Cold atmospheric pressure air plasma jet disinfection of table eggs: Inactivation of Salmonella enterica, cuticle integrity and egg quality.

Eggshell cuticles are first lines of defense against egg-associated pathogens, such as Salmonella enterica serovar Enteritidis (SE). Infections from eggs contaminated with this strain remain a significant risk. In addition, changes in the cuticle are closely related to changes in egg safety. The emerging non-thermal atmospheric pressure plasma technology enables a high rate of microbial inactivation at near-ambient temperatures, making it ideal for food safety applications. This study examines the effects of a cold atmospheric pressure air plasma jet (CAAP-J) on eggshell cuticle and egg quality whilst inactivating SE. Shell eggs inoculated with SE (7 log10 cfu/egg) were used as the samples to test the decontamination performance of the device. The tests were conducted using an industrial CAAP-J with different power levels (600-800 W), exposure times (60-120 s), at a fixeddistance of 20 mm from the plasma jet and an air flow rate of 3600 L/h. It was found that the best results were obtained after 120 s at maximum plasma power (800 W). Subsequent to the implementation of this plasma procedure, it was determined that no viable cells could be detected. After CAAP-J treatment, the temperature remains below 50.5 °C, thereby minimizing the risk of altering egg quality. All specific measurements (egg white pH, yolk pH, yolk color, HU, and eggshell breaking strength) have shown that CAAP-J treatment has no negative effect on egg quality. No changes in eggshell cuticle quality after CAAP-J treatment was confirmed through scanning electron microscope (SEM).

Effect of plasma-activated water on the biofilm-forming ability of Salmonella enterica serovar Enteritidis and expression of the related genes.

In recent years, microbial decontamination with plasma-activated water (PAW) has attracted a lot of research attention in the field of food industry. Despite several studies showing that PAW effectively inactivates planktonic bacteria, few studies have been conducted on biofilms. The present study was, therefore, designed to evaluate the effect of PAW on the biofilm formation characteristics of Salmonella Enteritidis. Comparing the expression patterns of biofilm-related genes in PAW-treated and non-treated planktonic and biofilm cells provided insight into how PAW regulates this process. The results showed that a 30-minute exposure to PAW at room temperature significantly reduced S. enteritidis planktonic cells. This exposure resulted in a decreased expression of the genes involved in the early stages of biofilm formation (csgD, agfA, fimA, lpfE, and rpoS), and an increased expression of the csrA gene in S. enteritidis planktonic cells. These results indicated the inhibitory effect of PAW on the biofilm formation process in S. enteritidis. Results of the initial attachment assay confirmed these findings, where, after 6 h, the number of PAW-treated cells attached to the stainless steel surfaces were significantly lower than non-treated ones. Furthermore, biofilm development assay revealed that the number of PAW-treated biofilm cells were significantly lower than non-treated ones after 24 h incubation at 37 °C. These findings were confirmed by measurements of the major components of biofilm i.e., extracellular DNA (eDNA), protein and carbohydrate. The amount of these components in 24-hour biofilms produced by PAW-treated S. enteritidis cells was significantly lower than that of non-treated cells. PAW's treatment on preformed 24-hour biofilms for 30 min led to a decrease in the expression of genes involved in quorum sensing and cellulose synthesis (csgD, bapA, adrA, luxS and sdiA) and an increase in the expression of the csrA gene. This treatment also reduced the number and metabolic activity of biofilm cells compared to non-treated biofilm cells. In total, the present study demonstrated that PAW has an inhibitory effect on the process of biofilm formation in S. enteritidis and hence, the food industry should pay special attention to PAW as a promising treatment to eliminate bacterial biofilms.

Modelling of inactivation kinetics of Escherichia coli, Salmonella Enteritidis and Bacillus subtilis treated with a multi-hollow surface dielectric barrier discharge plasma.

