bacteriostatic anti-vibrio parahaemolyticus activity of pseudoalteromonas sp. strains dit09, dit44...
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ORIGINAL PAPER
Bacteriostatic anti-Vibrio parahaemolyticus activityof Pseudoalteromonas sp. strains DIT09, DIT44 and DIT46isolated from Southern Chilean intertidal Perumytilus purpuratus
Carlos P. Aranda • Cristian Valenzuela •
Javier Barrientos • Javier Paredes • Pablo Leal •
Miguel Maldonado • Felix A. Godoy • Carlos G. Osorio
Received: 19 August 2011 / Accepted: 17 March 2012 / Published online: 30 March 2012
� Springer Science+Business Media B.V. 2012
Abstract We characterised the anti-Vibrio parahaemo-
lyticus (anti-V. parahaemolyticus) marine bacteria DIT09,
DIT44 and DIT46 isolated from the intertidal mussel Pe-
rumytilus purpuratus. The 16S rRNA gene sequences
identify a Pseudoalteromonas sp. that form a clade with P.
prydzensis and P. mariniglutinosa. The strains produced
bacteriostatic anti-V. parahaemolyticus agents during the
exponential growth phase, which were also active against
V. cholerae and V. anguillarum, but not on other Gram
positive and Gram negative bacteria. Bacteriostatic agents
could be permeated by analytic ultra-filtration with 3.5 kDa
cut-off, partially precipitated with 70 and 90 % ammonium
sulphate, but not extracted with ethyl acetate. Reverse-
phase HPLC revealed the production of a set of 5–6 active
compounds by each strain (elution from 20 to 40 % ace-
tonitrile), with similar but non identical HPLC patterns.
Additionally, V. parahaemolyticus was able to progres-
sively overcome the inhibition of antibiotics in trypticase
soy agar with Fe(III) 0.5 up to 2 mM, suggesting the
involvement of a set of novel siderophore or active mole-
cules targeted at different Fe-siderophore uptake systems.
The overall findings suggest that Pseudoalteromonas sp.
DIT strains produce a putatively novel class of bacterio-
static and probably amphiphilic anti-Vibrio agents, indi-
cating the need for further studies with chemical
purification followed by their structural and functional
characterization. Finally, the crude cell-free extracts, as
well as the strains incubated at 103 and 105 c.f.u./mL, did
not cause mortality in Artemia franciscana nauplii, sug-
gesting that these bacteria are serious candidates for further
probiotic evaluations with shellfish and fish cultures.
Keywords Vibrio parahaemolyticus �Pseudoalteromonas � Bacteriostatic � Probiotic
Introduction
Bacteria of the Vibrio genus are ubiquitous members of
the normal microbiota of coastal marine environments,
and are frequently the cause of disease (vibriosis) in
almost all cultured marine aquatic animals such as crus-
taceans, molluscs and fishes (V. anguillarum, V. algino-
lyticus, V. cholerae, V. fischeri, V. furnissii, V. harveyi, V.
ordalii, V. salmonicida, V. splendidus, V. vulnificus and V.
wodanis) (Gomez-Leon et al. 2005; McCann et al. 2003;
Lunder et al. 2000; Pedersen et al. 1998; Esteve et al.
1995). Members of this genus are the bacterial agents
principally responsible for outbreaks of human diarrhoeal
disease associated with faecal contaminated water (V.
cholerae) (Mridha et al. 2011; Luquero et al. 2011) or
raw molluscan shellfish around the world (V. cholerae, V.
parahaemolyticus) (Chao et al. 2011; Ansede-Bermejo
et al. 2010; DePaola et al. 2010; Jatapai et al. 2010).
Furthermore, shellfish are also a vehicle for the trans-
mission of a rapidly fatal septicaemia due to V. vulnificus,
that occurs mainly in North American populations
(DePaola et al. 2010).
Scientist have considered the use of marine probiotics as
a non antibiotic-based approach in preventing aquaculture
C. P. Aranda (&) � C. Valenzuela � J. Barrientos � J. Paredes �P. Leal � M. Maldonado � F. A. Godoy
Centro i*mar, Universidad de Los Lagos,
Camino a Chinquihue Km. 6, Puerto Montt, Chile
e-mail: [email protected]
C. G. Osorio
Programa de Microbiologıa y Micologıa, Instituto de Ciencias
Biomedicas (ICBM), Facultad de Medicina, Universidad de
Chile, Santiago, Chile
123
World J Microbiol Biotechnol (2012) 28:2365–2374
DOI 10.1007/s11274-012-1044-z
vibriosis (Desriac et al. 2010; Isnansetyo et al. 2009; Bal-
cazar et al. 2006; Verschuere et al. 2000) and shellfish food-
borne outbreaks (Teplitski et al. 2009). Based on this use, a
broad new definition of probiotics has arisen, which is
described as ‘‘a live microbial adjunct which has a benefi-
cial effect on a host by modifying the host-associated or
ambient microbial community, by ensuring improved use of
feed or enhancing its nutritional value by the enhancing of
the hosts response towards disease, or by improving the
quality of its ambient environment’’ (Verschuere et al.
