CFU Counts Research Articles

Pesticides and bacterial density - analysis of a dam situated in agricultural area

The present study aimed to detect the types and concentration of pesticides in the surface water of a dam located in an agricultural region, and relate them to aquatic bacterial density. 1L of water were collected six times, three in the dry season (D1, D2 and D3) and three in the rainy season (R1, R2 and R3). The pesticide analysis was performed using CG/MS. The bacterial density was analyzed by means of CFU counting. The most abundant colonies that were cultivated in all studied samples were isolated and identified by molecular methods. The bacterial density had a significant difference between the rainy and dry seasons. In the dry season, the average bacterial density (5.69x105±2.86 x105 UFC mL-1) was higher than in the rainy season (2.31x105±1.73 x105 UFC mL-1). In the dry season, lambda cyhalothrin (0.3 µg L -1) and endrin (0.23 µg L -1) were found in D1 collection and endosulfan sulfate in D1 and D2 (0.05 - 0.27 µg L -1). In the rainy season, DEA (0.02 - 0.09 µg L-1) and atrazine (0.02 - 0.06 µg L -1) were detected in all collections, metalachlor (0.2 µg L -1), chlorpyrifos (0.2 µg L -1) in R1 and R2 and endosulfan sulfate (0.1 µg L -1) in R1. In the dry season, the bacterial density in R1 was significantly lower than R2 and R3. High correlation among those pesticides and the bacterial density was observed. Endrin, lambda cyhalothrin and endosulfan sulphate are toxic and persistent pesticides, and may therefore have influenced the decrease in aquatic bacterial density. Molecular analyzes identified the bacterium Bacillus weihenstephanensis, known to be present in environments contaminated by pesticides and for its biodegradable potential.

983. Pathogen-specific PET/CT imaging for Gram negative implant-associated spinal infections

983. Pathogen-specific PET/CT imaging for Gram negative implant-associated spinal infections

What is essential remains invisible to the eyes? Blood pressure cuffs colonized by bacterial diversity.

Using sphygmomanometers to measure blood pressure is a common practice in the healthcare context. The disinfection and maintenance of these devices is essential in clinical practice to prevent the proliferation of microorganisms. The aim of this study was to determine the presence of pathogenic microorganisms in sphygmomanometer cuffs in the clinical setting. A cross-sectional study was carried out. Five types of healthcare centers, selected through convenience sampling, participated in this study. Samples were collected from the inside of sphygmomanometer cuffs, and labeled and delivered to the laboratory for analysis. The samples were incubated in an oven at 35.5°C for 24h. A total CFU count was carried out on the plates that were cataloged as positive. Colonies that showed growth were identified using the matrix-assisted laser desorption/ionization-mass spectrometry technology. Of the total sample, (N = 372), 69.1% were positive and were isolated. In 30.9% (n = 115), no bacterial development was found within 48h. A total of 257 microorganisms were found. The mean number of colony-forming units was 29.62 (SD = 32.33). The socio-health centers had the highest amount of bacterial contamination in the cuffs. In regards to the type of microorganisms, 31.5% (n = 81) found were Bacillus cereus, followed by 26.8% (n = 69) of Staphylococcus hominis and 9.7% (n = 25) were Pantoea agglomerans, among others. Statistically significant differences were found between the type of microorganism and the hours elapsed since the last disinfection (X2(19) = 44.582; p = 0.001). Statistically significant differences were found between the time elapsed since the last disinfection and the type of sphygmomanometer (X2 (2) = 117.752; p = 0.000). Despite the fact that most hospitals and health centers have established infection control policies and protocols, the results of this study indicate the presence of pathogenic microorganisms in blood pressure cuffs in the clinical setting.

In Vitro Activity of a Hypochlorous Acid-Generating Electrochemical Bandage against Yeast Biofilms.

The antibiofilm activity of a hypochlorous acid (HOCl)-producing electrochemical bandage (e-bandage) was assessed against 14 yeast isolates in vitro. The evaluated e-bandage was polarized at +1.5 VAg/AgCl to allow continuous production of HOCl. Time-dependent decreases in the biofilm CFU counts were observed for all isolates with e-bandage treatment. The results suggest that the described HOCl-producing e-bandage could serve as a potential alternative to traditional antifungal wound biofilm treatments.

