Journal of Fish Diseases 2016
12579
Search and analysis of genes involved in
antibiotic resistance in Chilean strains of Piscirickettsia
salmonis
~ oz1,2, A Yan
C Cartes1,2, A Isla1,2, F Lagos1,2, D Castro1,2, M Mun
and J Figueroa1,2
1 Faculty of Sciences, Institute of Biochemistry and Microbiology, Universidad Austral de Chile, Valdivia, Chile
2 FONDAP Centre: Interdisciplinary Centre for Aquaculture Research (INCAR), O’Higgins, Concepcion, Chile
3 Department of Basic Sciences, Faculty of Sciences, Universidad Santo Tomas, Valdivia, Chile
Abstract
Piscirickettsia salmonis is the pathogen causing Pis-
cirickettsiosis. For treatment, the industry mainly
uses oxytetracycline and florfenicol, so it is essen-
tial to understand the degree of susceptibility of
this pathogen to these drugs. But this is still
unknown for a large number of P. salmonis
strains, as are the molecular mechanisms responsi-
ble for greater or lesser susceptibility. However,
genes that confer resistance to these antimicrobials
have been reported and characterized for this and
other bacterial species, among which are mem-
brane proteins that take out the drug. Our results
identified differences in the degree of susceptibility
to both antibiotics among different Chilean iso-
lated of these bacteria. We analysed 10 available
genomes in our laboratory and identified ~140
genes likely to be involved in antibiotic resistance.
We analysed six specific genes, which suggests that
some of them would eventually be relevant in
conferring resistance to both antibiotics, as they
encode for specific transporter proteins, which
increase the number of transcripts when grown in
media with these antibiotics. Our results were cor-
roborated with EtBr permeability analysis, which
revealed that the LF-89 strain accumulates this
compound and has a reduced capacity to expulse
it compared with the field strains.
Correspondence J Figueroa, Institute of Biochemistry and
Microbiology, Universidad Austral de Chile, Casilla 567, Val-
divia, Chile (e-mail: jefigueroa@uach.cl)
Ó 2016
John Wiley & Sons Ltd 1
doi:10.1111/jfd.
~ ez1,2, D Haussmann1,2,3
Keywords: antibiotic resistance genes, Piscirickettsia
salmonis, salmonid rickettsial septicaemia.
Introduction
Piscirickettsia salmonis is a pathogenic bacterium
that causes salmonid rickettsial septicaemia (SRS)
or Piscirickettsiosis. It is able to infect different
species of salmonids and is currently considered
an endemic disease in Chile, causing significant
losses in salmon production in the south of Chile
(McCarthy et al. 2008; Rojas et al. 2009).
P. salmonis is a bacterium currently described as
intracellular facultative, due to its ability to grow
in cell-free artificial media (Mauel, Ware & Smith
2008; Mikalsen et al. 2008; Vera et al. 2012;
~ez et al. 2012). It is aerobic, Gram negative,
Yan
pleomorphic – although generally it has coccoid
or ring morphology – stationary, not encapsu-
lated, and has a diameter of between 0.2 and
1.5 lm (Bravo & Campos 1989; Rojas et al.
2007). P. salmonis has been classified as a
gammaproteobacteria (Fryer 2002), in the order
Thiotrichales, family Piscirickettsiacaea, genus Pis-
cirickettsia according to the sequencing of the 16S
rRNA gene (Fryer et al. 1992; Rozas & Enrıquez
2014).
In active cases of bacterial diseases, the adminis-
tration of antibiotics is still the main control tech-
nique. Among the existing treatments are
antimicrobials such as oxytetracycline, florfenicol,
 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

oxolinic acid and flumequine, the first two being

Materials and methods
the most used by the Chilean salmon industry.
Both oxytetracycline and florfenicol are broad- Bioinformatic analysis of P. salmonis genomes
spectrum bacteriostatic antimicrobials. Oxytetracy-
Genomes of P. salmonis corresponding to the LF-
cline’s action mechanism consists in reversibly
89 reference strain (ATCC VR-1361, Pulgar
inhibiting the synthesis of proteins, preventing the
et al. 2015), and Chilean strains IBM-009 iso-
union of aminoacyl-tRNA to the A site of the
lated from O. mikiss (Similar to LF-89, Man-
bacterial ribosome, joining directly to protein S7
dakovic et al. 2016) and IBM-034 isolated from
of the 30S subunit (Goldman et al. 1983). In the
O. mikiss (similar to EM-90, Mandakovic et al.
case of florfenicol, this also acts by inhibiting the
2016), were analysed using the bioinformatics
bacterium’s protein synthesis, by inhibiting pep-
tool GenDB, after having sequenced them en
tidyl transferase by reversible binding to the sub-
masse covering close to 85% with Roche 454
unit ribosomal 50S (Cannon, Hartford & Davies
and Illumina technology. The putative antibiotic
1990). However, the result of antimicrobial treat-
resistance genes found using this software were
ments is that their effectiveness has diminished
compared between strains, by using BLAST and
compared to their first uses for the control of
comparing these genes against NCBI databases in
P. salmonis (Branson & Diaz-Munoz 1991; Cvi-
order to see the presence or absence of these dif-
tanich, Garate & Smith 1991), which indicates
ferent analysed strains. After obtaining the coding
that the bacterium has developed resistance to
sequences of these genes, we studied the con-
these drugs (Henrıquez et al. 2016).
served domains of each amino acid level using
The rapid expression of genetic variation in
the bioinformatics tool online SMART, together
bacteria is favoured by their rapid multiplication
with the application Conserved Domains and Pro-
– which produces a large number of generations
tein Classification (NCBI). The identity percent-
in a short time – and also by the haploid
age of these sequences compared with those of
genetic material. Throughout the years, bacteria
other bacterial species was analysed using infor-
have developed genetic and biochemical mecha-
mation from the NCBI database (http://
nisms to avoid the inhibitory effects of antibi-
www.ncbi.nlm.nih.gov/), using the BLAST tool,
otics (Levin & Bergstrom 2000). Among these
determining the sequences with greatest amino
are drug transporter pumps, which are mem-
acid identity. We performed an in silico analysis
brane proteins that have been characterized in
of these genomes to identify the total number of
five families (Li & Nikaido 2009). There are
antibiotic resistance genes present using the tool
also resistance mechanisms by enzymatic inacti-
PSORTb v3.0.2.
vation and ribosomal protection proteins, among
others. Drug resistance mechanisms for some
fish pathogens have been studied; however, there
Cultivation of P. salmonis
is little information for the case of P. salmonis.
Therefore, the degree of resistance or susceptibil- Strains of P. salmonis grew to infect the SHK-1 cell
ity of different subtypes of P. salmonis present line with 70–80% confluence in Leibovitz’s L-15
to these antimicrobials, as well as which genes grow medium supplemented with SBF 10%. Cells
and mechanisms may be involved in conferring were maintained at a temperature of 20 °C for
it, is of fundamental importance. Therefore, the ~5 days depending on the bacterial strain and its
objective of the present study was to analyse infection speed. Upon showing cytopathic effects,
antimicrobial susceptibility in the two most fre- the bacteria were released to the medium, and ali-
quently used antimicrobials, in different strains quots of 150 and 200 lL were taken from the med-
of P. salmonis isolated in Chile, and compare ium and were added to 8 lL of modified CASO
them with the type LF-89 strain, as well as liquid culture (Vera et al. 2012). Each culture of
identify probable genes that could be involved liquid medium with bacteria was incubated in a sha-
in conferring this resistance and evaluate how ker at 18 °C, protected from light, at 150 rpm for
their expression varies in bacteria grown with ~4–5 days depending on the strain, until reaching
different concentrations of antibiotics at different an OD600 of 0.6–0.8. We certified P. salmonis
times. using Gram, IFAT and PCR (Mauel et al. 2008).

