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ARTICLE
Determination of MICING: a new assay for assessing minimal
inhibitory concentration for invasive growth
J. Zupan &Z. Tomičić&P. Raspor
Received: 17 July 2014 /Accepted: 12 January 2015 /Published online: 27 January 2015
#Springer-Verlag Berlin Heidelberg 2015
Abstract Ourworkwasfocusedonanewassayfor
characterising clinically important yeast. This assay was de-
veloped due to the need for new diagnostic methods for
recognising potentially virulent strains of increasingly impor-
tant non-albicans yeast pathogens, such as Saccharomyces
cerevisiae and Candida glabrata. With the great diversity
among strains for virulence and virulence factors, identifica-
tion to the species level is not sufficient; therefore, testing for
specific virulent traits remains the best option. We show here
that the proposed assay uncovers the relationships between the
three most important yeast virulence traits in a single test: the
ability of a strain to invade solid medium, while resisting the
presence of an antimycotic and high temperature (37 °C). We
combined the quantitative agar invasion assay with classical
antimycotic susceptibility testing into a single assay. Similarly
to the minimal inhibitory concentration (MIC) value, we de-
fined the MICING (minimal inhibitory concentration of
antimycotic for invasive growth) as the concentration of an
antimycotic above which the yeast invasive growth is signif-
icantly repressed. In this study, we tested three of the most
common antimycotics: fluconazole, itraconazole and
amphotericin B. The response of yeast strains invasion was
characteristic of each antimycotic, indicating their mecha-
nisms of action. In addition to MICING, the assay provides
quantitative information about the superficial and invasive
growth, and also about the relative invasion, which helps in
identifying clinically important yeast, such as azole-resistant
and/or invasive strains of S. cerevisiae and C. glabrata.
Introduction
The efficiency of yeast opportunistic pathogens relies on the
relationships between the deficiency of the host defence and
the effectiveness of the yeast virulence factors. These viru-
lence factors include different mechanisms of antimycotic re-
sistance (e.g. antimycotic-related efflux pumps), extracellular
enzymes, the ability to grow at higher temperatures and/or
other, less understood, virulence traits, such as dimorphism
and invasive growth [1–3]. Typical diagnostics in treating
yeast infections include yeast identificationto the species level
and testing of the yeast susceptibility to the most common
antimycotics, while the other virulence traits are not usually
considered. Indeed, other tests are usually not needed when a
well-known pathogen is identified, such as Candida albicans,
for which treatment procedures are well standardised.
On the other hand, non-albicans opportunistic pathogens
are increasingly implicated in human infections [4], which
often complicates treatment due to misidentification of the
causative agent and due to the unexpected occurrence of re-
sistance to the prescribed antimycotics. Indeed, Saccharomy-
ces cerevisiae has been reported in numerous cases of
candidiasis-like infections [5–8], which often occurs in con-
nection with its probiotic relative Saccharomyces boulardii
(nom. nud.) [9–11]. Symptoms can be atypical and easily
ascribed to other pathogens. It has also been shown that
S. cerevisiae is highly related to Candida glabrata, which
J. Zupan (*)
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101,
1000 Ljubljana, Slovenia
e-mail: jure.zupan@fvz.upr.si
Z. Tomičić
Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara
1, 21000 Novi Sad, Serbia
e-mail: zoricateh@yahoo.com
J. Zupan :P. Raspor
Faculty of Health Sciences, University of Primorska, Polje 42,
6310 Izola, Slovenia
P. Ra s p o r
e-mail: peter.raspor@fvz.upr.si
Eur J Clin Microbiol Infect Dis (2015) 34:1023–1030
DOI 10.1007/s10096-015-2324-y
has been the second most prevalent Candida species over the
last decade for invasive candidiasis cases, and has drawn a lot
of attention due to high mortality rates [4,12–16].
In contrast to C. albicans, the virulence traits of these two
opportunistic pathogens, S. cerevisiae and C. glabrata, are not
well known; namely, we still cannot explain satisfactorily why
some S. cerevisiae strains are highly virulent in a murine mod-
el and others are not [17,18]. What is known is that (i) low
susceptibility to azole antimycotics is regularly observed
[19–21], (ii) many strains are tolerant to high temperatures,
such as 42 °C [17,22], and some strains of S. cerevisiae and
C. glabrata (iii) show a dimorphic switch and (iv) invade solid
media [22–24].
