Amplified 16S Ribosomal DNA Research Articles

Microbial Sterility Testing for Mesenchymal Progenitor Cells Derived from Human Osteoarthritis and Rheumatoid Arthritis Cartilage.

The goal is to evaluate the microbial sterility of mesenchymal progenitor cells (MPCs)-derived from osteoarthritis (OA) and rheumatoid arthritis (RA) articular cartilages. Contaminants, including bacteria and fungi in MPC cultures were initially evaluated by inoculation culture methods and then affirmed through the amplification of 16S ribosomal DNA (rDNA) and internally transcribed spacer (ITS) regions, respectively, using polymerase chain reaction (PCR). Further, the mollicutes, if any, were identified by genus-specific 16S rDNA, and the positive samples were reamplified using species-specific primers. No bacteria or fungi were found to be compromising the sterility of MPCs (n = 20) assessed by both traditional culture methods and PCR. However, two in early passages and three in later passages of MPCs had the presence of mollicutes. Further rescreening for species of mollicutes indicated the presence of Mycoplasma hyorhinis, M. salivarium, and M. arginine. The PCR methods employed in this study could be beneficial as a rapid sterility testing of cell lines.

Isolation ofRickettsia,Rickettsiella, andSpiroplasmafrom Questing Ticks in Japan Using Arthropod Cells

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks.

Diversity and functional characterization of antifungal-producing Streptomyces-like microbes isolated from the rhizosphere of cajuput plants (Melaleuca leucodendron L.)

The study was undertaken to evaluate the diversity of actinomycetes from the rhizosphere of the cajuput plant (Melaleuca leucodendron L.) using ARDRA, and to examine their in vitro antifungal potency against selected fungi. A total of 78 Streptomyces-like microbes were isolated from the limestone rhizosphere of cajuput plants and cultured in SN agar medium. The ability to inhibit fungal growth was observed using a dual culture assay. The diversity of the isolates was examined by morphological and genotype profiling using ARDRA (Amplified 16S ribosomal DNA restriction analysis), following which they were assigned to eight groups. Seventeen (21.8%) strains showed the ability to produce the antifungal compound as evidenced by their antagonism in vitro against the tested fungi, namely Saccharomyces cerevisiae BY 47420, Candida albicans CGMCC 2538, Aspergillus flavus NRLL 3357, and Fusarium oxysporum KFCC 11363P in the dual culture assay. One isolate, GMR22, which showed potent antagonism against unicellular and filamentous fungi, displayed 97% 16S rRNA sequence similarity to Streptomyces vayuensis. Keywords:Antifungal, Streptomyces-like microbes, Melaleuca leucadendron L., limestone soil, rhizosphere

Characterizationofbacteriacausingbovinemastitisbyamplifiedribosomal DNA restriction analysis (ARDRA) method

Mastitis is an important constraint that accounts for high economic loss in dairy farms across the world. The prevalence and distribution of mastitis in dairy cows in Bangladesh is well-documented while most of thesefarms are confronted with problems of clinical and subclinical mastitis. This research was conducted to assess the rapid detection of bovine subclinical mastitis by DNA based ARDRA method and to validate its efficiency to identify other associated organisms. Traditional biochemical tests such as California mastitis test (CMT) was used for initial screening followed by direct DNA extraction from 146 CMT positive milk samples (n=196) collected from 5 different dairy farms in Chittagong. Using the extracted DNA as template, amplification of 16s ribosomal DNA by previously described universalprimers (27F and 1492R) was successfully achieved. Subsequent restriction digestion (with Hae-III) of PCR amplicons (n=51) revealed characteristic restriction pattern indicating six different groups of organisms in 38 cases where complete digestion was found. Further sequence analysis from corresponding PCR products and bioinformatic analysis revealed the identity of the responsible pathogens. Plasmid profile also was investigated to develop hypothesis associated with drug resistance patterns. The study shows that, in absence of sequencing facilities, ARDRA can be a useful approach to efficiently characterize the mastitis-causing bacteria indicating diagnostic implications.

