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1.
Nat Commun ; 12(1): 348, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441540

RESUMO

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.


Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Células CACO-2 , Bovinos , Células Cultivadas , Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Proteínas Quimiotáticas Aceptoras de Metil/genética , Camundongos Endogâmicos C57BL , Movimento/fisiologia , Mutação , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Fatores de Transcrição/genética
2.
Proc Natl Acad Sci U S A ; 117(33): 20235-20243, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32753384

RESUMO

All cells require Mg2+ to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+ starvation in host tissues. However, the Mg2+ transporter MgtB enables the facultative intracellular pathogen Salmonella enterica serovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, the Salmonella small protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How does Salmonella preserve MgtB to promote survival inside macrophages? We establish that the Salmonella small protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival in Slc11a1 +/+ macrophages, resistance to oxidative stress, and growth under Mg2+ limitation conditions. The Salmonella Mg2+ transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, the mgtB, mgtR, and mgtU genes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Macrófagos/microbiologia , Magnésio/metabolismo , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Macrófagos/fisiologia , Camundongos , Plasmídeos/genética , Salmonella typhimurium/genética , Virulência
3.
Appl Environ Microbiol ; 86(21)2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32859592

RESUMO

Salmonella enterica serovar Typhimurium is a prevalent incitant of enteritis in human beings and nonhuman animals. It has been proposed that host defense responses incited by Salmonella allow the bacterium to overcome colonization resistance. Piglets (n = 24) were orally inoculated with S. enterica serovar Typhimurium DT104 or buffer alone, and the host and microbial responses were temporally examined at the acute (2 days postinoculation [dpi]), subacute (6 dpi), and recovery (10 dpi) stages of salmonellosis. At the acute stage of disease, body temperatures were elevated, and feed consumption and weight gain were reduced. The densities of Salmonella associated with the gut mucosa decreased over time, with higher densities of the bacterium in the ileum and the large intestine. Moreover, substantive histopathological changes were observed as a function of time, with prominent epithelial injury and neutrophil infiltration observed at 2 dpi. Correspondingly, a variety of host metrics were temporally affected in piglets with salmonellosis (e.g., TNFα, IFNγ, PR39, ßD2, iNOS, IL8, REGIIIγ). The enteric microbiota was characterized using culture-independent and -dependent methods in concert, and taxon- and location-specific changes to the microbiota were observed in infected piglets. Bacteroides spp. (e.g., Bacteroides uniformis, Bacteroides fragilis), Streptococcus spp. (e.g., Streptococcus gallolyticus), and various Gammaproteobacteria were highly associated with inflamed tissues, while bacteria within the Ruminococcaceae and Veillonellaceae families were mainly associated with healthy mucosae. In conclusion, the study findings showed that S Typhimurium incited temporal and spatial modifications to the swine autochthonous microbiota, and to host defense responses, that were consistent with overcoming colonization resistance to incite salmonellosis in swine.IMPORTANCE Limited information is available on host and enteric microbiota responses incited by Salmonella enterica serovar Typhimurium in swine and on possible mechanisms by which the bacterium overcomes colonization resistance to incite salmonellosis. Temporal characterization of a variety of host metrics in piglets (e.g., physiological, histopathological, and immunological) showed the importance of studying the progression of salmonellosis. A number of host responses integrally associated with disease development were identified. Utilization of next-generation sequence analysis to characterize the enteric microbiota was found to lack sufficient resolution; however, culture-dependent and -independent methods in combination identified taxon- and location-specific changes to bacterial communities in infected piglets. The study identified bacterial and host responses associated with salmonellosis, which will be beneficial in understanding colonization resistance and in the development of effective alternatives to antibiotics to mitigate salmonellosis.


