The bioprinting of high-concentrated collagen bioinks is a promising technology for tissue engineering and regenerative medicine. Collagen is a widely used biomaterial for bioprinting because of its natural abundance in the extracellular matrix of many tissues and its biocompatibility. High-concentrated collagen hydrogels have shown great potential in tissue engineering due to their favorable mechanical and structural properties. However, achieving high cell proliferation rates within these hydrogels remains a challenge. In static cultivation, the volume of the culture medium is changed once every few days. Thus, perfect perfusion is not achieved due to the relative increase in metabolic concentration and no medium flow. Therefore, in our work, we developed a culture system in which printed collagen bioinks (collagen concentration in hydrogels of 20 and 30 mg/mL with a final concentration of 10 and 15 mg/mL in bioink) where samples flow freely in the culture medium, thus enhancing the elimination of nutrients and metabolites of cells. Cell viability, morphology, and metabolic activity (MTT tests) were analyzed on collagen hydrogels with a collagen concentration of 20 and 30 mg/mL in static culture groups without medium exchange and with active medium perfusion; the influence of pure growth culture medium and smooth muscle cells differentiation medium was next investigated. Collagen isolated from porcine skins was used; every batch was titrated to optimize the pH of the resulting collagen to minimize the difference in production batches and, therefore, the results. Active medium perfusion significantly improved cell viability and activity in the high-concentrated gel, which, to date, is the most limiting factor for using these hydrogels. In addition, based on SEM images and geometry analysis, the cells remodel collagen material to their extracellular matrix.
- Publikační typ
- časopisecké články MeSH
It is believed that 3D bioprinting will greatly help the field of tissue engineering and regenerative medicine, as live patient cells are incorporated into the material, which directly creates a 3D structure. Thus, this method has potential in many types of human body tissues. Collagen provides an advantage, as it is the most common extracellular matrix present in all kinds of tissues and is, therefore, very natural for cells and the organism. Hydrogels with highly concentrated collagen make it possible to create 3D structures without additional additives to crosslink the polymer, which could negatively affect cell proliferation and viability. This study established a new method for preparing highly concentrated collagen bioinks, which does not negatively affect cell proliferation and viability. The method is based on two successive neutralizations of the prepared hydrogel using the bicarbonate buffering mechanisms of the 2× enhanced culture medium and pH adjustment by adding NaOH. Collagen hydrogel was used in concentrations of 20 and 30 mg/mL dissolved in acetic acid with a concentration of 0.05 and 0.1 wt.%. The bioink preparation process is automated, including colorimetric pH detection and adjustment. The new method was validated using bioprinting and subsequent cultivation of collagen hydrogels with incorporated stromal cells. After 96 h of cultivation, cell proliferation and viability were not statistically significantly reduced.
- Publikační typ
- časopisecké články MeSH
Background: Antimicrobial submicrometer particles are being studied as promising interventions against a wide range of skin conditions, such as fungal or bacterial infections. Aims: To submicronize chloroxine, the crystalline compound 5,7-dichloro-8-hydroxyquinoline, by nanoprecipitation and characterize the resulting assemblies. Methods: The chloroxine particles were stabilized by a nonionic surfactant and were studied by a broth microdilution assay against 20 medically important bacteria and fungi. The intervention was studied using a murine model of skin irritation. Results & conclusion: Chloroxine nanoparticles with a diameter of 600-800 nm exhibit good tolerability in terms of skin irritation in vivo and good antimicrobial activity. Thus, the fabricated formulation shows great promise for interventions for both cutaneous infection control and prophylaxis.
Clostridioides (Clostridium) difficile infection (CDI) is the most common causative pathogen of health care-associated gastrointestinal infections; however, due to the overlap of clinical symptoms with those of other causes of acute gastroenteritis, the selection of the most appropriate laboratory test is difficult. From April to October 2018, 640 stool samples requested for CDI testing were examined using the mariPOC CDI and Gastro test (ArcDia), which allows the detection of C. difficile glutamate dehydrogenase (GDH) and toxin A/B, norovirus genogroups GI and GII.4, rotavirus, adenovirus, and Campylobacter spp. In parallel, the C. Diff Quik Chek Complete test (Alere) was used as a routine diagnostic assay, and C. difficile toxigenic culture was used as a reference method. The sensitivity of the mariPOC CDI and Gastro test was comparable to that of C. Diff Quik Chek Complete for the detection of GDH (96.40% [95% confidence interval {CI}, 91.81% to 98.82%] versus 95.68% [95% CI, 90.84 to 98.40%]; P = 1.00) and was higher for the detection of toxin A/B (66.67% [95% CI, 57.36 to 75.11%] versus 55.56% [95% CI, 46.08 to 64.74%]; P = 0.00). The specificity of the mariPOC CDI and Gastro test was lower than that of C. Diff Quik Chek Complete for GDH detection (95.21% [95% CI, 92.96% to 96.91%] versus 97.60% [95% CI, 95.85% to 98.76%]; P = 0.04) and comparable to that of C. Diff Quik Chek Complete for toxin A/B detection (99.24 [95% CI, 98.05% to 99.79%] versus 99.81% [95% CI, 98.94% to 100.0%]; P = 0.37). In 29 cases (4.53%), other causative agents of diarrhea were detected (Campylobacter spp. [n = 17], rotavirus [n = 7], and norovirus genogroup GII.4 [n = 5]).
