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ABSTRACT Klebsiella pneumoniae is a significant healthcare-associated pathogen, notable for its diverse virulence and antibiotic resistance profiles. This study aimed to characterize the genotypic and phenotypic diversity of K. pneumoniae isolates and evaluate their virulence using the Galleria mellonella model. Biomass production, metabolic activity, capsule formation, and siderophore production were assessed in 27 K. pneumoniae isolates from hospital-associated infections. Lethality curves were generated using the G. mellonella model, with survival monitored hourly from 16 to 48 hours. The most common sequence types (ST) identified were the high-risk clones ST307 (N = 10), ST512 (N = 8), ST101 (N = 7), and ST661 (N = 2). These STs were associated with distinct K-locus, including KL102, KL107, KL17, and KL39. Most isolates belonged to the O2afg locus (N = 18), with the K. pneumoniae carbapenemase genotype detected in 96.3% of strains. None of the isolates were classified as hypervirulent. Phenotypically, ST661 exhibited the highest biomass production despite showing similar metabolic activity to other STs. A positive correlation was observed between biomass and siderophore production, while capsule production was inversely correlated with biomass. In the G. mellonella model, ST661 demonstrated the highest virulence, resulting in 100% mortality by 48 hours, compared to survival rates of 21.4% for ST101, 38.0% for ST307, and 31.2% for ST512. These findings underscore the pathogenic potential of ST661 isolates with enhanced biofilm production. The G. mellonella model may serve as an effective in vivo system for evaluating the virulence of emerging K. pneumoniae lineages.IMPORTANCEWe demonstrate that the Galleria mellonella model is a useful tool to analyze the virulence of carbapenem-resistant Klebsiella pneumoniae strains. Our findings highlight the pathogenicity of carbapenem-resistant K pneumoniae isolates, particularly the role of the ST661 that, despite being a rare lineage, harbors the blaVIM gene and is associated with high biofilm production and the highest mortality rates.

Background: Oritavancin (ORT) is a new single-dose lipoglycopeptide showing in vitro activity against staphylococci and vancomycin-resistant enterococci. However, there is no data regarding its potential use as a catheter lock solution are scarce. We constructed an in vitro model to analyze the efficacy and stability of an ORT lock solution against the biofilm of staphylococci and enterococci over 7 days at 37 °C. Methods: We used Staphylococcus aureus, Staphylococcus epidermidis, and vancomycin-susceptible Enterococcus faecalis ATCC strains. We performed a metabolic activity assay using a 2-mg/ml solution of ORT over a 7-day incubation period at 37 °C. The solution was tested against 24-h biofilms of each strain at day 0 and 7. Metabolic activity was measured using the XTT assay, and median absorbance obtained at 490 nm in the spectrophotometer was compared between day 0 and day 7. Results: The percentage reduction in metabolic activity was 95.3 % between biofilms treated with ORT solution incubated for 7 days and biofilms treated with ORT solution before incubation. Conclusion: Ours is a proof-of-concept study that shows ORT to be a potential treatment as a catheter lock solution for eradication of staphylococcal and E. faecalis biofilms. It is needed to further test ORT against more clinical strains and to compare its activity with other antimicrobials in a biofilm model.

Background: Chemical debridement is a fundamental step during the surgical treatment of both acute and chronic periprosthetic joint infection (PJI). However, there is no consensus on the optimal solution, nor is there sufficient evidence on the optimal irrigation time and combination of solutions. In an in vitro study, our group recently demonstrated that sequential combination debridement (SCD) with 3% acetic acid (AA) followed by 10% povidone iodine (PI) and 5 mM hydrogen peroxide (H2O2) was the best strategy for reducing bacterial load. The present study aimed to validate these findings in an in vivo model. Results: The median (IQR) log CFU/mL was lower in the group of mice treated with SCD (2.85 [0.00–3.72]) than in the Bactisure™ group (4.02 [3.41–4.72], p = 0.02). While this reduction was also greater than in the PI group (3.99 [1.11–4.33]), the difference did not reach statistical significance (p = 0.19). Cell viability assays showed no differences between treatments. S. aureus bacteremia was detected in 10% of mice treated with SCD, compared to 30% in the PI group and 10% in the Bactisure™ group. The difference was not statistically significant (p = 0.36). Conclusion: Our findings confirm that SCD significantly reduced bacterial load in an in vivo S. aureus PJI model, showing superior anti-biofilm activity compared to Bactisure™ and comparable performance to PI alone. These results highlight SCD’s potential to serve as a standardized chemical debridement protocol, combining enhanced efficacy with clinical applicability. Methods: We tested SCD with 3% AA for 3 min, 10% PI for 3 min, and H2O2 for 3 min in a 7-day Staphylococcus aureus (ATCC29213)-based murine femur PJI model and compared the results with single treatments of 10% PI for 3 min or Bactisure™ solution for 3 min. A sterile steel implant with local administration of saline solution for 3 min was used as a non-infected control. After completing irrigation procedures, under anesthesia, mice were euthanized, and implants were analyzed for CFU/mL counts and cell viability rates. Blood cultures were obtained pre-euthanasia to detect bacteremia.

BackgroundSeveral studies have shown that tranexamic acid (TXA), an antifibrinolytic, reduces postoperative infection rates. Recent in vitro research showed that TXA alone and in combination with vancomycin and gentamicin had a synergistic effect against some staphylococcal strains. In the present study, this synergistic effect was validated in samples from patients with staphylococcal periprosthetic infection (PPI) and in an in vivo model.MethodsWe tested 19 clinical strains (5 Staphylococcus aureus and 14 coagulase-negative staphylococci [CoNS]) against 10 mg/ml TXA alone and in combination with serial dilutions of vancomycin and gentamicin. The standardized microtiter plate method was used. The minimal inhibitory concentration (MIC) were calculated using standard visualization of well turbidity. We also used an S. aureus (ATCC29213) murine subcranial PPI model to compare the synergistic effect of TXA and gentamicin with that of TXA or gentamicin alone after 4 days of monitoring. The mice were euthanized, and disks were removed for analysis of cfu/ml counts and cell viability rate. Biofilm structure of both in vitro and in vivo samples was also analyzed using scanning electron microscopy (SEM).ResultsWhen TXA was combined with vancomycin or gentamicin, the MIC decreased in 30% of the strains studied. According to species, the MIC50 for vancomycin and gentamicin alone and in combination with TXA against S. aureus strains was the same. This was also the case for CoNS with vancomycin and its corresponding combination, whereas with gentamicin and TXA, a reduction in MIC50 was observed (2 dilutions). In addition, in the in vivo model, the mean (SD) log cfu/ml and cell viability rate obtained from the implant was lower in the group of mice treated with TXA and gentamicin than in those treated only with TXA or gentamicin. SEM images also corroborated our findings in strains in which the MIC was reduced, as well as the in the mice implants, with the area occupied by biofilm being greater in samples treated only with gentamicin or TXA than in those treated with TXA+gentamicin.ConclusionWe confirm that combining TXA with vancomycin or gentamicin exerts a synergistic effect. However, this only occurs in selected strains.

Food safety control is a key issue in the food and agriculture industries. For such purposes, developing miniaturized analytical methods is critical for enabling the rapid and sensitive detection of food supplements, allergens, and pollutants. Here, a novel bioanalytical methodology based on DNA-functionalized gold nanoparticles (AuNPs) and colorimetric detection was developed to detect the presence of sesame (a major allergen) through sesame seed DNA as a target, in food samples. The presence of sesame DNA induces controlled nanoparticle aggregation/desegregation, resulting in a color change (from blue to red) proportional to sesame DNA concentration. The incorporation of multicomponent nucleic acid enzymes (MNAzymes) in this strategy has been carried out to perform an isothermal signal amplification strategy to improve the sensitivity of detection. Also, open-source software for color analysis was used to ensure an unbiased visual color-change detection, enhancing detection accuracy and sensitivity and opening the possibility of performing a simple and decentralized analyte detection. The method successfully detected the presence of sesame DNA in sesame seed, sesame oil, olive oil, and sunflower oil. In brief, the developed approach constitutes a simple and affordable alternative to perform a highly sensitive detection of DNA in food without complex methodologies or the requirement of expensive instrumentation.

ABSTRACTPlant chemodiversity is a helpful tool for disease prevention and a basis for adjuvant treatments to conventional therapies. In this regard, the extract of Polypodium leucotomos rhizomes, PLE, has shown benefits fighting inflammation and recurrent infections but its molecular mechanism is poorly undersood. This work shows that Plesinox 3A, containing PLE, Vitamins A, C, and selenium, helps modulate the initial inflammatory response triggered by bacterial LPS through the upregulation of IL8 and IL10, together with downregulation of COX2, IL1B and TNF, in a more efficient manner than PLE alone. Additionally, this formulation enhances the antiviral response through the upregulation of MX1, IFNA1 and IFNG in different cell types. Finally, the addition of vitamins and selenium to PLE in Plesinox 3A greatly boosts the anti-bacterial properties of PLE alone. Overall, these findings support the combined use of PLE, vitamins, and selenium, in the form of Plesinox 3A as an immune booster to prevent recurrent infections, highlighting a gene set potentially involved in its beneficial effect, as well as showing its direct anti-bacterial properties, which are greater than those of PLE alone.

Background. Escherichia coli commonly causes catheter-related bloodstream infection (C-RBSI) in specific populations. The differential time to positivity (DTTP) technique is the recommended conservative procedure for diagnosing C-RBSIs. Methods. We conducted a retrospective study of episodes in which E. coli was isolated from catheter lumens obtained using the DTTP technique. Microbiological and clinical data were obtained based on the DTTP technique as either catheter colonization, C-RBSI, or non-C-RBSI. Results. A total of 89 catheter blood cultures were included, classified as follows: catheter colonization, 33.7%; C-RBSI, 9.0%; and non-C-RBSI, 57.3%. Only 15.7% of the catheters were withdrawn, with no positive catheter-tip cultures. We found no statistically significant differences in catheter type, antibiotic treatment, or clinical outcome among the groups, except for the frequency of catheter lock therapy or in the frequency of successful treatment. Mortality was associated with C-RBSI in only one patient. Conclusion. E. coli bacteremia diagnosed by the DTTP technique was classified as non-catheter-related in most patients. As the majority of the catheters were retained, E. coli bacteremia could not be microbiologically confirmed as catheter-related by the catheter-tip culture. Future studies are needed to assess the profitability of the DTTP technique for diagnosing E. coli C-RBSIs.

Irrigation and debridement using an irrigation solution is a fundamental step during the surgical treatment of both acute and chronic periprosthetic joint infection (PJI). However, there is no consensus on the optimal solution, nor is there sufficient evidence on the optimal irrigation time and combination of solutions. Therefore, it is necessary to determine which solution or combination of solutions is most efficacious against biofilm, as well as the optimal irrigation time. We conducted an experimental in vitro model by inoculating stainless steel discs with ATCC strains of methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and a clinical strain of Staphylococcus epidermidis. The discs were all irrigated with commonly used antiseptic solutions (10% and 3% povidone iodine, hydrogen peroxide, 3% acetic acid, and Bactisure™) for 1 min, 3 min, and 5 min and their combinations for 9 min (3 min each) vs. sterile saline as a positive control. We evaluated the reduction in biofilm based on colony-forming unit (cfu) counts and in combination assays, also based on cell viability and scanning electron microscopy. All antiseptics alone reduced more than 90% of cfu counts after 1 min of irrigation; the worst results were for hydrogen peroxide and 3% acetic acid. When solutions were sequentially combined, the best results were observed for all those starting with acetic acid, in terms of both reduction of log cfu/mL counts and viable cells. We consider that a combination of antiseptic solutions, particularly that comprising the sequence acetic acid + povidone iodine + hydrogen peroxide, would be the best option for chemical debridement during PJI surgery.

The worldwide cement industry plays an important role in addressing the climate change challenge. Brazil’s cement industry currently has 91 cement plants with an installed production capacity of 94 million tons per year and has started to calculate the net CO2 emissions to achieve a carbon-neutral cement sector by 2050. Accordingly, the carbon dioxide uptake due to mortar and concrete carbonation is subtracted from the carbon dioxide emitted by the chemical reaction for the calcination of lime, i.e., the calcination process performed during clinker production. Now-adays, the Intergovernmental Panel on Climate Change (IPCC) Guidelines for National Greenhouse Gas (GHG) Inventories to report the GHG emissions do not include any calculation procedure to consider the mortar and concrete carbonation. However, the Intergovernmental Panel on Climate Change (IPCC)’s Sixth Assessment Report (AR6) recognizes the physico-chemical process known as carbonation. Brazilian net carbon dioxide emissions of cements produced from 1990 to 2019 are estimated considering the carbon dioxide uptake during the service-life and end-of-life and secondary usage stages (Tier 1). This is a fundamental scientific and technological novelty that changes the current approach to estimate the carbon dioxide emissions due to the Portland cement clinker production. Even considering the relative novelty of this approach, it should be promoted in the future and included in the national inventory report (NIR). The carbon dioxide uptake by mortar and concrete carbonation for 30 years is about 140 million tons. Within this thirty-year period about 483 million tons have been released due to the calcination process.

Background: Chemical debridement is a fundamental step during the surgical treatment of both acute and chronic periprosthetic joint infection (PJI). However, there is no consensus on the optimal solution, nor is there sufficient evidence on the optimal irrigation time and combination of solutions. In an in vitro study, our group recently demonstrated that sequential combination debridement (SCD) with 3% acetic acid (AA) followed by 10% povidone iodine (PI) and 5 mM hydrogen peroxide (H2O2) was the best strategy for reducing bacterial load. The present study aimed to validate these findings in an in vivo model. Results: The median (IQR) log CFU/mL was lower in the group of mice treated with SCD (2.85 [0.00–3.72]) than in the Bactisure™ group (4.02 [3.41–4.72], p = 0.02). While this reduction was also greater than in the PI group (3.99 [1.11–4.33]), the difference did not reach statistical significance (p = 0.19). Cell viability assays showed no differences between treatments. S. aureus bacteremia was detected in 10% of mice treated with SCD, compared to 30% in the PI group and 10% in the Bactisure™ group. The difference was not statistically significant (p = 0.36). Conclusion: Our findings confirm that SCD significantly reduced bacterial load in an in vivo S. aureus PJI model, showing superior anti-biofilm activity compared to Bactisure™ and comparable performance to PI alone. These results highlight SCD's potential to serve as a standardized chemical debridement protocol, combining enhanced efficacy with clinical applicability. Methods: We tested SCD with 3% AA for 3 min, 10% PI for 3 min, and H2O2 for 3 min in a 7-day Staphylococcus aureus (ATCC29213)-based murine femur PJI model and compared the results with single treatments of 10% PI for 3 min or Bactisure™ solution for 3 min. A sterile steel implant with local administration of saline solution for 3 min was used as a non-infected control. After completing irrigation procedures, under anesthesia, mice were euthanized, and implants were analyzed for CFU/mL counts and cell viability rates. Blood cultures were obtained pre-euthanasia to detect bacteremia. [ABSTRACT FROM AUTHOR]

