William Miller

Mechanisms of antibiotic resistance in enterococci.

Multidrug-resistant (MDR) enterococci are important nosocomial pathogens and a growing clinical challenge. These organisms have developed resistance to virtually all antimicrobials currently used in clinical practice using a diverse number of genetic strategies. Due to this ability to recruit antibiotic resistance determinants, MDR enterococci display a wide repertoire of antibiotic resistance mechanisms including modification of drug targets, inactivation of therapeutic agents, overexpression of efflux pumps and a sophisticated cell envelope adaptive response that promotes survival in the human host and the nosocomial environment. MDR enterococci are well adapted to survive in the gastrointestinal tract and can become the dominant flora under antibiotic pressure, predisposing the severely ill and immunocompromised patient to invasive infections. A thorough understanding of the mechanisms underlying antibiotic resistance in enterococci is the first step for devising strategies to control the spread of these organisms and potentially establish novel therapeutic approaches.

Whole-Genome Analyses of Enterococcus faecium Isolates with Diverse Daptomycin MICs

ABSTRACT Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a “last-resort” antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120→Ala and Trp73→Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.

Multicenter Evaluation of Ceftolozane/Tazobactam for Serious Infections Caused by Carbapenem-Resistant Pseudomonas aeruginosa

A multicenter, retrospective study of patients infected with carbapenem-resistant Pseudomonas aeruginosa who were treated with ceftolozane/tazobactam was performed. Among 35 patients, pneumonia was the most common indication and treatment was successful in 26 (74%). Treatment failure was observed in all cases where isolates demonstrated ceftolozane-tazobactam minimum inhibitory concentrations ≥8 μg/mL.

Mechanism of Action and Resistance to Daptomycin in Staphylococcus aureus and Enterococci

Lipopeptides are natural product antibiotics that consist of a peptide core with a lipid tail with a diverse array of target organisms and mechanisms of action. Daptomycin (DAP) is an example of these compounds with specific activity against Gram-positive organisms. DAP has become increasingly important to combat infections caused by Gram-positive bacteria because of the presence of multidrug resistance in these organisms, particularly in methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). However, emergence of resistance to DAP during therapy is a well-described phenomenon that threatens the clinical use of this antibiotic, limiting further the therapeutic options against both MRSA and VRE. This work will review the historical aspects of the development of DAP, as well as the current knowledge on its mechanism of action and pathways to resistance in a clinically relevant context.

Electrical and reliability characteristics of lead-zirconate-titanate (PZT) ferroelectric thin films for DRAM applications

The electrical and reliability characteristics of lead-zirconate-titanate (PZT) ferroelectric capacitors are presented. Thin Pb(Zr/sub 0.5/Ti/sub 0.5/)O/sub 3/ films of 350 nm were prepared using a solution-gelatin technique. Hysteresis loop and pulse measurement methods indicate that PZT films exhibit a large charge storage density of 7.3 mu C/cm/sup 2/ and an equivalent dielectric constant of approximately 577. In addition, good endurance behavior (no fatigue or aging wearout after >10/sup 13/ cycles under accelerated unipolar stressing), reasonable transient characteristics (with estimated write time of approximately 0.1 ns and refresh time of approximately 2.5 s), and high effective breakdown field suggest that the PZT ferroelectric material has good potential for applications in advanced DRAMs (dynamic RAMs).<<ETX>>

Vancomycin-Resistant Enterococci: Therapeutic Challenges in the 21st Century.

Vancomycin-resistant enterococci are serious health threats due in part to their ability to persist in rugged environments and their propensity to acquire antibiotic resistance determinants. Enterococci have now established a home in our hospitals and possess mechanisms to defeat most currently available antimicrobials. This article reviews the history of the struggle with this pathogen, what is known about the traits associated with its rise in the modern medical environment, and the current understanding of therapeutic approaches in severe infections caused by these microorganisms. As the 21st century progresses, vancomycin-resistant enterococci continue to pose a daunting clinical challenge.

