Full-length 16S rDNA Research Articles

Significant difference of the culturable anaerobic microbial species in GujingTribute pitmud with different ages

Most Chinese liquor belongs to strong-aroma type, and GujingTribute liquor with a history over 1800 years is one of this type. The flavor of strong-aroma Chinese liquor is largely determined by anaerobic bacteria embedded in the pits, especially the bottom area of the old pits. In this study, culturable anaerobic species in the old (50-100 years old) and young (5 years old) pitmuds were isolated with anaerobic cultivation and characterized by near full-length 16S rDNA sequencing. Ruminococcaceae bacterium CPB6 and Lactobacillus acetotolerans were found as the most culturable species in the freshly-sampled old and young pitmud samples, respectively. This result confirmed our previous findings by high throughput next generation sequencing that Ruminococcaceae bacterium CPB6 was more abundant in good quality pitmud while Lactobacillus acetotolerans was more abundant in ordinary or poor quality pitmud.

Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny.

Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (<500bp) 18S ribosomal DNA (rDNA) fragments, the use of full-length gene sequences (>2200bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15.

Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.

A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed ≥13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release.

An 18S rDNA Perspective on the Classification of Criconematoidea

In the nematode family Criconematidae, a taxonomy primarily based on cuticle characters has created classifications that are notoriously volatile. Molecular characters may lead to their stabilization. A phylogenetic tree of Criconematoidea was constructed using 166 new near full-length 18S rDNA sequences and 58 sequences from GenBank. Bayesian and maximum likelihood (ML) analyses produced trees with similar topologies. Major features include a strongly supported clade that includes Criconematidae and Hemicycliophoridae, excluding Paratylenchidae and Tylenchulidae. Another well-supported clade groups Criconema, Ogma, Crossonema, and Hemicriconemoides plus Xenocriconemella, combining nematodes with cuticular scales with those without scales at any life stage. Mesocriconema, Discocriconemella limitanea, Hemicaloosia, and Lobocriconema are recognized as monophyletic groups, but Criconemoides is paraphyletic. Both trees support an unexpected sister relationship between Bakernema and Hemicycliophora. The 18S rDNA dataset was insufficient for distinguishing genus boundaries between Criconema, Ogma, and Crossonema. The relationships depicted by the 18S rDNA phylogeny suggest that key morphological characters used in the classification of Criconematidae are not homologous.

Unique sequence characteristics account for good DGGE separation of almost full-length 18S rDNAs.

A major limiting factor for DGGE-based microbial community studies is that the fragments should not be much longer than 500bp for successful analysis. However, relatively high-resolution was achieved based on DGGE of the long 18S rDNA fragment (>1500bp), which might be surprising due to the known decrease in DGGE resolution of DNA molecules with large melted regions. A unique sequence characteristic was found in a specific region (ca. 275bp, named the NS1-end region) of 18S rDNAs, and fungal communities separated from Hong Qu glutinous rice wine brewing system was used to reveal the relationship between high resolution capacity and the unique sequence characteristics. The results showed that DGGE separation of the long 18S rDNA fragments depended on their NS1-end regions. The region is composed of a sequence-variable and short-length GC-poor region (ca. 160bp) and a GC-rich region (ca. 110bp), which contribute to the high resolution capacity achieved for DGGE of the long 18S rDNA fragments. Thus DGGE of the long 18S rDNA fragment is recommended as a target fragment for studies of fungal communities whose 18S rDNAs possess similar sequence characteristics. Good resolution and almost full-length 18S rDNA sequences can thus be obtained to provide more accurate and reliable analysis of fungal communities. Since more sequences are obtained directly from the PCR product through the long rDNA fragment approach, this is a convenient and effective approach for sequence-based analysis without using other complementary methods such as an rDNA clone library method.

Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer.

BackgroundThe miniaturised and portable DNA sequencer MinION™ has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinION™ system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences.ResultsUsing R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90 % of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using ‘universal’ primers.ConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinION™ DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.

