学会抄録

<p><b>Pod-IVR Pharmacokinetics in macaques</b> (A) <i>In vivo</i> release of TDF and FTC from each pod-IVR (N = 6/time point) over the course of the efficacy study determined by residual drug measurements from the pod-IVRs that were in place for 19 weeks with IVRs exchanged for new devices every 2 weeks. The top and bottom of the boxes show the 75th and 25th percentiles, respectively, and the line in the middle of the box is the median value. The dotted lines show the mean (N = 6) <i>in vivo</i> release from identical pod-IVRs obtained during the PK study preceding this efficacy study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157061#pone.0157061.ref026" target="_blank">26</a>]. (B) <i>In vivo</i> release profile for individual macaques (T1-T6) shows variability between animals. (C) TDF, TFV, TDF+TFV, and FTC levels in vaginal fluids collected at each ring exchange. Vaginal fluids were collected with Weck-Cel sponges proximal and distal to the pod-IVR placement. The dotted horizontal lines correspond to the medians from our previous PK study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157061#pone.0157061.ref026" target="_blank">26</a>]. Left panels-proximal; Right panels-distal; Dots-median.</p

mRNA analysis of PittGG biofilms.

<p>A. Principal component analysis of microarray gene expression of the PittGG biofilm bacteria (NTHi) exposed to 170 ng/mL ampicillin. The major variations (PC1, PC2, and PC3) are visualized in 3-dimensions. The three PCs together accounted for about 85% of the variations present in the entire data set. A distinguishable grouping difference can be seen between ampicillin treated (in blue) and non-treated (in red) samples. B. Hierarchical cluster of significantly differentially expressed genes in the PittGG biofilm bacteria (NTHi) treated with 170 ng/mL ampicillin. Heatmap visualization of 59 significantly regulated genes by ampicillin treatment. Results from the 3 replicate experiments (R1, R2 and R3) of ampicillin-treated and untreated biofilms are presented. A hierarchical clustering was analysed on the probe sets representing the 59 genes including 8 up and 51 down regulated gene transcripts with filter criteria of at least 1.5 folds change plus P≤0.05 and FDR less than 0.05. Rows: samples; columns: genes. The up-regulated genes relating to carbohydrate metabolism are indicated.</p

PittGG biofilm formation is stimulated by ampicillin in a narrow concentration range.

<p>Crystal violet assay of 1-day PittGG biofilms, formed in the presence of increasing amounts of ampicillin up 300 ng/mL, showed a stimulation of biofilm formation at 170 ng/mL concentration.</p

Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable <i>Haemophilus influenzae</i> by Up-Regulating Carbohydrate Metabolism

<div><p>Non-typeable <i>Haemophilus influenzae</i> (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.</p></div

Biofilm formation is variable between NTHi strains.

<p>Biofilm quantification using crystal violet assay showed the variability in biofilm formation between the NTHi strains in this study. Relative Biofilm Quantity was measured as OD₆₀₀ of crystal violet in DMSO; higher absorbance indicated more biofilm production. The PittAA and PittEE strains showed the least amount of biofilm attachment to the growth surface while PittII showed the most. The other three strains (2019, 9274 and PittGG) formed intermediate amounts of biofilm material (t-test analysis in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099204#pone.0099204.s002" target="_blank">Table S2</a>).</p

NTHi biofilms protect against a lethal dose of cefuroxime.

<p>Biofilms of NTHi strains 2019 and PittGG were formed overnight in the absence of antibiotic (Untreated), or in the presence of 170 ng/mL ampicillin (AMP), 230 ng/mL amoxicillin (AMOX) or 170 ng/mL cefuroxime (CEF). The amounts of antibiotic were chosen for their ability to stimulate biofilm formation in these two NTHi strains. After washing, the biofilms were exposed to 10 µg/mL of cefuroxime for a further 24 hr. The percentages of viable bacteria present in each biofilm are tabulated here. Not shown: planktonic NTHi bacteria are killed in the presence of 10 µg/mL cefuroxime. Although all of the formed biofilms (in the presence or absence of antibiotic) protected against cefuroxime, the amoxicillin-stimulated biofilm was able to protect bacteria from the lethal effects of the cefuroxime. p-values are documented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099204#pone.0099204.s003" target="_blank">Table S3</a>.</p

Significant genes identified by microarray transcriptomic analysis in the biofilm bacteria exposed to 170/mL ampicillin.

