High-resolution African HLA resource uncovers HLA-DRB1 expression effects underlying vaccine response.

How human genetic variation contributes to vaccine effectiveness in infants is unclear, and data are limited on these relationships in populations with African ancestries. We undertook genetic analyses of vaccine antibody responses in infants from Uganda (n = 1391), Burkina Faso (n = 353) and South Africa (n = 755), identifying associations between human leukocyte antigen (HLA) and antibody response for five of eight tested antigens spanning pertussis, diphtheria and hepatitis B vaccines. In addition, through HLA typing 1,702 individuals from 11 populations of African ancestry derived predominantly from the 1000 Genomes Project, we constructed an imputation resource, fine-mapping class II HLA-DR and DQ associations explaining up to 10% of antibody response variance in our infant cohorts. We observed differences in the genetic architecture of pertussis antibody response between the cohorts with African ancestries and an independent cohort with European ancestry, but found no in silico evidence of differences in HLA peptide binding affinity or breadth. Using immune cell expression quantitative trait loci datasets derived from African-ancestry samples from the 1000 Genomes Project, we found evidence of differential HLA-DRB1 expression correlating with inferred protection from pertussis following vaccination. This work suggests that HLA-DRB1 expression may play a role in vaccine response and should be considered alongside peptide selection to improve vaccine design.

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 3,960,704 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of March 2023, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. Phase 2 of our project will address the fact that the data in the public archives is heavily geographically biased towards the Global North. We therefore have contributed new raw data to ENA/SRA from many countries including Ghana, Thailand, Laos, Sri Lanka, India, Argentina and Singapore. We will incorporate these, along with all public raw data submitted between March 2023 and the current day, into an updated set of assemblies, and phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

SWGTS-a platform for Stream-Based host DNA depletion.

MOTIVATION: Microbial sequencing data from clinical samples is often contaminated with human sequences, which have to be removed prior to sharing. Existing methods for human read removal, however, are applicable only after the target dataset has been retrieved in its entirety, putting the recipient at least temporarily in control of a potentially identifiable genetic dataset with potential implications under regulatory frameworks such as the GDPR. In some instances, the ability to carry out stream-based host depletion as part of the data transfer process may be preferable. RESULTS: We present SWGTS, a client-server application for the transfer and stream-based host depletion of sequencing reads. SWGTS enforces a robust upper bound on the maximum amount of human genetic data from any one client held in memory at any point in time by storing all incoming sequencing data in a limited-size, client-specific intermediate processing buffer and by throttling the rate of incoming data if it exceeds the speed of host depletion carried out on the SWGTS server in the background. SWGTS exposes a HTTP-REST interface, is implemented using docker-compose, Redis and traefik, and requires less than 8 Gb of RAM for deployment. We demonstrate high filtering accuracy of SWGTS; incoming data transfer rates of up to 1.65 megabases per second in a conservative configuration; and mitigation of re-identification risks by the ability to limit the number of SNPs present on a popular population-scale genotyping array covered by reads in the SWGTS buffer to a low user-defined number, such as 10 or 100. AVAILABILITY: SWGTS is available on GitHub: https://github.com/AlBi-HHU/swgts (https://doi.org/10.5281/zenodo.10891052). The repository also contains a jupyter notebook that can be used to reproduce all the benchmarks used in this paper. All datasets used for benchmarking are publicly available. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Microbial Community Profiling Protocol with Full-length 16S rRNA Sequences and Emu.

16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high-throughput short-read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full-length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species-level resolution. This pipeline provides a low-cost solution for characterizing microbiome composition by exploiting real-time, long-read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full-length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu.

Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing.

The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.

HOGVAX: Exploiting epitope overlaps to maximize population coverage in vaccine design with application to SARS-CoV-2.