The efficacy of multi-hollow surface dielectric barrier discharge treatment against Escherichia coli, Salmonella Enteritidis and Bacillus subtilis was studied. Ambient air, O2, and N2 were used as working gas with a flow rate of 6 l/m. Power delivered into plasma was 30 W over an area of 2 × 2 cm2. The active species in plasma generated in different gases participating in the inactivation of microorganisms were evaluated by optical emission spectroscopy and Fourier transform infrared spectroscopy. Inactivation curves were fitted to the Bigelow log-linear, the biphasic, and Geeraerd models. According to the results, all plasma treatments inactivated tested microorganisms, depending on a working gas. The most sensitivity of bacteria was observed to the ambient air plasma. Inactivation up to 5 log for E. coli and S. Enteritidis could be achieved within 15 s of plasma treatment. Air plasma exposure of 25 s also led to log10 CFU/ml of B. subtilis from 7.98 to 4.39. S. Enteritidis was slight resistance to plasma treatment with N2. Within 180 s nitrogen plasma treatment, a 2.04 log10 CFU/ml reduction was recorded.

Oral Inoculation of Point-of-Lay Hens with the New South Wales Outbreak Strain of Salmonella Enteritidis Phage Type 12 Causes Infection, but Minimal Histopathology.

An outbreak of food poisoning in New South Wales (NSW) Australia in 2018, caused by Salmonella enterica serovar Enteritidis phage type 12 (PT12), was traced to eggs consumed from a NSW layer flock. This was the first report of Salmonella Enteritidis infection in NSW layer flocks, despite ongoing environmental monitoring. Clinical signs and mortalities were minimal in most flocks, although seroconversion and infection were demonstrated in some flocks. An oral dose-response challenge study with Salmonella Enteritidis PT12 was undertaken in commercial point-of-lay hens. Cloacal swabs collected at 3, 7, 10, and 14 days postinoculation and caeca, liver, spleen, ovary, magnum, and isthmus tissues collected at necropsy at either 7 or 14 days were processed for Salmonella isolation (AS 5013.10-2009 from ISO6579:2002). Histopathology was performed on the above tissues, as well as lung, pancreas, kidney, heart, and additional intestinal and reproductive tract tissues. Salmonella Enteritidis was consistently detected in cloacal swabs between 7 and 14 days postchallenge. The Salmonella Enteritidis PT12 isolate successfully colonized the gastrointestinal tract, liver, and spleen of all hens orally challenged with 107, 108, and 109 Salmonella Enteritidis, and less consistently colonized their reproductive tracts. On histopathology, mild lymphoid hyperplasia in the liver and spleen, along with hepatitis, typhlitis, serositis, and salpingitis, was observed at 7 and 14 days postchallenge, with a greater proportion of affected birds in the two higher dose groups. Diarrhea and culture of Salmonella Enteritidis from heart blood were not detected in challenged layers. The NSW isolate of Salmonella Enteritidis PT12 was able to invade and colonize the birds' reproductive tracts as well as a wide range of other tissues, indicating the potential for these naive commercial hens to contaminate their eggs.

