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The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook®, represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, “Adopt a Microorganism”, was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of São Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students’ responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students’ discourse richness in microbiology even without face-to-face traditional classes.

Background International clones of multidrug-resistant Escherichia coli have been a leading cause of human and animal infections worldwide. Microbial inactivation by blue light has been proposed as an effective treatment for superficial infections and surface contamination. Aim To evaluate the inactivation efficacy of blue light irradiation against high-risk multidrug-resistant strains of E. coli. Methods Blue LED light (λ = 410 nm) was used to inactivate in vitro suspensions of colistin- broad-spectrum cephalosporin-, or carbapenem-resistant E. coli strains belonging to sequence types (STs) ST10, ST131 and ST648, carrying mcr-1, blaCTX-M or blaKPC-2 genes. Results Our results showed that all E. coli strains were susceptible to blue light irradiation, independently of antibiotic resistance and virulence profiles. In addition, blue light irradiation induced a strain-specific and dose-dependent bacterial effect. Conclusion Our results support use of blue light as a promising antimicrobial option against MDR pathogens, including high-risk clones of E. coli displaying resistance to polymyxins or broad-spectrum β-lactam antibiotics.

Capsicum peppers have not been investigated as sources of quorum sensing (QS) inhibitors. This study aimed to identify compounds in pimenta-malagueta (Capsicum frutescens) and red pepper (Capsicum annuum) extracts and to evaluate their effect on violacein production in Chromobacterium violaceum ATCC 12472 and C. violaceum CV026, as well as biofilm formation (BF) in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1. Among the extracts, pimenta-malagueta methanolic extract (PMME) was chosen because it contained capsaicin, dihydrocapsaicin, and luteolin in greater amount than the other extracts. In general, PMME partially inhibited bacterial growth at 2.5 and 5.0 mg/mL, as well as capsaicin at 100 µg/mL and luteolin at 62.5, 125, and 250 µg/mL. At lower concentrations, PMME and luteolin reduced violacein production in C. violaceum ATCC 12472 without affecting growth, a result that was not observed with capsaicin. We show that violacein inhibition by PMME is likely due to luteolin. In silico docking evaluation showed that luteolin binds to the CviR QS regulator. Crystal violet staining and confocal microscopy revealed that BF was increased by PMME and capsaicin, being remarkably superior for P. aeruginosa PAO1 at 30 °C. Capsaicin is not an effective QS inhibitor, while luteolin should be further investigated for its potential effects in QS regulated phenotypes. PRACTICAL APPLICATION: Quorum sensing (QS) is a form of bacterial communication targeted for studies aiming to inhibit bacterial virulence. QS regulates phenotypes that influence microbial activities across many areas, including Food Science. Capsicum frutescens is a type of chili pepper consumed in Brazil, rich in bioactive compounds such as capsaicin (which gives its pungency) and luteolin (a phenolic compound). We show that C. frutescens extract and luteolin inhibit QS in a model bacterium, along with the possible molecular mechanism of inhibition. Capsaicin did not inhibit QS neither biofilm formation. Luteolin should be further investigated for its QS inhibition properties and biotechnological applications.

Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E. coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.

As EPEC típicas apresentam padrão de adesão localizada (LA) em células epiteliais, formando de microcolônias compactas. As EPEC atípicas aderem no padrão de adesão localizada-like (LAL), no qual não se observa microcolônias e que ocorre tardiamente em relação à LA. O objetivo deste estudo foi caracterizar a interação de EPEC atípica que apresenta o padrão LAL com a célula epitelial. A cinética da formação da lesão A/E, característica da patogênese de EPEC, demonstrou um atraso na adesão e na formação da lesão em EPEC atípica, que correspondeu à expressão tardia de Intimina, Tir e EspA. A complementação de EPEC atípica com o regulador perABC antecipou a expressão destes fatores e a formação da lesão A/E. Não foi observada relação entre outras adesinas descritas em DEC com o padrão LAL, bem como a mobilização dos receptores eucarióticos nucleolina e b1-Integrina para o foco de adesão. Observou-se a possível interação da Intimina com um peptídeo da membrana da célula epitelial de 300 kDa, podendo tratar-se de um provável receptor para essa adesina. Typical EPEC exhibit a localized adherence (LA) pattern in epithelial cells, where tight bacterial clusters are produced. Atypical EPEC produce a localized-like (LAL) adhesion pattern, in which the compact clusters are not formed and that occurs in a later stage of the infection. The aim of this study was to characterize the interaction of LAL-adherent atypical EPEC with the epithelial cell. The kinetics of the A/E lesion, the main feature of EPEC pathology, showed a clear delay in its formation by the atypical strains, probably a consequence of the delay observed in the expression of Intimin, Tir and EspA. The complementation of atypical EPEC with the perABC regulator revealed to be effective in not only advancing the expression of these factors, as well the formation of A/E lesion. No correlation was found between the presence of different adhesins already described in DEC and the LAL pattern. The analysis of the possible interaction of Intimin with epithelial cell receptors showed the presence of a 300 kDa peptide that it seems to interact with this adhesin.

