Invitro and Invivo Analysis of Human Milk Lactic Acid Bacteria Isolates for Their Anti-hypercholesterolemia Actions.

The aim of this study was to evaluate the cholesterol lowering ability of Lactic Acid Bacteria (LAB) isolated from human breast milk under in vitro and in vivo conditions. Six LAB isolates namely Lacticaseibacillus casei 1A, Lactobacillus gasseri 5A, Enterococcus faecium 2C, Limosilactobacillus fermentum 3D, Pediococcus acidilactici 1C, and Lactiplantibacillus plantarum 7A, were examined for their bile resistance, bile salt hydrolase activity, cholesterol assimilation and viability in cholesterol rich; DeMan Rogosa and Sharpe broth, simulated gastric, small and upper intestinal conditions. During in vivo experiments, two putative LAB isolates were orally gavage to BALB/c mice, fed with normal basal and cholesterol rich (HCD) diets, daily for a period of 4 weeks. Blood serum analysis including total serum cholesterol, triglycerides, high-density and low-density lipoprotein (LDL) cholesterol levels and total fecal LAB counts of the animals were determined. The isolates in study showed bile resistance and bile salt hydrolysis activity, while significant differences (P < 0.05) were seen in their cholesterol assimilation ability. L. gasseri 5A (195.67%) and L. plantarum 7A (193.78%) displayed highest cholesterol removal percentages, respectively. Animals in HCD, fed with L. gasseri 5A and L. plantarum 7A showed decreased levels of total cholesterol and LDL, compared to the control groups. In HCD group liver weight was increased, while fecal LAB counts were decreased. No changes were observed in behavior or body weight in all experimental groups. In conclusion, L. gasseri 5A and L. plantarum 7A isolated from human breast milk demonstrates significant hypocholesterolaemic actions in vitro and in vivo and might be considered a promising candidates for preventing hypercholesterolemia in man and animals.

Propolis: An update on its chemistry and pharmacological applications.

Propolis, a resinous substance produced by honeybees from various plant sources, has been used for thousands of years in traditional medicine for several purposes all over the world. The precise composition of propolis varies according to plant source, seasons harvesting, geography, type of bee flora, climate changes, and honeybee species at the site of collection. This apiary product has broad clinical applications such as antioxidant, anti-inflammatory, antimicrobial, anticancer, analgesic, antidepressant, and anxiolytic as well asimmunomodulatory effects. It is also well known from traditional uses in treating purulent disorders, improving the wound healing, and alleviating many of the related discomforts. Even if its use was already widespread since ancient times, after the First and Second World War, it has grown even more as well as the studies to identify its chemical and pharmacological features, allowing to discriminate the qualities of propolis in terms of the chemical profile and relative biological activity based on the geographic place of origin. Recently, several in vitro and in vivo studies have been carried out and new insights into the pharmaceutical prospects of this bee product in the management of different disorders, have been highlighted. Specifically, the available literature confirms the efficacy of propolis and its bioactive compounds in the reduction of cancer progression, inhibition of bacterial and viral infections as well as mitigation of parasitic-related symptoms, paving the way to the use of propolis as an alternative approach to improve the human health. However, a more conscious use of propolis in terms of standardized extracts as well as new clinical studies are needed to substantiate these health claims.

Designing of Western Blot Technique for Glanders Diagnosing in Iran.

