Research in Microbiology 165 (2014) 647e656

www.elsevier.com/locate/resmic

Original article

The Pseudomonas community in metal-contaminated sediments as revealed

by quantitative PCR: a link with metal bioavailability
St
ephanie Roosa a, Corinne Vander Wauven c, Gabriel Billon b, Sandra Matthijs c, Ruddy Wattiez a,
David C. Gillan a,*
a
Proteomics and Microbiology Lab, Research Institute for Biosciences, Universit
e de Mons, 20 Place du Parc, B-7000 Mons, Belgium
b
G
eosystemes Lab, UFR de Chimie, Lillee1 University, Sciences and Technologies, 59655 Villeneuve d'Ascq, France
c
Institut de Recherches Microbiologiques JMW, 1 Av. E. Gryzon, 1070 Bruxelles, Belgium
Received 21 July 2014; accepted 21 July 2014
Available online 4 August 2014

Abstract

Pseudomonas bacteria are ubiquitous Gram-negative and aerobic microorganisms that are known to harbor metal resistance mechanisms such
as efflux pumps and intracellular redox enzymes. Specific Pseudomonas bacteria have been quantified in some metal-contaminated environ-
ments, but the entire Pseudomonas population has been poorly investigated under these conditions, and the link with metal bioavailability was
not previously examined. In the present study, quantitative PCR and cell cultivation were used to monitor and characterize the Pseudomonas
population at 4 different sediment sites contaminated with various levels of metals. At the same time, total metals and metal bioavailability (as
estimated using an HCl 1 M extraction) were measured. It was found that the total level of Pseudomonas, as determined by qPCR using two
different genes (oprI and the 16S rRNA gene), was positively and significantly correlated with total and HCl-extractable Cu, Co, Ni, Pb and Zn,
with high correlation coefficients (>0.8). Metal-contaminated sediments featured isolates of the Pseudomonas putida, Pseudomonas fluorescens,
Pseudomonas lutea and Pseudomonas aeruginosa groups, with other bacterial genera such as Mycobacterium, Klebsiella and Methylobacterium.
It is concluded that Pseudomonas bacteria do proliferate in metal-contaminated sediments, but are still part of a complex community.
2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Pseudomonas; Sediments; Metals; Bioavailability; Resistance; Quantitative PCR

1. Introduction to antibiotics [6], leading to the expansion of nosocomial

Pseudomonas bacteria are ubiquitous Gram-negative and
aerobic microorganisms that are member of the g-Proteobac-
teria. These bacteria are found in numerous natural environ-
ments, including soils, sediments and water [1,2], and have
been found in association with various plants and animals [3].
Pseudomonas bacteria are also responsible for different human
infections [4e6] and some species have developed resistance
* Corresponding author.
E-mail addresses: stephanie.roosa@umons.ac.be (S. Roosa), cvdwauve@
ulb.ac.be (C.V. Wauven), gabriel.billon@univ-lille1.fr (G. Billon), slmatthi@
ulb.ac.be (S. Matthijs), ruddy.wattiez@umons.ac.be (R. Wattiez), david.
gillan@umons.ac.be (D.C. Gillan).
diseases [7]. The ubiquity of the genus is a direct consequence
of its high adaptability. For instance, Pseudomonas bacteria
are able to survive in hydrocarbon-, PCB- and PAH-
contaminated environments due to oxidative degradation
processes and biosurfactant production [8,9]. Pseudomonas
spp. are also known to degrade various pesticides [10] and
harbor metal resistance mechanisms such as efflux pumps and
intracellular redox enzymes [11e13]. It is therefore not sur-
prising to find specific Pseudomonas bacteria in metal-
contaminated rivers and sediments [14e16] and these apti-
tudes to react with metals have even led to proposing their use
as bioremediation tools [13,17,18].
Metals are frequent contaminants of aquatic sediments
[19,20]. However, the total concentration of metals in

