Evaluation of decontamination process of heart valve and artery tissues in European Homograft Bank (EHB): a retrospective study of 1,055 cases

To evaluate the efficiency of decontamination practice in European Homograft Bank (EHB), the data of the cardiovascular tissues received during recent 2?years were retrospectively analysed in this study. After initial assessment, the tissues were incubated in a 3-antibiotics?? cocktail at 4°C for 20?C48?h. The states of contamination were evaluated before and after incubation with the focus on the differences in donor type, tissue type, germ type and incubation time. Amongst 1,055 eligible tissues, 77.2% were hearts and 22.8% were arteries. 82.2% of the tissues were retrieved from the multi-organ donors (MOD), 15.4% from the recipients of heart transplantation (RHT) and 2.4% from the non-heart beating donors (NHBD). The initial contamination rate was 27.4% with a significantly higher incidence in arteries. The RHT tissues had the lowest contamination rate comparing to that of MOD and NHBD. Staphylococcus species was the major source of contamination. After antibiotic incubation, 76.8% of the contaminated tissues were disinfected, which was significantly higher for the hearts than the arteries. The RHT tissues had the highest decontamination rate than that of MOD and NHBD tissues. Propionibacterium acnes was detected in 48.1% of the remaining contaminated cases. The average incubation time of the Propionibacterium-positive tissues was significantly shorter than that of decontaminated tissues. In conclusion, the current decontamination protocol of EHB is sufficient for most of the initially contaminated bacteria, whereas it is inadequate for Propionibacterium acnes. This may be related to the slow-growing nature of this bacterium and thereby the relative shorter antibiotic incubation time.     

The effect of bacterial contamination on the growth and gas evolution ofIn vitro cultured apricot shoots

Summary Shoots of “San Castrese” and “Portici” apricots (Prunus armeniaca L.) free of cultivable bacteria, shoots of the same origin exhibiting bacterial contamination after repeated subcultures, and contaminated shoots treated with cefotaxime were compared for gas exchange, proliferation rate, and fresh and dry weight. Cultures of San Castrese contaminated byBacillus circulans andSphingomonas paucimobilis, and of Portici contaminated withStaphylococcus hominis andMicrococcus kristinae, including those treated with cefotaxime, showed comparable shoot weights and lower proliferation rates than healthy cultures. Bacteria, even if not visible until the end of subculture, markedly influenced the gaseous composition of the jar headspace. Healthy cultures clearly showed photosynthetic activity at 60 μM·m⁻²·s⁻¹ photosynthetically active radiation; in contrast, oxygen quickly decreased and carbon dioxide increased in contaminated cultures, including those treated with cefotaxime, in which bacteria became visible in the culture medium only after repeated subcultures.     

Residual Antibiotics in Decontaminated Human Cardiovascular Tissues Intended for Transplantation and Risk of Falsely Negative Microbiological Analyses

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.     

Cryopreservation of human brain tissue allowing timely production of viable adult human brain cells for autologous transplantation

BACKGROUND: Autologous transplantation is an attractive approach to treat some neurological diseases. A major obstacle is the capacity to produce cells for transplantation at the appropriate time. We describe a cryopreservation procedure for adult human brain tissue allowing the generation of cells in vitro. METHODS: Neurological resections were dissected to separate white and grey matter. Fractions were frozen in a specific cryopreservation medium containing a selected serum and stored in liquid nitrogen. Tissue was thawed, cells were mechanically dissociated, expanded in culture and characterized by immunochemistry. RESULTS: Adult human brain tissue cryopreserved for up to two years was successfully used to generate brain cells that could be maintained in culture for up to 100 days. Cells expressed a variety of neuroectodermal markers including GFAP, S100beta, and neurofilament. CONCLUSION: A successful procedure for cryopreservation of adult human brain tissue has been established that might facilitate future autologous transplantation strategies.     

