* Corresponding authors:
Shaokoon Cheng - School of Mechanical Engineering, Macquarie University, 44 Waterloo Rd., Macquarie Park, Sydney, NSW, 2113, Australia
Email: shaokoon.cheng@mq.edu.au
Hui Xin Ong - Woolcock Institute of Medical Research & Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, 2037, Australia
Email: ong.hui@sydney.edu.au
KEYWORDS: Biofilm, static culture, biofilm substrate, interfaces, dual-chamber microreactor, reversible bonding
ABSTRACT
Biofilms are ubiquitous and notoriously difficult to eradicate and control, complicating human infections, industrial and agricultural biofouling. Current biofilm studies are commonly performed with the biofilm cultured on mono-interfaces and generally have neglected to consider more realistic models or approaches, where diverse interfaces are involved. In our study, a reusable dual-chamber microreactor with interchangeable membranes was developed to establish multiple interfaces for biofilm culture and test. Protocol for culturing Pseudomonas aeruginosa  (PAO1) on the air-liquid interface (ALI) and liquid-liquid interface (LLI) under static environmental conditions for 48h was optimized using this novel device. This study shows that LLI model biofilms are more susceptible to physical disruption compared to ALI model biofilm. SEM images revealed a unique ’mushroom-shaped’ microcolonies morphological feature, which is more distinct on ALI biofilms than LLI. Furthermore, the study showed that ALI and LLI biofilms produced a similar amount of extracellular polymeric substances (EPS). As differences in biofilm structure and properties may lead to different outcomes when using the same eradication approaches, the antimicrobial effect of an antibiotic, Ciprofloxacin (CIP), was chosen to test the susceptibility of 48h-old P. aeruginosa biofilms grown on ALI and LLI. Our results show that the minimum eradication concentration (MBCE) of CIP using our dual-chamber device reached 1600μg/ml, which is significantly higher than the conventional microtiter plate method (64μg/ml). The results highlight the importance of having a model that can closely mimic in-vivo conditions to develop more effective biofilm management strategies.
Introduction
Biofilms are the dominant surviving model of bacteria that exist on earth [1]. It plays a crucial part in the ecosystem, either beneficial or detrimental depending on the microbial species and their growth. For example, crops can benefit from non-pathogenic biofilm growth-promoting rhizobacteria (PGPR) biofilm [2] but can also trigger foodborne illnesses in plant diseases caused by pathogenic microbial biofilms [3]. Also, while biofilms can help degrade pollutants in liquid and gaseous effluents in wastewater treatment plants [4], undesired biofilm formation in drinking water, oil pipelines, and ship hulls can lead to biofouling and biocorrosion, which undermine operation safety and loss of productivity [4]. Furthermore, biofilms are typically associated with clinical chronic, nosocomial, and medical device-related infections [5-8].
The formation of biofilm starts with planktonic bacteria adhering to the surface and encasing the proliferated colonies in self-produced extracellular plyometric substance and become matured biofilm [1]. Biofilms protect the microorganism from hostile physical and chemical environments such as altered pH, osmolarity, nutrients scarcity, mechanical and shear forces, and block bacterial biofilm communities’ access from antibiotics and host’s immune cells [9]. Their properties are determined by both inherent biological attributes of bacterial strains and external environmental factors. Among all the environmental factors, surfaces play a critical role since their properties directly affect bacteria’s initial attachment, biofilm maturation, and final detaching. Studies on the impact of surface roughness, charge, hydrophobicity, tension, wettability, and microtopography on biofilm growth [10, 11] have persistently modeled biofilm as solid-attached structures submerged in liquid. Other studies have explored the development of biofilm aggregates cultured in agarose have been used to mimic the intrinsic properties of human tissue in various fields [12, 13], such as wound microenvironment. However, the variations in terms of interface type have been neglected. Furthermore, antibiotics have been widely used to treat biofilm infection diseases with multiple delivery paths, including parenteral, enteral, transdermal, inhalation, oral, and topical. The interaction between antibiotics and biofilms also varies depending on interfaces. A greater understanding of the influence of interfaces on biofilms will help to develop more efficient and targeted eradication approaches.
For this purpose, we proposed a dual-chamber microreactor with interchangeable interfaces to study biofilm structure and susceptibility to drugs. Our model brings the advantages of a microfluidic device, which include reduced sample volume and reagent consumption and precise environmental control. Moreover, the curing approach and reversible bonding technique adopted for device fabrication and assembling, further helps to reduce experimental time and cost and brings greater flexibility for sample manipulation. Compared with conventional antimicrobial susceptibly testing methods, our device can be considered more physiobiologically relevant in mimicking the microenvironment that biofilm grows and interacts with antibiotics.