16S rRNA gene sequence based identification of Vibrio spp. in shrimp and tilapia hatcheries of Bangladesh

Presence of Vibrio spp., one of the deadliest fish and shrimp pathogens in aquaculture facilities worldwide for which hatchery owners often suffer hectic economic losses, were identified in shrimp and tilapia hatcheries of Cox’s Bazar and Mymensingh, Bangladesh. Thirty seven Vibrio isolates, selected on the basis of their morphological dissimilarities in thiosulphate citrate bile salt sucrose agar (TCBS) plate, were subjected to amplified 16S ribosomal DNA restriction analysis (ARDRA) by using AluI restriction enzyme following their DNA extraction and amplification of 16S rRNA (1450 bp). From this analysis, representative isolates of 8 ARDRA groups, named as ARH 1 to ARH 8, of different band patterns were sequenced and identified as Vibrio alginolyticus, Aeromonas veronii, A. hydrophila, Vibrio vulnificus, V. cholerae, Edwardsiella hoshinae, Bacillus methylotrophicus and Aeromonas veronii, respectively. Vibrio species identified in this study are pathogenic for human and aquatic organisms, and were found only in shrimp hatchery with the dominance of V. alginolyticus. Findings of this study indicate the poor quality of water treatment and management of the hatchery. It was also observed that all the three Vibrio species were present in the Artemia rearing tank which indicates the possible source of pathogens.
 Dhaka Univ. J. Biol. Sci. 26(1): 45-58, 2017 (January)

16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry.

BackgroundThe objective of our study is to evaluate the potential use of Amplified 16S Ribosomal DNA Restriction Analysis (16S-ARDRA) and MALDI-TOF mass spectrometry (MS) as methods for species identification of Lactobacillus strains in poultry.ResultsA total of 80 Lactobacillus strains isolated from the cloaca of chicken, geese and turkeys were identified to the species level by MALDI-TOF MS (on-plate extraction method) and 16S-ARDRA. The two techniques produced comparable classification results, some of which were additionally confirmed by sequencing of 16S rDNA. MALDI-TOF MS enabled rapid species identification but produced more than one reliable identification result for 16.25 % of examined strains (mainly of the species L. johnsonii). For 30 % of isolates intermediate log(scores) of 1.70–1.99 were obtained, indicating correct genus identification but only presumptive species identification. The 16S-ARDRA protocol was based on digestion of 16S rDNA with the restriction enzymes MseI, HinfI, MboI and AluI. This technique was able to distinguish 17 of the 19 Lactobacillus reference species tested and enabled identification of all 80 wild isolates. L. salivarius dominated among the 15 recognized species, followed by L. johnsonii and L. ingluviei.ConclusionsThe MALDI-TOF MS and 16S-ARDRA assays are valuable tools for the identification of avian lactobacilli to the species level. MALDI-TOF MS is a fast, simple and cost-effective technique, and despite generating a high percentage of results with a log(score) <2.00, the on-plate extraction method is characterized by high-performance. For samples for which Biotyper produces more than one reliable result, MALDI-TOF MS must be used in combination with genotypic techniques to achieve unambiguous results. 16S-ARDRA is simple, repetitive method with high power of discrimination, whose sole limitation is its inability to discriminate between species with very high 16S rDNA sequence homology, such as L. casei and L. zeae. The assays can be used for discrimination of Lactobacillus bacteria from different habitats.

Identification of NDM-1 in a Putatively Novel Acinetobacter Species ("NB14") Closely Related to Acinetobacter pittii.

In this study, we describe the molecular characterization of a plasmid-located blaNDM-1 harbored by an Acinetobacter clinical isolate recovered from a patient in Turkey that putatively constitutes a novel Acinetobacter species, as shown by its distinct ARDRA (amplified 16S ribosomal DNA restriction analysis) profile and molecular sequencing techniques. blaNDM-1 was carried by a conjugative plasmid widespread among non-baumannii Acinetobacter isolates, suggesting its potential for dissemination before reaching more clinically relevant Acinetobacter species.

