10-fold Dilutions Research Articles

P-2100. Comparison of Fosfomycin MIC Values between Parent and Inner Colonies (IC) of Escherichia coli Arising During Disk Diffusion (DD) Testing

Abstract Background Recent studies have documented uropathogenic E. coli displaying the presence of IC during fosfomycin DD testing. These IC often demonstrate increased minimal inhibitory concentrations (MIC) when compared to their parent isolates. Investigating the clinical relevance of these IC and the extent of their resistance is a first step in determining whether they are associated with fosfomycin failure. Methods E. coli isolates (n = 69) collected from the University of Minnesota Medical Center (UMMC) that produced ≥ 3 discrete IC during at least one DD test were included. Parent and IC (n = 69) isolates underwent broth microdilution (BMD) testing using fosfomycin concentrations of 0.5-64 µg/mL for parent isolates and 8-1024 µg/mL for IC with incubation at 37°C for 16-20 hours. All susceptibility testing was supplemented with 25 µg/mL glucose-6-phosphate and conducted in duplicate on separate days. E. faecalis ATCC 29212 and E. coli ATCC 25922 were included as quality control strains. The results were evaluated using both CLSI (susceptible [S]: 64 µg/mL) and EUCAST (S: 8 µg/mL) breakpoints. Categorical agreement between the two guidelines were calculated. Results E. coli parent isolates had an MIC range of 1 to >64 µg/mL with a MIC50/90 of 8/32 µg/mL (Table 1). The IC isolates had significantly higher MIC ranges (< 8 and >1024 µg/mL) and MIC50/90 values of 256/ >1024 µg/mL. The parent isolates had 94.2% (n = 65) and 69.6% (n = 48) susceptibility per CLSI and EUCAST breakpoints, respectively. Using the more stringent EUCAST breakpoints, all IC isolates were resistant whereas 65.2% (n = 45) were considered resistant per CLSI breakpoints. Categorical agreement among the parent collection was 75.4% and 65.2% in the IC collection (Table 2). Conclusion Our data indicate IC arising from E. coli during fosfomycin DD testing are notably more resistant than their parent isolates, with an MIC50 increase of five, 2-fold dilutions. Future directions for this work include expanding the isolate collection and examining the fitness of these IC in comparison to their parents. Improving the understanding of the frequency, resistance, and fitness of IC arising from fosfomycin DD testing will help determine an optimal interpretation of IC, which may in turn improve patient outcomes. Disclosures Elizabeth B. Hirsch, PharmD, FCCP, FIDSA, GSK: Advisor/Consultant|GSK: Honoraria

Practice of ABO antibody titration in a transplant center: From tube method to manual gel column testing to automated column agglutination technology.

High-quality ABO antibody titre testing is required for ABO-incompatible haematopoietic stem cell transplantation and kidney transplantation. To assess the feasibility of automated ABO titration as an alternative to manual and semi-automatic titration during the peri-transplant period, a comparative study was conducted internally in a transfusion medicine laboratory. This study was performed in two stages. Firstly, the differences in anti-A/B detecting using conventional tube test (CTT) and manual column agglutination technology (CAT) were compared in group O pregnant women. Then, manual and automated CAT were applied to simultaneously detect ABO antibody levels in non-AB-group adult haematological oncology patients. In the first stage, four groups of 192 comparable results were generated from 48 subjects, which showed a high correlation between CTT and manual CAT (p < 0.001). However, the detection sensitivity of the latter was at least 1 fold higher dilution than that of the former. Fifty-six patients were tested in the second stage by simultaneous manual and automated CAT, and the paired titers differed within an acceptable range (±1 fold dilution). However, the concordance was slightly higher in group IgM (85.4%) than in group IgG (80.5%). Blood group specificity of ABO titers was also observed in this population, and no association between titers and other factors was found. Automated CAT standardises ABO titre testing and saves labor costs, although manual review of weak positive reactions is still necessary. Due to the blood group specificity of ABO antibodies, special attention should be paid to the quality control and clinical relevance of titre testing for group O recipients in ABO-mismatched transplantation.

