Active Media Perfusion in Bioprinted Highly Concentrated Collagen Bioink Enhances the Viability of Cell Culture and Substrate Remodeling.

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.

A New Method for the Production of High-Concentration Collagen Bioinks with Semiautonomic Preparation.

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.

Acute Pneumonia Caused by Clinically Isolated Legionella pneumophila Sg 1, ST 62: Host Responses and Pathologies in Mice.

Legionnaires' disease is a severe form of lung infection caused by bacteria belonging to the genus Legionella. The disease severity depends on both host immunity and L. pneumophila virulence. The objective of this study was to describe the pathological spectrum of acute pneumonia caused by a virulent clinical isolate of L. pneumophila serogroup 1, sequence type 62. In A/JOlaHsd mice, we compared two infectious doses, namely, 104 and 106 CFU, and their impact on the mouse status, bacterial clearance, lung pathology, and blood count parameters was studied. Acute pneumonia resembling Legionnaires' disease has been described in detail.

Nanocrystalline chloroxine possesses broad-spectrum antimicrobial activities and excellent skin tolerability in mice.

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.

Evaluation of a Gastrointestinal Pathogen Panel Immunoassay in Stool Testing of Patients with Suspected Clostridioides (Clostridium) difficile Infection.

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]).

Rifampicin Nanoformulation Enhances Treatment of Tuberculosis in Zebrafish.

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.

Bloodstream infection caused by Bacteroides denticanum, a close relative of Bacteroides pyogenes, misidentified by MALDI TOF- mass spectrometry.

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.

The emergence of Clostridium difficile PCR ribotype 078 in piglets in the Czech Republic clusters with Clostridium difficile PCR ribotype 078 isolates from Germany, Japan and Taiwan.

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.

The recognition and characterisation of Finnish Clostridium difficile isolates resembling PCR-ribotype 027.

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.

Analysis of proteomes released from in vitro cultured eight Clostridium difficile PCR ribotypes revealed specific expression in PCR ribotypes 027 and 176 confirming their genetic relatedness and clinical importance at the proteomic level.

BACKGROUND: Clostridium difficile is the causative agent of C. difficile infection (CDI) that could be manifested by diarrhea, pseudomembranous colitis or life-threatening toxic megacolon. The spread of certain strains represents a significant economic burden for health-care. The epidemic successful strains are also associated with severe clinical features of CDI. Therefore, a proteomic study has been conducted that comprises proteomes released from in vitro cultured panel of eight different PCR ribotypes (RTs) and employs the combination of shotgun proteomics and label-free quantification (LFQ) approach. RESULTS: The comparative semi-quantitative analyses enabled investigation of a total of 662 proteins. Both hierarchical clustering and principal component analysis (PCA) created eight distinctive groups. From these quantifiable proteins, 27 were significantly increased in functional annotations. Among them, several known factors connected with virulence were identified, such as toxin A, B, binary toxin, flagellar proteins, and proteins associated with Pro-Pro endopeptidase (PPEP-1) functional complex. Comparative analysis of protein expression showed a higher expression or unique expression of proteins linked to pathogenicity or iron metabolism in RTs 027 and 176 supporting their genetic relatedness and clinical importance at the proteomic level. Moreover, the absence of putative nitroreductase and the abundance of the Abc-type fe3+ transport system protein were observed as biomarkers for the RTs possessing binary toxin genes (027, 176 and 078). Higher expression of selected flagellar proteins clearly distinguished RTs 027, 176, 005 and 012, confirming the pathogenic role of the assembly in CDI. Finally, the histidine synthesis pathway regulating protein complex HisG/HisZ was observed only in isolates possessing the genes for toxin A and B. CONCLUSIONS: This study showed the applicability of the LFQ approach and provided the first semi-quantitative insight into the proteomes released from in vitro cultured panel of eight RTs. The observed differences pointed to a new direction for studies focused on the elucidation of the mechanisms underlining the CDI nature.

System with embedded drug release and nanoparticle degradation sensor showing efficient rifampicin delivery into macrophages.

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.

Two Clusters of Fluoroquinolone and Clindamycin-Resistant Clostridium difficile PCR Ribotype 001 Strain Recognized by Capillary Electrophoresis Ribotyping and Multilocus Variable Tandem Repeat Analysis.

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.

Clostridium difficile PCR ribotypes 001 and 176 - the common denominator of C. difficile infection epidemiology in the Czech Republic, 2014.

In 2014, 18 hospitals in the Czech Republic participated in a survey of the incidence of Clostridium difficile infections (CDI) in the country. The mean CDI incidence was 6.1 (standard deviation (SD):7.2) cases per 10,000 patient bed-days and 37.8 cases (SD: 41.4) per 10,000 admissions. The mean CDI testing frequency was 39.5 tests (SD: 25.4) per 10,000 patient bed-days and 255.8 tests (SD: 164.0) per 10,000 admissions. A total of 774 C. difficile isolates were investigated, of which 225 (29%) belonged to PCR ribotype 176, and 184 isolates (24%) belonged to PCR ribotype 001. Multilocus variable-number tandem repeat analysis (MLVA) revealed 27 clonal complexes formed by 84% (190/225) of PCR ribotype 176 isolates, and 14 clonal complexes formed by 77% (141/184) of PCR ribotype 001 isolates. Clonal clusters of PCR ribotypes 176 and 001 were observed in 11 and 7 hospitals, respectively. Our data demonstrate the spread of two C. difficile PCR ribotypes within 18 hospitals in the Czech Republic, stressing the importance of standardising CDI testing protocols and implementing mandatory CDI surveillance in the country.

