International Journal of Medical Microbiology 310 (2020) 151413

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International Journal of Medical Microbiology

journal homepage: www.elsevier.com/locate/ijmm

Development of a novel MALDI-TOF MS-based bile solubility test for rapid T

discrimination of Streptococcus pneumoniae⋆
Evgeny A. Idelevicha, Andreas Schlattmanna, Markus Kostrzewab, Karsten Beckera,c,*
a
Institute of Medical Microbiology, University Hospital Münster, Münster, Germany
b
Bruker Daltonik GmbH, Bremen, Germany
c
Friedrich Loeffler-Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany

ARTICLE INFO ABSTRACT

Keywords: Differentiation of Streptococcus pneumoniae from other Streptococcus mitis group streptococci (SMGS) remains
Streptococcus pneumoniae challenging despite the introduction of matrix-assisted laser desorption/ionization time-of-flight mass spectro-
MALDI-TOF mass spectrometry metry (MALDI-TOF MS). While the bile solubility test (BST) provides most reliable discrimination of pneumo-
Bile solubility test cocci, its practical implementation is limited by subjective visual interpretation and frequent inconclusive re-
Rapid test
sults. We aimed to develop a rapid confirmation BST based on direct-on-target MALDI-TOF MS assay. After
establishment of optimal test conditions, test performance was evaluated on 36 consecutive clinical SMGS iso-
lates. Colony material was suspended and pipetted onto a MALDI target. After drying, sodium deoxycholate in
different concentrations (2%, 5%, and 10 %) was added. Incubation for 30 min (at room temperature or 35 °C)
was followed by liquid removal and spot washing. After adding 70 % formic acid, spots were overlaid with
matrix and measured (MALDI Biotyper smart, Bruker). The absence of microbial spectra (Biotyper score <1.7) in
samples with sodium deoxycholate indicated efficient removal of bacterial biomass due to bile solubility, thus,
identifying pneumococci. In contrast, scores ≥1.7 were interpreted as lack of bile solubility and confirmation as
viridans streptococci other than S. pneumoniae. Highest test accuracy was achieved applying 5% sodium deox-
ycholate at 35 °C and 10 % sodium deoxycholate at room temperature. These test conditions provided 100 %
sensitivity and 100 % specificity for discrimination of S. pneumoniae. The developed MALDI-TOF MS-based BST
is an easy-to-perform assay with minimum hands-on time and objective readout. The promising results of this
proof-of-principle study warrant confirmation with large collections of epidemiologically diverse strains.

1. Introduction
Discrimination of Streptococcus pneumoniae from other viridans
streptococci is clinically important due to their different pathogenic
potential (Spellerberg et al., 2019). However, reliable differentiation of
pneumococci from other S. mitis group streptococci (SMGS) remains
challenging despite the introduction of matrix-assisted laser deso-
rption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)
(Burckhardt et al., 2017; Ikryannikova et al., 2013; Slotved et al., 2017;
Spellerberg et al., 2019; van Prehn et al., 2016; Varghese et al., 2017).
Using combination of different methods, the bile solubility test (BST)
has been recommended for accurate species identification within SMGS
(Satzke et al., 2013; Slotved et al., 2017). In comparison studies, BST
provided most reliable discrimination of pneumococci from other SMGS
(Arbique et al., 2004; Richter et al., 2008; Wessels et al., 2012; Yahiaoui
et al., 2016). However, after initial method description by Neufeld in
1900 (Neufeld, 1900), difficulties with performing BST and result in-
terpretation have been reported (Greey, 1939; Howden, 1979) and a
number of test improvements and modifications have been suggested
(Greey, 1939; Hawn and Beebe, 1965; Howden, 1979). Nowadays,
there is still no unified standardized protocol on conducting this test
and recommended testing conditions vary among different sources
(Atlas and Snyder, 2019; Spellerberg et al., 2019). Subjective visual
interpretation, frequent inconclusive reading results, and limited re-
producibility in different laboratories have been pointed out (Slotved
et al., 2017; Yahiaoui et al., 2016).
Here, we developed an easy-to-perform BST based on direct-on-
target MALDI-TOF MS assay (Idelevich et al., 2018) for rapid and

Abbreviations: SMGS, Streptococcus mitis group streptococci; BST, bile solubility test; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry; DOT-MGA, direct-on-target microdroplet growth assay; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration
⋆
A part of this work was presented at the ASM Microbe Congress, 20th–24th June 2019, San Francisco, CA, USA [SUNDAY-CPHM-LB-2]
⁎
Corresponding author at: Friedrich Loeffler-Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.
E-mail address: karsten.becker@med.uni-greifswald.de (K. Becker).

