Frequency Of Conjugative Transfer Research Articles

Inhibited conjugative transfer of antibiotic resistance genes in antibiotic resistant bacteria by surface plasma

Antibiotic resistant bacteria (ARB) and resistance genes (ARGs) are emerging environmental pollutants with strong pathogenicity. In this study, surface plasma was developed to inactivate the donor ARB with Escherichia coli (AR E. coli) as a model, eliminate ARGs, and inhibit conjugative transfer of ARGs in water, highlighting the influences of concomitant inorganic ions. Surface plasma oxidation significantly inactivated AR E. coli, eliminated ARGs, and inhibited conjugative transfer of ARGs, and the presence of NO3−, Cu2+, and Fe2+ all promoted these processes, and SO42− did not have distinct effect. Approximately 4.5log AR E. coli was inactivated within 10 min treatment, and it increased to 7.4log AR E. coli after adding Fe2+. Integrons intI1 decreased by 3.10log (without Fe2+) and 4.43log (adding Fe2+); the addition of Fe2+ in the surface plasma induced 99.8% decline in the conjugative transfer frequency. The inhibition effects on the conjugative transfer of ARGs were mainly attributed to the reduced reactive oxygen species levels, decreased DNA damage-induced response, decreased intercellular contact, and down-regulated expression of plasmid transfer genes. This study disclosed underlying mechanisms for inhibiting ARGs transfer, and supplied a prospective technique for ARGs control.

Hormetic dose responses induced by antibiotics in bacteria: A phantom menace to be thoroughly evaluated to address the environmental risk and tackle the antibiotic resistance phenomenon

The environmental contamination of antibiotics caused by their over or inappropriate use is a major issue for environmental and human health since it can adversely impact the ecosystems and promote the antimicrobial resistance. Indeed, considering that in the environmental matrices these drugs are present at low levels, the possibility that bacteria exhibit a hormetic response to increase their resilience when exposed to antibiotic subinhibitory concentrations might represent a serious threat. Information reported in this review showed that exposure to different types of antibiotics, either administered individually or in mixtures, is capable of exerting hormetic effects on bacteria at environmentally relevant concentrations. These responses have been reported regardless of the type of bacterium or antibiotic, thus suggesting that hormesis would be a generalized adaptive mechanism implemented by bacteria to strengthen their resistance to antibiotics. Hormetic effects included growth, bioluminescence and motility of bacteria, their ability to produce biofilm, but also the frequency of mutation and plasmid conjugative transfer. The evaluation of quantitative features of antibiotic-induced hormesis showed that these responses have both maximum stimulation and dose width characteristics similar to those already reported in the literature for other stressors. Notably, mixtures comprising individual antibiotic inducing stimulatory responses might have distinct combined effects based on antagonistic, synergistic or additive interactions between components. Regarding the molecular mechanisms of action underlying the aforementioned effects, we put forward the hypothesis that the adoption of adaptive/defensive responses would be driven by the ability of antibiotic low doses to modulate the transcriptional activity of bacteria. Overall, our findings suggest that hormesis plays a pivotal role in affecting the bacterial behavior in order to acquire a survival advantage. Therefore, a proactive and effective risk assessment should necessarily take due account of the hormesis concept to adequately evaluate the risks to ecosystems and human health posed by antibiotic environmental contamination.

Two novel blaNDM-1-harbouring transposons on pPrY2001-like plasmids coexisting with a novel cfr-encoding plasmid in food animal source Enterobacteriaceae

