Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents. Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant phenotype. In this work, a series of 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus NorA EPIs. The 2-phenyl-4-hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains.

Four novel inhibitors of the NorA efflux pump of Staphylococcus aureus, discovered through a virtual screening process, are reported. The four compounds belong to different chemical classes and were tested for their in vitro ability to block the efflux of a well-known NorA substrate, as well as for their ability to potentiate the effect of ciprofloxacin (CPX) on several strains of S. aureus, including a NorA overexpressing strain. Additionally, the MIC values of each of the compounds individually are reported. A structure-activity relationship study was also performed on these novel chemotypes, revealing three new compounds that are also potent NorA inhibitors. The virtual screening procedure employed FLAP, a new methodology based on GRID force field descriptors.

BACKGROUND Carbapenem-resistant Enterobacteriaceae (CRE) are rapidly emerging in hospitals in the United States and are posing a significant threat. To better understand the transmission dynamics and the acquisition of resistant strains, a thorough analysis of epidemiologic and molecular characteristics was performed. METHODS CRE isolated at Detroit Medical Center were analyzed from September 2008 to September 2009. blaKPC genes were investigated by polymerase chain reaction (PCR), and repetitive extragenic palindromic PCR (rep-PCR) was used to determine genetic similarity among strains. Epidemiologic and outcomes analyses were performed. RESULTS Ninety-two unique patient CRE isolates were recovered. Sixty-eight strains (74%) were Klebsiella pneumoniae, 7 were Klebsiella oxytoca, 15 were Enterobacter species, and 2 were Escherichia coli. Fifteen isolates (16%) were resistant to colistin, 14 (16%) were resistant to tigecycline, and 2 were resistant to all antimicrobials tested. The mean ± standard deviation age of patients was 63 ± 2 years. Sixty patients (68%) were admitted to the hospital from long-term care facilities. Only 70% of patients received effective antimicrobial therapy when infection was suspected, with a mean time to appropriate therapy of 120 ± 23 hours following sample culturing. The mean length of hospitalization after sample culturing was 18.6 ± 2.5 days. Of 57 inpatients, 18 (32%) died in the hospital. Independent predictors for mortality were intensive care unit stay (odds ratio [OR], 15.8; P =.003) and co-colonization with CRE and either Acinetobacter baumannii or Pseudomonas aeruginosa (OR, 17.2; P =.006). Among K. pneumoniae CRE, rep-PCR revealed 2 genetically related strains that comprised 70% and 20% of isolates, respectively. CONCLUSIONS In this large U.S. cohort of patients with CRE infection, which reflects the modern continuum of medical care, co-colonization with CRE and A. baumannii or P. aeruginosa was associated with increased mortality. Two predominant clones of K. pneumoniae accounted for the majority of cases of CRE infection.

Multidrug resistance efflux pumps contribute to antimicrobial and biocide resistance in Staphylococcus aureus. The detection of strains capable of efflux is time-consuming and labor-intensive using currently available techniques. A simple and inexpensive method to identify such strains is needed. Ethidium bromide is a substrate for all but one of the characterized S. aureus multidrug-resistant (MDR) efflux pumps (NorC), leading us to examine the utility of simple broth microtiter MIC determinations using this compound in identifying efflux-proficient strains. Quantitative reverse transcription-PCR identified the increased expression of one or more MDR efflux pump genes in 151/309 clinical strains (49%). Ethidium bromide MIC testing was insensitive (48%) but specific (92%) in identifying strains with gene overexpression, but it was highly sensitive (95%) and specific (99%) in identifying strains capable of ethidium efflux. The increased expression of norA with or without other genes was most commonly associated with efflux, and in the majority of cases that efflux was inhibited by reserpine. Ethidium bromide MIC testing is a simple and straightforward method to identify effluxing strains and can provide accurate predictions of efflux prevalence in large strain sets in a short period of time.

Background Co-colonization of patients with carbapenem-resistant Enterobacteriaceae (CRE) and Acinetobacter baumannii (AB) or Pseudomonas aeruginosa (PA) is reported to be associated with increased antibiotic resistance and mortality. Methods CREs isolated between September 2008 and September 2009 were analyzed at Detroit Medical Center. Patients who had an additional isolation of AB or PA during the period spanning 7 days before to 7 days after CRE isolation were considered co-colonized. Molecular typing was used to determine genetic similarity among CRE strains. Results Eighty-six unique patient isolates of CREs were analyzed. Thirty-four patients (40%) were co-colonized, and 26 (79%) had AB or PA isolated on the same day as the CRE. High Charlson Comorbidity Index score was an independent predictor for co-colonization. Recent stay at a long-term acute-care facility and previous therapy with antimicrobials with activity only against gram-positive microorganisms also were associated with co-colonization, but did not remain significant independent predictors. Co-colonization was associated with higher levels of resistance to carbapenems among CREs and increased 90-day mortality. Molecular typing revealed CRE polyclonality in co-colonized patients. Conclusions Co-colonization is found in patients with the greatest disease burden in the hospital and occurs due to the dissemination of multiple CRE strains. This finding calls into question the practice of cohorting patients with CRE in close proximity to patients with AB or PA.

Objectives:Pancreatic insufficiency (PI) and malabsorption of fats lead to reduced caloric intake, inability to maintain weight, and increased gastrointestinal symptoms. Thus, enteral nutrition (EN) is used in patients with cystic fibrosis (CF) and poor nutritional status. The current study evaluated safety, tolerability, and improvement of fatty acid (FA) status in red blood cell (RBC) membranes, a marker of long-term FA absorption, with an in-line digestive cartridge (RELiZORB) that hydrolyzes fat in enteral formula.Methods:Patients with CF receiving EN participated in a multicenter, 90-day open-label study during which RELiZORB was used with overnight EN. The primary endpoint was change over time in RBC uptake of docosahexaenoic acid (DHA)+ eicosapentaenoic acid (EPA). Gastrointestinal symptoms were collected to evaluate safety and tolerability. Several clinical and anthropometric parameters were also assessed throughout the study.Results:A total of 36 subjects completed the study with a mean age of 13.8 years, body mass index of 17.7 and 6.2 years mean use of overnight EN. Fat absorption significantly improved as shown by increased RBC levels of DHA+EPA, improved ω-6/ω-3 ratio, and increased plasma levels of DHA+EPA. RELiZORB use was not associated with any unanticipated adverse events.Conclusions:RELiZORB use was found to be safe, well tolerated, and resulted in increased levels of FAs in RBCs and plasma. This is the first prospective study to show EN can improve FA abnormalities in CF. Because improvement in omega-3 levels has been shown to help pulmonary and inflammatory status as well as anthropometric parameters in CF, RELiZORB may have important long-term therapeutic benefits in patients with CF.

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