Multicenter evaluation of the Selux Next-Generation Phenotyping antimicrobial susceptibility testing system.

The Selux Next-Generation Phenotyping (NGP) system (Charlestown, MA) is a new antimicrobial susceptibility testing system that utilizes two sequential assays performed on all wells of doubling dilution series to determine MICs. A multicenter evaluation of the performance of the Selux NGP system compared with reference broth microdilution was conducted following FDA recommendations and using FDA-defined breakpoints. A total of 2,488 clinical and challenge isolates were included; gram-negative isolates were tested against 24 antimicrobials, and gram-positive isolates were tested against 15 antimicrobials. Data is provided for all organism-antimicrobial combinations evaluated, including those that did and did not meet FDA performance requirements. Overall very major error and major error rates were less than 1% (31/3,805 and 107/15,606, respectively), essential agreement and categorical agreement were >95%, reproducibility was ≥95%, and the average time-to-result (from time of assay start to time of MIC result) was 5.65 hours.

Exercise adherence: beliefs of adults with knee osteoarthritis over 2 years.

Exercise is an established treatment to alleviate pain and improve function among adults with knee osteoarthritis (KOA). However, long-term adherence to exercise is poor and effective approaches to support adherence are limited. Here, we report on an ancillary study to a randomized controlled trial (RCT) where the primary outcome was 2-year adherence to a home based strength-training program. The aims of this current study were to (i) explore experiences, feelings, and perspectives related to long-term adherence to exercise among adults with painful KOA participating in a 2-year RCT, and (ii) identify factors that influenced long-term adherence to exercise. Methods: We purposively recruited 25 subjects and conducted in-depth interviews at the 2-year RCT assessment. In the RCT participants completed a 6-week group exercise program followed by automated telephone calls. Findings: Three conceptual categories describing beliefs about exercise were identified: (1) monitoring; (2) knowledge of how to manage their exercise behaviors; and (3) benefits of exercise. Monitoring provided by peers and instructors during group exercise, and telephone technology were valued by participants. Participants who reported low adherence expressed ambivalence about the benefits of exercise and a desire for more social support. Those who reported high adherence exhibited self-determination and self-efficacy. Conclusions: A novel finding is the conceptual link of self-determination to high adherence to strength-training exercises over 2 years among adults with KOA. Implications for physical therapists include identifying patients' autonomy, competence, and relatedness needs to foster intrinsic control for exercise behavior.

Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics.

Synthetic peptidomimetics may be designed to mimic functions of antimicrobial peptides, including potentiation of antibiotics, yet possessing improved pharmacological properties. Pairwise screening of 42 synthetic peptidomimetics combined with the antibiotics azithromycin and rifampicin in multidrug-resistant (MDR) Escherichia coli ST131 and Klebsiella pneumoniae ST258 led to identification of two subclasses of α-peptide/ß-peptoid hybrids that display synergy with azithromycin and rifampicin (fractional inhibitory concentration indexes of 0.03-0.38). Further screening of the best three peptidomimetics in combination with a panel of 21 additional antibiotics led to identification of peptidomimetics that potentiated ticarcillin/clavulanate and erythromycin against E. coli, and clindamycin against K. pneumoniae. The study of six peptidomimetics was extended to Pseudomonas aeruginosa, confirming synergy with antibiotics for five of them. The most promising compound, H-(Lys-ßNPhe)8-NH2, exerted only a minor effect on the viability of mammalian cells (EC50 ≥ 124-210 µM), and thus exhibited the highest selectivity toward bacteria. This compound also synergized with rifampicin and azithromycin at sub-micromolar concentrations (0.25-0.5 µM), thereby inducing susceptibility to these antibiotics at clinically relevant concentrations in clinical MDR isolates. This peptidomimetic lead and its analogs constitute promising candidates for efficient repurposing of rifampicin and azithromycin against Gram-negative pathogens.

Repurposing azithromycin and rifampicin against Gram-negative pathogens by combination with peptide potentiators.

Gram-negative bacterial pathogens are intrinsically resistant to several antibiotics that are not able to penetrate the cell envelope barrier. The aim of this study was to identify peptides that at low concentrations induce susceptibility to these antibiotics in multidrug-resistant (MDR) Gram-negative bacterial strains of clinical relevance. Pairwise screening of 34 diverse peptides and four antibiotics (erythromycin, linezolid, rifampicin and vancomycin) with primary activity against Gram-positive bacteria identified 4 peptides that at submicromolar concentrations conferred susceptibility to rifampicin or erythromycin in Escherichia coli ATCC 25922. The identified peptides exhibited synergy with azithromycin and potentiated clindamycin in MDR E. coli ST131 and Klebsiella pneumoniae ST258. The low cytotoxicity toward eukaryotic cells (IC50 > 50 µM) observed for two of these peptides (KLWKKWKKWLK-NH2 and GKWKKILGKLIR-NH2) prompted synthesis and evaluation of the corresponding all-d analogues (D1 and D2), which retained similar synergistic antibacterial profiles. Low concentrations of D1 and D2 in combination with azithromycin and rifampicin inhibited growth of most clinical E. coli, K. pneumoniae and Acinetobacter baumannii strains tested. These data demonstrate that combinatorial screening at low peptide concentrations constitutes an efficient approach to identify clinically relevant peptide-antibiotic combinations. In vivo pharmacokinetic/pharmacodynamic and toxicity studies are needed to further validate the use of the peptides identified in this study for repurposing azithromycin and rifampicin against Gram-negative pathogens.

Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of ß-Lactamase-Producing Escherichia coli.

Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli strains producing ß-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

ExbB cytoplasmic loop deletions cause immediate, proton motive force-independent growth arrest.