Assuntos
Ceco/microbiologia , Colo/microbiologia , Microbioma Gastrointestinal , Interações entre Hospedeiro e Microrganismos/imunologia , Íleo/microbiologia , Salmonella typhimurium/fisiologia , Animais , Ceco/imunologia , Colo/imunologia , Íleo/imunologia , Masculino , Distribuição Aleatória , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Fatores de Tempo
5.
Appl Environ Microbiol ; 86(17)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32591379

RESUMO

The Food Safety Modernization Act (FSMA) includes a time-to-harvest interval following the application of noncompliant water to preharvest produce to allow for microbial die-off. However, additional scientific evidence is needed to support this rule. This study aimed to determine the impact of weather on the die-off rate of Escherichia coli and Salmonella on spinach and lettuce under field conditions. Standardized, replicated field trials were conducted in California, New York, and Spain over 2 years. Baby spinach and lettuce were grown and inoculated with an ∼104-CFU/ml cocktail of E. coli and attenuated Salmonella Leaf samples were collected at 7 time points (0 to 96 h) following inoculation; E. coli and Salmonella were enumerated. The associations of die-off with study design factors (location, produce type, and bacteria) and weather were assessed using log-linear and biphasic segmented log-linear regression. A segmented log-linear model best fit die-off on inoculated leaves in most cases, with a greater variation in the segment 1 die-off rate across trials (-0.46 [95% confidence interval {95% CI}, -0.52, -0.41] to -6.99 [95% CI, -7.38, -6.59] log10 die-off/day) than in the segment 2 die-off rate (0.28 [95% CI, -0.20, 0.77] to -1.00 [95% CI, -1.16, -0.85] log10 die-off/day). A lower relative humidity was associated with a faster segment 1 die-off and an earlier breakpoint (the time when segment 1 die-off rate switches to the segment 2 rate). Relative humidity was also found to be associated with whether die-off would comply with FSMA's specified die-off rate of -0.5 log10 die-off/day.IMPORTANCE The log-linear die-off rate proposed by FSMA is not always appropriate, as the die-off rates of foodborne bacterial pathogens and specified agricultural water quality indicator organisms appear to commonly follow a biphasic pattern with an initial rapid decline followed by a period of tailing. While we observed substantial variation in the net culturable population levels of Salmonella and E. coli at each time point, die-off rate and FSMA compliance (i.e., at least a 2 log10 die-off over 4 days) appear to be impacted by produce type, bacteria, and weather; die-off on lettuce tended to be faster than that on spinach, die-off of E. coli tended to be faster than that of attenuated Salmonella, and die-off tended to become faster as relative humidity decreased. Thus, the use of a single die-off rate for estimating time-to-harvest intervals across different weather conditions, produce types, and bacteria should be revised.


Assuntos
Irrigação Agrícola , Escherichia coli/fisiologia , Alface/microbiologia , Salmonella typhimurium/fisiologia , Spinacia oleracea/microbiologia , Águas Residuárias/microbiologia , Tempo (Meteorologia) , California , Microbiologia de Alimentos , New York , Folhas de Planta/microbiologia , Espanha
6.
Anticancer Res ; 40(5): 2515-2523, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366396

RESUMO

BACKGROUND/AIM: Dedifferentiated liposarcoma (DDLPS) is associated with a poor survival rate even with multi-modality treatment. In the present study, we evaluated the efficacy of recombinant methioninase (rMETase) combined with tumor-targeting Salmonella typhimurium (S. typhimurium) A1-R against a doxorubicin-resistant DDLPS in a patient-derived orthotopic xenograft (PDOX) mouse model. MATERIALS AND METHODS: A recurrent high-grade DDLPS from the right retroperitoneum of a patient was grown orthotopically in the retroperitoneum of nude mice to establish a PDOX model. The PDOX models were randomly divided into the following groups: Control, no treatment; doxorubicin monotherapy; rMETase monotherapy; S. typhimurium A1-R monotherapy; S. typhimurium A1-R and rMETase combination therapy. Tumor length and width were measured before and after treatment. RESULTS: On day 14 after treatment, all treatments significantly inhibited DDLPS PDOX tumor growth compared to the untreated control except for doxorubicin monotherapy. rMETase combined with S. typhimurium A1-R was significantly more effective and regressed tumor volume compared to either rMETase or S. typhimurium A1-R alone. The relative body weight did not significantly differ between days 0 and 14 for individual groups. CONCLUSION: The combination of rMETase and S. typhimurium A1-R has important clinical potential for this recalcitrant sarcoma.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Liases de Carbono-Enxofre/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lipossarcoma/patologia , Salmonella typhimurium/fisiologia , Animais , Terapia Combinada , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Lipossarcoma/tratamento farmacológico , Camundongos , Proteínas Recombinantes/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Poult Sci ; 99(5): 2645-2649, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359600