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 Journal of Fish Diseases 2016
Cultivation of E. coli ATCC 25922
Bacteria were grown on an LB agar plate from a
colony in 5 mL of modified Mueller–Hinton
medium and allowed to grow at 37 °C for 24 h
prior to its use, reaching an A600 nm of 0.8 (Miller
et al. 2005).
Design and standardization of MIC
Both oxytetracycline and florfenicol were prepared
in 8 mg mL 1 stock solution. Oxytetracycline was
dissolved in absolute methanol, and florfenicol
dissolved in 95% ethanol. Both antibiotics
(Sigma) were stored at 20 °C (protected from
light and no more than 2 weeks of preparation).
The technique was first mounted with E. coli
ATCC 25922, to validate the method. Once the
bacterium had grown, microdilutions of this strain
were made based on the microplate dilution
method to determine MIC in aquatic bacteria
~ez et al. 2014).
(Miller et al. 2005; Yan
We used sterile 96-well ELISA plates with lids
and evaluated concentrations of 0.02 lg mL 1 up
to 8 lg mL 1, as well as a drug-free positive con-
trol and a negative control, with only medium.
The cultures were grown at 37 °C for 19 h
~ez et al. 2014). Subsequently, we read absor-
(Yan
bance at 600 nm using a microplate reader, and
the analysed data were plotted using SigmaPlot
11.0 software. Once the technique was standard-
ized, we determined the MIC in the aforemen-
tioned strains of P. salmonis. The P. salmonis
strains were grown in supplemented CASO liquid
medium until reaching an A600 nm of 0.8, and the
plates were incubated at 18 °C for 120 h, for sub-
sequent determination in the microplate reader
and data analysis. Each test was carried out five-
fold for each strain and the MIC value was
defined as the lowest concentration of antibiotics
for which no bacterium growth was observed at
18 °C after 120 h of incubation. The effect that
the antibiotic solvents – methanol and 95% etha-
nol – could produce on the bacterium’s growth
was also evaluated, using the same system used to
determine the antibiotics’ MIC, but using distilled
water rather than antibiotics.
Expression kinetics
The P. salmonis strains were grown in liquid med-
ium as described above, and once the exponential
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C Cartes et al. Genic antibiotic resistance in SRS
phase was reached, they were subjected to antimi-
crobial treatments. The treatments were performed
in triplicate and separately with oxytetracycline
and florfenicol at two different concentrations,
which were evaluated as non-lethal to the bac-
terium (0.08 lg mL 1). For each strain, we per-
formed a kinetics treatment at 2, 4 and 6 h with
respect to the bacterium without treatment, to
ultimately evaluate expression changes over time
with different concentrations of antibiotics using
RT-qPCR.
Extraction of total RNA from P. salmonis
We extracted the total RNA from each strain of
P. salmonis at each point in the kinetics. We col-
lected 1 mL of the sample for each case, which
we centrifuged at 14 000 9 g for 3 min. Subse-
quently, we extracted the total RNA, using a Tri-
zol Kit (Ambion). The sediment was resuspended
in 1 mL of Trizol, and then we added 0.2 mL of
chloroform by gently mixing and incubating for
3 min at room temperature. The solution was
then centrifuged at 12 000 9 g for 15 min at
4 °C and the aqueous phase was precipitated with
0.6 mL of isopropanol (+glycogen). We then
incubated this for 5 min at room temperature and
centrifuged at maximum speed for 10 min at
4 °C. The sediment was washed with 70% etha-
nol and subsequently centrifuged at 7500 9 g for
5 min at 4 °C. Finally, the settled RNA was left
to dry for 15 min at room temperature to later be
resuspended with DEPC-treated water. The RNA
was quantified by spectrophotometry at 260 nm,
using NanoVue (General Electric). Before cDNA
synthesis, we treated the sample, eliminating geno-
mic DNA using DNAse (Ambion) for 1 h accord-
ing to the manufacturer’s instructions. To confirm
the elimination of the gDNA, we performed a
PCR, amplifying the groE gene as the constitutive
expression normalizing gene of P. salmonis by per-
forming a PCR pre- and post-treatment with
DNAse.
cDNA synthesis
We performed cDNA synthesis for the first strand
by reverse transcription, using M-MLV reverse
transcriptase (Promega) and random primer (Pro-
mega) according to the manufacturer’s instruc-
tions. This was done in a volume of 20 lL,
adding 1 lL of dNTP mix (10 mM), 2 lL of
 Journal of Fish Diseases 2016
random primer (500 lg mL 1), 1 lg of total
RNA and 14.5 lL nuclease-free water. The mix-
ture was incubated for 10 min at 60 °C and
cooled in ice. Then, we added 1 lL of reverse
transcriptase, 4 lL of 59 enzyme buffer and
0.5 lL of RNAse inhibitor (rRNAsin, Promega).
The mixture was incubated for 60 min at 37 °C
followed by 10 min at 70 °C and the cDNAs
were stored at 20 °C until usage.
Expression analysis of P. salmonis genes by
qPCR
Having obtained the sequences for the genes to be
analysed using the described bioinformatics tools,
we designed qPCR primers for these genes, using
the PerlPrimer software described in Table S1.
We performed the expression analysis of the genes
defined in the genomes of P. salmonis strains
using the cDNA from the samples taken at each
point of the kinetics described above as reference.
We used the real-time GoTaq qPCR Master Mix
kit (Promega) according to the manufacturer’s
instructions. The qPCR was performed in Strata-
gene MX3000P and MX3005 equipment. The
equipment was programmed with the following
cycles: initial denaturation at 95 °C for 10 min,
45 cycles at 94 °C for 20 s, 60 °C for 15 s and
72 °C for 20 s. We also added one step in each
run to produce the dissociation curve, with 95 °C
for 20 s, 63 °C for 10 s and 95 °C for 1 s. Fluo-
rescence was measured at the end of each of the
45 cycles and during the last temperature increase,
for the elaboration of the dissociation curve. We
used the rpoD gene from P. salmonis as the nor-
malizing gene. Each quantification was performed
in triplicate. We calculated the baseline for each
sample’s cycle threshold (ct) by the MxPRO pro-
gram (Stratagene) using the LMS (least mean
square) algorithm and the same program that
delivered the ct data. We performed the relative
quantification according to the ‘DDct’ formula
(Pfaffl 2001), plotted the data and performed a
Student’s t-test using the program Sigmaplot 11.0,
selecting P < 0.01 as highly significant (**) and
P < 0.05 as significant (*).
EtBr accumulation test
The P. salmonis strains grew in liquid medium
under the same conditions as above, until reaching
the exponential phase. Subsequently, they were
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C Cartes et al. Genic antibiotic resistance in SRS
diluted in fresh liquid medium until reaching an
OD600 nm of 0.2. Finally, the bacteria were incu-
bated with and without EtBr 0.5 lg mL 1 (a
concentration which does not affect viability or
cell function (Viveiros et al. 2008)), in darkness at
18 °C with moderate stirring. We collected ali-
quots of 1 mL at different times. Each aliquot
was centrifuged at 2500 9 g for 10 min. We
washed each sediment twice with PBS 1X, and
these were finally resuspended in 300 lL of PBS
1X and placed on black 96-well plates for quan-
tification (Synergy 2 microplate reader, BioTek),
by measuring EtBr fluorescence within the bacte-
ria at 485/620 excitation/emission. Afterwards, we
placed the same sample in a 96-well plate to read
OD600 nm in the same microplate reader.
The results were expressed using the fluorescence/
absorbance ratio.
Results
In silico prediction of antibiotic resistance
genes in Chilean isolates of P. salmonis
We used the LF-89 reference strain genome (Pul-
gar et al. 2015) and two sequenced (but not
closed) genomes from field strains of P. salmonis
isolated in the south of Chile. The total numbers
of antibiotic resistance genes present in each
strain were classified according to their subcellu-
lar locations, which were relatively similar among
the three analysed strains. For example, the genes
that are probably involved and encode for pro-
teins located in the cytoplasm vary between 42
and 44, whereas those located in the outer mem-
brane vary between 2 and 3. For their part, those
corresponding to proteins located in the inner
cell membrane fluctuate between 80 (LF-89) and
85 (IBM-034). We also identified one gene for a
protein located in the periplasm (LF-89 and
IBM-034) and three to four genes for proteins of
unknown function. Globally, we determined 133
genes for the LF-89 strain, 134 genes for the
IBM-009 strain and 137 genes for the IBM-034
strain (Table 1). Subsequently, we classified these
genes for each analysed strain, according to the
response they produce against the mechanisms
different antibiotics produced. This analysis was
performed using the ARDB tool and can be
summarized in the following way: interfere syn-
thesis of cell wall (49 genes for LF-89, 51 genes
for IBM-009 and 54 genes for the IBM-034
 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

