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1.
Food Microbiol ; 98: 103791, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-33875220

RESUMEN

The gene encoding LysSTG2, an endolysin from Salmonella-lytic bacteriophage STG2, was cloned, overexpressed, and characterized. LysSTG2 consists of a single domain belonging to the Peptidase_M15 superfamily. LysSTG2 showed strong lytic activity against chloroform-treated S. Typhimurium cells after incubation at 4-50 °C for 30 min, at pH ranging from 7.0 to 11.0, and in the presence of NaCl from 0 to 300 mmol/L. It also showed lytic activity against all the 14 tested Gram-negative strains treated with chloroform, including Salmonella, E. coli, and Pseudomonas aeruginosa, but not against the Gram-positive bacteria tested. In addition, LysSTG2 (100 µg/mL) reduced the viability of S. Typhimurium NBRC 12529 planktonic cells by 1.2 log and that of the biofilm cells after 1-h treatment. Sequential treatment of slightly acidic hypochlorous water (SAHW) containing 40 mg/L available chlorine and LysSTG2 (100 µg/mL) was effective on S. Typhimurium NBRC 12529 biofilm cells, removing more than 99% of biofilm cells. These results demonstrate that LysSTG2 alone can effectively kill S. Typhimurium cells after permeabilization treatment and successfully control S. Typhimurium in biofilms in combination with SAHW, suggesting that the combined use of LysSTG2 and SAHW might be a novel and promising method for combating S. Typhimurium in food industries.


Asunto(s)
Bacteriófagos/enzimología , Biopelículas , Cloro/farmacología , Endopeptidasas/metabolismo , Ácido Hipocloroso/farmacología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/virología , Proteínas Virales/metabolismo , Bacteriófagos/genética , Biopelículas/efectos de los fármacos , Endopeptidasas/genética , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Proteínas Virales/genética , Agua/química
2.
Int J Food Microbiol ; 347: 109189, 2021 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-33838479

RESUMEN

Salmonella enterica serovar Typhimurium can survive some extreme environment in food processing, and vanillin generally recognized as safe is bactericidal to pathogens. Thus, we need to explore the responses of S. Typhimurium to vanillin in order to apply this antimicrobial agent in food processing. In this study, we exposed S. Typhimurium to commercial apple juice with/without vanillin (3.2 mg/mL) at 45 °C for 75 min to determine the survival rate. Subsequently, the 10-min cultures were selected for transcriptomic analysis. Using high-throughput RNA sequencing, genes related to vanillin resistance and their expression changes of S. Typhimurium were identified. The survival curve showed that S. Typhimurium treated with vanillin were inactivated by 5.5 log after 75 min, while the control group only decreased by 2.3 log. Such a discrepancy showed the significant antibacterial effect of vanillin on S. Typhimurium. As a result, 265 differentially expressed genes (DEGs) were found when coping with vanillin, among which, 225 showed up-regulation and 40 DEGs were down-regulated. Treated with vanillin, S. Typhimurium significantly up-regulated genes involved in cell membrane, acid tolerance response (ATR) and oxidative stress response, cold shock cross-protection, DNA repair, virulence factors and some key regulators. Firstly, membrane-related genes, including outer membrane (bamE, mepS, ygdI, lolB), inner membrane (yaiY, yicS) and other proteins (yciC, yjcH), were significantly up-regulated because of the damaged cell membrane. Then, up-regulated proteins associated with arginine synthesis (ArgABCDIG) and inward transportation (ArtI, ArtJ, ArtP and HisP), participated in ATR to pump out the protons inside the cell in this scenario. Next, superoxide stress response triggered by vanillin was found to have a significant up-regulation as well, which was controlled by SoxRS regulon. Besides, NADH-associated (nuoA, nuoB, nuoK, nadE, fre and STM3021), thioredoxin (trxA, trxC, tpx and bcp) and glutaredoxin (grxC and grxD) DEGs led to the increase of the oxidative stress response. Cold shock proteins such as CspA and CspC showed an up-regulation, suggesting it might play a role in cross-protecting S. Typhimurium from vanillin stress. Furthermore, DEGs in DNA repair and virulence factors, including flagellar assembly, adhesins and type III secretion system were up-regulated. Some regulators like fur, rpoE and csrA played a pivotal role in response to the stress caused by vanillin. Therefore, this study sounds an alarm for the risks caused by stress tolerance of S. Typhimurium in food industry.


