16S rDNA PCR Research Articles

PCR-DGGE analysis of bacterial population attached to the bovine rumen wall

We isolated and amplified by PCR 16S rDNA from bacteria attached to the bovine rumen wall and analyzed it by denaturing gradient gel electrophoresis (DGGE) with subsequent sequence analysis. The attached bacterial community differed from the bacteria of rumen content; however, no differences were observed among the five epithelial sampling sites taken from each animal. The DGGE profile of the bacterial population attached to the rumen wall represented a high inter-animal variation.

Telonemia-specific environmental 18S rDNA PCR reveals unknown diversity and multiple marine-freshwater colonizations

BackgroundRecent surveys of eukaryote 18S rDNA diversity in marine habitats have uncovered worldwide distribution of the heterotrophic eukaryote phylum Telonemia. Here we investigate the diversity and geographic distribution of Telonemia sequences by in-depth sequencing of several new 18S rDNA clone libraries from both marine and freshwater sites by using a Telonemia-specific PCR strategy.ResultsIn contrast to earlier studies that have employed eukaryote-wide PCR design, we identified a large and unknown diversity of phylotypes and the first rigorous evidence for several freshwater species, altogether comprising 91 unique sequences. Phylogenies of these and publicly available sequences showed 20 statistically supported sub-clades as well as several solitary phylotypes with no clear phylogenetic affiliation. Most of these sub-clades were composed of phylotypes from different geographic regions.ConclusionsBy using specific PCR primers we reveal a much larger diversity of Telonemia from environmental samples than previously uncovered by eukaryote-wide primers. The new data substantially diminish the geographic structuring of clades identified in earlier studies. Nevertheless, since these clades comprise several distinct phylotypes we cannot exclude endemicity at species level. We identified two freshwater clades and a few solitary phylotypes, implying that Telonemia have colonized freshwater habitats and adapted to the different environmental and ecological conditions at independent occasions.

Metagenomic analysis of bronchoalveolar lavage samples from patients with idiopathic interstitial pneumonia and its antagonic relation with Pneumocystis jirovecii colonization

Idiopathic interstitial pneumonias are interstitial lung diseases of unknown etiology which prognosis is usually fatal. Microbiota associated to bronchoalveolar lavage from 20 patients with negative bacterial cultures was explored by 16S-rDNA PCR–DGGE, showing a clearly negative relation among the presence of P. jirovecii and bacterial colonization. This is the first report of in vivo antagonistic relation among fungi and bacteria.

First Report of "Candidatus Liberibacter psyllaurous" (synonym "Ca. L. solanacearum") Associated with 'Tomato Vein-Greening' and 'Tomato Psyllid Yellows' Diseases in Commercial Greenhouses in Arizona.