Enteropathogenic Escherichia coli (EPEC) is classified as typical (tEPEC) or atypical (aEPEC) based on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesions on the gut mucosa. We compared the kinetics of A/E lesion formation induced by aEPEC and tEPEC. The examination of infected HEp-2 cells clearly demonstrated delayed A/E lesion formation by aEPEC in comparison to tEPEC. This delay was associated with the expression of locus of enterocyte effacement (LEE)-encoded virulence factors (i.e., intimin and EspD). Indeed, the insertion of a plasmid containing perABC , a transcriptional regulator of virulence factors involved in A/E formation, into aEPEC strains increased and accelerated the formation of A/E lesions. Interestingly, the enhanced expression and translocation of LEE-encoded proteins, such as those expressed in LEE5 (intimin) and LEE4 (EspD), in aEPEC ( perABC ) was independent of bacterial adhesion. The secretion kinetics of these two proteins representing LEE5 and LEE4 expression correlated with A/E lesion formation. We conclude that the lack of Per in the regulation network of virulence genes is one of the main factors that delay the establishment of A/E lesions induced by aEPEC strains.

Autotransporter (AT) protein-encoding genes of diarrheagenic Escherichia coli (DEC) pathotypes ( cah , eatA , ehaABCDJ , espC , espI , espP , pet , pic , sat , and tibA ) were detected in typical and atypical enteropathogenic E. coli (EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

Aims: The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle-forming pilus (BFP) expression. Methods and Results: Anti-BFP antiserum was raised in rabbits and its reactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl2, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. Conclusion: The assay enables reliable identification of BFP-expressing isolates and contributes to the differentiation of typical and atypical EPEC. Significance and Impact of the Study: The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx(2v-ha) and stx(2vb-hb)). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

O queijo Minas Artesanal da Canastra é produzido na Serra da Canastra (MG), utilizando leite cru, coalho e pingo, que é uma cultura endógena natural de cada queijaria. Devido ao uso de leite cru, o produto pode veicular microrganismos causadores de doenças veiculadas por alimentos, como Staphylococcus aureus. A caracterização molecular é uma ferramenta importante para avaliar a população microbiana do alimento e direcionar a aplicação de medidas de controle na produção. Este estudo caracterizou a diversidade genética, o potencial de virulência e determinou o perfil de susceptibilidade a antimicrobianos de S. aureus isolados de queijos produzidos na Serra da Canastra. Para o estudo transversal foram analisados 248 isolados de queijos que tinham um tempo de maturação de 22 dias, provenientes de 83 propriedades. Por outro lado, no estudo longitudinal foram analisados outros 197 isolados coletados ao longo do processo de maturação, provenientes de três propriedades. Os isolados foram submetidos a testes bioquímicos para confirmação do gênero e para a confirmação da espécie de S. aureus, foi identificado o gene nuc por meio da técnica de PCR. Além disso, foi pesquisado o gene mecA para a detecção de S. aureus Resistente a Meticilina (MRSA). Após os testes de confirmação, 144 isolados do estudo transversal e 159 do estudo longitudinal foram positivos para o gene nuc, específico para S. aureus. Posteriormente, o perfil clonal foi determinado por Eletroforese de Campo Pulsado (PFGE) utilizando a enzima SmaI e tipagem do locus agr por PCR multiplex. A análise por PFGE foi realizada no programa BioNumerics. A técnica PCR foi realizada para identificar a presença de genes que codificam a produção de hemolisinas, toxina TSST-1, enterotoxinas SEs (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEO, SEM), formação de biofilme e Componentes Microbianos de Superfície que Reconhecem a Matriz de Moléculas Adesivas (MSCRAMMs). Os isolados foram submetidos ao teste de susceptibilidade a antimicrobianos por disco de difusão. Por último, a formação de biofilme em microplaca de 96 poços, em caldo TSB a 37°C, foi verificada pela metodologia de Cristal Violeta. O gene mecA foi detectado em 1,9% dos 445 isolados. A tipagem agrrevelou que 83 (27,4%) dos isolados são do tipo agr-I, 95 (31,4%) agr-II e 43 (14,2%) agr-III, sendo