Transfer Of Antibiotic Resistance Plasmids Research Articles

Fluorescent tag reveals the potential mechanism of how indigenous soil bacteria affect the transfer of the wild fecal antibiotic resistance plasmid pKANJ7 in different habitat soils

Plasmids have increasingly become a point of concern since they act as a vital medium for the dissemination of antibiotic resistance genes (ARGs). Although indigenous soil bacteria are critical hosts for these plasmids, the mechanisms driving the transfer of antibiotic resistance plasmids (ARPs) have not been well researched. In this study, we tracked and visualized the colonization of the wild fecal antibiotic resistance plasmid pKANJ7 in indigenous bacteria of different habitat soils (unfertilized soil (UFS), chemical fertilized soil (CFS), and manure fertilized soil (MFS)). The results showed that plasmid pKANJ7 mainly transferred to the dominant genera in the soil and genera that were highly related to the donor. More importantly, plasmid pKANJ7 also transferred to intermediate hosts which aid in the survival and persistence of these plasmids in soil. Nitrogen levels also raised the plasmid transfer rate (14th day: UFS: 0.09%, CFS: 1.21%, MFS: 4.57%). Lastly, our structural equation model (SEM) showed that dominant bacteria shifts caused by nitrogen and loam were the major driver shaping the difference in the transfer of plasmid pKANJ7. Overall, our findings enhance the mechanistic understanding of indigenous soil bacteria’s role in plasmid transfer and inform potential methods to prevent the transmission of plasmid-borne resistance in the environment.

Sequence analysis and plasmid mobilization of a 6.6-kb kanamycin resistance plasmid, pSNC3-Kan, from a Salmonella enterica serotype Newport isolate

Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. A series of diverse ColE-like kanamycin resistance plasmids (“KanR plasmids”) from Salmonella enterica were characterized previously. In this study, the 6.6-kb pSNC3-Kan from a Salmonella enterica serotype Newport isolate was investigated. It possessed highly conserved RNA I/II and Tn602 (IS903-aph-IS903) regions to two other KanR plasmids pSe-Kan and pSBardo-Kan, but carried a mobC-mobA/BD operon. The mobilization proteins encoded by the mob operon of pSNC3-Kan showed high sequence identity (~95%) to those of an E. coli plasmid pEC34B, except that MobE was not present; and were much less conserved to those of another KanR plasmid pSN11/00Kan (43% - 86% identity). Four structurally different KanR plasmids were investigated for their ability to be mobilized by the conjugal transfer (tra) genes from F and IncP plasmids. Transfer genes derived from IncP plasmids can efficiently mobilize KanR plasmids possessing the mob operons (mobC-mobA/BD), such as pSNC3-Kan and pSN11/00Kan, in bi-parental mating experiments. On the other hand, F tra genes were able to mobilize pU302S, pSNC3-Kan and pSe-Kan, but not pSN11/00Kan. A plasmid-borne mob operon was not required for mobilization of the oriT(F)-bearing pSe-Kan by the F tra genes. This study underscores the complexity of plasmid interaction and the importance of how small mobilizable plasmids may contribute to the spread of antibiotic resistance genes.

Transfer of Antibiotic Resistance Plasmid from Commensal E. coli Towards Human Intestinal Microbiota in the M-SHIME: Effect of E. coli dosis, Human Individual and Antibiotic Use.

Along with (in)direct contact with animals and a contaminated environment, humans are exposed to antibiotic resistant bacteria by consumption of food. The implications of ingesting antibiotic resistant commensal bacteria are unknown, as dose-response data on resistance transfer and spreading in our gut is lacking. In this study, transfer of a resistance plasmid (IncF), harbouring several antibiotic resistance genes, from a commensal E. coli strain towards human intestinal microbiota was assessed using a Mucosal Simulator of the Human Intestinal Ecosystem (M-SHIME). More specifically, the effect of the initial E. coli plasmiddonor concentration (105 and 107 CFU/meal), antibiotic treatment (cefotaxime) and human individual (n = 6) on plasmid transfer towards lumen coliforms and anaerobes was determined. Transfer of the resistance plasmid to luminal coliforms and anaerobes was observed shortly after the donor strain arrived in the colon and was independent of the ingested dose. Transfer occurred in all six simulated colons and despite their unique microbial community composition, no differences could be detected in antibiotic resistance transfer rates between the simulated human colons. After 72 h, resistant coliform transconjugants levels ranged from 7.6 × 104 to 7.9 × 106 CFUcefotaxime resistant/mL colon lumen. Presence of the resistance plasmid was confirmed and quantified by PCR and qPCR. Cefotaxime treatment led to a significant reduction (85%) in resistant coliforms, however no significant effect on the total number of cultivable coliforms and anaerobes was observed.

