Coexistence of a nonresistance-conferring IncI1 plasmid favors persistence of the blaCTX-M-bearing IncFII plasmid in Escherichia coli.

The interaction between coexisting plasmids can affect plasmid-carried resistance gene persistence and spread. However, whether the persistence of the blaCTX-M gene in clinical Enterobacteriaceae is related to the interaction of coresident nonresistance-conferring plasmids has not been reported. This study was initiated to elucidate how a nonresistance-conferring IncI1 plasmid affected the blaCTX-M-bearing IncFII plasmid colocated on the same cell. Herein, we constructed three isogenic derivatives of E. coli C600, designated as C600FII, C600I1, and C600FII+I1, which harbored the blaCTX-M-IncFII plasmid and/or the nonresistance-IncI1 one. We discovered that strain C600FII+I1 conferred higher fitness advantages than strain C600FII; also, the stability of the blaCTX-M-IncFII plasmid was noticeably improved in an antibiotic-free environment when it coexisted with the IncI1 plasmid. To further explore why the IncI1 plasmid enhanced the persistence of the blaCTX-M-IncFII plasmid, we assessed the blaCTX-M-IncFII plasmid's copy numbers, conjugation frequencies, and rep gene expressions in strains C600FII and C600FII+I1. The results demonstrated that the rep expressions of the blaCTX-M-IncFII plasmid in strain C600FII+I1 was greatly decreased, along with the plasmid's copy numbers and mating efficiencies, compared to those in strain C600FII. Moreover, further study revealed that the intracellular ATP levels of strain C600FII+I1 were far lower than those of strain C600FII. Our findings confirmed that coexistence of the nonresistance-IncI1 plasmid can keep the blaCTX-M-IncFII plasmid more stable by increasing the fitness advantages of the host bacteria, which will pose a threat to preventing the long-term presence of the plasmid-carried blaCTX-M gene in clinical Enterobacteriaceae. IMPORTANCE: So far, plasmid-carried blaCTX-M is still the most common extended-spectrum beta-lactamase (ESBL) genotype in clinical settings worldwide. Except for the widespread use of third-generation cephalosporins, the interaction between coexisting plasmids can also affect the long-term stable existence of the blaCTX-M gene; however, the study on that is still sparse. In the present study, we assess the interaction of coinhabitant plasmids blaCTX-M-IncFII and nonresistance-IncI1. Our results confirmed that the increased fitness advantages of strain C600FII+I1 were attributable to the cohabitant nonresistance-IncI1 plasmid, which largely reduced the intracellular ATP levels of host bacteria, thus decreasing the rep gene expression of the blaCTX-M-IncFII plasmid, its copy numbers, and mating efficiencies, while the higher fitness advantages of strain C600FII+I1 enhanced the persistence of the blaCTX-M-IncFII plasmid. The results indicate that the nonresistance-IncI1 plasmid contributes to the long-term existence of the blaCTX-M-IncFII plasmid, implying a potentially new strategy for controlling the spread of resistance plasmids in clinical settings by targeting nonresistance plasmids.

Genetic Diversity and Antimicrobial Resistance of Extraintestinal E. coli Populations Pre- and Post-Antimicrobial Therapy on Broilers Affected by Colisepticemia.

The aim of the present study was to investigate the genetic diversity and antimicrobial resistance (AMR) of E. coli during enrofloxacin therapy in broilers affected by colisepticemia. Three unrelated farms with ongoing colibacillosis outbreaks were sampled at day 1 before treatment and at days 5, 10 and 24 post-treatment. A total of 179 E. coli isolates were collected from extraintestinal organs and submitted to serotyping, PFGE and the minimum inhibitory concentration (MIC) against enrofloxacin. PFGE clusters shifted from 3-6 at D1 to 10-16 at D5, D10 and D24, suggesting an increased population diversity after the treatment. The majority of strains belonged to NT or O78 and to ST117 or ST23. PFGE results were confirmed with SNP calling: no persistent isolates were identified. An increase in resistance to fluoroquinolones in E. coli isolates was observed along the treatment. Resistome analyses revealed qnrB19 and qnrS1 genes along with mutations in the gyrA, parC and parE genes. Interestingly, despite a fluoroquinolone selective pressure, qnr-carrying plasmids did not persist. On the contrary, two conjugative AMR plasmid clusters (AB233 and AA474) harboring AMR genes other than qnr were persistent since they were identified in both D1 and D10 genomes in two farms. Further studies should be performed in order to confirm plasmid persistence not associated (in vivo) to antimicrobial selective pressure.