que não foram detectados isolados classificados como agr-IV. A tipagem por PFGE revelou um total de 54 perfis. Assim, um isolado representativo de cada perfil foi utilizado nos demais testes que mostraram a presença dos genótipos spa mais frequentes t127 e t605 (20,58%); t002 (14,70%), seguidos pelos tipos t267 (8,82%); t1234 e t693 (5,8%) e t021, t177, t306, t321, t359, t442, t521, t693 e t5493 (2,9%). Além disso, encontramos a presença dos genes do grupo SEs, sea 1 (1,8%), seh 11 (20,3%), sei 10 (18,5%), sej 7 (12,9%), seg e seo 14 (25,9%), sem 8 (14,8%), e os genes seb, sec, sed, see e tst não foram detectados. Para os genes das hemolisinas, hla foi positivo em todos os isolados e hlb foi positivo em 53 (98,1%) isolados. Os genes positivos para MSCRAMMS foram fnbA, fnbB 18 (33,3%), clfA, clfB e eno 53 (98,1%), fib 44 (81,4%), bbp 4 (7,4%), cna 17 (31,4%) e ebps 10 (18,5%). Por último, os genes de formação de biofilme icaA e icaD estiveram presentes em 38 (70,3%) e 25 (46,2%) dos isolados, respectivamente. Na avaliação de susceptibilidade a antibióticos dos 54 isolados escolhidos, 25 (46,3%) apresentaram maior resistência a penicilina e 13 (24,0%) a tetraciclina. Em menor porcentagem (1,8%), 1 isolado cada foi resistente a eritromicina, cefoxitina, clindamicina, gentamicina, cotrimazol, azitromicina e trimetropim. Além disso, 8 isolados (14,8%) apresentaram resistência intermediaria a tetraciclina, 3 (5,5%) a gentamicina e 1 (1,8%) a tobramicina. No teste para a determinação da formação de biofilme por cristal violeta, 13,7%, foram classificados em isolados não formadores, 60,8% em fracamente formadores, 25,5% moderadamente formadores e nenhum como fortemente formador. A alta diversidade de cepas de S. aureus observada neste estudo mostrou que existem vários tipos de linhagens circulando na região da Canastra. A caracterização revelou uma elevada frequência de genes de virulência e que mais estudos são necessários para avaliar o potencial de produção de enterotoxinas nos queijos artesanais. A melhora dos procedimentos de higienização durante todas as etapas de produção pode ser uma solução para a redução dos níveis de contaminação por S. aureus. Canastra Minas Artesanal cheese is produced in Serra da Canastra (MG), using raw milk, rennet and a natural endogenous culture called pingo. Due to the use of raw milk, the product can carry microorganisms that cause foodborne diseases, such as Staphylococcus aureus. Molecular characterization is an important tool to assess the microbial population of food and guide the application of control measures in production. This study characterized the genetic diversity, virulence potential and determined the antimicrobial susceptibility profile of S. aureus isolated from cheeses produced in Serra da Canastra. A total of 248 isolates from 22 days ripened cheeses were obtained from 83 properties (cross sectional study). Another 197 isolates were collected during maturation (longitudinal study), in three properties. The isolates were submitted to biochemical tests to confirm the genus and to confirm the S. aureus species, the nuc gene was identified by PCR. In addition, the detection of mecA gene was performed for the detection of Methicillin Resistant S. aureus (MRSA). After confirmation tests, 144 isolates from the cross-sectional study and 159 from the longitudinal study were positive for the nuc gene, specific for S. aureus. Subsequently, the clonal profile of the isolates was determined by Pulsed Field Gel Electrophoresis (PFGE) using the SmaI enzyme and typing of the agr locus by multiplex PCR. PFGE analysis was performed using the BioNumerics program. PCR was performed to identify the presence of genes encoding the production of hemolysins, TSST-1 toxin, enterotoxins SEs (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEO, SEM), biofilm formation and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The isolates were submitted to the antimicrobial susceptibility test by disc diffusion. Finally, biofilm formation in a 96-well microplate in TSB broth at 37°C was verified by the Cristal Violeta method. The mecA gene was detected in 1.9% of the 445 isolates. Agr typing revealed that 83 (27.4%) of the isolates are agr-I, 95 (31.4%) agr-II and 43 (14.2%) agr-III, and no isolate was classified as agr-IV. PFGE typing revealed a total of 54 profiles. Thus, a representative isolate of each profile was used in the other tests that showed the presence