Emergence of Mycobacterium canariasense infections in central Israel.

In 2018, Mycobacterium canariasense bloodstream infection was diagnosed in Israel. Further investigation had identified additional five cases in three medical centers, including isolates from blood (1), cornea (1), and sputum (3). Isolates were susceptible to all the antimicrobial tested. All but one isolate was related by whole-genome phylogeny.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA.

BACKGROUND: Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom's MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom's MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom's MLST scheme in clinical samples with low concentrations of Chlamydia DNA. RESULTS: In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom's MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. CONCLUSIONS: The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

Genetic diversity of avocado (Persea americana Mill.) germplasm using pooled sequencing.

BACKGROUND: Discovering a genome-wide set of avocado (Persea americana Mill.) single nucleotide polymorphisms and characterizing the diversity of germplasm collection is a powerful tool for breeding. However, discovery is a costly process, due to loss of loci that are proven to be non-informative when genotyping the germplasm. RESULTS: Our study on a collection of 100 accessions comprised the three race types, Guatemalan, Mexican, and West Indian. To increase the chances of discovering polymorphic loci, three pools of genomic DNA, one from each race, were sequenced and the reads were aligned to a reference transcriptome. In total, 507,917 polymorphic loci were identified in the entire collection. Of these, 345,617 were observed in all three pools, 117,692 in two pools, 44,552 in one of the pools, and only 56 (0.0001%) were homozygous in the three pools but for different alleles. The polymorphic loci were validated using 192 randomly selected SNPs by genotyping the accessions within each pool. The sensitivity of polymorphic locus prediction ranged from 0.77 to 0.94. The correlation between the allele frequency estimated from the pooled sequences and actual allele frequency from genotype calling of individual accessions was r = 0.8. A subset of 109 SNPs were then used to evaluate the genetic relationships among avocado accessions and the genetic diversity of the collection. The three races were distinctly clustered by projecting the genetic variation on a PCA plot. As expected, by estimating the kinship coefficient for all the accessions, many of the cultivars from the California breeding program were closely related to each other, especially, the Hass-like ones. The green-skin avocados, e.g., 'Bacon', 'Zutano', 'Ettinger' and 'Fuerte' were also closely related to each other. CONCLUSIONS: A framework for SNP discovery and genetically characterizing of a breeder's accessions was described. Sequencing pools of gDNA is a cost-effective approach to create a genome-wide stock of polymorphic loci for a breeding program. Reassessing the botanical and the genetic knowledge about the germplasm accessions is valuable for future breeding. Kinship analysis may be used as a first step in finding a parental candidates in a parentage analyses.

Mango (Mangifera indica L.) germplasm diversity based on single nucleotide polymorphisms derived from the transcriptome.

BACKGROUND: Germplasm collections are an important source for plant breeding, especially in fruit trees which have a long duration of juvenile period. Thus, efforts have been made to study the diversity of fruit tree collections. Even though mango is an economically important crop, most of the studies on diversity in mango collections have been conducted with a small number of genetic markers. RESULTS: We describe a de novo transcriptome assembly from mango cultivar 'Keitt'. Variation discovery was performed using Illumina resequencing of 'Keitt' and 'Tommy Atkins' cultivars identified 332,016 single-nucleotide polymorphisms (SNPs) and 1903 simple-sequence repeats (SSRs). Most of the SSRs (70.1%) were of trinucleotide with the preponderance of motif (GGA/AAG)n and only 23.5% were di-nucleotide SSRs with the mostly of (AT/AT)n motif. Further investigation of the diversity in the Israeli mango collection was performed based on a subset of 293 SNPs. Those markers have divided the Israeli mango collection into two major groups: one group included mostly mango accessions from Southeast Asia (Malaysia, Thailand, Indonesia) and India and the other with mainly of Floridian and Israeli mango cultivars. The latter group was more polymorphic (FS=-0.1 on the average) and was more of an admixture than the former group. A slight population differentiation was detected (FST=0.03), suggesting that if the mango accessions of the western world apparently was originated from Southeast Asia, as has been previously suggested, the duration of cultivation was not long enough to develop a distinct genetic background. CONCLUSIONS: Whole-transcriptome reconstruction was used to significantly broaden the mango's genetic variation resources, i.e., SNPs and SSRs. The set of SNP markers described in this study is novel. A subset of SNPs was sampled to explore the Israeli mango collection and most of them were polymorphic in many mango accessions. Therefore, we believe that these SNPs will be valuable as they recapitulate and strengthen the history of mango diversity.

