Genetic Clones Research Articles

Nosocomial dissemination of hypervirulent Klebsiella pneumoniae with high-risk clones among children in Shanghai.

ObjectivesAlthough hypervirulent Klebsiella pneumoniae (hvKp) is an increasing public health problem, there remains limited epidemiological information regarding hvKp infections in children. Here, we conducted a clinical, molecular and phenotypic surveillance of hvKp strains in a pediatric population.MethodsNon-repetitive K. pneumoniae (Kp) strains consecutively collected during 2019-2020 were screened for hypervirulence genes (prmpA, prmpA2, iucA, iroB, and peg344) using PCR. Positive strains were further characterized by four phenotypic assays (string test, serum killing assay, siderophore production, Galleria mellonella lethality assay), followed by murine sepsis model to determine virulence in vitro and in vivo. Also, capsular types, sequence types, plasmid replicon types, antimicrobial resistance determinants and susceptibility were analyzed.ResultsA total of 352 isolates were collected, wherein 83 (23.6%) were hypervirulence genes-positive Kp (hgKp). A significant increase in KPC-2-producing KL47-ST11 among hgKp strains was observed, from 5.3% (1/19) in 2019 to 67.6% (25/37) in 2020 (P<.0001), suggesting the potential dissemination of the hybrid virulence and carbapenem-resistance encoding plasmid among children. Further, hgKp isolates were classified into hvKp (n = 27) and hgKp-low virulence (hgKp-Lv) (n = 56) based on virulence phenotypic assays. In hvKp, diverse genetic clones were observed and K1-ST23 or K2-ST25 strains with sensitivity to multiple antibiotics were prevalent (25.9%, 7/27). Compared with hgKp-Lv, hvKp infection had a higher propensity to involve severe pneumonia (22.2% vs. 12.5%) in elder children and significant higher mortality in mice (P = 0.0086). Additionally, either hvKp or hgKp-Lv infections were mostly healthcare-associated and hospital-acquired (74.1% vs. 91.9%).ConclusionsThese data suggest that K1-ST23 and K2-ST25 are high-risk clones of hvKp, and the genetic convergence of virulence and carbapenem-resistance is increasing among children. Control measures are needed to prevent the dissemination in clinical settings.

Abstract 6397: Understanding the glioblastoma microenvironment with spatial resolution in PDX models

Abstract Glioblastoma multiforme (GBM) is the most common adult brain tumour, and despite aggressive treatment, it recurs fatally. GBM tumours include diverse populations of malignant and non-neoplastic cells with distinct molecular capabilities and with differential levels of sensitivity to treatment. Understanding the cell dynamics that occur during the development of GBM resistance to therapy could reveal key aspects of this process, including how resistance is acquired in time and how the diverse cell types of the tumour microenvironment (TME) contribute to this phenotype. In addition to the role of the TME, GBM exhibits significant tumour heterogeneity with diverse genetic clones coexisting in the same tumor, as well as cells with similar genetic backgrounds capable of adopting distinct transcriptional states and subtypes. The complex and dynamic interactions between tumor and TME remain to be fully studied. This work focuses on the in vivo spatial organization in GBM during disease progression. We generated spatial transcriptomic data from a set of adult GBM samples grown as patient-derived xenograft (PDX) models, profiled at different time points of the disease. Three PDX lines from one GBM patient (derived from tumor core, vascularized area, and infiltrating front) were used to recapitulate the genetic and phenotypic heterogeneity observed in the human disease. Two replicates from each of 8 PDX mice were collected from early, mid, and late time points of tumour growth and data was generated using the 10X Genomics Visium platform. We developed a robust computational pipeline capable of distinguishing admixture of human (tumour) and mouse cells (TME), using state-of-the-art tools. Human and mouse cell types and states were identified using pooled and separate single-cell references of human GBM states, and mouse brain cells from both normal and tumour conditions. With this approach we observe spatially distinct patterns of both (a) tumour infiltration patterns specific to the each PDX line that includes non-random distribution of GBM transcriptional states and genetic clones, and (b) spatially distinct infiltration of TME components including microglial and macrophage populations. Overall, our approach addresses the challenge of understanding the tumor-TME relationship by application of spatial profiling in PDX models, and provides a computational pipeline complex multi-species analysis in the spatial transcriptomic field. Citation Format: Aly O. Abdelkareem, Katalin Osz, Donna Senger, Jennifer A. Chan, Sorana Morrissy. Understanding the glioblastoma microenvironment with spatial resolution in PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6397.

