Current detection methods require at least one 24-48 hour enrichment step for the detection of Salmonella. This poses a problem because product often needs to be shipped before microbial contamination levels can be adequately ascertained. Therefore, the need for more rapid methods of Salmonella detection becomes apparent. The purpose of this thesis was to determine if an immunologically-based method, Immunomagnetic Capture(IMC) -ELISA and molecular-based detection methods, PCR and Taqman® PCR employing IMC without enrichment, could detect at least 102 cfu/ml of S. Typhimurium in broiler carcass rinse fluid (CRF) samples. IMC-ELISA, IMC-PCR, and IMC-Taqman® PCR were initially tested using 0 to 106 cfu/ml of pure culture S. Typhimurium. Each detection method was tested using artificially contaminated CRF samples. Finally, standardized IMC-ELISA, IMC-PCR, and IMC-Taqman® PCR methods were tested using commercial CRF samples. Salmonella concentrations were verified using a traditional plate method. IMC-ELISA produced consistent results when detecting at least 104 to 106 cfu/mL of pure culture S. Typhimurium. IMC-ELISA was not able to produce repeatable results when testing artificially contaminated CRF samples. S. Typhimurium was not detected in commercial CRF samples which by virtue of direct plating on XLT-4 were found to contain essentially no Salmonella (<1 cfu/ml). IMC-PCR was able to consistently detect 102 cfu/ml, whereas IMC-Taqman® PCR was able to detect 101 cfu/ml of pure culture S. Typhimurium. IMC-PCR, required four hours to complete, and it consistently detected 104 cfu/ml of S. Typhimurium in artificially contaminated CRF samples. IMC-Taqman® PCR took 3 hours to perform and was able to detect 103 cfu/ml S. Typhimurium in artificially contaminated CRF samples. The sensitivity, as well as the decreased time requirements of these detection methods, would suggest their usefulness in a commercial processing setting. / Master of Science

Greenhouse grown tomato fruits and tissues were tested for the presence of Salmonella after the plants had been treated with Salmonella contaminated irrigation water or grown from contaminated seeds. Greenhouse grown tomato plants were placed into eight different groups. Groups one through six consisted of five plants each and were treated with 350 ml of 10^6 Salmonella contaminated irrigation water over a course of 70 days; group one received one 350 ml 10^7 Salmonella treatment, group two received two treatments, and so on, the treatments were scheduled every 14 days. Group seven was the control that consisted of five plants and received no Salmonella treatment. Group eight was grown from seeds that had been contaminated with Salmonella by soaking the seeds in a 10^8 Salmonella suspension for 24 hours at room temperature, and received no Salmonella watering treatment. A total of 128 tomatoes were sampled from the tomato plants of all three groups and none tested positive for Salmonella. Tissue samples consisting of roots, leaves, and stems, and were collected from one plant per each of three replications. No leaves or stems contained Salmonella, however, five of the twenty-four root samples were positive for Salmonella. In a second study, Salmonella was tested for its ability to survive in three concentrated fertilizer stock solutions and 1.6% diluted solutions of the fertilizer. Fertilizer sample CF-S was a stock solution of commercial 20N-4.4P-16.6K fertilizer, US-S was a mix of 11.3 kg UltraSol, 4.5 kg Epsom Salts, and 2.3 kg 0N-0P-43.2K in 114 L water, Fertilizer CN-S is a mix of 11.3 kg Calcium Nitrate and 56.7 g Iron chelate (10%) to 30 L water; Fertilizers CF-1.6, US-1.6, and CN-1.6 were the 1.6% fertilizer dilutions respectively. There was no significant difference (p<0.05) between the survival of Salmonella in fertilizer groups CF-1.6, US-S, US-1.6, CN-1.6, and the sterile distilled water control; all but US-S yielded less than a one log reduction in Salmonella over a period of 72 hours. US-S yielded over a two log reduction in Salmonella and was not significantly different than CN-S which had over a four log reduction. CF-S was significantly different than all samples and led to over a 6 log reduction of Salmonella. The results of this study showed no evidence that Salmonella was able to internalize in Cultiver trust tomato fruit or tissues above the root line when irrigated with contaminated water into the pine medium under greenhouse conditions. There was also no evidence that Salmonella is able to internalize in any tissues or fruit from contaminated seeds. The results also show that Salmonella was not able to survive in the commercial fertilizer stock solution (CF-S), and had limited survival in CN-S tomato fertilizer solution. The diluted fertilizer solution and US-S stock solution showed no significance in survival of Salmonella when compared to the sterile water control. / Master of Science in Life Sciences

Chapter: 1 Introduction Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars, which belong to the Salmonella enterica species. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Salmonella infection occurs via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18+ macrophages present in the intestinal lumen or via a forced entry in the non phagocytic enterocytes. Upon entry Salmonella resides in an intracellular phagosomal compartment called the Salmonella containing vacuole (SCV). The SCV only transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7. It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position. The mechanisms by which phagocytes kill the virulent Salmonella are not completely understood, however the role of nicotinamide-adenine dinucleotide phosphate (NADPH) phagocytic oxidase system has been strongly implicated. The generation of reactive oxygen species (ROS) occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, p47phox, p67phox, and a GTP-binding Rac protein. Further, professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Chapter:2 Resistance to host Nitrosative stress in Salmonella by quenching L-arginine. Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of L-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate L-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor aminoguanidine reverts the attenuation in arginase blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of L-arginine. In the whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner. Chapter:3 Hrg (hydrogen peroxide resistant gene), a LysR type transcriptional regulator confers resistance to oxidative stress in Salmonella LysR type transcriptional regulators are one of the key players that help bacteria adapt to different environments. We have christened STM0952, a putative LysR type transcriptional regulator in Salmonella enterica serovar Typhimurium as the hydrogen peroxide resistance gene (hrg). By generating a knock out of the hrg gene, we demonstrate that the hrg mutant serovar Typhimurium is sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. The hrg mutant is profoundly attenuated in the murine model of infection and shows decreased intracellular proliferation in macrophages. It was also found to induce increased amount of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild type. An overproducing strain of this gene showed a survival advantage over the wild type Salmonella under hydrogen peroxide induced stress condition. Microarray analysis suggested the presence of a Hrg regulon, which is required for resistance to the toxic oxidative products of the reticulo-endothelial system. Chapter:4 Importance of the host oxidative stress in antigen presentation and its modulation by Salmonella: Role of TLR Synthetic CpG containing oligodeoxynucleotide TLR-9 agonist (CpG ODN) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. We report that Salmonella Typhimurium growth can be inhibited by the CpG ODN treatment in the murine dendritic cells. This inhibitory effect was shown to be mediated by an increased reactive oxygen species (ROS) production. We further show that the CpG ODN treatment of the dendritic cells during Salmonella infection leads to a ROS dependent increased antigen presentation. In addition, TLR-9 signaling inhibitor was able to inhibit the CpG ODN mediated increased antigen presentation, ROS production and pathogen killing. These data indicate that CpG ODN can improve the ability of the murine dendritic cells to contain the growth of the virulent Salmonella through ROS dependent killing and could as well be used as an effective adjuvant in vaccines against Salmonella infection.

Chapter I Introduction Salmonellae are facultative Gram-negative intracellular pathogens. Different serovars of it causes a variety of diseases in multiple hosts with different disease outcomes. Salmonella enterica serovar Enteritidis and Typhimurium (STM) can infect domestic animals causing gastroenteritis or typhoid like fever. Typhoid fever in humans which is actually caused by Salmonella enterica serovar Typhi still remains a significant health problem in many parts of the world with an estimated annual incidence of nearly 16 million cases and about 600,000 deaths. The infection begins via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18 macrophages present in the intestinal lumen or via a forced entry in the non-phagocytic enterocytes. Upon entry, Salmonella resides in an intracellular phagosomal compartment called Salmonella containing vacuole (SCV) and has several strategies to counteract the host defense mechanisms. Following phagocytosis and its compartmentalization into Salmonella containing vacuole (SCV), a series of defense mechanisms are initiated. These include toxic reactive oxygen species or super oxide production, nitric oxide production, phagosomal acidification and release of hydrolases and defensins through fusion of phagosome with lysosomes generating highly bactericidal environment. The SCV transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7 and resist killing by avoiding phagosomal maturation and vesicular trafficking of iNOS and NADPH oxidase vesicles. Moreover, Salmonella also uses acidic pH of the SCV (~ pH 4.5) to assemble the Salmonella Pathogenecity Island 2 (SPI-2) type three secretion system (TTSS) which is essential for survival inside the macrophages. Salmonella uses these hostile conditions inside the host as cues for regulating their virulence factors using global regulatory factors. Hfq is one such global regulator playing an important role in many physiological processes and stress responses. Understanding the importance of Hfq regulated genes which impart Salmonella survival advantage under hostile conditions for successful infection will be of particular significance. The host too recognizes pathogen using innate immune receptors present either on the cell surface like TLRs (Toll Like Receptors) or inside the cells like NLRs (Nod Like Receptors). Innate immune receptors recognize pathogen associated molecular patterns (PAMPs) such as Lipopolysacharide (LPS), peptidoglycon (PGN), or hypomethylated DNA or RNA. Recognition of PAMPs by innate receptors leads, via activation of transcription factors (NF-κB and IRF3), to the generation of pro and anti-inflammatory cytokines, chemokines. Vaccination has been practiced for many years and it is one of the most effective methods of controlling infectious diseases like typhoid. At present two licensed vaccines against Salmonella are in use globally namely, Vi polysaccharide subunit vaccine (Typhim Vi™) and live attenuated typhoid vaccine (Vivotif Berma™). Lack of immunological memory, low efficacy (55-75 % protection) and requirement of higher number of doses are the important practical shortcomings associated with the currently used vaccines. So there is a need for a safer and immunogenic vaccine to combat Salmonella infection. Chapter II Salmonella Typhimurium lacking hfq gene induces long term memory response and confers protective immunity Currently available vaccines for typhoid have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new improved ones. Of the various strategies used for the generation of vaccine strains, focus is on manipulation of virulence regulator genes for bacterial attenuation. Hfq is a RNA chaperon which mediates the binding of small RNA to the mRNA and assists in post-transcriptional gene regulation in bacteria. Salmonella hfq deletion mutant is highly attenuated in vitro as well as in vivo implying its role in bacterial virulence. In this study, we have evaluated the efficacy of the Salmonella Typhimurium hfq deletion mutant as a candidate for live oral vaccine against Salmonella infection in murine salmonellosis model. The hfq deletion mutant is not only able to confer protection when administered orally to the mice against oral challenge