RESUMEN
This study aimed to evaluate the effects of thyme (Thymus vulgaris L.), rosemary (Rosmarinus officinalis L.), and ammoides (Ammoidespusilla L.) aqueous extracts supplementation on the quality of heat-treated (55, 65, and 75 °C) raw milk (sample lots: A, B, and C) and cold-stored pasteurized milk (lot D). The three herbs have shown rich polyphenol contents (32.65−104.23 mgGAE/g), relevant antioxidant capacity, and high caffeic and rosmarinic acids and catechin contents. A significant reduction in psychrotrophic and total viable bacteria counts (PC and TVC) was attained following milk extract supplementation in both experiments. Monitoring PC and TVC counts in sample lots (A, B, and C) has revealed a significant improvement in extracts' effectiveness in reducing the TVC count with the increment of temperature. The highest reductions of PC and TVC counts were achieved, respectively, in samples treated with thyme and rosemary in (lots: A, B, and C) and in samples treated with ammoides and thyme in (lot D). Thyme extract showed the highest milk serum DPPH scavenging activity (74.84% at 0.1 mg/mL) and induced a significant Listeria monocytogenes growth inhibition (>1 Log cycle). The sensory evaluation of supplemented milk has shown good consumer acceptability of ammoides-supplemented milk, quite similar to the control sample.
Asunto(s)
Apiaceae , Rosmarinus , Thymus (Planta) , Animales , Antioxidantes/análisis , Antioxidantes/farmacología , Leche/química , Extractos Vegetales/análisis , Extractos Vegetales/farmacología , Rosmarinus/química , Thymus (Planta)/químicaRESUMEN
Clostridium difficile is responsible for a wide spectrum of infection from asymptomatic carriage to severe, relapsing colitis. Since 2003, C. difficile infections have increased with a higher morbidity and mortality due to the emergence of epidemic and hypervirulent C. difficile strains such as those of the epidemic lineage 027/BI/NAP1. To decipher the hypervirulence and epidemicity of 027 strains, we analyzed gene expression profiles of the R20291 027 strain using a monoxenic mouse model during the first 38h of infection. A total of 741 genes were differentially expressed during the course of infection. They are mainly distributed in functional categories involved in host adaptation. Several genes of PTS and ABC transporters were significantly regulated during the infection, underlying the ability of strain R20291 to adapt its metabolism according to nutrient availability in the digestive tract. In this animal model, despite the early sporulation process, sporulation efficiency seems to indicate that growth of R20291 vegetative cells versus spores were favored during infection. The bacterial mechanisms associated to adaptability and flexibility within the gut environment, in addition to the virulence factor expression and antibiotic resistance, should contribute to the epidemicity and hypervirulence of the C. difficile 027 strains.
Asunto(s)
Adaptación Fisiológica , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/microbiología , Transcriptoma , Animales , Clostridioides difficile/genética , Clostridioides difficile/aislamiento & purificación , Genes Bacterianos , Ratones , Virulencia/genéticaRESUMEN
Clostridium difficile is the main agent responsible for hospital acquired antibiotic associated diarrhoea. In recent years, epidemic strains have emerged causing more severe infections. Whilst C. difficile has two major virulence factors, toxins TcdA and TcdB, it is generally accepted that other virulence components of the bacterium contribute to disease. Previously, it has been suggested that flagella expression from pathogenic bacteria might be implicated in virulence. In a recent study, we observed an increased mortality in a gnotobiotic mouse model when animals were colonized with an isogenic fliC mutant constructed in the PCR-ribotype 027 (B1/NAP1) strain R20291, while animals survived when colonized by the parental strain or after colonization by other high-toxin-producing C. difficile strains. To understand the reasons for this increased virulence, we compared the global gene expression profiles between the fliC-R20291 mutant and its parental strain using an in vitro and in vivo transcriptomic approach. The latter made use of the gnotobiotic mouse model. Interestingly, in the fliC mutant, we observed considerable up-regulation of genes involved in mobility, membrane transport systems (PTS, ABC transporters), carbon metabolism, known virulence factors and sporulation. A smaller but significant up-regulation of genes involved in cell growth, fermentation, metabolism, stress and antibiotic resistance was also apparent. All of these genes may be associated with the increased virulence of the fliC-R20921 mutant. We confirmed that the fliC mutation is solely responsible for the observed changes in gene expression in the mutant strain since expression profiles were restored to that of the wild-type strain in the fliC-complemented strain. Thus, the absence of FliC is directly or indirectly involved in the high mortality observed in the fliC mutant infected animals. Therefore, we provide the first evidence that when the major structural component of the flagellum is neutralized, deregulation of gene expression can occur during infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidad , Flagelina/metabolismo , Animales , Proteínas Bacterianas/genética , Clostridioides difficile/genética , Enterocolitis Seudomembranosa/microbiología , Flagelina/genética , Regulación Bacteriana de la Expresión Génica , Pleiotropía Genética , Masculino , Ratones , Virulencia/genética , Virulencia/fisiología , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Clostridium difficile is a frequent cause of severe, recurrent post-antibiotic diarrhoea and pseudomembranous colitis. The surface layer (S-layer) is the predominant outer surface component of C. difficile which is involved in pathogen-host interactions critical to pathogenesis. In this study, we characterized the S-layer protein A (SlpA) of animal and human strains belonging to different PCR-ribotypes (PR) and compared the in vitro adherence and in vivo colonization properties of strains showing different SlpA variants. Since each SlpA variant has been recently associated with an S-layer cassette, we were able to deduce the cassette for