Exogenous Glucose Interferes with Antimicrobial-Mediated ROS Accumulation and Bacterial Death.

Glucose is widely used in the reconstitution of intravenous medications, which often include antimicrobials. How glucose affects antimicrobial activity has not been comprehensively studied. The present work reports that glucose added to bacteria growing in a rich medium suppresses the bactericidal but not the bacteriostatic activity of several antimicrobial classes, thereby revealing a phenomenon called glucose-mediated antimicrobial tolerance. Glucose, at concentrations corresponding to blood-sugar levels of humans, increased survival of Escherichia coli treated with quinolones, aminoglycosides, and cephalosporins with little effect on minimal inhibitory concentration. Glucose suppressed a ROS surge stimulated by ciprofloxacin. Genes involved in phosphorylated fructose metabolism contributed to glucose-mediated tolerance, since a pfkA deficiency, which blocks the formation of fructose-1,6-bisphosphate, eliminated protection by glucose. Disrupting the pentose phosphate pathway or the TCA cycle failed to alter glucose-mediated tolerance, consistent with an upstream involvement of phosphorylated fructose. Exogenous sodium pyruvate or sodium citrate reversed glucose-mediated antimicrobial tolerance. Both metabolites bypass the effects of fructose-1,6-bisphosphate, a compound known to scavenge hydroxyl radical and chelate iron, activities that suppress ROS accumulation. Treatment with these two compounds constitutes a novel way to mitigate the glucose-mediated antimicrobial tolerance that may exist during intravenous antimicrobial therapy, especially for diabetes patients.

Prophage-encoded methyltransferase drives adaptation of community-acquired methicillin-resistant Staphylococcus aureus.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a novel mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that Δagr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Fluoroquinolone heteroresistance, antimicrobial tolerance, and lethality enhancement.

With tuberculosis, the emergence of fluoroquinolone resistance erodes the ability of treatment to interrupt the progression of MDR-TB to XDR-TB. One way to reduce the emergence of resistance is to identify heteroresistant infections in which subpopulations of resistant mutants are likely to expand and make the infections fully resistant: treatment modification can be instituted to suppress mutant enrichment. Rapid DNA-based detection methods exploit the finding that fluoroquinolone-resistant substitutions occur largely in a few codons of DNA gyrase. A second approach for restricting the emergence of resistance involves understanding fluoroquinolone lethality through studies of antimicrobial tolerance, a condition in which bacteria fail to be killed even though their growth is blocked by lethal agents. Studies with Escherichia coli guide work with Mycobacterium tuberculosis. Lethal action, which is mechanistically distinct from blocking growth, is associated with a surge in respiration and reactive oxygen species (ROS). Mutations in carbohydrate metabolism that attenuate ROS accumulation create pan-tolerance to antimicrobials, disinfectants, and environmental stressors. These observations indicate the existence of a general death pathway with respect to stressors. M. tuberculosis displays a variation on the death pathway idea, as stress-induced ROS is generated by NADH-mediated reductive stress rather than by respiration. A third approach, which emerges from lethality studies, uses a small molecule, N-acetyl cysteine, to artificially increase respiration and additional ROS accumulation. That enhances moxifloxacin lethality with M. tuberculosis in culture, during infection of cultured macrophages, and with infection of mice. Addition of ROS stimulators to fluoroquinolone treatment of tuberculosis constitutes a new direction for suppressing the transition of MDR-TB to XDR-TB.

Moxifloxacin-Mediated Killing of Mycobacterium tuberculosis Involves Respiratory Downshift, Reductive Stress, and Accumulation of Reactive Oxygen Species.

Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on the Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A noninvasive redox biosensor and a reactive oxygen species (ROS)-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD+ ratio, and increased the labile iron pool in M. tuberculosis. Lowering the NADH/NAD+ ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and cotreatment with NAC potentiated the antimycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice, where NAC restricted the emergence of resistance. Thus, NADH-reductive stress contributes to moxifloxacin-mediated killing of M. tuberculosis, and the respiration stimulator (NAC) enhances lethality and suppresses the emergence of drug resistance.

A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Glutamine Is Required for M1-like Polarization of Macrophages in Response to Mycobacterium tuberculosis Infection.