2000). This broad definition allows for any possible positive
effect upon the target host, such as competitive exclusion,
enzymatic contribution to digestion or enhancement of the
immune response (Perez et al. 2010; Balcazar et al. 2006).
Furthermore, it includes the production of inhibitory sub-
stances against pathogens, a mode of probiotic action which
has received increased attention recently (Desriac et al.
2010; Gillor et al. 2008; Gillor and Ghazaryan 2007).
The development of marine bacteria as biological control
agents that may replace antibiotic based strategies in
aquaculture or assist marine-food processing or preserva-
tion is now a priority and the aim of this research. We
screened anti-Vibrio parahaemolyticus activity of cultivat-
able bacteria from a sample of Perumytilus purpuratus, a
common mussel found in the rocky intertidal along much of
the Chilean coast. Southern Chile has experienced V.
parahaemolyticus shellfish food-borne outbreaks since
2004 (Gonzalez-Escalona et al. 2005) and is also the centre
of aquacultural activity in the region (salmon-Salmo salar,
Oncorhynchus kisutch and Oncorhynchus tshawytscha-,
trout-Oncorhynchus mykiss and Salmo trutta-, mussel-My-
tilus chilensis- and oyster-Crassostrea gigas). We focused
on Pseudoalteromonas sp. as the only genus with active
bacteria detected in the P. purpuratus samples. These bac-
teria exhibited strong anti-V. parahaemolyticus activity in
the cell-free extracts. Our aim was to perform preliminary
studies investigating these potential biocontroling agents
by, conducting a phylogenetic analysis based on 16S rRNA
gene sequence, assessing antagonistic activity against some
human and aquaculture-pathogenic Vibrio species, deter-
mining the bacteriostatic versus bactericidal mode of
action, conducting a preliminary chemical characterization
of the active molecule(s) involved, and finally assessing its
safety for marine aquacultural applications.
Materials and methods
Culture media and chemicals
Marine agar 2216 (MA), trypticase soy agar (TSA), try-
pticase soy broth (TSB), peptone and yeast extract were
purchased from Difco. Marine broth 2216 (MB) was
purchased from HiMedia. The Vibrio selective medium
thiosulfate citrate bile salts sucrose agar (TCBS) was pur-
chased from Britania. Solvents for HPLC were all HPLC
grade, water and methanol were purchased from J.
T. Baker, while acetonitrile and trifluoroacetic acid (TFA)
were purchased from Merck. Glycerol (p.a.), ethyl acetate
(p.a.) and all salts (p.a.) were purchased from Merck.
Mussel sampling and bacterial isolations
Twenty specimens of the mussel P. purpuratus were col-
lected from the exposed intertidal zone in the Dalcahue
inlet, Chiloe Island, Southern Chile (42�21044.900S,
73�36020.300W), on May 6, 2009. Animals were transported
back to the laboratory at field temperature (approximately
13 �C) a period of approximately 4 h. The mussels were
pooled, the tissues were removed from the shells using
aseptic instruments, placed in a sterile blender, and broken
up at high speed for 90 s in accordance with current marine
bacteriological analyses methods (Bacteriological Analyt-
ical Manual, U.S. Food and Drug Administration). Culti-
vatable marine heterotrophic bacteria and Vibrio sp. counts
in Dalcahue-intertidal-mussel tissue (DIT) were conducted
using tenfold serial dilutions in phosphate buffered saline
(PBS) solution (g/L: NaCl 8, KCl 0.2, Na2HPO4 1.44,
KH2PO4 0.24, adjusted at pH 7.4 with HCl), followed by
incubation in either MA (20 �C for 48 h) or TCBS (35 �C
for 24 h). Sixty-four cultivatable marine bacteria were
collected from MA and 32 putative Vibrio sp. from TCBS.
Isolated bacteria were re-isolated by colony spreading in
MA to ensure axenic cultures, which were later incubated
in a deep 96-well microplate with 1.1 mL MB per well at
20 �C for 24 h on a microplate vibrator (GFL 3023).
Finally the isolated bacteria were cryopreserved at -80 �C
with 20 % glycerol.
