Detection and Drug Susceptibility Testing of Neisseria gonorrhoeae Using Isothermal Microcalorimetry

Abstract

Background: Gonorrhea is a frequently encountered sexually transmitted disease that results in urethritis and can further lead to pelvic inflammatory disease, infertility, and possibly disseminated gonococcal infections. Thus, it must be diagnosed promptly and accurately. In addition, drug susceptibility testing should be performed rapidly as well. Unfortunately, Neisseria gonorrhoea is a fastidious microorganism that is difficult to grow and requires culturing in an opaque medium. Methods: Here, we used isothermal microcalorimetry (IMC) to monitor the growth and the antimicrobial susceptibility of N. gonorrhoea. Results: Using IMC, concentrations of N. gonorrhoea between 2000 and 1 CFU·mL⁻¹ were detected within 12 to 33 h. In addition, drug susceptibility could be monitored easily. Conclusions: The use of isothermal microcalorimetry provides an interesting and useful tool to detect and characterize fastidious microbes such as N. gonorrhoea that require media incompatible with optical detection conventionally used in many commercial systems.

Full text

microorganisms
Article
Detection and Drug Susceptibility Testing of Neisseria
gonorrhoeae Using Isothermal Microcalorimetry
Anabel E. Grütter 1, †, Tecla Lafranca 1,† , Aurelia Pahnita Sigg 1, Max Mariotti 1, Gernot Bonkat 2
and Olivier Braissant 1, *


Citation: Grütter, A.E.; Lafranca, T.;
Sigg, A.P.; Mariotti, M.; Bonkat, G.;
Braissant, O. Detection and Drug
Susceptibility Testing of Neisseria
gonorrhoeae Using Isothermal
Microcalorimetry. Microorganisms
2021,9, 2337. https://doi.org/
10.3390/microorganisms9112337
Academic Editor: Karim Fahmy
Received: 20 September 2021
Accepted: 8 November 2021
Published: 11 November 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 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/).
1
Department of Biomedical Engineering, University of Basel, Gewerbestrasse 14, 4123 Allschwil, Switzerland;
anabel.gruetter@stud.unibas.ch (A.E.G.); tecla.lafranca@stud.unibas.ch (T.L.);
aureliapahnita.sigg@stud.unibas.ch (A.P.S.); max.mariotti@stud.unibas.ch (M.M.)
2alta uro AG, Centralbahnplatz 6, 4051 Basel, Switzerland; Bonkat@alta-uro.com
*Correspondence: olivier.braissant@unibas.ch
† These authors contributed equally to this publication.
Abstract:
Background: Gonorrhea is a frequently encountered sexually transmitted disease that
results in urethritis and can further lead to pelvic inflammatory disease, infertility, and possibly
disseminated gonococcal infections. Thus, it must be diagnosed promptly and accurately. In addition,
drug susceptibility testing should be performed rapidly as well. Unfortunately, Neisseria gonorrhoea is
a fastidious microorganism that is difficult to grow and requires culturing in an opaque medium.
Methods: Here, we used isothermal microcalorimetry (IMC) to monitor the growth and the antimi-
crobial susceptibility of N. gonorrhoea. Results: Using IMC, concentrations of N. gonorrhoea between
2000 and 1 CFU
·
mL
−1
were detected within 12 to 33 h. In addition, drug susceptibility could be
monitored easily. Conclusions: The use of isothermal microcalorimetry provides an interesting and
useful tool to detect and characterize fastidious microbes such as N. gonorrhoea that require media
incompatible with optical detection conventionally used in many commercial systems.
Keywords: isothermal microcalorimetry; Neisseria gonorrhoea; antimicrobials
1. Introduction
Gonorrhea is the second most common bacterial sexually transmitted disease (STI)
worldwide. According to the World Health Organization (WHO), there were around 87
million new infections in 2016 [
1
,
2
]. The causative pathogen of Gonorrhea, Neisseria gonor-
rhoea (Ng), is a Gram-negative diplococcus that infects the urogenital tract as well as the
rectum and the pharynx [
3
–
5
]. Men with such a urogenital infection are often symptomatic,
presented as urethritis [
3
,
4
]. On the contrary, most women are asymptomatic. Symptoms
(if any) include urethritis or cervicitis and are often non-specific. Rectal and pharyngeal
Gonorrhea are present in both genders and remain mostly asymptomatic [
4
,
6
,
7
]. Due often
non-specific symptoms or even asymptomatic disease progressions, Gonorrhea can extend
into an ascending infection. Complications include pelvic inflammatory disease (PID),
ectopic pregnancy, infertility and possibly disseminated gonococcal infections (DGIs) [
8
,
9
].
Unfortunately, because many patients remain asymptomatic and the increase in sexually
risky behavior, the incidence continues to rise.
Furthermore, the time and effort required for the detection of N. gonorrhoea and the
determination of its antimicrobial susceptibility are high. For these reasons, an empirical an-
timicrobial therapy is often chosen as first-line treatment. This treatment consists of a single
dose of ceftriaxone in combination with azithromycin (see details in EAU guidelines [
10
]).
As with other bacteria, N. gonorrhoea is also able to develop resistance to various antibiotics,
including the first-line ones and many others [
11
]. Due to the emergence of resistance and
the consequent ineffectiveness of the antibiotics used, antibacterial susceptibility testing
becomes crucial to ensure effective and targeted therapy. Minimizing inadequate treatment
Microorganisms 2021,9, 2337. https://doi.org/10.3390/microorganisms9112337 https://www.mdpi.com/journal/microorganisms