Perimeter and carvacrol-loading regulate angiogenesis and biofilm growth in 3D printed PLA scaffolds.

Carvacrol is a natural low-cost compound derived from oregano which presents anti-bacterial properties against both Gram-positive and Gram-negative bacteria. In this work, carvacrol-loaded PLA scaffolds were fabricated by 3D printing as platforms to support bone tissue regeneration while preventing biofilm development. Scaffolds were printed with or without a perimeter (lateral wall) mimicking the cortical structure of bone tissue to further evaluate if the lateral interconnectivity could affect the biological or antimicrobial properties of the scaffolds. Carvacrol incorporation was performed by loading either the PLA filament prior to 3D printing or the already printed PLA scaffold. The loading method determined carvacrol localization in the scaffolds and its release profile. Biphasic profiles were recorded in all cases, but scaffolds loaded post-printed released carvacrol much faster, with 50-80% released in the first day, compared to those containing carvacrol in PLA filament before printing which sustained the release for several weeks. The presence or absence of the perimeter did not affect the release rate, but total amount released. Tissue integration and vascularization of carvacrol-loaded scaffolds were evaluated in a chorioallantoic membrane model (CAM) using a novel quantitative micro-computed tomography (micro-CT) analysis approach. The obtained results confirmed the CAM tissue ingrowth and new vessel formation within the porous structure of the scaffolds after 7days of incubation, without leading to hemorrhagic or cytotoxic effects. The absence of lateral wall facilitated lateral integration of the scaffolds in the host tissue, although increased the anisotropy of the mechanical properties. Scaffolds loaded with carvacrol post-printing showed antibiofilm activity against Staphylococcus aureus and Pseudomonas aeruginosa as observed in a decrease in CFU counting after biofilm detachment, changes in metabolic heat measured by calorimetry, and increased contact killing efficiency. In summary, this work demonstrated the feasibility of tuning carvacrol release rate and the amount released from PLA scaffolds to achieve antibiofilm protection without altering angiogenesis, which was mostly dependent on the perimeter density of the scaffolds.

Cytotoxicity and anti-biofilm properties of novel hybrid-glass-based caries infiltrant.

To assess the cytotoxicity of an experimental hybrid-glass-based infiltrant and its effect on biofilm attachment, growth and metabolic activity, and to compare it to the resin-based infiltrant Icon. Cytotoxicity of hybrid-glass-based material (EXP) and resin-based infiltrant Icon (Icon) was tested in direct contact tests on freshly cured (direct_mat) and on materials kept for 24h in cell culture medium (direct_exmat), and extract test with materials 24-h extracts (extract). Cell viability of L929 mouse fibroblast cell line was measured with MTT assay, according to ISO10993-5:2009. Biofilm attachment (5h), growth (24h and 48h) and lactic-acid production (24h and 48h) on glass-disk specimens coated with EXP or Icon, or uncoated (control), were assessed using a microcosm biofilm model and Amsterdam Active Attachment system. At indicated time points, biofilms were harvested, plated, and CFU counts were determined, while lactic-acid production was measured colorimetrically. Cell viability reduction by EXP was below 30%-threshold in direct contact tests, while in extract test an increased cell viability was observed. Icon reduced cell viability substantially in all three tests. Significantly less bacteria attached to the surface of EXP after 5h compared to Icon and control. Biofilm growth was significantly lower on EXP than on Icon and control after 24h, but this difference was smaller and statistically insignificant after 48h. There was no difference in lactic-acid production among groups. Novel hybrid-glass-based infiltrant seems to have a better biocompatibility and accumulates on its surface less bacteria than resin-based infiltrant, which makes it an attractive resin-free alternative.

