Mechanisms and development of antibiotic resistance

Several reviews of the mode of action of antimycobacterial drugs and their biochemical mechanisms of resistance have been published recently (Blanchard 1996, Cole 1994, Musser 1995, Webb & Davies 1998). This information is summarized in Table 1 and Fig. 1. Briefly, for most antibiotics available for the

TABLE 1 Genetics of resistance to common antimycobacterial antibiotics

Drug

Target

Altered gene

Isonicotinic hydrazide

Mycolic acid synthesis

inhA, katG, aphC

Ethambutol

Arabinosyltransferase

embCAB

Pyrazinamide

?

pncA

Streptomycin

Translation

rpsT, rrnA

Manamycin

Translation

rrnA

Viomycin

Translation

rrnA +rrnB

Clarithromycin

Translation

rrnB, ?

Rifamycin (rifampin)

Transcription

rpoB, ?

Ciprofloxacin

Replication

gyrA, parC?, efflux

? indicates that uncharacterized mechanisms of resistance exist.

? indicates that uncharacterized mechanisms of resistance exist.

treatment of mycobacterial infections, there are several mechanisms of resistance that have been well characterized on a genetic and biochemical basis. For some agents whose mode of action remains incompletely characterized (such as pyrazinamide), the detailed resistance mechanisms have proved difficult to assign. A number of efflux pump systems have been identified in mycobacteria, and the role of some level of increased drug efflux in the overall development of antibiotic resistance appears to be important (Takiff et al 1996). It is possible that an initial low level resistance due to increased drug efflux during therapy provides a selective advantage (survival), which then increases the possibility of the appearance of mutations leading to resistance by other mechanisms, such as target modification.

In this brief review I would like to discuss and speculate on several aspects of antibiotic resistance and its phenotypic expression in pathogenic mycobacteria which set them apart from other bacteria. The current upswing in multidrug-resistant infections by Gram-negative and Gram-positive bacteria in hospitals and the community is due in the majority of cases to the acquisition of plasmid-determined antibiotic resistance genes. Resistance due to chromosomal mutation is the exception (although certain 'exceptions' are highly significant, such as methicillin-resistant staphylococci, and antibiotic-resistant meningococci and pneumococci). By contrast, antibiotic resistance in clinical isolates of mycobacteria is due almost exclusively to the mutational alteration of chromosomal genes. However, as described later, antibiotic resistance in clinical situations may be a more complex genotype than was once realized.

There are a few instances in which acquisition of an antibiotic resistance gene from an exogenous source has apparently occurred in mycobacteria. Martin et al

Aminoglycosides ß-lactams Rifampicin

Aminoglycosides ß-lactams Rifampicin

Isoniazid Mechanism Action Image

EFFLUX

Isoniazid Pyrazinamide

EFFLUX

Many drugs especially Fluoroquinolones

^ Ethambutol Aminoglycosides Sulfonamides Rifampicin Macrolides Peptides Fluoroquinolones

Isoniazid Pyrazinamide

FIG. 1. The cartoon represents the cellular mechanisms of resistance to antibiotics at various critical stages in antibiotic action.

(1990) have identified a defective type I integron that is highly resistant to sulfonamide drugs in the chromosome of Mycobacterium fortuitum, A composite transposon Tn610 carries (between two IS6100 elements) two genes similar to those in the type I antibiotic resistance integrons typically found in Gramnegative bacteria: a truncated integrase gene (intH) and a gene for sulfonamide resistance (dihydropteroate synthase, sul3), Since the IS6100 sequences are related to the widely dispersed IS6, especially as found in Gram-negative bacteria, it is highly likely that Tn610 acquisition by mycobacteria originated in a member of the Enterobacteriaceae, The interspecific vector involved has not been identified, but we can speculate that the transfer was probably mediated by bacteriophages, In addition, to date integrons and their associated antibiotic resistance gene cassettes have been identified in Enterobacteriaceae (Recchia & Hall 1995),

Pang et al (1994) may have uncovered another example of resistance gene transfer into mycobacteria, While examining several instances of coexistence of streptomycetes and mycobacteria in human infections they found that the genes for tetracycline resistance, normally associated with a tetracycline-producing streptomycete, were shared by both genera, Other examples of gene acquisition and mobile genetic elements in the mycobacteria have been noted, Plasmids have been identified in a number of mycobacterial species but they do not appear to be involved in clinically significant antibiotic resistance (Picardeau & Vincent 1997), Although the available evidence indicates that the mechanisms of antibiotic resistance are almost exclusively due to mutation, one cannot eliminate the contingency that horizontal resistance gene transfer could occur in the mycobacteria,

This prompts the question: what is different about mycobacteria? In the first place it is possible that transferable resistance has not occurred (yet) in mycobacteria because the antibiotics employed in treatment are not broad-spectrum agents and, with the exception of streptomycin, have not been used on a worldwide scale for non-human applications, as animal growth promoters, for example, Thus, prudent antibiotic usage for mycobacterial infections may have inadvertently reduced the selection pressure for resistance,

There are other distinct genetic and physiological characteristics that set mycobacteria apart, Mycobacterial infections are:

(a) due to species with slow growth rates;

(b) intracellular pathogens that successfully parasitize host cells;

(c) caused by large populations of pathogenic organisms in a specific host environment (e,g, in the lung);

(d) able to pass through a non-growing (dormant) phase and be reactivated many years later; and

(e) often intrinsically resistant to a number of antibiotics (Jarlier & Nikaido 1994).

These factors lead to chronic infection and corresponding difficulty in treatment. The necessity to maintain appropriate drug levels over many months of treatment predisposes to the development of resistance, and the inherent toxicity of most anti-mycobacterial agents contributes to the difficulty of achieving therapeutic success.

The genetic constitution of mycobacteria is also a factor in the development of resistance. In particular, while most bacterial pathogens, such as Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, etc., have multiple copies of the rRNA genes (up to seven; Keener & Nomura 1996), mycobacteria such as M. tuberculosis possess only single copies of 16S and 23S rRNA genes (Bottger 1994, Gonzalez-y-Merchand et al 1996, Suzuki et al 1987). As a result, a single rDNA alteration in mycobacteria generates a dominant phenotype. Thus, resistance to an antibiotic such as streptomycin, which inhibits protein biosynthesis by binding to the 30S ribosomal subunit (Cundliffe 1981), most often results from mutations in the gene for ribosomal protein S12 (rpsL) that lead to high level streptomycin resistance in many different bacterial genera (Cundliffe 1979). Some rpsL mutations even determine a streptomycin-dependent phenotype, and such have been found for M. tuberculosis (Honore et al 1995). In most bacterial genera mutation in an rRNA gene has no phenotype, although the over-expression of a mutated (altered) 16S rRNA gene (rrnA) has been shown to confer streptomycin resistance (Morgan et al 1988). However, in M. tuberculosis, and other slow-growing mycobacteria possessing a single copy of the rrnA gene, mutations conferring streptomycin resistance are dominant and are expressed phenotypically (Finken et al 1993, Meier et al 1996). The same is true for translation inhibitors that bind to the 50S subunit, such as clarithromycin. Mutation in either an r-protein (L22) or 18S rRNA (rrnB) can lead to high level resistance (Bottger 1994, Meier et al 1994,1996).

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