Nonculture-based identification of bacteria in milk by protein fingerprinting

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI SepsityperTM Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3)-10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (104 cfu mL-1), bacterial identification could be performed after initial incubation at 37 degrees C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.

A pyrosequencing assay for the rapid discrimination of mitochondrial lineages in the Salmo trutta species complex

The European trout (Salmo trutta species complex) is genetically very diverse consisting of five distinct mitochondrial lineages that probably originated in the Pleistocene. Here, we describe a novel pyrosequencing protocol to generate two short sequence reads from the mitochondrial control region, which allow the unambiguous identification of all five lineages. The approach was found to be easily transferable between laboratories and should be a valuable tool for the assessment of genetic diversity in trout. Pyrosequencing-based assays for molecular species identification are expected to be generally useful whenever multiple positions in a short DNA sequence need to be assessed.

Phylogenetic analysis of Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis clinical strains reveals a clear species clustering

Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis are oral pathogens from the family Bacteroidaceae, regularly isolated from cases of gingivitis and periodontitis. In this study, the phylogenetic variability of these three bacterial species was investigated by means of 16S rRNA (rrs) gene sequence comparisons of a set of epidemiologically and geographically diverse isolates. For each of the three species, the rrs gene sequences of 11 clinical isolates as well as the corresponding type strains was determined. Comparison of all rrs sequences obtained with those of closely related species revealed a clear clustering of species, with only a little intraspecies variability but a clear difference in the rrs gene with respect to the next related taxon. The results indicate that the three species form stable, homogeneous genetic groups, which favours an rrs-based species identification of these oral pathogens. This is especially useful given the 7% sequence divergence between Prevotella intermedia and Prevotella nigrescens, since phenotypic distinction between the two Prevotella species is inconsistent or involves techniques not applicable in routine identification.

Spatial Distribution of Cryptic Species Diversity in European Freshwater Amphipods (Gammarus fossarum) as Revealed by Pyrosequencing

In order to understand and protect ecosystems, local gene pools need to be evaluated with respect to their uniqueness. Cryptic species present a challenge in this context because their presence, if unrecognized, may lead to serious misjudgement of the distribution of evolutionarily distinct genetic entities. In this study, we describe the current geographical distribution of cryptic species of the ecologically important stream amphipod Gammarus fossarum (types A, B and C). We use a novel pyrosequencing assay for molecular species identification and survey 62 populations in Switzerland, plus several populations in Germany and eastern France. In addition, we compile data from previous publications (mainly Germany). A clear transition is observed from type A in the east (Danube and Po drainages) to types B and, more rarely, C in the west (Meuse, Rhone, and four smaller French river systems). Within the Rhine drainage, the cryptic species meet in a contact zone which spans the entire G. fossarum distribution range from north to south. This large-scale geographical sorting indicates that types A and B persisted in separate refugia during Pleistocene glaciations. Within the contact zone, the species rarely co-occur at the same site, suggesting that ecological processes may preclude long-term coexistence. The clear phylogeographical signal observed in this study implies that, in many parts of Europe, only one of the cryptic species is present.

Mapping species distributions : A comparison of skilled naturalist and lay citizen science recording

Acknowledgements We are grateful to Elaine O’Mahony, Imogen Pearce, Richard Comont, Anthony McCluskey and other BBCT staff for the many hours of BeeWatch species identification and for all people who submitted sightings to BeeWatch, OPAL, BWARS and the various local recording schemes and societies. We thank the NBN for allowing us to download the bumblebee records without strings attached, and the Essex, Greater London, Cumbria and Sussex based recording centres for providing records upon request. Finally, we are indebted to Tom August and two anonymous reviewers for their valuable critique on an earlier version of this work.

Butterflies and moths of Pacific Northwest forests and woodlands : rare, endangered, and management-sensitive species /

"Species identification"--Cover.