Asunto(s)
Benzaldehídos/farmacología , Conservantes de Alimentos/farmacología , Jugos de Frutas y Vegetales/microbiología , Malus/microbiología , Salmonella typhimurium/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Proteínas Bacterianas/genética , Benzaldehídos/análisis , Conservantes de Alimentos/análisis , Jugos de Frutas y Vegetales/análisis , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Salmonella typhimurium/genética , Transcriptoma/efectos de los fármacos
3.
Int J Food Microbiol ; 347: 109198, 2021 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-33894462

RESUMEN

The U.S. FDA Food Safety Modernization Act Preventive Controls for Human Food Rule underlines the importance of an effective environmental monitoring (EM) program. EM is used to determine harborage sites of microorganisms on processing equipment, assess effectiveness of sanitation programs, and prevent transmission of foodborne pathogens. This study characterizes commercially-available polyurethane foam (PUF) and cellulose (CELL) EM tools for their efficacy in the release of foodborne pathogens from their sponge matrices. Specifically, the objectives of this study were to 1) compare the ability of EM tools to release microorganisms into a recovery eluent, 2) characterize EM tool performance at decreasing inoculum concentrations, and 3) assess the impact of various operators during the processing of EM samples. Two bacteria (Listeria monocytogenes, Salmonella Typhimurium) and one human norovirus surrogate (Tulane virus [TV]) were compared at decreasing inoculum levels utilizing two elution techniques (mechanical stomacher, manually by operator), and across six operators. Data indicated that EM tool material composition impacted the release of microorganisms (p = 0.0001), where the PUF EM tool released TV more readily than the CELL EM tool. Conversely, the decreasing inoculum levels did not statistically differ in the release of microorganisms from the EM tool matrices. In addition, no significant difference was found between the machine stomacher and manual elution by human operator or between operators. Overall, the study provides a detailed characterization of two commercially-available EM tools, and the differences identified in this study can be used to improve the effectiveness of EM programs.


Asunto(s)
Celulosa/farmacología , Monitoreo del Ambiente/métodos , Listeria monocytogenes/aislamiento & purificación , Norovirus/aislamiento & purificación , Poliuretanos/farmacología , Salmonella typhimurium/aislamiento & purificación , Carga Bacteriana/métodos , Recuento de Colonia Microbiana , Microbiología de Alimentos , Inocuidad de los Alimentos , Humanos , Listeria monocytogenes/genética , Norovirus/genética , Salmonella typhimurium/genética , Carga Viral/métodos
4.
Int J Med Microbiol ; 311(4): 151501, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33866091

