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issn:1098-656
3.  Globally Expanding Carbapenemase Finally Appears in Spain: Nosocomial Outbreak of Acinetobacter baumannii Producing Plasmid-Encoded OXA-23 in Barcelona, Spain 
Antimicrobial Agents and Chemotherapy  2013;57(10):5155-5157.
Resistance of Acinetobacter baumannii clinical isolates to carbapenems is on the rise worldwide mainly in association with the production of OXA-23. Until recently, however, OXA-23 was absent in Spain. In this work, we report the molecular characterization of a hospital outbreak of OXA-23-producing A. baumannii in Barcelona caused by a multidrug-resistant (MDR) clone belonging to international clone IC-II/sequence type ST85 between October 2010 and May 2011. blaOXA-23 was carried in a plasmid of 90 kb and located within the composite transposon Tn2006.
doi:10.1128/AAC.01486-13
PMCID: PMC3811394  PMID: 23877694
4.  Systematic Review and Meta-Analysis of Linezolid and Daptomycin for Treatment of Vancomycin-Resistant Enterococcal Bloodstream Infections 
Antimicrobial Agents and Chemotherapy  2013;57(10):5013-5018.
Bloodstream infections due to vancomycin-resistant enterococci (VRE-BSI) result in substantial patient mortality and cost. Daptomycin and linezolid are commonly prescribed for VRE-BSI, but there are no clinical trials to determine optimal antibiotic selection. We conducted a systematic review for investigations that compared daptomycin and linezolid for VRE-BSI. We searched Medline from 1966 through 2012 for comparisons of linezolid and daptomycin for VRE-BSI. We included searches of EMBASE, clinicaltrials.gov, and national meetings. Data were extracted using a standardized instrument. Pooled odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using a fixed-effects model. Our search yielded 4,243 publications, of which 482 contained data on VRE treatment. Most studies (452/482) did not present data on BSI or did not provide information on linezolid or daptomycin. Among the remaining 30 studies, 9 offered comparative data between the two agents. None were randomized clinical trials. There was no difference in microbiologic (n = 5 studies, 517 patients; OR, 1.0; 95% CI, 0.4 to 1.7; P = 0.95) and clinical (n = 3 studies, 357 patients; OR, 1.2; 95% CI, 0.7 to 2.0; P = 0.7) cures between the two antibiotics. There was a trend toward increased survival with linezolid compared to daptomycin treatment (n = 9 studies, 1,074 patients; OR, 1.3; 95% CI, 1.1 to 1.8; I2 = 0 [where I2 is a measure of inconsistency]), but this did not reach statistical significance (P = 0.054). There are limited data to inform clinicians on optimal antibiotic selection for VRE-BSI. Available studies are limited by small sample size, lack of patient-level data, and inconsistent outcome definitions. Additional research, including randomized clinical trials, is needed before conclusions can be drawn about treatment options for VRE therapy.
doi:10.1128/AAC.00714-13
PMCID: PMC3811395  PMID: 23896468
5.  Antimicrobial Activity of High-Mobility-Group Box 2: a New Function to a Well-Known Protein 
Antimicrobial Agents and Chemotherapy  2013;57(10):4782-4793.
The human intestinal tract is highly colonized by a vast number of microorganisms. Despite this permanent challenge, infections remain rare, due to a very effective barrier defense system. Essential effectors of this system are antimicrobial peptides and proteins (AMPs), which are secreted by intestinal epithelial and lymphoid cells, balance the gut microbial community, and prevent the translocation of microorganisms. Several antimicrobial proteins have already been identified in the gut. Nonetheless, we hypothesized that additional AMPs are yet to be discovered in this setting. Using biological screening based on antimicrobial function, here we identified competent antibacterial activity of high-mobility-group box 2 (HMGB2) against Escherichia coli. By recombinant expression, we confirmed this biologically new antimicrobial activity against different commensal and pathogenic bacteria. In addition, we demonstrated that the two DNA-binding domains (HMG boxes A and B) are crucial for the antibiotic function. We detected HMGB2 in several gastrointestinal tissues by mRNA analysis and immunohistochemical staining. In addition to the nuclei, we also observed HMGB2 in the cytoplasm of intestinal epithelial cells. Furthermore, HMGB2 was detectable in vitro in the supernatants of two different cell types, supporting an extracellular function. HMGB2 expression was not changed in inflammatory bowel disease but was detected in certain stool samples of patients, whereas it was absent from control individuals. Taken together, we characterized HMGB2 as an antimicrobial protein in intestinal tissue, complementing the diverse repertoire of gut mucosal defense molecules.