with serovar Typhimurium virulent strain, but also elicits cross protective immune responses to other Salmonella serovars. The vaccine candidate appears to be safe for use in pregnant mice. This protection is partially mediated by the increase in the number of CD4+ T lymphocytes upon vaccination. STM hfq deletion mutant further exhibited significant increase in the lipopolysaccharide as well as outer membrane protein specific IgG in the serum as well as secretory S-IgA in the intestinal washes. In addition, vaccination led to an increased serum IFN-γ and IL-6. Taken together, our results suggest that the Salmonella Typhimurium hfq deletion mutant can be an excellent live oral vaccine candidate. Chapter III Acidic pH induced STM1485 gene governs intracellular replication and pathogenesis in Salmonella During the course of infection, Salmonella has to face several potentially lethal environmental conditions such as low pH both inside and outside the host. The ability to sense and respond to the acidic pH is crucial for survival and replication of Salmonella. Exposure to acidic pH results in the expression of large pool of virulence genes. One such gene highly up regulated inside the macrophage is STM 1485. In order to understand physiological role of STM 1485 in Salmonella pathogenesis, STM 1485 gene was deleted chromosomally and characterized in vitro and in vivo. In vitro the mutant did not show any growth defects at pH 4.5 and no difference in acid tolerance response. The 1485 deletion mutant was compromised in its capacity to proliferate inside the cells and is further lowered inside activated macrophages. We further showed that surface translocation of SPI-2 encoded translocon protein SseB was reduced at low pH in vitro in STM 1485 mutant and the mutant was found to colocalize with lysosomes higher than the wild type. In addition, the STM 1485 deletion mutant displayed decreased virulence in murine typhoid model when infected intragastrically. Based on our results, we hypothesize that the acid shock protein encoded by the STM 1485 might be involved in the formation of SPI-2 translocon at low pH and there by contributing to the virulence of Salmonella. Chapter IV Role of Nod1 in sensing vacuolar pathogen Salmonella in epithelial cells Nod1 and Nod2 are the archetypal members of the Nod like receptor family (NLR) and they recognize distinct peptidoglycan motifs of Gram-negative and Gram-positive bacteria respectively. Role of Nod1 and Nod2 in sensing bacterial pathogens have been elucidated. However, the role of Nod1 in sensing vacuolar pathogen Salmonella in epithelial cells is not understood. So in this study we investiged the role of Nod1 in the innate immune response against Salmonella in epithelial cells. We demonstrate that the recognition of Salmonella by Nod1 leads to NF-κB activation and this activation is diminished in epithelial cells expressing a dominant-negative Nod1 construct or Nod1 shRNA. Using a set of Salmonella mutants we show that the availability of ligand is higher when the bacteria were in cytosol rather than in vacuole. Further we also observed that the Nod1 mediated killing of Salmonella is mediated through the defensins. Based on our results we hypothesize that Salmonella uses its vacuolar niche to evade Nod1 mediated innate immune response.

The genus Salmonella includes facultative intracellular pathogens. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans killing about 2,00,000 people globally every year. Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) cause food poisoning in humans. Salmonellae also cause disease in animals of economic importance like poultry and cattle. Treatment of diseases caused by these notorious pathogens is becoming more and more difficult because of the emergence of drug resistant strains. Thus, it is vital to understand the virulence mechanisms of Salmonella which can lead us to potential drug targets and also help us design effective vaccines. Salmonella has evolved many strategies to enter the host, to evade intracellular and extracellular antimicrobial activities of the host and to extract nutrition in the stringent and hostile environment of the host. These strategies have enabled Salmonella to survive and multiply in the host making it a successful pathogen. Present study deals with four such survival strategies of Salmonella. S. Typhimurium causes a systemic disease in mice that is similar to typhoid fever caused by serovar Typhi in humans. This serves as a good model system to study and understand the pathogenesis of Salmonellae. This model system has been used throughout this study. In the present thesis attempts have been made to identify some novel survival strategies of Salmonella. The thesis is divided into five chapters. Chapter 1 gives an introduction into the basic biology of these notorious pathogens. The diseases caused by Salmonellae are introduced in this chapter. Typhoid fever is discussed in detail covering its epidemiology, clinical features, diagnosis, treatment and prevention. Next section covers the virulence determinants of Salmonella. In this section, Salmonella pathogenicity islands are discussed in detail. This chapter concludes with an overview of molecular pathogenesis of Salmonella covering its invasion strategy and its dangerous life inside the host cell. Salmonella stays and multiplies inside a specialized endosomal compartment of the host cell known as Salmonella-containing vacuole (SCV). It is believed that Salmonella multiplies inside SCV resulting in single big vacuole containing multiple bacteria. The results of Chapter 2 challenge this notion. Using transmission electron microscopy and confocal laser scanning microscopy we show that SCV also divides along with the division of Salmonella resulting in multiple SCVs containing single bacterium per vacuole. We also show that this division is mediated by the molecular motor dynein. This chapter concludes with a discussion on the advantages of SCV division with respect to Salmonella. Successful intracellular pathogens must have some strategy either to avoid lysosomal fusion or to endure the toxic molecules of lysosomes. In case of Salmonella, it is well accepted that SCV-lysosome fusion is blocked. However, the exact mechanism of this process is still unclear. The results of Chapter 3 enhance our understanding of this issue. This chapter explores an interesting possibility