References

Wilkins M, Hall-Stoodley L, Allan RN, Faust SN. New approaches to the treatment of biofilm-related infections. J Infect. 2014; 69:S47-S52 https://doi.org/10.1016/j.jinf.2014.07.014

Goeres DM, Hamilton MA, Beck NA A method for growing a biofilm under low shear at the air–liquid interface using the drip flow biofilm reactor. Nat Protoc. 2009; 4:(5)783-788 https://doi.org/10.1038/nprot.2009.59

Kaplan JB, Izano EA, Gopal P Low levels of β-lactam antibiotics induce extracellular DNA release and biofilm formation in Staphylococcus aureus. MBio. 2012; 3:(4)e00198-12 https://doi.org/10.1128/mBio.00198-12

Walker JN, Horswill AR. A coverslip-based technique for evaluating Staphylococcus aureus biofilm formation on human plasma. Front Cell Infect Microbiol. 2012; 2 https://doi.org/10.3389/fcimb.2012.00039

O'Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol. 1998; 28:(3)449-461 https://doi.org/10.1046/j.1365-2958.1998.00797.x

World Health Organization. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. https://tinyurl.com/3dx6vmuv (accessed 29 June 2021)

Vandevelde NM, Tulkens PM, Van Bambeke F. Antibiotic activity against naive and induced Streptococcus pneumoniae biofilms in an in vitro pharmacodynamic model. Antimicrob Agents Chemother. 2014; 58:(3)1348-1358 https://doi.org/10.1128/AAC.01858-13

Stepanović S, Cirković I, Ranin L, Svabić-Vlahović M. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett Appl Microbiol. 2004; 38:(5)428-432 https://doi.org/10.1111/j.1472-765X.2004.01513.x

Das MC, Sandhu P, Gupta P Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: a combinatorial study with azithromycin and gentamicin. Sci Rep. 2016; 6 https://doi.org/10.1038/srep23347

Stepanović S, Vuković D, Dakić I A modified microtitre-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods. 2000; 40:(2)175-179 https://doi.org/10.1016/S0167-7012(00)00122-6

Kristich CJ, Li YH, Cvitkovitch DG, Dunny GM. Esp-independent biofilm formation by Enterococcus faecalis. J Bacteriol. 2004; 186:(1)154-163 https://doi.org/10.1128/JB.186.1.154-163.2004

Al-Ahmad A, Wiedmann-Al-Ahmad M, Auschill TM Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro. Arch Oral Biol. 2008; 53:(8)765-772 https://doi.org/10.1016/j.archoralbio.2008.02.014

Ommen P, Zobek N, Meyer RL. Quantification of biofilm biomass by staining: Non-toxic safranin can replace the popular crystal violet. J Microbiol Methods. 2017; 141:87-89 https://doi.org/10.1016/j.mimet.2017.08.003

Standar K, Kreikemeyer B, Redanz S Setup of an in vitro test system for basic studies on biofilm behavior of mixed-species cultures with dental and periodontal pathogens. PLoS One. 2010; 5:(10) https://doi.org/10.1371/journal.pone.0013135

Lembke C, Podbielski A, Hidalgo-Grass C Characterization of biofilm formation by clinically relevant serotypes of group A streptococci. Appl Environ Microbiol. 2006; 72:(4)2864-2875 https://doi.org/10.1128/AEM.72.4.2864-2875.2006

Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slimeproducing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun. 1982; 37:(1)318-326 https://doi.org/10.1128/IAI.37.1.318-326.1982

Fessia SL, Griffin MJ. A method for assaying biofilm capacity on polyurethanecoated slides. Peritoneal Dialysis International: Journal of the International Society for Peritoneal Dialysis. 1991; 11:(2)144-146 https://doi.org/10.1177/089686089101100209

Rollefson JB, Stephen CS, Tien M, Bond DR. Identification of an extracellular polysaccharide network essential for cytochrome anchoring and biofilm formation in Geobacter sulfurreducens. J Bacteriol. 2011; 193:(5)1023-1033 https://doi.org/10.1128/JB.01092-10

Pécastaings S, Roques C. Production of L. pneumophila monospecies biofilms in a low-nutrientconcentration medium. Methods Mol Biol. 2013; 954:219-224 https://doi.org/10.1007/978-1-62703-161-5_12

Marks LR, Reddinger RM, Hakansson AP. High levels of genetic recombination during nasopharyngeal carriage and biofilm formation in Streptococcus pneumoniae. MBio. 2012; 3:(5)e00200-12 https://doi.org/10.1128/mBio.00200-12