Burkholderia mallei is the etiologic agent of glanders. It is difficult to diagnose this zoonotic disease in its early stages. Some methods such as the complement fixation test (CFT) cause some problems for veterinary authorities and financial losses to animal owners due to false-positive results. The mallein test requires appropriate laboratory equipment and skilled personnel. To quickly and accurately diagnose the disease, especially in areas where animals cannot be kept, new methods (such as the Western blot test [WBT]) should be used to identify the disease. This study designed and optimized the Western blot (immunoblot) test using sera from 84 glanderous equids, and the sensitivity and specificity of ELISA and CFT were compared with the WBT. ELISA tests are based on B. mallei antigens whereas a purified lipopolysaccharide-containing B. mallei antigen is used in the WBT. The sensitivity and specificity of the tests were estimated using the cut-off values recommended by the test developers. The WBT and ELISA were significantly more specific than the CFT. The ELISA based on B. mallei antigens was significantly less sensitive than the CFT. Given their comparable sensitivities and specificities, the CFT (95.7%, 98.5%), the WBT (95%, 100%) and the ELISA (85%, 100%) should be further developed. The CFT is still the prescribed technique for serological investigation of equids for trade purposes to certify individual animals without glanders. Therefore, more efforts should be made to further improve and optimize the WBT and ELISA tests.

Anti-mycotoxigenic properties of probiotic Bacillus spp. in Japanese quails.

The current study was conducted to evaluate the anti-mycotoxigenic effects of previously isolated Bacillus spp. in Japanese quails. A total of 240-day-old Japanese quails were assigned in to six treatments and four replicates. Dietary treatments included the following: negative control (basal diet), positive control (basal diet + 2.5 ppm afltatoxin B1), probiotic treatments (basal diet + 2.5 ppm afltatoxin B1), and 108 cfu/ml of different Bacillus spp. (B. megaterium, B. subtilis, or B. laterosporus) in drinking water and treatment P (basal diet + 2.5 ppm afltatoxin B1 and 2.5 ppm Polysorb®). Body weight gain, feed intake, and feed conversion ratio were not affected by dietary treatments (P > 0.05). Carcass yield significantly increased in B. megaterium and B. subtilis treatments compared with positive control. Supplementation of B. megaterium significantly increased testes, uterus and oviduct weights, skin response to 2,4-dinitro 1-chlorobenzene and phytohemagglutinin, and antibody production against sheep red blood cells (P < 0.05). B. megaterium could significantly increase bursa weight and decrease liver weight compared with positive control (P < 0.05). B. megaterium, B. laterosporus, and Polysorb treatments significantly decreased H:L and aspartate aminotransferase activity in aflatoxin B1 fed control (P < 0.05). B. megaterium and B. laterosporus significantly increased tibia weight, length, radius, index, and ash content compared with positive control (P < 0.05). All dietary additives significantly reduced meat oxidation, total aerobic bacteria, and spore forming bacteria of ileal content compared with positive control (P < 0.05). Ileal lactic acid bacteria significantly increased in B. megaterium treatment (P < 0.05). Totally, B. megaterium might be a promising probiotic with a comparable afltatoxin B1 removal potential to commercial toxin binder (Polysorb).

Detection and characterization of purified antigenic proteins from culture filtrate of Mycobacterium bovis strain AN5.

BACKGROUND AND OBJECTIVES: Bovine tuberculosis diagnosis is usually performed by various tests with specific limitations. Mycobacterium bovis culture filtrate contains antigenic proteins that could be used to improve the sensitivity of bovine tuberculosis diagnosis. The objective of this study was to identify and purify antigenic proteins from culture filtrates of M. bovis strain AN5 for use in immunological assays. MATERIALS AND METHODS: Secreted proteins were purified from the heat-treated culture filtrate of M. bovis strain AN5. Proteins were precipitated with ammonium sulfate, fractionated by Sephadex G50 chromatography. The protein concentrations and the approximate molecular weight were determined by lowry method and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Immunological methods, including dot-blotting and western blotting, assessed the quality of the isolated proteins. RESULTS: The quantity of antigenic proteins in the culture medium was measured at far more than 15% of the amount of proteins secreted into medium. Three main chromatographic fractions obtained and showed concentrations of proteins ranging from 14 to 60 µg/ µl with molecular weights in the 10 to 180 kDa range. The purified antigens showed positive reactions to the infected cattle serum throughout dot-blotting. Western blotting revealed a total of 15 to 70 kDa molecular weight proteins. CONCLUSION: Immunoblotting analysis made it possible to detect and recognize novel antigens that are useful for bovine tuberculosis diagnosis improvement. This is significant since non-specific reactions were not observed when we utilized serum of cattle experimentally infected with M. bovis as a polyclonal antibody.

