Isolation Of Clones Research Articles

Subclonal Populations Detected by Flow Cytometry in Chronic Lymphocytic Leukemia

Abstract Introduction Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a mature, small B-cell lymphoma that typically co-expresses CD5 and CD23. Molecular-targeted therapies have produced remarkable therapeutic benefits in CLL, yet many patients are resistant to treatment. This study aims to identify subpopulations in CLL by conventional flow cytometry and clustering analysis. Methods One-hundred-and-forty-one CLL cases were taken from a public database and analyzed by uniform manifold approximation and projection (UMAP). Differences in size, granularity, and intensities of CD19, CD20, CD79a, CD5, CD23, CD10, and FMC7 were plotted with BD FACSDiva Software. Eighteen cases with a new diagnosis of CLL at our institution between January 1, 2021 to December 31, 2022 were analyzed. Only peripheral blood samples were included. Markers including forward scatter, side scatter, and fluorescent intensities of CD19, CD20, CD5, CD23, CD10, surface kappa, and surface lambda were used to identify discrete CLL clusters in UMAP plots. Results: Public Dataset Clustering analysis of conventional flow cytometry data from 141 cases from a public database reveals discrete populations of cells exhibiting varying sizes, granularity and fluorescent intensities. Among the 141 cases, 33 (23.4%) had 1 cluster, 64 (45.4%) with 2 clusters, 42 (29.8%) with 3 clusters and 2 (1.4%) with 4 clusters. Among the cases with 2 clusters, a smaller population of cells deviated from the main neoplastic population. This smaller population had a higher forward and side scatter, and brighter intensities in CD45 and CD19, consistent with prolymphocytes. Among the cases with 3 clusters, a third population recurrently had a smaller size by forward scatter. A few cases additionally had varying intensities by CD23 and CD19. Among the 2 cases with 4 clusters, only slight deviations by UMAP were seen and may be artifactual. Institutional Dataset Clustering analysis of 18 cases at our institution revealed complex patterns of cell clusters exhibiting varying sizes, granularity and surface receptor expression intensity. Among the 18 cases, 1 (5.6%) had 1 cluster, 1 (5.6%) with 2 clusters, 10 (55.6%) with 3 clusters, 3 (16.7%) with 4 clusters, and 3 (16.7%) with 5 clusters. Similar to the public dataset, the third population, in addition to the predominant population and prolymphocytes, consistently varied by lower forward scatter. Additional clusters, such as a population with varying CD23 signal intensities, were identified in some cases. Conclusions In our study, multiple clonal subsets in CLL/SLL are detected by conventional flow cytometry analysis. These subclonal populations may result in therapy resistance for these patients, and identifying them by flow cytometry analysis may provide additional prognostic information in the clinical setting. Furthermore, the ability to detect these subclonal populations may allow for isolation of specific clones for future investigation in research settings.

Evolution recovers the fitness of Acinetobacter baylyi strains with large deletions through mutations in deletion-specific targets and global post-transcriptional regulators.

Organelles and endosymbionts have naturally evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have purposefully engineered streamlined microbial genomes to create more efficient cellular chassis and define the minimal components of cellular life. During natural or engineered genome streamlining, deletion of many non-essential genes in combination often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-free Acinetobacter baylyi strain ADP1-ISx that each had a deletion of a different 22- to 42-kilobase region and two strains with larger deletions of 70 and 293 kilobases. We evolved replicate populations of ADP1-ISx and each deletion strain for ~300 generations in a chemically defined minimal medium or a complex medium and sequenced the genomes of endpoint clonal isolates. Fitness increased in all cases that were examined except for two ancestors that each failed to improve in one of the two environments. Mutations affecting nine protein-coding genes and two small RNAs were significantly associated with one of the two environments or with certain deletion ancestors. The global post-transcriptional regulators rnd (ribonuclease D), csrA (RNA-binding carbon storage regulator), and hfq (RNA-binding protein and chaperone) were frequently mutated across all strains, though the incidence and effects of these mutations on gene function and bacterial fitness varied with the ancestral deletion and evolution environment. Mutations in this regulatory network likely compensate for how an earlier deletion of a transposon in the ADP1-ISx ancestor of all the deletion strains restored csrA function. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution and that recovery tends to occur through a combination of deletion-specific compensation and global regulatory adjustments.