Identification of the causal agent of pistachio dieback in Australia

Symptoms associated with pistachio dieback in Australia include decline (little or no current season growth), xylem staining in shoots two or more years old, trunk mu and limb lesions (often covered by black, superficial fungal growth), excessive exudation of resin, dieback and death of the tree. Bacteria belonging to the genus Xanthomonas have been suggested as the causal agent. To confirm the constant association between these bacteria and the disease syndrome, the absence of other pathogens and the identity of the pathogen, we performed a series of isolations and pathogenicity tests. The only microorganism consistently isolated from diseased tissue was a bacterium that produced yellow, mucoid colonies and displayed morphological and cultural characteristics typical of the genus Xanthomonas. Database comparisons of the fatty acid and whole-cell protein profiles of five representative pistachio isolates indicated that they all belonged to X. translucens, but it was not possible to allocate the isolates to pathovar. Pathogenicity tests on cereals and grasses supported this identification. However, Koch's postulates have been only partially fulfilled because not all symptoms associated with pistachio dieback were reproduced on inoculated two-year-old pistachio trees. While discolouration was observed, dieback, excessive resinous exudate and trunk and limb lesions were not produced; expression of these symptoms may be delayed, and long-term monitoring of a small number of inoculated trees is in progress.

Identification of three gene candidates for multicellular resistance in colon carcinoma

Solid tumours display elevated resistance to chemo- and radiotherapies compared to individual tumour derived cells. This so-called multicellular resistance (MCR) phenomenon can only be partly explained by reduced diffusion and altered cell cycle status; even fast growing cells on the surface of solid tumours display MCR. Multicellular spheroids (MCS) recapture this phenomenon ex vivo and here we compare gene expression in exponentially growing MCS with gene expression in monolayer culture. Using an 18,664 gene microarray, we identified 42 differentially expressed genes and three of these genes can be linked to potential mechanisms of MCR. A group of interferon response genes were also up-regulated in MCS, as were a number of genes that that are indicative of greater differentiation in three-dimensional cultures.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Resolving taxonomic uncertainty in vulnerable elasmobranchs: are the Madeira skate (Raja maderensis) and the thornback ray (Raja clavata) distinct species?

Skates and rays constitute the most speciose group of chondrichthyan fishes, yet are characterised by remarkable levels of morphological and ecological conservatism. They can be challenging to identify, which makes monitoring species compositions for fisheries management purposes problematic. Owing to their slow growth and low fecundity, skates are vulnerable to exploitation and species exhibiting endemism or limited ranges are considered to be the most at risk. The Madeira skate Raja maderensis is endemic and classified as ‘Data Deficient’ by the IUCN, yet its taxonomic distinctiveness from the morphologically similar and more wide-ranging thornback ray Raja clavata is unresolved. This study evaluated the sequence divergence of both the variable control region and cytochrome oxidase I ‘DNA barcode’ gene of the mitochondrial genome to elucidate the genetic differentiation of specimens identified as R. maderensis and R. clavata collected across much of their geographic ranges. Genetic evidence was insufficient to support the different species designations. However regardless of putative species identification, individuals occupying waters around the Azores and North African Seamounts represent an evolutionarily significant unit worthy of special consideration for conservation management.

Resolving taxonomic uncertainty in vulnerable elasmobranchs: are the Madeira skate (Raja maderensis) and the thornback ray (Raja clavata) distinct species?

Skates and rays constitute the most speciose group of chondrichthyan fishes, yet are characterised by remarkable levels of morphological and ecological conservatism. They can be challenging to identify, which makes monitoring species compositions for fisheries management purposes problematic. Owing to their slow growth and low fecundity, skates are vulnerable to exploitation and species exhibiting endemism or limited ranges are considered to be the most at risk. The Madeira skate Raja maderensis is endemic and classified as ‘Data Deficient’ by the IUCN, yet its taxonomic distinctiveness from the morphologically similar and more wide-ranging thornback ray Raja clavata is unresolved. This study evaluated the sequence divergence of both the variable control region and cytochrome oxidase I ‘DNA barcode’ gene of the mitochondrial genome to elucidate the genetic differentiation of specimens identified as R. maderensis and R. clavata collected across much of their geographic ranges. Genetic evidence was insufficient to support the different species designations. However regardless of putative species identification, individuals occupying waters around the Azores and North African Seamounts represent an evolutionarily significant unit worthy of special consideration for conservation management.

