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Research in Microbiology 165 (2014) 647e656

Original article

The Pseudomonas community in metal-contaminated sediments as revealed

by quantitative PCR: a link with metal bioavailability
Stephanie Roosa a, Corinne Vander Wauven c, Gabriel Billon b, Sandra Matthijs c, Ruddy Wattiez a,
David C. Gillan a,*
Proteomics and Microbiology Lab, Research Institute for Biosciences, Universite de Mons, 20 Place du Parc, B-7000 Mons, Belgium
Geosystemes Lab, UFR de Chimie, Lillee1 University, Sciences and Technologies, 59655 Villeneuve d'Ascq, France
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


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

diseases [7]. The ubiquity of the genus is a direct consequence
Pseudomonas bacteria are ubiquitous Gram-negative and of its high adaptability. For instance, Pseudomonas bacteria
aerobic microorganisms that are member of the g-Proteobac- are able to survive in hydrocarbon-, PCB- and PAH-
teria. These bacteria are found in numerous natural environ- contaminated environments due to oxidative degradation
ments, including soils, sediments and water [1,2], and have processes and biosurfactant production [8,9]. Pseudomonas
been found in association with various plants and animals [3]. spp. are also known to degrade various pesticides [10] and
Pseudomonas bacteria are also responsible for different human harbor metal resistance mechanisms such as efflux pumps and
infections [4e6] and some species have developed resistance 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-
* Corresponding author. tudes to react with metals have even led to proposing their use
E-mail addresses: (S. Roosa), cvdwauve@ as bioremediation tools [13,17,18]. (C.V. Wauven), (G. Billon), slmatthi@ (S. Matthijs), (R. Wattiez), david.
Metals are frequent contaminants of aquatic sediments (D.C. Gillan). [19,20]. However, the total concentration of metals in
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 with reference sites [14]. Unfortunately, metal bioavailability
are not always bioavailable [21e26]. For a unicellular or- was not assessed.
ganism, a compound may be defined as bioavailable when it is The aim of the present research was to use quantitative
able to cross the cellular envelope at a given time [27]. Once PCR with specific primers to estimate the biomass of the
inside the cell, metals may have various effects according to whole Pseudomonas community in 4 aquatic sediments
their concentration and chemical properties [28]. When con- showing contrasted metal bioavailability. At the same time,
centration levels are too high, bacteria usually react by the total metal levels and bioavailable metals were estimated in
expression of specific metal resistance systems such as P-type the sediments using, respectively, strong acids and 1 M HCl
ATPases, metallothioneins, RND efflux pumps and/or CDF treatment. We also used cell cultivation to test the metal
transporters [13,28,29]. It is therefore expected that bacteria resistance capacities of some Pseudomonas isolates obtained
displaying the most efficient resistance systems are selected in at the most contaminated station.
metal-contaminated environments where metals are
bioavailable. 2. Materials and methods
As a large panel of metal resistance systems was observed
in many Pseudomonas species [13], and given the ubiquity of 2.1. Description of the sites and sediment sampling
the genus and its numerous species, it is anticipated that
Pseudomonas bacteria will have an advantage in sedimentary Four sites were considered in this study: Station 130
environments where metals are bioavailable, an advantage that (St130) in seawater and MetalEurop (MET), Ferin (FER) and
may lead to higher biomasses. However, to the best of our R^aches (RAC) in freshwater (Fig. 1). Station 130 is situated in
knowledge, the biomasses of Pseudomonas and metal the North Sea, on the Belgian Continental Plate. It presents
bioavailability were never assessed in parallel in natural sed- low concentrations of metals in comparison to the other
iments. Pseudomonads have been quantified in the environ- freshwater sites [36]. The MetalEurop site is the most metal-
ment using most-probable-number (MPN) enumeration [30], contaminated site of the present study. It is located in front
fluorescent in situ hybridization [31] and quantitative PCR of a smelter in the River Deule (northern France). This plant
(qPCR) [14,32e35], but most of these previous studies were was closed in 2003 after decades of accidental discharges into
not performed in metal-contaminated sediments or did not the river that started in 1893; today, the contamination level of
assess metal bioavailability [32e35]. In a study restricted to the site still remains many orders of magnitude higher than
Pseudomonas aeruginosa living in a metal-contaminated river, background values [37,38]. The R^aches site is mildly
the quantification of three genes (a Mn-containing superoxide contaminated and Ferin is also a control site, supposedly un-
dismutase SodA, a heat shock protein HtpX and a metal- contaminated; these two sites are situated in the same river
lothionein) was performed and significantly higher copy basin as MetalEurop. All sites selected in this study have a
numbers were found at the more contaminated sites compared similar granulometry, oxygen penetration depth, organic

Fig. 1. Map of the sites and their coordinates: St130: Station 130; FER: Ferin; RAC: R^aches; MET: MetalEurop.
