The authors thank Lynne Foster for technical assistance in infection experiments, James Fitzpatrick at the Washington University Center for Celluar Imaging (WUCCI) for access to the SEM, Scott Hultgren for the UTI89kanR&#160;UPEC strain, and David Hunstad for critical reading of the manuscript.
This work was supported by the National Science Foundation (Graduate Research Fellowship to VPO#DGE - 1143954), by the Center for Women&#39;s Infectious Disease Research at Washington University School of Medicine (Pilot Research Award to NMG), by the American Heart Association: #12POST12050583 (NMG) and #14POST20020011 (NMG), and by the National Institutes of Health, NIAID: R01 AI114635 (ALL) and NIDDK: R21 DK092586 (ALL), P50 DK064540-11 (SJH, project II PI:ALL) and K01 DK110225-01A1 (NMG). Some of the animal studies were performed in a facility supported by NCRR grant C06 RR015502. The Washington University Center for Cellular Imaging (WUCCI; where SEM was performed) and MSJ were supported by the Washington University School of Medicine, the Children&#39;s Discovery Institute of Washington University and the St. Louis Children&#39;s Hospital (CDI-CORE-2015-505), the Foundation for Barnes-Jewish Hospital (3770), and the National Institute for Neurological Disorders and Stroke (NS086741). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that they have no conflicts of interest relating to this study.

The first critical step in this model to identify mice that have not cleared UPEC bacteriuria during the primary UTI phase. These mice must be removed from the experiment as they would otherwise confound the rates of UPEC bacteriuria after G. vaginalis exposure. After the initial UPEC inoculation, urine should be collected weekly to monitor bacterial clearance. Approximately 65-80% of C57BL/6 mice will clear a UTI89kanR infection within 4 weeks. Other inbred mouse strains have different propensities for UPEC clearance42,43 and reservoir formation and thus may not be suitable for this model. The second critical point is that empirical studies have determined that two sequential inoculations of G. vaginalis (either 12 h or 1 wk apart) are necessary to trigger significant reservoir emergence above the background spontaneous emergence that occurs in control mice exposed only to PBS. Other durations of time between the two sequential exposures have not been tested but could yield similar results. It is important to note that a reduction in UPEC bladder titers was only observed in the model in which G. vaginalis exposures were given 1 wk apart39. While more than two exposures can be administered, empirical evidence suggests that repeated catheterization alone increases emergence, which may confound the interpretation of the results or require larger numbers of animals to distinguish differences between exposure groups and controls. Finally, the in situ&#160;bladder fixation method has several critical steps. Some skill is required to ensure that the fixative remains inside the clamped bladders. Deflated bladders will be more difficult to image by SEM. It is also essential to be very gentle when inoculating the fixative into the bladder, as scraping the urothelium with the fixative-containing catheter can induce urothelial exfoliation independent of what is triggered by G. vaginalis. All concentrations mentioned in the fixative cocktail are final concentrations. Improper ratios of these can result in insufficient fixing and swelling or shrinkage of the cells. Fixatives should be warmed to physiological temperatures to avoid temperature shock in cells and tissues. Warming also provides a slight improvement to the diffusion rate of fixatives through plasma membranes. While osmium staining can often be omitted for samples prepared for SEM analysis, it is an essential step in this protocol to stabilize lipids and prevent cracking of cellular membranes during critical point drying.
This protocol can be modified to test other UPEC and/or G. vaginalis strains for their ability to form reservoirs and to trigger their emergence, respectively. Other experimental factors can also be added, such as exposure to other vaginal bacteria (e.g., Lactobacillus crispatus PVAS100) or heat-killed G. vaginalis, neither of which demonstrate pathology in this model39. When selecting other bacterial strains to test, it is important to demonstrate consistent growth such that a standard inoculum concentration can be used in all experiments. The growth of JCP8151BSmR has been optimized in an anaerobic chamber. This strain could likely be cultivated in an anaerobic GasPak system, but this would require optimization to ensure robust bacterial growth. Finally, it may be possible to modify the timing of certain steps in the model. For instance, urine can be collected at earlier timepoints during the UPEC reservoir formation phase to monitor CFU or host responses. An adverse effect of collecting urine samples at early timepoints (3, 6, 12 hpi) on the progression of infection or establishment of reservoirs has not been observed in this model. Emergence of UPEC reservoirs has been reported to occur after two JCP8151BSmR doses given 12 h or 1 wk, but other time intervals have not yet been tested. It also may be possible to reduce the overall length of time for the model by reducing the UPEC reservoir formation phase to 2 weeks (rather than 4 weeks), since many of the mice clear bacteriuria by this time. Previous studies examining UPEC emergence following bladder exposure to chemical exfoliants used a 1 or 2 wk UPEC reservoir formation phase17,18. However, decreasing the amount of time for UPEC bacteriuria clearance may come at the cost of requiring more animals to be culled from the experiment. Finally, SEM analysis of the bladder can be performed at additional time points to observe the duration of the effect of G. vaginalis on the urothelium.
