doi:10.1111/jfd.

12427

Journal of Fish Diseases 2015

Comprehensive antibiotic susceptibility profiling of

Chilean Piscirickettsia salmonis field isolates
P Henrquez1, M Kaiser2, H Bohle1, P Bustos1 and M Mancilla1
1 Laboratorio de Diagnostico y Biotecnologa, ADL Diagnostic Chile Ltda, Puerto Montt, Chile
2 Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Leipzig,
Germany

Abstract

Introduction

Antibiotics have been extensively used against

infections produced by Piscirickettsia salmonis, a
fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the
Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected.
With the aim of obtaining a landscape of the
antimicrobial resistance of P. salmonis in Chile,
the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different
hosts and collected over 5 years were assessed.
The results allowed for the determination of epidemiological cut-off values that were used to characterize the pathogen population. This work
represents the first large-scale field study addressing the antimicrobial susceptibility of P. salmonis,
providing evidence of the existence of resistant
types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the
issue of resistance to florfenicol and OTC is still
in the onset.

The Salmonid rickettsial septicaemia (SRS) or piscirickettsiosis is caused by the Gram-negative bacterium Piscirickettsia salmonis (Fryer & Hedrick
2003), a fastidious intracellular facultative pathogen
(Mauel, Ware & Smith 2008; Mikalsen et al.
2008). Piscirickettsiosis is by far the most serious
infectious disease for the salmon farming industry
in Chile, affecting fish during the seawater productive cycle stage with high mortality (Rozas & Enriquez 2014). Even though SRS was reported for the
first time in 1989 in coho salmon (Oncorhynchus
kisutch) by means of the isolation of the prototypical LF-89 strain (Bravo & Campos 1989; Fryer
et al. 1992), the disease is now disseminated in all
reared salmonid species in Chile. Data provided by
the Chilean National Fisheries and Aquaculture
Service (Servicio Nacional de Pesca y Acuicultura,
Sernapesca) recognized that 74.1% and 73.5% of
the mortality in 2014 ascribed to infectious diseases
in Atlantic salmon (Salmo salar) and rainbow trout
(Oncorhynchus mykiss), respectively, were caused by
piscirickettsiosis (Sernapesca 2015b). Direct economic losses caused by P. salmonis have been estimated around US $100 million annually (Cabezas
2006), but a latest assessment including costs of
therapies and vaccination allocated this number
over US $450 million in 2012 (Camussetti et al.
2015). Therefore, SRS is a serious concern for the
competitiveness and future development of the
Chilean salmon industry.
Due to its relevance, an active surveillance plan
against SRS was introduced in 2012. The strategy
includes microbiological and molecular testing,
which is voluntary complemented by vaccination

Keywords: antibiotic resistance, florfenicol, minimal inhibitory concentration, oxytetracycline,

Piscirickettsia salmonis.

Correspondence M Mancilla, Laboratorio de Diagno stico y

Biotecnologa, ADL Diagnostic Chile Ltda, Sector La Vara s/n,
camino a Alerce, casilla 160, Puerto Montt, Chile
(e-mail: mmancilla@adldiagnostic.cl)
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Journal of Fish Diseases 2015

and antibiotic treatments, the latter administered

mainly through fish feeds. As the efficacy of vaccination in the field have resulted moderate (Marshall & Tobar 2014), delaying the first outbreak
after transfer to the sea but not preventing the disease (Jakob et al. 2014), control of SRS has relied
heavily on antimicrobials to combat outbreaks and
to limit the propagation of the pathogen (Cabello
et al. 2013). According to the official statistics,
over 90% of 563 tons of antimicrobial compounds employed in Chilean aquaculture in 2014
were used for treating piscirickettsiosis (Sernapesca
2015a). Initially, quinolones (flumequine, oxolinic
acid) were the drugs of election, but to ensure
food safety and environment protection, their
industrial use has declined to less than 1% (Miranda, Tello & Keen 2013). In fact, quinolones
have been practically replaced by florfenicol (FFC)
and oxytetracycline (OTC), which nowadays represent 71% and 28% of antibiotic prescriptions to
treat SRS, respectively (Sernapesca 2015a). It is
important to note that a constant increase in
antibiotic use has been recorded since 2010,
which correlates with a similar trend in productivity after the sanitary crisis triggered by outbreaks
of infectious salmon anaemia, ISA, in 2009 (Sernapesca 2015a). In this scenario, the threat of
antibiotic resistance emergence can become real.
For the monitoring of the correct application of
antibiotics and to prevent resistances, it is essential
to standardize and validate protocols to evaluate
the susceptibility against antimicrobials. This has
been tough to apply for P. salmonis: As a nutritionally demanding pathogen, the bacterium is
able to slowly grow in enriched free-cell media
but not in those recommended for susceptibility
testing. Several formulations have been developed
in the last few years that have been shown to support the growth of this bacterium (Mauel et al.
2008; Mikalsen et al. 2008; Yanez et al. 2012,
2013; Henriquez et al. 2013). But a method for
the quantitative study of antibiotic resistance of
P. salmonis was just recently reported (Yanez et al.
2014). Based on this work, the Clinical and Laboratory Standards Institute, CLSI, has suggested the
AUSTRAL-SRS liquid medium for the assessment
of the minimal inhibitory concentration (MIC) of
P. salmonis, which entails the titration of a specific
antibiotic level required to inhibit the cell growth
in microplates (CLSI 2014).
The concept of resistance covers epidemiological, clinical and pharmacological aspects requiring
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P Henr quez et al. P. salmonis antibiotic resistance

