A revisited two-step microtiter plate assay: Optimization of in vitro multiplicity of infection (MOI) for Coliphage and Vibriophage

We investigate the role of hyaluronic acid (HA) onS. pneumoniae in vitrobiofilm formation and evaluate gene expressions of virulence and/or biofilm related genes. Biofilms were grown in medium supplied with HA derived from capsule ofStreptococcus equi. The biomasses of biofilms were detected by crystal-violet (CV) microtiter plate assay, and the morphology was viewed under scanning electron microscope (SEM). The gene expressions were assessed by relative quantitative RT-PCR. The results showed that the HA support pneumococcal growth in planktonic form and within biofilms. The CV-microtiter plate assay detected significantly increased biofilm growth in medium containing HA. The SEM analysis revealed thick and organized biofilms in positive control and HA supplemented medium. ThenanA,nanB,bgaA,strH,luxS,hysA,ugl, and PST-EIIA encoding genes were significantly upregulated in the planktonic cells grown in presence of HA, while thelytAandcomAgenes were downregulated. Similarly theluxS,hysA,ugl, and PST-EIIA encoding genes were significantly upregulated by more than 2-folds in HA biofilms. The results of this study indicate that the HA derived from capsule ofS. equisupports pneumococcal growth in planktonic state and within biofilms and upregulated virulence and biofilm related genes.

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Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.1.378-382.2003 ◽

2003 ◽

Vol 47 (1) ◽

pp. 378-382 ◽

Cited By ~ 55

Author(s):

Michael S. Scherman ◽

Katharine A. Winans ◽

Richard J. Stern ◽

Victoria Jones ◽

Carolyn R. Bertozzi ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Drug Targeting ◽

Enzyme Inhibitor ◽

Microtiter Plate ◽

Biosynthetic Enzyme ◽

Cell Wall Synthesis ◽

Chemical Library ◽

Microtiter Plate Assay ◽

Plate Assay

ABSTRACT A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

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Evaluation of the Role of Staphylococci in the Pathomechanism of Conjunctivitis

10.21203/rs.3.rs-191872/v1 ◽

2021 ◽

Author(s):

Ewa Jasińska ◽

Agnieszka Bogut ◽

Agnieszka Magryś ◽

Alina Olender

Keyword(s):

Antibiotic Resistance ◽

Epithelial Cells ◽

Biofilm Formation ◽

Methicillin Resistance ◽

Microtiter Plate ◽

Host Cells ◽

Diffusion Method ◽

Microtiter Plate Assay ◽

Low Prevalence

Abstract Purpose: Determination of the association between ica genes and phenotypic biofilm formation in staphylococcal isolates involved in conjunctivitis, their antibiotic resistance as well as detection of selected virulence characteristics: adhesion to epithelial cells and in vitro cytotoxicity.Methods: The study included 26 Staphylococcus aureus (SA) and 26 Staphylococcus epidermidis (SE) isolates. The presence of icaAD genes and ica operon was determined by the PCR assay. Phenotypic biofilm formation was verified using the microtiter plate assay. Antibiotic resistance was performed using the disc diffusion method. Staphylococcal ability to attach to host cells was assessed by flow cytometry. Cytotoxicity on epithelial cells was evaluated by LDH assay.Results: The ica genes were detected in 26.9% of SE and in 42.3% of SA isolates. Only 15.3% of isolates (SE) were positive for both the icaAD and the ica operon. Phenotypically, 19.2% of SE isolates were strong biofilm producers, among which three were both icaAD- and ica operon-positive. 26.9% of SA isolates were strong biofilm producers. Methicillin resistance (MR) was detected in 34.6% of SE and 26.9% of SA isolates. 75% of MR isolates were multidrug resistant. SA isolates adhered to host cells more extensively than SE. SA isolates released higher level of LDH than SE.Conclusions: Adherence abilities were commonly observed in staphylococci associated with conjunctivitis. However, low prevalence of isolates positive for a complete and functional ica locus and low prevalence of strong biofilm producers was detected. SA adhered to a greater extent to eukaryotic cells than SE and were more cytotoxic.

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