Analysis of fine-scale phylogeny of Burkholderia pseudomallei in relation to regional geography and drug susceptibility in Thailand

Melioidosis caused by Burkholderia pseudomallei (Bp) is a public health threat. Genomic-epidemiology research on this deadly disease is scarce. We investigated whole-genome sequences of Bp isolates in relation to environmental source and drug susceptibility. In total, 563 Bp isolates were collected from 11 Northeast Thai provinces during the period 2004–2021. Patients (n = 530 isolates), infected animals (n = 8), and environmental sources (n = 25) provided samples. Phylogenetic analysis revealed genetic diversity among the Bp isolates, including numerous well-supported clusters of varying sizes. Through in-depth analysis of 38 monophyletic clades (MCs), we found eleven associated with province of origin (p-value < 0.001). Closely related clusters (CRCs) within MCs resembled MLST-identified “sequence types” (STs). We found 102 known and 52 novel STs. ST-70 was the most prevalent in this area (n = 78; 13.85%). Sample type (human/environmental) and sampling time intervals were not correlated with genetic distance among clonal Bp isolates. Some members of 12 CRCs had acquired resistance to co-trimoxazole and one against amoxicillin-clavulanic acid. Within Northeast Thailand, there is an association between Bp genotype and geographical origin.

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) RdJ clone (CC5-ST105-SCCmecII-t002) in Santa Catarina, Brazil

The emergence of highly successful genetic lineages of methicillin-resistant Staphylococcus aureus (MRSA) poses a challenge in human healthcare due to increased morbidity and mortality rates. The RdJ clone (CC5-ST105-SCCmecII-t002 lineage), previously identified in Rio de Janeiro, Brazil, was linked to bloodstream infections and features a mutation in the aur gene (encoding aureolysin). Additionally, clinical isolates derived from this clone were more effective at evading monocytic immune responses. This study aimed to detect the RdJ clone among clinical MRSA isolated in Santa Catarina (SC) and examine its antimicrobial resistance and phagocytosis evasion capabilities. Our findings revealed the RdJ clone in 20 % of MRSA isolates, all exhibiting multiresistance. RdJ clone isolates from SC did not demonstrate a decreased rate of phagocytosis compared to CC5 non-RdJ isolates. Structural analysis suggests that the aur mutation is unlikely to significantly impact aureolysin activity. Genomic analysis of one isolate unveiled a genetic variant of the RdJ clone, sharing lineage and gene distribution but lacking the aur mutation. This study enhances the understanding of the clinical and epidemiologic risks associated with the RdJ clone and the biological mechanisms underlying its spreading in SC.

Multiple Horizontal Mini-chromosome Transfers Drive Genome Evolution of Clonal Blast Fungus Lineages.

Crop disease pandemics are often driven by asexually reproducing clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb accessory mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

Rewinding the Tape to Identify Intrinsic Determinants of Reprogramming Potential.

Via retrospective isolation of clones using Rewind, Jain et al. identified primed states of cells that reprogram to induced pluripotent stem cells. Examining clones, they find that cells retain memory of over several rounds of cell division. Moreover, they show that extrinsic factors change the number of primed cells, suggesting that there exist diverse paths of reprogramming and states of priming.

Genomic characterization and virulence gene profiling of Erysipelothrix rhusiopathiae isolated from widespread muskox mortalities in the Canadian Arctic Archipelago