La inoculación oral de gallinas ponedoras en el pico de postura con la cepa de Salmonella Enteritidis PT12 del brote en Nueva Gales del Sur causa infección, pero una histopatología mínima. Un brote de intoxicación alimentaria en Nueva Gales del Sur (NSW), Australia en 2018, causado por Salmonella enterica serovar Enteritidis fagotipo 12, se rastreó hasta los huevos consumidos de una parvada de ponedoras de NSW. Este fue el primer informe de infección por Salmonella Enteritidis en parvadas de ponedoras de NSW, a pesar del monitoreo ambiental continuo. Los signos clínicos y la mortalidad fueron mínimos en la mayoría de las parvadas, aunque se demostró seroconversión e infección en algunas parvadas. Se llevó a cabo un estudio de desafío oral para evaluar la dosis y su respuesta para Salmonella Enteritidis PT12 en gallinas ponedoras comerciales. Los hisopos cloacales recolectados a los tres, siete, diez y 14 días posteriores a la inoculación y los tejidos de ciego, hígado, bazo, ovario, magnum e istmos recolectados en la necropsia a los siete o 14 días se procesaron para el aislamiento de Salmonella (AS 5013.10-2009 del estándar ISO6579: 2002). Se realizó histopatología en los tejidos anteriormente mencionados, así como de pulmón, páncreas, riñón, corazón y tejidos intestinales y del tracto reproductivo adicionales. Salmonella Enteritidis se detectó consistentemente en hisopos cloacales entre los siete y 14 días después del desafío. El aislado de Salmonella Enteritidis PT12 colonizó con éxito el tracto gastrointestinal, el hígado y el bazo de todas las gallinas desafiadas por vía oral con dosis de 107, 108 y 109 de Salmonella Enteritidis, pero colonizó de manera menos consistente sus tractos reproductivos. En la histopatología, se observó hiperplasia linfoide leve en el hígado y el bazo, junto con hepatitis, tiflitis, serositis y salpingitis, a los siete y 14 días posteriores a la exposición, con una mayor proporción de aves afectadas en los dos grupos de dosis más altas. En las ponedoras desafiadas no se detectaron diarrea ni cultivo de Salmonella Enteritidis de sangre colectada del corazón. El aislamiento de Salmonella Enteritidis PT 12 de Nueva Gales del Sur pudo invadir y colonizar los tractos reproductivos de las aves, así como una amplia gama de otros tejidos, lo que indica el potencial de estas gallinas comerciales sin inmunidad para contaminar sus huevos.

Dose response for Salmonella Typhimurium and Enteritidis and other nontyphoid enteric salmonellae.

This dose response assessment combines data from 6 human challenge studies and 44 outbreaks to determine infectivity and pathogenicity of several serotypes of nontyphoid Salmonella. Outcomes focus on the major serotypes Salmonella Enteritidis and Typhimurium, showing that Typhimurium is less infectious and has a lower probability of causing acute illness in infected subjects. The dose response relation of Salmonella Enteritidis is less steep than that of Typhimurium, indicating greater heterogeneity in infectivity and pathogenicity. This study revisits an older study with less flexible methods that could not combine the widely different outcomes of challenge studies and outbreaks, and had limited capability for dealing with missing information. Reported outcomes are in a format that allows use in calculations of uncertainty for quantitative risk assessment.

HPMCAS-Coated Alginate Microparticles Loaded with Ctx(Ile21)-Ha as a Promising Antimicrobial Agent against Salmonella Enteritidis in a Chicken Infection Model.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) in poultry is most often transmitted by the fecal-oral route, which can be attributed to high population density. Upon encountering the innate immune response in a host, the pathogen triggers a stress response and virulence factors to help it survive in the host. The aim of this study was to evaluate the effect of hypromellose acetate/succinate (HPMCAS)-coated alginate microparticles containing the Ctx(Ile21)-Ha antimicrobial peptide (AMP) on both intestinal colonization and systemic infection of laying hens challenged with S. Enteritidis. The applied AMP microsystem reduced the bacterial load of S. Enteritidis in the liver, with a statistical significance between groups A (control, no Ctx(Ile21)-Ha peptide) and B (2.5 mg of Ctx(Ile21)-Ha/kg) at 2 days postinfection (dpi), potentially indicating the effectiveness of Ctx(Ile21)-Ha in the first stage of infection by S. Enteritidis. In addition, the results showed a significant decrease in the S. Enteritidis counts in the spleen and cecal content at 5 dpi; remarkably, no S. Enteritidis counts were observed in livers at 5, 7, and 14 dpi, regardless of the Ctx(Ile21)-Ha dosage (p-value <0.0001). Using the Chi-square test, the effect of AMP microparticles on S. Enteritidis fecal excretion was also evaluated, and a significantly lower bacterial excretion was observed over 21 days in groups B and C, in comparison with the untreated control (p-value <0.05). In summary, the use of HPMCAS-Ctx(Ile21)-Ha peptide microcapsules in laying hens drastically reduced the systemic infection of S. Enteritidis, mainly in the liver, indicating a potential for application as a feed additive against this pathogen.

Long-chain glucomannan supplementation modulates immune responsiveness, as well as intestinal microbiota, and impacts infection of broiler chickens with Salmonella enterica serotype Enteritidis.

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens.