Random Mating Between Two Widely Divergent Mitochondrial Lineages of Cryptolestes ferrugineus (Coleoptera: Laemophloeidae): A Test of Species Limits in a Phosphine-Resistant Stored Product Pest

Effective pest management relies on accurate delimitation of species and, beyond this, on accurate species identification. Mitochondrial COI sequences are useful for providing initial indications in delimiting species but, despite acknowledged limitations in the method, many studies involving COI sequences and species problems remain unresolved. Here we illustrate how such impasses can be resolved with microsatellite and nuclear sequence data, to assess more directly the amount of gene flow between divergent lineages. We use a population genetics approach to test for random mating between two 8 ± 2% divergent COI lineages of the rusty grain beetle, Cryptolestes ferrugineus (Stephens). This species has become strongly resistant to phosphine, a fumigant used worldwide for disinfesting grain. The possibility of cryptic species would have significant consequences for resistance management, especially if resistance was confined to one mitochondrial lineage. We find no evidence of restricted gene flow or nonrandom mating across the two COI lineages of these beetles, rather we hypothesize that historic population structure associated with early Pleistocene climate changes likely contributed to divergent lineages within this species.

PCR-Internal Transcribed Spacer (ITS) genes sequencing and phylogenetic analysis of clinical and environmental Aspergillus species associated with HIV-TB co infected patients in a hospital in Abeokuta, southwestern Nigeria.

Background: Aspergillosis has been identified as one of the hospital acquired infections but the contribution of water and inhouse air as possible sources of Aspergillus infection in immunocompromised individuals like HIV-TB patients have not been studied in any hospital setting in Nigeria. Objective: To identify and investigate genetic relationship between clinical and environmental Aspergillus species associated with HIV-TB co infected patients. Methods: DNA extraction, purification, amplification and sequencing of Internal Transcribed Spacer (ITS) genes were performed using standard protocols. Similarity search using BLAST on NCBI was used for species identification and MEGA 5.0 was used for phylogenetic analysis. Results: Analyses of sequenced ITS genes of selected fourteen (14) Aspergillus isolates identified in the GenBank database revealed Aspergillus niger (28.57%), Aspergillus tubingensis (7.14%), Aspergillus flavus (7.14%) and Aspergillus fumigatus (57.14%). Aspergillus in sputum of HIV patients were Aspergillus niger, A. fumigatus, A. tubingensis and A. flavus. Also, A. niger and A. fumigatus were identified from water and open-air. Phylogenetic analysis of sequences yielded genetic relatedness between clinical and environmental isolates. Conclusion: Water and air in health care settings in Nigeria are important sources of Aspergillus sp. for HIV-TB patients.

Assessment of MALDI-TOF MS as Alternative Tool for Streptococcus suis Identification

The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Streptococcus suis isolates obtained from pigs, wild animals, and humans was evaluated using a PCR-based identification assay as the gold standard. In addition, MALDI-TOF MS was compared with the commercial multi-tests Rapid ID 32 STREP system. From the 129 S. suis isolates included in the study and identified by the molecular method, only 31 isolates (24.03%) had score values ≥2.300 and 79 isolates (61.24%) gave score values between 2.299 and 2.000. After updating the currently available S. suis MALDI Biotyper database with the spectra of three additional clinical isolates of serotypes 2, 7, and 9, most isolates had statistically significant higher score values (mean score: 2.65) than those obtained using the original database (mean score: 2.182). Considering the results of the present study, we suggest using a less restrictive threshold score of ≥2.000 for reliable species identification of S. suis. According to this cut-off value, a total of 125 S. suis isolates (96.9%) were correctly identified using the updated database. These data indicate an excellent performance of MALDI-TOF MS for the identification of S. suis.