S. Roosa et al. / Research in Microbiology 165 (2014) 647e656 649

matter and pH (all sediments are muddy with a mean grain the molar concentration of AVS exceeds that of the metals
size < 50 mm and a POC content between 1 and 3%). (i.e., the metal/AVS ratio is less than unity), they exist pre-
Sediment sampling at St130 was performed using a Rein- dominantly as insoluble metal sulfides, which presumably are
eck corer ( 15 cm) onboard the Zeeleeuw research vessel not biologically available [44]. For AVS measurement [45],
(June 2011; depth: 11 m). For the freshwater sites, sediments wet sediments (1 g, anoxic) were mixed with 6 M HCl for 1 h
were collected in the middle of the river using a Plexiglass at 25  C. The H2S gas that was produced was then trapped in a
tube ( 7 cm) fixed to a stainless steel bar (June 2011; 1 M NaOH solution. The sulfide concentration was measured
depth: 3 m). For all sites, three separate cores were obtained with a sulfide-specific electrode (Orion). Results were found to
(n 3) and for each core, the wateresediment interface be reproducible with an analytical precision 15%. To
(0e1 cm) and an anoxic layer (4e6 cm) were immediately calculate the TI, concentrations of bioavailable Cd, Co, Cu,
isolated in sterile plastic vessels. Samples were transported to Ni, Pb and Zn were used.
the lab in dry ice and divided into two parts: one part was
stored at 80  C for biomolecular analyses and the other part 2.3. Quantification of Pseudomonas by quantitative PCR
at 20  C for chemical analyses. For bacterial cultures, sub-
samples of sediments were brought back to the laboratory at Before DNA extraction, sediments (24 samples) were
4  C and directly used. washed using three different buffers in order to remove a
maximum of PCR inhibitors, as described in [46]. For each
2.2. Quantification of metals sample, DNA was then extracted from 750 mg of sediment
using three PowerSoil DNA extraction columns (Mobio) in
2.2.1. Total metals (TE) parallel (3  250 mg). DNA extracted from these three col-
Weighed amounts of dried and sieved (63 mm) sediments umns was then pooled and a final purification step was per-
were digested with concentrated acids as described elsewhere formed with a Qiaquick purification kit (Qiagen) in order to
[39]. Total metal concentrations were determined using increase the quality of the DNA extracts (260/280 and 260/230
inductively coupled plasma mass spectrometry (ICP-MS, ratios above 1.8) and ensure reliable and reproducible qPCR
Thermo Elemental, X7 series). Only 12 metals or semi-metals quantification. DNA quality of the 24 extracts was assessed by
were investigated (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V the measurement of the 260/280 nm and 260/230 nm ratios on
and Zn). Standard reference materials (Canadian International a Nanodrop ND-1000. The concentration of the DNA prepa-
Sediment Standards HISS-1, MESS-3 and PACS-2) were rations was evaluated with Picogreen quantification (Invi-
analyzed in parallel and metal recovery was on an average trogen) using a Fluostar Optima microplate reader (BMG
better than 90% for HISS-1, 67% for PACS-2 and 83% for Labtech).
MESS-3. To compare the total metal content at different For quantitative PCR (qPCR) two different primer sets
sampling sites, a metal pollution index (MPI) was calculated were used. One primer set targets a 249 bp fragment of the
with the following equation [40,41]: Pseudomonas lipoprotein oprI gene [47,48]: forward primer
MPI Cm1 *Cm2 **Cmn =n primer PS2 (5'-CTTGCGGCTGGCTTTTTCCAG-3'). The
other primer set targets a 251 bp region of the 16S rRNA gene
where Cmi is the concentration for metal i in the sample and n located in the V3eV4 hypervariable region at position
is the number of metals considered. In this study, Al, As, Cd, 435e686 [35]: Pse435F (5'-ACTTTAAGTTGGGAG-
Co, Cr, Cu, Fe, Mn, Ni, Pb, V and Zn were computed in this GAAGGG-3') and Pse686R (5'-ACACAGGAAATTCCACC-
index. ACCC-3'). The real time PCR mixture (20 mL final volume)
contained 10 mL of Power SYBR Green PCR Master Mix
2.2.2. Bioavailable metals (BM) and acid volatile sulfides (ABI), 0.4 mL of each primer (final concentration 200 nM),
(AVS) 7.2 mL of sterile water and 2 mL of samples or standards.
