RESUMEN
Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum ß-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum ß-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae. In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16_3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.
Asunto(s)
Antibacterianos , Productos Biológicos , Escherichia coli , Klebsiella pneumoniae , Plásmidos , beta-Lactamasas , Plásmidos/genética , Antibacterianos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Productos Biológicos/farmacología , Farmacorresistencia Bacteriana/genética , Conjugación Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Pruebas de Sensibilidad Microbiana , Microbiología de Alimentos , Transferencia de Gen HorizontalRESUMEN
Horizontal gene transfer plays a key role in the global dissemination of antimicrobial resistance (AMR). AMR genes are often carried on self-transmissible plasmids, which are shared amongst bacteria primarily by conjugation. Antibiotic use has been a well-established driver of the emergence and spread of AMR. However, the impact of commonly used non-antibiotic compounds and environmental pollutants on AMR spread has been largely overlooked. Recent studies found common prescription and over-the-counter drugs, artificial sweeteners, food preservatives, and environmental pollutants, can increase the conjugative transfer of AMR plasmids. The potential mechanisms by which these compounds promote plasmid transmission include increased membrane permeability, upregulation of plasmid transfer genes, formation of reactive oxygen species, and SOS response gene induction. Many questions remain around the impact of most non-antibiotic compounds on AMR plasmid conjugation in clinical isolates and the long-term impact on AMR dissemination. By elucidating the role of routinely used pharmaceuticals, food additives, and pollutants in the dissemination of AMR, action can be taken to mitigate their impact by closely monitoring use and disposal. This review will discuss recent progress on understanding the influence of non-antibiotic compounds on plasmid transmission, the mechanisms by which they promote transfer, and the level of risk they pose.
RESUMEN
BACKGROUND: Resistance nodulation division (RND) family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance. Expression of these pumps is carefully controlled by a network of regulators that respond to different environmental cues. EnvR is a TetR family transcriptional regulator encoded upstream of the RND efflux pump acrEF. METHODS: Binding of EnvR protein upstream of acrAB was determined by electrophoretic mobility shift assays and the phenotypic consequence of envR overexpression on antimicrobial susceptibility, biofilm motility and invasion of eukaryotic cells in vitro was measured. Additionally, the global transcriptome of clinical Salmonella isolates overexpressing envR was determined by RNA-Seq. RESULTS: EnvR bound to the promoter region upstream of the genes coding for the major efflux pump AcrAB in Salmonella, inhibiting transcription and preventing production of AcrAB protein. The phenotype conferred by overexpression of envR mimicked deletion of acrB as it conferred multidrug susceptibility, decreased motility and decreased invasion into intestinal cells in vitro. Importantly, we demonstrate the clinical relevance of this regulatory mechanism because RNA-Seq revealed that a drug-susceptible clinical isolate of Salmonella had low acrB expression even though expression of its major regulator RamA was very high; this was caused by very high EnvR expression. CONCLUSIONS: In summary, we show that EnvR is a potent repressor of acrAB transcription in Salmonella, and can override binding by RamA so preventing MDR to clinically useful drugs. Finding novel tools to increase EnvR expression may form the basis of a new way to prevent or treat MDR infections.
