scholarly journals Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA)

BioTech ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 19
Author(s):  
Maria Grazia Aruta ◽  
Martina Carducci ◽  
Francesca Micoli ◽  
Francesca Necchi ◽  
Omar Rossi
Keyword(s):  
High Throughput ◽  
Bactericidal Activity ◽  
High Performance ◽  
Traditional Methods ◽  
Bactericidal Assay ◽  
Colony Forming Units ◽  
Serum Bactericidal Assay ◽  
Live Bacteria

Serum bactericidal assay (SBA) is the method to investigate in vitro complement-mediated bactericidal activity of sera raised upon vaccination. The assay is based on incubating the target bacteria and exogenous complement with sera at different dilutions and the result of the assay is represented by the sera dilution being able to kill 50% of bacteria present in the inoculum. The traditional readout of the assay is based on measurement of colony-forming units (CFU) obtained after plating different reaction mixes on agar. This readout is at low throughput and time consuming, even when automated counting is used. We previously described a novel assay with a luminescence readout (L-SBA) based on measurement of ATP released by live bacteria, which allowed to substantially increase the throughput as well as to reduce the time necessary to perform the assay when compared to traditional methods. Here we present a further improvement of the assay by moving from a 96-well to a 384-well format, which allowed us to further increase the throughput and substantially reduce costs while maintaining the high performance of the previously described L-SBA method. The method has been successfully applied to a variety of different pathogens.

scholarly journals Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella

2020 ◽  
Vol 9 (2) ◽  
pp. 14 ◽  
Author(s):  
Omar Rossi ◽  
Eleonora Molesti ◽  
Allan Saul ◽  
Carlo Giannelli ◽  
Francesca Micoli ◽  
...  
Keyword(s):  
High Throughput ◽  
Bactericidal Activity ◽  
High Specificity ◽  
Laboratory Evaluation ◽  
Effective Vaccine ◽  
Bactericidal Assay ◽  
Laboratory Characterization ◽  
Homologous Strain ◽  
Human Sera ◽  
Serum Bactericidal Assay

Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.

scholarly journals Intralaboratory evaluation of luminescence based high-throughput Serum Bactericidal Assay (L-SBA) to determine bactericidal activity of human sera against Shigella

2020 ◽  
Author(s):  
O. Rossi ◽  
E. Molesti ◽  
A. Saul ◽  
C. Giannelli ◽  
F. Micoli ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
High Throughput ◽  
Bactericidal Activity ◽  
High Specificity ◽  
Effective Vaccine ◽  
Correlate Of Protection ◽  
Bactericidal Assay ◽  
Immune Correlate ◽  
Human Sera ◽  
Serum Bactericidal Assay

ABSTRACTDespite the huge decrease in deaths caused by Shigella worldwide in the last decades, shigellosis is still causing over 200,000 deaths every year. No vaccine is currently available, and the morbidity of disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not been established yet, the demonstration of bactericidal activity of antibodies induced upon vaccination may provide one means of functionality of antibodies induced on protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA).Here we present the development and intra-laboratory characterisation of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against homologous strain without any heterologous aspecificity detected against species-related and not species-related strains. We assessed linearity, repeatability and reproducibility of L-SBA on human sera.This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine bactericidal activity of any non-clinical and clinical sera that rely on complement mediated killing.IMPORTANCEShigella is an important cause of diarrhoea worldwide and antimicrobial resistance is on rise, thus efforts by several groups are ongoing to produce a safe and effective vaccine against shigellosis. Although a clear immune correlate of protection has not been established, demonstration of bactericidal capacity of sera from patients immunised with Shigella vaccines may provide one means of protecting against shigellosis. We have developed and fully characterised a novel high-throughput L-SBA method for evaluation of functionality of antibodies raised against S. sonnei in human sera. This work will allow the clinical testing of human sera raised against GMMA-based and potentially all vaccines producing antibodies than can work via complement mediated manner.

scholarly journals Development, Interlaboratory Evaluations, and Application of a Simple, High-ThroughputShigellaSerum Bactericidal Assay

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Moon H. Nahm ◽  
Jigui Yu ◽  
Hailey P. Weerts ◽  
Heather Wenzel ◽  
Chitradevi S. Tamilselvi ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Shigella Flexneri ◽  
Binding Activity ◽  
Serum Samples ◽  
Bacterial Killing ◽  
Content Type ◽  
Bactericidal Assay ◽  
Serum Bactericidal Assay ◽  
Reference Serum

ABSTRACTShigellais an important cause of diarrhea worldwide, with serotypesShigella flexneri2a,S. flexneri3a, andShigella sonneidemonstrating epidemiological prevalence. Many development efforts are focused onShigellalipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization withShigellavaccines containing LPS can elicit antibodies capable of killingShigellain a serotype-specific manner. Thus, to facilitateShigellavaccine development, we have developed a serum bactericidal assay (SBA) specific for threeShigellaserotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies againstS. flexneri2a,S. flexneri3a, andS. sonnei. Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. TheShigellaSBA, combined with a reference serum, should facilitate the development ofShigellavaccines across the field.IMPORTANCEShigellais an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effectiveShigellavaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuringShigella-specific functional antibodiesin vitro. We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitateShigellavaccine development.

