Evidence for evolution, resistance and genetic drift

How antibiotic resistance arises

A bacterial population may contain resistance variants before treatment; additional variants can arise during replication or arrive by horizontal gene transfer. An antibiotic inhibits susceptible bacteria more strongly than resistant bacteria, so resistant cells leave a larger proportion of the descendants. The antibiotic selects existing or newly arising variation; it does not direct bacteria to acquire the required mutation.

The numbers make pre-existing variants likely. Bacteria divide quickly, and Escherichia coli can manage it in twenty minutes under favourable conditions. A low mutation rate per base per division, multiplied by a population of 10⁹ cells in a single infection, gives a high probability that some cells carry a mutation affecting a protein the drug acts on.

Describe the drawing

Three circular dishes of bacteria in a row. In the first, sixty cells are drawn and two of them are coral, marking cells that already carry a resistance allele before any antibiotic is given. In the second, the grey susceptible cells are shrunken and dying while the two coral cells are untouched. In the third the dish is full again and every cell is coral. Arrows run between the dishes and the allele frequency is printed underneath as rising from zero point zero three to one.

The mechanisms of resistance are named separately, since a question may ask how a stated mutation causes resistance. A mutation may change the shape of the drug's target protein so the drug no longer binds. This is how streptomycin and rifampicin resistance work, by altering the ribosome and RNA polymerase. It may produce an enzyme that destroys the drug: β-lactamase hydrolyses the β-lactam ring of penicillins. It may increase production of an efflux pump that removes the drug faster than it enters, or reduce the permeability of the cell envelope.

The selection that follows is severe: for a susceptible cell, relative fitness in the presence of the drug is close to zero. Susceptible cells are inhibited or killed while resistant cells are not, and because the susceptible cells were also competitors for nutrients, the survivors grow into space and resources previously shared. Within hours the population is rebuilt from the survivors and most cells carry the resistance allele. No cell changed its genotype in response to the drug.

Antibiotic

A substance produced by a microorganism, or a synthetic derivative of one, that kills or inhibits the growth of bacteria.

Antibiotic resistance

The ability of a bacterium to survive and reproduce in a concentration of an antibiotic that would kill or inhibit others of its species.

Vertical gene transfer

Passing genes from a parent cell to its daughter cells by binary fission.

Horizontal gene transfer

Passing genes between bacteria that are not parent and offspring, most often on a plasmid transferred by conjugation.

Resistance often carries a cost. A ribosome altered so that streptomycin cannot bind is usually less efficient, and a cell running efflux pumps expends ATP on them. In the absence of the drug, resistant strains are often at a small disadvantage, so resistance frequencies can fall when use of an antibiotic is reduced across a population. The effect is limited: compensatory mutations can restore the lost efficiency while retaining the resistance.

Bactericidal and bacteriostatic antibiotics

Antibiotics fall into two groups by what they do to the bacteria they reach. A bactericidal antibiotic kills bacteria, so the number of living cells falls while the drug is present. A bacteriostatic antibiotic stops bacteria growing and dividing without killing them, so the number of living cells stays roughly where it was, and the infection is cleared by the patient's phagocytes and antibodies. Pearson Edexcel Biology B names penicillin as the example of the first and tetracycline as the example of the second.

Describe the drawing

A graph of the number of living bacteria per cubic centimetre, on a logarithmic scale from ten cubed to ten to the ninth, against hours after an antibiotic is added. Three cultures start at ten to the sixth. The grey culture with no antibiotic rises towards ten to the ninth. The cyan culture given a bacteriostatic antibiotic stays level at ten to the sixth. The coral culture given a bactericidal antibiotic falls steadily to below ten cubed after eight hours.

Penicillin acts on the cell wall. The wall of a bacterium is peptidoglycan (murein): long polysaccharide chains joined to one another by short peptide cross-links, which give the wall its strength. Penicillin binds to and inhibits the enzymes, transpeptidases, that form those cross-links. A growing cell is constantly opening its wall to insert new material, so in the presence of penicillin it goes on building wall that cannot be cross-linked and is weak. The cytoplasm has a lower water potential than the fluid outside, water enters by osmosis, and with no strong wall to resist the pressure the cell bursts. That is osmotic lysis, and it is why penicillin is bactericidal. It also explains two limits on the drug: it affects only bacteria that are growing and making new wall, and it does not act on human cells, which have no cell wall.