Description of Lelenchus brevislitus n. sp. (Nematoda: Tylenchidae), an example of a cryptic species from Iran and its phylogenetic relationships with other species in the family

Lelenchus brevislitusn. sp. is described and illustrated based on morphological, morphometric and molecular data. The new species is characterised by having females with slender, 452-488 μm long, body lateral field vestigial as a plain simple band under scanning electron microscopy (SEM) in vulval region and absent in proximal and distal body ends, cephalic region short,ca2 μm high, conical and truncate in anterior end in lateral view under SEM, dorso-ventrally flattened, amphidial pouches pocket-shaped and large under light microscopy (LM), their apertures short (ca1 μm long in SEM) longitudinal sinuous slits remarkably wide near the small oral disc and significantly narrowing posteriad stylet, slender. 6.5-8.0 μm long, developed median bulb with distinct valvular plates, monodelphic-prodelphic reproductive system having a short post-vulval uterine sac (PUS), transverse vulval slit (V = 47.5-51.7) bearing small lateral membranes on each side, long filiform female tail (154-170 μm, c = 2.8-3.1, c′ = 21.5-28.3), and males unknown. The new species is considered to be a cryptic species inside the Tylenchidae. Morphological comparisons with all species of the genus are discussed. Phylogenetic studies were performed for further characterisation of the new species, and the results, using nearly full length 18S rDNA data, revealed it formed a clade with an isolate ofL. leptosoma, both of which are in a sister clade with an isolate ofSakia. In a reconstructed phylogenetic tree using partial 28S rDNA sequences, the new species formed a monophyletic group, again withL. leptosoma, a sister clade to someMalenchusspp.

CRYPTIC HAPLOTYPES OF “CANDIDATUS LIBERIBACTER AFRICANUS”

“Candidatus Liberibacter africanus” (Laf) has long been recognised as a causal agent of the devastating citrus disease huanglongbing (HLB) or citrus greening. This species is currently restricted to Africa, the Arabian Peninsula and some Indian Ocean islands and is vectored by the African citrus psyllid, Trioza erytreae. Blotchy mottle on citrus leaves is characteristic of the disease. Somewhat similar symptoms in the rutaceous tree Calodendrum capensis (Cape chestnut) resulted in the discovery of Laf outside commercial citrus crops in South Africa. This was classed as a subspecies of Laf (capensis, hence LafC). In subsequent surveys of commercial citrus crops and Calodendrum, both natural and ornamental specimens, LafC was not found in citrus, nor has Laf been found in C. capensis. HLB was reported from Madagascar in 1968 but no sequences from this source have so far been published. Until fairly recently, only the reference 16S rRNA gene sequences of Laf (L22533) and LafC (AF137368) had been deposited in GenBank. Both of these reference sequences contain a number of unresolved nucleotides. When these nucleotide positions are invstigated by aligning against more recently available sequences, it becomes evident that these unresolved positions represent one percentage point difference in similarity between Laf and LafC. The originally reported 97.4% similarity is therefore incorrect based on this new information. Recalculating the similarity on the full length 16S rDNA sequence results in 99.54% similarity, a value too high to justify a subspecies status. LafC should therefore be downgraded as a haplotype of Laf. Further, the six 16S rRNA gene sequences currently available in GenBank identified as Laf as a species separate into two haplotype groups. The three haplotypes of Laf are therefore: (i) LafA, designated as the first accession sequenced (L22533); (ii) LafC, i.e the former subspecies “capensis”, to recognise the priority in the use of this term; (iii) LafB, the third previously unrecognised haplotype. Thus the cryptic presence of three haplotypes is revealed by this review of the Laf 16S rDNA sequences.