http://dx.doi.org/10.1016/j.resmic.2014.07.011
0923-2508/ 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
648 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656
sediments is not indicative of any biological effect, as metals
are not always bioavailable [21e26]. For a unicellular or-
ganism, a compound may be defined as bioavailable when it is
able to cross the cellular envelope at a given time [27]. Once
inside the cell, metals may have various effects according to
their concentration and chemical properties [28]. When con-
centration levels are too high, bacteria usually react by the
expression of specific metal resistance systems such as P-type
ATPases, metallothioneins, RND efflux pumps and/or CDF
transporters [13,28,29]. It is therefore expected that bacteria
displaying the most efficient resistance systems are selected in
metal-contaminated environments where metals are
bioavailable.
As a large panel of metal resistance systems was observed
in many Pseudomonas species [13], and given the ubiquity of
the genus and its numerous species, it is anticipated that
Pseudomonas bacteria will have an advantage in sedimentary
environments where metals are bioavailable, an advantage that
may lead to higher biomasses. However, to the best of our
knowledge, the biomasses of Pseudomonas and metal
bioavailability were never assessed in parallel in natural sed-
iments. Pseudomonads have been quantified in the environ-
ment using most-probable-number (MPN) enumeration [30],
fluorescent in situ hybridization [31] and quantitative PCR
(qPCR) [14,32e35], but most of these previous studies were
not performed in metal-contaminated sediments or did not
assess metal bioavailability [32e35]. In a study restricted to
Pseudomonas aeruginosa living in a metal-contaminated river,
the quantification of three genes (a Mn-containing superoxide
dismutase SodA, a heat shock protein HtpX and a metal-
lothionein) was performed and significantly higher copy
numbers were found at the more contaminated sites compared
Fig. 1. Map of the sites and their coordinates: St130: Station 130; FER: F
erin; RAC: R^aches; MET: MetalEurop.
with reference sites [14]. Unfortunately, metal bioavailability
was not assessed.
The aim of the present research was to use quantitative
PCR with specific primers to estimate the biomass of the
whole Pseudomonas community in 4 aquatic sediments
showing contrasted metal bioavailability. At the same time,
total metal levels and bioavailable metals were estimated in
the sediments using, respectively, strong acids and 1 M HCl
treatment. We also used cell cultivation to test the metal
resistance capacities of some Pseudomonas isolates obtained
at the most contaminated station.
2. Materials and methods
2.1. Description of the sites and sediment sampling
Four sites were considered in this study: Station 130
(St130) in seawater and MetalEurop (MET), F
erin (FER) and
R^aches (RAC) in freshwater (Fig. 1). Station 130 is situated in
the North Sea, on the Belgian Continental Plate. It presents
low concentrations of metals in comparison to the other
freshwater sites [36]. The MetalEurop site is the most metal-
contaminated site of the present study. It is located in front
of a smelter in the River Deule (northern France). This plant
was closed in 2003 after decades of accidental discharges into
the river that started in 1893; today, the contamination level of
the site still remains many orders of magnitude higher than
background values [37,38]. The R^aches site is mildly
contaminated and F
erin is also a control site, supposedly un-
contaminated; these two sites are situated in the same river
basin as MetalEurop. All sites selected in this study have a
similar granulometry, oxygen penetration depth, organic
 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656
matter and pH (all sediments are muddy with a mean grain
size < 50 mm and a POC content between 1 and 3%).
Sediment sampling at St130 was performed using a Rein-
eck corer ( 15 cm) onboard the Zeeleeuw research vessel
(June 2011; depth: 11 m). For the freshwater sites, sediments
were collected in the middle of the river using a Plexiglass
tube ( 7 cm) fixed to a stainless steel bar (June 2011;
depth: 3 m). For all sites, three separate cores were obtained
(n 3) and for each core, the wateresediment interface
(0e1 cm) and an anoxic layer (4e6 cm) were immediately
isolated in sterile plastic vessels. Samples were transported to
the lab in dry ice and divided into two parts: one part was
stored at 80  C for biomolecular analyses and the other part
at 20  C for chemical analyses. For bacterial cultures, sub-
samples of sediments were brought back to the laboratory at
4  C and directly used.
2.2. Quantification of metals
2.2.1. Total metals (TE)
Weighed amounts of dried and sieved (63 mm) sediments
were digested with concentrated acids as described elsewhere
[39]. Total metal concentrations were determined using
inductively coupled plasma mass spectrometry (ICP-MS,
Thermo Elemental, X7 series). Only 12 metals or semi-metals
were investigated (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V
and Zn). Standard reference materials (Canadian International