Asunto(s)
Proteínas Bacterianas , Salmonella typhimurium , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Farmacorresistencia Microbiana , Salmonella typhimurium/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Transcripción GenéticaRESUMEN
Objectives: Cross-resistance between antibiotics and biocides is a potentially important driver of MDR. A relationship between susceptibility of Salmonella to quinolones and triclosan has been observed. This study aimed to: (i) investigate the mechanism underpinning this; (ii) determine whether the phenotype is conserved in Escherichia coli; and (iii) evaluate the potential for triclosan to select for quinolone resistance. Methods: WT E. coli, Salmonella enterica serovar Typhimurium and gyrA mutants were used. These were characterized by determining antimicrobial susceptibility, DNA gyrase activity and sensitivity to inhibition. Expression of stress response pathways (SOS, RpoS, RpoN and RpoH) was measured, as was the fitness of mutants. The potential for triclosan to select for quinolone resistance was determined. Results: All gyrase mutants showed increased triclosan MICs and altered supercoiling activity. There was no evidence for direct interaction between triclosan and gyrase. Identical substitutions in GyrA had different impacts on supercoiling in the two species. For both, there was a correlation between altered supercoiling and expression of stress responses. This was more marked in E. coli, where an Asp87Gly GyrA mutant demonstrated greatly increased fitness in the presence of triclosan. Exposure of parental strains to low concentrations of triclosan did not select for quinolone resistance. Conclusions: Our data suggest gyrA mutants are less susceptible to triclosan due to up-regulation of stress responses. The impact of gyrA mutation differs between E. coli and Salmonella. The impacts of gyrA mutation beyond quinolone resistance have implications for the fitness and selection of gyrA mutants in the presence of non-quinolone antimicrobials.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/genética , Desinfectantes/farmacología , Mutación/efectos de los fármacos , Quinolonas/farmacología , Triclosán/farmacología , Girasa de ADN/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Aptitud Genética , Humanos , Pruebas de Sensibilidad Microbiana , Fenotipo , Salmonella typhimurium , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genéticaRESUMEN
OBJECTIVES: Antibiotics that enhance host natural defences to infection offer an alternative approach to treating infections. However, mechanisms underlying such processes are poorly understood. The aim of this study was to investigate the effects of clinically relevant concentrations of two antibiotics on bacterial interactions with murine macrophages. METHODS: Adhesion of Salmonella Typhimurium SL1344 to and invasion by Salmonella Typhimurium SL1344 of antibiotic-treated or untreated J774 murine macrophages were measured using a tissue culture infection model. Expression of genes central to the Toll-like receptor (TLR) signalling pathway of macrophages infected with Salmonella was analysed using the RT(2) Profiler PCR Array. Cytokine production was measured by ELISA. RESULTS: Adhesion of Salmonella Typhimurium SL1344 to J774 macrophage monolayers was increased when macrophages were exposed to ciprofloxacin and ceftriaxone, while invasion was decreased by ciprofloxacin. Expression of IL-1ß and TNF-α mRNA was greater in SL1344-infected macrophages that had been treated with ciprofloxacin or ceftriaxone than in macrophages exposed to antibiotics alone or SL1344 alone. TLR mRNA was down-regulated by SL1344 infection, a response that was not altered by antibiotic pretreatment. CONCLUSIONS: Clinically relevant concentrations of two antibiotics differentially enhanced the response of immune cells and their interaction with bacteria, increasing bacterial adhesion to macrophages and increasing cytokine production. As increased expression of IL-1ß fosters apoptosis of Salmonella-infected macrophages and clearance by neutrophils, the immunomodulatory potential of these antibiotics may explain, in part, why these two drugs continue to be used to treat salmonellosis successfully.
