In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates

ABSTRACT Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Sonication as a tool for disrupting biofilms and recovering microorganisms in bladder catheters / Sonicação como uma ferramenta para romper biofilmes e recuperar microrganismos em cateteres vesicais

Abstract Introduction: Urinary catheter-related infection is commonly associated with bacterial biofilm. The impact of anaerobes is unknown, but their detection in the biofilm on this device has not been previously reported. This study aimed to evaluate the capability to recovery strict, facultative, and aerobic microorganisms in patients using bladder catheters from ICUs using conventional culture, sonication, urinary analysis, and mass spectrometry. Methods: Parallel, sonicated bladder catheters from 29 critically ill patients were compared with their routine urine culture. Identification was performed using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. Results: The positivity rate in urine (n = 2, 3.4%) was lower than that in sonicated catheters (n = 7, 13.8%). Conclusion: Bladder catheter sonication showed more positive culture results than urine samples for anaerobic and aerobic microorganisms. The role of anaerobes in urinary tract infection and catheter biofilm is discussed.

Resumo Introdução: A infecção relacionada ao cateter urinário é comumente associada ao biofilme bacteriano. O impacto dos anaeróbios é desconhecido, mas sua detecção no biofilme deste dispositivo não foi relatada anteriormente. Este estudo teve como objetivo avaliar a capacidade de recuperar microrganismos estritos, facultativos e aeróbios em pacientes que utilizam cateteres vesicais de UTIs utilizando cultura convencional, sonicação, análise urinária e espectrometria de massa. Métodos: Paralelamente, foram comparados cateteres vesicais sonicados de 29 pacientes gravemente enfermos com sua urocultura de rotina. A identificação foi realizada utilizando dessorção/ionização a laser assistida por matriz com espectrometria de massa por tempo de voo. Resultados: A taxa de positividade na urina (n = 2; 3,4%) foi inferior à dos cateteres sonicados (n = 7; 13,8%). Conclusão: A sonicação do cateter vesical apresentou resultados de cultura mais positivos do que as amostras de urina para microrganismos anaeróbios e aeróbios. É discutido o papel dos anaeróbios na infecção do trato urinário e no biofilme do cateter.

Sonication as a tool for disrupting biofilms and recovering microorganisms in bladder catheters.

INTRODUCTION: Urinary catheter-related infection is commonly associated with bacterial biofilm. The impact of anaerobes is unknown, but their detection in the biofilm on this device has not been previously reported. This study aimed to evaluate the capability to recovery strict, facultative, and aerobic microorganisms in patients using bladder catheters from ICUs using conventional culture, sonication, urinary analysis, and mass spectrometry. METHODS: Parallel, sonicated bladder catheters from 29 critically ill patients were compared with their routine urine culture. Identification was performed using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. RESULTS: The positivity rate in urine (n = 2, 3.4%) was lower than that in sonicated catheters (n = 7, 13.8%). CONCLUSION: Bladder catheter sonication showed more positive culture results than urine samples for anaerobic and aerobic microorganisms. The role of anaerobes in urinary tract infection and catheter biofilm is discussed.

Antimicrobial Treatment of Staphylococcus aureus Biofilms.

Staphylococcus aureus is a microorganism frequently associated with implant-related infections, owing to its ability to produce biofilms. These infections are difficult to treat because antimicrobials must cross the biofilm to effectively inhibit bacterial growth. Although some antibiotics can penetrate the biofilm and reduce the bacterial load, it is important to understand that the results of routine sensitivity tests are not always valid for interpreting the activity of different drugs. In this review, a broad discussion on the genes involved in biofilm formation, quorum sensing, and antimicrobial activity in monotherapy and combination therapy is presented that should benefit researchers engaged in optimizing the treatment of infections associated with S. aureus biofilms.

Comparison of amphotericin B lipid complex, deoxycholate amphotericin B, fluconazole, and anidulafungin activity against Candida albicans biofilm isolated from breakthrough candidemia.