Tetracycline acts on protein synthesis. It enters the bacterial cell and binds to the small (30S) subunit of the 70S ribosome, where it stops tRNA molecules carrying amino acids from binding. No further amino acids are added to the growing polypeptide, so the cell cannot make the enzymes and structural proteins it needs to grow and divide. The cell is not destroyed, and the binding is reversible: if the drug is removed before the immune system has cleared the bacteria, they start to grow again. Human cells are much less affected because their cytoplasmic ribosomes are the larger 80S type and bacteria take up the drug far more readily than human cells do.

Bactericidal antibiotic

An antibiotic that kills bacteria, so the number of living cells falls while it is present. Penicillin is an example.

Bacteriostatic antibiotic

An antibiotic that stops bacteria growing and dividing without killing them, leaving the immune system to remove them. Tetracycline is an example.

The difference matters clinically because a bacteriostatic drug only holds the population still. Clearing the infection is left to the patient's white blood cells, so where those are few or cannot reach the bacteria well, a bactericidal drug is normally preferred. Examples are a patient whose neutrophil count has been lowered by chemotherapy, and infections of the heart valves or of the membranes around the brain. There is a second consequence. Penicillin kills only growing cells, so giving a bacteriostatic drug alongside it can reduce its effect, since the cells it would have killed have stopped growing.

The two groups are not absolute. Whether a drug kills or only inhibits can depend on its concentration and on the species of bacterium, and a drug is classed by what it does at the concentrations used in treatment.

A clear zone round an antibiotic disc on an agar plate, the result of the core practical on the effect of different antibiotics, shows that growth was prevented but cannot show which kind of drug prevented it. To find out, sterile technique is used to transfer a sample from the clear zone to fresh agar or broth with no antibiotic and incubate it. Growth means the cells were alive and only held back, so the drug was bacteriostatic at that concentration; no growth means they had been killed. Counting viable cells at intervals after adding the drug, as in the figure, gives the same answer quantitatively.

Two processes behind a rise in resistance

A rise in the frequency of resistance involves two distinct processes, which a complete answer separates.

The first is vertical transfer: a resistant cell divides and both daughters are resistant. This makes the frequency of a resistance allele rise within a population, and it is ordinary natural selection running at the speed bacteria divide.

The second is horizontal transfer, which has no equivalent in sexual inheritance between parent and offspring. Resistance genes are very often carried on plasmids, small circular loops of DNA separate from the main chromosome, and a plasmid can be copied and passed to another bacterium through a conjugation tube, including one of a different species. A plasmid may carry resistance to several antibiotics at once, which is how multiple resistance appears in one step. Bacteria also take up free DNA by transformation, and bacteriophages move DNA between cells by transduction.

A rise in resistance therefore involves both an increase in the number of resistant lineages and movement of the genes between lineages and species. The second accounts for a resistance gene first recorded in a harmless gut bacterium later appearing in a pathogen. MRSA, meticillin-resistant Staphylococcus aureus, rests on an acquired gene, mecA, encoding a penicillin-binding protein that β-lactam antibiotics cannot inhibit.

Describe the drawing

A graph of bacterial numbers on a logarithmic scale from ten cubed to ten to the ninth against days after the first dose. Two curves lie on top of each other for the first three days as the population falls. At an amber dashed line marking day three the coral curve turns and climbs back to its starting height, while the cyan curve carries on falling until it leaves the bottom of the scale.

Treatment duration acts on the same distribution. The cells in an infection are not equally susceptible, and the most susceptible are inhibited first. Treatment stopped when symptoms resolve leaves a non-random sample of cells: the least susceptible, including any carrying partial resistance. Those cells can regrow, and the population that follows is less susceptible than the one that started.

The evidence on duration is more qualified than the usual summary suggests. Since around 2017 a number of clinicians have argued that unnecessarily long courses prolong selection on a patient's other bacteria, and shorter evidence-based courses are now prescribed for several infections. Patients should take antibiotics exactly as prescribed; the appropriate duration depends on the infection, the drug and current clinical evidence.

