Identifying and mathematically modeling the time-course of extracellular metabolic markers associated with resistance to ceftolozane/tazobactam in Pseudomonas aeruginosa.

Extracellular bacterial metabolites have potential as markers of bacterial growth and resistance emergence but have not been evaluated in dynamic in vitro studies. We investigated the dynamic metabolomic footprint of a multidrug-resistant hypermutable Pseudomonas aeruginosa isolate exposed to ceftolozane/tazobactam as continuous infusion (4.5 g/day, 9 g/day) in a hollow-fiber infection model over 7-9 days in biological replicates (n = 5). Bacterial samples were collected at 0, 7, 23, 47, 71, 95, 143, 167, 191, and 215 h, the supernatant quenched, and extracellular metabolites extracted. Metabolites were analyzed via untargeted metabolomics, including hierarchical clustering and correlation with quantified total and resistant bacterial populations. The time-courses of five (of 1,921 detected) metabolites from enriched pathways were mathematically modeled. Absorbed L-arginine and secreted L-ornithine were highly correlated with the total bacterial population (r -0.79 and 0.82, respectively, P<0.0001). Ribose-5-phosphate, sedoheptulose-7-phosphate, and trehalose-6-phosphate correlated with the resistant subpopulation (0.64, 0.64, and 0.67, respectively, P<0.0001) and were likely secreted due to resistant growth overcoming oxidative and osmotic stress induced by ceftolozane/tazobactam. Using pharmacokinetic/pharmacodynamic-based transduction models, these metabolites were successfully modeled based on the total or resistant bacterial populations. The models well described the abundance of each metabolite across the differing time-course profiles of biological replicates, based on bacterial killing and, importantly, resistant regrowth. These proof-of-concept studies suggest that further exploration is warranted to determine the generalizability of these findings. The metabolites modeled here are not exclusive to bacteria. Future studies may use this approach to identify bacteria-specific metabolites correlating with resistance, which would ultimately be extremely useful for clinical translation.

Ceftolozane-Tazobactam against Pseudomonas aeruginosa Cystic Fibrosis Clinical Isolates in the Hollow-Fiber Infection Model: Challenges Imposed by Hypermutability and Heteroresistance.

Pseudomonas aeruginosa remains a challenge in chronic respiratory infections in cystic fibrosis (CF). Ceftolozane-tazobactam has not yet been evaluated against multidrug-resistant hypermutable P. aeruginosa isolates in the hollow-fiber infection model (HFIM). Isolates CW41, CW35, and CW44 (ceftolozane-tazobactam MICs of 4, 4, and 2 mg/L, respectively) from adults with CF were exposed to simulated representative epithelial lining fluid pharmacokinetics of ceftolozane-tazobactam in the HFIM. Regimens were continuous infusion (CI; 4.5 g/day to 9 g/day, all isolates) and 1-h infusions (1.5 g every 8 hours and 3 g every 8 hours, CW41). Whole-genome sequencing and mechanism-based modeling were performed for CW41. CW41 (in four of five biological replicates) and CW44 harbored preexisting resistant subpopulations; CW35 did not. For replicates 1 to 4 of CW41 and CW44, 9 g/day CI decreased bacterial counts to <3 log10 CFU/mL for 24 to 48 h, followed by regrowth and resistance amplification. Replicate 5 of CW41 had no preexisting subpopulations and was suppressed below ~3 log10 CFU/mL for 120 h by 9 g/day CI, followed by resistant regrowth. Both CI regimens reduced CW35 bacterial counts to <1 log10 CFU/mL by 120 h without regrowth. These results corresponded with the presence or absence of preexisting resistant subpopulations and resistance-associated mutations at baseline. Mutations in ampC, algO, and mexY were identified following CW41 exposure to ceftolozane-tazobactam at 167 to 215 h. Mechanism-based modeling well described total and resistant bacterial counts. The findings highlight the impact of heteroresistance and baseline mutations on the effect of ceftolozane-tazobactam and limitations of MIC to predict bacterial outcomes. The resistance amplification in two of three isolates supports current guidelines that ceftolozane-tazobactam should be utilized together with another antibiotic against P. aeruginosa in CF.

Determination of the Viability of Lactic Acid Bacteria by Dynamic In Vitro Gastrointestinal Model in Household and Industrial-Type Kefir Samples.

This study presents results based on differences in the antioxidant activity and lactic acid bacteria counts in different parts of the digestive tract following simulated gastrointestinal digestion of kefir samples. Statistically significant differences were observed in Lactobacillus counts in different kefir types including industrial (IK), starter culture (SCK), and kefir grains (KG). These differences were observed between the initial and second min in the mouth region (T = 3.968; p < 0.05); and between the initial, 60th, and 120th min in the stomach region (R = 11.146; p < 0.05). Additionally, a statistically significant difference was noted in the initial Lactobacillus levels among the IK, SCK, and KG in the stomach region (H = 7.205; p < 0.05). Also, significant differences were identified between the Lactococcus counts of IK across 0, 60, and 120 min in the stomach region (R = 10.236; p < 0.05). Notably, a statistically significant difference was noted in the Lactococcus levels in the KG between the initial and second min in the mouth region (T = 3.101; p < 0.05) and between 0, 60, and 120 min in the stomach region (R = 25.771; p < 0.001). These findings highlight the differences between the physicochemical characteristics of different kefir types. A decrease in lactic acid bacteria counts in kefir samples was observed throughout the dynamic in vitro gastrointestinal tract to reveal the significance of the digestive process when determining probiotic product capacity.

