Access to this full-text is provided by MDPI.
Content available from Microorganisms
This content is subject to copyright.
Citation: Sigg, A.P.; Mariotti, M.;
Grütter, A.E.; Lafranca, T.; Leitner, L.;
Bonkat, G.; Braissant, O. A Method to
Determine the Efficacy of a
Commercial Phage Preparation
against Uropathogens in Urine and
Artificial Urine Determined by
Isothermal Microcalorimetry.
Microorganisms 2022,10, 845.
https://doi.org/10.3390/
microorganisms10050845
Academic Editor: Karim Fahmy
Received: 18 February 2022
Accepted: 4 April 2022
Published: 20 April 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
microorganisms
Article
A Method to Determine the Efficacy of a Commercial Phage
Preparation against Uropathogens in Urine and Artificial Urine
Determined by Isothermal Microcalorimetry
Aurelia Pahnita Sigg 1, †, Max Mariotti 1 ,† , Anabel E. Grütter 1, Tecla Lafranca 1, Lorenz Leitner 2, Gernot Bonkat 3
and Olivier Braissant 1, *
1
Department of Biomedical Engineering, University of Basel, Gewerbestrasse 14, 4123 Allschwil, Switzerland;
aureliapahnita.sigg@stud.unibas.ch (A.P.S.); max.mariotti96@gmail.com (M.M.);
anabel.gruetter@stud.unibas.ch (A.E.G.); tecla.lafranca@stud.unibas.ch (T.L.)
2Department of Neuro-Urology, Balgrist University Hospital, University of Zürich, 8008 Zürich, Switzerland;
lorenz.leitner@balgrist.ch
3Alta uro AG, Centralbahnplatz 6, 4051 Basel, Switzerland; bonkat@alta-uro.com
*Correspondence: olivier.braissant@unibas.ch
† These authors contributed equally to this work.
Abstract:
Background: Urinary tract infections are commonly encountered and often treated with
antibiotics. However, the inappropriate use of the latter has led to the appearance of resistant strains.
In this context we investigate the use of calorimetry to rapidly determine if a phage cocktail can
be used as alternative to antibiotics. Methods: We used a commercially available phage cocktail
from an online pharmacy and tested it against a strain of Escherichia coli and a strain of Proteus
mirabilis. We used isothermal microcalorimetry to follow the metabolic activity of the bacterial
culture treated with the phage cocktail. Results: Isothermal microcalorimetry was able to follow
the dynamic of the bacterial metabolic activity reduction by the phage cocktail. Both pathogens
were strongly inhibited; however, some regrowth was observed for E. coli in urine. Conclusions:
Isothermal microcalorimetry proved to be a valuable technique when investigating the efficacy of
phage cocktails against uropathogens. We foresee that isothermal microcalorimetry could be used to
obtain rapid phagograms.
Keywords: isothermal microcalorimetry; bacteriophages; phage cocktail; E. coli;P. mirabilis
1. Introduction
Urinary tract infections (UTI) are the second most common infectious diseases di-
agnosed after respiratory infections [
1
,
2
]. They can affect the entire urinary tract and
progress toward urosepsis and septic shock, a life-threatening condition [
3
]. The most
common uropathogen that causes between 80 and 85% of UTIs is Escherichia coli [
4
,
5
],
a gram-negative, facultative anaerobic bacterium that is part of the normal human gut
microbiota. Still, some strains can be virulent and are responsible for a wide range of
diseases that have an intestinal or an extra-intestinal origin [
6
,
7
]. Another commonly
encountered pathogen of the urinary tract that is responsible for up to 10% of all UTIs
is Proteus mirabilis.P. mirabilis is another gram-negative rod-shaped bacterium, usually
encountered in complicated UTIs (e.g., catheter-associated UTI and patients with abnormal-
ities of the lower urinary tract) and often associated with phosphate stone formation due to
its urease production [
8
,
9
]. In catheter-associated UTI, this can lead to crystal formation
(mostly struvite-NH4MgPO4·6H2O) and ultimately to catheter blockage.
The extensive and sometimes inappropriate use of antibiotics in the previous years has
led to the rise of antimicrobial resistance in both E. coli and P. mirabilis. Recent reports [
10
,
11
]
on resistance rates to commonly used antibiotics show proportions of resistant isolates
between 39.7% and 98% for E. coli [
10
,
11
] and 21% and 91% for P. mirabilis with variations
Microorganisms 2022,10, 845. https://doi.org/10.3390/microorganisms10050845 https://www.mdpi.com/journal/microorganisms
Microorganisms 2022,10, 845 2 of 13
depending on the geographic location. Finding an alternative to fight resistant strains
of these pathogens is therefore highly needed [
9
,
12
,
13
]. This is even more important
considering the interplay and correlations between the resistance observed [14,15].
Among other alternatives, the therapeutic use of bacteriophages (phages) is receiving
more attention [16,17]. These naturally existing viruses specifically kill bacteria, and were
first described by Félix D’Hérelle, who observed their antibacterial activity. Phages are
highly specific to one bacterial species or strain and do not infect eukaryotic cells making
them a valuable alternative to antibiotics [
16
,
18
]. Indeed, in 1919, Félix D’Hérelle suc-
cessfully applied phages to treat bacterial infections [
16
,
17
]. However, the discovery of
penicillin and other wide spectrum antimicrobials, as well as the inconsistent results of
phage therapy in early clinical trials diminished the interests in phages having a much
narrower spectrum of activity [
19
,
20
]. The research and use of phage thus rapidly de-
creased, except in eastern European and post-Soviet countries, in which phage therapy
is well accepted and where commercial products are available. Nowadays, an increase
in antimicrobial resistance has renewed the interest in phage therapy to treat bacterial
infections, including UTI [21–23].
As phages have a species-specific host range and can penetrate biofilms, one can use
their abilities to stop the spread of resistant pathogens [
20
,
24
]. However, the drawback
of the narrow host range is the necessity to find the right phage for the right infection.
Assessing the efficacy of different phages and phage-based products can be a complicated
and is, using conventional microbiology techniques, a work-intensive task. The most
commonly used technique to evaluate phage susceptibility is plaque assays, for example
the double layer agar where lysis area (plaque) is clearly visible in the host lawn [
25
]. With
this method, results can be available after 18 to 24 h, making it inapplicable for critically
ill patients.