Bioavailable metals (BMs) were estimated for 6 metals Absolute qPCR quantifications were achieved using a Ste-
(Co, Cu, Mn, Ni, Pb, Zn) using a dilute HCl treatment [42,43]. pOnePlus Real Time PCR System (Applied Biosystem) with
Although all metals extracted using this method are not the cycle as follows: the initial denaturation step (95  C,
entirely bioavailable, the term bioavailable will be used 10 min) was followed by 50 cycles of denaturation at 94  C for
below for facility. Briefly, metals were extracted from 1 g of 30 s, annealing at 58  C (oprI gene) or 60  C (Pseudomonas
wet sediment (anoxic) using 1 M HCl for 24 h (in triplicates). 16S rRNA gene) for 30 s, extension at 72 c for 30 s. A melting
Each sample was then filtrated through a 0.45 mm filter and curve analysis from 60  C to 95  C was performed on the
metal concentrations were measured using inductively coupled product to confirm that only one product was amplified. The
plasma atomic emission spectroscopy (ICP-AES; Varian, Vista standard curves were made in duplicate with a 10-fold dilution
Pro, axial view). The relative standard deviation of the results series of reference plasmids containing the relevant target gene
was better than 10%. (oprI or 16S rRNA genes). Each of the 24 DNA extracts was
Bioavailable metals were also used to calculate a toxicity analyzed in triplicate (technical replicates, 50-fold dilution).
index (TI). This index is simply the ratio bioavailable metals/ The template copy number in each standard dilution was
AVS (acid volatile sulfides), as described in [44]. Indeed, when 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 software. Pearson correlation coefficients were calculated
number of each sample was standardized with the DNA con- between Pseudomonas levels and metal contents using Sta-
centration measured by Picogreen. Samples with a CT equal or tistica 7.0. The sequences obtained in this study have been
above the negative control were treated as below the limit of deposited in GenBank (see Table 1).
detection. The mean assay efficiencies was around 93%,
99.5% and 88.6% for oprI, Pseudomonas 16S rRNA and 3. Results
bacterial 16S rRNA, respectively. The copy number of the
bacterial 16S rRNA gene was also quantified and used at a 3.1. Metal levels in the sediments
ratio oprI/16S rRNA and Pseudomonas 16S rRNA/16S rRNA.
Metal levels in the investigated sediments are indicated in
2.4. Isolation of metal-tolerant bacteria and strain Fig. 2 (in mg kg1 for 6 selected metals) and Table S1 (in
characterization mmol kg1 for 12 metals). Total metal levels were low in
St130 and Ferin, while the most impacted site was Metal-
These analyses were restricted to aerobic sediments in Europ, especially for Cd, Cu, Pb and Zn. R^aches had a higher
MetalEurop. Enrichments and isolations were based on a Cu level than MetalEurop. Total metal concentrations for the
modified 284 mineral salt medium (1.5% agar) [50]. This 0e1 cm layer in St130 reached 0.11 (Co), 0.15 (Cu), 0.11
modified medium differed from the original medium [51] by the (Pb) and 1.21 (Zn) mmol kg1. In the same sediment layer of
substitution of TriseHCl by MOPS-NaOH (pH 7.0) and of MetalEurop, total metal concentrations reached 0.15 (Co),
Na2HPO4 by beta-glycerol-phosphate. The carbon source was 1.57 (Cu), 4.41 (Pb) and 49.23 (Zn) mmol kg1. In this study,
also modified by the addition of 0.2% w/v gluconate and 0.2% metal bioavailability was estimated by an HCl 1 M treatment
w/v glucose. Solid media were plated with increasing concen- (Fig. 2, Table S1). It can be seen that the concentration of
trations of metals. For that, stock solutions (1 M) of NaAsO2, bioavailable metals was always inferior to the total metal
Cd(NO3)2, CoCl2 6H2O, Cu(NO3)2 3H2O, Pb(NO3)2, NiCl2 concentration and that a gradient of bioavailability was pre-
6H2O and ZnSO4 were added for a maximum theoretical con- sent from St130 to MetalEurop. In MetalEurop, 9% of the
centration of 5 mM for Co2, 10 mM for AsOe 2 2
2 , Cd , Cu , Ni
total Zn may be considered as bioavailable; percentages
2 2
and 15 mM for Pb and Zn . Fresh sediments were resus- reach 19% for Pb and 8% for Cu (Table S1). The metal
pended in sterile water (1:1, v/v) and sonicated 3 times for 30 s. pollution index (MPI), calculated with 12 metals, was 65e72
After centrifugation of 1 min at 1000 rpm, 50 mL of supernatant for St130 and Ferin (they were not significantly different),
were spread on the different solid media. The plates were 175 for R^aches and reached 261 for MetalEurop. Clearly, on
incubated at 30  C until distinct colonies appeared on the highest the basis of the MPI, the stations formed three separate
metal concentrations. Isolates were then taxonomically char- groups (St130 & Ferin, R^aches, MetalEurop). The toxicity
acterized. Primer pH (AAG GAG GTG ATC CAG CCG CA) index (TI), which takes into account the quantity of sulfides,
and a slightly shortened pA primer (AG AGT TTG ATC CTG was inferior to 1 for all stations. The TI was about 0.11, 0.17,
GCT CAG) [52] were used to amplify the almost complete 16S 0.41 and 0.84 for, respectively, St130, Ferin, R^aches and
rRNA gene (position 29 to 1522 in Escherichia coli). Gene MetalEurop.
fragments were sequenced by Beckman Coulter Genomics.
