Genome Wide Search for Biomarkers to Diagnose Yersinia Infections

Bacterial identification on the basis of the highly conserved 16S rRNA (rrs) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4–6 base cutters). Yersinia species have 6–7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences—carB, fadJ, gluM, gltX, ileS, malE, nusA, ribD, and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, Y. rohdei, Y. ruckeri, and Y. similis. This approach can be applied for rapid diagnostic applications.

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Searching Biomarkers in the Sequenced Genomes of <Emphasis Type="Italic">Staphylococcus</Emphasis> for their Rapid Identification

Bacterial identification using rrs (16S rRNA) gene is widely reported. Bacteria possessing multiple copies of rrs lead to overestimation of its diversity. Staphylococcus genomes carries 5–6 copies of rrs showing high similarity in their nucleotide sequences, which lead to ambiguous results. The genomes of 31 strains of Staphylococcus representing 7 species were searched for the presence of common genes. In silico digestion of 34 common genes using 10 restriction endonucleases (REs) lead to select gene-RE combinations, which could be used as biomarkers. RE digestion of recA allowed unambiguous identification of 13 genomes representing all the 7 species. In addition, a few more genes (argH, argR, cysS, gyrB, purH, and pyrE) and RE combinations permitted further identification of 12 strains. By employing additional RE and genes unique to a particular strain, it was possible to identify the rest 6 Staphylococcus aureus strains. This approach has the potential to be utilized for rapid detection of Staphylococcus strains.

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A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for <Emphasis Type="Italic">Streptococcus</Emphasis>

The use of rrs (16S rRNA) gene is widely regarded as the “gold standard” for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4–7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.

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Genome Wide Analysis for Searching Novel Markers to Rapidly Identify Clostridium Strains

Microbial classification is based largely on the 16S rRNA (rrs) gene sequence, which is conserved throughout the prokaryotic domain. The Ribosomal Database Project (RDP) has become a reference point for almost all practical purposes. The use of this gene is limited by the fact that it can be used to identify only to the extent to what has been known and is available in the RDP. In order to identify an organism whose rrs is not present in the RDP database, we need to generate novel markers to place the unknown on the evolutionary map. Here, sequenced genomes of 27 Clostridium strains belonging to 9 species have been used to identify two sets of genes: (1) common to most of the species, and (2) unique to a species. Combinations of genes (recN, dnaJ, secA, mutS, and/or grpE) and their unique restriction endonuclease digestion (AluI, BfaI and/or Tru9I) patterns have been established to rapidly identify Clostridium species. This strategy for identifying novel markers can be extended to all other organisms and diagnostic applications.

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Development of Genomic Tools for the Identification of Certain Pseudomonas up to Species Level

Pseudomonas is a highly versatile bacterium at the species level with great ecological significance. These genetically and metabolically diverse species have undergone repeated taxonomic revisions. We propose a strategy to identify Pseudomonas up to species level, based on the unique features of their 16S rDNA (rrs) gene sequence, such as the frame work of sequences, sequence motifs and restriction endonuclease (RE) digestion patterns. A species specific phylogenetic framework composed of 31 different rrs sequences, allowed us to segregate 1,367 out of 2,985 rrs sequences of this genus, which have been classified at present only up to genus (Pseudomonas) level, as follows: P. aeruginosa (219 sequences), P. fluorescens (463 sequences), P. putida (347 sequences), P. stutzeri (197 sequences), and P. syringae (141 sequences). These segregations were validated by unique 30–50 nucleotide long motifs and RE digestion patterns in their rrs. A single gene thus provides multiple makers for identification and surveillance of Pseudomonas.     

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m²) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.     

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.     

Resistance of Auto- and Allotetraploid Triticeae Species and Accessions to Meloidogyne chitwoodi based on Genome Composition

The Columbia root-knot nematode Meloidogyne chitwoodi parasitizes several plant species, including grasses that have been developed for semiarid environments, and substantially reduces the productivity of cereals and the longevity of perennial grasses growing under semiarid conditions throughout the intermountain region. Thirty-two auto- and allotetraploid (2n = 28) taxa in the perennial Triticeae were evaluated as possible sources of resistance to M. chitwoodi. Low levels of root galling were observed on roots of all accessions; root-gall indices ranged from 0 (no galls) to 1.95 in the grasses compared to 4.67 for the susceptible ''Ranger'' alfalfa check on a scale of 1 to 6. Even though the gall ratings were low, significant (P < 0.01) differences among accessions of the same species, among species, and among genera with different genomes were observed. Within the reproductive indices, which ranged from 0.01 to 1.20 in the grasses compared to 65.38 for the alfalfa check, there was no difference among genera with different genomes and accessions within the same species and genome; however, there was a significant (P < 0.05) difference among species with the same genomes. This variation can be traced to Thinopyrum nodosum (Jaaska-19), which was the only accession with a reproductive factor greater than 1.00. Based on the data, all auto- and allotetraploids are considered resistant to M. chitwoodi.     

Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas

The life cycle of Vibrio parahaemolyticus has been conventionally associated with estuarine areas characterized by moderate salinity and warm seawater temperatures. Recent evidence suggests that the distribution and population dynamics of V. parahaemolyticus may be shaped by the existence of an oceanic transport of communities of this organism mediated by zooplankton. To evaluate this possibility, the presence of V. parahaemolyticus in the water column of offshore areas of Galicia was investigated by PCR monthly over an 18-month period. Analysis of zooplankton and seawater showed that the occurrence of V. parahaemolyticus in offshore areas was almost exclusively associated with zooplankton and was present in 80% of the samples. The influence of environmental factors assessed by generalized additive models revealed that the abundance and seasonality of V. parahaemolyticus in zooplankton was favoured by the concurrence of downwelling periods that promoted the zooplankton patchiness. These results confirm that offshore waters may be common habitats for V. parahaemolyticus, including strains with virulent traits. Additionally, genetically related populations were found in offshore zooplankton and in estuaries dispersed along 1500 km. This finding suggests that zooplankton may operate as a vehicle for oceanic dispersal of V. parahaemolyticus populations, connecting distant regions and habitats, and thereby producing impacts on the local community demography and the spread of Vibrio-related diseases.     

Complete Mitochondrial Genome of Anoplocephala magna Solidifying the Species

The 2 species of the genus Anoplocephala (Anoplocephalidae), A. perfoliata and A. magna, are among the most important equine cestode parasites. However, there is little information about their differences at the molecular level. The present study revealed that the mitochondrial (mt) genome of A. magna was 13,759 bp in size and 700 bp shorter than that of A. perfoliata. The 2 species includes 2 rRNA, 22 tRNA, and 12 protein-coding genes each. The size of each of the 36 genes was the same as that of A. perfoliata, except for cox1, rrnL, trnC, trnS2(UCN), trnG, trnH, trnQ, and trnP. In the full mitochondrial genome, the sequence similarity was 87.1%. The divergence in the nucleotide and amino acid sequences of individual protein-coding genes ranged from 11.1% to 16% and 6.8% to 16.4%, respectively. The 2 noncoding regions of the mt genome of A. magna were 199 bp and 271 bp in length, while the equivalent regions in A. perfoliata were 875 bp and 276 bp, respectively. The results of this study support the proposal that A. magna and A. perfoliata are separate species, consistent with previous morphological analyses.     

A Polymerase Chain Reaction Method for Identification of Five Major Meloidogyne Species

A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3'' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.     

Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization

Coral disease outbreaks have increased over the last three decades, but their causal agents remain mostly unclear (e.g., bacteria, viruses, fungi, protists). This study details a 14‐month‐long survey of coral colonies in which observations of the development of disease was observed in nearly half of the sampled colonies. A bimonthly qPCR method was used to quantitatively and qualitatively evaluate Symbiodinium assemblages of tagged colonies, and to detect the presence of Vibrio spp. Firstly, our data showed that predisposition to disease development in general, and, more specifically, infection by Vibrio spp. in Acropora cytherea depended on which clades of Symbiodinium were harbored. In both cases, harboring clade D rather than A was beneficial to the coral host. Secondly, the detection of Vibrio spp. in only colonies that developed disease strongly suggests opportunistic traits of the bacteria. Finally, even if sporadic cases of switching and probably shuffling were observed, this long‐term survey does not suggest specific‐clade recruitment in response to stressors. Altogether, our results demonstrate that the fitness of the coral holobiont depends on its initial consortium of Symbiodinium, which is distinct among colonies, rather than a temporary adaptation achieved through acquiring different Symbiodinium clades.     

Taxonomic and Molecular Identification of Mesocriconema and Criconemoides Species (Nematoda: Criconematidae)

Populations of Mesocriconema curvatum, M. kirjanovae, M. onoense, M. ornatum, M. sphaerocephala, M. surinamense, M. vadense, M. xenoplax, and Criconemoides informis from different geographical areas in the continental United States were characterized morphologically and molecularly. A new ring nematode from Washington County, Arkansas, is also described and named Mesocriconema ozarkiense n. sp., This new species is characterized by females with small flattened submedian lobes, lower than or at the same level as the labial disc, vagina straight, very well developed spermatheca without sperm, no more than one anastomoses, L=379-512 μm, V=89-93, stylet length = 49-61 μm, R=107-119, annuli with slightly crenate margins on tail portion and a simple anterior vulval lip. The molecular characterization of M. ozarkiense n. sp. using the ITS rRNA gene sequence and the phylogenesis relationship of this new species with the ring nematodes included in this study are provided.     