of the most frequent spagenotypes t127, t605 (20.58%); t002 (14.70%), followed by types t267 (8.82%); t1234, t693 (5.8%) e t021, t177, t306, t321, t359, t442, t521, t693 and t5493 (2.9%). In addition, we found the presence of the genes of the SEs group: sea 1 (1.8%), seh 11 (20.3%), sei 10 (18.5%), sej 7 (12.9%), seg and seo 14 (25.9%), sem 8 (14.8%), while seb, sec, sed, see and tst genes were not detected. For hemolysin genes, hla was positive in all isolates and hlb was positive in 53 (98.1%) isolates. The positive genes for MSCRAMMS were: fnbA, fnbB 18 (33.3%), clfA, clfB e eno 53 (98.1%), fib 44 (81.4%), bbp 4 (7.4%), cna 17 (31.4%) and ebps 10 (18.5%). Finally, the biofilm formation genes icaA and icaD were present in 38 (70.3%) and 25 (46.2%) of the isolates, respectively. In the evaluation of antibiotic susceptibility of the 54 isolates, 25 (46.3%) showed greater resistance to penicillin and 13 (24.0%) to tetracycline. In a lower percentage (1.8%), 1 isolate each was resistant to erythromycin, cefoxitin, clindamycin, gentamicin, contrimazole, azithromycin and trimethoprim. In addition, 8 isolates (14.8%) showed intermediate resistance to tetracycline, 3 (5.5%) to gentamicin and 1 (1.8%) to tobramycin. In the test for the determination of biofilm formation by crystal violet, 13.7% were classified as non-forming isolates, 60.8% as weakly forming, 25.5% moderately forming and none as strongly forming. The high diversity of S. aureus strains observed in this study showed that there are several types of strains circulating in the Canastra region. The characterization revealed a high frequency of virulence genes and that further studies are needed to assess the potential for enterotoxin production in artisanal cheeses. The improvement of hygiene procedures during all stages of production can be a solution for reducing the levels of contamination by S. aureus.

Influência dos sistemas de Quorum Sensing AI-1/AI-2/AI-3 nos fatores de virulência de EPEC atípica de origem animal. A secreção de moléculas sinalizadoras de baixo peso molecular que se acumulam no meio extracelular até atingir um limite crítico de concentração para sua detecção, acarretando em sinalizações intracelulares e respostas efetoras, define o sistema de comunicação bacteriano chamado Quorum Sensing. Este sistema, que regula comportamentos coletivos mostrou-se um mecanismo disseminado em diversas espécies bacterianas. Diversos estudos descreveram a existência de pelo menos 3 sistemas relacionados ao Quorum Sensing em bactérias Gram-negativas, LuxIR/AI-1, LuxS/AI-2 e QseBC/AI-3/Epinefrina/Norepinefrina. Fatores de virulência como formação de biofilme, motilidade e adesão ao epitélio do hospedeiro devem ser controlados de maneira adequada para tirar o melhor proveito da situação em que a bactéria se encontra. Este trabalho teve como objetivo analisar a influência e a correlação dos genes luxS, qseC e sdiA, relacionados ao sistema de comunicação bacteriana Quorum Sensing, nos principais fatores de virulência de uma amostra de EPEC atípica de origem animal. Foram construídos mutantes pela metodologia baseada na recombinação homóloga mediada pelo sistema Lambda Red, que foram submetidos a ensaios fenotípicos. A sinalização por AI-2 e luxS desempenham papéis na motilidade, formação de biofilme e adesão a células epiteliais. QseC influencia a produção de biofilme pela regulação de componentes da matriz extracelular, e participa de processos de motilidade e adesão. O hormônio epinefrina contribui na alteração de processos de motilidade, formação de biofilme e desenvolvimento da lesão A/E. O gene sdiA também tem um papel importante na regulação de fatores de virulência, mesmo na ausência de AHL. Uma interação antagônica parece existir entre os genes qseC e luxS. A ausência de sistemas de Quorum Sensing promove a produção de um biofilme robusto na amostra AP155. The secretion of low molecular weight signaling molecules that accumulate in the extracellular medium until reaching a critical concentration limit for its detection, leading to intracellular signaling and effector responses, defines the bacterial communication system called Quorum Sensing. This system regulates collective behaviors and has been proven to be a widespread mechanism in several bacterial species. Several studies have described the existence of at least 3 Quorum Sensing-related systems in Gram-negative bacteria, LuxIR/AI-1, LuxS/AI-2 and