How does fluoroquinolones affect and destroy bacteria

The dominance of sensitivity over resistance in the two target enzymes also implies that quinolones with similar potency against both gyrase and topoisomerase IV in an organism may require mutations in both enzymes before the mutant bacterium shows a substantial resistance phenotype Pan and Fisher , ; Strahilevitz and Hooper Fluoroquinolones currently in clinical use generally have differences in potency between the two target enzymes, and single target mutations produce eight- to fold increases in resistance.

Accumulating mutations in both target enzymes have been shown to cause increasing quinolone resistance. In many species, high-level quinolone resistance is generally associated with mutations in both gyrase and topoisomerase IV Schmitz et al. In several species, Mycobacterium tuberculosis , Helicobacter pylori , and Treponema pallidum , there is no topoisomerase IV, and gyrase provides the functions of both enzymes and is the only quinolone target Hooper Thus, selection of mutations with substantial resistance phenotypes is predicted to occur readily in these pathogens, a prediction consistent with the frequent occurrence of resistance with clinical use of quinolones without use of other active agents to treat infections with M.

Quinolones must cross the bacterial envelope to interact with their cytoplasmic gyrase and topoisomerase IV targets. Active quinolone efflux, reductions in influx, or both can decrease cytoplasmic quinolone concentrations and confer resistance. In Gram-positive bacteria, reduced diffusion across the cytoplasmic membrane has not been found to cause resistance, but active efflux transporters that include quinolones in their substrate profiles have been shown to cause low-level resistance.

In contrast, in Gram-negative bacteria, reduced diffusion through outer membrane porin diffusion channels can contribute to resistance. Reduced influx often acts in concert with basal or increased expression of efflux transporters with both contributing additively to resistance Lomovskaya et al. Quinolones themselves generally do not induce expression of efflux pumps. With the exception of plasmid-mediated quinolone resistance discussed later, acquired quinolone resistance by altered drug permeation occurs largely by mutations in genes encoding regulatory proteins that control the transcription of efflux pump or porin genes Grkovic et al.

Uncommonly, mutations in efflux pump structural genes have caused changes in pump substrate profiles that add quinolones Blair et al. The levels of quinolone resistance because of regulatory mutation and pump overexpression are often limited to about four- to eightfold increases in inhibitory concentrations, likely because of counterbalancing regulatory factors and cellular toxicities of high levels of pump overexpression.

In Gram-positive bacteria, the major facilitator superfamily MFS of transporters contains the largest number of efflux transporters that include quinolones in their substrate profiles. These efflux pumps are transporters energized by the proton gradient across the bacterial membrane and are generally antiporters with exchange of substrate and protons in opposite directions.

NorA Ubukata et al. NorA confers resistance to hydrophilic quinolones, such as norfloxacin and ciprofloxacin, whereas NorB and NorC each confer resistance to both hydrophilic quinolones and hydrophobic quinolones, such as sparfloxacin and moxifloxacin Yu et al.

Although there are natural quinolone-like compounds Heeb et al. Regulation of expression of these transporters is complex and involves several transcriptional regulators. MgrA, the most studied, acts as a positive regulator of norA expression and a negative regulator of norB and norC expression Ingavale et al. Posttranslational phosphorylation of MgrA by the PknB kinase results in the loss of the ability of MgrA dimers to bind the norA promoter and an increase in their binding to the norB promoter Truong-Bolduc et al.

Acidic conditions alter the proportions of phosphorylated and unphosphorylated MgrA, and oxidative and aeration conditions also affect dimerization and promoter binding Chen et al.

Thus, relative levels of expression of NorA, NorB, and NorC are modified in response to a variety of environmental conditions. Notably, norB expression is selectively increased in an abscess environment in response to low-free iron conditions and contributes to fitness and bacterial survival in abscesses Ding et al. The natural substrate of NorB, transport of which may contribute to improving fitness in an abscess environment, is not known.

In addition, physiologic increased expression of NorB at the site of infection would suggest that susceptibility testing under clinical laboratory conditions may not fully reflect susceptibility at the site of infection. NorG, a member of the GntR-like transcriptional regulators, can also modulate pump expression and levels of quinolone resistance; it is a direct activator of norA and norB expression but a direct repressor of norC expression Truong-Bolduc and Hooper ; Truong-Bolduc et al.