Abstract 4007: Evolutionary history of transformation from chronic lymphocytic leukemia to Richter’s syndrome

Abstract Richter’s syndrome (RS) arising from chronic lymphocytic leukemia (CLL) is a striking example of an aggressive malignant histology that emerges from indolent cancer. RS is a major barrier to disease control in CLL and is associated with poor clinical outcomes and limited survival. The genetic basis of RS is poorly understood, and its relationship to the antecedent CLL remains incompletely characterized. To study RS, we performed whole-exome sequencing (WES) on samples collected from 52 patients with RS of diffuse large B cell lymphoma (DLBCL) histology. For this genomic characterization, samples from 42 patients were analyzed as ‘trios’ (matched germline, CLL and RS tissue DNA) and those from 10 as ‘duos’ (matched CLL and RS DNA). Beyond addressing contamination of CLL DNA in the germline sample, we developed methods for discriminating between the RS and CLL clones which often coexist in the same samples. The discovery cohort revealed that RS and CLL were clonally-related in 45/52 (87%) cases based on WES analysis, with a distinct RS clone emerging from a CLL subclone. The remaining 13% were determined to be clonally unrelated. RS clones presented ~3x higher rates of additional mutations than the ancestral CLL clones from which they developed. We identified novel RS somatic driver mutations (in IRF2BP2, SRSF1, B2M, DNMT3A and others), a high rate of copy number variations with recurrent deletions (e.g., del(17p) [TP53], del(13q14.3), del(7q36), and del(15q13.11) [MGA], del(9p21.3) [CDKN2A/B], del(16q12.2)), focal amplifications (amp(7q21.2) [CDK6], amp(8q24.2) [RECQL4, MYC], amp(13q31.2) [ERCC5], and frequent whole genome duplication. To further investigate RS and CLL clonal evolution, we performed single-cell RNA-sequencing on biopsies at the time of RS diagnosis in 5 individuals with clonally related transformation. Using our novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations, yielding cluster-specific copy number profiles that matched the WES results of individual subclones of the RS and CLL populations. This enabled mapping genetic clones to specific expression patterns. Finally, we devised and tested a methodology that uses cfDNA for early detection of emerging Richter’s disease and have successfully identified Richter‘s tumor DNA in the blood several months prior to the clinical diagnosis. Our study thus defines drivers, distinct molecular subtypes and evolutionary path to RS and suggests strategies for its improved detection. Citation Format: Erin M. Parry, Ignaty Leshchiner, Romain Guièze, Connor Johnson, Eugen Tausch, Sameer Parikh, Camilla Lemvigh, Conor Messer, Daniel Rosebrock, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew Davids, Raquel A. Jacobs, Kara Slowik, Julien Broseus, Shanye Yin, Shuqiang Li, Geoff Fell, Ziao Lin, Binyamin A. Knisbacher, Neil Ruthen, Dimitri Livitz, Christof Schneider, Jialin Ma, Julian Hess, Laura Z. Rassenti, Thomas J. Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E. Kay, Kenneth J. Livak, Brian P. Danysh, Chip Stewart, Donna Neuberg, Jennifer R. Brown, Laxmi Parida, Stephan Stilgenbauer, Gad Getz, Catherine J. Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter’s syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4007.

Defense against Feeding by Spring Aphid Parasitism in the Upper Leaf Parts of Host Plants

In a symbiosis, each participant gains more fitness benefits than is paid in maintenance costs for the symbiosis. The mugwort aphid, Macrosiphoniella yomogicola, is ant-associated, and the host mugwort Artemisia montana is a genet-producing plant that has clonal aboveground shoots. M. yomogicola infests most A. montana shoots from spring to midsummer, and attending ants also repel leaf-eaters of the host plant. However, most aphid colonies become extinct after budding of A. montana inflorescence after early August. A few surviving aphid colonies (1∼3 per genet) produce sexuparae in mid-October. The shoots on which the sexuparae emerged lost most of their fitness because the aphids strongly suppress budding and growth of inflorescence. However, as the shoots are genetic clones of each other, the appearance of stem mothers in the next spring may result in early spreading of the aphids and attending ants to clonal shoots, which would protect the host from leaf-eaters. Here, we show that all shoots on a genet with stem mothers are occupied by aphids and ants much faster than those on a genet without stem mothers. The attending ants repel leaf-eaters to unimportant leaves for the host. Our results suggest that, as the shoots of a genet are all clones, sustaining the aphids on a few shoots may be beneficial to a genet as a whole through kin selection.

Long-term drought adaptation of unirrigated grapevines (Vitis vinifera L.)