Birkenhauer E, Neethirajan S, Weese J. Collagen and hyaluronan at wound sites influence early polymicrobial biofilm adhesive events. BMC Microbiol. 2014; 14:(1) https://doi.org/10.1186/1471-2180-14-191

Condell O, Iversen C, Cooney S Efficacy of biocides used in the modern food industry to control salmonella enterica, and links between biocide tolerance and resistance to clinically relevant antimicrobial compounds. Appl Environ Microbiol. 2012; 78:(9)3087-3097 https://doi.org/10.1128/AEM.07534-11

Charles CA, Ricotti CA, Davis SC Use of tissue-engineered skin to study in vitro biofilm development. Dermatol Surg. 2009; 35:(9)1334-1341 https://doi.org/10.1111/j.1524-4725.2009.01238.x

Hard-to-heal wounds

Wound infections

Biofilms

Development and assessment of a high-throughput biofilm and biomass testing platform

02 July 2021

Research

02 July 2021

Volume 5 · Issue 3

ISSN (print): 0969-0700

ISSN (online): 2052-2916

Digital issue

Abstract

Objective:

To develop and evaluate a simple platform technology for developing static biofilms in a 96-well microtitre plate for various downstream applications. The technology allows monitoring of growth rate, biofilm formation and quantifying biofilm biomass by using crystal violet (CV) and safranin O (SO) staining over seven-day time periods for pathogens including clinical isolates most commonly associated with hard-to-treat wound infections.

Method:

A total of 157 bacteria including Acinetobacter, Enterobacter, Klebsiella, Pseudomonas and Staphylococcus spp.were used in the study. Bacterial growth was measured at 600nm optical density (OD). Biofilm formation was monitored and assessed quantitatively with CV at 570nm and SO staining at 492nm for one-, two-, three- and seven-day incubation periods.

Results:

Bacterial growth rate and static biofilm biomass in the 96-well plates varied for various strains tested. Both CV and SO staining showed similar results in the biomass, with SO assay displaying more reproducible data throughout the study. Most of the strains were metabolically active even at the seven-day incubation period. Microbial adherences of all bacterial strains on the plastic surface was assessed with CV staining: 28 Acinetobacter, 17 Staphylococcus, 12 Pseudomonas and four Enterobacter strains were strong biofilm producers. Moderate biofilm-producing strains included 27 Staphylococcus, 14 Acinetobacter, eight Pseudomonas and three Enterobacter. Weak biofilm-producing strains included: 33 Staphylococcus, six Enterobacter, two Pseudomonas and one Acinetobacter. Only one Pseudomonas aeruginosa strain did not develop biofilm.

Conclusion:

Our results demonstrate the feasibility of using 96-well microtitre plates as a high-throughput platform for quantitative measurement and assessment of biofilm development over time. Studying microbial adherence or biofilm biomass generated on various surfaces using a high-throughput system could provide valuable information for in vitro testing and developing therapeutics for biofilm infections. Employing the biofilm testing platform described in this study makes it possible to simultaneously develop different biofilms formed by specific pathogens, and study potential association between the quantity of bacterial biomass and strength of a biofilm formed by specific wound pathogens. In addition, the described testing approach could provide an optimal model for standardised and high-throughput screening of candidate antibiofilm therapeutics.

Bacteria causing chronic infections grow predominately on surfaces and build sessile communities known as biofilms. Biofilms can be found anywhere and are common in the industrial, medical and wound care fields. The biofilm-related infections are known to be highly resistant to conventional antimicrobials and often protected from host immune responses.¹

Scientists conducting biofilm research try to employ laboratory testing conditions that closely mimic the clinical or environmental conditions. Currently, there are many simple biofilm-generating models such as using a flask, beaker, glass tube, test tube and silicone tubing. Different types of biofilms can be developed under specific culture conditions, such as static (no shear force applied) or dynamic (low or high shear force applied). When researchers choose biofilm models for their studies, four biofilm growth conditions are usually considered:²

Microtitre plate static biofilm model is one of the first methods used for quantifying biofilm formation.³ This method is designed to quantify the extent to which microbes attach to an abiotic surface. Using short incubation times (1–2 hours), initial attachment to a surface can be assessed, while longer incubation times (~20 hours) allow for measurement of biofilm formation. The primary advantage of this method is its relatively high throughput and cost-effectiveness, thereby enabling the screening of multiple parameters and evaluation of the efficacy of different treatments or compounds on microbial attachment or biofilm formation; however, its relevance and translation in vivo sometimes remains questionable.⁴

Register now to continue reading

Thank you for visiting Wound Central and reading some of our peer-reviewed resources for wound care professionals. To read more, please register today. You’ll enjoy the following great benefits: