A new killing assay with the Caenorhabditis elegans PX627 mutant to assess the virulence of uropathogenic Escherichia coli strains.

The nematode Caenorhabditis elegans (C. elegans) is a prime invertebrate host model for studying uropathogenic Escherichia coli (UPEC) pathogenesis. The aim of this work was to develop a new C. elegans killing assay based on feeding bacteria by the nematode throughout its life from the egg. With this model, the lifespan of C. elegans rrf-3, temperature-sterile, mutant, and PX627, auxin-inducible infertile, mutant fed UPEC strains, was compared. The behavior of three clinical UPEC strains and the non-pathogenic Escherichia coli OP50 strain was analyzed. Survival curves were generated by the Kaplan-Meier method and compared by the log-rank test over 10 days of follow-up. There was no significant difference between the survival curves obtained with each of the two C. elegans mutants (PX627 and rrf-3) fed with each of the strains of E. coli (OP50, G1722, G1473 or ER41). The UPEC strains were classified according to their virulence in vivo in the C. elegans PX627 mutant. The most virulent strain was ER41 which harbored the virulence genes fimA, papC and hlyA, expressed hemolysis in vitro and showed no antibiotic resistance. The least virulent strain was G1722 which only harbored the two adhesion factor genes, was not hemolytic and was resistant to multiple antibiotics. The C. elegans PX627 mutant fed with UPEC bacteria from the egg stage is a simple and inexpensive invertebrate animal model for assessing the in vivo virulence of different strains. The early exposure of C. elegans to pathogenic bacteria at the egg stage, without the need to change the incubation temperature, is an advantage over previously described C. elegans killing assays.

Adherence of Uropathogenic Escherichia Coli in Dog Urine After Consumption of Food Supplemented with Cranberry (Vaccinium Macrocarpon).

Introduction: Escherichia coli is the most common pathogen isolated from the urine of dogs with urinary tract infections (UTIs). While there are many studies in humans investigating the potential for the prevention of UTIs by dietary consumption of cranberry, few analogous studies have been carried out in dogs. Material and Methods: Eight dogs, four male and four female, were successively fed two diets, first a control without cranberry, and then the second diet containing cranberry extracts. Naturally excreted urine was collected on the tenth day after the start of each diet for 24 h and used for bacterial growth. Madin-Darby canine kidney cell adherence by the uropathogenic E. coli G1473 strain expressing type 1 pili and positive for P pili and haemolysin gene markers was quantified after growth in urine samples. Results: Significant reductions in bacterial adherence to MDCK cells (from -16.5 to -73.4%, P < 0.05) were observed in the four females but not in the males after consumption of the cranberry extracts compared to the same animals consuming the control diet. Conclusion: Dietary supplementation with cranberry may provide some degree of protection to female dogs against adhesion of uropathogenic E. coli to urinary epithelial cells.

Presence of age-associated low-grade inflammation does not worsen the body response to bacterial infection in old male rats.

In the field of frailty, there is an underlying hypothesis that chronic low-grade inflammation contributes to bad outcomes in response to a stressor. The host response to an Escherichia coli infection was assessed in 24 month old male rats exhibiting a chronic low-grade inflammation and in non-inflamed control rats. Mortality, weight loss and sarcopenia were the main outcomes measured. The presence of chronic low-grade inflammation did not affect post-infection mortality, body weight loss and tissue mass decreases. Infection-induced modifications of plasma acute phase proteins concentrations were not higher in low-grade inflamed than non-inflamed rats. Absolute synthesis rates of tissue proteins were independent of the initial inflammatory status, except for liver 10 days after infection. Altogether, age-associated chronic low-grade inflammation in male rats did not worsen the body response to bacterial infection. These results suggest that chronic low-grade inflammation is not an aggravating factor of the spiraling process leading to frailty.

Cranberry (Vaccinium macrocarpon) dietary supplementation and fecal microbiota of Wistar rats.

Cranberry (Vaccinium macrocarpon) dietary supplementation can help prevention of urinary tract infections through the supply of proanthocyanidin-type polyphenols (PAC). The main uropathogenic bacteria are members of the intestinal microbiota. A randomized cross-over experiment was done to investigate whether cranberry dietary supplementation affects concentrations of thermotolerant coliforms, Enterococcus spp. and Lactobacillus spp. in rat faeces. Thirteen rats, housed in individual cages, received successively two diets as pellets during 7 days each: a standard diet without polyphenols and the standard diet supplemented with cranberry powder containing 10.9 mg/100 g of PAC. There was a 7 days wash-out period in between with standard diet without polyphenols. Body weight and feed intake were recorded. Faeces were collected on the last day of treatment, and crushed to count the different bacterial populations using the most probable number method. Thermotolerant coliforms were grown in BGBLB tubes and on MacConkey agar. Enterococcus spp. were grown in Rothe and Litsky broths and on KF Streptococcus agar. Lactobacillus spp. were grown in Man Rogosa Sharpe broth. Body mass gains were not affected by cranberry supplementation. This is consistent with equal food intake, cranberry powder not providing significant energy supplement. Cranberry dietary supplementation was associated with changes in fecal concentrations of thermotolerant coliforms, and Enterococcus spp. in some rats, but did not induce significant changes in bacterial fecal concentrations in a global population of 13 rats. In conclusion, we did not observe any significant effect of dietary cranberry supplementation on the fecal microbiota of Wistars rats for a 7-day diet.

