Identification Of Biotypes Research Articles

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now widely used for marker/multibiomarker detection in medical diagnosis. We tested a new protocol for bacterial identification from blood culture broths in hospital routine by using collection tubes with separator gels on 503 included samples examined over 3 months, where 1.5 mL was injected by a syringe into BD Vacutainer tubes from BACTEC-positive bottles, before processing for bacterial protein extraction. Samples were loaded in duplicate onto the MALDI MS target, allowing a series of 12 samples to be processed in duplicate within 80 min by using Biflex III and BioTyper 2.0 software (Bruker). Including polymicrobial samples, 193 of 213 of Gram-negative bacteria (91.08%) and 284 of 319 of Gram-positive bacteria (89.02%) were correctly identified at the species level. Enterobacteriaceae constituted 35.15% of all species found, Staphylococaceae 37.96%, Streptococaceae and Enterococaceae 20.85%, Pseudomonadaceae 1.69%, and anaerobes 2.44%. In most of the polymicrobial samples, one of the species present was identified (80.9%). Seven isolates remained misidentified as Streptococcus pneumoniae, all belonging to Streptococcus mitis. Staphylococcus aureus was identified better when grown on anaero-aerobic medium, and MALDI BioTyper identification scores as low as 1.4 were pertinent, provided that four successive proposals of the same species were given. This new protocol correlates with conventional microbiology procedures by up to 90%, and by >95% for only monomicrobial samples, and provides a decreased turn-around time for identification of bacteria isolated from blood cultures, making this technology suitable also for blood cultures, with less delay and cost decreases in bacterial diagnostics, and favouring better care of patients.

Inheritance and Variability of Winter Wheat (Triticum) Morphologic Characteristics and Their Expression in DUS- test

The article provides summary of the research on inheritance, stability and variability of the Wheat morphologic characteristics, genotypes identification and examination of distinctness, uniformity and stability. Proposal is made to use alter characteristics for identification of biotypes and new candidate varieties for reference collections.

Mass Spectrometry Biotyper System Identifies Enteric Bacterial Pathogens Directly from Colonies Grown on Selective Stool Culture Media

We evaluated the performance and cost-effectiveness of a matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based Biotyper system for the routine identification of common enteric bacterial pathogens seen in middle Tennessee from suspicious colonies grown on selective stool culture media. A total of 304 suspicious colonies were selected and further identified from 605 stool specimens. The suspicious colonies were analyzed by the Biotyper system, and the results were compared to those from routine phenotypic methods, which identified 22 Salmonella species, 39 Shigella species, 3 enterohemorrhagic Escherichia coli (EHEC) isolates, 2 Yersinia enterocolitica isolates, 2 Campylobacter species, and 236 gastrointestinal normal flora isolates. The Biotyper system correctly identified the Salmonella species, Yersinia enterocolitica, and Campylobacter species but failed to distinguish the Shigella species and EHEC isolates from E. coli. Among the 236 normal flora isolates, 233 (98.7%) and 228 (96.6%) agreed at the genus and species levels, respectively, between the phenotypic and Biotyper methods. Organism identification scores were insignificantly different between colonies directly from selective media and subsequently from pure subculture. The entire Biotyper identification procedure, from smear preparation to final result reporting, can be completed within 30 min. The Biotyper system provides a rapid and simple screening tool for identifying many, but not all, suspicious colonies grown on selective media within 24 h after inoculation, which shortens test turnaround time by 2 to 3 days.