RESUMO

Feed additives can be alternatives to antibiotics for routinely encountered pathogens in the poultry production. The objective of this study was to understand effects of organic acid mixture on growth parameters and Salmonella Typhimurium (ST) colonization in broilers. Organic acid mixture is a feed-grade buffered formic acid and sodium formate mixture (Amasil NA). A total of 800 1-day-old Cobb500 males were fed one of the five dietary treatments: a negative control diet without ST challenge (NC), positive control diet with ST challenge (PC), 0.3% organic acid mixture with ST, 0.6% organic acid mixture with ST, and 0.9% organic acid mixture with ST. Treatments were assigned to 20 pens with 40 chicks/pen and 4 replicates of each treatment. Chickens were challenged with 107 CFU/mL of nalidixic acid-resistant ST (STNAR) 4-D posthatch. In the grower phase, feed conversion rate was significantly reduced in the 9% organic acid mixture compared with the PC. The body weight and body weight gain (BWG) were not affected either in the starter or grower phases. However, in the finisher phase, the nonchallenged NC had higher BWG than the PC (P < 0.05), whereas there were no differences in BWG among the NC and organic acid mixture fed groups. In addition, there was a significant effect of organic acid mixture on the colonization of cecal STNAR. At 9 dpi, cecal STNAR was 3.28 log10 in the PC that was reduced to 2.65 log10 at 0.3%, 1.40 log10 at 0.6%, and 0.84 log10 in 0.9% organic acid mixture. At 24 dpi, cecal STNAR recovery was 0.81, 0.99, 0.53, and 0.33 log10 in the PC and 0.3, 0.6, and 0.9% organic acid mixture, respectively. Similarly, at 38 dpi, cecal STNAR was 0.26, 0.11, 0.33, and 0 log10 in the PC, 0.3, 0.6, and 0.9%, respectively. These results show that organic acid mixture can be one dietary strategy to control ST infection and maintain efficient growth performance.


Assuntos
Galinhas , Formiatos/metabolismo , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/fisiologia , Ração Animal/análise , Animais , Antibacterianos/farmacologia , Peso Corporal/efeitos dos fármacos , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Formiatos/administração & dosagem , Masculino , Ácido Nalidíxico/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Ganho de Peso/efeitos dos fármacos
8.
Appl Environ Microbiol ; 86(14)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32385086

RESUMO

The aim of this research was to investigate the efficacy of the duty ratio and applied voltage in the inactivation of pathogens in soybean curd by pulsed ohmic heating (POH). The heating rate of soybean curd increased rapidly as the applied voltage increased, although the duty ratio did not affect the temperature profile. We supported this result by verifying that electrical conductivity increased with the applied voltage. Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in soybean curd were significantly (P < 0.05) inactivated by more than 1 log unit at 80 Vrms (root mean square voltage). To elucidate the mechanism underlying these results, the membrane potential of the pathogens was examined using DiBAC4(3) [bis-(1,3-dibutylbarbituric acid)trimethine oxonol] on the basis of a previous study showing that the electric field generated by ohmic heating affected the membrane potential of cells. The values of DiBAC4(3) accumulation increased under increasing applied voltage, and they were significantly (P < 0.05) higher at 80 Vrms, while the duty ratio had no effect. In addition, morphological analysis via transmission electron microscopy showed that electroporation and expulsion of intracellular materials were predominant at 80 Vrms Moreover, electrode corrosion was overcome by the POH technique, and the textural and color properties of soybean curd were preserved. These results substantiate the idea that the applied voltage has a profound effect on the microbial inactivation of POH as a consequence of not only the thermal effect, but also the nonthermal effect, of the electric field, whereas the duty ratio does not have such an effect.IMPORTANCE High-water-activity food products, such as soybean curd, are vulnerable to microbial contamination, which causes fatal foodborne diseases and food spoilage. Inactivating microorganisms inside food is difficult because the transfer of thermal energy is slower inside than it is outside the food. POH is an adequate sterilization technique because of its rapid and uniform heating without causing electrode corrosion. To elucidate the electrical factors associated with POH performance in the inactivation of pathogens, the effects of the applied voltage and duty ratio on POH were investigated. In this study, we verified that a high applied voltage (80 Vrms) at a duty ratio of 0.1 caused thermal and nonthermal effects on pathogens that led to an approximately 4-log-unit reduction in a significantly short time. Therefore, the results of this research corroborate database predictions of the inactivation efficiency of POH based on pathogen control strategy modeling.


