A New Era: The Fight Against Antibiotic-Resistant Pathogenic Bacteria

Overview

Over 70 years ago, the discovery of antibiotic medications provided hope and much needed help for a population vulnerable to the burden of bacterial infections. However, heavy dependence on and unregulated use of antibiotics have led to the phenomenon of widespread antibiotic resistance. The emergence of infectious organisms with the ability to evolve complex mechanisms that promote resistance to medications has antiquated conventional treatment and produced a growing crisis of resilient infections. The present state and future fate of antimicrobial resistance was featured on October 12, 2011, at the symposium, The Fight Against Antibiotic-Resistant Pathogenic Bacteria: A New Era. Experts discussed the gravity of current circumstances and the difficulties of treating drug-resistant bacteria. They also discussed the potential to undertake novel strategies utilizing the bacteriophage system to produce efficient therapeutic compounds to which bacteria have limited resistance.

Barry Kreiswirth, from the University of Medicine and Dentistry of New Jersey, provided a brief history of the successful experimental application of penicillin in 1940 to treat Staphylococcus aureus, which led to its characterization as the "wonder drug." However, the wide spread use of this "wonder drug" incrementally led to the emergence of penicillin-resistant S. aureus, called Phage 80/81, which caused a global pandemic of infections. In 1959, a synthetic penicillin called methicillin was developed to combat resistant strains, but the appearance of methicillin-resistant S. aureus (MRSA) occurred two years later. The motif recurred when strains resistant to vancomycin, the current first-line therapy for MRSA, were reported in 2002.

Kreiswirth explained S. aureus's acquisition of antibiotic defense via three specific mechanisms: Mobile genetic elements such as plasmids encoding β-lactamases (which break open the β-lactam ring found in some antibiotics), movable chromosomal segments that alter the binding target site for penicillin, and complex transposable operons that activate resistance genes. The evolution of drug-resistant S. aureus has left the medical community critically shorthanded against MRSA infections, with only vancomycin, linezolid, and daptomycin as available therapeutic choices. More than 50% of nosocomial (hospital-acquired) S. aureus infections are known to be methicillin resistant.

This timeline illustrates the recurring motif of antibiotic resistance in S. aureus: Resistance appears after the introduction of penicillin, methicillin, and lastly vancomycin. S. aureus can produce resistant clones as a result of the mobile genetic elements that alter the susceptibility of the organism to the antibiotic penicillin. (Image courtesy of Barry Kreiswirth)

Kreiswirth then proceeded to his next case study of antibiotic resistance—air-borne Mycobacterium tuberculosis—and emphasized the significance of tuberculosis worldwide. Infecting 2 billion people and causing 1.6 million deaths a year, mostly in developing countries, drug-resistant tubercle bacillus (TB) is a global concern. Early on, researchers and clinicians observed that TB readily became resistant to streptomycin-only treatment and realized that combination therapy was a more effective method against drug resistance. Oddly, this combination therapy approach has not been optimized to treat other infectious diseases. Even with combination therapy, however, various classes of drug-resistant M. tuberculosis have emerged, and with them, decreasing cure rates. Unlike by S. aureus, the acquisition of resistance by M. tuberculosis relies on single mutations in chromosomal "housekeeping" genes (genes essential for cellular maintenance) rather than on interactions of mobile genetic elements.

In his presentation, Kreiswirth noted the co-localization of TB and human immunodeficiency virus (HIV) outbreaks: Untreatable HIV infections have led to co-infections and outbreaks of multi-drug-resistant TB, most notably in New York in the early 1990s and as recently as 2006 in South Africa. Moreover, treating resistant TB is cumbersome because of the time-consuming challenge of testing TB infections for their susceptibility (responsiveness) to available antibiotics in vitro, which can take months and can delay the use of standard antibiotics therapies that are effective for some patients. Interestingly, drug-resistance target genes have been elucidated for nearly all anti-tuberculosis agents, indicating that mutational analysis of the bacteria could help predict resistance profiles at a faster rate than does standard susceptibility testing. Currently, susceptibility is measured by comparing a bacterial cell culture's growth rate on a control medium with a culture's growth rate on a drug-containing medium. Efficient molecular methods for susceptibility testing, such as hybridization technology and mass spectrometry, would reduce processing time as well. However, these techniques are not widely available to clinics in the U.S., and they have few advocates in the pharmaceutical industry because of the small market for TB treatment innovations. In fact, there has been no new drug development for TB since ethambutol was developed in 1968.

Kreiswirth continued his presentation with the problem of Enterobacteriaceae resistance toward broad spectrum carbapenems, a class of anti-bacterial drugs of last resort. Members of the Klebsiella and Escherichia genera have developed resistance mechanisms such as carbapenemase enzymes that hydrolyze and destroy carbapenems, defective membrane porins that prevent antibiotic entry, and over-expressed efflux pumps that inhibit accumulation of carbapenem compounds within the cell. Most genes that confer resistance are on mobile elements, and conjugative plasmids may present with multiple resistance-conferring genes, making them difficult to study and manipulate. In addition, treatment-resistant strains are becoming an endemic in New York and New Jersey hospitals and are spreading to different parts of the world, rendering containment of antibiotic-resistant disease a pressing global challenge.

He closed his presentation by discussing the motivating factors for pharmaceutical corporations, who lack financial incentive to develop new antibiotics because treatments for chronic diseases have a wider margin for profit. Additionally, widespread use of antibiotics in cattle farming, rampant use by health care providers, lack of patient compliance, dependence on broad spectrum antibiotics, and failure to use combination therapy effectively are all factors that have hastened the development of antibiotic resistance. Kreiswirth emphasized the need for innovative strategies against drug-resistant infections, including tighter infection control standards in health care settings, improved rapid diagnostic and susceptibility testing, and better therapeutic vaccines and immunomodulators. Kreiswirth's discussion of the history and mechanisms of antibiotic resistance set the stage for a conversation about phage therapy as a novel approach to infectious disease management.

