PARP14 is regulated by the PARP9/DTX3L complex and promotes interferon γ-induced ADP-ribosylation.

Protein ADP-ribosylation plays important but ill-defined roles in antiviral signalling cascades such as the interferon response. Several viruses of clinical interest, including coronaviruses, express hydrolases that reverse ADP-ribosylation catalysed by host enzymes, suggesting an important role for this modification in host-pathogen interactions. However, which ADP-ribosyltransferases mediate host ADP-ribosylation, what proteins and pathways they target and how these modifications affect viral infection and pathogenesis is currently unclear. Here we show that host ADP-ribosyltransferase activity induced by IFNγ signalling depends on PARP14 catalytic activity and that the PARP9/DTX3L complex is required to uphold PARP14 protein levels via post-translational mechanisms. Both the PARP9/DTX3L complex and PARP14 localise to IFNγ-induced cytoplasmic inclusions containing ADP-ribosylated proteins, and both PARP14 itself and DTX3L are likely targets of PARP14 ADP-ribosylation. We provide evidence that these modifications are hydrolysed by the SARS-CoV-2 Nsp3 macrodomain, shedding light on the intricate cross-regulation between IFN-induced ADP-ribosyltransferases and the potential roles of the coronavirus macrodomain in counteracting their activity.

Glucuronoxylomannan intranasal challenge prior to Cryptococcus neoformans pulmonary infection enhances cerebral cryptococcosis in rodents.

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a "Trojan horse" mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB) to promote C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated ~200,000 deaths worldwide each year.

Role of Oxidative Stress on Insulin Resistance in Diet-Induced Obesity Mice.

Insulin resistance is the link between obesity and type 2 diabetes mellitus. The molecular mechanism by which obese individuals develop insulin resistance has not yet been fully elucidated; however, inconclusive and contradictory studies have shown that oxidative stress may be involved in the process. Thus, this study aimed to evaluate the effect of reactive species on the mechanism of insulin resistance in diet-induced obese mice. Obese insulin-resistant mice were treated with N-acetylcysteine (NAC; 50 mg/kg per day, for 15 days) by means of oral gavage. Twenty-four hours after the last NAC administration, the animals were euthanized and their tissues were extracted for biochemical and molecular analyses. NAC supplementation induced improved insulin resistance and fasting glycemia, without modifications in food intake, body weight, and adiposity. Obese mice showed increased dichlorofluorescein (DCF) oxidation, reduced catalase (CAT) activity, and reduced glutathione levels (GSH). However, treatment with NAC increased GSH and CAT activity and reduced DCF oxidation. The gastrocnemius muscle of obese mice showed an increase in nuclear factor kappa B (NFκB) and protein tyrosine phosphatase (PTP1B) levels, as well as c-Jun N-terminal kinase (JNK) phosphorylation compared to the control group; however, NAC treatment reversed these changes. Considering the molecules involved in insulin signaling, there was a reduction in insulin receptor substrate (IRS) and protein kinase B (Akt) phosphorylation. However, NAC administration increased IRS and Akt phosphorylation and IRS/PI3k (phosphoinositide 3-kinase) association. The results demonstrated that oxidative stress-associated obesity could be a mechanism involved in insulin resistance, at least in this animal model.

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.

Liposomal drug delivery systems for the treatment of leishmaniasis.

Human le ishmaniasis is a vector-borne, neglected infectious disease that is widely distributed in America, Africa, Europe, and Asia. Current therapy is based on old and toxic drugs, including antimonials, aminoglycosides, and amphotericin. As a neglected disease, investment in the development of new therapeutic molecules is scarce. Considering these aspects, the optimization of treatment through novel delivery systems for current therapeutic agents is an attractive alternative. The encapsulation into liposomes of drugs used in treating leishmaniasis increases the concentration of these molecules in macrophages, which may not only increase the chance of cure but also expand their therapeutic spectrum to include resistant Leishmania, as well as reducing toxicity since the drug is less exposed to healthy cells. The classical example is the liposomal formulation of amphotericin B, a well-established therapeutic option that uses liposomes to decrease the progression of renal failure in patients. However, loading other leishmanicidal drugs into liposomes, such as pentavalent antimonials, presents an opportunity for innovative and cheaper therapeutic options for the treatment of human leishmaniasis. This review aims to discuss liposomes as a drug delivery system for leishmanicidal drugs.

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.

The nasal microbiome, nasal transcriptome, and pet sensitization.