This study reports identification of the carbapenemase-encoding gene from carbapenem-resistant Enterobacterales from food animals. A total of 40 bacterial isolates recovered from 475 faecal swabs obtained on one farm were tested for the presence of the blaNDM-1 gene by PCR. Species identification of three blaNDM-1-positive strains was conducted by MALDI-TOF/MS. Antimicrobial susceptibility testing was performed by broth microdilution. Transferability of the blaNDM-1 and cfr genes was determined by filter mating. The genetic environment of blaNDM-1 and cfr was analysed by whole-genome sequencing. Two Proteus mirabilis (JPM24 and YPM35) and one Providencia rettgeri (YPR25) carried blaNDM-1. The blaNDM-1 genes were located on conjugable pPrY2001-like plasmids often reported to carry important antimicrobial resistance genes (ARGs). YPR25 and YPM35 shared two almost identical conjugable plasmids, one carrying blaNDM-1 and the other cfr. The blaNDM-1 gene in YPR25 (same as YPM35) and JPM24 was located in two novel transposons, designated Tn6922 and Tn6923, respectively. Tn6922 and Tn6923 carried 14 and 7 ARGs, respectively, and both contained multiple copies of IS26 in the same direction, with a high degree of similarity. Additionally, cfr was located on a plasmid with an unreported high frequency of conjugative transfer in YPR25 (same as YPM35). We identified two novel blaNDM-1-containing transposons (Tn6922 and Tn6923) present on pPrY2001-like plasmids. The pPrY2001-like blaNDM-1 plasmids coexisted with a novel cfr plasmid, and both could transfer at high frequency, highlighting the importance of continuous surveillance of multiresistant Enterobacterales of animal origin that can serve as a reservoir for ARGs.

Effect of dissolved biochar on the transfer of antibiotic resistance genes between bacteria

The spread of antibiotic resistance genes (ARGs) is a global environmental issue. Dissolved biochar is more likely to contact bacteria in water, producing ecological risks. This study explored the effects of dissolved biochar on ARGs transfer in bacteria. Conjugative transfer efficiency was significantly different following treatment with different types of dissolved biochar. Typically, humic acid-like substance in dissolved biochar can significantly improve the transfer efficiency of ARGs between bacteria. When the concentration of dissolved biochar was ≤10 mg biochar/mL, humic acid-like substance substantially promoted ARGs transfer. An increase in dissolved biochar concentration weakened the ARGs transfer from humic acid-like substance. The inhibitory effects of small-molecule matters dominated, decreasing conjugative transfer frequency. At a concentration of 100 mg biochar/mL, the conjugative transfer efficiency of all treatments was lower than that of control. Compared with corn straw dissolved biochar, there were more transconjugants in pine sawdust dissolved biochar. Following treatment with 10 mg biochar/mL pine sawdust dissolved biochar, the number of transconjugants was at its maximum; approximately 7.3 folds higher than the control. We also explored mechanisms by which dissolved biochar impacts conjugative transfer. Due to the complex composition of dissolved biochar, its effects on the expression of conjugative transfer-related genes were also dynamic. This study investigates the ecological risk of biochar and guides its scientific application.

Plasma induced efficient removal of antibiotic-resistant Escherichia coli and antibiotic resistance genes, and inhibition of gene transfer by conjugation

Antibiotic-resistant bacteria (ARB) and their resistance genes (ARGs) are emerging environmental pollutants that pose great threats to human health. In this study, a novel strategy using plasma was developed to simultaneously remove antibiotic-resistant Escherichia coli (AR bio-56954 E. coli) and its ARGs, aiming to inhibit gene transfer by conjugation. Approximately 6.6 log AR bio-56954 E. coli was inactivated within 10 min plasma treatment, and the antibiotic resistance to tested antibiotics (tetracycline, gentamicin, and amoxicillin) significantly decreased. Reactive oxygen and nitrogen species (RONS) including •OH, 1O2, O2•−, NO2−, and NO3− contributed to ARB and ARGs elimination; their attacks led to destruction of cell membrane, accumulation of excessive intracellular reactive oxygen substances, deterioration of conformational structures of proteins, and destroy of nucleotide bases of DNA. As a result, the ARGs (tet(C), tet(W), blaTEM-1, aac(3)-II), and integron gene intI1), and conjugative transfer frequency of ARGs significantly decreased after plasma treatment. The results demonstrated that plasma has great prospective application in removing ARB and ARGs in water, inhibiting gene transfer by conjugation.

Comparative genomic analyses of β-lactamase (blaCMY-42)-encoding plasmids isolated from wastewater treatment plants in Canada.

