Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.

Mutation screening of the medium-chain acyl-CoA dehydrogenase (MCAD) and the ornithine transcarbamylase (OTC) genes by multiplex PCR amplification and sequencing

BACKGROUND: Sequencing based mutation screening assays of genes encompassing large numbers of exons could be substantially optimized by multiplex PCR, which enables simultaneous amplification of many targets in one reaction. In the present study, a multiplex PCR protocol originally developed for fragment analysis was evaluated for sequencing based mutation screening of the ornithine transcarbamylase (OTC) and the medium-chain acyl-CoA dehydrogenase (MCAD) genes. METHODS: Single exon and multiplex PCR protocols were applied to generate PCR templates for subsequent DNA sequencing of all exons of the OTC and the MCAD genes. For each PCR protocol and using the same DNA samples, 66 OTC and 98 MCAD sequence reads were generated. The sequences derived from the two different PCR methods were compared at the level of individual signal-to-noise ratios of the four bases and the proportion of high-quality base-signals. RESULTS: The single exon and the multiplex PCR protocol gave qualitatively comparable results for the two genes. CONCLUSIONS: Many existing sequencing based mutation analysis protocols may be easily optimized with the proposed method, since the multiplex PCR protocol was successfully applied without any re-design of the PCR primers and other optimization steps for generating sequencing templates for the OTC and MCAD genes, respectively.

Bringing high-throughput techniques to orphan crop of Africa: Highlights from the Tef TILLING Project

Orphan- or understudied-crops are mostly staple food crops in developing world. They are broadly classified under cereals, legumes, root crops, fruits and vegetables. These under-researched crops contribute to the diet of a large portion of resource-poor consumers and at the same time generate income for small-holder farmers in developing countries, particularly in Africa. In addition, they perform better than major crops of the world under extreme soil and climatic conditions. However, orphan crops are not without problems. Due to lack of scientific investigation, most of them produce low yields while others have a variety of toxins that affect the health of consumers. Here, we present some highlights on the status and future perspectives of the Tef Biotechnology Project that employs modern improvement technique in order to genetically improve tef (Eragrostis tef), one of the most important orphan crop in Africa. A reverse genetics approach known as TILLING (Targeting Induced Local Lesions IN Genome) is implemented in order to tackle lodging, the major yield limiting factor in tef.Key words: Orphan crops, underresearched crops, Eragrostis tef, TILLING, semi-dwarf.

The Mycoplasma conjunctivae genome sequencing, annotation and analysis

BACKGROUND: The mollicute Mycoplasma conjunctivae is the etiological agent leading to infectious keratoconjunctivitis (IKC) in domestic sheep and wild caprinae. Although this pathogen is relatively benign for domestic animals treated by antibiotics, it can lead wild animals to blindness and death. This is a major cause of death in the protected species in the Alps (e.g., Capra ibex, Rupicapra rupicapra). METHODS: The genome was sequenced using a combined technique of GS-FLX (454) and Sanger sequencing, and annotated by an automatic pipeline that we designed using several tools interconnected via PERL scripts. The resulting annotations are stored in a MySQL database. RESULTS: The annotated sequence is deposited in the EMBL database (FM864216) and uploaded into the mollicutes database MolliGen http://cbi.labri.fr/outils/molligen/ allowing for comparative genomics. CONCLUSION: We show that our automatic pipeline allows for annotating a complete mycoplasma genome and present several examples of analysis in search for biological targets (e.g., pathogenic proteins).

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Tularemia in a common marmoset (Callithrix jacchus) diagnosed by 16S rRNA sequencing

We report a case of tularemia in a common marmoset (Callithrix jacchus) diagnosed by determination of the isolate's 16S ribosomal RNA (rRNA) gene sequence. Pathological examination of the animal revealed a multifocal acute necrotizing hepatitis, interstitial nephritis, splenitis, and lymphangitis of the mandibular, retropharyngeal, and cervical and mesenteric lymph nodes. Moreover, multiple foci of acute necrosis were found in the epithelium of the jejunum and the interstitium of the lung. Bacteriological investigations revealed a septicemia. The isolated infectious agent was uncommon, not routinely diagnosed in our laboratory and therefore difficult to identify by conventional tools in a reasonable time and effort. thus, we decided to perform a genetic analysis based on the 16S rRNA gene sequence. Thereby, an infection with Francisella tularensis, the causative agent of tularemia, was unambiguously diagnosed. This shows the great advantage 16S rRNA gene sequencing has as a general identification approach for unusual or rare isolates.