Here, we propose to use isothermal microcalorimetry as a rapid and less demanding
alternative method to plaque assay. Isothermal microcalorimetry can measure microbial
growth in many conditions [
26
–
28
]. The heat production rate (heatflow or thermal power)
is considered as a proxy for the microbial metabolic activity. When the heat production
rate is integrated over time, the resulting sigmoid heat curve is used as a conventional
growth curve similar to those resulting from plate counts or turbidity measurements [
26
].
Still, isothermal microcalorimetry is much less applied for the study of viruses, in partic-
ular, the study of the interactions between phages and their target bacteria [
29
–
33
], was
assessed only in conventional rich media. This study investigated the effect of a commercial
polyvalent phage product on the growth of two urinary tract pathogens directly in urine
and artificial urine.
2. Materials and Methods
2.1. Urine and Artificial Urine Preparation
Urine samples from three healthy donors were pooled and centrifuged shortly after to
remove sediments, if present. After centrifugation, the urine was filtered through 0.22
µ
m
pore size filters (Millipore, Stericup
®
, Billerica, MA, USA). Artificial urine was prepared by
adding the following components: 10.0 g urea, 5.2 g NaCl, 2.1 g NaHCO
3
, 1.4 g Na
2
SO
4
,
1.3 g NH
4
Cl, 1.2 g K
2
HPO
4
, 1.0 g KH
2
PO
4
, 1.0 g peptone, 0.5 g MgSO
4·
7H
2
O, 0.4 g citric
acid, 0.37 g CaCl
2·
2H
2
O, 0.1 g lactic acid, 70.0 mg creatinine, 10.0 mg FeSO
4·
7H
2
O, and
5.0 mg yeast extract to 1 L of deionized water. To enhance pathogen growth: 20 mg lactose,
20 mg saccharose, and 560 mg glucose were also added to the artificial urine solution.
The artificial urine was sterilized by using a 0.22
µ
m pore size filter (Millipore, Stericup
®
,
Billerica, MA, USA). The urine and artificial urine were then stored at 4
◦
C until use or at
−80 ◦C for longer storage.
2.2. Microorganisms and Culture Conditions
Bacterial strains E. coli DSM 10142 and P. mirabilis DSM 4479 were used in this study.
The two strains were stored in cryovials at −80 ◦C in 20% glycerol. Strains were grown at
Microorganisms 2022,10, 845 3 of 13
37
◦
C overnight in Tryptic Soy Broth (17.0 g tryptone, 5.0 g NaCl, 3.0 g soy peptone, 2.5 g
glucose, and 2.5 g K
2
HPO
4
per 1 L purified water). Before use, the culture was diluted to a
concentration of ca. 106colony forming units (CFU)·mL−1.
2.3. Phage Cocktail Description
The phage cocktail used in this study was an “E. coli-Proteus” bacteriophage so-
lution (NPO Microgen, Nizhny Novgorod, Russia) obtained from an online pharmacy.
The phage titer against E. coli DSM 10142 of this phage product was determined at
2.6 ×106±0.5 ×106
Plaque forming units (PFU)
·
mL
−1
by serial dilution and plating
using the double-layer agar technique using Luria agar. This phage cocktail was pre-
viously characterized in metagenomic studies and consists of a mixture of 18 phages
with higher numbers of T4-like and T7-like phages observed using transmission electron
microscopy [34]. In addition, it was already used in human clinical trials [35].
2.4. Comparison of Isothermal Microcalorimetry with CFU, PFU and OD Data
To compare microcalorimetry data to conventional microbiological data, a measure-
ment was performed with E. coli in artificial urine with or without the addition of phages.
For the measurements, 5 mL of artificial urine inoculated at 10
7
CFU
·
mL
−1
were placed in
20 mL microcalorimeter vials and phage were added at 0.5% phage cocktail concentration
(that is 10
4
PFU
·
mL
−1
phage) when required. Sterility controls were performed using
uninoculated artificial urine. For the calorimetry measurements, vials were then sealed
and introduced into the microcalorimeter previously set at 37
◦
C and equilibrated at this
temperature for at least 2 days. After sample introduction and thermal equilibration, the
heat flow corresponding to the microbial metabolic heat production rate was measured
until it returned to the baseline.
For conventional microbiological measurements, 36 additional vials per condition
were prepared in calorimetric vials and incubated at 37
◦
C in a separated incubator. CFU
and optical density (OD) were measured over time by sacrificing 2 vials for each treatment.
CFU were determined by serial dilution and plating on Luria agar. OD was read on an LLG-
uniSPEC 4 spectrophotometer at 600 nm. Samples for phage determination were filtered
using a 0.22
µ
m pore size syringe filter and kept in the fridge at 4
◦
C for no more than 24 h.
After storage, the phage concentration was determined by serial dilution and plating using
the double-layer agar technique with Luria agar. The experiment was repeated twice.
2.5. Phage Cocktail Efficacy
Both strains were tested against the polyvalent phage preparation described
above [34,35]
.
For the test, sterile 4 mL calorimetry glass ampoules were filled with 3 mL of inoculated
solution (urine or artificial urine). Phages were added at 1% (v/v-30
µ
L/vial) and 5%
(v/v-150
µ
L/vial) of original phage cocktail. Untreated samples (no phage added) were
used as controls and sterile medium as negative control. All samples were prepared in
quadruplicates, except sterility controls, which were performed in triplicates.
The ampoules were then sealed using metal lid with a silicon rubber seal and intro-
duced into the microcalorimeter previously set at 37
◦
C and equilibrated at this temperature
for at least 2 days. After sample introduction and thermal equilibration, the heat flow
corresponding to the microbial metabolic heat production rate was recorded for at least
96 h or until it returned to the baseline.
2.6. Combination with Trimethoprim/Sulfamethoxazole
In the case of P. mirabilis, growth was strongly reduced but not completely sup-
pressed (see Result Section for details). For P. mirabilis, we investigated the combination
of the phage cocktail with Trimethoprim/Sulfamethoxazole (TMP/SMX) (Nopil
®
forte,
Mepha Schweiz AG, Basel, Switzerland). A diluted overnight culture was added with
1% (v/v-30 µL/vial
) of phages in combination with increasing concentrations of TMP/SMX.