Resilience to drought stress is an important crop selection and breeding target particularly under an environment of increasing freshwater scarcities resulting from higher evapotranspiration rates and reduced precipitation. Identifying new genetic material and clones with superior drought tolerance would increase available genetic resources and germplasm for both grapevine breeders and propagators, providing genetic material that has greater water use efficiency thereby reducing reliance on supplemental irrigation. Towards this goal, we explored the long-term drought adaptation of field-grown, unirrigated (or dry-grown) and own-rooted grapevines of pre-clonal origin from shallow (SR) and deep (DR) soils representing low and high soil water availability, respectively, in a South Australian vineyard. Despite lower soil moisture available to SR vines, both deep and shallow-rooted vines had similar vine water status, based on measurements of midday stem water potential ({Psi }_{s}), and leaf net photosynthesis (An). Due to the lower stomatal conductance (gs), SR had higher intrinsic water use efficiency (WUEi) than DR, however the carbon isotope ratio ({delta }^{13}C) of the fruit at harvest was similar between the two groups. Our observations suggest a degree of drought adaptation in the SR vines resulting from multi-decadal cyclical droughts. Overall, we demonstrate that pre-clonal Cabernet Sauvignon grapevines dry-grown in shallow soils have an enhanced resilience to drought compared to dry-grown vines in deep soils. This study has implications for selection of crop genetic material in a changing climate.

Heterogeneity and tumor evolution reflected in liquid biopsy in metastatic breast cancer patients: a review.

Breast cancer is a spatially and temporally dynamic disease in which differently evolving genetic clones are responsible for progression and clinical outcome. We review tumor heterogeneity and clonal evolution from studies comparing primary tumors and metastasis and discuss plasma circulating tumor DNA as a powerful real-time approach for monitoring the clonal landscape of breast cancer during treatment and recurrence. We found only a few early studies exploring clonal evolution and heterogeneity through analysis of multiregional tissue biopsies of different progression steps in comparison with circulating tumor DNA (ctDNA) from blood plasma. The model of linear progression seemed to be more often reported than the model of parallel progression. The results show complex routes to metastasis, however, and plasma most often reflected metastasis more than primary tumor. The described patterns of evolution and the polyclonal nature of breast cancer have clinical consequences and should be considered during patient diagnosis and treatment selection. Current studies focusing on the relevance of clonal evolution in the clinical setting illustrate the role of liquid biopsy as a noninvasive biomarker for monitoring clonal progression and response to treatment. In the clinical setting, circulating tumor DNA may be an ideal support for tumor biopsies to characterize the genetic landscape of the metastatic disease and to improve longitudinal monitoring of disease dynamics and treatment effectiveness through detection of residual tumor after resection, relapse, or metastasis within a particular patient.

Phytogenotypic Anthocyanin Profiles and Antioxidant Activity Variation in Fruit Samples of the American Cranberry (Vaccinium macrocarpon Aiton).

In this study, we conducted an analysis of the qualitative and quantitative composition of anthocyanins and anthocyanidins in different cultivars and genetic clones of American cranberries grown in Lithuanian climatic conditions. Four anthocyanin compounds predominated in fruit samples of American cranberry cultivars: cyanidin-3-galactoside, cyanidin-3-arabinoside, peonidin-3-galactoside, and peonidin-3-arabinoside. They accounted for 91.66 ± 2.79% of the total amount of the identified anthocyanins. The total anthocyanin content detected via the pH differential method was found to be by about 1.6 times lower than that detected via the UPLC method. Hierarchical cluster analysis and principal component analysis showed that the ‘Woolman’ cultivar distinguished from other cranberry cultivars in that its samples contained two times the average total amount of anthocyanins (8.13 ± 0.09 mg/g). The group of American cranberry cultivars ‘Howes’, ‘Le Munyon’, and ‘BL-8’ was found to have higher than average levels of anthocyanidin galactosides (means 3.536 ± 0.05 mg/g), anthocyanidins (means 0.319 ± 0.01 mg/g), and total anthocyanins (means 6.549 ± 0.09 mg/g). The evaluation of the antioxidant effect of cranberry fruit sample extracts showed that the greatest radical scavenging activity of the cranberry fruit extracts was determined in the fruit samples of ‘Woolman’ (849.75 ± 10.88 µmol TE/g) and the greatest reducing activity was determined in ‘Le Munyon’ (528.05 ± 12.16 µmol TE/g). The study showed a correlation between the total anthocyanin content and the antiradical and reductive activity of the extracts in vitro (respectively, R = 0.635 and R = 0.507, p < 0.05).

Genetic structure in patchy populations of a candidate foundation plant: a case study of Leymus chinensis using genetic and clonal diversity.

