The data provided are the estimates that resulted from raw qPCR fluorescence data (available upon... more The data provided are the estimates that resulted from raw qPCR fluorescence data (available upon request). The data are those used in the analyses described in the associated manuscript. See the ReadMe file for column descriptions
Broad-host-range plasmids play a critical role in the spread of antibiotic resistance and other t... more Broad-host-range plasmids play a critical role in the spread of antibiotic resistance and other traits. In spite of increasing information about the genomic diversity of closely related plasmids, the relationship between sequence divergence and host range remains unclear. IncP-1 plasmids are currently classified into six subgroups based on the genetic distance of backbone genes. We investigated whether plasmids from two subgroups exhibit a different host range, using two IncP-1c plasmids, an IncP-1b plasmid and their minireplicons. Efficiencies of plasmid establishment and maintenance were compared using five species that belong to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. The IncP-1b plasmid replicated and persisted in all five hosts in the absence of selection. Of the two IncP-1c plasmids, both were unable to replicate in alphaproteobacterial host Sphingobium japonicum, and one established itself in Agrobacterium tumefaciens but was very unstable. In contrast, both IncP-1c minireplicons, which produced higher levels of replication initiation protein than the wild-type plasmids, replicated in all strains, suggesting that poor establishment of the native plasmids is in part due to suboptimal replication initiation gene regulation. The findings suggest that host ranges of distinct IncP-1 plasmids only partially overlap, which may limit plasmid recombination and thus result in further genome divergence.
Introduction : Pathogens resistant to multiple antibiotics are rapidly emerging and can acquire t... more Introduction : Pathogens resistant to multiple antibiotics are rapidly emerging and can acquire their resistance genes by conjugation from non-pathogenic bacteria inhabiting a multitude of environments. In spite of the concern that foodborne pathogens can become multi-drug resistant by acquiring plasmids from environmental strains, little is known about the success of that natural genetic exchange. This study investigated if the multiresistance plasmid pB10, isolated from a wastewater treatment plant and harbouring amoxicillin, streptomycin, sulfonamides and tetracycline resistance genes, can be transferred to the foodborne pathogens Salmonella and Escherichia coli O157:H7 and how this alters the phenotype of the recipients. Material and methods : The strains used in this study were the donor strain Pseudomonas putida SM1443 (pB10::gfp) and the recipient strains Salmonella spp. (n=10) and E. coli O157:H7 (n=5). The Salmonella spp. strains belonged to the serovars S. Enteritidis (n=3), S. Typhimurium (n=4), S. Virchow (n=1), S. Infantis (n=1) and S. Hadar (n=1). Conjugation experiments were performed by filter mating on 0.22 µm polycarbonate filters. The transfer ratio was determined by plating and by flow cytometry in which gfp was used as reporter gene [1]. Antibiotic resistance profiles were determined for both recipients and transconjugants using the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [2]. Results : For 14 of the 15 recipient strains, transconjugants were obtained by plating and/or by flow cytometry. Based on plating, transfer ratios were between 6.8 x 10-9 and 3.0 x 10-2 while using flow cytometry, transfer ratios were between <1.0 x 10-5 and 1.9 x 10-2. The highest transfer ratio was observed for one of the S. Enteritidis strains. No transconjugants were detected with either method for the S. Hadar strain. The transfer ratios detected in the E.coli strains were lower than those detected in the Salmonella spp. strains, except for one E. coli strain. With a few exceptions, the transconjugants showed phenotypically increased resistance. Discussion : We showed that an environmental plasmid can be transferred to foodborne pathogenic bacteria at high transfer ratios; however the transfer ratio seemed strain dependent. Moreover, the newly acquired resistance genes turned antimicrobial sensitive strains into resistant ones. This event can lead to treatment failure and thus compromise public health [3]. In future work, the possible role of food matrices on the transfer of this plasmid will be examined. References 1. 1. Sorensen S.J. et al. (2003). Current Microbiology 47: 129–133. 2. Clinical and Laboratory Standards Institute (CLSI) (2010). 3. Su L.H. et al. (2003). Antimicrob Agents Chemother 47(2): 563–565.
Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they ca... more Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they can spread antibiotic resistance to pathogens. Even though most pathogens form biofilms, little is known about how MDR plasmids persist and evolve in biofilms. We hypothesize that (i) biofilms act as refugia of MDR plasmids by retaining them in the absence of antibiotics longer than well-mixed planktonic populations, and that (ii) the evolutionary trajectories that account for the improvement of plasmid persistence over time differ between biofilms and planktonic populations. In this study, we evolved Acinetobacter baumannii with an MDR plasmid in biofilm and planktonic populations with and without antibiotic selection. In the absence of selection biofilm populations were better able to maintain the MDR plasmid than planktonic populations. In planktonic populations plasmid persistence improved rapidly but was accompanied by a loss of genes required for the horizonal transfer of plasmids. I...
The history of soil microbiology contains several important lessons about approaches and attempts... more The history of soil microbiology contains several important lessons about approaches and attempts to alter the indigenous microbial community. Currently, a number of novel and powerful strategies are available. Yet, it is important for the scientist to be pragmatic concerning their potential usages. Moreover, the validity of concepts such as ‘Isolating - controlling - monitoring’, ‘Environmentally Acceptable Endpoint’ and ‘Nonextractable Residues’ warrants delineation.
The rapid spread of antibiotic resistance is a global health threat. A range of environments have... more The rapid spread of antibiotic resistance is a global health threat. A range of environments have been identified as reservoirs of the antibiotic resistance genes (ARGs) found in pathogens, but we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to an inability to identify the bacterial hosts of ARGs and the mobile genetic elements that mediate horizontal gene transfer due to the loss of intra-cellular contiguity upon DNA extraction. In two recent studies we describe the application of proximity-ligation methods for the determination of the in situ host range of numerous ARGs, viruses, plasmids, and integrons within complex microbiome samples. This method forms physical junctions between sequences present within the same cell prior to DNA extraction. Subsequent sequencing generates a dataset that robustly connects mobile elements to their hosts and can assemble de novo genomes from mixed communities. Our application of t...
Nature Research wishes to improve the reproducibility of the work that we publish. This form prov... more Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.
The rapid spread of antibiotic resistance is a serious human health threat. A range of environmen... more The rapid spread of antibiotic resistance is a serious human health threat. A range of environments have been identified as reservoirs of the antibiotic resistance genes (ARGs) found in pathogens. However, we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrated that the in vivo proximity ligation method Hi-C can determine the in situ host range of ARGs, plasmids, and integrons in a wastewater sample by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile elements and host genomes, mostly validating this method. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
Multidrug resistant bacterial pathogens have become a serious global human health threat, and con... more Multidrug resistant bacterial pathogens have become a serious global human health threat, and conjugative plasmids are important drivers of the rapid spread of resistance to last-resort antibiotics. Whereas antibiotics have been shown to select for adaptation of resistance plasmids to their new bacterial hosts, or vice versa , a general evolutionary mechanism has not yet emerged. Here we conducted an experimental evolution study aimed at determining general patterns of plasmid-bacteria evolution. Specifically, we found that a large conjugative resistance plasmid follows the same evolutionary trajectories as its non-conjugative mini-replicon in the same and other species. Furthermore, within a single host-plasmid pair three distinct patterns of adaptive evolution led to increased plasmid persistence: i) mutations in the replication protein gene ( trfA1 ); ii) the acquisition by the resistance plasmid of a transposon from a co-residing plasmid encoding a putative toxin-antitoxin syste...
Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antib... more Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms, and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multi-drug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimentalmodeling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Broad-host-range (BHR) IncP-1 plasmids have the ability to transfer between and replicate in near... more Broad-host-range (BHR) IncP-1 plasmids have the ability to transfer between and replicate in nearly all species of the Alpha-, Beta-and Gammaproteobacteria, but surprisingly few data are available on the stability of these plasmids in strains within their host range. Moreover, even though molecular interactions between the bacterial host and its plasmid(s) exist, no systematic study to date has compared the stability of the same plasmid among different hosts. The goal of this study was to examine whether the stability characteristics of an IncP-1 plasmid can be variable between strains within the host range of the plasmid. Therefore, 19 strains within the Alpha-, Beta-or Gammaproteobacteria carrying the IncP-1b plasmid pB10 were serially propagated in non-selective medium and the fraction of segregants was monitored through replica-picking. Remarkably, a large variation in the stability of pB10 in different strains was found, even between strains within the same genus or species. Ten strains showed no detectable plasmid loss over about 200 generations, and in two strains plasmid-free clones were only sporadically observed. In contrast, three strains, Pseudomonas koreensis R28, Pseudomonas putida H2 and Stenotrophomonas maltophilia P21, exhibited rapid plasmid loss within 80 generations. Parameter estimation after mathematical modelling of these stability data suggested high frequencies of segregation (about 0.04 per generation) or high plasmid cost (i.e. a relative fitness decrease in plasmid-bearing cells of about 15 and 40 %), which was confirmed experimentally. The models also suggested that plasmid reuptake by conjugation only played a significant role in plasmid stability in one of the three strains. Four of the 19 strains lost the plasmid very slowly over about 600 generations. The erratic decrease of the plasmid-containing fraction and simulation of the data with a new mathematical model suggested that plasmid cost was variable over time due to compensatory mutations. The findings of this study demonstrate that the ability of a so-called 'BHR' plasmid to persist in a bacterial population is influenced by strain-specific traits, and therefore observations made for one strain should not be generalized for the entire species or genus.
Antibiotic resistant bacteria are a constant threat in the battle against infectious diseases. On... more Antibiotic resistant bacteria are a constant threat in the battle against infectious diseases. One strategy for reducing their effect is to temporarily discontinue the use of certain antibiotics in the hope that in the absence of the antibiotic the resistant strains will be replaced by the sensitive strains. An experiment where this strategy is employed in vitro [5] produces data which showed a slow accumulation of sensitive mutants. Here we propose a mathematical model and statistical analysis to explain this data. The stochastic model elucidates the trend and error structure of the data. It provides a guide for developing future sampling strategies, and provides a framework for long term predictions of the effects of discontinuing specific antibiotics on the dynamics of resistant bacterial populations.
The data provided are the estimates that resulted from raw qPCR fluorescence data (available upon... more The data provided are the estimates that resulted from raw qPCR fluorescence data (available upon request). The data are those used in the analyses described in the associated manuscript. See the ReadMe file for column descriptions
Broad-host-range plasmids play a critical role in the spread of antibiotic resistance and other t... more Broad-host-range plasmids play a critical role in the spread of antibiotic resistance and other traits. In spite of increasing information about the genomic diversity of closely related plasmids, the relationship between sequence divergence and host range remains unclear. IncP-1 plasmids are currently classified into six subgroups based on the genetic distance of backbone genes. We investigated whether plasmids from two subgroups exhibit a different host range, using two IncP-1c plasmids, an IncP-1b plasmid and their minireplicons. Efficiencies of plasmid establishment and maintenance were compared using five species that belong to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. The IncP-1b plasmid replicated and persisted in all five hosts in the absence of selection. Of the two IncP-1c plasmids, both were unable to replicate in alphaproteobacterial host Sphingobium japonicum, and one established itself in Agrobacterium tumefaciens but was very unstable. In contrast, both IncP-1c minireplicons, which produced higher levels of replication initiation protein than the wild-type plasmids, replicated in all strains, suggesting that poor establishment of the native plasmids is in part due to suboptimal replication initiation gene regulation. The findings suggest that host ranges of distinct IncP-1 plasmids only partially overlap, which may limit plasmid recombination and thus result in further genome divergence.