P-1078. Gentamicin-releasing polydopamine catheter coating to prevent urinary catheter-related infections

Abstract Background Catheter-associated urinary infections (CAUTIs) are among the most common healthcare-associated infections in the United States. The risk of CAUTI corresponds to the duration of catheterization; prolonged catheter uses impacts patient morbidity, mortality, and healthcare costs. Pseudomonas aeruginosa is a leading cause of CAUTI and is particularly adept at forming surface-associated biofilms that are difficult to treat. In the present study, we developed a catheter coating using polydopamine (PD) to embed gentamicin on the surface. Bacterial Viability of PAO1 when exposed to PD-gentamicin coating Colony-forming unit (CFU) count of PAO1 exposed to concentration series of PD-only and PD-Gent well plate coating. Methods Coating synthesis: PD-antibacterial coating was synthesized using dopamine hydrochloride salt and antibiotic drugs (i.e., gentamicin) in an alkaline buffer in the presence of polyvinyl chloride (PVC) tubing. A similar procedure was used for the well-plate study using 150 µL of solution per well, followed by heating at 60 °C. Biofilm Quantification: PAO1 was added to a 96-well plate or tubes and incubated statically for 24 h. Biofilms were stained with 0.1% crystal violet, solubilized with 33% glacial acetic acid, and measured at OD590 nm. Cell viability: Colony forming units were measured using 10-fold serial dilutions on TSA agar. Biofilm formation of PAO1 after incubation with PD-gentamicin coating in PVC tubes PAO1 biofilm concentration changes with changing the PD to gentamicin ratio during coating synthesis. Results PD-gentamicin coated tubes and 96-well plates showed elimination of PAO1 viability at PD concentrations of 0.25 mg/mL and above but had little bactericidal effect at lower concentrations (Fig. 1). There was no reduction in viability in PAO1 exposed to PD or gentamicin alone. PAO1 biofilm was reduced in the median PD-gentamicin concentrations 0.1-0.5 mg/mL, but this effect was lost at the extremes of concentration (Fig. 2). Conclusion The PD-gentamicin coating eliminated PAO1 viability and reduced biofilm at PD concentration 0.25 mg/mL. Similar results were observed in 96-well plates and PVC tubes, suggesting versatility in the adherence of the coating material. Additional studies are needed to assess the duration of these effects over time. Disclosures All Authors: No reported disclosures

P-1087. Novel Antisense Wide-Range Peptide Nucleic Acids Coated on Polystyrene Plates to Prevent Bacterial Biofilm

Abstract Background Biofilms are three dimensional communities of microorganisms that are encased in self-produced extracellular polymeric substances. Biofilms play a key role in device-associated infections, including catheter-associated urinary tract infections, largely because they protect microorganisms from standard antimicrobial therapies. Antisense synthetic peptide nucleic acids (PNAs) are an emerging approach to inhibiting specific components of gene expression. Several regulatory and motility genes are relatively conserved across biofilm forming Gram-negative bacilli, such as rpoS, amrZ, rsmA, and motA. In the present study, wide range PNAs (wrPNAs) were developed by our group to exploit these conserved targets, offering a mechanism for precise, yet broad, biofilm inhibition. The impact of wrPNAs on Gram-negative biofilms and bacterial viability were investigated. Methods wrPNAs: Antisense-wrPNAs (patent pending) were designed using NCBI Blast to select a region inclusive of the start codon. The wrPNAs ranged from 12 to 14 basepairs, conjugated with a cell-penetrating peptide and O-linker, and were synthesized by PNA Bio (Figure 1). Quantify biofilm biomass: Bacterial isolates were grown in a 96-well polystyrene plate in minimal salt medium with wrPNA treatment (10 µM each) and incubated at 30˚C for 24hr in a humid chamber. Biofilms were stained with 0.1% crystal violet, solubilized in 33% glacial acetic acid, and measured at OD590 nm. Quantify bacterial viability: 10-fold serial dilutions and direct stamping onto agar plates. Statistical analysis: P values were determined using unpaired t-tests or one-way ANOVA with followup tests comparing the mean of each column to the control column (alpha = 0.05). Results wrPNA treatment significantly reduced biofilm biomass for their intended species (Figure 2). A cocktail of PNAs had a bactericidal effect on Pseudomonas aeruginosa, but wrPNA treatment had minimal effect on bacterial viability for the other Gram-negative bacilli tested (Figure 2). Conclusion wrPNAs against global regulator genes and a motility regular gene reduced biofilm biomass for their intended species. Future work will look at combination therapy to improve bactericidal effects as well as methods for coating treatments onto catheter and device surfaces. Disclosures All Authors: No reported disclosures

Nanobody-based indirect competitive ELISA for the detection of aflatoxin M1 in dairy products

