In vitro evaluation of cell adhesion and immunomodulatory properties of five Lactobacillus rhamnosus strains isolated from infants

Aims: Five Lactobacillus rhamnosus strains (UBU03, UBU06, UBU09, UBU34 and UBU37) with good in vitro probiotic properties, isolated from breast-fed infants, were evaluated for in vitro adhesion, competitive adhesion and immunomodulatory properties. Knowledge of such properties is important when considering specific circumstances when these strains might be used clinically. Methodology and results: The Caco-2 cell line was used for adhesion assays and for competitive adhesion assays against Escherichia coli O157:H7. Lactobacillus rhamnosus GG was used as the reference strain for adhesion assays. The immunomodulatory activities of the five strains were evaluated by determining the levels of the inflammatory cytokines IL-6, IL-12 and TNF-α, and of the immunoregulatory cytokine IL-10, produced by bacterial-activated THP-1 cells after 6, 12 and 24 h of stimulation. In the cell-adhesion assays, all five strains showed high adhesion properties. For UBU09, UBU34 and UBU37, adhesive capacity was higher than that of the reference strain. All strains except UBU03 showed the ability to inhibit adhesion of E. coli O157:H7 to Caco-2 cells. All strains induced IL-6 production but not IL-12 production. UBU03 and UBU09 could induce only one cytokine IL-6. UBU06 and UBU34 could each induce two (IL-6/IL-10 and IL-6/TNF-α, respectively). UBU37 could induce three cytokines (IL-6/TNF-α /IL-10). Conclusion, significance and impact of study: These five probiotic L. rhamnosus strains with high adhesion properties and with different in vitro cytokine induction profiles should be investigated further in different immunological conditions to identify appropriate circumstances for their clinical use.

Probiotic characterization of lactic acid bacteria isolated from infants feces and its application for the expression of green fluorescent protein

Aims: In this study, lactic acid bacteria (LAB) were isolated from 42 healthy infants and determined for probiotic properties. Twelve LAB isolates with potential probiotic properties were selected and screened for their feasibility of heterologous protein expression by selection of erythromycin sensitive isolates. Methodology and results: One of eleven erythromycin-sensitive LAB isolates identified and designated as Lactobacillus fermentum 47-7 was able to acquire and stable maintain the Escherichia coli-Lactobacillus shuttle vector, pRCEID-LC13.9. Further electrotransformation of L. fermentum 47-7 with the recombinant pLC13.9:LDH-PRO1:GFPuv containing green fluorescent protein (GFP) gene found that recombinant L. fermentum can express GFP. Conclusion, significance and impact of study: The probiotic L. fermentum isolate can be used as host for expression of heterologous proteins and could possibly be further developed as the alternate oral delivery system for various biomolecules for biotechnological application.

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4+/-0.09degrees C and 85.9+/-0.08degrees C for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

Molecular Differentiation of Schistosoma japonicum and Schistosoma mekongi by Real-Time PCR with High Resolution Melting Analysis

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5+/-0.07degrees C and 85.7+/-0.07degrees C, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

Detection and Differentiation of Mycobacterium Species in Clinical Specimens by Real-Time Polymerase Chain Reaction

Mycobacterium tuberculosis complex and Mycobacterium avium complex are causative agents of tuberculosis which is a major cause of human morbidity and mortality. Rapid diagnosis and species differentiation of mycobactria in specimens are important to control the disease and use appropriate antimicrobial therapy. In this study, primers and probe were design based on 16S-23S rDNA spacer sequence by using LightCycler 2.0 software. One set of primer, Rank 9, was used to perform real-time PCR and conventional PCR for detection and differentiation of Mycobacterium spp. Sybr Green dye for signal detection, successfully amplified all extracted DNA of mycobacteria. Its amplicon can be visualized by gel electrophoresis with the corresponding length of 214 bp. The nonspecific products of about 300 bp, were found in conventional PCR. The result suggested that these primers are not appropriate for conventional PCR. A hybridization probe for strain differentiation and verification of the protocol in clinical specimens is needed to complete the protocol.

Identificarion of SHV beta - lactamases in ESBL-producing Enterobacteriaceae from Srinagarind Hospital collected between 1998 and 1999 and in 2003

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