Regarding troubleshooting, there are some important considerations specifically with respect to the bladder SEM analysis. Depending on the mouse background used and amount of inflammation present, some bladders will present with very thin walls. These bladders tend to curl more during critical point drying and can result in a cowrie shell-like shape. If this occurs, the best method is to cut the shell-shaped bladder in half along the curled interface and then a second time to remove the bulk of the overhanging tissue. Cutting works best with a PTFE-coated double-edged razor blade. Excess fat can sometimes solubilize during the osmium staining steps. This can result in unwanted insoluble fat droplets that may not wash off during the rinsing and dehydration steps and that can settle on the bladder surface during subsequent drying. These droplets can appear as either small spheres or disc-like structures scattered over the sample (Figure 4D). This can be mitigated by ensuring that as much adipose tissue is removed from around the bladder as possible. Platinum can be substituted for iridium coating, but thicknesses should be kept to a minimum to reduce the masking of fine structural details. The use of a rotating stage during coating is highly recommended.
One limitation of this model is that it requires a large number of mice. Only 65-80% of C57BL/6 mice will clear their UPEC bacteriuria and be suitable for subsequent G. vaginalis or PBS inoculation (see Figure 2C). To obtain 10-12 mice per group (G. vaginalis inoculation vs. PBS), ~30 mice should be initially infected with UPEC. Further, multiple experiments are likely required to achieve the biological replicates necessary to detect statistical significance. When exposures were given 1 wk apart, UPEC emergence occurred in 14% of mice exposed to PBS (Figure 3B). Thus, detecting a significant increase in UPEC rUTI in G. vaginalis exposed mice relative to PBS controls (powered at 0.8; alpha=0.05 [one sided]) requires testing a cumulative total of at least 40 mice for each exposure group. An additional consideration is that these experiments are expensive and labor-intensive. Mice must be monitored weekly for UPEC clearance and the experimental time course is 4-5 wk depending on whether G. vaginalis is given twice in a 12 h timeframe or twice 1 wk apart. SEM is labor-intensive and may be costly, depending on microscope availability and service charges. Preparing the entire bladder for SEM provides abundant material for analysis but the drawback is that analyzing each bladder can be time-consuming. Thus, it is likely that only a limited number of bladders can be analyzed by SEM compared to the higher animal numbers used for urine and tissue titers. In addition, obtaining high-quality images of the curved surfaces of the bladder &#34;cups&#34; requires skill due to shadows that can impede visibility. Although bladder SEM is a useful tool for visualizing urothelial exfoliation, this method is largely qualitative. Because the sample is fixed in a round shape, and due to the use of glutaraldehyde in the fixative, screening for fluorescently expressing bacteria via light microscopy is not possible. Immunostaining and chemical dyes are incompatible with this process due to the use of glutaraldehyde that will crosslink most antigens and osmium and that will mask antigen sites and darken the tissue. That said, the SEM technique is useful for parameters that can be evaluated quantitatively without the use of additional probes, such as cell size48,49.