scholarly journals 1559. Ertapenem Plus Ceftriaxone or Ceftaroline Dual Β-Lactam Combination for Enterococcus faecalis

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S569-S569
Author(s):  
Jaclyn A Cusumano ◽  
Kathryn E Daffinee ◽  
Kerry LaPlante
Keyword(s):  
Enterococcus Faecalis ◽  
Bactericidal Activity ◽  
Half Life ◽  
Wild Type Strain ◽  
Standard Of Care ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Initial Inoculum ◽  
Colony Forming Units

Abstract Background Ampicillin-ceftriaxone β-lactam therapy has become the standard of care for treating serious Enterococcus faecalis infections. Alternative regimens are of interest due to ceftriaxone’s association with C. difficile infections and VRE colonization, and ampicillin’s instability and inconvenient dosing schedule. Methods E. faecalis wild-type strain JH2-2 was utilized in a 48-hour in vitro pharmacodynamic model with a starting inoculum of 106 colony-forming units (CFU)/mL. Models were performed in duplicate to triplicate. Simulated doses of ertapenem 1g every 24 hours (fCmax 12.2 μg/mL; half-life 4 hours; MIC 4 μg/mL), ceftriaxone 2 g every 12 hours (fCmax 28.5 μg/mL; half-life 6.5 hours; MIC 512 μg/mL), and ceftaroline 600 mg every 8 hours (fCmax 27.1 μg/mL; half-life 2.7 hours; MIC 2 μg/mL) were tested. Ertapenem was also combined with ceftriaxone or ceftaroline. Bacterial counts were obtained at 0, 4, 8, 24, 32, and 48 hours. Bactericidal activity was defined as ≥ 3-log10 CFU/mL reduction from the initial inoculum. MICs were assessed at 0, 24, and 48 hours using E-tests in accordance with CLSI. Results Ertapenem plus ceftriaxone, and ertapenem plus ceftaroline demonstrated bactericidal activity at 24 hours, but bacterial regrowth was observed at 48 hours (Table 1). An ertapenem MIC increase was only noted in one set of the ertapenem plus ceftriaxone models to 16mcg/mL at 48 hours, from 4mcg/mL at 0 hours. All other models did not have an increase in MIC. Conclusion Bactericidal activity of ertapenem-based dual β-lactam combinations may prove to be an alternative treatment for severe E. faecalis infections. Mechanistic understanding of penicillin-binding protein (PBP) saturation and optimization of antimicrobial pharmacodynamics must be explored. Disclosures All authors: No reported disclosures.

scholarly journals Opsonophagocytosis of Fluorescent Polystyrene Beads Coupled to Neisseria meningitidis Serogroup A, C, Y, or W135 Polysaccharide Correlates with Serum Bactericidal Activity

2002 ◽  
Vol 9 (2) ◽  
pp. 485-488 ◽  
Author(s):  
Joseph Martinez ◽  
Tamara Pilishvili ◽  
Suzanne Barnard ◽  
Joseph Caba ◽  
Willie Spear ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Bactericidal Activity ◽  
Simultaneous Measurement ◽  
Meningococcal Vaccine ◽  
Specific Flow ◽  
Polystyrene Beads ◽  
Serum Bactericidal Activity ◽  
Bactericidal Assay ◽  
Flow Cytometric ◽  
Serum Bactericidal Assay

ABSTRACT We developed a polysaccharide-specific flow cytometric opsonophagocytic assay (OPA) for the simultaneous measurement of functional antibody to Neisseria meningitidis serogroups A, C, Y, and W135. OPA titers significantly correlated with serum bactericidal assay titers for all serogroups tested (mean r = 0.96; P < 0.001). OPA could be used in meningococcal vaccine evaluation.

scholarly journals Pharmacokinetic and pharmacodynamic integration for optimal dosage of cefquinome against Streptococcus equi subsp. equi in foals

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Dong-Ha Lee ◽  
Biruk Tesfaye Birhanu ◽  
Eon-Bee Lee ◽  
Seung-Jin Lee ◽  
Naila Boby ◽  
...  
Keyword(s):  
Bactericidal Activity ◽  
High Performance ◽  
Ex Vivo ◽  
Area Under The Curve ◽  
Pharmacokinetic Parameters ◽  
Optimal Dosage ◽  
Clinical Environment ◽  
Bacterial Killing ◽  
Streptococcus Equi

