bPlasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzae carrying the small plasmid pB1000 conferring resistance to -lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzae populations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.
ColE1 plasmids are small mobilizable replicons that play an important role in the spread of antibiotic resistance in Pasteurellaceae. In this study, we describe how a natural single nucleotide polymorphism (SNP) near the origin of replication of the ColE1-type plasmid pB1000 found in a Pasteurella multocida clinical isolate generates two independent plasmid variants able to coexist in the same cell simultaneously. Using the Haemophilus influenzae Rd KW20 strain as a model system, we combined antibiotic susceptibility tests, quantitative PCRs, competition assays, and experimental evolution to characterize the consequences of the coexistence of the pB1000 plasmid variants. This coexistence produced an increase of the total plasmid copy number (PCN) in the host bacteria, leading to a rise in both the antibiotic resistance level and the metabolic burden produced by pB1000. Using experimental evolution, we showed that in the presence of ampicillin, the bacteria maintained both plasmid variants for 300 generations. In the absence of antibiotics, on the other hand, the bacteria are capable of reverting to the single-plasmid genotype via the loss of one of the plasmid variants. Our results revealed how a single mutation in plasmid pB1000 provides the bacterial host with a mechanism to increase the PCN and, consequently, the ampicillin resistance level. Crucially, this mechanism can be rapidly reversed to avoid the extra cost entailed by the increased PCN in the absence of antibiotics. KEYWORDS ColE1, experimental evolution, Haemophilus influenzae, plasmid stability pB1000 is a small ColE1-like plasmid carrying the bla ROB-1 -lactamase gene and conferring high-level resistance to -lactams. pB1000 has been described in multiple pathogens from Pasteurellaceae, including Haemophilus influenzae (1-7).The replication of ColE1 plasmids is under the control of a preprimer RNA (RNAII) which is negatively regulated by a small antisense RNA (RNAI) (8). Mutations affecting these RNAs encoded near the origin of replication (oriV) of the plasmid alter both the plasmid copy number (PCN) and the compatibility of ColE1 plasmids. Single mutations can give rise to new compatible plasmids that are able to coexist in the same cell, increasing the global PCN (7,8). These mutations have been widely described in vitro (9) and arise naturally by chance due to the high plasmid copy number exhibited by ColE1-like plasmids (10), but to the best of our knowledge, there are no natural examples of cohabiting ColE1 plasmids differentiated by only a single nucleotide polymorphism (SNP).In this work we analyze a heterozygous pB1000 differentiated only by an SNP near the oriV and isolated from a high-level ampicillin-resistant clinical isolate of Pasteurella multocida (5). We show that this SNP creates a new variant of the plasmid, which
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.
ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.
17The coexistence of multicopy plasmids is a common phenomenon. However, the 18 evolutionary forces promoting these genotypes are poorly understood. In this study, we 19 have analyzed multiple ColE1 plasmids (pB1000, pB1005 and pB1006) coexisting 20 within Haemophilus influenzae RdKW20 in all possible combinations. When 21 transformed into the naïve host, each plasmid type presented a particular copy number 22 and produced a specific resistance profile and biological cost, whether alone or 23 coexisting with the other plasmids. Therefore, there was no fitness advantage associated 24 with plasmid coexistence that could explain these common plasmid associations in 25 nature. Using experimental evolution, we showed how H. influenzae Rd was able to 26 completely compensate the fitness cost produced by any of these plasmids. Crucially, 27 once the bacterium has compensated for a first plasmid, the acquisition of new 28 multicopy plasmid(s) did not produced any extra biological cost. We argue therefore 29 that compensatory adaptation pave the way for the acquisition of multiple coexisting 30 ColE1 plasmids. 31 Importance 32Antibiotic resistance is a major concern for human and animal health. Plasmids play a 33 major role in the acquisition and dissemination of antimicrobial resistance genes. In this 34 report we investigate, for the first time, how plasmids are capable to cohabit stably in 35 populations. This coexistence of plasmids is driven by compensatory evolution 36 alleviating the cost of a first plasmid, which potentiates the acquisition of further 37 plasmids at no extra cost. This phenomenon explains the high prevalence of plasmids 38 coexistance in wild type bacteria, which generates multiresistant clones and contributes 39 to the maintenance and spread of antibiotic resistance genes within bacterial 40 populations. 41 42Antibiotic resistance is a serious problem in animal and human health and bacterial 43 plasmids play an essential role in the dissemination of resistance (1). In last years, 44 numerous works have described the importance of small ColE1-like plasmids in the 45 dissemination of resistance genes (2-15). These plasmids replicate via two RNAs (16). 46Natural SNPs in these RNAs allow different ColE1-like plasmids to stably cohabit 47 within the same cell (17, 18). If the plasmids bear antibiotic resistance genes, this 48 cohabitation confers antibiotic multiresistance to the host bacteria (9). 49The acquisition of plasmids usually entails a biological cost to the host bacterium that 50 will generate a selection against plasmid-bearing clones (19, 20). Thus, it is reasonable 51 to assume that the accumulation of various plasmids will decrease the fitness of bacteria 52 and therefore clones bearing several plasmids will be outcompeted in bacterial 53 populations. Notwithstanding, ColE1 plasmids cohabitation is a common phenomenon 54 in nature (9, 11, 13, 14, 21). In this study we test two hypotheses that could explain the 55 high prevalence of plasmid coexistence in nature: i) Positive epi...
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