Asunto(s)
Ceftriaxona/farmacología , Ciprofloxacina/farmacología , Citocinas/metabolismo , Factores Inmunológicos/farmacología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Receptores Toll-Like/biosíntesis , Animales , Adhesión Bacteriana/efectos de los fármacos , Línea Celular , Endocitosis/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Modelos BiológicosRESUMEN
Antimicrobial resistance genes (ARG), such as extended-spectrum ß-lactamase (ESBL) and carbapenemase genes, are commonly carried on plasmids. Plasmids can transmit between bacteria, disseminate globally, and cause clinically important resistance. Therefore, targeting plasmids could reduce ARG prevalence, and restore the efficacy of existing antibiotics. Cobalt complexes possess diverse biological activities, including antimicrobial and anticancer properties. However, their effect on plasmid conjugation has not been explored yet. Here, we assessed the effect of four previously characterised bis(N-picolinamido)cobalt(II) complexes lacking antibacterial activity on plasmid conjugation in Escherichia coli and Klebsiella pneumoniae. Antimicrobial susceptibility testing of these cobalt complexes confirmed the lack of antibacterial activity in E. coli and K. pneumoniae. Liquid broth and solid agar conjugation assays were used to screen the activity of the complexes on four archetypical plasmids in E. coli J53. The cobalt complexes significantly reduced the conjugation of RP4, R6K, and R388 plasmids, but not pKM101, on solid agar in E. coli J53. Owing to their promising activity, the impact of cobalt complexes was tested on the conjugation of fluorescently tagged extended-spectrum ß-lactamase encoding pCTgfp plasmid in E. coli and carbapenemase encoding pKpQILgfp plasmid in K. pneumoniae, using flow cytometry. The complexes significantly reduced the conjugation of pKpQILgfp in K. pneumoniae but had no impact on pCTgfp conjugation in E. coli. The cobalt complexes did not have plasmid-curing activity, suggesting that they target conjugation rather than plasmid stability. To our knowledge, this is the first study to report reduced conjugation of clinically relevant plasmids with cobalt complexes. These cobalt complexes are not cytotoxic towards mammalian cells and are not antibacterial, therefore they could be optimised and employed as inhibitors of plasmid conjugation.
Asunto(s)
Antiinfecciosos , Infecciones por Klebsiella , Animales , Agar , Antibacterianos/farmacología , Antiinfecciosos/farmacología , beta-Lactamasas/genética , Escherichia coli/genética , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/genética , Mamíferos/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genéticaRESUMEN
Antimicrobial resistance (AMR) is a global threat, with evolution and spread of resistance to frontline antibiotics outpacing the development of novel treatments. The spread of AMR is perpetuated by transfer of antimicrobial resistance genes (ARGs) between bacteria, notably those encoded by conjugative plasmids. The human gut microbiome is a known 'melting pot' for plasmid conjugation, with ARG transfer in this environment widely documented. There is a need to better understand the factors affecting the incidence of these transfer events, and to investigate methods of potentially counteracting the spread of ARGs. This review describes the use and potential of three approaches to studying conjugation in the human gut: observation of in situ events in hospitalized patients, modelling of the microbiome in vivo predominantly in rodent models, and the use of in vitro models of various complexities. Each has brought unique insights to our understanding of conjugation in the gut. The use and development of these systems, and combinations thereof, will be pivotal in better understanding the significance, prevalence, and manipulability of horizontal gene transfer in the gut microbiome.
Asunto(s)
Microbioma Gastrointestinal , Humanos , Microbioma Gastrointestinal/genética , Farmacorresistencia Bacteriana/genética , Plásmidos/genética , Antibacterianos/farmacología , Bacterias/genética , Transferencia de Gen Horizontal , Conjugación GenéticaRESUMEN
