Antibiotic resistance

Antimicrobial resistance is a pressing healthcare issue, resulting in at least 23,000 direct deaths every year in the United States [1]. The problem is exacerbated by the spread of multidrug-resistant organisms such as Enterococcus spp., Klebsiella spp., andStaphylococcus aureus [2]. In addition, treating multidrug-resistant Enterobacteria e.g.,Pseudomonas aeruginosa and Acinetobacter baumannii, and other Gram-negative bacilli are increasingly common problems in healthcare settings. Both appropriate and inappropriate use of antibiotics in humans and animals are major drivers for the global spread of antibiotic-resistant microbes. In addition, inadequate measures to control the spread of infections and safety concerns may account for the far-reaching effects of antibiotic resistance [3].

Depending on the type of resistance and associated expenses, conservative estimates of the annual economic burden to the United States alone could be as high as $55 billion ($20 billion in health service costs and $35 billion in lost productivity) [4]. Moreover, antibiotics require 72 candidates to yield an approved agent at a cost of  up to $800 million (other drugs require 15 candidates to yield one medication approved by the United States Food and Drug Administration) [5]. However, a world without effective therapeutics to combat increasing infectious diseases, infectious complications associated with other diseases, and the inevitability of drug-resistant bacteria as therapeutic applications of antibiotics become more widespread is unthinkable [6]. The search for a “magic bullet” to treat bacterial resistance i.e., mutations attributable to heritable genetic elements, is a necessity and will have to encompass the development of new medicinal chemistries to eradicate persisters (phenotypic variants that survive prolonged exposure to a bactericidal antibiotic despite being genetically susceptible [7]) that are frequently responsible for recalcitrant infections [8].

Targeted removal of pathogens with the aid of the immune system requires developing new compounds or combinations of existing antibiotics and other therapeutic regimens to shut down or bypass known bacterial resistance pathways. Bacterial strategies include horizontal transfer of resistance-conferring genes [9, 10], cell wall destruction, membrane alterations, restricted drug penetrance, and the presence of multidrug efflux pumps [8, 9]. Milestones during the golden era of antibiotic discovery include the identification of the aminoglycoside, streptomycin, in 1943 and the discovery of the quinolone, ciprofloxacin, in 1961. Aminoglycosides and fluoroquinolones can kill quiescent cells, while β-lactams such as penicillin (discovered in 1928) inhibit growing cells [8]. While there are about 200 conserved essential bacterial proteins, only a few targets have historically been exploited for drug discovery [8]. Three resistance pathways are targeted by representatives from the most successful antibiotic classes i.e., fluoroquinolones, β-lactams, and aminoglycosides inhibit DNA gyrase or topoisomerase, cell wall synthesis, and the ribosome (composed of 30s and 50s subunits) respectively [11]. Moreover, biofilms – the predominant bacterial matrices found in hospitals [12]– exhibit increased resistance to antimicrobial agents compared with planktonic cells. The amplified presence of persisters, one of five known mechanisms associated with biofilm antibiotic tolerance [13], may contribute to bacterial recalcitrance and relapsing infections [14]. Cell-intrinsic mechanisms such as toxin-antitoxin modules or cell-extrinsic factors such as chemical signals or reactive oxygen species may also contribute to the physiological basis of persistence i.e., a transitory growth arrest [7].

To overcome bacterial survival strategies and improve discovery hit rates, scientists will need to develop new screening technologies, medicinal chemistries, and devise rapid diagnostic techniques for etiologic agents. Synthetic tailoring of core chemical structures – the mainstay of novel antibiotic prospecting [6] –led to the development and significant use of numerous antibiotic classes e.g., penicillins, cephalosporins, macrolides, glycopeptides, tetracyclines, and sulfa drugs. However, the pace of discovery has slowed, with only two antibiotic classes (fluoroquinolones and oxazolidinones) being developed in the last five decades [8]. While additional modifications of the tetracycline and quinolone classes as well as the introduction of a pro-drug scaffold hold promise [8], it is unlikely that tailoring of antibiotic scaffolds can continue in perpetuity. New synthetic compounds used in combination with standard therapies may minimize resistance or enable the destruction of more than one bacterial target [8].