Sediment Standards HISS-1, MESS-3 and PACS-2) were
analyzed in parallel and metal recovery was on an average
better than 90% for HISS-1, 67% for PACS-2 and 83% for
MESS-3. To compare the total metal content at different
sampling sites, a metal pollution index (MPI) was calculated
with the following equation [40,41]:
MPI Cm1 *Cm2 **Cmn =n
where Cmi is the concentration for metal i in the sample and n
is the number of metals considered. In this study, Al, As, Cd,
Co, Cr, Cu, Fe, Mn, Ni, Pb, V and Zn were computed in this
index.
2.2.2. Bioavailable metals (BM) and acid volatile sulfides
(AVS)
Bioavailable metals (BMs) were estimated for 6 metals
(Co, Cu, Mn, Ni, Pb, Zn) using a dilute HCl treatment [42,43].
Although all metals extracted using this method are not
entirely bioavailable, the term bioavailable will be used
below for facility. Briefly, metals were extracted from 1 g of
wet sediment (anoxic) using 1 M HCl for 24 h (in triplicates).
Each sample was then filtrated through a 0.45 mm filter and
metal concentrations were measured using inductively coupled
plasma atomic emission spectroscopy (ICP-AES; Varian, Vista
Pro, axial view). The relative standard deviation of the results
was better than 10%.
Bioavailable metals were also used to calculate a toxicity
index (TI). This index is simply the ratio bioavailable metals/
AVS (acid volatile sulfides), as described in [44]. Indeed, when
649
the molar concentration of AVS exceeds that of the metals
(i.e., the metal/AVS ratio is less than unity), they exist pre-
dominantly as insoluble metal sulfides, which presumably are
not biologically available [44]. For AVS measurement [45],
wet sediments (1 g, anoxic) were mixed with 6 M HCl for 1 h
at 25  C. The H2S gas that was produced was then trapped in a
1 M NaOH solution. The sulfide concentration was measured
with a sulfide-specific electrode (Orion). Results were found to
be reproducible with an analytical precision 15%. To
calculate the TI, concentrations of bioavailable Cd, Co, Cu,
Ni, Pb and Zn were used.
2.3. Quantification of Pseudomonas by quantitative PCR
Before DNA extraction, sediments (24 samples) were
washed using three different buffers in order to remove a
maximum of PCR inhibitors, as described in [46]. For each
sample, DNA was then extracted from 750 mg of sediment
using three PowerSoil DNA extraction columns (Mobio) in
parallel (3  250 mg). DNA extracted from these three col-
umns was then pooled and a final purification step was per-
formed with a Qiaquick purification kit (Qiagen) in order to
increase the quality of the DNA extracts (260/280 and 260/230
ratios above 1.8) and ensure reliable and reproducible qPCR
quantification. DNA quality of the 24 extracts was assessed by
the measurement of the 260/280 nm and 260/230 nm ratios on
a Nanodrop ND-1000. The concentration of the DNA prepa-
rations was evaluated with Picogreen quantification (Invi-
trogen) using a Fluostar Optima microplate reader (BMG
Labtech).
For quantitative PCR (qPCR) two different primer sets
were used. One primer set targets a 249 bp fragment of the
Pseudomonas lipoprotein oprI gene [47,48]: forward primer
PS1 (5'-ATGAACAACGTTCTGAAATTC-3') and reverse
primer PS2 (5'-CTTGCGGCTGGCTTTTTCCAG-3'). The
other primer set targets a 251 bp region of the 16S rRNA gene
located in the V3eV4 hypervariable region at position
435e686 [35]: Pse435F (5'-ACTTTAAGTTGGGAG-
GAAGGG-3') and Pse686R (5'-ACACAGGAAATTCCACC-
ACCC-3'). The real time PCR mixture (20 mL final volume)
contained 10 mL of Power SYBR Green PCR Master Mix
(ABI), 0.4 mL of each primer (final concentration 200 nM),
7.2 mL of sterile water and 2 mL of samples or standards.
Absolute qPCR quantifications were achieved using a Ste-
pOnePlus Real Time PCR System (Applied Biosystem) with
the cycle as follows: the initial denaturation step (95  C,
10 min) was followed by 50 cycles of denaturation at 94  C for
30 s, annealing at 58  C (oprI gene) or 60  C (Pseudomonas
16S rRNA gene) for 30 s, extension at 72 c for 30 s. A melting
curve analysis from 60  C to 95  C was performed on the
product to confirm that only one product was amplified. The
standard curves were made in duplicate with a 10-fold dilution
series of reference plasmids containing the relevant target gene
(oprI or 16S rRNA genes). Each of the 24 DNA extracts was
analyzed in triplicate (technical replicates, 50-fold dilution).
The template copy number in each standard dilution was
calculated using the DNA concentration determined with
650 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656
Picogreen by the equation mentioned in [49]. The gene copy
number of each sample was standardized with the DNA con-
centration measured by Picogreen. Samples with a CT equal or
above the negative control were treated as below the limit of
detection. The mean assay efficiencies was around 93%,
99.5% and 88.6% for oprI, Pseudomonas 16S rRNA and
bacterial 16S rRNA, respectively. The copy number of the
bacterial 16S rRNA gene was also quantified and used at a
ratio oprI/16S rRNA and Pseudomonas 16S rRNA/16S rRNA.
2.4. Isolation of metal-tolerant bacteria and strain
characterization
These analyses were restricted to aerobic sediments in
