Medical Mycology 2001, 39, 479±482 Accepted 22 January 2001

Short communication

Large-scale screening of selected Candida maltosa,

Debaryomyces hansenii and Pichia anomala killer toxin
activity against pathogenic yeasts
P. BUZZINI & A. MARTINI
Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Sezione di Microbiologia Applicata, UniversitaÁ di Perugia, Borgo
XX Giugno, I-06100, Italy
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The killer activity of selected crude toxins produced by nine Candida maltosa,
Debaryomyces hansenii and Pichia anomala strains was tested at 37 o C against 383
strains belonging to 19 pathogenic species of 10 genera (Candida, Clavispora,
Cryptococcus, Filobasidiella, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces,
Stephanoascus and Trichosporon). The broad killer spectra exhibited by all crude
toxins may lead to the development of new antimycotic agents against medically
important yeasts.
Keywords killer toxin, pathogenic yeasts, screening survey
For personal use only.

Introduction The use of selected toxins as antimycotic agents

against yeasts that cause systemic mycoses has already
The killer phenomenon was Žrst discovered in 1963 by
been suggested [15,16]. Unfortunately, with few excep-
Bevan & Makeover [1] within strains of Saccharomyces
tions [15,17], the toxins studied to date lose killer activity
cerevisiae. At present, killer activity has been observed in
at or near human body temperature (37 oC) [2,16,18].
more than 90 yeasts species, belonging both to ascomy-
Literature data on activity spectra of killer strains against
cetous and basidiomycetous taxa, and the number of
pathogenic yeasts were often obtained by using very few
species involved is continuously increasing [2,3]. All
sensitive or killer cultures, most often inactive at 37 oC
yeast toxins (mycocins) studied to date have been found
[16,19,20].
to be of a proteinaceous nature [2]. In terms of mode of
Current literature describes the large-scale screening
action, several mechanisms, involving either cell mem-
surveys on the killing properties of selected yeasts, but
brane pore formation in target strains (with subsequent
only against sensitive strains of environmental or
loss of potassium ions and ATP, as well as disruption of
industrial importance [6,21–25].
cellular electrochemical potential across the membrane)
In the present study we conducted a large-scale
or the arrest of the cell cycle at the G1 phase, have been
screening survey aimed at selecting killer toxins effective
elucidated [2].
at 37 oC against pathogenic yeasts.
Possible applications of the killer phenomenon include
the biocontrol of fermentation processes against con-
taminants derived from environmental sources [4–6], as Materials and methods
well as the use of a panel of selected toxins for
intraspeciŽc characterization of industrially and clinically A total of 383 cultures belonging to the pathogenic
interesting yeast cultures [7–14]. species Candida albicans, C. glabrata, C. haemulonii, C.
inconspicua, C. parapsilosis, C. tropicalis, C. viswanathii,
C. zeylanoides, Clavispora lusitaniae, Cryptococcus
Correspondence: Dr Pietro Buzzini, Dipartimento di Biologia laurentii, Filobasidiella neoformans, Issatchenkia orien-
Vegetale e Biotecnologie Agroambientali, Sezione di Microbiologia
` di Agraria, Universita
Applicata, Facolta ` di Perugia, Borgo XX
talis, Kluyveromyces marxianus, Pichia angusta, P.
Giugno, I-06100, Italy. Tel.: ‡39 075 5856455; fax: ‡39 075 5856470; guilliermondii, P. norvegensis, Saccharomyces cerevisiae,
e-mail: pbuzzini@unipg.it. Stephanoascus ciferrii and Trichosporon spp. were tested
ã 2001 ISHAM
ISHAM, Medical Mycology, 39, 479±482
 480 Buzzini & Martini
as sensitive strains. Several of these, although commonly
regarded as species of industrial interest, have in recent
years gained the status of emerging pathogens because
they have been implicated in causing opportunistic
infections [26,27].
Strains were obtained from the Industrial Yeasts
Collection DBVPG of the Dipartimento di Biologia
Vegetale e Biotecnologie Agroambientali of the Uni-
versity of Perugia (DBVPG). All had been identiŽ ed
according to Yarrow [28]. Thirty isolates of C. albicans
and 10 of C. glabrata were acquired from local hospital
laboratories and were identiŽ ed by clinical procedures
[29].
The following yeasts were included in the killer panel:
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Candida maltosa (strains F17 and G7A), Debaryomyces
hansenii (Di29 and P41) and Pichia anomala (C33, C85,
Di8, Di28 and DBVPG 3649). Stability of their killer
phenotypes was checked using acridine orange as curing
agent [7,30].
YEPG (10 g l¡1 yeast extract, 10 g l¡1 peptone, 20 g
l glucose, 20 g l¡1 agar [Difco, Detroit, MI, USA])
¡1
agar slants were used to store cultures at 4 oC. Killer
medium (KM; 10 g l¡1 yeast extract, 20 g l¡1 peptone,
For personal use only.
20 g l¡1 glucose, 0¢03 g l¡1 methylene blue, 20 g l¡1 agar
(Difco), buffered at pH 4¢5 with citrate-phosphate
buffer) was used to screen the killer activity of the
panel cultures.
Killer activities of crude toxin preparations (concen-
tration ranging 1¢5–3¢5 m g ml¡1 of protein), obtained
as previously reported [8] and conserved as frozen
(–20 oC) cell-free liquid Žltrates and as freeze-dried
preparations, were tested using the agar diffusion well
bioassay (ADWB) in dishes containing KM agar pre-
inoculated with the different sensitive cultures [8].
Dishes were incubated at 37 oC for 24–48 h. Previous
results indicated that all tested toxins were fungicidal in
their activity [8].
A clear inhibition zone around the well ( > 1 mm)
surrounded by a crown of dark blue stained cells was
considered as a positive indication of killer activity. A
narrow but clear inhibition zone ( < 1 mm), or simply a
blue zone, conventionally ascribed to the accumulation
of methylene blue in dead cells, was interpreted as a
weak killer action, whereas absence of a clear or blue
zone was recorded as a insensitive (negative) reaction.
When discrepant results were encountered in repeated
experiments, this was always recorded as a negative,
insensitive response.
Results and discussion
Table 1 shows that all killer strains possessed a broad
killing activity at 37 oC. In agreement with previous
reports [3,7,10], the efŽcacy of killer activity appears
to vary from strain to strain among the target
organisms (Table 1). In contrast to previous observa-
tions [16], the differences in sensitivity did not allow
the distinction of clinical isolates from those collected
at the DBVPG.
According to current literature [2], the diversity of
cell-wall receptors involved in the binding of killer toxins
could be the basis of different sensitivity reactions. The
strain-related nature of killer susceptibility could be
misinterpreted as the result of the extreme difŽculty in
discriminating between two hypothetical classes of
insensitivity to mycocins, preliminarily deŽned by
Golubev [2] as immunity and resistance, which have
different implications both for taxonomy and in terms of
the underlying mechanisms of regulation and expression
[2].
A broad killer activity was observed for several toxins.
For instance, D. hansenii P41 toxin killed 100% of C.
glabrata, C. haemulonii, C. inconspicua, C. parapsilosis,
P. angusta, P. norvegensis and S. cerevisiae strains.
Similarly, all cultures of C. tropicalis were sensitive to
the toxins produced by C. maltosa G7A, D. hansenii
Di29 and P. anomala C33, C85, Di8, Di28 and DBVPG
3649 (Table 1).
By considering the activity spectra of more than one
killer toxin it was possible to recognize combinations
with extended killing abilities. Certain combinations of
toxins allowed the inactivation of all strains of C.
zeylanoides, I. orientalis, K. marxianus and P. guillier-
mondii. In addition, toxin combinations could inhibit
signiŽcant percentages of C. albicans and Trichosporon
spp. strains. Effective combinations are shown in Table
1. Only a few strains of Cr. laurentii were killed (Table
1), whereas no toxin exhibited activity against F.
neoformans.
To our knowledge, this is the Žrst screening carried out
at 37 oC concerning the killer sensitivity of a large,
statistically valid, number of pathogenic yeasts. The
availability of yeast killer toxins exhibiting stable activity
at human body temperature opens new perspectives for
their exploitation. It would be appropriate to investigate
the possibility of topical application of concentrated
puriŽed toxin preparations in therapy against pathogenic
yeasts.
Acknowledgements
This paper was supported by a grant of MURST,
ScientiŽc research 60%, 2001.
ã 2001 ISHAM, Medical Mycology, 39, 479±482
 Med Mycol Downloaded from informahealthcare.com by Kainan University on 04/02/15
For personal use only.

