Resistance Due to Reduced Entry of Drug into Pathogen. The outer membrane of gram-negative bacteria is a permeable barrier that excludes large polar molecules from entering the cell. Small polar molecules, including many antibiotics, enter the cell through protein channels called porins. Absence of, mutation in, or loss of a favored porin channel can slow the rate of drug entry into a cell or prevent entry altogether, effectively reducing drug concentration at the target site. If the target is intracellular and the drug requires active transport across the cell membrane, a mutation or phenotypic change that slows or abolishes this transport mechanism can confer resistance. As an example, Trypanosoma brucei is treated with suramin and pentamidine during early stages, but with melarsoprol and eflornithine when CNS disease (sleeping sickness) is present. Melarsoprol is actively taken up by trypanosome P2 protein transporter. When the parasite either lacks the P2 transporter, or has a mutant form, resistance to melarsoprol and cross resistance to pentamidine occur due to reduced uptake (Ouellette, 2001).
Resistance Due to Drug Efflux. Microorganisms can overexpress efflux pumps and then expel antibiotics to which the microbes would otherwise be susceptible. There are five major systems of efflux pumps that are relevant to antimicrobial agents:
the multidrug and toxic compound extruder (MATE)
the major facilitator superfamily (MFS) transporters
the small multidrug resistance (SMR) system
the resistance nodulation division (RND) exporters
ATP binding cassette (ABC) transporters
Efflux pumps are a prominent mechanism of resistance for parasites, bacteria, and fungi. One of the tragic consequences of resistance emergence has been the development of drug resistance by Plasmodium falciparum. Drug resistance to most antimalarial drugs, specifically chloroquine, quinine, mefloquine, halofantrine, lumefantrine, and the artemether-lumefantrine combination is mediated by an ABC transporter encoded by Plasmodium falciparum multidrug resistance gene 1 (Pfmdr1) (Happi et al., 2009). Point mutations in the Pfmdr1 gene lead to drug resistance and failure of chemotherapy. Drug efflux sometimes works in tandem with chromosomal resistance, as is seen in Streptococcus pneumoniae, and perhaps, Myobacterium tuberculosis. In these situations, induction of efflux pumps occurs early, which increases the MIC only modestly. However, this MIC increase is enough to allow further microbial replication and an increased mutation frequency, which enable the development of resistance via more robust chromosomal mutations (Gumbo et al., 2007b; Jumbe et al., 2006).
Resistance Due to Destruction of Antibiotic. Drug inactivation is a common mechanism of drug resistance. Bacterial resistance to aminoglycosides and to β-lactam antibiotics usually is due to production of an aminoglycoside-modifying enzyme or β-lactamase, respectively.
Resistance Due to Reduced Affinity of Drug to Altered Target Structure. A common consequence of either single point or multiple point mutations is change in amino acid composition and conformation of target protein. This change leads to a reduced affinity of drug for its target, or of a prodrug for the enzyme that converts the prodrug to active drug. Such alterations may be due to mutation of the natural target (e.g., fluoroquinolone resistance), target modification (e.g., ribosomal protection type of resistance to macrolides and tetracyclines), or acquisition of a resistant form of the native, susceptible target (e.g., staphylococcal methicillin resistance caused by production of a low-affinity penicillin-binding protein) (Hooper, 2002; Lim and Strynadka, 2002; Nakajima, 1999). Similarly, in HIV resistance mutations associated with reduced affinity are encountered in protease inhibitors, integrase inhibitors, fusion inhibitors, and non-nucleoside reverse transcriptase inhibitors (Nijhuis et al., 2009). Similarly, benzimidazoles are used against myriad worms and protozoa and work by binding to the parasite's tubulin; point mutations in the β-tubulin gene lead to modification of the tubulin and drug resistance (Ouellette, 2001).
Incorporation of Drug. An uncommon situation occurs when an organism not only becomes resistant to an antimicrobial agent but subsequently starts requiring it for growth. Enterococcus, which easily develops vancomycin resistance, can, after prolonged exposure to the antibiotic, develop vancomycin-requiring strains.
Resistance Due to Enhanced Excision of Incorporated Drug. Nucleoside reverse transcriptase inhibitors such as zidovudine are 2′-deoxyribonucleoside analogs that are converted to their 5′-triphosphate form and compete with natural nucleotides. These drugs are incorporated into the viral DNA chain and cause chain termination. When resistance emerges via mutations at a variety of points in the reverse transcriptase gene, phosphorolytic excision of the incorporated chain-terminating nucleoside analog is enhanced (Arion et al., 1998).
Hetero-resistance and Viral Quasi Species. Hetero-resistance is said to be present when a subset of the total microbial population is resistant, despite the total population being considered susceptible on testing (Falagas et al., 2008; Rinder, 2001). In a way, this should not be a surprise given that chromosomal mutations are a stochastic process and there is a baseline mutation rate for each gene. Therefore, a subclone that has alterations in genes associated with drug resistance is expected to reflect the normal mutation rates and occur at between 10−6 and 10−5 colonies. In bacteria, hetero-resistance has been described especially for vancomycin in S. aureus, vancomycin in Enterococcus faecium, colistin in Acinetobacter baumannii-calcoaceticus, rifampin, isoniazid, and streptomycin in M. tuberculosis, and penicillin in S. pneumoniae (Falagas et al., 2008; Rinder, 2001). Increased therapeutic failures and mortality have been reported in patients with hetero-resistant staphylococci and M. tuberculosis (Falagas et al., 2008; Hofmann-Thiel et al., 2009). For fungi, hetero-resistance leading to clinical failure has been described for fluconazole in Cryptococcus neoformans and Candida albicans (Marr et al., 2001; Mondon et al., 1999).
Viral replication is more error prone than replication in bacteria and fungi. Viral evolution under drug and immune pressure occurs relatively easily, commonly resulting in variants or quasi species that may contain drug-resistant subpopulations. This is not often termed hetero-resistance, but the principle is the same as described for bacteria and fungi: A virus may be considered susceptible to a drug because either phenotypic or genotypic tests reveal "lack" of resistance, when there is a resistant subpopulation just below the limit of assay detection. These minority quasi species that are resistant to antiretroviral agents have been associated with failure of antiretroviral therapy (Metzner et al., 2009).