Sitagliptin Alleviates Excessive Autophagy in Macrophages in Immune Thrombocytopenia Patients to Promote Megakaryopoiesis Via the AMPK/mTOR Pathway

Introduction: The pathogenesis of ITP is complex and mainly manifests as autoantibody-mediated platelet destruction and impaired platelet production. Autophagy is a highly conserved cellular process that eliminates damaged organelles and promotes the degradation of misfolded proteins. Appropriate autophagy exerts a protective effect, while excessive autophagy may lead to cell death and increased inflammation. The specific role of bone marrow macrophages (BM MΦ) in regulating megakaryopoiesis in ITP patients has not been elucidated. Sitagliptin, a DPP-4 inhibitor, was reported to play a key role in the production and engraftment of hematopoietic cells by modulating SDF-1α and several colony-stimulating factors, thus enhancing hematopoietic stem and hematopoietic progenitor cell recovery. However, whether sitagliptin has the potential to promote megakaryocyte maturation and platelet production in ITP is unclear. Therefore, this article explores the effect of macrophage autophagy on megakaryopoiesis in ITP patients and investigates its underlying role in the pathogenesis of ITP in order to explore whether sitagliptin could be a potential treatment for ITP. Methods: BM MΦ and CD34+ cells were isolated from ITP patients and healthy control subjects. Excessive autophagy in bone marrow macrophages from ITP patients was confirmed by Western blotting and transmission electron microscopy. PCR and Western blotting were used to assess the expression of A20 and autophagy-related AMPK/mTOR/ULK1 pathway components in ITP-MΦ. Vectors carrying A20 cDNA/TNFAIP3-specific shRNA were transduced into the BM MΦ from either healthy control subjects or ITP patients. The different BM MΦ populations were separately cocultured with CD34+ cells. We compared the ability of BM MΦ to support megakaryocytopoiesis by determining the relative percentages of MKs at different stages using flow cytometry as well as CFU counts of megakaryocytes and platelets in culture. The mechanism by which sitagliptin normalized the impaired function of ITP-MΦ was evaluated in vitro. A prospective single-arm clinical cohort study was designed to explore the efficacy and safety of sitagliptin in the treatment of corticosteroid-resistant or relapsed ITP. Results: The expression of LC3-II/LC3-I and Beclin1 increased in ITP-MΦ, and the expression of P62 decreased. The number of autophagic vesicles also increased, indicating enhanced autophagy in ITP-MΦ. Transcriptome sequencing and untargeted metabolomics revealed that macrophage autophagy-related pathways were involved in the pathogenesis of ITP. Further studies confirmed that ITP-MΦ regulated megakaryopoiesis, which was dependent on the A20-mediated autophagy-related AMPK/mTOR/ULK1 pathway. Inhibition of A20 expression with A20-shRNA inhibited the AMPK/mTOR/ULK1 pathway and alleviated excessive autophagy in ITP-MΦ, thus increasing the number of megakaryocytes, the proportion of hyperploid megakaryocytes and the number of platelets produced in the ITP-MΦ coculture system. Inhibition of autophagy with the autophagy inhibitor 3-MA reversed the A20 overexpression-induced excessive autophagy in MΦ and normalized the differentiation and maturation of megakaryocytes. Inhibiting the AMPK/mTOR/ULK1 signaling pathway with the AMPK inhibitor Compound C reversed the A20 overexpression-induced increase in the expression of AMPK/mTOR/ULK1 autophagy pathway components in MΦ and normalized megakaryopoiesis. Sitagliptin was found to inhibit the expression of A20 and AMPK/mTOR/ULK1 autophagy pathway components in ITP-MΦ in vitro. Sitagliptin improved the ability of ITP-MΦ to promote the differentiation and development of megakaryocytes and platelet production. Sitagliptin was administered to a total of 56 patients with corticosteroid-resistant or relapsed ITP. Complete response and overall response were observed in 11 (19.6%) and 21 patients (37.5%), respectively. Conclusions: A20 mediated the AMPK/mTOR/ULK1 signaling pathway to regulate autophagy in ITP-MΦ and affected megakaryopoiesis and platelet formation. Sitagliptin could restore the proper function of the A20-mediated AMPK/mTOR/ULK1 signaling pathway in ITP-MΦ, thus promoting megakaryopoiesis. Sitagliptin resulted in a sustained response in patients with corticosteroid-resistant/recurrent ITP and exhibited good safety. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multifunctional modification of orthodontic adhesives with ZnO quantum dots.

