Differential impacts of DNA repair machinery on fluoroquinolone persisters with different chromosome abundances.

DNA repair machinery has been found to be indispensable for fluoroquinolone (FQ) persistence of Escherichia coli. Previously, we found that cells harboring two copies of the chromosome (2Chr) in stationary-phase cultures were more likely to yield FQ persisters than those with one copy of the chromosome (1Chr). Furthermore, we found that RecA and RecB were required to observe that difference, and that loss of either more significantly impacted 2Chr persisters than 1Chr persisters. To better understand the survival mechanisms of persisters with different chromosome abundances, we examined their dependencies on different DNA repair proteins. Here, we show that lexA3 and ∆recN negatively impact the abundances of 2Chr persisters to FQs, without significant impacts on 1Chr persisters. In comparison, ∆xseA, ∆xseB, and ∆uvrD preferentially depress 1Chr persistence to levels that were near the limit of detection. Collectively, these data show that the DNA repair mechanisms used by persisters vary based on chromosome number, and suggest that efforts to eradicate FQ persisters will likely have to take heterogeneity in single-cell chromosome abundance into consideration. IMPORTANCE: Persisters are rare phenotypic variants in isogenic populations that survive antibiotic treatments that kill the other cells present. Evidence has accumulated that supports a role for persisters in chronic and recurrent infections. Here, we explore how an under-appreciated phenotypic variable, chromosome copy number (#Chr), influences the DNA repair systems persisters use to survive fluoroquinolone treatments. We found that #Chr significantly biases the DNA repair systems used by persisters, which suggests that #Chr heterogeneity should be considered when devising strategies to eradicate these troublesome bacterial variants.

Cephalosporin resistance, tolerance, and approaches to improve their activities.

Cephalosporins comprise a ß-lactam antibiotic class whose first members were discovered in 1945 from the fungus Cephalosporium acremonium. Their clinical use for Gram-negative bacterial infections is widespread due to their ability to traverse outer membranes through porins to gain access to the periplasm and disrupt peptidoglycan synthesis. More recent members of the cephalosporin class are administered as last resort treatments for complicated urinary tract infections, MRSA, and other multi-drug resistant pathogens, such as Neisseria gonorrhoeae. Unfortunately, there has been a global increase in cephalosporin-resistant strains, heteroresistance to this drug class has been a topic of increasing concern, and tolerance and persistence are recognized as potential causes of cephalosporin treatment failure. In this review, we summarize the cephalosporin antibiotic class from discovery to their mechanisms of action, and discuss the causes of cephalosporin treatment failure, which include resistance, tolerance, and phenomena when those qualities are exhibited by only small subpopulations of bacterial cultures (heteroresistance and persistence). Further, we discuss how recent efforts with cephalosporin conjugates and combination treatments aim to reinvigorate this antibiotic class.

Amino acids can deplete ATP and impair nitric oxide detoxification by Escherichia coli.

Nitric oxide (·NO) is a prevalent antimicrobial that is known to damage iron-containing enzymes in amino acid (AA) biosynthesis pathways. With Escherichia coli, ·NO is detoxified in aerobic environments by Hmp, which is an enzyme that is synthesized de novo in response to ·NO. With this knowledgebase, it is expected that the availability of AAs in the extracellular environment would enhance ·NO detoxification, because AAs would foster translation of Hmp. However, we observed that ·NO detoxification by E. coli was far slower in populations grown and treated in the presence of AAs (AA+) in comparison to those grown and stressed in the absence of AAs (AA-). Further experiments revealed that AA+ populations had difficulty translating proteins under ·NO stress, and that ·NO activated the stringent response in AA+ populations. Additional work revealed significant ATP depletion in ·NO-stressed AA+ cultures that far exceeded that of ·NO-stressed AA- populations. Transcription, translation, and RelA were not found to be significant contributors to the ATP depletion observed, whereas AA import was implicated as a significant ATP consumption pathway. Alleviating ATP depletion while maintaining access to AAs partially restored ·NO detoxification, which suggested that ATP depletion contributed to the translational difficulties observed in ·NO-stressed AA+ populations. These data reveal an unexpected interaction within the ·NO response network of E. coli that stimulates a stringent response by RelA in conditions where AAs are plentiful.