QseBC/AI-3/Epinephrine/Norepinephrine. Virulence factors such as biofilm formation, motility and adhesion to the host epithelium should be adequately regulated to make the most of the situation in which the bacterium is found. The aim of this work was to analyze the influence and correlation of luxS, qseC and sdiA genes, related to the bacterial communication system Quorum Sensing, on the main virulence factors of an atypical EPEC strain isolated from an animal. Mutants were obtained through homologous recombination mediated by the Lambda Red system, and then were submitted to phenotypic assays. AI-2 signaling and luxS play roles in motility, biofilm formation, and adhesion to epithelial cells. QseC influences the production of biofilm by the regulation of components of the extracellular matrix, and participates in the processes of motility and adhesion as well. The epinephrine hormone alters the processes of motility, biofilm formation and development of the A/E lesion. The sdiA gene also plays an important role in the regulation of virulence factors, even in the absence of AHL. An antagonistic interaction seems to exist between the qseC and luxS genes. The absence of Quorum Sensing systems promotes the production of a robust biofilm in the AP155 strain.

A inibição do quorum sensing (QS) altera a comunicação bacteriana, reduzindo a expressão de fatores de virulência e a formação de biofilmes, o que pode conferir menor pressão seletiva em comparação aos antibióticos tradicionais. As frutas e hortaliças constituem uma fonte rica em compostos com propriedades potenciais de inibição do QS. Entretanto, há pouca referência sobre o potencial de pimentas do gênero Capsicum e de seus compostos isolados como inibidores do QS. Esse trabalho teve como objetivo avaliar o efeito de extratos orgânicos obtidos das variedades de pimenta-malagueta e pimentão vermelho sobre o sistema QS dependente do sinalizador AI-1 (acil homoserina lactona - AHL) em bactérias Gram-negativas. Os extratos foram obtidos por extração em fase sólida e separados em uma fração metanólica e outra amônica; sendo os compostos característicos identificados e quantificados por cromatografia líquida de alta eficiência (CLAE). A atividade antimicrobiana dos extratos foi avaliada pela determinação da concentração inibitória mínima (MIC) e pela curva de crescimento de Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 e Pseudomonas aeruginosa PAO1. O efeito anti-QS dos extratos foi avaliado pelos testes de difusão em ágar e quantificação da produção de violaceína em meio líquido por C. violaceum e sobre a formação de biofilme, avaliado pelo ensaio de cristal violeta e microscopia confocal, em S. liquefaciens e P. aeruginosa nas temperaturas 30 ºC e 37 ºC. Os resultados obtidos pela CLAE indicaram que o extrato metanólico de pimenta-malagueta (EMPM) continha capsaicinoides como a capsaicina e dihidrocapsaicina, luteolina e outros compostos não identificados; já o extrato amônico desta não continha os compostos capsaicinoides. Ambos os extratos de pimentão vermelho continham luteolina e compostos não identificados, mas não apresentaram capsaicinoides. Como o EMPM era representativo dos demais extratos, por conter tanto capsaicinóides quanto luteolina, o foco deste trabalho foi avaliar os efeitos do EMPM sobre fenótipos microbianos nas concentrações 5; 2,5; 1,25 e 0,625 mg/ml, além de utilizar a capsaicina como controle comparativo em concentrações equivalentes às do extrato (25, 50 e 100 µg/ml). Os resultados da atividade antimicrobiana mostraram inibição parcial do crescimento das bactérias nas concentrações sub-MIC (MIC >5 mg/ml) de 5 e 2,5 mg/ml de EMPM. A capsaicina também inibiu parcialmente o crescimento das bactérias a 100 µg/ml, com exceção de S. liquefaciens a 37 ºC, cujo crescimento foi induzido em 50 e 25 µg/ml. A produção de violaceína foi reduzida pelo EMPM a 1,25 e 0,625 mg/ml, sem afetar o crescimento de C. violaceum. Ensaios com C. violaceum CV026, estirpe biosensora capaz de produzir o pigmento na presença de AI-1 exógeno, sugerem que o possível mecanismo de atuação do extrato sobre o sistema QS em C. violaceum 12472 é sobre a síntese do sinalizador, já que não foi observada inibição da produção de violaceína em CV026 pelo extrato. Contrariamente, a capsaicina incrementou a produção do pigmento na estirpe 12472, mas ensaios com a estirpe CV026 indicaram que a capsaicina não atua como sinalizador do QS, uma vez que esta não induziu a