Marine Pollution Bulletin 110 (2016) 75–81

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Antibiotic and metal resistance in a ST395 Pseudomonas aeruginosa

environmental isolate: A genomics approach
Pedro Teixeira a, Marta Tacão a,⁎, Artur Alves b, Isabel Henriques a
a
Biology Department, CESAM & IBIMED, University of Aveiro, Aveiro, Portugal
b
Biology Department, CESAM, University of Aveiro, Aveiro, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: We analyzed the resistome of Pseudomonas aeruginosa E67, an epiphytic isolate from a metal-contaminated es-
Received 8 June 2016 tuary. The aim was to identify genetic determinants of resistance to antibiotics and metals, assessing possible co-
Accepted 22 June 2016 selection mechanisms.
Available online 30 June 2016
Identification was based on phylogenetic analysis and average nucleotide identity value calculation. MLST affili-
ated E67 to ST395, previously described as a high-risk clone. Genome analysis allowed identifying genes probably
Keywords:
Pseudomonas aeruginosa
involved in resistance to antibiotics (e.g. beta-lactams, aminoglycosides and chloramphenicol) and metals (e.g.
Resistome mercury and copper), consistent with resistance phenotypes. Several genes associated with efflux systems, as
Co-selection well as genetic determinants contributing to gene motility, were identified.
Opportunistic pathogen Pseudomonas aeruginosa E67 possesses an arsenal of resistance determinants, probably contributing to adapta-
Genome sequencing tion to a polluted ecosystem. Association to mobile structures highlights the role of these platforms in multi-
Environment drug resistance. Physical links between metal and antibiotic resistance genes were not identified, suggesting a
predominance of cross-resistance associated with multidrug efflux pumps.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction aeruginosa genome harbors a large number of undescribed drug efflux

Pseudomonas aeruginosa is a Gram-negative γ-proteobacterium that
has the capacity to easily adapt to distinct environments such as soil,
marshes, plants and animal tissues (Madigan, 2012; Stover et al.,
2000). Additionally, the species metabolic diversity and its ability to
form biofilms makes it a remarkable opportunistic pathogen causing
nosocomial infections mainly in patients suffering from predisposing
conditions such as cystic fibrosis, burn wounds and immunodeficiency
(Zhao and Hu, 2010; Schwartz et al., 2015).
The increasing morbidity and mortality associated with Pseudomo-
nas aeruginosa infections has become a worldwide problem that is ag-
gravated by the emergence of strains resistant to multiple classes of
antibiotics (Nouwens et al., 2000). Actually, only a few antibiotics are ef-
fective for treatment of these infections since this species is intrinsically
resistant to a large number of antibiotics, mainly due to its low outer
membrane permeability and active efflux of antibiotics (Stover et al.,
2000). This species is also known for being intrinsically resistant to
most β-lactam antibiotics due to the overproduction of chromosomally
encoded AmpC β-lactamases (Zhao and Hu, 2010). Moreover, P.
⁎ Corresponding author at: University of Aveiro, Biology Department, Campus
Universitário Santiago, 3810-193 Aveiro, Portugal.
E-mail address: martat@ua.pt (M. Tacão).
systems that can actively participate in the excretion of structurally un-
related antibacterial molecules such as fluoroquinolones, tetracycline,
chloramphenicol, some β-lactams and also other compounds like
metals and organic solvents (Stover et al., 2000; Piddock, 2006;
Nikaido and Pagès, 2012).
Besides intrinsic resistance, under selective pressure P. aeruginosa
can also acquire other resistance mechanisms through chromosomal
mutations or horizontal gene transfer (HGT) of resistance genes
(Slekovec et al., 2012). Together, these resistance determinants render
P. aeruginosa a common cause of multi-drug resistant (MDR) infections
that impose a huge limitation on the therapeutic choices available
(Nikaido, 2009).
The spread and dissemination of antibiotic resistance comes as a re-
sult of the over-prescription of antibiotics, which are used in high doses
in clinical settings. However, the exposure of environmental landscapes
to pollutants (e.g. antibiotics, metals, disinfectants) derived from an-
thropogenic activities may also contribute to bacteria adaptation,
which can ultimately lead to the appearance of MDR strains (Slekovec
et al., 2012; Berendonk et al., 2015). In the environment, the main
sources contributing to this co-selective force are industrial, municipal
and hospital wastewater, aquaculture and animal husbandry facilities.