Wastewater treatment plants (WWTPs) are useful environments for investigating the occurrence, diversity, and evolution of plasmids encoding clinically relevant antibiotic resistance genes (ARGs). Our objective was to isolate and sequence plasmids encoding meropenem resistance from bacterial hosts within Canadian WWTPs. We used two enrichment culture approaches for primary plasmid isolation, followed by screening for antibiotic resistance, conjugative mobility, and stability in enteric bacteria. Isolated plasmids were sequenced using Illumina MiSeq and Sanger sequencing methods. Bioinformatics analyses resolved a multi-resistance IncF/MOBF12 plasmid, pFEMG (209 357 bp), harbouring resistance genes to β-lactam (blaCMY-42, blaTEM-1β, and blaNDM-5), macrolide (mphA-mrx-mphR), tetracycline (tetR-tetB-tetC-tetD), trimethoprim (dfrA12), aminoglycoside (aadA2), and sulfonamide (sul1) antibiotic classes. We also isolated an IncI1/MOBP12 plasmid pPIMR (172 280 bp) carrying similar β-lactamase and a small multi-drug efflux resistance gene cluster (blaCMY-42-blc-sugE) to pFEMG. The co-occurrence of different ARGs within a single 24 552 bp cluster in pFEMG - interspersed with transposons, insertion sequence elements, and a class 1 integron - may be of significant interest to human and veterinary medicine. Additionally, the presence of conjugative and plasmid maintenance genes in the studied plasmids corresponded to observed high conjugative transfer frequencies and stable maintenance. Extensive investigation is required to further understand the fitness trade-offs of plasmids with different types of conjugative transfer and maintenance modules.

Hormetic dose-responses for silver antibacterial compounds, quorum sensing inhibitors, and their binary mixtures on bacterial resistance of Escherichia coli

Silver antibacterial compounds (SACs) and quorum sensing inhibitors (QSIs), as the potential antibiotic substitutes, have been recommended to prevent and treat microbial infections for the purpose of controlling the increasingly serious bacterial resistance induced by the abuse of antibiotics. However, there is little information regarding the resistance risk of these compounds, especially their mixtures. In this study, bacterial mutation and RP4 plasmid conjugative transfer among bacteria were used to characterize the bacterial endogenous and exogenous resistance, respectively. The effects of SACs (including silver nitrate (AgNO3) and silver nanoparticle (AgNP)), QSIs, and their binary mixtures on the bacterial resistance were investigated via setting the frequency of mutation and conjugative transfer in Escherichia coli (E. coli) as the test endpoints. The results indicated that these two endpoints exhibited hormetic dose-responses to each treatment. Furthermore, the joint resistance actions between SACs and QSIs were all judged to be antagonism. Correlation analysis suggested that the promotion of the bacterial resistance in each treatment was closely related to its toxicity. It was speculated that AgNO3 and AgNP might both release Ag+ ions to facilitate the E. coli resistance, while QSIs probably acted on LsrR and SdiA proteins to stimulate the bacterial mutation and accelerate the RP4 plasmid conjugative transfer, respectively. These findings imply that the bacteria may generate targeted stress response to the survival pressure from environmental compounds, displaying hormetic phenomenon in resistance-related test endpoints. This study provides a new insight into the resistance risk induced by SACs and QSIs, benefiting the environmental risk assessment of these compounds from the perspective of bacterial resistance.

Co-effect of cadmium and iron oxide nanoparticles on plasmid-mediated conjugative transfer of antibiotic resistance genes

Conjunctive transfer of antibiotic resistance genes (ARGs) among bacteria driven by plasmids facilitated the evolution and spread of antibiotic resistance. Heavy metal exposure accelerated the plasmid-mediated conjunctive transfer of ARGs. Nanomaterials are well-known adsorbents for heavy metals removal, with the capability of combatting resistant bacteria/facilitating conjunctive transfer of ARGs. However, co-effect of heavy metals and nanomaterials on plasmid-mediated conjunctive transfer of ARGs was still unknown. In this study, we investigated the effect of the simultaneous exposure of Cd2+ and nano Fe2O3 on conjugative transfer of plasmid RP4 from Pseudomonas putida KT2442 to water microbial community. The permeability of bacterial cell membranes, antioxidant enzyme activities and conjugation gene expression were also investigated. The results suggested that the combination of Cd2+ and high concentration nano Fe2O3 (10 mg/L and 100 mg/L) significantly increased conjugative transfer frequencies of RP4 plasmid (p < 0.05). The most transconjugants were detected in the treatment of co-exposure to Cd2+ and nano Fe2O3, the majority of which were identified to be human pathogens. The mechanisms of the exacerbated conjugative transfer of ARGs were involved in the enhancement of cell membrane permeability, antioxidant enzyme activities, and mRNA expression levels of the conjugation genes by the co-effect of Cd2+ and nano Fe2O3. This study confirmed that the simultaneous exposure to Cd2+and nano Fe2O3 exerted a synergetic co-effect on plasmid-mediated conjunctive transfer of ARGs, emphasizing that the co-effect of nanomaterials and heavy metals should be prudently evaluated when combating antibiotic resistance.