BACKGROUND: Pet allergies are common in children with asthma. Microbiota and host responses may mediate allergen sensitization. OBJECTIVE: We sought to uncover host-microbe relationships in pet allergen sensitization via joint examination of the nasal microbiome and nasal transcriptome. METHODS: We collected nasal samples from 132 children with asthma for parallel 16S rRNA and RNA sequencing. Specific IgE levels for cat and dog dander were measured. Analyses of the nasal microbiome, nasal transcriptome, and their correlations were performed with respect to pet sensitization status. RESULTS: Among the 132 children, 91 (68.9%) were cat sensitized and 96 (72.7%) were dog sensitized. Cat sensitization was associated with lower nasal microbial diversity by Shannon index (P = .021) and differential nasal bacterial composition by weighted UniFrac distance (permutational multivariate ANOVA P = .035). Corynebacterium sp and Staphylococcus epidermidis were significantly less abundant, and the metabolic process "fatty acid elongation in mitochondria" was lower in pet-sensitized versus unsensitized children. Correlation networks revealed that the nasal expression levels of 47 genes representing inflammatory processes were negatively correlated with the relative abundances of Corynebacterium sp and S epidermidis. Thus, these species were directly associated not only with the absence of pet sensitization but also with the underexpression of host gene expression of inflammatory processes that contribute to allergen sensitization. Causal mediation analyses revealed that the associations between these nasal species and pet sensitization were mediated by nasal gene expression. CONCLUSIONS: Higher abundances of nasal Corynebacterium sp and S epidermidis are associated with absence of pet sensitization and correlate with lower expression of inflammatory genes.

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.

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.

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.

Pseudozyma spp. human infections: A systematic review.

Pseudozyma spp. are described as environmental yeasts but have also been identified as rare human pathogens found in immunocompromised patients. This systematic review details the clinical manifestations, diagnostic methodology, and empirical anti-fungal therapy for this rare yeast. PubMed, LILACS, Scielo, and Web of Science databases were searched for articles about Pseudozyma spp. infections from inception to June 2019. Inclusion criteria were any published studies that included patients with Pseudozyma spp. infection. Infections were identified using criteria set forth by the European Organization for Research and Treatment of Cancer, and were further classified according to clinical, laboratory, or radiologic findings, microbiologic confirmation, and response to therapy. Eleven articles were included with 15 patients. Oncological and/or hematological disorders were the most reported risk factors. Nontraditional microbiological methods correctly identified Pseudozyma spp., whereas traditional methods failed to identify fungal genus. Species were identified by sequencing, and most demonstrated a higher minimal inhibitory concentration (MIC) for fluconazole and echinocandins. MICs for itraconazole, voriconazole, and posaconazole varied by species. All isolates were susceptible to amphotericin B, which was the most used treatment. Pseudozyma spp. infections usually present with fever and are diagnosed by blood culture. Most species studied appeared to be resistant to fluconazole and echinocandin. Voriconazole, posaconazole, and amphotericin were effective in treating P. aphidis. However, more studies are needed to evaluate voriconazole and posaconazole in species other than P. aphidis.

Cerebrospinal Fluid Penetration of Vancomycin During Continuous Infusion Therapy in Patients With Nosocomial Ventriculitis.

BACKGROUND: This study aimed to evaluate the utility of a commercial kit used to measure serum vancomycin concentrations to determine vancomycin concentrations in cerebrospinal fluid (CSF) samples and evaluate CSF penetration when administered as a continuous high-dose infusion in patients with nosocomial ventriculitis. METHODS: This study included patients with external ventricular drain infection who were admitted to the intensive care unit between January 2018 and September 2020. After validation, CSF samples from 33 patients were collected. All patients received 30 mg/kg of vancomycin as a loading dose followed by 60 mg/kg as a maintenance dose in continuous infusion; all CSF samples were collected at least 48 hours after the first dose. RESULTS: Thirty-three patients were enrolled in this study. The median serum creatinine level was 0.66 mg/dL (0.5-0.92; n = 30), and median creatinine clearance was 119.2 mL/min (64.6-138.4; n = 13). The median serum vancomycin 24-hour area under the curve (AUC24h) was 838 mg*h/L (515-1010). The median CSF vancomycin concentration was 5.20 mg/L (1.95-12.4). Median serum vancomycin concentration was 34.9 mg/L (21.47-42.1), and median CSF/serum ratio was 18.6% (8.4-41.5). Acute renal injury occurred in 21% (n = 7) of the patients by the end of the therapy. In addition, the vancomycin CSF/serum ratio was positively correlated with the median serum creatinine level (r = 0.670; P = 0.004). CONCLUSIONS: Commercial vancomycin kits used to measure serum samples may be used to evaluate vancomycin concentrations in the CSF. Vancomycin penetration into CSF was 18.6%.

Immobilization of ß-galactosidase from Bacillus licheniformis for application in the dairy industry.

The food industry has developed a wide range of products with reduced lactose to allow people with intolerance to consume dairy products. Although ß-galactosidase has extensive applications in the food, pharma, and biotechnology industries, the enzymes are high-cost catalysts, and their use makes the process costly. Immobilization is a viable strategy for enzyme retention inside a reactor, allowing its reuse and application in continuous processes. Here, we studied the immobilization of ß-galactosidase from Bacillus licheniformis in ion exchange resin. A central composite rotational design (CCRD) was proposed to evaluate the immobilization process in relation to three immobilization solution variables: offered enzyme activity, ionic strength, and pH. The conditions that maximized the response were offered enzyme activity of 953 U, 40 mM ionic strength, and pH 4.0. Subsequently, experiments were performed to provide additional stabilization for biocatalyst, using a buffer solution pH 9.0 at 25 °C for 24 h, and crosslinking with different concentrations of glutaraldehyde. The stabilization step drastically impacted the activity of the immobilized enzyme, and the reticulation with different concentrations of glutaraldehyde showed significant influence on the activity of the immobilized enzyme. In spite of substantially affecting the initial activity of the immobilized enzyme, higher reagent concentrations (3.5 g L-1) were effective for maintaining stability related to the number of cycles of the enzyme immobilized. The ß-galactosidase from Bacillus licheniformis immobilized in Duolite A568 is a promising technique to produce reduced or lactose-free dairy products, as it allows reuse of the biocatalyst, decreasing operational costs.Key Points• Immobilization of ß-galactosidase from Bacillus licheniformis in batch reactor• Influence of buffer pH and ionic concentration and offered enzyme activity on immobilization• Influence of glutaraldehyde on operational stability.

