Filamentous fungus Penicillium commune as a promising source of

bioactive molecules

Lorena Diblasia,b, Federico Arrighic, Alicia Bardóna,c, Julio Silvab, Elena Cartagenaa*
Instituto de Química Orgánica. bCátedra de Micología. Facultad de Bioquímica, Química y Farmacia.
a

Universidad Nacional de Tucumán.Ayacucho 471. Tucumán (4000). Argentina.

c
INQUINOA – CONICET. Ayacucho 471. Tucumán (4000). Argentina.

*
Corresponding Author: Elena Cartagena
Email: ecartagena@fbqf.unt.edu.ar
Abstract

Penicillium is a genus from ascomycetous fungi of major importance in the natural

environment as well as in food and drug production. Antibiotics and statins are important
drug groups containing pharmacological potential produced by fungal metabolism. In this
context, the aim of this work was to investigate if a P. commune strain produces statins, and
bioactive molecules against the bacterial biofilm, main virulence factor contributing to the
chronicity of infections.
From an ethyl acetate extract of a P. commune INTA1 culture, four interesting fractions from
a sensory point of view and chromatographic profiles were obtained. In addition, their anti-
pathogenic effects were analyzed on Staphylococcus aureus and Pseudomonas aeruginosa
strains with clinical significance. The fractions F2 and F4 isolated from the fungal extract
were bioactive against S. aureus biofilm, and when F2 was mixed with semi synthetic
oxacillin antibiotic improved its antibiofilm activity. S. aureus biofilm over 1 h to exposure of
F2 (as well as F4) was effectively reduced by 90% at 100 µg/ml; while the biofilm formation
inhibition over 24 h at 50 µg/ml was of 53% for S. aureus, and 10% for P. aeruginosa. The
mixture of F2 (6 µg/ml) and oxacillin (3 µg/ml) was significantly more effective than F2 and
oxacillin alone on both Gram positive and negative bacteria (91% and 38%, respectively). F2
and its association with oxacillin showed a promising activity against both Gram positive and
negative bacteria with a biofilm phenotype.
Volatile composition of odorant bioactive F2 was investigated by GC-MS. The main
compounds were 3-isobutylhexahydropyrrolo [1,2-a]pyrazine-1,4-dione (1) (49.34%) with a
known antibacterial activity, hexanedioic acid mono (2-ethylhexyl) ester (39.66%), and
betulin (3.31%). From F4 2 (16.38%) like analogue of 1 was identified, and their structures
are similar to the bacterial autoinducer [(3s,9s)-hexahydro-3-(phenyl)-pyrrolo-(1,2-a)-
pyrazine-1,4-dione] involved in host-microbial interactions.
Spectroscopic and spectrometric techniques allowed us the determination of lovastatin from
odorless crystalline fraction F1. The known statin was previously isolated from other fungi
species, and herein it reports lovastatin as a polyketide-derived natural product from P.
commune.

Keywords: Penicillium commune, lovastatin, volatile metabolites, GC-MS, DIP/EI/ITD,