Hospital-acquired infections and codes of practice

A hospital-acquired infection (HAI) is one a patient picks up while in hospital: it was neither present nor incubating when they were admitted, and for counting purposes it is usually one that appears 48 hours or more after admission. The two best-known examples are MRSA and Clostridioides difficile, still often written Clostridium difficile or C. diff.

C. difficile shows how antibiotic use itself causes infection. It is an anaerobic bacterium that forms spores and can live in the gut in small numbers, held down by competition from the much larger population of harmless gut bacteria. A broad-spectrum antibiotic kills many of those competitors while C. difficile survives, so it multiplies and releases toxins that damage the lining of the colon, causing diarrhoea and in severe cases life-threatening colitis. Its spores survive for long periods on surfaces and are not killed by alcohol hand gel.

The contributory causes explain why hospitals concentrate infections, and resistant ones in particular:

Heavy antibiotic use. Patients in hospital receive antibiotics far more often than people in the community, so selection for resistance is strong and continuous, and broad-spectrum drugs clear the way for C. difficile.

Vulnerable patients. Many patients are elderly, recovering from surgery or have immune systems weakened by illness or treatment, so a smaller dose of a pathogen is enough to cause disease.

Breaches of the body's barriers. Surgical wounds, urinary catheters, intravenous cannulas and ventilator tubes give bacteria a route past the skin and mucous membranes.

Transmission between patients. Patients are close together and staff move from one to the next, so bacteria are carried on hands, clothing and shared equipment. Some patients and staff carry Staphylococcus aureus, including MRSA, in the nose or on the skin without any symptoms, and patients are moved between wards and between hospitals.

Each cause has led to a part of the codes of practice hospitals now follow. These fall into two groups: rules on prescribing antibiotics, often called antimicrobial stewardship, and rules on infection prevention and control.

Prescribing codes reduce the selection pressure. Antibiotics are prescribed only where a bacterial infection is likely, never for a viral one. A sample is taken for culture and sensitivity testing before treatment where possible, so a broad-spectrum drug started on suspicion can be changed to a narrow-spectrum one that targets the organism found. Prescriptions are reviewed after two or three days and stopped or changed if they are not needed, which in England is the basis of the national guidance called Start Smart then Focus. Some antibiotics are held in reserve and used only on a microbiologist's advice, so resistance to them is not selected for while other drugs still work.

Infection prevention and control codes break the chain of transmission:

Surveillance ties the two together. In England hospitals must report cases of MRSA bloodstream infection and C. difficile infection, so a rise on one ward or in one hospital is noticed and investigated. Both fell substantially in the years after these codes were brought in, but the measures arrived together, so the data cannot show how much each one contributed.

Hospital-acquired infection

An infection that a patient acquires while in hospital, which was not present or incubating on admission. MRSA and Clostridioides difficile are examples.

Infection prevention and control

The hospital practices that stop pathogens spreading between patients, such as hand hygiene, screening, isolation and cleaning.

Pesticide and herbicide resistance

The same mechanism operates in agricultural populations. Each case has four components: variation already present, a severe and uniform selection pressure applied by people, a short generation time, and a large population.

Insecticide resistance in mosquitoes is one example. Anopheles carrying a mutation in the voltage-gated sodium channel, the target of pyrethroids, in a change called knockdown resistance, survive treated bed nets that kill their neighbours. Pyrethroid resistance is now widespread across sub-Saharan Africa, within a couple of decades of nets being distributed at scale.

Warfarin resistance in rats shows a different outcome. Warfarin blocks the enzyme VKORC1, which recycles vitamin K, so poisoned rats bleed to death, and a resistance allele spread rapidly through British rat populations after warfarin was introduced in the 1950s. But homozygous resistant rats need far more vitamin K than they can normally obtain, so where warfarin is used the heterozygote is the fittest genotype and the population settles at an intermediate allele frequency rather than going to fixation. That is heterozygote advantage, the same pattern as in the sickle-cell allele.