Antimicrobial resistance (AMR) is a growing problem, especially in Gram-negative Enterobacteriaceae such as Klebsiella pneumoniae. Horizontal transfer of conjugative plasmids contributes to AMR gene dissemination. Bacteria such as K. pneumoniae commonly exist in biofilms, yet most studies focus on planktonic cultures. Here we studied the transfer of a multi-drug resistance plasmid in planktonic and biofilm populations of K. pneumoniae. We determined plasmid transfer from a clinical isolate, CPE16, which carried four plasmids, including the 119-kbp blaNDM-1-bearing F-type plasmid pCPE16_3, in planktonic and biofilm conditions. We found that transfer frequency of pCPE16_3 in a biofilm was orders-of-magnitude higher than between planktonic cells. In 5/7 sequenced transconjugants (TCs) multiple plasmids had transferred. Plasmid acquisition had no detectable growth impact on TCs. Gene expression of the recipient and a transconjugant was investigated by RNA-sequencing in three lifestyles: planktonic exponential growth, planktonic stationary phase, and biofilm. We found that lifestyle had a substantial impact on chromosomal gene expression, and plasmid carriage affected chromosomal gene expression most in stationary planktonic and biofilm lifestyles. Furthermore, expression of plasmid genes was lifestyle-dependent, with distinct signatures across the three conditions. Our study shows that growth in biofilm greatly increased the risk of conjugative transfer of a carbapenem resistance plasmid in K. pneumoniae without fitness costs and minimal transcriptional rearrangements, thus highlighting the importance of biofilms in the spread of AMR in this opportunistic pathogen. IMPORTANCE Carbapenem-resistant K. pneumoniae is particularly problematic in hospital settings. Carbapenem resistance genes can transfer between bacteria via plasmid conjugation. Alongside drug resistance, K. pneumoniae can form biofilms on hospital surfaces, at infection sites and on implanted devices. Biofilms are naturally protected and can be inherently more tolerant to antimicrobials than their free-floating counterparts. There have been indications that plasmid transfer may be more likely in biofilm populations, thus creating a conjugation "hotspot". However, there is no clear consensus on the effect of the biofilm lifestyle on plasmid transfer. Therefore, we aimed to explore the transfer of a plasmid in planktonic and biofilm conditions, and the impact of plasmid acquisition on a new bacterial host. Our data show transfer of a resistance plasmid is increased in a biofilm, which may be a significant contributing factor to the rapid dissemination of resistance plasmids in K. pneumoniae.
Asunto(s)
Antiinfecciosos , Klebsiella pneumoniae , Plásmidos/genética , Carbapenémicos/farmacología , Antiinfecciosos/farmacología , BiopelículasAsunto(s)
Señales (Psicología) , Interacciones Huésped-Patógeno , Inmunidad Innata/inmunología , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Animales , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/fisiología , Concentración de Iones de Hidrógeno , Intestinos/inmunología , Intestinos/microbiología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Fagosomas/química , Fagosomas/inmunología , Fagosomas/microbiología , Salmonella typhimurium/genética , Receptores Toll-Like/deficiencia , Receptores Toll-Like/inmunología , Virulencia/genética , Virulencia/inmunologíaRESUMEN
The interplay between pathogens and their hosts has been studied for decades using targeted approaches, such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods, such as transcriptomics and proteomics, have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomic study of Salmonella enterica serovar Typhimurium infection. Our results revealed that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease and by the host to counter infection.
Asunto(s)
Interacciones Huésped-Parásitos/fisiología , Metabolómica/métodos , Salmonelosis Animal/metabolismo , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Análisis de Fourier , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Bacteria communicate through the production of diffusible signal molecules termed autoinducers. The molecules are produced at basal levels and accumulate during growth. Once a critical concentration has been reached, autoinducers can activate or repress a number of target genes. Because the control of gene expression by autoinducers is cell-density-dependent, this phenomenon has been called quorum sensing. Quorum sensing controls virulence gene expression in numerous micro-organisms. In some cases, this phenomenon has proven relevant for bacterial virulence in vivo. In this article, we provide a few examples to illustrate how quorum sensing can act to control bacterial virulence in a multitude of ways. Several classes of autoinducers have been described to date and we present examples of how each of the major types of autoinducer can be involved in bacterial virulence. As quorum sensing controls virulence, it has been considered an attractive target for the development of new therapeutic strategies. We discuss some of the new strategies to combat bacterial virulence based on the inhibition of bacterial quorum sensing systems.