The following solutions were prepared: 1% phages + 50% minimal inhibitory concentra-
Microorganisms 2022,10, 845 4 of 13
tion (MIC) TMP/SMX (4 mg
·
L
−1
), 1% phages + 25% MIC TMP/SMX (2 mg
·
L
−1
), and fi-
nally,
1% phages + 12.5% MIC
TMP/SMX (1 mg
·
L
−1
). TMP/SMX (50% MIC) only, phages
(
1% v/v
) only, and untreated cultures were used as controls. Uninoculated medium served
as sterility control. All samples were prepared in quadruplicates except sterility controls
that were done in triplicates.
2.7. Data Analysis
The raw microcalorimetric data were translated into usable microbiological data
as growth rate (
µ
), lag phase duration (
λ
), and maximum growth (i.e., maximum heat
produced-Q). To determine these parameters the integration of the heat flow data was
performed, and the resulting heat curve was used as a proxy for the growth curve. The
heat curve was fitted with the Gompertz growth model and the growth parameters were
calculated. The data extraction and conversion to a CSV file was accomplished with TAM
Assistant (TA Instruments, New Castle, DE, USA). All the remaining calculations and curve
fittings were performed with the R version 3.6.3 [36] and the grofit package [37].
3. Results
3.1. Comparison of Isothermal Microcalorimetry and Conventional Microbiology
For E. coli cultures in artificial urine without the addition of the phage cocktail. the
metabolic activity (i.e., the heat flow) rapidly rose and slowly decreased. When integrating
the heat flow to obtain the heat over time curve (comparable to a growth curve), the growth
pattern showed an s-shaped curved typically expected from bacterial growth. The heat
over time curve for untreated E. coli fits well with the CFU and the OD data obtained from
the parallel samples (Figure 1).
When the phage cocktail is added to E. coli in artificial urine, the metabolic activity
rapidly decreases and returns to very low values (close to baseline) within 4 h. This matches
well with the CFU counts performed that also show a strong decrease within the first hours.
After 5 and 6 h the E. coli count was below detection limit (i.e., <100 CFU
·
mL
−1
). On
the contrary, the OD remained at stable value but showed only a minimal decrease over
time (Figure 1). In artificial urine, this might be due to the formation of struvite or other
phosphate minerals that may nucleate on cell or cell debris, even in organisms that do not
produce urease [
38
–
41
]. Indeed, microscopic investigation showed crystals in the artificial
urine after culture with or without phages, thus, in such media, OD might overestimate the
cell number and hinder the detection of lysis.
Microorganisms 2022,10, 845 5 of 13
Microorganisms 2022, 10, x FOR PEER REVIEW 5 of 12
Figure 1. Growth of E. coli in artificial urine and with the addition of 0.5% v/v of phage cocktail (“E.
coli-Proteus” bacteriophage solution, NPO Microgen, Russia). (Top) Heat flow data (raw data);
(Middle) Heat over time data with matching CFU and OD600 data for the culture without phages.
(Bottom) Heat over time data with matching CFU, PFU, and OD600 data. For CFU, PFU, and OD,
points represent the raw measured data and the lines the fitted logistic model using those data. The
red arrows indicate the heat flow decrease and the matching CFU decrease. * indicates value detec-
tion limit (below 100 CFU·mL−1). CFU = Colony Forming Unit, OD600 = Optical Density at 600 nm,
PFU = Plaque Forming Units
3.2. Monitoring of Phage Cocktail Antibacterial Activity
Both bacterial strains were able to grow in both urine and artificial urine, as indicated
by the detection of metabolic heat production (Figures 2 and 3). Increasing concentrations
of phage cocktail resulted in a strong reduction in the growth of both pathogens, marked
Figure 1.
Growth of E. coli in artificial urine and with the addition of 0.5% v/v of phage cocktail
(“E. coli-Proteus” bacteriophage solution, NPO Microgen, Russia). (
Top
) Heat flow data (raw data);
(
Middle
) Heat over time data with matching CFU and OD
600
data for the culture without phages.
(
Bottom
) Heat over time data with matching CFU, PFU, and OD
600
data. For CFU, PFU, and OD,
points represent the raw measured data and the lines the fitted logistic model using those data. The
red arrows indicate the heat flow decrease and the matching CFU decrease. * indicates value detection
limit (below 100 CFU
·
mL
−1
). CFU = Colony Forming Unit, OD
600
= Optical Density at 600 nm,
PFU = Plaque Forming Units.
3.2. Monitoring of Phage Cocktail Antibacterial Activity
Both bacterial strains were able to grow in both urine and artificial urine, as indicated
by the detection of metabolic heat production (Figures 2and 3). Increasing concentrations of
phage cocktail resulted in a strong reduction in the growth of both pathogens, marked by a
rapid decrease in metabolic activity. The decrease in growth was dose-dependent, as shown
Microorganisms 2022,10, 845 6 of 13
by the variations in growth parameters (
µ
,
λ
, Q). In both cases, a decrease in the growth
rate
µ
and the heat produced Q (Tables 1and 2) was observed demonstrating that bacterial
growth was negatively affected by the phages. At higher phage concentration (
5% v/v
), a
marked increase in the lag phase was detected only for P. mirabilis. An increase in the lag
was also visible for E. coli but only in artificial urine and in the context of regrowth (see later
sections). The heat production of P. mirabilis was much higher compared to E. coli; this was
expected due to the presence of active urease (urea hydrolysis is an exothermic process with
a
∆
H of 119.2 kJ
·
mol
−1
). One main difference between the two pathogen was that
E. coli
activity was rapidly brought to baseline where no metabolic activity could be measured
anymore following the addition of phages. However, regrowth was observed in artificial
urine at several time points, meaning that some E. coli survived (note that the detection limit
of the instrument used is ca 30,000 E. coli
·
mL
−1
). This regrowth pattern was also observed
in previous studies (especially at lower initial phage concentration) with
E. coli
and other
pathogens [
30
,
42
]. On the contrary, although the phage preparation strongly decreased
the activity of P. mirabilis (>50% decrease in growth rate in urine
and >80%
decrease in
growth rate in artificial urine compared to the controls), the initial growth was not fully
suppressed (Figure 2).
Table 1.
Growth parameters of E. coli and P. mirabilis in urine when exposed to increasing amount of
phage cocktail (“E. coli-Proteus” bacteriophage solution, NPO Microgen, Nizhny Novgorod, Russia).