Herbicide resistance follows from the same mechanism, and glyphosate-resistant weed populations are established on farmland in North America and Australia. The agricultural response is designed around the biology: rotating chemicals with different modes of action so no single pressure is applied for long, and, for insect-resistant Bt crops, planting a refuge of non-Bt plants alongside. The refuge keeps susceptible insects breeding, so a rare resistant survivor is likely to mate with a susceptible one and produce heterozygous offspring, which the toxin still kills where resistance is recessive.

Four independent lines of evidence

Resistance observed in a bacterial population demonstrates the mechanism. The claim that the same mechanism produced the diversity of life rests on separate evidence, drawn from fields whose findings could have conflicted and do not.

Palaeontology. The fossil record puts organisms in an order: simple prokaryote-like forms in the oldest rocks, and major groups appearing in a sequence matching the order predicted by their anatomy. Transitional fossils exist where the argument most needs them: Tiktaalik between lobe-finned fish and tetrapods, Archaeopteryx between theropod dinosaurs and birds, and a series of whale ancestors with progressively reduced hind limbs. The record is incomplete, because fossilisation requires rapid burial and soft tissues rarely survive. State that limitation alongside the evidence.

Comparative anatomy. The pentadactyl limb is the standard case. The forelimb of a human, a bat, a whale and a horse is built on the same plan of bones: one upper arm bone, two forearm bones, wrist bones and digits, although in the horse the ulna is reduced and only one digit is fully formed. The limbs perform different functions. Independent origins would not predict the shared arrangement; descent with modification from a common ancestral limb does. Structures like this are homologous: same origin, different function, evidence of divergent evolution. Analogous structures, such as the wing of a bird and the wing of an insect, perform the same function with different underlying structures, and are evidence of convergent evolution rather than of relatedness.

Comparative biochemistry. Living organisms share core molecular systems: a nearly universal genetic code, the same set of twenty amino acids in proteins, and ATP as the immediate energy currency. A small number of variant genetic codes are known, in some mitochondria and some protoctists, and these are themselves derived from the standard code. Conserved molecules can be compared between species. Cytochrome c is identical in humans and chimpanzees, differs by about a dozen amino acids between humans and horses, by around twenty between humans and a tuna, and by more than forty between humans and yeast, a pattern matching the branching order the fossils and the anatomy already gave. Ribosomal RNA does the same job over greater distances, and it was rRNA comparison that separated the archaea from the bacteria. Where mutations accumulate at a steady rate, the number of differences estimates the time since two lineages diverged: a molecular clock.

Biogeography. The distribution of species across the land masses follows their history rather than their climates. Most of Australia's native land mammals other than bats are marsupials, filling niches that placentals fill elsewhere, because the continent became isolated before placental mammals spread there. Its native placentals are bats, which could fly in, and rodents that reached it from Asia far more recently. Oceanic islands hold endemic species closely related to those on the nearest mainland, among them the Galápagos finches and the Hawaiian Drosophila, and, before humans arrived, characteristically lacked whole groups such as native land mammals and amphibians that cannot cross an ocean.

Exam-board note. OCR Biology A asks explicitly for fossil, DNA and molecular evidence in module 4 (4.2.2(e)), alongside Darwin and Wallace. AQA covers the same material largely through DNA and amino acid sequence comparison in its classification content, and Cambridge International examines molecular evidence alongside classification. The argument in each case is that the lines of evidence are independent and agree, rather than that any one of them is decisive alone.

Darwin, Wallace and the formulation of the theory

Exam-board note: OCR Biology A requires the historical contributions of Darwin and Wallace to the formulation of the theory. Neither man originated the idea that species change. That had been in circulation for decades, and Jean-Baptiste Lamarck had published a mechanism for it in 1809, in which characteristics acquired during an organism's life were passed on. What was missing was a mechanism that worked, and both Darwin and Wallace arrived at the same one independently.

Charles Darwin sailed as naturalist on HMS Beagle from 1831 to 1836 and spent the voyage collecting and recording. The observations that mattered most came from South America and the Galápagos: fossils of extinct giant mammals that plainly resembled the living animals of the same continent, mockingbirds and finches that differed from island to island, and giant tortoises whose shell shape a resident could use to say which island an animal came from. Back in England he read Thomas Malthus on human population in 1838, and the missing piece arrived with it: populations produce far more offspring than can survive, so most of them must die before breeding, and which ones die is not random. Darwin then spent twenty years gathering evidence, including eight years on barnacles, breeding pigeons, and corresponding with animal breeders and gardeners across the country, and published nothing on natural selection.

