One unfortunate outcome of the use of antimicrobials is the development of microbial drug resistance, an adaptive response in which microorganisms begin to tolerate an amount of drug that would previously have been inhibitory. The development of mechanisms for resisting antimicrobial drugs is a result of the genetic versatility and adaptability of microbial populations. The property of drug resistance can be an intrinsic characteristic as well as acquired. Intrinsic drug resistance would be found in bacteria that must be resistant to any antibiotic that they produce or bacteria that have natural barriers that block the drug (the outer membrane of gram-negative bacteria, for example). This type of resistance is not the main problem with regard to antimicrobial chemotherapy. Of much greater importance is the acquisition of resistance by a microbe that was previously sensitive to the drug. In our context, the term drug resistance will refer to this type of acquired resistance.
How Does Drug Resistance Develop?
Contrary to popular belief, antibiotic resistance is not a recent phenomenon. Resistance to penicillin developed in some bacteria as early as 1940, 3 years before the drug was even approved for public use. The scope of the problem became apparent in the 1980s and 1990s when scientists and physicians observed treatment failures on a large scale.
Microbes become newly resistant to a drug after one of the following events occurs: (1) spontaneous mutations in critical chromosomal genes or (2) acquisition of entirely new genes or sets of genes via transfer from another species. Chromosomal drug resistance usually results from spontaneous random mutations in bacterial populations. The chance that such a mutation will be advantageous is minimal, and the chance that it will confer resistance to a specific drug is lower still. Nevertheless, given the huge numbers of microorganisms in any population and the constant rate of mutation, such mutations do occur. The end result varies from slight changes in microbial sensitivity, which can be overcome by larger doses of the drug, to complete loss of sensitivity.
Resistance through intermicrobial transfer originates from chromosomal genes and plasmids called resistance (R) factors being transferred through the processes we considered in chapter 9: conjugation, transformation, or transduction (figure 1). Studies have shown that plasmids encoded with drug resistance are naturally present in microorganisms before they have been exposed to the drug. Such traits are “lying in wait” for an opportunity to be ex pressed and to confer adaptability on the species. Many bacteria also maintain transposable drug-resistance sequences (transposons) that are duplicated and inserted from one plasmid to another or from a plasmid to the chromosome. Chromosomal genes and plasmids containing codes for drug resistance are faithfully replicated and inherited by all subsequent progeny. This sharing of resistance genes accounts for the rapid proliferation of drug-resistant species. A growing body of evidence points to the ease and frequency of gene transfers in biofilms between totally unrelated bacteria living in the body’s microbiota and the environment.
Fig1. Transfer of drug resistance. Bacteria transmit R factors, often containing numerous genes for drug resistance by means of conjugation, transduction, and transformation. The widespread occurrence of such exchanges accounts for the rapid spread of drug resistance.
Specific Mechanisms of Drug Resistance
Inside a bacterial cell, the net effect of these genetic transfers is one of the following, which actually causes the bacterium to be resistant (note that the numbers that follow correspond to the numbers seen in figure 2):
1. The development of alternative enzymes that inactivate the drug (occurs only when new genes are acquired).
2. Permeability or uptake of drug into bacterium is decreased or eliminated.
3. The microbe engages special drug transport pumps that re move the drug.
4. Binding sites for drug are decreased in number or affinity (can occur via mutation or acquisition of new genes).
5. An affected metabolic pathway is shut down or an alternate pathway is used (occurs due to mutation of original enzyme[s]).
Fig2. Examples of mechanisms of acquired drug resistance.
Drug Inactivation Mechanisms
Microbes inactivate drugs by producing enzymes that permanently alter drug structure. One example, bacterial exoenzymes called beta-lactamases, hydrolyze the beta-lactam ring structure of some penicillins and cephalosporins, rendering the drugs inactive. Two beta-lactamases—penicillinase and cephalosporinase—disrupt the structure of certain penicillin or cephalosporin molecules so their activity is lost (figure 2, part 1). So many strains of Staphylococcus aureus produce penicillinase that regular penicillin is rarely a possible therapeutic choice. Now that some strains of Neisseria gonorrhoeae, called PPNG,4 have also acquired penicillinase genes, alternative drugs are required to treat gonorrhea. A large number of other gram- negative species are inherently resistant to some of the penicillins and cephalosporins because of naturally occurring beta-lactamases.