This model offers several advantages beyond previously described methods. It allows the examination of mechanisms of UPEC rUTI caused by emergence from bladder reservoirs, as opposed to reintroduction into the bladder from an outside source. Other models of rUTI due to emergence from bladder reservoirs use chemical agents (protamine sulfate or chitosan) to cause urothelial exfoliation17,18, which would not be triggers of rUTI in women. G. vaginalis is a prevalent urogenital bacterium that has been detected in urine collected directly from the bladder via catheterization or suprapubic aspiration in some women23,26. This fact, coupled with the known association between BV (in which G. vaginalis overgrows in the vagina) and UTI, suggests that G. vaginalis is a clinically plausible trigger of rUTI. Finally, the in situ&#160;bladder fixation method preserves bladder ultrastructure and limits damage, ensuring that the bladder layers do not separate from one another. Previous methods for visualizing the urothelium traditionally have the user aseptically harvest, bisect, stretch, and pin the bladder onto a dissection tray before submerging the stretched bladder in fixative48. This method results in a very flat sample but does not ensure even or natural stretching of the tissue and can result in areas that are over and under stretched (resulting in highly wrinkled tissue) and can cause bladder layer separation. Additionally, these physical manipulations of the bladder to stretch and pin the tissue can cause damage, including urothelial exfoliation. Another method is to submerge intact bladders in fixative before embedding in paraffin and acquiring thin sections with a microtome. Thin sections are invaluable for immunohistochemistry experiments to examine bacteria and host protein localization but a thin section does not allow visualization of the urothelial surface. This&#160;SEM method allows the surface of the entire bladder to be examined at once.
As described, future applications of this model include testing other UPEC strains to determine whether they form intracellular reservoirs and testing&#160;other G. vaginalis strains to assess whether they elicit exfoliation and UPEC emergence to cause rUTI. Other mouse strains beyond C57BL/6 mice may also be tested, although mice with a high propensity for developing chronic cystitis (such as mice on the C3H background) are not recommended, since too many mice would need to be culled from the experiment. An additional advantage of C57BL/6 mice is that many genetic knockout strains are commercially available. Such strains provide an opportunity for interrogating the host factors involved in reservoir formation and/or emergence.

Urinary tract infections (UTI) impose a significant healthcare burden worldwide, impacting the quality of life of millions of people each year, especially women1. Uropathogenic Escherichia coli (UPEC) are the most frequent cause of UTI1. Many patients (approximately 20-30%) who develop UTI will experience a recurrent UTI (rUTI) within 6 months despite antibiotic-mediated clearance of the initial infection2. Unfortunately, as many as 5% of premenopausal women suffer from 3 or more rUTI each year3,4. Sequential episodes of rUTI can be caused by persistence of the same UPEC strain from the index case5,6,7,8. Data from human samples and mouse models suggest that same-strain rUTI could be caused by UPEC residing within quiescent reservoirs in the bladder. In humans, UPEC were detected in epithelial cells and bladder biopsies of patients with UTI9,10,11,12,13. Studies in C57BL/6 mice have demonstrated that some strains of UPEC can establish quiescent intracellular reservoirs in the bladder, as detected by fluorescence microscopy and by homogenization and culture of bladder tissue, that are maintained for months following resolution of bacteriuria14,15,16. Treatment of the bladder with agents that induce exfoliation of the bladder epithelium (urothelium), e.g. protamine sulfate17 or chitosan18, trigger emergence of UPEC from reservoirs to cause rUTI. These data suggest that in women harboring bladder UPEC reservoirs from a prior infection, bladder exposures that lead to urothelial exfoliation may trigger rUTI.
There is mounting evidence that the vaginal microbiota contributes to urinary tract infection19,20. Gardnerella vaginalis is a frequent member of both the vaginal and urinary microbiota21,22,23,24,25,26,27,28,29. In the vagina, the presence of high levels of G. vaginalis is associated with a microbial dysbiosis known as bacterial vaginosis (BV), which affects ~30% of women30,31,32. Women with BV are at a higher risk of experiencing UTI compared to women with a vaginal community dominated by Lactobacillus33,34,35,36,37. In mouse models, G. vaginalis causes epithelial exfoliation both in the vagina38 and in the bladder39. In C57BL/6 mice harboring UPEC bladder reservoirs, two sequential bladder exposures to G. vaginalis - but not to PBS - result in reemergence of UPEC from reservoirs to cause UPEC rUTI. The emergence is evidenced by the appearance of UPEC titers in urine from mice that had previously resolved UPEC bacteriuria and a subsequent decrease in UPEC bladder homogenate titers at sacrifice compared to PBS-exposed control animals39. Interestingly, there is not a lasting colonization by G. vaginalis in the bladder. In the vast majority of cases, two short exposures, each with less than 12 (h) of viable G. vaginalis in urine, are sufficient to elicit urothelial exfoliation and promote rUTI.
