Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

Intracellular bacterial communities, or IBCs, have been observed in mice that have been given experimental urinary tract infections, or UTIs. They've also been seen in the urine sediments of human UTI patients. The technique we're describing today is a way to isolate single IPCs from experimentally infected mice.

The mouth micropipetting technique is the only currently available technique for isolating intracellular bacteria from infected mouse bladders. To prepare glass mouth pipettes, firmly pinch both ends of a glass capillary tube and evenly rotate the middle of the tube back and forth along its axis over an open flame source. When the glass becomes soft, remove the capillary from the heat source and immediately pull the glass apart.

The ideal final length of the pulled capillary is three to five centimeters longer than an unpulled capillary to ensure an appropriate internal diameter for isolating single bladder epithelial cells. When the glass has cooled, check to see if the middle of the glass capillary is narrower and that the interior of the tube is still hollow. Picking up the large end of the pulled capillary with one hand, use forceps to grasp up the capillary at its narrowest point, making sure the forceps are wielded with enough force to grip the capillary firmly without crushing it.

Rapidly twist the forceps to snap the pulled capillary at the narrowest point. Then, place the mouth micropipette-sized capillary in a 100 millimeter Petri dish and UV sterilize the dish for 30 minutes. To harvest the bladder, place a mouse in the supine position and sterilize the abdominal area with 70%ethanol.

Using ethanol-sterilized forceps and surgical scissors, make an initial one-centimeter transverse skin incision above the urethral opening and expand the incision diagonally toward the upper limbs of the mouse to create a V-shaped cut along the entire anterior of the mouse that exposes the contents of the peritoneum. Using new sterilized tools, gently push down on the fat pads near the pelvic region of the mouse to cause the bladder to protrude and grasp the exposed bladder at the apex. Keeping a firm grip on the bladder, cut the ureters and urethra to free the bladder and insert the tip of one shaft of rounded forceps into the opening of the bladder where it was just incised.

Release the forceps gripping the apex and keeping the rounded forceps almost completely closed, use a second pair of forceps to gently pull the outside end of the mouth of the bladder away from the rounded forceps and around and over the other tip of the rounded forceps to turn the bladder inside out. Then, use the second pair of forceps to gently coax the inverted bladder from the tip of the forceps into one milliliter of cold PBS. And gently scrape the internal epithelial cell layer on the outside of the inverted bladder with two pairs of clean forceps.

When all of the cells have been scraped, insert the unpulled end of a pulled glass capillary into the rubber plug of an aspirator tube and tightly fit the narrow end of a one milliliter pipette tip into the other open end of the tube. Tightly fit the narrow end of a two milliliter aspirating pipette into the open, wider end of the one milliliter pipette tip and place the scraped cell suspension under a dissecting microscope. Using a 20 to 40X magnification, identify the IBCs as large fluorescent aggregates and dip the fine end of the glass capillary into a fresh tube of PBS for one second to reduce the uptake of unwanted volume through capillary action.

When an IBC of interest has been located, slowly bring the open end of the capillary tube toward the IBC and apply a very small suction force to the aspirating pipette to guide the IBC into the glass capillary. Then, move the capillary to an empty 1.5 milliliter centrifuge tube and apply a slight positive pressure to expel the droplet and the IBC into the tube. In addition to confirmation of the presence of a single isolated IBC in the collection tube via the dissecting microscope, the purity of the isolated IBC can be confirmed by confocal microscopy.

The isolated cells should stain for both E.coli and uroplakin and should be between 50 and 120 micrometers in size. After isolation, the presence and viability of the bacterial cells in the individual IBC can be confirmed through colony forming unit quantification or quantitative polymerase chain reaction for genomic equivalents. Notably, uninfected epithelial cells isolated with the same protocol do not demonstrate quantifiable amounts of bacteria.

Further, quantitative reverse transcription polymerase chain reaction analysis confirms the presence of bacterial genes in a range of individually isolated and pooled IBCs. Always ensure that a mouth micropipette can pick up and expel liquid easily. Do not hesitate to change the capillary or the entire apparatus if the mouth micropipette feels occluded.

This mouth micropipetting technique allows us to directly and specifically study the intracellular populations that form during an experimental UTI. Without this technique, those intracellular populations would be contaminated with and outnumbered by the extracellular bacteria. Some other infectious agents may be more easily aerosolized or transmitted than the E.coli we've used in this application.

In these cases, some additional controls may therefore be warranted, such as adding a filter or extending the tubing, depending on the application being used. The IBCs isolated using this mouth micropipetting technique may then be used in other downstream single cell analyses, such as QRTPCR or RNA sequencing.