Salmonella enterica serovar Enteritidis biofilm lifestyle induces lower pathogenicity and reduces inflammatory response in a murine model compared to planktonic bacteria.

Salmonellaenterica serovar Enteritidis (S. Enteritidis) is the most frequent serovar involved in human salmonellosis. It has been demonstrated that about 80% of infections are related to biofilm formation. There is scant information about the pathogenicity of S. Enteritidis and its relationship to biofilm production. In this regard, this study aimed to investigate the differential host response induced by S. Enteritidis biofilm and planktonic lifestyle. To this purpose, biofilm and planktonic bacteria were inoculated to BALB/c mice and epithelial cell culture. Survival studies revealed that biofilm is less virulent than planktonic cells. Reduced signs of intestinal inflammation and lower bacterial translocation were observed in animals inoculated with Salmonella biofilm compared to the planktonic group. Results showed that Salmonella biofilm was impaired for invasion of non-phagocytic cells and induces a lower inflammatory response in vivo and in vitro compared to that of planktonic bacteria. Taken together, the outcome of Salmonella-host interaction varies depending on the bacterial lifestyle.

Effects of Salmonella enterica ser. Enteritidis and Heidelberg on host CD4+CD25+ regulatory T cell suppressive immune responses in chickens.

Poultry infected with Salmonella mount an immune response initially, however the immune responses eventually disappear leading the bird to be a carrier of Salmonella. The hypothesis of this study is that Salmonella infection induces T regulatory cell numbers and cytokine production and suppress host T cells locally in the gut to escape the host immune responses. An experiment was conducted to comparatively analyze the effect of S. enterica ser. Enteritidis (S. Enteritidis) and S. enterica ser. Heidelberg (S. Heidelberg) infection on CD4+CD25+ T regulatory cell properties in chickens. A total of 144 broiler chicks were randomly distributed into three experimental groups of non-infected control, S. Enteritidis infected and S. Heidelberg infected groups. Chickens were orally inoculated with PBS (control) or 5x106 CFU/mL of either S. Enteritidis or S. Heidelberg at 3 d of age. Each group was replicated in six pens with eight chickens per pen. Chickens infected with S. Enteritidis had 6.2, 5.4, and 3.8 log10 CFU/g, and chickens infected with S. Heidelberg had 7.1, 4.8, and 4.1 log10 CFU/g Salmonella in the cecal contents at 4, 11, and 32 dpi, respectively. Both S. Enteritidis and S. Heidelberg were recovered from the liver and spleen 4 dpi. At 4, 11, and 32 dpi, chickens infected with S. Enteritidis and S. Heidelberg had increased CD4+CD25+ cell numbers as well as IL-10 mRNA transcription of CD4+CD25+ cells compared to that in the control group. CD4+CD25+ cells from S. Enteritidis- and S. Heidelberg-infected chickens and restimulated with 1 µg antigen in vitro, had higher (P < 0.05) IL-10 mRNA transcription than the CD4+CD25+ cells from the non-infected controls Though at 4dpi, chickens infected with S. Enteritidis and S. Heidelberg had a significant (P < 0.05) increase in CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, the CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, were comparable to that in the control group at 11 and 32dpi identifying that the host inflammatory response against Salmonella disappears at 11 dpi. It can be concluded that S. Enteritidis and S. Heidelberg infection at 3 d of age induces a persistent infection through inducing CD4+CD25+ cells and altering the IL-10 mRNA transcription of CD4+CD25+ cell numbers and cytokine production in chickens between 3 to 32 dpi allowing chickens to become asymptomatic carriers of Salmonella after 18 dpi.

Analysis of antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism.