A New Soybean Aphid (Hemiptera: Aphididae) Biotype Identified

Shortly after its arrival, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), became established as the most important insect pest of soybean, Glycine max L. (Merr.), in the northern part of the North American soybean production region. Soybean resistance is an environmentally sustainable method to manage the pest and new soybean aphid resistant cultivars are beginning to be deployed into production. However, an earlier study identifying a soybean aphid biotype that could colonize plants with the Rag1 resistance gene has raised concerns about the durability of soybean aphid resistance genes. Choice and nonchoice tests conducted in this study characterized the colonization of a soybean aphid isolate, collected from the overwintering host Frangula alnus P. Mill in Springfield Fen, IN, on different aphid resistant soybean genotypes. This isolate readily colonized plants with the Rag2 resistance gene, distinguishing it from the two biotypes previously characterized and indicating that it represented a new biotype named biotype 3. The identification of soybean aphid biotypes that can overcome Rag1 and Rag2 resistance, even before soybean cultivars with the resistance genes have been deployed in production, suggests that there is high variability in virulence within soybean aphid populations present in North America. Such variability in virulence gives the pest a high potential to adapt to and reduce the effective life of resistance genes deployed in production. The search for new soybean aphid resistance genes must, therefore, continue, along with the development of alternative sustainable strategies to manage the pest.

Identification ofYersinia enterocoliticaat the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy

Yersinia enterocolitica and other Yersinia species, such as Y. pseudotuberculosis, Y. bercovieri, and Y. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well defined Yersinia strains from Switzerland and Germany was used to create a method for FT-IR-based differentiation of Yersinia isolates at the species level. The isolates of Y. enterocolitica were also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and "non-O:3, O:5, and O:9"). For external validation of the constructed methods, independently obtained isolates of different Yersinia species were used. A total of 79.9% of Y. enterocolitica sensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of all Y. bercovieri, Y. intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.

Identification of three major Bemisia tabaci biotypes in China based on morphological and DNA polymorphisms

The sweet potato whitefly, Bemisia tabaci, is a complex species, and many of its biotypes are important agricultural pests. The B and Q biotypes are invasive and coexist with the native Cv biotype on vegetable and ornamental crops in China. In this study, these three biotypes were identified based on their morphological characteristics, RAPD–PCR analysis, and DNA sequences of the COI gene. The anterior and posterior wax fringes of the B, Q, and Cv biotypes significantly differed from each other. Based on this morphological characteristic, the three biotypes can easily be distinguished in greenhouses and fields. Genomic DNA RAPD–PCR band patterns also revealed differences between these biotypes using the H 16 primer. The B biotype has three bright DNA bands between 250 and 600 bp, the Q biotype only has one bright band at ∼300 bp, while the Cv biotype has no band between 250 and 500 bp. Both the Cv and Q biotypes have two bright bands at ∼750 and 1000 bp while the B biotype has only one band at ∼1000 bp. Based on the COI gene, the genetic identity between B and Cv, B and Q, and Q and CV was 85.8%, 94.7%, and 86.0%, respectively. The MP tree indicated that the phylogenetic relationship between the B and Q biotypes is much closer than that between the B and Cv or the Q and Cv biotypes. The uses of the morphological, RAPD–PCR, and DNA sequencing methods in biotype identification of B. tabaci are discussed.

Improved DNA Barcoding Method for <I>Bemisia tabaci</I> and Related Aleyrodidae: Development of Universal and <I>Bemisia tabaci</I> Biotype-Specific Mitochondrial Cytochrome c Oxidase I Polymerase Chain Reaction Primers