Assuntos
Escherichia coli O157/fisiologia , Temperatura Alta , Listeria monocytogenes/fisiologia , Potenciais da Membrana , Viabilidade Microbiana , Salmonella typhimurium/fisiologia , Soja/fisiologia , Fermentação , Calefação/métodos , Cinética
9.
Poult Sci ; 99(4): 1862-1874, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32241466

RESUMO

This study was conducted to investigate the effects of dietary arginine (Arg) supplementation on the inflammatory response and gut microbiota of broiler chickens subjected to Salmonella enterica serovar Typhimurium. One hundred and forty 1-day-old Arbor Acres male birds were randomly assigned to a 2 × 2 factorial arrangement including diet treatment (with or without 0.3% Arg supplementation) and immunological stress (with or without S. typhimurium challenge). Samples were obtained at 7 D after infection (day 23). Results showed that S. typhimurium challenge caused histopathological and morphological damages, but Arg addition greatly reduced these intestinal injuries. S. typhimurium challenge elevated the levels of serum inflammatory parameters, including diamine oxidase, C-reactive protein, procalcitonin, IL-1ß, IL-8, and lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITNF) homolog. However, Arg supplementation decreased the serum procalcitonin, IL-1ß, IL-8, and LITNF concentration. S. typhimurium challenge significantly increased jejunal IL-1ß, IL-8, IL-10, and IL-17 mRNA expression and tended to upregulate IL-22 mRNA expression, but Arg supplementation remarkably reduced IL-8 mRNA expression, tended to downregulate IL-22 mRNA expression, and dramatically elevated IFN-γ and IL-10 mRNA expression. In addition, sequencing data of 16S rDNA indicated that the population of Proteobacteria phylum; Enterobacteriaceae family; Escherichia-Shigella, and Nitrosomonas genera; and Escherichia coli and Ochrobactrum intermedium species were more abundant, but the population of Rhodocyclaceae and Clostridiaceae_1 families and Candidatus Arthromitus genus were less abundant in the ileal digesta of birds with only S. typhimurium infection when compared with the controls. Treatment with Arg in birds subjected to S. typhimurium challenge increased the abundances of Firmicutes phylum, Clostridiaceae_1 family, Methylobacterium and Candidatus Arthromitus genera but decreased the abundance of Nitrosomonas genus and Rhizobium cellulosilyticum and Rubrobacter xylanophilus species as compared with the only S. typhimurium-challenged birds. In conclusion, Arg supplementation can alleviate intestinal mucosal impairment by ameliorating inflammatory response and modulating gut microbiota in broiler chickens challenged with S. typhimurium.


Assuntos
Arginina/metabolismo , Fenômenos Fisiológicos Bacterianos , Galinhas/imunologia , Microbioma Gastrointestinal/fisiologia , Inflamação/veterinária , Salmonella typhimurium/fisiologia , Ração Animal/análise , Animais , Arginina/administração & dosagem , Galinhas/microbiologia , Dieta/veterinária , Suplementos Nutricionais/análise , Inflamação/tratamento farmacológico , Inflamação/microbiologia , Intestinos/anatomia & histologia , Intestinos/microbiologia , Intestinos/patologia , Masculino , Doenças das Aves Domésticas/microbiologia , Distribuição Aleatória , Salmonelose Animal/microbiologia , Estresse Fisiológico/imunologia
10.
Poult Sci ; 99(3): 1571-1580, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32115034