Screening for antagonistic activity
Cryopreserved bacteria were added with a 96-pin replicate
to fresh 1.1 mL MB per well and incubated at 20 �C for
24 h in the microplate vibrator. After incubation, the
microplate was centrifuged at 2,200g for 10 min and ali-
quots of supernatants (50 lL) were transferred to 6 mm
diameter wells (arranged in 4 9 2 format) on 100 mm
diameter Petri dishes containing 20 mL trypticase soy agar
plus NaCl 3 % (TSA-3) preinoculated with V. parahae-
molyticus tdh negative (ATCC 17802) (bacterial suspen-
sion at 0.5 McFarland spread over agar surface with a
cotton tip). Inhibition zones were observed after 24-48 h of
incubation at 20 �C. Antagonistic spectra in active extracts
were later studied using the human or aquaculture patho-
gens V. parahaemolyticus tdh positive (RIMD 2210633),
V. cholerae (environmental isolated), V. anguillarum
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123
(environmental isolated), Yersinia ruckeri (environmental
isolated) and Pseudomonas aeruginosa (ATCC 27853), with
Escherichia coli K12 and also with Staphylococcus aureus
(ATCC 6538-4), S. epidermidis and a beta-hemolytic
Streptococcus sp. (clinical isolates). These additional assays
were performed at 20 �C in TSA, with the exceptions of V.
anguillarum and V. parahaemolyticus tdh positive, which
used TSA amended with 2 or 3 % NaCl, respectively.
Preliminary morphological and physiological
characterization of active isolates
Temperature-dependant growth assays were conducted in MA
and MB at 5, 15, 20, 25, 30 and 33 �C. MA was used for
colony morphology observations and Gram staining. An
antibiotic susceptibility assay was conducted by the paper disk
method in 100 mm diameter Petri dishes containing 20 mL
TSA-3 pre-inoculated with a 0.5 McFarland suspension of
each active isolate, followed by a 24 h incubation at 20 �C.
The antibiotics assayed (in 6 mm-diameter discs) were:
erythromycin (15 mg/disc), florfenicol (30 mg/disc), strepto-
mycin (10 mg/disc), chloramphenicol (30 mg/disc), flumeq-
uine (30 mg/disc), oxolinic acid (2 mg/disc), oxytetracycline
(30 mg/disc) and amoxicillin (25 mg/disc). The NaCl ranges
for growth were determined in marine broth according to the
ZoBell 2216E formula (g/L: peptone 5, yeast extract 1, ferric
citrate 0.1, magnesium chloride 8.8, sodium sulfate 3.24,
calcium chloride 1.8, potassium chloride 0.55, sodium bicar-
bonate 0.16, potassium bromide 0.08, strontium chloride
0.034, boric acid 0.022, sodium silicate 0.004 sodium fluoride
0.0024, ammonium nitrate 1.6, disodium phosphate 0.008)
containing NaCl at 5, 10, 15, 20, 25 and 30 g/L.
Amplification of 16S rRNA genes and sequencing
Bacterial colonies grown overnight over TSA-3 were
homogenized en Milli Q water, boiled for 20 min and
centrifuged at 10,000g for 10 min. Supernatants were used
as DNA templates for PCR with the primers 1492R (50-GGT TAC CTT GTT ACG ACT T-30) (universal) and 27F
(50-AGA GTT TGA TCC TGG CTC AG-30) (specific for
Bacteria) (Lane 1991). The thermal profile consisted of
95 �C for 5 min followed by 35 cycles of 94 �C for 30 s,
55.6 �C for 90 s and 72 �C for 90 s and a final extension at
72 �C for 5 min. PCR products were purified by AxyPrep�
PCR Cleanup Kit (Axygen Biosciences) following the
manufacturers instructions. The sequencing reactions were
performed by Macrogen Inc (http://www.macrogen.com)
using the primer 1492R and later 27F (for nearly the full
length 16S rRNA gene sequence). The ABIRPRISMR Big
dye terminator v3.1 cycle sequencing kit (Applied Bio-
systems) was used following the manufacturers protocols.
The PCR cycle sequencing products were purified by the
ethanol precipitation method. The 16S rRNA gene
sequences were determined by a 3730XL DNA Analyzer
(Applied Biosystems).
Phylogenetic analysis based on 16S rRNA gene
sequence
Phred software (version 19.0) was used as base caller and
to assign quality values on sequences (Ewing et al. 1998).
Assembling and aligning of 16S rRNA gene sequence data
was performed with the program MEGA4 (Tamura et al.
2007). The retrieval of homologue sequences from Gene-
Bank/EMBL/DDBJ/PDB databases was performed during
March 2011 using the Basic Local Alignment Search Tool
(BLAST) with the MEGABLAST algorithm (Benson et al.
2000). Sequence affiliations were supported by phyloge-
netic analyses in PAUP 4.0b10 (Sinauer Associates, MA,
USA) with maximum likelihood (ML), maximum parsi-
mony (MP) and neighbour-joining (NJ) criteria (tree reli-
abilities supported by 1,000 bootstrap replicates calculated
for each criteria).
Sequence accession numbers
The sequences for 16S rRNA genes of Pseudoalteromonas
sp. strains DIT09, DIT44 and DIT46 obtained in this study
have been deposited in GenBank under the accession
numbers HQ199601, HQ199602 and HQ199603.