Whiteflies, heteropterans in the family Aleyrodidae, are globally distributed and severe agricultural pests. The mitochondrial cytochrome c oxidase I (mtCOI) sequence has been used extensively in whitefly phylogenetic comparisons and in biotype identification of the agriculturally important Bemisia tabaci (Gennadius) whitefly. Because of the economic importance of several whitefly genera, and the invasive nature of the B and the Q biotypes of Bemisia tabaci, mtCOI sequence data are continually generated from sampled populations worldwide. Routine phylogenetic comparisons and biotype identification is done through amplification and sequencing of an approximately 800-bp mtCOI DNA fragment. Despite its routine use, published primers for amplification of this region are often inefficient for some B. tabaci biotypes and especially across whitefly species. Through new sequence generation and comparison to available whitefly mtCOI sequence data, a set of polymerase chain reaction (PCR) amplification primers (Btab-Uni primers) were identified that are more efficient at amplifying approximately 748 bp of the approximately 800-bp fragment currently used. These universal primers amplify an mtCOI fragment from numerous B. tabaci biotypes and whitefly genera by using a single amplification profile. Furthermore, mtCOI PCR primers specific for the B, Q, and New World biotypes of B. tabaci were designed that allow rapid discrimination among these biotypes. These primers produce a 478-, 405-, and 303-bp mtCOI fragment for the B, New World, and Q biotypes, respectively. By combining these primers and using rapid PCR and electrophoretic techniques, biotype determination can be made within 3 h for up to 96 samples at a time.

Distribution of <I>Bemisia tabaci</I> (Hemiptera: Aleyrodidae) Biotypes in Florida–Investigating the Q Invasion

After the 2004 discovery of the Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Q biotype in the United States, there was an urgent need to determine its distribution. As part of a coordinated country-wide effort, an extensive survey of B. tabaci biotypes was conducted in Florida, with the cooperation of growers and state and federal agencies, to monitor the introduction and distribution of both the B and Q biotypes. The biotype status of submitted B. tabaci samples was determined by polymerase chain reaction (PCR) amplification and sequencing of a 700-800-bp mitochondrial cytochrome oxidase I small subunit (mtCOI) gene fragment, PCR amplification, and size determination of two unique microsatellite markers and esterase zymogram analysis. One hundred and eighty collections were sampled from 23 counties. Of these samples, 58% were from vegetables, 37% were from ornamentals, and 5% were from peanuts, alfalfa, and weeds. Eighteen percent of all collections were found to be the Q biotype that came from greenhouse grown ornamental and herbs located in six counties. Sequence comparison of the mtCOI gene identified three separate haplotypes within Florida that were defined as Q1, Q2, and Q3. Haplotypes could be used to associate populations known to be related by grower and plant type. For example, collections from five counties were made on hibiscus linked to the same grower and all samples contained only the Q1 haplotype. Other populations contained a mix of the Q2 and Q3 haplotypes, supporting the conclusion that the Q biotype must have entered Florida through at least two separate introductions. Our data also show that two microsatellite markers are a cost-effective diagnostic alternative for biotype identification with 100% concurrence with mtCOI sequence data.

Note: Biotype status ofBemisia tabaci from various crops in Cyprus

An extensive survey ofBemisia tabaci populations covering the southern half of the island of Cyprus was conducted in 2006 and 2007 in order to define the biotype status of the pest. Sampling was done both on protected and outdoor cultivations of vegetables and ornamental plants. Biotype identification was performed using molecular diagnostics based on the mitochondrial cytochrome oxydase I gene. Our results indicated the presence of only the biotype B in all 25 collections.

Doença das mucosas associada à dermatite generalizada em bovinos, Mato Grosso do Sul

