Objectives: Antimicrobial-resistant bacteria are a major global health threat. Mobile genetic elements (MGEs) have been crucial for spreading resistance to new bacterial species, including human pathogens. Understanding how MGEs promote resistance could be essential for prevention. Here we present an investigation of MGEs and their association with resistance genes in pathogenic bacteria collected from 59 diagnostic units during 2020, representing a snapshot of clinical infections from 35 counties worldwide. Methods: We analysed 3,095 whole-genome sequenced clinical bacterial isolates from over 100 species to study the relationship between resistance genes and MGEs. The mobiliome of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae were further examined for geographic differences, as these species were prevalent in all countries. Genes potentially mobilized by MGEs were identified by finding DNA segments containing MGEs and ARGs preserved in multiple species. Network analysis was used to investigate potential MGE interactions, host range, and transmission pathways. Results: The prevalence and diversity of MGEs and resistance genes varied among species, with E. coli and S. aureus carrying more diverse elements. MGE composition differed between bacterial lineages, indicating strong vertical inheritance. 102 MGEs associated with resistance were found in multiple species, and four of these elements seemed to be highly transmissible as they were found in different phyla. We identified 21 genomic regions containing resistance genes potentially mobilized by MGEs, highlighting their importance in transmitting genes to clinically significant bacteria. Conclusion: Resistance genes are spread through various MGEs, including plasmids and transposons. Our findings suggest that multiple factors influence MGE prevalence and their transposability, thereby shaping the MGE population and transmission pathways. Some MGEs have a wider host range, which could make them more important for mobilizing genes. We also identified 103 resistance genes potentially mobilised by MGEs, which could increase their transmissibility to unrelated bacteria.

Carbapenemase-producing Enterobacter spp. Serratia marcescens, Citrobacter freundii, Providencia spp., and Morganella morganii (CP-ESCPM) are increasingly identified as causative agents of nosocomial infections but are still not under systematic genomic surveillance. In this study, using a combination of whole-genome sequencing and conjugation experiments, we sought to elucidate the genomic characteristics and transferability of resistance genes in clinical CP-ESCPM isolates from Bulgaria. Among the 36 sequenced isolates, NDM-1 (12/36), VIM-4 (11/36), VIM-86 (8/36), and OXA-48 (7/36) carbapenemases were identified; two isolates carried both NDM-1 and VIM-86. The majority of carbapenemase genes were found on self-conjugative plasmids. IncL plasmids were responsible for the spread of OXA-48 among E. hormaechei, C. freundii, and S. marcescens. IncM2 plasmids were generally associated with the spread of NDM-1 in C. freundii and S. marcescens, and also of VIM-4 in C. freundii. IncC plasmids were involved in the spread of the recently described VIM-86 in P. stuartii isolates. IncC plasmids carrying bla_NDM-1 and bla_VIM-86 were observed too. bla_NDM-1 was also detected on IncX3 in S. marcescens and on IncT plasmid in M. morganii. The significant resistance transfer rates we observed highlight the role of the ESCPM group as a reservoir of resistance determinants and stress the need for strengthening infection control measures.

