Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a ?-Lactamase-Activated Antibacterial Prodrug.
ABSTRACT: Expression of ?-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to ?-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a ?-lactamase-cleavable motif. The prodrug is only bactericidal after activation by ?-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse ?-lactamases; bactericidal activity was not observed in strains without ?-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target ?-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce ?-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.
Project description:Understanding how antibiotic-producing bacteria deal with highly reactive chemicals will ultimately guide therapeutic strategies to combat the increasing clinical resistance crisis. Here, we uncovered a distinctive self-defense strategy featured by a secreted oxidoreductase NapU to perform extracellularly oxidative activation and conditionally overoxidative inactivation of a matured prodrug in naphthyridinomycin (NDM) biosynthesis from Streptomyces lusitanus NRRL 8034. It was suggested that formation of NDM first involves a nonribosomal peptide synthetase assembly line to generate a prodrug. After exclusion and prodrug maturation, we identified a pharmacophore-inactivated intermediate, which required reactivation by NapU via oxidative C-H bond functionalization extracellularly to afford NDM. Beyond that, NapU could further oxidatively inactivate the NDM pharmacophore to avoid self-cytotoxicity if they coexist longer than necessary. This discovery represents an amalgamation of sophisticatedly temporal and spatial shielding mode conferring self-resistance in antibiotic biosynthesis from Gram-positive bacteria.
Project description:To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis.Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K(+) efflux and Na(+) influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively.Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 μg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K(+) efflux and Na(+) influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect.Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections.
Project description:Gram-negative bacteria such as Escherichia coli commonly resist ?-lactam antibiotics using plasmid-encoded ?-lactamase enzymes. Bacterial strains that express ?-lactamases have been found to detoxify liquid cultures and thus to protect genetically susceptible strains, constituting a clear laboratory example of social protection. These results are not necessarily general; on solid media, for instance, the rapid bactericidal action of ?-lactams largely prevents social protection. Here, we tested the hypothesis that the greater tolerance of biofilm bacteria for ?-lactams would facilitate social interactions. We used a recently isolated E. coli strain, capable of strong biofilm formation, to compare how cooperation and exploitation in colony biofilms and broth culture drives the dynamics of a non-conjugative plasmid encoding a clinically important ?-lactamase. Susceptible cells in biofilms were tolerant of ampicillin-high doses and several days of exposure were required to kill them. In support of our hypothesis, we found robust social protection of susceptible E. coli in biofilms, despite fine-scale physical separation of resistant and susceptible cells and lower rates of production of extracellular ?-lactamase. In contrast, social interactions in broth were restricted to a relatively narrow range of ampicillin doses. Our results show that ?-lactam selection pressure on Gram-negative biofilms leads to cooperative resistance characterized by a low equilibrium frequency of resistance plasmids, sufficient to protect all cells.
Project description:We demonstrate that ciprofloxacin can be actively loaded into liposomes that contain small amounts of porphyrin-phospholipid (PoP). PoP renders the liposomes photoactivatable, so that the antibiotic is released from the carrier under red light irradiation (665 nm). The use of 2 molar % PoP in the liposomes accommodated active loading of ciprofloxacin. Further inclusion of 2 molar % of an unsaturated phospholipid accelerated light-triggered drug release, with more than 90 % antibiotic release from the liposomes occurring in less than 30 seconds. With or without laser treatment, ciprofloxacin PoP liposomes inhibited the growth of Bacillus subtilis in liquid media, apparently due to uptake of the liposomes by the bacteria. However, when liposomes were first separated from smaller molecules with centrifugal filtration, only the filtrate from laser-treated liposomes was bactericidal, confirming effective release of active antibiotic. These results establish the feasibility of remote loading antibiotics into photoactivatable liposomes, which could lead to opportunities for enhanced localized antibiotic therapy.
Project description:The already considerable global public health threat of multi-drug resistant Gram-negative bacteria has become even more of a concern following the emergence of New-Delhi metallo-?-lactamase (NDM-1) producing strains of Klebsiella pneumoniae and other Gram-negative bacteria. As an alternative approach to the traditional development of new bactericidal entities, we have identified a 2-aminoimidazole derived small molecule that acts as an antibiotic adjuvant and is able to suppress resistance of a NDM-1 producing strain of K. pneumoniae to imipenem and meropenem, in addition to suppressing resistance of other ?-lactam non-susceptible K. pneumoniae strains. The small molecule is able to lower carbapenem minimum inhibitory concentrations by up to 16-fold while exhibiting little bactericidal activity itself.
Project description:Intracellular bacterial infections localized to the lung alveolar macrophage (AM) remain one of the most challenging settings for antimicrobial therapy. Current systemic antibiotic treatment fails to deliver sustained doses to intracellular bacterial reservoirs, which necessitates prolonged treatment regimens. Herein, we demonstrate a new intracellular enzyme-cleavable polymeric prodrug with tailored ciprofloxacin release profiles in the lungs and AM. The targeted polymeric prodrug, termed "drugamers", incorporates (1) hydrophilic mannose residues to solubilize the antibiotic cargo and to target and enhance AM uptake and intracellular delivery, and (2) enzyme-cleavable linkage chemistry to provide high and sustained intracellular AM drug dosing. Prodrug monomers, derived from the antibiotic ciprofloxacin, were synthesized with either an intracellular protease cleavable dipeptide linker or a hydrolytic phenyl ester linker. RAFT polymerization was used to copolymerize the prodrug monomers and mannose monomer to synthesize well-defined drugamers without requiring a post-polymerization conjugation step. In addition to favorable in vivo safety profiles following intratracheal administration, a single dose of the drugamers sustained ciprofloxacin dosing in lungs and AMs above the minimum inhibitory concentration (MIC) over at least a 48?h period. The enzyme-cleavable therapeutic achieved a >10-fold increase in sustained ciprofloxacin in AM, and maintained a significantly higher whole lung PK as well. Ciprofloxacin dosed in identical fashion displayed rapid clearance with a half-life of approximately 30?min. Notably, inhalation of the mannose-targeted ciprofloxacin drugamers achieved full survival (100%) in a highly lethal mouse model of pneumonic tularemia, contrasted with 0% survival using free ciprofloxacin. These findings demonstrate the versatility of the drugamer platform for engineering the intracellular pharmacokinetic profiles and its strong therapeutic activity in treating pulmonary intracellular infections.