Microorganisms 2021,9, 2337 2 of 9
also reduces the emergence of new resistance [
12
]. Because of the growing number of cases,
the inefficiency of the currently applied diagnostic methods and the emergence of new
resistant strains, diagnostic methods that allow faster and targeted treatment are urgently
needed.
Currently, diagnosis of N. gonorrhoea relies mostly on a urethral swab followed by
microscopy, microbial culture or nucleic acid amplification test (NAAT) such as PCR or
LAMP. In high income settings the nucleic acid amplification test (NAAT) is the most
used test to diagnose Gonorrhea, whereas it is rarely used in low-income settings, due
to the high associated costs [
13
–
15
]. NAAT has a high specificity and sensitivity both
in urethral gonococcal infections and extragenital gonococcal infections. However, it is
recommended that a positive test result from an extragenital infection should be confirmed
with an alternative method due to the possibility of cross reactivity with commensal
or atypical Neisseria species [
16
–
19
]. In addition, NAAT may be superior to culture in
some respects, for example in sensitivity, ease, and speed of performance, as shown
in many studies. However, it lacks the possibility of direct antimicrobial susceptibility
testing [
20
–
22
]. Therefore, there is still the need for a new method, which can both detect
Neisseria and allows determination of antimicrobial resistance pattern in an easy and
practical manner.
In this context, we investigated the use of isothermal microcalorimetry (IMC) for
the detection and antimicrobial susceptibility testing of N. gonorrhoea. For microbiolo-
gists, N. gonorrhoea is considered to be a fastidious microorganism that requires specific
growth medium (such as chocolate agar, GC medium) that are solid and/or opaque due
to the presence of components such as agar (to make the medium solid), starch and
hemoglobin (making the medium cloudy or even opaque). Isothermal microcalorimetry
measures metabolic heat produced by active or replicating microorganisms in a given
medium [
23
,
24
]. Isothermal microcalorimetry (IMC) is a laboratory method for the mea-
surement and recording of real-time heat production rate (heatflow) at microwatt levels
(
µ
J/sec =
µ
W) during bacterial growth (i.e., heatflow curve) [
25
–
28
]. As only heat is mea-
sured, IMC does not require a transparent or liquid medium, making it suitable for the
detection of N. gonorrhoea in the medium. Previous studies have already shown promising
results in this field. For example, in 2012 a study using IMC demonstrated that this method
allows rapid detection of bacteriuria (in just 3.1 h), enables the pathogen to be targeted
through different heat flow patterns and that low colony counts (10
3
CFU/mL) are needed
for the detection. Bacterial strains of the most common urinary tract pathogens were used
in this study: Staphylococcus aureus,Enterococcus faecalis,Proteus mirabilis and Escherichia
coli [
28
]. Braissant and colleagues demonstrated that IMC could be used to find an ade-
quate antimicrobial therapy for urosepsis in just seven hours [
29
]. Regarding the drug
susceptibility, isothermal microcalorimetry has already been used for various species such
as MRSA [
30
], Aspergillus [
31
], Mycobacterium [
32
] and S. aureus and E. coli [
33
]. Finally,
the use of solid and opaque medium has already been shown to be possible with IMC,
especially in the context of tuberculosis diagnosis and drug susceptibility testing [
34
,
35
].
Given the reliability of these results, the aim of this study was to use this procedure in a
field that to our knowledge has not yet been explored, namely antimicrobial susceptibility
testing for N. gonorrhoea using IMC. Our aim was, therefore, to apply IMC to a bacterium
that has not attracted much attention until now and to provide a sensitive, rapid and
effective method of diagnosis and drug susceptibility testing that can ideally act as a means
of point-of-care testing (POCT) in the clinical world.
2. Materials and Methods
2.1. Organisms
The strains of N. gonorrhoea (ATCC 19424 and ATCC 43069) were obtained as dehy-
drated pellets (Microbiologics, St Cloud, MN, USA) and stored at 4
◦
C before use. The
pellets were rehydrated in sterile brain–heart infusion (BHI) medium previously auto-
claved at 121
◦
C for 15 min according to the manufacturer’s instructions. The pellets were