During the winter of 2006-2007, plants in commercial tomato greenhouses (GH-1 and GH-2; total 320 acres [129.5 ha]) in Arizona were infested with the potato psyllid Bactericera cockerelli (Sulc) and more than 60% and ~20% of the plants, respectively, exhibited leaf curling, chlorosis, and shortened internodes. In addition, some plants in GH-1 developed an unusual 'vein-greening' phenotype. Nucleic acids were isolated from 10 symptomatic and three asymptomatic plants from each greenhouse. PCR primers designed to amplify a phytoplasma-like 16S rDNA (850 bp) yielded the expected size product from GH-1 samples, whereas samples from GH-2 and the asymptomatic samples from both greenhouses did not. Several 16S rDNA PCR products (3 of 60) when cloned and sequenced, surprisingly shared 97% homology with 'Candidatus Liberibacter asiaticus' (GenBank No. GQ926917). PCR primers PSY680F 5'-GTTCGGAATAACTGGGCGTA-3' and PSY1R 5'-CCCATAAGGGCCATGAGGACT-3', based on the resultant 16S rDNA sequences, were used to amplify a 680-bp fragment from plant DNA extracts and psyllid lysates (1). A robust PCR product (~680 bp) was obtained from 10 of 10 GH-1 plant extracts (GQ926918) and from a GH-1-derived psyllid colony (28 of 35 adults) (GQ926919) and the tomato plants on which they were reared. In contrast, no 680-bp product was obtained from GH-1 asymptomatic plants (0 of 3), GH-2 plants (0 of 10 symptomatic; 0 of 3 asymptomatic), GH-2-derived psyllid colonies (0 of 35 adults), or psyllid colony tomato plants (data not shown). At least three 680-bp amplicons for each sample type were cloned and 8 to 10 inserts were sequenced for each. BLAST analysis revealed that all 680-bp sequences shared 99 to 100% nt identity with the analogous 16SrDNA from "Ca. Liberibacter psyllaurous" (2) and synonym "Ca. L. solanacearum" (3). A second molecular marker was obtained with the 1611F and 480R primers (2) to amplify the 16SrDNA-23S-ITS (980 bp) from >3 plant extracts and psyllid lysates that tested positive for liberibacter. Clustal W alignment of the 16S-23S-ITS sequences from GH-1 original tomato plants and psyllid colony plants (GQ926920) and psyllids (GQ926921) indicated they were 100% identical to each other and BLAST analysis indicated 99 to 100% shared identity with "Ca. L. psyllaurous" (EU812558) (synonym "Ca. L. solanacearum"). Transmission electron microscopy examination of GH-1 and GH-2 psyllids revealed rod and pleomorphic-shaped bacteria (0.5 to 2.0+ μm) at the brain-salivary gland interface in psyllids from the GH-1 liberibacter-positive colony. No such bacteria were observed in GH-2 liberibacter-negative psyllids. These results support an etiological role of a new liberibacter spp. in the development of the 'vein-greening' symptom phenotype. In contrast, the GH-2 'yellows' phenotype is reminiscent of 'psyllid toxicity' in tomato colonized by B. cockerelli (4). To our knowledge, this is the first report of distinct psyllid-associated diseases in greenhouse tomato in Arizona, one associated with a new 'Ca. Liberibacter' spp., manifest as 'vein-greening' disease, and the other associated with psyllid feeding, in which liberibacter is undetectable in plants and psyllids, and is manifest as the 'tomato psyllid yellows' disease.

Tropheryma whippleiin Patients with Pneumonia

Tropheryma whipplei is the etiologic pathogenic agent of Whipple disease (WD), characterized by various clinical signs, such as diarrhea, weight loss, lymphadenopathy, and polyarthritis. PCR-based methods for diagnosis of WD have been developed. T. whipplei has been identified in saliva and stool samples from patients with WD and from healthy persons. T. whipplei DNA has also been found in bronchoalveolar lavage (BAL) samples of a child with pneumonia. We detected DNA of T. whipplei in 6 (3%) of 210 BAL samples collected in intensive care units by using 16S rDNA and specific quantitative PCR. We identified 4 novel genotypes of T. whipplei. In 1 case, T. whipplei was the only bacterium; in 4 others, it was associated with buccal flora. We suggest that T. whipplei should be investigated as an etiologic agent of pneumonia.

Monitoring the Bacterial Community Dynamics in a Petroleum Refinery Wastewater Membrane Bioreactor Fed with a High Phenolic Load

The phenolic compounds are a major contaminant class often found in industrial wastewaters and the biological treatment is an alternative tool commonly employed for their removal. In this sense, monitoring microbial community dynamics is crucial for a successful wastewater treatment. This work aimed to monitor the structure and activity of the bacterial community during the operation of a laboratory-scale continuous submerged membrane bioreactor (SMBR), using PCR and RT-PCR followed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA libraries. Multivariate analyses carried out using DGGE profiles showed significant changes in the total and metabolically active dominant community members during the 4-week treatment period, explained mainly by phenol and ammonium input. Gene libraries were assembled using 16S rDNA and 16S rRNA PCR products from the fourth week of treatment. Sequencing and phylogenetic analyses of clones from 16S rDNA library revealed a high diversity of taxa for the total bacterial community, with predominance of Thauera genus (ca. 50%). On the other hand, a lower diversity was found for metabolically active bacteria, which were mostly represented by members of Betaproteobacteria (Thauera and Comamonas), suggesting that these groups have a relevant role in the phenol degradation during the final phase of the SMBR operation.

