Phenotypic and molecular characterisation of Salmonella spp. isolates in healthy poultry.

1. Epidemiological surveillance of Salmonella spp. serves as a primary tool for maintaining the health of poultry flocks. Characterising circulating serotypes is crucial for implementing control and prevention measures. This study conducted phenotypic and molecular characterisation of S. enterica Pullorum, S. enterica Heidelberg, and S. enterica Corvalis isolated from broiler chickens during slaughtering.2. All strains were susceptible to gentamicin, neomycin and norfloxacin. However, resistance rates exceeded 50% for ciprofloxacin and tiamulin, irrespective of the serotype. Approximately 64% of strains were classified as multidrug-resistant, with S. enterica Heidelberg strains exhibiting significantly higher overall resistance. The isolates demonstrated the ability to adhere and produce biofilm at a minimum of three temperatures, with S. enterica Pullorum capable of biofilm production at all temperatures encountered during poultry rearing.3. Each strain possessed between two and seven different virulence-associated genes. Genetic similarity, as indicated by pulsed field gel electrophoresis, exceeded 90% for all three serotypes and strains were classified in the R5 ribotype by PCR, regardless of serotype. Sequencing revealed high similarity among all strains, with homology ranging from 99.61 to 100% and all were classified to a single cluster.4. The results suggested a clonal relationship among the strains, indicating the possible circulation of a unique clonal group of S. enterica Pullorum in the southern region of Brazil.

Heat stress impairs egg production in commercial laying hens infected by fowl typhoid.

Salmonella Gallinarum (SG) is an avian-restricted pathogen that causes fowl typhoid in poultry. Although it has been reported frequently over many decades in poultry flocks worldwide, the microorganism is more commonly associated with poultry in developing countries, particularly those with high ambient temperatures, where the acute form of the disease results in considerable economic losses. A more detailed investigation of environmental factors that affect the course of disease may assist in identifying effective prevention and control measures. Heat stress is known to impair the immunological response to a variety of pathogens and clearly may be an important contributory factor in the prevalence of disease in countries with warm or hot climates. Thus, the objective of the present study was to evaluate the effects of heat stress on chickens infected with SG. For this, light and semi-heavy commercial laying hens were distributed randomly within four groups as follows: infected and non-infected groups in rooms held at ambient temperature, and infected and non-infected groups under heat stress. Clinical signs, egg production, and mortality were recorded daily. Bacteriological counts in liver and spleen samples were estimated at 2, 5, 7, and 14 days post-infection. The results showed that both SG infection and heat stress had similar effects on egg production and a synergistic effect of the two stressors was observed. The data show an interaction between disease and heat stress which could point towards environmental and biosecurity approaches to resolving the possible 30% fall in production observed in such countries.

An ordinal logistic regression approach to predict the variability on biofilm formation stages by five Salmonella enterica strains on polypropylene and glass surfaces as affected by pH, temperature and NaCl.

This study assessed the adhesion and formation of biofilm by five Salmonella enterica strains (S. Enteritidis 132, S. Infantis 176, S. Typhimurium 177, S. Heidelberg 281 and S. Corvallis 297) on polypropylene (PP) and glass (G) surfaces as affected by pH (4-7), NaCl concentration (0-10% w/v) and temperature (8-35 °C). Sessile counts <3 log CFU/cm2 were considered lack of adhesion (category 1), while counts ≥ 3 and < 5 log CFU/cm2 corresponded to adhesion (category 2) and counts ≥ 5 log CFU/cm2 corresponded biofilm formation (category 3). The obtained results categorized in these three responses were used to develop ordinal regression models to predict the probability of biofilm stages on PP- and G-surfaces. The experimental outcomes for lack of adhesion were >90% on PP- and G-surfaces. Generally, adhesion outcomes corresponded to approximately 36% of the total, whereas biofilm outcomes were close to 65% in both PP- and G-surfaces. The biofilm stages varied among the strains studied and with the material surface under the same experimental conditions. According to the generated ordinal models, the probability of adhesion and biofilm formation on PP-surface by the five S. enterica strains tested decreased at pH 4 or 5 in NaCl concentrations >4% and at a temperature <20 °C. On G-surface, the probability of adhesion increased pH 6 or 7, in the absence of NaCl and temperatures <20 °C, while, the probability of biofilm formation increased in the same pH, NaCl concentration up to 4% and temperatures ≥20 °C. This is the first study assessing the biofilm formation through categorical, ordinal responses and it shows that ordinal regression models can be useful to predict biofilm stages of S. enterica as a function of pH, NaCl, and temperature or their interactions.

