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11 pages, 1763 KiB  
Article
Persistence of Daptomycin-Resistant and Vancomycin-Resistant Enterococci in Hospitalized Patients with Underlying Malignancies: A 7-Year Follow-Up Study
by Lynn El Haddad, Georgios Angelidakis, Yuting Zhai, Layale Yaghi, Cesar A. Arias, Samuel A. Shelburne, Kwangcheol Casey Jeong and Roy F. Chemaly
Microorganisms 2024, 12(8), 1676; https://doi.org/10.3390/microorganisms12081676 (registering DOI) - 14 Aug 2024
Abstract
Vancomycin-resistant enterococci (VRE) commonly colonize the gut of individuals with hematologic malignancies or undergoing hematopoietic cell transplant (HCT) and may cause bacteremia. In 2012, we identified VRE isolates from patients and patients’ rooms and showed transmission networks of highly genetically related daptomycin-resistant (DR)-VRE [...] Read more.
Vancomycin-resistant enterococci (VRE) commonly colonize the gut of individuals with hematologic malignancies or undergoing hematopoietic cell transplant (HCT) and may cause bacteremia. In 2012, we identified VRE isolates from patients and patients’ rooms and showed transmission networks of highly genetically related daptomycin-resistant (DR)-VRE strains. This is a follow-up study performing whole-genome sequencing (WGS) and phylogenetic analyses on 82 clinical VRE strains isolated from stools and blood cultures of patients with leukemia and HCT between 2015 and 2019. Here, we observed transmission of highly genetically related strains between rooms on the same or on different floors, including a DR-VRE strain identified in 2012. Eleven of twenty-eight patients with DR-VRE were never exposed to daptomycin, suggesting horizontal transmission. Fifteen of the twenty-eight patients with DR-VRE died within 30 days of positive blood cultures. Amongst those, one DR-VRE strain belonging to ST1471 had the virulence gene bopD responsible for biofilm formation. Additionally, to our knowledge, this is the first report of a DR-VRE strain belonging to ST323 in the United States. In summary, our study demonstrated the emergence and persistence of VRE strains, especially DR-VRE, in our hospital. Adding WGS to routine infection control measures may timely identify potential horizontal VRE transmission including multi-drug-resistant isolates. Full article
(This article belongs to the Special Issue State-of-the-Art Medical Microbiology in the USA (2023, 2024))
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Figure 1
<p><b>Phylogenetic tree showing the genetic relatedness according to SNPs</b> found in the core genome of the 82 VRE isolates from 2016 to 2019. Transmissions between patients are shown. Phylogenetic trees were constructed using filtered core genome SNPs and the maximum likelihood tree search algorithm RAxML, and iTOL software was used to generate and visualize the tree. <span class="html-italic">Abbreviations:</span> ST, sequence type; DAP-R, daptomycin-resistant.</p>
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<p><b>Transmission networks of VRE isolates</b> differing by 5 or less SNPs in their core genomes during 2016 to 2019 in the hospital setting. Potential index cases of VRE are marked with a red triangle. The transmission time period ranged from 1 to 2 years with a median of 206 days. LKM, leukemia; HCT, hematopoietic cell transplant; ICU, intensive care unit. Note: There are 2 distinct clusters of ST80 VRE strains originating from the same room (different patients, different dates) and spread to patients on different floors, indicating possible recombination events and/or horizontal transmission.</p>
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18 pages, 2360 KiB  
Article
Antimicrobial Properties and Cytotoxicity of LL-37-Derived Synthetic Peptides to Treat Orthopedic Infections
by Vincenzo Pennone, Elisa Angelini, David Sarlah and Arianna B. Lovati
Antibiotics 2024, 13(8), 764; https://doi.org/10.3390/antibiotics13080764 - 14 Aug 2024
Viewed by 87
Abstract
Open fractures and prosthetic joints are prone to bacterial infections, especially those involving biofilms, and are worsened by antibiotic inefficacy and resistance. This highlights the need for targeted treatments against orthopedic infections. LL-37, a human cathelicidin, is known for its antimicrobial properties. This [...] Read more.
Open fractures and prosthetic joints are prone to bacterial infections, especially those involving biofilms, and are worsened by antibiotic inefficacy and resistance. This highlights the need for targeted treatments against orthopedic infections. LL-37, a human cathelicidin, is known for its antimicrobial properties. This study aimed to synthesize and evaluate LL-37-derived antimicrobial peptides (AMPs) for antibacterial efficacy and toxicity. Several truncated LL-37 analogues were created and tested against 18 bacterial strains, both ATCC and orthopedic clinical isolates, using MIC and MBC assays. Synergy with antibiotics and resistance development were also analyzed, alongside cytotoxicity on NIH-3T3 fibroblasts and hemolytic activity assessments. Six AMPs were synthesized, with FK-16 and GF-17 emerging as the most effective. The MIC values ranged from 4.69 to 18.75 µg/mL and 2.34 to 18.75 µg/mL, respectively, against S. epidermidis and S. aureus, with the MBC values matching the MIC values. Cytotoxicity tests showed no toxicity at concentrations below 75 µg/mL for GF-17 and 150 µg/mL for FK-16. Hemolytic activity was below 1% at 18.75 µg/mL for GF-17 and 75 µg/mL for FK-16. These AMPs showed no synergistic effects with antibiotics and no resistance development. FK-16 and GF-17 effectively removed biofilms, particularly against S. epidermidis. Incorporating these AMPs into surgical materials (hydrogels, cements, etc.) could enhance infection control in orthopedic procedures, warranting further in vivo studies. Full article
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Graphical abstract
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<p>Effect of selected AMPs at different concentrations on the viability of NIH-3T3 fibroblast compared to cells cultured in CM (NC) and CM supplemented with 0.1% Triton-X100 (PC) assessed by means of the MTT assay. Red dotted lines represent the NC mean value of Abs (0.23). Statistical significance: <span class="html-italic">p</span> &lt; 0.0001 (***).</p>
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<p>Hemolytic activity expressed as a percentage (%). Red dotted line reports the cut off range as ≤1%.</p>
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<p>MIC (<b>A</b>–<b>C</b>) and MBC (<b>D</b>–<b>F</b>) values comparing cLL-37, GF-17 and FK-16. Data were grouped per bacterial genera: Staphylococci (<b>A</b>,<b>D</b>), <span class="html-italic">P. aeruginosa</span> (<b>B</b>,<b>E</b>), <span class="html-italic">E. coli</span> (<b>C</b>,<b>F</b>) and reported as mean ± SEM. Significance, where present, is shown. * <span class="html-italic">p</span> ≤ 0.05; ** <span class="html-italic">p</span> ≤ 0.01; *** <span class="html-italic">p</span> ≤ 0.001; **** <span class="html-italic">p</span> ≤ 0.0001.</p>
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<p>Changes in the MIC values of FK-16 and GF-17 on different strains.</p>
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<p>CLSM images of 72-h-old biofilms produced by <span class="html-italic">S. epidermidis</span> ATCC 35984 and GOI1153754-03-14, <span class="html-italic">S. aureus</span> ATCC 49230 and Sau89. In the columns, the untreated biofilm and biofilms after treatments with FK-16, GF-17 and vancomycin are depicted. Scale bars: 100 μm. Red arrows highlight the biofilm disruption by vancomycin, while yellow arrows show the abundance of dead (red) bacteria in the case of FK-16 and GF-17 on <span class="html-italic">S. epidermidis</span> GOI1153754-03-04 biofilms.</p>
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<p>Histograms show the mean ± SD of relative mortality of each treatment in comparison with the controls (untreated). <span class="html-italic">p</span> values are shown where the differences were statistically significant.</p>
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20 pages, 4065 KiB  
Article
Laundry Isolate Delftia sp. UBM14 Capable of Biodegrading Industrially Relevant Aminophosphonates
by Ramona Riedel, Karsten Meißner, Arne Kaschubowski, Dirk Benndorf, Marion Martienssen and Burga Braun
Microorganisms 2024, 12(8), 1664; https://doi.org/10.3390/microorganisms12081664 - 13 Aug 2024
Viewed by 252
Abstract
Phosphonates such as ethylenediaminetetra (methylenephosphonic acid) (EDTMP) and aminotris (methylenephosphonic acid) (ATMP) are used every day in water treatment processes or in household products. Their consumption is still increasing, regardless of the debates on their environmental impact. Here, the microbial characterisation and determination [...] Read more.