doi:10.1128/AAC.00805-13
PMCID: PMC3811396  PMID: 23877675
6.  Characterization of the New AmpC β-Lactamase FOX-8 Reveals a Single Mutation, Phe313Leu, Located in the R2 Loop That Affects Ceftazidime Hydrolysis 
Antimicrobial Agents and Chemotherapy  2013;57(10):5158-5161.
A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.
doi:10.1128/AAC.00818-13
PMCID: PMC3811397  PMID: 23877692
7.  Dried Blood Spot Analysis Suitable for Therapeutic Drug Monitoring of Voriconazole, Fluconazole, and Posaconazole 
Antimicrobial Agents and Chemotherapy  2013;57(10):4999-5004.
Invasive aspergillosis and candidemia are important causes of morbidity and mortality in immunocompromised and critically ill patients. The triazoles voriconazole, fluconazole, and posaconazole are widely used for the treatment and prophylaxis of these fungal infections. Due to the variability of the pharmacokinetics of the triazoles among and within individual patients, therapeutic drug monitoring is important for optimizing the efficacy and safety of antifungal treatment. A dried blood spot (DBS) analysis was developed and was clinically validated for voriconazole, fluconazole, and posaconazole in 28 patients. Furthermore, a questionnaire was administered to evaluate the patients' opinions of the sampling method. The DBS analytical method showed linearity over the concentration range measured for all triazoles. Results for accuracy and precision were within accepted ranges; samples were stable at room temperature for at least 12 days; and different hematocrit values and blood spot volumes had no significant influence. The ratio of the drug concentration in DBS samples to that in plasma was 1.0 for voriconazole and fluconazole and 0.9 for posaconazole. Sixty percent of the patients preferred DBS analysis as a sampling method; 15% preferred venous blood sampling; and 25% had no preferred method. There was significantly less perception of pain with the DBS sampling method (P = 0.021). In conclusion, DBS analysis is a reliable alternative to venous blood sampling and can be used for therapeutic drug monitoring of voriconazole, fluconazole, and posaconazole. Patients were satisfied with DBS sampling and had less pain than with venous sampling. Most patients preferred DBS sampling to venous blood sampling.
doi:10.1128/AAC.00707-13
PMCID: PMC3811398  PMID: 23896473
8.  Plasmodium Dipeptidyl Aminopeptidases as Malaria Transmission-Blocking Drug Targets 
Antimicrobial Agents and Chemotherapy  2013;57(10):4645-4652.
The Plasmodium falciparum and P. berghei genomes each contain three dipeptidyl aminopeptidase (dpap) homologs. dpap1 and -3 are critical for asexual growth, but the role of dpap2, the gametocyte-specific homolog, has not been tested. If DPAPs are essential for transmission as well as asexual growth, then a DPAP inhibitor could be used for treatment and to block transmission. To directly analyze the role of DPAP2, a dpap2-minus P. berghei (Pbdpap2Δ) line was generated. The Pbdpap2Δ parasites grew normally, differentiated into gametocytes, and generated sporozoites that were infectious to mice when fed to a mosquito. However, Pbdpap1 transcription was >2-fold upregulated in the Pbdpap2Δ clonal lines, possibly compensating for the loss of Pbdpap2. The role of DPAP1 and -3 in the dpap2Δ parasites was then evaluated using a DPAP inhibitor, ML4118S. When ML4118S was added to the Pbdpap2Δ parasites just before a mosquito membrane feed, mosquito infectivity was not affected. To assess longer exposures to ML4118S and further evaluate the role of DPAPs during gametocyte development in a parasite that causes human malaria, the dpap2 deletion was repeated in P. falciparum. Viable P. falciparum dpap2 (Pfdpap2)-minus parasites were obtained that produced morphologically normal gametocytes. Both wild-type and Pfdpap2-negative parasites were sensitive to ML4118S, indicating that, unlike many antimalarials, ML4118S has activity against parasites at both the asexual and sexual stages and that DPAP1 and -3 may be targets for a dual-stage drug that can treat patients and block malaria transmission.