Growth and time course of antibiotic activity
Incubations of active bacteria DIT 09, DIT44 and DIT 46
were conducted in triplicate in 1,000 mL MB by inoculation
at 104 c.f.u./mL and incubation with orbital shaking
(150 rpm) at 20 �C for 170 h. Aliquots of 1.0 mL were taken
to follow growth using viable bacterial counts in MA and
photometric measurements (OD at 630 nm). Time course
antibacterial activities were monitored through relative anti-
V. parahaemolyticus (ATCC 17802) activity units (a.u.). To
this end the inhibition zones of 50 lL cell-free extracts
(obtained by centrifugation at 10,000g for 5 min) and their
twofold serial dilutions (until 1:64) in PBS were measured as
described above. Values of a.u. were defined as the maximal
twofold dilution able to induce an inhibition zone of at least
9 mm in diameter (an average value of the last twofold
dilutions was assigned if the detectable inhibition zone of the
more diluted sample was less than 9 mm in diameter).
Exploration of bactericidal or bacteriostatic mechanism
against V. parahaemolyticus
Unreplicated 24 h incubation in 1.0 L MB (20 �C,
150 rpm) of active bacteria DIT 09, DIT44 and DIT 46
World J Microbiol Biotechnol (2012) 28:2365–2374 2367
123
were performed. Cell-free extracts were collected by cen-
trifugation at 2,200g for 20 min, followed by pressure
driven filtration with a 0.22 lm MCE membrane (MS).
Minimal inhibitory concentrations (MIC) were determined
by dilution in TSB with NaCl 3 % (5 mL for final volume
with 50, 40, 30, 20, 10 and 5 % v/v of cell-free extracts)
followed by inoculations with V. parahaemolyticus (ATCC
17802) and incubations at 20 �C during 24 h. Minimal
bactericidal concentrations (MBC) for V. parahaemolyticus
were then determined after growth of 1:1,000 re-inoculated
negative MBC tubes in fresh 5 mL MB (48 h-incubations
at 20 �C).
Exploration of chemical nature of antibiotic
molecule(s)
The apparent molecular mass of active molecule(s) in
500 lL cell-free extracts of active bacteria DIT 09, DIT44
and DIT 46 were determined by ultra-filtration perme-
ability analysis on MicroconR centrifugal filter membranes
(Millipore) at 3, 10, 30 and 50 kDa cut-off according to
manufacturer specifications. Ultrafiltrations were con-
ducted until permeation of 320–360 lL (140–180 lL of
retentates). On the other hand, ammonium sulphate pre-
cipitation was performed in 5 mL crude extracts incubated
at 20 �C for 5 h with ammonium sulphate at 30, 50, 70 and
90 % saturation in independent experiments, followed by
centrifugation at 10,000g for 30 min and resuspension of
pellet in 500 lL PBS. The amounts of ammonium sulphate
added were calculated on line (http://www.encorbio.
com/protocols/AM-SO4.htm) (EnCor Biotechnology Inc,
Fla, USA). Additionally, cell-free extracts (5 mL) were
extracted with an equal volume of ethyl acetate (EtAc) to
verify partition coefficients for activities. Relative anti-V.
parahaemolyticus (ATCC 17802) activity units (a.u.) of
crude extracts, ultrafiltration retentates, ultrafiltration per-
meates, ammonium sulphate-precipitated and non-precipi-
tated fraction, as well as organic and aqueous extracts after
EtAc extraction, were all measured as described above
(except that activity of organic extracts was examined
through dry 50 lL impregnated paper disks).
Analytical bio-chromatography
Cell-free extracts of the tree selected active DITs isolates
were fractioned by HPLC and screened for anti-V. para-
haemolyticus activity. To this end, 200 mL of previously
collected cell-free extracts were twice extracted with
200 mL ethyl acetate. Aqueous fractions were freeze-dried
(Labconco lyophilizer) and then solubilized in 20 mL
methanol by vortexing and bath sonication. Non soluble
substances (probably salts from MB) were retained on filter
paper (Advantec 131) and washed with 10 mL methanol.
After vacuum evaporation (SpeedVac, Labconco), samples
were solubilized in 4 mL methanol and filtered on PDVF
membrane 0.22 lm (Millex, Millipore). The activity of
both crude and processed extracts were measured by a.u. as
previously described. HPLC (Waters 600 system and 2998
photodiode array detector) of methanolic extracts were
conducted in a reversed-phase C18 column (Atlantis dC18
3 lm, 4.8 9 150 mm) equilibrated with 0.1 % trifluoro-
acetic acid (TFA) at 35 �C. Samples (200 lL) were
injected at 0.5 mL/min (0–6 min) to optimize column
loadings. Elution of samples was made with acetonitrile-
0.1 % TFA through a concave gradient (0 to 100 %, curve
7) from 6 to 66 min at a flow rate of 1.5 mL/min. Fractions
were obtained every 0.5 min in a fraction collector (Gilson
FC204) on a deep 96-well microplate. Aliquots (100 lL)
were transferred (8-channel pipetting) to 6 mm filter disks
on a 96-well round bottom microplate, dried by vacuum
evaporation (SpeedVac Labconco with a microplate rotor)
and then deposited on targeting pre-inoculated plates to
measure the diameter of inhibition zones with V. para-
haemolyticus (ATCC 17802). Based on the results, bio-
chromatographies were repeated at 15 % acetonitrile-
0.1 % TFA as a starting condition in order to raise the
resolution of bioactive molecules.