BackgroundMuskoxen are important ecosystem components and provide food, economic opportunities, and cultural well-being for Indigenous communities in the Canadian Arctic. Between 2010 and 2021, Erysipelothrix rhusiopathiae was isolated from carcasses of muskoxen, caribou, a seal, and an Arctic fox during multiple large scale mortality events in the Canadian Arctic Archipelago. A single strain (‘Arctic clone’) of E. rhusiopathiae was associated with the mortalities on Banks, Victoria and Prince Patrick Islands, Northwest Territories and Nunavut, Canada (2010–2017). The objectives of this study were to (i) characterize the genomes of E. rhusiopathiae isolates obtained from more recent muskox mortalities in the Canadian Arctic in 2019 and 2021; (ii) identify and compare common virulence traits associated with the core genome and mobile genetic elements (i.e. pathogenicity islands and prophages) among Arctic clone versus other E. rhusiopathiae genomes; and iii) use pan-genome wide association studies (GWAS) to determine unique genetic contents of the Arctic clone that may encode virulence traits and that could be used for diagnostic purposes.ResultsPhylogenetic analyses revealed that the newly sequenced E. rhusiopathiae isolates from Ellesmere Island, Nunavut (2021) also belong to the Arctic clone. Of 17 virulence genes analysed among 28 Arctic clone isolates, four genes – adhesin, rhusiopathiae surface protein-A (rspA), choline binding protein-B (cbpB) and CDP-glycerol glycerophosphotransferase (tagF) – had amino acid sequence variants unique to this clone when compared to 31 other E. rhusiopathiae genomes. These genes encode proteins that facilitate E. rhusiopathiae to attach to the host endothelial cells and form biofilms. GWAS analyses using Scoary found several unique genes to be overrepresented in the Arctic clone.ConclusionsThe Arctic clone of E. rhusiopathiae was associated with multiple muskox mortalities spanning over a decade and multiple Arctic islands with distances over 1000 km, highlighting the extent of its spatiotemporal spread. This clone possesses unique gene content, as well as amino acid variants in multiple virulence genes that are distinct from the other closely related E. rhusiopathiae isolates. This study establishes an essential foundation on which to investigate whether these differences are correlated with the apparent virulence of this specific clone through in vitro and in vivo studies.

Occurrence of multi-carbapenemase-producing Enterobacterales in a tertiary hospital in Madrid (Spain): A new epidemiologic scenario

IntroductionMulti-carbapenemase-producing Enterobacterales (M-CPE) are increasingly described. We characterized the M-CPE isolates prospectively recovered in our hospital (Madrid, Spain) over two years (2021–2022). MethodsWe collected 796 carbapenem resistant Enterobacterales (CRE) from clinical and surveillance samples. Carbapenemase production was confirmed with phenotypic (immunochromatographic, disk diffusion) and molecular (PCR, WGS) techniques. Antimicrobial susceptibility was evaluated by a standard broth microdilution method. Clinical and demographic data were collected. ResultsOverall, 23 M-CPE (10 Klebsiella pneumoniae, 6 Citrobacter freundii complex, 3 Escherichia coli, 2 Klebsiella oxytoca, and 2 Enterobacter hormaechei) isolates were recovered from 17 patients (3% with CPE, 0.26-0.28 cases per 1000 admissions). OXA-48 + KPC-3 (7/23) and KPC-3 + VIM-1 (5/23) were the most frequent carbapenemase combinations. All patients had prior antibiotics exposure, including carbapenems (8/17). High resistance rates to ceftazidime/avibactam (14/23), imipenem/relebactam (16/23) and meropenem/vaborbactam (7/23) were found. Ceftazidime/avibactam + aztreonam combination was synergistic in all metallo-β-lactamase producers. Clonal and non-clonal related isolates were found, particularly in K. pneumoniae (5 ST29, 3 ST147, 3 ST307) and C. freundii (3 ST8, 2 ST125, 1 ST563). NDM-1 + OXA-48 was introduced with the ST147-K. pneumoniae high-risk clone linked to the transfer of a Ukrainian patient. We identified four possible nosocomial clonal transmission events between patients of the same clone with the same combination of carbapenemases (KPC-3 + VIM-1-ST29-K. pneumoniae, NDM-1 + OXA-48-ST147-K. pneumoniae and KPC-2 + VIM-1-ST145-K. oxytoca). Carbapenemase-encoding genes were located on different plasmids, except for VIM-1 + KPC-2-ST145-K. oxytoca. Cross-species transmission and a possible acquisition overtime was found, particularly between K. pneumoniae and E. coli producing OXA-48 + KPC-3. ConclusionM-CPE is an emerging threat in our hospital. Co-production of different carbapenemases, including metallo-β-lactamases, limits therapeutic options and depicts the need to reinforce infection control measures.

Genomic study of European Clostridioides difficile ribotype 002/sequence type 8.