of Salmonella reducing the lysosomal number and thereby reducing the chances of SCV-lysosome fusion. Using flowcytometry and confocal laser scanning microscopy, we show that Salmonella decreases the number of acidic lysosomes in murine macrophages. Thus, our results suggest that there is an imbalance in the ratio of vacuoles to acidic lysosomes which decreases the probability of SCV-lysosome fusion thereby helping Salmonella avoid lysosomes. Multicellular organisms use various defense strategies to protect themselves from microbial infections; production of antimicrobial peptides (AMPs) is one of them. Being cationic in nature, AMPs interact and cause pores in the bacterial membrane eventually killing the bacteria. Pathogenic micro-organisms like Salmonella have evolved many strategies to counteract the AMPs they encounter upon their entry into the host systems. S Typhimurium genome has a gene cluster consisting of yejA, yejB, yejE and yejF genes which encode a putative ABC transporter. Chapter 4 deals with the detailed characterization of these genes. Our study shows that these genes constitute an operon. We have deleted the yejF gene which encodes the ATPase component of this putative ABC transporter. The ΔyejF strain showed increased sensitivity to AMPs like protamine, melittin, polymyxin B and human defensins and was compromised to proliferate inside activated macrophages and epithelial cells. In murine typhoid model, the ΔyejF strain displayed decreased virulence when infected intragastrically. These findings suggest that the putative transporter encoded by the yejABEF operon is involved in counteracting AMPs and contributes to the virulence of Salmonella. An important biochemical property of Salmonella that distinguishes it from the closely related E. coli is its inability to ferment lactose. In E. coli, lactose fermentation is carried out by the products of lac operon which is regulated by a repressor encoded by lacI. Salmonella does not have the lac operon and lacI. It has been proposed that S.enterica has lost lac region (lacI and lacZYA) during its evolution. Chapter 5 deals with the evolutionary and physiological significance behind the loss of lac region by S.enterica. We show that expression of LacI in S. enterica suppresses its virulence by interfering with the expression of SPI-2 virulence genes. We also observed that the genome of S. bongori which does not have the virulence genes of SPI-2 has a homologue of LacI. Our results suggest that presence of lacI has probably hindered the acquisition of virulence genes of SPI-2 in S. bongori, whereas absence of lacI has facilitated the same in S. enterica making it a successful systemic pathogen. Thus, lacI has played a remarkable role in the evolution of Salmonella virulence. Brief summary of four studies that are not directly related to survival strategies of Salmonella are included in Appendix. First two studies analyze molecular evolution of SPIs to understand the mechanism of host specificity in Salmonella and the last two studies explore the signaling of lipopolysaccharide (LPS) derived from Salmonella.

Nous avons décrit la présence de tubules inter-cellulaires [inter-cellular tubules (ICTs)], qui apparaissent entre les deux cellules filles lors de la cytokinèse d’une cellule infectée. Nos données suggèrent que ces structures sont des vestiges de SITs qui connectaient les SCVs initialement présentes dans la cellule mère et qui ont été distribuées dans les cellules filles. Les effecteurs de T3SS2 sont nécessaires à la formation de ces tubules. De plus, nous avons établit une corrélation entre la formation des ICTs et la distribution asymétrique des vacuoles bactériennes entre les cellules filles. Les protéines effectrices de T3SS2 peuvent donc modifier la distribution des bactéries pendant la cytokinèse. Il a été démontré l’existence de différents types de SITs, de composition différentes et qui interagissent avec différents compartiments de la cellule hôte. Pendant la deuxième partie de ma thèse, nous avons caractérisé des tubules dépourvus de protéines de l’hôte mais riches en protéines effectrices [LAMP1-negative tubules (LNT)]. Nous avons montré que les effecteurs de T3SS2 SseF et SseG sont nécessaires à la formation de ces structures. L’inhibition de la formation des LNTs par la suppression de sseF/G est corrélée à un recrutement réduit de LAMP1 sur les SCVs. Cela suggère que la formation des tubules favorise la capture et le transport de LAMP1 vers les SCVs. Nous avons également observé une interaction indépendante de SKIP entre Arl8b et les deux domaines de l’effecteur SifA. En absence de SifA ou de Arl8b, les tubules ont une capacité limitée à capturer les protéines membranaires lysosomales. / We describe the presence of inter-cellular tubules (ICTs) that arise between daughter cells during cytokinesis of an infected cell. Our data suggest that these structures are remnants of SITs that connect bacterial vacuoles originally present in the parent cell and that have been distributed between daughters. T3SS-2 effectors are required for the formation of these tubules. Importantly, there is a correlation between the formation of ICTs and the asymmetric distribution of bacterial vacuoles in daughters. Thus, T3SS-2 effector proteins can manipulate the distribution of bacteria during cytokinesis. This may further increase bacterial spreading and the systemic character of the infection. Different kinds of SITs with diverse host protein contents have been characterised, suggesting the capacity of these tubules to interact with different host compartments. In the second part of my thesis, we performed a biochemical and functional characterization of LAMP1-negative tubules (LNT) that are decorated with effector proteins but essentially devoid of host proteins. We show that T3SS2 effectors SseF and SseG are required for the formation of these structures. The inhibition of LNTs formation by deletion of sseF/G is correlated with a reduced recruitment of LAMP1 to the SCVs. It suggests that formation of tubules favours the capture and the transport of LAMP1 towards the SCV to keep vacuole stable. An additional observation added to this study is that there is a SKIP-independent interaction between Arl8b and both domains of SifA. In the absence of SifA or Arl8b, tubules have a limited capacity to capture host membrane proteins from the late endosomal compartments.

