Bacterial resistance to antibiotics: No argument for evoluti

Figure 1. Mechanism of ciprofloxacin resistance.

(A) Ciprofloxacin interacts with gyrase, inhibiting its enzymatic activity. (B) A mutation in either of the genes, gyrA or gyrB, can change the conformational structure of gyrase, and reduce the binding affinity of the enzyme for ciprofloxacin. This results in an inability of the antibiotic to inhibit the gyrase, and the cell becomes resistant to the antibiotic.

Resistance to streptomycin can also result from spontaneous bacterial mutations. In this case, streptomycin blocks bacterial protein synthesis apparently by binding to the 16S rRNA segment of the ribosome and interfering with ribosome activity (Carter et al., 2000; Leclerc et al., 1991). Resistance to the antibiotic can occur by mutations in the 16S rRNA gene, which reduces the affinity of streptomycin for the 16S molecule (Springer et al., 2001). Reduction of specific oligopeptide transport activities also leads to spontaneous resistance of several antibiotics, including streptomycin (Kashiwagi et al., 1998). In these examples, resistance occurred as a result of the loss of a functional component/activity.

Loss of enzymatic activity can result in metronidazole resistance. Interacellular metronidazole must be enzymatically activated before it can serve as an antimicrobial agent. This activation is achieved by the enzyme, NADPH nitroreductase (Figure 2). If the metronidazole is not activated it has no inhibitory effect on the bacterium. Thus, if NADPH nitroreductase activity is absent in the cell metronidazole remains inactive. Loss of the reductase activity can occur by nonsense or deletion mutations in rdxA (Debets-Ossenkopp et al., 1999; Goodwin et al., 1998; Tankovic et al., 2000). In addition, NADPH nitroreductase activity can be severely reduced by a single missense mutation (a single amino acid change), which reduces its ability to activate metronidazole (Paul et al., 2001). All these mutations result in loss of the enzyme activity necessary for the drug to be effective in the cell, hence the cell becomes resistant to metronidazole. But, loss of enzymatic activity does not provide a genetic example of how that enzyme originally "evolved." Hence, mutations that provide resistance to metronidazole cannot be offered as true examples of "evolution in a Petri dish."

Several bacteria, including Escherichia coli, construct a mulitiple-antibiotic-resistance (MAR) efflux pump that provides the bacterium with resistance to multiple types of antibiotics, including erythromycin, tetracycline, ampicillin, and nalidixic acid. This pump expels the antibiotic from the cell's cytoplasm, helping to maintain the intracellular levels below a lethal concentration (Grkovic et al., 2002; Okusu et al., 1996) (Figure 3). The MAR pump is composed of the proteins MarA and MarB, whose synthesis is inhibited by the regulatory protein, MarR (Alekshun and Levy, 1999; Poole, 2000) (Figure 3). Mutations that reduce or eliminate the repression control of MarR result in over-production of the MarAB efflux pump, which enables the cell to expel higher concentrations of antibiotics or other antibacterial agents (Oethinger et al., 1998; Poole, 2000; Zarantonelli et al., 1999).



Figure 2. Activation of the antimicrobial agent, metronidazole.

After being transported into the cell, metronidazole requires structural modification to obtain its active, antimicrobial form. This activation is achieved by the enzyme, NADPH nitroreductase, which is a product of the rdxA gene. Mutations in rdxA can prevent synthesis of a functional NADPH nitroreductase activity, which prevents metronidazole from becoming activated.

The protein MarA also acts as a positive regulator by stimulating increased production of both MarA and MarB proteins (Alekshun and Levy, 1999) (Figure 3). In addition, the MarA protein indirectly inhibits the production of the porin, OmpF, a channel in the membrane that allows entry of some antibiotics into the cell (Cohen et al., 1988). Therefore, increased expression of MarA increases the efflux of antibiotics out of the cell, and reduces the transport of some antibiotics into the cell (Figure 3). Mutations of marR that reduce expression or activity of the MarR protein will thus enable over-expression of the MarAB efflux pump (Linde et al., 2000; Okusu et al., 1996), and provide an increased resistance of the bacterium to various antibiotics (Eaves et al., 2004; Hans-Jorg et al., 2000; Notka et al., 2002) (Figure 3). MarR defective mutants also possess increased bacterial tolerance to some organic chemical agents, such as cyclohexane (Aono et al., 1998).