RESUMEN
Antimicrobial resistance is an escalating threat with few new therapeutic options in the pipeline. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections globally and are prone to becoming recurrent and antibiotic resistant. We discovered and characterized six novel Autographiviridae and Guernseyvirinae bacterial viruses (phage) against uropathogenic Escherichia coli (UPEC), a leading cause of UTIs. The phage genomes were between 39,471 bp - 45,233 bp, with 45.0%-51.0% GC%, and 57-84 predicted coding sequences per genome. We show that tail fiber domain structure, predicted host capsule type, and host antiphage repertoire correlate with phage host range. In vitro characterisation of phage cocktails showed synergistic improvement against a mixed UPEC strain population and when sequentially dosed. Together, these phage are a new set extending available treatments for UTI from UPEC, and phage vM_EcoM_SHAK9454 represents a promising candidate for further improvement through engineering.
Asunto(s)
Bacteriófagos , Infecciones por Escherichia coli , Genoma Viral , Especificidad del Huésped , Terapia de Fagos , Infecciones Urinarias , Escherichia coli Uropatógena , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/virología , Infecciones Urinarias/microbiología , Infecciones Urinarias/virología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/virología , Humanos , Bacteriófagos/genética , Bacteriófagos/fisiologíaRESUMEN
Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.
Asunto(s)
Infecciones por Escherichia coli , Meningitis , Recién Nacido , Humanos , Escherichia coli/genética , Virulencia/genética , Células ClonalesRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion transporter required for epithelial homeostasis in the lung and other organs, with CFTR mutations leading to the autosomal recessive genetic disease CF. Apart from excessive mucus accumulation and dysregulated inflammation in the airways, people with CF (pwCF) exhibit defective innate immune responses and are susceptible to bacterial respiratory pathogens such as Pseudomonas aeruginosa. Here, we investigated the role of CFTR in macrophage antimicrobial responses, including the zinc toxicity response that is used by these innate immune cells against intracellular bacteria. Using both pharmacological approaches, as well as cells derived from pwCF, we show that CFTR is required for uptake and clearance of pathogenic Escherichia coli by CSF-1-derived primary human macrophages. CFTR was also required for E. coli-induced zinc accumulation and zinc vesicle formation in these cells, and E. coli residing in macrophages exhibited reduced zinc stress in the absence of CFTR function. Accordingly, CFTR was essential for reducing the intramacrophage survival of a zinc-sensitive E. coli mutant compared to wild-type E. coli. Ectopic expression of the zinc transporter SLC30A1 or treatment with exogenous zinc was sufficient to restore antimicrobial responses against E. coli in human macrophages. Zinc supplementation also restored bacterial killing in GM-CSF-derived primary human macrophages responding to P. aeruginosa, used as an in vitro macrophage model relevant to CF. Thus, restoration of the zinc toxicity response could be pursued as a therapeutic strategy to restore innate immune function and effective host defense in pwCF.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Macrófagos , Humanos , Antibacterianos/uso terapéutico , Fibrosis Quística/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Zinc/metabolismoRESUMEN
Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, with ~400 million cases across the globe each year. Uropathogenic Escherichia coli (UPEC) is the major cause of UTI and increasingly associated with antibiotic resistance. This scenario has been worsened by the emergence and spread of pandemic UPEC sequence type 131 (ST131), a multidrug-resistant clone associated with extraordinarily high rates of infection. Here, we employed transposon-directed insertion site sequencing in combination with metabolomic profiling to identify genes and biochemical pathways required for growth and survival of the UPEC ST131 reference strain EC958 in human urine (HU). We identified 24 genes required for growth in HU, which mapped to diverse pathways involving small peptide, amino acid and nucleotide metabolism, the stringent response pathway, and lipopolysaccharide biosynthesis. We also discovered a role for UPEC resistance to fluoride during growth in HU, most likely associated with fluoridation of drinking water. Complementary nuclear magnetic resonance (NMR)-based metabolomics identified changes in a range of HU metabolites following UPEC growth, the most pronounced being L-lactate, which was utilized as a carbon source via the L-lactate dehydrogenase LldD. Using a mouse UTI model with mixed competitive infection experiments, we demonstrated a role for nucleotide metabolism and the stringent response in UPEC colonization of the mouse bladder. Together, our application of two omics technologies combined with different infection-relevant settings has uncovered new factors required for UPEC growth in HU, thus enhancing our understanding of this pivotal step in the UPEC infection pathway. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) cause ~80% of all urinary tract infections (UTIs), with increasing rates of antibiotic resistance presenting an urgent threat to effective treatment. To cause infection, UPEC must grow efficiently in human urine (HU), necessitating a need to understand mechanisms that promote its adaptation and survival in this nutrient-limited environment. Here, we used a combination of functional genomic and metabolomic techniques and identified roles for the metabolism of small peptides, amino acids, nucleotides, and L-lactate, as well as the stringent response pathway, lipopolysaccharide biosynthesis, and fluoride resistance, for UPEC growth in HU. We further demonstrated that pathways involving nucleotide metabolism and the stringent response are required for UPEC colonization of the mouse bladder. The UPEC genes and metabolic pathways identified in this study represent targets for the development of innovative therapeutics to prevent UPEC growth during human UTI, an urgent need given the rapidly rising rates of global antibiotic resistance.