RESUMEN

BACKGROUND: Previous studies reported the prevalence of mcr-1 among clinical infected Salmonella isolates in China. However, the transmission dynamics of mcr-1 in different ecological niches were not well investigated. Our objective is to exhibit the transmission dynamics of mcr-1 in Salmonella. METHODS: 598 Salmonella isolates were recovered from ten hospitals; besides 936 pig faces and 167 pork samples were collected from January 2015 to December 2017 in Guangzhou, China. PCR and sequencing were used to identify mcr-1-positive Salmonella. Antimicrobial susceptibility testing was performed with 16 antimicrobials. Conjugation, S1-PFGE, and Southern blot were used to determine the transferability and location of mcr-1. Whole-genome sequencing was used to investigate pangenome, phylogeny, plasmid, and transposon. RESULTS: Eleven mcr-1-positive Salmonella isolates were identified from patients with infectious diarrhea. Five pig fecal samples and three pork samples contained mcr-1-positive Salmonella isolates. All isolates were multi-drug resistant. The mcr-1 genes were located on ∼210-250 kb IncHI2-pST3 plasmids, and 12 mcr-1 genes were transferable. All isolates were assigned to ST34 or its genetically closed STs. The distribution of the core-genome network was significantly correlated with source distributions. The accessory genes-based network demonstrated that the diverse clonal complexes could share highly similar accessory genomes. CONCLUSIONS: The prevalence of mcr-1-positive Salmonella among different sources was low. Clonal transmission could not be the main reason for the expansion of mcr-1-positive Salmonella, but be attributed to the horizontal transfer of IncHI2-pST3 plasmid. Continuous surveillance on Salmonella should be performed to investigate the response of colistin banning in food-producing animals by mcr-1-positive Salmonella populations.


Asunto(s)
Antibacterianos , Salmonella typhimurium , Animales , Antibacterianos/farmacología , China/epidemiología , Diarrea/epidemiología , Genómica , Humanos , Plásmidos/genética , Prevalencia , Salmonella typhimurium/genética , Porcinos
5.
Analyst ; 146(8): 2559-2566, 2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33899066

RESUMEN

There is significant demand for the development of rapid, sensitive, and specific methods for detecting bacterial pathogens in order to identify the causes of food poisoning. Nucleic acid amplification tests (NAATs) allow for the culture-free detection of bacterial pathogens and are not as labor intensive and time consuming as culture-based detection methods. However, suitable sample preparation methods must be developed for the realization of simple, rapid, and sensitive NAATs. To resolve this problem, we developed a new sample preparation method that integrates bacterial pathogen enrichment and DNA extraction. We engineered magnetic nanoparticles (MNPs) with a physicochemical probe (tryptamine) for single-tube sample preparation with minimal sample loss. The tryptamine-functionalized MNPs (Indole@MNPs) showed inherent hydrophobicity owing to the indole side chain and a change in their zeta potential with a decrease in the pH. Because of their physicochemical characteristics, the Indole@MNPs could adsorb bacterial pathogens, thus allowing sample enrichment and DNA binding and release through weak electrostatic interactions via pH control. We successfully detected Salmonella enterica serovar Typhimurium, a common cause of bacterial food poisoning, at a concentration of 10 CFU/10 mL in milk samples using quantitative PCR. Thus, the proposed method allows for the simple and sensitive detection of Salmonella typhimurium and can be used for nontyphoidal salmonella detection to ensure food safety.


Asunto(s)
Enfermedades Transmitidas por los Alimentos , Nanopartículas de Magnetita , Microbiología de Alimentos , Humanos , Magnetismo , Salmonella typhimurium/genética , Sensibilidad y Especificidad , Triptaminas
6.
J Dairy Sci ; 104(6): 6588-6597, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33715855

RESUMEN

In this study, we established a rapid and sensitive method for the detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk using biotin-exposure-based immunomagnetic separation (IMS) combined with sodium dodecyl sulfate (SDS), propidium monoazide (PMA), and multiplex real-time PCR (mRT-PCR). We used IMS to lessen the assay time for isolation of target bacteria. We then optimized the coupling conditions and immunomagnetic capture process. The immunoreaction and incubation times for 5 µg of mAb coupled with 500 µg of streptavidin-functionalized magnetic beads using a streptavidin-biotin system were 90 and 30 min, respectively. Treatment with SDS-PMA before mRT-PCR amplification eliminated false-positive outcomes from dead bacteria and identified viable target bacteria with good sensitivity and specificity. The limit of detection of IMS combined with the SDS-PMA-mRT-PCR assay for the detection of viable Salmonella Typhimurium, Staph. aureus, and L. monocytogenes in spiked milk matrix samples was 10 cfu/mL and remained significant even in the appearance of 106 cfu/mL of nontarget bacteria. The entire detection process was able to identify viable bacteria within 9 h. The combination of biotin-exposure-mediated IMS and SDS-PMA-mRT-PCR has potential value for the rapid and sensitive detection of foodborne pathogens.