https://doi.org/10.1016/j.ijmm.2020.151413
Received 2 October 2019; Received in revised form 13 January 2020; Accepted 12 February 2020
1438-4221/ © 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
E.A. Idelevich, et al.
objective confirmation of pneumococcal discrimination. We hypothe-
sized that lysed pneumococcal biomass would be efficiently removed
from the spot with liquid removal after lysis by sodium deoxycholate, a
commonly used bile salt reagent (Atlas and Snyder, 2019; Downie et al.,
1931; Spellerberg et al., 2019), resulting in the absence of a MALDI-
TOF microbial spectrum and, thus, identifying as S. pneumoniae. In
contrast, other SMGS would show characteristic microbial spectra de-
spite the treatment with sodium deoxycholate due to their bile in-
solubility. Efforts were made to design the assay as a rapid and easy-to-
perform test with minimum hands-on time, which can conveniently be
integrated into laboratory workflows.
2. Materials and methods
2.1. Bacterial strains
In preliminary experiments, four SMGS reference strains (S. pneu-
moniae ATCC 49619, S. mitis DSM12643, S. oralis DSM20627, and S.
pseudopneumoniae DSM18670) were used to establish optimal test
conditions.
Consecutive clinical blood culture SMGS isolates (n = 37) from
routine diagnostics of the Institute of Medical Microbiology (University
Hospital Münster) were used as challenge set for the evaluation of test
performance. Only one isolate per patient was eligible.
Species identification was confirmed by standard and molecular
methods. The methods of the bile solubility tube test and the bile so-
lubility test using direct application of the reagent to a colony on an
agar plate are described in the Supplementary text S1. Biochemical
identification was performed by the Vitek 2 instrument using the GP
card (REF 21342, bioMérieux, Marcy l’Etoile, France) in accordance
with the manufacturer’s instructions. Streptococcal isolates were con-
firmed by sodA gene sequencing. To distinguish between S. pneumoniae
and S. pseudopneumoniae, rpoB gene sequencing was performed.
Autolysin (lytA) and pneumolysin (ply) were detected using endpoint
PCR. Primer sequences and PCR amplification protocols are given in the
Supplementary text S2. Only isolates that could be unambiguously
identified as a species within the S. mitis group using NCBI’s Basic Local
Alignment Search Tool (Zhang et al., 2000) were included in this study.
2.2. MALDI-TOF MS-based bile solubility test
2.2.1. Investigation of optimal test conditions
Using four reference strains, multiple test conditions were in-
vestigated. For each strain, all test variants were studied simultaneously
on the same day from the same overnight culture on an agar plate.
Tested conditions included different processing methods for the in-
oculation of MALDI target, different concentrations of sodium deox-
ycholate, different incubation temperatures, different incubation times
as well as implementation or no implementation of a washing step
(Tables S2-S5). Each test condition was tested in triplicate, i.e. applied
to three spots of the MALDI target, and the median result was taken for
data analysis.
Bacteria were placed onto a spot of the disposable MALDI target
(MBT Biotargets-96, Bruker, Germany) and mixed with sodium deox-
ycholate solution in deionized water using six different processing
methods:
(i) Small amount of colony material was transferred and smeared onto
a spot using a toothpick. Subsequently, 6 μl sodium deoxycholate
were added.
(ii) 3 μl of a 4 McFarland suspension in water was pipetted onto a spot.
Subsequently, 3 μl sodium deoxycholate were added by pipetting.
(iii) 1 μl of a 4 McFarland suspension in water was pipetted onto a spot.
After the droplet has dried, 6 μl sodium deoxycholate were added
by pipetting.
(iv) The processing corresponded to the method (ii), but 30 μl of a 4
2
International Journal of Medical Microbiology 310 (2020) 151413
McFarland suspension in water and 30 μl sodium deoxycholate
were first mixed in a microtitre plate. Subsequently, 6 μl of the
mixture were pipetted onto a spot.
(v) A 1-μl loopful of colony material was suspended in 50 μl water in a
microtube (1.5 ml Eppendorf Safe-Lock Tube, Eppendorf,
Hamburg, Germany) and 3 μl of this suspension were pipetted onto
a spot. Subsequently, 3 μl sodium deoxycholate were added by
pipetting.
(vi) A 1-μl loopful of colony material was suspended in 50 μl water in a
microtube (1.5 ml Eppendorf Safe-Lock Tube, Eppendorf) and 1 μl
of this suspension was pipetted onto a spot. After the droplet had
dried, 6 μl sodium deoxycholate were added by pipetting.
With all processing methods, sodium deoxycholate solution in
deionized water was added in concentrations 2%, 5% or 10 %, as well
as water without sodium deoxycholate as control. Inoculated MALDI
targets were incubated for 30 min or for 3 h in a plastic box filled with 4
ml water (MALDI target transport box, Bruker) to avoid evaporation.
Incubation was accomplished at 35 °C as well as at room temperature.
Subsequently, the liquid was removed from spots by contact with ab-
sorptive paper strips (37 × 100 mm; GE Healthcare GmbH, Freiburg,
Germany) and the spots were washed by adding 6 μl water for 5 min
and again removal of liquid by absorption. For comparison, this
washing step was omitted in an additional set-up. Finally, 1 μl of 70 %
formic acid was added to the spot, overlaid with α-cyano-4-hydro-
xycinnamic acid (HCCA) containing internal standard for DOT-MGA
(Idelevich et al., 2018), and measured with the MALDI Biotyper smart
instrument (Bruker) using device settings as previously applied for
DOT-MGA (Idelevich et al., 2018). Bacterial test standard (Bruker) was
spotted onto each target and calibration performed. The acquired
spectra were evaluated by the MALDI Biotyper 4.1 software (Bruker),
which is usually used for standard microbial identification.
Test validity was defined as the appearance of characteristic mi-
crobial MALDI-TOF MS spectra with MALDI Biotyper score of ≥1.7
when water was used as control instead of sodium deoxycholate. The
absence of microbial spectrum (score <1.7) in samples with sodium
deoxycholate signified efficient removal of bacterial proteins from the
spot with the removal of liquid. Therefore, these score values were
interpreted as bile solubility and, hence, identification as S. pneumoniae.
Score values ≥1.7 achieved in samples with sodium deoxycholate were
interpreted as lack of bile solubility and confirmation as viridans
streptococci other than S. pneumoniae.
2.2.2. Evaluation of assay performance
Using a challenge set of consecutive clinical SMGS isolates, test
accuracy was evaluated. In this phase, test conditions were im-
plemented that demonstrated best performance during the preceding
investigation of optimal test conditions. When no pronounced differ-
ence in test performance was seen for a particular variable, e.g. con-
centration of sodium deoxycholate, all variants were again tested
during this investigation of test accuracy. This resulted in the final
experimental set-up (Fig. 1). As with the processing method “vi” in the
investigation of optimal test conditions, 1-μl loopful of colony material
was suspended in 50 μl water in a microtube and 1 μl of this suspension
was pipetted onto a spot. After the droplet has dried, 6 μl of sodium
deoxycholate in concentration 2%, 5% or 10 % (or 6 μl water as a
control) were added by pipetting. Inoculated targets were incubated for
30 min at room temperature or at 35 °C. Subsequently, the liquid was
removed from spots by contact with absorptive paper strips and the
spots were washed by adding 6 μl water for 5 min followed again by
removal of liquid. Thereafter, 1 μl of 70 % formic acid was added to the
spot, overlaid with HCCA matrix and measured with the MALDI Bio-
typer smart (Bruker) (Fig. 1).
As with the previous study part, all test variants were set-up si-
multaneously on the same day from the same overnight culture on an
agar plate. All tests were performed in triplicate, i.e. applied to three
E.A. Idelevich, et al.
Fig. 1. Workflow of the MALDI-TOF MS-based bile solubility test. The figure depicts testing conditions that enabled optimal assay performance.
spots of the MALDI target, and the median result was taken for data
analysis. Bacterial test standard (Bruker) was spotted on each target and
calibration was performed.
Determination of sodium deoxycholate minimum inhibitory con-
centration (MIC) and minimum bactericidal concentration (MBC) was
performed as given in Supplemental text S3.
3. Results
3.1. Standard identification methods
The initial collection comprised 14 S. pneumoniae and 23 non-S.
pneumoniae isolates (reported as S. mitis (n = 4), S. oralis (n = 6) and S.
mitis/oralis (n = 13)), according to the identification in routine diag-
nostics (Table S6). One isolate identified as S. mitis/oralis by routine
diagnostics, was identified as Streptococcus vestibularis by sodA se-
quencing. As this species does not belong to S. mitis group (Spellerberg
et al., 2019), this isolate was excluded from the study. Taken molecular
identification as reference method, the tested collection consisted of 14
S. pneumoniae and 22 non-S. pneumoniae isolates (12 S. mitis, 9 S. oralis
and 1 S. australis) (Table S6).
3.2. Minimum inhibitory concentration and minimum bactericidal
concentration
MIC and MBC values of sodium deoxycholate for the SMGS re-
ference strains were equal (Table S1). Vancomycin MICs determined for
S. pneumoniae ATCC 49619 for QC purpose, were in QC range.
3.3. Investigation of optimal test conditions
Characteristic microbial MALDI-TOF MS spectra with MALDI
Biotyper score (Bruker) of ≥1.7 were generated with all of four
streptococcal reference strains when water as control was used instead
of sodium deoxycholate (Fig. 2).
Results with reference strains demonstrated that the wash step is
indispensable (Tables S2-S5). Further, processing method “vi” for in-
oculation of MALDI targets showed more accurate results than other
3
International Journal of Medical Microbiology 310 (2020) 151413
methods (Tables S2-S5). Incubation time 30 min resulted in higher
accuracy than incubation for 3 h (Tables S2-S5). No unambiguous dif-
ference in test performance was observed for different concentrations of
sodium deoxycholate as well as for different incubation temperatures
(Tables S2-S5).
3.4. Evaluation of assay performance
Based on the preceding experiments with reference strains, test
conditions that resulted in higher accuracy were implemented for the
assay and its evaluation on clinical isolates comprising processing
method “vi”, the wash step, and incubation time 30 min. As no obvious
decision could be made based only on testing of reference strains re-
garding optimal concentration of sodium deoxycholate and optimal
incubation temperature, testing of clinical isolates included comparison
of these conditions (Table 1).
Characteristic microbial MALDI-TOF MS spectra with MALDI
Biotyper score (Bruker) of ≥1.7 were generated with water control
without sodium deoxycholate for all streptococcal strains at both in-
cubation temperatures tested, thus confirming test validity in 100 % of
isolates. The results for assay accuracy are presented in Table 1. Sodium
deoxycholate concentration of 5% in combination with incubation at 35
°C (sensitivity 100 %, specificity 100 %) as well as sodium deoxycholate
concentration of 10 % in combination with incubation at room tem-
perature (sensitivity 100 %, specificity 100 %) provided highest accu-
racy values.
4. Discussion
The introduction of MALDI-TOF MS into clinical microbiology has
considerably improved routine diagnostics and the majority of
European microbiology laboratories are now using MALDI-TOF MS for
identification of cultures (Idelevich et al., 2019a). For practical and cost
considerations, it would be advantageous to run necessary additional
diagnostic tests on the same MALDI-TOF MS instrument. Indeed, a
MALDI-TOF MS-based direct-on-target microdroplet growth assay
(DOT-MGA) was recently developed as a rapid universal antimicrobial
susceptibility testing method (Idelevich et al., 2018; Neonakis and
E.A. Idelevich, et al. International Journal of Medical Microbiology 310 (2020) 151413

