Antibiotic resistance and phage therapy

The development of resistance towards fitness-reducing agents often occurs among organisms that thrive in large numbers and have short generation lengths. The evolution of resistance has been observed over a range of lineages, including insects (Mallet et al., 1990), rodents (Ishizuka et al., 2008) and bacteria. The latter represents one of the major concerns in modern healthcare (Spellberg et al., 2013).

3.1.1 Antibiotic resistance

Antibiotics are compounds that kill or inhibit the growth of microorganisms or, from an evolutionary viewpoint, reduce their fitness. Although most commercial antibiotics are produced synthetically (Nussbaum et al., 2006), antibiotics have been abundant in nature for millions of years (Siettos and Russo, 2013; Spellberg et al., 2013). Not surprisingly, resistance towards antibiotics has been prevalent for an equal amount of time. The first signs of resistance to commercial antibiotics were discovered even before penicillin was widely in use (Koopman and Longini, 1994; Abraham and Chain, 1940). Soil bacteria are known to harbor resistance (Riesenfeld et al., 2004) and recently a bacterial strain isolated

in a cave for 4 million years was found to be resistant to 14 different “commercial”

antibiotics (Bhullar et al., 2012).

The use of antibiotics generates selection gradients, in which resistant mutants are favored over the wild types. Selection is most efficient in drug concentrations that settle within the “mutant selection window”, whose lower boundary marks the minimum inhibitory concentration and upper boundary the mutant prevention concentration (Drlica, 2003). Resistance may rise due to a random mutation in the bacterial genome or, more commonly, due to horizontal gene transfer (HGT) (Bennett, 2008). The workhorses of HGT, plasmids, are circular DNA elements that depend upon the cellular machinery of their host bacterium to replicate. Plasmids can survive in the extracellular medium as passive elements and occasionally become absorbed by bacteria by the process of transformation. Many plasmids grant their host antibiotic resistance and also the ability to perform inter-bacterial HGT. Such direct transfer of genetic material between bacteria is called conjugation. The host’s capability to conjugate vastly improves a plasmid’s chance of spreading in the bacterial population (Bennett, 2008).

Annually, hundreds of thousands of lives are being lost due to the declining strength of antibiotics (World Health Organization, 2012). A significant factor in the development of antimicrobial resistance is the overuse of antibiotics in medicine. Oftentimes antibiotics are prescribed to patients with symptoms stemming from viral infections – conditions against which antibiotic treatment is completely futile. It has been estimated that 60% of general antibiotic prescriptions are for the treatment of respiratory tract infections – a set of conditions that usually arise from viral infections (Lindbaek, 2006). In developing countries, antibiotics are usually obtainable without prescription (Hart and Kariuki, 1998).

Loose medicinal legislation in poor and overpopulated areas may be imperative for people with no possibility to consult a doctor. The resulting misuse of antibiotics in turn favors the emergence of resistance. For example, the use of the antibiotic ciprofloxacin in developing countries has been showing alarming rise in resistant strains since the 1990s (Green and Tillotson, 1997; Rahman et al., 2014).

In addition to consumption by humans, the farm industry employs antibiotics in the treatment of sick farm animals. In many countries antibiotics are also pre-emptively administered by integrating them in animals’ diets – a practice already banned in the European Union (Clark et al., 2012). Not only does careless agricultural use drive bacterial

evolution in the animals, it also releases large amounts of antimicrobials to the environment.

3.1.2 Phage therapy

Bacteriophages are bacteria-infecting viruses. Even though phage therapy (PT) has been the subject of increasing speculation during the last two decades or so, the treatment method itself dates back to pre-antibiotic times. The notion of using phages in battle against bacteria was hypothesized as soon as phages were first discovered (d' Hérelle, 1926) and put to practice a few years later (Eaton, 1934). This novel form of therapy drew interest from industry and research worldwide until the 1930s and 1940s. At this time, antibiotics were discovered. Their ease of use contrasted with difficulties involved in PT studies; soon enough antibiotics were embraced as the evident remedy for curing infections. Consequently, interest on PT declined. Currently, it subsists as an approved treatment method only in Russia and Georgia and as an ‘experimental treatment’ in Poland (Levin and Bull, 2004; Pirnay et al., 2010). Phages are also used in the food-industry in the United States as preservatives (Sillankorva et al., 2012). The modern interest in PT began when the efficacy of antibiotics became questioned due to the alarming rise in nosocomial, antibiotic resistant bacterial strains. Pharmaceutical companies have nevertheless shown little interest in developing the treatment, as monetary requirements are high with moderately minor benefits to be expected (Thiel, 2004).

Recently, Jalasvuori and colleagues studied the effects of confronting antibiotic resistant bacteria with plasmid-dependent phages (Jalasvuori et al., 2011). This specific class of phages only infects bearing cells. Unlike conventional phages, plasmid-dependent phages are capable of infecting a wide range of bacterial species, as long as the bacteria exhibit a plasmid-borne conjugation apparatus. Since plasmids often associate with antibiotic-resistance, this approach might prove useful in direct eradication of resistant cells. The study found that phage-dependent cells are highly effective in eradicating antibiotic resistance from a bacterial population. Some cells were also observed to lose the plasmid and consequently become resistant to phages and susceptible to antibiotics. Additionally, a small fraction of cells gained resistance towards phages while still retaining antibiotic resistance. Phage-resistance arose through the mutation of the conjugation apparatus, thus rendering the cell incapable of conjugation. The emergence of these mutants complicates population dynamics and gives rise to further questions. For

example, does the small population of phage-resistant bacteria still pose a threat to the patient? Is removing the ability to conjugate enough to tip the balance and eradicate the pathogen?

In light of the arising threat of antibiotic-resistance and promising recent discoveries in phage therapy, clinical trials following modern standards have become topical. Before such trials are initiated, in silico approaches may be used to review the role of phage therapy and other pre-emptive measures. The confined safety of computer circuitry allows us to test scenarios without having to account for the limitations of the real world.