Optimering av cellodlingsmedia genom kombinering av designade stamlösningar : med experimentell design och multivariat dataanalys

Syftet med projektet är att ta fram en metod för optimering av cellodlingsmedia som kan minska den tid och de resurser som dagens metoder kräver. Fem stamlösningar med adekvata komponenter designas och kombineras med olika flödeshastigheter till ett medium. Kombinationerna testas med experimentell design och analyseras med multivariat dataanalys för att få fram optimerade cellmedia. Odlingsmetoden som de fem stamlösningarna anpassas till är fed-batch, ett system där en av lösningarna är en baslösning och de resterande fyra designas till feedlösningar. De senare tillsätts i olika hastigheter till baslösningen anpassat efter cellernas näringsbehov. Strategin som har lagts upp för att designa stamlösningarna bygger på förhållandet mellan komponenternas förbrukningshastighet i de olika tillväxtfaserna. Under odlingen befinner sig cellerna i olika tillväxtfaser, i den exponentiella fasen ökar celldensiteten och i den stationära fasen sker större delen av produktionen av till exempel specifika antikroppar. Komponenterna i baslösningen förbrukas inte, eller i så liten mängd att de inte behöver tillsättas under odlingen, till skillnad från komponenterna i feedlösningarna som behöver tillsättas för att inte utarmas. I den exponentiella celltillväxtfasen, är behovet av de olika feedlösningarna flera gånger större än i den efterföljande stationära fasen. När önskad celldensitet är uppnådd så minskas flödeshastigheten från feedlösningarna och cellerna går in i stationärfas för att ge maximal produktion av önskad produkt. Projektet är uppbyggt av fyra delar: En litteraturstudie av nuvarande metoder för mediumutveckling, en beskrivning av framtagna stamlösningar, optimering av flödeshastigheten med experimentell design och multivariat dataanalys samt ett laborativt arbetsflöde av medias tillverkningsprocess

A Novel Microfluidic Assay for Rapid Phenotypic Antibiotic Susceptibility Testing of Bacteria Detected in Clinical Blood Cultures

Background Appropriate antibiotic therapy is critical in the management of severe sepsis and septic shock to reduce mortality, morbidity and health costs. New methods for rapid antibiotic susceptibility testing are needed because of increasing resistance rates to standard treatment. Aims The purpose of this study was to evaluate the performance of a novel microfluidic method and the potential to directly apply this method on positive blood cultures. Methods Minimum inhibitory concentrations (MICs) of ciprofloxacin, ceftazidime, tigecycline and/or vancomycin for Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus were determined using a linear antibiotic concentration gradient in a microfluidic assay. Bacterial growth along the antibiotic gradient was monitored using automated time-lapse photomicrography and growth inhibition was quantified by measuring greyscale intensity changes in the images. In addition to pure culture MICs, vancomycin MICs were determined for S. aureus from spiked and clinical blood cultures following a short centrifugation step. The MICs were compared with those obtained with the Etest and for S. aureus and vancomycin also with macrodilution. Results The MICs obtained with the microfluidic assay showed good agreement internally as well as with the Etest and macrodilution assays, although some minor differences were noted between the methods. The time to possible readout was within the range of 2 to 5 h. Conclusions The examined microfluidic assay has the potential to provide rapid and accurate MICs using samples from positive clinical blood cultures and will now be tested using other bacterial species and antibiotics

A Multiplex Fluidic Chip for Rapid Phenotypic Antibiotic Susceptibility Testing

Many patients with severe infections receive inappropriate empirical treatment, and rapid detection of bacterial antibiotic susceptibility can improve clinical outcome and reduce mortality. To this end, we have developed a multiplex fluidic chip for rapid phenotypic antibiotic susceptibility testing of bacteria. A total of 21 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were acquired from the EUCAST Development Laboratory and tested against amikacin, ceftazidime, and meropenem (Gram-negative bacteria) or gentamicin, ofloxacin, and tetracycline (Gram-positive bacteria). The bacterial samples were mixed with agarose and loaded in an array of growth chambers in the chip where bacterial microcolony growth was monitored over time using automated image analysis. MIC values were automatically obtained by tracking the growth rates of individual microcolonies in different regions of antibiotic gradients. Stable MIC values were obtained within 2 to 4 h, and the results showed categorical agreement with reference MIC values as determined by broth microdilution in 86% of the cases

Combination of polymyxin B and minocycline against multidrug-resistant Klebsiella pneumoniae : interaction quantified by pharmacokinetic/pharmacodynamic modelling from in vitro data

Lack of effective treatment for multidrug-resistant Klebsiella pneumoniae (MDR-Kp) necessitates finding and optimising combination therapies of old antibiotics. The aims of this study were to quantify the combined effect of polymyxin B and minocycline by building an in silico semi-mechanistic pharmacokinetic/pharmacodynamic (PKPD) model and to predict bacterial kinetics when exposed to the drugs alone and in combination at clinically achievable unbound drug concentration-time profiles. A clinical K. pneumoniae strain resistant to polymyxin B [minimum inhibitory concentration (MIC) = 16 mg/L] and minocycline (MIC = 16 mg/L) was selected for extensive in vitro static time-kill experiments. The strain was exposed to concentrations of 0.0625-48 ? MIC, with seven samples taken per experiment for viable counts during 0-28 h. These observations allowed the development of the PKPD model. The final PKPD model included drug-induced adaptive resistance for both drugs. Both the minocycline-induced bacterial killing and resistance onset rate constants were increased when polymyxin B was co-administered, whereas polymyxin B parameters were unaffected. Predictions at clinically used dosages from the developed PKPD model showed no or limited antibacterial effect with monotherapy, whilst combination therapy kept bacteria below the starting inoculum for 20 h at high dosages [polymyxin B 2.5 mg/kg + 1.5 mg/kg every 12 h (q12h); minocycline 400 mg + 200 mg q12h, loading + maintenance doses]. This study suggests that polymyxin B and minocycline in combination may be of clinical benefit in the treatment of infections by MDR-Kp and for isolates that are non-susceptible to either drug alone. (C) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/

Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments

Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa. We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (<= 10(6) CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal beta-lactams, ciprofloxacin, and amikacin. Genes encoding beta-lactamases (e.g., bla(PAO) and bla(OXA-50)) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations

The CellDirector 3D assay.

<p>a) Schematic overview of the CellDirector 3D assay setup for AST determination and b) close-up of the microfluidic growth chamber depicting details of the concentration gradient and MIC readout.</p

MIC values in mg/L obtained with the Etest and CellDirector 3D at bacterial inocula of 10⁵ CFU/mL.

<p>MIC values in mg/L obtained with the Etest and CellDirector 3D at bacterial inocula of 10⁵ CFU/mL.</p