Neutrophils are immune cells preferentially targeted by retinoic acid in elderly subjects.

BACKGROUND: The immune system gradually deteriorates with age and nutritional status is a major factor in immunosenescence. Of the many nutritional factors implicated in age-related immune dysfunction, vitamin A may be a good candidate, since vitamin A concentrations classically decrease during aging whereas it may possess important immunomodulatory properties via its active metabolites, the retinoic acids. This prompted us to investigate the immune response induced by retinoids in adults and elderly healthy subjects. Before and after oral supplementation with 13cis retinoic acid (0.5 mg/kg/day during 28 days), whole blood cells were phenotyped, and functions of peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) were investigated by flow cytometry and ELISA tests. RESULTS: In both young adults (n = 20, 25 ± 4 years) and older subjects (n = 20, 65 ± 4 years), retinoic acid supplementation had no effect on the distribution of leukocyte subpopulations or on the functions of PBMC (Il-2 and sIl-2R production, membrane expression of CD25). Concerning PMN, retinoic acid induced an increase in both spontaneous migration and cell surface expression of CD11b in the two different age populations, whereas bactericidal activity and phagocytosis remained unchanged. CONCLUSIONS: We demonstrated that retinoic acid induces the same intensity of immune response between adult and older subjects, and more specifically affects PMN functions, i.e. adhesion and migration, than PBMC functions.

Prognostic values of alpha2-macroglobulin, fibrinogen and albumin in regards to mortality and frailty in old rats.

The study aimed to determine if acute phase proteins (APP) are markers of frailty in old rats. We evaluated in male Wistar rats at 96 weeks of age (n=72) whether single measurements of alpha(2)-macroglobulin, fibrinogen and albumin are predictive of mortality, body weight loss and inflammatory status during a 10-week follow-up period. Rats were clustered depending on levels of these APP at baseline. Rats with extremely high levels of alpha(2)-macroglobulin or fibrinogen (upper quartiles), or extremely low level of albumin (lower quartile), had an 11.6, 8.1 and 5.3-fold higher risk of mortality, respectively, than other rats. Body weight loss was negatively correlated with alpha(2)-macroglobulin, a trend was observed with fibrinogen (P=0.08) but not with albumin. Rats with fibrinogen levels >4.0 g/L or alpha(2)-macroglobulin levels >91 mg/L (respective top halves) at 96 weeks of age had higher levels of alpha(2)-macroglobulin and fibrinogen and lower levels of albumin throughout the follow-up period and higher levels of sTNFR-1 and lipopolysaccharide-binding protein at 106 weeks of age. Highest levels of alpha(2)-macroglobulin, fibrinogen and lowest albumin were predictive of mortality, whereas moderate levels of alpha(2)-macroglobulin and fibrinogen were, according to body weight loss and inflammatory status, markers of frailty in old rats.

Presence of low-grade inflammation in old rats does not worsen skeletal muscle loss under an endotoxemic and dietary stress.

The study aimed to determine if age-associated low-grade inflammation aggravates the response to a stress, especially regarding to sarcopenia. Initial inflammatory status in 22-month-old rats was based on plasma alpha(2)-macroglobulin and fibrinogen concentrations. The stress applied was a single intra-peritoneal injection of lipopolysaccharide followed by a 23-day period of malnutrition, i.e. a 4% casein diet distributed in quantity limited to 50% of spontaneous food intake. The response to the stress was analyzed in non-inflamed and low-grade inflamed rats and compared to non-inflamed and low-grade inflamed rats, which received the control treatment (i.e. no lipopolysaccharide injection and an 18% casein diet). The stress-induced body weight loss was higher in inflamed than non-inflamed rats, but the decrease in muscle weight was not worsened. Muscle protein turnover was not affected by the stress. Plasma alpha(2)-macroglobulin levels increased after the stress, whatever the initial inflammatory status. However, fibrinogen levels decreased more in inflamed than non-inflamed rats and albumin levels were not affected by the stress. Independently of the initial inflammatory status, the liver glutathione content was strongly depleted by the stress. These results extend and support our previous findings by demonstrating that age-associated low-grade inflammation does not aggravate sarcopenia in old rats.