Genus affiliation of the sequences was then assessed from a 3.2. In situ quantification of Pseudomonas by qPCR and
BLAST analysis [53]. Sequences of the isolates were then correlations with metals
aligned with the type strains of the species as compiled into the
Euzeby database A neighbor- For oxic sediments, quantifications of the Pseudomonas
joining phylogenetic tree was constructed using the MEGA 5 oprI were efficient and specific, as demonstrated by the
software package [54] and the closest relative of the isolates was presence of a unique peak in the qPCR melt curve (data not
inferred from their clustering with type strains. The percentage shown). The oprI abundance varied between 6.8 103 4 103
of identity between an isolate and a type strain was calculated on copies per mg of DNA in St130 and 2.5 105 2.6 104 copies in
the basis of the number of identical residues in the two 16S MetalEurop (Fig. 3). In Ferin, values reached 1.7 104 1 104
rRNA gene sequences. copies and for R^aches 7.8 104 4.5 104 copies per mg of DNA.
Metal resistance was evaluated by plating isolates on me- The oprI gene was not detected in anoxic sediments (values
dium 869 [51] diluted 3 times and enriched with 1 mM Cu, Zn, were under the detection limit).
Co, Cd, Ni, Pb, As and 5 mM As. The strains that were not For Pseudomonas quantifications based on the 16S rRNA
inhibited by 1 mM Cd, Co, Cu, Ni, Pb and Zn [55e57] and in oxic sediments, the number of copies per mg of DNA was
5 mM As were regarded as being resistant. 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
2.5. Statistical analyses and GenBank accession copies per mg of DNA for St130, Ferin, R^aches and Metal-
numbers Europ, respectively (Fig. 3). It must be noted that a Pseudo-
monas 16S rRNA signal was also found for anoxic sediments,
The comparison between sites was done using ANOVA and but that values were up to 100 times lower and that no
post hoc multiple comparison pairwise t tests with the R 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
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- Similarly, low and/or non-significant values were observed for
domonas qPCR quantification methods (linear regression, total levels of Al, Fe, Mn and V.
r2 0.94). For HCl-extractable metals (6 metals investigated), Pseu-
Pseudomonas oprI gene levels were significantly and domonas oprI gene levels were significantly and positively
positively correlated (Pearson) with total levels of 8 metals correlated (Pearson) with levels of 5 metals (Cu 0.99, Co 0.90,
(As 0.94, Cd 0.85, Co 0.91, Cr 0.89, Cu 0.73, Ni 0.91, Pb 0.99 Ni 0.99, Pb 0.99, Zn 0.99). Low and/or non-significant values
and Zn 0.98). Low and/or non-significant values were were observed for Mn. Similarly, Pseudomonas 16S gene
observed for total levels of Al, Fe, Mn and V. Pseudomonas levels were significantly and positively correlated with levels
16S gene levels were significantly and positively correlated of 5 HCl-extractable metals (Cu 0.96, Co 0.93, Ni 0.99, Pb
with total levels of the same 8 metals (As 0.92, Cd 0.91, Co 0.94 an Zn 0.94). Similarly, low and/or non-significant values
0.95, Cr 0.89, Cu 0.82, Ni 0.95, Pb 0.97 and Zn 0.99). 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), Ferin (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

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-
Fig. 3. Quantification of the oprI gene (A) and the Pseudomonas 16S rRNA ized. The fact that the oprI gene was not found in deep layers of
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
the sediments is probably related to the behavior of Pseudo-
indicated by different letters above the bars (a, b, c, d). The graph (C) is a monas species that are mainly aerobic, and thus mainly located
linear regression between the two qPCR quantification approaches (16S rRNA in the top layers of the sediments. In addition, oprI genes are
of Pseudomonas versus the oprI gene of Pseudomonas); in this case, gene probably not multi-copy genes, contrary to 16S rRNA genes (7
levels were normalized by the total 16S rRNA gene signal, as determined with copies in the P. putida genome [59]). This may explain the fact
general primers targeting Bacteria.
that a faint signal for the Pseudomonas 16S rRNA gene was
found in deep layers of the sediments. Our results should, of
Finally, gene levels were also plotted against the toxicity course, be confirmed in future studies by other methods such as
index (TI) or the metal pollution index (MPI) and a very clear FISH and/or metagenomics. However, FISH is difficult to apply
linear relationship was observed in each case, with the r2 of when sediments contain low levels of cells, cells in biofilms and
the linear regression comprised between 0.89 and 0.96 (oxic autofluorescing particles, as in the present case [36].
