Microbial Transfer by Intermittent Catheters: An In Vitro Evaluation of Microbial Transfer in Catheter With Variable Protective Features.

PURPOSE: The purpose of this study was to evaluate the effects of various protective features (eg, catheter cap, introducer tip, and catheter sleeve) of hydrophilic intermittent catheters against contamination with urinary tract infection-associated microorganisms using an in vitro model. DESIGN: An in vitro study of microbial transfer. MATERIALS AND METHODS: Gloves were contaminated with uropathogenic microorganisms and used to simulate intermittent catheterization of male anatomical models with and without the protective features present in 5 commercially available hydrophilic catheters. Using this contaminated touch transfer method, both the meatus of the sterile male anatomical models and sterile surgical gloves of an operator were inoculated with a high level of microorganisms (107 and 109 colony-forming units [CFU], respectively). The operator then performed catheterization of the anatomical model. The most relevant segments of the catheter were sampled, and the level of microbial transfer and catheter contamination was quantified. Results from experimental and sample replicates from the 3 microbial species and 5 catheters (sleeved and unsleeved) were analyzed by pair-wise t tests and analysis of variance. RESULTS: Of the 5 commercially available sleeved intermittent catheters evaluated in this study, use of catheters with multiple protective components (ring cap, introducer tip, and catheter sleeve) resulted in significant improvement in protection against contamination with a 25- to 2500-fold lower level of microbial contamination (C1 segment) across all species as compared to catheters protected with only sleeves or un-sleeved catheters. CONCLUSIONS: The combination of a ring cap, protective introducer tip, and protective sleeve provides additional protection when compared to sleeve alone from transferring microbial contamination from the meatus to the advancing catheter. Additional research is needed to determine whether these design features result in fewer urinary tract infections among intermittent catheter users.

Copper-based dressing: Efficacy in a wound infection of ex vivo human skin.

This study aimed to evaluate the wound healing and antibacterial effects of two experimental copper dressings compared to a commercial silver dressing. Burn wounds were created in the ex vivo human skin biopsies, then were infected by Staphylococcus aureus. Tissues were treated with copper dressings, silver dressing, or a dressing without any antibacterial component. An infected wound tissue without treatment was considered as the control group. Three days after treatments, tissues were analyzed by bacterial count and histology staining, while their media was used to assess the expression of cytokines and chemokines. Histology staining confirmed the presence of second-degree burn wounds and colonization of bacteria in the surface and superficial layer of tissues. The results demonstrated a higher antibacterial effect, improved epithelium formation, and decreased wound area in one of the copper dressings compared to other dressings. Markers associated with infection control increased in both the copper and silver-treated groups. The cytokine profiling analysis revealed increased expression of markers related to angiogenesis and anti-inflammatory responses and decreased pro-inflammatory cytokine responses in the infected wound treated with one of the copper dressings. Our results confirmed the efficacy of the experimental copper dressing in reducing bacteria and promoting wound healing.

In vitro and Ex vivo Assessments of the Compatibility of Fibrin Sealant with Antimicrobial Compounds.

Background: Fibrin sealants are used as antimicrobial-releasing carriers for preventing surgical site infections; however, it is important to determine the release kinetics and antimicrobial effects of drugs added to fibrin sealants and the effects of drugs on clot/clotting properties. Materials and Methods: The antimicrobial and antibiofilm activity of cefazolin, colistin, gentamicin, oxacillin, tobramycin, and silver nitrate released from fibrin sealant were characterized using in vitro and ex vivo assays against bacteria commonly found on the skin. The effects of antimicrobial agents on the physical structure of the fibrin sealant were assessed with scanning electron microscopy (SEM) and on the clotting rate and strength of fibrin clots using run-off tests and rheology. Results: Generally, antibiotic agents were released gradually from fibrin sealant and were stable after release, with antimicrobial effects evident up to three days. Cefazolin, gentamicin, and oxacillin prevented biofilm formation of Staphylococcus aureus in porcine skin explants; gentamicin and colistin prevented biofilm formation of Pseudomonas aeruginosa. Gentamicin, cefazolin, colistin, and tobramycin did not affect the structural integrity or viscoelastic properties of fibrin sealant; changes were observed with oxacillin (SEM) and particularly silver nitrate (SEM and rheology). No antimicrobial agents caused deterioration of clotting time (run-off tests). Conclusions: From the antimicrobial agents tested, gentamicin and cefazolin showed prolonged release from fibrin sealant, sustained antimicrobial activity, and biofilm prevention properties against Staphylococcus aureus; similar results were observed for gentamicin and colistin against Pseudomonas aeruginosa. For each of these findings, the physical structure of the fibrin sealant, clotting rate, and strength of fibrin clots were unaffected.

Evaluation of supercritical CO2 sterilization efficacy for sanitizing personal protective equipment from the coronavirus SARS-CoV-2.