Asunto(s)
Escherichia coli/patogenicidad , Percepción de Quorum , Staphylococcus aureus/patogenicidad , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/metabolismo , Regulación Bacteriana de la Expresión Génica , Péptidos Cíclicos , VirulenciaRESUMEN
We show that dimethyl sulfoxide (DMSO) inhibits Salmonella hilA expression and that this inhibition is stronger under anaerobiosis. Because DMSO can be reduced to dimethyl sulfide (DMS) during anaerobic growth, we hypothesized that DMS was responsible for hilA inhibition. Indeed, DMS strongly inhibited the expression of hilA and multiple Salmonella pathogenicity island 1 (SPI-1)-associated genes as well as the invasion of cultured epithelial cells. Because DMSO and DMS are widespread in nature, we hypothesize that this phenomenon may contribute to environmental sensing by Salmonella.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Células Epiteliales/microbiología , Salmonella/efectos de los fármacos , Sulfuros/farmacología , Transactivadores/antagonistas & inhibidores , Anaerobiosis , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Salmonella/fisiologíaRESUMEN
Antimicrobial-resistant (AMR) infections pose a serious risk to human and animal health. A major factor contributing to this global crisis is the sharing of resistance genes between different bacteria via plasmids. The WHO lists Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae, producing extended-spectrum ß-lactamases (ESBL) and carbapenemases as "critical" priorities for new drug development. These resistance genes are most often shared via plasmid transfer. However, finding methods to prevent resistance gene sharing has been hampered by the lack of screening systems for medium-/high-throughput approaches. Here, we have used an ESBL-producing plasmid, pCT, and a carbapenemase-producing plasmid, pKpQIL, in two different Gram-negative bacteria, E. coli and K. pneumoniae Using these critical resistance-pathogen combinations, we developed an assay using fluorescent proteins, flow cytometry, and confocal microscopy to assess plasmid transmission inhibition within bacterial populations in a medium-throughput manner. Three compounds with some reports of antiplasmid properties were tested; chlorpromazine reduced transmission of both plasmids and linoleic acid reduced transmission of pCT. We screened the Prestwick library of over 1,200 FDA-approved drugs/compounds. From this, we found two nucleoside analogue drugs used to treat HIV, abacavir and azidothymidine (AZT), which reduced plasmid transmission (AZT, e.g., at 0.25 µg/ml reduced pCT transmission in E. coli by 83.3% and pKpQIL transmission in K. pneumoniae by 80.8% compared to untreated controls). Plasmid transmission was reduced by concentrations of the drugs which are below peak serum concentrations and are achievable in the gastrointestinal tract. These drugs could be used to decolonize humans, animals, or the environment from AMR plasmids.IMPORTANCE More and more bacterial infections are becoming resistant to antibiotics. This has made treatment of many infections very difficult. One of the reasons this is such a large problem is that bacteria are able to share their genetic material with other bacteria, and these shared genes often include resistance to a variety of antibiotics, including some of our drugs of last resort. We are addressing this problem by using a fluorescence-based system to search for drugs that will stop bacteria from sharing resistance genes. We uncovered a new role for two drugs used to treat HIV and show that they are able to prevent the sharing of two different types of resistance genes in two unique bacterial strains. This work lays the foundation for future work to reduce the prevalence of resistant infections.
Asunto(s)
Antibacterianos/farmacología , Fármacos Anti-VIH/farmacología , Proteínas Bacterianas/genética , Transferencia de Gen Horizontal/efectos de los fármacos , Plásmidos/genética , beta-Lactamasas/genética , Didesoxinucleósidos , Farmacorresistencia Bacteriana/efectos de los fármacos , Enterobacteriaceae/genética , Escherichia coli/genética , Infecciones por VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH , Klebsiella pneumoniae/genética , ZidovudinaRESUMEN
Viruses and bacteria colonize hosts by invading epithelial barriers. Recent studies have shown that interactions between the microbiota, pathogens and the host can potentiate infection through poorly understood mechanisms. Here, we investigated whether diverse bacterial species could modulate virus internalization into host cells, often a rate-limiting step in establishing infections. Lentiviral pseudoviruses expressing influenza, measles, Ebola, Lassa or vesicular stomatitis virus envelope glycoproteins enabled us to study entry of viruses that exploit diverse internalization pathways. Salmonella Typhimurium, Escherichia coli and Pseudomonas aeruginosa significantly increased viral uptake, even at low bacterial frequencies. This did not require bacterial contact with or invasion of host cells. Studies determined that the bacterial antigen responsible for this pro-viral activity was the Toll-Like Receptor 5 (TLR5) agonist flagellin. Exposure to flagellin increased virus attachment to epithelial cells in a temperature-dependent manner via TLR5-dependent activation of NF-ΚB. Importantly, this phenotype was both long lasting and detectable at low multiplicities of infection. Flagellin is shed from bacteria and our studies uncover a new bystander role for this protein in regulating virus entry. This highlights a new aspect of viral-bacterial interplay with significant implications for our understanding of polymicrobial-associated pathogenesis.