The distribution of genetic diversity on the landscape has critical ecological and evolutionary implications. This may be especially the case on a local scale for foundation plant species because they create and define ecological communities, contributing disproportionately to ecosystem function. We examined the distribution of genetic diversity and clones, which we defined first as unique multilocus genotypes (MLG), and then by grouping similar MLGs into multilocus lineages. We used 186 markers from inter-simple sequence repeats (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus chinensis. We examined the relationship between genetic and clonal diversities, their variation with patch size, and the effect of the number of markers used to evaluate genetic diversity and structure in this species. Every ramet had a unique MLG. Almost all patches consisted of individuals belonging to a single multilocus lineages. We confirmed this with a clustering algorithm to group related genotypes. The predominance of a single lineage within each patch could be the result of the accumulation of somatic mutations, limited dispersal, some sexual reproduction with partners mainly restricted to the same patch, or a combination of all three. We found strong genetic structure among patches of L. chinensis. Consistent with previous work on the species, the clustering of similar genotypes within patches suggests that clonal reproduction combined with somatic mutation, limited dispersal, and some degree of sexual reproduction among neighbors causes individuals within a patch to be more closely related than among patches.

Genetic Diversity within Leukemia-Associated Immunophenotype-Defined Subclones in AML

Acute myeloid leukemia (AML) is a heterogeneous clonal disease and in different patients, different combinations of mutations may be found that drive the disease. Also within patients, various leukemic clones with a different combination of mutations may co-exist, reflecting various evolutionary stages of the disease. At present, flowcytometry and mutational analyses are used to keep track of the malignant cells during and after treatment. Using panels of antibodies, abnormal cells can be identified when lineage markers are over- or underexpressed, or when abnormal differentiation leads to the co-expression of markers that normally are not expressed together on hematopoietic cells. These patient-specific leukemia-associated immunophenptypes (LAIPs) can be followed during and after treatment, and enable the early detection of resistant and recurring disease. Often, several LAIPs are present simultaneously within a patient, but their clonal relationship is not always clear. In addition, next-generation sequencing and mutation-specific PCR can be used to follow the leukemic cells. As the clinical consequences of MRD-positivity may consist of high-dose re-induction chemotherapy or stem cell transplantation, accurate results are of the highest importance. Although it has already been shown that flowcytometry and mutational analysis may have added value, in-depth information on the genetic make-up of immunophenotypically defined normal and leukemic subclones and their clonal relationship in AML is still scarce. To study the genetic similarities and differences between various normal and leukemic subclones defined by immunophenotyping, we performed an in-depth analysis of the genetic makeup of FACS-sorted immunophenotypically normal and aberrant subclones in 10 cases of AML. In total, 86 LAIPs and normal cell fractions were sorted and fully genotyped using error-corrected sequencing with a panel genes that are recurrently mutated in AML. In some cases, different aberrant populations from the same patient carried identical mutations, indicating that the leukemia was highly homogeneous, and that the phenotypically different LAIPs all were part of the same genetic clone. In these cases, monitoring the disease by following one of the present LAIPs would be sufficient, or, alternatively, following disease dynamics by molecularly monitoring one of the mutations would accurately show the dynamics of the disease. In other cases, different subclones harbored a different set of mutations. In addition, immunophenotypically normal populations frequently carried mutations, or, inversely, no mutations were found in immunophenotypically clearly aberrant clones. In these cases, following the disease by LAIPs or molecular markers would carry the risk that several subclones would be missed. In case these clones would be relevant for resistance or relapse, using either technique would be at risk to fail to detect MRD positivity. This work underscores that currently available, state-of-the-art immunophotyping and gene panels have added value and should be used simultaneously. We conclude that currently, MRD detection by molecular and immunophenotypic techniques are complementary, and should be used in parallel in order to obtain the most complete view on resistant disease and early detection of relapse. At the same time, both techniques can be further optimized with more monoclonal antibodies and genes that are screened for mutations, in order to increase their resolution. DisclosuresNo relevant conflicts of interest to declare.

Heterogeneity of Staphylococcus epidermidis in prosthetic joint infections: time to reevaluate microbiological criteria?

Prosthetic joint infection (PJI) is a feared and challenging to diagnose complication after arthroplasty, with Staphylococcus epidermidis as the major pathogen. One important criteria to define PJI is the detection of phenotypically indistinguishable microorganisms with identical antibiotic susceptibility pattern in at least two different samples. However, owing to phenotypical variation within genetic clones and clonal variation within a phenotype, the criteria may be ambiguous. We investigated the extent of diversity among coagulase-negative staphylococci (CoNS) in PJI and characterised S. epidermidis isolates from PJI samples, specifically multiple S. epidermidis isolates identified in individual PJI patients. We performed a retrospective cohort study on 62 consecutive patients with PJI caused by CoNS from two hospitals in Northern Sweden. In 16/62 (26%) PJIs, multiple S. epidermidis isolates were available for whole-genome analyses. Hospital-adapted multidrug-resistant genetic clones of S. epidermidis were identified in samples from 40/62 (65%) of the patients using a combination of pulsed-field gel electrophoresis and multilocus sequence typing. Whole-genome sequencing showed the presence of multiple sequence types (STs) in 7/16 (44%) PJIs where multiple S. epidermidis isolates were available. Within-patient phenotypical variation in the antibiotic susceptibility and/or whole-genome antibiotic resistance gene content was frequent (11/16, 69%) among isolates with the same ST. The results highlight the ambiguity of S. epidermidis phenotypic characterisation as a diagnostic method in PJI and call for larger systematic studies for determining the frequency of CoNS diversity in PJIs, the implications of such diversity for microbiological diagnostics, and the therapeutic outcomes in patients.