Abstract Cefquinome is administered in horses for the treatment of respiratory infection caused by Streptococcus equi subsp. zooepidemicus, and septicemia caused by Escherichia coli. However, there have been no attempts to use cefquinome against Streptococcus equi subsp. equi (S. equi), the causative agent of strangles. Hence the objective of this study was to calculate an optimal dosage of cefquinome against S. equi based on pharmacokinetics and pharmacodynamics integration. Cefquinome (1.0 mg/kg) was administered by intravenous and intramuscular routes to six healthy thoroughbred foals. Serum cefquinome concentrations were determined by high-performance liquid chromatography. The in vitro and ex vivo antibacterial activity were determined from minimum inhibitory concentrations (MIC) and bacterial killing curves. The optimal dosage was calculated from the integration of pharmacokinetic parameters and area under the curve (AUC24h/MIC) values. Total body clearance and volume of distribution of cefquinome after intravenous administration were 0.06 L/h/kg and 0.09 L/kg, respectively. Following intramuscular administration, a maximum concentration of 0.73 μg/mL at 1.52 h (Tmax) and a systemic bioavailability of 37.45% were observed. The MIC of cefquinome against S. equi was 0.016 μg/mL. The ex vivo AUC24h/MIC values representing bacteriostatic, and bactericidal activity were 113.11, and 143.14 h, respectively. Whereas the %T > MIC for bactericidal activity was 153.34%. In conclusion, based on AUC24h/MIC values and pharmacokinetic parameters, cefquinome when administered by intramuscularly at a dosage of 0.53 mg/kg every 24 h, would be effective against infection caused by S. equi in foals. Further studies may be necessary to confirm its therapeutic efficacy in a clinical environment.

scholarly journals Comparison of Live Bacteria Aerosolization during Removal of Dental Enamel Using a 9.3μm CO2 Laser, a 2.78μm Erbium Laser and a High-Speed Dental Drill

2021 ◽  
Vol 6 (4) ◽  
Author(s):  
Ali Badreddine
Keyword(s):  
Co2 Laser ◽  
High Speed ◽  
Dental Enamel ◽  
In Vitro Study ◽  
Vitro Study ◽  
Erbium Laser ◽  
Dental Procedures ◽  
Colony Forming Units ◽  
Live Bacteria

Purpose: To measure the amount of viable pathogens in the aerosol/splatter generated during dental procedures using lasers and high-speed drill. Methods: Three systems were used in this in vitro study: 9.3μm CO2 laser, 2.78μm erbium laser and a high-speed drill. 45 uncleaned human molars were randomly selected to be used for the test groups for the three systems. Bacteria ejected while cutting the buccal or lingual surfaces were collected on tryptic soy agar (TSA) plates in identical conditions for each measurement. On the opposite surface of each tooth, a non-cutting mist spray was applied. Results: The CO2 laser resulted in colony-forming units (CFU) with a mean of 1570 ± 3850 CFU/m3/s, which is statistically different (p < 0.001) relative to both the erbium laser and the drill with a mean of 185,000 ± 182,000 CFU/m3/s and 440,000 ± 496,000 CFU/m3/s, respectively. CFU measured from the non-cutting mist spray on the teeth was higher for the drill than for the lasers. Conclusion: The 9.3μm CO2 laser resulted in the lowest CFU in the aerosol/splatter during enamel removal as compared to that of the 2.78μm erbium laser and the traditional high-speed drill. Furthermore, the CO2 laser was the only system that did not increase aerosolization of bacteria while cutting compared to the non-cutting mist spray.

scholarly journals Detection of Complement-Mediated Antibody-Dependent Bactericidal Activity in a Fluorescence-Based Serum Bactericidal Assay for Group B Neisseria meningitidis

2000 ◽  
Vol 38 (8) ◽  
pp. 2878-2884 ◽  
Author(s):  
Kenneth T. Mountzouros ◽  
Alan P. Howell
Keyword(s):  
Neisseria Meningitidis ◽  
Bactericidal Activity ◽  
Fluorescent Dye ◽  
Bactericidal Assay ◽  
Viable Bacteria ◽  
Serum Bactericidal Assay ◽  
Functional Antibodies ◽  
Group B ◽  
Oxidative Respiration ◽  
Human And Mouse

Serum bactericidal assays (SBAs) for Group B meningococci are considered the methods of choice for the evaluation of functional antimeningococcal antibodies. Many investigators regard SBAs as time- and labor-intensive. Variations in SBA protocols among different laboratories make interpretation of results difficult. Here we describe a fluorescence-based serum bactericidal assay (fSBA) and compare the results obtained with the fSBA to the results obtained with a more conventional SBA. The results generated by both assays were dependent upon the surviving bacteria after incubation, and the assay mixtures contained identical components. Differences between assays lie in how the surviving bacteria are quantified. The fSBA described in the paper uses the fluorescent dye alamarBlue (M. V. Lancaster and R. D. Fields, U.S. patent 5501959, March 1996). The fluorescent signals generated in the fSBA correlate to the oxidative respiration of surviving bacteria. Viable bacteria were detected between 6 and 8 h directly from reaction mixtures in 96-well plates by the fSBA, whereas colonies isolated on semisolid media could be counted after 24 h of incubation. The bactericidal titers generated by both assays were nearly identical. The fSBA described here can be used as an assay for the screening of large quantities of individual sera as complement sources or as a method for the detection of functional antibodies directed against group B Neisseria meningitidisin both human and mouse antisera.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

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