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

IsolateR: an R package for generating microbial libraries from Sanger sequencing data.

Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species. isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections. isolateR is available at: https://github.com/bdaisley/isolateR.

EpiXact-ONT: Long-read whole genome sequencing for rapid outbreak detection and comprehensive plasmid transmission analysis

Background: Healthcare associated infections (HAIs) are a major contributor to patient morbidity and mortality. HAIs are increasingly important due to the rise of multidrug resistant pathogens which can lead to deadly nosocomial outbreaks. Traditional methods for investigating transmissions are slow, costly and have poor detection resolution. In addition, plasmid transmission which can horizontally transfer critical resistance and virulence genes is not part of routine infection control practice due to lack of comprehensive and cost-effective methods capable of identifying both pathogen and/or plasmid transmission. Here we demonstrate the utility of the Oxford Nanopore Technologies (ONT) platform for whole genome sequencing (WGS) based pathogen and plasmids transmission analysis. Methods: We developed a rapid end-to-end process that includes sample preparation, sequencing optimized for generating long-reads and bioinformatics workflow customized for error-prone ONT data. We use Flye to generate de novo assemblies and a secondary bioinformatics step to identify each circular sequence. Individual circular sequences with an Ori (≥1) are identified. For pathogen clonality analysis we perform a pairwise mapping-based chromosomal sequences comparison eliminating need for an external reference genome. Similarly, individual plasmids are separated and compared pairwise. We annotate both the circularized chromosomal and plasmid sequences for known resistance and virulence genes. Results: We performed ONT (and confirmatory Illumina) sequencing of the genomes of 20 bacterial isolates originating from 5 HAI investigations previously performed at Day Zero Diagnostics using epiXact®, our Illumina-based HAI sequencing and analysis lab service. ONT-based clonality determination had 100% agreement with the Illumina based pipeline. We also found that using the outbreak-specific assembled genomes instead of an external reference increased the SNP-calling resolution in the ONT pipeline. We also identified sets of clonal isolates with both identical plasmids and distinct plasmids; as well as sets of non-clonal isolates with identical plasmids and distinct plasmids. In one subset of 7 multi-species isolates, we identified 2-7 circularized plasmid sequences in each isolate, all harboring known resistance genes. 4 plasmids were found in multiple isolates, with each plasmid appearing in between 2 and 4 distinct isolates. Notably, blaNDM was identified in at least 1 plasmid in each isolate. Conclusion: We demonstrate the utility of ONT for comprehensive HAI investigations, establishing the potential to transform healthcare epidemiology with rapid outbreak determination covering pathogen and plasmid transmission in &lt; 2 4 hours from sample receipt.

An evaluation of a Stenotrophomonas maltophilia outbreak due to commercial arterial blood gas collection kit

BackgroundStenotrophomonas maltophilia is a gram-negative bacterium that can cause hospital infections and outbreaks within hospitals. This study aimed to evaluate an outbreak of Stenotrophomonas maltophilia, caused by ready-to-use commercial syringes containing liquid lithium and heparin for arterial blood gas collection in a university hospital.MethodsUpon detecting an increase in Stenotrophomonas maltophilia growth in blood cultures between 15.09.2021 and 19.11.2021, an outbreak analysis and a case-control study (52 patients for the case group, 56 patients for the control group) were performed considering risk factors for bacteremia. Samples from possible foci for bacteremia were also cultured. Growing bacteria were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The genetic linkage and clonal relationship isolates were investigated with pulsed-field gel electrophoresis (PFGE) in the reference laboratory.ResultsIn the case-control study, the odds ratio for the central venous catheter [3.38 (95% confidence interval [CI]: 1.444, 8.705 ; p = 0.006)], for surgery [3.387 (95% confidence interval [CI]: 1.370, 8.373 ; p = 0.008)] and for arterial blood gas collection history [18.584 (95% confidence interval [CI]:4.086, 84.197; p < 0.001)] were identified as significant risk factors. Stenotrophomonas maltophilia growth was found in ready-to-use commercial syringes used for arterial blood gas collection. Molecular analysis showed that the growths in the samples taken from commercial syringes and the growths from blood cultures were the same. It was decided that the epidemic occurred because the method for sterilization of heparinized liquid preparations were not suitable. After discontinuing the use of the kits with this lot number, the outbreak was brought under control.ConclusionsAccording to our results, disposable or sterile medical equipment should be included as a risk factor in outbreak analyses. The method by which injectors containing liquids, such as heparin, are sterilized should be reviewed. Our study also revealed the importance of the cooperation of the infection control team with the microbiology laboratory.