High-Throughput Screen Reveals the Structure-Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin.

The Burkholderia cepacia complex (Bcc) is a group of bacteria including opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. Ubonodin is a lasso peptide with promising activity against Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. We constructed a library of over 90 000 ubonodin variants with 2 amino acid substitutions and used a high-throughput screen and next-generation sequencing to examine the fitness of the entire library, generating the most comprehensive data set on lasso peptide activity so far. This screen revealed information regarding the structure-activity relationship of ubonodin over a large sequence space. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a submicromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia. Ubonodin and several of the variants identified in this study had lower MICs against certain Bcc strains than those of many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.

Genome-wide mapping of fluoroquinolone-stabilized DNA gyrase cleavage sites displays drug specific effects that correlate with bacterial persistence.

Bacterial persisters are rare phenotypic variants that are suspected to be culprits of recurrent infections. Fluoroquinolones (FQs) are a class of antibiotics that facilitate bacterial killing by stabilizing bacterial type II topoisomerases when they are in a complex with cleaved DNA. In Escherichia coli, DNA gyrase is the primary FQ target, and previous work has demonstrated that persisters are not spared from FQ-induced DNA damage. Since DNA gyrase cleavage sites (GCSs) largely govern the sites of DNA damage from FQ treatment, we hypothesized that GCS characteristics (e.g. number, strength, location) may influence persistence. To test this hypothesis, we measured genome-wide GCS distributions after treatment with a panel of FQs in stationary-phase cultures. We found drug-specific effects on the GCS distribution and discovered a strong negative correlation between the genomic cleavage strength and FQ persister levels. Further experiments and analyses suggested that persistence was unlikely to be governed by cleavage to individual sites, but rather survival was a function of the genomic GCS distribution. Together, these findings demonstrate FQ-specific differences in GCS distribution that correlate with persister levels and suggest that FQs that better stabilize DNA gyrase in cleaved complexes with DNA will lead to lower levels of persistence.

Causes of polymyxin treatment failure and new derivatives to fill the gap.

Polymyxins are a class of antibiotics that were discovered in 1947 from programs searching for compounds effective in the treatment of Gram-negative infections. Produced by the Gram-positive bacterium Paenibacillus polymyxa and composed of a cyclic peptide chain with a peptide-fatty acyl tail, polymyxins exert bactericidal effects through membrane disruption. Currently, polymyxin B and colistin (polymyxin E) have been developed for clinical use, where they are reserved as "last-line" therapies for multidrug-resistant (MDR) infections. Unfortunately, the incidences of strains resistant to polymyxins have been increasing globally, and polymyxin heteroresistance has been gaining appreciation as an important clinical challenge. These phenomena, along with bacterial tolerance to this antibiotic class, constitute important contributors to polymyxin treatment failure. Here, we review polymyxins and their mechanism of action, summarize the current understanding of how polymyxin treatment fails, and discuss how the next generation of polymyxins holds promise to invigorate this antibiotic class.

Fluoroquinolone Persistence in Escherichia coli Requires DNA Repair despite Differing between Starving Populations.