Table 1 In silico prediction of subcellular location of antibiotic Table 2 In silico prediction of antibiotic resistance genes
resistance genes
No. genes found in
Cellular Localization LF-89 IBM-009 IBM-034 Piscirickettsia salmonis
strains
Cytoplasm 44 44 42
Membrane external 2 2 3 General Action Mechanism LF-89 IBM-009 IBM-034
Extracellular 2 3 3
Membrane cytoplasmic 80 82 85 Interfere synthesis of cell wall 49 51 54
Periplasm 1 1 Inhibition of DNA gyrase 7 7 4
Unknown 4 3 3 Interfere synthesis of folic acid 2 2 2
Total 133 134 137 Interfere protein synthesis 45 47 43

The total number of genes per strain is indicated. Analysis with the tool Classification according to bacterial response against inhibition
PSORTb v3.0.2. mechanisms of various antibiotics. Analysis with ARDB tool.

Table 3 Oxytetracycline putative resistance genes in field

strain), inhibition of DNA gyrase (seven genes
for LF-89 and IBM-009; four genes for IBM-
034), interfere synthesis of folic acid (two genes
for each of the strains) and interfere protein syn-
thesis (45 genes for LF-89, 47 for IBM-009 and
43 for IBM-034, Table 2).
Subsequently, the in silico analysis (DB gene)
allowed us to identify the probable genes
involved in resistance to the antibiotics oxytetra-
cycline and florfenicol as well as multidrug resis-
tance genes. The genetic resistance to
oxytetracycline and florfenicol tracked in these
genomes is summarized in Table 3. Note that
not all of these genes were found present in the
three strains, indicating constituent differences in
the analysed genomes. The BLASTN alignment
of the genes’ nucleotide sequences with the
NCBI database assigned an annotation and we
established a nomenclature of the genes present
in P. salmonis and other bacterial species that
showed strong homology (Table 4). We analysed
amino acidic sequences delivered by the software
GenDB with SMART, which predicted two con-
served domains in all the analysed sequences, cor-
responding to the MFS1 domain (Major
facilitator superfamily domain), as well as a trans-
membrane domain located near the extreme car-
boxyl terminal. This indicates that membrane
proteins act as transporters, data that correlate
with those obtained via the BLASTN alignments,
which indicated strong homology with genes that
encode for membrane transporter proteins,
mainly for oxytetracycline and florfenicol
(Table 4). In this investigation, we studied genes
potentially involved in conferring antibiotic resis-
tance at genomic level in field strains of
P. salmonis IBM-034 and IBM-009, as well as
the strain LF-89, and we were able to identify
six putative genes resistant to these antibiotics.
strains of Piscirickettsia salmonis
tet-1 tet-2 bcr/ bcr2/ Bcr3/
Strains transp. tet-C transp. cfla cfla cfla
LF-89 + + + + +
IBM-009 + + + + + +
IBM-034 + + + + +
The putative genes possibly involved in conferring resistance to oxyte-
tracycline found by GenDB 2.2 software in Chilean isolate strains of
P. salmonis. Presence is indicated by (+) and absence by ( ).
Specific resistance genes for tetracyclines
From the bioinformatic analyses, we were able to
identify an encoding gene for a putative protein
resistant to tetracyclines and oxytetracyclines of
412 amino acids, which, by sequence homology,
corresponds to Tet-1 transporter, a transmem-
brane protein belonging to the Tet family, which
is responsible for conferring resistance to various
types of tetracycline (Chopra & Roberts 2001).
This transporter exports these molecules from the
bacterium, preventing them from reaching lethal
concentration. This gene was found to be present
in all analysed strains of P. salmonis. We also
identified an encoding gene for a protein resistant
to tetracycline and oxytetracycline of 411 amino
acids, to which we gave the annotation Tet-C.
Like Tet-C, this is a transmembrane protein
involved in conferring resistance to various types
of tetracycline (Chopra & Roberts 2001) and has
65% identity with proteins of the Tet of Legio-
nella and 41% of Coxiella (Table 4). Tet-2 trans-
porter was found in the IBM-009 strain, LF-89
strain (Pulgar et al. 2015) and IBM-034 strain
(Table 3). On the other hand, we identified a
putative encoding gene for a transporter pump of
417 amino acid, indicating that this protein could
be responsible for conferring resistance to

