Study of the Dynamic persistence of uropathogenic Escherichia coli in a human bladder-chip model of urinary tract infections

This short review is originally based on the research paper titled “Dynamic persistence of intracellular bacterial communities of uropathogenic Escherichia  coli in a human bladder-chip model of urinary tract infections”, authored by Kunal Sharma, Vivek V. Thacker, Neeraj Dhar, Thomas M. Simonet, François Signorino-Gelo, Graham Knott & John D. McKinney from Mckinney Lab. The research paper is under peer review, and the pre-print version is available here.

It explores the functioning of a human bladder-chip model.  This model includes the key features of bladder physiology that are important to early UPEC infection and using pressure driven flow-controlled microfluidics to apply a negative pressure within the bladder-chip.

Abstract

Uropathogenic Escherichia Coli (UPEC) observe a rapid growth within superficial bladder umbrella cells, and result in the formation of intracellular bacterial communities (IBCs) in the initial stages of urinary tract infections. Although, the dynamic response of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. Through this pressure-driven flow controlled microfluidics study, the researchers aimed to develop a human bladder-chip model in which umbrella cells and bladder microvascular endothelial cells are co-cultured under the flow of urine and nutritive media respectively. The filling of the bladder and subsequent emptying were mimicked mechanically by application and release of linear strain. Time-lapse microscopy was used to demonstrate the instant involvement of neutrophils from the vascular channel to regions where the infection has occurred. This led to the formation of neutrophil extracellular traps but could not stop IBC formation. Thereafter, the dynamic bacterial growth is tracked in individual IBCs through administration of antibiotics over two cycles that was merged with recovery periods. This section of the study demonstrated that the bacteria was not successfully eliminated from the IBCs at the time, and in a few cases did not work at all. In the course of recovery, spread from a significant fraction of IBC’s reseeded new sites of infection through bacterial shedding & host cell exfoliation. Findings like these strengthen the role of IBC’s as locations of bacterial persistence, that can result in a wide range of consequences if antibiotic regimens are not followed properly.

Introduction

Urinary Tract infections (UTIs), are the second most common cause of antibiotics being prescribed [1]. They are often characterized by a high frequency of recurrence, defined as a reappearance of infection within 12 months, in spite of the successful completion of antibiotic therapy. Cases of recurrence happens in approximately 25% of all UTIs [1] and pose a threat to the cost of healthcare and reduces the quality of life, more so because statistically 60% of all women are diagnosed with a UTI at least once within their lifetimes [2]. Uropathogenic Escherichia coli (UPEC), the primary causative agent for most UTIs, exhibit a complex behaviour within the bladder, with planktonic sub-populations within the urine exist alongside intracellular bacteria. UPEC exploration of the urinary bladder triggers substantial changes to the morphology of the bladder and a strong immune response.

Most of the current knowledge on early stages of UTI’s and the intracellular lifestyle is obtained from studies of the mouse model [3, 4, 5, 6, 7, 8, 9]. Studies of the mouse bladder explants with the help of microscopy have revealed formation of intracellular bacterial communities (IBC’s). These IBC’s contain thousands of bacteria within each superficial bladder cell [7]. IBC’s also act as a key component in clinical infection and are often harvested from the urine of cystitis patients (Robino et al., 2013; Rosen et al., 2007).

Over the last two decades, microfluidics has been increasingly used to develop in vitro and in vivo models as it presents many advantages from the reduction of volume solutions to the fine control over the biological media flows. This short review introduces how the authors employed pressure-driven flow controlled microfluidics to develop a human bladder-chip.

Aim & objectives

The objectives of this study are given below :

• The development & characterization of a bladder-chip model that mimics the architecture & physiological properties of the bladder
• To demonstrate that diapedesis of neutrophils to sites of infection on the epithelial side can lead to the formation of neutrophil swarms and neutrophil extracellular traps (NETs)
• To study how IBCs offer substantial protection to bacteria from antibiotic clearance.

Materials & methods

The bladder chip devices were made of Polydimethylsiloxane (PDMS). The microfluidic device dimensions are as follows: width of the channel – 1000 μm, height of the upper channel & lower channel – 1000 μm & 250 μm respectively.

Elveflow’s OB1 MK3 was the flow control system implemented in the experimental setup to regulate the negative pressure applied to the human bladder-chip. For this study, both bladder epithelial & endothelial cells were seeded on the respective sides of the microfluidic device.

The Elveflow OB1 flow controller was also used to mimic the bladder filling & voiding cycle. The microfluidic flow controller was connected to the compressed air line for the positive pressure & diaphragm vacuum pump for application of the negative pressure.

An image of the experimental setup for this study is shown in Figure 1, above.

Key findings

Reconstitution of bladder Uroepithelium and bladder vasculature

A bladder-chip infection model was established by co-culturing HTB9 bladder epithelial cells & HMVEC-Bd primary microvascular endothelial cells in (Fig 2).

Overall, from these examinations, it was confirmed that the bladder-chip is populated with cells that imitate the physiological conditions of the bladder vasculature and the superficial urothelial cell layer. Also, the design of the chip helped the flow of media with different compositions through epithelial & vascular channels.

Modelling the bladder filling & voiding on the bladder-chip

The bladder in humans experiences huge changes in volume and surface area over time. The construction of the bladder-chip device enables a linear stress to be applied to the porous membrane through a negative pressure adjacent to the main channel of the device. (Fig 2)

Overall the bladder chip model is used for the co-culture of 2 cell types in nutritionally different microenvironments with a level of stress that is physiologically pertinent and mimics bladder filling & voiding. The Elveflow OB1 is used to apply negative pressure on the side channels of the bladder chip device, showcased in Video 1 on the right.

Here are the key findings from this study:

• Results showcase the ability of the bladder-chip model to capture these interactions between mechanical function and physiology.
• Neutrophils that migrate to the site of infection are only partially able to control of the infection. This is most probably due to the unrestricted growth of a huge number of extracellular bacteria. Although, the model is able to capture most of the important aspects of the disease. This proves to be an important milestone in the use of organ on chip procedures to capture NET formation in infectious diseases.
• IBC formation starts immediately after infection and bacteria within IBCs are protected from neutrophil clearance. Observations from this study confirm the highly dynamic properties of these structures. Bacterial growth was seen to be asynchronous & heterogeneous and results include bacterial shedding, exfoliation and filamentation.
• Results showcase that bacteria within IBCs can continue to grow for a significant period of time after the start of the antibiotic treatment.

Conclusion

To summarize this research, the bladder-chip model includes the key features of bladder physiology that are important to early UPEC infection on a platform built using the pressure driven flow control mechanism. This platform is suitable for long-term live-cell imaging as well as for the administration of antibiotics. The findings clearly demonstrate the usability of this model for immunological & drug-delivery studies and show that IBCs are extremely dynamic structures that provide enough protection from antibiotic clearance for a considerable length of time.

These promising results were achieved by the researchers with the help of a highly accurate pressure driven flow control device in the Elveflow OB1 MK3+ flow controller. To gain an in-depth understanding of this study please refer to the pre-print version of the research paper here.

References