Transcriptional modifications enhance the persistence of resistant plasmids in the presence of tetracycline in an environmentally relevant concentration.

Antibiotic resistance genes (ARGs) have captured immense attention due to their widespread existence and remarkable ability to spread across species boundaries. Plasmid carriage promotes the persistence and spread of ARGs in the environment. To investigate the prevalence of plasmids, we conducted serial passage experiments on Pseudomonas putida KT2442 with multidrug-resistant plasmid RP4::gfp in the presence of tetracycline (TC) in an environmentally relevant concentration. The results showed that TC in environmental concentration compensated the fitness cost brought by the plasmid, prolonged the persistence time of the plasmid-bearing strain, and induced the reoccurrence of plasmids after the window time of plasmid loss. Transcriptome sequencing showed that plasmid recovery was compensated by the up-regulation of glyoxylic acid shunt and the down-regulation of ribosome biosynthesis. It is therefore hypothesized that transcriptional modifications may enhance the persistence of resistant plasmids within the population in the presence of TC in an environmentally relevant concentration. This work opens up an avenue for developing a technology based on the window time of plasmid loss to prevent the spread of ARGs.

Comparison of Fitness Cost, Stability, and Conjugation Frequencies of tet(X4)-Positive Plasmids in Chicken and Pig Escherichia coli.

The large-scale epidemic of the tet(X4) gene in the livestock and poultry industry is threatening public health; however, there is still a lack of comparative studies on tet(X4)-bearing plasmids in chicken and pig Escherichia coli. To evaluate the prevalence trend of tet(X4)-bearing plasmids and the factors influencing their persistence in the livestock and poultry industry, we examined the fitness cost, stability under tetracyclines pressure, and conjugation frequencies at various temperatures of six tet(X4)-bearing plasmids in four representative pig E. coli isolates and chicken E. coli isolates. Compared with pig E. coli, the plasmid in chicken E. coli showed lower fitness cost, and stronger ability to promote bacterial biofilm formation and motility. Meanwhile, the presence of tetracycline may favor the stability of tet(X4)-bearing plasmids, which was more common in chicken E. coli. Furthermore, the optimal temperature for IncX1 tet(X4)-bearing plasmid conjugation was 42 °C, and its conjugation frequency in chicken E. coli was higher than that in pig E. coli, whereas the optimal temperature for IncFII tet(X4)-bearing plasmid conjugation was 37 °C and it performed better in pig E. coli, suggesting the predominant plasmid types circulating in chicken E. coli and pig E. coli may be distinct. Collectively, although tet(X4) currently appears to be more prevalent in pig E. coli, this is probably independent of the fitness cost caused by tet(X4)-plasmids. To curb the future spread of the tet(X4) gene, reduced tetracyclines usage and tailored interventions should be applied in different breeding industries.

Plasmid carriage and the natural complexity of bacterial populations contributes to plasmid persistence / El transporte de plásmidos y la complejidad natural de las poblaciones bacterianas contribuyen a la persistencia del plásmido