To develop a multifunctional orthodontic adhesive (QDA) using ZnO quantum-dots (ZnQDs) as modifier and investigate the antibacterial capability, fluorescence property as well as biocompatibility and bonding property. ZnQDs were synthesized using sol-gel method. XPS, XRD, FT-IR, HRTEM, SAED, DLS and spectrofluorimetry were used to characterize ZnQDs. ZnQDs were incorporated into Transbond XT adhesive paste with 20 %, 30 %, 40 % mass fraction, respectively, to form the multifunctional adhesives (QDAs). Antibacterial capability was evaluated with MTT kit, CFU count and Live/Dead Bacterial Staining Kit. Ultraviolet photography and spectrofluorimetry were used to confirm the fluorescence property of QDAs. Biocompatibility assay was performed on gingival fibroblasts and subcutaneous tissue of rats. Softening in solvent rate, shear bond strength and degree of conversion (DC) were measured. The synthesized ZnQDs presented excellent crystallinity and fluorescence properties. MTT assay, CFU count and CLSM analysis indicated that QDAs had significant antibacterial activity compared with Transbond XT adhesive paste. CCK-8 assay and Live/Dead cell staining analysis denied the cytotoxicity of QDAs and histological analysis proved that QDAs all had no inflammatory irritation to subcutaneous tissue. Softening in solvent, shear bond strength and DC evaluations indicated that 20 % mixing ratio of ZnQDs could enhance the resistance to degradation without influencing the bond strength and DC. Ultraviolet photography and spectrofluorimetry analysis proved the fluorescence capability of QDAs. ZnQDs can impart antibacterial and fluorescence properties to orthodontic adhesives without affecting biocompatibility and bonding performance. QDAs can be multifunctional orthodontic adhesives to reduce bacterial adhesion around brackets and help orthodontists remove residual adhesives precisely when needed.

Mesoporous Calcium-Silicate Nanoparticles Loaded with Prussian Blue Promotes Enterococcus Faecalis Ferroptosis-Like Death by Regulating Bacterial Redox Pathway ROS/GSH.

Mesoporous calcium-silicate nanoparticles (MCSNs) are advanced biomaterials that have been used to control drug delivery for many years. Ultrasmall Prussian blue nanoparticles (UPBNPs) showed high peroxidase and catalase-like activities. This study evaluated the antibacterial and antibiofilm properties, mechanism and cytotoxicity of UPBNPs-MCSNs composites synthesized by both as precursors. UPBNPs-MCSNs were prepared and characterized. The antibacterial effect of UPBNPs-MCSNs was evaluated by the MTT assay and CFU counting method, and their biosafety was tested by CCK8. Then explore the antibacterial mechanism, including TEM observation of bacterial morphology, and detection of bacterial ROS, LPO and GSH levels. The antibiofilm activity of UPBNPs-MCSNs was tested by E. faecalis biofilm model in human roots. The roots were pretreated with materials and cultured with E. faecalis, and the survival of E. faecalis on the root canal wall was observed by SEM and CLSM. The results showed that UPBNPs-MCSNs had potent antibacterial and antibiofilm activities. They can aggregate on the dentin surface and significantly inhibit E. faecalis adhesion and colonization. Their antibacterial activity is as effective as NaClO and calcium hydroxide (CH), can significantly prolong the time of bacterial colonization than CH, but have lower cytotoxicity to normal cells. We found that UPBNPs-MCSNs trigger a like classic ferroptosis pathway in bacteria. UPBNPs-MCSNs can induce bacteria to produce ROS and LPO, and reduce GSH level. Moreover, we observed that the metal ions chelator and the antioxidant could block their antibacterial activity. These results reveal that UPBNPS-MCSNs have high antibacterial and antibiofilm, and can mediate the bacterial redox pathway ROS/GSH like the classical pathway of ferroptosis, providing a theoretical basis for them to develop into a safe and effective novel root canal disinfectant.

