Pilot-scale production and viability analysis of freeze-dried probiotic bacteria using different protective agents

The functional food industry requires an improvement of probiotic strain stability during storage, especially when they are stored at room temperature. In this study, the viability of freeze-dried Lactobacillus rhamnosus IMC 501^® and Lactobacillus paracasei IMC 502^® using different protective agents (i.e., glycerine, mannitol, sorbitol, inulin, dextrin, Crystalean^®) was determined and compared with semi skimmed milk (SSM) control. No significant differences were observed between the tested protectants and the control (SSM) during storage at refrigerated conditions. During storage at room temperature, only glycerine was found to stabilize viability better than other tested substances.     

Effect of micronutrient and probiotic fortified yogurt on immune-function of anti-retroviral therapy naive HIV patients

Background: Micronutrient supplementation has been shown to reduce the progression of HIV but does not have an effect on the intestinal barrier or the intestinal microbiota of HIV patients. Studies have suggested that probiotics could potentially complement micronutrients in preserving the immune-function of HIV patients. Objective: Assess the impact of micronutrient supplemented probiotic yogurt on the immune function of HIV patients. Design:We performed a randomized, double blind, controlled trial with CD4 count as primary outcome among HIV patients naïve to anti-retroviral treatment. Secondary outcomes included hematological parameters, incidence of diarrhea and clinical symptoms. A total of 112 HIV patients were randomized to receive a micronutrient fortified yogurt with (n = 55) or without additional probiotic Lactobacillus rhamnosus GR-1 (n = 57) for four weeks. Results:An average decline in CD4 count of −70 cells/μL (95% CI: −154 to −15) was observed in the micronutrient, probiotic group versus a decrease of −63 cells/μL (95% CI: −157 to −30) in the micronutrient control group (p = 0.9). Additional probiotic supplementation was well tolerated and not associated with adverse events. No difference between groups was detected in incidence of diarrhea or clinical symptoms. An improvement of hemoglobin levels was observed for all subjects, based upon a mean difference from baseline of 1.4 g/L (SD = 6) (p = 0.02). Conclusion:The addition of probiotics to a micronutrient fortified yogurt was well tolerated by HIV patients but was not associated with a further increase in CD4 count after one month.     

Beneficial effects of probiotic and food borne yeasts on human health

Besides being important in the fermentation of foods and beverages, yeasts have shown numerous beneficial effects on human health. Among these, probiotic effects are the most well known health effects including prevention and treatment of intestinal diseases and immunomodulatory effects. Other beneficial functions of yeasts are improvement of bioavailability of minerals through the hydrolysis of phytate, folate biofortification and detoxification of mycotoxins due to surface binding to the yeast cell wall.     

Probiotic food supplement reduces stress-induced gastrointestinal symptoms in volunteers: a double-blind,placebo-controlled,randomized trial

Stress plays an important role in the development of symptoms contributing to disease. Stress induces various disorders with gastrointestinal, physical, and psychological symptoms. Probiotics can help regulate or modulate gastrointestinal functions. The aim of the present study was to investigate the effects of a probiotic preparation (Probio-Stick) on stress-induced symptoms in volunteers. A double-blind, placebo-controlled, randomized study was conducted on volunteers with symptoms of stress. Subjects received a probiotic (Probio-Stick; Lallemand SAS, Saint-Simon, France) containing Lactobacillus acidophilus Rosell-52 and Bifidobacterium longum Rosell-175 (3 × 10⁹ colony-forming units per sachet stick) or a sensorially identical placebo without probiotics during a 3-week period. The consumption of probiotics significantly reduced 2 stress-induced gastrointestinal symptoms (abdominal pain and nausea/vomiting) for intention-to-treat or per-protocol populations. In contrast, the probiotics did not significantly modify the other physical and psychological symptoms and sleep problems induced by stressful life events for intention-to-treat or per-protocol populations. The results indicate that Probio-Stick can provide a beneficial effect on the gastrointestinal symptoms experienced by individuals affected by chronic stress.     

