Genetic Variability and the Potential Range of Darevskia rostombekowi in Transcaucasia

Abstract— The results of the analysis of the genetic variability of parthenogenetic Darevskia rostombekowi (Darevsky, 1957) species using four microsatellite-containing loci are presented. Based on 118 records with geographical coordinates of the presence of this species in Transcaucasia, the maps of potential range were created. The analysis of the genetic structure of populations demonstrated that despite the established multiclonality (seven clonal lines in four populations), D. rostombekowi was formed as a result of a single act of hybridization between closely related bisexual species. The predicted distribution of D. rostombekowi using the modelling of potential range revealed new suitable habitats, where the presence of the species has not been reported previously. The results of this study and the absence of multiple acts of hybridization during the formation of these clones may indicate a regression of population size of the species. Consequently, the estimation of the conservation status of this parthenogenetic species seems to be justified.

Heatwave-associated Vibrio infections in Germany, 2018 and 2019

Background Vibrio spp. are aquatic bacteria that prefer warm seawater with moderate salinity. In humans, they can cause gastroenteritis, wound infections, and ear infections. During the summers of 2018 and 2019, unprecedented high sea surface temperatures were recorded in the German Baltic Sea. Aim We aimed to describe the clinical course and microbiological characteristics of Vibrio infections in Germany in 2018 and 2019. Methods We performed an observational retrospective multi-centre cohort study of patients diagnosed with domestically-acquired Vibrio infections in Germany in 2018 and 2019. Demographic, clinical, and microbiological data were assessed, and isolates were subjected to whole genome sequencing and antimicrobial susceptibility testing. Results Of the 63 patients with Vibrio infections, most contracted the virus between June and September, primarily in the Baltic Sea: 44 (70%) were male and the median age was 65 years (range: 2–93 years). Thirty-eight patients presented with wound infections, 16 with ear infections, six with gastroenteritis, two with pneumonia (after seawater aspiration) and one with primary septicaemia. The majority of infections were attributed to V. cholerae (non–O1/non-O139) (n = 30; 48%) or V. vulnificus (n = 22; 38%). Phylogenetic analyses of 12 available isolates showed clusters of three identical strains of V. vulnificus, which caused wound infections, suggesting that some clonal lines can spread across the Baltic Sea. Conclusions During the summers of 2018 and 2019, severe heatwaves facilitated increased numbers of Vibrio infections in Germany. Since climate change is likely to favour the proliferation of these bacteria, a further increase in Vibrio-associated diseases is expected.

Fast and reliable sgRNA efficiency testing using HIReff

CRISPR/Cas9 is the method of choice for gene editing like the endogenous knock-in of sequences in order to investigate protein function, abundance or intracellular localization. One of the crucial steps in the preparation of CRISPR/Cas9-mediated knock-ins is the design of sgRNAs, which need to be tested carefully in order to minimize off-target binding and reach highest cleavage efficiency. Usually, sgRNA is evaluated via mismatch cleavage assays, like Surveyor or T7 endonuclease 1 assay. We demonstrate that these methods are often highly cost- and time-intensive with a low sensitivity and high fail rate. As an alternative, we present a new HITI-based sgRNA efficiency (HIReff) test to precisely evaluate sgRNA efficiency. HIReff is based on a sophisticated integration vector with on-site generation of a linear donor fragment that allows a comparably easy read-out via fluorescence signal and integrates several internal controls. Next to a quantifiable sgRNA assessment, HIReff provides additional information on the 'gene/protein to be studied' abundance, subcellular localization and promoter activity and allows derivation of fluorescence protein labeled clonal lines. We highlight benefits of HIReff in comparison to commonly used enzyme-based assays and demonstrate improved practicability and high sensitivity, while being less time-, labor- and cost-intensive at the same time. Our results suggest HIReff as a fast and easy-to-use alternative for sgRNA efficiency testing.

Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae

The aphid Myzus persicae is a destructive agricultural pest that displays an exceptional ability to develop resistance to both natural and synthetic insecticides. To investigate the evolution of resistance in this species we generated a chromosome-scale genome assembly and living panel of >110 fully sequenced globally sampled clonal lines. Our analyses reveal a remarkable diversity of resistance mutations segregating in global populations of M. persicae. We show that the emergence and spread of these mechanisms is influenced by host–plant associations, uncovering the widespread co‐option of a host-plant adaptation that also offers resistance against synthetic insecticides. We identify both the repeated evolution of independent resistance mutations at the same locus, and multiple instances of the evolution of novel resistance mechanisms against key insecticides. Our findings provide fundamental insights into the genomic responses of global insect populations to strong selective forces, and hold practical relevance for the control of pests and parasites.