µ
= growth rate,
λ
= lag phase duration, Q = maximum growth (i.e., maximum heat produced),
TTP = Time-to peak.
E. coli
Sample µ(h−1)λ(h) Q (J) TTP (h)
Control 0.18 ±0.01 2.08 ±0.18 1.93 ±0.11 5.67 ±0.12
1% 0.08 ±0.01 0.00 ±0.16 0.89 ±0.16 1.50 ±0.00
5% 0.04 ±0.01 0.00 ±2.69 0.57 ±0.02 1.52 ±0.04
P. mirabilis
Sample µ(h−1)λ(h) Q (J) TTP (h)
Control 1.28 ±0.05 0.0 ±0.3 28.89 ±0.3 4.00 ±0.3
1% 0.66 ±0.00 0.0 ±0.0 21.09 ±0.1 3.41 ±0.0
5% 0.59 ±0.08 4.6 ±0.4 19.6 ±1.6 12.5 ±0.2
Table 2.
Growth parameters of E. coli and P. mirabilis in artificial urine when exposed to increasing
amount of phage cocktail (“E. coli-Proteus” bacteriophage solution, NPO Microgen, Nizhny Novgorod,
Russia).
µ
= growth rate,
λ
= lag phase duration, Q = maximum growth (i.e., maximum heat produced).
E. coli First Peak Second Peak Third Peak
Sample µ(h−1)λ(h) Q (J) µ(h−1)λ(h) Q (J) µ(h−1)λ(h) Q (J)
Control 0.29 ±0.01 0.9 ±0.2 7.0 ±0.2
1% 0.04 ±0.02 17.8 ±3.6 0.7 ±0.5 0.09 ±0.04 63.4 ±12.1 1.6 ±1.0
5% 0.02 ±0.01 18.3 ±2.6 0.2 ±0.1 0.03 ±0.03 48.3 ±32.1 0.5 ±0.3 0.03 60.5 0.6
P. mirabilis
Sample µ(h−1)λ(h) Q (J) µ(h−1)λ(h) Q (J) µ(h−1)λ(h) Q (J)
Control 0.62 ±0.04 1.1 ±0.5 5.3 ±0.5
1% 0.11 ±0.01 4.0 ±1.1 2.9 ±0.2
5% 0.04 ±0.02 0.0 ±7.1 1.3 ±0.0
Microorganisms 2022,10, 845 7 of 13
Microorganisms 2022, 10, x FOR PEER REVIEW 7 of 12
Figure 2. Representative curve showing the heat flow and integrated heat over time of E. coli ex-
posed to increasing concentration of phage cocktail. (Left) Heat flow pattern in sterile filtered urine
(top) and associated heat over time curve (bottom). (Right) Heat flow pattern in sterile artificial
urine (top) and associated heat over time curve (bottom). Sterile controls are indicated in grey.
Figure 3. Representative curve showing the heat flow and integrated heat over time of P. mirabilis
exposed to increasing concentration of phage cocktail. (Left) Heat flow pattern in sterile filtered
urine (top) and associated heat over time curve (bottom). (Right) Heat flow pattern in sterile artifi-
cial urine (top) and associated heat over time curve (bottom). Sterile controls are indicated in grey.
Figure 2.
Representative curve showing the heat flow and integrated heat over time of E. coli
exposed to increasing concentration of phage cocktail. (
Left
) Heat flow pattern in sterile filtered
urine (top) and
associated heat over time curve (
bottom
). (
Right
) Heat flow pattern in sterile artificial
urine (top) and associated heat over time curve (bottom). Sterile controls are indicated in grey.
Microorganisms 2022, 10, x FOR PEER REVIEW 7 of 12
Figure 2. Representative curve showing the heat flow and integrated heat over time of E. coli ex-
posed to increasing concentration of phage cocktail. (Left) Heat flow pattern in sterile filtered urine
(top) and associated heat over time curve (bottom). (Right) Heat flow pattern in sterile artificial
urine (top) and associated heat over time curve (bottom). Sterile controls are indicated in grey.
Figure 3. Representative curve showing the heat flow and integrated heat over time of P. mirabilis
exposed to increasing concentration of phage cocktail. (Left) Heat flow pattern in sterile filtered
urine (top) and associated heat over time curve (bottom). (Right) Heat flow pattern in sterile artifi-
cial urine (top) and associated heat over time curve (bottom). Sterile controls are indicated in grey.
Figure 3.
Representative curve showing the heat flow and integrated heat over time of P. mirabilis
exposed to increasing concentration of phage cocktail. (
Left
) Heat flow pattern in sterile filtered
urine (top) and
associated heat over time curve (
bottom
). (
Right
) Heat flow pattern in sterile artificial
urine (top) and associated heat over time curve (bottom). Sterile controls are indicated in grey.
Microorganisms 2022,10, 845 8 of 13
The specific case of E. coli in artificial urine is of interest (Figure 2). Under these
conditions, several peaks can be seen in the heat flow pattern after the initial return to
the baseline induced by the phages. This indicates that E. coli metabolism was detected
and suggests a regrowth of this pathogen. The pattern suggests a possible prey–predator
interaction where some surviving E. coli might regrow until being re-infected by the phages.
3.3. Combination of Phages and Trimethoprim/Sulfamethoxazole
Neither phage solution alone nor TMP/SMX at 50% MIC succeeded in fully suppress-
ing P. mirabilis growth, although a strong reduction in growth was observed (Figure 3).
The heat flow pattern of the samples treated only with TMP/SMX generated a sharp peak
with a shape comparable to the control but with decreased overall activity (i.e., heat flow).
On the contrary, phage cocktail concentrations greater than 1% resulted in a smaller and
broader peak reaching its maximum activity with a delay of roughly 10 h compared to
the controls. When both phage and TMP/SMX were added together, the initial metabolic
activity peak was rather low. In addition, the activity followed a decrease comparable to
an exponential decay until activity returned to the baseline. In this case, no late peak or
regrowth was observed, thus suggesting that the combination of TMP/SMX and phage
succeeded in completely eradicating the pathogen. The growth parameters are shown in
Table 3and Figure 4.