However, there is a lack of knowledge regarding the relation of the en-
vironmental impact caused by anthropogenic pollution with co-

http://dx.doi.org/10.1016/j.marpolbul.2016.06.086
0025-326X/© 2016 Elsevier Ltd. All rights reserved.
76 P. Teixeira et al. / Marine Pollution Bulletin 110 (2016) 75–81
selection of antibiotic resistance and HGT events (Baker-Austin et al.,
2006), since the majority of the studies regarding antibiotic resistance
are focused in clinical isolates.
Here we report the analysis of an environmental Pseudomonas iso-
late, by adopting a whole genome sequencing approach. This epiphytic
isolate was retrieved from the plant Halimione portulacoides growing in
salt marshes strongly contaminated with a diversity of metal(oid)s. Our
aim was to unravel the genetic basis of the antibiotic and metal
resistome in this strain, getting insights into the adaptation to the envi-
ronmental contamination and into possible co-selection events.
2. Material and methods
2.1. Strain selection
Pseudomonas aeruginosa E67 was part of a collection of bacterial iso-
lates previously obtained from Halimione portulacoides (Henriques et al.,
2016), a small metal-accumulator halophyte shrub. Plant samples were
collected from Ria de Aveiro salt marshes, in an area contaminated with
high levels of metals both in sediments and accumulated in H.
portulacoides tissues (Fidalgo et al., 2016). Bacterial isolation and identi-
fication procedures were described previously (Henriques et al., 2016).
Briefly isolates were cultivated on MacConkey agar plates and incubated
at 37 °C for 16 h. Individual colonies were purified and stored in 20%
glycerol at −80 °C. The isolate was identified at the genus level by se-
quencing the 16S rRNA gene.
2.2. Phenotypic resistance profile and optimal growth conditions
In the scope of the previous study, the susceptibility profiles to some
antibiotics (amoxicillin, amoxicillin/clavulanic acid, ampicillin, aztreo-
nam, cefepime, cefotaxime, ceftazidime, cephalothin, ciprofloxacin,
chloramphenicol, gentamicin, imipenem, meropenem, kanamycin,
nalidixic acid, sulfamethoxazole/trimethoprim, rifampin), and metal(-
loid)s (As, Cr, Cu, Hg, Ni and Zn) were evaluated (Henriques et al.,
2016). In the present study additional substances were tested using an
identical methodology, namely piperacillin/tazobactam (30 μg/6 μg),
ticarcillin (75 μg), ticarcillin/clavulanic acid (75 μg/10 μg), and tetracy-
cline (30 μg) (Oxoid, United Kingdom). Briefly the profiles were deter-
mined by the agar disc diffusion method on Mueller–Hinton agar,
after 24 h of incubation at 37 °C, according to the Clinical Laboratory
Standards Institute guidelines (CLSI, 2012). Escherichia coli ATCC
25922 was used as quality control.
The isolate's optimal temperature, salinity and pH growth conditions
were determined by growing the isolate on LA medium under different
temperatures (25 °C, 30 °C and 37 °C), salt (NaCl) concentrations (0.5%,
1%, 2%, 4% and 8%) — and pH conditions (4.5, 6.0, 7.0 and 8.0).
2.3. Whole genome sequencing, assembly and annotation
Genomic DNA was isolated as previously described (Henriques et al.,
2006). Pseudomonas aeruginosa E67 genome was sequenced by the
company STAB VIDA (Portugal) using Ion torrent Sequencing Technolo-
gy and resulting reads were then subjected to a trimming process using
the CLC Genomic Workbench version 6.5. The quality of reads was de-
termined with the FastQC program (v3.4.1.1) (Andrews, 2010). CLC
Bio software package was used to assemble the genome sequence
data. The genome draft was annotated using the Rapid Annotation
using System Technology (RAST) (Aziz et al., 2008; Overbeek et al.,
2014). The tRNAscan-SE (Schattner et al., 2005) and RNAmmer 1.2
(Lagesen et al., 2007) were used to predict tRNA and rRNA genes, re-
spectively. Also, a secondary annotation was performed using the
DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)
(Huntemann et al., 2015), which allowed to refine the results obtained
from RAST annotation by performing a comparative analysis.