Can photocatalytic technology facilitate conjugative transfer of ARGs in bacteria at the interface of natural sphalerite under different light irradiation?

Antibiotic-resistance genes (ARGs) dissemination in water environment has raised concerns worldwide. Here, antibiotic-resistant bacteria (ARB) were exposed to the interface of natural sphalerite (NS) irradiated by different light sources (UV254nm, UV365nm, simulated sunlight (SS), and visible light (VL)) in water and their effects on ARG conjugative transfer were studied. The conjugative transfer frequency increased 2- to 10-fold under exposure to Photocatalysis (PC)NS+UV254nm or PCNS+VL and 1- to 2-fold under PCNS+UV365nm or PCNS+SS. During exposure, bacteria were directly stimulated by light irradiation or spontaneous photocatalytic inactivation, triggering oxidative stress, DNA repair, cell repair and transfer regulation of ARB. These results indicated that ARG transfer could be promoted once ARB involving in this environmental PC interface. This new viewpoint revealed that natural minerals with natural light irradiation play an important role in ARG dissemination. Besides, this study provided more detailed information for practical application of PC sterilization and control of ARG transfer.

Impact of Redox Conditions on Antibiotic Resistance Conjugative Gene Transfer Frequency and Plasmid Fate in Wastewater Ecosystems.

Wastewater is a common pathway for the spread of antibiotic resistance (AR) genes and bacteria into the environment. Biological treatment can mitigate this path, but horizontal gene transfer (HGT) between bacteria also occurs in such processes, although the influence of bioreactor habitat and ecology on HGT frequency is not well understood. Here, we quantified how oxidation-reduction (redox) conditions impact the fate of a Green fluorescent protein (Gfp)-tagged AR plasmid (pRP4-gfp) within an E. coli host (EcoFJ1) in the liquid phase and biofilms in bioreactors. Replicate reactors treating domestic wastewater were operated under stable aerobic (+195 ± 25 mV), anoxic (-15 ± 50 mV), and anaerobic (-195 ± 15 mV) conditions, and flow cytometry and selective plating were used to quantify donor strain, EcoFJ1(pRP4-gfp), and putative transconjugants over time. Plasmid pRP4-gfp-bearing cells disappeared rapidly in aerobic ecosystems (∼2.0 log reduction after 72 h), especially in the liquid phase. In contrast, EcoFJ1(pRP4-gfp) and putative transconjugants persisted much longer in anaerobic biofilms (∼1.0 log reduction, after 72 h). Plasmid transfer frequencies were also higher under anaerobic conditions. In parallel, protozoan abundances were over 20 times higher in aerobic reactors relative to anaerobic reactors, and protozoa numbers significantly inversely correlated with pRP4-gfp signals across all reactors (p < 0.05). Taken together, observed HGT frequency and plasmid retention are impacted by habitat conditions and trophic effects, especially oxygen conditions and apparent predation. New aerobic bioreactor designs are needed, ideally employing passive aeration to save energy, to minimize resistance HGT in biological wastewater treatment processes.

Effect of Biological Powdered Activated Carbon on Horizontal Transfer of Antibiotic Resistance Genes in Secondary Effluent