The efficacy of epitope vaccines depends on the included epitopes as well as the probability that the selected epitopes are presented by the major histocompatibility complex (MHC) proteins of a vaccinated individual. Designing vaccines that effectively immunize a high proportion of the population is challenging because of high MHC polymorphism, diverging MHC-peptide binding affinities, and physical constraints on epitope vaccine constructs. Here, we present HOGVAX, a combinatorial optimization approach for epitope vaccine design. To optimize population coverage within the constraint of limited vaccine construct space, HOGVAX employs a hierarchical overlap graph (HOG) to identify and exploit overlaps between selected peptides and explicitly models the structure of linkage disequilibrium in the MHC. In a SARS-CoV-2 case study, we demonstrate that HOGVAX-designed vaccines contain substantially more epitopes than vaccines built from concatenated peptides and predict vaccine efficacy in over 98% of the population with high numbers of presented peptides in vaccinated individuals.

The role of microbiome-host interactions in the development of Alzheimer´s disease.

Alzheimer`s disease (AD) is the most prevalent cause of dementia. It is often assumed that AD is caused by an aggregation of extracellular beta-amyloid and intracellular tau-protein, supported by a recent study showing reduced brain amyloid levels and reduced cognitive decline under treatment with a beta-amyloid-binding antibody. Confirmation of the importance of amyloid as a therapeutic target notwithstanding, the underlying causes of beta-amyloid aggregation in the human brain, however, remain to be elucidated. Multiple lines of evidence point towards an important role of infectious agents and/or inflammatory conditions in the etiology of AD. Various microorganisms have been detected in the cerebrospinal fluid and brains of AD-patients and have thus been hypothesized to be linked to the development of AD, including Porphyromonas gingivalis (PG) and Spirochaetes. Intriguingly, these microorganisms are also found in the oral cavity under normal physiological conditions, which is often affected by multiple pathologies like caries or tooth loss in AD patients. Oral cavity pathologies are mostly accompanied by a compositional shift in the community of oral microbiota, mainly affecting commensal microorganisms and referred to as 'dysbiosis'. Oral dysbiosis seems to be at least partly mediated by key pathogens such as PG, and it is associated with a pro-inflammatory state that promotes the destruction of connective tissue in the mouth, possibly enabling the translocation of pathogenic microbiota from the oral cavity to the nervous system. It has therefore been hypothesized that dysbiosis of the oral microbiome may contribute to the development of AD. In this review, we discuss the infectious hypothesis of AD in the light of the oral microbiome and microbiome-host interactions, which may contribute to or even cause the development of AD. We discuss technical challenges relating to the detection of microorganisms in relevant body fluids and approaches for avoiding false-positives, and introduce the antibacterial protein lactoferrin as a potential link between the dysbiotic microbiome and the host inflammatory reaction.

Characterisation of Type II DNA Methyltransferases of Metamycoplasma hominis.

Bacterial virulence, persistence and defence are affected by epigenetic modifications, including DNA methylation. Solitary DNA methyltransferases modulate a variety of cellular processes and influence bacterial virulence; as part of a restriction-modification (RM) system, they act as a primitive immune system in methylating the own DNA, while unmethylated foreign DNA is restricted. We identified a large family of type II DNA methyltransferases in Metamycoplasma hominis, comprising six solitary methyltransferases and four RM systems. Motif-specific 5mC and 6mA methylations were identified with a tailored Tombo analysis on Nanopore reads. Selected motifs with methylation scores >0.5 fit with the gene presence of DAM1 and DAM2, DCM2, DCM3, and DCM6, but not for DCM1, whose activity was strain-dependent. The activity of DCM1 for CmCWGG and of both DAM1 and DAM2 for GmATC was proven in methylation-sensitive restriction and finally for recombinant rDCM1 and rDAM2 against a dam-, dcm-negative background. A hitherto unknown dcm8/dam3 gene fusion containing a (TA) repeat region of varying length was characterized within a single strain, suggesting the expression of DCM8/DAM3 phase variants. The combination of genetic, bioinformatics, and enzymatic approaches enabled the detection of a huge family of type II DNA MTases in M. hominis, whose involvement in virulence and defence can now be characterized in future work.

An appeal for strengthening genomic pathogen surveillance to improve pandemic preparedness and infection prevention: the German perspective.