Background: Acquired 16S rRNA methyltransferases (16S-RMTases) confer high-level resistance to aminoglycosides and are often associated with β-lactam and quinolone resistance determinants. Methods: Using PCR, whole-genome sequencing and conjugation experiments, we conducted a retrospective genomic surveillance study of 16S-RMTase-producing Enterobacterales, collected between 2006 and 2023, to explore transmission dynamics of methyltransferase and associated antibiotic resistance genes. Results: Among the 10,731 consecutive isolates, 150 (1.4%) from 13 species carried armA (92.7%), rmtB (4.7%), and rmtF + rmtB (2.7%) methyltransferase genes. The coexistence of extended-spectrum β-lactamase (bla_CTX-M-3/15, bla_SHV-12, bla_SFO-1), carbapenemase (bla_NDM-1/5, bla_VIM-1/4/86, bla_OXA-48), acquired AmpC (bla_CMY-2/4/99, bla_DHA-1, bla_AAC-1), and plasmid-mediated quinolone resistance (qnrB, qnrS, aac(6′)-Ib-cr) genes within these isolates was also detected. Methyltransferase genes were carried by different plasmids (IncL/M, IncA/C, IncR, IncFIB, and IncFII), suggesting diverse origins and sources of acquisition. armA was co-transferred with bla_CTX-M-3/15, bla_NDM-1, bla_VIM-4/86, bla_OXA-48, bla_CMY-4, aac(6′)-Ib-cr, qnrB, and qnrS, while rmtF1 was co-transferred with bla_SFO-1, highlighting the multidrug-resistant nature of these plasmids. Long-read sequencing of ST6260 K. pneumoniae isolates revealed a novel resistance association, with rmtB1 and bla_NDM-5 on the chromosome, bla_OXA-232 on a conjugative ColKP3 plasmid, and rmtF1 with bla_SFO-1 on self-transmissible IncFIB and IncFII plasmids. Conclusions: The genetic plasticity of plasmids carrying methyltransferase genes suggests their potential to acquire additional resistance genes, turning 16S-RMTase-producing Enterobacterales into a persistent public health threat.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections represent a major public health concern and require comprehensive understanding of their genetic makeup. This study investigated the first occurrence of imipenemase (IMP)-carrying P. aeruginosa strains from Bulgaria. Whole genome sequencing identified a novel plasmid-mediated IMP-100 allele located in a a novel In4886 integron embedded in a putative Tn7700 transposon. Two other closely related chromosomal IMP variants, IMP-13 and IMP-84, were also detected. The IMP-producers were resistant to last-line drugs including cefiderocol (CFDC) (two out of three) and susceptible to colistin. The IMP-13/84 cassettes were situated in a In320 integron inserted in a Tn5051-like transposon as previously reported. Lastly, the p4782-IMP plasmid rendered the PA01 transformant resistant to CFDC, suggesting a transferable CFDC resistance. A variety of virulence factors associated with adhesion, antiphagocytosis, iron uptake, and quorum sensing, as well as secretion systems, toxins, and proteases, were confirmed, suggesting significant pathogenic potential consistent with the observed strong biofilm formation. The emergence of IMP-producing MDR P. aeruginosa is alarming as it remains unsusceptible even to last-generation drugs like CFDC. Newly detected IMP-100 was even located in a CFDC-resistant XDR strain.

Waste Water Treatment Plants (WWTP) aim to reduce contamination in effluent water; however, studies indicate antimicrobial resistance genes (ARGs) persist post-treatment, potentially leading to their spread from human populated areas into the environment. This study evaluated the impact of a large WWTP serving 125,000 people on the Iskar River in Bulgaria, by characterizing the spatial and short-term temporal dynamics in bacterial community dynamics and resistance profiles of the surface water. Pairs of samples were collected biweekly on four dates from two different locations, one about 800 m after the WWTP effluents and the other 10 km downstream. Taxonomic classification revealed the dominance of Pseudomonodota and Bacteriodota, notably the genera Flavobacterium, Aquirufa, Acidovorax, Polynucleobacter, and Limnohabitans. The taxonomic structure corresponded with both lentic and lotic freshwater habitats, with Flavobacterium exhibiting a significant decrease over the study period. Principal Coordinate Analysis revealed statistically significant differences in bacterial community composition between samples collected on different dates. Differential abundance analysis identified notable enrichment of Polynucleobacter and Limnohabitans. There were shifts within the enriched or depleted bacterial taxa between early and late sampling dates. High relative abundance of the genes erm(B), erm(F), mph(E), msr(E) (macrolides); tet(C), tet(O), tet(W), tet(Q) and tet(X) (tetracyclines); sul1 and sul2 (sulphonamides); and cfxA3, cfxA6 (beta-lactams) were detected, with trends of increased presence in the latest sampling dates and in the location closer to the WWTP. Of note, genes conferring resistance to carbapenems blaOXA-58 and blaIMP-33-like were identified. Co-occurrence analysis of ARGs and mobile genetic elements on putative plasmids showed few instances, and the estimated human health risk score (0.19) according to MetaCompare2.0 was low. In total, 29 metagenome-assembled genomes were recovered, with only a few harbouring ARGs. This study enhances our understanding of freshwater microbial community dynamics and antibiotic resistance profiles, highlighting the need for continued ARGs monitoring.

This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.