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 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

Table 4 In silico analysis of amino acidic identity of proteins putatively involved in oxytetracycline and florfenicol resistance with
other bacterial species

Amino acidic
Putative protein BLASTP Microorganism Seq. Identity Query Coverage Accession Number

Tet-1 Transporter MFS Transp. Piscirickettsia salmonis 99% 100% WP_016210134.1

Drug Transp. Legionella drancourtii 36% 91% WP_040535535.1
MFS Drug Transp. Legionella longbeachae 35% 99% WP_003631907.1
MFS Transp. Legionella cincinnatiensis 36% 98% WP_058465310.1
Tet Efflux transp. A. machinpongonensis 34% 92% WP_008201881.1
Tet-C MFS Transp. Piscirickettsia salmonis 100% 100% WP_017376863.1
MFS Transp. Legionella brunensis 65% 98% WP_058442504.1
MFS Transp. Legionella feelei 64% 98% WP_058443727.1
Drug Transp. Coxiella burnetti 41% 96% AIT63334.1
Tet-C Pseudovibrio axinellae 36% 89% KZL17046.1
Tet-2 Transporter MFS Transp. Piscirickettsia salmonis 100% 100% WP_016210850.1
MFS Transp. Photobacterium damselae 37% 96% WP_044175786.1
MFS Transp. Legionella lansingensis 26% 96% WP_051546195.1
MFS Transp. Photobacterim leiognathi 36% 95% WP_060988267.1
MFS Transp. Vibrio litoralis 36% 95% WP_027695523.1
Bcr/Cfla Bcr/Cfla drug transp. Piscirickettsia salmonis 100% 100% WP_016210940.1
Bcr/Cfla drug transp. P. acanthamoebae 32% 95% WP_006340377.1
Bcr/Cfla drug transp. Vibrio ordalii 34% 86% WP_029627046.1
Bcr/Cfla drug transp. Vibrio anguillarum 34% 86% WP_019282125.1
Bcr/Cfla drug transp. Vibrio owensii 32% 92% WP_038893731.1
Bcr2/Cfla Bcr/Cfla drug transp. Piscirickettsia salmonis 100% 100% WP_016209738.1
Bcr/Cfla drug transp. E. montiporae 39% 86% WP_034872832.1
Bcr/Cfla drug transp. E. elysicola 34% 86% WP_020583596.1
Bcr/Cfla drug transp. T. salexigens 34% 89% WP_028796317.1
Bcr/Cfla drug transp. Beggiatoa alba 35% 86% WP_002686147.1
Bcr3/Cfla Bcr/Cfla drug transp. Piscirickettsia salmonis 100% 100% WP_016209946.1
Bcr/Cfla drug transp. Aeromonas eucrenophila 34% 93% WP_042642495.1
Bcr/Cfla drug transp. Aeromonas media 35% 93% WP_005327414.1
Bcr/Cfla drug transp. Aeromonas salmonicida 35% 90% WP_058394676.1
Bcr/Cfla drug transp. Aeromonas tecta 34% 90% WP_050718350.1

The putative genes possibly involved in conferring resistance to florfenicol and tetracycline found by GenDB 2.2 software in Chilean isolate strains of
P. salmonis. The highest percentages of homology with respect to NCBI sequences are shown.

oxytetracycline, because it is present in all the
analysed strains of P. salmonis. Subsequently,
through domain analysis (SMART), we could
determine the transmembrane domains, two for
Tet-1 transporter, and one for Tet-C and the
tetracycline 2 transporter (Tet-2 transporter).
Additionally, we were able to determine a con-
served domain of the MFS family, to which the
Tet proteins Tet-C, Tet-1 transporter and the
Tet-2 transporter also belong (Bolhuis et al. 1997;
Schwarz et al. 2004), which corroborates that per-
haps membrane proteins confer resistance.
Another characteristic worth mentioning is that
the tet-1 transporter gene is found to be present in
a pathogenicity island, which is present in the two
analysed field strains of P. salmonis and in the LF-
89 strain which shows evidence of how the bac-
terium might have acquired this resistance gene
(data not shown).
In the case of the tet-C gene (strain IBM-009),
it was not observed that this gene be present in a
genomic or pathogenicity island; however, in its
genetic environment, the presence of transposases
was observed, which could provide evidence that
the gene is part of a transposon. Finally, we found
that the tet-2 transporter gene is not part of a
genomic island; however, we did observe other
putative resistance pumps located near this gene.
It could be seen that genetic environment, com-
prising a block of genetic homology analysed
through the software MAUVE, is the same in the
three analysed strains.
These results indicate that the three oxytetracy-
cline-specific resistance genes found in the gen-
omes of these strains are oxytetracycline
transporter pumps, something already seen in pre-
vious work on other bacteria, where it has been
described that in Gram-negative bacteria, the pre-
dominant resistance mechanisms to tetracyclines
are the drug carrier pumps (Poole 2005). In addi-
tion, this type of tetracycline resistance by trans-
porter pumps has also been observed in other fish