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.

Risk factors for plantar foot ulcer recurrence in patients with diabetes - A prospective pilot study.

INTRODUCTION: Diabetic foot ulcer (DFU) is a complication of diabetes mellitus (DM) with established recurrence risk factors evaluating patients from United States or Europe. There are scarce studies in developing countries about these risks. The aim of this study was to evaluate risk factors associated with DFU recurrence in a Brazilian prospective cohort. MATERIALS AND METHODS: A prospective cohort of patients with healed DFU followed from January 2014 to June 2017 in Curitiba, Brazil. Periodic home visits from a specialist nurse in DFU were performed during the period of the study to evaluate recurrence of ulcer. The presence of risk factors in the group of patients that developed an ulcer in the follow-up period was compared with the presence of these factors in the group of patients without recurrence. At enrollment, 35 subjects presented a previous ulcer distal with complete healing to follow-up. RESULTS: From 35 patients, 15 were male (43%) and the mean age was of 65.8 ± 10.9 years (48-85 year). Most patients were married with a low income (

Risk factors associated with contamination of allograft valves in a tissue bank.

The contamination of the transport solution used in cardiovascular allografts can occur from different sources. Risk factors associated with positive microbiological test of transport solution have not been reported previously. This study aimed to determine the risk factor for contamination of transport solution used in the heart valve allografts stored in a Brazilian tissue bank. This retrospective study was conducted on all donors of cardiovascular allografts stored in a tissue bank from December 2008 to December 2017. Microbiological cultures for aerobic and anaerobic bacteria, fungi/yeasts were carried out in TS. Clinical variables were included. From 1001 transport solution, 52% were contaminated. A total of 770 microorganisms were identified, and Staphylococcus spp. was identified in 248 isolates (32.2%). Skin bacteria from skin microbiota were the most commonly identified microorganisms (Staphylococcus spp., Cutibacterium spp., Corynebacterium spp., and Bacillus spp.), occurring in 49.6%. The presence of a diagnosis of healthcare-associated infection was not associated with skin contamination (odds ratio [OR] 0.62 [0.41-0.94]; p = 0.014). Conditions like fever, use of antibiotics, and leukocytosis were less likely associated with contamination of transport solution. A longer warm ischemic time was associated with higher frequency of contamination. In the multivariable analysis, warm ischemic time was independently associated with contamination, and antibiotic therapy was a factor that decreased the rate of contamination (p < 0.05). Contamination of transport solution is associated with modifiable risk factors, such as warm ischemic time. Measures to minimize contamination should be employed to avoid unnecessary tissue discharges.

Bioburden in transport solutions of human cardiovascular tissues: a comparative evaluation of direct inoculation and membrane filter technique.

All cardiac allograft tissues are under potential contamination, requiring a validated terminal sterilization process or a minimal bioburden. The bioburden calculation is important to determine the bacterial burden and further decontamination and disinfection strategies for the valve processing. The aim of this study was to determine the bioburden from transport solution (TS) of heart valves obtained from non-heart-beating and heart-beating donors in different culture methods. The bioburden from TS was determined in 20 hearts donated for valve allograft tissue using membrane filter (MF) and direct inoculation. Tryptic soy agar and Sabouraud plates were incubated and colonies were counted. Ninety-five percent of samples from this study were obtained from heart-beating donors. The warm ischemic time period for heart was 1.06 ± 0.74 h and the cold ischemic time period was 25.66 ± 11.16 h. The mean TS volume was 232.68 ± 96.67 mL (48.5-550 mL). From 20 samples directly inoculated on TSA agar plates, 2 (10%) were positive. However, when MF was used, from 20 samples in TSA, 13 (65%) were positive with a mean count of 1.36 ± 4.04 CFU/mL. In Sabouraud plates, the direct inoculation was positive in 5 samples (25%) with a mean count of 0.24 ± 0.56 CFU/mL. The use of MF increased the positivity to 50% (10 samples from a total of 20) with a mean of 0.28 ± 0.68 CFU/mL. The positivity was superior using MF in comparison with direct inoculation (p < 0.05). The bioburden of TS is low and MF is the technique of choice due to higher positivity.

Comparison of automated and conventional microbiological examination of donated human cardiac tissue in heart valve banking.

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (104 to 10-1 CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤ 0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p < 0.05). The positivity for growth in the automated method was higher in concentrated inoculum (> 102 CFU/mL) (p < 0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 103 CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.