Introduction: Plasmids carry and transport genes that assist their hosts to survive in many environments. Many studies have examined the conditions for plasmid persistence in bacterial populations. A limitation includes that a majority of the mathematical models for examining plasmid persistence only included bacteria from similar colonies. However, most bacterial cells inhabit complex communities where plasmids disseminate between varied bacterial host cells. Thus, there is a gap in knowledge concerning the persistence of plasmids in natural bacterial populations. To address a few of these gaps in knowledge, the present study attempted to examine the effects of plasmid carriage on intrinsic stages of bacterial populations in Bacillus subtilis co-cultures. Material and methods: B. subtilis cells were transformed with CRISPR-hCas-9 plasmid vectors where the natural phases of bacterial growth, biofilm production, and antibiotic resistance were examined in relation to plasmid carriage. These three natural phases were measured in relation to plasmid carriage through in vitro co-culture assays. Results: After calculating the CFU/mL, bacterial growth in the B. subtilis-Carrier with Escherichia coli (B. sub-C-E. coli) and Vibrio harveyi (B. sub-C-VH) co-cultures significantly decreased with a paired-t-test two-tailed P=0. The WT B. subtilis-V.H samples, the B. subtilis Carrier-V.H co-cultures, and the controls each scored a total of 40, 47, and 46 of crystal violet (CV) intensity of biofilm, respectively. Biofilm formation decreased after co-culturing E. coli with the B. subtilis-Carrier, yielding a P<0.001. The antibiotic resistance levels of the co-cultures increased by 3% for the B. sub-C-V.H samples while the B. sub-C-E. coli co-cultures decreased in antibiotic sensitivity by approximately 1.5%. Conclusions: Plasmid carriage contributes to plasmid persistence via altering the natural phases of bacterial populations (AU)

Introducción: Los plásmidos portan y transportan genes que ayudan a sus huéspedes a sobrevivir en muchos entornos. Muchos estudios han examinado las condiciones para la persistencia de plásmidos en poblaciones bacterianas. Una limitación incluye que la mayoría de los modelos matemáticos para examinar la persistencia de plásmidos solo incluyeron bacterias de colonias similares. Sin embargo, la mayoría de las células bacterianas habitan en comunidades complejas donde los plásmidos se diseminan entre diversas células huésped bacterianas. Por lo tanto, existe un vacío en el conocimiento sobre la persistencia de plásmidos en poblaciones bacterianas naturales. Para abordar algunas de estas lagunas en el conocimiento, el presente estudio intentó examinar los efectos del transporte de plásmidos en las etapas intrínsecas de las poblaciones bacterianas en cocultivos de Bacillus subtilis. Material y métodos: Células de B. subtilis se transformaron con vectores plasmídicos CRISPR-hCas-9 donde se examinaron las fases naturales de crecimiento bacteriano, producción de biopelículas y resistencia a los antibióticos en relación con el transporte del plásmido. Estas tres fases naturales se midieron en relación con el transporte de plásmidos a través de ensayos de cocultivo in vitro. Resultados: Después de calcular las UFC/mL, el crecimiento bacteriano en los cocultivos de B. subtilis-Carrier con Escherichia coli (B. sub-C-E. coli) y Vibrio harveyi (B. sub-C-VH) disminuyó significativamente con un -t-test de dos colas P=0. Las muestras WT B. subtilis-V.H, los cocultivos B. subtilis Carrier-V.H y los controles obtuvieron cada uno un total de 40, 47 y 46 de intensidad de biopelícula cristal violeta (CV), respectivamente. La formación de biopelículas disminuyó después de cocultivar E. coli con B. subtilis-Carrier, lo que arrojó un P<0,001 (AU)

Genomic analysis of qnr-harbouring IncX plasmids and their transferability within different hosts under induced stress.

BACKGROUND: Conjugative plasmids play a major role in the dissemination of antibiotic resistance genes. Knowledge of the plasmid characteristics and behaviour can allow development of control strategies. Here we focus on the IncX group of plasmids carrying genes conferring quinolone resistance (PMQR), reporting their transfer and persistence within host bacteria of various genotypes under distinct conditions and levels of induced stress in form of temperature change and various concentrations of ciprofloxacin supplementation. METHODS: Complete nucleotide sequences were determined for eight qnr-carrying IncX-type plasmids, of IncX1 (3), IncX2 (3) and a hybrid IncX1-2 (2) types, recovered from Escherichia coli of various origins. This data was compared with further complete sequences of IncX1 and IncX2 plasmids carrying qnr genes (n = 41) retrieved from GenBank and phylogenetic tree was constructed. Representatives of IncX1 (pHP2) and IncX2 (p194) and their qnrS knockout mutants, were studied for influence of induced stress and genetic background on conjugative transfer and maintenance. RESULTS: A high level of IncX core-genome similarity was found in plasmids of animal, environmental and clinical origin. Significant differences were found between the individual IncX plasmids, with IncX1 subgroup plasmids showing higher conjugative transfer rates than IncX2 plasmids. Knockout of qnr modified transfer frequency of both plasmids. Two stresses applied simultaneously were needed to affect transfer rate of wildtype plasmids, whereas a single stress was sufficient to affect the IncX ΔqnrS plasmids. The conjugative transfer was shown to be biased towards the host phylogenetic proximity. A long-term cultivation experiment pointed out the persistence of IncX plasmids in the antibiotic-free environment. CONCLUSIONS: The study indicated the stimulating effect of ciprofloxacin supplementation on the plasmid transfer that can be nullified by the carriage of a single PMQR gene. The findings present the significant properties and behaviour of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations.

