Current Pharmaceutical Design - Volume 19, Issue 2, 2013

Early after the introduction of the first (narrow spectrum) penicillins into clinical use, penicillinase-producing staphylococci replaced (worldwide) the previously susceptible microorganisms. Similarly, the extensive use of broad-spectrum, orally administered β- lactams (like ampicillin, amoxicillin or cefalexin) provided a favorable scenario for the selection of gram-negative microorganisms producing broad spectrum β-lactamases almost 45 years ago. These microorganisms could be controlled by the introduction of the so called “extended spectrum cephalosporins”. However, overuse of these drugs resulted, after a few years, in the emergence of extended-spectrum β-lactamases (ESBLs) through point mutations in the existing broad-spectrum β-lactamases, such as TEM and SHV enzymes. Overuse of extended-spectrum β-lactams also gave rise to chromosomal mutations in regulatory genes which resulted in the overproduction of chromosomal AmpC genes, and, in other regions of the world, in the explosive emergence of other ESBL families, like the CTX-Ms. Carbapenems remained active on microorganisms harboring these extended-spectrum β-lactamases, while both carbapenems and fourth generation cephalosporins remained active towards those with derepressed (or the more recent plasmidic) AmpCs. However, microorganisms countered this assault by the emergence of the so called carbapenemases (both serine- and metallo- enzymes) which, in some cases, are actually capable of hydrolyzing almost all β-lactams including the carbapenems. Although all these enzyme families (some of them represented by hundreds of members) are for sure pre-dating the antibiotic era in environmental and clinically significant microorganisms, it was the misuse of these antibiotics that drove their evolution. This paper describes in detail each major class of β-lactamase including epidemiology, genetic, and biochemical evaluations.

Treatment of serious P. aeruginosa infections becomes more challenging with each passing year. As this pathogen acquires more transferrable resistance mechanisms and continues to rapidly adapt and emerge resistant during the course of antimicrobial therapy, we face the growing threat of pan-resistance. This review has focused on those mechanisms that directly impact the future of β-lactam antibiotics, including the production of β-lactamases, porin-mediated resistance, and/or the overexpression of RND efflux pumps. With the pipeline of new anti-pseudomonal agents diminishing, it is essential that novel therapeutic strategies be explored. These include targeting biofilm formation and maintenance, virulence factors, and resistance mechanisms. Furthermore, we must continue to search for effective antibacterial combinations to not only prevent further emergence of resistance but also treat resistant strains already in the environment.

Acinetobacter spp. are Gram-negative bacteria that have become one of the most difficult pathogens to treat. The species A. baumannii, largely unknown 30 years ago, has risen to prominence particularly because of its ability to cause infections in immunocompromised patients. It is now a predominant pathogen in many hospitals as it has acquired resistance genes to virtually all antibiotics capable of treating Gram-negative bacteria, including the fluoroquinolones and the cephalosporins. Some members of the species have accumulated these resistance genes in large resistance islands, located in a "hot-spot" within the bacterial chromosome. The only conventional remaining treatment options were the carbapenems. However, A. baumannii possesses an inherent class D β-lactamase gene (blaOXA-51-like) that can have the ability to confer carbapenem resistance. Additionally, mechanisms of carbapenem resistance have emerged that derive from the importation of the distantly related class D β-lactamase genes blaOXA-23 and blaOXA-58. Although not inducible, the expression of these genes is controlled by mobile promoters carried on ISAba elements. It has also been found that other resistance genes including the chromosomal class C β-lactamase genes conferring cephalosporin resistance are controlled in the same manner. Colistin is now considered to be the final drug capable of treating infections caused by carbapenem-resistant A. baumannii; however, strains are now being isolated that are resistant to this antibiotic as well. The increasing inability to treat infections caused by A. baumannii ensures that this pathogen more than ranks with MRSA or Clostridium difficile as a threat to modern medicine.

