Construction of bioluminescent Salmonella strains for use in food microbiology.

Advances in genetic engineering allow different bacterial species that are normally non-luminescent can be developed to become bioluminescent organisms. Nowadays, living microorganisms such as Salmonella spp. are genetically engineered to produce this measurable signal. Many studies have been using the gene lux to modify micro-organisms in other to produce visible light. Besides, the use of the complete luxCDABE cassette allows the bioluminescence can be expressed continuously, without addition of exogenous substrates. Thus, bioluminescent bioreporter bacteria remain entirely self-sufficient in its ability to produce visible light. Moreover, an important advantage is that the bioluminescence measurements using bioreporter bacteria are not invasive and destructive to the cell, and also provide realtime results, which allows the opportunity to employ light emission with great success in several applications in Food Microbiology, as evaluating the antimicrobial efficacy to inactivate pathogens. Therefore, the objective of this study was to construct bioreporter bioluminescent strains of Salmonella and use them to evaluate the microbial resitant against natural antimicrobial compounds, as well as to investigate Lux protein expression and other proteins in different strains of recombinant bacteria using a light-scattering sensor.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESOs avanços na engenharia genética permitem que muitas espécies de bactérias que são normalmente não luminescentes sejam desenvolvidas, de modo que se tornem organismos bioluminescentes. Atualmente, micro-organismos como Salmonella spp. estão sendo geneticamente construídos a fim de produzir esse sinal mensurável. Desse modo, a utilização do cassete luxCDABE completo permite que a bioluminescência possa ser expressa de forma contínua, sem necessidade da adição de substratos exógenos. Assim, as bactérias biorrepórteres bioluminescentes permanecem inteiramente auto-suficientes em sua capacidade de produzir luz visível. Além disso, uma importante vantagem da bioluminescência é que as medições utilizando bactérias biorrepórteres não são invasivas e destrutivas para a célula, e ainda fornecem resultados em tempo real, o que permite a oportunidade de empregar a emissão de luz com muito sucesso em distintas aplicações em microbiologia de alimentos, como a avaliação da eficácia antimicrobiana para a inativação de agentes patogênicos. Portanto, o objetivo deste estudo foi construir cepas biorrepórteres bioluminescentes de Salmonella e utilizá-las para avaliar a resistência desse patógeno microbiano frente a compostos antimicrobianos naturais, bem como investigar a expressão da proteína Lux e de outras proteínas em diferentes cepas de bactérias recombinantes utilizando um sensor que capta imagens de dispersão

Listeria monocytogenes: monitoramento desse perigo biológico na cadeia produtiva de frangos do sul do Rio Grande do Sul

Listeria monocytogenes é uma bactéria patogênica que se tornou um grande desafio para as indústrias de alimentos, entre elas a de frangos, assim como para os órgãos de vigilância sanitária. Apesar da produção de frangos estar em expansão na região sul do Rio Grande do Sul, não há relatos sobre esse patógeno, dessa forma, objetivou-se avaliar a prevalência de L. monocytogenes e de seus sorotipos nos diversos segmentos dessa cadeia produtiva. Nos aviários isolou-se L. monocytogenes em 2,9% (1/35) das amostras de swabs cloacais, não se isolando o microrganismo em amostras provenientes das camas de aviários. No abatedouro, 11,7% (15/128) das amostras apresentaram contaminação por L. monocytogenes e nos frangos resfriados procedentes do comércio, a prevalência foi de 33,3% (15/45).Observou-se que 51,6% (16/31) das cepas de L. monocytogenes pertenciam ao sorotipo 1/2b; 22,5% (7/31) ao sorotipo 4e; 16,1% (5/31) ao sorotipo 1/2a; 6,4% (2/31) ao sorotipo 4b; e 3,2% (1/31) ao sorotipo 1/2c. Há disseminação de L. monocytogenes na cadeia produtiva de frangos da região sul do Rio Grande do Sul e a presença de sorotipos prevalentes em casos/surtos de listeriose traz preocupação à saúde pública

Fructose 1,6-Bisphosphate Aldolase, a Novel Immunogenic Surface Protein on <i>Listeria</i> Species

<div><p><i>Listeria monocytogenes</i> is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on <i>Listeria</i> would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in <i>Listeria</i> species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested <i>Listeria</i> species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of <i>Listeria</i> cells, including supernatant and the cell wall, setting <i>Listeria</i> spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope in FBA allowed distinguishing <i>Listeria</i> spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic <i>Listeria</i> isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of <i>Listeria</i> genus.</p></div

Cell localization and adhesion experiments for FBA characterization.