Biological Variability and Impact of Oral Contraceptives on Vitamins B6, B12 and Folate Status in Women of Reproductive Age

Vitamins B₆, B₁₂ and folate play crucial metabolic roles especially during the reproductive years for women. There is limited reporting of within-subject variability of these vitamins. This study aimed to determine the within and between subject variability in serum vitamins B₆, B₁₂, folate and erythrocyte folate concentrations in young women; identify factors that contribute to variability; and determine dietary intakes and sources of these vitamins. Data were obtained from the control group of a trial aimed at investigating the effect of iron on the nutritional status of young women (age 25.2 ± 4.2 year; BMI 21.9 ± 2.2 kg/m²). The coefficients of variability within-subject (CV_I) and between-subject (CV_G) for serum vitamins B₆, B₁₂ and folate, and erythrocyte folate were calculated. Food frequency questionnaires provided dietary data. CV_I and CV_G were in the range 16.1%–25.7% and 31.7%–62.2%, respectively. Oral contraceptive pill (OCP) use was associated (P = 0.042) with lower serum vitamin B₁₂ concentrations. Initial values were 172 ± 16 pmol/L and 318 ± 51 pmol/L for OCP and non-OCP users, respectively; with differences maintained at four time points over 12 weeks. BMI, age, physical activity, alcohol intake and haematological variables did not affect serum or erythrocyte vitamin concentrations. Vitamin B₁₂ intakes were derived from traditional and unexpected sources including commercial energy drinks. Young women using OCP had significantly lower serum vitamin B₁₂ concentrations. This should be considered in clinical decision making and requires further investigation.     

Diet,Microbiota and Immune System in Type 1 Diabetes Development and Evolution

Type 1 diabetes (T1D) is the second most frequent autoimmune disease in childhood. The long-term micro- and macro-vascular complications of diabetes are associated with the leading causes of disability and even mortality in young adults. Understanding the T1D etiology will allow the design of preventive strategies to avoid or delay the T1D onset and to help to maintain control after developing. T1D development involves genetic and environmental factors, such as birth delivery mode, use of antibiotics, and diet. Gut microbiota could be the link between environmental factors, the development of autoimmunity, and T1D. In this review, we will focus on the dietary factor and its relationship with the gut microbiota in the complex process involved in autoimmunity and T1D. The molecular mechanisms involved will also be addressed, and finally, evidence-based strategies for potential primary and secondary prevention of T1D will be discussed.     

Homocysteine Metabolism Gene Polymorphisms (MTHFR C677T,MTHFR A1298C,MTR A2756G and MTRR A66G) Jointly Elevate the Risk of Folate Deficiency

Folate deficiency is strongly associated with cardiovascular disease. We aimed to explore the joint effect of the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MTR) A2756G, and methionine synthase reductase (MTRR) A66G polymorphisms on folate deficiency in a Chinese hypertensive population. A total of 480 subjects aged 28–75 were enrolled in this study from September 2005–December 2005 from six hospitals in different Chinese regions. Known genotypes were detected by PCR-RFLP methods and serum folate was measured by chemiluminescence immunoassay. Our results showed that MTHFR 677TT and MTR 2756AG + GG were independently associated with a higher risk of folate deficiency (TT vs. CC + CT, p < 0.001 and AG + GG vs. AA p = 0.030, respectively). However, the MTHFR A1298C mutation may confer protection by elevating the serum folate level (p = 0.025). Furthermore, patients carrying two or more risk genotypes showed higher odds of folate deficiency than null risk genotype carriers, especially those carrying four risk genotypes. These findings were verified by generalized multifactor dimensionality reduction (p = 0.0107) and a cumulative effects model (p = 0.001). The results of this study have shown that interactions among homocysteine metabolism gene polymorphisms lead to dramatic elevations in the folate deficiency risk.     