Purulent Pericarditis due to Streptococcus pneumoniae Diagnosed by Pneumococcal Urinary Antigen Assay and 16S rDNA Sequence of the Pericardial Fluid

A 57-year old woman was admitted to our hospital with massive pericardial fluid. Culture of the pericardial fluid was negative, however, Binax NOW Streptococcus pneumoniae urinary antigen test was positive in pericardial fluid. 16S rDNA sequencing and PCR for lyt(A) gene of the pericardial fluid sample confirmed the microbiological diagnosis of S. pneumoniae. The patient was treated with surgical drainage and continuous intravenous infusion of penicillin G and its concentration in the serum and pericardial effusion was monitored. Incorporation of molecular methods such as antigen testing and nucleic acid sequencing would benefit the management of infectious diseases especially in culture negative cases.

Differing Prevalence and Diversity of Bacterial Species in Fetal Membranes from Very Preterm and Term Labor

BackgroundIntrauterine infection may play a role in preterm delivery due to spontaneous preterm labor (PTL) and preterm prolonged rupture of membranes (PPROM). Because bacteria previously associated with preterm delivery are often difficult to culture, a molecular biology approach was used to identify bacterial DNA in placenta and fetal membranes.Methodology/Principal findingsWe used broad-range 16S rDNA PCR and species-specific, real-time assays to amplify bacterial DNA from fetal membranes and placenta. 74 women were recruited to the following groups: PPROM <32 weeks (n = 26; 11 caesarean); PTL with intact membranes <32 weeks (n = 19; all vaginal birth); indicated preterm delivery <32 weeks (n = 8; all caesarean); term (n = 21; 11 caesarean). 50% (5/10) of term vaginal deliveries were positive for bacterial DNA. However, little spread was observed through tissues and species diversity was restricted. Minimal bacteria were detected in term elective section or indicated preterm deliveries. Bacterial prevalence was significantly increased in samples from PTL with intact membranes [89% (17/19) versus 50% (5/10) in term vaginal delivery p = 0.03] and PPROM (CS) [55% (6/11) versus 0% (0/11) in term elective CS, p = 0.01]. In addition, bacterial spread and diversity was greater in the preterm groups with 68% (13/19) PTL group having 3 or more positive samples and over 60% (12/19) showing two or more bacterial species (versus 20% (2/10) in term vaginal deliveries). Blood monocytes from women with PTL with intact membranes and PPROM who were 16S bacterial positive showed greater level of immune paresis (p = 0.03). A positive PCR result was associated with histological chorioamnionitis in preterm deliveries.Conclusion/SignificanceBacteria are found in both preterm and term fetal membranes. A greater spread and diversity of bacterial species were found in tissues of women who had very preterm births. It is unclear to what extent the greater bacterial prevalence observed in all vaginal delivery groups reflects bacterial contamination or colonization of membranes during labor. Bacteria positive preterm tissues are associated with histological chorioamnionitis and a pronounced maternal immune paresis.

640: 16S ribosomal DNA PCR identifies a greater number and broader diversity of microorganisms associated with intraamniotic infection than culture alone

To assess the diagnostic efficacy of amniotic fluid (AF) 16S ribosomal DNA PCR versus AF culture for diagnosis of intraamniotic infection (IAI) in women with preterm labor. 111 women with preterm labor, intact membranes, singleton, <37 weeks' gestation underwent amniocentesis as part of evaluation for IAI. Cultures were performed for eubacteria as well as Mycoplasma sp. and Ureaplasma sp. PCR was used to amplify highly-conserved 16S rRNA gene sequences to detect microbes across a broad taxonomic range. PCR amplicons positive for 16S rDNA were cloned and bidirectionally sequenced. Sequences were speciated using an rDNA library. Culture was positive in 12/111 cases (10.8%) and 16S rDNA PCR positive in 16/111 (14.4%). All 12 cases with positive cultures were also 16S rDNA PCR positive. The 4 culture-negative cases with a positive 16S rDNA had clinical characteristics consistent with IAI: latency < 48 hrs, IL-6 > 9 ng/ml, AF glucose < 20 mg/dL; organisms were observed on AF Gram stain in 3 of 4. Cultures recovered 6 species (most common Ureaplasma urealyticum), whereas 16S rDNA PCR with sequencing identified 12 distinct taxa. Sequencing detected microbial taxa not recovered by culture including Bergeyella, Sneathia, Leptotrichia and Bacteroides sp. Using a definition including results from both culture and 16S rDNA PCR, IAI was present in 14.4% of cases. Fastidious organisms not detected by traditional culture methods accounted for 25% of IAI cases and likely would not have been detected without the use of 16S rDNA PCR. The findings suggest that molecular evaluation of amniotic fluid may provide important insights into pathophysiology and nature of organisms responsible for IAI.