While susceptibility to antimycotics is regularly tested
using standard methods in either the Clinical and Laboratory
Standards Institute (CLSI) or the European Committee on
Antimicrobial Susceptibility Testing (EUCAST) format, these
three other known virulence traits, i.e. tolerance to high tem-
peratures, dimorphism and invasiveness, are not examined
routinely. Phenotypic switching, i.e. pseudohyphae formation,
is arguably indirectly involved in pathogenicity [25–27]andit
is difficult to quantify, while invasive growth, on the other
hand, appears to be a more easily measurable and directly
related virulence trait. In vivo models for the study of yeast
pathogenicity, such as experimental mice [4,18,28], are
scarce, and, therefore, in vitro methods need to be better
established. A method for the determination of yeast invasive
growth has not yet been implemented in diagnostic laborato-
ries, despite some major improvements for its conversion
from qualitative [29] to quantitative [23]. Consequently, we
believe that it is essential to push the development of new
methods that combine past experience with new possibilities
in this area, to provide the efficient and informative output that
is needed for both research and decision-making.
In the present study, we have striven to make progress
along these lines by combining a standard CLSI susceptibility
testing of antimycotics and a quantitative agar invasion assay,
to measure a new putative diagnostic property, the MICING:
the minimal inhibitory concentration of antimycotic for in-
vasive growth.
Materials and methods
Strains
Among the tested 76 S. cerevisiae and 57 C. glabrata clinical
and non-clinical strains that are deposited in the Collection of
Industrial Microorganisms (ZIM) at the Biotechnical Faculty
of the University of Ljubljana, Slovenia, ten S. cerevisiae and
two C. glabrata strains were selected for the development of
the MICING assay (Table 1). This selection was based upon
their ability to grow invasively, which was determined using a
quantitative agar-invasion assay. The invasion assay has been
described previously [23], except that the yeast extract pep-
tone dextrose medium was replaced by a medium containing
0.67 % yeast nitrogen base (YNB), 2 % glucose and 2 % agar
[30], as this stimulates invasive growth better due to its low
nitrogen content [22]. These 12 selected strains were used to
determine the MICING with three commonly used
antimycotics: the triazole antifungal agents, fluconazole and
itraconazole, and the polyene antifungal agent, amphotericin
B (all from Sigma, St. Louis, MO, USA). In addition, a
Table 1 Yeast strains used in the present study and their reported and tested virulence and invasion potential
Species/strain Origin Reported virulence or invasiveness Reference
Saccharomyces cerevisiae
ZIM 2266 Danish blue-veined cheese Highly invasive [23]
YJM 311 Man, bile duct Highly virulent/invasive [17,18,23]
YJM 128 Man with AIDS, lung Highly virulent/invasive [17,18,23]
YJM 309 Man, blood Highly virulent, moderately invasive [17,18,23]
YJM 273 Man, peritoneal fluid Moderately virulent/invasive [17,18,23]
YJM 308 Man, peritoneal fluid Moderately virulent/invasive [17,18,23]
YJM 222 Man Non-virulent/non-invasive [17,23]
ZIM 2261 Man, ascites fluid Highly invasive Present study
ZIM 2546 Man, sputum Moderately invasive Present study
ZIM 2547 Man, sputum Highly invasive Present study
Candida glabrata
ZIM 2344 Man, urine Highly invasive Present study
ZIM 2369 Man, bronchoalveolar lavage Moderately invasive Present study
Candida albicans
ATCC 10261 Man, nail, of case of paronychia Highly virulent/invasive [23,35]
1024 Eur J Clin Microbiol Infect Dis (2015) 34:1023–1030
C. albicans strain ATCC 10261 was used in the assay as a
positive control.
Preparation of test plates
The medium was prepared as follows: 4 % agar was
autoclaved, cooled to 70 °C and mixed with warmed, filter-
sterilised 2× YNB/glucose solution (pH 6.8) to the final con-
centration of 0.67 % YNB, 2 % glucose and 2 % agar. Imme-
diately before pouring the plates, an appropriate amount of
antimycotic stock solution was added to 5.5 ml of the cooled
medium mixture, followed by brief vortexing and then pipet-
ting of 4.5 ml of medium into 3.5-cm petri dishes (Golias,
Slovenia). The medium was left to solidify and dry for 24 h
at room temperature.
The selection of the antimycotics concentrations was based
on the minimal inhibitory concentrations (MICs) obtained in
the preliminarily microdilution modification assays by the ref-
erence method for broth dilution antifungal susceptibility test-
ing of yeast (CLSI standard M27-A3). From the range of ten
2-fold dilutions, five concentrations near the MIC value were
selected for each selected strain for the following MICING
assays. If the MICING differed significantly from the MIC,
the concentrations of antimycotics were increased or de-
creased accordingly and the assay was repeated.