Microorganisms 2022, 10, x FOR PEER REVIEW 8 of 12
The specific case of E. coli in artificial urine is of interest (Figure 2). Under these con-
ditions, several peaks can be seen in the heat flow pattern after the initial return to the
baseline induced by the phages. This indicates that E. coli metabolism was detected and
suggests a regrowth of this pathogen. The pattern suggests a possible prey–predator in-
teraction where some surviving E. coli might regrow until being re-infected by the phages.
3.3. Combination of Phages and Trimethoprim/Sulfamethoxazole
Neither phage solution alone nor TMP/SMX at 50% MIC succeeded in fully suppress-
ing P. mirabilis growth, although a strong reduction in growth was observed (Figure 3).
The heat flow pattern of the samples treated only with TMP/SMX generated a sharp peak
with a shape comparable to the control but with decreased overall activity (i.e., heat flow).
On the contrary, phage cocktail concentrations greater than 1% resulted in a smaller and
broader peak reaching its maximum activity with a delay of roughly 10 h compared to the
controls. When both phage and TMP/SMX were added together, the initial metabolic ac-
tivity peak was rather low. In addition, the activity followed a decrease comparable to an
exponential decay until activity returned to the baseline. In this case, no late peak or re-
growth was observed, thus suggesting that the combination of TMP/SMX and phage suc-
ceeded in completely eradicating the pathogen. The growth parameters are shown in Ta-
ble 3 and Figure 4.
Figure 4. Representative curve showing the growth of P. mirabilis in sterile artificial urine when
exposed to increasing 50% MIC TMP/SMX and/or 1% v/v phage cocktail (“E. coli-Proteus” bacterio-
phage solution, NPO Microgen, Nizhny Novgorod, Russia). (Top) Heat flow data; (bottom) inte-
grated heat over time data (proxy for the growth curve). Sterile controls are indicated in grey.
TMP/SMX = trimethoprim/sulfamethoxazole, MIC = minimal inhibitory concentration.
Figure 4.
Representative curve showing the growth of P. mirabilis in sterile artificial urine when
exposed to increasing 50% MIC TMP/SMX and/or 1% v/v phage cocktail (“E. coli-Proteus”
bacteriophage solution, NPO Microgen, Nizhny Novgorod, Russia). (
Top
) Heat flow data;
(
bottom
) integrated heat over time data (proxy for the growth curve). Sterile controls are indicated in
grey. TMP/SMX = trimethoprim/sulfamethoxazole, MIC = minimal inhibitory concentration.
Microorganisms 2022,10, 845 9 of 13
Table 3.
Growth parameters of P. mirabilis in artificial urine when exposed to 1% (v/v) phage
cocktail (“E. coli-Proteus” bacteriophage solution, NPO Microgen, Nizhny Novgorod Russia) and
added with increasing concentrations of TMP/SMX.
µ
= growth rate,
λ
= lag phase duration,
Q = maximum growth (i.e., maximum heat produced), TTP = Time to peak, TMP/SMX = trimetho-
prim/sulfamethoxazole, MIC = minimal inhibitory concentration.
P. mirabilis + TMP/SMX
Sample µ(h−1)λ(h) Q (J) TTP (h)
Control 0.54 ±0.01 0.0 ±0.0 5.2 ±0.1 3.2 ±0.1
1% phage 0.11 ±0.01 4.0 ±1.1 2.9 ±0.2 14.2 ±0.1
1% phage + 50% MIC 0.01 ±0.01 0.0 ±13.6 1.0 ±0.7 0.6 ±0.4
1% phage +25% MIC 0.01 ±0.01 0.0 ±11.9 0.8 ±0.7 0.7 ±0.5
TMP/SMX: 50% MIC 0.31 ±0.01 0.0 ±0.2 3.6 ±0.1 1.3 ±0.0
Blanks 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00
4. Discussion
UTIs are often a reason to prescribe antibiotics and inevitably lead to an increase in
antimicrobial resistance and the spread of multi-resistant bacteria [
5
,
10
]. Phage therapy
used as an antibacterial treatment is thus of medical and economic interest [
16
,
20
,
43
].
Although standardized and marketed products are not available in most of the world, the
current interest in phages suggest that such therapy may become more widespread. In this
study, using isothermal microcalorimetry, we showed that the commercial phage product
used induced bacterial lysis for E. coli and P. mirabilis in both types of urine. Our data are
consistent with previous measures performed with E. coli B and the T3 and T4 phages in
LB medium [
30
,
31
]. In addition, we also showed that the phage preparation used was
compatible with additional TMP/SMX treatment. Isothermal microcalorimetry being a
very sensitive real-time technique, it was also possible to see the speed of action and the
potential microbial regrowth. Therefore, using isothermal microcalorimetry, one rapidly
assesses which phage preparation could be of interest to treat a patient.
“Phage cocktails” containing multiple phage types have been used to counteract
the narrow host range of single phages and to control the growth of E. coli infectious
strains [
6
,
44
]. However, focusing on personalized phage therapy is possible as well. Such a
strategy relies on the isolation of the bacterial pathogen from the infection site and further
testing it against phages from a biobank. After this screening step, selected phages could be
transmitted to the patient to rapidly treat the UTI [
23
,
45
]. Considering the different studies
on rapid drug susceptibility testing using isothermal microcalorimetry [
46
] and previous
studies with phages and viruses [
29
–
32
], we believe that providing a phagogram or a
combined phagogram/antibiogram within 5 to 8 h is possible using patient urine directly,
as UTI are often characterized by a high pathogen load with only rare polymicrobial
infections. This would be of great interest, as it is expected to prevent the risk of mutant
appearance [
47
–
49
]. In order to further speed up the process, isothermal microcalorimetry
could be combined with a time-of-flight mass spectrometer MALDI-TOF [
46
,
50
,
51
] for
preliminary identification of the pathogen and thus narrowing the range of phage product
to be tested. Recent studies have shown that in acute UTI the MALDI-TOF identification
could be performed with urine directly as well.