This protocol describes a mouse model of rUTI caused by UPEC residing in intracellular bladder reservoirs, using G. vaginalis bladder inoculation to trigger the recurrence. The advance achieved by this model is that G. vaginalis is a clinically relevant biological trigger of rUTI compared to previously used chemical agents. Further, the relatively short-lived survival of G. vaginalis in the mouse urinary tract allows examination of the impact of transient microbial exposures on the urothelium, as might occur after sexual activity. In addition to outlining the rUTI model, this protocol also describes methods for urine cytology and in situ bladder fixation and imaging of the urothelium by scanning electron microscopy (SEM).
This protocol of G. vaginalis-induced recurrent UPEC UTI uses UPEC strain UTI89 bearing a kanamycin resistance cassette (UTI89kanR)40. Not all strains of UPEC tested were able to form intracellular bacterial communities during the acute infection stage in mice41 and it is not yet known if all strains of UPEC have the ability to form latent intracellular reservoirs. Reservoir formation should be confirmed prior to use of other UPEC strains in the model. This protocol uses a spontaneous streptomycin-resistant G. vaginalis isolate, JCP8151BSmR38. Induction of rUTI by JCP8151BSmR requires two sequential G. vaginalis inoculations, given either 12 h or 7 days (d) apart39. Whether or not other G. vaginalis strains induce exfoliation and/or UPEC rUTI remains to be determined with this model. It is essential to use UPEC and G. vaginalis strains with known antibiotic resistance (such as kanamycin or spectinomycin for UPEC and streptomycin for G. vaginalis) because antibiotics can be added to agar plates to prevent growth of endogenous mouse microbiota that could otherwise interfere with enumerating colony-forming units (CFU) to monitor infection. This is especially important for culturing urine specimens, because mouse urine frequently contains other bacteria that can overgrow on culture plates without antibiotics. The origin of these endogenous bacteria in mouse urine is unknown but likely reflects periurethral and urogenital bacteria picked up during urine collection.
G. vaginalis is a facultative anaerobic bacterium and, therefore, this protocol describes growing G. vaginalis JCP8151BSmR in an anaerobic chamber. If an anaerobic chamber is not available, other methods for maintaining anaerobic growth conditions (such as a GasPak pouch in an airtight container) can be utilized. Alternatively, some strains of G. vaginalis (including JCP8151BSmR) will grow in a standard tissue-culture incubator (5% CO2). Just as using G. vaginalis strains other than JCP8151BSmR requires testing to ensure that the bacteria behave similarly in this model, changing growth conditions requires empirical determination of ideal durations for culture (on plates and in liquid) and optical density (OD)600 equivalents to achieve desired viable inoculum concentrations. Moreover, it is not known whether growth conditions influence the pathobiology of G. vaginalis.
