Evolution of antibiotic resistant bacteria

Evolution of antibiotic
resistant bacteria
Tremendous quantities of antibiotics are produced and released into the environment.

90 – 180 million kg/year of antibiotics are used (enough for 25 BILLION full treatment courses ~ 4 per human/yr!)

About 10 X more antibiotics are used in agriculture than to treat people. (Levy 1997 estimated 30 X more in animals than in people).
“There are two major effects of an antibiotic: therapeutically, it treats the invading infectious organism, but it also eliminates other, or non-disease producing, bacteria in its wake. The latter do, in fact, contribute to the diversity of the ecosystem and the natural balance between susceptible and resistant strains.…
“The consequence of antibiotic use is, therefore, the disruption of the natural microbial ecology. This alteration may be revealed in the emergence of types of bacteria which are very different from those previously found there, or drug resistant variants of the same ones that were already present.”
Levy, 1997
Alexander Fleming (discoverer of penicillin) recognized the potential danger of antibiotic resistance.
In 1945, he warned that misuse of penicillin could lead to the selection and propagation of mutant forms of bacteria resistant to the drug.
The first penicillin-resistant bacteria appeared several years later. Their mutant gene encoded for a penicillin-destroying enzyme, penicillinase.
"... the mounting use of antibiotics, not only in people, but also in animals and in agriculture, has delivered a selection unprecedented in the history of evolution."
Levy, 1997
The development of penicillin resistance in gonococcal populations from 1980 through 1990 was incredibly rapid.
Figure 6.8 in Atlas and Bartha, 1998
Other examples:
In Japan, in 1953, only 0.2% of Shigella (causes bacillary dysentery) was resistant to antibiotics.

By 1965, 58% were resistant to sulfnilamide, streptomycin, chloramphenicol, and tetracycline.

Penicillin was extensively used in Hungary in the early 1970`s. By 1976,
> 50% of the strains of
Streptococcus pneumoniae were resistant to penicillin.

Intensive care units seem to have particularly high incidences of resistant microbes.
Figure 10.27 from Atlas, 1997
Bacteria become resistant through:
Adaptations (product of selection)

Acquisition and transmission of antibiotic resistance (horizontal gene transfer)
Evolution through natural selection can occur remarkably quickly when selection pressures are very strong and reproductive rates are very fast
(some bacteria generations are as short as 15-20 minutes!)
Genes that code for antibiotic resistance were in the gene pool before humans began to produce antibiotics over 50 years ago.
This was shown clearly by some experiments by Joshua Lederberg.
Fig 7.3 Volpe and Rosenbaum, 2000
Mutations furnish the source of genetic variability and natural selection acts upon that variability to generate adaptation (antibiotic resistance)
Mechanisms of resistance:
Decreased transport of the antibiotic into the cell membrane.

Production of enzymes that destroy the inhibitory capacity of the antibiotic (e.g. by hydrolyzing it so that it loses its inhibitory ability).
Modification of the antibiotic binding site so that the drug no longer binds to the target.

Production of alternate molecules that can replace those disrupted by the antibiotic.
Mechanisms of resistance:
Production of mechanisms to pump antibiotics out of cells

Production protective biofilms (upper layers protect lower layers)
Mechanisms of resistance:
Acquisition and transmission of antibiotic resistance
Bacteria often exchange resistance genes through R plasmids
Fig 7.4 Volpe and Rosengaum, 2000
Up to a thousand plasmid copies may exist in a cell and each one may carry as many as 300 different genes.
Transposons can move small DNA elements (including resistance genes) into bacterial chromosomes and/or bacteriophages.
Acquisition and transmission of antibiotic resistance
R plasmids spread easily from one bacterium to even across species because they are conjugative plasmids, meaning that they not only code for antibiotic resistance but also for mating, which increases the rate of transfer.
Acquisition and transmission of antibiotic resistance
Antibiotics are probably driving the evolution and spread of resistance plasmids
Fig 10.28 Atlas, 1997
Stuart Levy has conducted many studies of antibiotic resistance in E. coli associated with chickens.