The Conjugation Window in an Escherichia coli K-12 Strain with an IncFII Plasmid.

Many studies have examined the role that conjugation plays in disseminating antibiotic resistance genes in bacteria. However, relatively little research has quantitively examined and modeled the dynamics of conjugation under growing and nongrowing conditions beyond a couple of hours. We therefore examined growing and nongrowing cultures of Escherichia coli over a 24-h period to understand the dynamics of bacterial conjugation in the presence and absence of antibiotics with pUUH239.2, an IncFII plasmid containing multiantibiotic- and metal-resistant genes. Our data indicate that conjugation occurs after E. coli cells divide and before they have transitioned to a nongrowing phase. The result is that there is only a small window of opportunity for E. coli to conjugate with pUUH239.2 under both growing and nongrowing conditions. Only a very small percentage of the donor cells likely are capable of even undergoing conjugation, and not all transconjugants can become donor cells due to molecular regulatory controls and not being in the correct growth phase. Once a growing culture enters stationary phase, the number of capable donor cells decreases rapidly and conjugation slows to produce a plateau. Published models did not provide accurate descriptions of conjugation under nongrowing conditions. We present here a modified modeling approach that accurately describes observed conjugation behavior under growing and nongrowing conditions.IMPORTANCE There has been growing interest in horizontal gene transfer of antibiotic resistance plasmids as the antibiotic resistance crisis has worsened over the years. Most studies examining conjugation of bacterial plasmids focus on growing cultures of bacteria for short periods, but in the environment, most bacteria grow episodically and at much lower rates than in the laboratory. We examined conjugation of an IncFII antibiotic resistance plasmid in E. coli under growing and nongrowing conditions to understand the dynamics of conjugation under which the plasmid is transferred. We found that conjugation occurs in a narrow time frame when E. coli is transitioning from a growing to nongrowing phase and that the conjugation plateau develops because of a lack of capable donor cells in growing cultures. From an environmental aspect, our results suggest that episodic growth in nutrient-depleted environments could result in more conjugation than sustained growth in a nutrient rich environment.

Mouse Genetic Background Affects Transfer of an Antibiotic Resistance Plasmid in the Gastrointestinal Tract.

Dissemination of antibiotic resistance (AR) genes, often on plasmids, leads to antibiotic-resistant bacterial infections, which is a major problem for animal and public health. Bacterial conjugation is the primary route of AR gene transfer in the mammalian gastrointestinal tract. Significant gaps in knowledge about which gastrointestinal communities and host factors promote plasmid transfer remain. Here, we used Salmonella enterica serovar Kentucky strain CVM29188 carrying plasmid pCVM29188_146 (harboring streptomycin and tetracycline resistance genes) to assess plasmid transfer to Escherichia coli under in vitro conditions and in various mouse strains with a conventional or defined microbiota. As an initial test, the transfer of pCVM29188_146 to the E. coli strains was confirmed in vitro Colonization resistance and, therefore, a lack of plasmid transfer were found in wild-type mice harboring a conventional microbiota. Thus, mice harboring the altered Schaedler flora (ASF), or ASF mice, were used to probe for host factors in the context of a defined microbiota. To assess the influence of inflammation on plasmid transfer, we compared interleukin-10 gene-deficient 129S6/SvEv ASF mice (proinflammatory environment) to wild-type 129S6/SvEv ASF mice and found no difference in transconjugant yields. In contrast, the mouse strain influenced plasmid transfer, as C3H/HeN ASF mice had significantly lower levels of transconjugants than 129S6/SvEv ASF mice. Although gastrointestinal members were identical between the ASF mouse strains, a few differences from C3H/HeN ASF mice were detected, with C3H/HeN ASF mice having significantly lower abundances of ASF members 356 (Clostridium sp.), 492 (Eubacterium plexicaudatum), and 502 (Clostridium sp.) than 129S6/SvEv ASF mice. Overall, we demonstrate that microbiota complexity and mouse genetic background influence in vivo plasmid transfer.IMPORTANCE Antibiotic resistance is a threat to public health. Many clinically relevant antibiotic resistance genes are carried on plasmids that can be transferred to other bacterial members in the gastrointestinal tract. The current study used a murine model to study the transfer of a large antibiotic resistance plasmid from a foodborne Salmonella strain to a gut commensal E. coli strain in the gastrointestinal tract. We found that different mouse genetic backgrounds and a different diversity of microbial communities influenced the level of Escherichia coli that acquired the plasmid in the gastrointestinal tract. This study suggests that the complexity of the microbial community and host genetics influence plasmid transfer from donor to recipient bacteria.