Finally, when considering whether to utilize this model, researchers should be aware that it can require larger numbers of animals per group than do typical UTI mouse models. This is in part because induction of rUTI requires that the mice resolve the UPEC bacteriuria caused by the initial infection of the bladder. Thus, any mouse that fails to clear bacteriuria (a phenotype usually indicative of ongoing kidney infection) is not included in the rUTI phase of the protocol. The number of mice needed to power these studies is also influenced by the rate of &#34;spontaneous&#34; UPEC emergence into urine (12-14% on average). Finally, different mouse strains have different propensities for developing chronic bacteriuria versus intracellular reservoir formation42,43. If using mouse strains other than C57BL/6 in this model, it must be confirmed that the animals develop quiescent UPEC intracellular reservoirs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30G x 1/2 needles</td>
			<td>BD</td>
			<td>305106</td>
			<td>for catheters</td>
		</tr>
		<tr>
			<td>5 1/2&#34; straight forcep hemostat</td>
			<td>McKesson</td>
			<td>487377</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>ACE 600 Sputter coater</td>
			<td>Leica</td>
			<td></td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>aluminum SEM stub</td>
			<td>Ted Pella</td>
			<td>16111</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>EMS</td>
			<td>12340</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>conductive carbon adhesive tab&#160;</td>
			<td>Ted Pella</td>
			<td>16084-1</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Conductive silver paint</td>
			<td>Ted Pella</td>
			<td>16034</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>CPD 300 Critical Point Drier</td>
			<td>Leica</td>
			<td></td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Cytofunnel metal clip</td>
			<td>Simport</td>
			<td>M964B</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMS</td>
			<td>15050</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>EMS</td>
			<td>16320</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>Hema 3 staining kit</td>
			<td>Fisher</td>
			<td>23123869</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Cellgro&#160;</td>
			<td>25-060-Cl</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>iridium</td>
			<td>Ted Pella</td>
			<td>91120</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>isofluorane</td>
			<td></td>
			<td></td>
			<td>mouse anaesthesia</td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Gibco</td>
			<td>11815024</td>
			<td>add to UPEC LB selective plates (50 ug/mL)</td>
		</tr>
		<tr>
			<td>Luria-Bertani agar</td>
			<td>BD</td>
			<td>DF0445174</td>
			<td>UPEC growth plates</td>
		</tr>
		<tr>
			<td>Luria-Bertani broth</td>
			<td>BD</td>
			<td>DF0446173</td>
			<td>UPEC growth media</td>
		</tr>
		<tr>
			<td>Merlin FE-SEM</td>
			<td>Zeiss</td>
			<td></td>
			<td>scanning electron microscope</td>
		</tr>
		<tr>
			<td>Milli-Q Water Purifier</td>
			<td>Millipore</td>
			<td>IQ-7000</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>Olympus Vanox AHBT3 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>osmium tetroxide</td>
			<td>EMS</td>
			<td>19170</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>EMS</td>
			<td>15710</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427401</td>
			<td>for catheters</td>
		</tr>
		<tr>
			<td>Proteose Peptone #3&#160;</td>
			<td>Fisher&#160;</td>
			<td>DF-122-17-4</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>PTFE coated double edge razor blade</td>
			<td>EMS</td>
			<td>72000</td>
			<td>cutting bladders for SEM</td>
		</tr>
		<tr>
			<td>Shandon Cytocentrifuge</td>
			<td>Thermo Scientific</td>
			<td>A78300002</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Shandon cytofunnel filter</td>
			<td>Simport</td>
			<td>M965FWDV</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Shandon Double cytofunnel</td>
			<td>Simport</td>
			<td>M964-1D</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Shandon double cytoslides (coated)</td>
			<td>Thermo Scientific</td>
			<td>5991055</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>sodium cacodylate trihydrate</td>
			<td>EMS</td>
			<td>12310</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>spectrophotometer</td>
			<td>BioChrom</td>
			<td>80-3000-45</td>
			<td>measuring bacterial OD600</td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Gibco</td>
			<td>11860038</td>
			<td>add to G. vaginalis NYCIII selective plates (1 mg/mL)</td>
		</tr>
		<tr>
			<td>tuberculin slip tip syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>for catheters</td>
		</tr>
		<tr>
			<td>Yeast Extract&#160;</td>
			<td>Fisher</td>
			<td>DF0127-17-9</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
	</tbody>
</table>

recurrent escherichia coli urinary tract infection triggered by gardnerella vaginalis bladder exposure in mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Following inoculation, UPEC titers are detectable in urine (Figure 2B). Failure to plate urine samples on selective media containing kanamycin will likely result in overgrowth of endogenous mouse microbiota contaminating the urine. The level of UPEC bacteriuria will likely be high on day 1 and may increase during the first week before decreasing at later timepoints (Figure 2C). Approximately 65-80% of mice will have no detectable UPEC in the urine by 28 dpi (Figure 2C, green circle). These mice can be used in the subsequent steps of the model. Mice that remain bacteriuric (Figure 2C, red ellipse) should be eliminated from the experiment.
