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 10 000 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.
The ability of bacteria to horizontally transfer genetic material has turned antimicrobial resistance into one of the major sanitary crises of the 21st century. Plasmid conjugation is considered the main mechanism responsible for the mobilization of resistance genes, and its understanding is crucial to tackle this crisis.
Haemophilus 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.
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
bla
ROB-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.
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