This research was to explore antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism. Kirby-Bauer (K-B) disk method recommended by Clinical and Laboratory Standards Institute (CLSI) was used to test drug sensitivity of Salmonella enteritidis to 16 kinds of antibiotics including ß-lactams, aminoglycosides, quinolones, sulfonamides, chloramphenicols, and tetracyclines. Polymerase chain reaction (PCR) was performed to detect carrying of drug resistance genes of 29 kinds of antibiotics including ß-lactams, aminoglycosides, quinolones, sulfonamides, chloramphenicols, and tetracyclines of Salmonella enteritidis. The expressions of esp, ebpA, ge1E, and fsrB genes in biofilm group and plankton group were detected when Salmonella was induced, and difference of gene expression was detected by FQ-PCR. The drug resistance rates of Salmonella enteritidis to nalidixic acid, ampicillin, streptomyces, and cefoperazone were high, which were 94.5%, 75%, 67%, and 52%, respectively. 94 strains of Salmonella enteritidis formed 22 kinds of drug resistance spectrum, the strains were generally resistant to 4-5 antibiotics, and some strains formed fixed drug resistance spectrum as follows: AMP-CFP-STR-NA-TE (22.6,21.7%), AMP-STR-NA-TE (17,16%), and AMP-CFP-STR-NA (11.1,10.6%). During biofilm formation, fsr can increase expression of ge1E and decrease expression of esp and ebpA. Consequently, Salmonella enteritidis was generally resistant to nalidixic acid, ampicillin, and streptomycin, and the multidrug resistance was severe. The drug resistance genes sul2, sul3, blaTEM-1-like, tet(A), and tet(G) were highly carried in Salmonella enteritidis. Esp, ebpA, ge1E, and fsrB genes were closely related to biofilm formation of Salmonella enteritidis.

Interaction of Salmonella Gallinarum and Salmonella Enteritidis with peripheral leucocytes of hens with different laying performance.

Salmonella enterica ssp. enterica serovars Enteritidis (SE) and Gallinarum (SG) cause different diseases in chickens. However, both are able to reach the blood stream where heterophils and monocytes are potentially able to phagocytose and kill the pathogens. Using an ex vivo chicken whole blood infection model, we compared the complex interactions of the differentially host-adapted SE and SG with immune cells in blood samples of two White Leghorn chicken lines showing different laying performance (WLA: high producer; R11: low producer). In order to examine the dynamic interaction between peripheral blood leucocytes and the Salmonella serovars, we performed flow cytometric analyses and survival assays measuring (i) leucocyte numbers, (ii) pathogen association with immune cells, (iii) Salmonella viability and (iv) immune gene transcription in infected whole blood over a four-hour co-culture period. Inoculation of blood from the two chicken lines with Salmonella led primarily to an interaction of the bacteria with monocytes, followed by heterophils and thrombocytes. We found higher proportions of monocytes associated with SE than with SG. In blood samples of high producing chickens, a decrease in the numbers of both heterophils and Salmonella was observed. The Salmonella challenge induced transcription of interleukin-8 (IL-8) which was more pronounced in SG- than SE-inoculated blood of R11. In conclusion, the stronger interaction of monocytes with SE than SG and the better survivability of Salmonella in blood of low-producer chickens shows that the host-pathogen interaction and the strength of the immune defence depend on both the Salmonella serovar and the chicken line.

Global spread of Salmonella Enteritidis via centralized sourcing and international trade of poultry breeding stocks.

A pandemic of Salmonella enterica serotype Enteritidis emerged in the 1980s due to contaminated poultry products. How Salmonella Enteritidis rapidly swept through continents remains a historical puzzle as the pathogen continues to cause outbreaks and poultry supply becomes globalized. We hypothesize that international trade of infected breeding stocks causes global spread of the pathogen. By integrating over 30,000 Salmonella Enteritidis genomes from 98 countries during 1949-2020 and international trade of live poultry from the 1980s to the late 2010s, we present multifaceted evidence that converges on a high likelihood, global scale, and extended protraction of Salmonella Enteritidis dissemination via centralized sourcing and international trade of breeding stocks. We discovered recent, genetically near-identical isolates from domestically raised poultry in North and South America. We obtained phylodynamic characteristics of global Salmonella Enteritidis populations that lend spatiotemporal support for its dispersal from centralized origins during the pandemic. We identified concordant patterns of international trade of breeding stocks and quantitatively established a driving role of the trade in the geographic dispersal of Salmonella Enteritidis, suggesting that the centralized origins were infected breeding stocks. Here we demonstrate the value of integrative and hypothesis-driven data mining in unravelling otherwise difficult-to-probe pathogen dissemination from hidden origins.