Engineering the cellulolytic bacterium, Clostridium thermocellum, to co-utilize hemicellulose

Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different β-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the β-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.

Microevolution of Candida glabrata (Nakaseomyces glabrata) during an infection

Candida glabrata (Nakaseomyces glabrata) is an emergent and opportunistic fungal pathogen that colonizes and persists in different niches within its human host. In this work, we studied five clinical isolates from one patient (P7), that have a clonal origin, and all of which come from blood cultures except one, P7-3, obtained from a urine culture. We found phenotypic variation such as sensitivity to high temperature, oxidative stress, susceptibility to two classes of antifungal agents, and cell wall porosity. Only isolate P7-3 is highly resistant to the echinocandin caspofungin while the other four isolates from P7 are sensitive. However, this same isolate P7-3, is the only one that displays susceptibility to fluconazole (FLC), while the rest of the isolates are resistant to this antifungal. We sequenced the PDR1 gene which encodes a transcription factor required to induce the expression of several genes involved in the resistance to FLC and found that all the isolates encode for the same Pdr1 amino acid sequence except for the last isolate P7-5, which contains a single amino acid change, G1099C in the putative Pdr1 transactivation domain. Consistent with the resistance to FLC, we found that the CDR1 gene, encoding the main drug efflux pump in C. glabrata, is highly overexpressed in the FLC-resistant isolates, but not in the FLC-sensitive P7-3. In addition, the resistance to FLC observed in these isolates is dependent on the PDR1 gene. Additionally, we found that all P7 isolates have a different proportion of cell wall carbohydrates compared to our standard strains CBS138 and BG14. In P7 isolates, mannan is the most abundant cell wall component, whereas β-glucan is the most abundant component in our standard strains. Consistently, all P7 isolates have a relatively low cell wall porosity compared to our standard strains.These data show phenotypic and genotypic variability between clonal isolates from different niches within a single host, suggesting microevolution of C. glabrata during an infection.

Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry

BackgroundThe wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein.ResultsThe sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct.ConclusionsThe GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx- strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains' distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx-) evolutionary paths.

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display.

Gallbladder cancer (GBC) is a very aggressive malignant neoplasm of the biliary tract with a poor prognosis. There are no specific therapies for the treatment of GBC or early diagnosis tools; for this reason, the development of strategies and technologies that facilitate or allow an early diagnosis of GBC continues to be decisive. Phage display is a robust technique used for the production of monoclonal antibodies (mAbs) involving (1) the generation of gene libraries, (2) the screening and selection of isoforms related to an immobilized antigen, and (3) the in vitro maturation of the affinity of the antibody for the antigen. This research aimed to construct a human immune library from PBMCs of GBC patients and the isolation of scFv-phage clones with specificity against the larger extracellular loop belonging to claudin 18.2, which is an important biomarker overexpressed in GBC as well as gastric cancer. The immune-library-denominated GALLBLA1 was constructed from seven GBC patients and has a diversity of 6.12 × 1010pfu mL-1. After three rounds of panning, we were able to identify clones with specificity against claudin 18.2. GALLBLA1 can contribute to the selection, isolation, and recombinant production of new human mAbs candidates for the treatment of gastrointestinal cancers.

Development and validation of a random forest algorithm for source attribution of animal and human Salmonella Typhimurium and monophasic variants of S. Typhimurium isolates in England and Wales utilising whole genome sequencing data.