Project description:In response to antibiotics, bacteria activate regulatory systems that control the expression of genes that participate in detoxifying these compounds, like multidrug efflux systems. We previously demonstrated that the BaeSR two-component system from Salmonella enterica serovar Typhimurium (S. Typhimurium) participates in the detection of ciprofloxacin, a bactericidal antibiotic, and in the positive regulation of mdtA, an efflux pump implicated in antibiotic resistance. In the present work, we provide further evidence for a role of the S. Typhimurium BaeSR two-component system in response to ciprofloxacin treatment and show that it regulates sodA expression. We demonstrate that, in the absence of BaeSR, the transcript levels of sodA and the activity of its gene product are lower. Using electrophoretic mobility shift assays and transcriptional fusions, we demonstrate that BaeR regulates sodA by a direct interaction with the promoter region.
Project description:The environmental spread of antibiotic-resistant bacteria has been recognised as a growing public health threat for which hospitals play a significant role. The aims of this study were to investigate the prevalence of antibiotic resistance and antibiotic resistance genes (ARGs) in Escherichia coli isolates from hospital wastewater in Vietnam. Wastewater samples before and after treatment were collected using continuous sampling every month over a year. Standard disk diffusion and E-test were used for antibiotic susceptibility testing. Extended-spectrum beta-lactamase (ESBL) production was tested using combined disk diffusion. ARGs were detected by polymerase chain reactions. Resistance to at least one antibiotic was detected in 83% of isolates; multidrug resistance was found in 32%. The highest resistance prevalence was found for co-trimoxazole (70%) and the lowest for imipenem (1%). Forty-three percent of isolates were ESBL-producing, with the blaTEM gene being more common than blaCTX-M. Co-harbouring of the blaCTX-M, blaTEM and qepA genes was found in 46% of isolates resistant to ciprofloxacin. The large presence of antibiotic-resistant E. coli isolates combined with ARGs in hospital wastewater, even post-treatment, poses a threat to public health. It highlights the need to develop effective processes for hospital wastewater treatment plants to eliminate antibiotic resistant bacteria and ARGs.
Project description:Antibiotic-induced perturbation of the human gut flora is expected to play an important role in mediating the relationship between antibiotic use and the population prevalence of antibiotic resistance in bacteria, but little is known about how antibiotics affect within-host resistance dynamics. Here we develop a data-driven model of the within-host dynamics of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae. We use blaCTX-M (the most widespread ESBL gene family) and 16S rRNA (a proxy for bacterial load) abundance data from 833 rectal swabs from 133 ESBL-positive patients followed up in a prospective cohort study in three European hospitals. We find that cefuroxime and ceftriaxone are associated with increased blaCTX-M abundance during treatment (21% and 10% daily increase, respectively), while treatment with meropenem, piperacillin-tazobactam, and oral ciprofloxacin is associated with decreased blaCTX-M (8% daily decrease for all). The model predicts that typical antibiotic exposures can have substantial long-term effects on blaCTX-M carriage duration.
Project description:Combination therapy is recommended for infections with carbapenemase-producing Klebsiella pneumoniae. However, limited data exist on which antibiotic combinations are the most effective. The aim of this study was to find effective antibiotic combinations against metallo-beta-lactamase-producing K. pneumoniae (MBL-KP). Two VIM- and two NDM-producing K. pneumoniae strains, all susceptible to colistin, were exposed to antibiotics at clinically relevant static concentrations during 24-h time-kill experiments. Double- and triple-antibiotic combinations of aztreonam, ciprofloxacin, colistin, daptomycin, fosfomycin, meropenem, rifampin, telavancin, tigecycline, and vancomycin were used. Synergy was defined as a ?2 log10 decrease in CFU/ml between the combination and its most active drug after 24 h, and bactericidal effect was defined as a ?3 log10 decrease in CFU/ml after 24 h compared with the starting inoculum. Synergistic or bactericidal activity was demonstrated for aztreonam, fosfomycin, meropenem, and rifampin in double-antibiotic combinations with colistin and also for aztreonam, fosfomycin, and rifampin in triple-antibiotic combinations with meropenem and colistin. Overall, the combination of rifampin-meropenem-colistin was the most effective regimen, demonstrating synergistic and bactericidal effects against all four strains. Meropenem-colistin, meropenem-fosfomycin, and tigecycline-colistin combinations were not bactericidal against the strains used. The findings of this and other studies indicate that there is great potential of antibiotic combinations against carbapenemase-producing K. pneumoniae. However, our results deviate to some extent from those of previous studies, which might be because most studies to date have included KPC-producing rather than MBL-producing strains. More studies addressing MBL-KP are needed.