Effect of ferric ion addition upon antibacterial activity
Experiments were conducted in order to evaluate any
possible competitive effect of iron on antibacterial activity.
To this end, inhibition zones of cell-free extracts and their
twofold serial dilutions were evaluated against V. para-
haemolyticus (ATCC 17802) inoculated as described pre-
viously on TSA-3 alone and amended with FeCl3 6[H2O] at
0.1, 0.2, 0.4, 0.8, 1.0, 1.4 and 1.8 g/L. Incubations were
performed as described previously.
Bioassays for acute toxicity and pathogenic properties
of strains DIT09, DIT44 and DIT46
Short-term aquatic toxicity of cell-free extracts and long-
term pathogenic properties of strains DIT09, DIT44 and
DIT46 were performed on 12–24 h old nauplii of the brine
shrimp Artemia franciscana (Crustacea, Branchiopoda,
Anostraca) (Ruebhart et al. 2007). Acute toxicity tests on
nauplii (10 individuals per 10 mL) were performed in a
mixture of 5 mL cell-free extracts of active Pseudoaltero-
monas sp. strains (24 h cultures in MB) and 5 mL filtered
(0.2 lm) and sterilized (by autoclaving) sea water in 6-well
multiwell plates. After 24 and 48 h incubations (20 �C,
under gently orbital shaking) the nauplii were considered
dead if no movement of the appendages was observed after
15 s. Three replicates were used for each cell-free extract
and controls (50 % MB and pure filtered sterile sea water).
2368 World J Microbiol Biotechnol (2012) 28:2365–2374
123
Pathogenic properties of the bacteria were assayed at
*5 9 103 c.f.u./mL in filtered sterile sea water by 1:500
inoculation of 0.5 McFarland bacterial cultures (MB and
filtered sterile sea water were 1:500 inoculated as controls).
In this case, nauplii were also fed at the beginning of
incubation with the marine microalgae Dunaliella tertio-
lecta (5 9 104 cell/mL). Mortalitiy of nauplii were deter-
mined every 24 h over a period of 5 days. Possible growth
of strains were followed by viable bacterial counts at the
beginning and the end of incubations.
Results
Isolation and preliminary characterization of active
bacteria
The Dalcahue-intertidal mussel-tissue (DIT) exhibited
viable counts in marine agar (MA) and in TCBS of
1.8 9 105 c.f.u./g and 1.8 9 103 c.f.u./g, respectively. The
screening for antagonistic activity revealed that 28 % of
isolated bacteria from MA (18 isolated bacteria) exhibited
antagonistic activity against V. parahaemolyticus, while
TCBS-isolated bacteria (putatively Vibrio sp.) were all
inactive against this target. All active isolated bacteria
exhibited relatively narrow spectra of antagonistic activity,
resulting in the inhibition of all Vibrio sp. assayed (V.
parahaemolyticus tdh positive, V. parahaemolyticus tdh
negative, V. cholerae and V. anguillarum) but not of spe-
cies of other genera Yersinia ruckeri, Pseudomonas aeru-
ginosa, Escherichia coli, Staphylococcus aureus, S.
epidermidis and a beta-hemolytic Streptococcus sp. These
active bacteria all exhibited reddish brown-orange colonies
in MA, and were Gram negative, oxidase positive, able to
grow at temperatures ranging from 3 �C to 30 �C (with no
growth at 33 �C) and able to growth in all NaCl concen-
trations assayed (5 to 30 g/L). Finally, the tests for anti-
biotic susceptibility revealed that all active bacteria were
sensitive to the majority of antibiotics used on Chilean
salmon farms (Miranda and Zemelman 2002) with the
exception of erythromycin, for which all active strains
exhibited an intermediate resistance.
Identification of active isolated bacteria based on 16S
rRNA gene sequences
The eighteen active isolated bacteria were identified as
Pseudoalteromonas sp. according to the partial 16S rRNA
gene sequences. Length of accurate sequences (Phred
quality scores higher than 35) ranged from 120 to 860 pb
with almost no significant differences between their bp
assignments. Based on the best quality 16S rRNA gene
sequences, PCR products for strains DIT09, DIT44 and
DIT46 were selected for a near complete sequencing of the
16S rRNA gene, resulting in 99.5–99.8 % identical bp
assignment across the strains. The phylogenetic tree of
these three sequences indicates a well supported clade with
Pseudoalteromonas sp. isolated from deep sea sediment of
the south-west Pacific (EU823307) and Arctic seawater
(EUFJ889603, HQ882787) (unpublished), Pseudoaltero-
monas prydzensis (99 % identity) isolated from Antarctic
sea ice (Bowman 1998) and P. mariniglutinosa (98 %
identity) (Romanenko et al. 2003) (Fig. 1).