QTL mapping in multiple populations and development stages reveals dynamic quantitative trait loci for fruit size in cucumbers of different market classes.

KEY MESSAGE: QTL analysis in multi-development stages with different QTL models identified 12 consensus QTLs underlying fruit elongation and radial growth presenting a dynamic view of genetic control of cucumber fruit development. Fruit size is an important quality trait in cucumber (Cucumis sativus L.) of different market classes. However, the genetic and molecular basis of fruit size variations in cucumber is not well understood. In this study, we conducted QTL mapping of fruit size in cucumber using F2, F2-derived F3 families and recombinant inbred lines (RILs) from a cross between two inbred lines Gy14 (North American picking cucumber) and 9930 (North China fresh market cucumber). Phenotypic data of fruit length and diameter were collected at three development stages (anthesis, immature and mature fruits) in six environments over 4 years. QTL analysis was performed with three QTL models including composite interval mapping (CIM), Bayesian interval mapping (BIM), and multiple QTL mapping (MQM). Twenty-nine consistent and distinct QTLs were detected for nine traits from multiple mapping populations and QTL models. Synthesis of information from available fruit size QTLs allowed establishment of 12 consensus QTLs underlying fruit elongation and radial growth, which presented a dynamic view of genetic control of cucumber fruit development. Results from this study highlighted the benefits of QTL analysis with multiple QTL models and different mapping populations in improving the power of QTL detection. Discussion was presented in the context of domestication and diversifying selection of fruit length and diameter, marker-assisted selection of fruit size, as well as identification of candidate genes for fruit size QTLs in cucumber.

Gene expression profiling in juvenile and mature cuttings of Eucalyptus grandis reveals the importance of microtubule remodeling during adventitious root formation.

BACKGROUND: The ability to form adventitious roots (AR) is an economically important trait that is lost during the juvenile-to-mature phase change in woody plants. Auxin treatment, which generally promotes rooting in juvenile cuttings, is often ineffective when applied to mature cuttings. The molecular basis for this phenomenon in Eucalyptus grandis was addressed here. RESULTS: A comprehensive microarray analysis was performed in order to compare gene-expression profiles in juvenile and mature cuttings of E. grandis, with or without auxin treatment on days, 0, 1, 3, 6, 9 and 12 post AR induction. Under these conditions AR primordia were formed only in auxin-treated juvenile cuttings. However, clustering the expression profiles revealed that the time after induction contributed more significantly to the differences in expression than the developmental phase of the cuttings or auxin treatment. Most detected differences which were related to the developmental phase and auxin treatment occurred on day 6, which correlated with the kinetics of AR-primordia formation. Among the functional groups of transcripts that differed between juvenile and mature cuttings was that of microtubules (MT). The expression of 42 transcripts annotated as coding for tubulin, MT-associated proteins and kinesin motor proteins was validated in the same RNA samples. The results suggest a coordinated developmental and auxin dependent regulation of several MT-related transcripts in these cuttings. To determine the relevance of MT remodeling to AR formation, MTs were subjected to subtle perturbations by trifluralin, a MT disrupting drug, applied during auxin induction. Juvenile cuttings were not affected by the treatment, but rooting of mature cuttings increased from 10 to more than 40 percent. CONCLUSIONS: The data suggest that juvenile-specific MT remodeling is involved in AR formation in E. grandis.

Combining bulk segregation analysis and microarrays for mapping of the pH trait in melon.

The availability of sequence information for many plants has opened the way to advanced genetic analysis in many non-model plants. Nevertheless, exploration of genetic variation on a large scale and its use as a tool for the identification of traits of interest are still rare. In this study, we combined a bulk segregation approach with our own-designed microarrays to map the pH locus that influences fruit pH in melon. Using these technologies, we identified a set of markers that are genetically linked to the pH trait. Further analysis using a set of melon cultivars demonstrated that some of these markers are tightly linked to the pH trait throughout our germplasm collection. These results validate the utility of combining microarray technology with a bulk segregation approach in mapping traits of interest in non-model plants.