When faced with nutritional deprivation, bacteria undergo a range of metabolic, regulatory, and biosynthetic changes. Those adjustments, which can be specific or independent of the missing nutrient, often alter bacterial tolerance to antibiotics. Here, using fluoroquinolones, we quantified Escherichia coli persister levels in cultures experiencing starvation from a lack of carbon (C), nitrogen (N), phosphorous (P), or magnesium (Mg2+). Interestingly, persister levels varied significantly based on the type of starvation as well as fluoroquinolone used with N-starved populations exhibiting the highest persistence to levofloxacin, and P-starved populations exhibiting the highest persistence to moxifloxacin. However, regardless of the type of starvation or fluoroquinolone used, DNA repair was required by persisters, with ∆recA and ∆recB uniformly exhibiting the lowest persistence of the mutants assayed. These results suggest that while the type of starvation and fluoroquinolone will modulate the level of persistence, the importance of homologous recombination is consistently observed, which provides further support for efforts to target homologous recombination for anti-persister purposes.

Translational Fusion to Hmp Improves Heterologous Protein Expression.

Flavohemoglobins, which are widely distributed in prokaryotes and eukaryotes, play key roles in oxygen (O2) transport and nitric oxide (·NO) defense. Hmp is the flavohemoglobin of Escherichia coli, and here we report that the translational fusion of Hmp to the N-terminus of heterologous proteins increases their expression in E. coli. The effect required the fusion of the proteins, and was independent of both the O2-binding and catalytic activity of Hmp. Increased expression was at the translational level, likely to be downstream of initiation, and we observed that as little as the first 100 amino acids of Hmp were sufficient to boost protein production. These data demonstrate the potential of Hmp as an N-terminal fusion tag to increase protein yield, and suggest that the utility of bacterial hemoglobins to biotechnology goes beyond their O2 transport and ·NO detoxification capabilities.

Robustness of nitric oxide detoxification to nitrogen starvation in Escherichia coli requires RelA.

Reactive nitrogen species and nutrient deprivation are two elements of the immune response used to eliminate pathogens within phagosomes. Concomitantly, pathogenic bacteria have evolved defense systems to cope with phagosomal stressors, which include enzymes that detoxify nitric oxide (•NO) and respond to nutrient scarcity. A deeper understanding of how those defense systems are deployed under adverse conditions that contain key elements of phagosomes will facilitate targeting of those systems for therapeutic purposes. Here we investigated how Escherichia coli detoxifies •NO in the absence of useable nitrogen, because nitrogen availability is limited in phagosomes due to the removal of nitrogenous compounds (e.g., amino acids). We hypothesized that nitrogen starvation would impair •NO detoxification by E. coli because it depresses translation rates and the main E. coli defense enzyme, Hmp, is synthesized in response to •NO. However, we found that E. coli detoxifies •NO at the same rate regardless of whether useable nitrogen was present. We confirmed that the nitrogen in •NO and its autoxidation products could not be used by E. coli under our experimental conditions, and discovered that •NO eliminated differences in carbon and oxygen consumption between nitrogen-replete and nitrogen-starved cultures. Interestingly, E. coli does not consume measurable extracellular nitrogen during •NO stress despite the need to translate defense enzymes. Further, we found that RelA, which responds to uncharged tRNA, was required to observe the robustness of •NO detoxification to nitrogen starvation. These data demonstrate that E. coli is well poised to detoxify •NO in the absence of useable nitrogen and suggest that the stringent response could be a useful target to potentiate the antibacterial activity of •NO.

Counting Chromosomes in Individual Bacteria to Quantify Their Impacts on Persistence.

Persisters are phenotypic variants within bacterial populations that tolerate antibiotic treatments considerably better than the majority of cells. A phenotypic quality that varies within bacterial populations is the chromosome number of individual cells. One, two, four, or more chromosomes per cell have been observed previously, and the impact of genome copy number can range from gene dosage effects to an inability to perform specific DNA repair functions, such as homologous recombination. We hypothesize that chromosome abundance is an underappreciated phenotypic variable that could impact persistence to antibiotics. Here, we describe methodologies to segregate bacterial populations based on chromosome number, assess the purity of those subpopulations, and suggest assays that could be used to quantify the impacts of genome abundance on persistence.

Rifamycin antibiotics and the mechanisms of their failure.