A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens.

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Behavior of Salmonella Enteritidis and Shigella flexneri during induction and recovery of the viable but nonculturable state.

Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.

Salmonella Enteritidis foodborne infection induces altered placental morphometrics in the murine model.

INTRODUCTION: Salmonella foodborne disease during pregnancy causes a significant fetal loss in domestic livestock and preterm birth, chorioamnionitis and miscarriage in humans. These complications could be associated with alterations in placental structure. This study was aimed to determine how a low dose of Salmonella Enteritidis during late gestation affects placental histomorphometric in mice. METHODS: We used a self-limiting enterocolitis murine model. BALB/c pregnant animals received a low dose of Salmonella Enteritidis (3-4 x 102 CFU/mouse) on gestational day (GD) 15. At day 3 post infection bacterial loads, serum cytokines expression and placental histomorphometrics parameters were analyzed. RESULTS: We found that a sub-lethal infection with Salmonella induced a significant drop in fetal weight -to-placental weight-ratio and an increase in the placental coefficient. After bacterial inoculation maternal organs were colonized, inducing placental morphometric alterations, including increased placental thickness, reduced surface area, and diminished major and minor diameters. Also, foci of necrosis accompanied by acute leukocyte infiltration in decidual zone, reduction of vascular spaces and vascular congestion in labyrinth zone, were also evident in placentas from infected females on GD 18. Our data shows that placentas from infected mothers are phenotypically different from control ones. Furthermore, expression of IFN-gamma and IL-6 was up regulated in response to Salmonella in maternal serum. DISCUSSION: Our findings demonstrate that a low dose of Salmonella during late gestation alters the placental morphometry leading to negative consequences on pregnancy outcome such as significant reduction in fetal body weight.

Positive regulation of Type III secretion effectors and virulence by RyhB paralogs in Salmonella enterica serovar Enteritidis.

Small non-coding RNA RyhB is a key regulator of iron homeostasis in bacteria by sensing iron availability in the environment. Although RyhB is known to influence bacterial virulence by interacting with iron metabolism related regulators, its interaction with virulence genes, especially the Type III secretion system (T3SS), has not been reported. Here, we demonstrate that two RyhB paralogs of Salmonella enterica serovar Enteritidis upregulate Type III secretion system (T3SS) effectors, and consequently affect Salmonella invasion into intestinal epithelial cells. Specifically, we found that RyhB-1 modulate Salmonella response to stress condition of iron deficiency and hypoxia, and stress in simulated intestinal environment (SIE). Under SIE culture conditions, both RyhB-1 and RyhB-2 are drastically induced and directly upregulate the expression of T3SS effector gene sipA by interacting with its 5' untranslated region (5' UTR) via an incomplete base-pairing mechanism. In addition, the RyhB paralogs upregulate the expression of T3SS effector gene sopE. By regulating the invasion-related genes, RyhBs in turn affect the ability of S. Enteritidis to adhere to and invade into intestinal epithelial cells. Our findings provide evidence that RyhBs function as critical virulence factors by directly regulating virulence-related gene expression. Thus, inhibition of RyhBs may be a potential strategy to attenuate Salmonella.

Hydroethanolic Extracts of Haplopappus baylahuen Remy and Aloysia citriodora Palau Have Bactericide Activity and Inhibit the Ability of Salmonella Enteritidis to Form Biofilm and Adhere to Human Intestinal Cells.