Stochastic clonal dynamics and genetic turnover in exponentially growing populations

We consider an exponentially growing population of cells undergoing mutations and ask about the effect of reproductive fluctuations (genetic drift) on its long-term evolution. We combine first step analysis with the stochastic dynamics of a birth–death process to analytically calculate the probability that the parent of a given genotype will go extinct. We compare the results with numerical simulations and show how this turnover of genetic clones can be used to infer the rates underlying the population dynamics. Our work is motivated by growing populations of tumour cells, the epidemic spread of viruses, and bacterial growth.

Practical Considerations for Using RNA Sequencing in Management of B-Lymphoblastic Leukemia: Malaysia-Singapore Acute Lymphoblastic Leukemia 2020 Implementation Strategy

Despite the immense genetic heterogeneity of B-lymphoblastic leukemia [or precursor B-cell acute lymphoblastic leukemia (B-ALL)], RNA sequencing (RNA-Seq) could comprehensively interrogate its genetic drivers, assigning a specific molecular subtype in >90% of patients. However, study groups have only started to use RNA-Seq. Forbroader clinical use, technical, quality control, and appropriate performance validation are needed. Wedescribe the development and validation of an RNA-Seq workflow for subtype classification, TPMT/NUDT15/TP53 variant discovery, and immunoglobulin heavy chain (IGH) disease clone identification for Malaysia-Singapore acute lymphoblastic leukemia (ALL) 2020. We validated this workflow in 377 patients in our preceding Malaysia-Singapore ALL 2003/Malaysia-Singapore ALL 2010 studies and proposed the quality control measures for RNA quality, library size, sequencing, and data analysis using the International Organization for Standardization 15189 quality and competence standard for medical laboratories. Compared with conventional methods, we achieved >95% accuracy in oncogene fusion identification, digital karyotyping, and TPMT and NUDT15 variant discovery. We found seven pathogenic TP53 mutations, confirmed with Sanger sequencing, which conferred a poorer outcome. Applying this workflow prospectively to the first 21 patients in Malaysia-Singapore ALL 2020, we identified the genetic drivers and IGH disease clones in >90% of patients with concordant TPMT, NUDT15, and TP53 variants using PCR-based methods. The median turnaround time was 12 days, which was clinically actionable. In conclusion, RNA-Seq workflow could be used clinically in management of B-cell ALL patients.

Patients Who Are Refractory to Ida-FLAG As Initial Induction Chemotherapy for AML Have Persistence of the Dominant Genetic Mutations and a Poor Clinical Outcome