Stability, Toxicity, and Antibacterial Potential of Gallic Acid-Loaded Graphene Oxide (GAGO) Against Methicillin-Resistant Staphylococcus aureus (MRSA) Strains.

The impetuous usage of antibiotics has led to the perpetual rise of methicillin-resistant Staphylococcus aureus (MRSA), which has garnered the interest of potential drug alternatives, including nanomaterials. The present study investigates the stability, toxicity, and antibacterial potential of gallic acid-loaded graphene oxide (GAGO) on several MRSA strains. The stability of a synthesized and characterized GAGO was monitored in different physiological media. The toxicity profile of GAGO was evaluated in 3T3 murine fibroblast cells and the embryonic zebrafish model. The antibacterial activity of GAGO against MRSA, methicillin-susceptible S. aureus (MSSA), and community-acquired MRSA; with or without Panton-valentine leucocidin gene (MRSA-pvl+ and MRSA-pvl-) was investigated through disk diffusion, CFU counting method, time-kill experiment, and high-resolution transmission electron microscopy (HRTEM) observation. A stable GAGO nanocomposite has shown an improved toxicity profile in 3T3 murine fibroblast cells and zebrafish embryos, besides exhibiting normal ROS levels than graphene oxide (GO) and GA (gallic acid). The nanocomposite inhibited the growth of all bacterial strains employed. The effectiveness of the GAGO nanocomposite was comparable to cefoxitin (CFX), at ≥150 µg/mL in MRSA and MSSA. GAGO exhibited a significantly delayed response towards MRSA-pvl+ and MRSA-pvl-, with increased inhibition following 8 to 24 h of exposure, while comparable activity to native GA was only achieved at 24 h. Meanwhile, for MRSA and MSSA, GAGO had a comparable activity with native GA and GO as early as 2 h of exposure. HRTEM observation further reveals that GAGO-exposed cells were membrane compromised. In summary, the present study indicates the antibacterial potential of GAGO against MRSA strains, but further study is warranted to understand the mechanism of action of GAGO and its resistance in MRSA strains.

5-Aminolevulinic Acid and Red Led in Endodontics: A Narrative Review and Case Report.

The present study aims to discuss the main factors involving the use of 5-aminolevulinic acid together with red LED light and its application in endodontic treatment through a narrative review and a case report. Persistence of microorganisms remaining on chemical-mechanical preparation or intracanal dressing is reported as the leading cause of failure in endodontics. Photodynamic therapy has become a promising antimicrobial strategy as an aid to endodontic treatment. Being easy and quick to apply, it can be used both in a single session and in several sessions, as well as not allowing forms of microbial resistance. 5-aminolevulinic acid in combination with red LED light has recently been studied in many branches of medicine, with good results against numerous types of bacteria including Enterococuss faecalis. The case report showed how bacterial count of CFU decreased by half (210 CFU/mL), after 45 min of irrigation with a gel containing 5% of 5-aminolevulinic acid compared to the sample before irrigation (420 CFU/mL). The subsequent irradiation of red LED light for 7 min, the bacterial count was equal to 0. Thus, it is concluded that the use of 5-aminolevulinic acid together with red LED light is effective in endodontic treatment.

Investigation of Bacterial Persistence and Filaments Formation in Clinical Klebsiella pneumoniae

Bacterial persistence is recognized as a major cause of antibiotic therapy failure, causing biofilms, and chronic intractable infections. The emergence of persisters in Klebsiella pneumoniae isolates has become a worldwide public health concern. The goal of the present study is to investigate the formation of persister cells beside filaments in Iraqi K. pneumoniae isolates. A total of fifty clinical K. pneumoniae isolates were collected from different clinical specimens and identified using the genotypic identification by using specific primer (rpoB gene) from housekeeping genes. Persister cells investigation is performed by exposure of stationary phase K. pneumoniae isolates to a high concentration of ciprofloxacin (×10 MIC) and counting the number of viable persister cells by CFU counts. Bacterial filament formation is detected and measured by light microscope scanning electron microscope. The results show the bility of these pathogenic bacteria to form persister cells to survive the bactericidal antibiotics and to cause chronic infection.Furthermore, persistent isolates have the ability to change in shape and size extensively, about 4 times increase in cell length than their normal length. These phenomena are possibly the initial stages of bacterial resistance prevalence.