INTRODUCTION: Biofilm formation causes virulence and resistance in Candida albicans. However, little is known about breakthrough candidemia isolates. We evaluated the antifungal activity of fluconazole, anidulafungin, deoxycholate amphotericin B (dAMB), and amphotericin B lipid complex (ABLC) against biofilms of C. albicans isolated from patients with breakthrough candidemia. METHODS: The present study used strains of C. albicans isolated from breakthrough and non-breakthrough candidemia patients (control group). The susceptibility of planktonic cells to amphotericin B, anidulafungin, and fluconazole was determined by broth microdilution. Antifungal activity in sessile cells was evaluated using the minimum biofilm eradication concentration (MBEC), metabolic activity was estimated by reducing MTT, and biomass was estimated using crystal violet retention. RESULTS: The planktonic strains were susceptible to amphotericin B, anidulafungin, and fluconazole, with minimum inhibitory concentrations of 1, ≤0.03, and 2mg/L, respectively. However, fluconazole and anidulafungin did not exert an antifungal effect on biofilms. Additionally, dAMB and ABCL reduced the metabolic activity and biomass. However, eradication was only achieved using 16mg/L dAMB. C. albicans isolates of breakthrough candidemia exhibited strong biofilm production, and the in vitro activity of available therapeutic options was poor. CONCLUSION: In the present study, only dAMB and ABCL exhibited antibiofilm effects against sessile breakthrough candidemia isolates.

Different concentrations of vancomycin with gentamicin loaded PMMA to inhibit biofilm formation of Staphylococcus aureus and their implications.

BACKGROUND: This study aimed to evaluate different concentrations of vancomycin and/or gentamicin loaded polymethylmethacrylate (PMMA) against biofilm formation of Staphylococcus aureus. METHODS: Biofilm production of S. aureus in PMMA loaded with different concentrations of vancomycin and gentamicin were evaluated by quantitative analysis of biofilm cells, scanning electronic microscopy, viability assay, Fourier transform infrared spectroscopy, and checkerboard. Statistical analysis was performed by Mann Whitney test. The difference in colony forming units per mL was significant when p < 0.05. RESULTS: All loaded PMMA presented a reduction in the number of colony forming units per mL (p < 0.05). The gentamicin-loaded PMMA could inhibits the grown of sessile cells (p < 0.05), where the group vancomycin 4 g + gentamicin 500 mg presented a better result. The Fourier transform infrared spectra showed no significant differences, and checkerboard of vancomycin and gentamicin showed synergism. CONCLUSION: Effects against adherence and bacterial development in PMMA loaded with antibiotics were mainly seen in the group vancomycin 4 g + gentamicin 500 mg, and synergic effect can be applied in antibiotic-loaded cement.

A carbapenem-resistant Acinetobacter baumannii outbreak associated with a polymyxin shortage during the COVID pandemic: an in vitro and biofilm analysis of synergy between meropenem, gentamicin and sulbactam.

BACKGROUND: During the COVID-19 pandemic, the burden of nosocomial infections caused by MDR pathogens has caused a shortage of polymyxins. Thus, we evaluated the in vitro synergism and antibiofilm activity of antimicrobial combinations and propose a test kit for synergism against carbapenem-resistant Acinetobacter baumannii (CRAB). METHODS: Fifty-six CRAB isolates were tested for synergy between meropenem, gentamicin and ampicillin/sulbactam. MICs were determined by broth microdilution. Synergism was tested using chequerboard analysis, followed by a time-kill curve. Additionally, minimum biofilm eradication concentration was determined and the antibiofilm activity of the combinations was evaluated by MTT assay and biomass reduction. A test kit was developed for routine laboratory testing to detect synergism. RESULTS: All CRAB isolates were resistant to gentamicin and ampicillin/sulbactam. Chequerboard synergism occurred against 75% of the isolates. Meropenem + ampicillin/sulbactam was the most frequent combination with synergism (69%), followed by ampicillin/sulbactam + gentamicin (64%) and meropenem + gentamicin (51%). All combinations presented only bacteriostatic activity and no bactericidal or antibiofilm effects. The routine laboratory test showed 100% accuracy compared with other in vitro assays. CONCLUSIONS: Our study demonstrates the potential role of antibiotic combinations against planktonic bacteria. In vitro synergism is possible and can be an alternative treatment for patients with CRAB infection during a polymyxin shortage.

