Ureaplasma urealyticum Causes Hyperammonemia in an Experimental Immunocompromised Murine Model
Ureaplasma urealyticum Causes Hyperammonemia in an Experimental Immunocompromised Murine Model
Xiaohui Wang 0 2 3 4
Melissa J. Karau 0 2 3 4
Kerryl E. Greenwood-Quaintance 0 2 3 4
Darci R. Block 0 2 4
Jayawant N. Mandrekar 0 1 2 4
Scott A. Cunningham 0 2 3 4
Robin Patel 0 2 3 4 5
0 a Current address: Center of Infectious Diseases, West China Hospital, Sichuan University , Chengdu, Sichuan , China ¤b Current address: Division of Infectious Diseases, State Key Laboratory of Biotherapy , Chengdu, Sichuan , China
1 Department of Health Sciences Research, Mayo Clinic , Rochester, Minnesota , United States of America
2 Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester , MN. R.P. is supported by the National Institutes of Health [grant numbers R01 AR056647 and R01 AI91594]. X.W. is supported by the State Scholarship Fund from the China Scholarship Council as a Visiting Scientist at Mayo Clinic. The funders had no role in study design , data
3 Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester , Minnesota, United States of America, 2 Division of Clinical Core Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester, Minnesota , United States of America
4 Editor: Christopher James Johnson , US Geological Survey , UNITED STATES
5 Division of Infectious Diseases, Department of Medicine, Mayo Clinic , Rochester, Minnesota , United States of America
Hyperammonemia syndrome is an often fatal complication of lung transplantation which has been recently associated with Ureaplasma infection. It has not been definitely established that Ureaplasma species can cause hyperammonemia. We established a novel immunocompromised murine model of Ureaplasma urealyticum infection and used it to confirm that U. urealyticum can cause hyperammonemia. Male C3H mice were pharmacologically immunosuppressed with mycophenolate mofetil, tacrolimus and oral prednisone for seven days, and then challenged intratracheally (IT) and/or intraperitoneally (IP) with 107 CFU U. urealyticum over six days, while continuing immunosuppression. Spent U. urealyticum-free U9 broth was used as a negative control, with uninfected immunocompetent mice, uninfected immunosuppressed mice, and infected immunocompetent mice serving as additional controls. Plasma ammonia concentrations were compared using Wilcoxon ranks sum tests. Plasma ammonia concentrations of immunosuppressed mice challenged IT/IP with spent U9 broth (n = 14) (range 155-330 μmol/L) were similar to those of normal mice (n = 5), uninfected immunosuppressed mice (n = 5), and U. urealyticum IT/IP challenged immunocompetent mice (n = 5) [range 99-340 μmol/L, p = 0.60]. However, immunosuppressed mice challenged with U. urealyticum IT/IP (n = 20) or IP (n = 15) had higher plasma ammonia concentrations (range 225-945 μmol/L and 276-687 μmol/L, respectively) than those challenged IT/IP with spent U9 broth (p<0.001). U. urealyticum administered IT/IP or IP causes hyperammonemia in mice pharmacologically immunosuppressed with a regimen similar to that administered to lung transplant recipients.
Data Availability Statement; All relevant data are within the paper
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: Dr. Patel reports grants from
BioFire, Check-Points, Curetis, 3M, Merck, Hutchison
Biofilm Medical Solutions, Accelerate Diagnostics,
Allergan, and The Medicines Company. Dr. Patel is a
consultant to Curetis, Roche, Qvella, and Diaxonhit.
In addition, Dr. Patel has a patent on Bordetella
pertussis/parapertussis PCR with royalties paid by
TIB, a patent on a device/method for sonication with
royalties paid by Samsung to Mayo Clinic, and a
patent on an anti-biofilm substance issued. Dr. Patel
serves on an Actelion data monitoring board. Dr.
Patel receives travel reimbursement and an editor’s
stipend from ASM and IDSA, and honoraria from the
USMLE, Up-to-Date and the Infectious Diseases
Board Review Course.