Microorganisms 2021,9, 2337 3 of 9
rehydrated 1 h before use. Pellets where homogenized using a grinding tool and through
repeated vortexing. Following homogenization, the solution was used as inoculum for the
different experiments described below. In addition, the inoculum bacterial concentration
was determined by plating on Columbia agar with added 5% sheep blood (Becton Dickin-
son (BD), Franklin Lakes, NJ, USA) after serial 10-fold dilutions. An additional plate was
prepared by streaking 10
µ
L of the undiluted inoculum on the same medium. All plates
were incubated 48 h at 37 ◦C and visually inspected or counted after this time.
2.2. Detection in Various Medium
To test the effects of different supplement addition on the growth and thus further
detection in urine or medium, supplements were added to the urine or the GC base medium
(Condalab, Madrid, Spain) according to Table 1. GC medium base and hemoglobin solution
were autoclaved separately at 121
◦
C for 15 min and mixed after cooling down. All other
components were obtained sterile and added aseptically to obtain the final medium. Urine
was obtained from voluntary healthy donors and was rapidly centrifuged to remove
sediments (if any). A final filtration through a 0.2
µ
m filter (stericup) was performed to
ensure sterility and clarity. After filtration, the sterile urine was used directly or stored at
4◦C until use if required. All additions to urine were made aseptically.
Table 1.
Medium composition tested to measure growth parameters of N. gonorrhoea and define the
best possible liquid medium.
No Supplement Urine Urine +
Hemoglobin (1% w/v)GC Medium GC Medium +
Hemoglobin (1% w/v)
Isovitale X 1% v/v 1% v/v 1% v/v 1% v/v *
Sheep blood ** 1% v/v 1% v/v 1% v/v 1% v/v
* denotes the composition of the ATCC 814 medium. ** defibrinated.
In addition, using the medium providing the best growth (i.e., ATCC 814 medium—see
results section for details) we also performed a dilution series to determine the detection
speed. Detection was considered positive when the signal passed above a threshold of
10 µW.
2.3. Drug Susceptibility Testing
Measurements performed in the previous section showed that GC medium (ATCC
medium no. 814) containing hemoglobin (1% w/v) and Isovitale X (1% v/v) was the most
effective medium for the cultivation of the N. gonorrhoea strains used (see results section for
details). We therefore determined the drug susceptibility in this medium. For this proof
of principle, we used ceftriaxone at the following concentrations: 4.17, 1.25, 0.42, 0.13 and
0.04
µ
g
·
L
−1
. Sterile controls and controls without antimicrobials were subjected to the
same conditions.
2.4. Calorimetry Procedure
All the samples were prepared in sterile 4 mL calorimetric glass ampoules. Then, 3 mL
of sample was placed in the ampoule, and the ampoule was sealed. The ampoule was
introduced into the calorimeter (TAM III, Waters/TA, New Castle, DE, USA) following the
two-step thermal equilibration procedure recommended by the manufacturer. After 1 h of
thermal equilibration, heat production rates (heatflow) were recorded until they returned
to baseline or for at least 96 h. At the end of the experiment, data were resampled to obtain
an effective sampling rate of 1 data point every 5 min and exported as a CSV file.
2.5. Statistical Analysis
All the calculations were performed in the statistics program R. The raw data (i.e.,
heatflow curves) were integrated to obtain heat over time curves. All further curve fitting was
performed under the assumption that the heat produced during growth is proportional to the