Clinical features and characteristics of Clostridium difficile PCR-ribotype 176 infection: results from a 1-year university hospital internal ward study.

BACKGROUND: Clostridium difficile infection (CDI) is a major cause of antibiotic-associated diarrhoea. Given an increasing CDI incidence and global spread of epidemic ribotypes, a 1-year study was performed to analyse the molecular characteristics of C. difficile isolates and associated clinical outcomes from patients diagnosed with CDI in the Internal Medicine department at University Hospital Motol, Prague from February 2013 to February 2014. RESULTS: A total of 85 unformed stool samples were analysed and CDI was laboratory confirmed in 30 patients (6.8 CDI cases per 10,000 patient bed days and 50.6 CDI cases per 10,000 admissions). The CDI recurrence rate within 3 months of treatment discontinuation was 13.3% (4/30). Mortality within 3 months after first CDI episode was 26.7% (8/30), with CDI the cause of death in two cases. 51.9% of C. difficile isolates belonged to PCR-ribotype 176. MLVA of ribotype 176 isolates revealed two clonal complexes formed by 10/14 isolates. ATLAS scores and Horn's index were higher in patients with ribotype 176 infections than with non-ribotype 176 infections. CONCLUSION: This study highlights the clinical relevance of C. difficile PCR-ribotype 176 and its capacity to spread within a healthcare facility.

Antibiotic profiling of Clostridium difficile ribotype 176--A multidrug resistant relative to C. difficile ribotype 027.

Antibiotic profiling of twenty Czech Clostridium difficile PCR-ribotype 176 isolates revealed a high level of resistance to erythromycin, ciprofloxacin and moxifloxacin (n = 20) and to rifampicin (n = 13). Accumulation of resistance mechanisms to multiple antibiotics highlight that PCR-ribotype 176 belong to problematic epidemic strains.

Clostridium difficile ribotype 078 cultured from post-surgical non-healing wound in a patient carrying ribotype 014 in the intestinal tract.

Extra-intestinal infections caused by Clostridium difficile are rare. The risk of extra-intestinal infections associated with C. difficile may be particularly relevant in environments contaminated with C. difficile spores. This paper describes the case of a non-diarrheic patient colonized with C. difficile ribotype 014 in the intestinal tract who developed a post-surgical wound infection by C. difficile ribotype 078. The infection responded to metronidazole administered first intravenously and then orally. This case indicates that C. difficile may not only be related to diarrheic diseases, but also to infections of non-healing wounds, especially in situations when C. difficile is the only isolated pathogen.

A case of imported Clostridium difficile PCR-ribotype 027 infection within the Czech Republic which has a high prevalence of C. difficile ribotype 176.

The first case of Clostridium difficile RT027 infection in the Czech Republic (CZ) was identified. The patient had been hospitalised in Germany prior to moving to CZ. Multiple-Locus Variable number tandem repeat Analysis revealed a genetic relatedness between the patient's isolate and RT027 isolate collected in the German hospital.

C. difficile ribotype 027 or 176?

Clostridium difficile is a major nosocomial pathogen of present times. The analysis of 624 C. difficile strains from 11 hospitals in the Czech Republic in 2013 revealed that 40% of isolates belonged to ribotype 176. These results suggest that the incidence of CDI (C. difficile infection) in the Czech Republic has increased probably in connection with C. difficile ribotype 176. The molecular systems Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA) diagnoses toxigenic strains and supports C. difficile ribotype 027 determination based on three specific target places in the toxigenic C. difficile genome. Twenty-nine strains cultivated from stool specimens were evaluated by the Xpert systems as presumed C. difficile PCR ribotype 027 were confirmed as a C. difficile ribotype 176 based on ribotyping. A further 120 C. difficile strains of ribotype 176 were examined for the presence of genes tcdB, cdtB and deletion in position 117 in the tcdC gene. Our experience shows that due to the correspondence of the target places, C. difficile ribotype 176 may be interpreted as ribotype 027 by Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA). Further molecular analysis as ribotyping based on capillary electrophoresis is needed to differentiate between C. difficile ribotypes 027 and 176 for appropriate epidemiological situation control on local and national levels.

Pneumococcal urinary antigen positivity in healthy colonized children: is it age dependent?

BACKGROUND: Pneumococcal urinary antigen test is a valuable tool for diagnosing pneumococcal pneumonia and meningitis in adults. Its use in children is generally not accepted because of nonspecificity at this age. It is frequently positive in asymptomatic nasopharyngeal carriers. The aim of our study was to assess the age limit from which the test is no longer positive in asymptomatic healthy carriers. METHODS: A total of 197 children aged 36-83 months attending 9 day care centers in Prague were enrolled during February and March 2010. Nasopharyngeal swab specimens were collected from each participant and selectively cultivated. The presence of pneumococcal antigen in urine was detected by BinaxNOW® S. pneumoniae kit. RESULTS: Streptococcus pneumoniae was cultivated in 53.3 % of healthy children with the highest colonization rate (59.3 %) in children aged 48-59 months. The most frequently colonizing serotypes were: 19F, 23F, 3, 19A, 6B and 4. The presence of pneumococcal antigen in urine decreased with age from 39.0 % in 36-47 months to 17.9 % in 72-83 months old (p = 0.031). The antigen positivity was serotype-dependent and more frequent in nonvaccinated children. CONCLUSION: We demonstrated age-dependent linear decrease of pneumococcal antigen excretion into urine in healthy children. The positivity rate of the test in children aged 72-83 months was similar to that referred in healthy adults, irrespective of colonization. To confirm this age limit for use of this test in diagnostics of pneumococcal diseases, further study in school-age children is justified.