ArlRS, a two-component regulatory system, has been shown to affect expression of norA as well Fournier and Hooper ; Fournier et al. There are often hierarchies in regulatory networks, and other regulators can affect expression of MgrA and NorG. Such complex regulatory networks affecting pump expression imply the importance of modulation of pump functions in cellular physiology and may contribute to different bacterial responses to quinolones in different environments that affect pump expression.

Other MFS efflux transporters that can contribute to quinolone resistance in S. MFS transporters in other Gram-positive bacteria have also been shown to include quinolones in their substrate profiles. These transporters include Bmr, Bmr3, and Blt of B. MATE family pumps, like those of the MFS, are secondary transporters energized by the membrane electrochemical gradient.

MepA confers resistance to norfloxacin, ciprofloxacin, moxifloxacin, and sparfloxacin, as well as other antimicrobials and dyes Kaatz et al. Mutation in FepR causes FepA overexpression and resistance to norfloxacin and ciprofloxacin Guerin et al. Members of the ABC family of transporters are, in contrast to the other pump families discussed, energized by ATP hydrolysis. PatAB of S. In Gram-negative bacteria, the majority of efflux pumps that can effect quinolone resistance are members of the resistance—nodulation—division RND superfamily Li et al.

The RND pumps are secondary antiporters composed of a pump protein localized in the cytoplasmic membrane, an outer membrane channel protein, and a membrane fusion protein that links the pump and the outer membrane protein Du et al. Some outer membrane components may link to more than one pump—fusion protein pair, enabling export of substrates across both inner and outer membranes Li and Nikaido The best-studied systems have been in E.

Crystal structures of the complex have revealed a trimer of AcrB pump monomers that rotate around a central axis perpendicular to the membrane, with each monomer as its rotation position changes assuming a different conformation mediating different steps in substrate binding and extrusion through the channel Nikaido and Takatsuka Substrates enter the vestibule of AcrB from the periplasmic space between the inner and outer membranes or the outer leaflet of the inner membrane.

Binding sites for ciprofloxacin and other substrates of diverse chemical types have been identified in the central cavity of the periplasmic domain of AcrB Yu et al. Fluoroquinolones, which are zwitterionic, are presumed to cross the outer membrane through OmpF and OmpC porin diffusion channels, down-regulation or mutation of which may amplify resistance.

Mutations in the MarR regulator can result in both an increase in acrB expression as well as a decrease in ompF expression, dually contributing to quinolone resistance Alekshun and Levy Mutations in the E. Expression of AcrAB-TolC also confers resistance to bile salts and is induced by bile salts, likely one of its natural substrates Rosenberg et al.

Thus, AcrAB supports the ability of E. Mutations in mexA and oprM cause increased uptake of norfloxacin and increased susceptibility to fluoroquinolones Poole et al. Increased expression of MexAB-OprM because of mutations in the MexR negative regulator causes increased resistance to ciprofloxacin and nalidixic acid, and mexR mutants can be selected with exposure to fluoroquinolones Poole et al.

These pumps vary in expression levels in wild-type strains Li et al. Mutations in the global regulator MvaT, which affects quorum sensing and virulence, also causes increased expression of mexEF-oprM and resistance to norfloxacin Westfall et al. Mutations in the MexZ repressor cause increased expression of MexXY-OprM and resistance to fluoroquinolones, aminoglycosides, and other pump substrates Matsuo et al. Most quinolones in clinical use have a fluorine at position 6 and a positively charged substituent at position 7 e.

In contrast, quinolones lacking a positive charge at position 7 e. Additional RND pumps that cause quinolone resistance have been found in a broad range of Gram-negative bacteria.

Salmonella spp. Baucheron et al. The CmeABC RND pump of Campylobacter jejuni contributes to the resistance of mutants selected with enrofloxacin, a veterinary quinolone similar to ciprofloxacin Lin et al. Among nonenteric bacteria, in A.

Non-RND efflux pumps are much less common in Gram-negative bacteria. Among MFS pumps, in E. MdfA, originally termed CmlA, confers resistance to both chloramphenicol and fluoroquinolones Yang et al. There are additional examples in both Gram-positive and Gram-negative bacteria in which there is evidence of efflux in quinolone-resistant isolates determined by either reduction in resistance with addition of a broad efflux pump inhibitor or reduced quinolone accumulation in resistant cells, but the contributing pump or its regulator have not been identified Li and Nikaido ; Nikaido and Takatsuka Thus, efflux-mediated resistance to quinolones and many other antimicrobials is widespread.