Asunto(s)
Antígenos Bacterianos/metabolismo , Coinfección/inmunología , Flagelina/metabolismo , Interacciones Microbiota-Huesped/inmunología , Internalización del Virus , Células A549 , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Coinfección/microbiología , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/microbiología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Pulmón/citología , Permeabilidad , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 5/agonistas , Receptor Toll-Like 5/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Virosis/inmunología , Virosis/virologíaRESUMEN
Antimicrobial resistance (AMR) is a global problem hindering treatment of bacterial infections, rendering many aspects of modern medicine less effective. AMR genes (ARGs) are frequently located on plasmids, which are self-replicating elements of DNA. They are often transmissible between bacteria, and some have spread globally. Novel strategies to combat AMR are needed, and plasmid curing and anti-plasmid approaches could reduce ARG prevalence, and sensitise bacteria to antibiotics. We discuss the use of curing agents as laboratory tools including chemicals (e.g. detergents and intercalating agents), drugs used in medicine including ascorbic acid, psychotropic drugs (e.g. chlorpromazine), antibiotics (e.g. aminocoumarins, quinolones and rifampicin) and plant-derived compounds. Novel strategies are examined; these include conjugation inhibitors (e.g. TraE inhibitors, linoleic, oleic, 2-hexadecynoic and tanzawaic acids), systems designed around plasmid incompatibility, phages and CRISPR/Cas-based approaches. Currently, there is a general lack of in vivo curing options. This review highlights this important shortfall, which if filled could provide a promising mechanism to reduce ARG prevalence in humans and animals. Plasmid curing mechanisms which are not suitable for in vivo use could still prove important for reducing the global burden of AMR, as high levels of ARGs exist in the environment.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Genes Bacterianos/genética , Plásmidos/genética , Animales , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Microbiología Ambiental , Transferencia de Gen Horizontal , HumanosRESUMEN
The rapid dissemination of antimicrobial resistance (AMR) around the globe is largely due to mobile genetic elements, such as plasmids. They confer resistance to critically important drugs, including extended-spectrum beta-lactams, carbapenems, and colistin. Large, complex resistance plasmids have evolved alongside their host bacteria. However, much of the research on plasmid-host evolution has focused on small, simple laboratory plasmids in laboratory-adapted bacterial hosts. These and other studies have documented mutations in both host and plasmid genes which occur after plasmid introduction to ameliorate fitness costs of plasmid carriage. We describe here the impact of two naturally occurring variants of a large AMR plasmid (pKpQIL) on a globally successful pathogen. In our study, after pKpQIL plasmid introduction, no changes in coding domain sequences were observed in their natural host, Klebsiella pneumoniae However, significant changes in chromosomal and plasmid gene expression may have allowed the bacterium to adapt to the acquisition of the AMR plasmid. We hypothesize that this was sufficient to ameliorate the associated fitness costs of plasmid carriage, as pKpQIL plasmids were maintained without selection pressure. The dogma that removal of selection pressure (e.g., antimicrobial exposure) results in plasmid loss due to bacterial fitness costs is not true for all plasmid/host combinations. We also show that pKpQIL impacted the ability of K. pneumoniae to form a biofilm, an important aspect of virulence. This study used highly relevant models to study the interaction between AMR plasmids and pathogens and revealed striking differences from results of studies done on laboratory-adapted plasmids and strains.IMPORTANCE Antimicrobial resistance is a serious problem facing society. Many of the genes that confer resistance can be shared between bacteria through mobile genetic elements, such as plasmids. Our work shows that when two clinically relevant AMR plasmids enter their natural host bacteria, there are changes in gene expression, rather than changes to gene coding sequences. These changes in gene expression ameliorate the potential fitness costs of carriage of these AMR plasmids. In line with this, the plasmids were stable within their natural host and were not lost in the absence of selective pressure. We also show that better understanding of the impact of resistance plasmids on fundamental pathogen biology, including biofilm formation, is crucial for fighting drug-resistant infections.
