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6.  Population Pharmacokinetic-Pharmacodynamic Analysis of Voriconazole and Anidulafungin in Adult Patients with Invasive Aspergillosis 
To evaluate the exposure-response relationships for efficacy and safety of voriconazole and anidulafungin in adult patients with invasive aspergillosis (IA), a population pharmacokinetic-pharmacodynamic (PK-PD) analysis was performed with data from a phase 3, prospective, double-blind, comparative study evaluating voriconazole and anidulafungin combination therapy versus voriconazole (and placebo) monotherapy. Anidulafungin/placebo treatment duration was 2 to 4 weeks, and voriconazole treatment duration was 6 weeks. Efficacy (6-week all-causality mortality and 6-week global response [n = 176]) and safety (hepatic [n = 238], visual [n = 199], and psychiatric [n = 183] adverse events [AEs]) endpoints were analyzed separately using a binary logistic regression model. In IA patients receiving voriconazole monotherapy, no positive associations between voriconazole exposure and efficacy or safety were identified. In IA patients receiving combination therapy, no positive associations between voriconazole or anidulafungin exposures and efficacy were identified. The 6-week survival rate tended to increase as anidulafungin treatment duration increased; this finding should be considered with caution. Additionally, in IA patients receiving combination therapy, a positive association between voriconazole and anidulafungin exposures (area under the curve [AUC] and trough concentration [Cmin]) and hepatic AEs was established; a weak positive association between voriconazole exposure (AUC and Cmin) and psychiatric AEs was also established, but no association between voriconazole exposure and visual AEs was identified. Besides the drug exposures, no other covariates (i.e., CYP2C19 genotype status, age, weight, body mass index, sex, race, or neutropenia status) were identified as significant predictors of the efficacy and safety endpoints in IA patients. This study was registered on (NCT00531479).
PMCID: PMC4126689  PMID: 24914120
7.  Highly Sulfated K5 Escherichia coli Polysaccharide Derivatives Inhibit Respiratory Syncytial Virus Infectivity in Cell Lines and Human Tracheal-Bronchial Histocultures 
Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The interaction between RSV and HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was used to generate a collection of sulfated K5 derivatives with a backbone structure that mimics the heparin/heparan sulfate biosynthetic precursor. The screening of a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation revealed the highly sulfated K5 derivatives K5-N,OS(H) and K5-OS(H) to be inhibitors of RSV. Their 50% inhibitory concentrations were between 1.07 nM and 3.81 nM in two different cell lines, and no evidence of cytotoxicity was observed. Inhibition of RSV infection was maintained in binding and attachment assays but not in preattachment assays. Moreover, antiviral activity was also evident when the K5 derivatives were added postinfection, both in cell-to-cell spread and viral yield reduction assays. Finally, both K5-N,OS(H) and K5-OS(H) prevented RSV infection in human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. Together, these features put K5-N,OS(H) and K5-OS(H) forward as attractive candidates for further development as RSV inhibitors.
PMCID: PMC4135974  PMID: 24914125
8.  Deletion of the Uracil Permease Gene Confers Cross-Resistance to 5-Fluorouracil and Azoles in Candida lusitaniae and Highlights Antagonistic Interaction between Fluorinated Nucleotides and Fluconazole 
We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae. Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The function of each transporter was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport.
PMCID: PMC4135975  PMID: 24867971
9.  Editorial Board 
PMCID: PMC4135976
10.  Improved Detection of Emerging Drug-Resistant Mutant Cytomegalovirus Subpopulations by Deep Sequencing 
In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.