Two sequential G. vaginalis exposures given 12 h (Figure 3A) or 1 wk apart (Figure 3B) result in the emergence of UPEC from intracellular reservoirs to cause recurrent bacteriuria. Both the level of UPEC bacteriuria (Mann-Whitney test) and the fraction of mice displaying UPEC rUTI (Fisher&#39;s exact test) are significantly higher in mice exposed to G. vaginalis compared to the PBS control group. Urine cytology analysis detects PMNs in urine from G. vaginalis-exposed mice that&#160;displayed UPEC emergence&#160;(Figure 3C). In the model with two exposures given 1 wk apart, UPEC titers in bladder tissue are lower in G. vaginalis-exposed mice compared to PBS (Figure 3D), presumably due to emergence of UPEC from reservoirs and subsequent clearance.
Visualization of in situ-fixed bladder tissue by SEM reveals large superficial umbrella urothelial cells lining the bladder surface in control mice exposed only to PBS (Figure 4A). Urothelial exfoliation is evidenced by a loss of superficial umbrella cells, revealing smaller underlying transitional epithelial cells&#160;in mice exposed to G. vaginalis (Figure 4B). Early after UPEC inoculation during the establishment of intracellular reservoirs, UPEC are visible on the urothelium and filamenting out of exfoliating cells (Figure 4C).

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61967/61967fig02.jpg" /> 
Figure 2. Monitoring UPEC titers in urine during Phase 1 (reservoir formation). (A) Schematic of colony-forming units (CFU) plating. (B) Representative image of UPEC titers in urine on LB+kanamycin. Black circles indicate urine sample spots that should be counted to calculate CFU/mL. (C) Time course of UPEC bacteriuria in C57BL/6 mice. Each line represents an individual mouse, tracing the UPEC urine titers over time. Dotted line indicates the limit of detection (1000 CFU/mL). Red ellipse indicates four mice (out of 20) that failed to resolve UPEC bacteriuria and would therefore not be used for the G. vaginalis-induced rUTI model. Conversely, green circle indicates mice that resolved UPEC bacteriuria and proceeded to subsequent phases. (D) Table of data used to generate graph in panel C. Yellow, detectable CFU; green, no CFU. (E) Randomization of mice into exposure groups based on the time point at which UPEC CFU were no longer detected in urine (&#34;Day resolved&#34;). The mouse numbers in the left column of panel D are the same mouse numbers given in panel E. <a href="https://www.jove.com/files/ftp_upload/61967/61967fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61967/61967fig03.jpg" /> 
Figure 3. G. vaginalis triggers UPEC rUTI. UPEC titers in urine following two sequential urinary tract exposures to PBS (circles) or G. vaginalis (Gvag; squares) given 12 h (A) or 1 wk (B) apart. Each symbol represents an individual mouse. The highest CFU/mL UPEC detected from each mouse between 1-3 d following the second exposure are plotted. Mice with no detectable bacteriuria are plotted at the limit of detection (dotted line). (C) Urine cytology analysis showing UPEC (arrowheads) and polymorphonuclear (PMN) cells (arrows). Scale bar = 20 &#956;m. (D) UPEC titers in bladder tissues collected 3 d following two sequential urinary tract exposures given 1 wk apart. Each symbol represents a different mouse and zeros are plotted at the limit of detection (dotted line). In A, B, and D, boxes are at the first and third quartile with the median marked and whiskers from min to max. Mann-Whitney U tests * P &#60; 0.05; ** P &#60; 0.01; **** P &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/61967/61967fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61967/61967fig04.jpg" /> 
Figure 4. SEM analysis of bladders fixed in situ. Bladders were collected from mice 3 h after two exposures (12 h apart) to PBS (A) or G. vaginalis (C). Dotted lines illustrate a single urinary epithelial cell, which is smaller in G. vaginalis-exposed bladders because the large superficial cells have exfoliated away revealing the underlying transitional epithelium. (B) Bladder collected 6 h after initial inoculation with UPEC, during Phase 1 of the model, showing urothelial exfoliation and extracellular UPEC. (D) Example of insoluble fat droplets present on the bladder surface. Scale bars are 20 &#956;m in the main images and 2 &#956;m in the inset. <a href="https://www.jove.com/files/ftp_upload/61967/61967fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice at JoVE.com

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

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Immunology and Infection

5.7K Views.  Washington University School of Medicine in Saint Louis. A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Video: Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.