Frank M. Aarestrup’s research while affiliated with Technical University of Denmark and other places

In September 2023, an ongoing mpox outbreak emerged in South Kivu (DRC) which spread to other regions and countries. We describe the epidemiological and genomic evolution of the outbreak between September 2023 and June 2024. Samples were collected from hospitalized patients, along with data on residence and possible exposures. Employee numbers and locations were recorded for bars with sex workers. Where possible, exposures were linked to genomic sequencing data for cluster analysis. In total, 670 cases were admitted to Kamituga hospital from 17 health areas. Of the cases, 52,4% were in females, and 47,6% in males. The majority (83,4%) were linked to professional sexual interactions. Seven deaths occurred and three healthcare workers acquired mpox. Eight of fourteen pregnant women had fetal loss. Phylogenetic analysis revealed three Clade Ib clusters. Longer branches of a sequence clustering with sequences from Kenya, Uganda, Sweden and Thailand indicate more undocumented spread. Mutations were mostly APOBEC3-type mutations indicative of sustained human-to-human transmission. No clear link between sequence cluster, bar or health area was observed. These data suggest rapid spread mostly through sexual contact within densely populated areas. Spread to neighboring countries highlights the need for extended cross-border collaboration, health education strategies focusing on sex workers, contact tracing, clinical care and surveillance.

Single-cell sequencing may serve as a powerful complementary technique to shotgun metagenomics to study microbiomes. This emerging technology allows the separation of complex microbial communities into individual bacterial cells, enabling high-throughput sequencing of genetic material from thousands of singular bacterial cells in parallel. Here, we validated the use of microfluidics and semi-permeable capsules (SPCs) technology (Atrandi) to isolate individual bacterial cells from sewage and pig fecal samples. Our method involves extracting and amplifying single bacterial DNA within individual SPCs, followed by combinatorial split-and-pool single-amplified genome (SAG) barcoding and short-read sequencing. We tested two different sequencing approaches with different numbers of SPCs from the same sample for each sequencing run. Using a deep sequencing approach, we detected 1,796 and 1,220 SAGs, of which 576 and 599 were used for further analysis from one sewage and one fecal sample, respectively. In shallow sequencing data, we aimed for 10-times more cells and detected 12,731 and 17,909 SAGs, of which we used 2,456 and 1,599 for further analysis for sewage and fecal samples, respectively. Additionally, we identified the top 10 antimicrobial resistance genes (ARGs) in both sewage and feces samples and linked them to their individual host bacterial species.

Background WGS can potentially be routinely used in clinical microbiology settings, especially with the increase in sequencing accuracy and decrease in cost. Escherichia coli is the most common bacterial species analysed in those settings, thus fast and accurate diagnostics can lead to reductions in morbidity, mortality and healthcare costs. Objectives To evaluate WGS for diagnostics and surveillance in a collection of clinical E. coli; to examine the pool of antimicrobial resistance (AMR) determinants circulating in Denmark and the most frequent STs; and to evaluate core-genome MLST (cgMLST) and SNP-based clustering approaches for detecting genetically related isolates. Methods We analysed the genomes of 699 E. coli isolates collected throughout all Danish Clinical Microbiology Laboratories. We used rMLST and KmerFinder for species identification, ResFinder for prediction of AMR, and PlasmidFinder for plasmid identification. We used Center for Genomic Epidemiology MLST, cgMLSTFinder and CSI Phylogeny to perform typing and clustering analysis. Results Genetic AMR determinants were detected in 56.2% of isolates. We identified 182 MLSTs, most frequently ST-69, ST-73, ST-95 and ST-131. Using a maximum 15-allele difference as the threshold for genetic relatedness, we identified 23 clusters. SNP-based phylogenetic analysis within clusters revealed from 0 to 13 SNPs, except two cases with 111 and 461 SNPs. Conclusions WGS data are useful to characterize clinical E. coli isolates, including predicting AMR profiles and subtyping in concordance with surveillance data. We have shown that it is possible to adequately cluster isolates through a cgMLST approach, but it remains necessary to define proper interpretative criteria.

VanHAX-mediated glycopeptide resistance has been consistently high in one of the three main sewer systems in Copenhagen, Lynetten, for +20 years. To explore this for other glycopeptide resistance genes, and whether the colonization has resulted in establishment of multiple bacterial taxa, we mapped 505 shotgun metagenomic data sets from the inlet of three sewage treatment plants to 831 different glycopeptide resistance genes. Only vanHAX and vanHBX genes were differentially abundant in Lynetten. Analyses of eight contigs suggested limited variations in the flanking regions. Proximity ligation metagenomic analysis of 12 samples from Lynetten identified 441 and 5 paired reads mapping to vanHAX and vanHBX, respectively. The other end of these reads was mapped to generated metagenomic-assembled genomes and NCBI using BLAST. vanHBX could only be linked to the phylum level (Bacillota). Plasmid analysis of vanHBX Hi-C contigs showed that these were mainly located on plasmids reported found in enterococci species. Most vanHAX-linked reads could only be linked to phylum and class level, but some reads were assigned to Enterococcus faecium (7 reads), Enterococcus faecalis (4 reads), Paenibacillus apiarius (2 reads), and Paenibacillus thiaminolyticus (27 reads). Ten of the 20 Hi-C contigs-containing vanHAX were annotated as plasmid, all reported found in Enterococcus species. This study shows that while Hi-C technology is valuable for linking antimicrobial resistance genes to bacterial taxa, it suffers from challenges in reliably mapping the linked read to a genomic region with sufficient taxonomic information. Our results also suggest that over the +20 years of colonizing a sewer system, vanHAX has not become widespread across multiple taxa, remaining primarily in E. faecalis and E. faecium, with the exception of Paenibacillus. IMPORTANCE Long-term colonization of microbial communities with antimicrobial-resistant bacteria is expected to result in sharing of the resistance genes between several different bacterial taxa of the communities. We investigated microbiomes from a sewer, which have been colonized with glycopeptide-resistant bacteria harboring the mobile vanHAX gene cluster for a minimum of 20 years, using metagenomics sequencing and Hi-C. We found that despite the long-term presence in the sewer, the vanHAX genes have seemingly not disseminated widely.