Cultivable oral bacteriota dysbiosis in mechanically ventilated COVID-19 patients.

Potential interactions between the SARS-CoV-2 virus and the human oral microbiota are currently investigated widely. Patients with COVID-19 requiring mechanical ventilation in an intensive care unit (ICU) setting are at high risk of developing severe complications, including ventilator-associated pneumonia, thus making oral health management important. The aim of this study was to evaluate the oral health status and assess the dysbiosis of cultivable oral bacteriota in COVID-19 patients hospitalized in an ICU with acute respiratory distress within 36 h following intubation. In this prospective cohort study, we recruited 56 adult COVID-19 patients that qualified for mechanical ventilation in the Temporary ICU for COVID-19 Patients of the University Hospital in Krakow. On admission to the ICU, oral health of patients was assessed using the modified Beck Oral Assessment Score (BOAS). Four oral habitats were sampled, namely the buccal mucosa, tongue, buccal dental surface and gingival pocket. Microorganisms were identified by MALDI/TOF mass spectrometry. The mean age of the study population was 66.5 ± 12.7 years, there were 24 (42.9%) females. All patients included in this study were intubated and ventilated in the ICU, with a corresponding high mortality rate (76.8%). On admission to ICU, 76.8% subjects scored 11-20 on the BOAS scale (median 12 [IQR 10-14]), indicating moderate or severe dysfunction of oral health. Potentially pathogenic bacteria were identified in the oral microbiota samples, including Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae in 23.2%, 39.3%, 17.9%, and 19.6% of patients, respectively. Lactobacillus spp. were present in 57.1% subjects. The mean CFU counts of all bacteria strains in dental brushes were 9.3E+5 (1.4E+6) and in gingival pockets 7.6E+5 (1.4E+6). The highest CFU counts were observed for Enterococcus spp. and, Lactobacillus spp., although these did not differ significantly from CFU counts of Streptococcus spp. and Staphylococcus spp. In this report we comprehensively characterized the oral health condition and cultivable oral bacteriota in COVID-19 patients hospitalized in an ICU with acute respiratory distress within 36 h following intubation. The oral bacteriota showed significant qualitative and quantitative dysbiosis. Hospitalization in an ICU and mechanical ventilation are important factors leading to oral dysbiosis in SARS-CoV-2 patients.

Quantitative PCR Effectively Quantifies Triazole-Susceptible and Triazole-Resistant Aspergillus fumigatus in Mixed Infections.

Increasing resistance to triazole antifungals in Aspergillus fumigatus is worrisome because of the associated high mortality of triazole-resistant A. fumigatus (TRAF) infections. While most studies have focused on single triazole-susceptible (WT) or TRAF infections, reports of TRAF cases developing mixed WT and TRAF infections have been described in several studies. However, the prevalence of mixed infections and their responses to current recommended therapies are unknown and could be inappropriate, leading to poor clinical outcomes. To address the urgent need for tools to diagnose, monitor disease development and therapy efficacies in mixed infection settings where quantification of WT versus TRAF is key, this study developed a novel qPCR assay to differentiate WT and TRAF harboring the cyp51A-TR34/L98H mutation. The proposed assay successfully quantified A. fumigatus and discriminated TRAF-TR34 in vitro and in vivo, which was achieved by increasing the yield of extracted DNA through improved homogenization and specific primers targeting the WT-sequence or TR34-insertion and a TaqMan-probe directed to A. fumigatus. The here-developed qPCR assay overcomes sensitivity issues of methodologies such as CFU counts, providing specific, reproducible, and reliable quantitative information to study and follow up the (interplay and individual) effects of mixed A. fumigatus infections on disease development and treatment responses.