Worldwide Epidemiology of Salmonella Serovars in Animal-Based Foods: a Meta-analysis.

Salmonella spp. are among the most important foodborne pathogens and the third leading cause of human death among diarrheal diseases worldwide. Animals are the primary source of this pathogen, and animal-based foods are the main transmission route to humans. Thus, understanding the global epidemiology of Salmonella serovars is key to controlling and monitoring this bacterium. In this context, this study aimed to evaluate the prevalence and diversity of Salmonella enterica serovars in animal-based foods (beef, pork, poultry, and seafood) throughout the five continents (Africa, the Americas [North and Latin America], Asia, Europe, and Oceania). The meta-analysis consisted of a chemometric assessment (hierarchical cluster analysis and principal component analysis) to identify the main epidemiological findings, including the prevalence and diversity of the Salmonella serovars in each matrix. Regarding the serovar distribution, S Typhimurium presented a cosmopolitan distribution, reported in all four assessed matrices and continents; poultry continues to play a central role in the dissemination of the Enteritidis serovar to humans, and Anatum and Weltevreden were the most frequently found in beef and seafood, respectively. Additionally, we recommended careful monitoring of certain serovars, such as Derby, Agona, Infantis, and Kentucky. Finally, given the scientific data regarding the most frequently reported serovars and which matrices constitute the main vehicles for the transmission of this pathogen, control programs may be improved, and specific interventions may be implemented in an attempt to reduce the risk of this pathogen reaching humans.IMPORTANCE Salmonellosis is caused by Salmonella spp. and is the third leading cause of death among food-transmitted diseases. This pathogen is commonly disseminated in domestic and wild animals, and the infection's symptoms are characterized by acute fever, nausea, abdominal pain, and diarrhea. The animals are the primary source of salmonellae, and animal-based foods are the main transmission route to humans. Therefore, data collected from these sources could contribute to future global interventions for effective control and surveillance of Salmonella along the food chain. In light of this, the importance of our research is in identifying the prevalence of Salmonella serovars in four animal-based food matrices (pork, poultry, beef, and seafood) and to evaluate the importance that each matrix has as the primary source of this pathogen to humans.

Salmonella enterica persister cells form unstable small colony variants after in vitro exposure to ciprofloxacin.

Persistence phenotype and small colony variants (SCVs) can be part of a bacterial bet-hedging strategy for survival under environmental stresses, such as antimicrobial exposure. These phenotypes are of particular concern in persistent and relapsing infections, since cells resume to normal growth after cessation of the stressful condition. In this context, we found persisters and unstable SCVs as phenotypic variants of Salmonella enterica that were able to survive ciprofloxacin exposure. A high heterogeneity in persister levels was observed among S. enterica isolates grown under planktonic and biofilm conditions and exposed to ciprofloxacin or ceftazidime, which may indicate persistence as a non-multidrug-tolerant phenotype. Nevertheless, a comparable variability was not found in the formation of SCVs among the isolates. Indeed, similar proportions of SCV in relation to normal colony phenotype (NCP) were maintained even after three successive cycles of ciprofloxacin exposure testing colonies from both origins (SCV or NCP). Additionally, we found filamentous and dividing cells in the same scanning electron microscopy images from both SCV and NCP. These findings lead us to hypothesize that besides variability among isolates, a single isolate may generate distinct populations of persisters, where cells growing under distinct conditions may adopt different and perhaps complementary survival strategies.

Predicting adhesion and biofilm formation boundaries on stainless steel surfaces by five Salmonella enterica strains belonging to different serovars as a function of pH, temperature and NaCl concentration.