MetalEurop. Enrichments and isolations were based on a
modified 284 mineral salt medium (1.5% agar) [50]. This
modified medium differed from the original medium [51] by the
substitution of TriseHCl by MOPS-NaOH (pH 7.0) and of
Na2HPO4 by beta-glycerol-phosphate. The carbon source was
also modified by the addition of 0.2% w/v gluconate and 0.2%
w/v glucose. Solid media were plated with increasing concen-
trations of metals. For that, stock solutions (1 M) of NaAsO2,
Cd(NO3)2, CoCl2 6H2O, Cu(NO3)2 3H2O, Pb(NO3)2, NiCl2
6H2O and ZnSO4 were added for a maximum theoretical con-
centration of 5 mM for Co2, 10 mM for AsOe 2 2
2 , Cd , Cu , Ni
2
2 2
and 15 mM for Pb and Zn . Fresh sediments were resus-
pended in sterile water (1:1, v/v) and sonicated 3 times for 30 s.
After centrifugation of 1 min at 1000 rpm, 50 mL of supernatant
were spread on the different solid media. The plates were
incubated at 30  C until distinct colonies appeared on the highest
metal concentrations. Isolates were then taxonomically char-
acterized. Primer pH (AAG GAG GTG ATC CAG CCG CA)
and a slightly shortened pA primer (AG AGT TTG ATC CTG
GCT CAG) [52] were used to amplify the almost complete 16S
rRNA gene (position 29 to 1522 in Escherichia coli). Gene
fragments were sequenced by Beckman Coulter Genomics.
Genus affiliation of the sequences was then assessed from a
BLAST analysis [53]. Sequences of the isolates were then
aligned with the type strains of the species as compiled into the
Euzeby database http://www.bacterio.cict.fr. A neighbor-
joining phylogenetic tree was constructed using the MEGA 5
software package [54] and the closest relative of the isolates was
inferred from their clustering with type strains. The percentage
of identity between an isolate and a type strain was calculated on
the basis of the number of identical residues in the two 16S
rRNA gene sequences.
Metal resistance was evaluated by plating isolates on me-
dium 869 [51] diluted 3 times and enriched with 1 mM Cu, Zn,
Co, Cd, Ni, Pb, As and 5 mM As. The strains that were not
inhibited by 1 mM Cd, Co, Cu, Ni, Pb and Zn [55e57] and
5 mM As were regarded as being resistant.
2.5. Statistical analyses and GenBank accession
numbers
The comparison between sites was done using ANOVA and
post hoc multiple comparison pairwise t tests with the R
software. Pearson correlation coefficients were calculated
between Pseudomonas levels and metal contents using Sta-
tistica 7.0. The sequences obtained in this study have been
deposited in GenBank (see Table 1).
3. Results
3.1. Metal levels in the sediments
Metal levels in the investigated sediments are indicated in
Fig. 2 (in mg kg1 for 6 selected metals) and Table S1 (in
mmol kg1 for 12 metals). Total metal levels were low in
St130 and F
erin, while the most impacted site was Metal-
Europ, especially for Cd, Cu, Pb and Zn. R^aches had a higher
Cu level than MetalEurop. Total metal concentrations for the
0e1 cm layer in St130 reached 0.11 (Co), 0.15 (Cu), 0.11
(Pb) and 1.21 (Zn) mmol kg1. In the same sediment layer of
MetalEurop, total metal concentrations reached 0.15 (Co),
1.57 (Cu), 4.41 (Pb) and 49.23 (Zn) mmol kg1. In this study,
metal bioavailability was estimated by an HCl 1 M treatment
(Fig. 2, Table S1). It can be seen that the concentration of
bioavailable metals was always inferior to the total metal
concentration and that a gradient of bioavailability was pre-
sent from St130 to MetalEurop. In MetalEurop, 9% of the
total Zn may be considered as bioavailable; percentages
reach 19% for Pb and 8% for Cu (Table S1). The metal
pollution index (MPI), calculated with 12 metals, was 65e72
for St130 and F
erin (they were not significantly different),
175 for R^aches and reached 261 for MetalEurop. Clearly, on
the basis of the MPI, the stations formed three separate
groups (St130 & F
erin, R^aches, MetalEurop). The toxicity
index (TI), which takes into account the quantity of sulfides,
was inferior to 1 for all stations. The TI was about 0.11, 0.17,
0.41 and 0.84 for, respectively, St130, F
erin, R^aches and
MetalEurop.
3.2. In situ quantification of Pseudomonas by qPCR and
correlations with metals
For oxic sediments, quantifications of the Pseudomonas
oprI were efficient and specific, as demonstrated by the
presence of a unique peak in the qPCR melt curve (data not
shown). The oprI abundance varied between 6.8 103 4 103
copies per mg of DNA in St130 and 2.5 105 2.6 104 copies in
MetalEurop (Fig. 3). In F
erin, values reached 1.7 104 1 104
copies and for R^aches 7.8 104 4.5 104 copies per mg of DNA.
The oprI gene was not detected in anoxic sediments (values
were under the detection limit).
For Pseudomonas quantifications based on the 16S rRNA
in oxic sediments, the number of copies per mg of DNA was
higher and the same trend was observed: 1.2 106 1.8 106,
1.1 106 2.6 105, 1.7 107 4.9 106 and 3.7 107 1.3 106
copies per mg of DNA for St130, F
erin, R^aches and Metal-
Europ, respectively (Fig. 3). It must be noted that a Pseudo-
monas 16S rRNA signal was also found for anoxic sediments,
but that values were up to 100 times lower and that no
particular trend was observed (data not shown). As indicated
 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656 651

