N-Acetyl-L-cysteine and Cysteamine: New Strategies Against Mixed Biofilms of Non-encapsulated Streptococcus pneumoniae and Non-typeable Haemophilus influenzae [PublishAheadOfPrint]

Acute otitis media, a polymicrobial disease of the middle ear cavity of children, is a significant public health problem worldwide. It is most frequently caused by encapsulated Streptococcus pneumoniae and non-typeable Haemophilus influenzae, although the widespread use of pneumococcal conjugate vaccines is apparently producing an increase in carriage of non-encapsulated S. pneumoniae. Frequently, pneumococci and H. influenzae live together in the human nasopharynx forming a self-produced biofilm. Biofilms represent a global medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence. Here, we describe the development of an in vitro, non-encapsulated S. pneumoniae–non-typeable H. influenzae biofilm system using polystyrene or glass-bottom plates. Using confocal laser scanning microscopy and specific fluorescent labeling of pneumococcal cells with Helix pomatia agglutinin revealed an even distribution of both species within the biofilm. This simple and robust protocol of mixed biofilms has been used for testing the antimicrobial properties of two well known antioxidants that are widely used in the clinical setting, i.e., N-acetylcysteine and cysteamine. This repurposing approach showed the high potency of N-acetylcysteine and cysteamine against mixed biofilms of non-encapsulated S. pneumoniae and non-typeable H. influenzae. Decades of clinical use mean that these compounds are safe to use, which may accelerate their evaluation in humans.

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Genetic environment of the blaKPC-2 gene in Klebsiella pneumoniae isolate which may have been imported to Russia from Southeast Asia [PublishAheadOfPrint]

The nucleotide sequence of a bla_KPC-2-harboring plasmid (pKPCAPSS) from Klebsiella pneumoniae ST273 isolated in Saint Petersburg, Russia from a patient with history of recent travel to Vietnam is presented. This 127,970-bp plasmid possessed both IncFII and IncR replicons. bla_KPC-2 was localized on a hypothetical mobile element. This element was flanked by 38bp inverted Tn3 repeats and included a Tn3-specific transposase gene, macrolide resistance operon (mphA, mrx, mphR), and a fragment of bla_TEM with unique polymorphisms.

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Pharmacokinetic Evaluation of Darunavir Administered Once or Twice daily in Combination with Ritonavir or the Three Direct Acting Antiviral Regimen of Ombitasvir, Paritaprevir, Ritonavir and Dasabuvir in Adults Co-infected with Hepatitis C and Human Immunodeficiency Virus [PublishAheadOfPrint]

Background: The three-direct acting antiviral (3D) regimen containing ombitasvir, paritaprevir, ritonavir and dasabuvir ± ribavirin (RBV) is approved for treatment of HCV GT1/HIV-1 co-infection. Results of a pharmacokinetic substudy of 3D and darunavir are presented.

Methods: HCV/HIV-1 infected subjects were randomized to maintain a darunavir 800 mg once daily (QD) or switch to a darunavir 600 mg twice daily (BID) based antiretroviral regimen. On Study Day 1, subjects received 3D and RBV plus darunavir for 12 weeks. Pharmacokinetic parameters were compared for darunavir with and without 3D. Pharmacokinetic parameters of 3D were compared to historical data.

Results: Ten subjects received darunavir QD and 12 subjects received darunavir BID. The central value ratios (90% confidence interval [CI]) for darunavir C_max, AUC₂₄ and C₂₄ administered QD with 3D vs. alone were 0.92 (0.72, 1.18), 0.83 (0.71, 0.98) and 0.64 (0.44, 0.93), respectively. The ratios (90% CI) for darunavir C_max, AUC₁₂ and C₁₂ administered BID with 3D were 0.92 (0.76, 1.12), 0.88 (0.73, 1.05) and 0.73 (0.58, 0.92), respectively. Exposures of 3D were similar or slightly lower compared with historical data. All darunavir trough concentrations (C_trough) associated with an HIV-1 RNA >40 copies/mL were above the darunavir EC₅₀ of 550 ng/mL for resistant virus.

Conclusions: The 3D regimen with darunavir QD or BID did not affect darunavir C_max and AUC, whereas darunavir C_trough decreased. Changes in pharmacokinetic parameters of 3D were not considered clinically significant. Episodes of intermittent HIV-1 viremia were infrequent and not associated with darunavir C_trough values below 550 ng/mL. (This study has been registered at ClinicalTrials.gov under identifier NCT01939197.)

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Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-quinolone-3-diarylethers and Antimycin in Toxoplasma gondii [PublishAheadOfPrint]

Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochin-like-quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases including toxoplasmosis, malaria and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of the cytochrome bc₁ complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the mechanism of ELQ-316 remains unknown. Here we describe the selection and genetic characterization of T. gondii clones resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interaction of Qi site inhibitors with the apicomplexan cytochrome bc₁ complex.