Dissecting horizontal and vertical gene transfer of antibiotic resistance plasmid in bacterial community using microfluidics

The spread of antibiotic resistance genes (ARGs) has become an emerging threat to the global health. Although horizontal gene transfer (HGT) is regarded as one of the major pathways, more evidence has shown the significant involvement of vertical gene transfer (VGT). However, traditional cultivation-based methods cannot distinguish HGT and VGT, resulting in often contradictory conclusions. Here, single-cell microfluidics with time-lapse imaging has been successfully employed to dissect the contribution of plasmid-mediated HGT and VGT to ARG transmission in an environmental community. Using Escherichia coli with an ARG-coded plasmid pKJK5 with trimethoprim resistance as the donor, we quantified the effects of three representative antibiotics (trimethoprim, tetracycline and amoxicillin) on the ARG transfer process in an activated sludge bacterial community. It was found that HGT was influenced by the inhibitory mechanism of an antibiotic and its targets (donor, recipient alone or together), whereas VGT contributes significantly to the formation of transconjugants and consequently ARG spreading. Trimethoprim is highly resisted by the donor and transconjugants, and its presence significantly increased both the HGT and VGT rates. Although tetracycline and amoxicillin both inhibit the donor, they showed different effects on HGT rate as a result of different inhibitory mechanisms. Furthermore, we show the kinetics of HGT in a community can be described using an epidemic infection model, which in combination with quantitative measure of HGT and VGT on chip provides a promising tool to study and predict the dynamics of ARG spread in real-world communities.

Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

Transfer of antibiotic resistance plasmids in pure and activated sludge cultures in the presence of environmentally representative micro-contaminant concentrations

The presence of antibiotics in the natural environment has been a growing issue. This presence could also account for the influence that affects microorganisms in such a way that they develop resistance against these antibiotics. The aim of this study was to evaluate whether the antibiotic resistant gene (ARG) plasmid transfer can be facilitated by the impact of 1) environmentally representative micro-contaminant concentrations in ppb (part per billion) levels and 2) donor-recipient microbial complexity (pure vs. mixed). For this purpose, the multidrug resistant plasmid, pB10, and Escherichia coli DH5α were used as a model plasmid and a model donor, respectively. Based on conjugation experiments with pure (Pseudomonas aeruginosa PAKexoT) and mixed (activated sludge) cultures as recipients, increased relative plasmid transfer frequencies were observed at ppb (μg/L) levels of tetracycline and sulfamethoxazole micro-contaminant exposure. When sludge, a more complex community, was used as a recipient, the increases of the plasmid transfer rate were always statistically significant but not always in P. aeruginosa. The low concentration (10ppb) of tetracycline exposure led to the pB10 transfer to enteric bacteria, which are clinically important pathogens.

The Animal Food Supplement Sepiolite Promotes a Direct Horizontal Transfer of Antibiotic Resistance Plasmids between Bacterial Species

Animal fodder is routinely complemented with antibiotics together with other food supplements to improve growth. For instance, sepiolite is currently used as a dietary coadjuvant in animal feed, as it increases animal growth parameters and improves meat and derived final product quality. This type of food additive has so far been considered innocuous for the development and spread of antibiotic resistance. In this study, we demonstrate that sepiolite promotes the direct horizontal transfer of antibiotic resistance plasmids between bacterial species. The conditions needed for plasmid transfer (sepiolite and friction forces) occur in the digestive tracts of farm animals, which routinely receive sepiolite as a food additive. Furthermore, this effect may be aggravated by the use of antibiotics supplied as growth promoters.

Efficient transfer of antibiotic resistance plasmids by transduction within methicillin-resistant Staphylococcus aureus USA300 clone

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.

