Conjugative Transfer Of Antibiotic Resistance Genes Research Articles

Small RNA GadY in Escherichia coli enhances conjugation system of IncP-1 by targeting SdiA

Plasmid-mediated conjugation is a common mechanism for most bacteria to transfer antibiotic resistance genes (ARGs). The conjugative transfer of ARGs is emerging as a major threat to human beings. Although several transfer-related factors are known to regulate this process, small RNAs (sRNAs)-based regulatory roles remain to be clarified. Here, the Hfq-binding sRNA GadY in donor strain Escherichia coli (E. coli) SM10λπ was identified as a new regulator for bacterial conjugation. Two conjugation models established in our previous studies were used, which SM10λπ carrying a chromosomally integrated IncP-1α plasmid RP4 and a mobilizable plasmid pUCP24T served as donor cells, and P. aeruginosa PAO1 or E. coli EC600 as the recipients. GadY was found to promote SM10λπ-PAO1 conjugation by base-pairing with its target mRNA SdiA, an orphan LuxR-type receptor that responds to exogenous N-acylated homoserine lactones (AHLs). However, SM10λπ-EC600 conjugation was not affected due to EC600 lacking AHLs synthase. It indicates that the effects of GadY on conjugation depended on AHLs-SdiA signalling. Further study found GadY bound SdiA to negatively regulate the global RP4 repressors KorA and KorB. When under ciprofloxacin or levofloxacin treatment, GadY expression in donor strain was enhanced, and it positively regulated quinolone-induced SM10λπ-PAO1 conjugation. Thus, our study provides a novel role for sRNA GadY in regulating plasmid-mediated conjugation, which helps us better understand bacterial conjugation to counter antibiotic resistance.

Chlorite and bromate alter the conjugative transfer of antibiotic resistance genes: Co-regulation of oxidative stress and energy supply

The widespread use of disinfectants during the global response to the 2019 coronavirus pandemic has increased the co-occurrence of disinfection byproducts (DBPs) and antibiotic resistance genes (ARGs). Although DBPs pose major threats to public health globally, there is limited knowledge regarding their biological effects on ARGs. This study aimed to investigate the effects of two inorganic DBPs (chlorite and bromate) on the conjugative transfer of RP4 plasmid among Escherichia coli strains at environmentally relevant concentrations. Interestingly, the frequency of conjugative transfer was initially inhibited when the exposure time to chlorite or bromate was less than 24 h. However, this inhibition transformed into promotion when the exposure time was extended to 36 h. Short exposures to chlorite or bromate were shown to impede the electron transport chain, resulting in an ATP shortage and subsequently inhibiting conjugative transfer. Consequently, this stimulates the overproduction of reactive oxygen species (ROS) and activation of the SOS response. Upon prolonged exposure, the resurgent energy supply promoted conjugative transfer. These findings offer novel and valuable insights into the effects of environmentally relevant concentrations of inorganic DBPs on the conjugative transfer of ARGs, thereby providing a theoretical basis for the management of DBPs.

Per/polyfluoroalkyl substances modulate plasmid transfer of antibiotic resistance genes: A balance between oxidative stress and energy support

Emerging contaminants can accelerate the transmission of antibiotic resistance genes (ARGs) from environmental bacteria to human pathogens via plasmid conjugation, posing a great challenge to the public health. Although the toxic effects of per/polyfluoroalkyl substances (PFAS) as persistent organic pollutants have been understood, it is still unclear whether and how PFAS modulate the transmission of ARGs. In this study, we for the first time reported that perfluorooctanoic acid (PFOA), perfluorododecanoic acid (PFDoA) and ammonium perfluoro (2-methyl-3-oxahexanoate) (GenX) at relatively low concentrations (0.01, 0.1 mg/L) promoted the conjugative transfer of plasmid RP4 within Escherichia coli, while the plasmid conjugation was inhibited by PFOA, PFDoA and GenX at relatively high concentrations (1, 10 mg/L). The non-unidirectional conjugation result was ascribed to the co-regulation of ROS overproduction, enhanced cell membrane permeability, shortage of energy support as well as l-arginine pool depletion. Taking the well-known PFOA as an example, it significantly enhanced the conjugation frequency by 1.4 and 3.4 times at relatively low concentrations (0.01, 0.1 mg/L), respectively. Exposure to PFOA resulted in enhanced cell membrane permeability and ROS overproduction in donor cells. At high concentrations of PFOA (1, 10 mg/L), although enhanced oxidative stress and cell membrane permeability still occurred, the ATP contents in E. coli decreased, which contributed to the inhibited conjugation. Transcriptome analysis further showed that the expression levels of genes related to arginine biosynthesis (argA, argC, argF, argG, argI) and transport (artJ, artM, artQ) pathways were significantly increased. Intracellular l-arginine concentration deficiency were observed at high concentrations of PFOA. With the supplementary exogenous arginine, it was demonstrated that arginine upregulated conjugation transfer- related genes (trfAp, trbBp) and restores the cell number of transconjugants in PFOA-treated group. Therefore, the inhibited conjugation at high concentrations PFOA were attributed to the shortage of ATP and the depletion of L-arginine pool. These findings provide important insights into the effect environmental concentrations of PFAS on the conjugative transfer of ARGs, and update the regulation mechanism of plasmid conjugation, which is critical for the management of antibiotic resistance in aquatic environments.

Biochar Effectively Inhibits the Horizontal Transfer of Antibiotic Resistance Genes via Restraining the Energy Supply for Conjugative Plasmid Transfer.

Horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) through plasmid-mediated conjugation poses a major threat to global public health. Biochar, a widely used environmental remediation material, has remarkable impacts on the fate of ARGs. However, although biochar was reported being able to inhibit the HGT of ARGs via conjugation and transformation, little is known about the intracellular process that mediates the inhibition effects. On the other hand, as typical natural organic matter, fulvic acid is a common environmental influencer, and how it interferes with the effect of biochar on the HGT of ARGs is unknown. Therefore, this study investigated the effects on the conjugative transfer of ARGs between Escherichia coli MG1655 and E. coli HB101 carrying plasmid RP4, with biochars pyrolyzed at three temperatures and with the corresponding biochars coating with fulvic acid. Results showed that biochar with higher pyrolyzed temperature had a more substantial inhibitory effect on the conjugative transfer of the RP4 plasmid. The inhibitory effect of biochar was mainly attributed to (i) down-regulation of plasmid transfer gene expression, including the formation of conjugative transfer channel and plasmid replication, due to restrained adenosine triphosphate (ATP) energy supply and (ii) decreased cell membrane permeability. Conversely, the fulvic acid coating diminished this inhibition effect of biochar, mainly by providing more ATP and strengthening intracellular reactive oxygen species (ROS) defense. Our findings shed light on the intracellular process that mediates the effects of biochar on the conjugative transfer of ARGs, which would provide support for using biochar to reduce the spread of ARGs.

Presence of bromide and iodide promotes the horizontal transfer of antibiotic resistance genes during chlorination: A preliminary study

Chlorination was reported to have a great potential to increase horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs), which poses a great threat to global human health. Bromide (Br−) and iodide (I−) ions are widely spread ions in water and wastewater. In chlorination, Br− and I− can be oxidized to active bromine and iodine species. The influence of the co-existing different halogen oxidants (chlorine + bromine or iodine species) on HGT of ARGs were rarely investigated. In this study, the conjugative transfer of ARGs between a donor strain E. coli K12 and a recipient strain E. coli HB101 was investigated in chlorination without/with the presence of Br− or I−. Immediately after the addition of sodium hypochlorite, 53–88 % of the dosed chlorine was rapidly consumed, 10 %–42 % fast transformed into organic combined chloramines, and only low levels of free chlorine (0.02–0.8 mg/L as Cl2) left in the diluted cultural medium. Conjugative transfer mediated by the RP4 plasmid was not significantly enhanced in chlorination without the presence of Br− or I−. With the presence of Br− (0.5–5.0 mg/L) or I− (0.05–0.5 mg/L) in chlorination, the co-existing free halogen oxidants and their organic combined ones up-regulated the mRNA expression of the oxidative stress-regulatory gene (rpoS), outer membrane protein gene (ompC), and conjugation-relevant genes (trbBp and trfAp), and caused more damage to cell entirety. As a result, the co-existing reactive halogen oxidants enhanced the HGT of ARGs probably via conjugative transfer and transformation. This study showed that the presence of Br− and I− of common levels in aquatic environment promoted HGT of ARGs in chlorination, thus accelerating the transmission and prevalence of ARGs.

Biological Mitigation of Antibiotic Resistance Gene Dissemination by Antioxidant-Producing Microorganisms in Activated Sludge Systems.

Antibiotic resistance is the principal mechanism of an evergrowing bacterial threat. Antibiotic residues in the environment are a major contributor to the spread of antibiotic resistance genes (ARGs). Subinhibitory concentrations of antibiotics cause bacteria to produce reactive oxygen species (ROS), which can lead to mutagenesis and horizontal gene transfer (HGT) of ARGs; however, little is known about the mitigation of ARG dissemination through ROS removal by antioxidants. In this study, we examine how antioxidant-producing microorganisms inoculated in replicate activated sludge systems can biologically mitigate the dissemination of ARGs. Through quantitative polymerase chain reaction (qPCR), we showed that antioxidant-producing microorganisms could decrease the persistence of the RP4 plasmid and alleviate enrichment of ARGs (sul1) and class 1 integrons (intl1). Metagenomic sequencing identified the most diverse resistome and the most mutated Escherichia coli ARGs in the reactor that contained antibiotics but no antioxidant-producing microorganisms, suggesting that antioxidant-producing microorganisms mitigated ARG enrichment and mutation. Host classification revealed that antioxidant-producing microorganisms decreased the diversity of ARG hosts by shaping the microbial community through competition and functional pathway changes. Conjugative experiments demonstrated that conjugative transfer of ARGs could be mitigated by coculture with antioxidant-producing microorganisms. Overall, this is a novel study that shows how ARG enrichment and HGT can be mitigated through bioaugmentation with antioxidant-producing microorganisms.