PMCID: PMC4135977  PMID: 24890586
11.  Antagonism between Bacteriostatic and Bactericidal Antibiotics Is Prevalent 
Combination therapy is rarely used to counter the evolution of resistance in bacterial infections. Expansion of the use of combination therapy requires knowledge of how drugs interact at inhibitory concentrations. More than 50 years ago, it was noted that, if bactericidal drugs are most potent with actively dividing cells, then the inhibition of growth induced by a bacteriostatic drug should result in an overall reduction of efficacy when the drug is used in combination with a bactericidal drug. Our goal here was to investigate this hypothesis systematically. We first constructed time-kill curves using five different antibiotics at clinically relevant concentrations, and we observed antagonism between bactericidal and bacteriostatic drugs. We extended our investigation by performing a screen of pairwise combinations of 21 different antibiotics at subinhibitory concentrations, and we found that strong antagonistic interactions were enriched significantly among combinations of bacteriostatic and bactericidal drugs. Finally, since our hypothesis relies on phenotypic effects produced by different drug classes, we recreated these experiments in a microfluidic device and performed time-lapse microscopy to directly observe and quantify the growth and division of individual cells with controlled antibiotic concentrations. While our single-cell observations supported the antagonism between bacteriostatic and bactericidal drugs, they revealed an unexpected variety of cellular responses to antagonistic drug combinations, suggesting that multiple mechanisms underlie the interactions.
PMCID: PMC4135978  PMID: 24867991
12.  Carbapenem-Resistant Klebsiella pneumoniae Strains Exhibit Diversity in Aminoglycoside-Modifying Enzymes, Which Exert Differing Effects on Plazomicin and Other Agents 
We measured in vitro activity of plazomicin, a next-generation aminoglycoside, and other aminoglycosides against 50 carbapenem-resistant Klebsiella pneumoniae strains from two centers and correlated the results with the presence of various aminoglycoside-modifying enzymes (AMEs). Ninety-four percent of strains were sequence type 258 (ST258) clones, which exhibited 5 ompK36 genotypes; 80% and 10% of strains produced Klebsiella pneumoniae carbapenemase 2 (KPC-2) and KPC-3, respectively. Ninety-eight percent of strains possessed AMEs, including AAC(6′)-Ib (98%), APH(3′)-Ia (56%), AAC(3)-IV (38%), and ANT(2″)-Ia (2%). Gentamicin, tobramycin, and amikacin nonsusceptibility rates were 40, 98, and 16%, respectively. Plazomicin MICs ranged from 0.25 to 1 μg/ml. Tobramycin and plazomicin MICs correlated with gentamicin MICs (r = 0.75 and 0.57, respectively). Plazomicin exerted bactericidal activity against 17% (1× MIC) and 94% (4× MIC) of strains. All strains with AAC(6′)-Ib were tobramycin-resistant; 16% were nonsusceptible to amikacin. AAC(6′)-Ib combined with another AME was associated with higher gentamicin, tobramycin, and plazomicin MICs than AAC(6′)-Ib alone (P = 0.01, 0.0008, and 0.046, respectively). The presence of AAC(3)-IV in a strain was also associated with higher gentamicin, tobramycin, and plazomicin MICs (P = 0.0006, P < 0.0001, and P = 0.01, respectively). The combination of AAC(6′)-Ib and another AME, the presence of AAC(3)-IV, and the presence of APH(3′)-Ia were each associated with gentamicin resistance (P = 0.0002, 0.003, and 0.01, respectively). In conclusion, carbapenem-resistant K. pneumoniae strains (including ST258 clones) exhibit highly diverse antimicrobial resistance genotypes and phenotypes. Plazomicin may offer a treatment option against strains resistant to other aminoglycosides. The development of molecular assays that predict antimicrobial responses among carbapenem-resistant K. pneumoniae strains should be a research priority.
PMCID: PMC4135979  PMID: 24867988
13.  Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1 
Arylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellular Leishmania parasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raised Leishmania donovani axenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania, is dramatically reduced in DB766R parasites. In vitro susceptibility assays demonstrated that CYP5122A1 half-knockout L. donovani promastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellular L. donovani amastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.
PMCID: PMC4135980  PMID: 24890590
14.  Linezolid-Dependent Function and Structure Adaptation of Ribosomes in a Staphylococcus epidermidis Strain Exhibiting Linezolid Dependence 
Linezolid-dependent growth was recently reported in Staphylococcus epidermidis clinical strains carrying mutations associated with linezolid resistance. To investigate this unexpected behavior at the molecular level, we isolated active ribosomes from one of the linezolid-dependent strains and we compared them with ribosomes isolated from a wild-type strain. Both strains were grown in the absence and presence of linezolid. Detailed biochemical and structural analyses revealed essential differences in the function and structure of isolated ribosomes which were assembled in the presence of linezolid. The catalytic activity of peptidyltransferase was found to be significantly higher in the ribosomes derived from the linezolid-dependent strain. Interestingly, the same ribosomes exhibited an abnormal ribosomal subunit dissociation profile on a sucrose gradient in the absence of linezolid, but the profile was restored after treatment of the ribosomes with an excess of the antibiotic. Our study suggests that linezolid most likely modified the ribosomal assembly procedure, leading to a new functional ribosomal population active only in the presence of linezolid. Therefore, the higher growth rate of the partially linezolid-dependent strains could be attributed to the functional and structural adaptations of ribosomes to linezolid.