pathogenic bacteria, anti-biofilm activity.
Introduction

Fungi are known for their peculiar chemistry, differing from that of both animals and
higher plants. The secondary metabolites of fungi are produced to aid establishment in the
various ecological niches of different fungal life forms. Several of these metabolites have
played important roles as fungi-derived drugs revolutionizing the treatment of several serious
conditions in man. We still know relatively little about the general nature of fungal chemistry
and in addition to this we only seem to know a fraction of the estimated number of existing
fungal species on Earth. Today approximately 14,000 species of macrofungi are known to
science, but estimations show that this may well be only a tenth of the actual number of all
living macromycete species. For fungi in the widest sense, the total number of species on
Earth has been estimated to 1.1 million. Within the fungal kingdom lies a vast, and to a large
extent, still unexplored potential for future pharmaceutical drugs (Bohlin et al., 2010).
Penicillium is a genus from ascomycetous fungi of major importance in the natural
environment as well as in the food and drug production. Among fungal metabolites, statins
are a group with pharmacological interest as hypocholesterolemic drugs. Statins are a class of
drugs that inhibiting the enzyme HMG-CoA reductase, which plays a central role in the
production of cholesterol in liver. Increased cholesterol levels have been associated with
pathological conditions such as atherosclerosis, and cardiovascular diseases. Statins are
therefore used in the prevention of these diseases.
According to Endo (2004), the fungal production of statins would occur as a defense
mechanism against other microbes that required sterols and/or other mevalonate-derived
isoprenoid compounds to their growth. The first statin isolated was mevastatin from P.
citrinum broths, and then the discovery of lovastatin from soil fungus Aspergillus terreus,
xerophilc fungus Monascus ruber, and species of the genera Hypomyces, Doratomyces,
Phoma, Eupenicillium, Gymnoascus, and Trichoderma took place. The other statins were
obtained by a semi-synthetic process, involving the chemical modification of the lovastatin
side chain, as simvastatin or by the biotransformation of mevastatin by Streptomyces
carbophilus such as, pravastatin. Finally, fluvastatin and atorovastatin are fully synthetic
statins, derived from mevalonate and pyridine, respectively (Manzoni and Rollini, 2002;
Endo, 2004).
Numerous studies today indicate that human infections are, in large part, caused by the
ability of bacteria to develop surface attached poly-microbial communities known as biofilms.
Microbial biofilms consist of groups of bacterial cells adherent to a surface and enclosed
within a self produced extracellular matrix. Adaptation to surface attached growth within a
biofilm is accompanied by significant changes in gene and protein expression, as well as
metabolic activity which confers resistance to antimicrobial therapy. Bacterial biofilm
phenotype is a major virulence factor contributing to the chronicity of infections (Sanchez et
al., 2013).
The present research is focused on bioactive metabolites study from P. commune, such
as statins, and effective volatile compounds against pathogenic bacteria with a biofilm
phenotype.

Experimental part

Fungal strain and media

Penicillium commune INTA1 was isolated from the environment, and classified by
morphological criteria and a microscope method (Sanidad Aviar laboratory of INTA, Entre
Ríos, Argentina). The fungal strain was maintained in Petri dishes of PGA (potato, glucose,
agar). After incubation from the original slant, the dishes were incubated at 28 ºC for 5 days,
and subsequently stored at 5 ºC.
A suspension of spores (harvested from plates) was obtained with sterile saline
solution (NaCl, 7.8 g l-1) and concentrations adjusted to 107 conidia ml-1 (λ , DO = 0.3)
550 nm

(Chakravarti, 2002; CasasLópez, 2003).

The cultivation medium, contained per litre glucose: 20 g, glycerol: 24 ml, peptone: 8
g, NaNO3: 2 g, MgSO4: 1 g. All components were dissolved with soybean water (soybean: 40
g, sterile water: 1000 ml), and maintained for 6 days at 4 ºC, before fermentation. Then, the
liquid medium was sterilized at 121 ºC and ¾ de atm (Chakravarti, 2002). A second medium
was prepared in the same way, but containing 10 g glucose per liter.
The culture medium contained glucose and glycerol as carbon source, and peptone and
soybean meal as nitrogen source.

Fermentation and conditions

Fungal pellets were obtained by germination from spores suspended in shake flasks in
a preliminary fermentation stage, and used for further inoculation of corresponding
bioreactors. Seed cultures (1% , v/v) were carried out in a 500 ml flasks containing 150 ml
medium in duplicate, held on a rotary shaker at 110 rpm, 28 ºC and pH = 6.3 (Szakács, 1998;
Kumar et al., 2000; Casas López, 2004; Bizukojc, 2007). A medium control (without fungal
strain) was also performed in duplicate.
Fermentations lasted 10 days [Chakravarti, 2002], and thereafter the pH of each
culture was adjusted to 6.3 again.

Extraction and isolation of fungal metabolites

The biomasses obtained were removed by filtering with cellulose filter and vacuum,
the filtrate media were extracted with ethyl acetate twice a room temperature. The AcOEt
extracts were dried over anhydrous Na2SO4 and were evaporated in rotavapor (Büchi R-3000)
under vacuum at 40 ºC to give 0.4665 g of extract (E), and 0.0229 g of extract from the
second medium containing 50% of glucose (E2).
The isolation of the fungal metabolites from extracts was carried out by routine
chromatographic techniques.

