biofilm formation

Overview

Bacterial adhesion and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases.  Biofilms are resistant to innate host defences, mechanical removal and antibiotic treatments.  It is therefore important to understand the physiological environment and mechanisms that lead to the spread of bacteria.  Experimental conditions of biofilm formation on cardiovascular stents, tubing, different surfaces and in the intestines can easily be studied using Cellix's microfluidic pumps and biochips.  The dimensions of the biochips facilitate both high and low shear stress conditions.  The microcapillary walls of Cellix's biochips may be pre-coated with proteins of interest to promote adhesion and culture of biofilms under different shear stress conditions.  Once the biofilm is cultured, it is then possible to flow different substances (e.g. antibiotics) over the biofilm to investigate detachment.  There are a variety of different configurations which include the Vena8 biochip range, the Kima pump, the ExiGo pump and the Mirus Evo Nanopump.

Assays:  

  • high and low shear stress conditions easily obtained with Vena8 Glass Coverslip biochips.  These biochips can be supplied with/without the glass coverslip attached.  Researchers can attach their own material of interest (e.g. coupon) to the bottom of the chips, easily sealing the microchannels.
  • recirculation studies for biofilm formation and growth can easily be accomplished with the Kima pump - it can also sit inside a standard CO2 incubator.  This can be connected to Cellix's biochips to culture 8 channels in parallel or alternatively tubing connections for other consumables may be supplied / adapted.
  • larger channels sizes from the Vena8 Glass Coverslip range enable the growth of mature biofilms without channel blockages over longer periods of time; >48hrs.

 

Assay Examples:

 

E. Coli Adhesion and Biofilm Formation

  • Escherichia coli typically colonises the gastrointestinal tract of human infants within a few hours after birth.  Usually, E. coli and its human host coexist in good health and with mutual benefit for decades.  However, there are several highly adapted E. coli clones that have acquired specific virulence attributes, which confirm an increased ability to adapt to new niches and allow them to cause a broad spectrum of disease.  Three general clinical syndromes can result from E. coli infection:  enteric/diarrhoeal disease, urinary tract infections (UTIs) and sepsis/meningitis.
  • Aim:  To elucidate the importance of physiological shear stress environment required for E. coli adhesion, colonisation and biofilm formation using Cellix's microfluidic pumps and biochips.
  • E. coli K-12 strain expressing FimH-wt was supplied by Prof. Evgeni Sokurenko for accumulation and mobility/firm adhesion assays along with FimH-j96 (A 188D) for biofilm formation studies.
  • Adhesion assays:  Bacteria were infused into 1M (monomannose); 3M (trimannose) and BSA coated microchannels of the biochips at shear stresses of 0.1, 0.3, 1.0 and 8.0 dyne/cm2; 100s per shear stress level.
  • Adhesion Assay Results:  Bacteria adhered readily on the 1M and 3M surfaces at the lowest shear stresses tested and accumulation increased with increased shear stress (see figures below).  Inhibitors for 1M and 3M binding were used as controls.  It was also shown that increased shear stress resulted in a transition from rolling to stationary E. Coli adhesion and rolling was dominant at the lower shear stresses.
  • Biofilm Formation Results:  Low shear stress on accumulated FimH E. Coli resulted in a time-dependent biofilm formation with a 4-fold increase in bacterial surface coverage over the time course of 3hrs.

 

Shear stress enhances accumulation of E. Coli on surfaces coated with 1M and 3M ligands.

 

Increased shear tress results in a transition from rolling (figure on LHS) to stationary E. Coli adhesion (figure on RHS).

 

 Low shear stress on accumulated FimH E. Coli results in time-dependent biofilm formation with a 4-fold increase in bacterial surface coverage over the time course of 3hrs.

 


Staphylococcus Aureus and Staphylococcus Epidermis Biofilm Culture

  • Staphyloccus aureus and Staphylococcus Epidermis are the most common causes of medical device-associated infections.  Incidences of S. aureus infections range from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections.  S. epidermis infections are generally hospital-acquired.  S. epidermis infections are of particular concern for people with catheters or other surgical implants.
  • Microchannels of the Vena8 Fluoro+ biochip were coated with plasma for 1 hour; then 20μL of S. aureus bacteria was seeded for 1 hour (via pipette).  The biochip was then connected to the Kima pump and perfusion (BHI media with 1% glucose) was set at 200μL/min for 18 hours.
  • Microchannels of the Vena8 Endothelial+ biochip were coated with plasma for 1 hour; then 20μL of S. epidermis bacteria was seeded for 1 hour (via pipette).  The biochip was then connected to the Kima pump and perfusion (BHI media with 1% glucose) was set at 200μL/min for 6 hours.
  • Results: 

10X magnification:  S.aureus strain BH1CC (MRSA clinical isolate) grown under conditions of flow for 18hrs on chips precoated with plasma.

 

40X magnification:  S.aureus strain BH1CC (MRSA clinical isolate) grown under conditions of flow for 18hrs on chips precoated with plasma.

 

S.epidermis grown under conditions of flow for 6hrs on chips precoated with plasma.

 


How to add a coupon to Cellix's biochips for investigation of biofilm formation on different materials

 


How to recover bacteria from Cellix's biochips for further study