16S rDNA PCR: Five year experience from a Tertiary Referral Centre

16S rDNA PCR: Five year experience from a Tertiary Referral Centre

PCR rDNA 16S dans le diagnostic étiologique des endocardites infectieuses à hémocultures négatives

We report the case of a 55 year-old man presenting with a double aortic and mitral endocarditis for which resected valve culture was repeatedly negative. Specific PCR made on valves because of highly positive blood tests for Bartonella henselae remained negative. A molecular approach was made with 16S rDNA PCR, followed by sequencing. Bartonella quintana was identified as the etiology of endocarditis. B. quintana, “fastidious” bacteria, even if hard to identify in a laboratory, is often reported as a blood culture negative endocarditis (BCNE) agent. Molecular biology methods have strongly improved the diagnosis of BCNE. We propose a review of the literature focusing on the interest of broad-spectrum PCR on valve for the etiological diagnosis of BCNE.

Development and validation of a modified broad-range 16S rDNA PCR for diagnostic purposes in clinical microbiology

Broad-range PCR followed by sequencing identifies bacterial pathogens, even in challenging settings such as patients receiving antibiotics or infected with fastidious or non-cultivable organisms. The major problem with broad-range PCR is the risk of sample contamination. Risk is present at every step of the procedure, starting from sample collection. Contaminating bacterial DNA may be present not only in laboratory reagents but also at the surface of plastic consumables and containers used for specimen drawing and transport to the diagnostic laboratory. Contaminating DNA is amplified efficiently, leading to false-positive results. Thus, high specificity depends on eliminating such spurious targets, an awkward problem given the abundance of such targets and a highly sensitive method that detects very small numbers of molecules. Several investigators have reported strategies for eliminating the amplification of contaminating DNA sequences. So far, none of these methods has been entirely effective and reproducible. Here we describe a method that uses Exonuclease III ( ExoIII) to disable contaminating sequences from acting as templates, while maintaining the high sensitivity of PCR for pathogen DNA. We use this assay in 144 clinical specimens from normally sterile sites, identifying pathogens from 24 (17%). Conventional methods identified pathogens in only four of these specimens, all of which were positive for the same pathogen by PCR. Compared with conventional methods, broad-range PCR with ExoIII pre-treatment of reagents substantially improves the diagnostic yield of bacterial pathogen identification from normally sterile sites.

Effects of long-term CO 2 fumigation on fungal communities in a temperate forest soil

This study aimed at determining the impact of long-time elevated CO 2 fumigation on fungal communities in a temperate forest soil. In addition to the CO 2 concentration, both time and its interaction with the CO 2 affected the activity of 1,4-β-N-acetylglucosaminidase that is mainly of the fungal origin in the soil. No significant change in Shannon's indexes (from 18S rDNA-PCR–DGGE) was observed between the ambient and elevated CO 2 treatments. Analysis of time-course indicated that the succession of soil fungal community was altered by the elevated CO 2 fumigation, and the variations in the soil samples under Pinus koraiensis Sieb. et Zucc were larger than those under the Pinus sylvestriformis (Takenouchi) T. Wang ex Cheng samples. The results suggest that the increase in atmospheric CO 2 concentrations could alter the temporal patterning of soil fungal communities.

Trypanosoma irwini n. sp (Sarcomastigophora: Trypanosomatidae) from the koala (Phascolarctos cinereus)

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0.9% at the 18S rDNA and 10.7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38.2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.