Introduction : Pathogens resistant to multiple antibiotics are rapidly emerging and can acquire t... more Introduction : Pathogens resistant to multiple antibiotics are rapidly emerging and can acquire their resistance genes by conjugation from non-pathogenic bacteria inhabiting a multitude of environments. In spite of the concern that foodborne pathogens can become multi-drug resistant by acquiring plasmids from environmental strains, little is known about the success of that natural genetic exchange. This study investigated if the multiresistance plasmid pB10, isolated from a wastewater treatment plant and harbouring amoxicillin, streptomycin, sulfonamides and tetracycline resistance genes, can be transferred to the foodborne pathogens Salmonella and Escherichia coli O157:H7 and how this alters the phenotype of the recipients. Material and methods : The strains used in this study were the donor strain Pseudomonas putida SM1443 (pB10::gfp) and the recipient strains Salmonella spp. (n=10) and E. coli O157:H7 (n=5). The Salmonella spp. strains belonged to the serovars S. Enteritidis (n=3), S. Typhimurium (n=4), S. Virchow (n=1), S. Infantis (n=1) and S. Hadar (n=1). Conjugation experiments were performed by filter mating on 0.22 µm polycarbonate filters. The transfer ratio was determined by plating and by flow cytometry in which gfp was used as reporter gene [1]. Antibiotic resistance profiles were determined for both recipients and transconjugants using the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [2]. Results : For 14 of the 15 recipient strains, transconjugants were obtained by plating and/or by flow cytometry. Based on plating, transfer ratios were between 6.8 x 10-9 and 3.0 x 10-2 while using flow cytometry, transfer ratios were between <1.0 x 10-5 and 1.9 x 10-2. The highest transfer ratio was observed for one of the S. Enteritidis strains. No transconjugants were detected with either method for the S. Hadar strain. The transfer ratios detected in the E.coli strains were lower than those detected in the Salmonella spp. strains, except for one E. coli strain. With a few exceptions, the transconjugants showed phenotypically increased resistance. Discussion : We showed that an environmental plasmid can be transferred to foodborne pathogenic bacteria at high transfer ratios; however the transfer ratio seemed strain dependent. Moreover, the newly acquired resistance genes turned antimicrobial sensitive strains into resistant ones. This event can lead to treatment failure and thus compromise public health [3]. In future work, the possible role of food matrices on the transfer of this plasmid will be examined. References 1. 1. Sorensen S.J. et al. (2003). Current Microbiology 47: 129–133. 2. Clinical and Laboratory Standards Institute (CLSI) (2010). 3. Su L.H. et al. (2003). Antimicrob Agents Chemother 47(2): 563–565.
Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they ca... more Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they can spread antibiotic resistance to pathogens. Even though most pathogens form biofilms, little is known about how MDR plasmids persist and evolve in biofilms. We hypothesize that (i) biofilms act as refugia of MDR plasmids by retaining them in the absence of antibiotics longer than well-mixed planktonic populations, and that (ii) the evolutionary trajectories that account for the improvement of plasmid persistence over time differ between biofilms and planktonic populations. In this study, we evolved Acinetobacter baumannii with an MDR plasmid in biofilm and planktonic populations with and without antibiotic selection. In the absence of selection biofilm populations were better able to maintain the MDR plasmid than planktonic populations. In planktonic populations plasmid persistence improved rapidly but was accompanied by a loss of genes required for the horizonal transfer of plasmids. I...
The history of soil microbiology contains several important lessons about approaches and attempts... more The history of soil microbiology contains several important lessons about approaches and attempts to alter the indigenous microbial community. Currently, a number of novel and powerful strategies are available. Yet, it is important for the scientist to be pragmatic concerning their potential usages. Moreover, the validity of concepts such as ‘Isolating - controlling - monitoring’, ‘Environmentally Acceptable Endpoint’ and ‘Nonextractable Residues’ warrants delineation.
The rapid spread of antibiotic resistance is a global health threat. A range of environments have... more The rapid spread of antibiotic resistance is a global health threat. A range of environments have been identified as reservoirs of the antibiotic resistance genes (ARGs) found in pathogens, but we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to an inability to identify the bacterial hosts of ARGs and the mobile genetic elements that mediate horizontal gene transfer due to the loss of intra-cellular contiguity upon DNA extraction. In two recent studies we describe the application of proximity-ligation methods for the determination of the in situ host range of numerous ARGs, viruses, plasmids, and integrons within complex microbiome samples. This method forms physical junctions between sequences present within the same cell prior to DNA extraction. Subsequent sequencing generates a dataset that robustly connects mobile elements to their hosts and can assemble de novo genomes from mixed communities. Our application of t...
Nature Research wishes to improve the reproducibility of the work that we publish. This form prov... more Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.