Memoria para optar al Título Profesional de Médico Veterinario / Salmonella spp. es una bacteria Gram-negativa y agente etiológico de la salmonelosis, enfermedad transmitida por los alimentos (ETA). Es el principal agente bacteriano causante de enfermedad gastrointestinal en nuestro país. El objetivo de este estudio fue verificar el método analítico VIDAS® Easy Salmonella, para la detección de Salmonella spp. en diferentes matrices de alimentos, dentro del Laboratorio de Salud Pública Ambiental y Laboral de la SEREMI de Salud Región Metropolitana. Se analizaron 60 muestras correspondientes a cinco matrices de alimentos. Para cada matriz se analizaron 12 muestras, diez de las cuales fueron contaminadas artificialmente con una cepa de Salmonella enterica serovar Typhimurium ATCC® 14028 y dos fueron analizadas sin contaminar (control). Siguiendo el protocolo descrito por el fabricante, las muestras fueron pre-enriquecidas, enriquecidas y sometidas a detección inmunoenzimática presuntiva mediante el kit VIDAS® Easy Salmonella. Las muestras positivas y negativas, fueron sembradas en agar selectivo XLD y confirmadas a través del kit API 20E®. El criterio de aceptación de la verificación del método alternativo en cada matriz, fue la detección positiva del 100 % de las muestras contaminadas artificialmente. Para determinar la concordancia existente entre el método alternativo y la siembra en agar XLD, se utilizó el coeficiente estadístico Kappa (κ). Los datos obtenidos en todas las muestras (hortalizas (10/10), cecinas (10/10), mariscos (10/10), carne de ave (10/10) y mayonesas envasadas (10/10)), arrojaron un 100% de concordancia (κ = 1) entre el método alternativo y la siembra en agar XLD. Los resultados sugieren que el método alternativo VIDAS® Easy Salmonella, puede ser utilizado como método de screening en las matrices analizadas, dentro del Laboratorio de Salud Pública Ambiental y Laboral de la Secretaría Regional Ministerial de Salud Región Metropolitana

<p>Poznavanje i razumevanje adhezivne sposobnosti i formiranja biofilma patogenih bakterija, kao i njihovog odnosa prema faktorima koji mogu stimulisati ili inhibirati razvoj biofilma, je od fundamentalnog značaja za iznalaženje mera za njihovu efikasnu prevenciju i eliminaciju.<br />Imajući u vidu navedenu činjenicu kao i da je Salmonella enterica serotip Enteritidis epidemiolo&scaron;ki najfrekventniji serotip cilj ovog istraživanja je bio da se ispita: sposobnost različitih sojeva Salmonella Enteritidis izolovanih iz kliničkog materijala, hrane za životinje i odabranog referentnog soja da formiraju biofilm, adherentnost na povr&scaron;ine od stakla i nerđajućeg čelika, sposobnost preživljavanja odabranih biofilm produkujućih sojeva kao i mogućnost primene konfokalne laserske i skening elektronske mikroskopije u vizuelizaciji trodimenzionalne strukture biofilma.<br />Određivanjem morfotipa kolonija na Kongo red agaru na temperaturi inkubiranja od 25&deg;C među testiranim izolatima detektovana su tri morfotipa RDAR (red, dry and rough), BDAR (brown dry and rough) i SAW (smooth and white). Polovina testiranih izolata je eksprimirala RDAR morfotip. Izolati koji su eksprimirali karakterističan morfotip na ovoj temperaturi su formirali na vazduh tečnost međufazi isti tip pelikule.<br />Uporednom analizom rezultata primenjenih skrining testova za utvrđivanje sposobnosti formiranja biofilma pri temperaturi inkubiranja od 25&deg;C ustanovljena je korelacija između pojave određenog morfotipa na Kongo crvenom agaru i sposobnosti formiranja biofilma u kristal violet i pelikula testu. Međutim, sa povećanjem temperature inkubiranja na 37oC, ova korelacija nije ustanovljena, sa izuzetkom izolata SE8.<br />Svi testirani izolati su pokazali sposobnost adherencije na povr&scaron;inu stakla i nerđajućeg čelika, ali u različitoj meri. Na sposobnost adherencije povoljniji uticaj je imala temperatura inkubiranja od 25&deg;C (p&lt;0,05), sa izuzetkom izolata SE3 (p&gt;0,05). Između stepena adherencije izolata na povr&scaron;ine stakla i nerđajućeg čelika nisu ustanovljene statistički značajne razlike (p&gt;0,05).<br />Praćenjem stope preživljavanja tokom 28 dana u uslovima isu&scaron;ivanja evidentirana je znatno veća stopa preživljavanja ćelija izolata RDAR morfotipa u odnosu na stopu preživljavanja ćelija BDAR morfotipa (p&lt;0,05). Praćenjem stope preživljavanja tokom 90 dana u uslovima povremene dostupnosti hranljivih materija, zabeležena je veća stopa preživljavanja u odnosu na stopu preživljavanja u uslovima isu&scaron;ivanja. U uslovima povremene dostupnosti hranljivih materija nakon devedeset dana ispitivanja kod obe grupe izolata procenat vijabilnih ćelija je iznosio vi&scaron;e od 50%.<br />Primenjenim mikroskopskim tehnikama (CLSM i SEM) omogućena je detaljna vizualizacija formiranih biofilmova. Na modelu izolata SERDAR morfotipa, ustanovljeno je da se formiranje biofilma pod primenjenim eksperimentalnim uslovima, odvija u tri faze: 1) inicijalna adhezija za povr&scaron;inu i formiranje manjih ćelijskih agregata (24h); 2) formiranje većih ćelijskih agregata uz produkciju EPS (48h); 3) sazrevanje biofilma uz značajnu produkciju EPS &scaron;to omogućava formiranje stabilne trodimenzionalne strukture biofilma (96h).