Microorganisms 2021,9, 2337 4 of 9
growth curve (i.e., that the cost of producing a bacterial cell remains constant) [
36
,
37
], and
using the Gompertz growth model the maximal growth rate (
µ
), the lag phase (
λ
), and the
maximum heat (Q
max
) were calculated from the heat over time curve [
37
–
39
]. Additionally,
heatflow above 10
µ
W was considered to be the threshold for positive detection, and the
corresponding time was recorded.
3. Results
3.1. Effect of Medium and Supplements
N. gonorrhoea ATCC 19424 was used to test for different growth in the different media.
The results are summarized in Table 2. This strain of N. gonorrhoea was not cultivable in
any urine-based medium. We assume that the concentration of necessary nutrients is too
low even with morning urine. This means the detection of N. gonorrhoea with IMC in urine
or artificial urine is compromised. However, using GC-based medium showed growth
in all conditions. The growth was improved by the addition of Isovitale X supplement
and hemoglobin to a lesser extent. The use of sheep blood did not improve growth and
even showed a decrease (however, the decrease was not significant). Out of the used
mediums and additions, the conventional growth medium (GC medium, ATCC medium
no. 814) with hemoglobin and Isovitale X supplement performed the best since it showed
the highest growth rate with a low lag phase compared to the other combinations. In this
medium the growth pattern shows a main peak with additional smaller peaks that are
found before the maximum activity is reached (see Figure 1). A possible explanation for
the multiple peaks could be the use of different nutrients or terminal electron acceptors by
N. gonorrhoea during its growth as previously described [24,27,40].
Table 2.
Growth parameters of N. gonorrhoea in the different media used. The results are the mean of four replicates and the
associated standard deviation.
Growth Medium Growth Rate
(h−1)
Lag Phase
(h)
Total Heat
(J)
TTP
(h)
GC 0.095 ±0.010 39.4 ±16.2 3.74 ±1.30 67.5 ±10.7
GC + Isovitale X 0.307 ±0.015 28.8 ±1.8 4.83 ±0.38 40.8 ±0.8
GC + Isovitale X + Blood 0.234 ±0.025 19.6 ±1.3 5.34 ±0.14 39.6 ±3.6
GC + hemoglobin 0.119 ±0.016 16.7 ±1.2 4.65 ±0.17 36.2 ±5.2
GC + hemoglobin + Isovitale X 0.322 ±0.025 17.3 ±0.7 5.26 ±0.12 29.4 ±1.0
GC + hemoglobin + Isovitale X + blood 0.305 ±0.016 19.3 ±1.0 5.47 ±0.08 28.9 ±2.4
Sterile GC medium 0.002 ±0.001 ND 0.12 ±0.05 ND
Urine 0.007 ±0.001 ND 0.28 ±0.08 ND
Urine + Isovitale X 0.012 ±0.001 ND 0.59 ±0.08 ND
Urine + Isovitale X + blood 0.011 ±0.003 ND 0.47 ±0.08 ND
Urine + hemoglobin 0.004 ±0.003 ND 0.60 ±0.71 ND
Urine + hemoglobin + Isovitale X 0.006 ±0.003 ND 0.60 ±0.51 ND
Urine + hemoglobin + Isovitale X + blood 0.004 ±0.001 ND 0.36 ±0.06 ND
Sterile filtered urine 0.004 ±0.001 ND 0.62 ±0.45 ND
ND: Not determined, TTP: Time to peak.
3.2. Time to Detection
To determine the time to detection, we used a 10
µ
W threshold corresponding to
ca. 3.3
·
10
5
CFU
·
mL
−1
(assuming a 2 pW per cell heat production rate [
41
,
42
]). The serial
dilution with the two selected strains of N. gonorrhoea led to measured detection times
between 12 and 33 h depending on the strain and bacterial concentration used (that were
of rather low concentration but comparable to those found in patient urine- Table 3). Only
one sample with very low CFU counts of the ATCC 43069 strain did not pass the threshold
of 10
µ
W (although it came close). This emphasizes that the differences between the strains