Improved Plasmodium falciparum dilution cloning through efficient quantification of parasite numbers and c-SNARF detection

Background Molecular and genetic studies of blood-stage Plasmodium falciparum parasites require limiting dilution cloning and prolonged cultivation in microplates. The entire process is laborious and subject to errors due to inaccurate dilutions at the onset and failed detection of parasite growth in individual microplate wells. Methods To precisely control the number of parasites dispensed into each microplate well, parasitaemia and total cell counts were determined by flow cytometry using parasite cultures stained with ethidium bromide or SYBR Green I. Microplates were seeded with 0.2 or 0.3 infected cells/well and cultivated with fresh erythrocytes. The c-SNARF fluorescent pH indicator was then used to reliably detect parasite growth. Results Flow cytometry required less time than the traditional approach of estimating parasitaemia and cell numbers by microscopic examination. The resulting dilutions matched predictions from Poisson distribution calculations and yielded clonal lines. Addition of c-SNARF to media permitted rapid detection of parasite growth in microplate wells with high confidence. Conclusion The combined use of flow cytometry for precise dilution and the c-SNARF method for detection of growth improves limiting dilution cloning of P. falciparum. This simple approach saves time, is scalable, and maximizes identification of desired parasite clones. It will facilitate DNA transfection studies and isolation of parasite clones from ex vivo blood samples.

Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation

The event of cellular reprogramming into pluripotency is influenced by several factors, such as in vitro culture conditions (e.g., culture medium and oxygen concentration). Herein, bovine iPSCs (biPSCs) were generated in different levels of oxygen tension (5% or 20% of oxygen) and supplementation (bFGF or bFGF + LIF + 2i—bFL2i) to evaluate the efficiency of pluripotency induction and maintenance in vitro. Initial reprogramming was observed in all groups and bFL2i supplementation initially resulted in a superior number of colonies. However, bFL2i supplementation in low oxygen led to a loss of self-renewal and pluripotency maintenance. All clonal lines were positive for alkaline phosphatase; they expressed endogenous pluripotency-related genes SOX2, OCT4 and STELLA. However, expression was decreased throughout the passages without the influence of oxygen tension. GLUT1 and GLUT3 were upregulated by low oxygen. The biPSCs were immunofluorescence-positive stained for OCT4 and SOX2 and they formed embryoid bodies which differentiated in ectoderm and mesoderm (all groups), as well as endoderm (one line from bFL2i in high oxygen). Our study is the first to compare high and low oxygen environments during and after induced reprogramming in cattle. In our conditions, a low oxygen environment did not favor the pluripotency maintenance of biPSCs.

Parasite-driven cascades or hydra effects: susceptibility and foraging depression shape parasite-host-resource interactions

When epidemics kill hosts and increase their resources, should the density of hosts decrease (with a resource increase, this constitutes a trophic cascade) or increase (a hydra effect)? Seeking answers, we integrate trait measurements, a resource-host-parasite model, and experimental epidemics with plankton. This combination reveals how a spectrum from cascades to hydra effects can arise. It reflects tension between parasite-driven mortality (a density-mediated effect) and foraging depression upon contact with parasite propagules (a trait-mediated one). In the model, mortality rises when higher susceptibility to infection increases infection prevalence. Epidemics release resources while suppressing hosts (creating a cascade). In contrast, when hosts are less susceptible and parasites depress their foraging, a resource feedback can elevate host density during epidemics (creating a hydra effect), particularly at higher carrying capacity of resources. This combination elevates primary production relative to per-host consumption of resources (two key determinants of host density). We test these predictions of the qualitative effects of host traits and resource carrying capacity with trait measurements and a mesocosm experiment. Trait measurements show clonal lines of zooplankton hosts differ in their foraging depression and susceptibility. We seeded resource-host-parasite mesocosms with different host genotypes and provided different nutrient supplies to test model predictions. Hydra effects and trophic cascades arose under different conditions, as predicted by the model. Hence, tension between trait-mediated and density-mediated effects of parasites governs the fate of host density during epidemics, from cascades to hydra effects, via feedbacks with resources.