Phosphonates such as ethylenediaminetetra (methylenephosphonic acid) (EDTMP) and aminotris (methylenephosphonic acid) (ATMP) are used every day in water treatment processes or in household products. Their consumption is still increasing, regardless of the debates on their environmental impact. Here, the microbial characterisation and determination of the biodegradation potential of selected industrially relevant phosphonates for the isolate Delftia sp. UMB14 is reported. The opportunistic strain was isolated from a biofilm that was derived from a conventional washing machine using conventional detergents containing phosphonates. In antimicrobial susceptibility testing, the strain was only susceptible to sulfonamide, tetracycline, and chloramphenicol. Physiological and biochemical characteristics were determined using the BIOLOG EcoPlate assay. Most importantly, the strain was shown to convert D-malic acid and D-mannitol, as confirmed for strains of Delftia lacustris, and thus the new isolate could be closely related. Biodegradation tests with different phosphonates showed that the strain preferentially degrades ATMP and EDTMP but does not degrade glyphosate (GS) and amino (methylphosphonic acid) (AMPA). A specific gene amplification confirmed the presence of phnX (phosphonoacetaldehyde hydrolase) and the absence of PhnJ (the gene for the core component of C–P lyase). The presence of PhnCDE is strongly suggested for the strain, as it is common in Delftia lacustris species. Full article
(This article belongs to the Section Environmental Microbiology)
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Figure 1
<p>Chemical structures of the phosphonates used in the degradation tests.</p>
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<p>Biodegradation test of selected phosphonates with strain <span class="html-italic">Delftia</span> sp. UMB14. The phosphonates were dosed at 20 mgP L<sup>−1</sup> to ensure comparability, except of HEDP, to prevent the precipitation of the latter. (<b>A1</b>,<b>A2</b>) ATMP. (<b>B1</b>,<b>B2</b>) EDTMP. (<b>C1</b>,<b>C2</b>) IDMP. (<b>D1</b>,<b>D2</b>) HEDP. (<b>E1</b>,<b>E2</b>) DTPMP. (<b>F1</b>,<b>F2</b>) GS. (<b>G1,G2</b>) AMPA. (<b>H1</b>,<b>H2</b>) Positive control KH<sub>2</sub>PO<sub>4</sub>. The optical density was measured at 660 nm.</p>
Full article ">Figure 2 Cont.
<p>Biodegradation test of selected phosphonates with strain <span class="html-italic">Delftia</span> sp. UMB14. The phosphonates were dosed at 20 mgP L<sup>−1</sup> to ensure comparability, except of HEDP, to prevent the precipitation of the latter. (<b>A1</b>,<b>A2</b>) ATMP. (<b>B1</b>,<b>B2</b>) EDTMP. (<b>C1</b>,<b>C2</b>) IDMP. (<b>D1</b>,<b>D2</b>) HEDP. (<b>E1</b>,<b>E2</b>) DTPMP. (<b>F1</b>,<b>F2</b>) GS. (<b>G1,G2</b>) AMPA. (<b>H1</b>,<b>H2</b>) Positive control KH<sub>2</sub>PO<sub>4</sub>. The optical density was measured at 660 nm.</p>
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<p>Growth test with strain <span class="html-italic">Delftia</span> sp. UMB14 and different sole phosphorus sources. (<b>A</b>) KH<sub>2</sub>PO<sub>4</sub> as sole P source. (<b>B</b>) ATMP as sole P source. (<b>C</b>) HEDP as sole P source. Test was running in triplicates for each test condition. The optical density was measured at 660 nm.</p>
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<p>Adaptation test with inocula with preconditioned bacteria either grown on ATMP or EDTMP. (<b>A</b>) Growth test on different phosphonates with pre-inoculated bacteria on ATMP as P source. (<b>B</b>) Growth test on different phosphonates with pre-inoculated bacteria on EDTMP as P source. The optical density was measured at 660 nm.</p>
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16 pages, 1563 KiB  
Article
Enhancing Antibiotic Efficacy and Combating Biofilm Formation: Evaluating the Synergistic Potential of Origanum vulgare Essential Oil against Multidrug-Resistant Gram-Negative Bacteria
by Bilal Saoudi, Karim Bariz, Sarah Saci, Yousra Belounis, Hakima Ait Issad, Mohamed Abbaci, Mohamed Abou Mustapha, El-Hafid Nabti, Rawaf Alenazy, Mohammed Sanad Alhussaini, Abdulrahman A. I. Alyahya, Mohammed Alqasmi, Maryam S. Alhumaidi, Fawaz M. Almufarriji and Karim Houali
Microorganisms 2024, 12(8), 1651; https://doi.org/10.3390/microorganisms12081651 - 12 Aug 2024
Viewed by 384
Abstract
Multidrug-resistant (MDR) Gram-negative bacteria remain a global public health issue due to the barrier imposed by their outer membrane and their propensity to form biofilms. It is becoming imperative to develop new antibacterial strategies. In this context, this study aims to evaluate the [...] Read more.
Multidrug-resistant (MDR) Gram-negative bacteria remain a global public health issue due to the barrier imposed by their outer membrane and their propensity to form biofilms. It is becoming imperative to develop new antibacterial strategies. In this context, this study aims to evaluate the antibacterial efficacy of Origanum vulgare essential oil (OEO), alone and in combination with antibiotics, as well as its antibiofilm action against multidrug-resistant Gram-negative strains. OEO components were identified by gas chromatography-mass spectrometry (GC-MS), and antibacterial activity was assessed using the agar diffusion test and the microdilution method. Interactions between OEO and antibiotics were examined using the checkerboard method, while antibiofilm activity was analyzed using the crystal violet assay. Chemical analysis revealed that carvacrol was the major compound in OEO (61.51%). This essential oil demonstrated activity against all the tested strains, with inhibition zone diameters (IZDs) reaching 32.3 ± 1.5 mm. The combination of OEO with different antibiotics produced synergistic and additive effects, leading to a reduction of up to 98.44% in minimum inhibitory concentrations (MICs). In addition, this essential oil demonstrated an ability to inhibit and even eradicate biofilm formation. These results suggest that OEO could be exploited in the development of new molecules, combining its metabolites with antibiotics. Full article
(This article belongs to the Special Issue Healthcare-Associated Infections and Antimicrobial Therapy)
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Figure 1
<p>Checkerboard assays using OEO and antibiotics. Blue wells indicate growth inhibition. Pink wells indicate growth. H1 well is used for sterility control, and the 12th-column is for growth control. H2–H11 contains antibiotic alone, while G1–A1 contains the OEO alone. All the other wells contain combinations of antibiotic and OEO. (<b>A</b>) Synergistic combination between OEO and CIP against <span class="html-italic">A. baumannii</span> 14889 strain. (<b>B</b>) Additive effect observed between OEO and CTX against <span class="html-italic">E. coli</span> 45 strain.</p>
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<p>Percentages of inhibition of biofilm formation (blue) and eradication of preformed biofilm (orange) by OEO against tested bacterial strains.</p>
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15 pages, 3885 KiB  
Article
Green Synthesis of Cobalt-Doped CeFe2O5 Nanocomposites Using Waste Gossypium arboreum L. Stalks and Their Application in the Removal of Toxic Water Pollutants
by Saloni Koul, Mamata Singhvi and Beom Soo Kim
Nanomaterials 2024, 14(16), 1339; https://doi.org/10.3390/nano14161339 - 12 Aug 2024
Viewed by 365
Abstract
Currently, there is an increasing need to find new ways to purify water by eliminating bacterial biofilms, textile dyes, and toxic water pollutants. These contaminants pose significant risks to both human health and the environment. To address this issue, in this study, we [...] Read more.
Currently, there is an increasing need to find new ways to purify water by eliminating bacterial biofilms, textile dyes, and toxic water pollutants. These contaminants pose significant risks to both human health and the environment. To address this issue, in this study, we have developed an eco-friendly approach that involves synthesizing a cobalt-doped cerium iron oxide (CCIO) nanocomposite (NC) using an aqueous extract of Gossypium arboreum L. stalks. The resulting nanoparticles can be used to effectively purify water and tackle the challenges associated with these harmful pollutants. Nanoparticles excel in water pollutant removal by providing a high surface area for efficient adsorption, versatile design for the simultaneous removal of multiple contaminants, catalytic properties for organic pollutant degradation, and magnetic features for easy separation, offering cost-effective and sustainable water treatment solutions. A CCIO nanocomposite was synthesized via a green co-precipitation method utilizing biomolecules and co-enzymes extracted from the aqueous solution of Gossypium arboreum L. stalk. This single-step synthesis process was accomplished within a 5-h reaction period. Furthermore, the synthesis of nanocomposites was confirmed by various characterization techniques such as Fourier-transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), dynamic light scattering (DLS), and energy dispersive X-ray (EDX) technology. CCIO NCs were discovered to have a spherical shape and an average size of 40 nm. Based on DLS zeta potential analysis, CCIO NCs were found to be anionic. CCIO NCs also showed significant antimicrobial and antioxidant activity. Overall, considering their physical and chemical properties, the application of CCIO NCs for the adsorption of various dyes (~91%) and water pollutants (chromium = ~60%) has been considered here since they exhibit great adsorption capacity owing to their microporous structure, and represent a step forward in water purification. Full article
(This article belongs to the Special Issue Nanomaterials in Water Applications)
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Figure 1
<p>Illustration of the application of synthesized CCIO NCs in water pollutant removal.</p>
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<p>Characterization of CCIO NCs using (<b>A</b>) SEM, (<b>B</b>) TEM, (<b>C</b>) EDX, (<b>D</b>) FTIR, (<b>E</b>) zeta potential, and (<b>F</b>) XRD.</p>
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<p>UV-Vis spectra of: (<b>A</b>) SF at different concentrations (1–5 mg/mL), (<b>B</b>) MG at different concentrations (1–5 mg/mL), (<b>C</b>) MB at (1–5 mg/mL), and (<b>D</b>) the regeneration efficiency (%) of CCIO NCs using SF, MG, and MB dyes.</p>
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<p>UV-Vis spectra for analyzing the adsorption capacity of CCIO NPs against chromium (<span class="html-small-caps">VI</span>).</p>
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<p>UV-Vis spectra of SF, MG, and MB at 10 mg/mL concentration.</p>
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<p>Antioxidant activity of CCIO NCs at different concentrations (1–5 mg/mL).</p>
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<p>Antimicrobial activity of synthesized CCIO NPs against <span class="html-italic">E. coli</span> and <span class="html-italic">S. aureus</span>.</p>
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<p>Regeneration efficiency of reused CCIO NCs, using SF dye.</p>
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<p>Plausible mechanism of action of CCIO NCs in dye adsorption.</p>
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39 pages, 12019 KiB  
Review
“Stop, Little Pot” as the Motto of Suppressive Management of Various Microbial Consortia
by Elena Efremenko, Nikolay Stepanov, Olga Senko, Olga Maslova, Ilya Lyagin, Maksim Domnin and Aysel Aslanli
Microorganisms 2024, 12(8), 1650; https://doi.org/10.3390/microorganisms12081650 - 12 Aug 2024
Viewed by 266
Abstract
The unresolved challenges in the development of highly efficient, stable and controlled synthetic microbial consortia, as well as the use of natural consortia, are very attractive for science and technology. However, the consortia management should be done with the knowledge of how not [...] Read more.
