pH-taxis drives aerobic bacteria in duodenum to migrate into the pancreas with tumors

Abstract

As oral or intestinal bacteria have been found in pancreatic cystic fluid and tumors, understanding bacterial migration from the duodenum into the pancreas via hepato-pancreatic duct is critical. Mathematical models of migration of aerobic bacteria from the duodenum to the pancreas with tumors were developed. Additionally, the bacterial distributions under the pH gradient and those under flow were measured in double-layer flow based microfluidic device and T-shaped cylinders. Migration of aerobic bacteria from the duodenum into pancreas is counteracted by bile and pancreatic juice flow but facilitated by pH-taxis from acidic duodenum fluid toward more favorable slightly alkaline pH in pancreatic juice. Additionally, the reduced flow velocity in cancer patients, due to compressed pancreatic duct by solid tumor, facilitates migration. Moreover, measured distribution of GFP E. coli under the pH gradient in a microfluidic device validated pH-tactic behaviors. Furthermore, Pseudomonas fluorescens in hydrochloride solution, but not in bicarbonate solution, migrated upstream against bicarbonate flow of > 20 μm/s, with an advancement at approximately 50 μm/s.

Figure 1

This work aims to understand how environmental factors in hepato-pancreatic duct such as pH, carbon dioxide and oxygen concentration, and fluid flow contribute to migration of aerobic bacteria from the duodenum to the pancreas with tumors.

Figure 2

Simulated concentrations of carbon dioxide (blue), hydrogen ion (green), and bicarbonate (red) on the wall of hepato-pancreatic duct. Simulated pH increased greatly between the duodenal fluid and the pancreatic duct (green) since hydrogen ions (green) were neutralized by bicarbonate (red) with carbon dioxide as a byproduct (blue). Simulated ion concentration distribution in the hepatopancreatic duct of healthy individuals. Ion concentrations at a ductal radius of 2.6 mm $pH=-{log}_{10}([H^{{}_{+}}])$.

Figure 3

The simulated bacterial concentration in the healthy pancreas (blue) is lower than that in the literature (orange), but simulated bacterial amount in pancreas at 3.2 CFU agrees reasonably we the literature that 15% of healthy pancreas contained detectable bacteria. Simulated one in the pancreas with tumors (gray) agrees reasonably well with the literature (yellow). The literature value was calculated using the DNA weight of E. coli at 17 fg/cell. PDAC: pancreatic ductal adenocarcinoma.

Figure 4

Simulated bacterial concentrations in hepato-pancreatic duct and surrounding pancreatic tissues in cancer patients (a) and those on the wall of the duct (b). The major driving force of bacterial migration is pH-taxis from acidic to neutral pH. This migration is made easier by reduced pancreatic juice and bile flow rate due to obstructions of the pancreatic and bile duct by solid tumors.

Figure 5

Parametric sensitivity analysis for bacterial migration from the duodenum into the pancreas with tumors. The changes in simulated bacterial concentration in pancreas are shown with each parameter changed.

Figure 6

A steady pH gradient is generated in a microfluidic device, with pH at 5–6 at the top and 7–8 at the bottom (a). GFP E. coli were attracted away from the upper channel with a lower pH toward the lower part with neutral pH (b, c, e blue and orange), though bacteria migrated vertically little without gradient (d). a: pH was visualized in bromothymol blue. b, c: Bacteria were included in either the upper inlet (b) or the lower inlet (c). d: GFP E.coli migrated little without gradient. e: Distribution of GFP E. coli in the proximal (dotted) and distal (solid) channels. Photos in b–d were taken in black-and-white mode under black light at 350 nm.

Figure 7

Migration of P. fluorescens from hydrochloride solution against the bicarbonate flow of μl/min was measured in a simply fabricated PDMS four-millimeter diameter T-shaped cylinder. a: Distribution of pH in the T-shaped cylinder is visualized in bromocresol purple. The pH increased from 5–6 in dark yellow at the top to purple (neutral) at the right and the bottom of the cylinder. b: An example of bacterial distribution over time from bicarbonate solution as a control. c: P. fluorescens migrated upstream against flow under the pH gradient at the T-junction (a) with a penetration rate of approximately 50 μm/s. d: Average bacterial penetration velocity in three different experiments. e: A procedure for calculating a matrix of bacterial penetration depth over time (b, c). Bacterial distribution was quantified from the brightness of the image of each frame in the movies, under UV light in dark condition, which corresponds to the intrinsic fluorescence of P. fluorescens.

Figure 8

Environmental factors in the upper gastrointestinal tract affect migration of aerobic bacteria from the duodenum into the pancreas via bacterial taxes. The pH-taxis under the pH-gradient between acidic duodenum fluid and pancreatic juice at slightly alkaline pH is the major driving force for migration to pancreas. Aerotaxis away from higher carbon dioxide at the duodenum also increases migration slightly. This migration is counteracted by pancreatic and bile flow but a solid tumor on pancreatic duct at pancreatic head reduces the fluid flow, and thus facilitates migration.