This study aimed to assess the capability of 97 epidemic S. enterica strains belonging to 18 serovars to form biofilm. Five strains characterized as strong biofilm-producers, belonging to distinct serovars (S. Enteritidis 132, S. Infantis 176, S. Typhimurium 177, S. Heidelberg 281 and S. Corvallis 297) were assayed for adhesion/biofilm formation on stainless steel surfaces. The experiments were conducted in different combinations of NaCl (0, 2, 4, 5, 6, 8 and 10% w/v), pH (4, 5, 6 and 7) and temperatures (8 °C, 12 °C, 20 °C and 35 °C). Only adhesion was assumed to occur when S. enterica counts were ≥3 and <5 log CFU/cm2, whereas biofilm formation was defined as when the counts were ≥5 log CFU/cm2. The binary responses were used to develop models to predict the probability of adhesion/biofilm formation on stainless steel surfaces by five strains belonging to different S. enterica serovars. A total of 99% (96/97) of the tested S. enterica strains were characterized as biofilm-producers in the microtiter plate assays. The ability to form biofilm varied (P < 0.05) within and among the different serovars. Among the biofilm-producers, 21% (20/96), 45% (43/96), and 35% (34/96) were weak, moderate and strong biofilm-producers, respectively. The capability for adhesion/biofilm formation on stainless steel surfaces under the experimental conditions studied varied among the strains studied, and distinct secondary models were obtained to describe the behavior of the five S. enterica tested. All strains showed adhesion at pH 4 up to 4% of NaCl and at 20 °C and 35 °C. The probability of adhesion decreased when NaCl concentrations were >8% and at 8 °C, as well as in pH values ≤ 5 and NaCl concentrations > 6%, for all tested strains. At pH 7 and 6, biofilm formation for S. Enteritidis, S. Infantis, S. Typhimurium, S. Heidelberg was observed up to 6% of NaCl at 35 °C and 20 °C. The predicted boundaries for adhesion were pH values < 5 and NaCl ≥ 4% and at temperatures <20 °C. For biofilm formation, the predicted boundaries were pH values < 5 and NaCl concentrations ≥ 2% and at temperatures <20 °C for all strains. The secondary models obtained describe the variability in boundaries of adhesion and biofilm formation on stainless steel by five strains belonging to different S. enterica serovars. The boundary models can be used to predict adhesion and biofilm formation ability on stainless steel by S. enterica as affected by pH, NaCl and temperature.

Salmonella Gallinarum field isolates and its relationship to vaccine strain SG9R.

1. The aim of the present study was to determine if the 9R-strain of the Salmonella Gallinarum live vaccine was responsible for having fowl typhoid outbreaks in chicken flocks from both chicken and turkey breeders as well as to verify the antimicrobial resistance of the isolates from the outbreaks. 2. The triplex polymerase chain reaction, standard antimicrobial test, beta-lactamase genes identification and Ion Torrent PMG whole-genome sequence were used in the field isolates and in the vaccine strain of S. Gallinarum. 3. The 60 tested isolates were not from vaccine origin and manifested high resistance to drugs from macrolide and quinolone groups. Whole-genome sequencing (WGS) and single nucleotide polymorphism analysis on selected isolates for core genes from Salmonella enterica confirmed the wild origin of these isolates and showed two possible sources of S. Gallinarum in the studied outbreaks. 4. S. Gallinarum isolated from fowl typhoid outbreaks in the studied period were not caused by the use of the SG9R live vaccine. The source of strains sequenced was diverse.

Molecular characterisation of Salmonella strains isolated from outbreaks and sporadic cases of diarrhoea occurred in Paraná State, South of Brazil.

A total of 46 strains of Salmonella isolated from patients with sporadic diarrhoea or involved in foodborne outbreaks were analysed by PCR for genus identification and serotyping. Subtyping was performed using pulsed-field gel electrophoresis (PFGE) and multiple amplification of phage locus typing (MAPLT) for seven variable loci. Bacteria were identified as belonging to serotype Enteritidis (33 strains; 71·7%) or Typhimurium (13 strains; 28·3%). A high similarity coefficient (94·6%) was observed in the Salmonella Enteritidis group for which were found three related PFGE profiles and only one MAPLT; strains representing profile PA/P1/MI were prevalent (27; 81·8%). Two Salmonella Typhimurium isolates were untypeable by PFGE. The remaining 11 strains had eight PFGE and three MAPLT profiles. The discriminatory power of MAPLT was lower than that of PFGE. Salmonella Enteritidis of clonal nature is predominant in Paraná State, with the most prevalent profile PA/P1/M1 associated with sporadic diarrhoea and with seven of nine reported outbreaks. In conclusion, PFGE shows higher discriminatory power among Salmonella strains.