For isothermal microcalorimetry to become useful in this context, a defined and
reproducible procedure is required. To increase reproducibility, artificial urine seems to be
promising; still its use needs to be further discussed. Indeed, the addition of phages in urine
resulted in a decrease in microbial activity that ultimately returned to baseline indicating a
complete suppression of growth. However, in artificial urine two to three peaks could be
detected after the initial return to baseline. In human urine, the presence of antibacterial
enzymes/peptides, for example,
α
- and
β
-defensins or cathelicidin, probably contributes
Microorganisms 2022,10, 845 10 of 13
to inhibit the growth of E. coli and potentially P. mirabilis [
52
]. In addition, in artificial
urine, the different peaks interpreted as regrowth of the pathogens seems to indicate a
prey–predator behavior of the phages–host system [
53
]. Thus, there is a possibility that
phage efficacy might be slightly underestimated in artificial urine. In real world scenarios,
it is expected that if the growth rate is sufficiently reduced the surviving pathogens (if any)
will ultimately be flushed out of the bladder by urination. Therefore, we assume that such
approach would be safe for the patients.
Finally, we must recognize the limitation of the present study. Isothermal microcalorime-
ters do not allow shaking thus potentially limiting the contact between phages and host
cells especially at low phage concentration. This might also have allowed for some re-
growth observed. In addition, isothermal microcalorimetry measures active metabolism
(i.e., active bacteria) but only provides an indirect picture of the phage activity as phage are
using the microbial enzymatic machinery to replicate. However, considering the amount
of work required for assessing bacteria and phage concentrations over time isothermal
microcalorimetry remains a valuable proxy that strongly decreases the workload compared
to CFU and PFU counts, OD or metabolic assays. Similarly, this study has focused on two
strains only, thus making any generalization of the results too preliminary. Therefore, fur-
ther studies should include more clinically relevant strains obtained from several patients.
Further improvement could also include the optimization of the artificial urine composition.
Indeed, artificial urine can probably be added with vitamin supplements to further improve
the growth of uropathogens such as E. coli (in our study, P. mirabilis grew very well in
artificial urine) and make the assay even faster. Finally, with respect to P. mirabilis, we must
note that this pathogen is more commonly encountered in catheter-associated UTIs, where
it often forms biofilms on catheters. Therefore, future studies should include antibiofilm
assessment of the phage products. Similar assessment is possible and was performed for
single phage already [30].
5. Conclusions
Isothermal microcalorimetry has proven to be a valuable technique when investigating
the efficacy of phage preparation against various uropathogens
in vitro
. We foresee that
isothermal microcalorimetry could be used to obtain rapid phagograms to choose the best
phage or phage cocktail in the case of a urinary tract infection. The ease of measurement
and the low workload combined to fast result delivery makes this technique appealing for
future clinical studies. Alternatively, for the same reasons, isothermal microcalorimetry
could also be used as a quality control tool for phage preparations, thus decreasing the
workload for QA/QC control of such products in industrial settings.
Author Contributions:
Conceptualization, O.B. and G.B.; methodology, O.B.; formal analysis, A.P.S.,
M.M., A.E.G., T.L. and O.B.; original draft preparation, A.P.S., M.M., A.E.G., T.L., L.L., O.B. and G.B.
All authors have read and agreed to the published version of the manuscript.
Funding:
This research received no external funding. O.B. and the calorimetry lab at the University
of Basel are supported by the Merian Iselin Stiftung.
Institutional Review Board Statement: Not applicable.
Data Availability Statement:
Data presented in this study are available on request from the corre-
sponding author.
Acknowledgments:
The authors wish to thank Shawna McCallin (Department of Neuro-Urology,
Balgrist University Hospital, University of Zürich, Zürich, Switzerland) for her advice and the great
help in the revision of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Microorganisms 2022,10, 845 11 of 13
References
1.
Bonkat, G.; Braissant, O.; Widmer, A.F.; Frei, R.; Rieken, M.; Wyler, S.; Gasser, T.C.; Wirz, D.; Daniels, A.U.; Bachmann, A. Rapid
detection of urinary tract pathogens using microcalorimetry: Principle, technique and first results. BJU Int.
2012
,110, 892–897.
[CrossRef] [PubMed]
2.
McLellan, L.K.; Hunstad, D.A. Urinary Tract Infection: Pathogenesis and Outlook. Trends Mol. Med.
2016
,22, 946–957. [CrossRef]
[PubMed]
3. Nicolle, L.E. Urinary Tract Infection. Crit. Care Clin. 2013,29, 699–715. [CrossRef] [PubMed]
4.
Mathai, D.; Jones, R.N.; Pfaller, M.A. Epidemiology and frequency of resistance among pathogens causing urinary tract infections
in 1510 hospitalized patients: A report from the SENTRY Antimicrobial Surveillance Program (North America). Diagn. Microbiol.
Infect. Dis. 2001,40, 129–136. [CrossRef]
5.
Grabe, M.; Bjerklund-Johansen, T.E.; Botto, H.; European Association of Urology. Guidelines on Urological Infections 2013. Eur.
Assoc. Urol. 2013. Available online: https://uroweb.org/guidelines/urological-infections (accessed on 3 April 2022).
6.
Bolocan, A.S.; Callanan, J.; Forde, A.; Ross, P.; Hill, C. Phage therapy targeting Escherichia coli-a story with no end? FEMS
Microbiol. Lett. 2016,363, fnw256. [CrossRef] [PubMed]
7.
Cabal, A.; García-Castillo, M.; Cantón, R.; Gortázar, C.; Domínguez, L.; Álvarez, J. Prevalence of Escherichia coli virulence genes
in patients with diarrhea and a subpopulation of healthy volunteers in Madrid, Spain. Front. Microbiol.
2016
,7, 641. [CrossRef]
[PubMed]
8. Schaffer, J.N.; Pearson, M.M. Proteus mirabilis and Urinary Tract Infections. Microbiol. Spectr. 2015,3, 383–433. [CrossRef]
9.
Gomaa, S.; Serry, F.; Abdellatif, H.; Abbas, H. Elimination of multidrug-resistant Proteus mirabilis biofilms using bacteriophages.
Arch. Virol. 2019,164, 2265–2275. [CrossRef]
10.
Pormohammad, A.; Nasiri, M.J.; Azimi, T. Prevalence of antibiotic resistance in escherichia coli strains simultaneously isolated
from humans, animals, food, and the environment: A systematic review and meta-analysis. Infect. Drug Resist.
2019
,12, 1181–1197.
[CrossRef]
11.
Fasugba, O.; Gardner, A.; Mitchell, B.G.; Mnatzaganian, G. Ciprofloxacin resistance in community- and hospital-acquired
Escherichia coli urinary tract infections: A systematic review and meta-analysis of observational studies. BMC Infect. Dis.