PMCID: PMC4135981  PMID: 24890589
15.  Risk Factors and Outcomes for Patients with Bloodstream Infection Due to Acinetobacter baumannii-calcoaceticus Complex 
Identifying patients at risk for bloodstream infection (BSI) due to Acinetobacter baumannii-Acinetobacter calcoaceticus complex (ABC) and providing early appropriate therapy are critical for improving patient outcomes. A retrospective matched case-control study was conducted to investigate the risk factors for BSI due to ABC in patients admitted to the Detroit Medical Center (DMC) between January 2006 and April 2009. The cases were patients with BSI due to ABC; the controls were patients not infected with ABC. Potential risk factors were collected 30 days prior to the ABC-positive culture date for the cases and 30 days prior to admission for the controls. A total of 245 case patients were matched with 245 control patients. Independent risk factors associated with BSI due to ABC included a Charlson's comorbidity score of ≥3 (odds ratio [OR], 2.34; P = 0.001), a direct admission from another health care facility (OR, 4.63; P < 0.0001), a prior hospitalization (OR, 3.11; P < 0.0001), the presence of an indwelling central venous line (OR, 2.75; P = 0.011), the receipt of total parenteral nutrition (OR, 21.2; P < 0.0001), the prior receipt of β-lactams (OR, 3.58; P < 0.0001), the prior receipt of carbapenems (OR, 3.18; P = 0.006), and the prior receipt of chemotherapy (OR, 15.42; P < 0.0001). The median time from the ABC-positive culture date to the initiation of the appropriate antimicrobial therapy was 2 days (interquartile range [IQR], 1 to 3 days). The in-hospital mortality rate was significantly higher among case patients than among control patients (OR, 3.40; P < 0.0001). BSIs due to ABC are more common among critically ill and debilitated institutionalized patients, who are heavily exposed to health care settings and invasive devices.
PMCID: PMC4135982  PMID: 24890594
16.  Comparative Genomics of an IncA/C Multidrug Resistance Plasmid from Escherichia coli and Klebsiella Isolates from Intensive Care Unit Patients and the Utility of Whole-Genome Sequencing in Health Care Settings 
The IncA/C plasmids have been implicated for their role in the dissemination of β-lactamases, including gene variants that confer resistance to expanded-spectrum cephalosporins, which are often the treatment of last resort against multidrug-resistant, hospital-associated pathogens. A blaFOX-5 gene was detected in 14 Escherichia coli and 16 Klebsiella isolates that were cultured from perianal swabs of patients admitted to an intensive care unit (ICU) of the University of Maryland Medical Center (UMMC) in Baltimore, MD, over a span of 3 years. Four of the FOX-encoding isolates were obtained from subsequent samples of patients that were initially negative for an AmpC β-lactamase upon admission to the ICU, suggesting that the AmpC β-lactamase-encoding plasmid was acquired while the patient was in the ICU. The genomes of five E. coli isolates and six Klebsiella isolates containing blaFOX-5 were selected for sequencing based on their plasmid profiles. An ∼167-kb IncA/C plasmid encoding the FOX-5 β-lactamase, a CARB-2 β-lactamase, additional antimicrobial resistance genes, and heavy metal resistance genes was identified. Another FOX-5-encoding IncA/C plasmid that was nearly identical except for a variable region associated with the resistance genes was also identified. To our knowledge, these plasmids represent the first FOX-5-encoding plasmids sequenced. We used comparative genomics to describe the genetic diversity of a plasmid encoding a FOX-5 β-lactamase relative to the whole-genome diversity of 11 E. coli and Klebsiella isolates that carry this plasmid. Our findings demonstrate the utility of whole-genome sequencing for tracking of plasmid and antibiotic resistance gene distribution in health care settings.