Alfred Russel Wallace was a working collector who funded his own expeditions by selling specimens, first in the Amazon and then across the Malay Archipelago. He noticed the same regularities Darwin had, and rather more sharply on distribution. The faunal boundary now called the Wallace Line is his: it runs between Borneo and Sulawesi and on between Bali and Lombok, and puts an Asian fauna on one side of a strait a few tens of kilometres wide and a quite different one on the other. In 1858, ill with fever on Ternate, he wrote a short essay setting out natural selection and posted it to Darwin, whom he knew as a correspondent, asking him to pass it on if he thought it worth anything.

What happened next is why the theory carries both names. Darwin, recognising that Wallace had reached the same mechanism, put the matter to his friends Charles Lyell and Joseph Hooker, and on 1 July 1858 the Linnean Society of London heard Wallace's essay read alongside extracts from Darwin's unpublished work of 1844 and a letter he had written in 1857. Neither man was present. Darwin then wrote On the Origin of Species at speed and published it in November 1859, which is why it is his book that changed the argument. The two men remained on good terms, and it was Wallace who later published a book called Darwinism.

Two limitations of their account are examinable. Neither of them knew how inheritance worked: Mendel's paper appeared in 1866 and was ignored until 1900, so Darwin was left with a blending theory that should have destroyed variation within a few generations, and he never solved it. And neither had any idea where new variation came from, because mutation was not described until the twentieth century. The modern synthesis of the 1930s and 1940s, which married natural selection to Mendelian genetics and to population mathematics, produced the version of the theory taught today.

Changes in allele frequency that selection did not cause

Not every change in a gene pool is adaptive. A difference between populations requires evidence of a fitness effect before it can be described as an adaptation.

Genetic drift is change in allele frequency caused by chance alone. In any finite population the alleles reaching the next generation are a sample of those in this one, and a sample deviates from the population it is drawn from. Which individuals breed, which gametes combine and which juveniles die from causes unrelated to their genotype all change allele frequencies without any difference in fitness. The smaller the population, the larger the expected deviation, for the same statistical reason that ten coin tosses depart further from half than a thousand do.

Describe the drawing

Two graphs side by side, each plotting allele frequency from zero to one against fifty generations for seven separate populations that all started at one half. In the left panel, for ten breeding adults, the coral traces wander widely and some reach zero or one and stay there. In the right panel, for a thousand breeding adults, the cyan traces stay in a narrow band close to one half throughout.

Drift eventually leads to fixation, with one allele reaching a frequency of 1 while the other is lost, and a lost allele can only return by mutation or migration. And drift can fix a mildly harmful allele or lose a mildly beneficial one, because in a small population chance overwhelms a small fitness difference. Conservation genetics treats small population size as a threat for that reason.

Genetic drift

A change in allele frequencies from one generation to the next caused by chance sampling of gametes, rather than by selection. Its effect is greatest in small populations.

Genetic bottleneck

A sharp reduction in population size that leaves the survivors carrying only a fraction of the original genetic diversity.

Founder effect

The reduced and unrepresentative genetic diversity of a population established by a small number of individuals separated from a larger population.

A bottleneck is drift acting through a sharp reduction in population size. Northern elephant seals were hunted down to a few dozen animals in the 1890s; the species has recovered to hundreds of thousands but retains little genetic variation, because the alleles lost were not present in the survivors and later breeding cannot restore them. Cheetahs show the same pattern from an older event, to the point that unrelated individuals accept skin grafts from one another.

The founder effect is the same sampling process at the establishment of a population rather than during its history. When a few individuals colonise an island or found a settlement, the alleles they carry are a small sample of the parent gene pool, and any allele they happen to carry at unusual frequency will be at that frequency in all their descendants. Hence conditions far commoner in some human populations than in the world at large: Ellis-van Creveld syndrome among the Old Order Amish, descended from around two hundred eighteenth-century founders, and variegate porphyria among Afrikaners, traceable to a single seventeenth-century couple. No selection for those alleles is involved.