When chickens that hosted E. coli with multi-resistance plasmids were kept in a clean and isolated part of the barn, they did not lose the resistant bacterial strains over many months of the study.
 
But, when the chicken’s cages were relocated to different sites around the barn, then the incidence of antibiotic resistant E. coli was slowly reduced in the chicken’s microflora community.

In another study, 4 chickens excreting resistant flora were added to 10 chickens excreting susceptible flora. Resistance was lost, the susceptible flora won out.
 

What was the role of adding chickens that harbored susceptible microflora?
 

“For immediate change in resistance frequency, the result relies on numbers, not large differences in bacterial fitness. Moreover, there is no active counter-selective force which propels repopulation with susceptible strains.”
Levy 1997
 
Coevolution = An intimate and interactive evolutionary relationship between two or more species in which direct genetic change in one species is attributable to genetic change in the other(s).
The “Red Queen Hypothesis”
In 1973 VanValen referred to the difficulty faced by species locked in a coevolutionary arms race as the “Red Queen Problem.” Recall in Lewis Carroll’s Red Queen who had to run faster and faster just to stay in place.
This is an appropriate analogy: the environment constantly changes and populations must continue to evolve to survive.
Antibiotics are now everywhere in the environment, and humans and bacteria are engaged in an arms race.

Who is likely to win?
“…perhaps the very way we fight infection should be reconsidered. As in other aspects of our social behavior, we identify sometimes-annoying creatures as mortal enemies and are determined to annihilate them.”
Amabile-Cuevas, 2004
Therapies in the post-antibiotic era
May target virulence factors instead of the entire organism.
Develop vaccines to prevent infection in the first place.
Analogous to “biological control” and “integrated pest management” strategies used in agriculture and manipulate competitors or parasites of virulent organisms?
Therapies in the post-antibiotic era
May target virulence factors instead of the entire organism.
e.g. Design drugs that target the adhesion of virulent bacteria to a tissue. These drugs would have the advantage of slowing selection for resistance because they would not kill the bacteria.
Therapies in the post-antibiotic era
May target virulence factors instead of the entire organism.
e.g. Develop drugs that target the plasmids that contain the resistance genes. This would be appropriate in the treatment of Bacillus anthracis in which the virulence factor is contained on a plasmid.
Develop vaccines to prevent infection in the first place.
DNA and protein sequences can reveal potential drug targets and facilitate the production of vaccines.
e.g. The genome sequence of Neisseria meningitiidis is helping to identify candidates for a vaccine against this organism
Therapies in the post-antibiotic era
Therapies in the post-antibiotic era
Manipulate competitors or parasites of virulent organisms.
e.g. establish healthy communities of microorganisms in ears and gastrointestinal tracks
“... Some have talked about spraying hospital rooms with susceptible commensal organisms to replace and compete with the disease agents."
Levy, 1997
Phage Therapeutics International Inc. is a public Washington company formed to develop, manufacture, and acheive regulatory approval of phage pharmaceutical products for the treatment of antibiotic-resistant and other bacterial infections. They are currently performing studies to establish the safety and efficacy of phage treatments against Staphylococcus aureus and S. epidermis.

“Phage have several advantages over traditional antibiotics. One advantage is that phage multiply exponentially, just like bacteria. A small initial dose of phage will multiply as it infects cells, diminishing the need for repeated administrations. Phage can also mutate during replication, just as bacteria do…

… Thus, the same mechanism that may lead to antibiotic or phage resistant bacteria can produce new phage that recognize altered bacteria. One side-effect of traditional antibiotics is the killing of useful bacteria, such as those that help us digest our food or compete with more dangerous bacteria. The specificity of phage reduces the chance that useful bacteria are killed when fighting an infection.”
“Studies in mice show protection against otherwise lethal infections. Following an independent test of safety and efficacy under cGLP (current Good Laboratory Practices) guidelines, a financing round will precede Phase I clinical trials. Patent claims include phage compositions, production methods, and uses for treating diseases in humans and animals. Patents are also being prepared for novel producer bacterial strains for mass production of therapeutic phage product candidates.”