Time course production of antimicrobial agent
Anti-V. parahaemolyticus activity of Pseudoalteromonas
sp. DIT09, DIT44 and DIT46 reached average maximum
values at the end of exponential phase (24 h culture incu-
bation). Beyond that point, activity progressively declined
during the stationary phase to become undetectable in the
cell-free fraction after 4 days (Fig. 2).
Determination of bactericidal or bacteriostatic effects
Cell-free extracts of new unreplicated cultures were col-
lected after 24 h-incubations, with 24 a.u./50 lL for strains
DIT09 and DIT46, and 16 a.u./50 lL for strain DIT44.
These three extracts exhibited a minimal inhibitory con-
centration (CMI) of 20 % v/v on V. parahaemolyticus tdh
positive and tdh negative. No bactericidal effects were
detected (below 50 % v/v), indicating a bacteriostatic but
not a bactericidal effect on the target strains.
Chemical characterisation of bioactive molecules
The active agents of Pseudoalteromonas sp. strains DIT 09,
DIT44 and DIT 46 were mostly permeable through mem-
branes with 3 kDa cut-off. Relative activity units for per-
meates were no different to the values observed prior to
ultrafiltration (24 a.u./50 lL for strains DIT09 and DIT46,
and 16 a.u./50 lL for strains DIT44). However, a slight
increase in activity levels was detected in retentates (32
a.u./50 lL for strains DIT09 and DIT46, and 24 a.u./50 lL
for strains DIT44), suggesting that the apparent molecular
mass of the active agents in the three strains were close to
3 kDa. On the other hand, the active agent of strain DIT09
(240 a.u. in 500 lL of cell-free extract) was partially pre-
cipitated with ammonium sulphate at 70 and 90 % satu-
ration. At 70 % salt, activity recovered after precipitation
and pellet re-suspension reaching 8 % (20 a.u.), while
67 % of activity (160 u.a.) was recovered after precipita-
tion at 90 % salt. Similar observations were made with
strains DIT44 and DIT46. In addition, anti-Vibrio activity
with the three strains was not recorded with EtAc, as
100 % a.u. remained in the aqueous phase.
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Analytical HPLC of active extracts
Measured anti-Vibrio activities of methanol stabilized
extracts for Pseudoalteromonas sp. strains were 20–34 %
higher than values for unprocessed aqueous cell-free
extracts. These values were interpreted as an overestima-
tion of nearly 100 % recovery. These overestimations were
likely due to inhibition zone deviations between the
methods used for aqueous versus organic extracts (based on
50 lL wells for aqueous and 20 lL impregnated paper
disks for organic extracts). Reverse phase HPLC with
acetonitrile gradient from 0 to 100 % revealed the exis-
tence of 3 or 4 peaks associated with anti-Vibrio activity,
which eluted at the end of complex chromatograms while
acetonitrile-0.1 % TFA were pumped at approximately
40 % (not shown). When chromatographies were repeated
at higher resolutions (gradients from 15 to 100 %), patterns
with several bioactive molecules were eluted between 27
and 41 min, while acetonitrile-0.1 % TFA pumped ranged
between 23 and 43 %. These chromatograms were very
similar but not identical, with 3 clearly dominating
bioactive molecules for strain DIT09, 6 bioactive mole-
cules well differentiated for strain DIT44 and finally 5
clearly dominating bioactive molecules for strain DIT46
(Fig. 3). Ultraviolet absorption spectral analyses of active
peaks indicated that active compounds eluted were impure.
Taking this into account, UV spectra still suggest a low
aromaticity of the involved molecules, with hight absorp-
tion at 220 nm, minimal absorption at 260 nm, followed by
a weak broad spectral band at 280 nm, and no absorption
beyond 320 nm (not shown).
Effect of iron (III) on anti-V. parahaemolyticus activity
Experiments demonstrated an iron-dependent reversion of
anti-V. parahaemolyticus activity on cell-free extracts of
Pseudoalteromonas sp. DIT09 (Fig. 4), DIT44 and DIT 46
(not shown), which occurred as expected for a competitive
mechanism between iron (III) and the anti-Vibrio agents.
Experiments were also conducted in 1.4 and 1.8 g/L FeCl36[H2O] (5.2 and 6.7 mM), but these concentrations
Fig. 1 Neighbour-joining tree indicating the phylogenetic relation-
ships inferred from partial 16S rRNA gene sequences (E. coli position
38–1,434) of strains DIT09, DIT44 and DIT46 (boldface type), other
Pseudoalteromonas species (c subclass of Proteobacteria) and some
outlooks. Bootstrap support values for three phylogenetic criteria
(ML/MP/NJ) are shown adjacent to the corresponding node. A dash (-
) indicates bootstrap support of less than 50 %. GenBank accession
numbers are shown in parenthesis. The tree was rooted with the
Desulfobacter vibrioformis (d subclass of Proteobacteria)(NR_029177). Knuc value = 0.01
2370 World J Microbiol Biotechnol (2012) 28:2365–2374
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appeared to be toxic as V. parahaemolyticus was unable to
grow.