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 Journal of Fish Diseases 2016
pathogens such as Photobacterium, Vibrio, Pseu-
domonas, Alteromonas, Citrobacter and Salmonella
spp., seen in previous papers by Furushita, Shiba
& Maeda (2003).
Other transporters
The bioinformatic analysis identified an encoding
gene for a putative transporter protein of the Bcr/
Clfa family (395 amino acids), which corresponds
to a transmembrane protein (12 transmembrane
segments), a family of proteins that confers resis-
tance to various antibiotics, including molecules
such as chloramphenicol and florfenicol. This gene
was found to be present in all strains of
P. salmonis. According to the conserved domains
analysis with the software SMART, a conserved
MFS domain – characteristic of this superfamily –
was observed, which corresponds to the family of
drug carrier proteins Bcr/Cfla (Poole 2005) and a
transmembrane domain. In this genetic environ-
ment, other encoding genes to proteins of the
MFS family and other drug carriers were identi-
fied, indicating that these are very closely located
in the genome. This correlates with Poole’s find-
ings (2005), which explained that the exporters
specific to chloramphenicol and florfenicol of the
MFS family are generally encoded in mobile ele-
ments along with other resistance genes. However,
we did not observe that they form part of a
pathogenicity island (a characteristic present in the
three studied strains).
We also managed to identify another encoding
gene for a likely carrier protein of 416 amino
acids, to which the denomination bcr2/cfla was
given. This gene was found to be present in the
genomes of the three analysed strains. The analysis
of their conserved domains indicated a characteris-
tic domain of the MFS family, corroborating in
part the notation assigned by GenDB, as well as
the greatest identities with florfenicol carrier pro-
teins in species of Legionella, phylogenetically
close to P. salmonis. This genetic environment was
the same for the three analysed strains, with a
block of homology containing the same genes,
among which we found integrases and other heavy
metal carrier pumps. This correlates with the
report by Baker-Austin et al. (2006), which
showed an association between resistance to
antibiotics and resistance to heavy metals, and that
these genes are often found in the same mobile
genetic environment.
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C Cartes et al. Genic antibiotic resistance in SRS
Finally, we identified the chloramphenicol/flor-
fenicol transporter (bcr3/cfla) and found a gene
that encodes a transmembrane protein of 410
amino acids, present in all the analysed strains.
We also identified two transmembrane domains
and one domain characteristic of the family MFS,
which comprises numerous drug resistance pro-
teins. The highest percentages of amino acid iden-
tity correspond to a drug resistance protein in
various Aeromonas (~35%), another species from
aquatic bacteria. The genetic environment analysis
identified a block of homology for the three anal-
ysed strains, including this gene, and revealed the
presence of transposases.
Determining MIC for oxytetracycline and
florfenicol
The sensitivity to different antimicrobials that iso-
lated strains of P. salmonis present was expressed in
the form of minimum inhibitory concentration
(MIC), which was determined by microdilution
methods on plates recommended by CLSI (Clinical
and Laboratory Standards Institute) with some
modifications in the composition of the medium,
the incubation times and the temperature required
by P. salmonis (Yan~ez et al. 2014). The results show
that for both antimicrobials, the IBM-009 strain
was shown to be the most resistant compared to the
strains IBM-034 and LF-89, which showed the
same resistance (Fig. 1). In particular, we observed
differences in the resistance to these antimicrobials
between strains of P. salmonis, and in addition, the
IBM-009 strain showed to be more resistant to flor-
fenicol than to oxytetracycline, unlike the other two
strains, which proved to have the same degree of
resistance to both antimicrobials.
Expression of genes involved in probable
resistance to oxytetracycline and florfenicol
We evaluated the basal expression of six genes,
probably involved in conferring antibiotic resis-
tance in three strains of P. salmonis.
The analysis of the amount of transcripts of the
tet-1 transporter gene shows that the highest val-
ues are obtained in the LF-80 strain with about
35-fold over control, while field strains show a
much smaller expression (fewer than five times).
This at all times analysed with notable differences
between the LF-89 strain and field strains
(Fig. 2a). We also analysed the transcript levels
 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

Figure 1 Oxytetracycline and florfenicol MIC in strains of Piscirickettsia salmonis. Bacteria were grown in supplemented CASO liq-
uid medium, in 96-well ELISA plates with different antibiotic concentrations, incubated for 5 days. We measured their absorbance
at 600 nm. MICs for oxytetracycline are shown at the top of the figure; the LF-89 strain shows a value of 0.125 lg mL 1, while
the IBM-034 strain evinces the same value and the IBM-009 strain a markedly higher value (1 lg mL 1). Meanwhile, determining
CIM for florfenicol shows a similar situation, that is the LF-89 and IBM-034 strains show the same values as for oxytetracycline
(0.125 lg mL 1), whereas the IBM-009 strain showed a higher resistance value (4 lg mL 1).

for the gene tet-C and the results showed that the time points (Fig. 2b). The three analysed genes
strain with the highest quantity of transcripts was are present in the genomes of the three strains;
the reference strain LF-89 at the three analysed however, we also analysed genes which were not

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 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

Figure 2 Determination of the tetracycline transporter gene transcript levels. The relative expression of three genes involved in resis-
tance to tetracycline and florfenicol was assessed by RT-qPCR. (a) The results show that for the tet-1 transporter gene, we deter-
mined that the strain with highest transcript levels is the IBM-034 strain at 2 hpt at the three analysed time points. (b) The results
show that for the tet-C gene, the IBM-009 strain was determined to be the only strain that revealed transcripts for this gene, ratify-
ing the bioinformatic genome analysis. No significant changes in the transcripts levels were determined for any of the times analysed.
(c) Meanwhile, the determination for the tet-2 transporter gene showed that the strain with highest expression is LF-89 at all three
times post-treatment. We used rpoD as the normalizing gene. We used n = 3; the bars indicate standard error. We considered
P < 0.01 as highly significant (**) and P < 0.05 as significant (*) according to Student’s t-test.

found in all genomes in the bioinformatic and IBM-034 strains, only in the IBM-009 strain,
analysis, such as the gene tet-C (Table 3). The which showed slight increases in transcripts that
results of this analysis show that indeed, no tran- were not significant with respect to the control,
scripts were evinced for this gene in the LF-89 being only 1.8 times higher at 2 hpt (Fig. 2b).