Persistence and safety assessment of novel probiotic strain Lactobacillus plantarum 1 strain Lp86 and Lp36 in Salmonella typhi infected mice.

The species of Lactic acid bacteria are known to confer beneficial effects on the host by inhabiting in their gastrointestinal tract (GIT). They succeed in surviving the harsh conditions of the GIT by exhibiting strong tolerance against gastric acids, digestive enzymes and bile simultaneously antagonizing the pathogens by production of antimicrobials. This study has been conducted to elaborate these probiotic characteristics in vivo for which mice were intragastrically given a probiotic approved dose of 1011cfu/ml for 4 days to assess the persistence of two probiotic candidates Lactobacillus plantarum Lp36 and Lactobacillus plantarum Lp86. The fecal count of the test probiotic candidates were seen to persist well in the GIT for 15 days with a count ranging between 104-108cfu/ mg of feces (p>0.01). The safety assessment of L. plantarum Lp36 in healthy and S. typhi in infected mice showed an increase in cell count from (day zero of inoculation) 106cfu/100mg of feces to108cfu/100mg (p>0.01) which was maintained till day six, suggesting the persistence in the GIT. The sections of the mice intestinal lining under scanning electron microscope revealed the adherence of Lp36 and Lp86 to the intestinal epithelia. The mice did not show any adverse effect on its health. These findings make our strains promising probiotic candidates to be used to promote health benefits after further assessments.

Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021.

BACKGROUND AND OBJECTIVES: Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by autochthonous Lactobacillus casei TA0021 strain. MATERIALS AND METHODS: The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed. RESULTS: The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of >9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold. CONCLUSION: The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.

In vitro and in vivo safety analysis of Enterococcus faecium 2C isolated from human breast milk.

INTRODUCTION: Safety analysis of probiotic bacteria is an obligatory characteristic to be evaluated prior to application in food or pharmacological products. This study was designed to evaluate in vitro and in vivo safety parameters of Enterococcus faecium 2C strain, a probiotic candidate isolated from human breast milk. MATERIAL AND METHODS: E.faecium 2C was studied for its hemolytic activity and phenotypic antibiotics resistance profile. In vivo safety of the mentioned Enterococcus strain was studied by determining acute oral toxicity in Wistar Male rats. The animals were randomly divided into two groups of 3 animals each. The test group animals were gavaged daily with bacterial dose of 1 × 1011 CFU/kg of animal body weight for 21 consecutive days. The animals in control group received normal basal diet without any supplementations. Hematological and biochemical parameters, organ weight, body weight and common health features of the animals were recorded. RESULTS: E.faecium 2C appeared non-hemolytic and sensitive to the majority of the tested antibiotics. The Wistar male rats fed orally with the mentioned bacterial suspensions survived the test period, and showed normal growth and development. No adverse effects on the general health condition, behavior, and growth were seen in the treated animals compared to control group. Additionally, no significant changes in the hematological results, blood biochemistry, organ weights and histopathology of the rats in treatment groups were observed. None of the vital organs of the treated animals showed signs of bacteremia or infectivity. CONCLUSION: E.faecium 2C strain isolated from human breast milk might be considered safe for use in probiotic formulations intended for man and animals.

Molecular prevalence of bovine noroviruses and neboviruses in newborn calves in Iran.