Acute toxicity and pathogenic properties of active
strains
Cell-free extract of strains DIT09, DIT44 and DIT46
(diluted at 50 % in filtered sterile sea water) did not cause
any mortality of Artemia franciscana nauplii. Furthermore,
long term incubations with 3.3 9 103 ± 2.2 9 103 c.f.u./
mL of strains did not cause any mortality, even though
bacterial viable bacterial counts reached 3.3 9 104 ± 8.7
9 103 c.f.u./mL at day 5.
Discussion
Marine bacteria belonging to the genus Pseudoalteromonas
of the class Gammaproteobacteria have attracted particu-
lar interest due to their ability to produce a variety of
biologically active compounds that have antibacterial
(Hayashida-Soiza et al. 2008; Holmstrom and Kjelleberg
1999), antifouling (Yee et al. 2007), antibiofilm (Dheilly
et al. 2010) properties, in addition to a number of other
pharmaceutically relevant compounds (Bowman 2007).
Pseudoalteromonas spp. had been identified as marine
bacteria with the ability to produce antimicrobial agents
even before the genus was officially established (Nair and
Simidu 1987; Barja et al. 1989; Gauthier et al. 1995).
However, so far studies have been limited to a relatively
small subset of Pseudoalteromonas strains of the currently
much larger known diversity of the genus (Bowman 2007).
The unique group of strains producing antibacterial
compounds detected from the intertidal sample of P. pur-
puratus consisted of 18 Pseudoalteromonas sp., all highly
homogeneous in terms of morphology, physiology and
genetics. All these active strains exhibited identical anti-
biotic sensitivity patterns as well as identical antimicrobial
action spectra. The antibiotic properties observed were
targeted against V. parahaemolyticus tdh positive, V.
parahaemolyticus tdh negative, V. cholerae and V. an-
guillarum, and were inactive against Yersinia ruckeri,
Pseudomonas aeruginosa, Escherichia coli, Staphylococ-
cus aureus, S. epidermidis and a beta-hemolytic Strepto-
coccus sp. This finding suggests a relative specificity for
bacteria belonging to the Vibrio genus, however this
statement requires further confirmation with more Vibrio
spp. and ideally more strains of pathogenic Vibrio spp. In
reality, some degree of heterogeneity in the susceptibility
of the genus is to be expected, similar to that reported for
Pseudoalteromonas sp. strain S2V2, a potential Vibrio-
biocontroling agent isolated from Indonesian coastal sam-
ples, which exhibited antibacterial properties on several but
not all Vibrio species, even displaying some variability
within individual species such as V. fluvialis, V. anguilla-
rum and V. metschnikovii (Isnansetyo et al. 2009).
From our active bacteria collection, we selected the
three Pseudoalteromonas sp. strains DI09, DIT44 and
DIT46 based on a random criteria combined with their
availability for near full length 16S rRNA gene sequences.
These sequences were practically identical and belonging
to the previously well defined Pseudoalteromonas clade of
P. prydzensis and P. mariniglutinosa (Bowman 2007),
species which previously had been unrecognised as anti-
biotic producing species. The scarce nucleotide assignment
differences of the 16S rRNA gene sequences for the three
selected active strains suggests that they were derived from
the same bacterial population. However we decided to
continue studying the three selected strains because there
may have been differences between them and to provide
more significance to the results.
Antibiotics produced by strains DIT09, DIT44 and
DIT46 were released to the culture media and reached a
Fig. 2 Growth of strains DIT09 (upper), DIT44 (middle) and DIT46
(lower), measured by viable bacterial count and optical density (OD)
at 630 nm. Time course of antibiotic production was measured by
relative anti-V. parahaemolyticus (ATCC 17802) activity units (a.u.)
in 50 lL of cell-free extracts
World J Microbiol Biotechnol (2012) 28:2365–2374 2371
123
maximum at the end of the exponential phase. The active
agents were precipitable in the presence of 70 and 90 %
ammonium sulphate, were permeable through membranes
with 3 kDa cut-off and had good solubility in methanol.
Furthermore, the active agents were not extracted with
ethyl acetate though they were strongly retained with C18
columns, facts than indicate the presence of both hydro-
phobic and hydrophilic moieties in the active agents
(amphiphilic molecules). HPLC analyses revealed the
production of a set of active compounds by each strain,
which exhibited similar but non identical chromatograms
probably due to: (1) slight differences in the physiology of
strains, (2) normal culture variations of the same bacteria
or (3) variability in chromatographies due to any matrix
effect by the extracts loaded. Which of these alternative
explanations applies will only be determined after the
complete purification and structural characterisation of the
active molecules for the three strains, combined with
HPLC profiling at different culture times.
