Multiple Drug Resistance

MULTIPLE  DRUG  RESISTANCE

R-factors

Several issues of multiple drug resistance have already been raised in this chapter. Notable examples are MRSA, which can harbour both small cryptic plasmids and larger plasmids encoding resistance to antiseptics, disinfectants, trimethoprim, penicillin, gentamicin, tobramycin and kanamycin, and multidrug-resistant M. tuberculosis. Of equal concern are instances where isolates can become resistant to multiple, chemically distinct agents in a single biological event. One of the earliest examples was in Japan in 1959. Previously sensitive E. coli became resistant to multiple antibiotics through acquisition of a conjugative plasmid (R-factor) from resistant Salmonella and Shigella isolates. A number of R-factors have now been characterized including RP4, encoding resistance to ampicillin, kanamycin, tetracycline and neomycin, found in Ps. aeruginosa and other Gram-negative bacteria; R1, encoding resistance to ampicillin, kanamycin, sulphonamides, chloramphenicol and streptomycin, found in Gram-negative bacteria and pSH6, encoding resistance to gentamicin, trimethoprim and kanamycin, found in Staph. aureus.

Mobile   Gene   Cassettes   And   Integrons

Many Gram-negative resistance genes are located in gene cassettes. One or more of these cassettes can be integrated into a specific position on the chromosome termed an integron. More than 60 cassettes have been identified, each comprising only a promotor-less single gene (usually antibiotic resistance) and a 59-base element forming a specific recombination site. This recombination site confers mobility because it is recognized by specific recombinases encoded by integrons that catalyse integration of the cassette into a specific site within the integron. Thus, integrons are genetic elements that recognize and capture multiple mobile gene cassettes. As the gene typically lacks a promoter, expression is dependent on correct orientation into the integron to supply the upstream promoter. Four classes of integron have been identified, although only one member of class 3 has been described and class 4 integrons are limited to Vibrio cholerae. Analysis of the resistant Shigella strains isolated in Japan has shown that some of the conjugative plasmids included an integron with one or two integrated cassettes.

Chromosomal Multiple-Antibiotic Resistance (mar) Locus

The multiple-antibiotic resistance (mar) locus was first described in E. coli by Stuart Levy and colleagues at Tufts University and has since been recognized in other enteric bacteria. The locus consists of two divergently transcribed units, marC and marRAB. Little is known of marC and marB; however, marR encodes a repressor of the operon, and marA encodes a transcriptional activator affecting expression of more than 60 genes. Increased expression of the MarRAB operon resulting from mutations in marO or marR, or from inactivation of MarR following exposure to inducing agents such as salicylate, leads to the Mar phenotype. This phenotype is characterized by resistance to structurally unrelated antibiotics, organic solvents, oxidative stress and chemical disinfectants. A number of effector mechanisms have been identified, including increased expression of the acrAB-tolC multidrug efflux system  and the soxRS regulon.

Multidrug Efflux Pumps

Whereas some efflux pumps excrete only one drug or class of drugs, a multidrug efflux pump can excrete a wide range of compounds where there is often little or no chemical similarity between the substrates. One common characteristic may be agents with a significant hydrophobic domain. For this reason, hydrophilic compounds such as the aminoglycoside antibiotics are not exported by these systems. A distinction needs to be drawn between those efflux systems, typically in Gram-positive bacteria, that pump their substrate across the cytoplasmic membrane, such as the QacA and Smr pumps which both export quaternary ammonium compounds and basic dyes, and those which efflux across the cytoplasmic and outer membranes of Gram-negative bacteria. There are some examples of single membrane systems in Gramnegative bacteria, such as the EmrE protein in E. coli, but they are not of great clinical significance. The majority of Gram-negative pumps span both membranes and include the AcrAB-TolC system in E. coli and the MexABOprM system in Ps. aeruginosa. Genomic analyses are revealing numerous homologues. Using the MexABOprM system as the prototypic example (Figure 13.8), MexA is the linker protein and MexB is in the cytoplasmic membrane. MexB is a resistance-nodulation-division (RND) family member and is predicted to be a proton antiporter with 12 membrane-spanning α-helices. OprM shows homology with outer-membrane channels of systems thought to export such diverse molecules as nodulation signals and alkaline proteases. Mutations in regulatory genes such as nalB cause overexpression of MexAB-OprM and consequently multidrug resistance. MexB is a proton antiporter and efflux by this and other members of the RND family is energized by the proton motive force. This contrasts with mammalian multidrug efflux pumps (MDR) that are powered by ATP hydrolysis.