Hyperammonemia syndrome (HS) is a rare and potentially fatal complication of lung
]. Case series show that this syndrome may occur in as many as 4.1% of lung
transplant recipients, with 67% of affected patients dying within 30 days of lung
transplantation . Until recently, the etiology of HS was unclear, with potential etiologies suggested in
individual cases to include hepatic glutamine synthetase deficiency [
], and disseminated
Mycoplasma hominis infection [
]. Recently, using PCR and specialized culture, we found
evidence of Ureaplasma infection in lung transplant recipients with HS [
]. However, causality
had not yet been established. Herein, we established a new experimental pharmacologically
immunosuppressed murine model of U. urealyticum infection and used it to show that U.
urealyticum can cause hyperammonemia. The immunosuppression regimen studied was designed
to mimic that administered to lung transplant recipients.
This study was carried out in accordance with the recommendations in the Guide for the Care
and Use of Laboratory Animals of the National Institutes of Health, and was approved by
Mayo Clinic Institutional Animal Care and Use Committee (protocol number: A8115). Mayo
Clinic is AAALAC accredited (000717), registered with the USDA (41-R-0006), and has an
Assurance with OLAW (A3291-01). Mice were housed in a biosafety level 2,
specific-pathogen-free, AAALAC-accredited facility, where sentinel mice were tested quarterly for murine
pathogens; tested mice were negative for murine pathogens throughout the course of this
study. Mice had unrestricted access to irradiated rodent food (LabDiet formula 5053) and
water. The housing room was environmentally controlled (temperature 68–74°F, relative
humidity 30–70%, 12:12-hour light:dark cycle). All efforts were made to minimize suffering.
Mice were monitored twice daily, and anesthetized mice were monitored until awake. Animals
were monitored for decreased activity, decreased body temperature, hunched stature, distress,
and inability to eat and drink; if these findings were severe, animals were euthanized.
U. urealyticum ATCC 27618 (American Type Culture Collection, Manassas, VA) was used to
challenge the mice. For inoculum preparation, U. urealyticum was cultivated in U9 broth
(Hardy Diagnostics, Santa Maria, CA) at 37°C in air until a color change was observed (~7
hours). The culture broth was centrifuged at 4,000 rpm for 30 minutes (to concentrate U.
urealyticum). Cells were resuspended in spent U9 broth to prepare final concentrations of 2x108
CFU/mL. Fresh inoculum was immediately administered. For morphological identification
and quantitation, U. urealyticum was plated onto A8 agar (Hardy Diagnostics) and incubated
anaerobically at 37°C for five days. Colonies were enumerated by 100X microscopy.
Mice were pharmacologically immunosuppressed using intraperitoneal (IP) mycophenolate
mofetil (90 mg/kg) (Cellcept Intravenous, Roche Laboratories, Inc., Nutley, NJ), IP tacrolimus
(1.2 mg/kg) (Prograf, Astellas Pharma US, Inc., Northbrook, IL), and oral prednisone (6 mg/
kg) (Prednisone Intensol, Roxane Laboratories, Inc., Columbus, OH) administered daily for
seven days prior to challenge with U. urealyticum and continued over six days of microbial
challenge (i.e., until the day before sacrifice).
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Experimental Mouse Model
Immunocompetent C3H male mice (22–29 g, Charles River Laboratories, Wilmington, MA)
were studied. Plasma ammonia concentrations were assessed in uninfected,
non-immunosuppressed mice (n = 5), as well as uninfected mice administered the immunosuppression regimen
for 13 days (n = 5). Pharmacologically immunosuppressed mice were challenged with U.
urealyticum in U9 broth over six days by four routes: intratracheal (IT) challenge every other day
(n = 13), IP challenge every day (n = 15), intramuscular (IM) challenge every day (n = 15), or a
combination of IT challenge every other day and IP challenge every day (n = 20). For IT
challenge, mice were anesthetized with ketamine/xylazine (90/10 mg/kg), placed in a vertical
position, and 50 μl of U. urealyticum suspension placed into the trachea using a 22G curved gavage
needle. Mice remained vertical for two minutes and were monitored until awake. For IP and
IM challenge, 250 μl of U. urealyticum suspension was injected into the peritoneal cavity or
caudal thigh muscle, respectively. A vehicle negative control group (n = 14) was identically IT/
IP challenged with spent U9 broth (filtered through a 0.1 μm filter). To assess the effect of
pharmacologic immunosuppression alone, immunocompetent mice (n = 5) were also
challenged IT/IP with U. urealyticum. U. urealyticum challenge was administered at least six hours
after administration of immunosuppressive agents.