Objetivo: El bazedoxifeno es un SERM de 3ª generación con efectos agonistas sobre el hueso y sobre el útero y el tejido mamario. Nuestro objetivo ha sido estudiar los efectos del bazedoxifeno sobre la calidad ósea en un modelo experimental de ratas ovariectomizadas. Material y métodos: Se utilizaron 3 grupos de 15 ratas Wistar hembras de 6 meses de edad: uno control; otro de ratas ovariectomizadas; y un tercer grupo de ratas ovariectomizadas tratadas con bazedoxifeno (0,33 mg/kg/día). Tras 8 meses se estudiaron la densitometría ósea lumbar y femoral, los parámetros microtomográficos, los marcadores bioquímicos de remodelado y los parámetros biomecánicos del hueso. Resultados: La ovariectomía descendió la densidad ósea femoral y lumbar. La última se recuperó parcialmente con bazedoxifeno. El remodelado óseo aumentó, recuperando el bazedoxifeno los niveles de formación. El bazedoxifeno recuperó la fracción volumétrica ósea (BV/TV), la densidad de superficie ósea (BS/TV), el aumento en la separación trabecular (Tb.Sp), la disminución en el número de trabéculas (Tb.N), el aumento del factor de patrón trabecular (Tb.Pf) y el índice de modelo estructural (SMI). La superficie relativa cortical aumentó tras la ovariectomía, normalizándose con bazedoxifeno. También recuperó la deformación máxima antes de la rotura producida por la ovariectomía, y amortiguó parcialmente la ganancia de peso de las ratas ovariectomizadas. Conclusiones: Nuestro estudio muestra resultados positivos del bazedoxifeno sobre la calidad ósea. Este fármaco podría estar especialmente indicado para mujeres jóvenes postmenopáusicas con osteoporosis o en riesgo de padecerla.

Food safety control is a key issue in the food and agriculture industries. For such purposes, developing miniaturized analytical methods is critical for enabling the rapid and sensitive detection of food supplements, allergens, and pollutants. Here, a novel bioanalytical methodology based on DNA-functionalized gold nanoparticles (AuNPs) and colorimetric detection was developed to detect the presence of sesame (a major allergen) through sesame seed DNA as a target, in food samples. The presence of sesame DNA induces controlled nanoparticle aggregation/desegregation, resulting in a color change (from blue to red) proportional to sesame DNA concentration. The incorporation of multicomponent nucleic acid enzymes (MNAzymes) in this strategy has been carried out to perform an isothermal signal amplification strategy to improve the sensitivity of detection. Also, open-source software for color analysis was used to ensure an unbiased visual color-change detection, enhancing detection accuracy and sensitivity and opening the possibility of performing a simple and decentralized analyte detection. The method successfully detected the presence of sesame DNA in sesame seed, sesame oil, olive oil, and sunflower oil. In brief, the developed approach constitutes a simple and affordable alternative to perform a highly sensitive detection of DNA in food without complex methodologies or the requirement of expensive instrumentation. [ABSTRACT FROM AUTHOR]

Aflatoxin M1 (AFM1) is one of the most widespread aflatoxins that can be present in the milk of lactating mammals. It can cause carcinogenicity, mutagenesis, teratogenesis, genotoxicity and immunosuppression. The WHO recommends reducing the AFM1 concentration in food products, so the European Commission has set a maximum allowable limit of 0.05 µg L−1 in milk and its products. Thus, there is a need to develop new methodologies to satisfy the demand for reliable, cost-effective, robust and sensitive AFM1 routine controls. In the present work, a competitive phosphorescent immunosensor for AFM1 quantification in milk, based on antibody–antigen recognition and Mn:ZnS quantum dots (d-QDs) as photoluminescent labels, has been developed. Two different assay strategies based on the use of d-QDs as labels of secondary antibodies (direct assay), or of a derivative species of the antigen AFM1-Bovine Serum Albumin (indirect assay) were compared in terms of analytical performance for AFM1 quantification. The best analytical results were obtained with the immunoassay format that uses d-QDs as tags of secondary antibodies (direct assay), and said design was finally selected. The selected immunosensor provided a detection limit for AFM1 quantification of only 0.002 µg L−1, which greatly satisfied the maximum tolerable limit of AFM1 in milk of 0.05 µg L−1. The accuracy, calculated as recovery of AFM1 in fortified skimmed milk samples, ranged from 81 to 90%, with relative standard deviations from 3% to 14%. These results bring to light the good performance of such phosphorescent biosensors as simple and fast alternatives to conventional chromatographic analytical methods.