Antibiotic resistance monitoring in wastewater in the Nordic countries: A systematic review.

The Nordic countries (Denmark, Finland, Iceland, Norway, and Sweden) have effectively kept lower antibiotic-resistant bacterial (ARB) pathogen rates than many other countries. However, in recent years, these five countries have encountered a rise in ARB cases and challenges in treating infections due to the growing prevalence of ARB pathogens. Wastewater-based surveillance (WBS) is a valuable supplement to clinical methods for ARB surveillance, but there is a lack of comprehensive understanding of WBS application for ARB in the Nordic countries. This review aims to compile the latest state-of-the-art developments in WBS for ARB monitoring in the Nordic countries and compare them with clinical surveillance practices. After reviewing 1480 papers from the primary search, 54 were found relevant, and 15 additional WBS-related papers were included. Among 69 studies analyzed, 42 dedicated clinical epidemiology, while 27 focused on wastewater monitoring. The PRISMA review of the literature revealed that Nordic countries focus on four major WBS objectives of ARB: assessing ARB in the human population, identifying ARB evading wastewater treatment, quantifying removal rates, and evaluating potential ARB evolution during the treatment process. In both clinical and wastewater contexts, the most studied targets were pathogens producing carbapenemase and extended-spectrum beta-lactamase (ESBL), primarily Escherichia coli and Klebsiella spp. However, vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) have received more attention in clinical epidemiology than in wastewater studies, probably due to their lower detection rates in wastewater. Clinical surveillance has mostly used culturing, antibiotic susceptibility testing, and genotyping, but WBS employed PCR-based and metagenomics alongside culture-based techniques. Imported cases resulting from international travel and hospitalization abroad appear to have frequently contributed to the rise in ARB pathogen cases in these countries. The many similarities between the Nordic countries (e.g., knowledge exchange practices, antibiotic usage patterns, and the current ARB landscape) could facilitate collaborative efforts in developing and implementing WBS for ARB in population-level screening.

Untargeted Proteomics-Based Approach to Investigate Unintended Changes in Genetically Modified Maize for Environmental Risk Assessment Purpose.

Profiling technologies, such as proteomics, allow the simultaneous measurement and comparison of thousands of plant components without prior knowledge of their identity. The combination of these non-targeted methods facilitates a more comprehensive approach than targeted methods and thus provides additional opportunities to identify genotypic changes resulting from genetic modification, including new allergens or toxins. The purpose of this study was to investigate unintended changes in GM Bt maize grown in South Africa. In the present study, we used bi-dimensional gel electrophoresis based on fluorescence staining, coupled with mass spectrometry in order to compare the proteome of the field-grown transgenic hybrid (MON810) and its near-isogenic counterpart. Proteomic data showed that energy metabolism and redox homeostasis were unequally modulated in GM Bt and non-GM maize variety samples. In addition, a potential allergenic protein-pathogenesis related protein -1 has been identified in our sample set. Our data shows that the GM variety is not substantially equivalent to its non-transgenic near-isogenic variety and further studies should be conducted in order to address the biological relevance and the potential risks of such changes. These finding highlight the suitability of unbiased profiling approaches to complement current GMO risk assessment practices worldwide.

Revisiting Risk Governance of GM Plants: The Need to Consider New and Emerging Gene-Editing Techniques.

New and emerging gene-editing techniques make it possible to target specific genes in species with greater speed and specificity than previously possible. Of major relevance for plant breeding, regulators and scientists are discussing how to regulate products developed using these gene-editing techniques. Such discussions include whether to adopt or adapt the current framework for GMO risk governance in evaluating the impacts of gene-edited plants, and derived products, on the environment, human and animal health and society. Product classification or definition is one of several aspects of the current framework being criticized. Further, knowledge gaps related to risk assessments of gene-edited organisms-for example of target and off-target effects of intervention in plant genomes-are also of concern. Resolving these and related aspects of the current framework will involve addressing many subjective, value-laden positions, for example how to specify protection goals through ecosystem service approaches. A process informed by responsible research and innovation practices, involving a broader community of people, organizations, experts, and interest groups, could help scientists, regulators, and other stakeholders address these complex, value-laden concerns related to gene-editing of plants with and for society.

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses.