São descritos os aspectos epidemiológicos, clínicos, patológicos e diagnósticos de uma forma de dermatite associada à doença das mucosas (DM) em bovinos. Também são abordadas metodologias para a identificação de animais persistentemente infectados (PI) e o impacto nos índices zootécnicos no rebanho afetado. Os casos de dermatite associados com DM ocorreram em dois bovinos Nelore, de 12 e 24 meses de idade, pertencentes a uma fazenda de ciclo completo de bovinos de corte no estado de Mato Grosso do Sul. Os sinais clínicos nesses animais consistiam de emagrecimento lento e progressivo, formação de crostas difusas na pele de todo o corpo, pele ressecada, múltiplas ulcerações nas gengivas e face dorsal da língua, que evoluíram para fendas longitudinais, formação de projeções cornificadas e desprendimento dos cascos. Em um caso, também ocorreu diarréia no estágio final da doença. Na necropsia observaram-se ainda erosões longitudinais no esôfago. O exame histológico revelou focos de necrose de coagulação na mucosa do esôfago e língua, com infiltrado de neutrófilos e linfócitos. As lesões da pele consistiam de necrose de coagulação da epiderme associada com infiltrado de neutrófilos e hiperqueratose. Nos dois casos, a suspeita clínica foi confirmada pelo isolamento viral e identificação dos biótipos citopático e não-citopático do vírus da diarréia viral bovina (BVDV), além da detecção de antígenos virais em tecidos por imunoistoquímica. De um lote de 300 bovinos que tiveram contato com animais afetados, 38 foram testados e apresentaram altos títulos de anticorpos para o BVDV. Amostras de sangue coletadas de 1.025 animais jovens e 40 touros da propriedade foram submetidas a pesquisa de vírus para se identificarem possíveis animais persistentemente infectados (PI). O vírus foi isolado do sangue de três bezerros no teste inicial e, 12 meses depois, em dois deles que permaneceram na propriedade. Imunoistoquímica realizada em biópsia de orelhas identificou apenas um destes animais como positivo. O rebanho apresentou redução no índice de fertilidade e taxa de desmame no ano seguinte ao nascimento dos bezerros PI, mas estes indicadores retornaram posteriormente aos valores anteriores. Os resultados apresentados demonstram a presença da infecção por BVDV em rebanhos de corte no estado de Mato Grosso do Sul, e evidenciam a necessidade da inclusão dessa enfermidade no diagnóstico diferencial de causas de dermatites generalizadas.

The comparison of the methods for the identification of pathogenic serotypes and biotypes of Yersinia enterocolitica: Microbiological methods and PCR

In this study, pathogenic strains of Y. enterocolitica were identified by microbiological and PCR methods. The samples were collected from pigs, cattle, poultry, and slaughter houses. Three common techniques were used to isolate Y. enterocolitica from the samples – ITC, PSB, and direct on the CIN. Primers A1/A2, Y1/Y2, and rfbC 1/rfbC 2 were used for the specific detection of the pathogenic strains of Y. enterocolitica. Traditional microbiological methods were found to be insufficient for the specific identification of the Y. enterocolitica pathogen. In comparison with PCR which was able to detect 149 strains, the biochemical test could detect only 138 species. These results show that the use of biochemical methods of cultivation did not allow the identification of all Y. enterocolitica pathogens. In total, 149 strains of pathogenic Y. enterocolitica were examined of which 120 were from pigs, 19 from poultry, 8 were cattle strains, and 2 came from the environments of slaughterhouses.  

Biotype 2 Russian Wheat Aphid Resistance among Wheat Germplasm Accessions

The Russian wheat aphid [Diuraphis noxia (Mordvilko), RWA] is an important pest of wheat (Triticum aestivum L.) in the western Great Plains region of the United States. The recent identification of a RWA biotype (provisionally denoted as Biotype 2) that is virulent on currently deployed RWA‐resistant cultivars necessitates the rapid identification of resistance to the new biotype. The objective of this study was to identify Biotype 2 RWA resistance among a collection of germplasm accessions previously determined to be resistant to the original North American biotype of RWA (provisionally denoted as Biotype 1). A collection of 761 germplasm accessions was evaluated in standard seedling screening tests with Biotype 2 RWA. ‘TAM 107’ (carries no RWA resistance genes) and ‘Halt’ (Dn4 resistance gene) were used as susceptible checks while the germplasm line 94M370 (Dn7 resistance gene) was used as a resistant check. Evaluation of accessions showing at least a moderate level of resistance in unreplicated tests was repeated with three replications in a randomized complete block design. Forty‐four germplasm accessions were identified as having high to moderate levels of resistance to Biotype 2 RWA. Most of these accessions originated from areas of the world where RWA is endemic or were derived from germplasm accessions that originated from these areas. Ten accessions showed a level of resistance comparable with the 94M370 check. These accessions should prove useful in future genetic studies and for breeding for resistance to Biotype 2 RWA.