2.4. Phylogenetic analysis and MLST profile
The phylogenetic affiliation of the E67 isolate was confirmed using
16S rRNA and gyrB gene sequences and comparing them with the se-
quences of the closest 20 type strains included in the genus Pseudomo-
nas. The phylogenetic analysis was carried out using Molecular
Evolutionary Genetics Analysis (MEGA) software version 6.0 (Tamura
et al., 2013). Concatenated nucleotide sequences were initially aligned
using ClustalW (Thompson et al., 1994) and then used to construct a
phylogenetic neighbor joining tree, using bootstrap values (1000 repli-
cates) for accessing the reliability of the branches. The average nucleo-
tide identity (ANI) value was also calculated between the draft
genome obtained in this study and the available reference genome of
P. aeruginosa PAO1 (Goris et al., 2007).
Multilocus sequence typing (MLST) of E67 was performed according
to the protocol of Curran et al. (Curran et al., 2004), modified by Van
Mansfeld et al. (Mansfeld et al., 2009), using the internal fragments of
a group of seven housekeeping genes: acsA, aroE, guaA, mutL, nuoD,
ppsA and trpE.
2.5. Annotation of antibiotic and metal resistance genes
The E67 genome was screened for antimicrobial resistance genes
and mobile genetic elements by browsing the RAST subsystems related
to virulence and transposable elements. In addition, we used the IMG/
ER (Integrated Microbial Genomes/Expert Review) annotation and
also three web-based tools: Resfinder 2.1 (Zankari et al., 2012), the Re-
sistance Gene Identifier provided by CARD (Comprehensive Antibiotic
Resistance Database) (McArthur et al., 2013) and the Antibiotic Resis-
tance Genes Database (ARDB) (Liu and Pop, 2009). IslandViewer 3
(Dhillon et al., 2015) was used to predict and analyze the presence of
genomic islands in the E67 isolate genome using P. aeruginosa PAO1 as
a reference with the assistance of a BLAST based analysis.
2.6. Nucleotide sequence accession numbers
This Whole Genome Shotgun project has been deposited at DDBJ/
ENA/GenBank under the accession LXFB00000000. The version de-
scribed in this paper is version LXFB01000000.
3. Results and discussion
3.1. Strain phenotypic analysis
Pseudomonas aeruginosa E67 optimal temperature, pH and NaCl con-
centration for growth were determined as 37 °C, 6.0 and 0.5% (w/v), re-
spectively. These features are according to the ones previously reported
for this species (Klein et al., 2009; LaBauve and Wargo, 2005; Kumar
Deshwal and Kumar, 2013; Tsuji et al., 1982). Regarding the antibiotic
resistance phenotype, isolate E67 displayed resistance or intermediate
resistance to several antibiotics, namely amoxicillin, the combination
of amoxicillin with clavulanic acid, ampicillin, cefotaxime, kanamycin,
cephalothin, nalidixic acid, chloramphenicol, rifampicin, aztreonam,
imipenem, meropenem and tetracycline. However, it was susceptible
to cefepime, ceftazidime, ciprofloxacin, gentamicin, ticarcillin,
sulphamethoxazole with trimethoprim and piperacillin with tazobac-
tam. Phenotypic characteristics are summarized in Table 1.
Pseudomonas aeruginosa has intrinsic resistance to many antimicro-
bial agents including most penicillins, narrow-spectrum cephalospo-
rins, cefotaxime, and also other compounds such as tetracycline and
chloramphenicol, which in part explains the isolate's resistance pheno-
type obtained (Zhao and Hu, 2010; Lutz and Lee, 2011). However, E67
was found to have intermediate resistance to aztreonam, imipenem
and meropenem, antibiotics which are considered to be clinically rele-
vant antipseudomonal drugs (Magiorakos et al., 2012).
 P. Teixeira et al. / Marine Pollution Bulletin 110 (2016) 75–81
Table 1
Phenotypic characteristics of the isolate E67.
Characteristicsa
Antibiotic resistance phenotype AML AMC AMP ATM CTX KF IPM NA C K TE
SXT
Metal(oid) resistance phenotype As Cr Cu Hg Ni
Temperature optimum 37 °C
pH range for growth 6–8
NaCL concentration range for 0.5–4.0%
growth
a
AMP —ampicillin, AML — amoxicillin, AMC — amoxicillin/clavulanic acid, ATM — az-
treonam, KF — cephalotin, CTX — cefotaxime, IPM — imipenem, C — chloramphenicol, K —
kanamycin, TE — tetracycline, and SXT — sulfamethxazole-trimethoprim.