Table 1
Phylogenetic characterization of the bacterial strains isolated in MetalEurop sediments.
Class Genus Strain Closest species (16S rRNA alignment) % Identity Accession
(BLAST) (BLAST)
Firmicutes Bacillus SR58 B. aryabhattai EF114313.2 99 KF866096
Actinobacteria Mycobacterium SR32 M. vaccae AF480591.1/ 97 KF866111
SR33 Undetermined 99 KF866112
SR34 M. llatzerense AJ746070.2 99 KF866113
Rhodococcus SR42 R. erythreus X79289.1 99 KF866119
Streptomyces SR41 S. coelicoflavus AB184650.1 100 KF866120
a-Proteobacteria Sphingomonas SR30 S. xenophaga X94098 99 KF866134
Methylobacterium SR13 M. extorquens AB175631.1.1435 99 KF866106
SR24 M. radiotolerans AB175641.1. 99 KF866107
g-Proteobacteria Klebsiella SR15 K. oxytoca AF129440.1 99 KF866101
SR53 K. oxytoca AF129440.1 99 KF866102
SR55 K. oxytoca AF129440.1 99 KF866103
SR57 K. planticola AF129443.1 99 KF866105
SR69 K. ornithinolytica U78182.1 99 KF866104
Enterobacter SR16 E. minipressuralis Z96077.1 99 KF866098
SR65 E. mori EU721605.2 99 KF866100
Pseudomonas SR14 P. nitroreducens ATCC 33634T 99 KF866123
SR18 P. monteilli AF0644458 100 KF866124
SR44 P. putida D84020.1 98 KF866125
SR45 P. putida D84020.1 99 KF866126
SR54 P. lutea AY364537.1 98 KF866127
SR56 P. lutea AY364537.1 98 KF866128
SR64 P. lutea AY364537.1 98 KF866129
SR66 P. putida D84020.1 98 KF866130
SR67 P. arsenicoxydans FN645213.1 99 KF866131
SR68 P. putida D84020.1 98 KF866132
Aeromonas SR28 A. salmonicida AY987751.1 99 KF866086
b-Proteobacteria Delftia SR27 D. lacustris EU888308.1 99 KF866097
Undetermined Undetermined SR35 Undetermined / /