Antibiotic resistance genes (ARGs), as emerging environmental contaminants, have become a threat to human health. Studies have shown that secondary effluent from wastewater treatment plants was an important source of ARGs, and the horizontal transfer of ARGs that took place in secondary effluent has increased its pollution to the water environment; the conjugative transfer is the main form of horizontal transfer. Powdered activated carbon was widely used in advanced wastewater treatment, it would gradually be converted into biological powdered activated carbon (BPAC) during long-term use, there was still no related research on whether the microorganisms on BPAC could promote horizontal transfer of ARGs. In this article, the system between S17-1 lamp pir Escherichia coli and MG1655-chl E. coli was constructed to explore the effect of BPAC on the conjugative transfer of resistance plasmid pHS-AVC-LW1144. The main results were as follows: the conjugative transfer of resistance plasmid could take place without and with BPAC, the effect was better with added BPAC, and the BPAC effect of promoting the conjugative transfer was strongest at 20 mg/L. When incubation time was 24 h and the BPAC dosage was 20 mg/L, the zygote concentrations and conjugative transfer frequency were the highest. The zygote concentrations were higher at low temperature (4°C) than normal (25°C) and high temperature (50°C) conditions. The zygote concentrations under alkaline (pH = 9) were higher than acidic (pH = 5) and neutral (pH = 7). Therefore, within a certain range, the longer the incubation time, the better the effect of promoting the conjugative transfer of resistance plasmid, and in low temperature and alkaline conditions, the effect of promoting the conjugative transfer were also better. Based on this study, we hoped to provide basic support for advanced treatment of reclaimed water and risk control of ARGs.

New parameters for the quantitative assessment of the proliferation of antibiotic resistance genes dynamic in the environment and its application: A case of sulfonamides and sulfonamide resistance genes

Antibiotic resistance genes (ARGs) have been widely detected around the world and are generally viewed as emerging pollutants with environmental persistence. The proliferation of ARGs can be easily promoted by antibiotics. However, the dynamics of ARGs in the environment are still unable to be quantified using a single parameter, which is vital to evaluating the ability of ARGs to spread by antibiotics and effectively controlling ARGs. A new parameter, termed the relative area ratio of sample to control (ΔAR), was developed based on the quantitative features determined by ARG-time curves in soils contaminated with sulfonamides (SAs) and verified by quantitative structure-activity relationships (QSARs) models. The results showed that ΔAR can not only be used to accurately quantify the characteristics of SAs resistance genes (Suls) over time but also be applied to reveal the relationships between the proliferation of Suls and important factors (i.e., concentrations and chemical structures). Moreover, the ΔAR-based QSARs model indicated that bioavailability and the frequency of conjugative transfer, rather than the ability of induced mutations in bacteria, tend to be key processes of the characteristics of the proliferation of Suls. Therefore, ΔAR is a useful parameter to perform environmental risk assessments of ARG proliferation in the environment.

Natural sphalerite nanoparticles can accelerate horizontal transfer of plasmid-mediated antibiotic-resistance genes

Minerals and microorganisms are integral parts of natural environments, and they inevitably interact. Antibiotic-resistance genes (ARGs) significantly threaten modern healthcare. However, the effects of natural minerals on ARG propagation in aquatic systems are not fully understood. The present work studied the effects of natural sphalerite (NS) nanoparticles on the horizontal transfer of ARGs from Escherichia coli DH5α (CTX) (donor) to E. coli C600 (Sm) (recipient), and from E. coli DH5α (MCR) (donor) to E. coli C600 (Sm), and their underlying mechanisms. NS particles (0.5–50 mg L−1) induced an NS-concentration-dependent increase in conjugative transfer frequency. The underlying mechanisms associated with the facilitated ARG transfer included the production of intracellular reactive oxygen species, the SOS response, changes in bacterial cell morphology, and alteration of mRNA levels of bacterial cell membrane protein-related genes and genes associated with conjugative ARG transfer. The information herein offers new mechanistic understanding of risks of bacterial resistance resulting from NS.