doi:10.1128/AAC.02495-12
PMCID: PMC3811399  PMID: 23836185
9.  Combination of Pantothenamides with Vanin Inhibitors as a Novel Antibiotic Strategy against Gram-Positive Bacteria 
Antimicrobial Agents and Chemotherapy  2013;57(10):4794-4800.
The emergence of resistance against current antibiotics calls for the development of new compounds to treat infectious diseases. Synthetic pantothenamides are pantothenate analogs that possess broad-spectrum antibacterial activity in vitro in minimal media. Pantothenamides were shown to be substrates of the bacterial coenzyme A (CoA) biosynthetic pathway, causing cellular CoA depletion and interference with fatty acid synthesis. In spite of their potential use and selectivity for bacterial metabolic routes, these compounds have never made it to the clinic. In the present study, we show that pantothenamides are not active as antibiotics in the presence of serum, and we found that they were hydrolyzed by ubiquitous pantetheinases of the vanin family. To address this further, we synthesized a series of pantetheinase inhibitors based on a pantothenate scaffold that inhibited serum pantetheinase activity in the nanomolar range. Mass spectrometric analysis showed that addition of these pantetheinase inhibitors prevented hydrolysis of pantothenamides by serum. We found that combinations of these novel pantetheinase inhibitors and prototypic pantothenamides like N5-Pan and N7-Pan exerted antimicrobial activity in vitro, particularly against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes) even in the presence of serum. These results indicate that pantothenamides, when protected against degradation by host pantetheinases, are potentially useful antimicrobial agents.
doi:10.1128/AAC.00603-13
PMCID: PMC3811400  PMID: 23877685
10.  Clotrimazole Dampens Vaginal Inflammation and Neutrophil Infiltration in Response to Candida albicans Infection 
Antimicrobial Agents and Chemotherapy  2013;57(10):5178-5180.
The pathology of vulvovaginal candidiasis (VVC) caused by Candida albicans is associated with a nonprotective inflammatory response and is frequently treated with clotrimazole. We investigated the mechanisms by which clotrimazole resolves VVC. Low levels of clotrimazole, which do not block fungal growth, inhibit expression of a “danger response” transcription factor, c-Fos, block production of proinflammatory cytokines, and inhibit neutrophil infiltration to the site of infection.
doi:10.1128/AAC.01244-13
PMCID: PMC3811401  PMID: 23896471
11.  KPC-Producing, Multidrug-Resistant Klebsiella pneumoniae Sequence Type 258 as a Typical Opportunistic Pathogen 
Antimicrobial Agents and Chemotherapy  2013;57(10):5144-5146.
The virulence of a KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) strain representing those circulating in Greece was assessed in a mouse septicemia model. The strain was virtually avirulent (50% lethal dose, >108 and 5 × 107 CFU for immunocompetent and neutropenic animals, respectively). Also, it was highly susceptible to serum killing, rapidly phagocytosed in vitro, and classified as K41, which is not among the virulent capsular types. The findings indirectly support the notion that high ST258-associated mortality is largely due to inefficient antimicrobial treatment.
doi:10.1128/AAC.01052-13
PMCID: PMC3811402  PMID: 23856769
12.  Antimicrobial Peptide GL13K Is Effective in Reducing Biofilms of Pseudomonas aeruginosa 
Antimicrobial Agents and Chemotherapy  2013;57(10):4903-4910.