ESBL and AmpC β-Lactamase Encoding Genes in E. coli From Pig and Pig Farm Workers in Vietnam and Their Association With Mobile Genetic Elements

Animals are considered important sources of ESBL/AmpC-producing bacteria in humans. We analyzed indications of transfer of ESBL/AmpC genes between pigs and pig farmers in Vietnam by analyzing whole genome sequences of 114 ESBL/AmpC-producing E. coli isolated from the two hosts, and performed conjugation experiments and plasmid profiling to confirm that such transfer could have happened. ESBL-encoding genes detected in pigs and pig farmers included blaCTX–M-55, blaCTX-M-27, blaCTX-M-65, blaCTX-M-15, blaCTX-M-14, blaCTX-M-3, blaCTX-M-24, and blaCARB-2, and AmpC β-lactamases included blaCMY-2, blaDHA-1, and blaCMY-42. The most frequent ESBL gene, blaCTX-M-55, was carried on plasmid with replicons types IncF, IncX, IncH, IncN, IncR, and IncP. The insertion transposases downstream of the blaCTX-M-55 gene were different in plasmids carried by different strains. The second most detected gene, blaCTX-M-27, is found in a stable genetic arrangement with the same flanking transposons seen across strains, and the gene was located on similar conjugal IncF plasmid types, suggesting a horizontal spread of these plasmids. In three strains, we observed a novel blaCTX-M-27 harboring IncF type of plasmid which had not been reported before. Its closest reference in NCBI was the non-ESBL Salmonella Typhimurium plasmid pB71 that might have experienced an insertion of blaCTX-M-27. Our data also point to an emergence of plasmids co-carrying ESBL genes, mcr genes, quinolones and other antimicrobials resistance determinants, and such plasmids require special attention. Plasmids phylogeny confirmed that the blaCTX-M-55 encoding plasmids varied considerably, while those encoding blaCTX-M-27 were closely related. Plasmids harboring both ESBL genes were confirmed to be conjugative and not to differ in transfer efficacy. The isolates carrying the plasmids, even those with plasmids of similar types, showed wide genetic variation with high number of SNPs, suggesting horizontal spread of plasmids into different clonal lines. Their virulence profiles did not confirm to known pathotypes, suggesting that unrelated commensals are a main reservoir for ESBL and AmpC β-lactamases in both humans and pigs. Overall, despite evidence of transferability of plasmids in the analyzed strains, our findings do not support that ESBL-producing E. coli from pigs or their ESBL/AmpC encoding plasmids are commonly spread to workers in close contact with the animals.

Accumulation of somatic mutations leads to genetic mosaicism in Cannabis

Cannabis is typically propagated using stem cuttings taken from mother plants to produce genetically uniform propagules. However, producers anecdotally report that clonal lines deteriorate over time and eventually produce clones with less vigour and lower cannabinoid levels than the original mother plant. While the cause of this deterioration has not been investigated, one potential contributor is the accumulation of somatic mutations within the plant. To test this, we used deep sequencing of whole genomes (>50x depth of coverage) to compare the variability within an individual Cannabis sativa cv. Honey Banana plant sampled at the bottom, middle and top. Overall, we called over 6 million sequence variants based on a published reference genome (SNPs, MNPs, and indels) and found that that the top had the most by a sizable amount. We compared the variants among the samples and uncovered that nearly 600K (34%) were unique to the top while the bottom only contained 148K (12%) and middle with 77K (9%) unique variants. Bioinformatics tools were used to identify high impact mutations in critical cannabinoid/terpene biosynthesis pathways. While none were identified, some contained more than double the average level of nucleotide diversity (π) in or near the gene, including OLS, CBDAS, HMGR2 and CsTPS9FN. The first two genes code for essential enzymes required for the cannabinoid pathway while the other two are involved in the terpene pathways, demonstrating that mutations were accumulating within these pathways and could influence their function. Overall, these data identified a measurable number of intra-plant genetic diversity that could impact the long-term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigour and cannabinoid content.

Distinct clonal lineages and within-host diversification shape invasive Staphylococcus epidermidis populations

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.