Pseudomonas aeruginosa E67 exhibits a metal(oid) resistance pheno-
type that allows it to survive in metal contaminated environments, in-
cluding resistance to arsenic, chromium, copper, mercury and nickel.
Given the relevant resistance phenotypes detected, a Whole Ge-
nome Sequencing (WGS) approach was adopted to gain detailed infor-
mation on the genetic basis of P. aeruginosa E67 antibiotic and metal
resistance, which may explain co-selection of resistance to both classes
of compounds.
3.2. General genome analysis
The analysis of the WGS showed that the draft genome is
6,876,232 bp in length comprised in 316 contigs with an average GC
content of 66.0%, which is consistent with previously reported P.
aeruginosa genomes (Winsor et al., 2011). Also, a total of 6760 coding
sequences (CDS) were predicted together with 62 tRNA and 5 rRNA
genes, as featured in Table 2. Taking into account the average read
length and the total number of reads, a genome coverage value of 79×
was calculated and also, a N50 value of 102,009 kb was obtained. No
plasmid was found in the genome sequence of this bacterium.
From the RAST automatic annotation resulted 261 contigs contain-
ing protein-coding genes (PEGs) and 563 subsystems that cover 55.3%
of the genomic features, while 42.0% of the genes were not inserted in
none of the RAST subsystems. In Table S1, it is noticeable that among
the 563 subsystems identified, carbohydrates metabolism, cofactors
metabolism and pigment biosynthesis are the most represented.
These results are in concordance with metabolic studies of P. aeruginosa
since metabolites related to amino acid and sugar metabolism have
been shown to represent approximately half of the bacteria metabo-
lome (Frimmersdorf et al., 2010; Silby et al., 2011).
3.3. Phylogenetic and genotypic analysis
According to the phylogenetic tree (Fig. 1), isolate E67 is closely re-
lated to P. aeruginosa DSM50071 and the ANI value of 99.43% obtained
also corroborates the species identification inferred by the analysis of
the phylogenetic tree. Furthermore, using the RAST pipeline, it was
also possible to confirm the isolate's identification by comparison to
the closest phylogenetic neighbors (Overbeek et al., 2014). Isolate E67
was assigned to ST395 by the Curran scheme for MLST (Curran et al.,
Table 2
General genomic features of the whole genome sequence of P.
aeruginosa E67.
Feature
Accumulated length 6,876,232 bp
Average GC content 66.0%
Number of contigs 316
N50 102.01 bp
Number of CDS 6760
Number of tRNA 62
Number of rRNA 5
77
2004). This ST is considered to be a high-risk clone due to its AmpC-me-
diated resistance to beta-lactams and aminoglycoside resistance medi-
ated by aadB determinants (Libisch et al., 2009). Also, this ST is
associated with a MDR phenotype and has been reported in hospital
wastewaters, water treatment plants and rivers in Hungary (Libisch et
al., 2009), eastern France (Slekovec et al., 2012), Spain (Fernández-
Olmos et al., 2013) and also in the UK Martin et al., 2013. Some of
these P. aeruginosa ST395 strains obtained from water sources were
also associated with infections, which suggest that MDR P. aeruginosa
can be easily transferred from its natural environment to clinical set-
tings (Kos et al., 2015). In terms of its clinical occurrence, it has been as-
sociated with infections in cystic fibrosis patients (Fernández-Olmos et
al., 2013) and other hospital infections in the previously referred coun-
tries (Libisch et al., 2009; Fernández-Olmos et al., 2013; Martin et al.,
2013; Cholley et al., 2011).