Application of next-generation sequencing to study ascitic microbiome in cirrhotic patients with or without spontaneous bacterial peritonitis

Spontaneous bacterial peritonitis is an important cause of morbidity and mortality in cirrhotic patients with ascites. The key step in the pathogenesis of spontaneous bacterial peritonitis is bacterial translocation from intestinal lumen to mesenteric lymph nodes, and from there to the systemic circulation and ascitic fluid. We aimed to study the ascitic microbiota of cirrhotic patients with or without spontaneous bacterial peritonitis. Both the 16S polymerase chain reaction approach and the whole genome shotgun approach were adopted for the next-generation sequencing technology. We compared the results derived from the two methods. The bacterial culture-negative ascites in cirrhotic patients, which even failed for amplification of 16S ribosomal DNA, were found to contain much less bacterial DNA than the culture-positive ones, indicating that the paucity of bacteria, instead of the difficulty of bacterial culture, was possibly the main reason for negative culture result of the ascites. Escherichia coli was the predominant species in all samples, and the bacteria of low abundance were also identified by the next-generation sequencing technology. Whole genome shotgun-based next-generation sequencing is an appropriate method for depicting the microbiome of ascites or of other specimens with a low abundance of bacterial DNA.

Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil

Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses.

Biodegradation of benzoate by protoplast fusant via intergeneric protoplast fusion between Pseudomonas putida and Bacillus subtili

Intergeneric protoplast fusion was performed between Pseudomonas putida, a strain with moderate benzoate tolerance and degradation ability (<2000 mg l−1) while slow growth rate (specific growth rate μ = 4.5 × 10−3 h−1) and Bacillus subtilis, a strain with fast growth rate while intolerance to benzoate. A stable fusant (F29) with enhanced benzoate tolerance (as high as 10 000 mg l−1) and degradation ability (μ = 30.9 × 10−3 h−1) was obtained. The fusant is similar to P. putida in terms of amplified 16S ribosomal DNA restriction analysis (16S-ARDRA) and 16S rDNA clone sequencing analysis while alike to B. subtilis according to Gram staining. Possible biodegradation pathway of benzoate by F29 is benzene ring cleavage between two hydroxyl groups (ortho cleavage) with catechol being intermediate metabolite. However, the verification of benzoate biodegradation mechanism by this fusant requires further investigation.

Molecular Identification of Bacterial DNA in the Chorioretinal Scars of Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is an inherited disorder characterized by defects in phagocyte-derived nicotinamide adenine dinucleotide phosphate oxidase. It is typically diagnosed in childhood and leads to severe, recurrent bacterial or fungal infections. Chorioretinal lesions are the most common ocular manifestation. We sought to determine whether there are infectious agents in CGD-associated chorioretinopathy. Medical records and ocular histopathology from CGD cases from January 1983 to January 2012 at the National Institutes of Health were retrospectively reviewed. Chorioretinal cells from normal and lesional tissues of the same eye were microdissected. Primers for Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia sp., and a panbacterial 16S ribosomal DNA were used for polymerase chain reaction. Seventeen CGD patients had ocular tissues (16 autopsied cases and 1 chorioretinal biopsy) examined. Of these 17, 8 demonstrated CGD-associated chorioretinal lesions in at least one eye on histopathology. Of these 8, 7 showed amplification of 16S ribosomal DNA within the lesion; of these 7, two also amplified S. epidermidis and one P. aeruginosa. One had no bacterial DNA amplified. Importantly, no microbial DNA was amplified from the normal, non-lesional ocular tissues of these 8 cases. Furthermore, only 1 of the 9 eyes without chorioretinopathy had amplified Burkholderia DNA, that patient had a history of Burkholderia infection. We detected bacterial DNA in 7 of 8 (88%) cases with CGD-associated chorioretinopathy and only in 1 normal ocular tissue of 17 CGD cases. Bacterial infection may play a role in the pathogenesis of CGD-associated chorioretinal lesions.

