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Rifampicin resistance desensitizes elongating RNAP to ppGpp a, Same image from Fig.5e with b, quantification plots. In vitro transcription reactions of wild type and rpoB H526Y RNAP with or without ppGpp (100 μM). Stalled RNAP elongation complexes (20-mer, denoted with the red arrow) were chased to the end of the template (runoff, denoted with the blue arrow), and the labelled nascent RNA then separated using PAGE. Numbering corresponds to time points 0, 10, 20, 40 and 60-seconds following the chase reaction. Arrows (labelled 1 through 5) correspond to major pause sites. Source data
Source publication
Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) confer antibiotic resistance and often have global effects on transcription that compromise fitness and stress tolerance of resistant mutants. We suggested that the non-essential genome, through its impact on the bacterial transcription cycle, may represent an untapped source o...
Citations
... For example, E. coli Nissle 1917 was engineered to secrete three antimicrobial peptides, Enterocin A, Enterocin B, and Hiracin JM79 to reduce vancomycin-resistant Enterococcus in the intestinal tract [56]. Alternatively, identified bacteriocins can be reconstructed with minor transformation of amino acid, devoting to better antibacterial performance [99,100]. Second, the performance of bacteriocin-producing microbes in GIT is affected by multiple factors such as clearing out of the host due to weak colonization to the intestine, degradation of bacteriocin by gastrointestinal proteases, and absorption of bacteriocin by gastrointestinal matrix. ...
Gastrointestinal (GI) infection by intestinal pathogens poses great threats to human health, and the therapeutic use of antibiotics has reached a bottleneck due to drug resistance. The developments of antimicrobial peptides produced by beneficial bacteria have drawn attention by virtue of effective, safe, and not prone to developing resistance. Though bacteriocin as antimicrobial agent in gut infection has been intensively investigated and reviewed, reviews on that of bacteriocin-producing beneficial microbes are very rare. It is important to explicitly state the prospect of bacteriocin-producing microbes in prevention of gastrointestinal infection towards their application in host. This review discusses the potential of gut as an appropriate resource for mining targeted bacteriocin-producing microbes. Then, host-related factors affecting the bacteriocin production and activity of bacteriocin-producing microbes in the gut are summarized. Accordingly, the multiple mechanisms (direct inhibition and indirect inhibition) behind the preventive effects of bacteriocin-producing microbes on gut infection are discussed. Finally, we propose several targeted strategies for the manipulation of bacteriocin-producing beneficial microbes to improve their performance in antimicrobial outcomes. We anticipate an upcoming emergence of developments and applications of bacteriocin-producing beneficial microbes as antimicrobial agent in gut infection induced by pathogenic bacteria.
... There is increasing recognition of the collateral consequences of antibiotic resistance acquisitions being a viable target for combination antibiotic therapy. Detailed analysis of the fitness costs of antibiotic resistance can provide insight into unique genetic and phenotypical liabilities that can be therapeutically targeted (79). Identification of highly conserved pathways that can be therapeutically targeted to potentiate the activity of antibiotics, particularly against tolerant populations, provides avenues to expand the therapeu tic lifespan of current antibiotics (80). ...
The increasing prevalence of antibiotic resistant bacteria is a major global health concern. While many species have the potential to develop antibiotic resistance, understanding the barriers to resistance emergence in the clinic remains poorly understood. A prime example of this is fluroquinolone resistance in Streptococcus pneumoniae , whereby, despite continued utilization, resistance to this class of antibiotic remains rare. In this study, we found that the predominant pathways for developing resistance to this antibiotic class severely compromised the infectious capacity of the pneumococcus, providing a key impediment for the emergence of resistance. Using in vivo models of experimental evolution, we found that S. pneumoniae responds to repeated fluoroquinolone exposure by modulating key metabolic pathways involved in the generation of redox molecules, which leads to antibiotic treatment failure in the absence of appreciable shifts in resistance levels. These data underscore the complex pathways available to pathogens to evade antibiotic mediating killing via antibiotic tolerance.
... A similar global transcriptional response mediated by CedA is likely to underlie tolerance to other clinically relevant antibiotics we examined here and to oxidative stress, highlighting CedA as a new promising antimicrobial target, especially in light of its positive retention in uropathogenic E. coli 11 . 29 were used for tetracycline-inducible expression of CedA (paTc-cedA) and its truncated variant lacking first 11 amino acids (paTc-cedAΔ11N). Plasmids pVS10 30 and pSumoH10-cedA were used for the overexpression of E. coli RNAP and CedA, respectively. ...
Gene expression in Escherichia coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ⁷⁰ holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of β and σ⁷⁰ subunits to stabilize an open-promoter complex. CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis.
... DNA released as a result of the application of H 2 O 2 may contain antibiotic resistance genes, abandoning the previous bacterial cell and being taken up by a competent cell. As antibiotic resistance genes represent a burden for bacterial cells, once these genes are extracellularly released, the bacterial cell will gain more fitness [24]. ...