Candida krusei isolated from fruit juices ultrafiltration membranes promotes colonization of Escherichia coli O157:H7 and Salmonella enterica on stainless steel surfaces.

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm2, respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm2 and 0.55 log CFU cm2 when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm2, respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.

Physical and microbiological quality of opaque, sanitized, and chilled quail eggs experimentally contaminated with Salmonella enteric ser. Typhimurium / Qualidade física e bacteriológica de ovos opacos de codornas sanitizados, refrigerados e contaminados experimentalmente por Salmonella enterica ser. Typhimurium

The objective of this study was to verify the physical, chemical and microbiological quality of Japanese quail eggs artificially contaminated with Salmonella enterica ser. Typhimurium. The eggs were sanitized and stored at different temperatures (between 5 and 25 ºC) for 27 days. We used 768 eggs with opaque shells, typical pigments of the species, and average weight of 11 g. The experimental design was completely randomized in a 2x2x2 factorial arrangement (contamination x sanitation x cooling) with six replications and one egg per experimental unit. The eggs were contaminated by handling with 1.5 x 105 colony forming unit (CFU) of Salmonella. Typhimurium / mL and sanitized according to the treatments with a 5 ppm Cl solution. The data were subjected to analysis of variance and t test. Bacterial contamination has damaged the egg weight, Haugh unit, yolk index and albumen, and pH of yolk and albumen, from 18 days of storage. The egg storage time and storage temperature affected the internal quality of quail eggs in all variables. The worst internal quality was observed in eggs stored at 25 ºC. The sanitation and cooling reduced the growth of Salmonella in contaminated eggs. Eggs in opaque shell, when not refrigerated, should be consumed within 18 days after laying

Objetivou-se estudar a qualidade física, química e microbiológica de ovos de codornas contaminados artificialmente com Salmonella enterica ser. Typhimurium, sanitizados e armazenados a diferentes temperaturas (5 e 25 ºC), durante 27 dias. Foram utilizados 768 ovos com cascas opacas e peso médio de 11g. O delineamento foi inteiramente casualizado em esquema fatorial 2x2x2 (contaminação x sanitização x refrigeração) com seis repetições e um ovo por parcela. Os ovos foram contaminados pelo manuseio com 1,5 x 105 unidade formadoras de colônias (UFCs) e, de acordo com os tratamentos, foram sanitizados com solução com 5 ppm de Cl. Os dados foram submetidos à análise de variância e teste t. A contaminação bacteriana prejudicou o peso do ovo, uH, índice de gema e de albume e  pH de gema e de albume, a partir de 18 dias de armazenamento. O tempo de estocagem dos ovos e a temperatura de armazenamento influenciaram a qualidade interna dos ovos de codornas em todas as variáveis estudadas. A pior qualidade interna foi observada em ovos armazenados na temperatura de 25 ºC. A sanitização e a refrigeração reduziram o crescimento da Salmonella nos ovos contaminados. Ovos com casca opaca, quando não refrigerados, devem ser consumidos em até 18 dias após a postura

Physical and microbiological quality of opaque, sanitized, and chilled quail eggs experimentally contaminated with Salmonella enteric ser. Typhimurium / Qualidade física e bacteriológica de ovos opacos de codornas sanitizados, refrigerados e contaminados experimentalmente por Salmonella enterica ser. Typhimurium

The objective of this study was to verify the physical, chemical and microbiological quality of Japanese quail eggs artificially contaminated with Salmonella enterica ser. Typhimurium. The eggs were sanitized and stored at different temperatures (between 5 and 25 ºC) for 27 days. We used 768 eggs with opaque shells, typical pigments of the species, and average weight of 11 g. The experimental design was completely randomized in a 2x2x2 factorial arrangement (contamination x sanitation x cooling) with six replications and one egg per experimental unit. The eggs were contaminated by handling with 1.5 x 105 colony forming unit (CFU) of Salmonella. Typhimurium / mL and sanitized according to the treatments with a 5 ppm Cl solution. The data were subjected to analysis of variance and t test. Bacterial contamination has damaged the egg weight, Haugh unit, yolk index and albumen, and pH of yolk and albumen, from 18 days of storage. The egg storage time and storage temperature affected the internal quality of quail eggs in all variables. The worst internal quality was observed in eggs stored at 25 ºC. The sanitation and cooling reduced the growth of Salmonella in contaminated eggs. Eggs in opaque shell, when not refrigerated, should be consumed within 18 days after laying(AU)