The inhibitory effect of natural bioactives on the growth of pathogenic bacteria

The objective of this study was to evaluate the inhibitory activity of natural products, against growth of Escherichia coli (ATCC 25922) and Salmonella typhimurium (KCCM 11862). Chitosan, epigallocatechin gallate (EGCG), and garlic were used as natural bioactives for antibacterial activity. The testing method was carried out according to the disk diffusion method. All of chitosan, EGCG, and garlic showed inhibitory effect against the growth of E. coli and Salmonella typhi. To evaluate the antibacterial activity of natural products during storage, chicken skins were inoculated with 10⁶ of E. coli or Salmonella typhi. The inoculated chicken skins, treated with 0.5, 1, or 2% natural bioactives, were stored during 8 day at 4℃. The numbers of microorganisms were measured at 8 day. Both chitosan and EGCG showed significant decrease in the number of E. coli and Salmonella typhi in dose dependent manner (P < 0.05). These results suggest that natural bioactives such as chitosan, EGCG may be possible to be used as antimicrobial agents for the improvement of food safety.     

Dietary supplementation of milk fermented with probiotic Lactobacillus fermentum enhances systemic immune response and antioxidant capacity in aging mice

Although probiotics are known to enhance the host immune response, their roles in modulating immunosenescence, resisting infection, and improving redox homeostasis during aging remain unclear. Therefore, the present study was devised in aging mice to assess the antiimmunosenescence potential from the consumption of milk that is fermented with probiotic Lactobacillus fermentum MTCC 5898 (LF). We hypothesized that probiotic supplementation would boost immunity, improve antioxidant capacity, and resist severity of pathogenic infection in aging mice. To test this hypothesis, during a trial period of 2 months, 16-month-old male Swiss mice were kept on 3 experimental diets: basal diet (BD), BD supplemented with skim milk, and BD supplemented with probiotic LF-fermented milk. A concurrent analysis of several immunosenescence markers that include neutrophil functions, interleukins profile, inflammation and antibody responses in the intestine as well as analysis of antioxidant enzymes in the liver and red blood cells was performed. Neutrophil respiratory burst enzymes and phagocytosis increased significantly in probiotic LF-fed groups, whereas no exacerbation in plasma levels of monocyte chemotactic protein 1 and tumor necrosis factor α was observed. Splenocytes registered increased interferon-γ but decreased interleukin 4 and interleukin 10 production, whereas humoral antibodies registered decreases in immunoglobulin G1 (IgG1)/IgG2a ratio and IgE levels in the probiotic-fed groups. Antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in LF-fed groups showed increased activities, which were more pronounced in the liver than in red blood cell. An Escherichia coli–based infection model in aging mice was also designed to validate the protective attributes of LF. Administration of probiotic LF significantly reduced E coli population in organs (intestine, liver, spleen, and peritoneal fluid), as compared with control groups, by enhancing E coli–specific antibodies and inflammatory proteins. Based on these results, it appears that LF supplementation alleviated immunosenescence, enhanced antioxidant enzyme activities, and resisted E coli infection in aging mice; thereby, signifying its potential in augmenting healthy aging.     

Risk factors for colonization with extended-spectrum beta-lactamase-producing bacteria and intensive care unit admission

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are emerging pathogens. To analyze risk factors for colonization with ESBL-producing bacteria at intensive care unit (ICU) admission, we conducted a prospective study of a 3.5-year cohort of patients admitted to medical and surgical ICUs at the University of Maryland Medical Center. Over the study period, admission cultures were obtained from 5,209 patients. Of these, 117 were colonized with ESBL-producing Escherichia coli and Klebsiella spp., and 29 (25%) had a subsequent ESBL-positive clinical culture. Multivariable analysis showed the following to be statistically associated with ESBL colonization at admission: piperacillin-tazobactam (odds ratio [OR] 2.05, 95% confidence interval [CI] 1.36-3.10), vancomycin (OR 2.11, 95% CI 1.34-3.31), age > 60 years (OR 1.79, 95% CI 1.24-2.60), and chronic disease score (OR 1.15; 95% CI 1.04-1.27). Coexisting conditions and previous antimicrobial drug exposure are thus predictive of colonization, and a large percentage of these patients have subsequent positive clinical cultures for ESBL-producing bacteria.     