Background
Disinfectants are important in the food industry to prevent the transmission of pathogens. Excessive use of disinfectants may increase the probability of bacteria experiencing long-term exposure and consequently resistance and cross-resistance to antibiotics. This study aims to investigate the cross-resistance of multidrug-resistant, drug-resistant, and drug-susceptible isolates of Salmonella enterica serovar Typhimurium (S. Typhimurium) with different sequence types (STs) to a group of antibiotics after exposure to different food-grade disinfectants.
Methods
A panel of 27 S. Typhimurium strains with different antibiograms and STs were exposed to increasing concentrations of five food-grade disinfectants, including hydrogen peroxide (H2O2), benzalkonium chloride (BAC), chlorine dioxide (ClO2), sodium hypochlorite (NaClO), and ethanol. Recovered evolved strains were analyzed using genomic tools and phenotypic tests. Genetic mutations were screened using breseq pipeline and changes in resistance to antibiotics and to the same disinfectant were determined. The relative fitness of evolved strains was also determined.
Results
Following exposure to disinfectants, 22 out of 135 evolved strains increased their resistance to antibiotics from a group of 14 clinically important antibiotics. The results also showed that 9 out of 135 evolved strains had decreased resistance to some antibiotics. Genetic mutations were found in evolved strains. A total of 77.78% of ST34, 58.33% of ST19, and 66.67% of the other STs strains exhibited changes in antibiotic resistance. BAC was the disinfectant that induced the highest number of strains to cross-resistance to antibiotics. Besides, H2O2 induced the highest number of strains with decreased resistance to antibiotics.
Conclusions
These findings provide a basis for understanding the effect of disinfectants on the antibiotic resistance of S. Typhimurium. This work highlights the link between long-term exposure to disinfectants and the evolution of resistance to antibiotics and provides evidence to promote the regulated use of disinfectants.
... In addition to its importance in combating clinical resistance [2][3][4] , the study of antibiotic resistant mutants has also advanced our understanding of fundamental biological processes 5 . Because antibiotics function by targeting enzymes necessary for bacterial survival, resis tance mutations have been instrumental in the characterization of essential cellular machinery such as RNA polymerase [6][7][8][9][10] (RNAP) and the ribosome [11][12][13][14] . ...
... Collectively, resistance mutants manifest a fascinating breadth of phenotypes that are a consequence of changes in the properties of the mutant enzyme at each step of the transcription cycle 15,16,[21][22][23][24] . Although Rif is notorious for high-frequency resistance 4,25,26 , the known spectrum of resistant mutants makes less than 5% of the non-redundant mutational space at this region of the β-subunit of RNAP. Therefore, our functional understanding of the Rif binding site is still incomplete. ...
... Previous work has shown that polymerase speed determines the efficiency of Rho termination 32 . To test whether mutants sensitive to both 5FU and BCM have faster RNAP, we measured their elongation rate using a multiprobe qPCR assay along the lac operon and their pause frequency using native elongating transcript sequencing (NETseq) 4,43 . Indeed, these mutants have a higher elongation rate (Fig. 4d) and pause less frequently (Fig. 4e) than wild-type RNAP. ...
Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering ¹ to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug–enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription–replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.
... However, some RIF-resistance mutations in the RRDR not only decrease RIF binding but also lead to alterations in the cell wall (16). In E. coli, the clinically relevant H526Y RRDR mutant is very sensitive to cell wall inhibitors and to the deletion of genes encoding auxiliary functions related to cell wall synthesis and division (66). Similarly, we report here that B. subtilis RRDR mutations can lead to either sensitivity or resistance to an antibiotic (CEF) that inhibits PG synthesis. ...
Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance-determining region (RRDR) of rpoB that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the β-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single rpoB allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32 times more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP-N-acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. IMPORTANCE Rifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy and for multidrug-resistant strains may include β-lactams. Mutations in rpoB present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the β-lactam cefuroxime (CEF). One clinically relevant rpoB mutation conferred resistance to both RIF and CEF, whereas one other increased CEF sensitivity. We were able to link these CEF sensitivity phenotypes to accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc), which feedback regulates GlmS activity and thereby peptidoglycan synthesis. Further, we found that higher CEF concentrations precluded the emergence of high RIF resistance. Collectively, these results suggest that multidrug treatment regimens may limit the available pathways for the evolution of antibiotic resistance.
... A different approach to limit tolerance is to target bacterial detox systems with smallmolecule inhibitors, such as those for hydrogen sulfide production (19), that impart tolerance in WT cells across various bacterial pathogens. And, in cases of tolerant mutants that are very common in clinical settings, it may be possible to target them specifically (20). ...
... There is increasing recognition of the collateral consequences of antibiotic resistance acquisitions being a viable target for combination antibiotic therapy. Detailed analysis of the fitness costs of antibiotic resistance can provide insight into unique genetic and phenotypical liabilities that can be therapeutically targeted 86 . Identification of highly conserved pathways that can be therapeutically targeted to potentiate the activity of antibiotics, particularly against tolerant populations, provides avenues to expand the therapeutic lifespan of current antibiotics 87 . ...