Asunto(s)
Listeria monocytogenes , Animales , Azidas , Biotina , Separación Inmunomagnética/veterinaria , Leche , Propidio/análogos & derivados , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Salmonella typhimurium/genética , Dodecil Sulfato de Sodio , Staphylococcus aureus/genética
7.
Mutat Res ; 863-864: 503299, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33678240

RESUMEN

N-Acyloxy-N-alkoxyamides are direct-acting mutagens in S. typhimurium TA100 and TA98. A reliable QSAR for their activity in TA100 has been developed, which indicates reversible intercalation into the DNA helix through naphthalene substituents. In this paper, we show that fluorene as a substituent does not facilitate intercalation while fluorenone does, although the efficacy is determined by the position of substitution on the fluorenone as well as the N-acyloxy-N-alkoxyamide side chain. Where intercalation is evident, the increased binding to DNA is similar to that of naphthalene and is worth the equivalent of ca four LogP hydrophobicity units. 4-Substituted fluorenones, where the anomeric amide group is in the bay region do not intercalate, which is attributed to the requirement for a weaker edge-on, rather than an end-on intercalation. Mutagencity in S. typhimurium TA98, which detects frame shifts through intercalation, supports the findings. Fluorene appears not to intercalate, which points to the fact that the charge delocalised 2-fluorenylnitrenium ion, the ultimate metabolite from 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF) is the itercalating agent responsible for frameshift mutations leading to their carcinogenicity.


Asunto(s)
Sustancias Intercalantes , Mutagénesis , Mutágenos , Salmonella typhimurium , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Pruebas de Mutagenicidad , Mutágenos/química , Mutágenos/farmacología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
8.
Nat Commun ; 12(1): 1546, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33750771

RESUMEN

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte de Proteínas/fisiología , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/química , Sistemas de Secreción Tipo III/metabolismo , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica en Hélice alfa , Salmonella enterica/metabolismo , Salmonella typhimurium/genética , Sistemas de Secreción Tipo III/genética
9.
Food Res Int ; 141: 110117, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33641984

RESUMEN

Despite food safety recommendations, raw egg-based foods, such as mayonnaise, are frequently identified as the source of Salmonella during outbreaks. Acidification and storage temperature have been linked with reduced bacterial culturability. Raw egg-based sauces stored at 25 °C have historically been linked with faster decline of Salmonella culturability than preparations stored at 5 °C. This study aimed to determine whether reduced culturability in acidified mayonnaise correlated with reduced in vitro bacterial motility, invasiveness and viability as well as disease-causing capacity in BALB/c mice. Acidification of mayonnaise and incubation at 25 °C for 4 h significantly reduced culturability of Salmonella Typhimurium DT9 but was dependent on initial bacterial load. Bacteria recovered from acidified mayonnaise exhibited reduced invasiveness into polarized cultured intestinal epithelial cells and 12 h post inoculation were no longer invasive suggesting a reduced capacity to cause disease. To confirm this, BALB/c mice were inoculated with Salmonella Typhimurium contaminated mayonnaise stored at 5 °C or 25 °C for 12, 24, 48, 72, and 96 h. Mice inoculated with mayonnaise incubated at 5 °C for 12 and 24 h exhibited mild to moderate disease symptoms; all other mayonnaise treatment groups did not exhibit disease symptoms. In acidified mayonnaise, Salmonella Typhimurium DT9 exhibited a global downregulation of metabolism, stress response, and virulence genes upon addition to mayonnaise. After 4 h of incubation at both 5 °C and 25 °C, however, the vast majority of genes were upregulated which was maintained over the 96-hour experiment suggesting that bacteria were severely stressed. Salmonella Typhimurium DT9 cells were isolated from mayonnaise samples and ATP production was quantified. At both 5 °C and 25 °C, ATP production decreased in acidified mayonnaise preparations. At 25 °C, ATP production decreased more rapidly than at 5 °C. After 24 h, ATP production of bacteria in mayonnaise stored at 25 °C was not significantly different from the dead control group. Thus, the current recommendation of only serving freshly prepared raw egg-sauces or refrigerating immediately after preparation, could be placing consumers at higher risk for contracting salmonellosis.