Genomic, phenotypic and demographic characterization of Mycobacterium tuberculosis in Israel in 2021.

According to World Health Organization WHO, Tuberculosis (TB) is the second cause of death from infectious disease worldwide. During 2021, 10.6 million people were infected with TB, and 1.6 million people died. TB is caused by pathogens belonging to the Mycobacterium tuberculosis complex (MTBC), mainly by Mycobacterium tuberculosis (M.tb). Members of this complex are acid-fast bacilli, which can cause intrapulmonary and extra pulmonary TB, and can be divided into various lineages, based on genomic markers. The main public health threat comes from drug resistant M.tb strains, which are responsible for about 25% of TB death and treatment failure worldwide. Treating drug resistant TB patients significantly raises the costs of TB treatment. This study provides an overview of the demographic and drug susceptibility characteristics of newly diagnosed TB patients in Israel in 2021. The State of Israel has a very low level of TB endemicity and is at a pre-elimination phase. Notably, only 11.7% of the newly diagnosed TB patients were born in Israel. In this report, of the 154 new laboratory-confirmed TB patients, 66.7% had pulmonary TB, while 16% had extrapulmonary TB. Males accounted for 52% of the patients, with the most prevalent age group being 21-40. Most patients were citizens of Israel (53.9%), while 37.7% had no Israeli citizenship. Among non-citizens, there was a predominance of males and patients aged 21-40. The susceptibility profile showed a high resistance rate to streptomycin (18.2%) and to a lower extent to isoniazid (13.6%), pyrazinamide (8.4%), rifampicin (7.8%), and ethambutol (3.2%). Only 2 cases of XDR-TB and 10 MDR-TB strains were detected in Israel in 2021, with both XDR strains and 5 out of 10 MDR strains belonging to the Beijing lineage. Most of Beijing isolates were resistant to at least one tested drug. Genomic sequencing of 134 out of 156 strains and bioinformatics analysis using the MTBseq program and WHO mutation catalogue shows a good match with only 9 discrepancies between phenotypic and genotypic susceptibility profiles in first line drugs. The most common lineage is Delhi-Cas (23%) followed by the Beijing lineage (17%). Most patients from the Delhi-Cas lineage were born in Africa, while patients with Beijing isolates were born in different countries. Minimum spanning tree analysis identified 15 clusters. The study highlights the need for ongoing surveillance of TB using molecular and phenotypic tools to further decreasing the spreading level of the disease and develop effective treatment strategies.

Prolonged survival of a patient with active MDR-TB HIV co-morbidity: insights from a Mycobacterium tuberculosis strain with a unique genomic deletion.

Coinfection of HIV and multidrug-resistant tuberculosis (MDR-TB) presents significant challenges in terms of the treatment and prognosis of tuberculosis, leading to complexities in managing the disease and impacting the overall outcome for TB patients. This study presents a remarkable case of a patient with MDR-TB and HIV coinfection who survived for over 8 years, despite poor treatment adherence and comorbidities. Whole genome sequencing (WGS) of the infecting Mycobacterium tuberculosis (Mtb) strain revealed a unique genomic deletion, spanning 18 genes, including key genes involved in hypoxia response, intracellular survival, immunodominant antigens, and dormancy. This deletion, that we have called "Del-X," potentially exerts a profound influence on the bacterial physiology and its virulence. Only few similar deletions were detected in other non-related Mtb genomes worldwide. In vivo evolution analysis identified drug resistance and metabolic adaptation mutations and their temporal dynamics during the patient's treatment course.