Inheritance of IncP-9 Catabolic Plasmids in Pseudomonas Bacteria

This work investigated the capability of the conjugative transfer of epsilon-caprolactam biodegradation (CAP) plasmids, belonging to incompatibility (Inc) groups P-2, P-7, P-9, and IncP-9 naphthalene biodegradation (NAH) plasmids to various species of Pseudomonas bacteria, including strains of plant growth-promoting rhizobacteria (PGPR), and the character of plasmid inheritance in these strains. The frequency of the conjugative transfer of all CAP and NAH plasmids (irrespective of their Inc groups) into Pseudomonas PGPR was shown to be lower than that into P. putida soil strains by more than an order of magnitude. The low frequency of conjugative transfer was found not to be caused by secretion of antibiotically active metabolites by Pseudomonas PGPR strains. Stability of inheritance of catabolic plasmids under nonselective conditions was established to depend on their Inc group and bacterial host strain. Thus, all investigated plasmids were stably maintained in P. putida KT2442 and BS394 strains for at least 100 generations. However, only CAP plasmids of IncP-2 and IncP-7 groups were stably maintained in Pseudomonas PGPR cells. CAP and NAH plasmids of different IncP-9 subgroups were eliminated from cells of rhizosphere pseudomonads within several generations. Thus, the work first revealed inheritance features of IncP-9 catabolic plasmids in Pseudomonas PGPR cells.

The effect and mechanism of urban fine particulate matter (PM2.5) on horizontal transfer of plasmid-mediated antimicrobial resistance genes

Fine particulate matter (PM2.5) and antimicrobial resistance are two major threats to public health worldwide. Current air pollution studies rely heavily on the assessment of PM2.5 chemistry and toxicity. However, whether and how PM2.5 affects the proliferation and transfer of antimicrobial resistance genes (ARGs) in various environments has remained unanswered. This study investigated the effects and potential mechanisms of urban PM2.5 on the horizontal transfer of ARGs between opportunistic Escherichia coli (E. coli) strains. The results showed that urban PM2.5 samples collected from Xi'an (XA), Shanghai (SH), and Shijiazhuang (SJZ) in China induced location- and concentration-dependent promotion of conjugative transfer frequencies compared to the control group. The relevant mechanisms were also explored, including the formation of intracellular reactive oxygen species (ROS) and the subsequent induction of oxidative stress, SOS response, changes in membrane permeability, and alternations in mRNA expression of genes involved in horizontal transfer. This study highlights the effect of PM2.5 on promoting the horizontal transfer of ARGs and elucidates the mechanism of the antimicrobial-resistance risks posed by urban PM2.5. These findings are of great value in understanding the transmission of antimicrobial resistance in various environments and provide valuable information for re-evaluating air quality assessment practices.

QSAR-based investigation on antibiotics facilitating emergence and dissemination of antibiotic resistance genes: A case study of sulfonamides against mutation and conjugative transfer in Escherichia coli

Antibiotic resistance genes (ARGs), which are emerging environmental contaminants, have posed great threats to global public health. Although extensive efforts have been undertaken to investigate ARG pollution, little attention has been paid to the structural information of antibiotics when exploring their impact on the emergence and dissemination of ARGs. In this study, setting Escherichia coli (E. coli) as the test organism, the effects of sulfonamides (SAs) on growth, mutation frequency and conjugative transfer frequency were tested, and quantitative structure-activity relationship (QSAR) was used to quantitatively analyze the promotion of SAs on these biological effects and explore their possible mechanism. The constructed QSAR models reveal that SAs may increase expression of the FtsZ protein and pili in E. coli via binding to the SdiA protein, ultimately leading to SAs facilitation of growth, mutation frequency and conjugative transfer frequency. The results indicate that SAs can produce selective pressure on E. coli to promote the emergence and dissemination of ARGs. This study provides reference data for further investigation of the emergence and dissemination of ARGs under antibiotic exposure and a new perspective for the mechanistic exploration of ARG pollution.

Antibiotic resistant bacteria survived from UV disinfection: Safety concerns on genes dissemination

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are the emerging contaminants leading to a serious worldwide health problem. Although disinfection like ultraviolet (UV) irradiation could remove part of ARB and ARGs, there still are residual ARB and ARGs in the effluent of wastewater treatment plants.Conjugative transfer is main concern of the risk of ARGs and little is known about the effects of UV disinfection on the transfer ability of the non-inactivated ARB in the effluent which will enter the environment. Hence the influences of UV irradiation and reactivation on ARB conjugative transfer ability were studied under laboratory condition, focusing on the survival bacteria from UV irradiation and the reactivated bacteria, as well as their descendants. The experimental results imply that even 1 mJ/cm2 UV disinfection can significantly decrease the conjugative transfer frequency of the survival bacteria. However, viable but not culturable state cells induced by UV can reactivate through both photoreactivation and dark repair and retain the same level of transfer ability as the untreated strains. This finding is essential for re-considering about the post safety of UV irradiated effluent and microbial safety control strategies were required.