Asunto(s)
Salmonella typhimurium , Animales , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Salmonella typhimurium/genética , Temperatura , Virulencia
11.
J Vis Exp ; (167)2021 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-33586704

RESUMEN

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336ΔmicC by the λ Red-based recombination system and the complemented mutant 50336ΔmicC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336ΔmicC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336ΔmicC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336ΔmicC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336ΔmicC and 50336ΔmicC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas de la Membrana/genética , ARN no Traducido/genética , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidad , Animales , Proteínas Bacterianas/metabolismo , Pollos/microbiología , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Plásmidos/genética , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinación Genética/genética , Salmonelosis Animal/microbiología , Salmonella typhimurium/genética , Virulencia/genética
12.
Nucleic Acids Res ; 49(4): 2357-2374, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33638994

RESUMEN

RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.


Asunto(s)
Proteínas Bacterianas/química , Regiones Promotoras Genéticas , Regulón , Salmonella typhimurium/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Modelos Moleculares , Mutación , Fosforilación , Conformación Proteica , Salmonella typhimurium/metabolismo , Transcripción Genética
13.
Science ; 371(6527): 400-405, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33479153

RESUMEN

Key to the success of intracellular pathogens is the ability to sense and respond to a changing host cell environment. Macrophages exposed to microbial products undergo metabolic changes that drive inflammatory responses. However, the role of macrophage metabolic reprogramming in bacterial adaptation to the intracellular environment has not been explored. Here, using metabolic profiling and dual RNA sequencing, we show that succinate accumulation in macrophages is sensed by intracellular Salmonella Typhimurium (S. Tm) to promote antimicrobial resistance and type III secretion. S Tm lacking the succinate uptake transporter DcuB displays impaired survival in macrophages and in mice. Thus, S Tm co-opts the metabolic reprogramming of infected macrophages as a signal that induces its own virulence and survival, providing an additional perspective on metabolic host-pathogen cross-talk.


Asunto(s)
Interacciones Huésped-Patógeno , Macrófagos/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Ácido Succínico/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Supervivencia Celular , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Modelos Animales de Enfermedad , Femenino , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , RNA-Seq , Salmonella typhimurium/genética , Virulencia
14.
Food Microbiol ; 95: 103671, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33397606

RESUMEN

The lack of proper gastrointestinal models assessing the inter-strain virulence variability of foodborne pathogens and the effect of the vehicle (food matrix) affects the risk estimation. This research aimed to propose a dynamic and integrated in vitro/ex vivo gastrointestinal model to evaluate the probability and severity of infection of foodborne pathogens at different matrices. An everted gut sac was used to determine the adhesion and invasion of Salmonella enterica and tissue damage. S. Typhimurium ATCC 14028 was used as a representative bacterium, and two matrices (water and cheese) were used as vehicles. No differences (p > 0.05) in the probability of infection (Pinf) were found for intra-experimental repeatability. However, the Pinf of cheese-vehiculated S. Typhimurium was different compared to water- vehiculated S. Typhimurium, 7.2-fold higher. The histological analysis revealed Salmonella-induced tissue damage, compared with the control (p < 0.05). In silico proposed interactions between two major Salmonella outer membrane proteins (OmpA and Rck) and digested peptides from cheese casein showed high binding affinity and stability, suggesting a potential protective function from the food matrix. The results showed that the everted gut sac model is suitable to evaluate the inter-strain virulence variability, considering both physiological conditions and the effect of the food matrix.