Antimicrobial-free graphene nanocoating decreases fungal yeast-to-hyphal switching and maturation of cross-kingdom biofilms containing clinical and antibiotic-resistant bacteria

Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA) synergize in cross-kingdom biofilms to increase the risk of mortality and morbidity due to high resistance to immune and antimicrobial defenses. Biomedical devices and implants made with titanium are vulnerable to infections that may demand their surgical removal from the infected sites. Graphene nanocoating (GN) has promising anti-adhesive properties against C. albicans. Thus, we hypothesized that GN could prevent fungal yeast-to-hyphal switching and the development of cross-kingdom biofilms. Herein, titanium (Control) was coated with high-quality GN (coverage > 99%). Thereafter, mixed-species biofilms (C. albicans combined with S. aureus or MRSA) were allowed to develop on GN and Control. There were significant reductions in the number of viable cells, metabolic activity, and biofilm biomass on GN compared with the Control (CFU counting, XTT reduction, and crystal violet assays). Also, biofilms on GN were sparse and fragmented, whereas the Control presented several bacterial cells co-aggregating with intertwined hyphal elements (confocal and scanning electronic microscopy). Finally, GN did not induce hemolysis, an essential characteristic for blood-contacting biomaterials and devices. Thus, GN significantly inhibited the formation and maturation of deadly cross-kingdom biofilms, which can be advantageous to avoid infection and surgical removal of infected devices.

Long-Term Effects of Single-Dose Cephalosporin or Macrolide Use on the Prevalence of AmpC and Extended-Spectrum β-Lactamase Producing Escherichia coli in the Feces of Beef Cattle.

Extended-spectrum-β-lactamase (ESBL) and AmpC-lactamase-producing Enterobacteriaceae are serious public health threats. Due to an increasing number of reports of ESBL and AmpC producing Escherichia coli in agricultural settings, it is critical to understand the relationship between the use of two of the highest priority critically important human antibiotics (e.g., third generation cephalosporins [3GC] and macrolides) in food animals and their potential contribution to the selection of ESBL/AmpC E. coli. The objective of our randomized controlled feedlot trial was to measure the effects of ceftiofur crystalline-free acid and tulathromycin on 3GC resistant fecal E. coli populations in cattle before and at various time points after treatment up to and including at slaughter. Multi-level mixed-effects linear regression showed no effect of ceftiofur and tulathromycin on 3GC E. coli CFU counts at slaughter (Day 99); however, a significant (p < 0.05) population shift was observed from susceptible to 3GC resistant E. coli immediately after ceftiofur administration (Day 7). Among 799 fecal samples screened using selective media, 17.7% were ESBL/AmpC E. coli positive, which were further tested for phenotypic antibiotic susceptibility. The majority of the isolates from these plates were multidrug-resistant (94.3%) and expressed either AmpC (78.1%) or ESBL (28.1%) phenotype. A subset of isolates was whole-genome sequenced (n = 20) and identified to harbor chromosomal and/or plasmidal bla genes such as CMY-2, CTX-M, and TEM. Our findings show a time-dependent selection of antibiotics on 3GC-resistant E. coli. High prevalence of multidrug-resistant ESBL/AmpC E. coli found in cattle feces highlights the importance of prudent use of antibiotics in livestock.

Iron-Modified Blood Culture Media Allow for the Rapid Diagnosis and Isolation of the Slow-Growing Pathogen Francisella tularensis.

ABSTRACTThe life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays.IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

A novel universal adhesive for improved dentin remineralization with antibiofilm potential against Streptococcus mutans and other cariogenic-pathogens