We analysed whether the hydroethanolic extracts from leaves of Haplopappus baylahuen Remy (bailahuen) and Aloysia citriodora Palau (cedron) inhibit the growth and ability of Salmonella Enteritidis to form biofilms and to adhere to human intestinal epithelial cells. Herein, we first determined the total phenolic content and antioxidant and antibacterial activities of the extracts. Then, Salmonella Enteritidis was treated with the extracts to analyse biofilm formation by scanning electronic microscopy and the violet crystal test. We also measured the efflux pump activity of Salmonella Enteritidis since biofilm formation is associated with this phenomenon. Furthermore, the human intestinal cell line Caco-2 was infected with Salmonella Enteritidis pretreated with the extracts, and 30 min later, the number of bacteria that adhered to the cell surface was quantified. Finally, we determined by qPCR the expression of genes associated with biofilm formation, namely, the diguanilate cyclase AdrA protein gene (adrA) and the BapA protein gene (bapA), and genes associated with adhesion, namely, the transcriptional regulator HilA (hilA). The phenolic content and antioxidant and bactericide activities were higher in bailahuen than in the cedron extract. Biofilm formation was inhibited by the extracts in a dose-dependent manner, while the activity of efflux pumps was decreased only with the cedron extract. Adhesion to Caco-2 cells was also inhibited without differences between doses and extracts. The extracts decreased the expression of adrA; with the cedron extract being the most efficient. The expression of hilA is affected only with the cedron extract. We concluded that hydroethanolic extracts of bailahuen and cedron differentially inhibit the growth of Salmonella Enteritidis and affect its the ability to form biofilms and to adhere to human intestinal epithelial cells. These results highlight the presence of molecules in bailahuen and cedron with a high potential for the control of the Salmonella Enteritidis pathogenesis.

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya.

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003-2010. Nine isolates were identified as S. Typhimurium sequence type 313 while the other two were S. Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S. Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.

Phenotypic analyses of Salmonella enterica serovar Enteritidis strains isolated in the pre- and post-epidemic period in Brazil.

Salmonella Enteritidis has caused, since the 1980s, a sustained epidemic of human infections in many countries. This study analyzed S. Enteritidis strains isolated before and after the epidemic period in Brazil regarding their capacities to survive to acid, oxidative, and high-temperature stresses, and capacity to grow in egg albumen. Moreover, the ability to invade human epithelial cells (Caco-2) and to survive inside human (U937) and chicken (HD11) macrophages was checked. Post-epidemic strains showed a better ability to survive after 10 min under acid stress at 37 °C (P ≤ 0.05). However, both groups of strains showed similar ability to survive after 1 h under acid stress at 37 °C and at 42 °C independently of the time of exposure. Similar ability was verified in both groups of strains regarding oxidative stress, growth in egg albumen, high-temperature stress, invasion to Caco-2 cells, and invasion and survival in macrophages. In conclusion, post-epidemic S. Enteritidis strains showed a better ability to survive under the acid stress found in the stomach, which might be an advantage to reach the intestine and colonize chickens and humans. However, both groups of strains did not differ significantly in the majority of the phenotypic tests analyzed in this study.

Influence of low water activity on the thermal resistance of Salmonella Enteritidis PT30 and Enterococcus faecium as its surrogate in egg powders.

Egg powders are increasingly popular ingredients, due to their functionality and compactness, in industrial food production and preparation at homes. However, there is a lack of studies that evaluate the thermal resistance of Salmonella Enteritidis PT30 and its potential surrogate Enterococcus faecium NRRL B-2354 in egg powders. This study examined the log-linear relationship between the thermal resistance of Salmonella Enteritidis (D-value) and the water activity (aw) of egg powders. The changes of aw in the egg powders with temperature were measured using a Vapor Sorption Analyzer and a high-temperature cell. The D80 ℃-value of S. Enteritidis PT30 and E. faecium inoculated in the egg powders preconditioned to three aw levels (0.3, 0.45, and 0.6) at 20 ℃ were determined using aluminum thermal death test cells. The aw values increased (P < 0.05) in all three egg powders when the temperature of the samples was raised from room temperature to 80 ℃. The D80 ℃-values ranged from 5.3 ± 0.1 to 25.9 ± 0.2 min for S. Enteritidis while 10.4 ± 0.4 to 43.8 ± 0.4 for E. faecium in samples of the three different aw levels. S. Enteritidis PT30 showed a log-linear relationship between D80 ℃-values and aw80 ℃ for the egg powders. This study contributes to our understanding of the impact of aw on the development of thermal treatments for low-moisture foods.