in Fig. 3, there was good agreement between the two Pseu-
domonas qPCR quantification methods (linear regression,
r2 0.94).
Pseudomonas oprI gene levels were significantly and
positively correlated (Pearson) with total levels of 8 metals
(As 0.94, Cd 0.85, Co 0.91, Cr 0.89, Cu 0.73, Ni 0.91, Pb 0.99
and Zn 0.98). Low and/or non-significant values were
observed for total levels of Al, Fe, Mn and V. Pseudomonas
16S gene levels were significantly and positively correlated
with total levels of the same 8 metals (As 0.92, Cd 0.91, Co
0.95, Cr 0.89, Cu 0.82, Ni 0.95, Pb 0.97 and Zn 0.99).
Similarly, low and/or non-significant values were observed for
total levels of Al, Fe, Mn and V.
For HCl-extractable metals (6 metals investigated), Pseu-
domonas oprI gene levels were significantly and positively
correlated (Pearson) with levels of 5 metals (Cu 0.99, Co 0.90,
Ni 0.99, Pb 0.99, Zn 0.99). Low and/or non-significant values
were observed for Mn. Similarly, Pseudomonas 16S gene
levels were significantly and positively correlated with levels
of 5 HCl-extractable metals (Cu 0.96, Co 0.93, Ni 0.99, Pb
0.94 an Zn 0.94). Similarly, low and/or non-significant values
were observed for total levels of Mn.

