A recurrent case of left-sided endocarditis caused by high-level aminoglycoside-resistant Enterococcus faecalis was successfully treated with ceftaroline and daptomycin. This combination demonstrated excellent in synergy in vitro. Mechanistically, ceftaroline enhanced binding of daptomycin to the cell membrane and sensitized E. faecalis to killing by human cathelicidin LL-37, a cationic innate host defense peptide. Daptomycin plus ceftaroline may be considered in salvage therapy in E. faecalis endovascular infections and requires further study.

Isoniazid resistance is highly prevalent in Vietnam. We investigated the molecular and epidemiological characteristics and the association with first-line treatment outcomes of the main isoniazid resistance mutations in Mycobacterium tuberculosis in codon 315 of the katG and in the promoter region of the inhA gene.Mycobacterium tuberculosis strains with phenotypic resistance to isoniazid from consecutively diagnosed smear-positive tuberculosis patients in rural Vietnam were subjected to Genotype® MTBDRplus testing to identify katG and inhA mutations. Treatment failure and relapse were determined by sputum culture.In total 227/251 isoniazid-resistant strains (90.4%) had detectable mutations: 75.3% in the katG315 codon and 28.2% in the inhA promoter region. KatG315 mutations were significantly associated with pre-treatment resistance to streptomycin, rifampicin and ethambutol, but not with the Beijing genotype, and predicted both unfavorable treatment outcome (treatment failure or death) and relapse inhA promoter region mutations were only associated with resistance to streptomycin and relapse.In tuberculosis patients, M. tuberculosis katG315 mutations but not inhA mutations are associated with unfavorable treatment outcome. InhA mutations do however increase the risk of relapse, at least with treatment regimens that contain only isoniazid and ethambutol in the continuation phase.

Acinetobacter baumannii is an important emerging pathogen among healthcare-acquired infections and is responsible for severe nosocomial and community-acquired pneumonia. Currently available mouse models of A. baumannii pneumonia show poor colonization with little to no extrapulmonary dissemination. Here, we describe a mouse model of A. baumannii pneumonia using a clinical isolate (LAC-4 strain) that reliably reproduces the most relevant features of human pulmonary A. baumannii infection and pathology. Using this model, we have shown that LAC-4 infection induced rapid bacterial replication in the lungs, significant extrapulmonary dissemination, and severe bacteremia by 24 h post intranasal inoculation. Infected mice showed severe bronchopneumonia and dilatation and inflammatory cell infiltration in the perivascular space. More significantly, 100% of C57BL/6 and BALB/c mice succumbed to 10(8) CFU of LAC-4 inoculation within 48 hours. When this model was used to assess the efficacy of antimicrobials, all mice treated with imipenem and tigecycline survived a lethal intranasal challenge, with minimal clinical signs and body weight loss. Moreover, intranasal immunization of mice with formalin-fixed LAC-4 protected 40% of mice from a lethal (100 xLD100) intraperitoneal challenge. Thus, this model offers a reproducible acute course of A. baumannii pneumonia without requiring additional manipulation of host immune status, which will facilitate the development of therapeutic agents and vaccines against A. baumannii pneumonia in humans.

Background.

Numerous studies have reported low concentrations of antituberculosis drugs in tuberculosis (TB) patients, but few studies have examined whether low drug concentrations affect TB treatment response.Patients and methods. We examined steady-state plasma concentrations of isoniazid, rifampicin, and pyrazinamide at two hours after administration of drugs (C2h) among 181 patients with pulmonary tuberculosis in Indonesia and related these to bacteriological response during treatment.

Results.

C2h values below reference values for either isoniazid, rifampicin, or pyrazinamide were found in 91% of patients; 60% had at least two low C2h concentrations. The isoniazid C2h was noticeably lower in fast versus slow acetylators (0.9 mg/L vs. 2.2 mg/L, p<0.001). At the end of treatment, 82% of the patients were cured, while 30 patients (17%) had dropped out during the study, and 2 patients (1%) failed treatment. No association was found between C2h concentrations and sputum culture results at 8 weeks of treatment. Post-hoc analysis showed that patients with low pyrazinamide C2h (p=0.01) and patients with large extensive lung lesions (p=0.01) were at risk of at least one positive culture at week 4, 8 or 24/32.

Conclusion.