sediments, Fig. 4). For five of the metals investigated (Cu, Co, Ni, Pb and Zn)
gene levels were positively correlated with both total metals
3.3. Metal resistance of bacterial isolates in MetalEurop and HCl-extractable metals (the potentially bioavailable
metals), with high correlation coefficients. We may conclude
A series of bacterial isolates were obtained in MetalEurop, that Pseudomonas levels are affected by levels of these five
the most contaminated station, using a minimum mineral metals in sediments, either in the easily exchangeable/labile
medium. Then, only the colonies appearing on plates with the fraction (HCl extractions) or deeply occluded in the minerals
highest metal concentration (29 colonies in total) were (total extractions). This is also illustrated by the good and
selected for further analysis. The highest metal concentration positive correlations obtained between total extractions and
at which colonies appeared on the plates was 3 mM for Pb and HCl extractions, particularly for Co, Ni, Pb, Zn (data not
Ni, 2 mM for Zn and Co, 1 mM for Cd and 0.5 mM for Cu. shown). However, it must be noted that no conclusion can be
These 29 metal-resistant colonies were then characterized by drawn for the other elements such as As, Cd and Cr (as they
sequencing their 16S rRNA gene (Table 1). These bacteria were not quantified after HCl extraction). Despite the fact that
were essentially g-Proteobacteria (58.6%) with Pseudomonas both metal extraction protocols produced the same result in the
(10 isolates, 34%) and Klebsiella (5 isolates, 17.2%). present study, it must be emphasized that this is not always the
Five isolates grouped with Firmicutes (Mycobacterium, 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, the Pseudomonas species that proliferate are members of the
total and HCl-extractable Co produced different results and it P. putida, P. fluorescens, P. lutea and P. aeruginosa groups
was concluded that adsorbed/labile Co was correlated with (since only 29 isolates were characterized, it is possible that
czcA levels, contrary to total Co (in other words, occluded Co other Pseudomonas groups do occur); and finally (4) Pseu-
probably had limited biological effects). Other studies have domonas bacteria proliferate in a complex microbial com-
also concluded that it is important to extract metals from munity featuring other bacterial members such as Bacillus,
sediments using various protocols [25,26]. Mycobacterium, Rhodococcus, Streptomyces, Sphingomonas,
The present study indicated that Pseudomonas bacteria do Methylobacterium, Klebsiella, Enterobacter and Delftia.
proliferate in metal-contaminated environments, particularly
in sediments of MetalEurop. It is first important to note that Conflict of interest
other isolates were obtained, such as Klebsiella, Mycobacte-
rium, Methylobacterium and Delftia (Table 1). Pseudomonas The authors declare no conflict of interest.
bacteria are therefore part of a more complex community in
MetalEurop, as confirmed by another study [26], and are Acknowledgments
probably not the most adapted to metals. Four groups of
Pseudomonas isolates were found in MetalEurop, among This work was supported by a F.R.I.A. (Fund for Research
which the P. putida group with 5 isolates. This is not sur- in Industry and Agriculture) grant to S.R. and a F.N.R.S.
prising, as the genome of P. putida KT2440 is known to (Fonds National De La Recherche Scientifique) grant to
encode many genes involved in tolerance to metals and met- D.C.G. and R.W. (FRFC Nr 2.4577.12). We thank Max Mer-
alloids [59]. For instance, P. putida was found to bear two geay for his help and ideas, as well as Andre Cattrijsse and all
systems for arsenic, one for chromate, four to six systems for the crew of the RV Zeeleeuw.
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 Appendix A. Supplementary data
the P. putida isolates obtained here (SR44, SR45, SR66, SR68)
are probably the same strain, as their 16S rRNA sequence is Supplementary data related to this article can be found at
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 References
metal stresses, as shown for a marine strain of P. fluorescens
that adapted to metals by inducing seven defense mechanisms [1] Isnansetyo A, Kamei Y. Bioactive substances produced by marine
[12]. The P. lutea group (three strains found here) is not isolates of Pseudomonas. J Ind Microbiol Biotechnol 2009;36:1239e48.
particularly known for its metal resistance capacities [60]. [2] Cho JC, Tiedje JM. Biogeography and degree of endemicity of fluores-
cent Pseudomonas strains in soil. Appl Environ Microbiol 2000;66:
However, these bacteria are able to solubilize phosphate and
such a capacity might be used by the cell to tolerate high metal [3] Berthe FCJ, Michel C, Bernardet J-F. Identification of Pseudomonas
levels. Indeed, some bacteria are known to immobilize metals anguillispetica isolated from several fish species in France. Dis Aquat
with phosphate [61e63], a process that may be termed bio- Org 1995;21:151e5.
mineralization. Finally, bacteria in the P. aeruginosa group [4] Mena KD, Gerba CP. Risk assessment of Pseudomonas aeruginosa in
water. Rev Environ Contam Toxicol 2009;201:71e115.
(one strain in MetalEurop) are also well adapted to metal
[5] Hancock REW, Speert DP. Antibiotic resistance in Pseudomonas aeru-
stresses either through adsorption to the cell surface [64] or by ginosa: mechanisms and impact on treatment. Drug Resist Uptake
metal efflux [65,66]. All the Pseudomonas isolates obtained in 2000;3:247e55.
this study were able to tolerate metals, particularly Cu, Zn and [6] Lambert PA. Mechanisms of antibiotic resistance in Pseudomonas aer-
As. These metals may reach high levels in MetalEurop sedi- uginosa. J R Soc Med 2002;95:22e6.