Human parotid secretory protein (PSP; BPIF2A) is predicted to be structurally similar to bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. Based on the locations of known antimicrobial peptides in the latter two proteins, potential active peptides in the PSP sequence were identified. One such peptide, GL13NH2 (PSP residues 141 to 153) was shown previously to interfere with LPS binding and agglutinate bacteria without bactericidal activity. By introducing three additional positively charged lysine residues, the peptide was converted to the novel bactericidal cationic peptide GL13K (MIC for Pseudomonas aeruginosa, 8 μg/ml [5.6 μM]). We investigated the antibiofilm activity of GL13K against static, monospecies biofilms of P. aeruginosa PAO1. Two-hour exposure of a 24-h biofilm to 64 μg/ml (44.8 μM) GL13K reduced biofilm bacteria by 102, and 100 μg/ml (70 μM) GL13K reduced bacteria by 103. Similar results could be achieved on 48-h-old biofilms. Lower concentrations of GL13K (32 μg/ml [22.4 μM]) were successful in reducing biofilm cell numbers in combination with tobramycin. This combination treatment also achieved total eradication of the biofilm in a majority (67.5%) of tested samples. An alanine scan of GL13K revealed the importance of the leucine residue in position six of the peptide sequence, where replacement led to a loss of antibiofilm activity, whereas the impact of replacing charged residues was less pronounced. Bacterial metalloproteases were found to partially inactivate GL13K but not a d amino acid version of the peptide.
doi:10.1128/AAC.00311-13
PMCID: PMC3811403  PMID: 23917321
13.  Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial Activity In Vivo 
Antimicrobial Agents and Chemotherapy  2013;57(10):4945-4955.
The emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapy in vivo. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activity in vivo. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs [with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand the in vivo relevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activities in vivo against Salmonella infection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes.
doi:10.1128/AAC.00152-13
PMCID: PMC3811404  PMID: 23877702
14.  DNA Microarray for Genotyping Antibiotic Resistance Determinants in Acinetobacter baumannii Clinical Isolates 
Antimicrobial Agents and Chemotherapy  2013;57(10):4761-4768.
In recent decades, Acinetobacter baumannii has emerged as an organism of great concern due to its ability to accumulate antibiotic resistance. In order to improve the diagnosis of resistance determinants in A. baumannii in terms of lead time and accuracy, we developed a microarray that can be used to detect 91 target sequences associated with antibiotic resistance within 4 h from bacterial culture to result. The array was validated with 60 multidrug-resistant strains of A. baumannii in a blinded, prospective study. The results were compared to phenotype results determined by the automated susceptibility testing system VITEK2. Antibiotics considered were piperacillin-tazobactam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, amikacin, gentamicin, tobramycin, ciprofloxacin, and tigecycline. The average positive predictive value, negative predictive value, sensitivity, and specificity were 98, 98, 99, and 94%, respectively. For carbapenemase genes, the array results were compared to singleplex PCR results provided by the German National Reference Center for Gram-Negative Pathogens, and results were in complete concordance. The presented array is able to detect all relevant resistance determinants of A. baumannii in parallel. The short handling time of 4 h from culture to result helps to provide fast results in order to initiate adequate anti-infective therapy for critically ill patients. Another application would be data acquisition for epidemiologic surveillance.
doi:10.1128/AAC.00863-13
PMCID: PMC3811405  PMID: 23856783
15.  A Peptide Inhibitor of Cytomegalovirus Infection from Human Hemofiltrate 
Antimicrobial Agents and Chemotherapy  2013;57(10):4751-4760.
Naturally occurring substances with antimicrobial activity can serve as a starting point for the rational design of new drugs to treat infectious diseases. Here, we screened a library of peptides derived from human hemofiltrate for inhibitory effects on human cytomegalovirus (CMV) infection. We isolated a previously unknown derivative of the neutrophil-activating peptide 2, which we termed CYVIP, for CMV-inhibiting peptide. The peptide blocked infection with human and mouse CMV as well as with herpes simplex virus type 1 in different cell types. We found that CYVIP interferes with virus attachment to the cell surface, and structure-activity relationship studies revealed that positively charged lysine and arginine residues of CYVIP are essential for its inhibitory activity. The N-terminal 29 amino acids of the peptide were sufficient for inhibition, and substitution with an acidic residue further improved its activity. The target structure of CYVIP on the cell surface seems to be the sulfate residues of heparan sulfate proteoglycans, which are known to serve as herpesvirus attachment receptors. Our data suggest that O-sulfation of heparan sulfate is required for binding of CYVIP, and furthermore, that the initial interaction of CMV particles with cells takes place preferentially via 6-O-linked sulfate groups. These findings about CYVIP's mode of action lay the basis for further development of antivirals interfering with attachment of CMV to cells, a crucial step of the infection cycle.