<br />Nasuprot karakteristikama koje bakterije pokazuju tokom rasta u medijumima koji obiluju hranljivim materijama, bakterije u biofilmovima pokazuju drugačije osobine u pogledu ekspresije gena i karakteristika rasta. Zahvaljujući ovim razlikama, bakterije u biofilmovima pokazuju povećanu rezistenciju na antibiotike i dezinficijense, zbog čega se konstantno razvijaju nove kontrolne strategije u cilju iznalaženja potencijalnih biolo&scaron;kih re&scaron;enja koja pored različitih enzima, faga, antimikrobnih jedinjenja proizvedenih od strane mikroorganizama uključuju i antimkrobna jedinjenja biljnog porekla kao &scaron;to su biljni ekstrakti, etarska ulja i različiti začini. Stoga, je u okviru drugog segmenta ovog istraživanja ispitivan hemijski sastav etarskih ulja, antimikrobni efekat etarskih ulja (O. heracleoticum , O. vulgare , Th. vulgaris i Th. serpyllum) i pojedinačnih komponenti etarskog ulja (karvakrola i timola) na bujonske kulture testiranih sojeva Salmonella Enteritidis kao i uticaj odabranih koncentracija etarskih ulja na inicijalnu adheziju i već formirani biofilm odabranih sojeva Salmonella Enteritidis.<br />Etarska ulja je karakterisao visok zbirni udeo glavnih fenolnih komponenti karvakrola i timola: O. heracleoticum (71,6%), O. vulgare (63,6%), Th. vulgaris (59,77%), Th. serpyllum (40,04%). Etarska ulja su ispoljila antimikrobni efekat sledećim redosledom: O. heracleoticum &gt;O. vugare =Th. vulgaris &gt;Th. serpyllum. Antimikrobni efekat etarskih ulja je bio direktno srazmeran zbiru fenolnih komponenti (karvakrola i timola) u etarskom ulju. U odgovoru na tretman etarskim uljima između izolata S. Enteritidis nisu ustanovljene razlike.<br />Etarska ulja, karvakrol i timol su pokazali inhibitorni efekat na inicijalnu adheziju i posledično na formiranje biofilma testiranih izolata S. Enteritidis na dozno zavisan način. Upoređivanjem uticaja etarskih ulja na inhibiciju inicijalne adhezije i metabolitičke aktivnosti ćelija između izolata RDAR i BDAR morfotipa ustanovljene razlike nisu bile statistički značajne (p&gt;0,05).<br />Ispitivanjem uticaja etarskih ulja, karvakrola i timola na ukupnu biomasu biofilma i metabolitičku aktivnost ćelija dokazano je da etarska ulja u primenjenim koncentracijama ispoljavaju uticaj na redukciju ukupne biomase formiranog biofilma i metaboličke aktivnosti bakterijskih ćelija na dozno zavisan način u funkciji vremena. Znatno veća efikasnost primenjenih tretmana je pokazana u slučaju njihove primene na biofilmove formirane od strane izolata BDAR morfotipa (p&lt;0,05).</p> / <p><span style="font-size:10px;">Knowledge and understanding ability of the pathogenic bacteria that adhere to surface and form biofilm, as well as their relationship between these abilities and factors that stimulate or inhibit biofilm development, are essential to develop strategies for their prevention and elimination.<br />Considering also the fact that Salmonella enterica serotype Enteritidis has been epidemiologically the most frequently found serotype, the aims of this study were to evaluate: biofilm forming ability of several Salmonella Enteritidis strains isolated from clinical material, feed and selected control strain, their ability to adhere to glass and stainless steel surfaces, survival of selected biofilm-producing strains, as well as the possibility of applying confocal laser scanning (CLSM) and scanning electron microscopy (SEM) for visualization of biofilm three-dimensional structure.<br />Determination of colony morphotype on Congo red agar at incubation temperature of 25&deg;C revealed that among all tested isolates three morphotypes were detected: RDAR (red, dry and rough), BDAR (brown dry and rough) and SAW (smooth and white). Half of all tested isolates expressed RDAR morphotype. All isolates that expressed specific morphotype at this incubation temperature also formed the corresponding type of pellicle at air-liquid interface.<br />Comparing the results of the applied assays was ascertained the correlation between specific morphotype on Congo red agar and biofilm forming ability in Cristal violet and pellicle tests, at incubation temperature of 25&ordm;C. In the case of assays conducted at 37&deg;C, this correlation was not established, except for the isolate SE8.<br />All tested isolates showed varying degree of the ability to adhere to glass and stainless steel surfaces. Incubation temperature of 25&ordm;C had more favorable effect on the adherence, with the exception of isolate SE3 (p&gt;0.05). There were no statistically significant differences between adherence ability of all isolates to glass and stainless steel surfaces (p&gt;0.05).<br />Accompaniment of the survival rate during 28 days in the conditions of desiccation, the significantly higher survival rate was</span> <span style="font-size:10px;">obtained for RDAR than BDAR morphotype isolates (p&lt;0.05). Accompaniment of the survival rate during 90 days in the conditions of occasional availability of nutrients, it was detected the higher survival rate than in condition of desiccation. Under these conditions, after 90 days, there were more than 50% of viable cells among both groups of isolates.<br />Applied microscopic techniques (CLSM and SEM) provided detailed visualization of formed biofilms. On model of SERDAR morphotype isolate, it was established that biofilm formation under this experimental conditions has three phases: 1) initial adhesion to the surface and formation of small cell aggregates (24h); 2) formation of large cell aggregates followed with production of extracellular polymer substance (EPS) (48h); 3) maturation of biofilm followed with significant EPS production, which allows formation of stabile three dimensional structure of the biofilm (96h).<br />Contrary to characteristics that bacteria expressed during their growth in the nutrient media, bacteria in biofilms show different properties in terms of genes expression and growth characteristics. Due to these differences, bacteria in biofilms showed higher resistance to antibiotics and disinfectants. For these reasons are being constantly developed new potential biological control strategies that aim at finding the potential biological solutions that besides different enzymes, phages, antimicrobial compounds produced by microorganisms, also include antimicrobial compounds of plant origin, such as extracts, essential oils and different spices.