Variant Identification in Multi-Sample Pools by Illumina Genome Analyzer Sequencing

Multi-sample pooling and Illumina Genome Analyzer (GA) sequencing allows high throughput sequencing of multiple samples to determine population sequence variation. A preliminary experiment, using the RET proto-oncogene as a model, predicted ≤30 samples could be pooled to reliably detect singleton variants without requiring additional confirmation testing. This report used 30 and 50 sample pools to test the hypothesized pooling limit and also to test recent protocol improvements, Illumina GAIIx upgrades, and longer read chemistry. The SequalPrep^TM method was used to normalize amplicons before pooling. For comparison, a single ‘control’ sample was run in a different flow cell lane. Data was evaluated by variant read percentages and the subtractive correction method which utilizes the control sample. In total, 59 variants were detected within the pooled samples, which included all 47 known true variants. The 15 known singleton variants due to Sanger sequencing had an average of 1.62±0.26% variant reads for the 30 pool (expected 1.67% for a singleton variant [unique variant within the pool]) and 1.01±0.19% for the 50 pool (expected 1%). The 76 base read lengths had higher error rates than shorter read lengths (33 and 50 base reads), which eliminated the distinction of true singleton variants from background error. This report demonstrated pooling limits from 30 up to 50 samples (depending on error rates and coverage), for reliable singleton variant detection. The presented pooling protocols and analysis methods can be used for variant discovery in other genes, facilitating molecular diagnostic test design and interpretation.     

Vibrio bacteria in raw oysters: managing risks to human health

The human-pathogenic marine bacteria Vibrio vulnificus and V. parahaemolyticus are strongly correlated with water temperature, with concentrations increasing as waters warm seasonally. Both of these bacteria can be concentrated in filter-feeding shellfish, especially oysters. Because oysters are often consumed raw, this exposes people to large doses of potentially harmful bacteria. Various models are used to predict the abundance of these bacteria in oysters, which guide shellfish harvest policy meant to reduce human health risk. Vibrio abundance and behaviour varies from site to site, suggesting that location-specific studies are needed to establish targeted risk reduction strategies. Moreover, virulence potential, rather than simple abundance, should be also be included in future modeling efforts.     

Coral-mucus-associated Vibrio integrons in the Great Barrier Reef: genomic hotspots for environmental adaptation

Integron cassette arrays in a dozen cultivars of the most prevalent group of Vibrio isolates obtained from mucus expelled by a scleractinian coral (Pocillopora damicornis) colony living on the Great Barrier Reef were sequenced and compared. Although all cultivars showed >99% identity across recA, pyrH and rpoB genes, no two had more than 10% of their integron-associated gene cassettes in common, and some individuals shared cassettes exclusively with distantly-related members of the genus. Of cassettes shared within the population, a number appear to have been transferred between Vibrio isolates, as assessed by phylogenetic analysis. Prominent among the mucus Vibrio cassettes with potentially inferable functions are acetyltransferases, some with close similarity to known antibiotic-resistance determinants. A subset of these potential resistance cassettes were shared exclusively between the mucus Vibrio cultivars, Vibrio coral pathogens and human pathogens, thus illustrating a direct link between these microbial niches through exchange of integron-associated gene cassettes.     

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors

Background

The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.

Results

The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed.

Conclusions

Strains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.

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New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities

Bleaching of Oculina patagonica has been extensively studied in the Eastern Mediterranean Sea, although no studies have been carried out in the Western basin. In 1996 Vibrio mediterranei was reported as the causative agent of bleaching in O. patagonica but it has not been related to bleached or healthy corals since 2003, suggesting that it was no longer involved in bleaching of O. patagonica. In an attempt to clarify the relationship between Vibrio spp., seawater temperature and coral diseases, as well as to investigate the putative differences between Eastern and Western Mediterranean basins, we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. Two sampling points located in the Spanish Mediterranean coast were chosen for this study: Alicante Harbour and the Marine Reserve of Tabarca. A complex and dynamic assemblage of Vibrio spp. was present in O. patagonica along the whole year and under different environmental conditions and coral health status. While some Vibrio spp. were detected all year around in corals, the known pathogens V. mediteranei and V. coralliilyticus were only present in diseased specimens. The pathogenic potential of these bacteria was studied by experimental infection under laboratory conditions. Both vibrios caused diseased signs from 24 °C, being higher and faster at 28 °C. Unexpectedly, the co-inoculation of these two Vibrio species seemed to have a synergistic pathogenic effect over O. patagonica, as disease signs were readily observed at temperatures at which bleaching is not normally observed.