Insights into conjugative transfer of antibiotic resistance genes affected by soil minerals

AbstractWidespread antibiotic resistance genes (ARGs) have caused critical threats to public health on a global scale. Soil, composed mainly of minerals, acts as a source of resistance determinants, playing a considerable role in the development and dissemination of ARGs. The great abundance of ARGs in the soil environment raises concern about the effect of minerals on the spread of ARGs. Herein, the horizontal transfer of a model plasmid pMP2463 containing ARGs from Escherichia coli S17‐1 to Pseudomonas putida KT2440 was monitored following exposure to four common soil minerals, namely, kaolinite, montmorillonite, goethite and birnessite. Birnessite resulted in concentration‐dependent increases in conjugative transfer of plasmid pMP2463 by 1.3–4.3 fold, compared with that in the control group. However, no obvious laws were found in the change of conjugative transfer rate at different concentrations of kaolinite and montmorillonite. As for goethite, the conjugative transfer rate increased firstly and then decreased as the concentration increased. The possible mechanisms underlying birnessite‐induced conjugative transfer of plasmid were explored; birnessite is capable of initiating intracellular reactive oxygen species (ROS) formation, inducing the oxidative stress response. Additionally, birnessite notably facilitated the mRNA expression of the outer membrane protein genes, which contributed to cellular membrane pore formation and horizontal gene transfer, and altered the mRNA expression of conjugative‐related genes that are responsible for conjugative transfer of mobile genetic elements between bacteria. This study triggers questions regarding the potential role of soil minerals in the global dissemination of antimicrobial resistance and provides insights into the interactions between bacteria and minerals in the natural soil environment.Highlights The effect of soil minerals on conjugative transfer of ARGs was explored. Birnessite induced the concentration‐dependent increase of conjugative transfer. Mechanisms underlying ROS formation, membrane permeability increase, gene expression alteration were revealed. Our results provide evidence for the effect of birnessite on antibiotic resistance dissemination.

Exposure to Al2O3 nanoparticles facilitates conjugative transfer of antibiotic resistance genes from Escherichia coli to Streptomyces

The spread of antibiotic resistance genes (ARGs) has become a global environmental issue; it has been found that nanoparticles (NPs) can promote the transfer of ARGs between bacteria. However, it remains unclear whether NPs can affect this kind of conjugation in Streptomyces, which mainly conjugate with other bacteria via spores. In the present study, we demonstrated that Al2O3 NPs significantly promote the conjugative transfer of ARGs from Escherichia coli (E. coli) ET12567 to Streptomyces coelicolor (S. coelicolor) M145 without the use of heat shock method. The number of transconjugants induced by Al2O3 particles was associated with the size and concentration of Al2O3 particles, exposure time, and the ratio of E. coli and spores. When nanoparticle size was 30 nm at a concentration of 10 mg/L, the conjugation efficiency reached a peak value of 182 cfu/108 spores, which was more than 60-fold higher than that of the control. Compared with nanomaterials, bulk particles exhibited no significant effect on conjugation efficiency. We also explored the mechanisms by which NPs promote conjugative transfer. After the addition of NPs, the intracellular ROS content increased and the expression of the classical porin gene ompC was stimulated. In addition, ROS enhanced the mRNA expression levels of conjugative genes by inhibiting global regulation genes. Meanwhile, expression of the conjugation-related gene intA was also stimulated, ultimately increasing the number of transconjugants. Our results indicated that Al2O3 NPs significantly promoted the conjugative transfer of ARGs from bacteria to spores and aggravated the diffusion of resistance genes in the environment.

Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera

Antibiotic resistance is a worldwide public health concern. Conjugative transfer between closely related strains or species of bacteria is an important method for the horizontal transfer of multidrug-resistance genes. The extent to which nanomaterials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic-resistance genes in bacteria, especially across genera, is still unknown. Here we show that nanomaterials in water can significantly promote the horizontal conjugative transfer of multidrug-resistance genes mediated by the RP4, RK2, and pCF10 plasmids. Nanoalumina can promote the conjugative transfer of the RP4 plasmid from Escherichia coli to Salmonella spp. by up to 200-fold compared with untreated cells. We also explored the mechanisms behind this phenomenon and demonstrate that nanoalumina is able to induce oxidative stress, damage bacterial cell membranes, enhance the expression of mating pair formation genes and DNA transfer and replication genes, and depress the expression of global regulatory genes that regulate the conjugative transfer of RP4. These findings are important in assessing the risk of nanomaterials to the environment, particularly from water and wastewater treatment systems, and in the estimation of the effect of manufacture and use of nanomaterials on the environment.