Phenotypic changes in DNS <i>S</i>. <i>aureus</i> strains.

<p>(A) Comparison of CW thickness and septation between DAP-S R6837 and DNS R6838 strains. TEM was used to measure the cell thickness and cell septation. N = total number of bacterial cells counted. (B) Membrane fluidity was calculated spectrofluorometrically and polarization index (<i>p</i>) were determined. Lower <i>p</i> value indicates higher membrane fluidity associated with DAP non-susceptibility. The differences in CW thickness (A) and polarization values (B) between DAP-S and DNS <i>S</i>. <i>aureus</i> strains are not statistically significant. (C) Determination of cell surface charge of the DAP-S and DNS <i>S</i>. <i>aureus</i> strains by cytochrome C binding assay. The data were analyzed by paired t test and P values were determined. *P<0.05.</p

DNS <i>S</i>. <i>aureus</i> strains exhibit differential expression of genes involved in CW synthesis.

<p>Expression profile of (A) <i>vraSR</i> and (B) <i>walKR</i> genes at the indicated times of bacterial growth by qRT-PCR. 16S rRNA was used as an internal control. Results are representative of at least two independent experiments performed. Statistical analysis was carried out using one-way ANOVA and a <i>P</i> value of 0.05 or less was considered significant. ***<i>P</i><0.001.</p

DAP-S or DNS <i>S</i>. <i>aureus</i> strains do not exhibit altered expression of <i>femA</i> and <i>femB</i> genes involved in CW synthesis.

<p>Expression profile of (A) <i>femA</i> and (B) <i>femB</i> genes at the indicated times of bacterial growth by qRT-PCR. 16S rRNA was used as an internal control. Results are representative of at least two independent experiments performed.</p

DNS <i>S</i>. <i>aureus</i> strains exhibit upregulated expression of genes involved in autolysis.

<p>Expression profile of (A) <i>atlA</i> and (B) <i>lytM</i> genes at the indicated times of bacterial growth by qRT-PCR. 16S rRNA was used as an internal control. Results are representative of at least two independent experiments performed. Statistical analysis was carried out using one-way ANOVA and a <i>P</i> value of 0.05 or less was considered significant. **<i>P</i><0.01; ***<i>P</i><0.001.</p

DNS <i>S</i>. <i>aureus</i> strain exhibits alterations in expression of genes involved in maintaining CM charge.

<p>Expression profile of <i>mprF</i> gene at the indicated times of bacterial growth by qRT-PCR. 16S rRNA was used as an internal control. Results are representative of at least two independent experiments. Statistical analysis was carried out using one-way ANOVA and a <i>P</i> value of 0.05 or less was considered significant. ***<i>P</i><0.001.</p

Mutational differences between DNS and DAP-S isogenic strain pairs identified by whole genome sequencing.

<p>Mutational differences between DNS and DAP-S isogenic strain pairs identified by whole genome sequencing.</p

Growth characteristics and DAP susceptibility of DAP-S and DNS <i>S</i>. <i>aureus</i> strains.

<p>Bacterial growth of the indicated isogenic pairs of DAP-S and DNS <i>S</i>. <i>aureus</i> strains was monitored by measuring OD₆₀₀ at 1-hour interval (A) and quantifying bacteria by plating on sheep blood agar plates. The colonies were counted and the results are expressed as Log₁₀ CFU/mL (B). Minimum Inhibitory Concentrations (MICs) of DAP for isogenic DAP-S and DNS isogenic pairs as determined by microdilution method and E-test. The DNS <i>S</i>. <i>aureus</i> strains are represented in bold letters (C). The data shown are representative of two independent experiments conducted with similar results.</p

DNS <i>S</i>. <i>aureus</i> strains exhibit alterations in expression of genes involved in maintaining CM charge.

<p>Expression profile of (A) <i>dltA</i>; (B) <i>dltB;</i> (C) <i>dltC</i> and (D) <i>dltD</i> genes at the indicated times of bacterial growth by qRT-PCR. 16S rRNA was used as an internal control. Results are representative of at least two independent experiments performed. Statistical analysis was carried out using one-way ANOVA and a <i>P</i> value of 0.05 or less was considered significant. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

List of primer sequences used for transcriptional analysis.

<p>List of primer sequences used for transcriptional analysis.</p

<i>In Vivo</i> Dissolution and Systemic Absorption of Immediate Release Ibuprofen in Human Gastrointestinal Tract under Fed and Fasted Conditions

<i>In vivo</i> drug dissolution in the gastrointestinal (GI) tract is largely unmeasured. The purpose of this clinical study was to evaluate the <i>in vivo</i> drug dissolution and systemic absorption of the BCS class IIa drug ibuprofen under fed and fasted conditions by direct sampling of stomach and small intestinal luminal content. Expanding current knowledge of drug dissolution <i>in vivo</i> will help to establish physiologically relevant <i>in vitro</i> models predictive of drug dissolution. A multilumen GI catheter was orally inserted into the GI tract of healthy human subjects. Subjects received a single oral dose of ibuprofen (800 mg tablet) with 250 mL of water under fasting and fed conditions. The GI catheter facilitated collection of GI fluid from the stomach, duodenum, and jejunum. Ibuprofen concentration in GI fluid supernatant and plasma was determined by LC–MS/MS. A total of 23 subjects completed the study, with 11 subjects returning for an additional study visit (a total of 34 completed study visits). The subjects were primarily white (61%) and male (65%) with an average age of 30 years. The subjects had a median [min, max] weight of 79 [52, 123] kg and body mass index of 25.7 [19.4, 37.7] kg/m². Ibuprofen plasma levels were higher under fasted conditions and remained detectable for 28 h under both conditions. The AUC_0–24 and <i>C</i>_max were lower in fed subjects vs fasted subjects, and <i>T</i>_max was delayed in fed subjects vs fasted subjects. Ibuprofen was detected immediately after ingestion in the stomach under fasting and fed conditions until 7 h after dosing. Higher levels of ibuprofen were detected in the small intestine soon after dosing in fasted subjects compared to fed. In contrast to plasma drug concentration, overall gastric concentrations remained higher under fed conditions due to increased gastric pH vs fasting condition. The gastric pH increased to near neutrality after feedingbefore decreasing to acidic levels after 7 h. Induction of the fed state reduced systemic levels but increased gastric levels of ibuprofen, which suggest that slow gastric emptying and transit dominate the effect for plasma drug concentration. The finding of high levels of ibuprofen in stomach and small intestine 7 h post dosing was unexpected. Future work is needed to better understand the role of various GI parameters, such as motility and gastric emptying, on systemic ibuprofen levels in order to improve <i>in vitro</i> predictive models