Vincent A. Fischetti, principal investigator of the Laboratory of Bacterial Pathogenesis and Immunology at the Rockefeller University, presented his work on bacteriophage lysins as promising anti-infectives for controlling gram-positive pathogens. Bacteriophages, viruses that infect bacteria, have been subject to million years of evolution and have developed mechanisms for infiltrating bacteria, surviving within the bacterial cell, and ultimately destroying of the host to release replicated viral progeny. The last step is primarily facilitated by bacteriophage lytic enzymes or lysins, which consist of a catalytic domain and a binding domain connected by a linker peptide. The lysin molecule binds with high affinity and specificity to cell wall-associated substrates in the bacteria and catalyzes the hydrolysis of important bonds within the peptidoglycan layer of that cell wall, leading to the wall's weakening and eventual degradation. Interestingly, Fischetti and colleagues have found externally introduced lysins effective against gram-positive bacteria but not against gram-negative bacteria, whose peptidoglycan layer the lysins cannot easily reach. Applying these results to the problem of bacterial infections, Fischetti's group proposed targeting gram-positive bacteria in mucus membranes, blood, and infected tissue using specific recombinant bacteriophage lysins, a technique known as phage therapy. Fischetti emphasized that colonizing bacteria such as S. pyogenes, S. pneumoniae, S. aureus, and Group B streptococci are responsible for many diseases, ranging from common ear infections to pneumonia, which could be curtailed if indigenous bacteria are eliminated.

Fischetti's group conducted experiments in mouse models, successfully demonstrating the ability of small amounts of intravenously administered lysin to eliminate streptococci in the oral cavity and to increase survivability against pneumococcal bacteremia. Similarly, nasally-delivered lysins have been shown to reduce the occurrence of S. pneumoniae-induced otitis media in the presence of concurrent influenza infection. In a pneumococcal pneumonia mouse model, the intraperitoneal infusion of lysin resulted in a survival percentage of 100%. By contrast, no mice in the control group survived longer than six days after infection. Fischetti expressed his amazement with these results and explained that since lysin molecules have extremely short half-lives, ranging from 20 minutes to 30 minutes, their potency and efficacy at acting on their targets must be very high. In addition, continuous infusion of lysin reduced bacterial vegetation and load by degrading the biofilm present on heart valves in rat models of endocarditis. This presents the possibility of combination therapy, where lysins can be used to break down biofilms and reduce vegetation, thus allowing antibiotics to reach their targets more effectively.

In the above experiment, mice were inoculated with pneumococci intranasally, and they developed pneumonia. The two experimental groups consisted of those receiving intraperitoneal injection of Cpl-1 lysin or those treated with amoxicillin, while the control group received buffer. The Cpl-1 group showed greatest survival percentage in the given time period, slightly better than the group receiving amoxicillin treatment. (Image courtesy of Vincent Fischetti)

Fischetti's group developed MRSA-specific lysins and injected them into rat wounds infected with MRSA, demonstrating improved prognosis and wound healing. Keeping clinical situations in mind, Fischetti proposed the use of lysins as prophylaxis to prevent staphylococcal infection during the irrigation of surgical sites before closure or when prosthetic devices are placed internally. Revisiting the concept of combination therapy, Fischetti's group conducted kinetic experiments to assess the potential synergistic effect of lysins on the efficacy of other antibiotic therapies. They found that with concurrent use of lyins, antibiotics can be used in smaller amounts than are usually needed and can be more effective against drug-resistant organisms than traditional antibiotics alone. In addition, Fischetti's group determined that phage lysins are not vulnerable to resistance, that their side effects are limited to minor allergic reactions, that they are not neutralized by specific antibodies, and that they are therefore able to be used for chronic therapy. Lysin therapy is being harnessed by ContraFect, a startup company in New York, to produce lysins specific for hospital-acquired MRSA infections. Clinical trials are scheduled to begin in July of 2012.

The second part of Fischetti's presentation was dedicated to profiling the binding of lysin enzymes to essential cell wall substrates to identify novel pathways in gram-positive organisms that could be used to produce antibiotics against which bacteria are less able to develop resistance. The reason bacteria are less resistant to lysins lies in the ability of lysins to act rapidly on carbohydrate targets within the cell wall. These targets are critical for bacterial viability and, hence, cannot be easily altered by the organism. Furthermore, through an evolutionary process, lysins are able to select their targets and to bind tightly to them. Fischetti advocates capitalizing on the phage lysins' natural target identification process by delineating cell wall-binding receptors and elucidating the pathway for these receptors' synthesis within the peptidoglycan layer. His group applied this approach to B. anthracis, distinguished a unique polysaccharide lysin target within the cell wall, and recognized an epimerase enzyme necessary for its synthesis. They subsequently crystallized the enzyme in the presence of its substrate and developed an inhibitor compound, termed Epimerox, to block its allosteric site. Initial experiments with Epimerox have demonstrated improved survivability in mice infected B. anthracis and have shown no evidence of resistance emerging. In conclusion, targets identified by phage lysins present promising opportunities for the development of new antibiotics directed at disrupting the cell wall structure.

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Presentations available from:
Vincent A. Fischetti, PhD (The Rockefeller University)
Barry Kreiswirth, PhD (UMDNJ — New Jersey Medical School)