The Escherichia coli TonB system consists of the cytoplasmic membrane proteins TonB, ExbB, and ExbD and multiple outer membrane active transporters for diverse iron siderophores and vitamin B12. The cytoplasmic membrane proteins harvest and transmit the proton motive force (PMF) to outer membrane transporters. This system, which spans the cell envelope, has only one component with a significant cytoplasmic presence, ExbB. Characterization of sequential 10-residue deletions in the ExbB cytoplasmic loop (residues 40 to 129; referred to as Δ10 proteins) revealed that it was required for all TonB-dependent activities, including interaction between the periplasmic domains of TonB and ExbD. Expression of eight out of nine of the Δ10 proteins at chromosomal levels led to immediate, but reversible, growth arrest. Arrest was not due to collapse of the PMF and did not require the presence of ExbD or TonB. All Δ10 proteins that caused growth arrest were dominant for that phenotype. However, several were not dominant for iron transport, indicating that growth arrest was an intrinsic property of the Δ10 variants, whether or not they could associate with wild-type ExbB proteins. The lack of dominance in iron transport also ruled out trivial explanations for growth arrest, such as high-level induction. Taken together, the data suggest that growth arrest reflected a changed interaction between the ExbB cytoplasmic loop and one or more unknown growth-regulatory proteins. Consistent with that, a large proportion of the ExbB cytoplasmic loop between transmembrane domain 1 (TMD1) and TMD2 is predicted to be disordered, suggesting the need for interaction with one or more cytoplasmic proteins to induce a final structure.

Mutations in Escherichia coli ExbB transmembrane domains identify scaffolding and signal transduction functions and exclude participation in a proton pathway.

The TonB system couples cytoplasmic membrane proton motive force (pmf) to active transport of diverse nutrients across the outer membrane. Current data suggest that cytoplasmic membrane proteins ExbB and ExbD harness pmf energy. Transmembrane domain (TMD) interactions between TonB and ExbD allow the ExbD C terminus to modulate conformational rearrangements of the periplasmic TonB C terminus in vivo. These conformational changes somehow allow energization of high-affinity TonB-gated transporters by direct interaction with TonB. While ExbB is essential for energy transduction, its role is not well understood. ExbB has N-terminus-out, C-terminus-in topology with three TMDs. TMDs 1 and 2 are punctuated by a cytoplasmic loop, with the C-terminal tail also occupying the cytoplasm. We tested the hypothesis that ExbB TMD residues play roles in proton translocation. Reassessment of TMD boundaries based on hydrophobic character and residue conservation among distantly related ExbB proteins brought earlier widely divergent predictions into congruence. All TMD residues with potentially function-specific side chains (Lys, Cys, Ser, Thr, Tyr, Glu, and Asn) and residues with probable structure-specific side chains (Trp, Gly, and Pro) were substituted with Ala and evaluated in multiple assays. While all three TMDs were essential, they had different roles: TMD1 was a region through which ExbB interacted with the TonB TMD. TMD2 and TMD3, the most conserved among the ExbB/TolQ/MotA/PomA family, played roles in signal transduction between cytoplasm and periplasm and the transition from ExbB homodimers to homotetramers. Consideration of combined data excludes ExbB TMD residues from direct participation in a proton pathway.

Quadriceps weakness and its relationship to tibiofemoral and patellofemoral knee osteoarthritis in Chinese: the Beijing osteoarthritis study.

OBJECTIVE: Weakness has been documented as a feature of tibiofemoral knee osteoarthritis (OA) and may cause disease in this compartment by shock absorption during impulse loading at heel strike, when the patellofemoral joint is not engaged. Our objective was to determine the association of muscle weakness with compartment-specific knee OA, to evaluate sex-specific differences in this relationship, and to determine, by evaluating asymptomatic individuals with OA, whether symptoms may produce the weakness seen in OA. METHODS: This cross-sectional study involved 2,472 subjects (1,475 women and 997 men) ages 60 years or older from 4 central districts of Beijing, China. For all subjects, a skyline view of each knee and an anteroposterior (AP) or posteroanterior (PA) radiograph of both knees were obtained during weight bearing. Radiographs were read by one reader for Kellgren/Lawrence (K/L) grade, joint space narrowing (JSN), and osteophytes. We defined a subject as having tibiofemoral OA when the K/L grade was > or =2 on AP/PA view, patellofemoral OA on skyline view when the osteophyte score was > or =2 (or when the JSN score was > or =2 and the osteophyte score was > or =1), and mixed OA when the knee had both patellofemoral and tibiofemoral radiographic OA. Strength was measured isometrically for each leg separately, and knee pain was evaluated by questionnaire. RESULTS: In women, quadriceps weakness was associated with tibiofemoral OA (odds ratio [OR] 0.7, 95% confidence interval [95% CI] 0.4-1.0), patellofemoral OA (OR 0.6, 95% CI 0.4-0.9), and mixed OA (OR 0.4, 95% CI 0.3-0.6). In men, weakness was associated with mixed OA (OR 0.5, 95% CI 0.3-0.8), and the ORs suggesting an association of patellofemoral OA with weakness were the same as those in women, although in men this trend did not reach statistical significance (P = 0.12). In men, isolated tibiofemoral disease was not associated with weakness; however, the sample size in this analysis was limited. When subjects with knee symptoms were excluded, the relationship of quadriceps weakness to OA was attenuated, with only the relationship between muscle weakness and mixed OA remaining significant. CONCLUSION: There is a relationship between quadriceps weakness and knee OA in all compartments, with the strongest association in mixed disease. Pain may contribute to some of this weakness.