Understanding How Commensal Obligate Anaerobic Bacteria Regulate Immune Functions in the Large Intestine

The human gastrointestinal tract is colonised by trillions of commensal bacteria, most of which are obligate anaerobes residing in the large intestine. Appropriate bacterial colonisation is generally known to be critical for human health. In particular, the development and function of the immune system depends on microbial colonisation, and a regulated cross-talk between commensal bacteria, intestinal epithelial cells and immune cells is required to maintain mucosal immune homeostasis. This homeostasis is disturbed in various inflammatory disorders, such as inflammatory bowel diseases. Several in vitro and in vivo studies indicate a role for Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, Bacteroides fragilis, Akkermansia muciniphila and segmented filamentous bacteria in maintaining intestinal immune homeostasis. These obligate anaerobes are abundant in the healthy intestine but reduced in several inflammatory diseases, suggesting an association with protective effects on human health. However, knowledge of the mechanisms underlying the effects of obligate anaerobic intestinal bacteria remains limited, in part due to the difficulty of co-culturing obligate anaerobes together with oxygen-requiring human epithelial cells. By using novel dual-environment co-culture models, it will be possible to investigate the effects of the unstudied majority of intestinal microorganisms on the human epithelia. This knowledge will provide opportunities for improving human health and reducing the risk of inflammatory diseases.     

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples

Background

Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples.

Methods

A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar''s χ² test, with consideration of a P-value of <0.05 as indication of significant difference.

Results

In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture.

Conclusion

Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.     

Infection by Ralstonia species in cystic fibrosis patients: identification of R. pickettii and R. mannitolilytica by polymerase chain reaction

The frequency of respiratory tract infections caused by Ralstonia species in persons with cystic fibrosis (CF) and the role of these species in CF pulmonary disease are not well documented. In part, this lack of documentation may be attributed to the difficulty in accurately identifying Ralstonia species; R. mannitolilytica and R. pickettii in particular may be misidentified as other closely related species, particularly those of the Burkholderia cepacia complex. We used polyphasic analyses to identify 42 Ralstonia isolates from sputum cultures from 38 CF patients. Several isolates that could not be identified to the species level may belong to novel Ralstonia species. We demonstrated chronic colonization by using genotyping of serial isolates recovered from the same patient. To facilitate identification of R. mannitolilytica and R. pickettii, we developed 16S ribosomal DNA-based polymerase chain reaction assays that allow sensitive and specific identification of these species.     

Disseminated Leishmaniasis Caused by Leishmania tropica in a Puppy from Karaj,Central Iran

A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was examined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large number of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT). DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran.     

Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

Background:

Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.

Methods:

A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.

Results:

Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.

Conclusion:

The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.     

Transovarial Transmission of Babesia ovis by Rhipicephalus sanguineus and Hyalomma marginatum

Background

Rhipicephalus sanguineus and Hyalomma marginatum are the most common species in sheep herds in Northeast of Iran. There is preliminary evidence that these species may be the vectors of Babesia ovis in Iran. We carried out two experiments in Mashhad area, Khorasan Razavi Province to determine whether B. ovis could be transovarially transmitted by R. sanguineus and H. marginatum.

Methods

In experiment 1, adults of laboratory reared H. marginatum and R.sanguineus were infected with B. ovis isolated from naturally infected sheep in Mashhad area by feeding the ticks on the sheep inoculated intravenously by infected blood samples. The inoculated sheep showed clinical signs with parasitaemia while the adult ticks were engorging on them. The engorged females were collected and kept at 28°C and 85% relative humidity in incubator. Then, larval, nymphal and adult stages derived from engorged females were used to infest the clean sheep. In experiment 2, two splenectomized sheep were infested only with the same adult ticks of two species.

Results

Examination of smears and PCR of blood samples to detect of B. ovis in infested sheep in two experiments were negative.

Conclusion

It seems that R. sanguineus and H. marginatum can not transovarially transmit B. ovis in sheep.     