Predicting plasmid persistence in microbial communities by coarse-grained modeling.

Plasmids are a major type of mobile genetic elements (MGEs) that mediate horizontal gene transfer. The stable maintenance of plasmids plays a critical role in the functions and survival for microbial populations. However, predicting and controlling plasmid persistence and abundance in complex microbial communities remain challenging. Computationally, this challenge arises from the combinatorial explosion associated with the conventional modeling framework. Recently, a plasmid-centric framework (PCF) has been developed to overcome this computational bottleneck. This framework enables the derivation of a simple metric, the persistence potential, to predict plasmid persistence and abundance. Here, we discuss how PCF can be extended to account for plasmid interactions. We also discuss how such model-guided predictions of plasmid fates can benefit from the development of new experimental tools and data-driven computational methods.

Contagious Antibiotic Resistance: Plasmid Transfer among Bacterial Residents of the Zebrafish Gut.

By characterizing the trajectories of antibiotic resistance gene transfer in bacterial communities such as the gut microbiome, we will better understand the factors that influence this spread of resistance. Our aim was to investigate the host network of a multidrug resistance broad-host-range plasmid in the culturable gut microbiome of zebrafish. This was done through in vitro and in vivo conjugation experiments with Escherichia coli as the donor of the plasmid pB10::gfp When this donor was mixed with the extracted gut microbiome, only transconjugants of Aeromonas veronii were detected. In separate matings between the same donor and four prominent isolates from the gut microbiome, the plasmid transferred to two of these four isolates, A. veronii and Plesiomonas shigelloides, but not to Shewanella putrefaciens and Vibrio mimicus When these A. veronii and P. shigelloides transconjugants were the donors in matings with the same four isolates, the plasmid now also transferred from A. veronii to S. putrefaciensP. shigelloides was unable to donate the plasmid, and V. mimicus was unable to acquire it. Finally, when the E. coli donor was added in vivo to zebrafish through their food, plasmid transfer was observed in the gut, but only to Achromobacter, a rare member of the gut microbiome. This work shows that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome depends on the donor-recipient species combinations and therefore their spatial arrangement. It also suggests that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food.IMPORTANCE To understand how antibiotic resistance plasmids end up in human pathogens, it is crucial to learn how, where, and when they are transferred and maintained in members of bacterial communities such as the gut microbiome. To gain insight into the network of plasmid-mediated antibiotic resistance sharing in the gut microbiome, we investigated the transferability and maintenance of a multidrug resistance plasmid among the culturable bacteria of the zebrafish gut. We show that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome can depend on which species are involved, as some are important nodes in the plasmid-host network and others are dead ends. Our findings also suggest that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food.

Mobile Compensatory Mutations Promote Plasmid Survival.