Measurement of Plasma Ammonia Concentrations
Mice were euthanized with CO2 asphyxiation 24 hours after the last IP, IM, or IT/IP challenge
or 48 hours after the last IT challenge. Blood was collected via cardiac puncture and placed into
1.5 ml EDTA collection tubes. EDTA blood was immediately centrifuged at 8000 rpm for 3
minutes and plasma frozen at -80°C. Plasma ammonia concentrations were determined within
24 hours using a Vitros 350 (Ortho Clinical Diagnostics, Inc., Raritan, NJ).
U. urealyticum Culture and Real-time PCR Detection
Lung tissue, thigh muscle tissue and cardiac blood were cultured for U. urealyticum in U9
broth at 37°C for five days. Whole lung and thigh muscle were crushed in sterile stomacher
bags with 3 ml MicroTest M5 transport media (Remel, Lenexa, KS); 200 μl of tissue
homogenate was cultured. Positive cultures were confirmed by plating to A8 agar and by real-time
PCR performed as described previously [
], using a LightCycler 1.5 real-time PCR instrument
(Roche Diagnostic Gmbh, Mannheim, Germany) with probe dye Red-640. Additionally, U.
urealyticum was directly assayed in lung tissue, thigh muscle tissue, and cardiac blood by
realtime PCR after total DNA extraction using the DNeasy Blood and Tissue kit (Qiagen, Valencia,
Plasma ammonia concentrations between groups were compared using Wilcoxon ranks sum
tests. All tests were two sided; p-values less than 0.05 were considered statistically significant.
No adjustment for multiple comparisons was made due to the small sample sizes. Analysis was
performed using SAS version 9.4 (SAS Inc. Cary, NC).
Plasma Ammonia Concentrations
The plasma ammonia concentrations of normal (non-immunosuppressed, uninfected) C3H
mice (n = 5) were 160–280 μmol/L. The plasma ammonia concentrations of uninfected
immunosuppressed mice (n = 5) were 171–340 μmol/L. The range of plasma ammonia
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Fig 1. Plasma ammonia concentrations in the eight groups of experimental mice. The median of each
immunosuppressed and infected group is shown by a short dash. Groups labeled by stars represent those with
significantly elevated ammonia concentrations compared with group D, immunosuppressed mice inoculated with
spent U. urealyticum-free U9 broth intratracheally (IT) every other day and intraperitoneally (IP) every day
(P<0.001). Groups from left to right: A). Immunocompetent and uninfected mice (n = 5). B). Immunosuppressed but
uninfected mice (n = 5). C). Immunocompetent mice challenged with U. urealyticum IT every other day and IP every
day (n = 5). D). Immunosuppressed mice challenged with spent U9 broth without bacteria IT every other day and IP
every day (n = 14). E). Immunosuppressed mice challenged with U. urealyticum IT every other day and IP every
day (n = 20). F). Immunosuppressed mice challenged with U. urealyticum IT every other (n = 13). G).
Immunosuppressed mice challenged with U. urealyticum IP every day (n = 15). H). Immunosuppressed mice
challenged with U. urealyticum intramuscularly every day (n = 15).
concentrations of immunocompetent mice challenged IT/IP with U. urealyticum (n = 5) was
99–212 μmol/L (median, 178 μmol/L). The range of plasma ammonia concentrations of
pharmacologically immunosuppressed mice challenged with spent U9 vehicle IT/IP (n = 14)
was 155–330 μmol/L (median, 209 μmol/L). There was no difference in the plasma ammonia
concentrations in this group compared with the other three control groups (median 212 μmol/
L, range 99–340 μmol/L, p = 0.60).
Mice in the IT/IP challenge group (n = 20) had elevated plasma ammonia concentrations
(median 328 μmol/L, range 225–945 μmol/L) compared to vehicle IT/IP negative control mice
(p<0.001). Mice in the IT challenge group (n = 13) had similar plasma ammonia concentrations
(median 206 μmol/L, range 110–306 μmol/L) to vehicle IT/IP negative control mice (p = 0.98).