Aflatoxin M1 (AFM1) is known to be carcinogenic, mutagenic, and teratogenic and poses a serious threat to food safety and human health, which makes its surveillance critical. In this study, an indirect competitive ELISA (icELISA) based on a nanobody (Nb M4) was developed for the sensitive and rapid detection of AFM1 in dairy products. In our previous work, Nb M4 was screened from a Bactrian-camel-immunized phage-displayed library. It exhibits VH-like features, possesses higher thermal stability than monoclonal antibody (mAb 1E6) and tightly binds to AFM1–BSA with a KD value of 2.5 nM. Under the optimal conditions, its half-maximal inhibitory concentration was 0.338 ng/mL, the limit of detection was 0.051 ng/mL, and linearity was noted in the range of 0.168–0.679 ng/mL. Nb M4 displayed almost no cross-reactivity with other mycotoxins. No matrix effect was observed in milk and milk powder samples, and the matrix effect in yogurt samples could be weakened by 2-fold dilution. Furthermore, validation studies in spiked samples (milk, yogurt, and milk powder) resulted in good recoveries of 95.40–111.33%, with a low coefficient of variation (2.89–6.78%). High-performance liquid chromatography was used to evaluate the accuracy and reliability of the developed icELISA, which indicated a satisfactory consistent correlation (R2 = 0.9722). This study highlights the potential of Nb M4 as a promising component for detecting AFM1 in dairy products.

Sensitivity of Primary Human Glioblastoma Cell Lines to Mumps Virus Vaccine Strain

The sensitivity of human glioblastoma cells to virus-mediated oncolysis was investigated on five patient-derived cell lines. Primary glioblastoma cells (Gbl13n, Gbl16n, Gbl17n, Gbl25n, and Gbl27n) were infected with 10-fold serial dilutions of the Leningrad-3 strain of mumps virus, virus reproduction and cytotoxicity were monitored for 96–120 hours. Immortalized human non-tumor NKE cells were used as controls to determine virus specificity. Four out of the five glioblastoma cell lines examined were susceptible to mumps virus infection, whereas no virus reproduction was observed in the non-tumor cell line. Moreover, the level of proapoptotic caspase-3 activity was increased in all infected cells 48 hours after infection. The kinetics of viral RNA accumulation in the studied glioblastoma cell lines was comparable with the rate of cell death. The data suggest that glioblastoma cell lines are permissive for mumps virus. Glioblastoma cell lines differed in type I IFN production in response to mumps virus infection. In addition, it was shown that MV infection was able to induce immunogenic death of glioblastoma cells.

Anti-dermatophytic activities and time-kill kinetics of the methanol extracts of Napoleona imperialis

Background: Dermatophytosis is one of the most common cutaneous infections in the world. This is a superficial fungal infection that pose public health challenges to man and animals. The objective of this study is to evaluate the anti-dermatophytic activities of the different parts of Napoleona imperialis extract against selected clinical dermatophytes isolated from school children in Anambra State, Nigeria.Methodology: The pulverized materials of the authenticated plant parts were extracted using cold maceration method for 48 hours in methanol. Stock solutions of the extracts were prepared by dissolving 1000mg of the extract in 2ml of dimethylsulphoxide (DMSO) to obtain a final concentration of 500mg/ml, that was used to primarily screen the plant extracts for their anti-dermatophytic activities on the selected dermatophytes by the agar well diffusion method. For determining the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), stock solution of the plant extracts was prepared by dissolving 10000mg of the extract in 2 ml of DMSO to attain a final concentration of 5,000 mg/ml, which was used to prepare different concentrations of the extract by 2-fold serial dilution. The extracts were then tested on selected dermatophytes consisting of two isolates of Trichophyton mentagrophytes, one isolate of Microsporum audouinii, one isolate of Microsporum canis, and two isolates of Microsporum ferrugineum. The time kill kinetics was also performed using standard method.Results: The mean (±SD) inhibition zone diameter produced by the stem bark extract against the different dermatophytes at concentration of 500mg/ml was significantly higher than all other plant extracts used (p&lt;0.05) with M. ferrugineum as the most susceptible. The MICs and MFCs were the same for the leaf, root, stem bark and seed extracts of the plant, which ranged from 31.25-250 mg/ml for M. audouinii; 62.5-250 mg/ml for M. canis, and 0.978-62.5 mg/ml for M. ferrugineum 2. Similarly, the MICs and MFCs were the same for the root extract of the plant against T. mentagrophyte 2 (62.5 mg/ml), stem bark extract against T. mentagrophyte 1 (3.912 mg/ml) and against T. mentagrophyte 2 (1.956 mg/ml). The seed extract against T. mentagrophyte 1 and against M. ferrugineum 1 also had the same MIC and MFC (15.625 mg/ml). In the time -kill assay, there was drastic reduction in the number of viable cells count within 0-1 hour, and at 8-hour time period, there were no viable cells.Conclusion: Crude methanol extracts of N. imperialis stem bark exhibited higher fungicidal effect when compared to the other plant parts. This extract can be a complementary source of novel antifungal agents. The study provides evidence for the use of this plant in traditional settings for the treatment of dermatophytosis.