First report of Tragopogon dubius witches' broom disease associated with a subgroup 16SrI‐B phytoplasma in Iran

Tragopogon dubius (yellow salsify, Asteraceae) is a perennial herb in Iran. In a survey conducted for phytoplasma diseases in 2014, T. dubius witches' broom disease was observed in vineyards and green areas of Bajgah and Zarghan localities (Fars province, Iran). The characteristic symptoms of T. dubius witches' broom were flower virescence and phyllody, crown proliferation and witches' broom (Fig. 1). Since the symptoms were suggestive of phytoplasma infection, plants were assayed for presence of phytoplasma by PCR amplification. Total DNA was extracted from 0.3 g of midrib tissue of four witches' broom affected and two symptomless T. dubius plants (two plants per location), using the procedure of Zhang et al. (6). Total DNA samples were tested by both direct and nested PCR assays using phytoplasma universal 16S rDNA primer pairs P1/P7 (Deng & Hiruki, 1; Schneider et al., 4) and R16F2n/R16R2 (Gundersen & Lee, 2) that amplify fragments of 1800 bp and 1250 bp, respectively. Expected fragments were amplified following direct and nested PCR from all symptom-bearing but not from symptomless plants. P1/P7-primed PCR products amplified from four witches' broom-affected T. dubius plants were separately cloned and sequenced. The full-length 16S rDNA sequences displayed 100% sequence identity with each other. A consensus sequence corresponding to the Bajgah T. dubius witches' broom was deposited in GenBank (Accession No. KR262955). A Blast search showed that this sequence had 100% identity with Oenothera phytoplasma (‘Candidatus Phytoplasma asteris‘) (M30790), representative of subgroup 16SrI-B. Virtual RFLP analysis using iPhyClassifier online tool (http://www.ba.ars.usda.gov/data/mppl/iphyclassifier), carried out with 17 restriction enzymes, indicated the presence of a 16SrI-B phytoplasma in witches' broom-affected T. dubius plants (Fig. 2). Phylogenetic analysis of full length 16S rDNA sequences using MEGA version 5 (Tamura et al., 5) grouped T. dubius witches' broom phytoplasma with onion yellows phytoplasma (GenBank Accession No. D12569), a member of 16SrI-B subgroup (Fig. 3). To our knowledge this is the first report of natural occurrence and partial molecular characterisation of a phytoplasma associated with T. dubius witches' broom disease. A 16SrI-B subgroup phytoplasma has previously been reported as causal agent of rapeseed phyllody disease in Zarghan (Salehi et al., 3). The significance of diseased T. dubius as natural reservoir of rapeseed phyllody phytoplasma remains to be determined. Aster yellows phytoplasmas were reported in grapevine in several countries. However, possible transmission of T. dubius witches' broom phytoplasma to grapevine is yet to be tested.

Comparative Analysis of Vaginal Bacterial Diversity in Northern-Chinese Women Associated With or Without Bacterial Vaginosis

Background: Bacterial Vaginosis (BV) is the common vaginal infection in women and it has been linked to enhanced risks for pre-term birth, pelvic inflammatory disease and sexually transmitted diseases. BV is caused by a disorder of vaginal microbiota which changes from the normal lactobacillus dominated community to a more diverse community of non-lactobacillus bacteria. Several previous reports analyzed the overall vaginal microbial communities of volunteers from limited sampling area and they suggested a possible link between the vaginal microbial contents and the ethnicity of women. Here, we analyzed the diversity of vaginal microbiota in 10 subjects associated with BV (BV+) and 10 subjects without BV (BV-) from the metropolitan area of Herbing in Northern China using full-length 16S rDNA. Results: The vaginal bacterial communities detected in subjects with BV were much more taxon rich and diverse than those without BV. At a 97% sequence similarity cutoff, the number of operational taxonomic units (OTUs) per 10 subjects with BV was nearly three times greater than 10 subjects without BV by 29.4 ± 9.3 versus 11.7 ± 7.8 (Mean ±SD). Our data confirmed that there is a shift in the abundance of bacterial species present in the vaginal environment when BV and non-BV groups were compared. Each sequence read was assigned to a genus or a species when possible. Principal component analysis was performed at genus levels. Most BV+ samples clustered together while there were two clusters among BV- samples. Several bacteria have been found to be associated with BV, including Gardnerella, Atopobium, Peptoniphilus, Leptotrichia/Sneathia, Prevotella, Parvimonas and Dialister, Based on result of classification, four possible novel phylotype microorganisms were found. Conclusions: The data presented here on the composition and richness of the vaginal microbial ecosystem in BV and health state will provide the depth insight in the etiology of BV.