Streptococcus pneumoniae is a major human pathogen of global health concern, causing a range of mild to severe infections, including acute otitis media, pneumonia, sepsis, and meningitis. The rapid emergence of antibiotic resistance among S. pneumoniae isolates poses a serious public health problem worldwide. Resistant pneumococcal strains have rendered the mainstay treatment with beta-lactams, fluoroquinolones, and macrolides, ineffective. Antibiotic resistance in S. pneumoniae has spread globally via the emergence of de novo mutations and horizontal transfer of resistance. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage, as is often the case with other classes of antibiotics. In this study, we demonstrated that deleterious fitness costs constrain the emergence of individual fluoroquinolone resistance mutations in either topoisomerase IV or gyrase A in S. pneumoniae . Generation of double point mutations in the target enzymes in topoisomerase IV and gyrase A conferred high-level fluoroquinolone resistance while restoring fitness comparable to the sensitive wild-type. During an in vivo model of antibiotic resistance evolution, S. pneumoniae was able to circumvent deleterious fitness costs imposed by resistance determinants through development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, an effect that could be recapitulated pharmacologically. The metabolic mutants conferring tolerance resulted in a fitness benefit during infection following antibiotic treatment with fluroquinolones. These data suggest that emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by host fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes may be an important contributor to the evasion of antibiotic mediated killing.
... Combination therapy, defined as the application of a combination of drugs, is one of the most attractive and promising medical strategies, and it aims to strengthen the therapeutic efficiency and reduce the side effects of drugs. For instance, different types of antibacterial drugs have been used together in the clinic to expand the antimicrobial spectrum [43], and more than one kind of drug is generally prescribed to patients to treat hypertension or cancer [44,45]. Particularly for navitoclax, the drug combination strategy in tumor-related research has yielded positive results. ...
Compounds with senolysis activity are discovered in recent years, featuring by their capacity to specifically eliminate senescent cells in vitro or in vivo. These compounds, referring to as Senolytics, provide a new method for aging counteraction and probably for geriatric disease amelioration. However, their clinical application is unpractical still, mainly because of the safety issue. In fact, the effective dose range even of the most potent senolytic cannot guarantee the safety requirements application for human being. Here, we report a study which investigated the combinational application of one potential senolytic molecule navitoclax, a Bcl-2 inhibitor with several mTOR inhibitors, to assess the influence of this combination on the senolytic outcome. Our results reveal that pan-mTOR inhibitors can reduce the dosage or timespan of navitoclax necessary for reaching IC50 and LT50 in senescent cells, also extend the lifespan of premature-aged Drosophila and mitigate the aging-related phenotype. Our results also confirmed that mTOR inhibitor sensitized senolytic cell death is apoptotic and pan-mTOR inhibitors PP242 and AZD8055 works more effectively than mTORC1 inhibitor Rapamycin. Mechanically, we verified the crucial role of mTORC2 inhibition contributes sensitization by increasing the expression of the pro-apoptotic protein Bim. In summary, this study firstly exposes the sensitization effect of pan-mTOR inhibitors on navitoclax-induced senolytic apoptosis, therefore providing novel evidence to show the advantage of drug combination on setting senotherapy. It also provides an intriguing clue to demonstrate the value of mTORC2 inhibition for apoptotic death of senescent cells.
... Evolved antibiotic resistance is a costly affair for many species of bacteria and often results in decreased competitive fitness in the absence of selection pressure [44,45]. Nevertheless, it has also been exhibited that S. aureus often tackles such trade-offs by way of mutations that compensate for changes [46]. ...
Staphylococcus haemolyticus has emerged to be a frequently encountered late-onset sepsis pathogen among newborn infants. Critical care of neonates involves substantial usage of antibiotics and these pathogens are often exposed to sub-optimal doses of antibiotics which can augment maintenance of selection determinants and a range of physiological effects, prime among them being biofilm formation. Therefore, in this study, the outcome of a sub-inhibitory dosage of a commonly prescribed third-generation antibiotic, cefotaxime (CTX), on multidrug resistant (MDR) S. haemolyticus, was investigated. A total of 19 CTX-resistant, MDR and 5 CTX-susceptible strains isolated from neonates were included. Biofilm-forming abilities of S. haemolyticus isolates in the presence of sub-optimal CTX (30 μg/mL) were determined by crystal violet assays and extracellular DNA (eDNA) quantitation. CTX was found to significantly enhance biofilm production among the non-susceptible isolates (p-valueWilcoxintest-0.000008) with an increase in eDNA levels (p-valueWilcoxintest-0.000004). Further, in the absence of antibiotic selection in vitro, populations of MDR isolates, JNM56C1 and JNM60C2 remained antibiotic non-susceptible after >500 generations of growth. These findings demonstrate that sub-optimal concentration of CTX induces biofilm formation and short-term non-exposure to antibiotics does not alter non-susceptibility among S. haemolyticus isolates under the tested conditions.
