However, the absence of post-commercialization treatments raises concerns about their microbiological safety. This study investigated the processing practices of 28 Brazilian MPV plants and compared the microbiological quality of these products with fresh counterparts in the city of Sao Paulo, Brazil. Through cluster analysis, the processing plants were categorized into two groups: group 1 (nineteen plants) primarily uses chemical substances in the washing step, while group 2 (nine plants) avoids chemical use but employs similar rinsing practices. Microbiological analysis of 100 samples (49 unprocessed and 51 MPVs) revealed no significant differences in microbial group counts (Enterobacteriaceae, coliforms, and E. coli) between the in natura (unprocessed) and MPV products. However, the prevalence of E. coli was higher in natura vegetables than in MPVs. The results indicated the presence of Salmonella DNA (from either dead or live cells or residual DNA) in 4 samples (3 in natura and 1 MPV) using conventional PCR, suggesting the presence of the pathogen in these samples. Listeria monocytogenes was absent, but Listeria innocua was found in two unprocessed products. The study suggests that certain MPVs have microbial loads similar to unprocessed vegetables, potentially serving as carriers for pathogen transmission. These findings emphasize the importance of understanding practices in Brazilian MPV processing plants, informing the implementation of control measures to improve MPV safety and shelf-life, thus ensuring microbiological safety.

Investigating processing practices and microbiological quality of minimally processed vegetables in Brazil.

Bacteria coordinate gene expression in a cell density-dependent manner using a communication process called quorum sensing (QS). The expression of virulence factors, biofilm formation and enzyme production are examples of QS-regulated phenotypes that can interfere with food quality and safety. Due to the importance of these phenotypes, the inhibition of bacterial communication as an anti-virulence strategy is of great interest. This work aimed to evaluate the effect of phenolic compounds on the inhibition of biofilm formation by Pseudomonas aeruginosa PAO1, using concentrations that do not interfere in bacterial growth. The synergistic effect of rosmarinic acid, baicalein, curcumin and resveratrol with tobramycin and between the phenolics themselves was evaluated. The tested combinations proved to be a good strategy for reducing the dose of antibiotics used in treatments and obtaining satisfactory results against P. aeruginosa biofilms. The combination of the four compounds at the highest concentration (500 μM) completely inhibited biofilm formation. The obtained results contribute to understanding the effect of phenolic compounds on QS inhibition, which may help to define the mechanism of inhibition, in addition to expanding the biotechnological potential of these compounds for future applications in the food, pharmaceutical and medical fields.

Synergistic effect of the combination of phenolic compounds and tobramycin on the inhibition of Pseudomonas aeruginosa biofilm.

Bacteria coordinate gene expression in a cell density-dependent manner in a communication process called quorum sensing (QS). The expression of virulence factors, biofilm formation and enzyme production are QS-regulated phenotypes that can interfere in human health. Due to this importance, there is great interest in inhibiting QS, comprising an anti-virulence strategy. This work aimed to evaluate the effect of selected phenolic compounds on the inhibition of QS-regulated phenotypes in Pseudomonas aeruginosa PAO1, using concentrations that do not interfere in bacterial growth. This is one of the main premises for studying the effect of compounds on QS. Firstly, an in-silico study with the LasR and RhlR proteins of P. aeruginosa by molecular docking of 82 phenolic compounds was performed. Then, a screening with 13 selected phenolic compounds was performed, using biosensor strains P. aeruginosa lasB-gfp and P. aeruginosa rhlA-gfp, which emit fluorescence when the QS system is activated. From this assay, eight compounds were selected and evaluated for inhibition of pyocyanin, rhamnolipids, proteases, elastase, and motility. The compounds variably inhibited the evaluated virulence factors. The greatest inhibitions were observed for swarming motility, achieving inhibition rates of up to 50% for baicalein (500 µM) and curcumin (50 µM). Notably, curcumin showed satisfactory inhibition for all phenotypes even at lower concentrations (12.5 to 50 µM) compared to the other compounds (125 to 500 µM). Four compounds - rosmarinic acid, baicalein, curcumin, and resveratrol - were finally tested against biofilm formation observed by optical microscopy. This study demonstrated that phenolic compounds exhibit strong in silico binding to P. aeruginosa LasR and RhlR proteins and variably inhibit QS-regulated phenotypes in vitro. Although no biofilm inhibition was observed, future studies combining compounds and exploring molecular mechanisms are recommended. These findings highlight the biotechnological potential of phenolic compounds for future applications in the food, clinical, and pharmaceutical fields.

Article	Year
Quorum Quenching and Microbial Control through Phenolic Extract of Eugenia Uniflora Fruits. Journal of food science\| PubMed ID: 27603708	2016
Dual-species biofilm of Listeria monocytogenes and Escherichia coli on stainless steel surface. World journal of microbiology & biotechnology\| PubMed ID: 29651554	2018
Challenges of teaching food microbiology in Brazil. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]\| PubMed ID: 31267440	2020
Overview of Foodborne Disease Outbreaks in Brazil from 2000 to 2018. Foods (Basel, Switzerland)\| PubMed ID: 31547589	2019
Genomic characterization of Staphylococcus aureus from Canastra Minas Artisanal Cheeses. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]\| PubMed ID: 37594655	2023
Investigating processing practices and microbiological quality of minimally processed vegetables in Brazil. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]\| PubMed ID: 38472699	2024
Synergistic effect of the combination of phenolic compounds and tobramycin on the inhibition of Pseudomonas aeruginosa biofilm. Microbial pathogenesis\| PubMed ID: 39454803	2024
Exploring the antivirulence potential of phenolic compounds to inhibit quorum sensing in Pseudomonas aeruginosa. World journal of microbiology & biotechnology\| PubMed ID: 39794611	2025

Uelinton Manoel Pinto

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