RESUMO

The effects of 3 ethanol levels (30, 50, and 70%) with and without thiamine dilaurylsulfate (TDS; 1,000 ppm) were evaluated for the reduction of natural mesophilic aerobic bacteria (MAB), coliforms, and inoculated Salmonella Typhimurium (S. Typhimurium) in chicken skin. The chicken skin was inoculated with a 7 log cfu/mL suspension of S. Typhimurium. Loosely, intermediately, and tightly attached cells were recovered from chicken skin through shaking at 200 rpm for 5 min, stomaching for 1 min, and blending for 1 min, respectively. Increasing the ethanol concentration reduced the number of MAB, coliforms, and S. Typhimurium on the chicken skin, whereas TDS treatment without ethanol was not effective. Intermediately and tightly attached microorganisms (total MAB, coliforms, and S. Typhimurium) were more resistant to chemical disinfectants than loosely attached microorganisms. The combination of 70% ethanol with TDS was most effective than the combination of TDS with lower concentrations of ethanol in reducing populations of loosely, intermediately, and tightly attached MAB (by 1.88 log cfu/g, 1.21 log cfu/g, and 0.84 log cfu/g, respectively), coliforms (by 1.14 log cfu/g, 1.04 log cfu/g, and 0.67 log cfu/g, respectively), and S. Typhimurium (by 1.62 log cfu/g, 1.72 log cfu/g, and 1.27 log cfu/g, respectively). However, the chicken skin treated with higher concentrations of ethanol was tougher (P < 0.05) and more yellow and less red (P < 0.05) than that treated with lower concentrations of ethanol or with water (control). On the other hand, a combination of 30% ethanol and TDS yielded the best results, showing the reduction greater than 0.5 log cfu/g in S. Typhimurium, with no negative effect on chicken skin color or texture. Thus, a combination of 30% ethanol and TDS appears to be the optimal treatment for reducing microbial contamination of skin-on chicken products to enhance poultry safety without decreasing food quality, and this treatment could be applied in the poultry industry.


Assuntos
Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Etanol/farmacologia , Manipulação de Alimentos , Microbiologia de Alimentos , Tiamina/farmacologia , Animais , Antibacterianos/administração & dosagem , Bactérias Aeróbias/efeitos dos fármacos , Bactérias Aeróbias/fisiologia , Galinhas , Relação Dose-Resposta a Droga , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/fisiologia , Etanol/administração & dosagem , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Pele/microbiologia , Tiamina/administração & dosagem
11.
Res Vet Sci ; 130: 161-169, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32193003

RESUMO

Non-typhoidal Salmonella is considered a major public health concern. The growing relevance of pigs as reservoir of Salmonella spp. has prompted several countries to set up surveillance and control programs to fight Salmonella infection in swine and reduce public health risk. In the last decade, pork production in Córdoba increased significantly to become one of the most important pig production provinces in Argentina. The aim of this study was to estimate Salmonella spp. prevalence and associated risk factors in large scale-farms in this province. Mesenteric lymph nodes (MLN) of 580 pigs from 20 finishing large-scale farms were collected between 2014 and 2015 to estimate Salmonella infection. A prevalence of 41.5% (95%CI: 37.6-45.6%) was observed. Two major risk factors were significantly associated with Salmonella infection, both related to the pre-slaughter period (distance from the farm to the slaughterhouse and lairage time), highlighting the need to pay special attention to pre-slaughter practices in the province. Shortening transport times and complying with national regulations for lairage time at slaughter may help to reduce the prevalence of infection. Sixteen different serovars were identified, being S. Anatum and S. Typhimurium the most prevalent ones. Moreover, two isolate of the monophasic variant of Salmonella Typhimurium (I 4,5,12:i:-) resistant to enrofloxacin and which also displayed multidrug resistance was isolated for first time from pigs in Córdoba. The moderate to high levels of antimicrobial resistance detected for antibiotics commonly used in the pig sector suggested the need for implementing a plan to limit their use in the province.


Assuntos
Farmacorresistência Bacteriana , Salmonelose Animal/epidemiologia , Salmonella/efeitos dos fármacos , Doenças dos Suínos/epidemiologia , Criação de Animais Domésticos , Animais , Antibacterianos/farmacologia , Argentina/epidemiologia , Prevalência , Fatores de Risco , Salmonella/fisiologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Sus scrofa , Suínos , Doenças dos Suínos/microbiologia
12.
Appl Environ Microbiol ; 86(8)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32033951