The SARS-CoV-2 pandemic has highlighted the importance of viable infection surveillance and the relevant infrastructure. From a German perspective, an integral part of this infrastructure, genomic pathogen sequencing, was at best fragmentary and stretched to its limits due to the lack or inefficient use of equipment, human resources, data management and coordination. The experience in other countries has shown that the rate of sequenced positive samples and linkage of genomic and epidemiological data (person, place, time) represent important factors for a successful application of genomic pathogen surveillance. Planning, establishing and consistently supporting adequate structures for genomic pathogen surveillance will be crucial to identify and combat future pandemics as well as other challenges in infectious diseases such as multi-drug resistant bacteria and healthcare-associated infections. Therefore, the authors propose a multifaceted and coordinated process for the definition of procedural, legal and technical standards for comprehensive genomic pathogen surveillance in Germany, covering the areas of genomic sequencing, data collection and data linkage, as well as target pathogens. A comparative analysis of the structures established in Germany and in other countries is applied. This proposal aims to better tackle epi- and pandemics to come and take action from the "lessons learned" from the SARS-CoV-2 pandemic.

Complete sequences of six major histocompatibility complex haplotypes, including all the major MHC class II structures.

Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina, ultra-long Nanopore, and PacBio HiFi sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (GRCh38/hg38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.06%-0.49% in a 1000 Genomes Project read remapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

[Establishment of genomic pathogen surveillance to strengthen pandemic preparedness and infection prevention in Germany]. / Etablierung der Genomischen Erreger-Surveillance zur Stärkung des Pandemie- und Infektionsschutzes in Deutschland.

The SARS-CoV­2 pandemic has shown a deficit of essential epidemiological infrastructure, especially with regard to genomic pathogen surveillance in Germany. In order to prepare for future pandemics, the authors consider it urgently necessary to remedy this existing deficit by establishing an efficient infrastructure for genomic pathogen surveillance. Such a network can build on structures, processes, and interactions that have already been initiated regionally and further optimize them. It will be able to respond to current and future challenges with a high degree of adaptability.The aim of this paper is to address the urgency and to outline proposed measures for establishing an efficient, adaptable, and responsive genomic pathogen surveillance network, taking into account external framework conditions and internal standards. The proposed measures are based on global and country-specific best practices and strategy papers. Specific next steps to achieve an integrated genomic pathogen surveillance include linking epidemiological data with pathogen genomic data; sharing and coordinating existing resources; making surveillance data available to relevant decision-makers, the public health service, and the scientific community; and engaging all stakeholders. The establishment of a genomic pathogen surveillance network is essential for the continuous, stable, active surveillance of the infection situation in Germany, both during pandemic phases and beyond.

Rapid Selection of Sotrovimab Escape Variants in Severe Acute Respiratory Syndrome Coronavirus 2 Omicron-Infected Immunocompromised Patients.

BACKGROUND: Monoclonal antibodies (mAbs) that target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are predominantly less effective against Omicron variants. Immunocompromised patients often experience prolonged viral shedding, resulting in an increased risk of viral escape. METHODS: In an observational, prospective cohort, 57 patients infected with Omicron variants who received sotrovimab alone or in combination with remdesivir were followed. The study end points were a decrease in SARS-CoV-2 RNA <106 copies/mL in nasopharyngeal swabs at day 21 and the emergence of escape mutations at days 7, 14, and 21 after sotrovimab administration. All SARS-CoV-2 samples were analyzed using whole-genome sequencing. Individual variants within the quasispecies were subsequently quantified and further characterized using a pseudovirus neutralization assay. RESULTS: The majority of patients (43 of 57, 75.4%) were immunodeficient, predominantly due to immunosuppression after organ transplantation or hematologic malignancies. Infections by Omicron/BA.1 comprised 82.5%, while 17.5% were infected by Omicron/BA.2. Twenty-one days after sotrovimab administration, 12 of 43 (27.9%) immunodeficient patients had prolonged viral shedding compared with 1 of 14 (7.1%) immunocompetent patients (P = .011). Viral spike protein mutations, some specific for Omicron (e.g., P337S and/or E340D/V), emerged in 14 of 43 (32.6%) immunodeficient patients, substantially reducing sensitivity to sotrovimab in a pseudovirus neutralization assay. Combination therapy with remdesivir significantly reduced emergence of escape variants. CONCLUSIONS: Immunocompromised patients face a considerable risk of prolonged viral shedding and emergence of escape mutations after early therapy with sotrovimab. These findings underscore the importance of careful monitoring and the need for dedicated clinical trials in this patient population.