Rifamycins are a class of antibiotics that were first discovered in 1957 and are known for their use in treating tuberculosis (TB). Rifamycins exhibit bactericidal activity against many Gram-positive and Gram-negative bacteria by inhibiting RNA polymerase (RNAP); however, resistance is prevalent and the mechanisms range from primary target modification and antibiotic inactivation to cytoplasmic exclusion. Further, phenotypic resistance, in which only a subpopulation of bacteria grow in concentrations exceeding their minimum inhibitory concentration, and tolerance, which is characterized by reduced rates of bacterial cell death, have been identified as additional causes of rifamycin failure. Here we summarize current understanding and recent developments regarding this critical antibiotic class.

Toxin Induction or Inhibition of Transcription or Translation Posttreatment Increases Persistence to Fluoroquinolones.

Toxin-antitoxin modules are widespread in prokaryotes, and the capacity of toxin accumulation to increase the tolerances of bacteria to antibiotics has been well documented. The conventional model for this functionality implies that an overabundance of toxin arrests bacterial growth, which inhibits processes targeted by antibiotics and thereby limits their corruption and the lethal damage that would ensue. Implicit in this model is that toxins exert their influence on antibiotic lethality before and/or during treatment, even though they are also present and functional after treatment concludes. Given recent evidence establishing that the period following antibiotic treatment (recovery) is important for the survival of nongrowing bacterial populations treated with fluoroquinolones (FQs), we assayed to what extent toxins influence bacterial survival during the recovery period. With both LdrD and MazF, toxins of type I and II systems, respectively, controlling accumulation to occur only after FQ treatment of nongrowing cultures resulted in significant increases in persisters. Further genetic investigation revealed important roles for homologous recombination and nucleotide excision repair machinery. Focusing on the wild type, we did not observe any SOS-induced toxin functioning in this manner; however, an analogous phenomenon was observed for wild-type Escherichia coli as well as uropathogenic E. coli (UPEC) when transcription or translation was inhibited during the post-FQ recovery period. Collectively, these data reveal the capacity of toxins to thwart FQ killing even after the treatment has concluded and show that FQ treatment of nongrowing bacteria can be rendered largely ineffective if bacteria cannot readily resume translation and growth at the conclusion of treatment. IMPORTANCE Overabundances of toxins have been shown to increase the antibiotic tolerances of bacteria. Largely, these effects have been attributed to the abilities of toxins to inhibit bacterial growth before and during antibiotic exposure. In this study, we assessed to what extent toxins can influence bacterial survival following antibiotic treatment, rather than before or during. Using two mechanistically distinct toxins, we show that their accumulations after antibiotic exposure have the capacity to increase the abundances of fluoroquinolone persisters from nongrowing populations. Further, we show with wild-type and uropathogenic E. coli that chemical inhibition of growth, not just that induced by toxins, produces analogous results. These observations reveal another dimension of how toxins influence antibiotic tolerance and highlight the importance of postantibiotic physiology on bacterial survival.

Pseudomonas aeruginosa prioritizes detoxification of hydrogen peroxide over nitric oxide.

OBJECTIVE: Bacteria are exposed to multiple concurrent antimicrobial stressors within phagosomes. Among the antimicrobials produced, hydrogen peroxide and nitric oxide are two of the most deleterious products. In a previous study, we discovered that when faced with both stressors simultaneously, Escherichia coli prioritized detoxification of hydrogen peroxide over nitric oxide. In this study, we investigated whether such a process was conserved in another bacterium, Pseudomonas aeruginosa. RESULTS: P. aeruginosa prioritized hydrogen peroxide detoxification in a dose-dependent manner. Specifically, hydrogen peroxide detoxification was unperturbed by the presence of nitric oxide, whereas larger doses of hydrogen peroxide produced longer delays in nitric oxide detoxification. Computational modelling revealed that the rate of nitric oxide consumption in co-treated cultures was biphasic, with cells entering the second phase of detoxification only after hydrogen peroxide was eliminated from the culture.