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 Journal of Fish Diseases 2016
Once basal expression had been quantified, the
quantity of transcripts (expression) of these genes
was measured after incubation of the bacterium
with both antibiotics. It can be noted that the
gene tet-2 transporter, likely candidate as the
gene responsible for conferring resistance to
oxytetracycline, is inducible with respect to its
basal expression after treatment with this antimi-
crobial. Increased expression of this gene is
observed in the three analysed strains, still signif-
icant after 2-h treatment with oxytetracycline in
the LF-89 strain, but with greatest increase in
the IBM-034 strain with an increase of close to
300 times greater than the control (defined with
a value of 1). The gene decays at 4 h of treat-
ment with oxytetracycline. For its part, the
IBM-009 strain showed a significant increase in
expression at 4 h of treatment with this antimi-
crobial (Fig. 2c).
We evaluated the expression of an encoding
gene for a transmembrane transporter of bcr/cfla
type, which would eventually confer resistance to
chloramphenicol and florfenicol. After performing
treatments with florfenicol, a significant increase
was observed in this gene’s expression in the three
analysed strains after 2 h post-treatment (hpt).
The IBM-009 strain was shown to have a greater
increase in this gene’s expression (close to 12
times) more than the other two strains; this
increase slowed with time (Fig. 3a). For its part,
the analysis of the gen bcr2/cfla was very surpris-
ing, insofar as it was not amplified in the RT-
qPCR experiments, despite the bioinformatic anal-
ysis revealing the presence of these genes in all
strains. Therefore, it was only possible to demon-
strate transcript levels for field isolates IBM-034
and IBM-009, showing an increase of nearly three
times at 2 hpt, which then decays to basal levels
at 4 hpt, with a further increase to 6 hpt and an
increase for the IBM-034 strain more than five
times greater than the 009 strain, which remains
at basal levels (Fig. 3b).
Finally, the analysis of the bcr3/cfla trans-
porter, which according to the bioinformatic
analysis, showed the presence in the three strains
with sequenced genomes, whereas after the RT-
qPCR analysis, only transcripts in the IBM-009
strain were evinced. A significant increase in
the quantity of transcripts at 6 hpt can be seen
– almost six times greater than the control –
which then decays to nearly three times at 6 hpt
(Fig. 3c).
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C Cartes et al. Genic antibiotic resistance in SRS
EtBr accumulation test
To measure differences in permeability between
strains of P. salmonis, an assay of intracellular
EtBr accumulation was performed, a fluorescent
compound described as a substrate for a large
number of carrier pumps (Efflux pumps; Viveiros
et al. 2008; Sandoval et al. 2016). The strains
were exposed to non-lethal concentrations of EtBr
(0.5 lg mL 1) per 24 h. The analysis showed a
significantly higher internal accumulation of EtBr
in the LF-89 type strain with respect to the other
analysed strains, particularly between 6 and
12 hpt, and occasionally 4 or 5 times more. EtBr
uptake was shown to have a significant increase
over time, reaching its peak at 12 hpt (Fig. 4).
Discussion
Antibiotic resistance has evolved due to its genetic
variation, produced by bacterial DNA through
phenomena such as mutations, the exchange of
genetic material between bacteria and the subse-
quent selection of these variants due to the pres-
ence of antibiotics in their surroundings (Levin &
Bergstrom 2000; Courvalin & Trieu-Cuot 2001;
Summers 2002). These evolutionary changes in
bacteria are usually not subject to change, and
therefore prevention to avoid these arrays and
genetic transfers should focus on modifying this
process’s selective pressures and, principally, the
indiscriminate use of antibiotics in various human
activities (Levy 2001; Swartz 2002). Thus, it is
important to use antibiotics properly, which
means a safe alternative to measure drug resistance
focuses on testing minimal inhibitory concentra-
tion (MIC) and thus determining the potentially
least susceptible bacterial strains.
Currently, oxytetracycline and florfenicol – two
broad-spectrum bacteriostatic agents – are the
most commonly used in the Chilean aquaculture
industry to combat outbreaks of P. salmonis (SER-
NAPESCA, 2014, http://www.sernapesca.cl/).
However, since their use began, antimicrobial
treatments have proved inconsistent or have failed
in effective control of P. salmonis (Branson &
Diaz-Munoz 1991; Cvitanich et al. 1991). This
indicates that the bacterium has been able to gen-
erate drug resistance mechanisms that have not yet
been clarified for P. salmonis. Therefore, we stud-
ied Chilean strains of the pathogen which have
potentially developed resistance to these
 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

Figure 3 Determination of bcr/cfla transporter family transcript levels. The relative expression of three genes involved in resistance
to tetracycline and florfenicol was assessed by RT-qPCR. (a) In the case of the bcr/cfla gene, the three strains show similar transcript
levels at the three analysed times, with higher expression at 2 hpt. (b) In the case of the bcr2/cfla gene, only transcripts from the field
strains and not from the reference strain LF-89 could be visualized. At all times, the IBM-034 strain had the higher levels of tran-
scripts at 2 and 6 hpt. (c) Finally, the bcr3/cfla transporter showed that the IBM-009 strain was the only one to generate transcripts,
with a peak at 4 hpt. We used rpoD as the normalizing gene. We used n = 3; the bars indicate standard error. We considered
P < 0.01 as highly significant (**) and P < 0.05 as significant (*) according to Student’s t-test.

antimicrobials, comparing them with the refer- (Walbaum). In this study, genes described as
ence strain LF-89 (ATCC VR-1361) originally potentially conferring antibiotic resistance were
isolated from coho salmon, Oncorhynchus kisutch analysed at genomic level in field strains of

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 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

Figure 4 EtBr accumulation assay in strains of Piscirickettsia salmonis. Three strains of P. salmonis were cultivated in the presence of
EtBr (0.5 lg mL 1). Intracellular EtBr accumulation was evaluated over time between 1 and 24 h, measuring emitted fluorescence.
The optical density (OD) was measured at 600 nm for each sample in order to normalize the fluorescence value. We used n = 3;
the bars indicate standard error. We considered P < 0.01 as highly significant (**) and P < 0.05 as significant (*) according to Stu-
dent’s statistical t-test.

P. salmonis (IBM-034 and IBM-009 isolated from
O. mikiss), as well as the strain LF-89, and we
were able to identify six putative genes resistant to
these antibiotics.
Bioinformatic analyses of nucleotide and ami-
noacid sequences showed that the various analysed
genes showed high nucleotidic conservation
between strains. However, through the multiple
alignment of amino acid sequences, the presence
of SNPs in the genes in question was determined
(data not shown). The in silico analyses of these
genomes indicate that these genes are present in
the three analysed strains of P. salmonis, which
was ratified by RT-qPCR analysis, wherein we
observed these genes’ expressions.
The MIC was determined for the type LF-89
strain, which showed values ranging from 0.08 to
0.25 lg mL 1 for oxytetracycline and florfenicol.
Previous studies for the type LF-89 strain made
~ez et al. (2014) showed MIC values equiva-
by Yan
lent to those obtained in this study. Smith et al.
(1999) studied four strains of P. salmonis, classify-
ing the type LF-89 strain as susceptible, with
MIC ranges for oxytetracycline of 2.5 and
0.5 lg mL 1 for chloramphenicol (florfenicol
analogue). However, that study was performed,
growing the bacteria in CHSE-214 cells (a liquid
culture to grow bacteria had not yet been
developed), which implies important methodologi-
cal differences. The IBM-034 strain showed the
same MIC values as the type strain for both
antimicrobials. Finally, the IBM-009 strain
showed more resistance to both antibiotics, with a
range of 0.5–2 lg mL 1 for oxytetracycline and
2–8 lg mL 1 for florfenicol. The data obtained
for the IBM-009 strain indicate that this strain
has a greater range of florfenicol resistance than
for oxytetracycline, data comparable with Yan ~ez
et al. (2014), who observed that other field iso-
lates of P. salmonis were also largely resistant to
this antimicrobial. Similar data were obtained by
Henrıquez et al. (2016), who analysed various
Chilean isolates of P. salmonis, and obtained
oxytetracycline and florfenicol resistance ranges
similar to those obtained in this study. Note that
in the same analysis, they also determined that
florfenicol resistance ranges were slightly higher
than those for oxytetracycline in some field iso-
lates of P. salmonis.
The MIC values correlate with RT-qPCR data,
where the IBM-009 strain expresses a larger num-
ber of genes potentially involved in antibiotic
resistance than that of reference strain LF- 89. It
bears mentioning that the MIC values were
determined using the microdilution method in
liquid medium (Broth microdilution method)