Fig. 2. MALDI-TOF mass spectra generated with MALDI-TOF MS-based bile solubility test for S. pneumoniae and non-S. pneumoniae reference strains. Baseline
subtraction and smoothing of spectra were performed with flexAnalysis 4.0 (Bruker). Y-axis (intensity) of each reference strain’s spectrum with sodium deoxycholate
is scaled corresponding to the spectrum of the same reference strain’s control with water.

Table 1 concentrations. This is in accordance with the data of Mellroth et al.

Accuracy of the MALDI-TOF MS-based bile solubility test for rapid dis- who observed effective killing after deoxycholate treatment of the S.
crimination of Streptococcus pneumoniae from other mitis group streptococci, n pneumoniae T4ΔlytA mutant without lytic phenotype (Mellroth et al.,
= 36. 2012). In contrast, lytic action of sodium deoxycholate is highly specific
Sodium deoxycholate Room temperature 35 °C for S. pneumoniae due to the presence of autolysin LytA (Richter et al.,
concentration 2008; Yahiaoui et al., 2016; Mellroth et al., 2012) – the property on
Sensitivity Specificity Sensitivity Specificity which BST is based.
2% 50.0 % 100 % 28.6 % 100 %
Processing method “vi” not only had the best accuracy (Tables S2-
5% 92.9 % 100 % 100 % 100 % S5), but also was one of the most convenient and practical methods to
10 % 100 % 100 % 100 % 86.4 % inoculate the MALDI target. It appears that the rest of sodium deox-
ycholate remaining on a spot after liquid removal may disturb MALDI-
TOF MS measurement. Comparative testing of reference strains de-
Spandidos, 2019). MALDI-TOF MS-based DOT-MGA was also suggested monstrated that the wash step was essential (Tables S2–S5), remarkably
as optochin susceptibility testing method for differentiation of S. improving spectra quality. It can be assumed that water removal within
pneumoniae from other SMGS (Idelevich et al., 2019b), since conven- this wash step can occur much earlier than after 5 min without com-
tional MALDI-TOF identification of these bacteria remains challenging promising washing efficiency and spectra quality. Further optimization
(Slotved et al., 2017; Spellerberg et al., 2019; Varghese et al., 2017; studies should investigate this issue. Also, reproducibility experiments
Burckhardt et al., 2017; Ikryannikova et al., 2013; van Prehn et al., should explore whether measurement of only one or two spots instead
2016). of three provides reliable results.
This study focused on the development of a MALDI-TOF MS-based The protocols for the standard bile solubility test are diverse and
bile solubility test for the reliable discrimination of pneumococci from lacking a uniform recommendation (Atlas and Snyder, 2019; Slotved
other SMGS. As sodium deoxycholate causes unspecific inhibition and et al., 2017; Spellerberg and Brandt, 2015). For the tube test, re-
killing of S. pneumoniae and other mitis group streptococci, growth- commended inoculum size varies from McFarland 0.5–1.0 to McFarland
based susceptibility testing methods, such as MALDI-TOF MS DOT-MGA 4 or “heavy suspension” (Atlas and Snyder, 2019; Slotved et al., 2017;
(Idelevich et al., 2019b, 2018), cannot directly be applied for differ- Spellerberg et al., 2019). On the one hand, initial inoculum of McFar-
entiation between these microorganisms based on bile action. MIC and land 0.5–1.0 appears not turbid enough to allow optimal visual eva-
MBC of sodium deoxycholate were similar for S. pneumoniae and other luation of decrease in turbidity over time, according to our experience.
SMGS (Table S1), indicating that sodium deoxycholate inhibits and kills On the other hand, preparation of a “heavy suspension” does not allow
both S. pneumoniae and other mitis group streptococci at similar

4
E.A. Idelevich, et al.
optimal standardization. We therefore decided to use McFarland 4 in-
oculum for the standard bile solubility test. According to different
sources, the recommended final concentration of sodium deoxycholate
in a tube test considerably varies, as does the recommended incubation
time from 10 min to 3 h (Atlas and Snyder, 2019; Slotved et al., 2017;
Spellerberg et al., 2019).
There was no pronounced difference in test accuracy between in-
cubation at room temperature or at 35 °C (Table 1), which is in line
with the observations of Neufeld (Neufeld, 1900). Later studies de-
monstrated that lysis takes place slightly more rapidly with a tem-
perature rise from 21 to 37 °C (Downie et al., 1931). Furthermore, it
appears reasonable to choose an incubation temperature of 35 °C for
our assay because it corresponds to current recommendations for
standard BSTs (Atlas and Snyder, 2019; Spellerberg et al., 2019).
Particular difficulties have previously been reported with dis-
crimination of S. pneumoniae and S. pseudopneumoniae (Wessels et al.,
2012). The biochemical system was not able to identify the S. pseu-
dopneumoniae reference strain using Vitek 2 GP card. While no clinical
isolates of S. pseudopneumoniae were among the clinical collection, our
MALDI-TOF MS-based test was able to discriminate the S. pseudopneu-
moniae reference strain as bile-insoluble. Other studies have similarly
reported that S. pseudopneumoniae is not soluble in bile (Arbique et al.,
2004; Keith et al., 2006; Wessels et al., 2012). Wessels et al. emphasized
that the development of an easily implementable assay able to differ-
entiate S. pseudopneumoniae would provide valuable insight into its
clinical importance (Wessels et al., 2012).
The developed MALDI-TOF MS bile solubility test is an easy-to-
perform confirmation assay with optimal integration into laboratory
workflow that requires minimum hands-on time and allows objective
readout. Standardization and automated software evaluation would
increase test performance. The promising results of our proof-of-prin-
ciple study warrant confirmation with large collections of epidemiolo-
gically diverse strains.
Acknowledgements
We are thankful to Damayanti Kaiser, Daniela Kuhn, Annkatrin Bibo
and Martina Schulte for excellent technical assistance.
This study was funded in part by grants from the German Federal
Ministry of Education and Research (BMBF) to EAI and KB (16GW0150)
and MK (16GW0149K).
E.A.I. and K.B. are inventors of pending patents, which are owned
by the University of Münster and licensed to Bruker. K.B. received
speaker and consultation honoraria from Becton Dickinson,
bioMérieux, Bruker Daltonik, Hain Lifescience, Roche Molecular
Systems and ThermoFisher. E.A.I. received a speaker honorarium from
Bruker. M.K. is employee of Bruker. AS has nothing to declare.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.ijmm.2020.151413.
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