Mycobacterium intracellulare subsp. chimaera from Cardio Surgery Heating-Cooling Units and from Clinical Samples in Israel Are Genetically Unrelated.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens that cause illness primarily in the elderly, in the immunocompromised or in patients with underlying lung disease. Since 2013, a global outbreak of NTM infection related to heater-cooler units (HCU) used in cardio-thoracic surgery has been identified. This outbreak was caused by a single strain of Mycobacterium intracellulare subsp. chimaera. In order to estimate the prevalence of this outbreak strain in Israel, we sampled Mycobacterium intracellulare subsp. chimaera from several HCU machines in Israel, as well as from patients, sequenced their genomes and compared them to the outbreak strain. The presence of mixed mycobacteria species in the samples complicated the analysis of obtained sequences. By applying a metagenomic binning strategy, we were able to obtain, and characterize, genomes of single strains from the mixed samples. Mycobacterium intracellulare subsp. chimaera strains were compared to each other and to previously reported genomes from other countries. The strain causing the outbreak related to the HCU machines was identified in several such machines in Israel but not in any clinical sample.

HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene.

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Draft Genome Sequences of Cronobacter muytjensii Cr150, Cronobacter turicensis Cr170, and Cronobacter sakazakii Cr611.

The sequencing and bioinformatics analyses of isolates Cr150, Cr170, and Cr611 from powdered infant formula indicate that the three strains represent new members in the Cronobacter muytjensii, Cronobacter turicensis, and Cronobacter sakazakii groups, respectively.

Draft Genome Sequence of Cronobacter sakazakii Cr268, Isolated from Infant Powder Formula.

Cronobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in premature infants. Strain Cr268 was isolated from imported powdered infant formula in 2009 during routine microbial examination according to ISO-22964 ("Microbiology of the food chain-horizontal method for the detection of Cronobacter spp."). Isolate Cr268 was confirmed to be C. sakazakii by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and standard biochemical analysis. Here, we announce its genome, which represents a new member in the C. sakazakii group.

Ultrahigh-density linkage map for cultivated cucumber (Cucumis sativus L.) using a single-nucleotide polymorphism genotyping array.

Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequence-related amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ~2 Kb and have expected genotype calls in parents/F1 hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F1 hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a '9930' × 'Gy14' recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R2 = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F1 hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs.

Single-nucleotide polymorphism markers from de-novo assembly of the pomegranate transcriptome reveal germplasm genetic diversity.

Pomegranate is a valuable crop that is grown commercially in many parts of the world. Wild species have been reported from India, Turkmenistan and Socotra. Pomegranate fruit has a variety of health-beneficial qualities. However, despite this crop's importance, only moderate effort has been invested in studying its biochemical or physiological properties or in establishing genomic and genetic infrastructures. In this study, we reconstructed a transcriptome from two phenotypically different accessions using 454-GS-FLX Titanium technology. These data were used to explore the functional annotation of 45,187 fully annotated contigs. We further compiled a genetic-variation resource of 7,155 simple-sequence repeats (SSRs) and 6,500 single-nucleotide polymorphisms (SNPs). A subset of 480 SNPs was sampled to investigate the genetic structure of the broad pomegranate germplasm collection at the Agricultural Research Organization (ARO), which includes accessions from different geographical areas worldwide. This subset of SNPs was found to be polymorphic, with 10.7% loci with minor allele frequencies of (MAF<0.05). These SNPs were successfully used to classify the ARO pomegranate collection into two major groups of accessions: one from India, China and Iran, composed of mainly unknown country origin and which was more of an admixture than the other major group, composed of accessions mainly from the Mediterranean basin, Central Asia and California. This study establishes a high-throughput transcriptome and genetic-marker infrastructure. Moreover, it sheds new light on the genetic interrelations between pomegranate species worldwide and more accurately defines their genetic nature.

Peptidic modulators of protein-protein interactions: progress and challenges in computational design.

With the decline in productivity of drug-development efforts, novel approaches to rational drug design are being introduced and developed. Naturally occurring and synthetic peptides are emerging as novel promising compounds that can specifically and efficiently modulate signaling pathways in vitro and in vivo. We describe sequence-based approaches that use peptides to mimic proteins in order to inhibit the interaction of the mimicked protein with its partners. We then discuss a structure-based approach, in which protein-peptide complex structures are used to rationally design and optimize peptidic inhibitors. We survey flexible peptide docking techniques and discuss current challenges and future directions in the rational design of peptidic inhibitors.