In this study, bovine enteric caliciviruses (BECs) were detected in 49.4% of a total of 253 stool specimens for diarrheic calves collected from 42 industrial dairy farms from March 2010 to February 2012. Genogroup III norovirus (NoVsGIII) were more prevalent (39.5%) than neboviruses (NBs) (15%), and coinfections were observed in 5.1% of the samples tested. Sequence analysis of the partial polymerase gene from 13 NoVsGIII samples indicated the circulation of both genotype 1 and genotype 2 strains. Among the six NB strains sequenced, five were related to the Bo/Nebraska/80/US strain, while one was related to the Bo/Newbury1/76/UK strain.

Bacteriocinogenic potential and virulence traits of Enterococcus faecium and E. faecalis isolated from human milk.

BACKGROUND AND OBJECTIVES: Human milk is a continuous supply of Lactic Acid bacteria (LAB), including enterococci with probiotic potentials. The aim of this study was to analyze two Enterococcus species, isolated from human milk for their probiotic potential, bacteriocin producing ability and virulence traits. MATERIALS AND METHODS: Enterococcus faecium TA0033 and E. faecalis TA102 were tested for acid and bile tolerance, survival in simulated gastric and intestinal conditions. The antibacterial spectrum of the isolates was tested by agar well diffusion assay. The antagonistic agent was characterized by physico-chemical methods. The enterocin structural genes, virulence determinants, vancomycin resistance and biogenic amine genes, such as hdc1, hdc2, tdc, ldc and odc were also determined. RESULTS: The tested isolates survived acidic conditions, high bile salt (1%), simulated gastric and intestinal conditions. The culture supernatant fluids of the two isolates inhibited the growth of Escherichia coli, Listeria monocytogenes, Salmonella typhi, Staphylococcus aureus, Shigella dysenteriae and Streptococcus agalactiae. The antagonistic activity was lost in the presence of proteolytic enzymes but tolerated the action of catalase, lysozyme and lipase. In contrast to enterocin TA102, enterocin TA0033 possessed bactericidal mode of action. Bacteriocin structural genes, entA and entB were present in the genome of the two isolates, while E. faecalis TA102 additionally harboured entP and bac31 genes. The phenotypic and genotypic virulence assessment studies indicated hyaluronidase (hyl) production and vancomycin resistance in E. faecalis TA102 while, none of the isolates harboured the biogenic amine genes. CONCLUSION: The presence of virulence genes in E. faecalis TA102 calls for careful monitoring of Enterococcus isolates for their safety parameters.

Strain differentiation of 13 indigenous Mycobacterium bovis isolates from infected cattle by restriction fragment length polymorphism analysis.

Mycobacterium bovis is mainly detected in cattle throughout the world. This bacterium is considered the main causative agent of tuberculosis in man and animals. M. bovis is also reported to be endemic in badgers and in farmed and feral deer. The disease caused by M. bovis is a slow progressive disease with clinical signs not apparent until late in the disease process. key factors for effective control of tuberculosis includes rapid detection, adequate therapy, and contact tracing to halt further transmission. However, in order to locate the source and route of transmission of M. bovis infection, a thorough epidemiological analysis is routinely carried out, that could lead to the control of disease in the herds and avoid economic losses. Recent developments in DNA technology and molecular biology have led to methods for the rapid detection of mycobacterium DNA. Among a number of described molecular methods, IS6110 fingerprinting is the recommended standard primary genotyping method and the most widely used worldwide. In this study, we used restriction fragment length polymorphism analysis with probes derived from the insertion element IS6110, the polymorphic GC-rich sequence (PGRS), and the direct repeat (DR) sequence which proved to be a useful method for differentiating M. bovis strains. A total of 13M. bovis samples from infected cattle in the West Azerbaijan Province of Iran were included in the study. The samples were submitted to the Tuberculosis Reference Laboratory at the Razi Vaccine and Serum Research Institute, Karaj. All isolates were cultivated on Lowenstein Jensen media with pyruvate (pyruvic acid), and then identified according to The World Organization for Animal Health (OIE) recommendations. The extracted genomic DNA samples of the isolates in the study were subjected to IS6110 primers, digested with restriction enzyme PvuII, and hybridized by oligonucleotide probes PGRS and DR. Polymorphic banding patterns obtained after hybridization discriminated the M. bovis strains and a database of strain types was established. Based on our results, the 13 isolates showed five different DNA patterns with a PGRS probe and similarly five patterns were obtained with the DR probe. PP-1 pattern was found almost among all the isolates while a distinct DNA pattern PP-3 was seen specifically from the West Azerbaijan Province.