<br />Therefore, the other segment of this research was investigation of the chemical composition and antimicrobial properties of different essential oils (O. heracleoticum, O. vulgare, Th. vulgaris and Th. serpyllum) and their components (carvacrol and thymol), against broth cultures of Salmonella Enteritidis. Also, selected concentrations of essential oils were tested against initial adhesion and preformed biofilm of selected Salmonella Enteritidis isolates.<br />Essential oils were characterized by high amount of phenol compounds carvacrol and thymol: O. heracleoticum (71.6%), O. vulgare (63.6%), Th. vulgaris (59.77%) and Th. serpyllum (40.04%). Essential oils showed antimicrobial potential as follows: O. heracleoticum &gt; O. vugare = Th. vulgaris &gt; Th. serpyllum. Antimicrobial effect was directly proportional to the total content of phenolic components (carvacrol and thymol) in essential oil. Between responses of different S. Enteritidis isolates to essential oil treatment, there was no significant difference.<br />Essential oils, carvacrol and thymol demonstrated inhibitory effect on initial adhesion and consequently, on biofilm formation of S. Enteritidis isolates, in a dose-dependent manner. Comparing influence of essential oil on the inhibition of initial cell adhesion and metabolic activity of cells RDAR and BDAR morphotype, no statistically significant differences were established (p&gt;0.05).<br />Examination of the influence of essential oils, carvacrol and thymol on total biomass of preformed biofilms and metabolic activity of cells, it was revealed that essential oils in applied concentrations cause reduction of total biomass of preformed biofilm and metabolic activity</span> <span style="font-size:10px;">of bacterial cells in a time and dose dependent manner. Applied treatments demonstrated significantly higher efficiency on BDAR morphotype biofilms (p&lt;0.05).</span></p>

La producción de cobayos es una actividad que cada vez cobra más importancia en nuestro país, debido al aumento de la demanda de la carne de esta especie en el mercado nacional e internacional. Sin embargo, los conocimientos en sanidad de cobayos son aún escasos, haciéndola vulnerable a las diversas enfermedades. En este contexto sobresale la salmonelosis enfermedad que causa altos porcentajes de mortalidad y morbilidad en producción de cobayos. El presente estudio identificó las lesiones anatomopatológicas más frecuentes que predominaron en órganos de cobayos infectados con Salmonella spp. Para ello se realizó el estudio retrospectivo de 125 protocolos de necropsia de cobayos, obtenidos del Laboratorio de Histología, Embriología y Patología Veterinaria de la Facultad de Medicina Veterinaria de la Universidad Nacional Mayor de San Marcos, en un período comprendido entre el 2001 al 2007, el diagnóstico de la salmonelosis fue confirmada por el Laboratorio de Microbiología Veterinaria de dicha Universidad, resultando 81 cobayos positivos a Salmonella spp. y 44 negativos. Los resultados muestran que la mayoría de órganos se encontraron lesionados, con una mediana de 5 órganos por animal, siendo el hígado el órgano que frecuentemente presentó lesiones patológicas con un 87.7 % ± 0.07 (71/81), siendo la imagen patomorfológica que predominó la hepatitis necrótica con un 44.4 % (36/81). Así mismo, se realizó la clasificación de las lesiones anatomopatológicas en procesos inflamatorios, trastornos circulatorios, degenerativos y de adaptación siendo la inflamación el trastorno patológico más frecuente, representando el 43.4 % ± 4.8 (177/408) del total de órganos lesionados encontrados en los 81 cobayos infectados con Salmonella spp. Palabras Claves: Cobayos, Salmonella spp., Lesiones anatomopatológicas / --- The guinea pig production is an activity that becomes increasingly more important in our country due to increased demand for the meat of this species in domestic and international markets. However, health knowledge in guinea pigs are still scarce, making it vulnerable to various diseases. In this context stands salmonellosis, disease that causes high mortality and morbidity rates of production of guinea pigs. This study identified the most common pathologic lesions predominated in organs of guinea pigs infected with Salmonella spp. This retrospective study was conducted on necropsy of 125 guinea pigs, obtained from the Laboratory of Histology, Embryology and Veterinary Pathology, College of Veterinary Medicine of the Universidad Nacional Mayor de San Marcos, in a period between 2001 to 2007, the diagnosis of salmonellosis was confirmed by the Veterinary Microbiology Laboratory of the University, resulting in 81 guinea pigs positive for Salmonella spp. and 44 negatives. The results show that most organs were injured, with a median of 5 organs per animal, the liver being the organ that pathological lesions often presented with a 87.7% ± 0.07 (71/81) being the prevailing pathomorphological image necrotic hepatitis in 44.4% (36/81). Likewise, it was made the classification of pathological lesions in inflammatory processes, circulatory disorders, degenerative and adaptive, inflammation being the most common pathological disorder, accounting for 43.3% ± 4.8 (177/408) of total injured organs found in the 81 guinea pigs infected with Salmonella spp. Key Words: Guinea pigs, Salmonella spp., anatomopathological lesions.