Asunto(s)
Proteínas Bacterianas/genética , Replicación del ADN , Metabolismo Energético , Klebsiella pneumoniae/genética , Plásmidos , Transcripción Genética , beta-Lactamasas/genética , Aptitud GenéticaRESUMEN
For over 20 years, bacterial multidrug resistance (MDR) efflux pumps have been studied because of their impact on resistance to antimicrobials. However, critical questions remain, including why produce efflux pumps under non-antimicrobial treatment conditions, and why have multiple pumps if their only purpose is antimicrobial efflux? Salmonella spp. possess five efflux pump families, including the resistance-nodulation-division (RND) efflux pumps. Notably, the RND efflux pump AcrD has a unique substrate profile, distinct from other Salmonella efflux pumps. Here we show that inactivation of acrD results in a profoundly altered transcriptome and modulation of pathways integral to Salmonella biology. The most significant transcriptome changes were central metabolism related, with additional changes observed in pathogenicity, environmental sensing, and stress response pathway expression. The extent of tricarboxylic acid cycle and fumarate metabolism expression changes led us to hypothesize that acrD inactivation may result in motility defects due to perturbation of metabolite concentrations, such as fumarate, for which a role in motility has been established. Despite minimal detectable changes in flagellar gene expression, we found that an acrD mutant Salmonella enterica serovar Typhimurium isolate was significantly impaired for swarming motility, which was restored by addition of fumarate. The acrD mutant outcompeted the wild type in fitness experiments. The results of these diverse experiments provide strong evidence that the AcrD efflux pump is not simply a redundant system providing response resilience, but also has distinct physiological functions. Together, these data indicate that the AcrD efflux pump has a significant and previously underappreciated impact on bacterial biology, despite only minor perturbations of antibiotic resistance profiles. IMPORTANCE: Efflux pumps in Gram-negative bacteria are studied because of their important contributions to antimicrobial resistance. However, the role of these pumps in bacterial biology has remained surprisingly elusive. Here, we provide evidence that loss of the AcrD efflux pump significantly impacts the physiology of Salmonella enterica serovar Typhimurium. Inactivation of acrD led to changes in the expression of 403 genes involved in fundamental processes, including basic metabolism, virulence, and stress responses. Pathways such as these allow Salmonella to grow, survive in the environment, and cause disease. Indeed, our data show that the acrD mutant is more fit than wild-type Salmonella under standard lab conditions. We hypothesized that inactivation of acrD would alter levels of bacterial metabolites, impacting traits such as swarming motility. We demonstrated this by exogenous addition of the metabolite fumarate, which partially restored the acrD mutant's swarming defect. This work extends our understanding of the role of bacterial efflux pumps.