Molecular Characterization of a Phytoplasma Associated with Coconut Yellow Decline (CYD) in Malaysia

Problem statement: Molecular methods have been used to detect phytoplasma in Malayan Red Dwarf (MRD) ecotype of coconut for the first time in Malaysia. Detect phytoplasma from coconut showing yellowing symptoms by nested PCR and Classify phytoplasma associated with disease of coconut palms, based on analysis of 16S rRNA gene operon sequences and virtual RFLP. Approach: Twenty MRD symptomatic palms were tested. Leaf spear, inflorescence and wood shavings from trunks of coconut palms showing yellowing symptoms were harvested from Serdang located in Selangor state. PCR assays and sequence analysis were carried out. Results: Nested PCR with primer pairs R16F2n/R16R2 and fU5/rU3 resulted in amplification of products of approximately 1.2 kb and 890 bp respectively, from 8 out of 20 MRD symptomatic palms tested. Sequence analysis of the 16S rDNA PCR products determined that the phytoplasma strain associated with Coconut Yellow Decline (CYD) in MRD ecotype belongs to the 'Candidatus Phytoplasma cynodontis' (16SrXIV) group of phytoplasmas. In addition, nested R16F2n/R16R2 PCR products from 6 spear leaves and 2 inflorescences from MRD palms showed high sequence similarity to the 16S rRNA gene from coconut chloroplasts, with a similar size (approximately 1.3 kb) and a further 5 R16F2n/R16R2 PCR products from MRD inflorescences showed high sequence similarities to Bacillus spp. 16S rRNA gene sequences. Conclusion: These results indicate that sequencing is a reliable method for the detection. Furthermore, trunk borings are the most reliable source of DNA for phytoplasma detection in coconuts using 16S rRNA gene primers, since there is less co-amplification of PCR products from other organisms when compared to spear leaves and inflorescences, nor from the spear leaves and inflorescences of MRD palms when primers fU5/rU3 were used in nested PCR.

Bacterial phylogenetic diversity in a Spartina marsh in China

The bacterial phylogenetic diversities of the bacterial communities in the salt marsh colonized by Spartina alterniflora (SA) and uncultured marsh (UM) along the Yellow Sea of China were analyzed based on the 16S rDNA PCR techniques. Two libraries containing 251 clones of 16S rRNA genes from the Spartina colonized marsh and 283 from UM were constructed by PCR using a bacteria-specific primer 8f and the common primer 1542r. Forty-seven clones from SA and 55 from UM were selected for partial sequencing. A phylogenetic tree was constructed by the alignment analysis of the total environmental DNA sequences. Clones were clustered into 8 divisions of bacteria: (1) Proteobacteria containing 12 genera in beta, gamma, delta and epsilon subdivision; (2) Bacteroidetes containing 7 genera; (3) Planctomycetes containing 2 genera; (4) Firmicutes containing 1 genus; (5) Spirochaetes containing 1 genus; (6) Acidobacteria containing 1 genus; (7) Actinobacteria containing 1 genus; and (8) BRC1 containing 1 genus, as well as 3 groups of bacteria that could not be clustered into any recognized bacterial divisions or candidate divisions. BLAST searches of the GenBank database confirmed that 79.7% of the clones in the two libraries were closely related to the 16S rDNA sequences of the uncultured bacteria with the similarity ≥90%. Among them, Proteobacteria (36.7% and 54.4% of the total clones from the SA and UM libraries, respectively) and Bacteroidetes (30.0% and 18.3%, respectively) are two dominant groups in both the sites. However, some phyla, such as Firmicutes and Actinobacteria, were found at SA site but not at UM site, and vice versa, some phyla, such as Spirochaetes and BRC1, were found at UM, but not at SA. Moreover, the dominant species in each phylum were varied, dependent on whether or not the marsh was colonized by Spartina. For instance, in Bacteroidete, 44.4% clones at SA library were affiliated to Gelidibacter, but 32.7% clones were affiliated to Flexibacter at UM library. In Proteobacteria, the clones at UM library affiliated to δ, γ, ɛ and β-subdivisions were 38.8%, 34.7%, 23.5% and 3.1%, respectively, but at SA library were 30.3%, 47.0%, 13.6% and 9.1%, correspondingly. These results indicated that the bacterial diversity in the salt marsh along the Yellow Sea of China was greatly changed after Spartina colonization.