The global dissemination of plasmids encoding antibiotic resistance represents an urgent issue for human health and society. While the fitness costs for host cells associated with plasmid acquisition are expected to limit plasmid dissemination in the absence of positive selection of plasmid traits, compensatory evolution can reduce this burden. Experimental data suggest that compensatory mutations can be located on either the chromosome or the plasmid, and these are likely to have contrasting effects on plasmid dynamics. Whereas chromosomal mutations are inherited vertically through bacterial fission, plasmid mutations can be inherited both vertically and horizontally and potentially reduce the initial cost of the plasmid in new host cells. Here we show using mathematical models and simulations that the dynamics of plasmids depends critically on the genomic location of the compensatory mutation. We demonstrate that plasmid-located compensatory evolution is better at enhancing plasmid persistence, even when its effects are smaller than those provided by chromosomal compensation. Moreover, either type of compensatory evolution facilitates the survival of resistance plasmids at low drug concentrations. These insights contribute to an improved understanding of the conditions and mechanisms driving the spread and the evolution of antibiotic resistance plasmids. IMPORTANCE Understanding the evolutionary forces that maintain antibiotic resistance genes in a population, especially when antibiotics are not used, is an important problem for human health and society. The most common platform for the dissemination of antibiotic resistance genes is conjugative plasmids. Experimental studies showed that mutations located on the plasmid or the bacterial chromosome can reduce the costs plasmids impose on their hosts, resulting in antibiotic resistance plasmids being maintained even in the absence of antibiotics. While chromosomal mutations are only vertically inherited by the daughter cells, plasmid mutations are also provided to bacteria that acquire the plasmid through conjugation. Here we demonstrate how the mode of inheritance of a compensatory mutation crucially influences the ability of plasmids to spread and persist in a bacterial population.

Reconsidering plasmid maintenance factors for computational plasmid design.

Plasmids are genetic parasites of microorganisms. The genomes of naturally occurring plasmids are expected to be polished via natural selection to achieve long-term persistence in the microbial cell population. However, plasmid genomes are extremely diverse, and the rules governing plasmid genomes are not fully understood. Therefore, computationally designing plasmid genomes optimized for model and nonmodel organisms remains challenging. Here, we summarize current knowledge of the plasmid genome organization and the factors that can affect plasmid persistence, with the aim of constructing synthetic plasmids for use in gram-negative bacteria. Then, we introduce publicly available resources, plasmid data, and bioinformatics tools that are useful for computational plasmid design.

Conjugative plasmids enable the maintenance of low cost non-transmissible plasmids.

Some plasmids can be transferred by conjugation to other bacterial hosts. But almost half of the plasmids are non-transmissible. These plasmid types can only be transmitted to the daughter cells of their host after bacterial fission. Previous studies suggest that non-transmissible plasmids become extinct in the absence of selection of their encoded traits, as plasmid-free bacteria are more competitive. Here, we aim to identify mechanisms that enable non-transmissible plasmids to persist, even if they are not beneficial. For this purpose, an individual-based model for plasmid population dynamics was set up and carefully tested for structural consistency and plausibility. Our results demonstrate that non-transmissible plasmids can be stably maintained in a population, even if they impose a substantial burden on their host cells growth. A prerequisite is the co-occurrence of an incompatible and costly conjugative plasmid type, which indirectly facilitates the preservation of the non-transmissible type. We suggest that this constellation might be considered as a potential mechanism maintaining plasmids and associated antibiotic resistances. It should be investigated in upcoming laboratory experiments.

Genome Dynamics of Escherichia coli during Antibiotic Treatment: Transfer, Loss, and Persistence of Genetic Elements In situ of the Infant Gut.

Elucidating the adaptive strategies and plasticity of bacterial genomes in situ is crucial for understanding the epidemiology and evolution of pathogens threatening human health. While much is known about the evolution of Escherichia coli in controlled laboratory environments, less effort has been made to elucidate the genome dynamics of E. coli in its native settings. Here, we follow the genome dynamics of co-existing E. coli lineages in situ of the infant gut during the first year of life. One E. coli lineage causes a urinary tract infection (UTI) and experiences several alterations of its genomic content during subsequent antibiotic treatment. Interestingly, all isolates of this uropathogenic E. coli strain carried a highly stable plasmid implicated in virulence of diverse pathogenic strains from all over the world. While virulence elements are certainly beneficial during infection scenarios, their role in gut colonization and pathogen persistence is poorly understood. We performed in vivo competitive fitness experiments to assess the role of this highly disseminated virulence plasmid in gut colonization, but found no evidence for a direct benefit of plasmid carriage. Through plasmid stability assays, we demonstrate that this plasmid is maintained in a parasitic manner, by strong first-line inheritance mechanisms, acting on the single-cell level, rather than providing a direct survival advantage in the gut. Investigating the ecology of endemic accessory genetic elements, in their pathogenic hosts and native environment, is of vital importance if we want to understand the evolution and persistence of highly virulent and drug resistant bacterial isolates.