The purpose of this research is to evaluate the supercritical carbon dioxide (scCO2) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks, and N95 respirators. Preliminarily, Bacillus atrophaeus were inoculated into different environments (dry, hydrated, and saliva) to imitate coughing and sneezing and serve as a "worst-case" regarding challenged PPE. The inactivation of the microbes by scCO2 sterilization with NovaKill or H2O2 sterilant was investigated as a function of exposure times ranging from 5 to 90 min with a goal of elucidating possible mechanisms. Also, human coronavirus SARS-CoV-2 and HCoV-NL63 were inoculated on the respirator material, and viral activity was determined post-treatment. Moreover, we investigated the reprocessing ability of scCO2-based decontamination using wettability testing and surface mapping. Different inactivation mechanisms have been identified in scCO2 sanitization, such as membrane damage, germination defect, and dipicolinic acid leaks. Moreover, the viral sanitization results showed a complete inactivation of both coronavirus HCoV-NL63 and SARS-CoV-2. We did not observe changes in PPE morphology, topographical structure, or material integrity, and in accordance with the WHO recommendation, maintained wettability post-processing. These experiments establish a foundational understanding of critical elements for the decontamination and reuse of PPE in any setting and provide a direction for future research in the field.

In Vitro Evaluation of Common Antimicrobial Solutions Used for Breast Implant Soaking and Breast Pocket Irrigation-Part 1: Efficacy Against Planktonic Bacteria.

BACKGROUND: Planktonic bacteria can be inadvertently introduced during breast surgery procedures, which are hypothesized to lead to complications such as infection, capsular contracture, breast implant-associated anaplastic large cell lymphoma, and a prolonged local inflammatory response. The utilization of antimicrobial solutions such as triple antibiotic solution (TAB) and/or 10% povidone-iodine (PI) in breast pocket irrigation or implant soaking has been proposed to reduce planktonic bacterial attachment and potential complications. OBJECTIVES: A series of in vitro assessments were performed to evaluate the antimicrobial utility of TAB and PI, either alone or in combination, against planktonic bacteria. METHODS: Planktonic gram-positive and gram-negative bacterial strains were exposed to TAB and PI ± TAB for up to 10 minutes in a bacterial time-kill assay. The efficacy of various dilutions of PI as well as the effects of serum protein on PI efficacy were also investigated. RESULTS: TAB was ineffective at the timeframes tested (≤10 minutes) when utilized alone; however, when utilized with PI, significant log reduction of all tested planktonic species was achieved. PI alone was also effective, even including dilute concentrations (eg, 0.5% PI), although the presence of serum proteins required higher concentrations of PI (eg, 2.5%) to eradicate the bacterial load. CONCLUSIONS: Our data suggest PI-containing solutions may be preferred over either saline or TAB without PI for primary breast pocket irrigation and implant soaking in primary breast surgeries as a means to significantly reduce planktonic bacteria. These data provide an impetus for surgeons to re-evaluate the efficacy of TAB solution in these clinical settings.

Lab-on-a-bubble: synthesis, characterization, and evaluation of buoyant gold nanoparticle-coated silica spheres.

This paper describes the development and preparation of a new class of materials for surface-enhanced Raman scattering (SERS) consisting of gold nanoparticles coated onto hollow, buoyant silica microspheres. These materials allow for a new type of molecular assay designated as a lab-on-a-bubble (LoB). LoB materials serve as a convenient platform for the detection of analytes in solution and offer several advantages over traditional colloidal gold and planar SERS substrates, such as the ability to localize and concentrate analytes for detection. An example assay is presented using the LoB method and cyanide detection. Cyanide binds to SERS-active, gold-coated LoBs and is detected directly from the corresponding SERS signal. The abilities of LoBs and a gold colloid to detect cyanide are compared, and in both cases, a detection limit of ~170 ppt was determined. Differences in measurement error using LoBs versus gold colloid are also described, as well as an assay for 5,5'-dithiobis(2-nitrobenzoic acid) that shows the benefit of using LoBs over SERS analyses in colloids, which are often plagued by particle aggregation.

PAA-derived gold nanorods for cellular targeting and photothermal therapy.

A single-step LbL procedure to functionalize CTAB-capped GNRs via electrostatic self-assembly is reported. This approach allows for consistent biomolecule/GNR coupling using standard carboxyl-amine conjugation chemistry. The focus is on cancer-targeting biomolecule/GNR conjugates and selective photothermal destruction of cancer cells by GNR-mediated hyperthermia and NIR light. GNRs were conjugated to a single-chain antibody selective for colorectal carcinoma cells and used as probes to demonstrate photothermal therapy. Selective targeting and GNR uptake in antigen-expressing SW 1222 cells were observed using fluorescence microscopy. Selective photothermal therapy is demonstrated using SW 1222 cells, where >62% cell death was observed after cells are treated with targeted A33scFv-GNRs.

Multiplex single nucleotide polymorphism genotyping utilizing ligase detection reaction coupled surface enhanced Raman spectroscopy.

Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high specificity. We present the first multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The platform has the advantage in that the diagnostic peaks of Raman are more distinct than fluorescence, and in theory, a clinically significant number of markers can be multiplexed in a single sample using different SERS reporters. Here we report LDR-SERS multiplex SNP genotyping of K-Ras oncogene alleles at 10 pM detection levels, optimization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Genomic DNA from typed cells lines was obtained and scored for the K-Ras genotype. These advances are significant as we have further developed our new SNP genotyping platform and have demonstrated the ability to correlate genotype ratios directly to diagnostic Raman peak signal intensity.

In vitro self-assembly of gold nanoparticle-coated poly(3-hydroxybutyrate) granules exhibiting plasmon-induced thermo-optical enhancements.

Polyhydroxyalkanoate (PHA) synthase attached to gold nanoparticles (AuNP) produce poly(3-hydroxybutyrate) (PHB) upon the addition of 3-hydroxybutyrate-CoA, and then coalesce to form micrometer-sized AuNP-coated PHB granules. These AuNP-coated PHB granules are potential theranostic agents that have enhanced imaging capabilities and are capable of heating upon near-infrared laser irradiation. The AuNP-coated PHB exhibited 11-fold enhancement in surface-enhanced Raman scattering over particles prior polymerization. Stained AuNP-coated PHB exhibited a 6-fold enhancement in fluorescence intensity as well as a 1.3-fold decrease in photobleaching rate compared to PHB granules alone. The granules were also shown to emit heat when illuminated at 808 nm with a 3.9-fold increase in heating rate compared to particles alone.

Detection of carbendazim by surface-enhanced Raman scattering using cyclodextrin inclusion complexes on gold nanorods.

Surface-enhanced Raman scattering (SERS) was used for the detection of carbendazim using gold nanorods derivatized with a beta-cyclodextrin derivative to bind this fungicide. Gold nanorods were synthesized with an aspect ratio of 3.3 to match the 785 nm excitation wavelength used in the SERS studies. A thiolated cyclodextrin-based sensor molecule was synthesized, and its inclusion complex with carbendazim was formed and studied using SERS spectroscopy. SERS analysis of the inclusion complex at different concentrations in the presence of gold nanorods afforded good quality Raman spectra of carbendazim at micromolar concentrations. Quantitative analysis was preformed using partial least-squares (PLS), and a calibration plot for these data was generated. Results suggest that carbendazim concentrations as low as 50 microM can be accurately detected using the described SERS assay.

Surface-enhanced Raman scattering based ligase detection reaction.

Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced Raman scattering (SERS) to the ligase detection reaction (LDR). The reaction uses two LDR primers, one of which contains a Raman enhancer and the other a reporter dye. In LDR, one of the primers is designed to interrogate the SNP. When the SNP being interrogated matches the discriminating primer sequence, the primers are ligated and the enhancer and dye are brought into close proximity enabling the dye's Raman signature to be detected. By detecting the Raman signature of the dye rather than its fluorescence emission, our technique avoids the problem of spectral overlap which limits number of reactions which can be carried out in parallel by existing systems. We demonstrate the LDR-SERS reaction for the detection of point mutations in the human K-ras oncogene. The reaction is implemented in an electrokinetically active microfluidic device that enables physical concentration of the reaction products for enhanced detection sensitivity and quantization. We report a limit of detection of 20 pM of target DNA with the anticipated specificity engendered by the LDR platform.

Optimized linkage and quenching strategies for quantum dot molecular beacons.

Quantum dot (QD) molecular beacons were explored for sequence-specific DNA detection. The effectiveness of multiple linkage strategies and fluorescence quenchers were compared in hybridization-based assays. To compare linkage strategies, covalent amide linkage and streptavidin-biotin binding were used to link semiconductor QDs to molecular beacon DNA. Amide-linked beacons showed a 57% greater fluorescence increase than streptavidin-linked beacons when hybridized to 200 pmol of target DNA. The specificity of the molecular beacons, however, was similar for both linkage methods. Hybridization of both QD molecular beacons with non-complementary target DNA resulted in approximately 50% lower fluorescence intensity than hybridization with complementary DNA. The effectiveness of different quencher moieties was also evaluated. Iowa Black and 1.4 nm Nanogold-quenched molecular beacons exhibited approximately 2-fold greater fluorescence increases than dabcyl-quenched beacons when hybridized to complementary target. Specificity for target DNA was also confirmed through hybridization assays with non-complementary DNA. To provide insight into differences between the QD molecular beacons and the linkage strategies used, the hydrodynamic radius of each was measured. These measurements indicated that the larger radius of the streptavidin QDs (13.5 nm) than the carboxyl QDs (7 nm) could have a negative effect on FRET-based quenching for QD molecular beacons. These data outline the importance of choosing proper linkage methods and quencher moieties for creating high-quality QD molecular beacons.