ã
Table 1 Activity spectrum at 37 o C of selected killer toxins against pathogenic yeasts

No of killer toxins (Number of killed strains)

No of No of
tested DBVPG killed

2001 ISHAM, Medical Mycology, 39, 479±482

Species strains F17 G7A Di29 P41 C33 C85 Di8 Di28 3649 Combination of killer toxins strains

Candida albicans 72 36 15 13 30 36 24 C33, Di8, Di28, Di29, DBVPG 3649 45

Candida glabrata 38 24 38 8 15 18 25 22
Candida haemulonii 3 1 3 1 1 1
Candida inconspicua 5 1 5 2 1
Candida parapsilosis 20 1 1 14 20 6 9 12 15 13
Candida tropicalis 13 12 13 13 13 13 13 13 13
Candida viswanathii 1 1 1 1 1 1 1 1 1
Candida zeylanoides 18 9 10 16 13 8 11 15 16 15 Di29, F17, P41 18
Clavispora lusitaniae 3 1 1 3 3 3 3 3 3
Cryptococcus laurentii 25 1 2 1 1 Di8, Di28, Di29, P41 4
Issatchenkia orientalis 41 10 5 12 39 10 12 11 14 14 Di29, P41 41
Kluyveromyces marxianus 53 1 1 50 52 42 47 48 50 48 Di29, P41 53
Pichia angusta 5 5 5 5 5 5 5 5
Pichia guilliermondii 26 7 8 20 21 15 15 19 20 16 Di28, Di29, P41 26
Pichia norvegensis 3 2 3 2 2 2 2 2
Saccharomyces cerevisiae 15 13 15 6 5 13 13 9
Stephanoascus ciferrii 3 1 3
Trichosporon spp. 19 1 9 1 1 1 1 1 Di29, P41 11
Killer activity against pathogenic yeasts
481
 482 Buzzini & Martini
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