Changes in gut microbiota in predigested Hibiscus sabdariffa L calyces and Agave (Agave tequilana weber) fructans assessed in a dynamic in vitro model (TIM-2) of the human colon.

Hibiscus sabdariffa (Hb) calyces are a source of dietary fiber (DF) and phenolic compounds. Agave fructans (AF) and oligofructans (OF) are considered as soluble DF. The aim of the study was to investigate changes in gut microbiota upon feeding predigested Hb, AF, OF or Mix (Hb/AF) to a dynamic, validated in vitro model of the human colon (TIM-2), using sequencing of the V3-V4 regions of the 16S rRNA gene. A pooled human fecal microbiota was used. Production of short-chain fatty acids (SCFAs), branched-chain fatty acids (BSCFAs) and ammonia was also assessed. Samples were taken after 0, 24, 48 and 72 h. Principal component (PC) analysis of fermentation metabolites and relative abundance of genera was carried out, and extracted factors were based on eigenvalues >1.0 and explained >60% of variance. Fermentation of samples resulted in different SCFAS concentrations. The highest butyric acid production was on AF and OF, while the molar ratio of SCFAS on Hb was 63:18:18 for acetic, propionic and butyric acid, respectively. BSCFAS were also produced upon feeding the studied substrates, but in much lower concentrations. About 45 bacteria genera were identified and 10 of these were the most abundant changing during the fermentation time, amongst which a high relative abundance in Bifidobacterium, Bacteroides and Catenibacterium, that changed during the fermentation time depending of substrate. Hb feeding after 48 h led to Bifidobacterium being the most abundant genus. Two PCs were identified: after 24 h of fermentation PC1 was highly influenced by Bifidobacterium and Prevotella, which was related with Hb and SIEM feeding. Evaluation of the changes in metabolites and gut microbiota composition during colonic fermentation in a validated in vitro model provides a complete and reliable view of the potential prebiotic effect of different dietary fibers.

A new dynamic model for in vitro evaluation of intravascular devices.

INTRODUCTION:: A dynamic model to evaluate thrombus formation on intravascular catheters in vitro is presented. The model enables fluid infusion, variation in the catheter orientation, and variable flow conditions. It was applied on a catheter used to shunt cerebrospinal fluid to a vein, a dural venous sinus, for the treatment of hydrocephalus. METHODS:: Fresh human blood-filled circuits were circulated in a non-occlusive roller pump. A catheter infused either with cerebrospinal fluid, Ringer's lactate, or no fluid (control) was inserted through each circuit's wall. Sixteen circuits (six cerebrospinal fluid, six Ringer's lactate, four control) ran for 60 min. Qualitative assessment was performed by measuring viscoelastic properties of blood at the start and end of the experiment; quantitative evaluation of clot formation by scanning electron microscope. RESULTS:: Average blood velocity was 79 mm/s, with a pressure wave between 5 and 15 mm Hg. At the experiment's end, the infused fluid represented 5.88% of the blood/infusion volume in the circuit. The control circuits showed no statistical difference between the start and end for viscoelastic testing, whereas both Ringer's lactate and cerebrospinal fluid enhanced coagulation, most pronounced for the latter. Most thrombus material was observed on catheters in the cerebrospinal fluid group. Clot formation was less pronounced on the surface of the catheter facing the blood flow. DISCUSSION:: A dynamic model for intravascular catheter testing mimics better clinical conditions when evaluating blood-material interaction. Catheter position, blood flow around the catheter, and infusion fluid all have a potential impact on the hemocompatibility of a given catheter.

Behaviour of citrus pectin during its gastrointestinal digestion and fermentation in a dynamic simulator (simgi®).

The behaviour of citrus pectin during digestion and its potential prebiotic properties were examined using a Dynamic Gastrointestinal Simulator (simgi®) model for the human gut, which simulates processes in the stomach, small intestine, ascending, transverse and descending colon. A remarkable non-digestibility of pectin in the upper gastrointestinal tract was observed by HPLC-ELSD analysis, where ∼88% of citrus pectin remained intact during its transit through the stomach and small intestine. Fermentation of pectin stimulated the growth of beneficial bacteria such as Bifidobacterium spp, Bacteroides spp and Faecalobacterium prausnitzii. High increases of short-chain fatty acids (SCFA) were observed, especially in acetate and butyrate concentration due to direct fermentation of pectin or by cross-feeding interaction between bacteria. This is the first study on the digestibility and fermentation of pectin carried out in a complex dynamic gastrointestinal simulator, being of special relevance the results obtained for F. prausnitzii.