RESUMO

Salmonella enterica is a foodborne pathogen often leading to gastroenteritis and is commonly acquired by consumption of contaminated food of animal origin. However, frequency of outbreaks linked to the consumption of fresh or minimally processed food of nonanimal origin is increasing. New infection routes of S. enterica by vegetables, fruits, nuts, and herbs have to be considered. This leads to special interest in S. enterica interactions with leafy products, e.g., salads, that are mainly consumed in a minimally processed form. The attachment of S. enterica to salad is a crucial step in contamination, but little is known about the bacterial factors required and mechanisms of adhesion. S. enterica possesses a complex set of adhesive structures whose functions are only partly understood. Potentially, S. enterica may deploy multiple adhesive strategies for adhering to various salad species and other vegetables. In this study, we systematically analyzed the contributions of the complete adhesiome, of lipopolysaccharide (LPS), and of flagellum-mediated motility of S. enterica serovar Typhimurium (STM) in adhesion to Valerianella locusta (corn salad). We deployed a reductionist, synthetic approach to identify factors involved in the surface binding of STM to leaves of corn salad, with particular regard to the expression of all known adhesive structures, using the Tet-on system. This work reveals the contribution of Saf fimbriae, type 1 secretion system-secreted BapA, an intact LPS, and flagellum-mediated motility of STM in adhesion to corn salad leaves.IMPORTANCE Transmission of gastrointestinal pathogens by contaminated fresh produce is of increasing relevance to human health. However, the mechanisms of contamination of, persistence on, and transmission by fresh produce are poorly understood. We investigated the contributions of the various adhesive structures of STM to the initial event in transmission, i.e., binding to the plant surface. A reductionist system was used that allowed experimentally controlled surface expression of individual adhesive structures and analyses of the contribution to binding to leave surfaces of corn salad under laboratory conditions. The model system allowed the determination of the relative contributions of fimbrial and nonfimbrial adhesins, the type 3 secretion systems, the O antigen of lipopolysaccharide, the flagella, and chemotaxis of STM to binding to corn salad leaves. Based on these data, future work could reveal the mechanism of binding and the relevance of interaction under agricultural conditions.


Assuntos
Aderência Bacteriana , Microbiologia de Alimentos , Salmonella typhimurium/fisiologia , Valerianella/microbiologia , Lipopolissacarídeos/metabolismo
13.
Nat Commun ; 11(1): 1007, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081907

RESUMO

Bacterial biofilms, especially those associated with implanted medical devices, are difficult to eradicate. Curli amyloid fibers are important components of the biofilms formed by the Enterobacteriaceae family. Here, we show that a human monoclonal antibody with pan-amyloid-binding activity (mAb 3H3) can disrupt biofilms formed by Salmonella enterica serovar Typhimurium in vitro and in vivo. The antibody disrupts the biofilm structure, enhancing biofilm eradication by antibiotics and immune cells. In mice, 3H3 injections allow antibiotic-mediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms.


Assuntos
Amiloide/imunologia , Proteínas de Bactérias/imunologia , Biofilmes/crescimento & desenvolvimento , Salmonella typhimurium/imunologia , Salmonella typhimurium/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Infecções Relacionadas a Cateter/prevenção & controle , Epitopos/imunologia , Humanos , Macrófagos/imunologia , Camundongos , Infecções por Salmonella/prevenção & controle
14.
Pathog Dis ; 78(1)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32083661

RESUMO

Current study aims to characterize the influence of sub-minimum inhibitory concentration (sub-MIC) of ciprofloxacin on Salmonella intracellular survival and host virulence. Herein, Salmonella resistance patterns to various antibiotics were in agreement with those reported in previous studies. Moreover, intracellular survival of both ciprofloxacin-sensitive and -resistant Salmonella was markedly reduced upon treatment with sub-MIC of ciprofloxacin as determined by gentamicin protection assay. These findings were further confirmed using immunostaining indicating an inhibitory effect of sub-MIC of ciprofloxacin on Salmonella intracellular survival. RT-qPCR revealed that expression of genes encoding Salmonella type three secretion system (TTSS) decreased upon bacterial exposure to sub-MIC of ciprofloxacin. Furthermore, bacterial exposure to sub-MIC of ciprofloxacin significantly reduced expression of both sifA and sifB, which are important for Salmonella filaments formation within the host. Treatment of Salmonella with sub-MIC of ciprofloxacin reduced bacterial capacity to kill mice infection models. A lower mortality rate was observed in mice injected with Salmonella treated with sub-MIC of ciprofloxacin as compared with mice inoculated with untreated bacteria. Collectively, current findings indicate that, in addition to its bactericidal potential, sub-MIC of ciprofloxacin could inhibit Salmonella intracellular survival, virulence genes expression as well as host pathogenesis, providing another mechanism for ciprofloxacin in limiting Salmonella host infection.