Decreased Drug Permeability or Increased Drug Elimination
The resistance of some bacteria can be due to a mechanism that prevents the drug from entering the cell and acting on its target. For example, the outer membrane of the cell wall of certain gram-negative bacteria is a natural blockade for some of the penicillin drugs. Aminoglycoside resistance is known to develop through changes in drug permeability caused by point mutations in proteins of the transport system or outer membrane (figure 2, parts 2 and 3).
Many bacteria possess multidrug-resistant (MDR) pumps that actively transport drugs and other chemicals out of cells. These pumps are proteins encoded by plasmids or chromosomes. They are stationed in the cell membrane and expel molecules by a proton motive force similar to ATP synthesis (figure 2, part 3). They confer drug resistance on many gram-positive pathogens (Staphylococcus, Streptococcus) and gram-negative pathogens (Pseudomonas, E. coli). Because MDRs lack selectivity, one type of pump can expel a broad array of antimicrobial drugs, detergents, and other toxic substances.
Change of Drug Receptors
As most drugs act on a specific target such as protein, RNA, DNA, or membrane structure, microbes can circumvent drugs by altering the nature of this target. Bacteria can become resistant to aminoglycosides when point mutations in ribosomal proteins arise (figure 2, part 4). Erythromycin and clindamycin resistance is associated with an alteration on the 50S ribosomal binding site. Penicillin resistance in Streptococcus pneumoniae and methicillin resistance in Staphylococcus aureus are related to an alteration in the binding proteins in the cell wall. Several species of enterococci have acquired resistance to vancomycin through a similar alteration of cell wall proteins. Some bacteria may become resistant by shedding their cell wall entirely, con verting to a cell-wall-deficient form (L-form) that is unaffected by penicillin. Fungi can become resistant by decreasing their syn thesis of ergosterol, the principal receptor for certain antifungal drugs.
Changes in Metabolic Patterns
The action of antimetabolites can be circumvented if a microbe develops an alternative metabolic pathway or enzyme (figure 2, part 5). Sulfonamide and trimethoprim resistance develops when microbes deviate from the usual patterns of folic acid synthesis. Fungi can acquire resistance to flucytosine by completely shutting off certain metabolic activities.
Biofilms and Drug Resistance
It is estimated that about 60% of infections involve biofilms, com plex communities of microorganisms growing within an extracellular matrix (first discussed in chapter 4). Biofilms add another level of complexity to thoughts of bacterial resistance. Always a problem on natural tissues—for example, bacterial infections of the heart valves, middle ear, and teeth—the increased use of indwelling medical devices has created yet another habitat in which biofilms may become tenaciously attached. Catheters, artificial valves, and prosthetic devices can harbor persistent biofilms that are difficult to treat or remove.
Microbes in infectious biofilms may be hundreds of times more drug resistant than the same free, unattached microbes. Most obviously, microbes are protected by the impenetrable nature of the extracellular matrix—drugs may simply be blocked from entry. We also know that microbes within the biofilm communicate with one another and are more likely to engage in the transfer of drug- resistant plasmids and genes. And of course, if one microbe in a biofilm can inactivate a particular drug, nearby microbes may benefit from the drug-free environment.
Natural Selection and Drug Resistance
So far we have been considering drug resistance at the cellular and molecular levels, but its full impact is felt only if this resistance occurs throughout the cell population. Let us examine how this might happen and its long-term therapeutic consequences.
Any large population of microbes is likely to contain a few individual cells that are already drug resistant because of prior mutations or transfer of plasmids. As long as the drug is not present in the habitat, the numbers of these resistant forms will remain low because they have no particular growth advantage. But if the population is subsequently exposed to this drug, sensitive individuals are inhibited or destroyed, and resistant forms survive and proliferate. As the population continues to grow, offspring of these resistant microbes will inherit this drug resistance. In time, the replacement population will have more of the drug-resistant forms and can eventually become completely resistant (process figure 3). In ecological and evolutionary terms, the environ mental factor (in this case, the drug) has put selection pressure on the population, allowing the better adapted microbe (the drug-resistant one) to survive and grow. The end result is a population that has evolved to a condition of drug resistance.
Fig3. The development and spread of antibiotic resistance.
Natural selection for drug-resistant forms is a common phenomenon. It takes place in natural habitats, medical environments, and within the bodies of humans and animals during drug therapy. See 12.1 Making Connections to find out some of the current implications of drug resistance. Table 1 summarizes a number of actions that are being considered to slow its development.
Table1. Strategies to Limit Drug Resistance of Microorganisms
