High plasmidome diversity of extended-spectrum beta-lactam-resistant Escherichia coli isolates collected during one year in one community hospital

Plasmid-encoded antibiotic resistance encompasses many classes of currently used antibiotics. In globally distributed Escherichia coli lineages plasmids, which spread via horizontal gene transfer, are responsible for the dissemination of genes encoding extended-spectrum β-lactamases (ESBL). In this study, we combined 2nd and 3rd generation sequencing techniques to reconstruct the plasmidome of overall 97 clinical ESBL-E. coli isolates. Our results highlight the enormous plasmid diversity in respect to size, replicon-type and genetic content. Furthermore, we emphasize the diverse plasmid distribution patterns among the clinical isolates and the high intra- and extracellular mobility potential of resistance conferring genes. While the majority of resistance conferring genes were located on large plasmids of known replicon type, small cryptic plasmids seem to be underestimated resistance gene vectors. Our results contribute to a better understanding of the dissemination of resistance-conferring genes through horizontal gene transfer as well as clonal spread.     

High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4

Horizontal dissemination of the genes encoding extended spectrum beta-lactamases (ESBLs) via conjugative plasmids is facilitating the increasingly widespread resistance of pathogens to beta-lactam antibiotics. However, there is relatively little known about the regulatory factors and mechanisms that govern the spread of these plasmids. Here, we carried out a high-throughput, transposon insertion site sequencing analysis (TnSeq) to identify genes that enable the maintenance and transmission of pESBL, an R64 (IncI1)-related resistance plasmid that was isolated from Escherichia coli O104:H4 linked to a recent large outbreak of gastroenteritis. With a few exceptions, the majority of the genes identified as required for maintenance and transmission of pESBL matched those of their previously defined R64 counterparts. However, our analyses of the high-density transposon insertion library in pESBL also revealed two very short and linked regions that constitute a previously unrecognized regulatory system controlling spread of IncI1 plasmids. In addition, we investigated the function of the pESBL-encoded M.EcoGIX methyltransferase, which is also encoded by many other IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in expanding the plasmid''s host range. Collectively, our work illustrates the power of the TnSeq approach to enable rapid and comprehensive analyses of plasmid genes and sequences that facilitate the dissemination of determinants of antibiotic resistance.     

FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium –copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium -copy number plasmid vectors in E. coli.     

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.Subject terms: Antibiotics, Microbial ecology, Phylogenomics     

Essential gene acquisition destabilizes plasmid inheritance

Extra-chromosomal genetic elements are important drivers of evolutionary transformations and ecological adaptations in prokaryotes with their evolutionary success often depending on their ‘utility’ to the host. Examples are plasmids encoding antibiotic resistance genes, which are known to proliferate in the presence of antibiotics. Plasmids carrying an essential host function are recognized as permanent residents in their host. Essential plasmids have been reported in several taxa where they often encode essential metabolic functions; nonetheless, their evolution remains poorly understood. Here we show that essential genes are rarely encoded on plasmids; evolving essential plasmids in Escherichia coli we further find that acquisition of an essential chromosomal gene by a plasmid can lead to plasmid extinction. A comparative genomics analysis of Escherichia isolates reveals few plasmid-encoded essential genes, yet these are often integrated into plasmid-related functions; an example is the GroEL/GroES chaperonin. Experimental evolution of a chaperonin-encoding plasmid shows that the acquisition of an essential gene reduces plasmid fitness regardless of the stability of plasmid inheritance. Our results suggest that essential plasmid emergence leads to a dose effect caused by gene redundancy. The detrimental effect of essential gene acquisition on plasmid inheritance constitutes a barrier for plasmid-mediated lateral gene transfer and supplies a mechanistic understanding for the rarity of essential genes in extra-chromosomal genetic elements.     

Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.     

Gene pool transmission of multidrug resistance among Campylobacter from livestock,sewage and human disease

The use of antimicrobials in human and veterinary medicine has coincided with a rise in antimicrobial resistance (AMR) in the food-borne pathogens Campylobacter jejuni and Campylobacter coli. Faecal contamination from the main reservoir hosts (livestock, especially poultry) is the principal route of human infection but little is known about the spread of AMR among source and sink populations. In particular, questions remain about how Campylobacter resistomes interact between species and hosts, and the potential role of sewage as a conduit for the spread of AMR. Here, we investigate the genomic variation associated with AMR in 168 C. jejuni and 92 C. coli strains isolated from humans, livestock and urban effluents in Spain. AMR was tested in vitro and isolate genomes were sequenced and screened for putative AMR genes and alleles. Genes associated with resistance to multiple drug classes were observed in both species and were commonly present in multidrug-resistant genomic islands (GIs), often located on plasmids or mobile elements. In many cases, these loci had alleles that were shared among C. jejuni and C. coli consistent with horizontal transfer. Our results suggest that specific antibiotic resistance genes have spread among Campylobacter isolated from humans, animals and the environment.     