In response to Mycobacterium tuberculosis infection, macrophages mount proinflammatory and antimicrobial responses similar to those observed in M1 macrophages activated by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). A metabolic reprogramming to hypoxia-inducible-factor 1 (HIF-1)-mediated uptake of glucose and its metabolism by glycolysis is required for M1-like polarization, but little is known about other metabolic programs driving the M1-like polarization during infection. We report that glutamine serves as a carbon and nitrogen source for the metabolic reprogramming to M1-like macrophages. Widely targeted metabolite screening identified an association of glutamine and/or glutamate with highly affected metabolic pathways of M1-like macrophages. Moreover, stable isotope-assisted metabolomics of U13C glutamine and U13C glucose revealed that glutamine, rather than glucose, is catabolized in both the oxidative and reductive tricarboxylic acid (TCA) cycles of M1-like macrophages, thereby generating signaling molecules that include succinate, biosynthetic precursors such as aspartate, and itaconate. U15N glutamine-tracing metabolomics further revealed participation of glutamine nitrogen in synthesis of intermediates of purine and pyrimidine metabolism plus amino acids, including aspartate. These findings were corroborated by diminished M1 polarization from chemical inhibition of glutaminase (GLS), the key enzyme in the glutaminolysis pathway, and by genetic deletion of GLS in infected macrophages. Thus, the catabolism of glutamine is an integral component of metabolic reprogramming in activating macrophages and it coordinates with elevated cytosolic glycolysis to satisfy the cellular demand for bioenergetic and biosynthetic precursors of M1-like macrophages. Knowledge of these new immunometabolic features of M1-like macrophages should advance the development of host-directed therapies for tuberculosis. IMPORTANCE Macrophages play essential roles in determining the progression and final outcome of human infection by Mycobacterium tuberculosis. While upregulation of hypoxia-inducible-factor 1 (HIF-1) and a metabolic reprogramming to the Warburg Effect-like state are known to be critical for immune cell activation in response to M. tuberculosis infection, our overall knowledge about the immunometabolism of M1-like macrophages is poor. Using widely targeted small-metabolite screening, stable isotope tracing metabolomics, and pharmacological and genetic approaches, we report that, in addition to enhanced glucose catabolism by glycolysis, glutamine is utilized as an important carbon and nitrogen source for the generation of biosynthetic precursors, signaling molecules, and itaconate in M. tuberculosis-induced M1-like macrophages. Recognizing this novel contribution of glutamine to the immunometabolic properties of M. tuberculosis-infected macrophages may facilitate the development of treatments for tuberculosis and stimulate comparable studies with other pathogen-macrophage interactions.

Diabetes as a potential compounding factor in COVID-19-mediated male subfertility.

Recent work indicates that male fertility is compromised by SARS-CoV-2 infection. Direct effects derive from the presence of viral entry receptors (ACE2 and/or CD147) on the surface of testicular cells, such as spermatocytes, Sertoli cells, and Leydig cells. Indirect effects on testis and concentrations of male reproductive hormones derive from (1) virus-stimulated inflammation; (2) viral-induced diabetes, and (3) an interaction between diabetes and inflammation that exacerbates the deleterious effect of each perturbation. Reproductive hormones affected include testosterone, luteinizing hormone, and follicle-stimulating hormone. Reduction of male fertility is also observed with other viral infections, but the global pandemic of COVID-19 makes demographic and public health implications of reduced male fertility of major concern, especially if it occurs in the absence of serious symptoms that would otherwise encourage vaccination. Clinical documentation of COVID-19-associated male subfertility is now warranted to obtain quantitative relationships between infection severity and subfertility; mechanistic studies using animal models may reveal ways to mitigate the problem. In the meantime, the possibility of subfertility due to COVID-19 should enter considerations of vaccine hesitancy by reproductive-age males.

Bacterial death from treatment with fluoroquinolones and other lethal stressors.