produção de violaceína nesta estirpe. Já a formação de biofilme foi incrementada na presença do EMPM, sendo consideravelmente maior em P. aeruginosa a 30 ºC. Igualmente, observou-se indução da formação de biofilme por capsaicina em S. liquefaciens (37 ºC) e P. aeruginosa (30 ºC). Porém, a capsaicina não teve efeito sobre a formação de biofilme de S. liquefaciens quando cultivada a 30 ºC, nem P. aeruginosa a 37 ºC. Os resultados revelam que a produção de violaceína em C. violaceum ATCC 12472 é inibida pelo EMPM, mas não pela capsaicina. Já, o EMPM e a capsaicina, de forma geral, não inibem a formação de biofilme de S. liquefaciens MG1 nem P. aeruginosa PAO1. Outros estudos são necessários para elucidar os mecanismos pelos quais o EMPM e a capsaicina agem sobre os fenótipos avaliados neste trabalho. Quorum sensing inhibition alters bacterial communication by reducing virulence factors expression and biofilm formation, exerting less selective pressure compared to antibiotics. Fruits and vegetables are rich sources of compounds with potential QS-inhibition properties. However, there are few references about the potential of peppers belonging to the genus Capsicum and its isolated compounds as QS inhibitors. This study aimed to assess the effect of organic extracts obtained from Capsicum varieties, pimenta-malagueta (red chili) and pimentão vermelho (red bell pepper), on the AI-1 dependent QS system. The extracts were obtained by solid phase extraction and split into a methanolic and an ammonic fraction. Characteristic compounds were identified and quantified by high performance liquid chromatography (HPLC). The antimicrobial activity of the extracts was assessed by determining the minimal inhibitory concentration (MIC) and the growth curve of Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 and Pseudomonas aeruginosa PAO1. The anti-QS effect of the extracts was evaluated by the agar diffusion assay and the quantification of violacein production was assessed in liquid medium by C. violaceum, as well as in the biofilm formation test determined by the crystal violet assay and confocal microscopy with S. liquefaciens and P. aeruginosa at 30 ºC and 37 ºC. HPLC results showed that the methanolic extract of pimenta-malagueta (EMPM) contained capsaicinoids such as capsaicin and dihidrocapsaicin, luteolin and other unidentified compounds in lower concentrations; while its ammonic extract did not have capsaicinoids. Both pimentão vermelho extracts contained luteolin and other unidentified compounds in low concentrations, but they did not contain capsaicinoids. As EMPM was representative among the extracts because it contained capsaicinoids and luteolin, the focus of this work was to assess the effect of EMPM over microbial phenotypes at concentrations of 5, 2.5, 1.25 and 0.625 mg/ml, using capsaicin as a comparative control at equivalent concentrations to those in EMPM (25, 50 and 100 µg/ml). Antimicrobial activity assays showed a partial inhibition growth of bacteria at sub-MIC concentrations (MIC >5 mg/ml) of EMPM at 5 and 2.5 mg/ml. Similarly, capsaicin partially inhibited bacterial growth at 100 µg/ml, except for S. liquefaciens at 37 ºC in which growth was induced at 50 and 25 µg/ml. Violacein production was reduced by EMPM at 1,25 and 0,625 mg/ml without affecting C. violaceum growth. Assays with C. violaceum CV026, a biosensor strain that produces violacein in the presence of exogenous AI-1, suggest that EMPM reduced violacein production in C. violaceum 12472 by interfering with the AI-1 synthesis. In contrast, capsaicin incremented violacein synthesis in strain 12472, but experiments with strain CV026 revealed that capsaicin does not function as an analog of AI-1. Biofilm formation was increased in EMPM presence, being remarkably superior in P. aeruginosa cultivated at 30 ºC, as opposed to cultivation at 37 ºC. Similarly, capsaicin induced biofilm formation in S. liquefaciens (37 ºC) and P. aeruginosa (30 ºC). However, capsaicin did not affect biofilm formation on S. liquefaciens cultured at 30 ºC, neither on P. aeruginosa at 37 ºC. These results show that violacein production in C. violaceum ATCC 12472 is inhibited by EMPM, but not by capsaicin. In general, EMPM and capsaicin did not inhibit biofilm formation in S. liquefaciens MG1 neither in P. aeruginosa PAO1. More studies are necessary to elucidate the mechanisms by which EMPM and capsaicin affect the studied phenotypes in this work.

