Eficácia de películas edíveis suplementadas com componentes de óleos essenciais no controlo de Listeria monocytogenes e Salmonella enterica Typhimurium

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Detecção de Salmonella spp. a partir de carcaças de bovinos obtidas durante o processamento em abatedouros-frigoríficos em Mato Grosso do Sul: resultados preliminares

2015

Detecção de Salmonella em tomates (Lycopersicon esculentum mill) pelos métodos fda-bam e mini vidas-biomérieux.

Resumo: Diversos surtos de Salmonella ocasionados pelo consumo de tomate contaminados com este micro-organismo têm sido relatados ultimamente, o que torna primordial a investigação sobre a presença desse patógeno nesse alimento. Métodos que permitam a avaliação rápida da presença de Salmonella em alimentos são de suma importância. O objetivo desse estudo foi comparar o método tradicional da Food and Drug Administration - Bacteriologycal Analytical Manual (FDA-BAM) com um método rápido da mini Vitek Immuno Diagnostic System Assay (Mini?Vidas-SLM)-bioMérieux, para detecção de Salmonella Brazil inoculada artificialmente na superfície de tomates. Foram analisadas 215 amostras de tomates inoculadas artificialmente com Salmonella Brazil com níveis de inóculos variando de 0,4 a 940 UFC/tomate. Os resultados obtidos mostraram que os métodos estudados apresentaram uma ótima concordância entre si, para todas as faixas de inóculo analisadas.

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota.

Salmonella Typhimurium exploits inflammation to its own advantage in piglets

Salmonella Typhimurium (S. Typhimurium) is responsible for foodborne zoonotic infections that, in humans, induce self-limiting gastroenteritis. The aim of this study was to evaluate whether the wild-type strain S. Typhimurium (STM14028) is able to exploit inflammation fostering an active infection. Due to the similarity between human and porcine diseases induced by S. Typhimurium, we used piglets as a model for salmonellosis and gastrointestinal research. This study showed that STM14028 is able to efficiently colonize in vitro porcine mono-macrophages and intestinal columnar epithelial (IPEC-J2) cells, and that the colonization significantly increases with LPS pre-treatment. This increase was then reversed by inhibiting the LPS stimulation through LPS antagonist, confirming an active role of LPS stimulation in STM14028-intracellular colonization. Moreover, LPS in vivo treatment increased cytokines blood level and body temperature at 4 h post infection, which is consistent with an acute inflammatory stimulus, capable to influence the colonization of STM14028 in different organs and tissues. The present study proves for the first time that in acute enteric salmonellosis, S. Typhimurium exploits inflammation for its benefit in piglets.

Association of extended-spectrum β-lactamase VEB-5 and 16S rRNA methyltransferase armA in Salmonella enterica from the United Kingdom

Aminoglycosides and beta-lactams are used for the treatment of a wide range of infections due to both Gram-negative and Gram-positive. An emerging aminoglycoside resistance mechanism, methylation of the aminoacyl site of the 16S rRNA, confers high-level resistance to clinically important aminoglycosides such as amikacin, tobramycin and gentamicin. Eight 16S rRNA methyltransferase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF and npmA, have been identified in several species of enterobacteria worldwide (2, 6, 7, 9, 11, 13, 14). Resistance to extended spectrum β-lactams remains additionally an important clinical problem. Apart from the large TEM, SHV, and CTX-M families, several other extended-spectrum β-lactamases (ESBLs) have been identified, including VEB enzymes, which confer high-level resistance to cephalosporins and monobactams. Although 16S rRNA methyltransferases have been frequently identified associated with different ESBLs, there has been no report of association of a 16S rRNA methyltransferase with a VEB enzyme, except for the identification of rmtC with blaVEB-6 (14)

ArmA methyltransferase in a monophasic Salmonella enterica isolate from food

The 16S rRNA methyltransferase ArmA is a worldwide emerging determinant that confers high-level resistance to most clinically relevant aminoglycosides. We report here the identification and characterization of a multidrug-resistant Salmonella enterica subspecies I.4,12:i:- isolate recovered from chicken meat sampled in a supermarket on February 2009 in La Reunion, a French island in the Indian Ocean. Susceptibility testing showed an unusually high-level resistance to gentamicin, as well as to ampicillin, expanded-spectrum cephalosporins and amoxicillin-clavulanate. Molecular analysis of the 16S rRNA methyltransferases revealed presence of the armA gene, together with bla(TEM-1), bla(CMY-2), and bla(CTX-M-3). All of these genes could be transferred en bloc through conjugation into Escherichia coli at a frequency of 10(-5) CFU/donor. Replicon typing and S1 pulsed-field gel electrophoresis revealed that the armA gene was borne on an ~150-kb broad-host-range IncP plasmid, pB1010. To elucidate how armA had integrated in pB1010, a PCR mapping strategy was developed for Tn1548, the genetic platform for armA. The gene was embedded in a Tn1548-like structure, albeit with a deletion of the macrolide resistance genes, and an IS26 was inserted within the mel gene. To our knowledge, this is the first report of ArmA methyltransferase in food, showing a novel route of transmission for this resistance determinant. Further surveillance in food-borne bacteria will be crucial to determine the role of food in the spread of 16S rRNA methyltransferase genes worldwide.

Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new pandemic strain?

A marked increase in the prevalence of S. enterica serovar 4,[5],12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,[5],12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,[5],12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,[5],12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.

Salmonella Enterica serovar Typhimurium Infection in Mouse Pregnancy

Salmonella are Gram-negative, intracellular food-borne pathogens that cause pregnancy complications. In pregnant mice, Salmonella enterica serovar Typhimurium (S.Tm) infection results in placental bacterial replication, inflammation, necrosis, and fetal loss by unknown mechanisms. Necroptosis, or programmed necrosis mediated by RIPK3 (receptor-interacting protein kinase 3), an inflammatory cell death pathway, is implicated in the pathogenesis of S.Tm in non-pregnant mice. This goal of this thesis was to investigate the role of necroptosis in the pathogenesis of S.Tm infection during mouse pregnancy. I hypothesized that elimination of the key necroptotic cell death protein RIPK3 would decrease placental inflammation and trophoblast cell death, and increase conceptus survival compared to controls. Mice expressing a functional Slc11a1 (encodes the natural resistance-associated macrophage protein 1, NRAMP1) gene with or without RIPK3 function (Ripk3-/-Slc11a1+/+ compared to Slc11a1+/+) were infected with 103 S.Tm by tail vein injection on gestational day (GD) 12. Mice were euthanized on GD 14 (48h post-infection) or GD 15 (72h post-infection) and implantation sites (IS) and maternal serum were harvested for analyses. In nearly all challenged mice (except one outlier), S.Tm were detected in most IS within a litter but there was limited immune cell infiltration, placental damage or cell death in Slc11a1 competent mice regardless of Ripk3 gene deletion. Maternal serum cytokine analyses confirmed lack of maternal immune responses to S.Tm infection. IS amongst the litter of a single dam (Ripk3-/-Slc11a1+/+ at 72h postinfection) displayed heavy but not universal placental S.Tm infection of decidual tissues and spongiotrophoblast, associated with elevated maternal serum pro-inflammatory cytokines. S.Tm infection of the fetal yolk sac (YS) was observed in 54.5% of IS from this dam. YS infection was confirmed in archival samples in mice expressing Ripk3 with intact Slc11a1 and in mice lacking functional Slc11a1. In Slc11a1 incompetent mice, S.Tm were detected in placental labyrinthine trophoblast. Based on the available data, this thesis suggests that Ripk3 and necroptosis have no significant roles in either promotion or prevention of progressive Salmonella infection during mouse pregnancy. It also provides pilot data that NRAMP1 controls placental localization and lethality due to YS infection.

Resistência antimicrobiana em salmonella spp. isoladas de aviários de frangos de corte: dados parciais.

2016

Ocratossina A, metalli, insetticidi e fitoestratti: valutazione degli effetti e degli aspetti residuali nel pollame e in alimenti destinati all'uomo.

La presente tesi di Dottorato di Ricerca descrive le attività di ricerca riguardanti diversi ambiti della sicurezza alimentare, della farmacologia e tossicologia veterinaria. Sono presentati i dati relativi alla ricerca di ocratossina A (OTA) in campioni di bile, rene e fegato di polli, salami artigianali e diversi tipi di formaggio; le concentrazioni di diversi metalli pesanti in uova di galline rurali e 5 tipologie di uova industriali; i dati preliminari ottenuti dalle analisi dei primi campioni di uova e penne provenienti da allevamenti amatoriali di pollame, analizzati per la ricerca del fipronil e metaboliti. Sempre per fipronil, e la molecola foxim, sono presentati dati inerenti a studi di potenziale farmaco-resistenza di popolazioni naturali di Dermanyssus gallinae, e dell’attività insetticida di alcuni fitocomposti a base di tannini. Riguardo all’uso di fitoestratti, sono presentati i dati preliminari ottenuti nei primi mesi di una prova sperimentale in galline di razza Livornese allevata con metodo free-range, finalizzata allo studio della attività anti-infiammatoria e immuno-stimolante, nonché alla valutazione della sicurezza della integrazione nella dieta di Boswellia serrata e Salix alba. Sono stati condotti anche studi traslazionali con altri settori scientifici, relativamente la prevalenza sierologica e microbiologica per Salmonella enterica var. enteritidis e var. thiphymurium in allevamenti ornamentali e da auto-consumo di pollame, e di prevalenza e caratterizzazione di potenziali varianti virali e batteriche (incluse zoonosiche) di alcune malattie infettive del pollame ornamentale. In specie avicole ornamentali è riportato anche uno studio di campo sul trattamento off-label con Fenbendazolo e Flubendazolo in infestazioni massive da Cyathostoma bronchialis in oche esotiche ornamentali. A seguito di una collaborazione con l’Università di Firenze, sono presentati anche i risultati ottenuti da uno studio su modelli murini, dell’azione e funzionamento dei recettori Beta3, espressi nelle neoplasie più frequenti che si verificano nelle donne in gravidanza.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Influence Of The Major Nitrite Transporter Nirc On The Virulence Of A Swollen Head Syndrome Avian Pathogenic E. Coli (apec) Strain.