Chromatography techiniques
Analytical thin layer chromatography (TLC) was performed on Merck precoated silica
gel 60 F254 plates to analyze each fungal extracts, and controls. Different mobile phases were
employed for development of method. UV irradiation (λ: 254 nm and 366 nm), and oxidative
solution of Godin were employed as physical and chemical revealing, respectively. The
lovastatin, simvastatin, and mevastatin (MP Biomedicals LLC) were used as standards, and an
extract from culture medium was employed as negative control.
A portion of the acetate ethyl extract from P. commune (87.7 mg) was separated on
silica gel column chromatography (1:100, extract/ silica gel relation) using CHCl 3:AcOEt (0-
100%), as mobile phase. The eluted fractions were monitored by TLC employing statins as
standards.
The fractions F2-4, with particular aroma, were analyzed by GC-MS (EI) technique.
The analysis was carried out using a Thermo electron TraceTM Ultra couple with split-split less
injector and Polaris Q ion trap mass spectrometer equipped with a DB-5 capillary column (30
m x 0.25 mm, film thickness 0.25 µm). The initial temperature of the column was 60 C (0
min). A temperature programming was applied from 60 °C to 246 C at a rate flow of 3
C/min, and finally 246 C for 3 min. Carrier gas was helium (flow 0.3 ml/min). Injection
mode split-less with surge (30 s, surge pressure 100 kPa). The main volatile constituents were
determined by comparison of their mass spectra with standard data of NIST GC/MS library.
Fourier transformed infrared spectroscopy (FT-IR)
The FT-IR spectra of extracts from the fungal culture, medium control, and the
fraction F1 were recorded with a Perkin Elmer spectrophotometer 1600 FT-IR, in order to
determine the δ-lactone ring absorption (~ 1735 cm-1 ), and absorption bands associated with
C=O bond stretching at 1180 cm-1of δ or γ-lactones (Pretsch et al., 1998).

Mass spectrometry (DIP / EI / ITD)

The extracts, F1, and standards of statins were dissolved in CH2Cl2 to determine their
fragmentation patterns at 70 eV by direct introduction into a mass spectrometer (DIP: Direct
Introduction Probe/EI: 70eV-Electron Ionization / ITD: Ion Trap Detector Polaris Q Thermo
Electron; gas carrier He; ion trap detector; ion source temperature 200 °C; acquisition range
from 50 to 500 uma / DIP: temperature program 60-450 °C, temperature gradient 100
°C/min).The identification of lovastatin was easily achieved by comparison with the
fragmentation pattern of authentic standards of statins.

Antibacterial and antibiofilm activities of volatiles from P. commune

Fungal metabolites and antibiotics
Solutions of F1-4 from P. commune, lovastatin standard, and antibiotics: azithromycin
and oxacillin were screened.

Bacterial strains and media

Staphylococcus aureus ATCC 6538 P, and Pseudomonas aeruginosa ATCC 27853
from the American Type Culture Collection were grown in Müller Hinton (MH, Britania),
and Luria-Bertani media (LB, Cabeo, Rockville, MD, USA), respectively, and employed for
the bioassays.

Bacterial growth assay

Overnight cultures of each strain were diluted to reach 10 5 CFU/ml in LB or MH
medium. The diluted culture (190 µl) was placed in each of the 96 wells of a microtitre
polystyrene plate. Solutions containing 50, and 25 µg/ml of F1-4, and mixture of F1 (6 µg/ml)
with oxacillin (3 µg/ml) in DMSO-distilled water (50:50) were prepared separately and 10 µl
of each one was pipetted to the plastic microtitre plate wells individually (eight replicates).
Control wells (eight replicates) contained the diluted culture (190 µl) and 10 µl of a solution
of DMSO-H2O (50:50). Medium control was prepared using sterile LB or MH. Bacteria grew
in liquid medium at 37 °C, and growth was detected as turbidity (600 nm) using a microtitre
plate reader (Power Wave XS2, Biotek, VT, USA). The maximum level of DMSO to which
the cells were exposed was 2.5 %. The negative control was oxacillin as cell wall synthesis
inhibitor. Then, it was quantified the biofilm formed after 24 h of incubation using the crystal
violet dye (O'Toole and Kolter, 1998).