Objetivou-se estudar a qualidade física, química e microbiológica de ovos de codornas contaminados artificialmente com Salmonella enterica ser. Typhimurium, sanitizados e armazenados a diferentes temperaturas (5 e 25 ºC), durante 27 dias. Foram utilizados 768 ovos com cascas opacas e peso médio de 11g. O delineamento foi inteiramente casualizado em esquema fatorial 2x2x2 (contaminação x sanitização x refrigeração) com seis repetições e um ovo por parcela. Os ovos foram contaminados pelo manuseio com 1,5 x 105 unidade formadoras de colônias (UFCs) e, de acordo com os tratamentos, foram sanitizados com solução com 5 ppm de Cl. Os dados foram submetidos à análise de variância e teste t. A contaminação bacteriana prejudicou o peso do ovo, uH, índice de gema e de albume e  pH de gema e de albume, a partir de 18 dias de armazenamento. O tempo de estocagem dos ovos e a temperatura de armazenamento influenciaram a qualidade interna dos ovos de codornas em todas as variáveis estudadas. A pior qualidade interna foi observada em ovos armazenados na temperatura de 25 ºC. A sanitização e a refrigeração reduziram o crescimento da Salmonella nos ovos contaminados. Ovos com casca opaca, quando não refrigerados, devem ser consumidos em até 18 dias após a postura(AU)

A temporal study of Salmonella enterica serotypes from broiler farms in Brazil.

The present study analyzes the characteristics of Salmonella spp. from broiler chicken farms in Brazil. In total, 82 Salmonella spp. strains were characterized by serotyping, determining susceptibility to antimicrobials, and using pulsed-field gel electrophoresis (PFGE). Fifteen Salmonella serotypes were identified, among which Minnesota (40.24%), Infantis (14.63%), Heidelberg (7.31%), Senftenberg (6.09%), and Mbandaka (6.09%) were the most frequent. Salmonella Minnesota occurred mostly in the state of Mato Grosso do Sul and in one of the broiler companies surveyed. Approximately 60% of the strains were resistant to at least one of the antimicrobials tested. From these isolates, 17.07% were resistant to only one antimicrobial (tetracycline or streptomycin), and 9.75% were resistant to 3 or more antimicrobial classes. Thirteen resistance profiles were characterized, the most frequent of which were the resistance to tetracycline (15.85%); to the combination of trimethroprim with sulfamethoxazole, and tetracycline (10.97%); and to the combination of streptomycin and tetracycline (9.75%). Multiple correspondence analysis revealed that susceptibility or resistance of the analyzed strains and also particular Salmonella serotypes were associated with broiler-producing companies where the samples were collected. Strains presented high intraserotype genetic variability, as shown by the 64 PFGE profiles, suggesting the existence of several contamination sources in the surveyed farms.

Fowl typhoid (Salmonella Gallinarum) outbreak in Japanese quail (Coturnix coturnix japonica).

A fowl typhoid (FT) outbreak is reported in a flock of 400 Japanese quail (Coturnix coturnix japonica) at 91 days of age. Of these, 222 died suddenly, and necropsy revealed swollen liver and spleen with off-white to yellowish granules and reddish small intestine mucosa. Histopathology showed severe multifocal necrosis of liver and spleen (5/5), pulmonary congestion with macrophage infiltration in air capillaries (5/5), discrete interstitial nephritis (2/2), superficial necrosis of the intestinal mucosa with large numbers of coccobacilli (2/2), moderate peritonitis (2/2), and discrete airsacculitis (1/1). Anti-Salmonella immunohistochemistry (IHC) stained the cytoplasm of macrophages or free in the liver (5/5), spleen (5/5), lungs (4/5), kidneys (2/2) small intestine mucosa (2/2), cecum (1/1), bone marrow (1/1), air sacs (1/1), and ovary (1/1). In the heart (5/5), brain (2/2), esophagus (2/2), pancreas (2/2), proventriculus (2/2), gizzard (1/1), bursa of Fabricius (1/1), oviduct (1/1), and skeletal muscle (1/1) staining was observed only in the lumen of blood vessels. Salmonella Gallinarum was isolated in pure cultures of liver, spleen, lung, intestine, and blood samples of two birds.