Fate of some types of bacteria in hydrocortisone acetate ointment

The survival of Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538 and Bacillus pumilus ATCC 14884 in hydrocortisone acetate ointment was investigated. When the ointment was contaminated and packed in metal tubes all bacteria except B. pumilus were killed in a matter of days, but when the contaminated ointment was packed in jars, all bacteria survived provided atmospheric oxygen was allowed at intervals to pass into the containers.     

Inhibition of Vancomycin and High-Level Aminoglycoside-Resistant Enterococci Strains and Listeria monocytogenes by Bacteriocin-Like Substance Produced by Enterococcus faecium E86

Three hundred and thirty nine lactic bacteria strains isolated from food samples were screened for antimicrobial activity. Only one strain isolated from meat pie and identified as Enterococcus faecium produced a bacteriocin-like inhibitory substance (BLIS) showing activity against Enterococcus, Leuconostoc, Lactobacillus, Listeria, Corynebacterium and Staphylococcus aureus. The BLIS produced was resistant to acid and alkali treatment and 121oC for 15 min. The addition of BLIS in BHI contaminated with Listeria monocytogenes decreased the contamination in 4.8 log cycles in 24 h. The inhibition of listeria was also obtained in milk. Forty multiresistant enterococci strains were inhibited in the well-diffusion test. Two vancomycin resistant strains tested in liquid with BLIS were also inhibited. The BLIS producer showed no pathogenicity marker.     

A generally applicable cryopreservation method for nitrite-oxidizing bacteria

Nitrite-oxidizing bacteria are key members of the global nitrogen cycle but their study is hampered by their limited availability in culture, mostly due to laborious cultivation procedures and the lack of stable preservation methods. In this study, it was demonstrated that long-term cryopreservation of nitrite-oxidizing bacteria assigned to the genera Nitrobacter, Nitrospina, Nitrococcus, Nitrotoga and Nitrospira was possible using a simple and rapid protocol. Their survival was tested with different cryoprotecting agents, DMSO and Hatefi, and in various carbon-rich preservation media, ten-fold diluted TSB, and ten-fold diluted TSB supplemented with 1% trehalose, and 1% sucrose. Optimal preservation conditions were strain-dependent and marine strains appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide with or without ten-fold diluted trypticase soy broth as a preservation medium allowed successful preservation of all tested strains.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results: A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10³ CFU ml^?1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.     

Ovarian tissue cryopreservation by stepped vitrification and monitored by X-ray computed tomography

Ovarian tissue cryopreservation is, in most cases, the only fertility preservation option available for female patients soon to undergo gonadotoxic treatment. To date, cryopreservation of ovarian tissue has been carried out by both traditional slow freezing method and vitrification, but even with the best techniques, there is still a considerable loss of follicle viability. In this report, we investigated a stepped cryopreservation procedure which combines features of slow cooling and vitrification (hereafter called stepped vitrification). Bovine ovarian tissue was used as a tissue model. Stepwise increments of the Me₂SO concentration coupled with stepwise drops-in temperature in a device specifically designed for this purpose and X-ray computed tomography were combined to investigate loading times at each step, by monitoring the attenuation of the radiation proportional to Me₂SO permeation. Viability analysis was performed in warmed tissues by immunohistochemistry. Although further viability tests should be conducted after transplantation, preliminary results are very promising. Four protocols were explored. Two of them showed a poor permeation of the vitrification solution (P1 and P2). The other two (P3 and P4), with higher permeation, were studied in deeper detail. Out of these two protocols, P4, with a longer permeation time at −40 °C, showed the same histological integrity after warming as fresh controls.     

Preservation of female genetic resources of common carp through oogonial stem cell manipulation

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (−1 °C/min) and short-term storage (−80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me₂SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at −80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.     

Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, that is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (−196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5–1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes. The technique was highly efficient in cryopreserving meristem shoot tips of different pineapple genotypes, and was also less laborious than techniques previously reported. This is a first report on application of the droplet-vitrification technique to diverse genotypes of cultivated and wild pineapples and the first report on histological changes occurring in cryopreserved Ananas tissue.

     

Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100

A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.     