IM-TORNADO: a tool for comparison of 16S reads from paired-end libraries.

Motivation16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads.ResultsWe created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity.Availability and ImplementationIM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq.

The feline oral microbiome: A provisional 16S rRNA gene based taxonomy with full-length reference sequences

The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species.The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria.Clone libraries were produced using “universal” and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees.The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as “Propionibacterium sp. feline oral taxon FOT-327” is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

Host-associated bacterial taxa from Chlorobi, Chloroflexi, GN02, Synergistetes, SR1, TM7, and WPS-2 Phyla/candidate divisions

Background and objectiveIn addition to the well-known phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Spirochaetes, Fusobacteria, Tenericutes, and Chylamydiae, the oral microbiomes of mammals contain species from the lesser-known phyla or candidate divisions, including Synergistetes, TM7, Chlorobi, Chloroflexi, GN02, SR1, and WPS-2. The objectives of this study were to create phyla-selective 16S rDNA PCR primer pairs, create selective 16S rDNA clone libraries, identify novel oral taxa, and update canine and human oral microbiome databases.Design16S rRNA gene sequences for members of the lesser-known phyla were downloaded from GenBank and Greengenes databases and aligned with sequences in our RNA databases. Primers with potential phylum level selectivity were designed heuristically with the goal of producing nearly full-length 16S rDNA amplicons. The specificity of primer pairs was examined by making clone libraries from PCR amplicons and determining phyla identity by BLASTN analysis.ResultsPhylum-selective primer pairs were identified that allowed construction of clone libraries with 96–100% specificity for each of the lesser-known phyla. From these clone libraries, seven human and two canine novel oral taxa were identified and added to their respective taxonomic databases. For each phylum, genome sequences closest to human oral taxa were identified and added to the Human Oral Microbiome Database to facilitate metagenomic, transcriptomic, and proteomic studies that involve tiling sequences to the most closely related taxon. While examining ribosomal operons in lesser-known phyla from single-cell genomes and metagenomes, we identified a novel rRNA operon order (23S-5S-16S) in three SR1 genomes and the splitting of the 23S rRNA gene by an I-CeuI-like homing endonuclease in a WPS-2 genome.ConclusionsThis study developed useful primer pairs for making phylum-selective 16S rRNA clone libraries. Phylum-specific libraries were shown to be useful for identifying previously unrecognized taxa in lesser-known phyla and would be useful for future environmental and host-associated studies.

Effect of antimicrobial growth promoter administration on the intestinal microbiota of beef cattle