doi:10.1128/AAC.00854-13
PMCID: PMC3811406  PMID: 23856778
16.  Azole Heteroresistance in Cryptococcus neoformans: Emergence of Resistant Clones with Chromosomal Disomy in the Mouse Brain during Fluconazole Treatment 
Antimicrobial Agents and Chemotherapy  2013;57(10):5127-5130.
We have previously reported that Cryptococcus neoformans strains are innately heteroresistant to fluconazole in vitro, producing minor, highly resistant subpopulations due to adaptive formation of disomic chromosomes. Using a mouse model, we assessed the emergence of heteroresistant clones in the brain during fluconazole treatment and found that the occurrence of heteroresistant clones in vivo with chromosomal disomy is strain dependent. Interestingly, emergence of heteroresistant clones in vivo was unrelated to the strain's MIC to fluconazole.
doi:10.1128/AAC.00694-13
PMCID: PMC3811407  PMID: 23836187
17.  Complete Nucleotide Sequence of a blaKPC-Harboring IncI2 Plasmid and Its Dissemination in New Jersey and New York Hospitals 
Antimicrobial Agents and Chemotherapy  2013;57(10):5019-5025.
Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains have spread worldwide and become a significant public health threat. blaKPC, the plasmid-borne KPC gene, was frequently identified on numerous transferable plasmids in different incompatibility replicon groups. Here we report the complete nucleotide sequence of a novel blaKPC-3-harboring IncI2 plasmid, pBK15692, isolated from a multidrug-resistant K. pneumoniae ST258 strain isolated from a New Jersey hospital in 2005. pBK15692 is 78 kb in length and carries a backbone that is similar to those of other IncI2 plasmids (pR721, pChi7122-3, pHN1122-1, and pSH146-65), including the genes encoding type IV pili and shufflon regions. Comparative genomics analysis of IncI2 plasmids reveals that they possess a conserved plasmid backbone but are divergent with respect to the integration sites of resistance genes. In pBK15692, the blaKPC-3-harboring Tn4401 was inserted into a Tn1331 element and formed a nested transposon. A PCR scheme was designed to detect the prevalence of IncI2 and pBK15692-like plasmids from a collection of clinical strains from six New Jersey and New York hospitals isolated between 2007 and 2011. IncI2 plasmids were found in 46.2% isolates from 318 clinical K. pneumoniae strains. Notably, 59 pBK15692-like plasmids (23%) have been identified in 256 KPC-bearing K. pneumoniae strains, and all carried KPC-3 and belong to the epidemic ST258 clone. Our study revealed that the prevalence of IncI2 plasmids has been considerably underestimated. Further studies are needed to understand the distribution of this plasmid group in other health care regions and decipher the association between IncI2 plasmids and blaKPC-3-bearing ST258 strains.
doi:10.1128/AAC.01397-13
PMCID: PMC3811408  PMID: 23896467
18.  The FDA Reboot of Antibiotic Development 
Antimicrobial Agents and Chemotherapy  2013;57(10):4605-4607.
doi:10.1128/AAC.01277-13
PMCID: PMC3811409  PMID: 23896479
19.  Rapid Emergence of Resistance to Linezolid and Mutator Phenotypes in Staphylococcus aureus Isolates from an Adult Cystic Fibrosis Patient 
Antimicrobial Agents and Chemotherapy  2013;57(10):5186-5188.
Linezolid has emerged as an important therapeutic option for the treatment of Staphylococcus aureus in patients with cystic fibrosis. We report the rapid emergence, upon treatment with linezolid, of linezolid-resistant S. aureus clinical isolates through the accumulation of resistance-associated 23S rRNA mutations, together with acquisition of an altered mutator phenotype.
doi:10.1128/AAC.01392-13
PMCID: PMC3811410  PMID: 23917314
20.  Head-to-Head Comparison of Inhibitory and Fungicidal Activities of Fluconazole, Itraconazole, Voriconazole, Posaconazole, and Isavuconazole against Clinical Isolates of Trichosporon asahii 
Antimicrobial Agents and Chemotherapy  2013;57(10):4841-4847.