an antibiotic concentration value or breakpoint to

distinguish between strain categories (Turnidge &
Paterson 2007). The clinical breakpoint refers to a
threshold value that splits strains with a high
probability to be treated from those where the
treatment likely will fail. The epidemiological
breakpoint addresses the issue of resistance mechanisms, separating wild-type (WT) bacteria from
those non-wild-type (NWT) that have acquired or
selected a mechanism, and requires the study of a
large number of isolates from diverse geographical
and host origins, collected during an extended
period of time. The pharmacological breakpoint
considers
pharmacokinetic/pharmacodynamic
parameters derived from animal models, dealing
with the efficacy in vivo. At present, there are not
established criteria to determine whether a
P. salmonis isolate is resistant to a specific antibiotic due to the lack of studies considering a representative number of isolates. Therefore, there is a
need for determining those values that allow for a
more accurate description of the P. salmonis population regarding the susceptibility to diverse
antimicrobials.

Material and methods

Bacterial isolation, culture medium

composition and growth conditions
Tissue samples (pool of kidney, liver and brain)
from fish were aseptically collected for colony isolation during isolation field campaigns or laboratory routine analyses. Escherichia coli ATCC
25922 was used as a control for antimicrobial susceptibility testing. The P. salmonis LF-89 type
strain from the American Type Collection Culture
(ATCC VR-1361) was included for comparison.
The information associated with all 292
P. salmonis isolates is shown in Table S1. Bacteria
were recovered from three different salmonid species in samples collected during SRS outbreaks
between 2010 and 2014 and were phenotypically
characterized for susceptibility to quinolones (oxolinic acid, OA; flumequine, FLU), FFC and
OTC (all purchased from Sigma-Aldrich). The
isolates were streaked onto ADL-PSA plates, a
solid derivative of ADL-PSB growth medium,
which is formulated with a composition equivalent to that reported by others (Yanez et al.
2012), but with a different proportion in some
components (Table S2). For isolation, plates were

Journal of Fish Diseases 2015

maintained at 19  2 C for 78 days. The isolates were kept frozen at 80 C in appropriate

cryovials containing a mixture of 80% ADL-PSB
and 20% DMSO (Merck).
Minimal inhibitory concentration assessment
For antibiotic susceptibility, MICs for quinolones,
FFC and OTC were assessed according to the
instructions given by the CLSI, guide M49-A
(CLSI 2006), but introducing the ADL-PSB medium for P. salmonis growth and some modifications. Briefly, stock solutions (1280 lg mL 1) of
antibiotics were prepared in 500 lL of ethanol
95% (FFC) or methanol (OTC), adjusting to a
final volume of 10 mL with sterile distilled water.
Quinolones were prepared in water, using drops
of 1 M NaOH to aid dissolution. Solutions were
maintained at 20 or 80 C. All microplates
included growth and solvent control wells. 96-well
microplates containing twofold serial dilutions of
0.00864 lg mL 1 of quinolones or FFC, and
0.008512 lg mL 1 for OTC were inoculated
with 5.0 9 105 CFU well 1 prepared with fresh
bacteria previously grown on ADL-PSB for 48 h
at 19  2 C. Microplates were incubated statically at 19  2 C for 57 days. The absorbance
at 580 nm for each well was measured with an
Absorbance Microplate Reader ELx800 (BioTek
Instruments, Inc). The lowest concentration where
bacterial growth was not detected corresponded to
the MIC value. All experiments were performed
in duplicate.
Determination of WT MIC range and cut-off
values
We essentially applied a method previously
described (Turnidge, Kahlmeter & Kronvall 2006)
to define the WT MIC range of P. salmonis. Any
computation was based on the assumption that
log-transformed MIC values of P. salmonis WT
follow normal Gaussian distribution. For each
antibiotic tested, we defined a first subset of samples corresponding to the three lower MIC values;
for further subsets, the samples of the respective
next higher MIC were included. Multiple subsets
were then subjected to nonlinear least squares
regression to fit their log2-transformed MIC data
to a normal distribution. Regression analysis and
Microsoft Excels NORMDIST function enabled
us to calculate an estimated number of samples to
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P Henr quez et al. P. salmonis antibiotic resistance