Biofilm assays
Bacterial biofilm in the presence of fractions was quantified by the technique
described by O'Toole and Kolter (1998) modified. The biofilm quantification assay is based
on the ability of bacteria to form biofilms on polystyrene. The technique requires the addition
of violet crystal solution which stains the cells but not the polystyrene.
A volume of 180 µL of MH (S. aureus) or LB (P. aeruginosa) broth and 10 µl of each
fractions and controls were placed in each well of a 96-well polystyrene microtiter plate.
Then, each well was inoculated with 10 µl of an overnight culture with biofilm phenotype
(108 CFU ml-1). The microplates were incubated at 37 °C for 1 h in a moist chamber. Control
experiments were performed in octuplicate using DMSO-H2O (without fractions). The
negative controls were azithromycin as a known quorum-sensing inhibitor, and oxacillin.
After this time period, 25 μl of violet crystal solution (1%) were added to the wells,
incubated 15 min at room temperature and then rinsed thoroughly and repeatedly with water
to remove planktonic cells and unattached dye. Biomass-attached dye was solubilized with
ethanol; and the absorbance was then measured at 540 nm in a microplate spectrophotometer
(Biotek- Power Wave XS2 with GEN5 data analysis software).

Statistical analysis
Differences in the mean values were evaluated by analysis of variance (ANOVA). The
Tukey test was used for all pair wise multiple comparisons of groups. In all analyses, values
of P < 0.05 were considered statistically different (Statistix 7.1, 2002).

Results and discussion

Fermentation and conditions

 Submerged cultures of filamentous fungi are widely used to produce commercially
important metabolites including many antibiotics and the cholesterol lowering drugs (statins).
Microorganisms are able to produce lovastatin in submerged culture during the secondary
phase (idiophase) of fungi growth (Gupta et al., 2007). The β-hydroxyl lovastatin is the more
active form of this drug, but is unstable (Jaivel and Marimuthu, 2010). For this reason, it was
optimized the fermentation conditions (28 °C and pH = 6.3), according to Kumar et al.
(2000), who demonstrated that this incubation temperature and pH (between 5.8 to 6.3)
provided optimum conditions for lovastatin production by A. terreus, in the batch process.

Extraction and isolation

The lovastatin was extracted as the lactone form. Because of the hydroxyl form of
lovastatin is not stable, the lactone form is normally the primary lovastatin detected in the
fermented products. The extraction yield was of 1.56 g/l (E), and 0.076 g/l (E2, from the P.
commune culture containing a 50% of glucose).
The chromatographic separation of acetate ethyl extract from P. commune (87.7 mg)
gave four fractions that were collected according to chromatographic profiles. The fraction F1
was eluted by CHCl3-AcOEt 85:15 (yield: 3.53% in relation to processed extract), and it was
isolated as colorless needle crystals. While, F2 (CHCl 3-AcOEt 75:25, 3.76%), F3 (CHCl3-
AcOEt 60: 40, 7.3%), and F4 (AcOEt, 19.3%) with peculiar aroma, were analyzed by GC-
MS, and the main constituents are shown in Table 1.

Analytical TLC
P. commune extracts (E and E2), F1, and controls were chromatographyed by TLC
using CHCl3/AcOEt 4:3 (57:43) as good developing solvents. The eluted statins were then
visualized using UV light at 254 nm, and when the plates were sprayed with the Godin
reagent, violet spots were determined with Rf similar to lovastatin standart (Rf = 0.41). These
spots were absent in the extract from the broth culture (Fig.1). A 50% of glucose in the
formula of the culture medium decreases the yield of the extraction process.