Pseudomonas aeruginosa is a ubiquitous pathogen that could colonize multiple environments and establish a biofilm within 24 h [14]. It can cause a variety of infections, such as chronic lung infection [15] and urinary tract infections (UTIs) [16], and its accentuated antibiotic resistance during biofilm growth poses a significant threat to the medical community. The biofilm developed in the respiratory system can be modeled as biofilm formed on the air-liquid interface (ALI), while biofilm developed in the urinary tract can be best characterized by liquid-liquid interface (LLI) model biofilm. In this study, we used this novel device to optimize a protocol for culturingPseudomonas aeruginosa (PAO1) on ALI and LLI, respectively, under static environmental conditions for 48h. Biofilm susceptibility was also evaluated after exposure to ciprofloxacin from the substrate side to mimic systemic circulation exposure, which permeates through a physical barrier to reach the biofilm site.
Materials and Methods
Materials
Polydimethylsiloxane monomer and curing agent (Sylgard 184, Dow Corning) were purchased from Revolution Industrial (Australia). Formlab® Clear resin V4 was obtained from Core Electronics (Australia). Isopropyl alcohol (IPA) (≥99%) and ethanol (80% v/v) were obtained from Chem-supply Pty Ltd (Australia). Ambersil® polymer remover was purchase from RS components Pty Ltd (Australia). Hydrophilic porous polyester (PETE) membrane (pore size 0.2μm, pore density 3 x 108 (pores/cm2), open area 9.4%, thickness 10μm, bubble point 20psi, water flow rate 10mL/min/cm2, airflow rate 3L/min/cm2) was purchased from Sterlitech (USA). Translucent silicon rubber (TRANSIL®) was obtained from Barnes Products Pty Ltd (Australia). Tygon tubing (1.59mm OD x 0.51mm ID), 23G stainless steel couplers (0.63mm OD x 0.33mm ID), and razor-sharp stainless-steel biopsy punches (0.5mm and 1.25mm OD) were purchased from Darwin Microfluidics (France). Stainless steel screws and nuts were obtained from Small Parts & Bearings (Australia). Blunt needles (22G) and sterile disposable syringes were obtained from Livingstone (Australia). Pseudomonas aeruginosa (PAO1 ATCC 15692) was purchased from the American Type Culture Collection (ATCC, Rockville, USA). Cation-adjusted Mueller-Hinton Broth (CAMHB) was purchased from BD Biosciences (Australia). Phosphate-buffered saline (PBS), LB Broth with agar (Lennox), Alcian blue (1% w/v in acetic acid, 3% v/v pH 2.5), AeraSeal® Film, and formaldehyde solution (≥36.0% in water) were purchased from Sigma Aldrich (Australia). FilmTracer™ LIVE/DEAD® Biofilm viability kit was obtained from Thermo Fisher Scientific (Australia). Ciprofloxacin hydrochloride was supplied by MP (Biomedical Australasia Pty Limited, Australia). Milli-Q water was obtained from Biopak® Polisher system (Merck KGaA, Germany).
Device design and fabrication
The microreactor (Fig.1) consists of a dual-chamber design (basal and apical) physically separated by a porous hydrophilic PETE membrane, used as the substrate for biofilm growth. Each device comprised three identical dual-chamber flow cells allowing for the testing of multiple experimental setups in parallel. The dimensions of the chamber were: 1mm (width) x 4mm (length) x 0.5mm (height). The flow chambers (Fig. 1-B) were fabricated using PDMS by 3D printing microfluidic fabrication technique [17]. The molds for PDMS casting were printed using a clear resin on a Form2 3D printer (Formlabs), washed for 15 min with IPA (Formlabs, Form wash), and cured under UV light for 90 mins at 65°C (Formlabs, Formcure). Before pouring the PDMS, the mold was spray-coated with polymer remover to protect the mold surface and to assist with the PDMS removal after the curing process. PDMS monomer and curing agent were mixed (10:1 w/w), cast into the mold, degassed, and cured at 65°C for 12h. After cooling, the PDMS chamber was peeled off from the mold, and the inlet and outlet holes were punched using a 0.5mm biopsy puncher. Two pieces of thin silicon gaskets (0.3mm) were placed between the membrane and the PDMS layers to prevent leakage and protect the membrane from the mechanical bounding force. The silicone gaskets (Fig. 1-C) were fabricated by pouring silicone on the plastic sheet and pressing the 3D printed template into the uncured silicone mix and against the plastic sheet. The silicone gaskets were cured at room temperate for 25min and then peeled from the plastic sheet.
Device assembly
Reversible mechanical bonding of the multiple layers (Fig. 1-A) was obtained using two pieces of acrylic Plexiglas covers (76mm × 25 mm x 3mm) with holes aligned with the position of the inlet, outlet and bonding holes in the PDMS layers. The mechanical bonding was achieved by twelve screws and nuts positioned around the perimeter of the flow chamber to ensure the alignment of the layers and the interface area between the two chambers. The flow into the channels was supplied through microbore tubing connected to stainless-steel couplers (23G), inserted into the inlet and outlet ports. Before the bacteria inoculation, 1 mL sterilized water was injected into two chambers to ensure no leaking occurs.