Bacterial Analysis of Breast Milk: A Tool to Differentiate Raynaud’s Phenomenon from Infectious Mastitis During Lactation

Lactational Raynaud's syndrome may be misdiagnosed as infectious mastitis on the basis of the breast pain. The objective of this work was to elucidate if microbiological analysis of milk may contribute to the differentiation of both conditions. Ten lactating women clinically diagnosed by Spanish lactation consultants were included in the study. Of these, five suffered from mastitis and the remaining five suffered from Raynaud's syndrome. Breast milk samples were inoculated on diverse culture media. Seventy isolates were selected and identified by 16S rDNA PCR sequencing. Parallel, PCR-DGGE and quantitative real-time PCR were used to assess the presence of bacterial DNA in the samples. Neither bacteria nor yeasts could be detected in the milk samples provided by the women suffering from Raynaud's syndrome. In contrast, large numbers of bacteria were isolated from those with infectious lactational mastitis. Globally, the levels of bacterial DNA were significantly higher in the milk of mastitis-suffering women. Bacteriological analysis of milk can be an useful tool to facilitate the differential diagnosis between the infectious mastitis and Raynaud's syndrome during lactation.

Microbial population profiles of the microflora associated with pre- and postharvest tomatoes contaminated withSalmonella typhimuriumorSalmonella montevideo

To determine the microflora profiles of pre- and postharvest tomatoes contaminated with Salmonella montevideo or S. typhimurium DT104. Salmonella montevideo or S. typhimurium was inoculated onto the flowers of tomato plants with the microflora of the subsequent fruit examined using a combination of Source Carbon Utilization and 16S rDNA-PCR profiling. From 16S rDNA profiles it was evident that tomatoes derived from Salmonella inoculated plants harboured a different microbial population compared to nontreated controls. The same result was observed for tomatoes inoculated at postharvest and subsequently stored for 14 days at 15 degrees C. From sequencing analysis it was found that tomatoes derived from Salmonella inoculated plants but testing negative for the enteric pathogen, frequently harboured Enterobacter and Bacillus spp. In contrast, both bacterial types were not found associated with tomatoes testing positive for Salmonella. Salmonella introduced onto tomatoes at pre- or postharvest alters the composition of the microbial community. The presence of Enterobacter and Bacillus spp negatively affects the persistence of Salmonella on preharvest tomatoes. Salmonella appears to modify rather than become integrated into the microbial communities associated with tomatoes. Yet, the presence of antagonistic bacteria appears to reduce the persistence of the enteric pathogen.

Méningite néonatale à Listeria monocytogenes après traitement maternel de 3 semaines pendant la grossesse

We report the case of a pregnant woman with listeriosis at 26 gestational weeks followed by premature labor at 30 gestational weeks. Bacterial meningitis was suspected in the neonate with ventriculitis on sonography, a high level of protein in the cerebrospinal fluid (CSF), and an identified specific bacterial genome of Listeria monocytogenes (PCR 16S rDNA and sequencing and specific amplification of L. monocytogenes hly gene) in CSF. Neonatal meningitis was complicated with cerebral venous sinus thrombosis and ventriculomegaly. Listeriosis during pregnancy can lead to severe complications in the neonate. Thus, listeriosis should be a diagnostic concern in febrile pregnant women at any stage of pregnancy. First-line treatment is based on high-dose amoxicillin (> or =6g/day) and must be used for at least 3 weeks for treatment of listeriosis during pregnancy. If the fetus survives, longer therapy until delivery can be discussed.

The Effect of Lactic Acid Bacteria Isolates on the Urinary Tract Pathogens to Infants In Vitro

Urinary tract infections are common clinical problems in children, even though lots of treatment strategies have been tried. Many studies of the application of probiotics for urinary tract infection in female adults exist, but there is a lack of studies in children. The aims of this study were to screen probiotic strains for inhibiting the uropathogens in vitro, to find candidates for in vivo study. Nine strains of E. coli were isolated from children with urinary tract infection and six uropathogens were obtained from Korean Colletion for Type Cultures and American Type Culture Collection. Also 135 lactic acid bacteria (LAB) strains were isolated from healthy children, and were identified through physiologic, biochemical methods, 16S rDNA PCR, and data analysis. And with agar disk diffusion assay technique the antimicrobial activities of these LAB strains against those uropathogens were examined. Three strains of separated LAB strains demonstrated major antimicrobial activity against all the uropathogens. In the agar disk diffusion assay technique, antimicrobial activities increased most in the 4th day culture broth with separated Lactobacillus. In summary, some LAB can be used as candidates to develop the probiotic microorganisms that inhibit uropathogens in children, and are expected to be applied to treatment and prevention of pediatric urinary tract infection.