FT-IR spectra
Only P. commune extracts, and F1, exhibited infrared absorption bands at 3400 (OH),
1750 (C=O), and 1280 (C-O) cm-1 clearly assignable to the hydroxyl, and ester functionality,
respectively. It is important to note that typical δ-lactones’ absorptions at 1735 (C=O), and
1180 cm-1 (C-O) were detected.
DIP/EI/ITD technique
The presence of lovastatin in the extracts, and F1 from P. commune was confirmed by
the correspondence of the molecular ion, and EI-MS fragmentation patterns with its standard
at the same conditions. It is important to remark that both mass spectra showed an intense
signal corresponding to the protonated molecular ion or adduct ion represented by [M + H] +,
formed by the interaction of a lovastatin molecule with a proton. This phenomenon called
self-chemical ionization can occur in the ion trap mass spectrometer (McLuckey et al., 1988;
Pannell et al., 1989).
A good correlation between main MS peaks of F1 and lovastatin standard was found
by DIP/EI/ITD (Figure 2).

Antibacterial and antibiofilm activities of odorant volatiles from P. commune

The biofilm of S. aureus over 1 h to exposure of F2 (as well as F4) at 100 µg/ml was
effectively reduced by 90% (Fig. 3). While, the biofilm formation inhibition over 24 h to
exposure of F2 (50 µg/ml) was of 53% for S. aureus, and 10% for P. aeruginosa (Fig. 4). The
mixture of F2 (6 µg/ml) and oxacillin (3 µg/ml) was significantly more effective than F2 and
oxacillin alone on both Gram positive and negative bacteria (91% and 38%, respectively).
Thus, F2 improved the oxacillin activity via cell wall inhibition. F2 and its association with
oxacillin showed promising activity against both bacteria Gram positive and negative with
biofilm phenotypes.
The effects of F2 at 50 µg/ml against S. aureus biofilm formation was independent of
bacterial growth (Fig. 5). As shown in figure 4, the moderate reduction of cell growth of
mixture M (39%) could explain partially the strong effect observed on S. aureus biofilm
formation (91%). Therefore, another mechanism would be involved as it was supposed in a
previous report on antipathogenic volatiles from Aspergillus parasiticus (Cartagena et al.,
2014), which does not kill bacteria or stop their growth. Rather, they control bacterial
virulence factors, such as biofilm, and prevent the development of resistant strains (Otto,
2004).
Fungal metabolites effects on P. aeruginosa were more important on cell growth than
biofilm formation. The largest growth reduction was of 37% for F2 at 50 µg/ml.
In all experiments there were no significant differences between F1 and lovastatin, and
they possessed a weak activity at 50 µg/mL (Fig. 4). The coherence of their biological and
chemical data was key for determining of lovastatin as main metabolite from F1.
Determination of volatile bioactive from P. commune by GC-MS
The GC-MS analysis of odorant F2 and F3 allowed us to determine, in different
percentage, pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- (1), an
antibacterial volatile substance, previously isolated from Vibrio parahaemolyticus (Pandey et
al., 2010), and from an endophytic fungus Penicillium sp. (Devi and Wahab, 2012). The
molecule 1 was reported as ergot peptide alkaloids (van Mansvelt et al., 1978). The bioactive
fraction F4, contained pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- (2), an analogue of chain
shorter than 1 (Table 1).
The chemical structures found are similar to autoinducer (cFP) [(3s,9s)-hexahydro-3-
(phenyl)-pyrrolo-(1,2-a)-pyrazine-1,4-dione] (Fig. 6) which is involved in the host-microbe
interactions (Park et al., 2006). The active compound cFP was detected from Gram negative
bacteria V. fischeri, V. vulnificus, V. harveyi and Pseudomonas aeruginosa, which induced the
expression of V. fischeri lux reporter system (Holden et al., 1999). cFP was considerate as a
signal molecule controlling the expression of genes important for the pathogenicity of bacteria
(Park et al., 2006). To our surprise, bioactive cFP was also produced by Gram positive
bacteria as Lactobacillus plantarum (Ström et al., 2002). Hence, the mechanism by which F2,
and F4 reduces the bacterial biofilm would be the disruption of cell to cell communication.
Nevertheless, further studies are needed to confirm that F2 would act as cFP antagonists
(structural competitives or functionals).
The present study provides evidence of the production of lovastatin and health
promising metabolites from a P. commune INTA1 strain and their pharmacological potential.