The broad substrate profiles of these pumps link quinolone resistance to multidrug resistance and constitute mechanisms by which use of non-quinolone antimicrobials can also increase quinolone resistance. A similar linkage to multidrug resistance occurs with plasmid-mediated quinolone resistance, which is discussed in the next section. Transferable nalidixic acid resistance had been sought unsuccessfully in the s Burman , and plasmid-mediated resistance was thought unlikely to exist because quinolones are synthetic compounds, and adequate resistance can arise by chromosomal mutations Courvalin When the responsible gene, named qnr and later qnrA , was cloned and sequenced facilitating its identification by PCR Tran and Jacoby , qnr was soon found at low-frequency on plasmids in Gram-negative isolates around the world.

In the last decade, PMQR genes have been found in bacterial isolates worldwide. They reduce bacterial susceptibility to quinolones, usually not to the level of clinical nonsusceptibility, but facilitate the selection of mutants with higher level quinolone resistance and promote treatment failure.

Cloning and sequencing qnrA revealed that it encoded a residue protein with a tandemly repeating unit of five amino acids that indicated membership in the many thousand-member pentapeptide repeat family of proteins Tran and Jacoby Further searches led to the discovery of related genes for plasmid-mediated pentapeptide repeat proteins qnrS Hata et al. The first pentapeptide-repeat protein to have its structure determined by X-ray crystallography was MfpA, which is encoded on the chromosome of M.

In vitro, MfpA inhibits DNA supercoiling by gyrase and, although it fails to block gyrase inhibition by quinolone, it can still confer quinolone resistance to whole cells by competing with DNA to reduce the number of lethal double-strand breaks produced by quinolone Hegde et al. Like MfpA, they form rod-like dimers but have additional structural features. The structure of QnrB1 is shown in Figure 1.

Hydrogen bonding between backbone atoms of neighboring coils stabilizes the helix. The rod-like structure of the QnrB1 dimer is shown above with the sequence of the monomer below. Face names and color are shown at the top along with the naming convention for the five residues of the pentapeptide repeats. Loops A and B are indicated by one and two asterisks, respectively, with their sequences indicated below and the loops shown as black traces on the diagram.

The molecular twofold symmetry is indicated with a black diamond. Type II turn containing faces are shown as spheres and type IV-containing faces as strands. N-term, Amino terminal; C-term, carboxy terminal. From Jacoby et al. The monomers of QnrB1 and AhQnr have projecting loops of eight and 12 amino acids that are important for their activity.

Deletion of the small A loop reduces quinolone protection, whereas deletion of the larger B loop or both loops destroys protective activity Vetting et al. Removal of even a single amino acid in the larger loop compromises protection. EfsQnr lack loops, but EfsQnr differs from MfpA in having a amino-acid flexible extension required for full protective activity Hegde et al. In vitro, more Qnr is required to protect DNA gyrase as the inhibiting concentration of quinolone is increased Tran and Jacoby In a gel-displacement assay Tran et al.

Binding to GyrA is reduced by the same amino- and carboxy-terminal and loop B deletions in QnrB that destroy its protective activity, whereas subinhibitory concentrations of ciprofloxacin reduce binding to GyrA but not to GyrB, suggesting that Qnr protects gyrase by blocking access of quinolone to GyrA sites essential for its lethal action. Many naturally occurring antibiotics and synthetic agents also target DNA gyrase.

Qnr protects against compounds with a somewhat quinolone-like structure Jacoby et al. Qnr, however, does not block agents acting on the GyrB subunit, and it also does not block simocyclinone D8, which, like quinolones, binds to the amino terminus of GyrA and blocks DNA binding Hearnshaw et al.

Aquatic bacteria are especially well represented, including species of Aeromonas , Photobacterium , Shewanella , and Vibrio Poirel et al. QnrB homologs, on the other hand, are encoded on the chromosome of members of the Citrobacter freundii complex Jacoby et al.

The small, nonconjugative plasmids that carry qnrD are especially likely to be found in Proteeae , such as Proteus mirabilis , P. The worldwide distribution of qnr suggests an origin well before quinolones were discovered.

Indeed, qnrB genes and pseudogenes have been discovered on the chromosome of C. What the native function of Qnr may have been is an as-yet unanswered question. The role of TrpArg is to position the Tyr face for optimal interaction Vetting et al.

Several alleles have been described Quiroga et al. The gene has been found worldwide in a variety of Enterobacteriaceae and even in P. OqxAB was first identified as a plasmid-mediated efflux pump conferring resistance to the olaquindox, a food additive enhancing growth in pigs Sorensen et al.

