CITATION: Baquero B, Negri MC, Morosini MI, Blazquez J. 1993. The role of selective antibiotic concentrations on the evolution of antimicrobial resistance. APUA Newsletter 11(4): 4.

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The role of selective antibiotic concentrations on the evolution of antimicrobial resistance
Bernando Baquero, Maria Cristina Negri, Maria Isabel Morosini, Jesus Blazquez
Department of Microbiology, Ramon y Cajal Hospital, National Institute of Health (INSALUD), Madrid, Spain

An unexpectedly rapid evolution of bacterial genes involved in antibiotic resistance is occurring under our eyes. A single gene encoding the widespread TEM-1 (or TEM-2) beta-lactamase, hydrolysing ampicillin, was changed in different ways so that now the enzyme is now able to inactivate third generation cephalosporins or monobactams. Modifications in a gene encoding a penicillin-binding protein (PBP2) in Streptococcus pneumoniae has provoked the frightening threat of beta-lactam resistance in the most common bacterial pathogen in the respiratory tract. Interestingly, when the "new" TEM or PBP genes involved in resistance were sequenced, it was frequently found that several mutations were present in the gene, suggesting that a cryptic evolution had occurred. That implies that each one of the 'previous' mutational events was in fact selected, and the resulting enrichment of the harboring bacterial clone favored the appearance of new, selectable mutations. In most cases, conventional antibiotic susceptibility tests failed to detect early mutations increasing only in a very modest amount the minimal inhibitory concentration (MIC) of the organism. In such a way, the use of the selecting antibiotic was non-discontinued and the mutation was selected.

Not only clinicians, but also microbiologists have frequently disregarded the importance of "low-level resistance," as it was assumed that the mutants exhibiting low MICs were unselectable, considering the high antibiotic concentrations attainable during treatments. From our point of view, that was a tragic mistake.

At any dosage, antibiotics create concentration gradients, resulting from pharmacokinetic factors such as the elimination rate of the tissue distribution. Most probably, the bacterial populations are facing a wide range of antibiotic concentrations after each administration of the drug. On the other hand, the spontaneous variability of microbial populations may provide a wide possibility of potentially-selectable resistant variants. Which is the antibiotic concentration able to select one of these resistant variants?

The answer is simple: any antibiotic concentration is potentially selective of a resistant variant if it is able to inhibit the susceptible population but not the variant harboring a mechanism of resistance. In other words, a selective antibiotic concentration (SAC) is such if exceed the minimal inhibitory concentration (MIC) of the susceptible population, but not that corresponding (even if it is very close) to the variant population. If the MICs of both the susceptible and the variant populations is surpassed, no selection takes place; and the same is true if the antibiotic concentration is below the MICs of both populations. Therefore, the selection of a particular variant may occur in a very narrow range of concentrations.

The continuous variation of antibiotic concentrations may resemble a tuning device which 'selects' at a particular radio frequency a particular emission. Under or over such a frequency the emission is lost. The 'valley' between the MICs of the susceptible and the resistant variant populations is the 'frequency signal' recognized by the SAC.

Because of the natural competition of bacterial populations in a closed habitat, the 'signal' is immediately amplified. The fit mutant shows an intensive, distinctive reproduction rate at the expense of the more susceptible population, leading to a quantum modification of the culture, as could be predicted by the 'periodic selection.'

The above proposed model on the effect of SACs was tested in the laboratory using mixed cultures of susceptible and resistant bacterial populations. A dense culture of an Escherichia coli strain harboring a wild TEM-1 beta-lactamase was mixed with their homogenic derivatives (obtained by directed mutagenesis) harboring the beta-lactamases TEM-12 (a single amino acid replacement with respect to the TEM-1 enzyme) and TEM-10 (a single amino acid replacement with respect to TEM-12). The relative proportions of the three strains were 90:9:1 of the total population. The mixture was incubated during 4 h with different antibiotic concentrations, and the composition of the total population was then analyzed by subculture. At a very low cefotaxime concentration, 0.008 mcg/ml, TEM-12 (conventional MIC=0.06 mcg/ml) began to be selected against TEM-1 (MIC=0.03 mcg/ml), reached a maximal selection (nearly 80% of total population) at 0.03 mcg/ml and was again displaced by TEM-1 at 0.06 mcg/ml. At this turn, TEM-1 was displaced by TEM-10 (MIC=0.25 mcg/ml) at 0.12 mcg/ml. As predicted, TEM-12 was only selected within a narrow concentration range. Therefore, low antibiotic concentrations efficiently select low-level resistant mutants. As far as this population is enriched, it may serve as a new source of secondary genetic variants (for instance TEM-10 can give rise to TEM-12), which were subsequently selected against the predominant population in new (higher) concentration intervals. Similar results were obtained when mixed susceptible and resistant Streptococcus pneumoniae populations were challenged with different low beta-lactam concentrations: intermediate resistant strains were selected over the predominant susceptible population only at discrete low-level concentrations.

Selection with high antibiotic concentrations will only give rise to high-level resistant variants. But during treatments, low-level concentrations, particularly in the so-called long-acting drugs, occurs with a higher frequency than the high-level ones, both in terms of time (duration) and space (different colonized locations in the human body), and therefore its overall selective power is certainly higher. Any treatment produces a low-level potentially selective antibiotic concentration for resistant bacteria.

Microbiology has been more concerned about the mechanisms of resistance than for population genetics or the evolutionary processes leading to the appearance and spread of antimicrobial resistance. The time is arrived to propose evolutionary models, serving the scientifically support measures against the environmental health damage produced by antibiotics.

The resistant population (R) will be efficiently selected over the susceptible one (S) in a short range of selective concentrations (in this case, 0.1-0.2 mcg/ml). Concentrations below or over this range are non-selective. A low concentration (0.01 mcg/ml) will "select" both S and R populations; a higher concentration (2 mcg/ml) will "counterselect" both S and R. In both cases, the selective power for R populations is very low (left figure). Selection takes place at particular concentrations (selective antibiotic concentrations or SACs).


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