Development and evaluation of a real‐time PCR assay targeting chromosomal DNA of Erwinia amylovora

Aims:  To develop and evaluate a new and reliable real‐time PCR detection protocol on chromosomal DNA of the contagious plant pathogenic bacterium Erwinia amylovora, the causal agent of fire blight. Methods and Results:  A Taqman^® minor‐groove‐binder real‐time PCR assay targeting a hypothetical protein coding gene of Erw. amylovora has been developed. Colony PCR of 113 bacterial strains from different taxa was performed to prove specificity. Serial decimal dilutions of Erw. amylovora showed a consistent detection sensitivity of 2 bacterial units per μl. All strains of Erw. amylovora could be identified, and there were no cross‐reactions with matrices or other bacteria also testing naturally contaminated samples. Conclusions:  Rapid, reliable and sensitive detection of Erw. amylovora is important to avoid the spread of the disease within orchards, and the distribution by contaminated plant material or vectors carrying the pathogen. The selected conserved target gene allows relative quantitative detection of Erw. amylovora from different sources and host taxa. The newly developed protocol also enables the detection of recently found natural strains that lack the species‐specific plasmid pEA29, which was so far widely used as target for detection and identification of this plant pathogen by PCR. Significance and Impact of the Study:  This study demonstrates that the newly developed and evaluated real‐time assay can specifically be used for identifying all known strains of the EU quarantine plant pathogen Erw. amylovora. Low concentrations of the bacteria can be detected and relatively quantified using a different target area than other real‐time PCRs designed so far.     

The effects of β-tricalcium phosphate 3D scaffold in-situ cryopreservation on the migration rate and osteogenic ability of mesenchymal stem cells

To readily supply seed cells for tissue engineering and ensure their constant availability for experiments, it is imperative to establish an in-situ cryopreservation method for cell storage. We investigated the effects of a β-tricalcium phosphate (β-TCP) 3D scaffold in-situ cryopreservation method on the migration rate and osteogenic ability of mesenchymal stem cells (MSCs). Compared to using a 2D plate culture and trypsinized cryopreservation, MSCs on β-TCP 3D scaffolds demonstrated a higher amplification rate, and the harvest and survival rates (HSR) increased from 55.9 to 81.3% when the 3D in-situ cryopreservation method was applied. The cell migration rate and alkaline phosphatase (ALP) activity were unaffected after in-situ cryopreservation, and unexpectedly, the Specific ALP activity of migrating cells was higher than that of non-cryopreserved cells, suggesting that the cell-scaffold combination could be cryopreserved using the present protocol without loss of proliferative or osteogenic potential. These findings highlight a methodology for 3D scaffold in-situ cryopreservation and passage for MSC production in bone tissue engineering, and present the possibility of designing a perfusion cells/scaffold factory for scale-up production.     

In Vivo Magnetic Enrichment,Photoacoustic Diagnosis,and Photothermal Purging of Infected Blood Using Multifunctional Gold and Magnetic Nanoparticles

Bacterial infections are a primary cause of morbidity and mortality worldwide. Bacteremia is a particular concern owing to the possibility of septic shock and the development of metastatic infections. Treatment of bacteremia is increasingly compromised by the emergence of antibiotic resistant strains, creating an urgent need for alternative therapy. Here, we introduce a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of Staphylococcus aureus in tissue and blood. We show that this method could be applicable for label-free diagnosis and treatment of in the bloodstream using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of circulating bacteria cells (CBCs), two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of S. aureus surface-associated markers such as protein A and lipoprotein. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of CBCs with threshold sensitivity as low as 0.5 CBCs/mL, in vivo magnetic enrichment of CBCs, PT eradication of CBCs, and real-time monitoring of therapeutic efficacy by CBC counting. Our PA-PT nano-theranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biological tool to gain insights on dissemination pathways of CBCs, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans, especially using bypass schematic.     