Genes for oqxAB are commonly found on the chromosome of K. The newer fluoroquinolones have an extended antimicrobial spectrum compared to their older congeners, and are highly active against most Gram-negative pathogens including the Enterobacteriaceae and Pseudomonas aeruginosa.

While Staphylococcus aureus and coagulase-negative staphylococci are usually susceptible to the fluoroquinolones, streptococci are generally more resistant and enterococci are resistant. All of the newer fluoroquinolones may be administered orally and most of them have been administered parenterally. They are widely distributed in the body, attaining therapeutic concentrations in most tissues. It helped to trigger a wave of reports on websites such as the Quinolone Antibiotics Adverse Reaction Forum, which by hosted more than 5, posts.

The late Jay Cohen, then a psychiatrist and medical researcher at the University of California, San Diego, contacted patients through the sites and published 45 case studies 1.

Cohen warned that after taking fluoroquinolones, some people had developed serious problems in multiple organs. These effects came on rapidly and lasted for months or years. But complaints and patient petitions continued. The FDA says that the reports of adverse events it receives — sent in by drug manufacturers, by doctors and directly by consumers — cannot be used to reach conclusions about the severity of problems associated with drugs.

Still, the fluoroquinolones have attracted more complaints than other more widely used antibiotics. Bennett is also director of the Southern Network on Adverse Reactions, a state-funded pharmaceutical-safety watchdog, which has been working with people affected by fluoroquinolones since Such warnings are placed inside a black box on drug labels, and call attention to serious or life-threatening risks.

As alerts mounted, patients launched lawsuits against manufacturers of the drugs, claiming they had not been adequately informed of risks. These cases have been variously won, lost or settled for undisclosed sums, and many are still in progress; manufacturers argue that they handled risks appropriately, and work with the FDA to update safety labels. In November , the FDA voted to recognize FQAD as a syndrome on the basis of cases that the agency regarded as clear-cut: otherwise healthy people who took fluoroquinolones for minor ailments and then developed disabling and potentially irreversible conditions 2.

The FDA also noted a disturbing pattern: fluoroquinolones had a much higher percentage of disabilities among their serious-adverse-event reports than did other antibiotics. Beatrice Golomb at the University of California, San Diego, has been working for a decade with people affected by fluoroquinolones, beginning with David Melvin, a police officer and keen cyclist who had to use a wheelchair after he was given levofloxacin for suspected epididymitis in Accumulating evidence, Golomb says, suggests that fluoroquinolones are damaging mitochondria, the power packs inside human cells that evolved from symbiotic, bacteria-like cells billions of years ago.

This kind of harm can affect every cell in the body, explaining why a wide range of symptoms can appear and get worse over time. Mitochondrial toxicity is a problem with many classes of drug, says Mike Murphy, who studies the biology of mitochondria at the University of Cambridge, UK. But because mitochondria retain some similarities to their bacterial ancestors, antibiotics can pose a particular threat to them.

Researchers have shown, for example, that aminoglycoside antibiotics can cause deafness by damaging mitochondria in the hair cells of the ear 3. Isolated studies from the s onwards have suggested that fluoroquinolones impair mitochondrial function, but a study 4 by Collins and his colleagues is the most convincing, researchers say. They reported that antibiotics in several classes triggered oxidative stress — a build-up of reactive, oxygen-containing molecules — in mitochondria, inhibiting their function across a range of mammalian cells, as well as in mice.

Pharmaceutical researchers had spotted the issue, too: in , toxicologist Yvonne Will and her colleagues at Pfizer in Groton, Connecticut, reported an assay to detect mitochondrial damage early in drug development 5.

One problem is that there is still no reliable biomarker that researchers can use to test for mitochondrial damage in people, tying cell-line research to clinical experience. Nor is it known precisely how the fluoroquinolones are damaging human cells. But Neil Osheroff, a biochemist at Vanderbilt University in Nashville, Tennessee, who studies fluoroquinolones, is doubtful about that result.

He has done his own lab tests, and found that, at therapeutic concentrations, the fluoroquinolones prescribed by doctors have very little effect on human DNA 7. At a conference last September, Bennett reported preliminary data that might hint at why only some people develop serious side effects from fluoroquinolones. But this antibiotic does not affect the DNA gyrases of humans and thus, again, bacteria die while the host remains unharmed.

Many other compounds can kill both bacterial and human cells. It is the selective action of antibiotics against bacteria that make them useful in the treatment of infections while at the same time allowing the host to live another day.

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