[7] Mesaros N, Nordmann P, Plesiat P, Roussel-Delvallez M, Van Eldere J,
ments (Table 2). Surprisingly, the Pseudomonas isolates ob-
Glupczynski Y, et al. Pseudomonas aeruginosa: resistance and thera-
tained in this study did not tolerate Cd and Co at the peutic options at the turn of the new millennium. Clin Microbiol Infect
concentration of 1 mM. It is possible that the metal level used 2007;13:560e78.
in the test (1 mM) was too high for these isolates. This level [8] Furukawa K. Biochemical and genetic bases of microbial degradation of
was indeed higher than the total Cd (0.71 mM) and total Co polychlorinated biphenyls (PCBs). J Gen Appl Microbiol 2000;46:
(0.15 mM) observed in the field in MetalEurop, and much
[9] Ron EZ, Rosenberg E. Biosurfactants and oil bioremediation. Curr Opin
higher than the bioavailable Cd (0.05 mM) and bioavailable Co Biotechnol 2002;13:249e52.
(0.01 mM) observed in the sediments. [10] Foster LJR, Kwan BH, Vancov T. Microbial degradation of the organo-
We conclude that (1) Pseudomonas bacteria do proliferate phosphate pesticide. FEMS Microbiol Lett 2004;240:49e53.
in muddy sediments contaminated with Pb (up to [11] Gonzales-Guerrero M, Raimunda D, Cheng X, Arguello JM. Distinct
950 mg kg1), Zn (up to 3300 mg kg1), Cd (up to functional roles of homologuous Cu efflux ATPases in Pseudomonas
aeruginosa. Mol Microbiol 2010;78:1246e58.
46 mg kg1) and Cu (up to 140 mg kg1); (2) at least for the [12] Poirier I, Hammann P, Kuhn L, Bertrand M. Strategies developed by the
sediments investigated, correlations with total and HCl- marine bacterium Pseudomonas fluorescens BA3SM1 to resist metals: a
extractable metals were both good (>0.8) and positive; (3) proteome analysis. Aquat Toxicol 2013;128/129:215e32.
S. Roosa et al. / Research in Microbiology 165 (2014) 647e656 655

[13] Aguilar-Barajas E, Ramrez-Daz MI, Riveros-Rosas H, Cervantes C. [33] Deschaght P, Van Daele S, De Baets F, Vaneechoutte M. PCR and
Heavy metal resistance in pseudomonads. In: Ramos JL, Filloux A, ed- detection of Pseudomonas aeruginosa in respiratory samples of CF pa-
itors. Pseudomonas, vol. 6: molecular microbiology, infection and tients. A literature review. J Cyst Fibros 2011;10:293e7.
biodiversity. Dordrecht: Springer; 2010. [34] Saha R, Donofrio RS, Bagley ST. Development of a real-time TaqMan
[14] Bouskill NJ, Barnhart EP, Galloway TS, Handy RD, Ford TE. Quanti- assay to detect mendocina sublineage Pseudomonas species in contam-
fication of changing Pseudomonas aeruginosa soda, htpX and mt gene inated metalworking fluids. J Ind Microbiol Biotechnol 2010;37:843e8.
abundance in response to trace metal toxicity: a potential in situ [35] Bergmark L, Poulsen PHB, Abu Al-Soud W, Norman A, Hansen LH,
biomarker of environmental health. FEMS Microbiol Ecol Srensen SJ. Assessment of the specificity of burkholderia and Pseudo-
2007;60:276e86. monas qPCR assays for detection of these genera in soil using 454
[15] Pepi M, Volterrani M, Renzi M, Marvasi M, Gasperini S, Franchi E, et al. pyrosequencing. FEMS Microbiol Lett 2012;333:77e84.
Arsenic-resistant bacteria isolated from contaminated sediments of the [36] Gillan DC, Pernet P. Adherent bacteria in heavy metal-contaminated
Orbetello Lagoon, Italy, and their characterization. J Appl Microbiol marine sediments. Biofouling 2007;23:1e13.
2007;103:2299e308. [37] Boughriet A, Proix N, Billon G, Recourt P, Ouddane B. Environmental
[16] Li K, Ramakrishna W. Effect of multiple metal resistant bacteria from impacts of heavy metal discharges from a smelter in Deule-canal sedi-
contaminated lake sediments on metal accumulation and plant growth. J ments (Northern France): concentration levels and chemical fraction-
Hazard Mater 2011;189:531e9. ation. Water Air Soil Pollut 2007;180:83e95.
[17] Vullo DL, Ceretti HM, Daniel MA, Ramirez SAM, Zalts A. Cadmium, [38] Lesven L, Lourino-Cabana B, Billon G, Recourt P, Ouddane B,
zinc and copper biosorption mediated by Pseudomonas veronii 2E. Mikkelsen O, et al. On metal diagenesis in contaminated sediments of the
Bioresour Technol 2008;99:5574e81. Deule river (northern France). Appl Geochem 2010;25:1361e73.
