Study of the impact of cultivation conditions and peg surface modification on the in vitro biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis in a system analogous to the Calgary biofilm device
Introduction.Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.
Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.
Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.
Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.
Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.
Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).
ArcherNK,
MazaitisMJ,
CostertonJW,
LeidJG,
PowersME et al.Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Virulence2011; 2:445–459 [View Article][PubMed]
KırmusaoğluS.
Staphylococcal biofilms: pathogenicity, mechanism and regulation of biofilm formation by quorum-sensing system and antibiotic resistance mechanisms of biofilm-embedded microorganisms. Microbial Biofilms-Importance and Applications Intech2016 189–209 [View Article]
KhatoonZ,
McTiernanCD,
SuuronenEJ,
MahTF,
AlarconEI.
Bacterial biofilm formation on implantable devices and approaches to its treatment and prevention. Heliyon2018; 4:e01067 [View Article][PubMed]
ChristensenGD,
SimpsonWA,
YoungerJJ,
BaddourLM,
BarrettFF et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol1985; 22:996–1006 [View Article][PubMed]
StepanovićS,
VukovićD,
HoláV,
Di BonaventuraG,
DjukićS et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS2007; 115:891–899 [View Article][PubMed]
LeeJY,
MonkIR,
da SilvaAG,
SeemannT,
ChuaKY et al. Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis
. Nat Microbiol2018; 3:1175–1185 [View Article]
FigueiredoAMS,
FerreiraFA,
BeltrameCO,
CôrtesMF.
The role of biofilms in persistent infections and factors involved in ica-independent biofilm development and gene regulation in Staphylococcus aureus
. Crit Rev Microbiol2017; 43:602–620 [View Article][PubMed]
AllkjaJ,
BjarnsholtT,
CoenyeT,
CosP,
FallareroA et al. Minimum information guideline for spectrophotometric and fluorometric methods to assess biofilm formation in microplates. Biofilm2020; 2:100010 [View Article][PubMed]
SinghAK,
PrakashP,
AchraA,
SinghGP,
DasA et al. Standardization and classification of in vitro biofilm formation by clinical isolates of Staphylococcus aureus
. J Glob Infect Dis2017; 9:93–101 [View Article][PubMed]
KwasnySM,
OppermanTJ.
Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery. Curr Protoc Pharmacol2010; 13: [View Article]
HongQ,
DongX,
ChenM,
SunH,
HongL et al. An in vitro and in vivo study of plasma treatment effects on oral biofilms. J Oral Microbiol2019; 11:1603524 [View Article][PubMed]
ShanksRM,
MeehlMA,
BrothersKM,
MartinezRM,
DoneganNP et al. Genetic evidence for an alternative citrate-dependent biofilm formation pathway in Staphylococcus aureus that is dependent on fibronectin binding proteins and the GraRS two-component regulatory system. Infect Immun2008; 76:2469–2477 [View Article]
ArciolaCR,
CampocciD,
SpezialeP,
MontanaroL,
CostertonJW.
Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials. Biomaterials2012; 33:5967–5982 [View Article]
ReffuveilleF,
JosseJ,
ValléQ,
GangloffCM,
GangloffSC.
Staphylococcus aureus biofilms and their impact on the medical field. The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus2017; 11:187 [View Article]
BeenkenKE,
MrakLN,
GriffinLM,
ZielinskaAK,
ShawLN et al. Epistatic relationships between sarA and agr in Staphylococcus aureus biofilm formation. PLoS One2010; 5:e10790 [View Article][PubMed]
KucharíkováS,
VeldeGV,
HimmelreichU,
Van DijckP.
Candida albicans biofilm development on medically-relevant foreign bodies in a mouse subcutaneous model followed by bioluminescence imaging. J Vis Exp2015; 95:e52239 [View Article]
SinghS,
SinghSK,
ChowdhuryI,
SinghR.
Understanding the mechanism of bacterial biofilms resistance to antimicrobial agents. Open Microbiol J20171153–6253 [View Article][PubMed]
CroesS,
DeurenbergRH,
BoumansMLL,
BeisserPS,
NeefC et al.Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage. BMC Microbiol2009; 9:1–9 [View Article][PubMed]
LeonhardM,
ZatorskaB,
MoserD,
TanY,
Schneider-SticklerB.
Evaluation of combined growth media for in vitro cultivation of oropharyngeal biofilms on prosthetic silicone. J Mater Sci Mater Med2018; 29:4545 [View Article][PubMed]
CeriH,
OlsonME,
StremickC,
ReadRR,
MorckD et al. The calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol1999; 37:1771–1776 [View Article][PubMed]
KipangaPN,
LuytenW.
Influence of serum and polystyrene plate type on stability of Candida albicans biofilms. J Microbiol Methods2017; 139:8–11 [View Article][PubMed]
ZapotocznaM,
McCarthyH,
RudkinJK,
O’GaraJP,
O’NeillE.
An essential role for coagulase in Staphylococcus aureus biofilm development reveals new therapeutic possibilities for device-related infections. J Infect Dis2015; 212:1883–1893 [View Article][PubMed]
Study of the impact of cultivation conditions and peg surface modification on the in vitro biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis in a system analogous to the Calgary biofilm device