Assuntos
Proteínas de Bactérias/genética , Ciprofloxacino/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Virulência/genética
15.
Curr Microbiol ; 77(5): 836-845, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31932998

RESUMO

Amoebic bacterial interactions are the most ancient form of host pathogen interactions. Here, we investigate the fate of Salmonella typhimurium and Acanthamoeba castellanii T4 genotype upon mutual interactions in a nutrition free environment. The role of type 1 fimbriae and motility of S. typhimurium during interactions with A. castellanii has also been investigated. Deletion of genes encoding the type 1 fimbriae subunit FimA, type 1 fimbriae tip protein FimH, chemotaxis regulatory proteins CheA and CheY and major flagella subunits FliC and FljB was performed through homologous recombination. In vitro association, invasion and survival assays of S. typhimurium wild-type and mutant strains were performed upon co-incubation of bacteria with A. castellanii trophozoites in a nutrition free environment. The deletion gene encoding type 1 fimbriae subunit FimA reduced, whereas the deletion of genes encoding flagella subunits FliC and FljB of flagella enhanced the association capability of S. typhimurium with A. castellanii. Invasion of A. castellanii by Salmonella was significantly reduced upon the loss of type 1 fimbriae subunit FimA and type 1 fimbriae tip protein FimH. Co-incubation of S. typhimurium with A. castellanii in phosphate buffered saline medium stimulated the growth of S. typhimurium wild-type and mutant strains. Viable A. castellanii trophozoites count became significantly reduced upon co-incubation with S. typhimurium within 48 h. Type 1 fimbriae play a pivotal role in the adherence of S. typhimurium to the A. castellanii cell surface. Subsequently, this interaction provides S. typhimurium an advantage in growth.


Assuntos
Acanthamoeba castellanii/microbiologia , Acanthamoeba castellanii/fisiologia , Fímbrias Bacterianas/metabolismo , Interações Hospedeiro-Patógeno , Salmonella typhimurium/fisiologia , Acanthamoeba castellanii/genética , Aderência Bacteriana , Fímbrias Bacterianas/genética , Deleção de Genes , Genótipo , Mutação , Salmonella typhimurium/genética
16.
J Vis Exp ; (155)2020 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-31984953

RESUMO

The dimeric transcription factor NF-κB regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-κB is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα). Activation of NF-κB requires the degradation of IκBα, which then exposes a nuclear localization signal on NF-κB and promotes its trafficking to the nucleus. Once in the nucleus, NF-κB binds to the promotor region of NF-κB target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression. The activation of NF-κB occurs independently of transcription or translation. Therefore, the activation state of NF-κB must be measured either by quantifying NF-κB specifically in the nucleus, or by quantifying expression of NF-κB target genes. In this protocol, cells stably transfected with an NF-κB::luciferase reporter construct are assayed for NF-κB activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-κB, which traffics to the nucleus and binds to κB sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-κB activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.


Assuntos
Regulação da Expressão Gênica , Luciferases/metabolismo , NF-kappa B/metabolismo , Infecções por Salmonella/genética , Salmonella typhimurium/fisiologia , Técnicas de Cultura de Tecidos , Ativação Enzimática , Células HeLa , Humanos , Luciferases/genética , Luminescência , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
17.
Res Microbiol ; 171(3-4): 143-152, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31991172

RESUMO

Polyamines are small cationic amines required for modulating multiple cell process, including cell growth and DNA and RNA stability. In Salmonella polyamines are primarily synthesized from L-arginine or L-ornithine. Based on a previous study, which demonstrated that polyamines affect the expression of virulence gene in S. Typhimurium, we investigated the role of polyamines in the global gene and protein expression in S. Typhimurium. The depletion of polyamine biosynthesis led to down-regulation of genes encoding structural components of the Type Three Secretion system 1 (TTSS1) and its secreted effectors. Interestingly, Expression of HilA, which is the master regulator of Salmonella Pathogenicity Island 1 (SPI1), was only reduced at the post-transcriptional in the polyamine mutant. Enzymes related to biosynthesis and/or transport of several amino acids were up-regulated, just as the Mg2+-transport systems were three to six-fold up-regulated at both the transcriptional and protein levels. Furthermore, in the polyamine depletion mutant, proteins related to stress response (IbpA, Dps, SodB), were 2-5 fold up-regulated. Together our data provide strong evidence that polyamine depletion affects expression of proteins linked with virulence and stress response of S. Typhimurium. Furthermore, polyamines positively affected translation of HilA, the major regulator of SPI1.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Poliaminas/metabolismo , Biossíntese de Proteínas , Salmonella typhimurium/fisiologia , Estresse Fisiológico , Transativadores/genética , Mutação , Proteômica/métodos , Infecções por Salmonella/microbiologia , Virulência/genética , Fatores de Virulência/genética
18.
J Appl Microbiol ; 128(1): 2-14, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31038778