Antibiotic-resistance gene transfer in antibiotic-resistance bacteria under different light irradiation: Implications from oxidative stress and gene expression

Due to the significant public health risks, there is substantial scientific interest in the increasing abundance of antibiotic-resistance bacteria (ARB) and the spread of antibiotic-resistance genes (ARGs) in aquatic environments. To clearly understand the mechanism of ARG transfer, this study examined the conjugative transfer of genes encoding resistance to cephalosporin (blaCTX) and polymyxin (mcr-1) from two antibiotic-resistant donor strains, namely E. coli DH5α (CTX) and E. coli DH5α (MCR), and to a streptomycin-resistant receptor strain (E. coli C600 (Sm)). Conjugative transfer was specifically studied under different light irradiation conditions including visible light (VL), simulated sunlight (SS) and ultraviolet light (UV254nm). Results show that the conjugative transfer frequency was not affected by VL irradiation, while it was slightly improved (2–10 fold) by SS irradiation and extremely accelerated (up to 100 fold) by UV irradiation. Furthermore, this study also explored the link between ARG transfer and stress conditions. This was done by studying physiological and biochemical changes; oxidative stress response; and functional gene expression of co-cultured AR-E. coli strains under stress conditions. When correlated with the transfer frequency results, we found that VL irradiation did not affect the physiological and biochemical characteristics of the bacteria, or induce oxidative stress and gene expression. For SS irradiation, oxidative stress occurred slowly, with a slight increase in the expression of target genes in the bacterial cells. In contrast, UV irradiation, rapidly inactivated the bacteria, the degree of oxidative stress was very severe and the expression of the target genes was markedly up-regulated. Our study could provide new insight into the underlying mechanisms and links between accelerated conjugative transfer and oxidative stress, as well as the altered expression of genes relevant to conjugation and other stress responses in bacterial cells.

The first high frequency of recombination-like conjugal transfer from an integrated origin of transfer sequence in Bacillus subtilis 168

Bacillus subtilis 168 was developed as a genome vector to manipulate large DNA fragments. The system is based on the inherent natural transformation (TF) activity. However, DNA size transferred by TF is limited up to approximately 100kb. A conjugal transfer system capable of transferring DNA fragments considerably larger than those transferred by TF was developed. A well-defined oriT110 sequence and a cognate relaxase gene from the pUB110 plasmid were inserted into the xkdE gene of the B. subtilis genome. Transfer of antibiotic resistance markers distant from the oriT110 locus to the recipient B. subtilis occurred only in the presence of pLS20, a helper plasmid that provides a type IV secretion system. Marker transmission was consistent with the orientation of oriT110 and required a recA-proficient recipient. The first conjugal transfer system of genomic DNA should provide a valuable alternative genetic tool for editing the B. subtilis genome.

Instability of the IncFII-Type Plasmid Carrying blaNDM-5 in a Klebsiella pneumoniae Isolate.

In this study, we characterized the blaNDM-5-bearing plasmid in a Klebsiella pneumoniae isolate that had lost the plasmid during serial passage. We determined the complete sequences of the plasmid pCC1410-2, which was extracted from a K. pneumoniae ST709 isolate collected at a Korean hospital from which two NDM-5-producing K. pneumoniae isolates were subsequently isolated. As a result, the pCC1410-2 plasmid had a backbone structure that was similar to those of two plasmids previously reported from the same hospital, but lacked some antibiotic resistance genes (blaTEM-1, rmtB, mphR(A), mrx(A), and mph(A)). A 9-bp repeating unit encoding three amino acids (Gln-Gln-Pro) was inserted in TraD in pCC1410-2. Thus, the pCC1410-2 plasmid might be transferred from the previously identified carbapenem-resistant K. pneumoniae, but some delections and inversions might have occurred during the process. We compared the transfer frequency and stability of the plasmids. The relative frequency of conjugative transfer and stability in the host were significantly lower in pCC1410-2 than in previously reported blaNDM-5-bearing plasmids in Korea. A low transfer frequency and instability in the host may cause underestimation of carbapenemase-producing Enterobacteriaceae in the clinical setting and in surveillance studies.