Bacterial assemblages shifts from healthy to yellow band disease states in the dominant reef coral Montastraea faveolata

Descriptions of microbial diversity in healthy and diseased corals are necessary first steps before further investigating the mechanisms that lead to coral pathology. This is the first study that characterizes the microbial associates from healthy corals to yellow band disease (YBD) lesions using two complementary screening techniques of bacterial 16S rRNA genes [amplified 16S ribosomal DNA restriction analysis (ARDRA) of clone libraries and denaturing gradient gel electrophoresis (DGGE)]. Both these techniques showed similar trends, namely a significant difference in the bacterial community and an increase in diversity from healthy to YBD diseased lesions. There was an increase in the number of sequences retrieved of potentially pathogenic bacteria in diseased tissues compared with healthy samples, most notably from the genus Vibrio. Furthermore, we also detected a number of known pathogenic bacteria within the natural healthy microbiota such as Vibrio carchariae and Vibrio harveyi, a result supporting previous studies, showing healthy corals have the ability to harbour these species.

Pulmonary Actinomycosis Caused by &lt;i&gt;Actinomyces cardiffensis&lt;/i&gt;

We herein present the first case of pulmonary actinomycosis caused by Actinomyces cardiffensis (A. cardiffensis). A computed tomography (CT) examination revealed a nodule with cavitation in the left upper lobe of the lung. One month later, the lesion had almost disappeared, but a new nodule with peripheral consolidation had appeared in the right middle lobe. Because organizing pneumonia was suspected, prednisolone was begun and improvement was seen. However, two months after the initiation of corticosteroid administration, a chest CT scan showed a lung abscess. The patient underwent surgical resection of the abscess. A. cardiffensis was identified by an amplified 16S ribosomal DNA restriction analysis of a pus sample.

Prosthetic Joint Infection Caused by Mycobacterium alvei in an Elderly Patient

We report the first case of prosthetic joint infection caused by Mycobacterium alvei, which was identified by PCR-restriction fragment length polymorphism and verified by analysis of nucleotide sequences of its amplified 16S ribosomal DNA. The pathogen was susceptible to linezolid, amikacin, ciprofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. The clinical implications are discussed.

Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. &amp; Zucc.) in different ecoregions of China

A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S-23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study.

Identification of Several Mycobacterium Species by using Amplified 16S Ribosomal DNA

Identification of Several Mycobacterium Species by using Amplified 16S Ribosomal DNA

Fate of Chlortetracycline- and Tylosin-Resistant Bacteria in an Aerobic Thermophilic Sequencing Batch Reactor Treating Swine Waste

Antibiotics have been added to animal feed for decades. Consequently, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria. The objective of this work was to characterize the impact of an aerobic thermophilic biotreatment on aerobic, antibiotic-resistant bacteria in swine waste. The proportion of tylosin- and chlortetracycline-resistant bacteria grown at 25 degrees C, 37 degrees C, and 60 degrees C decreased after treatment, but they were still abundant (10(2) to 10(8) most probable number ml(-1)) in the treated swine waste. The presence of 14 genes conferring resistance to tylosin and chlortetracycline was assessed by polymerase chain reaction in bacterial populations grown at 25 degrees C, 37 degrees C, and 60 degrees C, with or without antibiotics. In 22 cases, genes were detected before but not after treatment. The overall gene diversity was wider before [tet(BLMOSY), erm(AB)] than after [tet(LMOS), erm(B)] treatment. Analysis by denaturing gradient gel electrophoresis of amplified 16S ribosomal DNA (rDNA) fragments generally showed a reduction of the bacterial diversity, except for total populations grown at 60 degrees C and for tylosin-resistant populations grown at 37 degrees C. The latter were further investigated by cloning and sequencing their 16S rDNA. Phylotypes found before treatment were all closely related to Enterococcus hirae, whereas six different phylotypes, related to Pseudomonas, Alcaligenes, and Pusillimonas, were found after treatment. This work demonstrated that the aerobic thermophilic biotreatment cannot be considered as a means for preventing the dissemination of aerobic antibiotic-resistant bacteria and their resistance genes to the environment. However, since pathogens do not survive the biotreatment, the effluent does not represent an immediate threat to animal or human health.