INTRODUCTION: Lethal stressors, including antimicrobials, kill bacteria in part through a metabolic response proposed to involve reactive oxygen species (ROS). The quinolone anti-bacterials have served as key experimental tools in developing this idea. AREAS COVERED: Bacteriostatic and bactericidal action of quinolones are distinguished, with emphasis on the contribution of chromosome fragmentation and ROS accumulation to bacterial death. Action of non-quinolone antibacterials and non-antimicrobial stressors is described to provide a general framework for understanding stress-mediated, bacterial death. EXPERT OPINION: Quinolones trap topoisomerases on DNA in reversible complexes that block DNA replication and bacterial growth. At elevated drug concentrations, DNA ends are released from topoisomerase-mediated constraint, leading to the idea that death arises from chromosome fragmentation. However, DNA ends also stimulate repair, which is energetically expensive. An incompletely understood metabolic shift occurs, and ROS accumulate. Even after quinolone removal, ROS continue to amplify, generating secondary and tertiary damage that overwhelms repair and causes death. Repair may also contribute to death directly via DNA breaks arising from incomplete base-excision repair of ROS-oxidized nucleotides. Remarkably, perturbations that interfere with ROS accumulation confer tolerance to many diverse lethal agents.

Gain-of-Function Mutations in Acid Stress Response (evgS) Protect Escherichia coli from Killing by Gallium Nitrate, an Antimicrobial Candidate.

Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDA-approved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, and kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.

Susceptibility of Cutibacterium acnes to topical minocycline foam.

FMX101 4% minocycline foam (FMX101 4%) is a novel, topical minocycline formulation for treatment of acne vulgaris. We report that FMX101 4% had an MIC90 of 0.25 µg/ml and was ≥4-fold more active than comparator antimicrobials against a panel of 98 clinical Cutibacterium acnes isolates. The panel was diverse by clonal complex and sequence type, having 20 novel multi-locus sequence types including clonal complexes and sequence types associated with acne (CC1, CC3, and CC4; ST1 and ST3). Some isolates were phenotypically resistant to clindamycin (6.1%), erythromycin (14.3%), and tetracycline (2.0% intermediate resistance). Six isolates (6.4%) carried a mutation in the quinolone resistance-determining region of gyrA. With C. acnes, spontaneous resistance to FMX101 4% occurred at frequencies ranging from ≤5 × 10-9 to <1 × 10-8; mutations were identified in rpsJ, a gene encoding 30S ribosomal protein S10. No mutant exhibited a minocycline MIC above 0.5 µg/ml. No second-step mutation in previously isolated mutants or strains containing rpsJ ± 16S rRNA mutations was detected following minocycline challenge. Minocycline retained antibacterial activity against C. acnes over 15 multiple passages; thus, no selective growth advantage for minocycline-resistant mutants occurred under the experimental conditions. FMX101 4% has the potential to retain the favorable resistance profile of minocycline in diverse C. acnes isolates while providing the benefits of a topical formulation for treatment of acne vulgaris.

Reactive oxygen species play a dominant role in all pathways of rapid quinolone-mediated killing.

BACKGROUND: Quinolones have been thought to rapidly kill bacteria in two ways: (i) quinolone-topoisomerase-DNA lesions stimulate the accumulation of toxic reactive oxygen species (ROS); and (ii) the lesions directly cause lethal DNA breaks. Traditional killing assays may have underestimated the ROS contribution by overlooking the possibility that ROS continue to accumulate and kill cells on drug-free agar after quinolone removal. METHODS: Quinolone-induced, ROS-mediated killing of Escherichia coli was measured by plating post-treatment samples on agar with/without anti-ROS agents. RESULTS: When E. coli cultures were treated with ciprofloxacin or moxifloxacin in the presence of chloramphenicol (to accentuate DNA-break-mediated killing), lethal activity, revealed by plating on quinolone-free agar, was inhibited by supplementing agar with ROS-mitigating agents. Moreover, norfloxacin-mediated lethality, observed with cells suspended in saline, was blocked by inhibitors of ROS accumulation and exacerbated by a katG catalase deficiency that impairs peroxide detoxification. Unlike WT cells, the katG mutant was killed by nalidixic acid or norfloxacin with chloramphenicol present and by nalidixic or oxolinic acid with cells suspended in saline. ROS accumulated after quinolone removal with cultures either co-treated with chloramphenicol or suspended in saline. Deficiencies in recA or recB reduced the protective effects of ROS-mitigating agents, supporting the idea that repair of quinolone-mediated DNA lesions suppresses the direct lethal effects of such lesions. CONCLUSIONS: ROS are the dominant factor in all modes of quinolone-mediated lethality, as quinolone-mediated primary DNA lesions are insufficient to kill without triggering ROS accumulation. ROS-stimulating adjuvants may enhance the lethality of quinolones and perhaps other antimicrobials.

Post-stress bacterial cell death mediated by reactive oxygen species.