Source attribution has traditionally involved combining epidemiological data with different pathogen characterisation methods, including 7-gene multi locus sequence typing (MLST) or serotyping, however, these approaches have limited resolution. In contrast, whole genome sequencing data provide an overview of the whole genome that can be used by attribution algorithms. Here, we applied a random forest (RF) algorithm to predict the primary sources of human clinical Salmonella Typhimurium (S. Typhimurium) and monophasic variants (monophasic S. Typhimurium) isolates. To this end, we utilised single nucleotide polymorphism diversity in the core genome MLST alleles obtained from 1,061 laboratory-confirmed human and animal S. Typhimurium and monophasic S. Typhimurium isolates as inputs into a RF model. The algorithm was used for supervised learning to classify 399 animal S. Typhimurium and monophasic S. Typhimurium isolates into one of eight distinct primary source classes comprising common livestock and pet animal species: cattle, pigs, sheep, other mammals (pets: mostly dogs and horses), broilers, layers, turkeys, and game birds (pheasants, quail, and pigeons). When applied to the training set animal isolates, model accuracy was 0.929 and kappa 0.905, whereas for the test set animal isolates, for which the primary source class information was withheld from the model, the accuracy was 0.779 and kappa 0.700. Subsequently, the model was applied to assign 662 human clinical cases to the eight primary source classes. In the dataset, 60/399 (15.0%) of the animal and 141/662 (21.3%) of the human isolates were associated with a known outbreak of S. Typhimurium definitive type (DT) 104. All but two of the 141 DT104 outbreak linked human isolates were correctly attributed by the model to the primary source classes identified as the origin of the DT104 outbreak. A model that was run without the clonal DT104 animal isolates produced largely congruent outputs (training set accuracy 0.989 and kappa 0.985; test set accuracy 0.781 and kappa 0.663). Overall, our results show that RF offers considerable promise as a suitable methodology for epidemiological tracking and source attribution for foodborne pathogens.

Exploring Rhodospirillum rubrum response to high doses of carbon monoxide under light and dark conditions

Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and gasification. Their main final product is synthesis gas (syngas) composed of CO, CO2, H2, and methane. Syngas can be converted into various products using CO-tolerant microorganisms. Among them, Rhodospirillum rubrum is highlighted for its biotechnological potential. However, the extent to which high doses of CO affect its physiology is still opaque. For this reason, we have studied R. rubrum behavior under high levels of this gas (up to 2.5 bar), revealing a profound dependence on the presence or absence of light. In darkness, the key variable affected was the lag phase, where the highest levels of CO retarded growth to more than 20 days. Under light, R. rubrum ability to convert CO into CO2 and H2 depended on the presence of an additional carbon source, such as acetate. In those conditions where CO was completely exhausted, CO2 fixation was unblocked, leading to a diauxic growth. To enhance R. rubrum tolerance to CO in darkness, a UV-accelerated adaptive laboratory evolution (UVa-ALE) trial was conducted to isolate clones with shorter lag phases, resulting in the isolation of clones 1.4-2B and 1.7-2A. The adaptation of 1.4-2B was mainly based on mutated enzymes with a metabolic function, while 1.7-3A was mostly affected at regulatory genes, including the anti-repressor PpaA/AerR. Despite these mutations having slight effects on biomass and pigment levels, they successfully provoked a significant reduction in the lag phase (−50%).Keypoints• CO affects principally R. rubrum lag phase (darkness) and growth rate (light)• CO is converted to CO2/H2during acetate uptake and inhibits CO2fixation (light)• UVa-ALE clones showed a 50% reduction in the lag phase (darkness)Graphical

Comparative analysis of Argentinian and European populations of Ramularia collo‐cygni on barley

AbstractRamularia collo‐cygni (Rcc) is a major barley pathogen that causes yield and grain quality losses worldwide. The main sources of Rcc inoculum are the seed and asexual airborne spores. In Argentina, Rcc is considered to be an emerging threat to barley crops, especially as most varieties are susceptible to Rcc and have a European genetic background. Here, we describe the population genetic diversity and structure of the Argentinian Rcc population, based on 10 simple‐sequence repeat (SSR) markers, in order to compare it with Rcc populations from the Czech Republic and Scotland. The Argentinian Rcc population showed lower genetic diversity, higher level of structuring and higher number of clonal isolates than European populations. Significant differentiation at population origin (country) and region (Europe and South America) level suggests the occurrence of a genetic bottleneck and/or a founder effect on Rcc entry to Argentina and that this population could still be in a state of establishment and emergence. Further research on Rcc genetic structure at local and global scale will be crucial for the understanding of Rcc population dynamics for disease management.