each of our strains. In this study, an identity of 99-100 % was found among the SlpA of isolates belonging to PR 012, 014/020, 045 and 078. One exception was the SlpA of a poultry isolate, PR 014/020, which showed 99 % identity with that of strain 0160, another PR 014/020 which contains an S-layer cassette 6. Interestingly, this cassette has also been found in a PR 018 strain, an emerging virulent type currently predominant in Italy. Five other SlpA variants (v014/020a-e) were identified in strains PR 014/020. In vitro adherence assays and in vivo colonization experiments were performed on five PR 014/020 strains: human 1064 (v014/020e), human 4684/08 (v014/020b), human IT1106 (v078a), poultry P30 (v014/020d) and poultry PB90 (v014/020b) strains. Adhesion assays indicate that C. difficile strains vary in their capacity to adhere to cells in culture and that adhesion seems to be independent of the SlpA variant. Colonization properties were assessed in vivo using a dixenic mouse model of colonization. The kinetics of faecal shedding and caecal colonization were similar when human 4684/08 (v014/020b) strain was compared with human 1064 (v014/020e) and poultry PB90 (v014/020b) strain. In contrast, poultry P30 (v014/020d) strain outcompeted both human 4684/08 (v014/020b) and IT1106 (v078a) strains and its adherence to caeca at day 7 was significantly higher. The peculiar characteristics of C. difficile P30 seem to advantage it in colonizing the intestinal mice niche, increasing its ability to compete and adapt. The results obtained underline the need of an increased attention to the genetic evolution of C. difficile to prevent and limit the consequences of the emergence of increasingly virulent strains.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Clostridioides difficile/fisiología , Células Epiteliales/microbiología , Intestinos/microbiología , Adaptación Fisiológica , Secuencia de Aminoácidos , Animales , Clostridioides difficile/clasificación , Clostridioides difficile/genética , Enterocolitis Seudomembranosa/microbiología , Variación Genética , Humanos , Ratones , Ratones Endogámicos C3H , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Filogenia , Aves de Corral/microbiologíaRESUMEN
Clostridium difficile is a major cause of healthcare-associated infection and inflicts a considerable financial burden on healthcare systems worldwide. Disease symptoms range from self-limiting diarrhoea to fatal pseudomembranous colitis. Whilst C. difficile has two major virulence factors, toxin A and B, it is generally accepted that other virulence components of the bacterium contribute to disease. C. difficile colonises the gut of humans and animals and hence the processes of adherence and colonisation are essential for disease onset. Previously it has been suggested that flagella might be implicated in colonisation. Here we tested this hypothesis by comparing flagellated parental strains to strains in which flagella genes were inactivated using ClosTron technology. Our focus was on a UK-outbreak, PCR-ribotype 027 (B1/NAP1) strain, R20291. We compared the flagellated wild-type to a mutant with a paralyzed flagellum and also to mutants (fliC, fliD and flgE) that no longer produce flagella in vitro and in vivo. Our results with R20291 provide the first strong evidence that by disabling the motor of the flagellum, the structural components of the flagellum rather than active motility, is needed for adherence and colonisation of the intestinal epithelium during infection. Comparison to published data on 630Δerm and our own data on that strain revealed major differences between the strains: the R20291 flagellar mutants adhered less than the parental strain in vitro, whereas we saw the opposite in 630Δerm. We also showed that flagella and motility are not needed for successful colonisation in vivo using strain 630Δerm. Finally we demonstrated that in strain R20291, flagella do play a role in colonisation and adherence and that there are striking differences between C. difficile strains. The latter emphasises the overriding need to characterize more than just one strain before drawing general conclusions concerning specific mechanisms of pathogenesis.
Asunto(s)
Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/microbiología , Flagelos/fisiología , Toxinas Bacterianas/metabolismo , Clostridioides difficile/clasificación , Clostridioides difficile/metabolismo , Enterocolitis Seudomembranosa/epidemiología , Enterocolitis Seudomembranosa/fisiopatología , Humanos , Mucosa Intestinal/microbiología , Especificidad de la EspecieRESUMEN
Clostridium difficile is a frequent cause of severe, recurrent, post-antibiotic diarrhoea and pseudomembranous colitis. Its pathogenicity is mediated mainly by two toxins, TcdA and TcdB. However, different adhesins have also been described as important colonization factors which are implicated in the first step of the intestinal infection. In this study, we focused our interest on one of these adhesins, fibronectin-binding protein A (FbpA), and on its role in the intestinal colonization process. A mutant of FbpA (CDΔFbpA) was constructed in C. difficile strain 630Δerm by using ClosTron technology. This mutant was characterized in vitro and in vivo and compared to the isogenic wild-type strain. Adhesion of the CDΔFbpA mutant to the human colonic epithelial cell line Caco-2 and to mucus-secreting HT29-MTX cells was examined. Surprisingly, the CDΔFbpA mutant adhered more than the wild-type parental strain. The CDΔFbpA mutant was also analysed in three different mouse models by following the intestinal implantation kinetics (faecal shedding) and caecal colonization (7 days post-challenge). We showed that in monoxenic mice, CDΔFbpA shed C. difficile in faeces at the same rate as that of the isogenic wild-type strain but its colonization of the caecal wall was significantly reduced. In dixenic mice, the shedding rate was slower for the CDΔFbpA mutant than for the isogenic wild-type strain during the first days of infection, but no significant difference was observed in caecal colonization. Similar rates of intestinal implantation and caecal colonization were observed for both strains in assays performed in human microbiota-associated mice. Taken together, our data suggest that FbpA plays a role in intestinal colonization by C. difficile.