Lynn Zechiedrich

Mechanisms Accounting for Fluoroquinolone Resistance in Escherichia coli Clinical Isolates

ABSTRACT Fluoroquinolone MICs are increased through the acquisition of chromosomal mutations in the genes encoding gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), increased levels of the multidrug efflux pump AcrAB, and the plasmid-borne genes aac(6′)-Ib-cr and the qnr variants in Escherichia coli. In the accompanying report, we found that ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs for fluoroquinolone-resistant E. coli clinical isolates were very high and widely varied (L. Becnel Boyd, M. J. Maynard, S. K. Morgan-Linnell, L. B. Horton, R. Sucgang, R. J. Hamill, J. Rojo Jimenez, J. Versalovic, D. Steffen, and L. Zechiedrich, Antimicrob. Agents Chemother. 53:229-234, 2009). Here, we sequenced gyrA, gyrB, parC, and parE; screened for aac(6′)-Ib-cr and qnrA; and quantified AcrA levels in E. coli isolates for which patient sex, age, location, and site of infection were known. We found that (i) all fluoroquinolone-resistant isolates had gyrA mutations; (ii) ∼85% of gyrA mutants also had parC mutations; (iii) the ciprofloxacin and norfloxacin MICs for isolates harboring aac(6′)-Ib-cr (∼23%) were significantly higher, but the gatifloxacin and levofloxacin MICs were not; (iv) no isolate had qnrA; and (v) ∼33% of the fluoroquinolone-resistant isolates had increased AcrA levels. Increased AcrA correlated with nonsusceptibility to the fluoroquinolones but did not correlate with nonsusceptibility to any other antimicrobial agents reported from hospital antibiograms. Known mechanisms accounted for the fluoroquinolone MICs of 50 to 70% of the isolates; the remaining included isolates for which the MICs were up to 1,500-fold higher than expected. Thus, additional, unknown fluoroquinolone resistance mechanisms must be present in some clinical isolates.

Expression of Multidrug Efflux Pump Genes acrAB-tolC, mdfA, and norE in Escherichia coli Clinical Isolates as a Function of Fluoroquinolone and Multidrug Resistance

ABSTRACT In a single quantitative study, we measured acrA, acrB, tolC, mdfA, and norE expression in Escherichia coli clinical isolates by using real-time PCR. acrA and acrB overexpression strongly correlated with fluoroquinolone and multidrug resistance; tolC, mdfA, and norE expression did not. The order of abundance of efflux pump transcripts in all fluoroquinolone-susceptible isolates was tolC (highest), then acrA and acrB, and then mdfA and norE. Our findings suggest acrAB overexpression is an indicator of multidrug resistance.

The why and how of DNA unlinking

The nucleotide sequence of DNA is the repository of hereditary information. Yet, it is now clear that the DNA itself plays an active role in regulating the ability of the cell to extract its information. Basic biological processes, including control of gene transcription, faithful DNA replication and segregation, maintenance of the genome and cellular differentiation are subject to the conformational and topological properties of DNA in addition to the regulation imparted by the sequence itself. How do these DNA features manifest such striking effects and how does the cell regulate them? In this review, we describe how misregulation of DNA topology can lead to cellular dysfunction. We then address how cells prevent these topological problems. We close with a discussion on recent theoretical advances indicating that the topological problems, themselves, can provide the cues necessary for their resolution by type-2 topoisomerases.

Relationships among Ciprofloxacin, Gatifloxacin, Levofloxacin, and Norfloxacin MICs for Fluoroquinolone-Resistant Escherichia coli Clinical Isolates