Microorganisms 2021,9, 2337 5 of 9
of the same organism should be taken into account. In addition, we assume that it is likely
that after sedimentation of the medium, only a micro-colony could develop, being trapped
in the sediment. Increasing growth would have probably required stirring or shaking that
is not possible with the used instrument.
Figure 1.
Growth of N. gonorrhoea ATCC 19424 (
A
,
B
) and ATCC 43069 (
C
,
D
) monitored using IMC. A: Heatflow of N.
gonorrhoea ATCC 19424 and insert showing details of the initial 25 h. B: Heat produced by of N. gonorrhoea ATCC 19424
over time showing a curve similar to a growth curve. C: Heatflow of N. gonorrhoea ATCC 19424. D: Heat produced by of N.
gonorrhoea ATCC 19424 over time showing a curve similar to a growth curve.
Table 3.
Table showing the detection time with respect to the number of cells in the medium. The
threshold for positive detection was set at 10
µ
W (10
µ
W is approximately 3.3
×
10
5
CFU
·
mL
−1
; see
main text).
ATCC 19424 ATCC 43069
CFU·mL−1Time to
Detection (h) CFU·mL−1Time to
Detection (h)
~2400 12.0 ±0.3 ~460 18.9 ±0.4
~240 14.6 ±0.2 ~46 26.0 ±0.4
~24 16.9 ±0.4 ~5 33.6 ±3.3
~2 21.9 ±3.2 ~0.5 ** ND *
0 ND 0 ND
Sterile controls ND Sterile controls ND
* Growth was detected, but the signal did not reach the threshold of 10
µ
W. ** At this concentration only one out
of the three replicates showed growth. See figure for details.
3.3. Drug Susceptibility Testing
When increasing concentrations of ceftriaxone were added up to 3
×
MIC, inhibition
of Neisseria growth was clearly visible (Figure 2). The growth rate decreased from 0.29 to
0.00 h
−1
at MIC, as the lag phase duration remained roughly similar for all the samples
between 29.9
±
1.5 and 36.1
±
1.5. The MIC measured for both strains is consistent with