Antimicrobial efficacy, which is central to many aspects of medicine, is being rapidly eroded by bacterial resistance. Since new resistance can be induced by antimicrobial action, highly lethal agents that rapidly reduce bacterial burden during infection should help restrict the emergence of resistance. To improve lethal activity, recent work has focused on toxic reactive oxygen species (ROS) as part of the bactericidal activity of diverse antimicrobials. We report that when Escherichia coli was subjected to antimicrobial stress and the stressor was subsequently removed, both ROS accumulation and cell death continued to occur. Blocking ROS accumulation by exogenous mitigating agents slowed or inhibited poststressor death. Similar results were obtained with a temperature-sensitive mutational inhibition of DNA replication. Thus, bacteria exposed to lethal stressors may not die during treatment, as has long been thought; instead, death can occur after plating on drug-free agar due to poststress ROS-mediated toxicity. Examples are described in which (i) primary stress-mediated damage was insufficient to kill bacteria due to repair; (ii) ROS overcame repair (i.e., protection from anti-ROS agents was reduced by repair deficiencies); and (iii) killing was reduced by anti-oxidative stress genes acting before stress exposure. Enzymatic suppression of poststress ROS-mediated lethality by exogenous catalase supports a causal rather than a coincidental role for ROS in stress-mediated lethality, thereby countering challenges to ROS involvement in antimicrobial killing. We conclude that for a variety of stressors, lethal action derives, at least in part, from stimulation of a self-amplifying accumulation of ROS that overwhelms the repair of primary damage.

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.

Fluoroquinolone-Gyrase-DNA Cleaved Complexes.

The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. These ternary complexes, called cleaved complexes because the DNA moiety is broken, block replication, transcription, and bacterial growth. Cleaved complexes readily form in vitro when gyrase, plasmid DNA, and quinolone are combined and incubated; complexes are detected by the linearization of plasmid DNA, generally assayed by gel electrophoresis. The stability of the complexes can be assessed by treatment with EDTA, high temperature, or dilution to dissociate the complexes and reseal the DNA moiety. Properties of the complexes are sensitive to quinolone structure and to topoisomerase amino acid substitutions associated with quinolone resistance. Consequently, studies of cleaved complexes can be used to identify improvements in quinolone structure and to understand the biochemical basis of target-based resistance. Cleaved complexes can also be detected in quinolone-treated bacterial cells by their ability to rapidly block DNA replication and to cause chromosome fragmentation; they can even be recovered from lysed cells following CsCl density-gradient centrifugation. Thus, in vivo and cell-fractionation tests are available for assessing the biological relevance of work with purified components.

Suppression of Reactive Oxygen Species Accumulation Accounts for Paradoxical Bacterial Survival at High Quinolone Concentration.

When bacterial cells are exposed to increasing concentrations of quinolone-class antibacterials, survival drops, reaches a minimum, and then recovers, sometimes to 100%. Despite decades of study, events underlying this paradoxical high-concentration survival remain obscure. Since reactive oxygen species (ROS) have been implicated in antimicrobial lethality, conditions generating paradoxical survival were examined for diminished ROS accumulation. Escherichia coli cultures were treated with various concentrations of nalidixic acid, followed by measurements of survival, rate of protein synthesis, and ROS accumulation. The last measurement used a dye (carboxy-H2DCFDA) that fluoresces in the presence of ROS; fluorescence was assessed by microscopy (individual cells) and flow cytometry (batch cultures). High, nonlethal concentrations of nalidixic acid induced lower levels of ROS than moderate, lethal concentrations. Sublethal doses of exogenous hydrogen peroxide became lethal and eliminated the nalidixic acid-associated paradoxical survival. Thus, quinolone-mediated lesions needed for ROS-executed killing persist at high, nonlethal quinolone concentrations, thereby implicating ROS as a key factor in cell death. Chloramphenicol suppressed nalidixic acid-induced ROS accumulation and blocked lethality, further supporting a role for ROS in killing. Nalidixic acid also inhibited protein synthesis, with extensive inhibition at high concentrations correlating with lower ROS accumulation and paradoxical survival. A catalase deficiency, which elevated ROS levels, overcame the inhibitory effect of chloramphenicol on nalidixic acid-mediated killing, emphasizing the importance of ROS. The data collectively indicate that ROS play a dominant role in the lethal action of narrow-spectrum quinolone-class compounds; a drop in ROS levels accounted for the quinolone tolerance observed at very high concentrations.