Dental demineralization is a widespread problem and the main responsible for dental caries and dentin hypersensitivity. Dental caries is a biofilm-driven bacterial infection that causes mineral depletion in enamel and is associated with an increased prevalence of medical rehabilitative polymeric implants. Since existing tissue-engineering methods are incapable of manufacturing synthetic dentin adhesive, modern therapeutic interventions address the remineralization of caries-infected dentin to prevent its inevitable removal. In recent years, magnetic nanoparticle-coated adhesives were known to deliver drugs to the pulp and get better adhesive penetration into dentin. In the present study, we prepared and assessed the effectiveness of antimicrobial peptide (cecropin A) coated Fe3O4-nanoparticle on dentin remineralization, bond strength, and its antibiofilm potential against Streptococcus mutans, is associated with dental caries in humans. Prior to the experiment, the synthesized [email protected]3O4 nanoparticle was characterized and assessed its antibiofilm potential against S. mutans. The transcriptomic approach with qPCR analysis stated that [email protected]3O4 nanoparticle significantly downregulated the expression of biofilm-regulated genes such as GtfB, GtfC, GtfD, VicR, ComDE, and Smu0630 in S. mutans, which validates the outcome of in vitro results. Interestingly, the prepared [email protected]3O4-nanoparticle coated adhesive effectively increased the dentin remineralization and bond strength. Fascinatingly, [email protected]3O4 adhesive significantly hindered the biofilm formation of S. mutans on the dentin surface. Notably, the [email protected]3O4 adhesive accomplished reduced biofilm CFU count, lactic acid production, and biofilm metabolic activity. In addition, the [email protected]3O4 adhesive also reduced the biofilm formation of other caries-associated bacterial pathogens, including S. sobrinus, S. sanguinis, and S. mitis. Overall, the current research found that the [email protected]3O4 adhesive was more effective in remineralizing dentin lesions with antibiofilm potential in order to avoid Streptococcus-derived dental caries.

Effect of a Hygiene Protocol on Denture-Related Stomatitis Remission, Local Inflammatory Factors, and Hemodynamic Responses by Arterial Pressure.

Denture-related stomatitis (DRS) is frequent oral inflammation in complete denture wearers. This study evaluated the effect of a hygiene protocol on DRS remission, local inflammatory factors, and hemodynamic responses. Thirty-three individuals were enrolled in the study. The outcomes were measured before and after 10 days of a hygiene protocol treatment consisting of brushing the palate with a soft brush and water and denture brushing with a denture-specific brush and mild soap, as well as immersion of the denture for 20 min in a 0.25% sodium hypochlorite solution. Data were analyzed by paired Wilcoxon for biofilm removal and CFU count of microorganisms. The paired T test was used to assess salivary MUC 1, cytokines, and arterial pressure (p < 0.05). A significant difference was found in the DRS degree (p < 0.001), biofilm (p < 0.001), microbial load of Candida spp. (p < 0.001), Gram-negative (p < 0.004), Staphylococcus spp. (p < 0.001), and S. mutans (p < 0.001) of the denture, and S. mutans (p < 0.001) of the palate after use of the protocol. The salivary flow (p = 0.2) and pH (p = 0.97) did not change; there was an increase of MUC 1 (p = 0.049) and a decrease in IL-6 (p = 0.038), IL-2 (p = 0.04), IL-10 (p = 0.041), and IFNγ (p = 0.04). There was also a decrease in systolic (p = 0.012) and mean arterial pressure (p = 0.02). The current hygiene protocol reduced the inflammation degree of DRS and promoted an improvement of local inflammatory factors and a reduction in the systolic arterial pressure of the patients.

Dual antibacterial and anti-inflammatory efficacy of a chitosan-chondroitin sulfate-based in-situ forming wound dressing

None of the currently available wound dressings exhibit combined antibacterial and anti-inflammatory activity. Using polyelectrolyte complexation (PEC) between a cationic polysaccharide chitosan (CH) and an anionic glycosaminoglycan chondroitin sulfate (CS), we have developed a unique in-situ forming scaffold (CH-CS PEC), which develops at the wound site itself to influence the function of the wound bed cells. The current study demonstrated that CH-CS PEC could induce bacterial cell death through membrane pore formation and increased ROS production. Moreover, possibly due to its unique material properties including medium-soft viscoelasticity, porosity, and surface composition, CH-CS PEC could modulate macrophage function, increasing their phagocytic ability with low TNF-α and high IL-10 production. Faster wound closure and decreased CFU count was observed in an in-vivo infected wound model, with reduced NF-κB and increased VE-cadherin expression, indicating reduced inflammation and enhanced angiogenesis. In summary, this study exhibited that CH-CS PEC has substantial antibacterial and immunomodulatory properties.