Comparisons of Salmonella conjugation and virulence gene hyperexpression mediated by rumen protozoa from domestic and exotic ruminants

Recent studies have identified a phenomenon in which ciliated protozoa engulf Salmonella and the intra-protozoal environment hyperactivates virulence gene expression and provides a venue for conjugal transfer of antibiotic resistance plasmids. The former observation is relegated to Salmonella bearing the SGI1 multiresistance integron while the latter phenomenon appears to be a more generalized event for recipient Salmonella. Our previous studies have assessed virulence gene hyperexpression only with protozoa from the bovine rumen while conjugal transfer has been demonstrated in rumen protozoa from cattle and goats. The present study examined virulence gene hyperexpression for Salmonella exposed to rumen protozoa obtained from cattle, sheep, goats, or two African ruminants (giraffe and bongo). Conjugal transfer was also assessed in these protozoa using Salmonella as the recipient. Virulence gene hyperexpression was only observed following exposure to the rumen protozoa from cattle and sheep while elevated virulence was also observed in these animals. Conjugal transfer events were, however, observed in all protozoa evaluated. It therefore appears that the protozoa-based hypervirulence is not universal to all ruminants while conjugal transfer is more ubiquitous.

The Mechanism of Plasmid Curing in Bacteria

Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic compounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli, Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the populations studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds. The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar ring system with substitution in the L-molecular region. A symmetrical pi-electron conjugation at the highest occupied molecular orbitals favours the antiplasmid effect. The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the DNA to which they bind. In this manner "extrachromosomal" plasmid DNA that exists in a superhelical state binds more compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the energy of HOMO-orbitals. Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a new perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance. The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing mutations.

Infectious Plasmid Resistance and Efflux Pump Mediated Resistance

Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.

The Relationship between the Transferability of Enterotoxin Plasmids and the Simultaneous Transfer of Antibiotic Resistance Plasmids

SummaryEstablished donors of enterotoxin plasmids were used in a study to determine the relationship between the transferability of Ent plasmids and the simultaneous transfer of antibiotic resistance plasmids. The transfer of Ent plasmids from R plasmid‐containing E. coli was not detected when drug‐resistant transconjugants were selected. The addition of a sub‐inhibitory concentration of an antibiotic to the mating mixture did not result in increased formation of Ent/R plasmid‐containing transconjugants.ZusammenfassungDas Verhältnis zwischen der Transferabilität von Enterotoxinplasmiden und simultaner Übertragung von AntibiotikaresistenzplasmidenDefinierte Donoren von Enterotoxinplasmiden wurden verwendet für Versuche das Verhältnis zwischen der Transferabilität von Ent‐Plasmiden und der simultanen Übertragung von Antibiotikaresistenz zu bestimmen. Die Übertragung der Ent‐Plasmide von R‐Plasmide aufweisenden E. coli war nicht festzustellen, wenn arzneimittelresistente Transkonjuganten selektiert wurden. Der Zusatz einer unterschwelligen Hemmkonzentration eines Antibiotikums zu der Matingmischung hatte keine erhöhte Bildung von Ent/R‐Plasmid‐tragenden Transkonjuganten zur Folge.RésuméRapport entre le caractère transférable des plasmides d'entérotoxines et la transmission simultanée des plasmides de résistance aux antibiotiqueDes donneurs définis de plamides d'entérotoxines ont été utilisés pou déterminer par des essais le rapport entre la caractère transférable de plasmides d'entérotoxines et la transmission simultanée d'une résistance au: antibiotiques. La transmission de plasmides d'entérotoxines à partir d'E. col présentant des plasmides r n'a pas été démontrée lorsque des transconjugué résistant aux médicaments furent sélectionnés. L'addition d'une sous‐dose d concentration inhibitrice d'un antibiotique au mélange de maturation n'*** provoqué aucune augmentation de la formation de transconjugués porteurs d plasmides entérotoxines‐R.ResumenLa relación entre la transferabilidad de plasmidos enterotoxínicos y la transmisión simultánea de plasmidos resistentes a los antibióticosSe utilizaron donores definidos de plasmidos enterotoxínicos par; determinar en ensayos la relación existente entre la transfer a bilidad de plasmi dos ent y la transmisión simultánea de la resistencia frente a antibióticos. N*** se pudo comprobar la transmisión del plasmide ent de E. coli que presentab*** plasmide R, cuando se seleccionaron transconjugantes fármacorresistentes. L*** adición de una concentración inhibidora subumbral de un antibiótico a l*** mezcla de apareamiento no tenía por resultado formación aumentada algun*** de transconjugados portadores de plasmido ent/R.