Performing the assay
The medium was inoculated as described previously [23],
with minor modifications. Briefly, cells were transferred
from pre-grown colonies and spotted precisely in equally
distant triplicates onto the agar surface. With intention to
produce single colonies with the best reproducibility, we
aimed to inoculate as small an area as possible. This was
achieved by manual inoculation, which, in our original
quantitative agar invasion assay, proved to be rapid and
very useful, even in comparison with a micromanipulator
[23]. Namely, a sterile plastic loop was used to form a
biomass into a ‘needle’shape, by which the plates were
inoculated. After 4 days of incubation at 37 °C, greyscale
tiff images of the plates (tiff format) were generated using
the Gel Doc 2000 gel documentation system (Bio-Rad).
Colonies above the agar surface were then washed off
with a gentle stream of deionised water and the plates
were recorded again (Fig. 1).
The amount of surface and invasive colony biomass
was quantified densitometrically, the same as which
DNA or protein is usually quantified in electrophoretic
gels; the volume of the analysed spot is calculated as
the product of area and pixel intensity of the analysed
spot, which was, in our case, surface or invasive colony.
For this purpose, the Quantity One v.4.6.5 software
(Bio-Rad) was used. Transformation to exact units
(mm
3
) was performed as described previously using a
calibration curve [23]. Finally, the resulting parameters
obtained with this quantification were: surface colony
volume, invasive colony volume and relative invasion,
which is defined as the portion of the invasive colony
volume relative to the whole (invasive and surface) col-
ony volume [23].
Fig. 1 The minimal inhibitory concentration of antimycotic for invasive
growth (MICING) assay platform. Representative images using the Can-
dida glabrata strain ZIM 2344 on itraconazole plates, before and after
washing the colonies off the agar surface. For clearer presentation, only
the control plate (0 μg/ml itraconazole) is shown in full frame (a), with the
others showing only single representative colonies (b). The densitometric
simulations are shown beneath each colony, from which the colony vol-
umes were calculated (c)
Eur J Clin Microbiol Infect Dis (2015) 34:1023–1030 1025
Results
The selection of strains
To test the new assay and to reliably determine the
MICINGs, we aimed to select at least moderately invasive
strains. They were initially selected from among 76
S. cerevisiae strains (40 clinical and 36 non-clinical) and
57 C. glabrata strains (all clinical), according to their
previous data [22,23], and according to the additional
tests performed using the quantitative agar invasion assay.
In the full S. cerevisiae collection, the invasive strains
represented 30 % (12/40) of the clinical strains and only
8 % (3/36) of the non-clinical strains. Among the full
C. glabrata collection, 7 % (4/57) of the strains were
agar-invasive under selected conditions.
The MICING assay was performed for the nine selected
invasive S. cerevisiae strains, the one non-invasive
S. cerevisiae (control) strain, the two invasive C. glabrata
strains and the one C. albicans strain, which represented a
positive control (Table 1), with three of the most common
antimycotics used: fluconazole, itraconazole and
amphotericin B. Figure 1illustrates the setup of the MICING
assay.
The invasiogram: the MICING, invasive and superficial
colony volume, and relative invasion
The data from this new MICING method are shown in the form
of ‘invasiograms’(Figs. 2,3and 4). The minimal concentra-
tions of fluconazole, itraconazole and amphotericin B at which
the invasive growth of the tested strain was inhibited is named
the MICING. The MICING thresholds for the three
antimycotics were chosen to be equivalent to the CLSI standard
M27-A3, as follows: for fluconazole and itraconazole, the low-
est concentration that inhibited more than 50 % of the control
invasive growth (i.e. the growth on the plates with no
antimycotic added); for amphotericin B, the lowest concentra-
tion that completely inhibited invasive growth. As well as these
MICINGs, the MICs are also shown for comparison, which
were obtained using the microdilution modification of the ref-
erence method for broth dilution for antifungal susceptibility
testing of yeast (CLSI standard M27-A3). In the invasiograms,
only the invasive strains are included; the negative control
(strain YJM 222) was non-invasive in all of the tests.