Os venenos de serpentes contém uma mistura complexa de toxinas (proteínas e enzimas) que destroem os processos bioquímicos ou fisiológicos da presa. A crotamina é um dos principais componentes do veneno da cascavel Sul Americana Crotalus durissus terrificus. Essa proteína pertence à família dos polipeptídeos miotóxicos de baixo peso molecular (SBMPs). Uma importante atividade da crotamina é a habilidade de penetrar rapidamente em células proliferamente ativas, podendo ser usada como nanocarreadora, levando DNA plasmidial e drogas para dentro de células tumorais. Outra atividade interessante desta toxina é a indução de morte celular dose dependente em células cancerígenas proliferamente ativas sem prejudicar células normais, demonstrando promissora atividade anticâncer. A crotamina apresenta forte atividade antifúngica e moderada atividade contra procariotos, age bloqueando canais de potássio dependente de voltagem, sendo seletiva para os canais Kv1.1, Kv1.2 e Kv1.3, e induz paralisia dos membros posteriores de camundongo. Dada a importância das atividades da crotamina, a toxina foi usada para produzir na forma recombinante. De forma geral, as toxinas animais são peptídeos secretados para o lúmen da glândula de veneno e são ricos em pontes dissulfeto, comumente expressas em sistemas procarióticos na forma de corpúsculos de inclusão ou em sistemas de expressão periplasmática. Foram construídos três genes sintéticos, um para a expressão em citoplasma e dois para a expressão em periplasma bacteriano utilizando o vetor pRSET-A (Invitrogen ®). Para a expressão de crotamina em citoplasma bacteriano, a região do DNA que codifica a região do peptídeo maduro foi clonada em fusão com uma cauda simples de 6 x His separada por uma seqüência codificante do sítio de clivagem de enterokinase em substituição à proteína de fusão do vetor comercial pRSETA. Para a expressão em periplasma a região codificante da crotamina madura foi clonada sob o controle da seqüência sinal da OmpA de E. coli. Todos os códons raros da crotamina madura foram otimizados de acordo com os códons preferenciais de E. coli. Em uma das construções para a expressão em periplasma, além da crotamina madura, o peptídeo sinal de OmpA também teve seus códons raros otimizados. Os plasmídeos desenhados para a expressão da crotamina madura em citoplasma e periplasma foram transformados em bactéria competente BL21(DE3) e BL21-AI e a indução da proteína foi feita por IPTG e arabinose, respectivamente. A expressão foi analisada por SDS-PAGE e Western Blotting, mostrando que foi possível expressar as três construções em BL21-AI. Snake venoms contain a complex cocktail of toxins (proteins and enzymes) which disrupt physiological or biochemical processes of the pray. Crotamine is one of the major components present in the venom of the South American rattlesnake Crotalus durissus terrificus. It belongs to the small basic myotoxins peptide SBMPs. An important activity of crotamine is the ability to rapidly penetrate proliferative cells, acting as a nanocarrier, carrying plasmid DNA and drugs into tumor cells. Another interesting activity of this toxin is the ability to promote cell death of actively proliferating cancer cells in a dose dependent manner. Crotamine shows strong antifungical activity and modest activities against prokaryotes, it acts by blocking voltage-dependent potassium channels being selective for channels Kv1.1, Kv1.2 e Kv1.3 and inducing paralysis of the hind limbs of mice. Given the importance of the activities of crotamine, the toxin was chosen for production in the recombinant form. Generally, animal toxins are peptides secreted into the lumen of the venom gland and are rich in disulfide bridges. When expressed in prokaryotic system, animal toxins are commonly found in the form of inclusion bodies. They can also be expressed in periplasmic sytem. Three synthetic genes were constructed, one for expression in the cytoplasm and two for expression in the bacterial periplasm using pRSET-A (Invitrogen ®) system. For cytoplasm expression, the DNA region encoding the mature peptide was cloned in fusion with a 6 x His tag separated by a site. For expression in the periplasm, the mature crotamine coding region was cloned in fusion with E. coli OmpA signal sequence. All crotamine and OmpA signal peptide rare codons were optimized according to E. coli preferred codon usage. The plasmids designed for crotamine expression in periplasm and cytoplasm have been transformed in strains BL21(DE3) and BL21-AI were used as hosts, and the induction of recombinant protein was done by IPTG and arabinose, respectively. The induced culture products were analyzed by SDSPAGE and Western Blot, showing that it was possible to express the three constructs in BL21-AI.