Antituberculosis drug concentrations were often low, but treatment response was nevertheless good. No association was found between drug concentrations and 8 weeks culture conversion, but low pyrazinamide drug concentrations may be associated with a less favourable bacteriological response. The use of higher doses of pyrazinamide may warrant further investigation.

Azoles are among the most successful classes of antifungals. They act by inhibiting α-14 lanosterol demethylase in the ergosterol biosynthesis pathway. Oropharyngeal candidiasis (OPC) occurs in about 90% of HIV infected individuals, and 4-5% are refractory to current therapies, including azoles, due to the formation of resistant biofilms produced in the course of OPC. We reasoned that compounds affecting a different target may potentiate azoles to produce increased killing and an anti-biofilm therapeutic. 2-adamantanamine (AC17) was identified in a screen for compounds potentiating the action of miconazole against biofilms of Candida albicans. AC17, a close structural analog to the antiviral amantadine, did not affect the viability of C. albicans, but caused the normally fungistatic azoles to become fungicidal. Transcriptome analysis of cells treated with AC17 revealed that the ergosterol and filamentation pathways were affected. Indeed, cells exposed to AC17 had decreased ergosterol content and were unable to invade agar. In vivo, the combination of AC17 and fluconazole produced significant reduction in fungal tissue burden in a guinea pig model of cutaneous candidiasis, while each treatment alone did not have a significant effect. The combination of fluconazole and AC17 also showed improved efficacy (P-value of 0.018) compared to fluconazole alone when fungal lesions were evaluated. AC17 is a promising lead in the search for more effective antifungal therapeutics.

Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, TP10 and MAP, with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX Green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane-permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis; as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicate the possibility to design peptide-based therapies for infectious diseases.

Linezolid plays an increasingly important role in the treatment of multidrug-resistant tuberculosis. However, patients should be carefully monitored due to time- and dose-dependent toxicity. Clarithromycin plays a more modest role. Therapeutic drug monitoring may contribute to assessing treatment regimens, helping to reduce toxicity whilst maintaining adequate drug exposure. Oral fluid sampling could provide a welcome alternative in cases where conventional plasma sampling is not possible or desirable. The aim of this study was to clinically validate the analysis of linezolid and clarithromycin and its metabolite hydroxyclarithromycin in oral fluid of patients with multidrug-resistant tuberculosis.Serum and oral fluid samples were simultaneously obtained and analyzed using validated methods, after extensive cross-validation between the two matrices. Passing-Bablok regressions and Bland-Altman analysis showed that oral fluid analysis of linezolid and clarithromycin appeared suitable for therapeutic drug monitoring in MDR-TB patients. No correction factor is needed for the interpretation of linezolid oral fluid concentration with a linezolid serum/oral fluid ratio of 0.97 (95% CI, 0.92-1.02). However, the clarithromycin serum/oral fluid concentration ratio is 3.07 (95% CI, 2.45-3.69). Analysis of hydroxyclarithromycin in oral fluid was not possible in this study due to a non linear relationship between concentration in serum and oral fluid. In conclusion, the analysis of linezolid (no correction factor) and clarithromycin (correction factor *3) in oral fluid is applicable for therapeutic drug monitoring in multidrug-resistant tuberculosis as an alternative to conventional serum sampling. Easy sampling, using a non-invasive technique may facilitate therapeutic drug monitoring in specific patient categories.

Naturally occurring quinolone and quinolone-like compounds, such as quinine, 2-hydroxyquinoline, 4-hydroxyquinoline, and 2-heptyl-3-hydroxy-4(1H)-quinolone increased expression of qnrS1 in Escherichia coli 2.3- to 11.2-fold similar to a synthetic quinolone, ciprofloxacin. In contrast, chromosomal qnrVS1 of Vibrio splendidus was not induced by these compounds. Molecules associated with quorum-sensing, such as N-3-hydroxybutyryl-homoserine lactone (HSL), N-hexanoyl-HSL, and N-3-(oxododecanoyl)-HSL did not show an induction effect for either qnrS1 or qnrVS1 at tested concentrations..

With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs as compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at 0.5 PPM concentration, while LLKKK-18 showed antibacterial activity only with NP-2 at 0.5 PPM dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with an anti-tuberculosis drug rifampicin. The size and shape of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, while treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed FITC-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template.

Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited, and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents which inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents which are urgently needed.