From the invasiograms, it can be seen that the relative
invasion trends, which are indicated by the arrows in Figs. 2,
3and 4, are characteristic for each antimycotic. When com-
pared to the MICs, the MICINGs are generally higher, which
Fig. 2 Invasiogram for evaluation of the MICINGs for fluconazole of the
nine Saccharomyces cerevisiae and two Candida glabrata invasive
strains. The minimal inhibitory concentrations (MICs) are also shown
in brackets [as indicated; determined by Clinical and Laboratory
Standards Institute (CLSI) standard M27-A3]. The arrows indicate the
relative invasion trend typical for the fluconazole action. The error bars
represent standard deviations of three replicates
1026 Eur J Clin Microbiol Infect Dis (2015) 34:1023–1030
is most probably due to the longer incubation time (4 days for
MICING vs. 2 days for MIC). However, for both of the azole
antimycotics, the average increase of the MICING relative to
the MIC is 4-fold, while for amphotericin B, the MICINGs
are, on average, 53-fold of the MICs. The median of the MICs
for fluconazole, itraconazole and amphotericin B were 1, 0.75
and 0.125, respectively, while the median of the MICINGs for
fluconazole, itraconazole and amphotericin B were 6, 1.5 and
2, respectively.
Regarding the resistance on the three tested antimycotics
expressed with the MICING value, the following two strains
can be exposed: the strain C. glabrata ZIM 2369 had an
MICING of 64 μg/ml of fluconazole and 8 μg/ml of
itraconazole, while the clinical S. cerevisiae strain YJM 309
resisted amphotericin B with an MICING of 8 μg/ml, which is
very high for this antimycotic. The most sensitive strain in the
test, taking into account all three antimycotics, was the
C. albicans strain ATCC 10261.
Discussion
Compared with the CLSI standard method, the proposed
MICING method offers more informative data, with four main
parameters provided: the surface colony volume, the invasive
colony volume, the relative invasion and the MICING value
itself. The surface and invasive colony volumes describe the
absolute level of the strain’s invasiveness, while the relative
invasion could indicate an important environmental signal
which induces mechanisms for invasive growth into the solid
surface. As presented in this study, this signal can also be an
antimitotic. Practically speaking, a high relative invasion can,
thus, provide important information for a physician when an
antimycotic treatment is being chosen, while the MICING
value could have similar importance to the MIC value regard-
ing the resistance of strains to antimycotics.
We observed significant discrepancies between MICINGs
and MICs, which can be attributed to longer incubation or to
the differences in the medium matrix. This difference puts the
information from such standard liquid-medium-based tests in
a different perspective, as it is known that, especially for hy-
drophobic drugs like amphotericin B, drug performance is
highly dependent on tissue distribution [31]. Thus, this poses
the question of how the current tests reflect susceptibility and
response of yeast to antimycotics in vivo.
Our survey on invasiveness in S. cerevisiae showed that
there are more invasive strains among clinical strains when
compared to other natural isolates (results not yet published).
This correlation between S. cerevisiae invasiveness and
Fig. 3 Invasiogram for evaluation of the MICINGs for itraconazole of
the nine S. cerevisiae and two C. glabrata invasive strains. The MICs are
also shown in brackets (as indicated; determined by CLSI standard M27-
A3). The arrows indicate the relative invasion trend typical for the
itraconazole action. The error bars represent standard deviations of three
replicates
Eur J Clin Microbiol Infect Dis (2015) 34:1023–1030 1027
clinical origin appears to support the hypothesis that invasive-
ness is a virulence trait in S. cerevisiae.However,ithastobe
emphasisedthat clinical origin does not assure the virulence of
a strain, and, similarly, on the other hand, non-clinical origin
does not assure the avirulence of a strain. On the other hand,
the number of C. glabrata invasive strains was expected to be
higher, although, unfortunately, there are no data relating to
the virulence of these strains, and, therefore, it is not possible,
at this level, to determine whether invasiveness is a virulence
trait in C. glabrata or not. Nevertheless, the rationale behind
the invasiveness seems to entitle this ability as virulence traits
for several reasons; this pointed growth helps to find nutrients,
anchors colonies, hides from the immune system of a host and
protects from various unfavourable extrinsic conditions, such
as low pH, peristalsis, bile salts, micro flora etc., which give
invasive strains an important competitive advantage [32].
A possible use of invasiograms in diagnostics
From the invasiogram, virulence traits of the isolate can be
estimated; it shows the ability of strains to grow at elevated
temperature, invasiveness and their susceptibility to
antimycotics. Moreover, we noticed that each of the used
antimycotics showed a specific invasiogram, which can be
connected to the mechanisms of resistance against a particular
antimycotic. For fluconazole, the trend here appears to
characteristically promote increased relative invasion when
its concentration approaches the MICING (Fig. 2). This char-
acteristic is of particular importance, because it can have an
important impact on the efficiency of this antimycotic treat-
ment. This trend towards increased relative invasion can be
explained considering the active excretion of fluconazole by
the efflux pumps that have been identified in S. cerevisiae,and
which represent an important virulence trait [33,34]; this
presence of fluconazole seems to represent a stress against
which the yeast cells can resist with an intensity initially pro-
portional to the fluconazole concentration, until they reach a
point where they fail resisting, i.e. above the MICING.