Screening

Boosting the antibiotic arsenal requires screening existing chemical libraries for overlooked compounds, identifying cryptic biosynthetic pathways in antibiotic-producing strains that may yield useful metabolites [8, 15], evaluating novel synthetic and natural sources for potentially successful antibiotics, and developing reliable innovative discovery platforms geared towards targeted therapies with minimal effects to host cells [8]. Rapid identification of the etiologic agent is a prerequisite for the development of such targeted therapies e.g., a rapid diagnostic test developed for methicillin-resistant Staphylococcus aureus will enable the identification of selective agents against this clinically important pathogen [8]. Chemical analysis of a lead compound’s mode of action and potential resistance mechanisms would be the next step. Reference databases of proprietary targets will be useful in assigning mechanisms of action to lead compounds. However, a limitation of this approach is the number of compounds required for successful mechanism-of-action classifications ─ a minimum of 5 to 6 compounds per class [16]. Furthermore, an understanding of interactions between drugs and resistance mutations  [10] could guide the selection of combination therapies aimed at increasing the number of hits in order to eradicate a targeted pathogen. Ideally, any screening technology should also identify non-essential pathways that may contribute to resistance such as drug efflux pumps or enzymes that could destroy the compound.

Currently the paucity of medications that can effectively penetrate bacterial cells (Gram-negative bacteria are very effective at keeping out drugs [8]) may necessitate rescreening existing chemical libraries of promising lead compounds with modified “Lipinski’s rule of 5”(physicochemical properties that influence oral bioavailability) guidelines. Traditional libraries were typically derived synthetically or from natural sources and screened using rules that are not applicable to antimicrobials and focused only on assessing molecular properties pertaining to oral bioavailability [8]. Rules of bacterial penetration could conceivably be developed based on in vitro tests using compounds known to effectively permeate bacteria. Appropriate tests of focused libraries in situ could also eliminate compounds with poor serum binding, instability or poor tissue distribution in vivo [8].

A screen performed by nature outnumbers currently available synthetic compounds. Lipopeptide (daptomycin), glycylcycline (tegicycline), streptogramin  (pristinamycin), glycopeptide (vancomycin), macrolide (erythromycin),, aminoglycoside (streptomycin), phenylpropanoid (chloramphenicol), β-lactam (penicillins, cephalosporins, carbapenems, monobactams),  and polyketides (tetracycline) core structures have all been constructed from natural sources [17]. Uncultivated organisms (99% of bacterial species are not cultivable by traditional methods [18]), marine [17], mammalian [19], reptilian [20], fungi and plant sources are among the important reservoirs for ideal antibiotics i.e., pro-drugs converted by intra-cellular bacterial enzymes into reactive compounds capable of killing persisters and actively dividing cells [8].

The probabilities of discovering new compounds in old libraries were estimated to be around 10-5 to 10-4 [8]. Leads for combination therapies could potentially emerge from pre-designed bacterial silencer RNA (siRNA) libraries as in the case of the discovery of platensimycin and platensin in the Merck 245 siRNA collection [6, 8]. Although the clinical utility of platensin and platensimycin have been questioned [6, 21], it is conceivable that lead compounds discovered in gene-knockdown collections could re-sensitize bacteria to approved antibiotics when used synergistically in selected combination therapies [6]. Metagenomics of cultivable and uncultivable organisms may yield further lead compounds and predict the emergence and epidemiology of antibiotic resistance e.g., the comprehensive antibiotic resistance database is a unifying bioinformatics compilation of 3411 resistance genes from disparate sources designed to exploit the power and falling costs of next-generation DNA sequencing technologies [22].

A significant problem stalling the drug discovery pipeline is the question of which compounds are worth further characterization and purification. Sensitive bioassays using feasible test organisms could be effective in identifying novel antibiotics e.g., the differential susceptibility of Bacillus subtilis test and parent strains provided an important secondary screen for the evaluation of potential bacterial RNA polymerase inhibitors from a primary screening of actinomycete isolates [15].

Other strategies

Ongoing challenges in the treatment of infectious diseases include the specter of secondary infections e.g., Clostridium difficile-associated diarrhea and candidiasis and collateral damage to bacterial mutualists [23]. Thus, alternative antimicrobials or therapies that augment the host response to infectious diseases are also currently under investigation.

Antimicrobial peptides (AMPs), typically 20 to 50 amino acid residues long, are important components of the innate immune system and may be used to overcome resistance challenges from different microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and viruses [5]. Natural AMPs from bacteria usually kill target pathogens with a high potency (pico- to nanomolar range) and specificity [24] via protein- or non-protein-mediated mechanisms [5]. Since resistant strains have been observed in preclinical trials, it will be important to undergo an analysis of the co-evolution of bacterial resistant strains during the development of AMPs [5]. Other strategies currently being studied include phage and photodynamic therapies, probiotics, honey therapy, maggot therapy, herbal medicines, statins, vaccines, and fecal transplants [23, 25]. In the latter case, the objective is to shift the diseased microbiome of patients to a “healthier” alternative stable state [23]. Fecal transplantation has been successful in resolving the symptoms of recurrent C. difficile-associated diarrhea [26]. General use of active microbial repopulation strategies will, however, require proof of efficacy and pharmaceutical-grade products [23].

References

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