BackgroundAntimicrobial growth promoters (AGPs) are antimicrobial agents administered to livestock in feed for prolonged periods to enhance feed efficiency. Beef cattle are primarily finished in confined feeding operations in Canada and the USA, and the administration of AGPs such as chlortetracycline and sulfamethazine (Aureo S-700 G) is the standard. The impacts of AGPs on the intestinal microbiota of beef cattle are currently uncertain; it is documented that AGPs administered to beef cattle pass through the rumen and enter the intestine. To ascertain the impacts of Aureo S-700 G on the small and large intestinal microbiota of beef cattle (mucosa-associated and within digesta), terminal restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR) for total bacteria were applied. Beef cattle were maintained in an experimental feedlot (five replicate pens per treatment), and AGP treatment cattle were administered Aureo S-700 G in feed, whereas control cattle were administered no antimicrobials. As the intestinal microbiota of beef cattle has not been extensively examined, clone library analysis was applied to ascertain the primary bacterial constituents of the intestinal microbiota.ResultsComparative T-RFLP and qPCR analysis (n = 122 samples) revealed that bacterial community fingerprints and bacterial load within digesta differed from those associated with mucosa. However, the administration of Aureo S-700 G did not affect bacterial community fingerprints or bacterial load within the small and large intestine relative to control cattle. Analysis of >1500 near full length 16S rDNA clones revealed considerably greater bacterial diversity in the large relative to the small intestine of beef cattle. Mucosa-associated bacterial communities in the jejunum were dominated by Proteobacteria, and differed conspicuously from those in the ileum and large intestine. Although the ileum contained bacterial clones that were common to the jejunum as well as the cecum, Firmicutes clones associated with mucosa dominated in the ileum, cecum, and descending colon. In the descending colon, clone library analysis did not reveal a difference in the richness or diversity of bacterial communities within digesta relative to those associated with mucosa. However, T-RFLP analysis indicated a significant difference in T-RF relative abundance (i.e. difference in relative taxon abundance) between mucosa-associated and digesta communities attributed in part to the differential abundance of Bacteriodes, Alistipes, Oscillibacter, and unclassified Clostridiales.ConclusionsThese data demonstrate that there was no significant difference in the composition of the predominant intestinal bacteria constituents within animals administered Aureo S-700 G and those not administered AGPs after a 28 day withdrawal period.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Ageratum conyzoides in China.

Ageratum conyzoides L. is believed to act as reservoir host for many plant diseases. In June 2011, a 30% incidence of bacterial wilt on A. conyzoides was observed in a field of Rhizoma kaempferiae in Yangchun city of Guangdong province. The initial symptoms were wilting of the apical leaves during the day, which recovered at night. After 4 to 6 days, the leaves became totally necrotic. The basal stems of the diseased plants were blackened and the vascular tissue turned brown. To investigate the disease etiology before understanding the disease link between A. conyzoides and R. kaempferiae, 10 plants with typical wilting symptoms were collected from the field. A total of 10 bacterial isolates were isolated from the vascular tissue of each diseased plant on tripheny tetrazolium chloride (TZC) medium. After incubation at 30°C for 2 days, the plates had large, irregular round, fluidal, white colonies with a pink center. Thirty healthy A. conyzoides plants at the four- to six-leaf growth stage were inoculated by injuring the roots and soaking them in a bacterial suspension (1 × 108 cfu/ml) for 20 min with the 10 bacterial isolates separately, and planted in 10-cm pots with sterile gardening soil in a glasshouse (28 to 35°C). Sterile water was used as a negative control. Five days after inoculation, a few leaves of the inoculated plants began to exhibit wilting. The inoculated plants eventually showed the same symptoms as those in the field. The same bacterium was reisolated from inoculated plants. The 30 negative control plants did not have wilt symptoms. With the same inoculation procedure, the bacterium also caused wilting on tomato (25 of 30), pepper (10 of 30), eggplant (2 of 30), ginger (11 of 15), and R. kaempferiae (8 of 15). Using the universal bacterial 16S rDNA primer set 27f/1541R (3), approximately 1,400 bp-fragments were amplified from the 10 isolates, respectively. The sequences for the 10 fragments (GenBank Accession Nos. JX294065 to JX294074) were identical and had 100% sequence identity with 16S rDNA of R. solanacearum GMI1000 (AL646052). The 10 isolates were able to oxidize disaccharides (lactose, maltose, and cellobiose) and hexose alcohols (mannitol, dulcitol, and sorbitol). According to Hayward's classification, all isolates were biovar 3 (2). Based on the pathogenicity tests, carbohydrate utilization, and near full-length 16S rDNA sequences, the bacterial isolates from the diseased A. conyzoides belonged to race 4 and biovar 3 of R. solanacearum. Furthermore, the specific 280-bp and 140-bp fragments were respectively amplified from all 10 isolates by using the multiplex PCR (1). In addition, specific 165-bp fragments were amplified from all the isolates using the specific primers AKIF/AKIR (3), which indicates the bacterium belongs to R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on A. conyzoides in China.