Pathogenesis of the Pseudomonas aeruginosa Biofilm: A Review.

Pseudomonas aeruginosa is associated with several human infections, mainly related to healthcare services. In the hospital, it is associated with resistance to several antibiotics, which poses a great challenge to therapy. However, one of the biggest challenges in treating P. aeruginosa infections is that related to biofilms. The complex structure of the P. aeruginosa biofilm contributes an additional factor to the pathogenicity of this microorganism, leading to therapeutic failure, in addition to escape from the immune system, and generating chronic infections that are difficult to eradicate. In this review, we address several molecular aspects of the pathogenicity of P. aeruginosa biofilms.

Collagen matrices are preserved following decellularization of a bovine bone scaffold.

The decellularization of bovine bone has emerged as a strategy for the repair, replacement, and regeneration of bone defects. To evaluate the effects of a new protocol of bone decellularization and its impact on the structure and collagen scaffold. Cancellous bone from bovine femur was dissected in fragments and decellularized based on protocol of multiple steps. The residual protein levels, histological, morphometric, and scanning electronic microscopy analyses were carried out to evaluate the effects of decellularization and the impact on the structure and collagen scaffold. A cytotoxicity assay was performed. Residual protein analysis showed an important removal of bone marrow components and cell debris from the bone. Sections revealed that collagen fibers presented integrity and absence of cells in the decellularized bone. Sirius Red-stained sections of collagen fiber collagen matrix were maintained after decellularization. Scanning electron microscopy revealed that the main bone structure, despite being irregular, was maintained in both groups, with no significant visual differences between the surface characteristics according to the groups. Decellularized bovine bone demonstrated a degree of toxicity of 3, indicating moderate reactivity. The present data demonstrate that the main bone structure was maintained. Additionally, the chemical and physical treatments were able to remove cellular debris, and extracellular matrix architecture and collagen were preserved. However, the tissue showed moderate toxicity.

Comparative study of decellularization techniques to obtain natural extracellular matrix scaffolds of human peripheral-nerve allografts.

BACKGROUND: We hypothesize that adding sonication cycles to the process of decellularization of cadaveric human peripheral nerves will increase the removal of cell debris and myelin sheath, increasing their utility as allografts. METHODS: Our aim of this study was to develop a decellularization protocol that allows the removal of cells and myelin sheath without detrimental effects on nerve architecture. Segments of ulnar and median nerves from human donors, isolated both before and after cardiac arrest, were subjected to two methods of decellularization: two-detergent-based (M1) and the same method with sonication added (M2). We evaluated the histology of unprocessed and decellularized nerves (n = 24 per group) for general morphology, presence of cell nuclei, nuclear remnants, collagen fibers, and myelin. We performed immunohistochemistry to verify the removal of Schwann cells associated with histomorphometry. We used scanning electron microscopy (EM) to evaluate the ultrastructure of both native and decellularized nerves. The efficacy of decellularization was assessed by analysis of genomic DNA. RESULTS: Histology confirmed that both decellularization protocols were adequate and maintained natural nerve architecture. Scanning EM showed that 3D ultrastructural architecture also was maintained. Histomorphometric parameters showed a more complete removal of the myelin with the M2 protocol than with M1 (p = 0.009). Fiber diameter and density were not modified by decellularization methods. CONCLUSIONS: Sonication can be a complementary method to decellularization of peripheral nerve allografts with sonication increasing the effectiveness of detergent-based protocols for the removal of unwanted cellular components from peripheral nerve allografts.

Evaluation of Staphylococcus aureus and Candida albicans biofilms adherence to PEEK and titanium-alloy prosthetic spine devices.

