Extensive antimicrobial resistance mobilization via Multicopy Plasmid Encapsidation mediated by temperate phages

Objectives: To investigate the relevance of multicopy plasmids in antimicrobial resistance and assess their mobilization mediated by phage particles Methods: Several databases with complete sequences of plasmids and annotated genes were analysed. The 16S methyltransferase gene armA conferring high-level aminoglycoside resistance was used as a marker in eight different plasmids, from different incompatibility groups, and with differing sizes and plasmid copy numbers. All plasmids were transformed into Escherichia coli bearing one of four different lysogenic phages. Upon induction, encapsidation of armA in phage particles was evaluated using qRT-PCR and Southern blotting. Results: Multicopy plasmids carry a vast set of emerging clinically important antimicrobial resistance genes. However, 60% of these plasmids do not bear mobility (MOB) genes. When carried on these multicopy plasmids, mobilization of a marker gene armA into phage capsids was up to 10000 times more frequent than when it was encoded by a large plasmid with a low copy number. Conclusions: Multicopy plasmids and phages, two major mobile genetic elements (MGE) in bacteria, represent a novel high-efficiency transmission route of antimicrobial resistance genes that deserves further investigation

Explorando la Transducción y la Adaptación de Plásmidos en la Resistencia a los Antimicrobianos

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, leída el 30-01-2024Antimicrobial resistance (AMR) has become a growing public health threat worldwide. The increasing use and misuse of antibiotics since their introduction into the clinical setting hasled to the emergence of bacteria resistant to multiple antimicrobial agents, resulting in infections that are increasingly difficult to treat. Plasmids are an important vehicle for AMR genes and can serve as reservoirs and facilitate their spread. Therefore, plasmid biology is being studied thoroughly to understand the mechanisms of plasmid adaptation, transmission, and persistence to use the gained knowledge to appropriately address this issue. ColE1-likeplasmids form a widespread plasmid family that is frequently associated with the dissemination of resistance genes and has been shown to give rise to multidrug-resistant pathogens...La resistencia a los antimicrobianos (RAM) se ha convertido en una amenaza creciente parala salud pública en todo el mundo. El aumento del uso y el abuso de los antibióticos desde su introducción en el ámbito clínico ha provocado la aparición de bacterias resistentes a múltiples agentes antimicrobianos, lo que da lugar a infecciones cada vez más difíciles de tratar. Los plásmidos son un vehículo importante para los genes de resistencia y pueden servir de reservorio y facilitar su propagación. Por ello, se está estudiando a fondo la biología de los plásmidos para comprender los mecanismos de adaptación, transmisión y persistencia de los mismos y utilizar estos conocimientos para abordar adecuadamente este problema. Los plásmidos del tipo ColE1 forman una familia de plásmidos muy extendida que se asocia con frecuencia a la propagación de genes de resistencia y que ha demostrado dar lugar a patógenos multirresistentes...Fac. de VeterinariaTRUEunpu

Insertion Sequences Determine Plasmid Adaptation to New Bacterial Hosts

ABSTRACT Plasmids facilitate the vertical and horizontal spread of antimicrobial resistance genes between bacteria. The host range and adaptation of plasmids to new hosts determine their impact on the spread of resistance. In this work, we explore the mechanisms driving plasmid adaptation to novel hosts in experimental evolution. Using the small multicopy plasmid pB1000, usually found in Pasteurellaceae, we studied its adaptation to a host from a different bacterial family, Escherichia coli. We observed two different mechanisms of adaptation. One mechanism is single nucleotide polymorphisms (SNPs) in the origin of replication (oriV) of the plasmid, which increase the copy number in E. coli cells, elevating the stability, and resistance profile. The second mechanism consists of two insertion sequences (ISs), IS1 and IS10, which decrease the fitness cost of the plasmid by disrupting an uncharacterized gene on pB1000 that is harmful to E. coli. Both mechanisms increase the stability of pB1000 independently, but only their combination allows long-term maintenance. Crucially, we show that the mechanisms have a different impact on the host range of the plasmid. SNPs in oriV prevent the replication in the original host, resulting in a shift of the host range. In contrast, the introduction of ISs either shifts or expands the host range, depending on the IS. While IS1 leads to expansion, IS10 cannot be reintroduced into the original host. This study gives new insights into the relevance of ISs in plasmid-host adaptation to understand the success in spreading resistance. IMPORTANCE ColE1-like plasmids are small, mobilizable plasmids that can be found across at least four orders of Gammaproteobacteria and are strongly associated with antimicrobial resistance genes. Plasmid pB1000 carries the gene blaROB-1, conferring high-level resistance to penicillins and cefaclor. pB1000 has been described in various species of the family Pasteurellaceae, for example, in Haemophilus influenzae, which can cause diseases such as otitis media, meningitis, and pneumonia. To understand the resistance spread through horizontal transfer, it is essential to study the mechanisms of plasmid adaptation to novel hosts. In this work we identify that a gene from pB1000, which encodes a peptide that is toxic for E. coli, and the low plasmid copy number (PCN) of pB1000 in E. coli cells are essential targets in the described plasmid-host adaptation and therefore limit the spread of pB1000-encoded blaROB-1. Furthermore, we show how the interplay of two adaptation mechanisms leads to successful plasmid maintenance in a different bacterial family

Multiplatform Metabolomics Characterization Reveals Novel Metabolites and Phospholipid Compositional Rules of Haemophilus influenzae Rd KW20

2023 Descuento MDPIHaemophilus influenzae is a gram-negative bacterium of relevant clinical interest. H. influenzae Rd KW20 was the first organism to be sequenced and for which a genome-scale metabolic model (GEM) was developed. However, current H. influenzae GEMs are unable to capture several aspects of metabolome nature related to metabolite pools. To directly and comprehensively characterize the endometabolome of H. influenzae Rd KW20, we performed a multiplatform MS-based metabolomics approach combining LC-MS, GC-MS and CE-MS. We obtained direct evidence of 15–20% of the endometabolome present in current H. influenzae GEMs and showed that polar metabolite pools are interconnected through correlating metabolite islands. Notably, we obtained high-quality evidence of 18 metabolites not previously included in H. influenzae GEMs, including the antimicrobial metabolite cyclo(Leu-Pro). Additionally, we comprehensively characterized and evaluated the quantitative composition of the phospholipidome of H. influenzae, revealing that the fatty acyl chain composition is largely independent of the lipid class, as well as that the probability distribution of phospholipids is mostly related to the conditional probability distribution of individual acyl chains. This finding enabled us to provide a rationale for the observed phospholipid profiles and estimate the abundance of low-level species, permitting the expansion of the phospholipidome characterization through predictive probabilistic modelling.Ministerio de Ciencia e Innovación (España)European CommissionDepto. de Sanidad AnimalCentro de Vigilancia Sanitaria Veterinaria (VISAVET)Fac. de VeterinariaTRUEpubDescuento UC