Serial Passage and the Origin of Resistance
A student performs serial passage of Escherichia coli K-12 for 12 transfers, each 24 hours, in LB broth containing ampicillin. The ampicillin concentration doubles every two transfers, from 2 µg/mL to 128 µg/mL. A control population is passaged in LB broth without ampicillin. After passage, the treated population grows at 256 µg/mL, while the frozen ancestral strain does not grow above 4 µg/mL. The student streaks the evolved population on ampicillin plates and finds three colony morphologies; one isolate carries a plasmid with a beta-lactamase gene. The student writes, "The bacteria became resistant because they were exposed to ampicillin, which proves that exposure causes beneficial mutations." The teacher asks the student to make a more precise claim and to propose a test that could distinguish selection of pre-existing variants from mutation induced by the antibiotic.
1. Critique the student's claim. What does the experiment demonstrate, and what does it not demonstrate?
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The experiment demonstrates that the treated population acquired heritable resistance to a much higher ampicillin concentration than the ancestor, and that resistance can be selected under antibiotic exposure. It does not demonstrate that ampicillin caused the beneficial mutation. The more precise claim is that genetic variation for resistance existed or arose in the population, and ampicillin exposure selected the resistant variants. The plasmid carrying beta-lactamase may have been present at low frequency before passage, or it may have been acquired during passage, but the experiment does not establish which.
The phrase 'exposure causes beneficial mutations' is a Lamarckian claim that the data cannot support. Selection does not create mutations; it changes allele frequencies. The control population is useful because it shows that passage in LB alone did not produce the same high-level resistance, but the control does not prove that the antibiotic induced the mutation. The three colony morphologies also raise the possibility of multiple genotypes or contamination. The student should avoid claiming a mechanism until a test separates pre-existing variation from induced mutation.
2. Describe an experiment that could distinguish selection of pre-existing resistant variants from mutation induced by ampicillin.
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One approach is a fluctuation test. The student would set up many independent small cultures from the same ancestral population, grow them without ampicillin, and then plate each entire culture on ampicillin. If resistant variants arise randomly before selection, the number of resistant colonies per culture will vary widely, with occasional 'jackpot' cultures. If resistance is induced only by exposure, the counts should be more evenly distributed, approximately Poisson. A related approach is replica plating: grow a lawn of the ancestor on nonselective medium, replica-plate it onto ampicillin, and see whether resistant colonies appear at positions that were already present before exposure. That demonstrates pre-existing resistant variants.
The student could also freeze samples at each transfer, sequence the plasmid and chromosomal genes, and test whether the same resistance allele is present at low frequency in the ancestor. Another test is to passage the evolved population without ampicillin for several generations and check whether resistance is stable; stable resistance is consistent with a heritable genetic change, though it does not by itself distinguish selection from induction. Combining a fluctuation test with molecular detection of the resistance determinant would be strongest.
3. What do the control population and the three colony morphologies tell you, and what should the student do next?
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The control population shows that passaging in LB without ampicillin does not by itself produce growth at 256 µg/mL. That strengthens the inference that the antibiotic selection, rather than ordinary laboratory passage, was responsible for the high-level resistance. It does not prove that the antibiotic caused a mutation, but it rules out a simple effect of time in culture.
The three colony morphologies indicate that the evolved population is not homogeneous. It may contain multiple independent resistant mutants, a mixture of resistant and sensitive cells, or a contaminant. Before making claims, the student should streak for single colonies, confirm purity and identity, and measure the minimum inhibitory concentration for each isolate separately. Plasmid profiles, transformation of the plasmid into a sensitive strain, and sequencing of the beta-lactamase gene would test whether the plasmid is sufficient for resistance. The student should also check whether the three morphologies differ in resistance level; if only one carries the plasmid, the others may use a different mechanism. These steps would turn a mixed population result into a testable genetic claim.