RESUMO

Use of bacteria in cancer therapy, despite being considered as a potent strategy, has not really picked up the way other methods of cancer therapies have evolved. However, in recent years, the interest on use of bacteria to kill cancer cells has renewed considerably. The standard and widely followed strategies of cancer treatment often fail either due to the complexity of tumour biology or because of the accompanying side effects. In contrast, these limitations can be easily overcome in a bacteria-mediated approach. Salmonella is a bacterium, which is known for its ability to colonize solid or semisolid tumours more efficiently than any other bacteria. Among more than 2500 serovars of Salmonella, S. Typhimurium has been widely studied for its antagonistic effects on cancer cells. Here in, we review the current status of the preclinical and the clinical studies with a focus on the mechanisms that attribute the anticancer properties to nontyphoidal Salmonella.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/terapia , Salmonella/fisiologia , Animais , Humanos , Neoplasias/microbiologia , Salmonella/crescimento & desenvolvimento , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/fisiologia
19.
Int J Food Microbiol ; 317: 108422, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-31756646

RESUMO

This study was conducted to validate a simulated commercial baking process for plain muffins against E. coli O121 (isolated from the recent illness outbreak associated with flour), and compare the thermal inactivation parameters (D- and z-values) of cocktails of four isolates of E. coli O121 and three serovars of Salmonella (Newport, Typhimurium, and Senftenberg) in muffin batter. Flour samples were spray inoculated with the E. coli O121 or Salmonella cocktails, dried back to the pre-inoculation weight to achieve ~7 log10 CFU/g, and used to prepare muffin batter. For the muffin baking validation study using E. coli O121, muffin batter was baked at 375 °F (190.6 °C) oven temperature for 21 min followed by 30 min of ambient cooling. The E. coli O121 population decreased by >7 log10 CFU/g in muffins by 17 min of baking, and was completely eradicated after 21 min of baking and ambient cooling. The D-values of E. coli O121 and Salmonella cocktails in muffin batter at 60, 65 and 70 °C were 42.0 and 38.4, 7.5 and 7.2, and 0.4 and 0.5 min, respectively; whereas the z-values of E. coli O121 and Salmonella were 5.0 and 5.2 °C, respectively.


Assuntos
Pão/microbiologia , Escherichia coli O157/fisiologia , Farinha/microbiologia , Temperatura Alta , Salmonella typhimurium/fisiologia , Temperatura Baixa , Contagem de Colônia Microbiana , Culinária/métodos , Manipulação de Alimentos , Microbiologia de Alimentos
20.
Nat Microbiol ; 5(1): 192-205, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31792428

RESUMO

MicroRNAs (miRNAs) are increasingly recognized for their role in infection by bacterial pathogens, although the effect of each individual miRNA remains largely unknown. Here, we used a comparative genome-wide microscopy-based functional screening approach to identify miRNAs controlling infection by two bacterial pathogens-Salmonella enterica serovar Typhimurium and Shigella flexneri. Despite the similarities between these pathogens, we found infections to be controlled by largely non-overlapping subsets of miRNAs, seemingly reflecting different requirements prompted by their distinct intracellular lifestyles. By characterizing a small subset of miRNAs chosen among the strongest inhibitors of Shigella infection, we discovered that miR-3668, miR-4732-5p and miR-6073 exert a selective effect on Shigella infection by impairing bacterial actin-based motility by downregulating N-WASP. Additionally, by identifying let-7i-3p miRNA as a strong inhibitor of Salmonella replication and performing in-depth analysis of its mechanisms of action, we showed that this miRNA specifically inhibits Salmonella infection via modulation of endolysosomal trafficking and the vacuolar environment by targeting the host RGS2 protein. These findings illustrate two paradigms underlying miRNA-mediated regulation of bacterial infection, acting as part of the host response to infection, or as part of bacterial strategies to modulate the host environment and favour pathogenesis.


Assuntos
Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/microbiologia , MicroRNAs/genética , Salmonella typhimurium/fisiologia , Shigella flexneri/fisiologia , Animais , Regulação da Expressão Gênica , Genômica , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/metabolismo , Especificidade da Espécie , Suínos
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