be found in the particular subset, and the subset

with the minimum difference between the estimated number of samples and the true number of
samples was considered the optimum fit. Once we
obtained the WT MIC range, epidemiological
cut-off values (ECOFFs) were computed using the
log2 estimated mean and standard deviation of the
optimum fit in Microsoft Excels NORMINV
function and are reported here as rounded to the
nearest standard two-fold dilution.
PCR identification
DNA samples were obtained from bacteria cultured in agar plates using E.Z.N.ATM Bacterial
DNA Kit (Omega-Biotek) following the instructions given by the manufacturer. P. salmonis identification was confirmed by a PCR method
previously described (Mauel, Giovannoni & Fryer
1996). PCR rounds were carried out in a final
reaction volume of 25 lL. The reaction mixture
contained 10 pmol of each primer, 0.1 nmol of
each deoxynucleotide triphosphate, 2 mM MgCl2,
1.0 U of Taq DNA polymerase (Invitrogen) and
2.0 lL of template. The thermal profile for PCR
was as follows: preheating at 95 C for 5 min; 30
cycles of denaturation at 95 C for 20 s, annealing at 55 C for 30 s, and extension at 72 C for
40 s; and a final extension step at 72 C for
5 min, using a thermal cycler 2720 (Applied
Biosystems).
Results

Our strain collection was established in 2008

when no well-proven liquid medium for MIC
assessments of P. salmonis was available. Therefore, we developed a medium that facilitated
P. salmonis growth and isolation as well as phenotypic characterization. This medium resulted suitable for diagnostic purposes, and we tested its
performance in the microdilution method used to
determine the MIC values presented in this study.
MICs for the E. coli ATCC 25922 control strain
registered in MuellerHinton broth resulted identical to those observed in ADL-PSB medium
when using the same growth conditions (Table 1).
Thus, MICs for the P. salmonis LF-89 strain were
comparable to those previously obtained for FFC
and OTC by others (Yanez et al. 2014).
For this study, a comprehensive collection of
292 P. salmonis isolates encompassing eight

P Henr quez et al. P. salmonis antibiotic resistance

Journal of Fish Diseases 2015

Table 1 MIC values (lg mL 1) observed for control strains in

ADL-PSB medium (19  2 C)
Strain
E. coli ATCC 25922
P. salmonis LF-89b

OA

FLU

FFC

OTC

0.25
0.06

0.125
0.06

4.0
0.5

0.5
0.5

OA, oxolinic acid; FLU, flumequine; FFC, florfenicol; OTC, oxytetracycline; MIC, minimal inhibitory concentration.
a
Identical values were observed in cation-adjusted MuellerHinton
broth using similar growth conditions (2448 h).
b
Incubation time: 57 days. Values reported by Yanez et al. 2014 were
0.25 for FFC and OTC using the same strain.

geographical macrozones (five from Region de Los

Lagos and three from Region de Aysen), representative of about 90% of the Chilean salmon marine
farms, was considered (Table S1). Histograms of
MIC frequencies obtained from this collection
revealed a typical bimodal distribution and
allowed for a clear distinction between WT and
NWT groups for the quinolones tested (Fig. 1).
While the first peak represented MIC values
obtained from WT and ranged from 0.031
0.125 lg mL 1 for OA and FLU, MICs for
NWT fell within 2.08.0 lg mL 1. For OTC,
the majority of isolates presented a WT MIC
between 0.125 and 1.0 lg mL 1 of the antibiotic,
whereas the NWT isolates exhibited MICs as high

as 256.0 lg mL 1. On the contrary, MICs for

FFC displayed an overlapped bimodal distribution, with the WT subpopulation apparently
tolerating a wide range of this antimicrobial
(0.1252 lg mL 1), suggesting the presence of a
small number of isolates with impaired susceptibility in the range of 4.08.0 lg mL 1. These
isolates also depicted resistance to quinolones.
Optimum fits were used to define the WT MIC
range for all antibiotics tested (Fig. 1). ECOFFs
determined by calculating the upper 0.1% of the
Gaussian distribution and rounding to the nearest
twofold dilution were 0.125, 0.250, 2.0 and
4.0 lg mL 1 for FLU, OA, FFC and OTC,
respectively. The nearest twofold standard dilution
corresponded to the next higher standard dilution
for all antibiotics except FLU, and >99% of calculated MIC values for FLU fell within the indicated cut-off range.
An appraisal of the prevalence of resistant
phenotypes can be deduced from Table 2. The
proportion of resistant types reached 54.1%
(158/292) of the entire collection, which resulted
from the combination of isolates exhibiting
QUI-resistant, QUI-resistant/FFC-intermediate and
OTC-resistant phenotypes. The most prevalent
resistant phenotype was related to quinolones