Short communication. Identification of the Poncirus biotype of Tylenchulus semipenetrans in Valencia, Spain

High population densities of Tylenchulus semipenetrans Cobb were detected on mature Poncirus trifoliate (L.) Raf. rootstocks in a commercial citrus orchard in Rafelguaraf, Valencia, Spain. Until now, this rootstock was considered resistant to populations of T. semipenetrans. The biotype of the nematode was characterised according to its host range. Under greenhouse conditions, it proved to be very capable of reproduction on the Bennecke and Rubidoux cultivars of P. trifoliate and on rough lemon (Citrus jambhiri Lush) at seven months post-infestation. It was also able to infest grapevine 141-R (Vitis berlandieri xV. rupestris), but not cv. Arbequina olive (Olea europaea L.). These results suggest this Valencia population of T. semipenetrans behaves similarly to the Poncirus biotype.

Genetic diversity of Striga hermonthica and Striga asiatica populations in Kenya

SummaryThe parasitic angiosperms, Striga hermonthica and Striga asiatica, severely constrain cereal production in sub‐Saharan Africa by causing huge losses in grain yield. Understanding the diversity of Striga populations is important because it allows identification of races or biotypes thus improving chances of breeding success. Amplified fragment length polymorphism (AFLP) analysis was used to study genetic diversity among 17 populations of S. asiatica and 24 populations of S. hermonthica from Kenya. A total of 349 DNA fragments ranging from 51 to 500 bp were obtained from four EcoRI and MseI primer combinations. Genetic distances for S. asiatica populations ranged from 0.009 to 0.116 with a mean of 0.032. S. hermonthica populations had a genetic distance that ranged from 0.007 to 0.025 with a mean of 0.015. Only two clusters were found in S. asiatica populations whereas no apparent structure was evident in S. hermonthica populations. There was no evidence of isolation by distance for the two species. Although the low genetic diversity suggests Striga is relatively uniform across the populations studied, it is possible that pathogenicity and virulence genes may be located in genomic regions that were not sampled. The data, however, does not provide evidence to support diversification of both Striga species in the region where the study was conducted.

Identification of Brucella Species and Biotypes using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP)

Brucellosis is a worldwide zoonosis causing reproductive failures in livestock and a severe multi-organ disease in humans. The genus Brucella is divided into seven species and various biotypes differing in pathogenicity and host specificity. Although Brucella spp. represent a highly homogenous group of bacteria, RFLPs of selected genes display sufficient polymorphism to distinguish Brucella species and biovars. PCR-RFLP analysis shows excellent typeability, reproducibility, stability, and epidemiological concordance. Consequently, PCR-RFLP assays of specific gene loci can serve as tools for diagnostic, epidemiological, taxonomic, and evolutionary studies. Various PCR-RFLPs used for the identification of Brucella species and biotypes are reviewed.

EP002 A proposal for three biotypes of the pathogenic alga Prototheca zopfii, and standards for their indentification

Colorless algae of the genus Prototheca out of the family of Chlorellaceae have been identified to be the only known plant causative agents of infections in humans and animals.Recently, the recognition of distinct phenotypic clusters have been reported as assigned as variants'. A representative selection of Prototheca (P.) zopfii strainsisolated from different environmental habitats or clinical cases was characterised in a polyphasic approach in order to assess their intraspecies taxonomic position.The P. zopfii- strains were compared by a number of phenotypic and genetic criteria, including growth characteristics, biochemical reactions, and serotypingresults. Based on emended standards for biotype identification P, zopfii strains showed auxanographical differences and distinct assimilation patterns withrespect to utilization of amino acids and glycerol.

Emended phenotypic characterization of Prototheca zopfii: a proposal for three biotypes and standards for their identification.