- MeSH
- bakteriální proteiny genetika MeSH
- bakteriální toxiny genetika MeSH
- Clostridioides difficile * enzymologie genetika imunologie MeSH
- diagnostické testy rutinní MeSH
- dítě MeSH
- dospělí MeSH
- enterotoxiny genetika MeSH
- feces mikrobiologie MeSH
- glutamátdehydrogenasa MeSH
- imunoanalýza * metody normy MeSH
- klostridiové infekce diagnóza imunologie mikrobiologie MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- management nemoci MeSH
- mladiství MeSH
- mladý dospělý MeSH
- předškolní dítě MeSH
- pseudomembranózní enterokolitida diagnóza imunologie mikrobiologie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of alveolar macrophages. These cells avidly take up nanoparticles, even without the use of specific targeting ligands, making the use of nanotherapeutics ideal for the treatment of such infections. Methoxy poly(ethylene oxide)- block-poly(ε-caprolactone) nanoparticles of several different polymer blocks' molecular weights and sizes (20-110 nm) were developed and critically compared as carriers for rifampicin, a cornerstone in tuberculosis therapy. The polymeric nanoparticles' uptake, consequent organelle targeting and intracellular degradation were shown to be highly dependent on the nanoparticles' physicochemical properties (the cell uptake half-lives 2.4-21 min, the degradation half-lives 51.6 min-ca. 20 h after the internalization). We show that the nanoparticles are efficiently taken up by macrophages and are able to effectively neutralize the persisting bacilli. Finally, we demonstrate, using a zebrafish model of tuberculosis, that the nanoparticles are well tolerated, have a curative effect, and are significantly more efficient compared to a free form of rifampicin. Hence, these findings demonstrate that this system shows great promise, both in vitro and in vivo, for the treatment of tuberculosis.
- MeSH
- dánio pruhované MeSH
- lidé MeSH
- makrofágy * metabolismus mikrobiologie MeSH
- modely nemocí na zvířatech MeSH
- Mycobacterium tuberculosis růst a vývoj MeSH
- myši MeSH
- nanočástice * chemie terapeutické užití MeSH
- nosiče léků * chemie farmakokinetika farmakologie MeSH
- RAW 264.7 buňky MeSH
- rifampin * chemie farmakokinetika farmakologie MeSH
- tuberkulóza farmakoterapie metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bacteroides pyogenes can cause infections in humans. We describe a case of bloodstream infection caused by Bacteroides denticanum that probably originated from a dog bite. MALDI-TOF MS misidentified this new species as B. pyogenes. Subsequent analysis using the 16S rRNA sequencing approach identified the species as B. denticanum.
- MeSH
- Bacteroides chemie klasifikace genetika izolace a purifikace MeSH
- bakteriemie diagnóza mikrobiologie MeSH
- infekce bakteriemi rodu Bacteroides diagnóza mikrobiologie MeSH
- lidé MeSH
- psi MeSH
- senioři MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- techniky typizace bakterií MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Clostridium difficile is a major nosocomial pathogen in humans with an increasing incidence in the community. The "one-health" approach of research is needed to investigate possible reservoirs of C. difficile and route of its transmission. The objective of this study is to investigate the occurrence of C. difficile in pigs in the Czech Republic with characterisation of the isolates to determine their genetic relatedness to C. difficile isolates from European and Asian pigs. A total of 198 pig faeces samples from 23 farms were investigated and of those 57 samples (55 piglets, 2 sows) from 11 farms were confirmed as C. difficile positive. The majority of C. difficile isolates belonged to the sequence type 11 and clade 5. The predominant ribotypes were 078 (n = 23), 078-variant (n = 5), 033 (n = 10) followed by RTs 150 (n = 7), 011 (n = 5), 045 (n = 4), 126, 014, 002 (n = 1, each). All isolates were susceptible to metronidazole, vancomycin and tetracycline. Isolates of RTs 150 and 078-variant were moxifloxacin resistant (MIC≥32 mg/L) and carried the amino acid substitution Thr82Ile in the GyrA. A multi-locus variable number tandem-repeats analysis (MLVA) revealed a clonal relatedness of isolates within individual farms and in C. difficile RT078 isolates between two Czech farms. Czech C. difficile RT078 isolates clustered with German C. difficile RT078 isolates and Czech C. difficile 078-variant isolates clustered with C. difficile RT078 isolates from Japan and Taiwan. This study found an emergence of C. difficile RT078 in Czech piglets that was related genetically to C. difficile RT078 isolates from Germany, Japan and Taiwan.
