Biofilm assays

Fig. 21 Top Schematic setup of an ultrasonically vibrating micropitted silicon surface. The cavitation chamber is filled with pure liquids or a cell cultivation liquid for biological assays. Bottom Temporal recording of a biofilm removed by microbubbles. The grey area with black dots is the zone covered by biofilm. The pit is indicated with a large black dot. Microbubbles can be identified as the blurred dark region surrounding the pit. Reproduced with permission from [110]. Copyright 2012 AIP Publishing...

A crystal violet (CV) assay, which stains the total bacterial biomass, was used to study the efficacy of peptoids against biofilms. The absorbance of the CV stain is measured at = 590nm, and a decrease in absorbance (pale purple) compared to imtreated control (dark purple) implies a decrease in biomass. At 12.5 pM, six out of the seven peptoids tested in this study were able to prevent the formation of P. aeruginosa biofilms by 40-70%, with peptoid 1 being the most active (70% redurtion) and comparable in efficacy to ciprofloxacin and tobramycin (Table 3). However, AMPs only caused 10% reduaion. This was probably because the peptoids were able to kill the planktonic cells which in turn reduced the number of bacterial cells that could have led to the formation of biofilms. 

The CV assay is a good measure to screen the efficacy of antimicrobials against biofilms, except it does not provide information regarding the bacterial cell viability. To ensure that the peptoids were killing bacteria rather than just physically removing them from the stuface, the deaease in the cell viability was measured by bacterial plating for prevention of biofilm formation and reduction of existing biofilms. At 12.5 pM, peptoids 1 and showed similar effects to... 

A h input to a library of 17,500 small molecules identified two 2-amino-3-acyl-tetrahydrobenzothiophene derivatives as hit compounds against pill-dependent biofilm formation in a uropathcgenic Escherichia coli strain UT189. Several 2-amino-3-acyl-tetrahydrobenzothiophene derivatives were prepared via a Gewald reaction and their antibaaerial and antivirulence activity evaluated (140BC194). Of these, the hit compound and one of its derivatives, shown below, were found to prevent pill formation in a hemaglutination (HA) titer assay. 

It is important to note that antimicrobial and biofilm resistance are two different characteristics though some materials show both properties at the same time. Antimicrobial materials do not automatically prevent biofilm formation and vice versa. Antimicrobial surfaces could kill bacteria on contact but if dead bacteria cell debris blocks the active biocidal surface, biofilm formation could eventually occur. For example, quaternary anunonium polymers can effectively kill bacteria but when the surface is fouled with dead bacteria debris, biofilm formation is inevitable [188]. Materials with antibiofilm properties will repel the bacterial adhesion very effectively but may not kill the bacteria when they do colonize the surface. PEG surfaces are well known to repel bacteria adhesion. However, PEG surfaces show little antimicrobial activity. Quantitative antibiofilm efficacy tests can be divided into two categories static (minimum biofilm eradication concentration assay, MBEC) and dynamic (flow cell assay). In addition, SEM is a semiquantitative assay, which is discussed in Section 2.5. 

Figure 2.34 MBEC assay, (a) Biofilms form on the polyst5rene pegs of the MBEC device when planktonic bacteria adsorb to the surface. These bacteria become irreversibly attached and grow to form mature biofihns. Biofilms are encased in sUme, which is sometimes visible to the naked eye. Planktonic cells are also shed from the surface of biofilms, which serves as the inoculum for CA determinations, (b) The peg lid has 96 identical plastic pegs. This hd fits into a trough with channels designed to guide an inoculum across the surface of the pegs. The peg lid fits into a standard 96-well microplate as weU, which is used to set up serial dilutions of antimicrobials.

Bardounitis, E., Huddleston, W., Ceri, H. and Olson, M. E., 2001. Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system. FEMS Microbiology Letters 203,... 

Vikram A, Jesudhasan PR, Jayaprakasha GK, Pillai BS, Patil BS (2010) Grapefruit bioactive limonoids modulate E. coli 0157 H7 TTSS and biofilm. Int J Food Microbiol 140 109-116 Wang J, Zhao H, Kong W, Jin C, Zhao Y, Qu Y, Xiao X (2010) Microcalorimetric assay on the antimicrobial property of five hydroxyanthraquinone derivatives in rhubarb (Rheum palmatum L.) to Bifidobacterium adolescentis. Phytomedicine 17 684-689 Weinmann I (1997) History of the development and application of coumarin and coumarin-related compounds. In O Kennedy R, Thornes RD (eds) Coumarins biology, applications and mode of action. Wiley Press, Chichester... 

The potential to inhibit binding of cell surface fucose to LecB and thus disrupt biofilms has also been investigated by the group of Consoli and Geraci, [53] using a calix[4]arene fixed in the cone conformation with four a-L-C-fucosyl units at the upper rim 58 (Fig. 22.18). Cell viability assays with planktonic Pseudomonas aeuruginosa (wild type PAOI) confirmed the molecule has no direct antimicrobial... 

Since bacteria are capable of forming biofilms on most surfaces, future tests should be focused on biofilm quantification. In assaying biocide efficacy, tests should be conducted based on biofilm populations rather than on liquid culture efficacy (Gu et al. 1998c, 2000c). Planktonic cells are not representative of conditions on surfaces of materials. 

Sanford BA, Thomas VL, Mattingly SJ, Ramsay MA, MiEer MM (1995) Lectin-biotin assay for slime present in in situ biofilm produced by Staphylococcus epidermidis using transmission electron microscopy (TEM). J Ind Microbiol... 

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