3.4. Resistance genetic determinants
By accessing the annotation of the resistance determinants, it is clear
that genes encoding for components of efflux pumps occupy a signifi-
cant part of the isolate's resistance arsenal (Table S2), which empha-
sizes these pumps contribution to a reduced susceptibility towards
antibiotics (Bambeke et al., 2000; Vila and Martínez, 2008). RND efflux
pumps that have been previously characterized in this species
(Piddock, 2006), namely MexAB-OprM, MexXY-OprM, MexCD-OprJ,
MexEF-OprN and MexHI-opmD were identified, as well as the regulator
genes merR, nefxB, mexT and mexG. The overexpression of these pumps
in P. aeruginosa is a recognized cause of antibiotic cross-resistance, being
responsible for the exportation of fluoroquinolones, tetracycline, chlor-
amphenicol, some β-lactams and also other unrelated compounds in-
volved in the quorum sensing process (Schwartz et al., 2015; Piddock,
2006). Genes related to other efflux pumps superfamilies such as MFS,
ABC, MATE, DMT and SMR were also well represented in the isolate's ge-
nome (Table S2) and are known to play a predominant role in the resis-
tance to certain antibiotics (e.g. tetracycline, fluoroquinolones,
erythromycin and macrolides, among others) and other unrelated com-
pounds such as organic solvents (Piddock, 2006; Tian et al., 2009; He et
al., 2004). We also detected a high number of genes coding for ABC
transporter proteins that are involved in the secretion of several viru-
lence factors in addition to antimicrobial compounds efflux (Piddock,
2006).
Genes conferring resistance to specific antibiotics were identified
and are summarized in Table 3. Regarding β-lactam resistance, four
genes encoding for hydrolases belonging to the beta-lactamase class C
were annotated by RAST and confirmed with a second annotation
based on IMG/ER. This is a characteristic feature described in ST395
Pseudomonas isolates (Slekovec et al., 2012; Libisch et al., 2009). One
of these genes has high similarity (98.7% in terms of deduced amino
acid sequence) with the chromosomal drug-inducible gene blaAmpC,
which encodes a wide-spectrum class C beta-lactamase that contributes
to the intrinsic β-lactam resistance of P. aeruginosa (Zhao and Hu, 2010).
Also, directly upstream from blaAmpC, the HTH-type transcriptional acti-
vator ampR was detected. When overproduced as a result of mutations,
AmpC expression may become a major cause of resistance to widely
used antipseudomonal penicillins (ticarcillin and piperacillin),
monobactams (aztreonam), and third-generation (ceftazidime) and
fourth-generation (cefepime) cephalosporins (Berrazeg et al., 2015).
Other genes identified encoded putative class C beta-lactamases that ac-
cording to the ARDB database displayed from 27.5% to 35.0% amino acid
similarity with previously described beta-lactamases. The hydrolytic
pattern of these proteins has never been investigated and future studies
are needed to assess their possible contribution to the pseudomonads
resistance profiles. Additionally, a gene encoding a putative class B
beta-lactamase was identified and the deduced amino acid sequence
displayed 23.4% of similarity (according to the ARDB database) with a
previously described metallo-beta-lactamase. Also, a naturally
78 P. Teixeira et al. / Marine Pollution Bulletin 110 (2016) 75–81

Fig. 1. Phylogenetic tree based on 16S rDNA and gyrB sequences of 20 type strains of Pseudomonas species and E67 isolate, generated by the neighbor-joining method. The numbers at the
nodes represent levels (%) of bootstrap support from 1000 resampled datasets.

occurring oxacillinase gene in P. aeruginosa, blaOXA-50, was detected and
the encoded protein displayed 99.0% amino acid similarity with the one
encoded in the genome of P. aeruginosa PAO1. The expression of this
gene has been previously described to promote decreased susceptibility
to ampicillin, ticarcillin, moxalactam and meropenem (Girlich et al.,
2004), though its contribution to the resistance phenotype has low rel-
evance (Courvalin et al., 2010).
Pseudomonas aeruginosa E67 resistome also harbors other relevant
antibiotic resistance determinants such as genes putatively expressing
phosphotransferases (24.8% to 99.3% similarity in terms of amino acid

Table 3
Genes conferring antibiotic resistance found in P. aeruginosa E67.