Treatment of disseminated Trichosporon infections still remains difficult. Amphotericin B frequently displays inadequate fungicidal activity and echinocandins have no meaningful antifungal effect against this genus. Triazoles are currently the drugs of choice for the treatment of Trichosporon infections. This study evaluates the inhibitory and fungicidal activities of five triazoles against 90 clinical isolates of Trichosporon asahii. MICs (μg/ml) were determined according to Clinical and Laboratory Standards Institute microdilution method M27-A3 at 24 and 48 h using two endpoints, MIC-2 and MIC-0 (the lowest concentrations that inhibited ∼50 and 100% of growth, respectively). Minimum fungicidal concentrations (MFCs; μg/ml) were determined by seeding 100 μl of all clear MIC wells (using an inoculum of 104 CFU/ml) onto Sabouraud dextrose agar. Time-kill curves were assayed against four clinical T. asahii isolates and the T. asahii ATCC 201110 strain. The MIC-2 (∼50% reduction in turbidity compared to the growth control well)/MIC-0 (complete inhibition of growth)/MFC values that inhibited 90% of isolates at 48 h were, respectively, 8/32/64 μg/ml for fluconazole, 1/2/8 μg/ml for itraconazole, 0.12/0.5/2 μg/ml for voriconazole, 0.5/2/4 μg/ml for posaconazole, and 0.25/1/4 μg/ml for isavuconazole. The MIC-0 endpoints yielded more consistent MIC results, which remained mostly unchanged when extending the incubation to 48 h (98 to 100% agreement with 24-h values) and are easier to interpret. Based on the time-kill experiments, none of the drugs reached the fungicidal endpoint (99.9% killing), killing activity being shown but at concentrations not reached in serum. Statistical analysis revealed that killing rates are dose and antifungal dependent. The lowest concentration at which killing activity begins was for voriconazole, and the highest was for fluconazole. These results suggest that azoles display fungistatic activity and lack fungicidal effect against T. asahii. By rank order, the most active triazole is voriconazole, followed by itraconazole ∼ posaconazole ∼ isavuconazole > fluconazole.
doi:10.1128/AAC.00850-13
PMCID: PMC3811412  PMID: 23877683
21.  Discovery of Novel Small-Molecule HIV-1 Replication Inhibitors That Stabilize Capsid Complexes 
Antimicrobial Agents and Chemotherapy  2013;57(10):4622-4631.
The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTD site 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid.
doi:10.1128/AAC.00985-13
PMCID: PMC3811413  PMID: 23817385
22.  Association of Fluoroquinolone Resistance, Virulence Genes, and IncF Plasmids with Extended-Spectrum-β-Lactamase-Producing Escherichia coli Sequence Type 131 (ST131) and ST405 Clonal Groups 
Antimicrobial Agents and Chemotherapy  2013;57(10):4736-4742.
The global increase of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli is associated with the specific clonal group sequence type 131 (ST131). In order to understand the successful spread of ESBL-producing E. coli clonal groups, we characterized fluoroquinolone resistance determinants, virulence genotypes, and plasmid replicons of ST131 and another global clonal group, ST405. We investigated 41 ST131-O25b, 26 ST131-O16, 41 ST405, and 41 other ST (OST) ESBL-producing isolates, which were collected at seven acute care hospitals in Japan. The detection of ESBL types, fluoroquinolone resistance-associated mutations (including quinolone resistance-determining regions [QRDRs]), virulence genotypes, plasmid replicon types, and IncF replicon sequence types was performed using PCR and sequencing. blaCTX-M, specifically blaCTX-M-14, was the most common ESBL gene type among the four groups. Ciprofloxacin resistance was found in 90% of ST131-O25b, 19% of ST131-O16, 100% of ST405, and 54% of OST isolates. Multidrug resistance was more common in the ST405 group than in the ST131-O25 group (56% versus 32%; P = 0.045). All ST131-O25b isolates except one had four characteristic mutations in QRDRs, but most of the isolates from the other three groups had three mutations in common. The ST131-O25b and ST405 groups had larger numbers of virulence genes than the OST group. All of the ST131-O25b and ST405 isolates and most of the ST131-O16 and OST isolates carried IncF replicons. The most prevalent IncF replicon sequence types differed between the four clonal groups. Both the ST131-O25b and ST405 clonal groups had a fluoroquinolone resistance mechanism in QRDRs, multidrug resistance, high virulence, and IncF plasmids, suggesting the potential for further global expansion and a need for measures against these clonal groups.