Phagosome-Bacteria Interactions from the Bottom Up.

When attempting to propagate infections, bacterial pathogens encounter phagocytes that encase them in vacuoles called phagosomes. Within phagosomes, bacteria are bombarded with a plethora of stresses that often lead to their demise. However, pathogens have evolved numerous strategies to counter those host defenses and facilitate survival. Given the importance of phagosome-bacteria interactions to infection outcomes, they represent a collection of targets that are of interest for next-generation antibacterials. To facilitate such therapies, different approaches can be employed to increase understanding of phagosome-bacteria interactions, and these can be classified broadly as top down (starting from intact systems and breaking down the importance of different parts) or bottom up (developing a knowledge base on simplified systems and progressively increasing complexity). Here we review knowledge of phagosomal compositions and bacterial survival tactics useful for bottom-up approaches, which are particularly relevant for the application of reaction engineering to quantify and predict the time evolution of biochemical species in these death-dealing vacuoles. Further, we highlight how understanding in this area can be built up through the combination of immunology, microbiology, and engineering.

Ploidy is an important determinant of fluoroquinolone persister survival.

Genetic mutants have demonstrated the importance of homologous recombination (HR) to fluoroquinolone (FQ) persistence, which suggests that single-cell chromosome (Chr) abundance might be a phenotypic variable of importance to persisters. Here, we sorted stationary-phase E. coli based on ploidy and subjected the subpopulations to tolerance assays. Subpopulations sorted to contain diploid cells harbored up to ∼40-fold more FQ persisters than those sorted to contain monoploid cells. This association was observed with distinct FQs, in independent environmental conditions, and with more than one strain of E. coli (MG1655; uropathogenic CFT073) but was abolished in HR-deficient strains (ΔrecA and ΔrecB). It was observed that the persister level of monoploid subpopulations exceeded those of ΔrecA and ΔrecB by 10-fold or more, and subsequent high-purity sorting confirmed that observation. Those data suggested the existence of distinct FQ persister subtypes: those that are and are not proficient with HR. Time-lapse microscopy revealed significant differences in initial size and growth dynamics during the post-antibiotic recovery period for persisters from monoploid- and diploid-enriched subpopulations. In addition, non-persisters in monoploid-enriched subpopulations elongated minimally following FQ treatment, resembling previous observations of HR-deficient strains, whereas non-persisters in diploid-enriched subpopulations on average filamented extensively. Together, these results identify a phenotypic variable with a significant impact on FQ persistence, establish the existence of more than one type of persister to the same antibiotic in an isogenic culture, and reveal roles for RecA and RecB in FQ persistence, even in the absence of homologous chromosomes.

Metabolites Potentiate Nitrofurans in Nongrowing Escherichia coli.

Nitrofurantoin (NIT) is a broad-spectrum bactericidal antibiotic used in the treatment of urinary tract infections. It is a prodrug that once activated by nitroreductases goes on to inhibit bacterial DNA, RNA, cell wall, and protein synthesis. Previous work has suggested that NIT retains considerable activity against nongrowing bacteria. Here, we have found that Escherichia coli grown to stationary phase in minimal or artificial urine medium is not susceptible to NIT. Supplementation with glucose under conditions where cells remained nongrowing (other essential nutrients were absent) sensitized cultures to NIT. We conceptualized NIT sensitivity as a multi-input AND gate and lack of susceptibility as an insufficiency in one or more of those inputs. The inputs considered were an activating enzyme, cytoplasmic abundance of NIT, and reducing equivalents required for NIT activation. We systematically assessed the contribution of each of these inputs and found that NIT import and the level of activating enzyme were not contributing factors to the lack of susceptibility. Rather, evidence suggested that the low abundance of reducing equivalents is why stationary-phase E. coli are not killed by NIT and catabolites can resensitize those cells. We found that this phenomenon also occurred when using nitrofurazone, which established generality to the nitrofuran antibiotic class. In addition, we observed that NIT activity against stationary-phase uropathogenic E. coli (UPEC) could also be potentiated through metabolite supplementation. These findings suggest that the combination of nitrofurans with specific metabolites could improve the outcome of uncomplicated urinary tract infections.