Repression of flagella is a common trait in field isolates of Salmonella enterica serovar Dublin and is associated with invasive human infections.

The nontyphoidal Salmonella enterica serovar Dublin is adapted to cattle but infrequently infects humans, very often resulting in invasive infections with high levels of morbidity and mortality. A Salmonella-induced intestinal acute inflammatory response is postulated as a mechanism to prevent bacterial dissemination to systemic sites. In S. enterica serovar Typhimurium, flagella contribute to this response by providing motility and FliC-mediated activation of pattern recognition receptors. In this study, we found 4 Salmonella enterica isolates, with the antigenic formula 9,12:-:-, that, based on fliC sequence and multilocus sequence type (MLST) analyses, are aflagellate S. Dublin isolates. Interestingly, all were obtained from human bloodstream infections. Thus, we investigated the potential role of flagella in the unusual invasiveness exhibited by S. Dublin in humans by analyzing flagellation and proinflammatory properties of a collection of 10 S. Dublin human clinical isolates. We found that 4 of 7 blood isolates were aflagellate due to significantly reduced levels of fliC expression, whereas all 3 isolates from other sources were flagellated. Lack of flagella correlated with a reduced ability of triggering interleukin-8 (IL-8) and CCL20 chemokine expression in human intestinal Caco-2 cells and with reduced early inflammation in the ceca of streptomycin-pretreated C57/BL6 mice. These results indicate that flagella contribute to the host intestinal inflammatory response to Salmonella serovar Dublin and suggest that their absence may contribute to its systemic dissemination through dampening of the gut immune response. Analysis of FliC production in a collection of cattle isolates indicated that the aflagellate phenotype is widely distributed in field isolates of S. Dublin.

[Therapeutic intervention alternatives in cancer, using attenuated live bacterial vectors: Salmonella enterica as a carrier of heterologous molecules]. / Alternativas de intervención terapéutica en cáncer usando vectores bacterianos vivos atenuados: Salmonella enterica como acarreador de moléculas heterólogas.

Salmonella enterica is a facultative anaerobic bacteria, whose ability to colonize antigen-presenting cells (APCs) such as dendritic cells and macrophages, has allowed its successful use as an alive, attenuated bacterial vector for vaccination. Salmonella enterica elicits efficient cellular, humoral and mucosal immune responses, against heterologous antigens including viruses, parasites, other bacterial species and tumor-associated antigens, since it is capable of delivering these antigens to cells of the immune system. The extracellular expression of heterologous antigens on the surface of Salmonella enterica via its type I, III and V secretion systems, and their delivery into infected cells is essential for its stimulation of immune responses against these antigens. Moreover, Salmonella enterica is a promising therapeutic agent against cancer, as demonstrated by reports of pre-clinical and clinical studies indicating that, after systemic administration, Salmonella enterica preferentially localizes in solid tumors and metastases as compared to normal tissues. In this review, we focus on novel prophylactic and therapeutic anti-cancer approaches using Salmonella enterica as a delivery system of heterologous molecules with the aim of inhibiting tumor growth.

The type VI secretion system encoded in Salmonella pathogenicity island 19 is required for Salmonella enterica serotype Gallinarum survival within infected macrophages.

Salmonella enterica serotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported that S. Gallinarum harbors a type VI secretion system (T6SS) encoded in Salmonella pathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observed in vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced under in vitro bacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins. In vitro bacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon after Salmonella uptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpV and vgrG) revealed that SPI-19 T6SS contributes to S. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked to Salmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used by Salmonella to survive within host cells.

A glance at Listeria and Salmonella cell invasion: different strategies to promote host actin polymerization.

The facultative intracellular bacterial pathogens Listeria monocytogenes and Salmonella enterica have evolved multiple strategies to invade a large panel of mammalian cells. These pathogens use the host cell actin system for invasion and became a paradigm for the study of host-pathogen interactions and bacterial adaptation to mammalian hosts. The key signaling component that these pathogens use to orchestrate actin remodeling is the Arp2/3 complex, which is related to polymerization of actin filaments. These bacterial pathogens are able to trigger distinct invasion mechanisms. On the one hand, L. monocytogenes invade a host cell in a way dependent on the specific interactions between bacterial and host cell proteins, which in turn activate the host cell actin polymerizing machinery that culminates with bacterial internalization. Also, Listeria escapes from the newly formed parasitophorous vacuole and moves among adjacent cells by triggering actin polymerization. On the other hand, Salmonella invades a host cell by delivering into the cytoplasm virulence factors which directly interact with host regulators of actin polymerization which leads to bacterial uptake. Moreover, Salmonella avoids vacuole lyses and modulates the early and late endosomal markers presented in the vacuole membrane. This mini-review focuses on the different pathways that L. monocytogenes and S. enterica activate to modulate the actin cytoskeleton in order to invade, to form the parasitophorous vacuole, and to migrate inside host cells.