Some genetically modified (GM) plants have transgenes that confer tolerance to abiotic stressors. Meanwhile, other transgenes may interact with abiotic stressors, causing pleiotropic effects that will affect the plant physiology. Thus, physiological alteration might have an impact on the product safety. However, routine risk assessment (RA) analyses do not evaluate the response of GM plants exposed to different environmental conditions. Therefore, we here present a proteome profile of herbicide-tolerant maize, including the levels of phytohormones and related compounds, compared to its near-isogenic non-GM variety under drought and herbicide stresses. Twenty differentially abundant proteins were detected between GM and non-GM hybrids under different water deficiency conditions and herbicide sprays. Pathway enrichment analysis showed that most of these proteins are assigned to energetic/carbohydrate metabolic processes. Among phytohormones and related compounds, different levels of ABA, CA, JA, MeJA and SA were detected in the maize varieties and stress conditions analysed. In pathway and proteome analyses, environment was found to be the major source of variation followed by the genetic transformation factor. Nonetheless, differences were detected in the levels of JA, MeJA and CA and in the abundance of 11 proteins when comparing the GM plant and its non-GM near-isogenic variety under the same environmental conditions. Thus, these findings do support molecular studies in GM plants Risk Assessment analyses.

Levels of DNA methylation and transcript accumulation in leaves of transgenic maize varieties.

BACKGROUND: Prior to their release in the environment, transgenic crops are examined for their health and environmental safety. In addition, transgene expression needs to be consistent in order to express the introduced trait (e.g. insecticidal and/or herbicide tolerance). Moreover, data on expression levels for GM events are usually required for approval, but these are rarely disclosed or they are considered insufficient. On the other hand, biosafety regulators do not consider epigenetic regulation (e.g. DNA methylation, ncRNAs and histone modifications), which are broadly known to affect gene expression, within their risk assessment analyses. Here we report the results of a DNA methylation (bisulfite sequencing) and transgene transcript accumulation (RT-qPCR) analysis of four Bt-expressing single transgenic maize hybrids, under different genetic backgrounds, and a stacked transgenic hybrid expressing both insecticidal and herbicide tolerance traits. RESULTS: Our results showed differences in cytosine methylation levels in the FMV promoter and cry2Ab2 transgene of the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids under the same genetic background showed differences in the 35S promoter sequence. The results of transgene transcript accumulation levels showed differences in both cry1A.105 and cry2Ab2 transgenes among the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids showed difference for the cry2Ab2 transgene only. CONCLUSIONS: Overall, our results show differences in DNA methylation patterns in all varieties, as well as in transgene transcript accumulation levels. Although the detection of changes in DNA methylation and transgenic accumulation levels does not present a safety issue per se, it demonstrates the need for additional studies that focus on detecting possible safety implications of such changes.

Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice.

BACKGROUND: In genetically modified (GM) crops there is a risk that the inserted genes may introduce new allergens and/or adjuvants into the food and feed chain. The MON810 maize, expressing the insecticidal Cry1Ab toxin, is grown in many countries worldwide. In animal models, intranasal and intraperitoneal immunisations with the purified Cry1Ab proteins have induced immune responses, and feeding trials with Cry1Ab-containing feed have revealed some altered immune responses. Previous investigations have primarily measured antibody responses to the protein, while investigations of clinical food allergy symptoms, or allergy promotion (adjuvant effect) associated with the Cry1Ab protein are largely missing. We aimed to investigate immunogenic, allergenic and adjuvant properties of purified Cry1Ab toxin (trypCry1Ab, i.e., trypsin activated Cry1Ab) in a mouse model of food allergy. METHOD: Female C3H/HeJ mice were immunized by intragastric gavage of 10 µg purified, trypsin activated Cry1Ab toxin (trypCry1Ab) alone or together with the food allergen lupin. Cholera toxin was added as a positive control for adjuvant effect to break oral tolerance. Clinical symptoms (anaphylaxis) as well as humoral and cellular responses were assessed. RESULTS: In contrast to results from previous airway investigations, we observed no indication of immunogenic properties of trypCry1Ab protein after repeated intragastric exposures to one dose, with or without CT as adjuvant. Moreover, the results indicated that trypCry1Ab given by the intragastric route was not able to promote allergic responses or anaphylactic reactions against the co-administered allergen lupin at the given dose. CONCLUSION: The study suggests no immunogenic, allergenic or adjuvant capacity of the given dose of trypCry1Ab protein after intragastric exposure of prime aged mice.

Transgene expression and Bt protein content in transgenic Bt maize (MON810) under optimal and stressful environmental conditions.

Bt protein content in transgenic insect resistant (Bt) maize may vary between tissues within plants and between plants growing under different environmental conditions. However, it is unknown whether and how Bt protein content correlates with transgene expression, and whether this relationship is influenced by stressful environmental conditions. Two Bt maize varieties containing the same transgene cassette (MON 810) were grown under optimal and stressful conditions. Before and during stress exposure, the upper leaves were analysed for transgene expression using quantitative RT-PCR and for Bt content using ELISA. Under optimal conditions there was no significant difference in the transgene expression between the two investigated Bt maize varieties whereas Bt protein content differed significantly. Transgene expression was correlated with Bt protein content in only one of the varieties. Under stressful environmental conditions we found similar transgene expressions as under optimal conditions but Bt content responded differently. These results suggest that Bt content is not only controlled by the transgene expression but is also dependent on the genetic background of the maize variety. Under stressful conditions the concentration of Bt protein is even more difficult to predict.