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Figure 1. TLC profiles of P. commune INTA1 extracts, statins, and control of broth culture

E and E2: P. commune INTA1 extracts, standards: Mev: mevastatin,

Lov: lovastatin, Sim: simvastatin, and control BC: broth culture extract.
Figure 2. Lovastain EI-MS spectrum, chemical structure and main MS peak from F1

HO O

O
O
H

H3C O
H H
CH3 CH3

H3C

Fig. 2.A. Lovastatin EI-MS spectrum and chemical structure.

Fig. 2.B. Main EI-MS peaks of F1 and their assignments

F1 [m/z]: 405 [M+H] +, 404 [M] +•, 387 [MH-H2O] +, 303 [C19H27O3]+,
199 [C13H11O2]+, 198 [C13H10O2] +•, 159 [C12H15] +, 157 [C12H13] +, 143 [C7H11O3] +
Figure 3. Biofilms of S. aureus ATCC 6538 P after 1 h of incubation

1,2

0,8
OD [540 nm]

0,6

0,4

0,2

0
S. aureus F2 100 F3 100 F4 100 AZT OXA

F 2-4 100: Odorant fractions 2-4 [100 µg/ml], AZT: Azithromycin [25 µg/ml], OXA: Oxacillin [6 µg/ml].
Strain: S. aureus ATCC 6538 P. All experiments showed significant differences with the S. aureus control (P <
0.05).

Figure 4. Biofilm of S. aureus and P. aeruginosa strains after 24 h of incubation

4,5
4
* * * *
3,5
3
OD [540 nm]

2,5
2
1,5
1
0,5
0

F1-2: Fractions 1 (50 µg/ml) and 2 (25, 50 µg/ml). LOV 50: lovastatin (50 µg/ml). ATB: Oxacillin (6 µg/ml). M:
F2 (6 µg/ml) + OXA (3 µg/ml). Strains: S. aureus ATCC 6538 P and P. aeruginosa ATCC 27853. *Not
significant differences were observed between these culture conditions.
 Figure 5. Cell growth of S. aureus and P. aeruginosa strains after 24 h of incubation
2

1,8

1,6

1,4

1,2
OD [600 nm]

0,8

0,6

0,4

0,2

0
50

25

50

50

25
50

50

50
B

B
M

M
sa
us

AT

AT
re

no
F2

F2

F1

F2

F2

1
V

V
au

LO

LO
gi

F
ru
S.

ae
P.

F1, 2: Fractions 1 (50 µg/ml) and 2 (25, 50 µg/ml). LOV 50: lovastatin (50 µg/ml). ATB: Oxacillin (6 µg/ml).
M: F1 (6 µg/ml) + OXA (3 µg/ml). Strains: S. aureus ATCC 6538 P and P. aeruginosa 27853. All experiments
showed significant differences with their respective control.

Table 1. GC-MS of the odorant fractions F2-4 from P. commune INTA1

Molecular Retention time Area

formula
Compounds of F2
Pyrrolo[1,2-a]pyrazine-1,4-dione,
C11H18N2O2
hexahydro-3-(2-methylpropyl)- 44 min 49.34 %

C30H50O2
Betulin 56 min 3.31 %
Hexanedioic acid, mono (2-ethylhexyl)
C14H26O4
ester 57 min 39.66 %

Compounds of F3
Pyrrolo[1,2-a]pyrazine-1,4-dione,
C11H18N2O2
hexahydro-3-(2-methylpropyl)- 44 min 21.81 %

Hexanedioic acid, mono (2-ethylhexyl) C14H26O4

57 min 33.43 %
ester
Dihydroergotamine C33H37N5O5
57.6 min 4.8 %
Compounds of F4
Pyrrolo[1,2-a]pyrazine-1,4-dione,
C7H10N2O2
hexahydro- 38 min 16.38 %

Hexanedioic acid, mono (2-ethylhexyl)

C14H26O4
ester 57 min 83.62 %
Figure 6. Chemical structures of fungal molecules 1, 2, and bacterial autoinducer cFB

O O O

N N N

NH NH HN
HO

O O O
1 2 cFB