2015
,
15, 1–16. [CrossRef]
12.
Kot, B.; Gru ˙
zewska, A.; Szweda, P.; Wicha, J.; Parulska, U. Antibiotic resistance of uropathogens isolated from patients hospitalized
in district hospital in central Poland in 2020. Antibiotics 2021,10, 447. [CrossRef] [PubMed]
13.
Adamus-Bialek, W.; Zajac, E.; Parniewski, P.; Kaca, W. Comparison of antibiotic resistance patterns in collections of Escherichia
coli and Proteus mirabilis uropathogenic strains. Mol. Biol. Rep. 2013,40, 3429–3435. [CrossRef] [PubMed]
14.
Gajdács, M.; Bátori, Z.; Burián, K. Interplay between phenotypic resistance to relevant antibiotics in gram-negative urinary
pathogens: A data-driven analysis of 10 years’ worth of antibiogram data. Life 2021,11, 1059. [CrossRef] [PubMed]
15.
Gajdács, M.; Urbán, E. Comparative epidemiology and resistance trends of Proteae in urinary tract infections of inpatients and
outpatients: A 10-year retrospective study. Antibiotics 2019,8, 91. [CrossRef] [PubMed]
16. Summers, W.C. Bacteriophage therapy. Annu. Rev. Microbiol. 2001,55, 437–451. [CrossRef]
17. Deresinski, S. Bacteriophage therapy: Exploiting smaller fleas. Clin. Infect. Dis. 2009,48, 1096–1101. [CrossRef]
18.
Monk, A.B.; Rees, C.D.; Barrow, P.; Hagens, S.; Harper, D.R. Bacteriophage applications: Where are we now? Lett. Appl. Microbiol.
2010,51, 363–369. [CrossRef]
19.
Gill, J.; Hyman, P. Phage Choice, Isolation, and Preparation for Phage Therapy. Curr. Pharm. Biotechnol.
2010
,11, 2–14. [CrossRef]
20.
Kutter, E.; De Vos, D.; Gvasalia, G.; Alavidze, Z.; Gogokhia, L.; Kuhl, S.; Abedon, S. Phage Therapy in Clinical Practice: Treatment
of Human Infections. Curr. Pharm. Biotechnol. 2010,11, 69–86. [CrossRef]
21.
Leitner, L.; Ujmajuridze, A.; Chanishvili, N.; Goderdzishvili, M.; Chkonia, I.; Rigvava, S.; Chkhotua, A.; Changashvili, G.;
McCallin, S.; Schneider, M.P.; et al. Intravesical Bacteriophages for Treating Urinary Tract Infections in Patients Undergoing
Transurethral Resection of the Prostate: A Randomised, Placebo-Controlled, Double-Blind Clinical Trial. Lancet Infect. Dis.
2021
,
21, 427–436. [CrossRef]
22.
Leitner, L.; Kessler, T.M.; Klumpp, J. Bacteriophages: A Panacea in Neuro-Urology? Eur. Urol. Focus
2020
,6, 518–521. [CrossRef]
[PubMed]
23.
Pirnay, J.P.; Kutter, E. Bacteriophages: It’s a medicine, Jim, but not as we know it. Lancet Infect. Dis.
2021
,21, 309–311. [CrossRef]
24.
Buckling, A.; Rainey, P.B. Antagonistic coevolution between a bacterium and a bacteriophage. Proc. R. Soc. B Biol. Sci.
2002
,269,
931–936. [CrossRef] [PubMed]
25. Clokie, M.R.J.; Kropinski, A.M. Bacteriophages: Methods and Protocols; Humana: Louisville, KY, USA, 2009.
26.
Braissant, O.; Wirz, D.; Göpfert, B.; Daniels, A.U. Use of isothermal microcalorimetry to monitor microbial activities. FEMS
Microbiol. Lett. 2010,303, 1–8. [CrossRef]
27. Wadsö, I. Isothermal microcalorimetry in applied biology. Thermochim. Acta 2002,394, 305–311. [CrossRef]
28.
Maskow, T.; Harms, H. Real time insights into bioprocesses using calorimetry: State of the art and potential. Eng. Life Sci.
2006
,6,
266–277. [CrossRef]
Microorganisms 2022,10, 845 12 of 13
29.
Tkhilaishvili, T.; Wang, L.; Tavanti, A.; Trampuz, A.; Di Luca, M. Antibacterial efficacy of two commercially available bacterio-
phage formulations, staphylococcal bacteriophage and PYO bacteriophage, against methicillin-resistant Staphylococcus aureus:
Prevention and eradication of biofilm formation and control of a systemic infection of Galleria mellonella larvae. Front. Microbiol.
2020,11, 110. [CrossRef] [PubMed]
30.
Tkhilaishvili, T.; Di Luca, M.; Abbandonato, G.; Maiolo, E.M.; Klatt, A.B.; Reuter, M.; Möncke-Buchner, E.; Trampuz, A. Real-time
assessment of bacteriophage T3-derived antimicrobial activity against planktonic and biofilm-embedded Escherichia coli by
isothermal microcalorimetry. Res. Microbiol. 2018,169, 515–521. [CrossRef]
31.
Guosheng, L.; Yi, L.; Xiangdong, C.; Peng, L.; Ping, S.; Songsheng, Q. Study on interaction between T4 phage and Escherichia coli
B by microcalorimetric method. J. Virol. Methods 2003,112, 137–143. [CrossRef]
32.
Morais, F.M.; Buchholz, F.; Hartmann, T.; Lerchner, J.; Neu, T.R.; Kiesel, B.; Harms, H.; Maskow, T. Chip-calorimetric monitoring
of biofilm eradication with bacteriophages reveals an unexpected infection-related heat profile. J. Therm. Anal. Calorim.
2014
,115,
2203–2210. [CrossRef]
33. Tan, A.M.; Lu, J.H. Microcalorimetric study of antiviral effect of drug. J. Biochem. Biophys. Methods 1999,38, 225–228. [CrossRef]
34.
McCallin, S.; Sarker, S.A.; Sultana, S.; Oechslin, F.; Brüssow, H. Metagenome analysis of Russian and Georgian Pyophage cocktails
and a placebo-controlled safety trial of single phage versus phage cocktail in healthy Staphylococcus aureus carriers. Environ.