Learner preferences regarding integrating, sequencing and aligning virtual patients with other activities in the undergraduate medical curriculum: A focus group study

CONTEXT: E-learning resources, such as virtual patients (VPs), can be more effective when they are integrated in the curriculum. To gain insights that can inform guidelines for the curricular integration of VPs, we explored students' perceptions of scenarios with integrated and non-integrated VPs aimed at promoting clinical reasoning skills. METHODS: During their paediatric clerkship, 116 fifth-year medical students were given at least ten VPs embedded in eight integrated scenarios and as non-integrated add-ons. The scenarios differed in the sequencing and alignment of VPs and related educational activities, tutor involvement, number of VPs, relevance to assessment and involvement of real patients. We sought students' perceptions on the VP scenarios in focus group interviews with eight groups of 4-7 randomly selected students (n = 39). The interviews were recorded, transcribed and analysed qualitatively. RESULTS: The analysis resulted in six themes reflecting students' perceptions of important features for effective curricular integration of VPs: (i) continuous and stable online access, (ii) increasing complexity, adapted to students' knowledge, (iii) VP-related workload offset by elimination of other activities, (iv) optimal sequencing (e.g.: lecture--1 to 2 VP(s)--tutor-led small group discussion--real patient) and (V) optimal alignment of VPs and educational activities, (vi) inclusion of VP topics in assessment. CONCLUSIONS: The themes appear to offer starting points for the development of a framework to guide the curricular integration of VPs. Their impact needs to be confirmed by studies using quantitative controlled designs.

Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.

Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04-0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9-2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.

CAP Reform vs WTO: Crop Diversification or Crop Rotation? : Legal opinion prepared by Dr. Christian Häberli at the request of APRODEV, the Association of World Council of Churches related Development Organisations in Europe

The European Commission’s proposals for the Legislative Framework of the Common Agricultural Policy (CAP) in the period 2014-2020 include, inter alia, the introduction of a “strong greening component”. For the first time, all EU farmers in receipt of support are to “go beyond the requirements of cross compliance and deliver environmental and climate benefits as part of their everyday activities crop diversification as a contribution to all EU farmers in receipt of support go beyond the requirements of cross compliance and deliver environmental and climate benefits as part of their everyday activities.” In a legal opinion prepared at the request of APRODEV, the Association of World Council of Churches related Development Organisations in Europe (www.aprodev.eu), Christian Häberli examines the WTO implications of this proposal, as compared with an alternative proposal to rather link direct payments to crop rotation. The conclusions are twofold: 1. Crop rotation is at least as likely to be found Green Box-compatible as crop diversification. Moreover, it will be more difficult to argue that crop diversification is “not more than minimally production-distorting” because it entails for most farmers less cost and work. 2. Even if (either of the two cropping schemes) were to be found “amber”, the EU would not have to relinquish this conditionality. This is because the direct payments involved would in all likelihood not, together with the other price support instruments, exceed the amount available under the presently scheduled maximum.

The prospect of applying chemical elicitors and plant strengtheners to enhance the biological control of crop pests

An imminent food crisis reinforces the need for novel strategies to increase crop yields worldwide. Effective control of pest insects should be part of such strategies, preferentially with reduced negative impact on the environment and optimal protection and utilization of existing biodiversity. Enhancing the presence and efficacy of native biological control agents could be one such strategy. Plant strengthener is a generic term for several commercially available compounds or mixtures of compounds that can be applied to cultivated plants in order to ‘boost their vigour, resilience and performance’. Studies into the consequences of boosting plant resistance against pests and diseases on plant volatiles have found a surprising and dramatic increase in the plants' attractiveness to parasitic wasps. Here, we summarize the results from these studies and present new results from assays that illustrate the great potential of two commercially available resistance elicitors. We argue that plant strengtheners may currently be the best option to enhance the attractiveness of cultivated plants to biological control agents. Other options, such as the genetic manipulation of the release of specific volatiles may offer future solutions, but in most systems, we still miss fundamental knowledge on which key attractants should be targeted for this approach.

Tracking a tuberculosis outbreak over 21 years: strain-specific single nucleotide polymorphism-typing combined with targeted whole genome sequencing.

BACKGROUND  Whole genome sequencing (WGS) is increasingly used in molecular-epidemiological investigations of bacterial pathogens, despite cost- and time-intensive analyses. We combined strain-specific single nucleotide polymorphism (SNP)-typing and targeted WGS to investigate a tuberculosis cluster spanning 21 years in Bern, Switzerland. METHODS  Based on genome sequences of three historical outbreak Mycobacterium tuberculosis isolates, we developed a strain-specific SNP-typing assay to identify further cases. We screened 1,642 patient isolates, and performed WGS on all identified cluster isolates. We extracted SNPs to construct genomic networks. Clinical and social data were retrospectively collected. RESULTS  We identified 68 patients associated with the outbreak strain. Most were diagnosed in 1991-1995, but cases were observed until 2011. Two thirds belonged to the homeless and substance abuser milieu. Targeted WGS revealed 133 variable SNP positions among outbreak isolates. Genomic network analyses suggested a single origin of the outbreak, with subsequent division into three sub-clusters. Isolates from patients with confirmed epidemiological links differed by 0-11 SNPs. CONCLUSIONS  Strain-specific SNP-genotyping allowed rapid and inexpensive identification of M. tuberculosis outbreak isolates in a population-based strain collection. Subsequent targeted WGS provided detailed insights into transmission dynamics. This combined approach could be applied to track bacterial pathogens in real-time and at high resolution.