PH-Adjusted Liquid SERS Approach: Toward a Reliable Plasma-Based Early Stage Lung Cancer Detection.

Surface-enhanced Raman spectroscopy (SERS) provides a rapid and nondestructive method for biological plasma analysis, offering unparalleled sensitivity and specificity. However, most current studies predominantly employ the drop-cast method, where liquid samples are dried on the SERS substrate for spectral recording. While effective, this method is both time-consuming and inconsistent. Additionally, the Raman spectral signal of uric acid in plasma often dominates, overshadowing other crucial biomarkers necessary for disease differentiation. In this study, we present the application of label-free SERS detection for distinguishing early stage lung cancer (LC) from healthy controls (HC) using plasma samples. Plasma was diluted, adjusted to a weakly acidic pH, and combined with silver nanoparticles (Ag NPs) for SERS analysis. To maintain the plasma in a liquid state and prevent evaporation, liquid paraffin was employed. Our findings indicate that a 10-fold dilution of plasma under weakly acidic conditions significantly enhanced diagnostic performance, achieving an area under the receiver operating characteristic curve (AUC) of 0.975. This method is featured by its simplicity and high efficiency, presenting a promising strategy for the minimally invasive diagnosis of early stage LC using SERS technology.

Comparative survival of five porcine reproductive and respiratory syndrome virus strains on six fomites.

Despite the availability of vaccines, porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause disease outbreaks in pigs worldwide. One of the reasons for this problem is the frequent mutation of the virus, which creates new variants. This study was conducted to determine the survival of five PRRSV strains on four non-porous and two porous fomites at 22-25°C (room temperature). Five strains of PRRSV (1-7-4, 1-8-4, VR 2332, 1-4-4 MN, and 1-4-4 SD) were used in this study. Circular pieces of aluminum, boot material, polyvinyl chloride, stainless steel, cardboard, and concrete were used as fomites. A small volume of each virus strain was placed on the fomite, followed by incubation at room temperature. The virus surviving at different time points was eluted in an eluent solution. Serial 10-fold dilutions of the eluate were inoculated in MARC-145 cells for virus titration. Multivariate analysis of variance (MANOVA) was used for statistical analysis, and post hoc analysis was used for multiple pairwise comparisons. Three of the five strains were inactivated within 36 h on non-porous fomites; the remaining two survived for 72 h. On porous fomites, all five strains were inactivated within 12 h. MANOVA at p < 0.05 indicated that the inactivation of strains 1-7-4 and 1-4-4 SD was significant compared with the other strains. In addition, the number of virus titers was significantly reduced on stainless steel compared to other fomites. Our findings illustrate how the interaction between the PRRSV strain and fomite material affect viral stability over time. The results also provide an understanding of fomites' role in PRRSV epidemiology as indirect transmitters of the virus.

Identification of IspD as a novel target for tuberculosis treatment using compound M6.

Tuberculosis (TB) is a serious infectious disease that endangers human health, and TB becomes more difficult in eradiation due to its multidrug resistance (MDR). The objective of this research was to identify novel targets for treating TB. A 2-fold serial dilution method was used to determine minimal inhibitory concentrations (MIC) of compound M6 against Mycobacterium smegmatis (M. smegmatis). Compound M6 was subjected to reverse molecular docking with seven Mycobacterium tuberculosis proteins, and the best binding protein with the highest LibDock score was evaluated. The target protein with the highest score was purified through prokaryotic expression. Isolated target proteins were investigated for the enzyme activities and for the kinetic effect of compound M6 by absorbance detection. Subsequently, the CRISPR/Cas9 technology was employed to inhibit target gene expression for detecting MIC changes. Finally, potential targets were evaluated for the effect of the compound M6 in bacteria. The MIC values of compound M6 against M. smegmatis were 32 μg/mL. The results from reverse molecular docking show that IspD has the highest LibDock score of 142.50, followed by Rv0674, IspF, and Dxr, with docking scores of 110.762, 71.6955, and 57.7446, respectively. IspD is a key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway of MTB. The aKi and Ki values of M6 for the substrate MEP are 609.58 μM and 81.33 μM. For CTP, the aKi and Ki values are 657.89 μM and 40.07 μM. With tetracycline inducing CRISPR/Cas9 to suppress the expression of IspD, the MIC value of M6 against IspD went down significantly from 32 μg/mL to 4 μg/mL. IspD is a novel target of the compound M6 for treating TB.