doi:10.1128/AAC.00641-13
PMCID: PMC3811414  PMID: 23856781
23.  Echocardiogram Study To Evaluate the Effect of the Novel Hepatitis C Virus NS5A Inhibitor GSK2336805 on Cardiac Contractility in Healthy Subjects 
Antimicrobial Agents and Chemotherapy  2013;57(10):5141-5143.
GSK2336805 is a hepatitis C virus NS5A inhibitor in clinical development for the treatment of chronic hepatitis C virus infection. This was a single-center, randomized, double-blind, placebo-controlled, two-period crossover study in healthy adults to evaluate the effects of a single 150-mg dose of GSK2336805 on echocardiographic measures of contractility. GSK2336805 had no effect on ejection fraction, and there was no significant correlation between GSK2336805 plasma concentration and ejection fraction. (This study has been registered at Clinicaltrials.gov under registration no. NCT01424540.)
doi:10.1128/AAC.00668-13
PMCID: PMC3811415  PMID: 23856771
24.  Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1 
Antimicrobial Agents and Chemotherapy  2013;57(10):4743-4750.
Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.
doi:10.1128/AAC.00309-13
PMCID: PMC3811417  PMID: 23856780
25.  Amphotericin B- and Voriconazole-Echinocandin Combinations against Aspergillus spp.: Effect of Serum on Inhibitory and Fungicidal Interactions 
Antimicrobial Agents and Chemotherapy  2013;57(10):4656-4663.
Antifungal combination therapy with voriconazole or amphotericin B and an echinocandin is often employed as primary or salvage therapy for management particularly of refractory aspergillosis. The pharmacodynamic interactions of amphotericin B- and voriconazole-based combinations with the three echinocandins caspofungin, micafungin, and anidulafungin in the presence of serum were tested against 15 Aspergillus fumigatus complex, A. flavus complex, and A. terreus complex isolates to assess both their growth-inhibitory and fungicidal activities. The in vitro activity of each drug alone and in combination at a 1:1 fixed concentration ratio was tested with a broth microdilution colorimetric method, and interactions were assessed by isobolographic analysis. Synergy was found for all amphotericin B- and voriconazole-based combinations, with amphotericin B-based combinations showing strong inhibitory synergistic interactions (interaction indices of 0.20 to 0.52) and with voriconazole-based combinations demonstrating strong fungicidal synergistic interactions (interaction indices of 0.10 to 0.29) (P < 0.001). Drug- and species-specific differences were found, with caspofungin and the A. fumigatus complex exhibiting the weakest synergistic interactions. In the presence of serum, the synergistic interactions were reduced in the order (from largest to smallest decrease) micafungin > anidulafungin > caspofungin, and A. flavus complex > A. fumigatus complex > A. terreus complex, resulting in additive interactions, particularly for inhibitory activities of amphotericin B-echinocandin combinations and fungicidal activities of voriconazole-echinocandin combinations. Drug- and species-specific differences were found in the presence of serum for inhibitory activities of antifungal drugs, with the lowest interaction indices being observed for amphotericin B-caspofungin (median, 0.77) and for the A. terreus complex (median, 0.56). The present in vitro data showed that serum had a major impact on synergistic interactions of amphotericin B-echinocandin and voriconazole-echinocandin combinations, resulting in additive interactions and explaining the indifferent outcomes usually observed in vivo.
doi:10.1128/AAC.00597-13
PMCID: PMC3811418  PMID: 23856768

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