Escherichia coli enteropatogênica atípica (aEPEC) faz parte de um grupo de patógenos capazes de formar um tipo de lesão característica em cultura de tecidos epiteliais, denominada attaching and effacing (A/E). Os genes que são necessários para produção da lesão A/E estão localizados em uma ilha de patogenicidade denominada região LEE (locus of enterocyte effacement). A transcrição de genes da região LEE está sujeita a regulação por vários fatores, entre eles quorum sensing, termo utilizado para designar um mecanismo de regulação gênica dependente da concentração celular. Esse mecanismo é usado por bactérias Gram-positivas e Gram-negativas e em ambos os casos envolve a produção e detecção de moléculas sinalizadoras extracelulares, denominadas autoindutores. Até o momento, pelo menos quatro sistemas de quorum sensing foram descritos, entre eles o sistema de autoindutor AI-3 encontrado em bactérias Gram-positivas e Gram-negativas. Diversos mecanismos celulares, entre eles a expressão de fatores de virulência em amostras de EPEC e EHEC, são regulados por esse fenômeno. O principal objetivo deste estudo foi verificar se existe uma possível regulação por quorum sensing na interação in vitro de uma amostra de E. coli da microbiota intestinal com amostras de aEPEC. Após a confirmação da produção de AI-3 por amostras de E.coli da microbiota intestinal foram realizados ensaios de adesão e quantificação utilizando meio pré-condicionado com esta amostra, epinefrina e bloqueadores que confirmaram que os padrões de adesão de aEPEC obtidos em menor tempo são devidos a presença de AI-3 no meio pré-condicionado, indicando a participação de quorum sensing nessa interação. Além disso, foi observado um fenômeno citotóxico nas células que não é produzido pelo AI-3. Atypical Enteropathogenic Escherichia coli (aEPEC) are part of a group of pathogens capable of forming a type of lesion characteristic of epithelial tissues in culture, called attaching and effacing (A/E). The genes that are required for production of A/E lesion are located in a pathogenicity island called LEE region (locus of enterocyte effacement). The transcription of LEE genes in the region is subject to regulation by various factors, including quorum sensing, a term used to describe a mechanism of gene regulation dependent on cell concentration. This mechanism is used by Gram-positive and Gram-negative and in both cases involves the production and detection of extracellular signaling molecules, called autoinducers. So far, four systems of quorum sensing have been described, including the system of autoinducers AI-3 found in Gram-positive and Gram-negative bacteria. Several cellular mechanisms, including expression of virulence factors in EPEC and EHEC are regulated by this phenomenon. The main objective of this study was to determine whether there is a possible regulation by quorum sensing in the in vitro interaction of a strains of E. coli of the intestinal microbiota with strains aEPEC. After confirming the production of AI-3 in E. coli of the intestinal microbiota were performed adhesion assays and quantification using means preconditioned with this strains, epinephrine, and blockers who confirmed that patterns of adherence of aEPEC obtained in less time are due to the presence of AI-3 in the preconditioned means, indicating the involvement of quorum sensing in this interaction. Furthermore, we observed a phenomenon that cytotoxic cells is not produced by AI-3.