HPLC analysis and antimicrobial activity of Vitex altissima leaves extracts

The exploration of ethnomedicinal plants for phytochemical and pharmacological properties is crucial for developing novel therapeutics for chronic diseases. Vitex altissima, known as the Peacock chaste tree, is a significant member of the Verbenaceae family, is widely utilized in traditional medicine. This study investigates the phytochemical composition and antibacterial properties of V. altissima, a plant recognized for its medicinal use by various indigenous communities, including the Malayali tribes of Servarayan hills. The study involved extracting phytochemicals from the leaves of V. altissima using hexane, ethyl acetate and methanol through soxhlet extraction. The extracts were analyzed for secondary metabolites, including carbohydrates, proteins, amino acids, steroids, glycosides, alkaloids, tannins, phenolics, flavonoids, terpenoids, saponins, anthraquinones, oils and resins, diterpenes, phlobatannins and coumarins. Quantitative assessments of phenolic compounds, flavonoids and alkaloids were performed. Column chromatography was employed to fractionate the methanolic extract, resulting in 12 distinct fractions. These fractions were screened for antibacterial activity against E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aureus using the agar well diffusion method. The minimal inhibitory concentration (MIC) was determined through a 2-fold serial dilution technique. High-performance liquid chromatography (HPLC) was utilized to identify the polyphenolic compounds responsible for antibacterial activity. Phytochemical screening revealed a diverse array of bioactive compounds in the extracts, with methanol extracts showing the highest total phenolic content (218 ± 7.21 mg GAE/g of extract) and significant antibacterial activity. The ethyl acetate extract exhibited the highest total flavonoid content (40.67 ± 6.65 mg QE/g of extract). Among the fractions, the column fraction from the chloroform-methanol gradient (VACF) demonstrated superior antimicrobial activity, particularly against S. mutans and P. aureginosa. The MIC values for VACF were 427 µg/mL for P. aeruginosa and 400 µg/mL for S. aureus, indicating potent antibacterial properties. HPLC analysis identified key polyphenolic compounds, including p-coumaric acid, ferulic acid and elagic acid, as the primary contributors to the antibacterial activity. The presence of these compounds aligns with the observed antimicrobial efficacy and highlights the potential of V. altissima extracts as a source of natural antibacterial agents.

8801 Towards Safe and Efficacious Medical Therapy for Patients with Clinically Non-Functioning Pituitary Tumors

Abstract Disclosure: D. Zhang: None. M. Bergsneider: None. W. Kim: None. M.B. Wang: None. H. Vinters: None. A.P. Heaney: None. Clinically non-functioning pituitary tumors (CNFTs) account for one-third of all pituitary adenomas. They do not cause clinical and/or biochemical evidence of tumor-related hormone hypersecretion, grow insidiously and typically present with mass effect symptoms such as headache and visual field deficits. Surgical resection is the current standard of care for CNFTs but complete resection is infrequently achieved due to their size and invariable invasion of locally adjacent structures. CNFT remnant tumor regrowth rates approach 50% and these patients may need repeat surgery and/or radiotherapy (RT). The latter is quite effective in achieving tumor control, but carries a risk of hypopituitarism of ∼ 40% at 10 years. Although dopamine agonists and somatostatin receptor ligands have been explored as medical therapy, there are currently no consistently effective medical treatments for CNFTs. There is a clear unmet need for novel safe and effective medical therapies for these comparatively common tumors. In a highly innovative approach we have used patient-derived ex vivo 3D tumoroid human CNFTs in an automated high throughput compound screen (HTS) to identify novel tumoricidal and growth inhibitory compounds for CNFTs. To date, we have screened ∼4,000 compounds and have identified 13 potential hits that inhibited tumor cell proliferation (a reduction of 76±15%) and induced apoptosis (induction rate of 8.2 ± 6.6) in the CNFT tumoroids respectively. The hit compounds to date fall into 3 categories based on their targets, namely Na+/K+-ATPase inhibitors (n=6), reactive oxygen species (ROS) regulators (n=4), and cell cycle inhibitors (n=3). Representative “hit” compounds from each group identified in our primary screen were rescreened in triplicate in 20 concentrations from 25µM to 50pM at 2-fold dilutions. Dose-response curves characterized compound doses that inhibited cell viability by 50% (IC50) and ranged from 13nM to 103nM. Two of these 3 compounds also induced 2-3-fold apoptosis at 0.1uM with the final compound displaying a potent 5-10-fold pro-apoptotic effect at 0.15uM concentration. In parallel, we used single cell RNA sequencing to validate targets that emerged from our HTS and to characterize the mechanism of action (MOA) of these hit compounds. As proof of concept, several of the known drug targets were noted to be amongst the highest differentially expressed genes in the CNFT tumor cell population. This approach validating a candidate target in conjunction with demonstrable activity of a drug for that same target in CNFTs may simultaneously provide us with a back-bone molecule to further develop with structural activity relationship analysis and/or allow us to individualize drug choice in patients based on immunocytochemical tumor target expression. Presentation: 6/1/2024