Construcción de mutantes de salmonella enterica por inactivación de los genes invG/invE y ssaJ/ssaK de las islas de patogenicidad 1 y 2 / Construction of mutants of salmonella enterica for inactivation of invG/invE y ssaJ/ssaK genes of pathogenicity islands 1 and 2

Para la comprensión de las bases genéticas de los mecanismos de patogenicidad de Salmonella se han descrito diversas metodologías para manipular el ADN genómico y generar mutantes con características particulares. En este estudio se reporta la construcción de mutantes a partir de varios serotipos de S. enterica, por sustitución e inactivación de los genes invG/invE en SPI-1 y de los genes ssaJ/ssaK en SPI-2 mediante la técnica de recombinasa Red del fago λ descrita por Datsenko y Wanner (2000). Los genes delecionados en las SPI-1 y SPI-2 codifican para las proteínas que participan en la formación de los sistemas de secreción tipo III, responsables de la invasión y supervivencia intracelular de S. enterica en las células hospedadoras. Los resultados de este trabajo permitirán realizar estudios futuros in vivo para evaluar la posible atenuación de la virulencia de las cepas mutantes, así como aportar nuevos conocimientos sobre los mecanismos genéticos involucrados en la fisiopatogenia de las enfermedades producidas por los serovares estudiados. Además, esta técnica se recomienda para generar de manera eficiente mutantes de diferentes serotipos de S. enterica con la finalidad de estudiar los genes cromosómicos y sus productos.

To understand the genetic basis of Salmonella pathogenicity mechanisms, various methods have been described to manipulate and generate mutant genomic DNA with specific characteristics. In this study we report the construction of mutants from several serotypes of S. enterica, substitution and inactivation of genes invG/invE in SPI-1 gene and ssaJ/ssaK in SPI-2 by the technique of phage λ Red recombinase, as described by Datsenko and Wanner (2000). The gene deletion in SPI-1 and SPI-2 encodes proteins involved in the formation of type III secretion systems responsible for the invasion and intracellular survival of S. enterica in the host cells. The results of this work will allow in vivo studies to evaluate the possible attenuation of virulence of the mutant strains, as well as to provide new insights into the genetic mechanisms involved in the pathogenesis of diseases caused by these bacteria. Moreover, this technique is recommended to efficiently generate mutants of different serotypes of S. enterica in order to study the chromosomal genes and their products.

Dam and its role in pathogenicity of Salmonella enterica.

Dam methylation is an essential factor involved in the virulence of an increasing number of bacterial pathogens including Salmonella enterica. Lack of Dam methylation causes severe attenuation in animal models. It has been proposed that dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. In this review, we focus our attention on the role of methylation by Dam protein in regulating bacterial gene expression and virulence in Salmonella enterica.

T cell immunity evasion by virulent Salmonella enterica.

Salmonella enterica are Gram-negative bacteria that cause systemic disease in their specific hosts. One of the recently appreciated features of Salmonella pathogenicity is the capacity of the bacteria to impair host adaptive immunity by interfering with DC function and T cell activation. It is likely that this feature of virulent Salmonella is needed to promote systemic dissemination in the host. Recent studies have suggested explanations for some of the molecular mechanisms developed by virulent Salmonella to impair DC and T cell function. Several of these mechanisms require the expression of virulence genes encoded within Salmonella pathogenicity islands. Targeted deletion of these genes diminishes Salmonella pathogenicity and leads to efficient activation of T cells by Salmonella-infected DCs. In this review, recent data that support the subversion of DC function by Salmonella as a means to evade host adaptive immunity and cause systemic infection are discussed. These new findings suggest a new pathogenesis model with DCs as key targets for Salmonella virulence factors.