Mice in the IP challenge group (n = 15) had elevated plasma ammonia concentrations (median
408 μmol/L, range 276–687 μmol/L) compared to vehicle IT/IP negative control mice (p<0.001).
Mice in IM challenge group (n = 15) had similar plasma ammonia concentrations (median
222 μmol/L, range 189–320 μmol/L) to vehicle IT/IP negative control mice (p = 0.15). A
comparison of plasma ammonia concentrations for each group is shown in the Fig 1.
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U. urealyticum Culture and Real-time PCR
Culture and PCR results are shown in the Table 1. Uninfected immunosuppressed mice
(n = 5), IT/IP challenged immunocompetent mice (n = 5), and spent U9 IT/IP vehicle control
mice (n = 14) had negative cultures and PCR results. Among immunosuppressed mice
challenged with U. urealyticum IT, IP, IM or IT/IP, all cardiac blood cultures and PCR results were
negative, that is, no mice had detectable U. urealyticum bacteremia or DNAemia. In
immunosuppressed mice challenged with IT U. urealyticum, 30% (10/33) of lung tissues were
cultureor PCR-positive for U. urealyticum. All mice in the IP challenge group had culture- and
PCRnegative lung tissues. All 15 immunosuppressed mice challenged with IM U. urealyticum
(100%) had culture- or PCR-positive thigh muscle tissues.
Seven mice died prior to the blood and tissue collection. In the vehicle IT/IP negative
control group, two died on day 10 because of anesthesia administered for IT challenge. In the IT/
IP challenge group, one died on day 10, just after receiving immunosuppression; autopsy
showed ascites. And, another died unexpectedly just before sacrifice on day 14. In the IT
challenge group, one died on day 12 after anesthesia, while another died unexpectedly on day 14
just before sacrifice. In the IP challenge group, one died shortly after inoculation on day 8.
Because of rapid coagulation of blood in deceased mice, their blood was not collected for
ammonia determination. Beyond the seven mice described above, no other mice met criteria
for humane endpoints requiring euthanasia prior to the experimental endpoint. The outcome
of mortality was acknowledged as a possibility as part of our Institutional Animal Care and
Use Committee approval, because hyperammonemia may lead to death, as can anesthesia and
Hyperammonemia syndrome is a previously-unexplained condition wherein lung transplant
recipients develop progressive elevations in plasma ammonia concentrations within the first
postoperative month, mostly within the first ten days [
]. The majority develop cerebral
edema, which causes mental status changes, and even seizures and/or coma. This condition
can result in death. We recently reported an association between U. urealyticum or U. parvum
and HS in lung transplant recipients [
]. Here, we have shown that U. urealyticum is sufficient
to cause hyperammonemia in an immunocompromised experimental animal model.
IT, intratracheal; IP, intraperitoneal; IM, intramuscular; NA, not applicable.
*: Groups with significantly elevated ammonia concentrations compared with vehicle IT/IP negative control (P<0.001).
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Ureaplasma species, which belong to the class Mollicutes, are part of the normal genital
tract flora. They lack a cell wall and do not grow on routine culture media, but can be isolated
using specialized media containing urea. They produce large amounts of urease which
hydrolyses urea to generate ATP [
]. In adults, Ureaplasma species have been associated with
urogenital infections, while in neonates, they can cause invasive diseases, including pneumonia and
Herein, we have confirmed that U. urealyticum can cause hyperammonemia;
immunosuppressed mice challenged with U. urealyticum IT/IP or IP had higher plasma ammonia levels (up
to 945 μmol/L) than vehicle-challenged controls (p<0.001). This particular control was included
to ensure that exogenous ammonia, present in the inoculated broth, would not yield
hyperammonemia; we found that immunosuppressed mice challenged IT and IP with spent U9 broth did
not have higher plasma ammonia concentrations than the other three control groups studied. As
far as we know, immunocompetent adults infected with Ureaplasma species, with urethritis for
example, do not develop hyperammonemia [
]. Consistent with this, immunocompetent mice
not receiving pharmacologic immunosuppression, but challenged IT/IP with U. urealyticum, did
not develop elevated plasma ammonia levels. This suggests that immunocompromised status per
se may be important in the pathogenesis of Ureaplasma-related HS, and that
Ureaplasma-associated HS may be an opportunistic infection-associated syndrome.