[18] Cycon M, Wojcik M, Piotrowska-Seget Z. Biodegradation of the [39] Lourino-Cabana B, Lesven L, Billon G, Proix N, Recourt P, Ouddane B,
organophosphorus insecticide daizinon by Serratia sp. and Pseudomonas et al. Impacts of metal contamination in calcareous waters of Deule river
sp. and their use in bioremediation of contaminated soil. Chemosphere (France): waterquality and thermodynamic studies on metallic mobility.
2009;76:494e501. Water Air Soil Pollut 2010;206:187e201.
[19] Calmano W, F orstner U. Sediments and toxic substances. Berlin, Ger- [40] Usero J, Gonzalez-Regalado E, Gracia I. Trace metals in the bivalve
many: Springer-Verlag; 1996. mollusks Ruditapes decussates and Ruditapes philippanarum from the
[20] Eggleton J, Thomas KV. A review of factors affecting the release and Atlantic Coast of Southern Spain. Environ Int 1997;23:291e8.
bioavailability of contaminants during sediment disturbance events. En- [41] Caeiro S, Costa MH, Ramos TB, Fernandes F, Silevira N, Coimbra A,
viron Int 2004;30:973e80. et al. Assessing heavy metal contamination in Sado Estuary sediment: an
[21] Turner A, Olsen YS. Chemical versus enzymatic digestion of contami- index analysis approach. Ecol Indic 2005;5:151e69.
nated estuarine sediment: relative importance of iron and manganese [42] Sutherland RA. Comparison between non-residual Al, Co, Cu, Fe, Mn,
oxides in controlling trace metal bioavailability. Estuar Shelf Coast Sci Ni, Pb and Zn released by a three-step sequential extraction procedure
2000;51:717e28. and a dilute hydrochloric acid leach for soil and road deposited sediment.
[22] Kahru A, Ivask A, Kasemets K, Pollumma L, Kurvet I, Franois M, et al. Appl Geochem 2002;17:353e65.
Biotests and biosensors in ecotoxicological risk assessment of filed soils [43] Snape I, Scouller RC, Stark SC, Stark J, Riddle MJ, Gore DB. Characterisation
polluted with zinc, lead, and cadmium. Environ Toxicol Chem of the dilute HCl extraction method for the identification of metal contami-
2005;34:1439e47. nation in Antarctic marine sediments. Chemosphere 2004;57:491e504.
[23] Devesa-Rey R, Daz-Fierros F, Barral MT. Trace metals in river bed [44] Ankley GT. Evaluation of metal/acid-volatile sulfide relationships in the
sediments: an assessment of their partitioning and bioavailability by prediction of metal bioaccumulation by benthic macroinvertebrates.
using multivariate exploratory analysis. J Environ Manag Environ Toxicol Chem 1996;15:2138e46.
2010;91:2471e7. [45] Allen HE, Fu G, Deng B. Analysis of acid volatile sulfide (AVS)
[24] Ianni C, Magi E, Soggia F, Rivaro P, Frache R. Trace metal speciation in and simultaneously extracted metals (SEM) for the estimation of
coastal and off-shore sediments from Ross Sea (Antarctica). Microchem potential toxicity in aquatic sediments. Environ Toxicol Chem
J 2010;96:203e12. 1993;12:1441e54.
[25] Gillan DC, Baeyens W, Bechara R, Billon G, Denis K, Grosjean P, et al. [46] Fortin N, Beaumier D, Lee K, Greer CW. Soil washing improves the
Links between bacterial communities in marines sediments and trace recovery of total community DNA from polluted and high organic con-
metal geochemistry as measured by in situ DET/DGT approaches. Mar tent sediments. J Microbiol Meth 2004;56:181e91.
Pollut Bull 2012;64:353e62. [47] De Vos D, Lim A, De Vos P, Sarniguer A, Kersters K, Cornelis P.
[26] Roosa S, Wattiez R, Prygiel E, Lesven L, Billon G, Gillan DC. Bacterial Detection of the outer membrane lipoprotein I and its gene in fluorescent
metal resistance genes and metal bioavailability in contaminated sedi- and non-fluorescent pseudomonads: implications for taxonomy and
ments. Environ Pollut 2014;189:143e51. diagnosis. J Gen Microbiol 1993;139:2215e23.
[27] Semple KT, Doick KJ, Jones KC, Burauel P, Craven A, Harms H. [48] Matthijs S, Coorevits A, Gebrkidan TT, Tricot C, VanDer Wauven C,
Defining bioavailability and bioaccessibility of contaminated soil and Pirnay JP, et al. Evaluation of oprI and oprL genes as molecular markers
sediments is complicated. Environ Sci Technol 2004;38:228Ae31A. for the genus Pseudomonas and their use in studying the biodiversity of a
[28] Bruins MR, Kapil S, Oehme FW. Microbial resistance to metals in the small Belgian River. Res Microbiol 2012;164:254e61.
environment. Ecotoxicol Environ Saf 2000;45:198e207. [49] Whelan JA, Russell NB, Whelan MA. A method for the absolute quan-
[29] Nies DH. Efflux mediated heavy metal resistance in prokaryotes. FEMS tification of cDNA using real-time PCR. J Immun Meth
Microbiol Rev 2003;27:313e39. 2003;278:261e9.