Identification of pathogenic bacteria in blood cultures: Comparison between conventional and PCR methods

Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae were found to be the most prevalent bacteremia-causing bacteria in a survey in a medical center. A PCR method for identification of these five most common pathogens in blood cultures was developed. A unique sequence was chosen for each pathogen and used for primer design. Sixty-one blood samples (from hospitalized patients) in which bacterial growth was detected were processed in parallel by conventional microbiological methods and by the PCR method. The results obtained by PCR were identical to those obtained by conventional methods in 93.4% of the cases. PCR failed to identify bacteria which were found conventionally in only 6.6% of the cases (mostly bacteria not included in the PCR cassette). Another group of eighty-eight blood samples from patients were processed immediately upon their arrival at the laboratory by taking aliquots for the PCR method. The blood sample bottles were processed in parallel by conventional methods. In 78.4% of the cases the results of both methods were identical. In 12.5% of the cases, PCR afforded identification of bacteria but conventional methods showed no bacteria in the sample. On the other hand, PCR afforded 9.1% negative results while conventional methods identified bacteria not included in the PCR cassette. It is concluded that the molecular method appears to be a specific and precise method for identifying pathogenic bacteria in blood samples.     

Abundance and Diversity of <Emphasis Type="Italic">n</Emphasis>-Alkane-Degrading Bacteria in a Forest Soil Co-Contaminated with Hydrocarbons and Metals: A Molecular Study on <Emphasis Type="Italic">alkB</Emphasis> Homologous Genes

Unraveling functional genes related to biodegradation of organic compounds has profoundly improved our understanding of biological remediation processes, yet the ecology of such genes is only poorly understood. We used a culture-independent approach to assess the abundance and diversity of bacteria catalyzing the degradation of n-alkanes with a chain length between C₅ and C₁₆ at a forest site co-contaminated with mineral oil hydrocarbons and metals for nearly 60 years. The alkB gene coding for a rubredoxin-dependent alkane monooxygenase enzyme involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism was used as biomarker. Within the area of study, four different zones were evaluated: one highly contaminated, two intermediately contaminated, and a noncontaminated zone. Contaminant concentrations, hydrocarbon profiles, and soil microbial respiration and biomass were studied. Abundance of n-alkane-degrading bacteria was quantified via real-time PCR of alkB, whereas genetic diversity was examined using molecular fingerprints (T-RFLP) and clone libraries. Along the contamination plume, hydrocarbon profiles and increased respiration rates suggested on-going natural attenuation at the site. Gene copy numbers of alkB were similar in contaminated and control areas. However, T-RFLP-based fingerprints suggested lower diversity and evenness of the n-alkane-degrading bacterial community in the highly contaminated zone compared to the other areas; both diversity and evenness were negatively correlated with metal and hydrocarbon concentrations. Phylogenetic analysis of alkB denoted a shift of the hydrocarbon-degrading bacterial community from Gram-positive bacteria in the control zone (most similar to Mycobacterium and Nocardia types) to Gram-negative genotypes in the contaminated zones (Acinetobacter and alkB sequences with little similarity to those of known bacteria). Our results underscore a qualitative rather than a quantitative response of hydrocarbon-degrading bacteria to the contamination at the molecular level.     

Sterilization of sealed PVDF pouches containing decellularized scaffold by electrical stimulation

A terminal sterilization process for tissue engineering products, such as allografts and biomaterials is necessary to ensure complete removal of pathogenic microorganisms such as the bacteria, fungi, and viruses. However, it can be difficult to sterilize allografts and artificial tissue models packaged in wet conditions without deformation. In this study, we investigated the sterilization effects of electrical stimulation (ES) and assessed its suitability by evaluating sterility assurance levels in pouches at a constant current. Stability of polyvinylidene fluoride pouches was determined by a sterility test performed after exposure to five microorganisms (Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans) for 5 days; the sterility test was also performed with decellularized human dermal tissues inoculated with the five microorganisms. Sterilization using ES inactivated microorganisms both inside and outside of sealed pouches and caused no damage to the packaged tissue. Our results support the development of a novel system that involves ES sterilization for packaging of implantable biomaterials and human derived materials.