The unresolved challenges in the development of highly efficient, stable and controlled synthetic microbial consortia, as well as the use of natural consortia, are very attractive for science and technology. However, the consortia management should be done with the knowledge of how not only to accelerate but also stop the action of such “little pots”. Moreover, there are a lot of microbial consortia, the activity of which should be suppressively controlled. The processes, catalyzed by various microorganisms being in complex consortia which should be slowed down or completely cancelled, are typical for the environment (biocorrosion, landfill gas accumulation, biodegradation of building materials, water sources deterioration etc.), industry (food and biotechnological production), medical practice (vaginitis, cystitis, intestinal dysbiosis, etc.). The search for ways to suppress the functioning of heterogeneous consortia in each of these areas is relevant. The purpose of this review is to summarize the general trends in these studies regarding the targets and new means of influence used. The analysis of the features of the applied approaches to solving the main problem confirms the possibility of obtaining a combined effect, as well as selective influence on individual components of the consortia. Of particular interest is the role of viruses in suppressing the functioning of microbial consortia of different compositions. Full article
(This article belongs to the Special Issue Latest Review Papers in Antimicrobial Agents and Resistance 2024)
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Figure 1
<p>Scheme of different types of polymicrobial biofilms containing various fungi: microorganisms can coexist having a layer-by-layer arrangement (<b>A</b>), they can be mixed partially (<b>B</b>) or deeply (<b>C</b>) when one type of cell is located in the upper layer, and another type in the lower (<b>D</b>); the fungal spores can appear in the biofilm matrix (<b>E</b>), or fungi can cover the layers formed by other cells (<b>F</b>).</p>
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<p>Basic methods and means of suppressive effects on polymicrobial consortia containing filamentous fungi or yeasts.</p>
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<p>Chemical structures of some low-weight organic effectors investigated for suppression of mixed consortia.</p>
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<p>Representation of some AMPs mentioned in <a href="#microorganisms-12-01650-t002" class="html-table">Table 2</a>. Structures were obtained from PDB (4MGP and 5NNM) and Uniprot (P01507) and visualized using PyMol (ver. 1.7.6, Schrödinger, LLC, New York, NY, USA).</p>
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<p>Structures of some metal-containing NPs mentioned in <a href="#microorganisms-12-01650-t004" class="html-table">Table 4</a>. The corresponding crystal structures were obtained from Cambridge Crystallographic Data Centre (CCDC 1741252, 1744485, 1763757, 1612598, 1481933 and 13950), expanded to around 1.2–1.5 nm in Mercury (v. 4.2.0, CCDC, Cambridge, UK) and visualized in PyMol. In this regard, many researchers propose their use to suppress the metabolism of pathogens in biofilms, but it has been found that consortia formed in the presence of heavy metals are tolerant to their negative effects [<a href="#B7-microorganisms-12-01650" class="html-bibr">7</a>].</p>
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<p>Structures of some enzymes mentioned in <a href="#microorganisms-12-01650-t006" class="html-table">Table 6</a>: hyaluronoglucosaminidase (PDB 1FCV) (<b>A</b>), protease (PDB 3PVK) (<b>B</b>), chitinase (PDB 6BT9) (<b>C</b>), β-glucuronidase (PDB 5C70) (<b>D</b>), RNAse (PDB 5ARK) (<b>E</b>), DNAse (PDB 1DNK) (<b>F</b>).</p>
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<p>Structures of some representative viruses mentioned in <a href="#microorganisms-12-01650-t009" class="html-table">Table 9</a>: <span class="html-italic">Penicillium stoloniferum</span> partitivirus F (PDB 3ES5) (<b>A</b>), <span class="html-italic">Cryphonectria nitschkei</span> chrysovirus 1 (EMD-2062) (<b>B</b>), <span class="html-italic">Fusarium poae</span> virus 1 (EMD-5171) (<b>C</b>).</p>
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<p>Network analysis of microbial consortia susceptible to antimicrobial treatment. The lines connect the microorganisms from different consortia discussed in the <a href="#microorganisms-12-01650-t001" class="html-table">Table 1</a>, <a href="#microorganisms-12-01650-t002" class="html-table">Table 2</a>, <a href="#microorganisms-12-01650-t003" class="html-table">Table 3</a>, <a href="#microorganisms-12-01650-t004" class="html-table">Table 4</a>, <a href="#microorganisms-12-01650-t005" class="html-table">Table 5</a>, <a href="#microorganisms-12-01650-t006" class="html-table">Table 6</a> and <a href="#microorganisms-12-01650-t007" class="html-table">Table 7</a>. Names of bacterial, fungal and yeast strains are labeled by black, pink and magenta color, respectively. The size of nodes and their labels is proportional to the ranking degree; the thickness of edges is proportional to their weight. Nodes belonging to different modularity classes are colored in groups accordingly. The analysis was realized in Gephi (v.0.10.1, available at <a href="https://gephi.org/users/download/" target="_blank">https://gephi.org/users/download/</a>, accessed on 12 May 2024).</p>
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<p>Network analysis of microbial consortia resistant to antimicrobial treatment. Names of bacterial, fungal and microalgal strains are labeled by black, pink and green color, respectively. The size of nodes and their labels is proportional to the ranking degree; the thickness of edges is proportional to their weight. Nodes belonging to different modularity classes are colored in groups accordingly. The analysis was realized in Gephi (v.0.10.1, accessible at <a href="https://gephi.org/users/download/" target="_blank">https://gephi.org/users/download/</a>, accessed on 12 May 2024).</p>
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<p>Percentage distribution of modern developments based on various approaches (I—non-peptide antimicrobial compounds of plant and animal origin, II—AMPs, III—enzymes, IV—microorganisms and their metabolites, V—various antimicrobial agents and disinfectants, VI—heavy metal ions and metal nanoparticles, VII—physical and chemical methods of influence, VIII—combined methods for inhibition of polymicrobial consortia) by areas of proposed application: <span style="color:#33CCCC">■</span>—medicine, <span style="color:blue">■</span>—preservation of cultural heritage sites, <span style="color:yellow">■</span>—fight against biocorrosion, <span style="color:#FF99CC">■</span>—food industry, <span style="color:lime">■</span>—agriculture, <span style="color:#FFCC00">■</span>—water purification and soil bioremediation. The figure was constructed using the data presented in <a href="#microorganisms-12-01650-t001" class="html-table">Table 1</a>, <a href="#microorganisms-12-01650-t002" class="html-table">Table 2</a>, <a href="#microorganisms-12-01650-t003" class="html-table">Table 3</a>, <a href="#microorganisms-12-01650-t004" class="html-table">Table 4</a>, <a href="#microorganisms-12-01650-t005" class="html-table">Table 5</a>, <a href="#microorganisms-12-01650-t006" class="html-table">Table 6</a> and <a href="#microorganisms-12-01650-t007" class="html-table">Table 7</a>.</p>
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20 pages, 5019 KiB  
Article
Enhancing Deer Sous Vide Meat Shelf Life and Safety with Eugenia caryophyllus Essential Oil against Salmonella enterica
by Miroslava Kačániová, Stefania Garzoli, Anis Ben Hsouna, Zhaojun Ban, Joel Horacio Elizondo-Luevano, Maciej Ireneusz Kluz, Rania Ben Saad, Peter Haščík, Natália Čmiková, Božena Waskiewicz-Robak, Ján Kollár and Alessandro Bianchi
Foods 2024, 13(16), 2512; https://doi.org/10.3390/foods13162512 - 12 Aug 2024
Viewed by 437
Abstract
Modern lifestyles have increased the focus on food stability and human health due to evolving industrial goals and scientific advancements. Pathogenic microorganisms significantly challenge food quality, with Salmonella enterica and other planktonic cells capable of forming biofilms that make them more resistant to [...] Read more.
Modern lifestyles have increased the focus on food stability and human health due to evolving industrial goals and scientific advancements. Pathogenic microorganisms significantly challenge food quality, with Salmonella enterica and other planktonic cells capable of forming biofilms that make them more resistant to broad-spectrum antibiotics. This research examined the chemical composition and antibacterial and antibiofilm properties of the essential oil from Eugenia caryophyllus (ECEO) derived from dried fruits. GC-MS analyses identified eugenol as the dominant component at 82.7%. Additionally, the study aimed to extend the shelf life of sous vide deer meat by applying a plant essential oil and inoculating it with S. enterica for seven days at 4 °C. The essential oil demonstrated strong antibacterial activity against S. enterica. The ECEO showed significant antibiofilm activity, as indicated by the MBIC crystal violet test results. Data from MALDI-TOF MS analysis revealed that the ECEO altered the protein profiles of bacteria on glass and stainless-steel surfaces. Furthermore, the ECEO was found to have a beneficial antibacterial effect on S. enterica. In vacuum-packed sous vide red deer meat samples, the anti-Salmonella activity of the ECEO was slightly higher than that of the control samples. These findings underscore the potential of the ECEO’s antibacterial and antibiofilm properties in food preservation and extending the shelf life of meat. Full article
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<p>GC-MS chromatogram of the ECEO.</p>
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<p>Representative MALDI-TOF mass spectra of <span class="html-italic">S. enterica</span>: (<b>A</b>) 3rd day; (<b>B</b>) 5th day; (<b>C</b>) 7th day; (<b>D</b>) 9th day; (<b>E</b>) 12th day; (<b>F</b>) 14th day. SE = <span class="html-italic">S. enterica</span>; G = glass; S = stainless-steel; and PC = planktonic cells.</p>
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<p>Representative MALDI-TOF mass spectra of <span class="html-italic">S. enterica</span>: (<b>A</b>) 3rd day; (<b>B</b>) 5th day; (<b>C</b>) 7th day; (<b>D</b>) 9th day; (<b>E</b>) 12th day; (<b>F</b>) 14th day. SE = <span class="html-italic">S. enterica</span>; G = glass; S = stainless-steel; and PC = planktonic cells.</p>
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<p>Representative MALDI-TOF mass spectra of <span class="html-italic">S. enterica</span>: (<b>A</b>) 3rd day; (<b>B</b>) 5th day; (<b>C</b>) 7th day; (<b>D</b>) 9th day; (<b>E</b>) 12th day; (<b>F</b>) 14th day. SE = <span class="html-italic">S. enterica</span>; G = glass; S = stainless-steel; and PC = planktonic cells.</p>
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<p>Dendrogram of <span class="html-italic">S. enterica</span> generated using MSPs of the planktonic cells and the control. SE = <span class="html-italic">S. enterica</span>; C = glass; S = stainless-steel; and PC = planktonic cells.</p>
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<p>Total viable count (log CFU/g) of sous vide deer meat samples after 1 and 7 days of storage, treated in a water bath at temperatures between 50 and 65 °C for 5 to 20 min. Data are the mean (bars indicate ± SD) of 3 deer meat samples. Control: deer meat samples placed in polyethylene bags without vacuum. Control vacuum: deer meat samples vacuum-packed in polyethylene bags. Essential oil: deer meat samples treated with 1% ECEO and vacuum-packed. <span class="html-italic">Salmonella enterica</span>: deer meat samples inoculated with <span class="html-italic">S. enterica</span> and vacuum-packed. Essential oil + <span class="html-italic">Salmonella enterica:</span> deer meat samples treated with 1% ECEO and inoculated with <span class="html-italic">S. enterica</span> and vacuum-packed.</p>
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<p>Total coliform bacteria (log CFU/g) of sous vide deer meat samples after 1 and 7 days of storage, treated in a water bath at temperatures between 50 and 65 °C for 5 to 20 min. Data are the mean (bars indicate ± SD) of 3 deer meat samples. Control: deer meat samples placed in polyethylene bags without vacuum. Control vacuum: deer meat samples vacuum-packed in polyethylene bags. Essential oil: deer meat samples treated with 1% ECEO and vacuum-packed. <span class="html-italic">Salmonella enterica</span>: deer meat samples inoculated with <span class="html-italic">S. enterica</span> and vacuum-packed. Essential oil + <span class="html-italic">Salmonella enterica:</span> deer meat samples treated with 1% ECEO and inoculated with <span class="html-italic">S. enterica</span> and vacuum-packed.</p>
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<p><span class="html-italic">Salmonella enterica</span> count (log CFU/g) of sous vide deer meat samples after 1 and 7 days of storage, treated in a water bath at temperatures between 50 and 65 °C for 5 to 20 min. Data are the mean (bars indicate ± SD) of 3 deer meat samples. Control: deer meat samples placed in polyethylene bags without vacuum. Control vacuum: deer meat samples vacuum-packed in polyethylene bags. Essential oil: deer meat samples treated with 1% ECEO and vacuum-packed. <span class="html-italic">Salmonella enterica</span>: deer meat samples inoculated with <span class="html-italic">S. enterica</span> and vacuum-packed. Essential oil + <span class="html-italic">Salmonella enterica:</span> deer meat samples treated with 1% ECEO and inoculated with <span class="html-italic">S. enterica</span> and vacuum-packed.</p>
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<p>Krona chart: Isolated species, genera, and families from deer sous vide meat at 1 day.</p>
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<p>Krona chart: Isolated species, genera, and families from deer sous vide meat after 7 days.</p>
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11 pages, 3094 KiB  
Article
Isolation of Electrochemically Active Bacteria from an Anaerobic Digester Treating Food Waste and Their Characterization
by Daichi Yoshizu, Soranosuke Shimizu, Miyu Tsuchiya, Keisuke Tomita, Atsushi Kouzuma and Kazuya Watanabe
Microorganisms 2024, 12(8), 1645; https://doi.org/10.3390/microorganisms12081645 - 11 Aug 2024
Viewed by 414
Abstract
Studies have used anaerobic-digester sludge and/or effluent as inocula for bioelectrochemical systems (BESs), such as microbial fuel cells (MFCs), for power generation, while limited studies have isolated and characterized electrochemically active bacteria (EAB) that inhabit anaerobic digesters. In the present work, single-chamber MFCs [...] Read more.
Studies have used anaerobic-digester sludge and/or effluent as inocula for bioelectrochemical systems (BESs), such as microbial fuel cells (MFCs), for power generation, while limited studies have isolated and characterized electrochemically active bacteria (EAB) that inhabit anaerobic digesters. In the present work, single-chamber MFCs were operated using the anaerobic-digester effluent as the sole source of organics and microbes, and attempts were made to isolate EAB from anode biofilms in MFCs by repeated anaerobic cultivations on agar plates. Red colonies were selected from those grown on the agar plates, resulting in the isolation of three phylogenetically diverse strains affiliated with the phyla Bacillota, Campylobacterota and Deferribacterota. All these strains are capable of current generation in pure-culture BESs, while they exhibit different electrochemical properties as assessed by cyclic voltammetry. The analyses of their cell-free extracts show that cytochromes are abundantly present in their cells, suggesting their involvement in current generation. The results suggest that anaerobic digesters harbor diverse EAB, and it would be of interest to examine their ecological niches in anaerobic digestion. Full article
(This article belongs to the Section Microbial Biotechnology)
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<p>Enrichment of EAB in MFCs. (<b>A</b>) Time courses of cell voltages. The external resistor of MFC5 was changed to 500 Ω on day 21. (<b>B</b>) Metabarcoding of bacteria in anode biofilm (<b>A</b>,<b>B</b>) and planktonic microbes (PM) in MFCs. OTUs were assigned to family-level taxonomic groups, and major families were compared. When an OTU is not affiliated with a known family, it is assigned to a taxonomic group of a higher rank.</p>
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<p>Colonies of strains ADMFC1 (<b>A</b>), ADMFC2 (<b>B</b>) and ADMFC3 (<b>C</b>) formed on DSM826 ager plates.</p>
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<p>Phylogenetic trees based on 16S rRNA gene sequences showing relationships between the isolated strains (<b>A</b>, ADMFC1; <b>B</b>, ADMFC2; <b>C</b>, ADMFC3) and close relatives. Accession numbers for the sequences retrieved from the databases are given in parentheses. The numbers at branch nodes are bootstrap values (per 100 trials); only values greater than 50 are shown. The scale bars indicate 0.02 substitution per sites.</p>
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<p>Current generation by strains ADMFC1 (<b>A</b>), ADMFC2 (<b>B</b>) and ADMFC3 (<b>C</b>) in BESs with 10 mM acetate as the electron donor. Anode potentials were poised at +0.4 V vs. SHE.</p>
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<p>Electrochemical properties of strains ADMFC1, ADMFC2 and ADMFC3, as assessed by comparative CV analyses in the presence of acetate as the electron donor. Curves obtained in the second cycles are shown.</p>
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<p>Differential spectra (reduced minus oxidized forms) for detergent-solubilized cell-free extracts of strains ADMFC1 (<b>A</b>), ADMFC2 (<b>B</b>) and ADMFC3 (<b>C</b>). For comparison, a spectrum for <span class="html-italic">G. sulfurreducens</span> is also shown in panel <b>D</b>.</p>
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10 pages, 2763 KiB  
Communication
Long-Term Stability and Efficacy of NCT Solutions
by Gabriel J. Staudinger, Zach M. Thomas, Sarah E. Hooper, Jeffrey F. Williams and Lori I. Robins
Int. J. Mol. Sci. 2024, 25(16), 8745; https://doi.org/10.3390/ijms25168745 - 10 Aug 2024
Viewed by 336
Abstract
To realize the potential for the use of N-chlorotaurine (NCT) in healthcare, a better understanding of the long-term stability of the compound in water is needed. An array of analytical procedures is required that can measure changes in NCT concentration over time [...] Read more.
To realize the potential for the use of N-chlorotaurine (NCT) in healthcare, a better understanding of the long-term stability of the compound in water is needed. An array of analytical procedures is required that can measure changes in NCT concentration over time and allow for the detection and identification of contaminants and likely degradation end products. We used UV-Vis and NMR spectroscopy, HPLC, and LCMS to establish the stability of NCT in solutions subjected to prolonged ambient and elevated temperatures. Stability proved to be dependent on concentration with half-lives of ~120 days and ~236 days for 1% and 0.5% solutions of NCT at ~20 °C. Regardless of initial pH, all solutions shifted toward and maintained a pH of ~8.3 at 20 °C and 40 °C. NCT at 500 µg/mL and 250 µg /mL inhibited biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus but did not disperse established biofilms. NCT exposure to the biofilms had profound effects on the viability of both bacteria, reducing live organisms by >90%. Exposure of Interleukin-6 (IL-6) to 11 µM NCT reduced the binding of IL-6 to an immobilized specific antibody by ~48%, which is 5× the amount required for HOCl to bring about the same effect in this test system. Our data demonstrate the potency of the compound as an antimicrobial agent with potential benefits in the management of infected chronic wounds and suggest that NCT may contribute to anti-inflammatory processes in vivo by direct modification of cytokine mediators. Full article
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<p>UV-Vis spectroscopy, IR spectroscopy, and <sup>13</sup>C NMR spectroscopy of NCT. (<b>A</b>) UV–Visible spectroscopy wavelength scans of NCT (blue), N,N-dichlorotaurine (black), and taurine (teal). (<b>B</b>) IR spectrum of taurine (black) and NCT (blue). (<b>C</b>) <sup>13</sup>C NMR spectrum of NCT (black, singlets at 50.74 and 48.96 ppm) and taurine (gray, singlets at 47.42 and 35.38 ppm).</p>
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<p>HPLC and LCMS analysis of NCT. (<b>A</b>) HPLC spectrum of NCT (1 mg/mL) at 252 nm. The area under the curve was calculated and purity was determined to be &gt;95%. (<b>B</b>) UV-Vis wavelength trace of NCT detected by the LCMS. (<b>C</b>) TIC data for the corresponding LCMS peak at 252 nm. (<b>D</b>) MS data for the corresponding TIC peak at 252 nm.</p>
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<p>Stability of aqueous NCT solutions at 1%, 0.5%, and 0.25% at ambient and elevated temperatures. (<b>A</b>) Starting pH 9.5 at room temperature. (<b>B</b>) Starting pH 7 at 40 °C. (<b>C</b>) Starting pH 8 at 40 °C. (<b>D</b>) Starting pH 9.5 at 40 °C. Each condition was repeated three times. Additional stability data for these trials are available in <a href="#app1-ijms-25-08745" class="html-app">Figure S1</a>. All 1% solutions are in blue; 0.5% in green; and 0.25% in black. A single-factor ANOVA was used to test for differences in starting pH values and for differences in concentrations.</p>
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<p>pH of aqueous NCT solutions at 1% (circle), 0.5% (square), and 0.25% (triangle). (<b>A</b>) Solutions starting at a pH value of 7. (<b>B</b>) Solutions starting at a pH value of 8. (<b>C</b>) Solutions starting at a pH value of 9.5. Additional pH data for the three trials are available in <a href="#app1-ijms-25-08745" class="html-app">Figure S2</a>.</p>
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<p>ELISA test results for IL-6 binding to an IL-6 specific antibody after treatment with concentrations of NCT ranging from 0 to 2.76 mM. A single-factor ANOVA was used to test for differences in binding at all concentrations of NCT tested.</p>
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<p>Viability of remaining <span class="html-italic">P. aeruginosa</span> (black) and <span class="html-italic">S. aureus</span> (gray) biofilm biomass after treatment with various NCT concentrations. Significant reductions in viability were calculated using ANOVA and Tukey’s post hoc analysis. The significant threshold concentrations were 0.004% for <span class="html-italic">S. aureus</span> and 0.06% for <span class="html-italic">P. aeruginosa</span>.</p>
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15 pages, 4285 KiB  
Article
Particulate 3D Hydrogels of Silk Fibroin-Pluronic to Deliver Curcumin for Infection-Free Wound Healing
by Azin Khodaei, Narges Johari, Fatemeh Jahanmard, Leonardo Cecotto, Sadjad Khosravimelal, Hamid Reza Madaah Hosseini, Reza Bagheri, Ali Samadikuchaksaraei and Saber Amin Yavari
Biomimetics 2024, 9(8), 483; https://doi.org/10.3390/biomimetics9080483 - 10 Aug 2024
Viewed by 529
Abstract
Skin is the largest protective tissue of the body and is at risk of damage. Hence, the design and development of wound dressing materials is key for tissue repair and regeneration. Although silk fibroin is a known biopolymer in tissue engineering, its degradation [...] Read more.
Skin is the largest protective tissue of the body and is at risk of damage. Hence, the design and development of wound dressing materials is key for tissue repair and regeneration. Although silk fibroin is a known biopolymer in tissue engineering, its degradation rate is not correlated with wound closure rate. To address this disadvantage, we mimicked the hierarchical structure of skin and also provided antibacterial properties; a hydrogel with globular structure consisting of silk fibroin, pluronic F127, and curcumin was developed. In this regard, the effect of pluronic and curcumin on the structural and mechanical properties of the hydrogel was studied. The results showed that curcumin affected the particle size, crystallinity, and ultimate elongation of the hydrogels. In vitro assays confirmed that the hydrogel containing curcumin is not cytotoxic while the diffused curcumin and pluronic provided a considerable bactericidal property against Methicillin-resistant Staphylococcus aureus. Interestingly, presence of pluronic caused more than a 99% reduction in planktonic and adherent bacteria in the curcumin-free hydrogel groups. Moreover, curcumin improved this number further and inhibited bacteria adhesion to prevent biofilm formation. Overall, the developed hydrogel showed the potential to be used for skin tissue regeneration. Full article
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Graphical abstract
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<p>SEM micrographs of SF, SFP, SFP-A, and SFP-A-Cur in two magnifications (<b>a</b>), size distribution histograms of SFP (<b>b</b>), SFP-A (<b>c</b>), and SFP-A-Cur (<b>d</b>) extracted from the analysis of SEM images using ImageJ software. The cross-section of SFP-A-Cur with gradient pore size indicated with yellow circles in different sizes (<b>e</b>) which can be explained through the schematic of the solution after casting on the salt crystals (<b>f</b>). It shows how salt leaching is applied only in the bottom of the sample jar, while on the surface, a denser layer with a smaller pore size is cast (<b>f</b>).</p>
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<p>FTIR spectrum of SFP-A-Cur sample and all the compounds individually (<b>a</b>), and non-reversing heat flow vector extracted from TMDSC test of the samples containing pluronic, emphasizing on recrystallization temperature (<b>b</b>). The stress-strain plots of experimental groups were extracted from the uniaxial compression test (<b>c</b>) and extracted mechanical properties: compression modulus (<b>d</b>) and ultimate elongation *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 (<b>e</b>).</p>
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<p>OM images of degredated residues in DMEM media (<b>a</b>), degradation profile based on weight loss (<b>b</b>) and pH variations (<b>c</b>) during 14 days of immersion in PBS for different experimental groups, and cumulative release of curcumin during 14 days calculated based on the absorption intensity of 400 nm wavelength, fitted with Korsmeyer–Peppas (<b>d</b>) and one-phase association (<b>e</b>) models.</p>
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<p>The viability of fibroblast cells cultured on the surface of four main experimental groups (<b>a</b>) and different groups with SF/P ratios between 2 and 6 (<b>b</b>). The reported values were normalized based on the control group at each time point—the CLSM images of cultured fibroblast cells stained with cytoskeleton assay. Actins are presented in red, and nuclei are colored in blue (the hydrogel is also colored in some parts with DAPI). Scale bar is equal to 100 µm (<b>c</b>).</p>
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<p>CFU results extracted from counted planktonic, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 (<b>a</b>) and adhesion (<b>b</b>) <span class="html-italic">S. aureus</span>, cultured on the surface of different experimental groups. The OD variations of bacteria suspension cultured in the curcumin release supernatant (<b>c</b>), showing the changes in bacterial growth. The CLSM images of the fluorescent <span class="html-italic">S. aureus</span> adhered to the surface (<b>d</b>), and the bright colonies are detected by ImageJ 1.54g based on prominence &gt;8 in the lower row. Attached colony number extracted by ImageJ analysis.</p>
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13 pages, 2670 KiB  
Review
Advances in Regenerative and Reconstructive Medicine in the Prevention and Treatment of Bone Infections
by Leticia Ramos Dantas, Gabriel Burato Ortis, Paula Hansen Suss and Felipe Francisco Tuon
Biology 2024, 13(8), 605; https://doi.org/10.3390/biology13080605 - 10 Aug 2024
Viewed by 260
Abstract
Reconstructive and regenerative medicine are critical disciplines dedicated to restoring tissues and organs affected by injury, disease, or congenital anomalies. These fields rely on biomaterials like synthetic polymers, metals, ceramics, and biological tissues to create substitutes that integrate seamlessly with the body. Personalized [...] Read more.
Reconstructive and regenerative medicine are critical disciplines dedicated to restoring tissues and organs affected by injury, disease, or congenital anomalies. These fields rely on biomaterials like synthetic polymers, metals, ceramics, and biological tissues to create substitutes that integrate seamlessly with the body. Personalized implants and prosthetics, designed using advanced imaging and computer-assisted techniques, ensure optimal functionality and fit. Regenerative medicine focuses on stimulating natural healing mechanisms through cellular therapies and biomaterial scaffolds, enhancing tissue regeneration. In bone repair, addressing defects requires advanced solutions such as bone grafts, essential in medical and dental practices worldwide. Bovine bone scaffolds offer advantages over autogenous grafts, reducing surgical risks and costs. Incorporating antimicrobial properties into bone substitutes, particularly with metals like zinc, copper, and silver, shows promise in preventing infections associated with graft procedures. Silver nanoparticles exhibit robust antimicrobial efficacy, while zinc nanoparticles aid in infection prevention and support bone healing; 3D printing technology facilitates the production of customized implants and scaffolds, revolutionizing treatment approaches across medical disciplines. In this review, we discuss the primary biomaterials and their association with antimicrobial agents. Full article
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<p>A diagram demonstrating multiple options for doping bone grafts or polymers for 3D printing using metal nanoparticles or antibiotics in bone reconstruction.</p>
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<p>Silver nanoparticles on bone surface used for orthopedic graft.</p>
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<p>Antibiotic-impregnated PLA models with <span class="html-italic">Staphylococcus aureus</span> test.</p>
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<p>Implants with PLA impregnated with antibiotics tested during surgery for hip replacement.</p>
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15 pages, 6620 KiB  
Article
Lactoferrin Affects the Viability of Bacteria in a Biofilm and the Formation of a New Biofilm Cycle of Mannheimia haemolytica A2
by Lucero Ruiz-Mazón, Gerardo Ramírez-Rico and Mireya de la Garza
Int. J. Mol. Sci. 2024, 25(16), 8718; https://doi.org/10.3390/ijms25168718 - 9 Aug 2024
Viewed by 317
Abstract
Respiratory diseases in ruminants are responsible for enormous economic losses for the dairy and meat industry. The main causative bacterial agent of pneumonia in ovine is Mannheimia haemolytica A2. Due to the impact of this disease, the effect of the antimicrobial protein, bovine [...] Read more.
Respiratory diseases in ruminants are responsible for enormous economic losses for the dairy and meat industry. The main causative bacterial agent of pneumonia in ovine is Mannheimia haemolytica A2. Due to the impact of this disease, the effect of the antimicrobial protein, bovine lactoferrin (bLf), against virulence factors of this bacterium has been studied. However, its effect on biofilm formation has not been reported. In this work, we evaluated the effect on different stages of the biofilm. Our results reveal a decrease in biofilm formation when bacteria were pre-incubated with bLf. However, when bLf was added at the start of biofilm formation and on mature biofilm, an increase was observed, which was visualized by greater bacterial aggregation and secretion of biofilm matrix components. Additionally, through SDS-PAGE, a remarkable band of ~80 kDa was observed when bLf was added to biofilms. Therefore, the presence of bLf on the biofilm was determined through the Western blot and Microscopy techniques. Finally, by using Live/Dead staining, we observed that most of the bacteria in a biofilm with bLf were not viable. In addition, bLf affects the formation of a new biofilm cycle. In conclusion, bLf binds to the biofilm of M. haemolytica A2 and affects the viability of bacteria and the formation a new biofilm cycle. Full article
(This article belongs to the Special Issue New Insights into Lactoferrin)
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<p><b>Viability of <span class="html-italic">M. haemolytica</span> A2 with bLf.</b> <span class="html-italic">M. haemolytica</span> incubated with different concentrations of bLf at different times. The concentrations of 3.5 and 6 μM were sub-inhibitory until the 48th hour of incubation. Concentrations of 8 and 9 μM were inhibitory since they showed a significant difference regarding bacteria grown without bLf (NT) <span class="html-italic">p</span> &lt; 0.05. Representative results of three independent experiments.</p>
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<p><b>Inhibitory and stimulatory effect of bLf on the biofilm formation of <span class="html-italic">M. haemolytica</span> A2.</b> (<b>a</b>) Bacteria were pre-incubated overnight with 3.5 and 6 μM bLf, then transferred to a microplate for biofilm formation; after 48 h, bacteria that had previous contact with bLf were no longer able to form a biofilm as bacteria without treatment do, since there was a reduction of 38 and 51% with incubation of 3.5 and 6 μM bLf, respectively. (<b>b</b>) Bacteria culture was transferred to a microplate for biofilm formation, and 3.5 and 6 μM bLf were added; after 48 h, an increase in biofilm was observed compared to the biofilm of untreated bacteria. These results were dependent on bLf concentration, as a significant difference was obtained between the different bLf concentrations used; bovine Lf alone was added to the microplate without biofilm to discard adherence of bLf to the microplate. Statistically significant differences between ratios are indicated (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001). Representative results of three independent experiments.</p>
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<p><b>Stimulatory effect of bLf on a mature biofilm of <span class="html-italic">M. haemolytica</span> A2</b>. Bovine Lf (3.5 and 6 μM) was added to a 48 h biofilm of <span class="html-italic">M. haemolytica</span> A2. After 24 h, an increase in biofilm with bLf was observed, compared to a biofilm of bacteria with no treatment. These results were dependent on bLf concentration, as a significant difference was obtained between both bLf concentrations used, respectively; Lf alone was added to the microplate without biofilm to discard adherence of bLf to the microplate. Statistically significant differences between ratios are indicated (* <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001). Representative results of three independent experiments.</p>
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<p><b>Biofilm observation under a Scanning Electron Microscope.</b> Biofilm of <span class="html-italic">M. haemolytica</span> A2 with no treatment (NT) at a magnification of 500× (<b>a</b>) and 10,000× (<b>b</b>). The blank space shows the surface to which it is attached; biofilm with 3.5 μM bLf at a magnification of 500× (<b>c</b>) and 10,000× (<b>d</b>). The biofilm was observed to a greater extent and structures on the bacterial surface were noticed, compared to the biofilm with no treatment (red arrow). Biofilm with 6 μM bLf at a magnification of 500× (<b>e</b>) and 10,000× (<b>f</b>). A remarkable increase in biofilm was observed, indicated by the lack of blank space, as well as an increase in the structures on the bacterial surface and a mesh-like coating (red arrows). Representative images of three independent samples.</p>
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<p><b>Proteins and carbohydrates of the biofilm matrix of <span class="html-italic">M. haemolytica</span> A2, observed under a laser confocal microscope.</b> Images show proteins of the biofilm without bLf (NT) with the Sypro Ruby stain (<b>a</b>–<b>c</b>), and carbohydrates of the biofilm with the Red Texas stain (<b>d</b>–<b>f</b>); proteins and carbohydrates from biofilm incubated with 3.5 μM bLf (<b>b</b>,<b>e</b>) and 6 μM bLf (<b>c</b>,<b>f</b>) are shown. The intensity of the fluorescence (white arrows) in the biofilms with bLf shows a major amount of these components compared to the biofilm with no treatment. Image magnification: 40×. Representative images of three independent samples.</p>
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<p><b>Results of 10% SDS-PAGE of <span class="html-italic">M. haemolytica</span> A2 biofilm proteins incubated or unincubated with bLf.</b> The protein pattern was analyzed by SDS-PAGE, using samples of 48 h biofilms with or without 3.5 and 6 μM bLf. With this assay, we did not observe differences in the biofilm pattern when bLf was added, in comparison to the biofilm without bLf. In addition, a remarkable band corresponding to the molecular weight of bLf (~80 kDa) was observed where it was added (red asterisks). Representative results of three independent samples.</p>
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<p><b>Presence of bLf in the biofilm of <span class="html-italic">M. haemolytica</span> A2 shown through laser confocal microscopy and Western blot</b>. Images show the distribution of bLf labeled with FITC (green fluorescence) at 3.5 μM (<b>b</b>) and 6 μM (<b>c</b>) throughout the biofilm of <span class="html-italic">M. haemolytica</span> A2, and a negative control where FITC-bLf was added to the plate with no biofilm (<b>a</b>). Western blot using anti-bLf in a biofilm without bLf (NT), with 3.5 or 6 μM bLf and a secondary anti-rabbit-HRP antibody alone as a negative control (<b>d</b>). Representative results of three independent samples.</p>
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<p><b>Bovine Lf affects the viability of bacteria in biofilms with bLf, visualized by Confocal Laser Microscopy.</b> CLM images of bacteria in biofilm for 48 h without bLf (NT) (<b>a</b>–<b>c</b>) and with 3.5 (<b>d</b>–<b>f</b>) or 6 μM (<b>g</b>–<b>i</b>) bLf, showing live bacteria with the Sypro stain (<b>a</b>,<b>d</b>,<b>g</b>) and dead bacteria with the propidium iodide exclusion stain (<b>b</b>,<b>e</b>,<b>h</b>). The merge shows a greater distribution of dead bacteria (visualized by the distribution of yellow fluorescence) in a biofilm with bLf (<b>f</b>,<b>i</b>) than in a biofilm without treatment, where a greater distribution of live bacteria was observed (visualized by the distribution of green fluorescence) (<b>c</b>). Representative images of three independent samples.</p>
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<p><b>Formation of a new biofilm cycle of <span class="html-italic">M. haemolytica</span> A2.</b> Bacteria of a 48 h biofilm with or without bLf were transferred to a new microplate to form a new biofilm cycle for another 48 h without bLf. A decrease in biofilm formation was observed in bacteria from a biofilm with bLf compared to bacteria from a biofilm with no treatment. Statistically significant differences between ratios are indicated (** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001). Representative results of three independent experiments.</p>
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26 pages, 4433 KiB  
Article
Characterization and Anti-Biofilm Activity of Lytic Enterococcus Phage vB_Efs8_KEN04 against Clinical Isolates of Multidrug-Resistant Enterococcus faecalis in Kenya
by Oumarou Soro, Collins Kigen, Andrew Nyerere, Moses Gachoya, Martin Georges, Erick Odoyo and Lillian Musila
Viruses 2024, 16(8), 1275; https://doi.org/10.3390/v16081275 - 9 Aug 2024
Viewed by 411
Abstract
Enterococcus faecalis (E. faecalis) is a growing cause of nosocomial and antibiotic-resistant infections. Treating drug-resistant E. faecalis requires novel approaches. The use of bacteriophages (phages) against multidrug-resistant (MDR) bacteria has recently garnered global attention. Biofilms play a vital role in E. [...] Read more.
Enterococcus faecalis (E. faecalis) is a growing cause of nosocomial and antibiotic-resistant infections. Treating drug-resistant E. faecalis requires novel approaches. The use of bacteriophages (phages) against multidrug-resistant (MDR) bacteria has recently garnered global attention. Biofilms play a vital role in E. faecalis pathogenesis as they enhance antibiotic resistance. Phages eliminate biofilms by producing lytic enzymes, including depolymerases. In this study, Enterococcus phage vB_Efs8_KEN04, isolated from a sewage treatment plant in Nairobi, Kenya, was tested against clinical strains of MDR E. faecalis. This phage had a broad host range against 100% (26/26) of MDR E. faecalis clinical isolates and cross-species activity against Enterococcus faecium. It was able to withstand acidic and alkaline conditions, from pH 3 to 11, as well as temperatures between −80 °C and 37 °C. It could inhibit and disrupt the biofilms of MDR E. faecalis. Its linear double-stranded DNA genome of 142,402 bp contains 238 coding sequences with a G + C content and coding gene density of 36.01% and 91.46%, respectively. Genomic analyses showed that phage vB_Efs8_KEN04 belongs to the genus Kochikohdavirus in the family Herelleviridae. It lacked antimicrobial resistance, virulence, and lysogeny genes, and its stability, broad host range, and cross-species lysis indicate strong potential for the treatment of Enterococcus infections. Full article
(This article belongs to the Special Issue Bacteriophages and Biofilms 2.0)
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<p>Plaque morphology of phage vB_Efs8_KEN04.</p>
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<p>Phage stability test of Enterococcus phage vB_Efs8_KEN04. (<b>A</b>) Thermal stability test. (<b>B</b>) pH stability test. Experiments were performed in triplicate. The triangle symbols represent individual data points and the error bars represent the standard deviation.</p>
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<p>Lytic characteristics of phage vB_Efs8_KEN04. (<b>A</b>) Phage yield using different multiplicity of infections (MOIs) to determine the optimal MOI. (<b>B</b>) Phage vB_Efs8_KEN04 lysis dynamics against <span class="html-italic">E. faecalis</span> EFS8.</p>
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<p>(<b>A</b>) Adsorption kinetics of the phage to its host. (<b>B</b>) One-step growth curve of phage vB_Efs8_KEN04 at a MOI of 1. Experiments were performed in triplicate, and the error bars represent the standard deviation.</p>
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<p>Determination of phage vB_Efs8_KEN04 receptor nature through adsorption rate measurements at different incubation temperatures. RT: room temperature (22−30 °C), NC: negative control, PC: positive control. Experiments were performed in triplicate, and the error bars represent the standard deviation.</p>
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<p>The biofilm formation profile of <span class="html-italic">Enterococcus faecalis</span> isolates. The biofilm formation experiment was performed in triplicate, and the error bars represent the standard deviation. The horizontal grid line represents the <span class="html-italic">E. faecalis</span> isolates that met the OD threshold of 0.0551 for biofilm-forming isolates.</p>
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<p>(<b>A</b>) Inhibition of biofilm by Enterococcus phage vB_Efs8_KEN04. (<b>B</b>) Biofilm disruption by phage vB_Efs8_KEN04. Biofilm inhibition and disruption experiments were performed in triplicate, and the error bars represent the standard deviation. Significance level: *, <span class="html-italic">p</span> &lt; 0.05 significant; **, <span class="html-italic">p</span> &lt; 0.01 very significant; ***, <span class="html-italic">p</span> &lt; 0.001 highly significant; ns, not statistically significant.</p>
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<p>(<b>A</b>) Inhibition of biofilm by Enterococcus phage vB_Efs8_KEN04. (<b>B</b>) Biofilm disruption by phage vB_Efs8_KEN04. Biofilm inhibition and disruption experiments were performed in triplicate, and the error bars represent the standard deviation. Significance level: *, <span class="html-italic">p</span> &lt; 0.05 significant; **, <span class="html-italic">p</span> &lt; 0.01 very significant; ***, <span class="html-italic">p</span> &lt; 0.001 highly significant; ns, not statistically significant.</p>
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<p>Circular genome map of Enterococcus phage vB_Efs8_KEN04 constructed using CGView.</p>
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<p>Phylogenetic analysis of Enterococcus phage vB_Efs8_KEN04 and other related Enterococcus bacteriophages based on the similarity of whole genome sequences. The phylogenetic tree was generated using the online Virus Classification and Tree Building Online Resource (VICTOR) platform with the formula d0 [<a href="#B65-viruses-16-01275" class="html-bibr">65</a>]. Color code legend from left to right: green squares in the first column correspond to the phages’ family cluster; green squares in the second column correspond to the phages’ genus cluster; the third column corresponds to the phages’ species cluster; the fourth column corresponds to the GC content (36-37%); and the fifth column corresponds to the genome length (min: 130,952 bp; max: 156,952 bp). The first and second column color codes and shapes indicate that all the phages are classified in the same family and genus, respectively. In the third and fourth columns, if the shape, color, and color intensity are the same, it means that the phages shared the same characteristics (species or GC content) but if they differed, it means that the phages are different species or have different GC contents.</p>
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<p>Heatmap of the average nucleotide identity values between phage vB_Efs8_KEN04 and the top 20 most similar Enterococcus bacteriophages. In the right half, the numbers represent the similarity values for each genome pair. In the left half, three indicator values are presented for each genome pair, in the order from top to bottom: aligned fraction genome 1 (for the genome found in this row), genome length ratio (for the two genomes in this pair), and aligned fraction genome 2 (for the genome found in this column). The vertical and horizontal red boxes indicate the intergenomic similarities between phage vB_Efs8_KEN04 and the most similar Enterococcus phages. The vertical and horizontal red text refer to the phage vB_Efs8_KEN04 isolated in this study and its GenBank accession number.</p>
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21 pages, 5142 KiB  
Article
A Novel Kunitz Trypsin Inhibitor from Enterolobium gummiferum Seeds Exhibits Antibiofilm Properties against Pathogenic Yeasts
by Matheus M. da Silva, Caio F. R. de Oliveira, Claudiane V. Almeida, Ismaell A. S. Sobrinho and Maria L. R. Macedo
Molecules 2024, 29(16), 3777; https://doi.org/10.3390/molecules29163777 - 9 Aug 2024
Viewed by 265
Abstract
Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using [...] Read more.
Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10−9 mol·L−1. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 μmol·L−1, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research. Full article
(This article belongs to the Section Medicinal Chemistry)
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<p>Gel filtration chromatography using a Sephadex G-100 column resulted in three peaks. The peak labelled EgPI G-100, which exhibited inhibitory activity against trypsin, is specified with an arrow.</p>
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<p>Hydrophobic chromatography using a Phenyl HP column results in five peaks. The gradient of (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> is depicted with a blue line. An arrow marks the identification of the peak of EgPI, showing signs of inhibiting trypsin.</p>
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<p>The 12.5% SDS-PAGE shows the results of the purification steps: 1: high-range molar mass marker (kDa); 2: crude extract; 3: EgPI-G100; 4: EgPI; and 5: EgPI reduced with 0.1 mol·L<sup>−1</sup> DTT.</p>
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<p>Stoichiometry of the inhibition of bovine trypsin by EgPI, demonstrating the relationship between inhibitor and enzyme molar ratios.</p>
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<p>CD spectra of EgPI (black) were incubated with 1 mmol·L<sup>−1</sup> DTT for 30 min at 30 °C.</p>
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<p>CD spectra of EgPI at various temperatures: 20 °C (grey); 30 °C (yellow); 40 °C (dark red); 50 °C (green); 60 °C (blue); 70 °C (red); 80 °C (purple); and 90 °C (pink). A notable reduction in β-sheet content and increased α-helix characteristics are observed starting at 70 °C (dashed lines).</p>
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<p>CD spectra of EgPI at various pH levels: pH 2 (green), pH 4 (red), pH 6 (yellow), pH 8 (blue), and pH 10 (black).</p>
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<p>Effects of EgPI on the biofilm mass of <span class="html-italic">C. albicans</span> ATCC 5314. This experiment examined how EgPI and amphotericin B affected the formation (<b>A</b>) and mature biofilm (<b>B</b>). The data show the viable mass of biofilm attached to a flat-bottom well stained with crystal violet. The letters denote significant differences between the groups. These differences were determined using one-way ANOVA followed by post hoc analysis (<span class="html-italic">p</span> ≤ 0.05).</p>
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<p>An investigation into the effects of EgPI and amphotericin B on the formation (<b>A</b>) and mature biofilm (<b>B</b>) of <span class="html-italic">C. tropicalis</span> ATCC 750 was conducted. The results present the viable biofilm adhered to a flat-bottom well stained with crystal violet. The letters denote significant differences between groups, determined using one-way ANOVA followed by post hoc analysis (<span class="html-italic">p</span> ≤ 0.05).</p>
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<p>Fluorescence microscopy of the effect of EgPI on the formation (<b>A</b>) and mature (<b>B</b>) <span class="html-italic">C. albicans</span> biofilm. Viable cells are stained green, and non-viable cells are stained red.</p>
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<p>Fluorescence microscopy of the effect of EgPI on the formation (<b>A</b>) and mature (<b>B</b>) <span class="html-italic">C. albicans</span> biofilm. Viable cells are stained green, and non-viable cells are stained red.</p>
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<p>Fluorescence microscopy of the effect of EgPI on the formation (<b>A</b>) and mature (<b>B</b>) <span class="html-italic">C. tropicalis</span> biofilm. Viable cells are stained green, and non-viable cells are stained red.</p>
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<p>Fluorescence microscopy of the effect of EgPI on the formation (<b>A</b>) and mature (<b>B</b>) <span class="html-italic">C. tropicalis</span> biofilm. Viable cells are stained green, and non-viable cells are stained red.</p>
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22 pages, 3224 KiB  
Article
Phenotypic and Genotypic Characterization of Resistance and Virulence Markers in Candida spp. Isolated from Community-Acquired Infections in Bucharest, and the Impact of AgNPs on the Highly Resistant Isolates
by Viorica Maria Corbu, Ana-Maria Georgescu, Ioana Cristina Marinas, Radu Pericleanu, Denisa Vasilica Mogos, Andreea Ștefania Dumbravă, Liliana Marinescu, Ionut Pecete, Tatiana Vassu-Dimov, Ilda Czobor Barbu, Ortansa Csutak, Denisa Ficai and Irina Gheorghe-Barbu
J. Fungi 2024, 10(8), 563; https://doi.org/10.3390/jof10080563 - 9 Aug 2024
Viewed by 312
Abstract
Background: This study aimed to determine, at the phenotypic and molecular levels, resistance and virulence markers in Candida spp. isolated from community-acquired infections in Bucharest outpatients during 2021, and to demonstrate the efficiency of alternative solutions against them based on silver nanoparticles (AgNPs). [...] Read more.
Background: This study aimed to determine, at the phenotypic and molecular levels, resistance and virulence markers in Candida spp. isolated from community-acquired infections in Bucharest outpatients during 2021, and to demonstrate the efficiency of alternative solutions against them based on silver nanoparticles (AgNPs). Methods: A total of 62 Candida spp. strains were isolated from dermatomycoses and identified using chromogenic culture media and MALDI-TOF MS, and then investigated for their antimicrobial resistance and virulence markers (VMs), as well as for metabolic enzymes using enzymatic tests for the expression of soluble virulence factors, their biofilm formation and adherence capacity on HeLa cells, and PCR assays for the detection of virulence markers and the antimicrobial activity of alternative solutions based on AgNPs. Results: Of the total of 62 strains, 45.16% were Candida parapsilosis; 29.03% Candida albicans; 9.67% Candida guilliermondii; 3.22% Candida lusitaniae, Candia pararugosa, and Candida tropicalis; and 1.66% Candida kefyr, Candida famata, Candida haemulonii, and Candida metapsilosis. Aesculin hydrolysis, caseinase, and amylase production were detected in the analyzed strains. The strains exhibited different indices of adherence to HeLa cells and were positive in decreasing frequency order for the LIP1, HWP1, and ALS1,3 genes (C. tropicalis/C. albicans). An inhibitory effect on microbial growth, adherence capacity, and on the production of virulence factors was obtained using AgNPs. Conclusions: The obtained results in C. albicans and Candida non-albicans circulating in Bucharest outpatients were characterized by moderate-to-high potential to produce VMs, necessitating epidemiological surveillance measures to minimize the chances of severe invasive infections. Full article
(This article belongs to the Special Issue Fungal Biofilms, 2nd Edition)
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<p>The experimental design (created with Biorender.com; accessed on 11 June 2024).</p>
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<p>The distribution of arbitrary units by isolation sources of <span class="html-italic">Candida</span> spp. strains. Legend: AU1 = 1 arbitrary unit, AU2 = 2 arbitrary units, and AU0 = 0 arbitrary units.</p>
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<p>Average MIC values for <span class="html-italic">Candida</span> spp. strains.</p>
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<p>Adherence inhibition percentage (PICA%) values for AgNPs compared to the <span class="html-italic">Candida</span> spp. strains (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001) (Dunnett’s multiple comparisons test).</p>
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<p>AgNPs’ effects on <span class="html-italic">Candida</span> spp. strains’ virulence factors (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.001, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001) (Dunnett’s multiple comparisons test). (<b>A</b>) Caseinase production of <span class="html-italic">Candida</span> sp. strains, (<b>B</b>) Hemolysis production of <span class="html-italic">Candida</span> sp. strains, (<b>C</b>) Amylase production of <span class="html-italic">Candida</span> sp. strains, (<b>D</b>) Esculin Hydrolysis production of <span class="html-italic">Candida</span> sp. strains.</p>
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<p>Extracellular NO content determined by the Griess reaction for AgNPs in the presence of <span class="html-italic">C. albicans</span> strains (Tukey’s method, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001).</p>
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<p>Pearson correlation among extracellular NO content, adhesion inhibition percentage (PICA%), caseinase activity (%), amylase activity (%), and hemolysin (%) for <span class="html-italic">C. albicans</span> (<b>A</b>) and <span class="html-italic">C. parapsilosis</span> (<b>B</b>).</p>
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