Characterization of indigenous lactobacillus strains for probiotic properties.

BACKGROUND: Probiotics are defined as adequate amount of live microorganisms able to confer health benefits on the host. Currently, most commercially available probiotic products in the market belong to genera Lactobacillus. Traditional dairy products are usually rich source of Lactobacillus strains with significant health benefits. In order to evaluate the probiotic potential of these bacteria, it is essential to assess their health benefits, efficacy, and safety. OBJECTIVES: The probiotic efficacy of two Lactobacillus strains namely Lactobacillus pentosus LP05 and L. brevis LB32 was evaluated. They were previously isolated from ewes' milk in a rural area in East Azerbaijan, Iran. MATERIALS AND METHODS: The selected isolates were tested for certain phenotypic characters and identified to genus and species level by 16S rRNA gene sequencing and species specific primers. Further analysis included acid and bile resistance, antagonistic activity, cholesterol removing ability, survival in simulated gastric and upper intestine contents, aggregation and coaggregation properties. Finally, the adhering ability of the selected Lactobacillus strains to epithelial cells was tested using Caco-2 cell lines. RESULTS: The selected isolates tolerated bile salt concentrations ranging from 0.5% to 3%, however their coefficient of inhibition were varied. Both isolates hydrolyzed bile and grew at pH values of 3, 4, and 5, while isolate LP05 was not able to hydrolyze arginine. Based on 16s rRNA gene sequencing and species-specific primers, the isolates were identified as L. brevis LB32 and L. pentosus LP05. In contrast to simulated gastric conditions, the growth rate of the isolates in alkaline conditions of upper intestine increased significantly with the passage of time reaching its maximum in 24 hours. These 2 isolates inhibited the growth of Listeria monocytogenes, Salmonella enteritidis, Shigella dysenteriae, Staphylococcus aureus, and Streptococcus pneumonia. Furthermore, L. brevis LB32 was able to reduce approximately 86% of cholesterol compared to L. pentosus LP05, which showed only 69% of reduction. Higher aggregation and coaggregation percentage and adherence to Caco-2 cell line was observed in L. pentosus LP05 compared to L. brevis LB32. CONCLUSIONS: This research study proved the presence of viable probiotic LAB microflora in the ewe milk with enhanced health benefits. The 2 selected Lactobacillus strains could be exploited in dairy or pharmaceutical industry in future.

Biotyping, capsular typing, and antibiotic resistance pattern of Haemophilus influenzae strains in Iran.

The aim of this study was to determine the capsular types of Haemophilus influenzae isolated from clinical specimens by slide agglutination serotyping (SAST) and PCR capsule typing methods. All the isolates were biotyped and their antibiotic resistance patterns also determined. Thirteen isolates of serotype b, 2 of serotype e, 4 of serotype f, and 19 nontypeable (NT) isolates were identified by SAST method in 38 H. influenzae culture-positive samples. Capsule typing by PCR increased the proportion of all invasive cases from 34.2% (by SAST) to 60.5%, and 6 culture-negative samples were identified as invasive H. influenzae (Hib) by this method. The discrepancy rate between SAST and PCR results were 41%. Biotypes I, II, and III were the prevalent biotypes whereas biotypes VI and VII were not found. The majority of capsule type b belonged to biotype II. The isolates were resistant to cotrimoxazole (47.1%) and ampicillin (43.6%). Multidrug resistance was observed in 7 of the isolates.