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Safety and Pharmacokinetics of CD101 IV, a Novel Echinocandin, in Healthy Adults [PublishAheadOfPrint]

CD101 IV is a novel echinocandin with distinctive pharmacokinetic properties that is being developed as a once-weekly treatment for candidemia and invasive candidiasis. CD101 has potent in vitro activity and in vivo efficacy against a broad range of Candida and Aspergillus species. The primary objective of two randomized, double-blind, placebo-controlled, dose-escalation studies in healthy adults was to determine the safety and tolerability of CD101 IV. Sequential cohorts of 8 subjects (n=6, active; n=2, placebo) were administered single (50, 100, 200, 400 mg) or multiple once-weekly (100 mg x2, 200 mg x2, 400 mg x3) doses of CD101 IV infused over 1 hour. There were no deaths, serious adverse events (SAEs), severe adverse events (AEs), or withdrawals from the study due to an AE. The majority of AEs were mild and all completely resolved. There was a higher incidence of total AEs and mild transient infusion reactions in the 400 mg x3 dose group. There were no clinically meaningful trends in postbaseline laboratory abnormalities and no safety issues related to electrocardiograms, vital signs, or physical exams. CD101 showed dose-proportional plasma exposures, minor accumulation (30%-55%), low apparent clearance (<0.28 L/hour), long half-life (t_1/2 >80 h), and minimal urine excretion. CD101 IV was safe and well tolerated at single and multiple doses up to 400 mg once weekly for 3 weeks, exhibited a long t_1/2, minimal accumulation over several weeks, negligible renal excretion, and high plasma exposures enabling once-weekly dosing.

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High-throughput screening and prediction models building for novel hemozoin inhibitors using physicochemical properties [PublishAheadOfPrint]

It is essential to continue the search for novel antimalarial drugs due to current spread of resistance against artemisinin by Plasmodium falciparum parasites. In this study, we developed in silico models to predict hemozoin inhibitors as a potential first-step screening for novel antimalarials. The in vitro colorimetric high throughput screening assay of hemozoin formation was used to identify hemozoin inhibitors from 9600 structurally diverse compounds. Physicochemical properties of positive hits and randomly selected compounds were extracted from ChemSpider database; they were used for developing prediction models to predict hemozoin inhibitors using two different approaches, i.e. traditional multivariate logistic regression, and Bayesian Modeling Average. Our results showed that a total of 224 positive hits exhibited the ability to inhibit the hemozoin formation with IC₅₀ ranging from 3.1 μM to 199.5 μM. The "best" model according to traditional multivariate logistic regression included three variables: octanol-water partition coefficient, number of hydrogen bond donors, and number of atoms of hydrogen. Whereas, the "best" model according to Bayesian Modeling Average was octanol-water partition coefficient, number of hydrogen bond donors, and index of refraction. Both models had a good discriminatory power with the area under curve values were 0.736, and 0.781 for the traditional multivariate model, and the Bayesian Modeling Average model respectively. In conclusion, the prediction models can be a new, useful and cost-effective approach for the first screen of hemozoin inhibition based antimalarial drug discovery.

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Genotyping and prevalence of pyrazinamide- and moxifloxacin-resistant tuberculosis in China, 2000-2010: findings from the national survey data [PublishAheadOfPrint]

We investigated the prevalence, trends and risk factors for pyrazinamide (PZA) and moxifloxacin (MOX) resistance among TB cases in China, and also analyzed the population structure of Mycobacterium tuberculosis strains.All the M. tuberculosis strains enrolled in this study were collected from the national TB prevalence surveys. Each strain was genotyped by analyzing the regions of RD105 and the IS6110 in the NTF region. The BACTEC MGIT 960 system was used to detect the drug susceptibility of M. tuberculosis isolates against PZA and MOX. Based on the genotyping results, 241 (66.4%) strains were classified as Beijing genotype in 2000, which was significantly lower than in 2010 (76.2%, P<0.01). The proportion of the modern Beijing genotype increased significantly from 49.6% in 2000 to 68.1% in 2010 (P<0.01), while no significant difference was observed in the rate of ancient Beijing genotype between 2000 and 2010 (P=0.676). In addition, we found that the proportion of PZA-resistance in 2010 (15.0%) was significantly higher than that in 2000 (9.6%, P=0.04). For MOX, there were more MOX-resistant isolates detected in 2010 (7.7%) than in 2000 (3.0%). In conclusion, our data demonstrate that the Beijing genotype was the predominant M. tuberculosis lineage during the past decade. The proportion of Beijing genotype significantly increased from 2000 to 2010, largely due to an increase in the modern Beijing sublineage. In addition, resistance to PZA and MOX increased significantly in China between 2000 and 2010.

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