- MeSH
- antibakteriální látky farmakologie MeSH
- Clostridioides difficile klasifikace genetika izolace a purifikace MeSH
- DNA gyráza genetika MeSH
- klostridiové infekce mikrobiologie přenos veterinární MeSH
- metronidazol farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- moxifloxacin farmakologie MeSH
- multilokusová sekvenční typizace MeSH
- prasata MeSH
- ribotypizace MeSH
- substituce aminokyselin genetika MeSH
- tetracyklin farmakologie MeSH
- vankomycin farmakologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Japonsko MeSH
- Německo MeSH
- Taiwan MeSH
PURPOSE: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. METHODS: Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. RESULTS: Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. CONCLUSION: Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.
- MeSH
- ADP-ribosatransferasy genetika MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- bakteriální proteiny genetika MeSH
- bakteriální toxiny analýza genetika MeSH
- Clostridioides difficile klasifikace účinky léků genetika izolace a purifikace MeSH
- DNA bakterií analýza MeSH
- DNA gyráza genetika MeSH
- dospělí MeSH
- enterotoxiny genetika MeSH
- fluorochinolony farmakologie MeSH
- genotyp MeSH
- klostridiové infekce epidemiologie mikrobiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- minisatelitní repetice genetika MeSH
- mladý dospělý MeSH
- molekulární epidemiologie MeSH
- moxifloxacin MeSH
- multilokusová sekvenční typizace metody MeSH
- polymerázová řetězová reakce metody MeSH
- represorové proteiny genetika MeSH
- ribotypizace metody MeSH
- sekvence nukleotidů MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- techniky typizace bakterií * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Finsko MeSH
AIM: To perform a retrospective analysis of the high occurrence of Clostridium difficile infection in the surgical department of a Czech tertiary care hospital and to identify weaknesses in C. difficile infection (CDI) prevention and control policies. METHODS: Clinical and epidemiological data on eleven CDI cases were collected. C. difficile isolates were characterized by capillary electrophoresis ribotyping, multilocus variable tandem repeat analysis (MLVA), gyrA gene fragment sequencing, and erm(B) fragment PCR amplification. Antibiotic susceptibility to metronidazole, vancomycin, ciprofloxacin, moxifloxacin, and clindamycin was tested. FINDINGS: Eleven CDI cases were caused by C. difficile PCR ribotype 001 strains. These strains revealed two different MLVA profiles with 11 tandem repeat differences. All isolates were susceptible to metronidazole and vancomycin and resistant to ciprofloxacin (MIC ≥32 mg/L), moxifloxacin (MIC ≥32 mg/L), and clindamycin (MIC ≥256 mg/L). All isolates revealed amino acid substitution Thr82Ile, in the GyrA and were erm(B) negative. CONCLUSION: Two fluoroquinolone and clindamycin-resistant C. difficile PCR ribotype 001 strain clusters occurred at one of the surgical departments of a tertiary care hospital. Ineffective decontamination with suboptimal concentration and time of exposure of sporicidal disinfectants may have resulted in C. difficile transmission.
- MeSH
- antibakteriální látky farmakologie MeSH
- centra terciární péče MeSH
- ciprofloxacin farmakologie MeSH
- Clostridioides difficile klasifikace účinky léků genetika izolace a purifikace MeSH
- DNA bakterií genetika metabolismus MeSH
- DNA gyráza genetika metabolismus MeSH
- elektroforéza kapilární MeSH
- exprese genu MeSH
- fluorochinolony farmakologie MeSH
- klindamycin farmakologie MeSH
- klostridiové infekce farmakoterapie mikrobiologie MeSH
- lidé MeSH
- methyltransferasy genetika metabolismus MeSH
- metronidazol farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- minisatelitní repetice MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- multilokusová sekvenční typizace MeSH
- retrospektivní studie MeSH
- ribotypizace MeSH
- senioři MeSH
- substituce aminokyselin MeSH
- vankomycin farmakologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
We have developed a biodegradable, biocompatible system for the delivery of the antituberculotic antibiotic rifampicin with a built-in drug release and nanoparticle degradation fluorescence sensor. Polymer nanoparticles based on poly(ethylene oxide) monomethyl ether-block-poly(ε-caprolactone) were noncovalently loaded with rifampicin, a combination that, to best of our knowledge, was not previously described in the literature, which showed significant benefits. The nanoparticles contain a Förster resonance energy transfer (FRET) system that allows real-time assessment of drug release not only in vitro, but also in living macrophages where the mycobacteria typically reside as hard-to-kill intracellular parasites. The fluorophore also enables in situ monitoring of the enzymatic nanoparticle degradation in the macrophages. We show that the nanoparticles are efficiently taken up by macrophages, where they are very quickly associated with the lysosomal compartment. After drug release, the nanoparticles in the cmacrophages are enzymatically degraded, with half-life 88±11 min.
- MeSH
- antituberkulotika aplikace a dávkování MeSH
- biokompatibilní materiály chemie MeSH
- makrofágy účinky léků metabolismus MeSH
- myši MeSH
- nanočástice chemie MeSH
- polyestery chemie MeSH
- polyethylenglykoly chemie MeSH
- RAW 264.7 buňky MeSH
- rezonanční přenos fluorescenční energie MeSH
- rifampin aplikace a dávkování MeSH
- systémy cílené aplikace léků * MeSH
- uvolňování léčiv * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