PMCID: PMC4135983  PMID: 24914121
17.  Pharmacological Study of Cefoxitin as an Alternative Antibiotic Therapy to Carbapenems in Treatment of Urinary Tract Infections Due to Extended-Spectrum-β-Lactamase-Producing Escherichia coli 
Cefoxitin could be an alternative to carbapenems in extended-spectrum-beta-lactamase-producing Escherichia coli (ESBL-EC) infections. However, pharmacological and clinical data regarding cefoxitin are limited. Using a recent pharmacological model and the MICs of ESBL-EC collected from pyelonephritis, we determined the probabilities to reach four pharmacological targets: free cefoxitin concentrations above the MIC during 50% and 100% of the administration interval (T>MIC = 50% and T>MIC = 100%, respectively) and free cefoxitin concentrations above 4× MIC during 50% and 100% of the administration interval (T>4MIC = 50% and T>4MIC = 100%, respectively). Cefoxitin could be used to treat ESBL-EC pyelonephritis, but administration modalities should be optimized according to MICs in order to reach pharmacological targets.
PMCID: PMC4135984  PMID: 24777104
18.  A Novel Ketolide, RBx 14255, with Activity against Multidrug-Resistant Streptococcus pneumoniae 
We present here the novel ketolide RBx 14255, a semisynthetic macrolide derivative obtained by the derivatization of clarithromycin, for its in vitro and in vivo activities against sensitive and macrolide-resistant Streptococcus pneumoniae. RBx 14255 showed excellent in vitro activity against macrolide-resistant S. pneumoniae, including an in-house-generated telithromycin-resistant strain (S. pneumoniae 3390 NDDR). RBx 14255 also showed potent protein synthesis inhibition against telithromycin-resistant S. pneumoniae 3390 NDDR. The binding affinity of RBx 14255 toward ribosomes was found to be more than that for other tested drugs. The in vivo efficacy of RBx 14255 was determined in murine pulmonary infection induced by intranasal inoculation of S. pneumoniae ATCC 6303 and systemic infection with S. pneumoniae 3390 NDDR strains. The 50% effective dose (ED50) of RBx 14255 against S. pneumoniae ATCC 6303 in a murine pulmonary infection model was 3.12 mg/kg of body weight. In addition, RBx 14255 resulted in 100% survival of mice with systemic infection caused by macrolide-resistant S. pneumoniae 3390 NDDR at 100 mg/kg four times daily (QID) and at 50 mg/kg QID. RBx 14255 showed favorable pharmacokinetic properties that were comparable to those of telithromycin.
PMCID: PMC4135985  PMID: 24550341
19.  Different Interaction Profiles of Direct-Acting Anti-Hepatitis C Virus Agents with Human Organic Anion Transporting Polypeptides 
Simeprevir (SMV), asunaprevir (ASV), daclatasvir (DCV), and sofosbuvir (SFV), which are newly developed direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) infection, are among the key components of anti-HCV regimens. Preclinical studies have identified inhibitory properties for some of these DAAs against organic anion transporting polypeptide 1B (OATP1B) functions. However, their details remain mostly uncharacterized. Because OATP1B1 and OATP1B3 play determinant roles in the pharmacokinetics of various drugs via their uptake into human hepatocytes, it is plausible that the inhibition of these OATP1Bs by a DAA would create a potential risk of drug-drug interaction, which has been an emerging concern in anti-HCV therapy. Accordingly, in the present study, we intended to clarify the inhibitory characteristics of newly developed DAAs toward OATP1B1 and -1B3 functions. The results of our coincubation inhibition assays have shown that all tested DAAs could inhibit OATP1B1 functions and that SMV, ASV, and DCV (to a lesser extent), but not SFV, exhibited long-lasting preincubation inhibitory effects on OATP1B1 functions. It was also found that the preincubation inhibitory effects of SMV and ASV could augment their coincubation inhibition potency. Furthermore, significant, but differential, inhibitory effects of the DAAs on the OATP1B3 function were identified. To summarize, our results clearly show that the newly developed DAAs are newly identified OATP1B1 and OATP1B3 inhibitors with distinctive interaction properties. It is believed that these inhibition profiles will provide essential information to all concerned parties with respect to the clinical significance of DAA-mediated inhibition of OATP1Bs in anti-HCV therapy.