ABSTRACT Fluoroquinolones are some of the most prescribed antibiotics in the United States. Previously, we and others showed that the fluoroquinolones exhibit a class effect with regard to the CLSI-established breakpoints for resistance, such that decreased susceptibility (i.e., an increased MIC) to one fluoroquinolone means a simultaneously decreased susceptibility to all. For defined strains, however, clear differences exist in the pharmacodynamic properties of each fluoroquinolone and the extent to which resistance-associated genotypes affect the MICs of each fluoroquinolone. In a pilot study of 920 clinical Escherichia coli isolates, we uncovered tremendous variation in norfloxacin MICs. The MICs for all of the fluoroquinolone-resistant isolates exceeded the resistance breakpoint, reaching 1,000 μg/ml. Approximately 25% of the isolates (n = 214), representing the full range of resistant norfloxacin MICs, were selected for the simultaneous determinations of ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs. We found that (i) great MIC variation existed for all four fluoroquinolones, (ii) the ciprofloxacin and levofloxacin MICs of >90% of the fluoroquinolone-resistant isolates were higher than the resistance breakpoints, (iii) ciprofloxacin and levofloxacin MICs were distributed into two distinct groups, (iv) the MICs of two drug pairs (ciprofloxacin and norfloxacin by Kendalls Tau-b test and gatifloxacin and levofloxacin by paired t test) were similar with statistical significance but were different from each other, and (v) ∼2% of isolates had unprecedented fluoroquinolone MIC relationships. Thus, although the fluoroquinolones can be considered equivalent with regard to clinical susceptibility or resistance, fluoroquinolone MICs differ dramatically for fluoroquinolone-resistant clinical isolates, likely because of differences in drug structure.

Structural diversity of supercoiled DNA

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

Bullied no more: When and how DNA shoves proteins around

The predominant protein-centric perspective in protein-DNA-binding studies assumes that the protein drives the interaction. Research focuses on protein structural motifs, electrostatic surfaces and contact potentials, while DNA is often ignored as a passive polymer to be manipulated. Recent studies of DNA topology, the supercoiling, knotting, and linking of the helices, have shown that DNA has the capability to be an active participant in its transactions. DNA topology-induced structural and geometric changes can drive, or at least strongly influence, the interactions between protein and DNA. Deformations of the B-form structure arise from both the considerable elastic energy arising from supercoiling and from the electrostatic energy. Here, we discuss how these energies are harnessed for topology-driven, sequence-specific deformations that can allow DNA to direct its own metabolism.

Contributions of the Combined Effects of Topoisomerase Mutations toward Fluoroquinolone Resistance in Escherichia coli

ABSTRACT In defined, isogenic strains, at least three mutations, two of which must be in gyrA, were required to exceed the CLSI breakpoint for fluoroquinolone resistance. Strains with double mutations in both gyrA and parC had even higher MICs of fluoroquinolones than strains with totals of three mutations.

Temporal Interplay between Efflux Pumps and Target Mutations in Development of Antibiotic Resistance in Escherichia coli

ABSTRACT The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop.

In the absence of writhe, DNA relieves torsional stress with localized, sequence-dependent structural failure to preserve B-form

To understand how underwinding and overwinding the DNA helix affects its structure, we simulated 19 independent DNA systems with fixed degrees of twist using molecular dynamics in a system that does not allow writhe. Underwinding DNA induced spontaneous, sequence-dependent base flipping and local denaturation, while overwinding DNA induced the formation of Pauling-like DNA (P-DNA). The winding resulted in a bimodal state simultaneously including local structural failure and B-form DNA for both underwinding and extreme overwinding. Our simulations suggest that base flipping and local denaturation may provide a landscape influencing protein recognition of DNA sequence to affect, for examples, replication, transcription and recombination. Additionally, our findings help explain results from single-molecule experiments and demonstrate that elastic rod models are strictly valid on average only for unstressed or overwound DNA up to P-DNA formation. Finally, our data support a model in which base flipping can result from torsional stress.

Hin-mediated DNA knotting and recombining promote replicon dysfunction and mutation.

BackgroundThe genetic code imposes a dilemma for cells. The DNA must be long enough to encode for the complexity of an organism, yet thin and flexible enough to fit within the cell. The combination of these properties greatly favors DNA collisions, which can knot and drive recombination of the DNA. Despite the well-accepted propensity of cellular DNA to collide and react with itself, it has not been established what the physiological consequences are.ResultsHere we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system. We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid.ConclusionThese results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.