Antibiotics Genes Contig location Products

Aminoglycosides aph 87_6184 Aminoglycoside phosphotransferase

aph 92_6490 Aminoglycoside 3′-phosphotransferase type IIb
aph 65_5285 Predicted aminoglycoside phosphotransferase
aph 65_5286 Predicted aminoglycoside phosphotransferase
Beta-lactams bla 3_2989 Beta-lactamase class C and other penicillin binding proteins
blaOxa-50 3_3019 Beta-lactamase OXA-50-like
bla 21_2044 Beta-lactamase class C
bla 3_2898 Beta-lactamase Class B
bla 48_4456 Beta-lactamase class C
ampG 12_768 Permease
bla 65_5322 Beta-lactamase class C
ampR 84_6081 HTH-type transcriptional activator ampR
ampC 84_6082 Beta-lactamase class C
Chloramphenicol catb4 23_2260 Type-B-Chloramphenicol O-acetyltransferase
qac 3_2983 Arabinose ABC transporter permease
rarD 42_4113–4114 Chloramphenicol-sensitive protein
rarD 93_6551 Chloramphenicol-sensitive protein
Lincosamide uup 91_6459 ABC transporter ATP-binding protein
Polymyxin arnT 35_3580 Undecaprenyl phosphate-alpha-4-amino-4-deoxy-L-arabinose arabinosyl transferase
arnD 35_3581 4-deoxy-4-formamido-L-arabinose-phosphoundecaprenol deformylase
arnA 35_3582 UDP-4-amino-4-deoxy-L-arabinose formylase
arnC 35_3583 Glycosyl transferase
eptA 52_4839 Phosphoethanolamine transferase
arnC 43_4225 Glycosyl transferase
arnT 43_4226 Undecaprenyl phosphate-alpha-L-Ara4N transferase
Streptogramin vgb1 42_4095 virginiamycin B lyase
Tetracycline tetA 36_3666 Tetracycline resistance protein
Vancomycin vanW 13_878 Vancomycin B-type resistance protein
 P. Teixeira et al. / Marine Pollution Bulletin 110 (2016) 75–81
sequence with aminoglycoside O-phosphotransferases present in P.
aeruginosa PA01genome, according to the ARDB database) and a chlor-
amphenicol acetyltransferases (98.7% similarity in terms of amino acid
sequence with a group B acetyltransferase found in other members of
the Pseudomonas genus), responsible for both enzymatic modification
and inactivation of aminoglycosides and chloramphenicol respectively.
These genes have been previously characterized by Schwartz and his
co-workers in P. aeruginosa (Schwartz et al., 2015). Also, genes were
identified whose expression may be responsible for polymyxin resis-
tance (e.g. genes 3580 to 3583 and 4225/4226 in contigs 35 and 43, re-
spectively) through the induction of modifications in the LPS structure
that results in reduced binding to polymyxins (Olaitan et al., 2014). Fi-
nally, a gene coding for a Virginiamycin B lyase, an enzyme responsible
for the inactivation of type B streptogramin antibiotics (Korczynska et
al., 2007), was identified (gene 4095 in contig 42).
Additionally, a gene coding for a putative vancomycin B-type resis-
tance protein was identified, which displayed 32.3% similarity with a
gene present in a VanG type vancomycin resistance operon characteris-
tic of Enterococcus and Eubacterium (Boyd et al., 2006; Courvalin, 2006).
This type of resistance in Pseudomonas is still poorly described in the lit-
erature since this antibiotic is not a choice for treatment in cases of P.
aeruginosa infections. However, the presence of this gene may be relat-
ed to functions other than antibiotic resistance.
In order to survive in a metal-contaminated environment, P.
aeruginosa E67 genome contains a diverse arsenal of genes putatively
contributing to the metal resistance phenotypes, whereas most of
them code for components of efflux systems. Besides actively expelling
antibiotics, some specific systems such as the cop system (Fig. 2) and P-
type ATPase's are responsible for conferring tolerance to metals like
copper, zinc, cobalt, chromium and cadmium (Teitzel and Parsek,
2003). Two mer operons, one complete operon located in the PAGI-2 ge-
nomic island (Fig. 2) and an incomplete operon missing the merA gene
were also identified. These structures are responsible for the reduction
of toxic Hg2+ to volatile Hg0 (Osborn, 1997), and were detected in the
isolate's genome without the merB gene, which is a common feature
in some organisms (Bruins et al., 2000). Also, mobile genetic determi-
nants were located next to both operons.
The complete ars operon, which is responsible for arsenic resistance
(Bruins et al., 2000), was also detected (Fig. 2). These resistance deter-
minants have already been identified in other metal resistant isolates,
mainly environmental isolates that are found in agricultural soils irrigat-
ed with wastewater and other metal contaminated sites (Schwartz et
al., 2015; Teitzel and Parsek, 2003; Malik and Aleem, 2011). Nonethe-
less, these metal resistant isolates have also been identified in clinical
settings, although the prevalence of metal resistance is considerably
lower comparatively to environmental isolates (Deredjian et al., 2011).