Tuning of the Lethal Response to Multiple Stressors with a Single-Site Mutation during Clinical Infection by Staphylococcus aureus.

The agr system of Staphylococcus aureus promotes invasion of host tissues, and as expected, agents that block agr quorum sensing have anti-infective properties. Paradoxically, agr-defective mutants are frequently recovered from patients, especially those persistently infected with S. aureus We found that an agr deficiency increased survival of cultured bacteria during severe stress, such as treatment with gentamicin, ciprofloxacin, heat, or low pH. With daptomycin, deletion of agr decreased survival. Therefore, agr activity can be either detrimental or protective, depending on the type of lethal stress. Deletion of agr had no effect on the ability of the antimicrobials to block bacterial growth, indicating that agr effects are limited to lethal action. Thus, the effect of an agr deletion is on bacterial tolerance, not resistance. For gentamicin and daptomycin, activity can be altered by agr-regulated secreted factors. For ciprofloxacin, a detrimental function was downregulation of glutathione peroxidase (bsaA), an enzyme responsible for defense against oxidative stress. Deficiencies in agr and bsaA were epistatic for survival, consistent with agr having a destructive role mediated by reactive oxygen species. Enhanced susceptibility to lethal stress by wild-type agr, particularly antimicrobial stress, helps explain why inactivating mutations in S. aureus agr commonly occur in hospitalized patients during infection. Moreover, the agr quorum-sensing system of S. aureus provides a clinically relevant example in which a single-step change in the response to severe stress alters the evolutionary path of a pathogen during infection.IMPORTANCE When phenotypes produced in response to an environmental stress are inadequate to buffer against that stress, changes that do buffer may become genetically encoded by natural selection. A clinically relevant example is seen with S. aureus mutants that are deficient in the key virulence regulator agr Paradoxically, defects in agr are selected during serious hospital infection and have been associated with worse outcome. The current work helps resolve this paradox: agr mutants are often less readily killed by lethal stressors without affecting MIC, a phenomenon known as tolerance. Our results indicate that tolerance, which would not be detected as resistance, can be selected in clinical settings. The data also support the ideas that (i) S. aureus broadly hedges against environmental change and stress through genome plasticity, (ii) reactive oxygen can be involved in the self-destructive response in bacteria, and (iii) therapeutic targeting of agr and virulence can be counterproductive.

Contribution of reactive oxygen species to thymineless death in Escherichia coli.

Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.

Fluoroquinolones as imaging agents for bacterial infection.

Diagnosis of deep-seated bacterial infection is difficult, as neither standard anatomical imaging nor radiolabeled, autologous leukocytes distinguish sterile inflammation from infection. Two recent imaging efforts are receiving attention: (1) radioactive derivatives of sorbitol show good specificity with Gram-negative bacterial infections, and (2) success in combining anatomical and functional imaging for cancer diagnosis has rekindled interest in 99mTc-fluoroquinolone-based imaging. With the latter, computed tomography (CT) would be combined with single-photon-emission-computed tomography (SPECT) to detect 99mTc-fluoroquinolone-bacterial interactions. The present minireview provides a framework for advancing fluoroquinolone-based imaging by identifying gaps in our understanding of the process. One issue is the reliance of 99mTc labeling on the reduction of sodium pertechnetate, which can lead to colloid formation and loss of specificity. Specificity problems may be reduced by altering the quinolone structure (for example, switching from ciprofloxacin to sitafloxacin). Another issue is the uncharacterized nature of 99mTc-ciprofloxacin binding to, or sequestration in, bacteria: specific interactions with DNA gyrase, an intracellular fluoroquinolone target, are unlikely. Labeling with 68Ga rather than 99mTc enables detection by positron emission tomography, but with similar biological uncertainties. Replacing the C6-F of the fluoroquinolone with 18F provides an alternative to pertechnetate and gallium that may lead to imaging based on drug interactions with gyrase. Gyrase-based imaging requires knowledge of fluoroquinolone action, which we update. We conclude that quinolone-based probes show promise for the diagnosis of infection, but improvements in specificity and sensitivity are needed. These improvements include the optimization of the quinolone structure; such chemistry efforts can be accelerated by refining microbiological assays.