Concept: Bacterial conjugation
ABSTRACT Horizontal gene transfer (HGT) is largely responsible for increasing the incidence of antibiotic-resistant infections worldwide. While studies have focused on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Escherichia coli, virulent clone ST131, and Klebsiella pneumoniae harboring extended-spectrum-β-lactamase (ESBL) bla(CTX-M-15) and metallo-β-lactamase bla(NDM-1), respectively, exhibited prolonged survival on stainless steel, with approximately 10(4) viable cells remaining from an inoculum of 10(7) CFU per cm(2) after 1 month at 21°C. HGT of bla to an antibiotic-sensitive but azide-resistant recipient E. coli strain occurred on stainless steel dry touch surfaces and in suspension but not on dry copper. The conjugation frequency was approximately 10 to 50 times greater and occurred immediately, and resulting transconjugants were more stable with ESBL E. coli as the donor cell than with K. pneumoniae, but bla(NDM-1) transfer increased with time. Transconjugants also exhibited the same resistance profile as the donor, suggesting multiple gene transfer. Rapid death, inhibition of respiration, and destruction of genomic and plasmid DNA of both pathogens occurred on copper alloys accompanied by a reduction in bla copy number. Naked E. coli DNA degraded on copper at 21°C and 37°C but slowly at 4°C, suggesting a direct role for the metal. Persistence of viable pathogenic bacteria on touch surfaces may not only increase the risk of infection transmission but may also contribute to the spread of antibiotic resistance by HGT. The use of copper alloys as antimicrobial touch surfaces may help reduce infection and HGT. IMPORTANCE Horizontal gene transfer (HGT) conferring resistance to many classes of antimicrobials has resulted in a worldwide epidemic of nosocomial and community infections caused by multidrug-resistant microorganisms, leading to suggestions that we are in effect returning to the preantibiotic era. While studies have focused on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Here we show prolonged (several-week) survival of multidrug-resistant Escherichia coli and Klebsiella pneumoniae on stainless steel surfaces. Plasmid-mediated HGT of β-lactamase genes to an azide-resistant recipient E. coli strain occurred when the donor and recipient cells were mixed together on stainless steel and in suspension but not on copper surfaces. In addition, rapid death of both antibiotic-resistant strains and destruction of plasmid and genomic DNA were observed on copper and copper alloy surfaces, which could be useful in the prevention of infection spread and gene transfer.
Multidrug resistance (MDR) plasmids frequently encode antibiotic resistance genes conferring qualitatively different mechanisms of resistance. We show that the antibiotic concentrations selecting for the RK2 plasmid in Escherichia coli depend upon the sociality of the drug resistance: Selection for a selfish drug resistance (efflux-pump) occurred at very low drug concentrations, just 1.3% of the sensitive’s MIC, whereas selection for a cooperative drug resistance (modifying-enzyme) occurred at drug concentrations exceeding the MIC of the plasmid-free strain.
Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation.
Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.
Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo.
Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, a genome editing technology, was shown to be versatile in treating several antibiotic-resistant bacteria. In the present study, we applied the CRISPR/Cas9 technology to kill extended spectrum beta-lactamase (ESBL)-producing Escherichia coli. ESBL bacteria are mostly multi-drug resistant (MDR), and have plasmid-mediated antibiotic resistance genes that can be easily transferred to other members of the bacterial community by horizontal gene transfer. To restore sensitivity to antibiotics in these bacteria, we searched for a CRISPR/Cas9 target sequence that was conserved among > 1,000 ESBL mutants. There was only one target sequence for each TEM- and SHV-type ESBL, with each of these sequences found in ~200 ESBL strains of each type. Furthermore, we showed that these target sequences can be exploited to re-sensitize MDR cells in which resistance is mediated by genes that are not the target of CRISPR/Cas9 system, but by genes that are present on the same plasmid as target genes. We believe our Re-Sensitization to Antibiotics from Resistance (ReSAFR) technology, which enhances the practical value of the CRISPR/Cas9 system, will be an effective method of treatment against plasmid-carrying MDR bacteria.
Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs.
Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F “sex” pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.
Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs.
In the absence of antibiotic-mediated selection, sensitive bacteria are expected to displace their resistant counterparts if resistance genes are costly. However, many resistance genes persist for long periods in the absence of antibiotics. Horizontal gene transfer (primarily conjugation) could explain this persistence, but it has been suggested that very high conjugation rates would be required. Here, we show that common conjugal plasmids, even when costly, are indeed transferred at sufficiently high rates to be maintained in the absence of antibiotics in Escherichia coli. The notion is applicable to nine plasmids from six major incompatibility groups and mixed populations carrying multiple plasmids. These results suggest that reducing antibiotic use alone is likely insufficient for reversing resistance. Therefore, combining conjugation inhibition and promoting plasmid loss would be an effective strategy to limit conjugation-assisted persistence of antibiotic resistance.