Regarding mobile genetic elements, we explored the RAST subsys-
tem related to transposable elements and phage elements and we de-
tected fourteen genes coding for integrases, and twenty-one for
transposases, as well as eleven genes coding for site-specific
recombinases, four for relaxases and five for other widespread coloniza-
tion island factors. In order to understand if detected mobile genetic el-
ements are organized in specific chromosomal regions, we screened the
E67 isolate's genome for genomic islands (Table S3). This analysis re-
vealed that one of the mer operons (Fig. 2) as well as genes coding for
Fig. 2. Diagram of the mer (A), cop (B) and ars (C) operons present in P. aeruginosa E67. In B) the HP stands for hypothetical protein. The mer operon (A) was present in the PAGI-2.
79
proteins associated with multidrug-resistance efflux pumps (such as
membrane fusion proteins and membrane transporters belonging to
the RND family) are located in the mobile structure PAGI-2 (Kung et
al., 2010). Additionally, the full-length or nearly full-length of other ge-
nomic islands previously described in P. aeruginosa were also identified,
namely PAGI-3, PAGI-4, PAGI-6, PAGI-7, PAGI-8 and PAGI-9 (Kung et al.,
2010; Battle et al., 2009). Most of these structures comprise genes relat-
ed to virulence, metabolic and transport functions (Dobrindt et al.,
2004). Various integrative conjugative elements (ICE's) resembling the
ones present in the PFGI-1 genomic island described in Pseudomonas
fluorescens Pf-5 were detected mainly in a PAPI-1/PAGI-5-like genomic
island and also in PAGI-2 (Mavrodi et al., 2009). Some of these ICE's be-
longing to the PFGI-1 genomic island are known to contribute to the
survival of Pseudomonas isolates by providing protection from environ-
mental stress (Seth-smith and Croucher, 2009). A genomic island con-
taining CRISPR repeat sequences belonging to the I-E CRISPR-Cas
subtype was also detected (Fig. 3). Besides their well-known function
as a bacterial adaptive immune system, CRISPR systems have also
been proved to play an important role in controlling HGT and, conse-
quently, the dynamics of antibiotic resistance in P. aeruginosa (van
Belkum et al., 2015). Genomic islands are known to contribute to the
evolution of microbial genomes, conferring rapid changes in virulence
potential and influencing traits such as antibiotic resistance, symbiosis,
fitness and adaptation in general (Hacker and Carniel, 2001). More in-
formation on the location and composition of the identified genomic
islands is shown in Table S3.
Multi-resistant Pseudomonas isolates have already been described
both in clinical and environmental settings. However, reports of MDR
isolates prevenient from hospitalized patients are more abundant, due
to the selective pressures that result from the use of antibiotics for the
treatment of P. aeruginosa infections (Santos-medellín et al., 2014).
Nonetheless, selective pressures present in natural environments such
as rivers and lakes as a result of human practices, are leading to the oc-
currence of an increasing number of MDR environmental Pseudomonas
isolates (Berendonk et al., 2015). Thus, despite being an environmental
isolate, P. aeruginosa E67 owns a vast arsenal of resistance genes that
contributes to its survival in a heavily polluted ecosystem. Also, the as-
sociation of some of these genes to mobile genetic elements helps to
confirm that the acquisition of resistance genes by HGT plays an impor-
tant role in the proliferation of this species and is often the cause of
MDR.
There is no evidence of a physical link between metal and antibiotic
resistance genes, and instead there seems to exist a predominance of
cross-resistance mechanisms associated with reduced multidrug efflux
pumps expression.
Role of the funding source
This work was supported by Fundação para a Ciência e Tecnologia
through project StARE (WaterJPI/0002/2013) and grants IF/00492/
2013 (I. Henriques) and IF/00835/2013 (A. Alves). Managers of the
funding source had no involvement on the study design, or in data col-
lection and interpretation, and neither on this manuscript's preparation
and submission. Such decisions are of the entire responsibility of the
authors.
80 P. Teixeira et al. / Marine Pollution Bulletin 110 (2016) 75–81
Fig. 3. Schematic representation of the CRISPR-Cas type I–E structure identified in the genome of P. aeruginosa E67.
Acknowledgments
The authors wish to acknowledge COST-European Cooperation in
Science and Technology, through COST Action ES1403: New and emerging
challenges and opportunities in wastewater reuse (NEREUS). Also we
wish to thank Jaqueline Rocha for assistance during the work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.marpolbul.2016.06.086.
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