Low-level environmental cadmium exposure is associated with DNA hypomethylation in Argentinean women

Background: Cadmium, a common food pollutant, alters DNA methylation in vitro. Epigenetic effects might therefore partly explain cadmium’s toxicity, including its carcinogenicity; however, human data on epigenetic effects are lacking.Objective: We evaluated the effects of dietary cadmium exposure on DNA methylation, considering other environmental exposures, genetic predisposition, and gene expression.Methods: Concentrations of cadmium, arsenic, selenium, and zinc in blood and urine of nonsmoking women (n = 202) from the northern Argentinean Andes were measured by inductively coupled mass spectrometry. Methylation in CpG islands of LINE-1 (long interspersed nuclear element-1; a proxy for global DNA methylation) and promoter regions of p16 [cyclin-dependent kinase inhibitor 2A (CDKN2A)] and MLH1 (mutL homolog 1) in peripheral blood were measured by bisulfite polymerase chain reaction pyrosequencing. Genotyping (n = 172) for the DNA (cytosine-5-)-methyltransferase 1 gene (DNMT1 rs10854076 and rs2228611) and DNA (cytosine-5-)-methyltransferase 3 beta gene (DNMT3B rs2424913 and rs2424932) was performed with Sequenom iPLEX GOLD SNP genotyping; and gene expression (n = 90), with DirectHyb HumanHT-12 (version 3.0).Results: Cadmium exposure was low: median concentrations in blood and urine were 0.36 and 0.23 µg/L, respectively. Urinary cadmium (natural log transformed) was inversely associated with LINE-1 methylation (β = –0.50, p = 0.0070; β = –0.44, p = 0.026, adjusted for age and coca chewing) but not with p16 or MLH1 methylation. Both DNMT1 rs10854076 and DNMT1 rs2228611 polymorphisms modified associations between urinary cadmium and LINE-1 (p-values for interaction in adjusted models were 0.045 and 0.064, respectively). The rare genotypes demonstrated stronger hypomethylation with increasing urinary cadmium concentrations. Cadmium was inversely associated with DNMT3B (r_S = –0.28, p = 0.0086) but not with DNMT1 expression (r_S = –0.075, p = 0.48).Conclusion: Environmental cadmium exposure was associated with DNA hypomethylation in peripheral blood, and DNMT1 genotypes modified this association. The role of epigenetic modifications in cadmium-associated diseases needs clarification.     

Resistance to Fluoroquinolone by a Combination of Efflux and Target Site Mutations in Enteroaggregative Escherichia coli Isolated in Korea

ObjectivesEnteroaggregative Escherichia coli (EAEC) was recently reported as a major diarrheagenic pathogen in infant and adult travelers, both in developing and developed countries. EAEC strains are known to be highly resistant to antibiotics including quinolones. Therefore in this study we have determined the various mechanisms of quinolone resistance in EAEC strains isolated in Korea.MethodsFor 26 EAEC strains highly resistant to fluoroquinolone, minimal inhibitory concentrations for fluoroquinolones were determined, mutations in the quinolone target genes were identified by PCR and sequencing, the presence of transferable quinolone resistance mechanism were identified by PCR, and the contribution of the efflux pump was determined by synergy tests using a proton pump inhibitor. The expression levels of efflux pump-related genes were identified by relative quantification using real-time PCR.ResultsApart from two, all tested isolates had common mutations on GyrA (Ser83Leu and Ser87Gly) and ParC (Ser80Gln). Isolates EACR24 and EACR39 had mutations that have not been reported previously: Ala81Pro in ParC and Arg157Gly in GyrA, respectively. Increased susceptibility of all the tested isolates to ciprofloxacin and norfloxacin in the presence of the pump inhibitor implies that efflux pumps contributed to the resistance against fluoroquinolones. Expression of the efflux pump-related genes, tolC, mdfA, and ydhE, were induced in isolates EACR 07, EACR 29, and EACR 33 in the presence of ciprofloxacin.ConclusionThese results indicate that quinolone resistance of EAEC strains mainly results from the combination of mutations in the target enzyme and an increased expression of efflux pump-related genes. The mutations Ala81Pro in ParC and Arg157Gly in GyrA have not been reported previously; the exact influence of these mutations should be investigated further.