BACKGROUND: Titanium and polyether-ether-ketone (PEEK) interbody cages are commonly used for spine fusion. Few data are known about bacterial and yeast biofilms formation in these implants. The aim of this study was to compare Staphylococcus aureus and Candida albicans biofilm formation in the surface of two different interbody devices used routinely in spine surgery. METHODS: Six bodies of proof specimens of PEEK and titanium alloy were used for microbiological tests, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. Experimental biofilm was produced with Staphylococcus aureus and Candida albicans, followed by quantitative analysis of planktonic cells and sessile cells. The comparison between the medians of biofilm quantification between the two models was performed using the Mann-Whitney test and considered the statistical difference for a p < 0.05. RESULTS: In the S. aureus model, in both planktonic and sessile cell counts, titanium-alloy samples showed lower values for colony forming units per milliliter (UFC/mL) (p < 0.05). The evaluation through the optic density of planktonic and sessile cells showed lower values in the titanium-alloy samples, however, only statistically significant in planktonic cell count (p < 0.05). The count of planktonic yeast cells in PEEK was similar to titanium-alloy samples, while the count of sessile yeast cells in titanium alloy was lower when compared to PEEK (p < 0.05). CONCLUSION: Titanium-alloy models were associated with less staphylococcal and Candida biofilm formation when compared with PEEK.

Disinfection protocol for human musculoskeletal allografts in tissue banking using hydrogen peroxide 30.

Musculoskeletal allografts are used in reconstructive procedures, however, the risk of contamination with potential pathogens is possible, and safe transplantation requires multiple processing considerations. Hydrogen peroxide (H2O2) has commonly been used in bone washing because it can remove donor cells and eliminate antigens, pathogens, or cytotoxic agents from the matrix. The aim of this study was to evaluate the quantitative activity of H2O2 in a model of bone contamination with a high bacterial load to define the bioburden reduction. Twelve bone disc models were artificially contaminated with Staphylococcus aureus. The bones were treated with a washing process composed by antibiotics, 30% hydrogen peroxide, and 70% alcohol. Tryptic Soy Agar plates were directly inoculated with 100µL of each step of the washing process and colonies were counted in CFU/mL. Scanning electron microscopy was used for bone structural analysis before and after the washing process. After antibiotics, there was a drop of less than 1 log for cancellous bone and almost 1 log for cortical bone. However, after H2O2, there as a drop of 3 logs for cortical (p = 0.007), and 2 logs for cancellous bone (p = 0.063). The use of alcohol did not change the bioburden following H2O2 in cancellous and cortical bone. Despite the important drop of bacterial load, H2O2 was not enough to completely eradicate bacterial with this model of bioburden. H2O2 is useful in decontamination, but antibiotics have little activity, and alcohol is useless. The process is useful in decontamination up to 3 logs of bioburden.

Direct detection of microorganisms in sonicated orthopedic devices after in vitro biofilm production and different processing conditions.

BACKGROUND: The gold standard for microbial detection in prosthetic joint infections is the multiple culture of the peri-prosthetic tissue. The fluid cultures after sonication can improve the recovery of the microorganisms. OBJECTIVE: The aim of this study was to evaluate the sonication technique with a plastic bag and the effect of refrigeration on microorganism detection with conventional culturing, MALDI-TOF MS and qPCR assay on an orthopedic screw model. METHODS: We produced biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans on orthopedic screws, which were stored under different conditions and temperatures before sonication. After sonication, the mass spectrometry by MALDI-TOF, qPCR and culture protocols was performed using the sonicated fluid, for detecting the microorganisms involved in the biofilm. RESULTS: The bacterial bioburden decreased by approximately one log after the refrigeration period, in the screws containing P. aeruginosa and S. aureus biofilms. All the microorganisms involved in the screw biofilms were detected with MALDI-TOF and qPCR. Significant reductions in CFU counts occurred only in groups stored in the plastic bag, indicating that changes in temperature and humidity may favor cell death. However, this variation is not important for this model as it did not affect the detection owing to the high counts obtained. CONCLUSION: Microbial identification by MALDI-TOF in sonicated fluid is feasible. With qPCR, there were no differences between the detection in the screws processed immediately or after refrigeration. It is necessary to consider whether or not the refrigeration period would affect microbial recovery in an explanted prosthesis.