Empirical Advanced Orthokeratology Through Corneal Topography: The University of Houston Clinical Study

Purpose. Traditionally, orthokeratology has used diagnostic lenses to determine the best fit. The purpose of this study was to determine the efficacy of fitting empirically from corneal topography, without the use of diagnostic lenses. Methods. Twenty-nine subjects, 18 to 37 years old, with myopia of 1.00 to 4.00 diopters (D) and astigmatism of no more than 1.50 D, were entered into this 6-month study. Corneal topography, scanning slit topography and corneal thickness (Orbscan), confocal microscopy, ultrasound corneal thickness, aberrometry, and biomicroscopy were used to assess corneal changes. Unaided logMAR high-contrast visual acuity, subjective refraction, and questionnaires were used to monitor vision and symptoms. Follow-up visits were scheduled after 1 day, 1 week, 2 weeks, 1 month, 3 months, and 6 months. Results. For 6-month data, unaided logMAR acuity improved from 0.78 ± 0.26 in the right eye and 0.75 ± 0.22 in the left eye to 0.06 ± 0.18 in the right eye and 0.04 ± 0.16 in the left eye. Myopia decreased from –2.55 ± 0.87 D in the right eye and −2.47 ± 0.89 D in the left eye to +0.45 ± 0.74 D in the right eye and −0.17 ± 0.69 D in the left eye. Shape factor, using corneal topography, increased from 0.85 ± 0.13 in the right eye and 0.85 ± 0.15 in the left eye to 1.28 ± 0.32 in the right eye and 1.30 ± 0.29 in the left eye. Both eyes showed a decrease in lower-order aberrations (i.e., defocus) and an increase in higher-order aberrations (i.e., spherical aberrations and coma). Conclusions. Myopia reduction after 1 week was clinically insignificant from the 1-month results, indicating that the full effect is achieved by 1 week. Neither total nor epithelial corneal thickness varied significantly from baseline measurements.

Deletion of liaR reverses daptomycin resistance in Enterococcus faecium independent of the genetic background

ABSTRACT We have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain of Enterococcus faecalis reversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved in Enterococcus faecium, we investigated the role of LiaR in a variety of clinical E. faecium strains representing the most common DAP-R genetic backgrounds. Deletion of liaR in DAP-R E. faecium R446F (DAP MIC of 16 μg/ml) and R497F (MIC of 24 μg/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 μg/ml, respectively). Moreover, DAP at concentrations of 13 μg/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, the liaR deletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR in E. faecium involves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5× the MIC), deletion of liaR not only markedly decreased the DAP MICs (0.064 and 0.047 μg/ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 μg/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.

Ceftaroline-Resistant, Daptomycin-Tolerant, and Heterogeneous Vancomycin-Intermediate Methicillin-Resistant Staphylococcus aureus Causing Infective Endocarditis

ABSTRACT We report a case of infective endocarditis (IE) caused by ceftaroline-resistant, daptomycin-tolerant, and heterogeneous vancomycin-intermediate methicillin-resistant S. aureus (MRSA). Resistance to ceftaroline emerged in the absence of drug exposure, and the E447K substitution in the active site of PBP2a previously associated with ceftaroline resistance was identified. Additionally, we present evidence of patient-to-patient transmission of the strain within the same unit. This case illustrates the difficulties in treating MRSA IE in the setting of a multidrug-resistant phenotype.

The Cefazolin Inoculum Effect Is Associated With Increased Mortality in Methicillin-Susceptible Staphylococcus aureus Bacteremia

Abstract Background Recent studies have favored the use of cefazolin over nafcillin for the treatment of methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. The clinical influence of the cefazolin inoculum effect (CzIE) in the effectiveness of cephalosporins for severe MSSA infections has not been evaluated. Methods We prospectively included patients from 3 Argentinian hospitals with S. aureus bacteremia. Cefazolin minimum inhibitory concentrations (MICs) were determined at standard (105 colony-forming units [CFU]/mL) and high (107 CFU/mL) inoculum. The CzIE was defined as an increase of MIC to ≥16 µg/mL when tested at high inoculum. Whole-genome sequencing was performed in all isolates. Results A total of 77 patients, contributing 89 MSSA isolates, were included in the study; 42 patients (54.5%) had isolates with the CzIE. In univariate analysis, patients with MSSA exhibiting the CzIE had increased 30-day mortality (P = .034) and were more likely to have catheter-associated or unknown source of bacteremia (P = .033) compared with patients infected with MSSA isolates without the CzIE. No statistically significant difference between the groups was observed in age, clinical illness severity, place of acquisition (community vs hospital), or presence of endocarditis. The CzIE remained associated with increased 30-day mortality in multivariate analysis (risk ratio, 2.65; 95% confidence interval, 1.10–6.42; P = .03). MSSA genomes displayed a high degree of heterogeneity, and the CzIE was not associated with a specific lineage. Conclusions In patients with MSSA bacteremia where cephalosporins are used as firstline therapy, the CzIE was associated with increased 30-day mortality. Clinicians should be cautious when using cefazolin as firstline therapy for these infections.