Fig. 3 Analytical reversed phase-HPLC of cell-free extracts for strains DIT09 (upper), DIT44 (middle) and DIT46 (lower)
Fig. 4 Apparent anti-V. parahaemolyticus activity (a.u.) of Pseud-oalteromonas sp. DIT09 cell-free extract in trypticase soy agar 3 %
NaCl amended with FeCl3 at different concentrations
2372 World J Microbiol Biotechnol (2012) 28:2365–2374
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The sets of antibiotic molecules produced by Pseud-
oalteromonas sp. strains DIT09, DIT44 and DIT46 exhib-
ited bacteriostatic but not bactericidal effects on V.
parahaemolyticus. This kind of antibacterial antibiotic
activity against Vibrio bacteria, including weak activity
against V. parahaemolyticus, has been described with the
marine Pseudoalteromonas sp. A1-J11 (Del Castillo et al.
2008b). Bacteriostatic antibiotics that we have described
should have a different chemical structure as the bacte-
riostatic agent produced by Pseudoalteromonas sp. A1-J11
was extracted by the organic solvent EtAc (Del Castillo
et al. 2008a) unlike those described here. The antibiotic
described for strain A1-J1 was structurally identified as
2-n-pentyl-4-quinolol and its mode of action remains
unknown, except that the length of the alkyl side chain of
the compound played an important role in the anti-bacterial
activity (Del Castillo et al. 2008b). Furthermore, the UV
spectra of the active chromatographic peaks of the active
extracts of DITs strains looked similar to those expected
for a peptide containing some aromatic amino acids, and
were very different to those measured for 2-n-pentyl-4-
quinolol (Del Castillo et al. 2008a, b).
On the other hand, the bacteriostatic mode of action of
the antibiotics suggests a possible siderophore-like struc-
ture for produced anti-Vibrio agents (Vraspir and Butler
2009). This hypothesis was supported with the clear
decline in apparent anti-V. parahaemolyticus activity (a.u.)
in TSA-3 amended with different FeCl3 concentrations,
which resembled the competitive mechanism between iron
(III) and the anti-Vibrio agents. However, the hypothesis of
a siderophore like structure implies that the active agents
can selectively out-compete ferric chelating molecules
produced by the sensitive targets detected, such as the
weak iron chelator vibrioferrin in V. parahaemolyticus
(Amin et al. 2009) and, by extension vibriobactin in V.
cholerae and anguibactin in V. anguillarum (Miethke and
Marahiel 2007). Furthermore, they should be unable to
outcompete iron uptaking molecules of the insensitive
targets assayed (Yersinia ruckeri, Pseudomonas aerugin-
osa, Escherichia coli or the Gram positive Staphylococcus
aureus, S. epidermidis and Streptococcus sp.). An alter-
native hypothesis explaining the decline in apparent anti-V.
parahaemolyticus activity in the presence of FeCl3 is that
the set of active molecules detected target at Fe-sidero-
phore uptake system probably being down-regulated inside
V. parahaemolyticus incubated in an excess of this metal.
Whatever the explanation, the production of bacteriostatic
agents by these DIT strains represents a putatively novel
class of antibiotic molecules produced by Pseudoaltero-
monas sp. Based on our findings, the expected chemical
nature of the active agents will encompass small amphi-
philic peptides probably containing some aromatic amino
acids as well as possible peptide-like amphiphilic
siderophores produced by bacterial nonribosomal peptide
synthetases (Vraspir and Butler 2009; Martinez and Butler
2007; Miethke and Marahiel 2007).
Bacteria belonging to Pseudoalteromonas genus have
been widely recognized as active against pathogenic bac-
teria belonging to the Vibrio genus (Vynne et al. 2011;
Dheilly et al. 2010; Gram et al. 2009; Isnansetyo et al.
2009; Del Castillo et al. 2008a; Longeon et al. 2004).
Based on the urgency of the search for alternative antibi-
otics to control Vibrio it is in our interest to continue
studying the use of Pseudoalteromonas DIT strains as
potential biocontrol agents of V. anguillarum in marine
aquaculture. In addition, the use of these strains as potential
biocontrol agents of human pathogenic V. parahaemolyti-
cus or V. cholerae, and possibly V. vulnificus if it proves to
be sensitive should also be explored. The fact that the
active DIT strains did not cause any mortality in Artemia
franciscana nauplii and neither did the antibiotic contain-
ing extracts, is also positive.
In conclusion, we report Pseudoalteromonas sp. strains
DIT09, DIT44 and DIT 46 as potential Vibrio-biocontrol-
ling agents. These strains are producers of a set of a pos-
sibly novel class of bacteriostatic anti-Vibrio antibiotics
and are phylogenetically closely related to P. prydzensis
and P. mariniglutinosa. Research will continue through
antibiotic purification followed by their structural and
functional characterisation.
Acknowledgments This work was supported by Chilean ‘‘Fondo de
Fomento al Desarrollo Cientıfico y Tecnologico’’ (FONDEF
D06I1054). The authors thanks to Matthew Lee for his critical review
of this manuscript.
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