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 Journal of Fish Diseases 2016
recommended by the Clinical and Laboratory
Standards Institute, CLSI (http://clsi.org/stan-
dards/); however, we made some modifications to
the medium in terms of composition, time and
incubation temperature as described by Yan ~ez
et al. (2012), due to the fact that this pathogen
has not been reported to grow in the recom-
mended medium (Mueller–Hinton). However,
our results show that this is an appropriate med-
ium to determine in vitro MIC in strains of
P. salmonis. It has been reported that antimicro-
bial resistance mechanisms may be accomplished
by inducing expression for multiple drug resis-
tance genes (Kohanski, DePristo & Collins 2010;
Sandoval et al. 2016). It has also been found that
subinhibitory concentrations of antibiotics can
induce expression for the genes that encode oxyte-
tracycline carriers (Berens & Hillen 2003) and
florfenicol analogues such as chloramphenicol
(George & Hall 2002). Thus, we evaluated
expression for the putative oxytetracycline resis-
tance gene, after performing treatments with
subinhibitory concentrations of this antimicrobial
on the bacterium. Expression for the gene tet-C
increased significantly after 2 hpt in the reference
strain LF-89 and IBM-034 strain. A smaller
increase was observed in the IBM-009 strain,
which proved to be more resistant to this antimi-
crobial; this is probably due to the fact that this
strain, according to the in silico analyses, has a
greater number of resistance genes, so the intracel-
lular antibiotic concentration could remain lower
than in the other two strains. In the case of the
florfenicol putative resistance gene bcr/Cfla, there
was a significant increase in expression at 2 hpt
with this antimicrobial in all three analysed
strains, which descended with time. It could also
be noted that the higher the antibiotic concentra-
tion, the higher the number of this gene’s tran-
scripts detected. This increased expression of
resistance genes could be correlated with the resis-
tance induction observed by Yan ~ez et al. (2014)
for these antimicrobials. They reported that Chi-
lean isolates of P. salmonis increased their resis-
tance range after previously being exposed to
subinhibitory concentrations of these antimicro-
bials. Finally, studies to evaluate whether these
genes are functional in the bacterium and whether
they are relevant in conferring resistance to one or
multiple antibiotics should be made. The EtBr
accumulation assay determined that the LF-89
strain is significantly the strain which most
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C Cartes et al. Genic antibiotic resistance in SRS
accumulated intracellular EtBr, compared with the
two other studied strains. Previous studies have
shown that this strain accumulated a significantly
greater quantity of this compound when com-
pared to another field strain of P. salmonis, and
the ability to expel EtBr increased if it was previ-
ously stimulated with subinhibitory concentrations
of florfenicol, indicating that this ability is due to
the action of membrane carriers that are stimu-
lated in the presence of antibiotics (Sandoval et al.
2016). This result supports the MIC results,
which effectively show the LF-89 strain to be
more susceptible to the studied antibiotics than
the 009 strain. The fact that the accumulation of
this compound is significantly less in the 009 and
034 strains may be due to the fact proposed
above, as these strains possess a greater number of
membrane carriers – possibly MDR (multidrug
resistance) type, like the MFS family analysed in
this study – giving these strains a higher capacity
to expel toxic compounds such as antibiotics or
EtBr (Viveiros et al. 2008) among others.
Acknowledgements
This work was supported by the grants Innova-
Corfo 013CN IBM-259, FONDECYT 1130069
and FONDAP 15110027, awarded to the Inter-
disciplinary Centre for Aquaculture Research
(INCAR).
References
Baker-Austin C., Wright M., Stepanauskas R. & Mcarthur J.
(2006) Co-selection of antibiotic and metal resistance.
Trends in Microbiology 14, 176–182.
Berens C. & Hillen W. (2003) Gene regulation by
tetracyclines. Constraints of resistance regulation in bacteria
shape TetR for application in eukaryotes. European Journal
of Biochemistry 270, 3109–3121.
Bolhuis H., van Veen H., Poolman B., Driessen A. & Konings
W. (1997) Mechanisms of multidrug transporters. FEMS
Microbiology Reviews 21, 55–84.
Branson E. & Diaz-Munoz D. (1991) Description of a new
disease condition occurring in farmed coho salmon,
Oncorhynchus kisutch (Walbaum), in South America. Journal
of Fish Diseases 14, 147–156.
Bravo S. & Campos M. (1989) Coho salmon syndrome in
Chile. Fish Health Section Newsletter, American Fisheries
Society 17, 3.
Cannon M., Hartford S. & Davies J. (1990) A comparative
study on the inhibitory actions of chloramphenicol,
thiamphenicol and some fluorinated derivatives.
Antimicrobiology Chemotherapy 26, 307–317.
 Journal of Fish Diseases 2016
Chopra I. & Roberts M. (2001) Tetracycline antibiotics: mode
of action, applications, molecular biology, and epidemiology
of bacterial resistance. Microbiology and Molecular Biology
Reviews 65, 232–260.
Courvalin P. & Trieu-Cuot P. (2001) Minimizing potential
resistance: the molecular view. Clinical Infectious Diseases 33
(Suppl. 3), S138–S146.
Cvitanich J., Garate N. & Smith E. (1991) The isolation of a
rickettsia-like organism causing disease and mortality in
Chilean salmonids and its confirmation by Koch’s postulate.
Journal of Fish Diseases 14, 121–145.
Fryer J. (2002) Clarification of the systematics of Piscirickettsia
salmonis. Fish Health Section Newsletter, American Fisheries
Society 30, 23.
Fryer J., Lannan C., Giovannoni S. & Wood N. (1992)
Piscirickettsia salmonis gen., sp. nov., the causative agent of a
epizootic disease in the Salmonid Fishes. International Journal
of Systematic and Evolutionary Microbiology 42, 120–126.
Furushita M., Shiba T. & Maeda T. (2003) Similarity of
tetracycline resistance genes isolated from fish farm bacteria
to those from clinical isolates. Applied and Environmental
Microbiology 69, 5336–5342.
George A. & Hall R. (2002) Efflux of chloramphenicol by the
CmlA1 protein. FEMS Microbiology Letters 209, 209–213.
Goldman R., Hasan T., Hall C., Strycharz W. & Cooperman
B. (1983) Photoincorporation of tetracycline into Escherichia
coli ribosomes. Identification of the major proteins
photolabeled by native tetracycline and tetracycline
photoproducts and implications for the inhibitory action of
tetracycline on protein synthesis. Biochemistry 22, 359–368.
Henrıquez P., Kaiser M., Bohle H., Bustos P. & Mancilla M.
(2016) Comprehensive antibiotic susceptibility profiling of
Chilean Piscirickettsia salmonis field isolates. Journal of Fish
Diseases 39, 441–448.
Kohanski M., DePristo M. & Collins J. (2010) Sublethal
antibiotic treatment leads to multidrug resistance via radical-
induced mutagenesis. Molecular Cell 37, 311–320.
Levin B. & Bergstrom C. (2000) Bacteria are different:
observations, interpretations, speculations, and opinions
about the mechanisms of adaptive evolution in prokaryotes.
Proceedings of the National Academy of Sciences (USA) 97,
6981–6985.
Levy S. (2001) Antibiotic resistance: consequences of inaction.
Clinical Infectious Diseases 33(Suppl. 3), S124–S129.
Li X. & Nikaido H. (2009) Efflux-mediated drug resistance in
bacteria: an update. Drugs 69, 1555–1623.
Mandakovic D., Glasner B., Maldonado J., Aravena P.,
Gonzalez M., Cambiazo V. & Pulgar R. (2016) Genomic-
based restriction enzyme selection for specific detection of
Piscirickettsia salmonis by 16S rDNA PCR-RFLP. Frontiers
in Microbiology 9, 643. doi:10.3389/fmicb.2016.00643.
Mauel M., Ware C. & Smith P. (2008) Culture of
Piscirickettsia salmonis on enriched blood agar. Journal of
Veterinary Diagnostic Investigation 20, 213–214.
McCarthy U., Bron J., Brown L., Pourahmad F., Bricknell I.,
Thompson K., Adams A. & Ellis A. (2008) Survival and
Ó 2016
John Wiley & Sons Ltd 14
C Cartes et al. Genic antibiotic resistance in SRS
replication of Piscirickettsia salmonis in rainbow trout
head kidney macrophages. Fish Shellfish Immunology 25,
477–484.
Mikalsen J., Skjaervik O., Wiik-Nielsen J., Wasmuth M. &
Colquhoun D. (2008) Agar culture of Piscirickettsia salmonis,
a serious pathogen of farmed salmonid and marine fish.
FEMS Microbiology Letters 278, 43–47.
Miller R., Walker R., Carson J., Coles M., Coyne R.,
Dalsgaard I., Gieseker C., Hsu H., Mathers J.,
Papapetropoulou M., Petty B., Teitzel C. & Reimschuessel
R. (2005) Standardization of a broth microdilution
susceptibility testing method to determine minimum
inhibitory concentrations of aquatic bacteria. Diseases of
Aquatic Organisms 64, 211–222.
Pfaffl M. (2001) A new mathematical model for relative
quantification in real-time RT-PCR. Nucleic Acids Research
29, e45.
Poole K. (2005) Efflux-mediated antimicrobial resistance.
Journal of Antimicrobial Chemotherapy 56, 20–51.
Pulgar R., Travisany D., Zu~niga A., Maass A. & Cambiazo V.
(2015) Complete genome sequence of Piscirickettsia
salmonis LF-89 (ATCC VR-1361) a major pathogen
of farmed salmonid fish. Journal of Biotechnology 212,
30–31.
Rojas M., Olivares J., Del Rıo R. & Marshall S. (2007)
Characterization of a novel and genetically different small
infective form of Piscirickettsia salmonis. Microbial
Pathogenesis 44, 370–378.
Rojas V., Galanti N., Bols N. & Marshall S. (2009)
Productive infection of Piscirickettsia salmonis in
macrophages and monocyte-like cells from rainbow trout, a
possible survival strategy. Journal of Cellular Biochemistry
108, 631–637.
Rozas M. & Enrıquez R. (2014) Piscirickettsiosis and
Piscirickettsia salmonis in fish: a review. Journal of Fish
Diseases 37, 163–188.
Sandoval R., Oliver C., Valdivia S., Valenzuela K., Haro R.,
Sanchez P., Olavarria V., Valenzuela P., Avenda~ no-Herrera
R., Romero A., Carcamo J., Figueroa J. & Ya~ nez A. (2016)
Resistance-nodulation-division efflux pump acrAB is
modulated by florfenicol and contributes to drug resistance
in the fish pathogen Piscirickettsia salmonis. FEMS
Microbiology Letters 363, doi:10.1093/femsle/fnw102.
Schwarz S., Kehrenberg C., Doublet B. & Cloeckaert A.
(2004) Molecular basis of bacterial resistance to
chloramphenicol and florfenicol. FEMS Microbiology Reviews
28, 519–542.
Smith P., Pizarro P., Ojeda P., Contreras J., Oyanedel S. &
Larenas J. (1999) Routes of entry of Piscirickettsia salmonis
in rainbow trout Oncorhynchus mykiss. Diseases of Aquatic
Organisms 37, 165–172.
Summers A. (2002) Generally overlooked fundamentals of
bacterial genetics and ecology. Clinical Infectious Diseases 34
(Suppl. 3), S85–S92.
Swartz M. (2002) Human diseases caused by foodborne
pathogens of animal origin. Clinical Infectious Diseases 34
(Suppl. 3), S111–S122.
 Journal of Fish Diseases 2016 C Cartes et al. Genic antibiotic resistance in SRS