Asunto(s)
Infecciones por Escherichia coli , Infecciones Urinarias , Escherichia coli Uropatógena , Humanos , Escherichia coli/genética , Fluoruros/metabolismo , Lipopolisacáridos/metabolismo , Infecciones Urinarias/microbiología , Infecciones por Escherichia coli/microbiología , Genómica , Nucleótidos/metabolismo , Lactatos/metabolismo , Escherichia coli Uropatógena/genéticaRESUMEN
Bacteria adapt to selective pressure in their immediate environment in multiple ways. One mechanism involves the acquisition of independent mutations that disable or modify a key pathway, providing a signature of adaptation via convergent evolution. Extra-intestinal pathogenic Escherichia coli (ExPEC) belonging to sequence type 95 (ST95) represent a global clone frequently associated with severe human infections including acute pyelonephritis, sepsis, and neonatal meningitis. Here, we analysed a publicly available dataset of 613 ST95 genomes and identified a series of loss-of-function mutations that disrupt cellulose production or its modification in 55.3% of strains. We show the inability to produce cellulose significantly enhances ST95 invasive infection in a rat model of neonatal meningitis, leading to the disruption of intestinal barrier integrity in newborn pups and enhanced dissemination to the liver, spleen and brain. Consistent with these observations, disruption of cellulose production in ST95 augmented innate immune signalling and tissue neutrophil infiltration in a mouse model of urinary tract infection. Mutations that disrupt cellulose production were also identified in other virulent ExPEC STs, Shigella and Salmonella, suggesting a correlative association with many Enterobacteriaceae that cause severe human infection. Together, our findings provide an explanation for the emergence of hypervirulent Enterobacteriaceae clones.
Asunto(s)
Infecciones por Escherichia coli , Proteínas de Escherichia coli , Meningitis , Ratones , Animales , Ratas , Humanos , Virulencia/genética , Infecciones por Escherichia coli/microbiología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Factores de Virulencia/genética , FilogeniaRESUMEN
Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Humanos , Plásmidos/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Enterobacteriaceae/genética , Secuencia de Bases , Conjugación GenéticaRESUMEN
The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.
Asunto(s)
Antiinfecciosos , Triaje , Especies Reactivas de Oxígeno/metabolismo , NADP/metabolismo , Macrófagos/metabolismo , Antiinfecciosos/metabolismo , Vía de Pentosa Fosfato/fisiologíaRESUMEN
Antibiotic resistance is an urgent threat to global health. Antidepressants are consumed in large quantities, with a similar pharmaceutical market share (4.8%) to antibiotics (5%). While antibiotics are acknowledged as the major driver of increasing antibiotic resistance, little attention is paid to the contribution of antidepressants in this process. Here, we demonstrate that antidepressants at clinically relevant concentrations induce resistance to multiple antibiotics, even following short periods of exposure. Antibiotic persistence was also enhanced. Phenotypic and genotypic analyses revealed the enhanced production of reactive oxygen species following exposure to antidepressants was directly associated with increased resistance. An enhanced stress signature response and stimulation of efflux pump expression were also associated with increased resistance and persistence. Mathematical modeling also predicted that antidepressants would accelerate the emergence of antibiotic-resistant bacteria, and persister cells would help to maintain the resistance. Overall, our findings highlight the antibiotic resistance risk caused by antidepressants.