Although β-lactams remain a cornerstone of veterinary therapeutics, only a restricted number are actually approved for use in food-producing livestock in comparison to companion animals and wildlife. Nevertheless, both registered and off-label use of third and fourth-generation cephalosporins in livestock may have influenced the emergence of plasmid-encoded AmpC β-lactamases (pAmpC) (mainly CMY-2) and CTX-M extended-spectrum β-lactamases (ESBLs) in both Gram-negative pathogens and commensals isolated from animals. This presents a public health concern due to the potential risk of transfer of β-lactam-resistant pathogens from livestock to humans through food. The recent detection of pAmpC and ESBLs in multidrug-resistant Enterobacteriaceae isolated from dogs has also confirmed the public health importance of β-lactam resistance in companion animals, though in this case, human-to-animal transmission may be equally as relevant as animal-to-human transmission. Identification of pAmpC and ESBLs in Enterobacteriaceae isolated from wildlife and aquaculture species may be evidence of environmental selection pressure arising from both human and veterinary use of β- lactams. Such selection pressure in animals could be reduced by the availability of reliable alternative control measures such as vaccines, bacteriophage treatments and/or competitive exclusion models for endemic production animal diseases such as colibacillosis. The global emergence and pandemic spread of extraintestinal pathogenic E. coli O25-ST131 strains expressing CTX-M-15 ESBL in humans and its recent detection in livestock, companion animals and wildlife is a major cause for concern and goes against the paradigm that Gramnegative pathogens do not necessarily have to lose virulence in compensation for acquiring resistance.

Pathogens that produce extended spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, and carbapenemases may appear falsely susceptible to β-lactam antibiotics in the laboratory. Infected patients may be treated with inappropriate antibiotics if laboratories do not perform accurate tests to detect these resistance mechanisms. Furthermore the resistant pathogens may spread undetected to amplify the therapeutic and infection control challenge. This review makes a case for why and how laboratories should perform tests to detect β-lactamase-mediated resistance in gram-negative pathogens.

Escherichia coli remains one of the most frequent causes of nosocomial and community-acquired bacterial infections including urinary tract infections, enteric infections, and systemic infections in humans. Extra-intestinal pathogenic E. coli or ExPEC had emerged during the 2000s as an important player in the resistance to antibiotics, especially to the cephalosporins and fluoroquinolones. Most importantly among ExPEC, is the increasing recognition of isolates producing “newer β-lactamases” that consist of plasmidmediated AmpC β-lactamases (e.g. CMY), extended-spectrum β-lactamases (e.g. CTX-M), and carbapenemases (e.g. NDM, KPC and OXA-48). Since the mid 2000's, E. coli that produce CTX-M enzymes (especially CTX-M-15), have emerged worldwide as important causes of community-associated urinary tract (UTIs) and blood stream infections. Community-associated acquisition and infections due to enterobacteria with plasmid-mediated AmpC β-lactamases are a relatively recent phenomenon and have been described in Canada and USA. Empiric antibiotic coverage for these resistant organisms should be considered in community patients presenting with sepsis involving the urinary tract especially if a patient recently traveled to a high-risk area. If this emerging public health threat is ignored, it is possible that the medical community may be forced in the near future to use the carbapenems as the first choice for the empirical treatment of serious infections associated with urinary tract infections originating from the community.

Although reports of antimalarial drug resistance emerged as early as 1910 from South America, the first event that really had a major impact on malaria control and drug development was the emergence of chloroquine resistance in the 2nd half of the 20th century. The appearance of resistance to chloroquine has marked the onset of a race between the development of ever new generations of antimalarial drugs and the emergence of resistance to these antimalarials, finally culminating in the emergence of clinical artemisinin resistance which was first reported in 2008. The potentially devastating impact of resistance to a drug that has been adopted as the first line drug for the treatment of uncomplicated falciparum malaria by virtually all malaria control programs throughout the malaria-endemic world, and for which there currently is no successor in sight should it truly fall victim to widespread drug resistance, calls for strategies to extend the useful life spans of currently available antimalarial drugs while at the same time stepping up efforts to develop novel combination therapies not based on artemisinins.

