Resistance To Other Antibiotics

RESISTANCE TO OTHER ANTIBIOTICS

Resistance To Tetracycline Antibiotics    

Chlortetracycline and oxytetracycline were discovered in the late 1940s and studies of representative populations before their widespread use suggests that emergence of resistance is a relatively modern event. More than 60% of Shigella flexneri isolates are resistant to tetracycline; resistant isolates of Salmonella enterica serovar Typhimurium are becoming more common and among Gram-positive species, approximately 90% of MRSA strains and 60% of multiply resistant Strep. pneumoniae are now tetracycline-resistant. The major mechanisms of resistance are efflux and ribosomal protection. One exception is the tet(X) gene that encodes an enzyme which modifies and inactivates the tetracycline molecule, although this does not appear to be clinically significant. The Tet efflux proteins belong to the major facilitator superfamily (MFS). These proteins exchange a proton for a tetracycline–cation (usually Mg²⁺) complex, reducing the intracellular drug concentration and protecting the target ribosomes in the cell. In Gram-negative bacteria, the efflux determinants comprise divergently oriented efflux and repressor proteins that share overlapping promoter and operator regions. In the absence of a tetracycline–Mg²⁺ complex, the repressor protein binds and blocks transcription of both genes. Drug binding alters the conformation of the repressor so that it can no longer bind the DNA operator region and block transcription. This method of regulation probably applies to all of the Gram-negative efflux systems including tet(A), tet(C), tet(D), tet(E), tet(G) and tet(H).

No repressor proteins have been identified in the Gram-positive tet(K) or tet(L) genes and regulation of plasmid-borne tetracycline resistance appears to be by translational attenuation, involving stem-loop mRNA structures and tetracycline-induced unmasking of the ribosome binding site permitting translation of the efflux protein. Regulation of chromosomal tet(L) expression involves tetracycline-promoted stalling of the ribosomes during translation of early codons of the leader peptide, which allows re-initiation of translation at the ribosome binding site for the structural gene. Ribosomal protection is mediated by cytoplasmic proteins that inhibit tetracycline and also confer resistance to doxycycline and minocycline. These proteins share homology with the elongation factors EF-Tu and EF-G, and expression of Tet(M) and Tet(O) proteins appears to be regulated. A 400-bp region upstream from the coding region for tet(O) is needed for full expression, but the mechanism(s) has not been characterized. The widespread emergence of effluxand ribosome protection-based resistance to firstand second-generation tetracyclines has prompted the development of the 9-glycinyltetracyclines (9glycylcyclines). 9-Amino-acylamido derivatives of minocycline have similar activity to earlier compounds; however, when the acyl group is modified to include an N,N-dialkylamine or 9-t-butyl-glycylamido moiety (Figure 13.6), antimicrobial activity is retained and the compounds are active against strains containing tet genes responsible for resistance by efflux and ribosomal protection.

Resistance  To Fluoroquinolone Antibiotics

Fluoroquinolones bind and inhibit two bacterial topoisomerase enzymes: DNA gyrase (topoisomerase II) which is required for DNA supercoiling, and topoisomerase IV which is required for strand separation during cell division. DNA gyrase tends to be the major target in Gram-negative bacteria, whereas both topoisomerases are inhibited in Gram-positive bacteria. Each topoisomerase is termed a hetero-tetramer, being composed of two copies of two different subunits designated A and B. The A and B subunits of DNA gyrases are encoded by gyrA and gyrB, respectively, whilst topoisomerase IV is encoded by parC and parE (grlA and grlB in Staph. aureus). Mutations in gyrA, particularly involving substitution of a hydroxyl group with a bulky hydrophobic group, induce conformational changes such that the fluoroquinolone can no longer bind. Mutations have also been detected in the B subunit, but these are probably less important. Alterations involving Ser80 and Glu84 of Staph. aureus grlA and Ser79 and Asp83 of Strep. pneumoniae parC have led to quinolone resistance. Like GyrB, mutations in ParE leading to resistance are not common. While changes in GyrA and ParC give resistance to the older fluoroquinolones, MIC values do not always rise above clinically defined breakpoints for newer agents such as gemi-floxacin and moxifloxacin.