Patients with AML who do not achieve remission with conventional 7+3 induction chemotherapy can frequently achieve CR with an intensive reinduction regimen such as Ida-FLAG. However, when Ida-FLAG is used as the initial induction regimen, the outcome of patients who do not achieve remission and the success of reinduction therapy is unknown.We identified 100 patients with newly diagnosed AML who received Ida-FLAG as their initial induction chemotherapy between November 2012 and September 2016. The median age was 59 (range 18-77); 49% were male. Twenty had therapy-related AML, 15 had AML following an MPN, 16 following MDS and 10 following an MDS/MPN overlap syndrome; 39 had de novo disease. One had low, 38 intermediate, 53 high risk and 8 unknown/inconclusive cytogenetics. Seventy-two achieved CR/CRi with FLAG-Ida induction. Nine died during induction prior to response assessment, and 19 were refractory. Survival, censored at the time of allogeneic transplantation, was longer for patients who achieved CR/CRi compared to those who did not (median OS 388 days vs 153 days, two-tailed log-rank p<0.001).Forty-four patients had DNA sequencing for mutations in 54 genes commonly mutated in AML using the TruSight Myeloid Sequencing Panel (TMSP; Illumina, San Diego, CA) on the Illumina MiSeq platform at the time of diagnosis. TP53 mutations were associated with disease refractoriness (p<0.05, corrected for multiple comparisons), as were high risk cytogenetics (p<0.05).Of the 19 patients whose disease was refractory to initial induction with Ida-FLAG, nine received intensive re-induction chemotherapy with NOVE-HiDAC (n=7), Ida-FLAG (n=1) and HiDAC, mitoxantrone, asparaginase, ATRA, vinblastine, valproate and imatinib (n=1). The remaining 10 patients received a low intensity experimental therapy on a clinical trial (n=1), low intensity/palliative therapy (n=2) or supportive care alone (n=7). Age, gender, presence of secondary AML and cytogenetic risk did not differ between those who did/did not receive reinduction.Of the 9 patients who received reinduction chemotherapy, only 1 achieved CR. This patient had secondary AML with high-risk cytogenetics and 70% blasts in the marrow at diagnosis. Following initial induction with Ida-FLAG, the patient had 6-7% marrow blasts with no recovery of their peripheral blood counts. The patient received a second course of Ida-FLAG as reinduction and achieved CR 51 days after the start of reinduction chemotherapy. The patient received an allotransplant but died of GVHD 69 days post-transplant. No other patient receiving reinduction chemotherapy responded and none achieved a PR/prolonged aplasia. No patients died during reinduction. No patients receiving experimental agents or low intensity therapy achieved CR or PR. Median OS for those receiving reinduction was similar to patients treated with palliative chemotherapy or clinical trial (182 v 153 days, p=0.2).We next assessed how Ida-FLAG and the reinduction regimens affected the genetic clones and subclones in the patients who did not achieve remission with Ida-FLAG. Paired blood or marrow samples from diagnosis and post-treatment were available for 4 consenting patients who did not achieve remission with Ida-FLAG or reinduction. For 2 patients, the second sample was available after Ida-FLAG induction and for 2 patients the second sample was available after reinduction. DNA was extracted and sequenced using the TMSP. All 4 patients had variants identified at diagnosis; 3 patients had recognized relevant somatic variants. All the predominant variants found at diagnosis were detected at follow-up. The variant allele fraction (VAF) of the predominant variants from diagnosis was similar or higher at the time of followup as shown in the figure. In one sample, variants found at low level in the diagnostic sample were reduced following reinduction but the dominant variant remained. No new variants were detected in follow-up samples.Thus, patients who do not achieve remission with Ida-FLAG as an initial induction regimen have a poor outcome and very low probability of achieving remission with a conventional reinduction regimen. Inferring clonal architecture by using VAF, reinduction chemotherapy may eliminate minor genetic subclones but the dominant clone persists. New therapeutic agents are needed for these patients. [Display omitted] DisclosuresKamel-Reid:BMS: Research Funding. Gupta:Incyte: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Schimmer:Medivir: Research Funding; Takeda Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Prevalence, antibiotic resistance, virulence and genetic diversity of Staphylococcus aureus isolated from bulk tank milk samples of U.S. dairy herds

BackgroundColonization of dairy cows by Staphylococcus aureus (S. aureus), especially those which are multi-drug resistant and toxin producing, is a concern for animal health and well-being as well as public health. The objective of this study was to investigate the prevalence, antibiotic resistance, gene content and virulence determinants of S. aureus in bulk tank milk samples (BTM) from U.S. dairy herds.ResultsBTM samples were collected, once in winter and once in summer, from 189 U.S. dairy herds. Of 365 BTM samples cultured, the sample and herd prevalence of S. aureus in BTM was 46.6% (170 of 365 samples) and 62.4% (118 of 189 herds), respectively. Among a subset of 138 S. aureus isolates that were stored for further analysis, 124 were genome sequenced after being confirmed as S. aureus using phenotypic tests. The most commonly identified antimicrobial resistance-associated gene was norA (99.2%) and mecA gene responsible for methicillin resistance (MRSA) was identified in one isolate (0.8%). The most frequently detected putative virulence genes were aur (100%), hlgB (100%), hlgA, hlgC, hlb (99.2%), lukE (95.9%) and lukD (94.3%). In the 53 staphylococcal enterotoxin positive isolates, sen (37.9%), sem (35.5%), sei (35.5%) and seg (33.1%) were the most frequently detected enterotoxin genes. Among the 14 sequence types (ST) and 18 spa types identified, the most common was ST2187 (20.9%) and t529 (28.2%), respectively. The most predominant clone was CC97 (47.6%) followed by CC unknown (36.3%). The single MRSA isolate belonged to ST72-CC8, spa type t126 and was negative for the tst gene but harbored all the other virulence genes investigated.ConclusionOur findings indicated a high prevalence of S. aureus in BTM of U.S. dairy herds, with isolates showing little evidence of resistance to antibiotics commonly used to treat mastitis. However, isolates often carried genes for the various enterotoxins. This study identified predominant genetic clones. Despite lower prevalence, the presence of MRSA and multi-drug resistant strains in BTM poses a significant risk to animal and public health if their number were to increase in dairy environment. Therefore, it is necessary to continuously monitor the use of antibiotics in dairy cows.