De acordo com a Food and Agriculture Organization of the United Nations (FAO, 2013), o consumo mundial de carne de frango tem aumentado de maneira significativa nas últimas décadas. Por outro lado, a preocupação dos produtores de alimentos, com a inocuidade de seus produtos também tem aumentado na mesma proporção. Durante as etapas da operação de abate de maneira geral, as contaminações cruzadas são as principais causas de disseminação de microrganismos patogênicos nos produtos obtidos e causadores de gastroenterites no consumidor. Espécies da enterobactéria Salmonella se enquadram como um dos maiores riscos desse tipo de doença devido a sua associação com os inúmeros surtos ocorridos a nível mundial, após o consumo desses produtos. Algumas enterobactérias possuem um gene blaKPC, que codifica a enzima carbapenemase, que confere resistência a antibióticos carbapenêmicos, agravando mais a situação. Alguns fatores de virulência encontrados nesse gênero de bactéria podem ainda estar associados a capacidade de adesão e formação de biofilmes em superfícies inertes, dificultando operações de higienização nas linhas de processamento. Assim sendo, a presente pesquisa objetivou a verificação da capacidade de estirpes de Salmonella spp isoladas de aviário e linha de abate de frangos de um frigorífico no estado do Rio Grande do Sul produzirem biofilmes e apresentarem resistência a antibióticos carbapenêmicos. Na avaliação da produção de biofilme, foi empregada a técnica de microplacas de polietileno e produção de cápsula segundo Stepanovic et al. (2004) e Rodrigues et al. (2006), respectivamente.Foram pesquisados os fatores de virulência de salmonelas, representados pelos genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH,e spvC, utilizando o método de Reação de cadeia de Polimerase (PCR), descrita por Borges et al. (2013). As estirpes foram submetidas ao teste de resistência a antibióticos carbapenêmicos pelo método de disco difusão com carbapenêmicos segundo Clinical and Laboratory Standards Institute-CLSI (2010) e pesquisa do gene de resistência a carbapenêmicos blaKCP, pela técnica de PCR, segundo NAAS et al. (2007). Obteve-se quatro perfis genéticos das estirpes de Salmonella spp.: perfil 1: genes ifpA, agfA, invA e avrA; perfil2: genes ifpA, agfA, sefA, invA e avrA; perfil 3: genes invA e avrA; perfil4: genes ifpA,agfA e invA. Observou-se a resistência das estirpes somente ao antibiótico imipenen. Entre as 36 estirpes de Salmonella spp. isoladas, todas foram consideradas produtoras de biofilme in vitro, sendo que 69% destas, apresentaram-se como fortes produtoras, 25% como moderadas, e apenas 6% como fracas produtoras. O método Agar Vermelho Congo não se mostrou eficiente para teste presuntivo de produção de biofilme para estirpes de Salmonella spp. Não foi evidenciado o gene blaKPC nas estirpes de Salmonella spp. isoladas na presente pesquisa. / According to Food and Agriculture Organization of the United Nations (FAO, 2013), the world consumption of meat of chicken has been higher significantly in the last decades. On the other hand, the concern of the food producers with the safety of their products also has increased in the same proportion. During the steps of the slaughter operation in general, cross contamination are the main causes of pathogenic microorganisms dissemination in the obtained products and the causes of gastroenteritis in the consumer. Species of the Enterobacter Salmonella fall as the highest risks of this kind of disease, due to their association with countless outbreaks which occurred worldwide, after the consumption of these products. Some enterobacter have a blaKPC gene, which codes for the carbapenemase enzyme that confers resistance to carbapenems antibiotics, aggravating the situation. Some virulence factors found in this bacteria genes can also be associated to the ability of adherence and the formation of biofilms in inert surfaces, making it difficult the operations of sanitation in the processing lines. Thus, the present research aimed to verify the capacity of Salmonella spp. strains isolated from aviary and slaughter line of chicken in a fridge of Rio Grande do Sul state to produce biofilms and be resistant to carbapenems antibiotics. In the evaluation of biofilm production, it was used the polyethylene microplates and capsule production technique according to Stepanovic et al. (2004) and Rodrigues et al. (2006), respectively. The virulence factors of salmonella were researched, represented by the genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, e spvC, using the Polymerase Chain Reaction (PCR) method described by Borges et al. (2013). The lineages were submitted to the carbapenems antibiotic resistence test by the disk diffusion method with carbapenems according to the Clinical and Laboratory Standards Institute-CLSI (2010), and the research of the resistance to carbapenems gene blaKCP, by the strains Salmonella spp.: profile 1: genes ifpA, agfA, invA and avrA.; profile 2: genes ifpA, agfA, sefA, invA and avrA; profile 3: genes invA and avrA; profile 4: genes ifpA, agfA and invA. It was observed the resistance of the strains only to the imipenen antibiotic. Among the other 36 cultures of Salmonella spp. isolated, all were considered to produce biofilm in vitro, of which 69% were strong producers, 25% moderate, and only 6% were low producers. The method Congo Red Agar was not efficient to the presumptive test of biofilm production for the Salmonella spp. strains. It was not evidenced the gene blaKPC in Salmonella spp. strains isolates in this research.