Vera T., Isla A., Cuevas A. & Figueroa J. (2012) Un nuevo Supporting Information
medio de cultivo para el patogeno Piscirickettsia salmonis.
Archivos de Medicina Veterinaria 44, 273–277. Additional Supporting Information may be found
Viveiros M., Martins A., Paixao L., Rodrigues L., Martins M., in the online version of this article:
Couto I., F€ahnrich E., Kern W. & Amaral L. (2008) Table S1. Sequence of primers used for RT-
Demonstration of intrinsic efflux activity of Escherichia coli qPCR and expected product.
K-12 AG100 by an automated ethidium bromide
Figure S1. Oxytetracycline and Florfenicol
method. International Journal of Antimicrobial Agents 31,
458–462. MIC for E. coli (ATCC 25922).
~ez A., Valenzuela K., Silva H., Retamales J., Romero A.,
Yan
Figure S2. Effect of methanol and ethanol on
Enriquez R., Figueroa J., Claude A., Gonzalez J., Avenda~ no- the growth of P. salmonis.
Herrera R. & Carcamo J. (2012) Broth medium for the
successful culture of the fish pathogen P. salmonis. Diseases of Received: 28 July 2016
Aquatic Organisms 97, 197–205. Revision received: 5 October 2016
~ez A., Valenzuela K., Matzner C., Olavarrıa V., Figueroa
Yan Accepted: 11 October 2016
J., Avenda~no-Herrera R. & Carcamo J. (2014) Broth
microdilution protocol for minimum inhibitory
concentration (MIC) determinations of the intracellular
salmonid pathogen Piscirickettsia salmonis to
florfenicol and oxytetracycline. Journal of Fish Diseases 37,
505–509.

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