<p>Genes that were either up or down regulated (≥1.5-fold plus P≤0.05, T-test and FDR≤0.05) when biofilms were formed in the presence of 170 ng/mL ampicillin are listed. Eight genes were up-regulated and 51 were down-regulated. Of the eight up-regulated genes, 5 were involved in carbohydrate metabolism and were enzymes involved in glycogen processing. The down-regulated genes were involved in a wide range of metabolic processes and 18 genes were unannotated (hypothetical).</p

Ultrastructural visualization of glycogen in PittGG biofilms.

<p>Resin-embedded thin sections of PittGG biofilms formed in the presence or absence of ampicillin (+/− Am) and treated with a glycogen stain or a no stain control (+/− gly). A–D Biofilms with ampicillin, sections stained for glycogen (periodic acid and sodium chlorite). The glycogen stain reacted with an extracellular granular substance (arrows) that was associated with biofilm bacteria. Exposure to antibiotic resulted in a bacterial size increase with some bacteria (A–C). Extracellular areas that were not in close proximity to bacteria cells (asterisk) did not contain the extracellular granular substance (B–D). E. Biofilm with antibiotic but only treated with sodium chlorite did not reveal extracellular granular substance. Large bacterial cells were present. F. Biofilm with no antibiotic, sections stained for glycogen. No extracellular granular substance was detected and bacterial cells had a normal diameter (approx. 500 nm). Some lysed cells were detected. G. Biofilm with no antibiotic, treated with sodium chlorite (no periodic acid). Bacterial cells appear normal with a few dying cells present (arrowhead). Scale bars = 500 nm.</p

Biofilm formation is stimulated by beta-lactam antibiotics.

<p>Crystal violet assays of 1-day NTHi biofilms formed in the presence of amoxicillin or amoxicillin. Strains 2019, 9274 and PittEE reacted to inhibitory concentrations of antibiotic (grey vertical bars, Biomass OD₆₀₀) by producing more crystal violet stainable biofilm (line graph). This stimulatory effect was different for each bacterial strain. PittGG did not react with amoxicillin and produced an ambiguous reaction to ampicillin. PittAA and PittII were not affected by amoxicillin or ampicillin in the concentration ranges studied. Cefuroxime (0–950 ng/mL range) showed a biofilm-stimulatory effect on all the NTHi strains under study. Each strain exhibited biofilm stimulation in different concentrations of antibiotic. Strain 2019 was maximally stimulated at 100 ng/mL cefuroxime, PittGG and PittII at 220 ng/mL, 9274, PittAA at 300 ng/mL and PittEE at 525 ng/mL.</p

Prevention of vaginal and rectal HIV transmission by antiretroviral combinations in humanized mice

<div><p>With more than 7,000 new HIV infections daily worldwide, there is an urgent need for non-vaccine biomedical prevention (nBP) strategies that are safe, effective, and acceptable. Clinical trials have demonstrated that pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) can be effective at preventing HIV infection. In contrast, other trials using the same ARVs failed to show consistent efficacy. Topical (vaginal and rectal) dosing is a promising regimen for HIV PrEP as it leads to low systematic drug exposure. A series of titration studies were carried out in bone marrow/liver/thymus (BLT) mice aimed at determining the adequate drug concentrations applied vaginally or rectally that offer protection against rectal or vaginal HIV challenge. The dose-response relationship of these agents was measured and showed that topical tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) can offer 100% protection against rectal or vaginal HIV challenges. From the challenge data, EC₅₀ values of 4.6 μM for TDF and 0.6 μM for FTC for HIV vaginal administration and 6.1 μM TDF and 0.18 μM for FTC for rectal administration were obtained. These findings suggest that the BLT mouse model is highly suitable for studying the dose-response relationship in single and combination ARV studies of vaginal or rectal HIV exposure. Application of this sensitive HIV infection model to more complex binary and ternary ARV combinations, particularly where agents have different mechanisms of action, should allow selection of optimal ARV combinations to be advanced into pre-clinical and clinical development as nBP products.</p></div