Different probiotic properties for Lactobacillus fermentum strains isolated from swine and poultry

Systematic procedures were used to evaluate the probiotic properties of Lactobacillus fermentum (L. fermentum) strains isolated from swine and poultry. The major properties included their capabilities to adhere to the intestinal epithelium of swine and poultry, the inhibition on pathogenic bacteria, and their tolerance to the gastric juice and bile salts. Results showed that L. fermentum strains from poultry digestive tract showed better adherence to the swine intestine and chicken crop epithelial cells as compared to those strains from the swine origin. In addition, six strains from poultry and one strain from swine showed adhesion specificity to their own intestinal epithelium. Four poultry isolates and one swine isolate were able to adhere to the epithelial cells from both swine and chicken. For gastric juice and bile tolerance, most of the strains isolated from swine or poultry were acid tolerant but less strains were bile intolerant. The spent culture supernatant (SCS) of these L. fermentum strains showed antagonistic effect against the indicator bacteria, such as Escherichia coli, Salmonella spp., Shigella sonnei and some enterotoxigenic Staphylococcus aureus. From the above studies, some L. fermentum strains isolated from poultry were found to have the probiotic properties required for use in animal feed supplement. This study suggested that poultry digestive tract may serve as potential source for the isolation of probiotic lactic acid bacteria.     

The presence of major world-wide clones for phage type 4 and 8 Salmonella enterica serovar Enteritidis and the evaluation of their virulence levels by invasiveness assays in vitro and in vivo

Seventy-seven animal isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from the United States were analyzed by phage typing and pulsed field gel electrophoresis (PFGE). Thirty-nine strains were found with phage types (PT) 4, 8, and 13a. When the chromosomal DNA of these 39 isolated strains with PT4, 8, and 13a were digested with XbaI, SpeI and NotI, followed by PFGE analysis, 28 strains were found with a pattern combination of X4S4N4, which was the major subtype. When PFGE patterns of the US isolates with PT 4 and 8 were compared with those of the Taiwanese and German isolates, pattern X3S3N3 was confirmed to be the world-wide subtype shared by PT 4 isolates, as previously reported, while pattern X4S4N4 was newly found to be the most common subtype shared by PT 8 strains. The presence of such major world-wide clones, however, does not necessarily mean that these clones are highly virulent, at least not according to the results of invasiveness assays using cultured human intestinal epithelium cell line Int-407 and living BALB/mice.     

Optimization of medium composition for improving biomass production of Lactobacillus plantarum Pi06 using the Taguchi array design and the Box-Behnken method

An immune-enhancing strain, Lactobacillus plantarum Pi06, isolated from a healthy infant was used for biomass production following optimization of the medium in shake-flask culture. Preliminary studies showed that commercial MRS medium and cultivation under static conditions generated higher biomass production than four other tested media with or without a shaking condition. The selected medium composition, consisting of glucose, yeast extract, soy peptone, ammonium citrate, and corn steep liquor, was further optimized using a systematic method that integrated the Taguchi array design and the Box-Behnken method. The response effects of these factors were first investigated using Taguchi design under an L ₁₆ (4⁵) array. The suggested medium composition, derived from Statistica 7.1 using the Taguchi design, was applied to cultivate cells and a biomass of 7.16 g dry cell weight (DCW)/L was obtained. Response surface methodology based on the Box-Behnken method for the three response variables of glucose, yeast extract, and corn steep liquor was then used to further increase the biomass level to 8.94 g DCW/L. The resulting optimum medium consisted of 35 g/L glucose, 35 g/L yeast extract, and 40 mL/L corn steep liquor. Compared with the initial medium, the biomass yield was improved from 4.31 to 8.94 g DCW/L, an enhancement of approximately 107%.     

Lactobacillus acidophilus LAP5 able to inhibit the Salmonella choleraesuis invasion to the human Caco-2 epithelial cell

The mechanisms for lactic acid bacteria (LAB) to inhibit Salmonella invasion appear to be multifactorial and include the adhesion of LAB to host intestine epithelium, the production of organic acids, or bacteriocin by LAB cells. Previously, we found a strain of Lactobacillus acidophilus isolated from swine, i.e. strain LAP5, was with antagonistic effect against Salmonella typhimurium. This strain LAP5 was also found to meet the requirements for probiotic use. In this study, we evaluate the potential of LAP5 strain to protect the human or swine from infection by Salmonella choleraesuis. We present evidence that the culture of LAP5 was able to inhibit the invasion of S. choleraesuis to human Caco-2 cell line. The LAP5 cell culture showed a higher inhibitory effect on the invasion of S. choleraesuis to Caco-2 cells than the spent culture supernatant (SCS) of LAP5 did. Also, the pH, organic acids or the bacteriocin, which act at low pH conditions, may play the role of antagonistic effect. The addition, adhesion of LAP5 cells to Caco-2 cell line may also play roles to reduce the invasion of S. choleraesuis.     