Salmonella Typhi (S. Typhi) is an important pathogen causing typhoid fever worldwide. The emergence of antibiotic resistance, including that of blaTEM genes encoding to TEM $\:\beta\:$-lactamases has been observed. This study aimed to investigate the dynamics of blaTEM genes in S. Typhi by analyzing the phylogeny and flanking region patterns and phylogenetic associating them with metadata (year, country) and genomic data (genotypes, antibiotic resistance genes (ARGs), plasmids). Genomic sequences of publicly available S. Typhi harboring blaTEM (n = 6079), spanning from 1983 to 2023, were downloaded and analyzed using CSIPhylogeny for phylogeny, Flankophile for identifying genetic contexts around blaTEM genes and GenoTyphi for determining genotypes, ARGs and plasmid replicons. We found that blaTEM-positive isolates occurred most commonly in specific location, especially in Asia and Africa and clustered among a limited number of genotypes. Flankophile identified 740 isolates (12.2%) with distinct flanking region patterns, which were categorized into 13 patterns. Notably, 7 patterns showed a predominantly phylogenetic association with genotypes. Additionally, these 7 patterns exhibited relation to the country, ARGs and plasmid replicons. Further examination of the flanking region patterns provided association with mobile genetic elements (MGEs). Taken together, this study suggests that blaTEM has been acquired by S. Typhi isolates a limited number of times and subsequently spread clonally with specific genotypes.

We describe cases with monkeypox virus (MPXV) Clade Ib in Burundi from their first detection in July until 20 August 2024. Testing 442 people with vesicular lesions confirmed 170 cases (98 male; 72 female), 82 (48%) being < 15 years old. Differential diagnosis of the first 30 individuals testing MPXV negative revealed chickenpox in 20. Cases occurred in 26 of 49 Burundi health districts, but mostly in Bujumbura Nord (88/170; 67%). Case-derived MPXV genetic sequences from Burundi and South-Kivu (Democratic Republic of the Congo), clustered together in phylogenetic analysis.

Background In September 2023, an mpox outbreak was reported in the eastern part, South Kivu Province, of Democratic Republic of the Congo. This outbreak is still ongoing and expanding to other regions and countries. Here, we describe the epidemiological and genomic evolution of the outbreak from September 2023 to June 2024. Methods Consenting patients with mpox-like symptoms admitted to the Kamituga and the Kamanyola hospitals were recruited to the study. Samples from throat, lesions, breast milk and placenta were collected for PCR testing and sequencing. For the patients from Kamituga hospital, data on place of residence and possible exposures were collected by interviews. The location and numbers of employees were collected for all bars with sex workers. Where possible, exposures were linked to the genomic sequencing data for cluster analysis. Findings In total, 670 (suspected) mpox cases were admitted to the Kamituga hospital. There were slightly more female than male cases (351/670 [52,4%] versus 319/670, [47,6%], and cases were reported from 17 different health areas. The majority of cases were reported in Mero (205/670 [30,6%]), followed by Kimbangu (115/670 [17,2%]), Kabukungu (105/670 [16,7%]), and Asuku (73/670 [10.9%]). During this period, 7 deaths occurred and 8 out of 14 women who were pregnant had fetal loss. Three healthcare workers acquired mpox infection when caring for patients. In depth case ascertainment showed that 83,4% of patients reported recent visits to bars for (professional) sexual interactions as a likely source of infection. Whole genome sequencing resulted in the generation of 58 genome sequences. Three main clusters characterized by specific mutations were identified and several miniclusters of 2 or more sequences with over two shared mutations. No clear link between sequence cluster, bar or health area was observed. The more recent sequences from Kamanyola were related to the sequences in Kamituga and confirmed to be Clade Ib. However, relatively long branches were observed and one of the sequences clustered with publicly released sequences from travelers in Kenya, Uganda, Sweden and Thailand, indicating more undocumented ongoing spread for cluster A than for the other clusters. Most observed mutations were APOBEC-3 related mutations indicative of ongoing human-to-human transmission. Interpretation These data suggests that the rapid transmission of monkeypox virus until June 2024 was mostly related to interactions with professional sex workers (PSW) within densely populated health areas. The expanding number of cases and the recent expansion to 29 other nearby health zones of South -Kivu as well as Rwanda, Burundi, Uganda and Kenya stresses the need for cross border surveillance and collaboration. Urgent enhanced response action is needed, including case finding, diagnostic capacity building, health education programmes focussing on sex workers, and possibly vaccination to limit further escalation and stop this outbreak.