The rapid spread of antibiotic resistance is a serious human health threat. A range of environmen... more The rapid spread of antibiotic resistance is a serious human health threat. A range of environments have been identified as reservoirs of the antibiotic resistance genes (ARGs) found in pathogens. However, we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrated that the in vivo proximity ligation method Hi-C can determine the in situ host range of ARGs, plasmids, and integrons in a wastewater sample by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile elements and host genomes, mostly validating this method. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
Multidrug resistant bacterial pathogens have become a serious global human health threat, and con... more Multidrug resistant bacterial pathogens have become a serious global human health threat, and conjugative plasmids are important drivers of the rapid spread of resistance to last-resort antibiotics. Whereas antibiotics have been shown to select for adaptation of resistance plasmids to their new bacterial hosts, or vice versa , a general evolutionary mechanism has not yet emerged. Here we conducted an experimental evolution study aimed at determining general patterns of plasmid-bacteria evolution. Specifically, we found that a large conjugative resistance plasmid follows the same evolutionary trajectories as its non-conjugative mini-replicon in the same and other species. Furthermore, within a single host-plasmid pair three distinct patterns of adaptive evolution led to increased plasmid persistence: i) mutations in the replication protein gene ( trfA1 ); ii) the acquisition by the resistance plasmid of a transposon from a co-residing plasmid encoding a putative toxin-antitoxin syste...
Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antib... more Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms, and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multi-drug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimentalmodeling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Broad-host-range (BHR) IncP-1 plasmids have the ability to transfer between and replicate in near... more Broad-host-range (BHR) IncP-1 plasmids have the ability to transfer between and replicate in nearly all species of the Alpha-, Beta-and Gammaproteobacteria, but surprisingly few data are available on the stability of these plasmids in strains within their host range. Moreover, even though molecular interactions between the bacterial host and its plasmid(s) exist, no systematic study to date has compared the stability of the same plasmid among different hosts. The goal of this study was to examine whether the stability characteristics of an IncP-1 plasmid can be variable between strains within the host range of the plasmid. Therefore, 19 strains within the Alpha-, Beta-or Gammaproteobacteria carrying the IncP-1b plasmid pB10 were serially propagated in non-selective medium and the fraction of segregants was monitored through replica-picking. Remarkably, a large variation in the stability of pB10 in different strains was found, even between strains within the same genus or species. Ten strains showed no detectable plasmid loss over about 200 generations, and in two strains plasmid-free clones were only sporadically observed. In contrast, three strains, Pseudomonas koreensis R28, Pseudomonas putida H2 and Stenotrophomonas maltophilia P21, exhibited rapid plasmid loss within 80 generations. Parameter estimation after mathematical modelling of these stability data suggested high frequencies of segregation (about 0.04 per generation) or high plasmid cost (i.e. a relative fitness decrease in plasmid-bearing cells of about 15 and 40 %), which was confirmed experimentally. The models also suggested that plasmid reuptake by conjugation only played a significant role in plasmid stability in one of the three strains. Four of the 19 strains lost the plasmid very slowly over about 600 generations. The erratic decrease of the plasmid-containing fraction and simulation of the data with a new mathematical model suggested that plasmid cost was variable over time due to compensatory mutations. The findings of this study demonstrate that the ability of a so-called 'BHR' plasmid to persist in a bacterial population is influenced by strain-specific traits, and therefore observations made for one strain should not be generalized for the entire species or genus.
Antibiotic resistant bacteria are a constant threat in the battle against infectious diseases. On... more Antibiotic resistant bacteria are a constant threat in the battle against infectious diseases. One strategy for reducing their effect is to temporarily discontinue the use of certain antibiotics in the hope that in the absence of the antibiotic the resistant strains will be replaced by the sensitive strains. An experiment where this strategy is employed in vitro [5] produces data which showed a slow accumulation of sensitive mutants. Here we propose a mathematical model and statistical analysis to explain this data. The stochastic model elucidates the trend and error structure of the data. It provides a guide for developing future sampling strategies, and provides a framework for long term predictions of the effects of discontinuing specific antibiotics on the dynamics of resistant bacterial populations.
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