Use of the polymerase chain reaction for specific detection of type A,D and E enterotoxigenic Staphylococcus aureus in foods

Summary By comparison with the DNA sequences coding for Staphylococcus aureus enterotoxins (ents) A, B, C, D and E, oligonucleotides unique to the entA, entD and entE genes were synthesized and used as polymerase-chain-reaction (PCR) primers for the specific detection of type A, D or E enterotoxigenic S. aureus. The relative molecular weights of the PCR products amplified with these primers were 210, 333 and 456 bp, respectively. Despite the high relatedness among these S. aureus enterotoxin genes, each primer pair allows specific detection with total discrimination from other types of enterotoxigenic S. aureus. DNA from non-enterotoxigenic S. aureus or from non-S. aureus would not interfere with the PCR results either. Primers designed for entE detection allow the discrimination of entE strains that when assayed by a serological method might be classified as entA-producing strains. Study of the detection sensitivity showed that by using these primers, DNA from 10⁰ cells of enterotoxigenic S. aureus could be detected unambiguously. When these oligonucleotide primers were used for the detection of S. aureus in foods, 10⁰-10¹ cells per gram of food could be detected and the naturally contaminating microflora in the food sample did not interfere with the detection. Correspondence to: H.-Y. Tsen     

Improvement of strain discrimination by combination of RAPD with PFGE for the analysis of the swine isolates of Salmonella enterica serovar Choleraesuis

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) can cause salmonellosis in pigs and humans. Currently, the most common method used for the subtyping of this Salmonella serovar is pulsed field gel electrophoresis (PFGE) using XbaI as a DNA digestion enzyme. In this study, we compared and combined PFGE with the randomly amplified polymorphic DNA method, for the typing of 95 S. Choloraesuis strains isolated from diseased pigs. Using PFGE with XbaI, with AvrII, and with SpeI digested DNA, 29, 74, and 40 patterns, respectively, were obtained. Also, 53, 15, and 35 strains, respectively, belong to the major patterns X1, A1, and S1. When these three digestion patterns were combined, 83 PFGE pattern combinations were obtained. On the other hand, using RAPD with selected primer alone generated 76 patterns, and 11 strains which fell within a single X1A1S1 PFGE combination pattern were discriminated into 10 patterns. Thus, for S. Choloraesuis, PFGE with AvrII allowed higher discrimination than PFGE with XbaI, and some of the PFGE groupings obtained by combining the XbaI, AvrII and SpeI digestion patterns were further subdivided by the RAPD method.     

Lipid peroxidation dysregulation in ischemic stroke: Plasma 4-HNE as a potential biomarker?

4-hydroxynonenal (4-HNE) is a major aldehyde produced during the lipid peroxidation of ω-6 polyunsaturated fatty acids. Recently, 4-HNE has been reported to contribute to the pathogenesis of neuronal diseases such as Alzheimer's disease. However, the role of 4-HNE in ischemic stroke is unclear yet. In this study, we found that plasma 4-HNE concentrations were higher in the genetic stroke-prone rats (stroke-prone spontaneously hypertensive rats) and experimental stroke rats with middle cerebral artery occlusion (MCAO). Moreover, administration of 4-HNE via intravenous injection before MCAO surgery not only enlarged cerebral ischemia-induced infarct area, but also increased oxidative stress in brain tissue, which was evidenced by the enhanced ROS/MPA levels, and the reduced GSH/GSSG ratio and MnSOD levels. Overexpression of aldehyde dehydrogenasesbcl-2 (ALDH2), an enzyme catalyses 4-HNE, rescued neuronal survival against 4-HNE treatment in PC12 cells. The plasma 4-HNE concentrations in patients with ischemic stroke were higher than those in control subjects. In a small sample population (N=60), the plasma 4-HNE concentration was positively correlated with the plasma homocysteine concentration, a risk factor for ischemic stroke. Taken together, our study suggests that the plasma 4-HNE level is a potential biomarker for ischemic stroke.     

Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples

Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10(0) target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis.     

Use of specific primers based on the 16S-23S internal transcribed spacer (ITS) region for the screening Bifidobacterium adolescentis in yogurt products and human stool samples

Effective methods for the identification and enumeration of lactic acid producing bacteria (LAB) cells are important for the quality control and assurance of probiotic products. In this study, we designed a polymerase chain reaction (PCR) primer set from the sequence in 16S-23S internal transcribed spacer (ITS) region and used it for the specific detection of Bifidobacterium adolescentis, one of the Bifidobacterium species used in probiotics. Specificity of the PCR primers, i.e., bits-1/bits-2, was assured by assay strains of B. adolescentis, other Bifidobacterium species, and strains of non-Bifidobacterium spp. Coupled with the use of a known primer set specific for Bifidobacterium species, Bifidobacterium strains and B. adolescentis could be identified from LAB strains in fermented dairy products and human fecal samples.