Evaluation of lactic acid bacteria dynamics during plaa-som fermentation using amplified 16S ribosomal DNA restriction analysis

Evaluation of lactic acid bacteria dynamics during plaa-som fermentation using amplified 16S ribosomal DNA restriction analysis

Molecular diversity and phylogeny of rhizobia associated with wild legumes native to Xinjiang, China

A total of 111 rhizobial strains were isolated from wild legumes in Xinjiang, an isolated region of northwest China. Nine genomic species belonging to four genera of Rhizobium, Mesorhizobium, Ensifer, and Bradyrhizobium were defined among these strains based on the characterization of amplified 16S ribosomal DNA restriction analysis (ARDRA), restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA intergenic spacers (IGS), 16S rRNA gene sequencing and multilocus sequence analysis (MLSA). Twenty-five nodC types corresponding to eight phylogenetic clades were divided by RFLP and sequence analysis of the PCR-amplified nodC gene. The acid-producing Rhizobium and Mesorhizobium species were predominant, which may be related to both the local environments and the hosts sampled. The present study also showed the limitation of using nod genes to estimate the host specificity of rhizobia.

Change in the Ileal Bacterial Population of Turkeys Fed Different Diets and After Infection with Salmonella as Determined with Denaturing Gradient Gel Electrophoresis of Amplified 16S Ribosomal DNA

Changes in ileal bacterial populations of Salmonella-infected turkeys fed different diets were analyzed by using 16S-V3 PCR denaturing gradient gel electrophoresis. Turkeys raised on litter flooring were fed wheat- and corn-based diets with and without enzyme preparations (XY1 and XY2, respectively) from 0 to 126 d. Preparation XY1 contained exclusively endoxylanase, whereas preparation XY2 contained endoxylanase, protease, and α-amylase (Danisco, Wiltshire, UK). The dietary activity levels of XY1 and XY2 were 2,500 and 650 endo-1,4-β-xylanase units/kg of feed, respectively. Microbial DNA was extracted from the ileal content of 16-wk-old turkeys, and the 16S rDNA gene was amplified by PCR and analyzed by denaturing gradient gel electrophoresis. Diversity indexes, including richness (number of species, S), evenness (relative distribution of species, EH), diversity (using Shannon's index, H′), and Sorenson's pairwise similarities coefficient (measures the species in common between different habitats, Cs) were calculated. Additionally, diversity indexes were associated with Salmonella prevalence determined from fresh fecal droppings collected from each pen. On the basis of contrast analysis, the wheat-based diets resulted in higher microbial diversity indexes than the corn-based diets (S = 10 vs. 12; EH = 0.9 vs. 0.8; H′ = 2.2 vs. 1.9, P < 0.05). Likewise, enzyme supplementation stimulated growth of the microbiota and increased the diversity indexes in comparison with unsupplemented treatments (S = 13 vs. 10; EH = 0.9 vs. 0.8; H′ = 2.2 vs. 1.9, P < 0.05). Salmonella prevalence was higher (P < 0.05) at 15 wk in turkeys fed the corn-based diet (Salmonella prevalence = 50%) than in turkeys fed the corn-enzyme (Salmonella prevalence = 13%) and wheat-based (Salmonella prevalence = 0%) dietary treatments. Therefore, contrast analysis showed that birds fed the corn control diet had lower microbiota diversity but higher Salmonella prevalence than birds fed the enzyme-supplemented and wheat-based diets. In contrast, birds fed the wheat-based diets had higher diversity but lower Salmonella prevalence than birds fed the corn-based diets. High dietary nonstarch polysaccharides from wheat and dietary exogenous enzyme supplementation promoted microbial community diversity and apparently discouraged Salmonella colonization through competitive exclusion. Nonstarch polysaccharides and dietary exogenous enzyme supplementation may be practical tools to control enteric pathogens and benefit the intestinal health and food safety of the birds.