Asunto(s)
Enfermedades Transmitidas por los Alimentos/microbiología , Tracto Gastrointestinal/microbiología , Salmonella typhimurium/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Queso/microbiología , Agua Dulce/microbiología , Humanos , Modelos Biológicos , Probabilidad , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Virulencia
15.
Appl Microbiol Biotechnol ; 105(4): 1563-1573, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33496815

RESUMEN

As a primary cause of food contamination and human diseases, Salmonella Typhimurium can easily form a biofilm that is difficult to remove from food surfaces, and often causes significant invisible threats to food safety. Although berberine has been widely used as an anti-infective drug in traditional medicine, some basic principles underlying its mechanism, especially the interaction between berberine and type I fimbriae genes, has not been verified yet. In this study, two strains of major fimbrial gene mutants (ΔfimA and ΔfimH) were constructed to demonstrate the possible action of berberine on type I fimbriae genes. The broth microdilution method was used to determine the antibacterial activity of berberine against selected strains (WT, ΔfimA, and ΔfimH). Cell agglutination experiments revealed that the number of S. Typhimurium type I fimbriae reduced after berberine treatment, which was consistent with transmission electron microscopy results. Quantitative real-time PCR experiments also confirmed that berberine reduced fimA gene expression, indicating a certain interaction between berberine and fimA gene. Furthermore, confocal laser scanning microscopy imaging of biofilm clearly revealed that berberine prevents biofilm formation by reducing the number of type I fimbriae. Overall, it is well speculated for us that berberine could be an excellent combating-biofilm drug in clinical microbiology and food preservation. KEY POINTS: • Reduce the number of fimbriae. • Berberine targeting fimA. • Effective biofilm inhibitor.


Asunto(s)
Berberina , Salmonella typhimurium , Berberina/farmacología , Biopelículas , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Humanos , Salmonella typhimurium/genética
16.
Nat Commun ; 12(1): 328, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436566

RESUMEN

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.


Asunto(s)
Sistema de Lectura Ribosómico/genética , Genoma Bacteriano , ARN de Transferencia/genética , Salmonella typhimurium/genética , Aminoácidos/metabolismo , Aminoacilación , Anticodón/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Codón/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Guanosina Trifosfato/metabolismo , Hidrólisis , Metilación , Modelos Moleculares , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/metabolismo
17.
Nat Commun ; 12(1): 348, 2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33441540

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiotaxis/fisiología , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Salmonella typhimurium/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/genética , Células CACO-2 , Bovinos , Células Cultivadas , Quimiotaxis/genética , Regulación Bacteriana de la Expresión Génica , Células HeLa , Humanos , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Ratones Endogámicos C57BL , Movimiento/fisiología , Mutación , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Factores de Transcripción/genética
18.
Nucleic Acids Res ; 49(2): 832-846, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33406256

RESUMEN

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDCSGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriVSGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter PS004. This replicon transiently maintains SGI1 as a 4-8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.


Asunto(s)
Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Conjugación Genética/genética , Farmacorresistencia Bacteriana Múltiple/genética , Secuencias Repetitivas Esparcidas/genética , Salmonella typhimurium/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica/genética , Genes Reporteros , Integrasas/metabolismo , Operón/genética , Filogenia , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas , Recombinasas/metabolismo , Replicón/genética , Alineación de Secuencia , Transactivadores/genética , Transactivadores/metabolismo
20.
Biosens Bioelectron ; 178: 113001, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33493900

RESUMEN

Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive/negative/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/virología , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Prueba de Ácido Nucleico para COVID-19/instrumentación , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Microbiología de Alimentos , Humanos , Magnetismo , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Polipropilenos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Salmonella typhimurium/genética , Salmonella typhimurium/aislamiento & purificación , Autoevaluación
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