Determination of antibiotics and detergent residues in decellularized tissue-engineered heart valves using LC-MS/MS.

Residual chemicals that are presented during tissue processing in human tissue banks can be a risk for the allograft recipient. Determine the residual concentrations of the antibiotics and detergent used in the process of human decellularized tissue-engineered heart valves stored in isotonic saline solution up to 18 months. A total of 24 human decellularized allografts were stored in sterile sodium chloride and analyzed immediately after the decellularization process (0 months) and after storage for 6, 12, and 18 months, which includes the use of sodium dodecyl sulfate (SDS) and antibiotics (cefoxitin, vancomycin hydrochloride, lincomycin hydrochloride, polymyxin B sulfate). These valves were used for suitability tests, the zone of inhibition evaluation, and direct contact cytotoxicity assay. The stock solution from 32 valves was used for LC-MS/MS analysis of antibiotics and SDS. Tissue samples from decellularized valves showed a zone of inhibition formation for S. aureus and B. subtilis, suggesting the presence of an inhibitory molecule in the tissue. Cytotoxicity tests were negative. Polymyxin B, vancomycin, and SDS were detected and quantified in human decellularized aortic and pulmonary allografts during all periods of the study. There were no traces of residual cefoxitin and lincomycin in the tissue stock solution. We found residual concentrations of the antibiotics and detergent used in the process of human decellularized tissue-engineered heart valves stored in isotonic saline solution up to 18 months.

Histological and Biomechanical Characteristics of Human Decellularized Allograft Heart Valves After Eighteen Months of Storage in Saline Solution.

Background: The best storage preservation method for maintaining the quality and safety of human decellularized allograft heart valves is yet to be established. Objective: The aim of the present study was to evaluate the stability in terms of extracellular matrix (ECM) integrity of human heart valve allografts decellularized using sodium dodecyl sulfate-ethylenediaminetetraacetic acid (SDS-EDTA) and stored for 6, 12, and 18 months. Methods: A total of 70 decellularized aortic and pulmonary valves were analyzed across different storage times (0, 6, 12, and 18 months) for solution pH measurements, histological findings, cytotoxicity assay results, biomechanical test results, and microbiological suitability test results. Continuous data were analyzed using one-way analysis of variance comparing the follow-up times. Results: The pH of the stock solution did not change during the different time points, and no microbial growth occurred up to 18 months. Histological analysis showed that the decellularized allografts did not present deleterious outcomes or signs of structural degeneration in the ECM up to 12 months. The biomechanical properties showed changes over time in different aspects. Allografts stored for 18 months presented lower tensile strength and elasticity than those stored for 12 months (p < 0.05). The microbiological suitability test suggested no residual antimicrobial effects. Conclusion: Changes in the structure and functionality of SDS-EDTA decellularized heart valve allografts occur after 12 months of storage.

Detection and quantification of human immunodeficiency virus and hepatitis C virus in cadaveric tissue donors using different molecular tests.

BACKGROUND: Tissues from cadaveric donors are used in several clinical circumstances, and the transmission of infectious diseases has been reported. Cadaveric donor (CD) blood sample analysis is challenging due to its poor quality. However, studies have demonstrated the usefulness of molecular based methods, and the lack of studies using available commercial molecular tests was reported. OBJECTIVE: The aim of this study was to evaluate the performance, specificity, sensitivity, and accuracy of different commercial molecular tests for HIV and HCV detection and quantification in CD through spiked samples. STUDY DESIGN: 20 CD and 20 blood donor samples were tested using 1,000 copies/mL and 1,000 IU/mL of lyophilized standards of HIV and HCV, respectively. Samples were analyzed by different molecular kits: XPERT HCV Viral Load and HIV-1 (Cepheid), COBAS® TaqMan® HIV-1 and COBAS® TaqMan® HCV Test, v2.0 (Roche), and artus® HI Virus-1 QS-RGQ and artus® HCV RG RT-PCR Kit (Qiagen). RESULTS: HIV and HCV in CD were detected by RT-PCR-based quantitative kits. The tests performed by the Cepheid and the Roche kits showed the most accurate, sensitive and specific results, however, a wide variability between the assays and kits was observed. The Qiagen kits did not demonstrate satisfactory results. CONCLUSIONS: CD evaluation showed great variability. The Cepheid and Roche kits were more sensitive for detecting HIV on CD and Cepheid was the most efficient kit for HCV quantification in CD. The Roche and Cepheid kits can be used to screen tissue donors for HIV and HCV.