A representative selection of Prototheca zopfii strains isolated from different environmental habitats or clinical cases was characterized in a polyphasic approach in order to assess their intraspecies taxonomic position. Recently, the recognition of distinct phenotypic clusters has been reported as the assignment of 'variants'. In the present study, 11 strains were compared by a number of phenotypic and genetic criteria, including growth characteristics, biochemical reactions and serotyping results. Based on emended standards for biotype identification, P. zopfii strains showed auxanographic differences and distinct assimilation patterns with respect to utilization of amino acids and glycerol. Serotyping by means of immunoblotting revealed that all isolates of variant II obtained from clinical cases, i.e. isolates from bovine mastitis or from human enteropathia, showed specific antigen patterns. They were found to be different from strains assigned to the other two variants with respect to their immunogenic antigens. Furthermore, comparison of partial 18S rDNA sequences confirmed distinct differences between the former variants. Based on these results, it is proposed that P. zopfii merits classification as a species comprising three biotypes.

Cronología de la colonización bacteriana en grandes quemados: ¿es el aislamiento estricto necesario?

Infection is still one of the main causes of mortality in severe burn patients. Strict isolation has been used for the prevention of infection, but the efficacy of this measure is debatable. The aim of this study was to determine the timing of bacterial colonization in these patients and to ascertain whether strict isolation is indicated. Thirty consecutive children with severe burns were studied. Patients were only barrier-nursed during dressing changes. On admission and twice weekly over the entire hospital stay, burn, sputum, gastric aspirates, feces, and blood samples were obtained for culture. All isolates were tested for specific biotypes. Results were studied with linear regression and repeated measures ANOVA to determine the timing of colonization and cross-colonization between patients. On admission, normal cutaneous flora were isolated from burn cultures of all patients. The remaining cultures were negative. After one week, gastric aspirates were found to be colonized by gram-negative bacteria and fungi. This was followed by colonization of feces, burn, and sputum cultures. Biotype identification showed unidirectional colonization from the gastrointestinal tract to burns and upper airway. There were no cross infections between patients. Microbial colonization in severe burn patients was endogenous in nature and there were no cross infections. Thus, strict isolation is not necessary in burn centers, except during outbreaks of multi-resistant microorganisms.

Molecular tools for clone identification: the case of the grapevine cultivar ‘Traminer’

AbstractIn viticulture, biotype identification problems have traditionally been solved using ampelography, ampelometry and chemical traits analysis. However, these tools have resulted in several false attributions, in particular when used at the clonal level. The availability of relatively cheap, reliable and reproducible tools to identify genetic differences at the clonal level would greatly facilitate the work of clonal patenting. In this work, 24 accessions of ‘Traminer’ cultivars were characterized using molecular markers. Three different approaches were applied: simple sequence repeats (SSR), amplified fragment length polymorphism (AFLP) and methyl‐sensitive amplified length polymorphism (MSAP). Results showed that SSRs were not a powerful tool for clonal distinction. In contrast, the AFLP technique was able to distinguish 16 out of the 24 cultivars, even though the average similarity was high (97.1%). The MSAP technique was used to evaluate qualitative differences in the degree of DNA methylation among clones. Results suggest that morphological differences among clones are probably due to the synergetic effect of genetic and epigenetic modifications, and that clonal identification could be greatly improved using molecular tools such as AFLP and MSAP.

Identification of Streptococcus bovis biotype I strains among S. bovis clinical isolates by PCR.

Streptococcus bovis causes 24% of all streptococcal infective endocarditis cases. There are many reports linking both S. bovis bacteremia and endocarditis with various forms of gastrointestinal disease (primarily colonic cancers). S. bovis is divided into two biotypes: I and II. The biotype I strain is much more frequently isolated from patients with endocarditis, gastrointestinal disease, or both. We describe here the isolation of biotype I-specific DNA sequences and the development of a PCR test which can identify S. bovis biotype I strains among S. bovis clinical isolates.