Diverse Broad-Host-Range Plasmids from Freshwater Carry Few Accessory Genes

Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid''s ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities.     

Genetic Drift Suppresses Bacterial Conjugation in Spatially Structured Populations

Conjugation is the primary mechanism of horizontal gene transfer that spreads antibiotic resistance among bacteria. Although conjugation normally occurs in surface-associated growth (e.g., biofilms), it has been traditionally studied in well-mixed liquid cultures lacking spatial structure, which is known to affect many evolutionary and ecological processes. Here we visualize spatial patterns of gene transfer mediated by F plasmid conjugation in a colony of Escherichia coli growing on solid agar, and we develop a quantitative understanding by spatial extension of traditional mass-action models. We found that spatial structure suppresses conjugation in surface-associated growth because strong genetic drift leads to spatial isolation of donor and recipient cells, restricting conjugation to rare boundaries between donor and recipient strains. These results suggest that ecological strategies, such as enforcement of spatial structure and enhancement of genetic drift, could complement molecular strategies in slowing the spread of antibiotic resistance genes.     

Host-specific factors determine the persistence of IncP-1 plasmids

Conjugative plasmids play a very important role in bacterial adaptation through the dissemination of useful traits. Incompatibility group P-1 (IncP-1) plasmids exhibit an extreme broad-host-range among Gram-negative bacteria and known to be one of the major agents to disseminate various phenotypic traits such as antibiotic resistance and xenobiotic degradation. Although the plasmids are believed to be very stable in most Gram-negative bacteria, little is known about the factors that affect their stability in various hosts, allowing their persistence in bacterial population. Here we show that the stability of the cryptic IncP-1β plasmid pBP136 differed greatly in four different Escherichia coli K12 host backgrounds (MG1655, DH5α, EC100, and JM109), whereas the closely related plasmid pB10 was stable in all four strains. The supply of the kleF gene, which is involved in the stability of IncP-1 plasmids but absent in pBP136, did not improve the stability of the plasmid. Our findings suggest that persistence of IncP-1 plasmids in the absence of selection is affected by strain-specific factors.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

Genetic Drift Suppresses Bacterial Conjugation in Spatially Structured Populations

Conjugation is the primary mechanism of horizontal gene transfer that spreads antibiotic resistance among bacteria. Although conjugation normally occurs in surface-associated growth (e.g., biofilms), it has been traditionally studied in well-mixed liquid cultures lacking spatial structure, which is known to affect many evolutionary and ecological processes. Here we visualize spatial patterns of gene transfer mediated by F plasmid conjugation in a colony of Escherichia coli growing on solid agar, and we develop a quantitative understanding by spatial extension of traditional mass-action models. We found that spatial structure suppresses conjugation in surface-associated growth because strong genetic drift leads to spatial isolation of donor and recipient cells, restricting conjugation to rare boundaries between donor and recipient strains. These results suggest that ecological strategies, such as enforcement of spatial structure and enhancement of genetic drift, could complement molecular strategies in slowing the spread of antibiotic resistance genes.     

Increased Abundance and Transferability of Resistance Genes after Field Application of Manure from Sulfadiazine-Treated Pigs

Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes sul1 and sul2 in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both sul genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of sul genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the sul gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into Escherichia coli. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance.     

Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis

Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis.     

Factors That Affect Transfer of the IncI1 β-Lactam Resistance Plasmid pESBL-283 between E. coli Strains

The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these can contribute to transmission of resistance genes through the food chain.     

Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration,and Acquisition of the Plasmid Results in a Variable Cost of Fitness

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P < 0.001) and increased movement of the IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.     

Construction of arsenic-resistant Thiobacillus ferrooxidans recombinant plasmids and the expression of autotrophic plasmid genes in a heterotrophic cell-free system

Recombinant plasmids between Thiobacillus ferrooxidans and Escherichia coli which contain arsenic and antibiotic resistance genes and unique restriction sites have been constructed. The expression of autotrophic T. ferrooxidans genes in a heterotrophic E. coli cell-free system was demonstrated.