The malaria parasite has been allowed to get perilously close to winning the upper hand in the race between new drugs and resistance development. Today, just one class of drugs is left to avoid a public health disaster of global proportions, the artemisinins, and even they are showing signs of a possible impending failure. Rational approaches to overcoming antimalarial drug resistance are difficult for several reasons. Resistance mechanisms are varied and imperfectly known across Plasmodium species and often there is not a good correlation between in vitro drug susceptibility, molecular markers of resistance and therapeutic failure, except for antimalarials acting on well defined molecular targets such as atovaquone and the antifolates. Drugs with more complex modes of action are expected to have correspondingly complex resistance mechanisms. Molecular markers of resistance for the most widely used quinoline, chloroquine, have been identified, but they are not applicable to all parasite species and perhaps not even to all strains. Analyses of drug resistance in vitro are also limited by the fact that only one malaria parasite species, Plasmodium falciparum, is amenable to long term culture. Nevertheless, reducing the risk of premature therapeutic failure due to quick resistance emergence needs to be considered from the earliest stages of drug discovery. In the present review we attempt to summarize the main mechanisms of resistance to current antimalarials and provide information on already available assays to estimate the propensity of a new molecule to select for resistant parasites.

Drug development often seeks to find “magic bullets” which target microbiologic proteins while not affecting host proteins. Paul Ehrlich tested methylene blue as an antimalarial but this dye was not superior to quinine. Many successful antimalarial therapies are “magic shotguns” which target many Plasmodium pathways with little interference in host metabolism. Two malaria drug classes, the 8- aminoquinolines and the artemisinins interact with cytochrome P450s and host iron protoporphyrin IX or iron, respectively, to generate toxic metabolites and/or radicals, which kill the parasite by interference with many proteins. The non 8-amino antimalarial quinolines like quinine or piperaquine bind heme to inhibit the process of heme crystallization, which results in multiple enzyme inhibition and membrane dysfunction. The quinolines and artemisinins are rapidly parasiticidal in contrast to metal chelators, which have a slower parasite clearance rate with higher drug concentrations. Iron chelators interfere with the artemisinins but otherwise represent a strategy of targeting multiple enzymes containing iron. Interest has been revived in antineoplastic drugs that target DNA metabolism as antimalarials. Specific drug targeting or investigation of the innate immunity directed to the more permeable trophozoite or schizont infected erythrocyte membrane has been under explored. Novel drug classes in the antimalarial development pipeline which either target multiple proteins or unchangeable cellular targets will slow the pace of drug resistance acquisition.

The most common treatments for infectious diseases target the invading pathogen. The efficacy of such an approach may, however, be countered by the possibility of the development of resistance to a pharmacophore, through mutation(s) in pathogen molecules required for activity. Given the fact that pathogens exploit host factors in order to grow in an otherwise hostile environment, one possible way to circumvent the emergence of resistance is to develop drugs that target non-essential host factors hijacked by the pathogen, rather than the pathogen's own molecules. Such solutions are already being developed for various viral and bacterial pathogens, but much less has been achieved with infections caused by protozoan parasites, as is the case of Plasmodium. Here, we highlight recent progress in host target-based anti-viral and anti-bacterial approaches and discuss possible host targets that may be used for anti-malarial interventions. Host molecules that play a role during either the liver or the blood stage of Plasmodium infection are outlined and their potential merits as anti-malarial targets are discussed.

Antimalarial drugs have in the past fallen prey to resistance and this problem is likely to continue in the future. One approach to developing drugs that might be less prone to resistance might be to target the disease rather than the parasite itself. The rationale for this idea, which has been somewhat developed in antibacterial chemotherapy, is that drugs that can alleviate disease pathogenesis while not compromising the survival, growth or transmission of the pathogen should not exert selective pressure that would encourage the emergence and spread of resistance. This review considers (concentrating on possible interventions at the parasite level) whether such ‘anti-disease’ therapy could be developed for severe Plasmodium falciparum malaria, and if so whether it might be less prone to resistance. Several anti-adhesive treatments, aiming to reduce the tissue sequestration of P. falciparum-parasitised erythrocytes that is associated with cerebral malaria and other complications, have been investigated as ‘adjunctive’ therapies. These therapies are however unlikely to be ‘resistance proof’ because sequestration appears to enhance parasite survival in the host. Severe malarial anaemia is another potentially fatal complication of malaria that results not only from lysis of host erythrocytes by intracellular parasites but to a greater extent from lysis of unparasitised erythrocytes and impaired erythropoiesis. The possibility of therapy interfering with the last of these processes, which may be more ‘resistance proof’, is discussed in detail.