[30] Canion A, Prakash O, Green SJ, Jahnke L, Kuypers MM, Kostka JE. [50] Taghavi S, Lesaulnier C, Monchy S, Wattiez R, Mergeay M, van der
Isolation and physiological characterization of psychrophilic denitrifying Lelie D. Lead (II) resistance in Cupriavidus metallidurans CH34:
bacteria from permanently cold Arctic fjord sediments (Svalbard, Nor- interplay between plasmid and chromosomally-located functions. Ant
way). Environ Microbiol 2013;15:1606e18. Van Leeuwen 2009;96:171e82.
[31] Gougoulias C, Shaw LJ. Evaluation of the environmental specificity of [51] Mergeay M, Nies D, Schlegel HG, Gerits J, Charles P, Van Gijsegem F.
fluorescence in situ hybridization (fish) using fluorescence-activated cell Alcaligenes eutrophus CH34 is a facultative chemolithotroph with
sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizo- plasmid-bound resistance to heavy metals. J.Bacteriol 1985;162:328e34.
sphere soil. Syst Appl Microbiol 2012;35:533e40. [52] Edwards U, Rogall T, Blocker H, Emde M, Bottger EC. Isolation and
[32] Gruntzig V, Nold SC, Zhou J, Tiedje JM. Pseudomonas stutzeri nitrite direct complete nucleotide determination of entire genes. Characteriza-
reductase gene abundance in environmental samples measured by real- tion of a gene coding for 16S ribosomal RNA. Nucl Acids Res
time PCR. Appl Environ Microbiol 2001;67:760e8. 1989;17:7843e53.
656 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656

[53] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local phosphate-solubilizing bacterium isolated from the rhizosphere of
alignment search tool. J Mol Biol 1990;215:403e10. grasses. Int J Syst Evol Microbiol 2004;54:847e50.
[54] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. [61] Macaskie LE, Empson RM, Cheetham AK, Grey CP, Skarnulis AJ.
MEGA5: molecular evolutionary genetics analysis using maximum Uranium bioaccumulation by a Citrobacter sp. as a result of
likelihood, evolutionary distance, and maximum parsimony methods. enzymatically-mediated growth of polycrystalline HUO2PO4. Science
Mol Biol E 2011;28:2731e9. 1992;257:782e4.
[55] Smith DH. R-factors mediated resistance to mercury, nickel, and cobalt. [62] Boswell CD, Dick RE, Macaskie LE. The effect of heavy metals and
Science 1967;156:1114e6. other environmental conditions on the anaerobic phosphate metabolism
[56] Sabry SA, Ghozian HA, Abou-Zeid D-M. Metal tolerance and antibiotic of Acinetobacter johnsonii. Microbiology 1999;145:1711e20.
resistance patterns of a bacterial population isolated from sea water. J [63] Martinez RJ, Beazley ML, Taillefert M, Arakaki AK, Skolnick J,
Appl Microbiol 1997;82:245e52. Sobecky PA. Aerobic uranium (VI) bioprecipitation by metal-resistant
[57] Abou-Shanab RA, Ghozlan H, Ghanem K, Moawad H. Behavior of bacteria isolated from radionuclide and metal-contaminated subsurface
bacterial populations isolated from rhizopshere of diplachne fusca soils. Environ Microbiol 2007;9:3122e33.
dominant in industrial sites. World J Microb Biotechnol 2005;21: [64] Singh R, Bishnoi NR, Kirrolia A. Evaluation of Pseudomonas aerugi-
1095e101. nosa an innovative bioremediation tool in multi metals ions from
[58] Mulet M, Lalucat J, Garca-Valdes E. DNA sequence-based analysis of simulated system using multi response methodology. Bioresour Technol
the Pseudomonas species. Environ Microbiol 2010;12:1513e30. 2013;138:222e34.
[59] Canovas D, Cases I, de Lorenzo V. Heavy metal tolerance and metal [65] Caille O, Rossier C, Perron K. A copper-activated two-component sys-
homeostasis in Pseudomonas putida as revealed by complete genome tem interacts with zinc and imipenem resistance in Pseudomonas aeru-
analysis. Environ Microbiol 2003;5:1242e56. ginosa. J Bacteriol 2007;189:4561e8.
[60] Peix A, Rivas R, Santa-Regina I, Mateos PF, Martinez-Molina E, [66] Zeng X, Tang J, Liu X, Jiang P. Response of P. aeruginosa E1 gene
Rodriguez-Barrueco C, et al. Pseudomonas lutea sp. nov., a novel expression to cadmium stress. Curr Microbiol 2012;65:799e804.