Asunto(s)
Antibacterianos , Antidepresivos , Antibacterianos/farmacología , Mutación , Antidepresivos/farmacología , Farmacorresistencia Microbiana , BacteriasRESUMEN
The ability of bacterial pathogens to adapt to host niches is driven by the carriage and regulation of genes that benefit pathogenic lifestyles. Genes that encode virulence or fitness-enhancing factors must be regulated in response to changing host environments to allow rapid response to challenges presented by the host. Furthermore, this process can be controlled by preexisting transcription factors (TFs) that acquire new roles in tailoring regulatory networks, specifically in pathogens. However, the mechanisms underlying this process are poorly understood. The highly conserved Escherichia coli TF YhaJ exhibits distinct genome-binding dynamics and transcriptome control in pathotypes that occupy different host niches, such as uropathogenic E. coli (UPEC). Here, we report that this important regulator is required for UPEC systemic survival during murine bloodstream infection (BSI). This advantage is gained through the coordinated regulation of a small regulon comprised of both virulence and metabolic genes. YhaJ coordinates activation of both Type 1 and F1C fimbriae, as well as biosynthesis of the amino acid tryptophan, by both direct and indirect mechanisms. Deletion of yhaJ or the individual genes under its control leads to attenuated survival during BSI. Furthermore, all three systems are up-regulated in response to signals derived from serum or systemic host tissue, but not urine, suggesting a niche-specific regulatory trigger that enhances UPEC fitness via pleiotropic mechanisms. Collectively, our results identify YhaJ as a pathotype-specific regulatory aide, enhancing the expression of key genes that are collectively required for UPEC bloodstream pathogenesis.
Asunto(s)
Infecciones por Escherichia coli , Proteínas de Escherichia coli , Sepsis , Infecciones Urinarias , Escherichia coli Uropatógena , Animales , Ratones , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones Urinarias/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Factores de Virulencia/genética , Escherichia coli Uropatógena/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
Cyclic adenosine diphosphate (ADP)-ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD+) hydrolysis. We show that v-cADPR (2'cADPR) and v2-cADPR (3'cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2'cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3'cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3'cADPR in bacteria as an antiviral and plant immunity-suppressing signaling molecule.
Asunto(s)
ADP-Ribosil Ciclasa , Proteínas Adaptadoras del Transporte Vesicular , Bacterias , Proteínas Bacterianas , ADP-Ribosa Cíclica , Inmunidad de la Planta , Receptores Toll-Like , ADP-Ribosil Ciclasa/química , ADP-Ribosil Ciclasa/genética , ADP-Ribosil Ciclasa/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADP-Ribosa Cíclica/biosíntesis , ADP-Ribosa Cíclica/química , Isomerismo , NAD/metabolismo , Dominios Proteicos , Receptores de Interleucina-1/química , Transducción de Señal , Receptores Toll-Like/química , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Triptófano/química , Triptófano/genéticaRESUMEN
Antibiotic resistance is a global concern threatening public health. Horizontal gene transfer (HGT) between bacterial species contributes greatly to the dissemination of antibiotic resistance. Conjugation is one of the major HGT pathways responsible for the spread of antibiotic resistance genes (ARGs). Antidepressant drugs are commonly prescribed antipsychotics for major depressive disorders and are frequently detected in aquatic environments. However, little is known about how antidepressants stress bacteria and whether such effect can promote conjugation. Here, we report that commonly prescribed antidepressants, sertraline, duloxetine, fluoxetine, and bupropion, can promote the conjugative transfer of plasmid-borne multidrug resistance genes carried by environmentally and clinically relevant plasmids. Noteworthy, the transfer of plasmids across bacterial genera is significantly enhanced by antidepressants at clinically relevant concentrations. We also reveal the underlying mechanisms of enhanced conjugative transfer by employing flow cytometric analysis, genome-wide RNA sequencing and proteomic analysis. Antidepressants induce the production of reactive oxygen species and the SOS response, increase cell membrane permeability, and upregulate the expression of conjugation relevant genes. Given the contribution of HGT in the dissemination of ARGs, our findings highlight the importance of prudent prescription of antidepressants and to the potential connection between antidepressants and increasing antibiotic resistance.