Asunto(s)
Antibacterianos/metabolismo , Farmacorresistencia Bacteriana , Proteínas de Transporte de Membrana/metabolismo , Salmonella typhimurium/metabolismo , Transporte Biológico Activo , Eliminación de Gen , Perfilación de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Salmonella typhimurium/genéticaRESUMEN
15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory downstream product of the cyclooxygenase enzymes. It has been implicated to play a protective role in a variety of inflammatory mediated diseases, including rheumatoid arthritis, neural damage, and myocardial infarctions. Here we show that 15d-PGJ2 also plays a role in Salmonella infection. Salmonella enterica Typhimurium is a Gram-negative facultative intracellular pathogen that is able to survive and replicate inside phagocytic immune cells, allowing for bacterial dissemination to systemic sites. Salmonella species cause a wide range of morbidity and mortality due to gastroenteritis and typhoid fever. Previously we have shown that in mouse models of typhoid fever, Salmonella infection causes a major perturbation in the prostaglandin pathway. Specifically, we saw that 15d-PGJ2 production was significantly increased in both liver and feces. In this work we show that 15d-PGJ2 production is also significantly increased in macrophages infected with Salmonella. Furthermore, we show that the addition of 15d-PGJ2 to Salmonella infected RAW264.7, J774, and bone marrow derived macrophages is sufficient to significantly reduce bacterial colonization. We also show evidence that 15d-PGJ2 is reducing bacterial uptake by macrophages. 15d-PGJ2 reduces the inflammatory response of these infected macrophages, as evidenced by a reduction in the production of cytokines and reactive nitrogen species. The inflammatory response of the macrophage is important for full Salmonella virulence, as it can give the bacteria cues for virulence. The reduction in bacterial colonization is independent of the expression of Salmonella virulence genes SPI1 and SPI2, and is independent of the 15d-PGJ2 ligand PPAR-γ. 15d-PGJ2 also causes an increase in ERK1/2 phosphorylation in infected macrophages. In conclusion, we show here that 15d-PGJ2 mediates the outcome of bacterial infection, a previously unidentified role for this prostaglandin.
Asunto(s)
Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Prostaglandina D2/análogos & derivados , Salmonella typhimurium/crecimiento & desarrollo , Animales , Ácido Araquidónico/metabolismo , Recuento de Colonia Microbiana , Citocinas/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/genética , Células HeLa , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inmunidad/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , PPAR gamma/metabolismo , Fosforilación/efectos de los fármacos , Prostaglandina D2/farmacología , Especies de Nitrógeno Reactivo/metabolismo , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Virulencia/efectos de los fármacos , Virulencia/genéticaRESUMEN
Salmonella enterica serovars are Gram-negative bacterial pathogens responsible for human diseases including gastroenteritis and typhoid fever. After ingestion, Salmonella cross the intestinal epithelial barrier, where they are phagocytosed by macrophages and dendritic cells, which then enables their spread to systemic sites during cases of typhoid fever. Salmonella use two type 3 secretion systems encoded by Salmonella pathogenicity islands (SPI) 1 and 2 to inject virulence proteins into host cells to modify cellular functions. SPI1 is involved in host cell invasion and inflammation, whereas SPI2 is required for intracellular survival and replication within phagocytes, and systemic spread. In this study the contribution of nearly all known SPI2 effectors was examined in an in vivo model of murine typhoid fever and cell culture models of macrophage and epithelial cell infection. Unmarked, in-frame deletions of SPI2 effectors were engineered in S. enterica serovar Typhimurium and the ability of the 16 different mutants to colonize and replicate was examined. In the typhoid model, we found that ΔspvB and ΔspiC mutants were attenuated for colonization of intestinal and systemic sites, while the ΔsseF mutant was attenuated in systemic organs. In epithelial cells, all mutants replicated to the same extent as the wild-type. In macrophages, ΔspiC, ΔsteC, ΔspvB, ΔssseK1/K2/K3, ΔsifA, and ΔsifB strains replicated poorly in comparison to wild-type Salmonella. This study provides a thorough screen of the majority of the known SPI2 effectors evaluated under the same conditions in various models of infection, providing a foundation for comparative examination of the roles and interactions of these effectors.