Combination of medicinal plants with antibiotics against Klebsiella pneumoniae and Acinetobacter baumannii

Background and aims: One essential plant-based strategy to deal with infections is antimicrobial synergism, which can make antimicrobials more efficient. The aim of this study was to investigate the interaction between these extracts and two widely used antibiotics, meropenem and gentamicin, on two multidrug-resistant (MDR) bacteria, K. pneumoniae and A. baumannii, in vitro. Methods: Different concentrations of Rosa damascena Mill., Malva sylvestris L., and Zataria multiflora Boiss. hydroalcoholic extracts (2-fold serial dilution from 131072 to 256 μg/mL) were administered against two MDR bacteria, and their combination with gentamicin and meropenem (serial dilution from 32 to 0.015 μg/mL) was investigated by the resazurin-based microdilution and the checkerboard method. The phytochemical properties of the extracts were also examined, and the total phenolic content (TPC), total flavonoid content (TFC), anthocyanin content, and antioxidant capacity of the extracts were determined. Results: Z. multiflora and R. damascena showed high antibacterial activity, and their minimal inhibitory concentrations on Acinetobacter baumannii were 1024 and 2048 μg/mL, respectively. Z. multiflora also had high TFC, TPC, and antioxidant activity and demonstrated additive interaction with meropenem and gentamicin with fractional inhibitory concentrations of 1 and 0.75, respectively. Conclusion: We suggest the potency of Z. multiflora-antibiotic combinations in treating MDR A. baumannii after future clinical studies.

Synergistic Effect of Metronidazole and Chlorhexidine against Porphyromonas gingivalis Growth: An In Vitro Study.

Aim: To evaluate the potential synergistic activity of metronidazole (MTZ) and chlorhexidine (CHX) against Porphyromonas. gingivalis (P. gingivalis) growth. Methods: Antimicrobial susceptibility tests of P. gingivalis to MTZ and CHX were performed on in vitro serial 2-fold dilutions of MTZ (from 1 mg/mL to 0.015 mg/mL) and CHX (from 1 mg/mL to 0.03 mg/mL) in thioglycollate medium broth in a 96-well plate. The turbidity of each sample was analyzed by absorbance spectrophotometry at 450 nm wavelengths by using an enzyme-linked immunosorbent assay (ELISA) reader. The MIC50 (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were assessed. To investigate the potential synergism between MTZ and CHX, bacterial cells were treated with MTZ or CHX, as described above, either alone or in combination. Results: The MIC50 of MTZ was 0.03 mg/mL while that of CHX ranged from 0.12 to 0.06 mg/mL. MTZ and CHX exerted a significant inhibitory effect on P. gingivalis growth in a dose-dependent manner. MTZ at a low and ineffective concentration of 0.015 mg/mL, associated with a suboptimal concentration of CHX (0.03 mg/mL), exhibited a significant synergistic inhibitory effect on bacterial growth (50% inhibition vs. control) (p < 0.001), and the effect was more remarkable with 0.06 mg/mL CHX (75% inhibition vs. control). Conclusions: CHX and MTZ showed a significant synergistic effect against P. gingivalis growth. A non-effective concentration of MTZ (0.015 mg/mL) combined with suboptimal concentrations of CHX (0.03 mg/mL and 0.06 mg/mL) were related to a 50% growth in the inhibition and 99.99% death of P. gingivalis, respectively. The applicability of the clinical use of these concentrations should be tested in randomized controlled trials.

DdPCR for the Detection and Absolute Quantification of Oropouche Virus.

Oropouche virus (OROV) is a segmented RNA virus belonging to the genus Orthobunyavirus in the family Peribunyaviridae. Herein, an in-house droplet digital PCR (ddPCR) assay was used for the detection and quantification of OROV. The ddPCR reaction was assessed as duplex assay using the human housekeeping gene RPP30. Limit of detection (LoD) analysis was performed in whole blood, serum, and urine. The assay was executed on a total of 28 clinical samples (whole blood n = 9, serum n = 11, and urine n = 8), of which 16 specimens were tested positive at the routine molecular diagnostics (endpoint and real-time PCRs). The LoD of the ddPCR performed using 10-fold serial dilution of OROV detected up to 1 cp/µL in all the biological matrices. Compared to the routine molecular diagnostics, the ddPCR assay showed 100% sensitivity for whole blood and serum and 75% for urine, highlighting higher positive rate of ddPCR. We have established a quantitative RNA detection method of OROV with high sensitivity and specificity based on ddPCR. This test is capable of quantitatively monitoring the viral load of OROV and can contribute, in addition to laboratory diagnosis, to shed light on the pathogenesis, filling in the knowledge gaps of this neglected disease and to the vector control programs.