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Trastorno Depresivo Mayor , Proteómica , Humanos , Trastorno Depresivo Mayor/genética , Farmacorresistencia Microbiana/genética , Transferencia de Gen Horizontal , Plásmidos/genética , Antibacterianos/farmacología , Bacterias/genética , Antidepresivos/farmacología , Genes BacterianosRESUMEN
Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli Patógena Extraintestinal , Adhesinas Bacterianas/metabolismo , Adhesinas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/metabolismo , Escherichia coli Patógena Extraintestinal/genética , Escherichia coli Patógena Extraintestinal/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Enfermedades Intestinales , Polisacáridos/metabolismoRESUMEN
The formation of aggregates and biofilms enhances bacterial colonisation and infection progression by affording protection from antibiotics and host immune factors. Despite these advantages there is a trade-off, whereby bacterial dissemination is reduced. As such, biofilm development needs to be controlled to suit adaptation to different environments. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the assembly of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 variants from different Escherichia coli pathotypes. We show that specific interactions between amino acids on the contacting interfaces of adjacent Ag43 proteins drives a common mode of trans-association that leads to cell clumping. Furthermore, subtle variation of these interactions alters aggregation kinetics and the degree of compacting within cell clusters. Together, our structure-function investigation reveals an underlying molecular basis for variations in the density of bacterial communities.
Asunto(s)
Adhesinas de Escherichia coli , Proteínas de Escherichia coli , Adhesinas de Escherichia coli/química , Adhesión Bacteriana , Biopelículas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Many antibiotic resistant uropathogenic Escherichia coli (UPEC) strains belong to clones defined by their multilocus sequence type (ST), with ST131 being the most dominant. Although we have a good understanding of resistance development to fluoroquinolones and third-generation cephalosporins by ST131, our understanding of the virulence repertoire that has contributed to its global dissemination is limited. Here we show that the genes encoding Afa/Dr fimbriae, a group of adhesins strongly associated with UPEC that cause gestational pyelonephritis and recurrent cystitis, are found in approximately one third of all ST131 strains. Sequence comparison of the AfaE adhesin protein revealed a unique allelic variant carried by 82.9% of afa-positive ST131 strains. We identify the afa regulatory region as a hotspot for the integration of insertion sequence (IS) elements, all but one of which alter afa transcription. Close investigation demonstrated that the integration of an IS1 element in the afa regulatory region leads to increased expression of Afa/Dr fimbriae, promoting enhanced adhesion to kidney epithelial cells and suggesting a mechanism for altered virulence. Finally, we provide evidence for a more widespread impact of IS1 on ST131 genome evolution, suggesting that IS dynamics contribute to strain level microevolution that impacts ST131 fitness. IMPORTANCE E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tract and bloodstream infections. Understanding the features of ST131 that have driven its global dissemination remains a critical priority if we are to counter its increasing antibiotic resistance. Here, we utilized a large collection of ST131 isolates to investigate the prevalence, regulation, and function of Afa/Dr fimbriae, a well-characterized UPEC colonization and virulence factor. We show that the afa genes are found frequently in ST131 and demonstrate how the integration of IS elements in the afa regulatory region modulates Afa expression, presenting an example of altered virulence capacity. We also exploit a curated set of ST131 genomes to map the integration of the antibiotic resistance-associated IS1 element in the ST131 pangenome, providing evidence for its widespread impact on ST131 genome evolution.
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Farmacorresistencia Bacteriana Múltiple , Infecciones por Escherichia coli , Infecciones Urinarias , Escherichia coli Uropatógena , Humanos , Adhesinas Bacterianas/metabolismo , Antibacterianos/metabolismo , Células Clonales , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Escherichia coli/genética , Infecciones Urinarias/genética , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/patogenicidad , Virulencia/genéticaRESUMEN
Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients, and antibiotic treatment is compromised by multidrug-resistant strains resistant to ß-lactams, carbapenems, cephalosporins, polymyxins, and tetracyclines. Among COVID-19 patients receiving ventilator support, a multidrug-resistant A. baumannii secondary infection is associated with a 2-fold increase in mortality. Here, we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break the resistance of A. baumannii to tetracycline class antibiotics. In vitro, the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multidrug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. PBT2 and zinc disrupted metal ion homeostasis in A. baumannii, increasing cellular zinc and copper while decreasing magnesium accumulation. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multidrug-resistant A. baumannii infections. IMPORTANCE Within intensive care unit settings, multidrug-resistant (MDR) Acinetobacter baumannii is a major cause of ventilator-associated pneumonia, and hospital-associated outbreaks are becoming increasingly widespread. Antibiotic treatment of A. baumannii infection is often compromised by MDR strains resistant to last-resort ß-lactam (e.g., carbapenems), polymyxin, and tetracycline class antibiotics. During the on-going COVID-19 pandemic, secondary bacterial infection by A. baumannii has been associated with a 2-fold increase in COVID-19-related mortality. With a rise in antibiotic resistance and a reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. Rescuing the efficacy of existing therapies for the treatment of MDR A. baumannii infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.