Anaerobic bioburden in transport solution of human cardiovascular tissues.

Although reports of infections caused by anaerobes after tissue transplantation are uncommon, contamination of allografts may result in substantial complications. Anaerobic incubation and testing of organ transport solution (TS) are not routine. The aim of this study was to determine the bioburden of strict anaerobic bacteria and oxygen tension of heart-TS. Forty TS from different donors were evaluated cultured using membrane filtration (MF), direct inoculation on broth and automated blood culture bottle (ABCB). Bacterial identification was performed by MALDI-TOF. The transport conditions were simulated to verify the bacterial recovery. A sterile bag fulfilled with 250 ml-1 of sterile saline was spiked with 100 CFU ml-1 of Clostridium perfringens and the fluid recovered 0 h, 1 h, 2 h, 6 h, 12 h, 24 h and 48 h for culture and oxygen measurement. Strict anaerobic bacteria were not isolated in heart-TS. The recovery of C.perfringens spiked in heart-TS was 100% using automated blood culture bottles. MF method detected >100 CFU only after 6 h of spiking. The manual culture was not able to recover C.perfringens after the process. The percentage of O2 measures varied from 77.6 to 87.9%. MF or ABCB are better than direct inoculation for recovery of anaerobes from heart-TS. Although all samples from heart donors were negative for anaerobes (probably due to low incidence of contamination), C.perfringens were all recovered in the simulated transport condition.

Decellularization as a method to reduce calcification in bovine pericardium bioprosthetic valves.

OBJECTIVES: Decellularization is an alternative method for processing biological tissues with decreased antigenicity and resistance to calcification. The aim of this study was to characterize the properties of decellularized (dCell) bovine pericardium fixed with 0.1% glutaraldehyde (GA) and to evaluate outcomes of bioprosthetic valves constructed with this tissue when implanted in the mitral position of juvenile sheep. METHODS: Bioprosthetic mitral valves were constructed with fresh bovine pericardium fixed in 0.5% GA (control group) or dCell bovine pericardium fixed in 0.1% GA (study group). Before implantation, samples were submitted to histological (haematoxylin-eosin, Movat and 4',6-diamidino-2-phenylindole), biochemical (residual deoxyribonucleic acid and α-gal epitopes) and biomechanical characterization. Valves were implanted (n = 8 in each group) as a mitral valve replacement for 180 days in sheep and explants were re-evaluated histologically and for calcification with radiological studies and calcium content determination. RESULTS: Unimplanted dCell pericardia exhibited a well-preserved extracellular matrix with absence of cells, a 77% reduction in deoxyribonucleic acid levels and with no detectable α-gal epitopes. When compared to controls, they had lower ultimate tensile strength (7.3 ± 5.4 vs 10.2 ± 3.0 mPa, P = 0.04) and greater percentage elongation in the longitudinal direction (29 ± 6.5% vs 23.8 ± 5.1%, P = 0.02). After 180 days in mitral position, dCell valves showed pliable leaflets without macroscopic signs of calcification. Histologically, dCell leaflets had intact collagen fibres, better tissue remodelling and a significant 89% reduction in calcium content. CONCLUSIONS: This study demonstrates that bioprosthetic valves constructed with dCell bovine pericardium fixed in low GA concentration were resistant to calcification and may thereby improve long-term durability of the tissue.