Investigation on the Distribution of Antibiotic Resistant Bacteria In Dry Fish of Chalan Beel Area From Northwestern Part of Bangladesh

Dried fish, a valuable animal protein source, may pose health risks due to microbial contamination influenced by water quality, hygiene practices, handling, packaging, and transportation. This research focuses on conducting an in-depth analysis of the occurrence and antibiotic resistance patterns of bacteria, identified through 16S rRNA sequencing, in different dried fish varieties obtained from the Chalan beel area in Northwestern Bangladesh. For the purpose of this study, homogenization of collected samples was carried out and resulting homogenates were refrigerated at a temperature of -20°C. Then the samples were subjected to a series of 10-fold dilutions and spread over 1.5% nutrient agar supplemented with Tetracycline. Thereafter, plates were incubated at 37°C for a duration of 24 hours to form bacterial colonies. Twelve aseptically collected dried fish samples had a total viable count ranging from 2.0 × 10² to 6.5 × 10⁵ CFU/ml, with tetracycline influence at 32 µg/ml (7 × 10² to 5.4 × 10³ CFU/ml) and 64 µg/ml (3.0 × 10² to 2.1 × 10³ CFU/ml). Notably, Tengra fish (Mystus sp.) exhibited the highest viable count 6.5 × 105 CFU/ml (Control), while Darikana (Esomus danricus), Chela (Salmostoma phulo), Moa (Amblypharyngodon mola), and Guchi (Mastacembelus pancalus) fish showed the lowest count (3.0 × 10² CFU/ml) at a Tetracycline concentration of 64 µg/ml. Moreover, microbial analysis using 16S rRNA gene sequencing identified twelve unique bacteria belong to genera. The most prevalent genus appears to be Enterobacter, identified by 6 strains. These include Enterobacter hormaechei strains UCHE20, RB13, AUH-ENM30 and 20710, Enterobacter ludwigii strain JCR-38 and Enterobacter sp. M3. Other identified genera included Klebsiella (with Klebsiella variicola and Klebsiella pneumoniae), Proteus (Proteus mirabilis), Macrococcus (Macrococcus caseolyticus), Acinetobacter (Acinetobacter johnsonii), and Bacillus (Bacillus sp.). This study provides valuable insights into the complex microbial dynamics in dried fish, emphasizing the need for enhanced food safety and public health measures. J. Bio-Sci. 32(2): 61-69, 2024

Cell Extracts Derived from Cypress and Cedar Show Antiviral Activity against Enveloped Viruses.

The antiviral efficacy of cell-extracts (CEs) derived from cypress (Chamaecyparis obtusa (Siebold & Zucc.) Endl., C. obtusa) and cedar (Cryptomeria japonica (Thunb. ex. L.) D.Don, C. japonica) was assessed using phi6 and MS2 bacteriophages, which are widely accepted surrogate models for enveloped and non-enveloped viruses, in order to verify their potential use as antiviral agents. Our results indicate that CEs derived from C. obtusa are dominantly composed of terpinen-4-ol (18.0%), α-terpinyl acetate (10.1%), bornyl acetate (9.7%), limonene (7.1%), and γ-terpinene (6.7%), while CEs derived from C. japonica are dominantly composed of terpinen-4-ol (48.0%) and α-pinene (15.9%), which exhibited robust antiviral activity against phi6 bacteriophage. Both CEs successfully inactivated the phi6 bacteriophage below the detection limit (10 PFU/mL) within a short exposure time of 30 s (log reduction value, LRV > 4). Through exposure experiments utilizing CEs with content ratios prepared via 2-fold serial dilutions (ranging from 3.13% to 100%), we demonstrated that the antiviral effect could be sustained up to a concentration of 25% (C. obtusa LRV = 3.8, C. japonica LRV > 4.3 at a 25% CE content ratio for each species). However, CEs with content ratios below 12.5% did not produce a significant reduction in bacteriophage concentration and consequently lost their antiviral effects. Conversely, both CEs did not exhibit antiviral activity against MS2 bacteriophage, a non-enveloped virus. Our findings reveal for the first time the potential of CEs derived from C. obtusa and C. japonica for use as antiviral agents specifically targeting enveloped viruses.