Infectious Clones Produce SARS-CoV-2 That Causes Severe Pulmonary Disease in Infected K18-Human ACE2 Mice.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has caused extensive mortality and morbidity and wreaked havoc on socioeconomic structures. The urgent need to better understand SARS-CoV-2 biology and enable continued development of effective countermeasures is aided by the production of laboratory tools that facilitate SARS-CoV-2 research. We previously created a directly accessible SARS-CoV-2 toolkit containing user-friendly reverse genetic (RG) infectious clones of SARS-CoV-2. Here, using K18-human ACE2 (hACE2) mice, we confirmed the validity of RG-rescued SARS-CoV-2 viruses to reproduce the infection profile, clinical disease, and pathogenesis already established in mice infected with natural SARS-CoV-2 isolates, often patient derived. RG-rescued SARS-CoV-2-infected K18-hACE2 mice developed substantial clinical disease and weight loss by day 6 postinfection. RG-rescued SARS-CoV-2 was recovered from the lungs and brains of infected K18-hACE2 mice, and infection resulted in viral pneumonia with considerable changes in lung pathology, as seen previously with natural SARS-CoV-2 infection. In mice infected with RG-rescued SARS-CoV-2-mCherry, mCherry was detected in areas of lung consolidation and colocalized with clinically relevant SARS-CoV-2-assocated immunopathology. RG-rescued SARS-CoV-2 viruses successfully recapitulated many of the features of severe COVID-19 associated with the K18-hACE2 model of SARS-CoV-2 infection. With utility in vivo, the RG-rescued SARS-CoV-2 viruses will be valuable resources to advance numerous areas of SARS-CoV-2 basic research and COVID-19 vaccine development.IMPORTANCE To develop COVID-19 countermeasures, powerful research tools are essential. We produced a SARS-COV-2 reverse genetic (RG) infectious clone toolkit that will benefit a variety of investigations. In this study, we further prove the toolkit's value by demonstrating the in vivo utility of RG-rescued SARS-CoV-2 isolates. RG-rescued SARS-CoV-2 isolates reproduce disease signs and pathology characteristic of the K18-hACE2 mouse model of severe COVID-19 in infected mice. Having been validated as a model of severe COVID-19 previously using only natural SARS-CoV-2 isolated from patients, this is the first investigation of RG-rescued SARS-CoV-2 viruses in K18-hACE2 mice. The RG-rescued SARS-CoV-2 viruses will facilitate basic understanding of SARS-CoV-2 and the preclinical development of COVID-19 therapeutics.

Genomic surveillance of methicillin-resistant Staphylococcus aureus in the Philippines, 2013-2014.

Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the leading causes of both nosocomial and community infections worldwide. In the Philippines, MRSA rates have remained above 50% since 2010, but resistance to other antibiotics, including vancomycin, is low. The MRSA burden can be partially attributed to pathogen-specific characteristics of the circulating clones, but little was known about the S. aureus clones circulating in the Philippines.We sequenced the whole genomes of 116 S. aureus isolates collected in 2013–2014 within the Antimicrobial Resistance Surveillance Program. The multilocus sequence type, spa type, SCCmec type, presence of antimicrobial resistance (AMR) determinants and virulence genes and relatedness between the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.The MRSA population in the Philippines comprised a limited number of genetic clones, including several international epidemic clones, such as CC30-spa-t019-SCCmec-IV-PVL+, CC5-SCCmec-typeIV and ST239-spa-t030-SCCmec-typeIII. The CC30 genomes were related to the South-West Pacific clone but formed a distinct, diverse lineage, with evidence of global dissemination. We showed independent acquisition of resistance to sulfamethoxazole/trimethoprim in various locations and genetic clones but mostly in paediatric patients with invasive infections. The concordance between phenotypic and genotypic resistance was 99.68% overall for eight antibiotics in seven classes.We have made the first comprehensive genomic survey of S. aureus in the Philippines, which bridges the gap in genomic data from the Western Pacific Region and will constitute the genetic background for contextualizing prospective surveillance.

Establishment of a Reverse Genetic System from a Bovine Derived Influenza D Virus Isolate.

The ruminant-associated influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5′ and 3′ rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5′ and 3′ NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5′ and 3′ non-coding regions (NCR) highlighted that the terminal 3′ end of all segments harbours an uracil instead of a cytosine nucleotide, similar to other influenza viruses. Subsequent analysis on the functionality of the 5′ and 3′ NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5′ and 3′ NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin, as well as by using an in vitro model recapitulating the natural replication site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.