It is also interesting to note that the food strain, ZIM 2266,
which originated from cheese, did not follow the same behav-
iour in the presence of fluconazole. However, whether or how
this trend might be connected to the clinical origin of a strain is
out of the framework of the present manuscript, and will re-
quire additional studies.
Itraconazole, however, had different effects on these yeast
(Fig. 3), although both of these antimycotics (i.e. fluconazole
and itraconazole) belong to the same, azole, group. The trend
for the relative invasion with itraconazole was again character-
istic for this antimycotic. At low concentrations, itraconazole
induced invasive growth in some strains, but not to the same
extent as fluconazole. The relative invasion was also mainly
reduced at higher itraconazole concentrations, which means
Fig. 4 Invasiogram for evaluation of the MICINGs for amphotericin B of
the nine S. cerevisiae and two C. glabrata invasive strains. The MICs are
also shown in brackets (as indicated; determined by CLSI standard M27-
A3). The arrows indicate the relative invasion trend typical for the
amphotericin B action. The error bars represent standard deviations of
three replicates
1028 Eur J Clin Microbiol Infect Dis (2015) 34:1023–1030
that only weak invasive growth was seen, and, instead, the
surface growth remained strong. If this observation is again to
be explained through the efflux pump theory, it would appear
that the efflux of itraconazole is less efficient than that of flu-
conazole. In addition, the MICINGs for itraconazole were gen-
erally lower than those for fluconazole. The explanation for the
differences in fluconazole and itraconazole effectiveness could
lie in a significant difference in their chemical structures and
pharmacokinetics. Both inhibit 14α-demethylase, which pro-
duces ergosterol, but itraconazole is a much larger and more
lipophilic molecule. Both parameters could play an important
part in the inhibition of 14α-demethylase or in the efflux of
both antimycotics as part of a resistance strategy.
Amphotericin B, as expected, acted as a fungicide rather
than a fungistatic. The invasive growth under this
amphotericin B treatment was, in most cases, vigorously sup-
pressed. This again resulted in a characteristic steep decrease
in the relative invasion at increased concentrations of
amphotericin B (Fig. 4).
Strain YJM 311 was of particular interest, as it shows the
highest virulence potential in mice[18], strong invasiveness at
elevated temperatures [22] and high resistance to all three of
these antimycotics (Figs. 2,3and 4). It shows the highest
MICs for itraconazole (2 μg/ml) and fluconazole (8 μg/ml),
and very high MICINGs for these two antimycotics, 4 μg/ml
and 16 μg/ml, respectively. However, as there are little data
available relating to the virulence of S. cerevisiae strains, these
data do not yet allow the claim of a significant correlation
between virulence and invasion. Confirmation of this hypoth-
esis would require additional in vivo tests with large numbers
of strains, as the clinical status of a strain does not assure its
virulence, as already discussed above.
As expected, the C. albicans strain showed the strongest
invasive growth. However, this was true only for controls, i.e.
plates without added antimycotics. Its MICINGs were very
low, namely, 0.125, 0.25 and 0.125 μg/ml for fluconazole,
itraconazole and amphotericin B, respectively, which is in
accordance with its status of azole-sensitive species.
In conclusion, the MICING assay provides a basis for a
good decision-making tool, with great potential for future
improvements, such as automation using a microplate for-
mat and modification of medium in terms of its structure
and composition to further resemble the host environment
for human pathogens. We have tested the microplate for-
mat, but the manual application needed when using this
format is too impractical. However, automation and medi-
um formation should not present a problem when the
MICING assay reaches this stage of development. Together
with rapid and stimulative progress in three-dimensional
support matrices, like self-assembled peptide hydrogels,
the assay could evolve in the direction to simulate epithelial
surfaces and replace cell cultures or, if being highly specu-
lative, the use of laboratory animals.
Acknowledgements We thank Lene Jespersen from KVL University,
Copenhagen, Denmark, Tadeja Matos from University of Ljubljana, Slo-
venia, and NežaČadež, the Curator of the Collection of Industrial Micro-
organisms (ZIM) at the Biotechnical Faculty, University of Ljubljana,
Slovenia, for providing us with the S. cerevisiae,C. glabrata and
C. albicans strains. We also thank NežaČadežfor the critical review of
the manuscript and constructive suggestions. The experimental work and
English correction of this manuscript were financially supported by the
Slovenian Ministry of Higher Education, Science and Technology (grant
no. Z7-4248).
Conflict of interest The authors declare that they have no conflict of
interest.
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