Integrated genomic surveillance enables tracing of person-to-person SARS-CoV-2 transmission chains during community transmission and reveals extensive onward transmission of travel-imported infections, Germany, June to July 2021.

BackgroundTracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective.AimWe demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters.MethodsGenomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021.ResultsCluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf.ConclusionIGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.

Emu: species-level microbial community profiling of full-length 16S rRNA Oxford Nanopore sequencing data.

16S ribosomal RNA-based analysis is the established standard for elucidating the composition of microbial communities. While short-read 16S rRNA analyses are largely confined to genus-level resolution at best, given that only a portion of the gene is sequenced, full-length 16S rRNA gene amplicon sequences have the potential to provide species-level accuracy. However, existing taxonomic identification algorithms are not optimized for the increased read length and error rate often observed in long-read data. Here we present Emu, an approach that uses an expectation-maximization algorithm to generate taxonomic abundance profiles from full-length 16S rRNA reads. Results produced from simulated datasets and mock communities show that Emu is capable of accurate microbial community profiling while obtaining fewer false positives and false negatives than alternative methods. Additionally, we illustrate a real-world application of Emu by comparing clinical sample composition estimates generated by an established whole-genome shotgun sequencing workflow with those returned by full-length 16S rRNA gene sequences processed with Emu.

Pangenome-based genome inference allows efficient and accurate genotyping across a wide spectrum of variant classes.

Typical genotyping workflows map reads to a reference genome before identifying genetic variants. Generating such alignments introduces reference biases and comes with substantial computational burden. Furthermore, short-read lengths limit the ability to characterize repetitive genomic regions, which are particularly challenging for fast k-mer-based genotypers. In the present study, we propose a new algorithm, PanGenie, that leverages a haplotype-resolved pangenome reference together with k-mer counts from short-read sequencing data to genotype a wide spectrum of genetic variation-a process we refer to as genome inference. Compared with mapping-based approaches, PanGenie is more than 4 times faster at 30-fold coverage and achieves better genotype concordances for almost all variant types and coverages tested. Improvements are especially pronounced for large insertions (≥50 bp) and variants in repetitive regions, enabling the inclusion of these classes of variants in genome-wide association studies. PanGenie efficiently leverages the increasing amount of haplotype-resolved assemblies to unravel the functional impact of previously inaccessible variants while being faster compared with alignment-based workflows.

RNA transcription and degradation of Alu retrotransposons depends on sequence features and evolutionary history.

Alu elements are one of the most successful groups of RNA retrotransposons and make up 11% of the human genome with over 1 million individual loci. They are linked to genetic defects, increases in sequence diversity, and influence transcriptional activity. Still, their RNA metabolism is poorly understood yet. It is even unclear whether Alu elements are mostly transcribed by RNA Polymerase II or III. We have conducted a transcription shutoff experiment by α-amanitin and metabolic RNA labeling by 4-thiouridine combined with RNA fragmentation (TT-seq) and RNA-seq to shed further light on the origin and life cycle of Alu transcripts. We find that Alu RNAs are more stable than previously thought and seem to originate in part from RNA Polymerase II activity, as previous reports suggest. Their expression however seems to be independent of the transcriptional activity of adjacent genes. Furthermore, we have developed a novel statistical test for detecting the expression of quantitative trait loci in Alu elements that relies on the de Bruijn graph representation of all Alu sequences. It controls for both statistical significance and biological relevance using a tuned k-mer representation, discovering influential sequence features missed by regular motif search. In addition, we discover several point mutations using a generalized linear model, and motifs of interest, which also match transcription factor-binding motifs.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection Clusters Based on Integrated Genomic Surveillance, Outbreak Analysis and Contact Tracing in an Urban Setting.