Fig. 2. Total (: above the bars) and bioavailable (black bars) metal concentrations in the top layer (0e1 cm) of Station 130 (St130), F
erin (FER), R^aches (RAC)
and MetalEurop (MET). HCl-extractable metals (mean SD; left Y-axis) were estimated using 1 M HCl extraction for 24 h. Total metals (mean SD; right Y-axis)
were determined by complete digestion in strong acids. Significant differences are indicated by different letters (a, b, c, d).
652 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656
Fig. 3. Quantification of the oprI gene (A) and the Pseudomonas 16S rRNA
gene (B) in sediments of the four stations (mean SD). All gene levels were
normalized by the total amount of extracted DNA. Significant differences are
indicated by different letters above the bars (a, b, c, d). The graph (C) is a
linear regression between the two qPCR quantification approaches (16S rRNA
of Pseudomonas versus the oprI gene of Pseudomonas); in this case, gene
levels were normalized by the total 16S rRNA gene signal, as determined with
general primers targeting Bacteria.
Finally, gene levels were also plotted against the toxicity
index (TI) or the metal pollution index (MPI) and a very clear
linear relationship was observed in each case, with the r2 of
the linear regression comprised between 0.89 and 0.96 (oxic
sediments, Fig. 4).
3.3. Metal resistance of bacterial isolates in MetalEurop
A series of bacterial isolates were obtained in MetalEurop,
the most contaminated station, using a minimum mineral
medium. Then, only the colonies appearing on plates with the
highest metal concentration (29 colonies in total) were
selected for further analysis. The highest metal concentration
at which colonies appeared on the plates was 3 mM for Pb and
Ni, 2 mM for Zn and Co, 1 mM for Cd and 0.5 mM for Cu.
These 29 metal-resistant colonies were then characterized by
sequencing their 16S rRNA gene (Table 1). These bacteria
were essentially g-Proteobacteria (58.6%) with Pseudomonas
(10 isolates, 34%) and Klebsiella (5 isolates, 17.2%).
Five isolates grouped with Firmicutes (Mycobacterium,
Streptomyces, Rhodococcus), three with a-Proteobacteria
(Sphingomonas and Methylobacterium) and only one with b-
Proteobacteria (Delftia) (Table 1).
Metal resistance of the 29 isolates was then investigated
further on 869 medium (diluted 3) containing 1 mM of an
individual metal ion (Table 2). Resistances to Cu, Pb and As
were the most frequent, and colony formation on these metals
was observed for most of the 29 isolates (89%). Resistances to
Co and Cd were the least frequent and concerned only 5
isolates (17%). For Zn, 11 isolates grew on 1 mM Zn.
The 10 Pseudomonas isolates obtained in this study
belonged to 4 phylogenetic groups, as defined by Mulet et al.
[58]: the Pseudomonas putida group (SR18, SR44, SR45,
SR66, SR68), the P. aeruginosa group (SR14), the Pseudo-
monas lutea group (SR64, SR54, SR56) and the Pseudomonas
fluorescens group (SR67) (Fig. S1).
4. Discussion
The main result of the present study was the finding that the
abundance of Pseudomonas, as determined by quantitative PCR
in the top layer of the sediments, is positively correlated with
sediment metal levels (using either total metals, HCl-
extractable metals, the metal pollution index or the toxicity
index). Such a result was probably not a qPCR bias because two
different primer sets were used in parallel, targeting two
different Pseudomonas genes. Similarly, it was probably not an
artefact of DNA extraction, because all sediments were treated
with the same protocol and DNA levels were always normal-
ized. The fact that the oprI gene was not found in deep layers of
the sediments is probably related to the behavior of Pseudo-
monas species that are mainly aerobic, and thus mainly located
in the top layers of the sediments. In addition, oprI genes are
probably not multi-copy genes, contrary to 16S rRNA genes (7
copies in the P. putida genome [59]). This may explain the fact
that a faint signal for the Pseudomonas 16S rRNA gene was
found in deep layers of the sediments. Our results should, of
course, be confirmed in future studies by other methods such as
FISH and/or metagenomics. However, FISH is difficult to apply
when sediments contain low levels of cells, cells in biofilms and
autofluorescing particles, as in the present case [36].
For five of the metals investigated (Cu, Co, Ni, Pb and Zn)
gene levels were positively correlated with both total metals
and HCl-extractable metals (the potentially bioavailable
metals), with high correlation coefficients. We may conclude
that Pseudomonas levels are affected by levels of these five
metals in sediments, either in the easily exchangeable/labile
fraction (HCl extractions) or deeply occluded in the minerals
(total extractions). This is also illustrated by the good and
positive correlations obtained between total extractions and
HCl extractions, particularly for Co, Ni, Pb, Zn (data not
shown). However, it must be noted that no conclusion can be
drawn for the other elements such as As, Cd and Cr (as they
were not quantified after HCl extraction). Despite the fact that
both metal extraction protocols produced the same result in the
present study, it must be emphasized that this is not always the
case. A good example is Co and the czcA metal resistance gene
 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656 653