Comparative proteomic analysis of genetically modified maize grown under different agroecosystems conditions in Brazil.

BACKGROUND: Profiling technologies allow the simultaneous measurement and comparison of thousands of cell components without prior knowledge of their identity. In the present study, we used two-dimensional gel electrophoresis combined with mass spectrometry to evaluate protein expression of Brazilian genetically modified maize hybrid grown under different agroecosystems conditions. To this effect, leaf samples were subjected to comparative analysis using the near-isogenic non-GM hybrid as the comparator. RESULTS: In the first stage of the analysis, the main sources of variation in the dataset were identified by using Principal Components Analysis which correlated most of the variation to the different agroecosystems conditions. Comparative analysis within each field revealed a total of thirty two differentially expressed proteins between GM and non-GM samples that were identified and their molecular functions were mainly assigned to carbohydrate and energy metabolism, genetic information processing and stress response. CONCLUSIONS: To the best of our knowledge this study represents the first evidence of protein identities with differentially expressed isoforms in Brazilian MON810 genetic background hybrid grown under field conditions. As global databases on outputs from "omics" analysis become available, these could provide a highly desirable benchmark for safety assessments.

Sexual isolation in Acinetobacter baylyi is locus-specific and varies 10,000-fold over the genome.

Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to approximately 10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.

The molecular evolution and structural organization of group I introns at position 1389 in nuclear small subunit rDNA of myxomycetes.

The number of nuclear group I introns from myxomycetes is rapidly increasing in GenBank as more rDNA sequences from these organisms are being sequenced. They represent an interesting and complex group of intervening sequences because several introns are mobile (or inferred to be mobile) and many contain large and unusual insertions in peripheral loops. Here we describe related group I introns at position 1389 in the small subunit rDNA of representatives from the myxomycete family Didymiaceae. Phylogenetic analyses support a common origin and mainly vertical inheritance of the intron. All S1389 introns from the Didymiaceae belong to the IC1 subclass of nuclear group I introns. The central catalytic core region of about 100 nt appears divergent in sequence composition even though the introns reside in closely related species. Furthermore, unlike the majority of group I introns from myxomycetes the S1389 introns do not self-splice as naked RNA in vitro under standard conditions, consistent with a dependence on host factors for folding or activity. Finally, the myxomycete S1389 introns are exclusively found within the family Didymiaceae, which suggests that this group I intron was acquired after the split between the families Didymiaceae and Physaraceae.

Obligatory group I introns with unusual features at positions 1949 and 2449 in nuclear LSU rDNA of Didymiaceae myxomycetes.

Myxomycetes (plasmodial slime molds) belonging to the order Physarales contain obligatory group I introns at positions 1949 and 2449 in their large subunit ribosomal RNA gene. Here, we report 36 group I introns from the Didymiaceae family (order Physarales) from 18 isolates representing three genera and seven species, and have reconstructed both host and intron phylogenies. The introns, named L1949 and L2449, were found in all isolates analyzed, consistent with an obligatory distribution in Didymiaceae. The introns fold at the RNA-level into typical group I ribozyme core structures that are relatively conserved, but contain large and highly variable extension sequences in peripheral domains without any detectable protein coding capacities. Furthermore, the L1949 and L2449 introns have probably become dependent on host factors for folding or activity. This assumption is based on that all introns tested for self-splicing in vitro failed to ligate the flanking exon regions. Phylogenies based on LSU rDNA and intron sequences are consistent with that the L1949 and L2449 introns follow a strict vertical inheritance within Didymiaceae. We suggest that the Didymiaceae L1949 and L2449 introns are well suited as high-resolution markers in genetic assessments at various taxonomic levels, from closely related strains of a single species to separating genera.

Short-term sequence evolution and vertical inheritance of the Naegleria twin-ribozyme group I intron.

BACKGROUND: Ribosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516) has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2) and a homing endonuclease gene (NaHEG). NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA. RESULTS: To elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, contains hallmarks of a degenerated NaHEG. Phylogenetic analyses performed on separate data sets representing NaGIR1, NaGIR2, NaHEG, and ITS1-5.8S-ITS2 ribosomal DNA are consistent with an overall vertical inheritance pattern of the intron within the Naegleria genus. CONCLUSION: The Nae.S516 twin-ribozyme intron was gained early in the Naegleria evolution with subsequent vertical inheritance. The intron was lost in the majority of isolates (70%), leaving a widespread but scattered distribution pattern. Why the apparent asexual Naegleria amoebae harbors active intron homing endonucleases, dependent on sexual reproduction for its function, remains a puzzle.

The recent transfer of a homing endonuclease gene.

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.