To our knowledge, this is the first model established to verify that U. urealyticum can cause
hyperammonemia. In the model studied, the route of U. urealyticum challenge appeared to be
important. This may be because the organisms live in or are absorbed through the peritoneum.
Interestingly, in the IM-challenged group, all 15 thigh muscle tissues were culture- or
PCRpositive for U. urealyticum, but no hyperammonemia was observed. Additional work is needed
to clarify the exact role of route of challenge and specific immunosuppression in U.
urealyticum-associated hyperammonemia. In lung transplant patients with HS, overt pneumonia
(such as infiltrates on radiographic images or inflammatory lesions in tissue biopsy specimens)
has not been reported, suggesting that although the lung may be infected, patients may not
have classic manifestations of pneumonia. Due to the inability of the mice to tolerate
IT-challenge more than every other day, we were unable to definitely establish whether IT challenge
alone can cause hyperammonemia in our model.
There are several limitations to our study. We studied a type strain of U. urealyticum; we
have anecdotally noted that different strains replicate at different rates and may produce
different amounts of urease. Therefore, a clinical isolate may have resulted in differential virulence
compared to that observed. In addition, there are two species of Ureaplasma associated with
HS in lung transplant patients, and we only studied one of them. In future studies, it would be
interesting to study U. parvum in the model described. IM challenge was included as this was
model development and there is a prior Ureaplasma bacteremia model that was established in
two-day-old mice via IM injection [
]; that there were no positive blood cultures or DNAemia
was unexpected based on findings from the neonatal mouse study [
]. Whether the difference
relates to mouse age, strain, or immune status, bacterial strain, culture or PCR methods, or
other factors, remains to be determined. Considering that not every mouse in the IT/IP and IT
challenge groups had positive lung cultures and/or PCR, some adult mice studied may have
cleared Ureaplasma species. We only assessed the animals at a single time point, and may have
missed documenting active Ureaplasma infection.
The pathogenesis of Ureaplasma-associated hyperammonemia remains to be determined.
Specific factors (beyond exposure to the organisms) may predispose to Ureaplasma
species-associated HS in lung transplant recipients. Previous studies showed that surfactant protein-A
deficiency and host immune response to multiple banded antigens of U. urealyticum related to
Ureaplasma-cidal activity [
], and Beeton et al. showed that antibody mediated clearance is
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required for killing of certain serovars of U. parvum . A recent article identified a single
nucleotide polymorphism in toll-like receptor 6 associated with a decreased risk for Ureaplasma
respiratory tract colonization and bronchopulmonary dysplasia in preterm infants [
The findings reported herein raise the intriguing hypothetical possibility that Ureaplasma
species may be associated with unexplained hyperammonemia in patients other than lung
transplant patients. A study evaluating host immune response in preterm neonates at risk of
developing bronchopulmonary dysplasia showed that Ureaplasma DNA could be detected at
low levels and decreased over time [
], raising the hypothetical possibility that Ureaplasma
species could explain some cases of transient hyperammonemia of the preterm infant [
Unexplained hyperammonemia also occurs in intensive care unit patients, raising the
hypothetical possibility that it too could relate to Ureaplasma species.
In conclusion, by establishing a novel experimental immunocompromised murine model
and measuring plasma ammonia concentrations, we verified that U. urealyticum can cause
hyperammonemia. Further research on pathogenesis, treatment, and host susceptibility may
be studied in this new model. This work provides the impetus to determine whether
Ureaplasma species might be associated with hyperammonemia in patient populations other than
lung transplant recipients.
We are grateful to Suzannah M. Schmidt Malan and Javier Fernandez Dominguez for their
Conceptualization: XW KG RP.
Data curation: XW MK KG DRB JNM SAC RP.
Formal analysis: XW MK.
Funding acquisition: XW MK KG DRB JNM SAC RP.
Investigation: XW MK KG DRB SAC RP.
Methodology: XW MK KG DRB JNM SAC RP.
Project administration: RP.
Resources: XW MK KG DRB JNM SAC RP.
Validation: XW MK KG DRB JNM SAC RP.
Visualization: XW MK KG DRB JNM SAC RP.
Writing - original draft: XW KG RP.
Writing - review & editing: XW KG RP.
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