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Infecciones por Acinetobacter , Acinetobacter baumannii , COVID-19 , Neumonía Asociada al Ventilador , Humanos , Animales , Ratones , Tigeciclina/farmacología , Neumonía Asociada al Ventilador/tratamiento farmacológico , Neumonía Asociada al Ventilador/microbiología , Tetraciclina/farmacología , Pandemias , Infecciones por Acinetobacter/microbiología , Farmacorresistencia Bacteriana Múltiple , Antibacterianos/farmacología , Carbapenémicos/farmacología , beta-Lactamas/farmacología , Pruebas de Sensibilidad Microbiana , Zinc/farmacologíaRESUMEN
Escherichia coli ST131 is a recently emerged antibiotic resistant clone responsible for high rates of urinary tract and bloodstream infections. Despite its global dominance, the precise mechanisms that have driven the rapid dissemination of ST131 remain unknown. Here, we show that the plasmid-associated resistance gene encoding the AAC(6')-Ib-cr enzyme that inactivates the fluoroquinolone (FQ) antibiotic ciprofloxacin is present in >70% of strains from the most rapidly expanding subgroup of multidrug resistant ST131. Using a series of genome-edited and plasmid-cured isogenic strains, we demonstrate that the aac(6')-Ib-cr gene confers a selective advantage on ST131 in the presence of ciprofloxacin, even in strains containing chromosomal GyrA and ParC FQ-resistance mutations. Further, we identify a pattern of emerging carbapenem resistance in other common E. coli clones carrying both aac(6')-Ib-cr and chromosomal FQ-resistance mutations, suggesting this dual resistance combination may also impart a selective advantage on these non-ST131 antibiotic resistant lineages.
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Infecciones por Escherichia coli , Escherichia coli , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Plásmidos/genéticaRESUMEN
The administration of prohibited substances has been used in agricultural show competitions and animal racing industries to gain unfair competitive advantages. We report the first large prospectively designed descriptive study of drug testing in four species (n = 1,598) over a 23 year period. 4.7% of tested exhibits returned positive results. Commonly detected substances included legitimate veterinary therapeutics such as the sedative acepromazine and the non-steroidal anti-inflammatory phenylbutazone. Targeted testing was more likely to return a positive result than random screening (50 vs 4.7% respectively) although numbers in this targeted sample were small (n = 12). Random drug testing programs were successful in detecting the minority of exhibits using prohibited substances although a wide variety of drugs were found to be used. Further vigilance and research is required in an ever-changing competitive climate to remain at the forefront of detecting new medications in animal show competitions.
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Hipnóticos y Sedantes , Detección de Abuso de Sustancias , Animales , Preparaciones FarmacéuticasRESUMEN
Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two ß-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Proteínas de Unión al ADN/farmacología , Péptidos Cíclicos/farmacología , Animales , Células de la Médula Ósea , Membrana Celular/efectos de los fármacos , Células Cultivadas , Eritrocitos , Cangrejos Herradura/metabolismo , Humanos , Ratones Endogámicos C57BL , Cultivo Primario de CélulasRESUMEN
Antibiotic resistance represents a global health challenge. The emergence of multidrug-resistant (MDR) bacteria such as uropathogenic Escherichia coli (UPEC) has attracted significant attention due to increased MDR properties, even against the last line of antibiotics. Bacteriophage, or simply phage, represents an alternative treatment to antibiotics. However, phage applications still face some challenges, such as host range specificity and development of phage resistant mutants. In this study, using both UPEC and non-UPEC hosts, five different phages were isolated from wastewater. We found that the inclusion of commensal Escherichia coli as target hosts during screening improved the capacity to select phage with desirable characteristics for phage therapy. Whole-genome sequencing revealed that four out of five phages adopt strictly lytic lifestyles and are taxonomically related to different phage families belonging to the Myoviridae and Podoviridae. In comparison to single phage treatment, the application of phage cocktails targeting different cell surface receptors significantly enhanced the suppression of UPEC hosts. The emergence of phage-resistant mutants after single phage treatment was attributed to mutational changes in outer membrane protein components, suggesting the potential receptors recognized by these phages. The findings highlight the use of commensal E. coli as target hosts to isolate broad host range phage with infectivity against MDR bacteria.