Topoisomerases are located in the cytoplasm and thus fluoroquinolones must cross the cell envelope to reach their target. Changes in outer-membrane permeability have been associated with resistance in Gram-negative bacteria, but permeability does not appear to be an issue with Gram-positive species. Efflux, however, does make a contribution to resistance, mainly low level, in both Gram-positive and Gram-negative bacteria. The NorA mediated efflux system in Staph. aureus was characterized in 1990. It is expressed weakly in wild-type strains and resistance is thought to occur via mutations leading to increased expression of norA. NorA is a member of the MFS and homologues are also present in Streptococcus pneumoniae and Bacillus sp. There is a tendency for it to be more effective for hydrophilic fluoroquinolones, but there is no strict correlation. Fluoroquinolones are now being used for treating M. avium and multidrug-resistant M. tuberculosis and efflux-mediated resistance has been identified. A number of efflux pumps have been identified among Gram-negative bacteria, including AcrA in E.coli, which is regulated in part by the multiple-antibiotic resistance (Mar) operon.

Resistance To Macrolide, Lincosamide And Streptogramin Antibiotics

Although chemically distinct, members of the macrolide, lincosamide and streptogramin (MLS) group of antibiotics all inhibit bacterial protein synthesis by binding to a target site on the ribosome. Gram-negative bacteria are intrinsically resistant due to the permeability barrier of the outer membrane, and three resistance mechanisms have been described in Gram-positive bacteria. Target modification, involving adenine methylation of domain V of the 23S ribosomal RNA, is the most common mechanism. The adenine-N⁶-methyltransferase, encoded by the erm gene, results in resistance to erythromycin and other macrolides (including the azalides), as well as the lincosamides and group B streptogramins. Streptogramin A-type antibiotics are unaffected and streptogramin A/B combinations remain effective. Expression of the erm gene may be constitutive or inducible. When expression is inducible, resistance is seen only against 14and 15-membered macrolides; lincosamide and streptogramin antibiotics remain active. Telithromycin (Figure 13.7), the first of a new class of ketolide agents in the MLS family, does not induce MLS resistance and also retains activity against domain V-modified ribosomes and inhibition of protein synthesis through strong interaction with domain II. The second resistance mechanism is efflux. Expression of the mef gene confers resistance to macrolides only, whereas msr expression results in resistance to macrolides and streptogramins. Efflux-mediated resistance of Staph. aureus to streptogramin A antibiotics is also conferred by vga and vgaB gene products. A third resistance mechanism, involving ribosomal mutation, has been reported in a small number of clinical isolates of Strep. pneumoniae.

Resistance To Chloramphenicol

Chloramphenicol inhibits protein synthesis by binding the 50S ribosomal subunit and preventing the peptidyltransferase step. Decreased outer-membrane permeability and active efflux have been identified in Gram-negative bacteria; however, the major resistance mechanism is drug inactivation by chloramphenicol acetyltransferase. This occurs in both Gram-positive and Gram-negative species, but the cat genes, typically found on plasmids, share little homology.

Resistance To The Oxazolidinone Antibiotics

Linezolid is the first of a new class of oxazolidinone antimicrobials with a novel target in protein synthesis. Linezolid does not interfere with translation initiation at the stage of mRNA binding or formation of 30S preinitiation complexes; rather, it involves binding the 50S rRNA. Its affinity for 50S rRNA from Gram-positive bacteria is twice that for the corresponding molecule in Gram negative bacteria and as such linezolid has been approved for treating various Gram-positive infections, including MRSA. Resistance is appearing, although rare at present. Mutation in the central loop of domain V of the component 23S rRNA subunit appears to be the main mechanism, including a G2576T mutation in three isolates of linezolid-resistant MRSA.