BACKGROUND: Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing. METHODS: In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform. RESULTS: Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population. CONCLUSIONS: Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population.

pmrCAB Recombination Events among Colistin-Susceptible and -Resistant Acinetobacter baumannii Clinical Isolates Belonging to International Clone 7.

Acinetobacter baumannii is a successful nosocomial pathogen due to its genomic plasticity. Homologous recombination allows genetic exchange and allelic variation among different clonal lineages and is one of the mechanisms associated with horizontal gene transfer (HGT) of resistance determinants. The main mechanism of colistin resistance in A. baumannii is mediated through mutations in the pmrCAB operon. Here, we describe two A. baumannii clinical isolates belonging to International Clone 7 (IC7) that have undergone recombination in the pmrCAB operon and evaluate the contribution of mobile genetic elements (MGE) to this phenomenon. Isolates 67569 and 72554 were colistin susceptible and resistant, respectively, and were submitted for short- and long-read genome sequencing using Illumina MiSeq and MinION platforms. Hybrid assemblies were built with Unicycler, and the assembled genomes were compared to reference genomes using NUCmer, Cortex, and SplitsTree. Genomes were annotated using Prokka, and MGEs were identified with ISfinder and repeat match. Both isolates presented a 21.5-kb recombining region encompassing pmrCAB. In isolate 67659, this region originated from IC5, while in isolate 72554 multiple recombination events might have happened, with the 5-kb recombining region encompassing pmrCAB associated with an isolate representing IC4. We could not identify MGEs involved in the mobilization of pmrCAB in these isolates. In summary, A. baumannii belonging to IC7 can present additional sequence divergence due to homologous recombination across clonal lineages. Such variation does not seem to be driven by antibiotic pressure but could contribute to HGT mediating colistin resistance. IMPORTANCE Colistin resistance rates among Acinetobacter baumannii clinical isolates have increased over the last 20 years. Despite reports of the spread of plasmid-mediated colistin resistance among Enterobacterales, the presence of mcr-type genes in Acinetobacter spp. remains rare, and reduced colistin susceptibility is mainly associated with the acquisition of nonsynonymous mutations in pmrCAB. We have recently demonstrated that distinct pmrCAB sequences are associated with different A. baumannii International Clones (IC). In this study, we identified the presence of homologous recombination as an additional cause of genetic variation in this operon, which, to the best of our knowledge, was not mediated by mobile genetic elements. Even though this phenomenon was observed in both colistin-susceptible and -resistant isolates, it has the potential to contribute to the spread of resistance-conferring alleles, leading to reduced susceptibility to this last-resort antimicrobial agent.

Emergence of the E484K mutation in SARS-COV-2-infected immunocompromised patients treated with bamlanivimab in Germany.

BACKGROUND: Monoclonal antibodies (mAb) have been introduced as a promising new therapeutic approach against SARS-CoV-2. At present, there is little experience regarding their clinical effects in patient populations underrepresented in clinical trials, e.g. immunocompromised patients. Additionally, it is not well known to what extent SARS-CoV-2 treatment with monoclonal antibodies could trigger the selection of immune escape viral variants. METHODS: After identifying immunocompromised patients with viral rebound under treatment with bamlanivimab, we characterized the SARS-CoV-2-isolates by whole genome sequencing. Viral load measurements and sequence analysis were performed consecutively before and after bamlanivimab administration. FINDINGS: After initial decrease of viral load, viral clearance was not achieved in five of six immunocompromised patients treated with bamlanivimab. Instead, viral replication increased again over the course of the following one to two weeks. In these five patients, the E484K substitution - known to confer immune escape - was detected at the time of viral rebound but not before bamlanivimab treatment. INTERPRETATION: Treatment of SARS-CoV-2 with bamlanivimab in immunocompromised patients results in the rapid development of immune escape variants in a significant proportion of cases. Given that the E484K mutation can hamper natural immunity, the effectiveness of vaccination as well as antibody-based therapies, these findings may have important implications not only for individual treatment decisions but may also pose a risk to general prevention and treatment strategies. FUNDING: All authors are employed and all expenses covered by governmental, federal state, or other publicly funded institutions.