PMCID: PMC4135986  PMID: 24867984
20.  Efficacy of Repeated Intravenous Administration of Peramivir against Highly Pathogenic Avian Influenza A (H5N1) Virus in Cynomolgus Macaques 
Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor α, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.
PMCID: PMC4135987  PMID: 24913156
21.  Treatment of Murine Cerebral Malaria by Artemisone in Combination with Conventional Antimalarial Drugs: Antiplasmodial Effects and Immune Responses 
The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.
PMCID: PMC4135990  PMID: 24913162
22.  Epinecidin-1 Has Immunomodulatory Effects, Facilitating Its Therapeutic Use in a Mouse Model of Pseudomonas aeruginosa Sepsis 
Antimicrobial peptides (AMPs) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of epinecidin-1 against a multidrug-resistant clinical isolate of P. aeruginosa (P. aeruginosa R) and a P. aeruginosa strain from ATCC (P. aeruginosa ATCC 19660) in vivo. The MICs of epinecidin-1 against P. aeruginosa R and P. aeruginosa ATCC 19660 were determined and compared with those of imipenem. Epinecidin-1 was found to be highly effective at combating peritonitis infection caused by P. aeruginosa R or P. aeruginosa ATCC 19660 in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that epinecidin-1 enhances the rate of survival of mice infected with the bacterial pathogen P. aeruginosa through both antimicrobial and immunomodulatory effects.
PMCID: PMC4135991  PMID: 24820078
23.  Interaction between Mutations and Regulation of Gene Expression during Development of De Novo Antibiotic Resistance 
Bacteria can become resistant not only by horizontal gene transfer or other forms of exchange of genetic information but also by de novo by adaptation at the gene expression level and through DNA mutations. The interrelationship between changes in gene expression and DNA mutations during acquisition of resistance is not well documented. In addition, it is not known whether the DNA mutations leading to resistance always occur in the same order and whether the final result is always identical. The expression of >4,000 genes in Escherichia coli was compared upon adaptation to amoxicillin, tetracycline, and enrofloxacin. During adaptation, known resistance genes were sequenced for mutations that cause resistance. The order of mutations varied within two sets of strains adapted in parallel to amoxicillin and enrofloxacin, respectively, whereas the buildup of resistance was very similar. No specific mutations were related to the rather modest increase in tetracycline resistance. Ribosome-sensed induction and efflux pump activation initially protected the cell through induction of expression and allowed it to survive low levels of antibiotics. Subsequently, mutations were promoted by the stress-induced SOS response that stimulated modulation of genetic instability, and these mutations resulted in resistance to even higher antibiotic concentrations. The initial adaptation at the expression level enabled a subsequent trial and error search for the optimal mutations. The quantitative adjustment of cellular processes at different levels accelerated the acquisition of antibiotic resistance.
PMCID: PMC4135992  PMID: 24841263
24.  Multidisciplinary Analysis of a Nontoxigenic Clostridium difficile Strain with Stable Resistance to Metronidazole 
Stable resistance to metronidazole in a nontoxigenic Clostridium difficile strain was investigated at both the genomic and proteomic levels. Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were found, suggesting that reduction of metronidazole, required for its activity, may be less efficient in this strain. Proteomic studies also showed a cellular response to oxidative stress.
PMCID: PMC4135993  PMID: 24913157
25.  Complete Sequencing of IncI1 Sequence Type 2 Plasmid pJIE512b Indicates Mobilization of blaCMY-2 from an IncA/C Plasmid 
Sequencing of pJIE512b, a 92.3-kb IncI1 sequence type 2 (ST2) plasmid carrying blaCMY-2, revealed a blaCMY-2 context that appeared to have been mobilized from an IncA/C plasmid by the insertion sequence IS1294. A comparison with published plasmids suggests that blaCMY-2 has been mobilized from IncA/C to IncI1 plasmids more than once by IS1294-like elements. Alignment of pJIE512b with the only other available IncI1 ST2 plasmid revealed differences across the backbones, indicating variability within this sequence type.
PMCID: PMC4135994  PMID: 24890591

Results 1-25 (24605)