Biopolymer generation from sweet sorghum juice: screening, isolation, identification, and fermentative polyhydroxyalkanoate production by Bacillus aryabhattai

This study aims to use sweet sorghum juice (SSJ) to produce an environmentally friendly biopolymer of polyhydroxyalkanoates (PHAs) by bacteria isolated from soil. Among 18 isolates, only 3 isolates, coded as S4, S6, and S11, were chosen to produce PHA via batch fermentation compared to 3 strains of Alcaligenes eutrophus, A. latus, and Hydrogenophaga sp., respectively, under controlled conditions at pH 7, 35 °C, and 20 g/L of initial total sugar in the SSJ. The results clearly showed that the isolate S4 can produce maximum PHAs of 1.74 g/L with 57.62% dry cell weight and yield maximum hourly productivity at 0.097 g/L. S4 was further identified by full-length 16S rDNA gene sequence homology, and the findings showed that it was closely related to Bacillus aryabhattai (99.70% similarity). Interestingly, there has been as of yet no report on the use of this strain to produce an added-value biopolymer of PHA.

Molecular characterization of a phytoplasma of the ash yellows group occurring in strawberry (Fragaria x ananassa Duch.) plants in Argentina

Strawberry (Fragaria x ananassa Duch. cv. Camino Real) plants showing phyllody symptoms were detected in production fields in Bella Vista (Corrientes province, Argentina). DNA from all symptomatic samples analyzed yielded fragments of the expected size in PCR reactions using phytoplasma universal primers. DNA from asymptomatic plants yielded no products. The associated phytoplasma was designated as Argentinean Strawberry Phyllody (ASP). The ASP sequence of the 16S rRNA gene showed 99–98% homology with members of the 16SrVII ash yellows group. The putative RFLP profile was indistinguishable from the Argentinean alfalfa witches’ broom (ArAWB) phytoplasma. The phylogenetic analysis of nearly full-length 16S rDNA sequence and 16S-23S spacer region yielded a consensus tree wherein ASP clustered into the ash yellows group (16Sr VII) with high confidence values (95), generating a separated branch (100 bootstrap value) together with ArAWB (16Sr VII-C). In Argentina, the 16Sr VII group was also detected in alfalfa (Medicago sativa L.) crops, and weeds [Artemisa annua L. and Conyza bonariensis (L.) Cronquist]. To our knowledge, this is the first report of a phytoplasma from the 16Sr VII group affecting strawberries in Argentina and worldwide.

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 108 cfu/ml of Escherichia coli and 105 cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.

Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15μm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99–100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.

Production of conjugated linoleic acids by Lactobacillus plantarum strains isolated from naturally fermented Chinese pickles.

Naturally fermented pickles harbour many lactic acid bacteria (LAB). Forty-three LAB strains with conjugated linoleic acid (CLA)-producing ability were isolated from three naturally fermented pickle brines. Of these isolates, lp15 identified as Lactobacillus plantarum by API 50 CHL system and full-length 16S rDNA sequence analysis exhibited the highest CLA-producing ability (26.1% conversion) at 48 h in de Man Rogosa Sharpe (MRS) broth in the presence of 100 µg/ml of linoleic acid (LA). Compared to other strains, L. plantarum strain lp15 showed the highest tolerance upon increased levels of LA in the medium, i.e., up to 600 µg/ml. This strain converted about 25% of LA into CLA isomers [predominantly cis-9, trans-11 CLA (9-CLA) and trans-10, cis-12 CLA (10-CLA)], of which 75% was 9-CLA. Interestingly, though the conversion rate of LA into CLA by lp15 remained stable between 100 to 600 µg/ml LA levels in the medium, it dropped sharply at 1000 µg/ml. Taken together, the lp15 strain displayed relatively high LA tolerance with higher conversion rate, which implies that this strain is a valuable candidate for enhancing the CLA content in food-sources like pickles.