Microbiol. 2018,20, 3278–3293. [CrossRef] [PubMed]
35.
Sarker, S.A.; Sultana, S.; Reuteler, G.; Moine, D.; Descombes, P.; Charton, F.; Bourdin, G.; McCallin, S.; Ngom-Bru, C.; Neville,
T.; et al. Oral Phage Therapy of Acute Bacterial Diarrhea with Two Coliphage Preparations: A Randomized Trial in Children
From Bangladesh. EBioMedicine 2016,4, 124–137. [CrossRef] [PubMed]
36.
R Development Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing:
Vienna, Austria, 2016; ISBN 3-900051-07-0. [CrossRef]
37.
Kahm, M.; Hasenbrink, G.; Lichtenberg-Fraté, H.; Ludwig, J.; Kschischo, M. Grofit: Fitting Biological Growth Curves with R.J.
Stat. Softw. 2010,33, 1–21. [CrossRef]
38.
Cosmidis, J.; Benzerara, K.; Guyot, F.; Skouri-Panet, F.; Duprat, E.; Férard, C.; Guigner, J.M.; Babonneau, F.; Coelho, C. Calcium-
phosphate biomineralization induced by alkaline phosphatase activity in Escherichia coli: Localization, kinetics, and potential
signatures in the fossil record. Front. Earth Sci. 2015,3, 84. [CrossRef]
39.
Cohen, M.S.; Davis, C.P.; Czerwinski, E.W.; Warren, M.M. Calcium phosphate crystal formation in Escherichia coli from human
urine: An in vitro study. J. Urol. 1982,127, 184–185. [CrossRef]
40.
Edin-Liljegren, A.; Hedelin, H.H.; Grenabo, L.; Pettersson, S.; McLean, R.J.C. Impact of Escherichia coli on urine citrate and
urease-induced crystallization. Scanning Microsc. 1995,9, 24.
41.
Rivadeneyra, M.A.; Gutierrez-Calderón, A.; Rivadeneyra, A.M.; Ramos-Cormenzana, A. A study of struvite precipitation and
urease activity in bacteria isolated from patients with urinary infections and their possible involvement in the formation of renal
calculi. Urol. Int. 1999,63, 188–192. [CrossRef]
42.
Gelman, D.; Yerushalmy, O.; Alkalay-Oren, S.; Rakov, C.; Ben-Porat, S.; Khalifa, L.; Adler, K.; Abdalrhman, M.; Coppenhagen-
Glazer, S.; Aslam, S.; et al. Clinical Phage Microbiology: A suggested framework and recommendations for the in-vitro matching
steps of phage therapy. Lancet Microbe 2021,2, e555–e563. [CrossRef]
43. McCallin, S.; Brüssow, H. Phage therapy: An alternative or adjunct to antibiotics? Emerg. Top. Life Sci. 2017,1, 105–116.
44.
Chadha, P.; Katare, O.P.; Chhibber, S.
In vivo
efficacy of single phage versus phage cocktail in resolving burn wound infection in
BALB/c mice. Microb. Pathog. 2016,99, 68–77. [CrossRef] [PubMed]
45.
Friman, V.P.; Soanes-Brown, D.; Sierocinski, P.; Molin, S.; Johansen, H.K.; Merabishvili, M.; Pirnay, J.P.; De Vos, D.; Buckling,
A. Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with
Pseudomonas aeruginosa cystic fibrosis bacterial isolates. J. Evol. Biol. 2016,29, 188–198. [CrossRef] [PubMed]
46.
Braissant, O.; Müller, G.; Egli, A.; Widmer, A.; Frei, R.; Halla, A.; Wirz, D.; Gasser, T.C.; Bachmann, A.; Wagenlehner, F.; et al. Seven
hours to adequate antimicrobial therapy in urosepsis using isothermal microcalorimetry. J. Clin. Microbiol.
2014
,52, 624–626.
[CrossRef] [PubMed]
47.
Górski, A.; Borysowski, J.; Mi˛edzybrodzki, R. Phage therapy: Towards a successful clinical trial. Antibiotics
2020
,9, 827. [CrossRef]
[PubMed]
48.
Jeon, G.; Ahn, J. Assessment of phage-mediated inhibition of Salmonella Typhimurium treated with sublethal concentrations of
ceftriaxone and ciprofloxacin. FEMS Microbiol. Lett. 2020,367, fnaa159. [CrossRef]
49.
Luscher, A.; Simonin, J.; Falconnet, L.; Valot, B.; Hocquet, D.; Chanson, M.; Resch, G.; Köhler, T.; van Delden, C. Combined
Bacteriophage and Antibiotic Treatment Prevents Pseudomonas aeruginosa Infection of Wild Type and cftr- Epithelial Cells. Front.
Microbiol. 2020,11, 1947. [CrossRef] [PubMed]
50.
Bonkat, G.; Braissant, O.; Rieken, M.; Solokhina, A.; Widmer, A.F.; Frei, R.; van der Merwe, A.; Wyler, S.; Gasser, T.C.; Bachmann,
A. Standardization of isothermal microcalorimetry in urinary tract infection detection by using artificial urine. World J. Urol.
2013
,
31, 553–557. [CrossRef] [PubMed]
51.
Ferreira, L.; Sánchez-Juanes, F.; González-Ávila, M.; Cembrero-Fuciños, D.; Herrero-Hernández, A.; González-Buitrago, J.M.;
Muñoz-Bellido, J.L. Direct identification of urinary tract pathogens from urine samples by matrix-assisted laser desorption
ionization-time of flight mass spectrometry. J. Clin. Microbiol. 2010,48, 2110–2115. [CrossRef]
Microorganisms 2022,10, 845 13 of 13
52.
Nielsen, K.L.; Dynesen, P.; Larsen, P.; Jakobsen, L.; Andersen, P.S.; Frimodt-Møller, N. Role of urinary cathelicidin LL-37 and
human β-defensin 1 in uncomplicated escherichia coli urinary tract infections. Infect. Immun. 2014,82, 1572–1578. [CrossRef]
53. Stern, A.; Sorek, R. The phage-host arms race: Shaping the evolution of microbes. BioEssays 2011,33, 43–51. [CrossRef]
Available via license: CC BY 4.0
Content may be subject to copyright.