An Adaptive Immune Ant Colony Optimization for Reducing Energy Consumption of Automatic Inspection Path Planning in Industrial Wireless Sensor Networks

Industrial wireless sensor networks (IWSNs) are usually fixedly deployed in industrial environments, and various sensor nodes cooperate with each other to complete industrial production tasks. The efficient work of each sensor node of IWSNs will improve the efficiency of the entire network. Automated robots need to perform timely inspection and maintenance of IWSNs in an industrial environment. Excessive inspection distance will increase inspection costs and increase energy consumption. Therefore, shortening the inspection distance can reduce production energy consumption, which is very important for the efficient operation of the entire system. However, the optimal detection path planning of IWSNs is an N‐P problem, which can usually only be solved by heuristic mathematical methods. This paper proposes a new adaptive immune ant colony optimization (AIACO) for optimizing automated inspection path planning. Moreover, novel adaptive operator and immune operator are designed to prevent the algorithm from falling into the local optimum and increase the optimization ability. In order to verify the performance of the algorithm, the algorithm is compared with genetic algorithm (GA) and immune clone algorithm (ICA). The simulation results show that the inspection distance of IWSNs using AIACO is lower than that of GA and ICA. In addition, the convergence speed of AIACO is faster than that of GA and ICA. Therefore, the AIACO proposed in this paper can effectively reduce the inspection energy consumption of the entire IWSN system.

Unique Mode of Cell Division by the Mycobacterial Genetic Resister Clones Emerging De Novo from the Antibiotic-Surviving Population.

The emergence of antibiotic genetic resisters of pathogenic bacteria poses a major public health challenge. The mechanism by which bacterial antibiotic genetic resister clones formed de novo multiply and establish a resister population remained unknown. Here, we delineated the unique mode of cell division of the antibiotic genetic resisters of Mycobacterium smegmatis and Mycobacterium tuberculosis formed de novo from the population surviving in the presence of bactericidal concentrations of rifampicin or moxifloxacin. The cells in the rifampicin/moxifloxacin-surviving population generated elevated levels of hydroxyl radical-inflicting mutations. The genetic mutants selected against rifampicin/moxifloxacin became multinucleated and multiseptated and developed multiple constrictions. These cells stochastically divided multiple times, producing sister-daughter cells phenomenally higher in number than what could be expected from their generation time. This caused an abrupt, unexpectedly high increase in the rifampicin/moxifloxacin resister colonies. This unique cell division behavior was not shown by the rifampicin resisters formed naturally in the actively growing cultures. We could detect such abrupt increases in the antibiotic resisters in others' and our earlier data on the antibiotic-exposed laboratory/clinical M. tuberculosis strains, M. smegmatis and other bacteria in in vitro cultures, infected macrophages/animals, and tuberculosis patients. However, it went unnoticed/unreported in all those studies. This phenomenon occurring in diverse bacteria surviving against different antibiotics revealed the broad significance of the present study. We speculate that the antibiotic-resistant bacillary clones, which emerge in patients with diverse bacterial infections, might be using the same mechanism to establish an antibiotic resister population quickly in the continued presence of antibiotics.IMPORTANCE The bacterial pathogens that are tolerant to antibiotics and survive in the continued presence of antibiotics have the chance to acquire genetically resistant mutations against the antibiotics and emerge de novo as antibiotic resisters. Once the antibiotic resister clone has emerged, often with compromise on growth characteristics, for the protection of the species, it is important to establish an antibiotic-resistant population quickly in the continued presence of the antibiotic. In this regard, the present study has unraveled multinucleation and multiseptation followed by multiple constrictions as the cellular processes used by the bacteria for quick multiplication to establish antibiotic-resistant populations. The study also points out the same phenomenon occurring in other bacterial systems investigated in our laboratory and others' laboratories. Identification of these specific cellular events involved in quick multiplication offers additional cellular processes that can be targeted in combination with the existing antibiotics' targets to preempt the emergence of antibiotic-resistant bacterial strains.

Gamma Knife Radiosurgery does not alter the copy number aberration profile in sporadic vestibular schwannoma

IntroductionIonizing radiation is a known etiologic factor in tumorigenesis and its role in inducing malignancy in the treatment of vestibular schwannoma has been debated. The purpose of this study was to identify a copy number aberration (CNA) profile or specific CNAs associated with radiation exposure which could either implicate an increased risk of malignancy or elucidate a mechanism of treatment resistance.Methods55 sporadic VS, including 18 treated with Gamma Knife Radiosurgery (GKRS), were subjected to DNA whole-genome microarray and/or whole-exome sequencing. CNAs were called and statistical tests were performed to identify any association with radiation exposure. Hierarchical clustering was used to identify CNA profiles associated with radiation exposure.ResultsA median of 7 (0–58) CNAs were identified across the 55 VS. Chromosome 22 aberration was the only recurrent event. A median aberrant cell fraction of 0.59 (0.25–0.94) was observed, indicating several genetic clones in VS. No CNA or CNA profile was associated with GKRS.ConclusionGKRS is not associated with an increase in CNAs or alteration of the CNA profile in VS, lending support to its low risk. This also implies that there is no major issue with GKRS treatment failure being due to CNAs. In agreement with previous studies, chromosome 22 aberration is the only recurrent CNA. VS consist of several genetic clones, addressing the need for further studies on the composition of cells in this tumor.