Synergy Screening Identifies a Compound That Selectively Enhances the Antibacterial Activity of Nitric Oxide.

Antibiotic resistance poses a serious threat to global health. To reinforce the anti-infective arsenal, many novel therapeutic strategies to fight bacterial infections are being explored. Among them, anti-virulence therapies, which target pathways important for virulence, have attracted much attention. Nitric oxide (NO) defense systems have been identified as critical for the pathogenesis of various bacteria, making them an appealing therapeutic target. In this study, we performed chemical screens to identify inhibitors of NO detoxification in Escherichia coli. We found that 2-mercaptobenzothiazole (2-MBT) can potently inhibit cellular detoxification of NO, achieving a level of inhibition that resembled the effect of genetically removing Hmp, the dominant detoxification enzyme under oxygenated conditions. Further analysis revealed that in the presence of NO, 2-MBT impaired the catalysis of Hmp and synthesis of Hmp and other proteins, whereas in its absence there were minimal perturbations to growth and protein synthesis. In addition, by studying the structure-activity relationship of 2-MBT, we found that both sulfur atoms in 2-MBT were vital for its inhibition of NO detoxification. Interestingly, when 2-mercaptothiazole (2-MT), which lacked the benzene ring, was used, differing biological activities were observed, although they too were NO dependent. Specifically, 2-MT could still prohibit NO detoxification, though it did not interfere with Hmp catalysis; rather, it was a stronger inhibitor of protein synthesis and it reduced the transcript levels of hmp, which was not observed with 2-MBT. Overall, these results provide a strong foundation for further exploration of 2-MBT and 2-MT for therapeutic applications.

Quantitative Modeling Extends the Antibacterial Activity of Nitric Oxide.

Numerous materials have been developed to try and harness the antimicrobial properties of nitric oxide (NO). However, the short half-life and reactivity of NO have made precise, tunable delivery difficult. As such, conventional methodologies have generally relied on donors that spontaneously release NO at different rates, and delivery profiles have largely been constrained to decaying dynamics. In recent years, the possibility of finely controlling NO release, for instance with light, has become achievable and this raises the question of how delivery dynamics influence therapeutic potential. Here we investigated this relationship using Escherichia coli as a model organism and an approach that incorporated both experimentation and mathematical modeling. We found that the best performing delivery mode was dependent on the NO payload, and developed a mathematical model to quantitatively dissect those observations. Those analyses suggested that the duration of respiratory inhibition was a major determinant of NO-induced growth inhibition. Inspired by this, we constructed a delivery schedule that leveraged that insight to extend the antimicrobial activity of NO far beyond what was achievable by traditional delivery dynamics. Collectively, these data and analyses suggest that the delivery dynamics of NO have a considerable impact on its ability to achieve and maintain bacteriostasis.

Quantifying Nitric Oxide Flux Distributions.

Nitric oxide (NO) is a radical that is used as an attack molecule by immune cells. NO can interact and damage a range of biomolecules, and the biological outcome for bacteria assaulted with NO will be governed by how the radical distributes within their biochemical reaction networks. Measurement of those NO fluxes is complicated by the low abundance and transience of many of its reaction products. To overcome this challenge, we use computational modeling to translate measurements of several biochemical species (e.g., NO, O2, NO2-) into NO flux distributions. In this chapter, we provide a detailed protocol, which includes experimental measurements and computational modeling, to estimate the NO flux distribution in an Escherichia coli culture. Those fluxes will have uncertainty associated with them and we also discuss how further experiments and modeling can be employed for flux refinement.