<p><b>(A)</b> Western blot using mAb-3F8 show that FBA protein is present in the cell wall and intracellular (membrane and cytoplasm) of three different <i>Listeria</i> species tested. The top band shows reaction with anti-InlA mAb-2D12. <b>(B)</b> When using only mAb-3F8 in Western blot, it is possible to observe that FBA is also present in the culture supernatant of two different <i>Listeria</i> species. <b>(C)</b> Due to the presence of FBA in the cell wall, a <i>L</i>. <i>monocytogenes</i> mutant (Δ<i>secA2</i>) was used to inquire the secretion pathway of FBA. However, no difference in band intensity was observed in the cell wall (CW) or intracellular (Intra) fractions between the wild type (WT) and mutant strains, indicating that the secretory pathway of FBA is not SecA2-dependent. <b>(D)</b> Adhesion experiments <i>in vitro</i> using HCT-8 cells show that mAb-3F8 has no inhibition activity, since it’s pre-incubation with <i>L</i>. <i>monocytogenes</i> cells shows similar levels of adhered bacteria as the negative controls with PBS and mAb-C11E9. The positive control mAb-2D12 (anti-InlA) was the only one to show significant reduction in adhesion.</p

SDS-PAGE and Western blot of bacteria from artificially contaminated cheese.

<p><b>(A)</b> SDS-PAGE (12%-acrylamide) showing the protein extracts from the bacteria after enrichment step using stomacher bags. Protein extracts from pure cultures were used as control. <b>(B)</b> Western blot using both mAb-3F8 and -2D12 on these protein extracts shows the expected detection bands: both InlA (88 kDa) and FBA (30 kDa) for the pathogenic strain; and only FBA (30 kDa) for the non-pathogenic. Purified rInlA and rFBA, and the pure cultures of <i>L</i>. <i>monocytogenes</i> and <i>L</i>. <i>innocua</i> were used as positive control for the antibodies.</p

Western and dot blots showing the presence of FBA in <i>Listeria</i> spp. only.

<p><b>(A)</b> SDS-PAGE 12% showing the protein preparations of two <i>Listeria</i> species and many related bacteria (upper part). In Western blot using mAb-3F8, only those extracts from <i>Listeria</i> showed a 30-kDa band corresponding to FBA. <b>(B)</b> Western blot using mAb-3F8 showing the presence of FBA in other <i>Listeria</i> species, but not in <i>E</i>. <i>coli</i> O157:H7 used as negative control. In this blot, mAb-2D12 (anti-InlA) was used together with mAb-3F8, showing an 88-kDa band corresponding to InlA, which is present only in <i>L</i>. <i>monocytogenes</i>. <b>(C)</b> The results found in the Western blot were same as the dot blot, in which only <i>Listeria</i> spp. (either live or heat-killed) could be detected when using mAb-3F8. <b>(D)</b> Western blot using both mAb-3F8 and -2D12 allows detection of three different serotypes of <i>L</i>. <i>monocytogenes</i> (4b, 1/2a, and 1/2b), no matter the medium used for their growth (LEB or FB).</p

Western blot assay with recombinant rCodY and rFBA proteins to determine the target of mAb-3F8.

<p><b>(A)</b> Western blot using mAb-3F8 as primary antibody shows that this mAb reacts with purified rFBA, as well as protein extract of <i>L</i>. <i>monocytogenes</i>, but not with rCodY. <b>(B)</b> The reaction using Anti-His primary antibody as control shows that both rFBA and rCodY were present in detectable amounts on the membrane.</p