EVALUATION OF THE TOXIC POTENTIAL OF GRAMOXONE HERBICIDE ON THE WEIGHT AND LIVER OF ALBINO RATS.

This research was carried out to evaluate the effect of diluted gramoxone herbicide at a dilution factor of 104 in a 10-fold serial dilution in graded doses of 0.25, 0.5 and 0.75 mg/kg b.w on male and female rats. Forty male and female rats of weights 79g to 119g were divided into group A, B, C and D. Each group contains 5 males and 5 females. Control group A and 0.25,0.5 and 0.75 mg/kg b.w orally treated rats in group B, C, and D were fed with pelletized feeds and water ad libitum for 28 days respectively. The rats were allowed to starve a day and weighed prior to sacrifice. Blood samples were collected in EDTA bottles. Result showed a decrease in AST, ALT, ALP in all treated groups in male rats, a decrease in AST in all treated groups, in ALT in group B and C (p&gt;0.05) but an increase in ALT in group D and an increase in ALP in all treated group in female rats (p&lt;0.05) compared with control. TP showed a decrease in all treated group in male rats (p&lt;0.05) measured with control. Albumin decreased/increased in group B/C in male rats, increased in group C and equal values in group B and D in male and female rats compared with control. There was an increased in weight in all treated groups and mild inflammatory effects on the liver tissue in group B and D. The results further registered gramoxone herbicide having paraquat as active ingredient toxic to the liver even at a much diluted state and at low doses.

Comparative evaluation of real-time polymerase chain reaction, conventional PCR and microscopy for detection of Babesia bigemina in bovines of Punjab (India).

Babesia bigemina is a major cause of bovine babesiosis, an economically devastating tick-borne disease that needs timely diagnosis for precise treatment. In present investigation, the detection efficacy of real-time PCR (qPCR) in comparison to conventional PCR and microscopy targeting 18S ribosomal gene was evaluated on 95 bovines (70 cattle and 25 buffaloes) suspected for babesiosis. Real-time PCR was standardized with the 10-fold serial dilutions in duplication of the given positive control (2 × 106 copy number) ranging from 106 to 100 copy number/µL and mean Ct value of each dilution was taken to extrapolate the curve. The samples with Ct value 36.92 of 10 copy number/µL were considered as positive. Out of 95 samples, 5 (5.26%), 21 (22.10%) and 49 (51.58%) positive by microscopy, conventional PCR and real-time PCR were in corresponding range of > 106-104, 104-103, and 103-<10 copy number/µL, respectively. The concordance of real-time PCR with conventional PCR and microscopy was moderate (Kappa = 0.523) and slight (Kappa = 0.09), respectively. The cows were at four times risk than the buffaloes for B. bigemina infection (Odds ratio:3.85, CI:1.4255-10.4370). This pioneer report from Punjab state (India) of application of real-time PCR to detect B. bigemina in bovines was found to be more sensitive than conventional PCR and microscopy that needs further investigations on a greater number of random samples.

Experimental Investigation of Tannery wastewater by Electrocoagulation method

Abstract Treating tannery wastewater poses a significant challenge due to its composition, comprising biogenic materials from hides and a diverse range of organic and inorganic chemicals. Experimental procedures were conducted using an electrochemical reactor employing sacrificial electrodes—mild steel as both anode and cathode—in a unceasing process. Numerous working parameters such as current density and electrolysis duration were examined to achieve optimal reduction in presence of turbidity, total solids, COD, BOD, TDS, and Chromium content. Enhanced reductions in COD, BOD, TDS, and chromium content were observed at higher flow rates. The applied current density also notably impacted the reduction efficiency of COD, BOD, TDS, and chromium content. Investigation into the electro coagulation method employing short cell current (1A) and solvable mild steel electrodes revealed their efficacy compared to aluminium electrodes, particularly in chromium removal, achieving over 90% removal efficiency. However, the use of mild steel electrodes resulted in the formation of a black precipitate characteristic of iron (II) sulphides during the treatment, although effectively reducing effluent coloration. The removal efficiency of chloride, however, remained lower, at less than 12%. A comprehensive discussion on the mechanisms involved in the removal of COD, chromium, total dissolved salts, total solids, chloride and coloration using soluble mild steel electrodes was presented. The predominant role of the electrode was highlighted by treating tannery wastewater with mild steel electrodes, followed by filtration, resulting in removal rates of 68.0% for chemical oxygen demand, 43.1% for chromium, 55.1% for total dissolved salts and 84.3% for coloration, considering initial concentrations of 2850 mg/L, 230 mg/L, 100 mg/L, 100 mg/L, and a 256- fold dilution, respectively.