Fig. 4. Representation of the oprI and Pseudomonas 16S rRNA quantifications against the toxicity index and the metal pollution index. Linear regressions are
computed for each plot and r2 are reported.

Table 2
Characterization of the metal resistance of the isolated strains and theoretical metal concentrations used for isolation. Resistance was assessed by growth with metal
compared to growth without metal. , colony formation identical with or without metal; e, no formation of colonies; nd, not determined.
Genus Strain Growth with metal (869 media diluted 3) Metal levels used
Cu 1 mM Zn 1 mM Co 1 mM Cd 1 mM Ni 1 mM Pb 1 mM As 1 mM As 5 mM for isolation

Bacillus SR58 +     nd + + 3 mM Pb
Mycobacterium SR32      + + nd 0.2 mM Cu
SR33     +   nd 0.2 mM Cu
SR34 +    + + + nd 0.5 mM Cu
Rhodococcus SR42 + + + + nd + + + 3 mM Ni
Streptomyces SR41 +    nd + + + 3 mM Ni
Sphingomonas SR30 + +   + + + + 0.2 mM Cu
Methylobacterium SR13 + + nd  + + + + 0.5 mM Cd
SR24 + + +   +  nd 2 mM Co
Klebsiella SR15 +     + + + 0.5 mM Cd
SR53 + +  + + nd + + 3 mM Pb
SR55 +    + nd + + 3 mM Pb
SR57 +    + nd + + 3 mM Pb
SR69 + nd  + + + + nd 2 mM Zn
Enterobacter SR16 +     + + + 0.5 mM Cd
SR65 + nd   + + + + 2 mM Zn
Pseudomonas SR14 + +   + + + + 0.5 mM Cd
SR18 + +   + + + + 1 mM Cd
SR44 + +   nd + + + 3 mM Ni
SR45 + +   nd + + + 3 mM Ni
SR54 +    + nd +  3 mM Pb
SR56 + +   + nd + + 3 mM Pb
SR64 + nd   + + + + 2 mM Zn
SR66 + nd   + + + + 2 mM Zn
SR67 + nd    + + + 2 mM Zn
SR68 + nd   + + + nd 2 mM Zn
Aeromonas SR28   nd   + + + 2 mM Co
Delftia SR27 + + nd + + + + + 2 mM Co
Undetermined SR35 nd +   + + + nd 2 mM Cu
654 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656
quantified by qPCR in sediments [26]. In the latter example,
total and HCl-extractable Co produced different results and it
was concluded that adsorbed/labile Co was correlated with
czcA levels, contrary to total Co (in other words, occluded Co
probably had limited biological effects). Other studies have
also concluded that it is important to extract metals from
sediments using various protocols [25,26].
The present study indicated that Pseudomonas bacteria do
proliferate in metal-contaminated environments, particularly
in sediments of MetalEurop. It is first important to note that
other isolates were obtained, such as Klebsiella, Mycobacte-
rium, Methylobacterium and Delftia (Table 1). Pseudomonas
bacteria are therefore part of a more complex community in
MetalEurop, as confirmed by another study [26], and are
probably not the most adapted to metals. Four groups of
Pseudomonas isolates were found in MetalEurop, among
which the P. putida group with 5 isolates. This is not sur-
prising, as the genome of P. putida KT2440 is known to
encode many genes involved in tolerance to metals and met-
alloids [59]. For instance, P. putida was found to bear two
systems for arsenic, one for chromate, four to six systems for
divalent cations, two systems for monovalent cations, two
operons for Cu chelation, one metallothionein for metal(loid)
binding and one system for Te/Se methylation [59]. Four of
the P. putida isolates obtained here (SR44, SR45, SR66, SR68)
are probably the same strain, as their 16S rRNA sequence is
almost identical, together with their metal resistance pattern.
One Pseudomonas isolate was found in the P. fluorescens
group (SR67). Members of this group are also well adapted to
metal stresses, as shown for a marine strain of P. fluorescens
that adapted to metals by inducing seven defense mechanisms
[12]. The P. lutea group (three strains found here) is not
particularly known for its metal resistance capacities [60].
However, these bacteria are able to solubilize phosphate and
such a capacity might be used by the cell to tolerate high metal
levels. Indeed, some bacteria are known to immobilize metals
with phosphate [61e63], a process that may be termed bio-
mineralization. Finally, bacteria in the P. aeruginosa group
(one strain in MetalEurop) are also well adapted to metal
stresses either through adsorption to the cell surface [64] or by
metal efflux [65,66]. All the Pseudomonas isolates obtained in
this study were able to tolerate metals, particularly Cu, Zn and
As. These metals may reach high levels in MetalEurop sedi-
ments (Table 2). Surprisingly, the Pseudomonas isolates ob-
tained in this study did not tolerate Cd and Co at the
concentration of 1 mM. It is possible that the metal level used
in the test (1 mM) was too high for these isolates. This level
was indeed higher than the total Cd (0.71 mM) and total Co
(0.15 mM) observed in the field in MetalEurop, and much
higher than the bioavailable Cd (0.05 mM) and bioavailable Co
(0.01 mM) observed in the sediments.
We conclude that (1) Pseudomonas bacteria do proliferate
in muddy sediments contaminated with Pb (up to
950 mg kg1), Zn (up to 3300 mg kg1), Cd (up to
46 mg kg1) and Cu (up to 140 mg kg1); (2) at least for the
sediments investigated, correlations with total and HCl-
extractable metals were both good (>0.8) and positive; (3)
the Pseudomonas species that proliferate are members of the
P. putida, P. fluorescens, P. lutea and P. aeruginosa groups
(since only 29 isolates were characterized, it is possible that
other Pseudomonas groups do occur); and finally (4) Pseu-
domonas bacteria proliferate in a complex microbial com-
munity featuring other bacterial members such as Bacillus,
Mycobacterium, Rhodococcus, Streptomyces, Sphingomonas,
Methylobacterium, Klebsiella, Enterobacter and Delftia.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by a F.R.I.A. (Fund for Research
in Industry and Agriculture) grant to S.R. and a F.N.R.S.
(Fonds National De La Recherche Scientifique) grant to
D.C.G. and R.W. (FRFC Nr 2.4577.12). We thank Max Mer-
geay for his help and ideas, as well as Andr
e Cattrijsse and all
the crew of the RV Zeeleeuw.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.resmic.2014.07.011.
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