Resistance To Trimethoprim        

Trimethoprim competitively inhibits dihydrofolate reductase (DHFR) and resistance can be caused by overproduction of host DHFR, mutation in the structural gene for DHFR and acquisition of the dfr gene encoding a resistant form. There are at least 15 DHFR enzyme types based on sequence homology and acquisition of dfr genes encoding alternative DHFR of type I, II or V is the most common mechanism of trimethoprim resistance among the Enterobacteriaceae.

Resistance  To  Mupirocin

Nasal carriage of MRSA strains has been identified as an important target for infection control protocols aimed at reducing spread and acquisition. Mupirocin (pseudomonic acid A) is an effective topical antimicrobial used in MRSA eradication. It is an analogue of isoleucine that competitively binds isoleucyl-tRNA synthetase (IRS) and inhibits protein synthesis. Low-level resistance (MIC 4–256 μg/ml) is usually due to mutation of the host IRS, whereas high-level resistance (MIC >512 μg/ml) is due to acquisition of a distinct IRS that is less sensitive to inhibition. The mupA gene, typically carried on transferable plasmids, is found in Staph. aureus and co-agulasenegative staphylococci, and encodes an IRS with only 30% homology to the mupirocin-sensitive form.

Resistance To Peptide Antibiotics—Polymyxin

Many peptide antibiotics have been described and can be broadly classified as non-ribosomally synthesized peptides; they include the polymyxins, bacitracins and gramicidins as well as the glycopeptides (section 5) and the ribosomally synthesized peptides such as the antimicrobial peptides of the innate immune system. Polymyxins and other cationic antimicrobial peptides have a self-promoted uptake across the cell envelope and perturb the cytoplasmic membrane barrier. Addition of a 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate groups on the lipid A component of Gram-negative lipopolysaccharide has been implicated in resistance to polymyxin. Details of the pathway for l-Ara4N biosynthesis from UDP glucuronic acid, encoded by the pmr operon, are emerging.

Resistance To Antimycobacterial Therapy

The nature of mycobacterial infections, particularly tuberculosis, means that chemotherapy differs from other infections. Organisms tend to grow slowly (long generation time) in a near dormant state with little metabolic activity. Hence, a number of the conventional antimicrobial targets are not suitable. Isoniazid is bactericidal, reducing the count of aerobically growing organisms. Pyrazinamide is active only at low pH, making it well suited to killing organisms within necrotic foci early in infection, but less useful later on when these foci have reduced in number. Rifampicin targets slow-growing organisms. Resistance mechanisms have now been described and multiple resistance poses a serious threat to health. Current treatment regimens result in a high cure rate and the combination of agents makes it highly unlikely that there will be a spontaneous resistant isolate to all the components. Problems most commonly occur in patients who receive inadequate therapy, which provides a serious selection advantage. Resistance can occur to single agents and subsequently to multiple agents. Resistance to rifampicin arises from mutation in the β subunit of RNA polymerase encoded by rpoB and resistant isolates show decreased growth rates. Modification of the catalase gene katG results in resistance to isoniazid, mainly by reduced or absent catalase activity. Catalase activity is absolutely required to convert isoniazid to the active hydrazine derivative. Interestingly, animal model studies suggest that M. tuberculosis strains in which the katG gene is inactivated are attenuated compared with wild-type strains. Low-level rifampicin resistance can be obtained by point mutations in inhA leading to its overexpression. Pyrazinamide is a prodrug requiring pyrazinamidase to produce the active pyrazinoic acid. Most cases of resistance are due to mutations in the pyrazinamidase gene (pncA), but gene inactivation by the insertion sequence IS6110 has been reported. Streptomycin resistance can arise through mutations in rrs and rpsL which affect streptomycin binding. However, these account for only half of the resistant isolates, so further resistance mechanisms await definition. Ethambutol resistance has been noted in M. tuberculosis and other species such as M. smegmatis. Ethambutol inhibits the polymerization of arabinan in the arabinogalactan and lipo-arabinomannan of the mycobacterial cell wall and one of its likely targets is the family of arabinosyl-transferases encoded by the emb locus. Missense mutations in the embB gene in this locus confer resistance to ethambutol.