Avian Pathogenic Escherichia coli (APEC) strains are extra-intestinal E. coli that infect poultry and cause diseases. Nitrite is a central branch-point in bacterial nitrogen metabolism and is used as a cytotoxin by macrophages. Unlike nitric oxide (NO), nitrite cannot diffuse across bacterial membrane cells. The NirC protein acts as a specific channel to facilitate the transport of nitrite into Salmonella and E. coli cells for nitrogen metabolism and cytoplasmic detoxification. NirC is also required for the pathogenicity of Salmonella by downregulating the production of NO by the host macrophages. Based on an in vitro microarray that revealed the overexpression of the nirC gene in APEC strain SCI-07, we constructed a nirC-deficient SCI-07 strain (ΔnirC) and evaluated its virulence potential using in vivo and in vitro assays. The final cumulative mortalities caused by mutant and wild-type (WT) were similar; while the ΔnirC caused a gradual increase in the mortality rate during the seven days recorded, the WT caused mortality up to 24h post-infection (hpi). Counts of the ΔnirC cells in the spleen, lung and liver were higher than those of the WT after 48 hpi but similar at 24 hpi. Although similar number of ΔnirC and WT cells was observed in macrophages at 3 hpi, there was higher number of ΔnirC cells at 16 hpi. The cell adhesion ability of the ΔnirC strain was about half the WT level in the presence and absence of alpha-D-mannopyranoside. These results indicate that the nirC gene influences the pathogenicity of SCI-07 strain.

Estabilidade oxidativa e microbiológica em carne de galinha mecanicamente separada e adicionada de antioxidantes durante período de armazenamento a -18 °C

Com o objetivo de acompanhar a estabilidade físico-química e microbiológica da carne mecanicamente separada (CMS) de diferentes origens e estocada durante 99 dias a -18 °C, foi realizada prévia mistura de conservante (nitrito de sódio) e antioxidante (eritorbato de sódio) em CMS obtida de duas linhagens de aves: galinhas matrizes de corte e galinhas poedeiras comerciais brancas. Na CMS de cada linhagem foram realizados três diferentes tratamentos: 1) controle (sem aditivos); 2) adição de 150 ppm de nitrito; e 3) adição de 150 ppm de nitrito e 500 ppm de eritorbato. Os resultados encontrados demonstraram que a adição de nitrito isoladamente não impediu a oxidação lipídica, avaliada através do índice de TBARS, nem a alteração na cor, avaliada em colorímetro. Por outro lado, a adição de nitrito juntamente com eritorbato foi efetiva na redução dos problemas de oxidação lipídica na CMS de galinhas matrizes, e em menor grau, na CMS de poedeiras. A adição de nitrito e eritorbato na CMS também melhorou a preservação da cor vermelha desejável (a*) ao longo do tempo. A avaliação da estabilidade microbiológica da CMS, realizada no primeiro e último dia de estocagem congelada, para microrganismos mesófilos, Escherichia coli, Staphylococcus aureus, Clostridium perfringens e Pseudomonas spp., e quinzenalmente para microrganismos psicrotróficos, indicou que não houve uma variação significativa nas contagens em função do tratamento utilizado (diferentes aditivos adicionados). Não foi detectada Salmonella spp. em nenhuma das amostras analisadas. Em função da melhoria da estabilidade oxidativa, recomenda-se a adição de nitrito (150 ppm) e eritorbato (500 ppm) em CMS de galinhas matrizes a ser estocada congelada por um período prolongado.

Microbiological quality of hamburgers sold in the streets of Cuiabá - MT, Brazil and vendor hygiene-awareness

This study evaluated the microbiological quality of hamburgers and the microbe community on the hands of vendors in Cuiabá, Mato Grosso, Brazil, in relation to vendors´ awareness as to what constitute acceptable food-handling practices as part of a broad-spectrum research programme on street foods in Brazil . Sale of the hamburger known as the 'baguncinha' is common and widespread in urban Cuiabá, Mato Grosso, Brazil. Food inspectors encounter various difficulties in carrying out inspections. One hundred and five hamburgers samples were evaluated using conventional methods including tests for facultative aerobic and/or anaerobic mesophytic bacteria, coliform counts at 45 °C, the coagulase test for Staphylococcus, Gram-staining for the presence of Bacillus cereus, Clostridium sulphite reductase and Salmonella spp. The hamburgers were categorized as unsuitable for human consumption in 31.4% of samples, with those testing positive for coliforms and Staphylococcus at unacceptably high levels by Brazilian standards. High levels of microbiological contamination were detected on the hands of the food handlers and mesophytic bacterial counts reached 1.8 × 10(4) CFU/hand. Interviews were carried out by means of questionnaires to evaluate levels of awareness as to acceptable food handling practices and it was found that 80,1% of vendors had never participated in any kind of training.