Microorganisms 2021,9, 2337 6 of 9
literature values for these well characterized susceptible strains. Please note that at MIC
level the strain ATCC 19424 shows considerably delayed growth and was clearly visible
(although very slow) only after 100 h (data not shown). This shows that the methodology
previously developed for urosepsis using a single concentration of antimicrobial to rapidly
determine if a strain is susceptible or resistant is also applicable with N. gonorrhoea. How-
ever, due to the slow growth and fastidious nature of Neisseria species, it is likely that at
least 12 to 15 h could be needed for such a discrimination.
Figure 2.
Heatflow (
A
) and heat over time (
B
) during the growth of N. gonorrhoea ATCC 43069 exposed to increasing
concentrations of ceftriaxone.
4. Discussion
Overall, the use of the IMC is easier than culturing, and there is little preparation
time. There is also the possibility of measuring antimicrobial susceptibility and rapidly
discriminating between susceptible and resistant strains. Once an optimal medium has
been chosen, detection is rather simple. In the development of calorimetric methods for the
detection of this fastidious pathogens, other media such as Thayer–Martin agar or modified
chocolate agar could be of interest [
43
]. However, in this study we focused on the use of
liquid media as they are more suited for a practical use considering diagnostic applications
and calorimetry. Still, the above-mentioned agar media could be of interest in the study of
the formation of biofilm as these structures have been shown to form on epithelial cells
and cervical cells during Neisseria infections [
44
]. In addition, future studies should also
include the use of selective antimicrobial supplements for the direct isolation of Neisseria
species [43].
The detection of N. gonorrhoea using isothermal microcalorimetry is slower than with
NAAT, but it allows more evaluations once the IMC measurements are completed. Indeed,
since the sealed ampoule still contains almost undisturbed sample with higher amounts
of Neisseria, those bacteria can be recovered for additional identification or confirmation
using MALDI-TOF or serological testing, for example. This is not possible with many
NAAT-based techniques at this point but is still required as NAAT can be sensitive to other
non-pathogenic but closely related Neisseria species [16,17,19].
In addition, we must emphasize that this study is very preliminary work and that
many improvements are possible when considering the nature and composition of the
medium. Indeed, as with non-motile microbial cells, hemoglobin (provided as dried red
blood cells) and starch (insoluble) tend to sediment and are found at the bottom of the
vial after the experiment. Therefore, it is very likely that the conditions for the growth
of N. gonorrhoea are not homogenous throughout the vial and thus might not be optimal.
Previous studies have shown that the use of Percoll or Ficoll that increase the density of
the medium is a good solution to avoid such sedimentation, thus providing much more
homogenous conditions for growth [
45
]. This; however, was not in the scope of the present
study but will be considered and further investigated using similar media. There are also
many other supplements such as Vitox or Yeast autolysate that could be used to make the

Microorganisms 2021,9, 2337 7 of 9
growth of N. gonorrhoea and other Neisseria species faster. This is indeed of interest as IMC
has been used for a long time to optimize growth condition for industrial purposes but
also for human cell research [
46
–
48
]. Finally, we did not investigate selective supplements
for direct determination and isolation from urine samples or swabs. This will also become
of interest as the technique progresses.
With respect to antimicrobial susceptibility testing, our results show that with rather
low concentration susceptibility could be determined within 12 h. At this point isolation
would be needed before running a calorimetry measurement. As isolation takes 24 h and
testing takes another 12 h, results can be expected within 36 h. Overall this is still 12 h faster
than conventional methods [
49
,
50
]. Use of a higher initial inoculum is expected to speed
up the detection, and we estimate that with an inoculum of 10
6
CFU
·
mL
−1
the time to
results could be lowered to 28 h. This would be similar to drug susceptibility testing using
direct qPCR [
51
]. Finally, using additional preparation steps such as magnetic antibodies,
it might be possible to skip the initial isolation, as a high amount of Neisseria would likely
outgrow other microbes (if any) present after the purification step.
In conclusion, the use of isothermal microcalorimetry provides an interesting and
useful tool to investigate fastidious microbes that require media incompatible with op-
tical detection conventionally used in many commercial systems. Detection and drug
susceptibility testing of N. gonorrhoea was rapidly performed. Further optimization with
respect to the microbial culture condition will surely further decrease the time to detection
and the time required for drug susceptibility testing. Therefore, we expect isothermal mi-
crocalorimetry to become a valuable tool for diagnostics and research of so-called fastidious
microorganisms, including N. gonorrhoea.
Author Contributions:
Conceptualization, O.B. and G.B.; methodology, O.B.; formal analysis, A.E.G.,
T.L., O.B.; original draft preparation, A.E.G., T.L., A.P.S., M.M., 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. is supported by the Merian Iselin Stiftung.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement:
Data presented in this study are available on request from the corre-
sponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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