Figure 1 Distribution of minimal inhibitory concentration values of P. salmonis isolates (N = 292) for three classes of antimicrobials (grey bars). Black curves represent optimum fits obtained after least square regression analysis and correspond to the expected
wild-type population for each antibiotic.
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P Henr quez et al. P. salmonis antibiotic resistance

Journal of Fish Diseases 2015

Table 2 Distribution of
between 2010 and 2014

antibiotic

susceptibility patterns

Phenotype
Year

SUS

QUIR

QUIR/FFCI

OTCR

2010
2011
2012
2013
2014
Totala

4
14
54
50
12
134 (45.9)

11
24
41
49
19
144 (49.3)

1
7
9 (3.1)

1
4
1
5 (1.7)

SUS, susceptible to quinolones, FFC and OTC; QUIR, resistant to quinolones; QUIR/FFCI, resistant to quinolones/intermediate susceptibility
to FFC; OTCR, oxytetracycline resistant.
a
Numbers between brackets represent percentages.

(49.3%), followed by QUI-resistant/FFC-intermediate isolates (3.9%), and OTC-resistant ones

(1.7%). Regarding a temporal analysis, quinoloneresistant isolates were present in our collection
since 2010. By contrast, isolates with an impaired
susceptibility to FFC or OTC did not emerge
until 2012 and 2013.
Although the isolates from Region de Aysen are
underrepresented in the dataset (62 out of 292), it
is possible to infer the distribution of antimicrobial susceptibility profiles by location and by host
(Fig. 2). As shown in Fig. 2a, resistant isolates
were most prevalent in Region de Los Lagos
(64.8% vs. 14.5%). This finding agreed with the
observed dissemination of resistant isolates for
quinolones only, with a prevalence of 60.9% in
that region, while only 8.1% were found to carry
this phenotype in Region de Aysen. A remarkable

Figure 2 Distribution of P. salmonis

phenotypes by location (a) and by host
species (b).
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finding was that the QUI-resistant/FFC-intermediate isolates were located in Region de Los
Lagos, while the OTC-resistant types were constrained to Region de Aysen. Surprisingly, both
phenotypes represented a small proportion of the
isolates (3.9% and 6.4% for QUI-resistant/FFCintermediate and OTC-resistant isolates, respectively). Interestingly, the isolates recovered from
Atlantic salmon shown to be more susceptible to
those found affecting rainbow trout (73.1% vs.
4%), which were in their majority resistant to quinolones (Fig. 2b). The isolates derived from coho
salmon samples (13) resulted to be QUI-resistant.
Discussion

Antibiotic resistance is a threat not only for

human, but also for animal health worldwide.
The above situation has drawn the attention of
the Chilean regulatory authority, which has implemented restrictions to avoid misuse and ruled veterinary prescriptions for pharmacological drugs
utilized for hydrobiological species mandatory.
However, a lack of consensus on the reduction in
their utilization remains as it appears that the only
effective measure for controlling outbreaks of SRS
so far is the antibiotic therapy (Rozas & Enriquez
2014). To guarantee sustainability of the industry,
amounts and frequency of antimicrobial SRS therapies have to be reduced and alternatives such as
changes in the production model, the introduction
of genetically resistant fish and new vaccines are
urgently needed.

Journal of Fish Diseases 2015

An early study performed with four P. salmonis

strains determined MICs for different antimicrobials (Smith et al. 1996). The discordance with
the values presented here can be explained by the
fact that Smith et al. used CHSE-214 cell monolayers as a substrate for bacterial growth. As
ECOFF values are protocol specific, a comparison
of our results with MICs obtained from infected
cell cultures is not feasible. Noteworthy, the MICs
for FFC and OTC for the control strains in our
study resulted to be similar to those previously
obtained using the CLSI suggested medium
(Yanez et al. 2014).
Description of subpopulations based on MICs
for antimicrobials has been well documented, and
there are institutions and committees that deal with
periodical updates of rules for the harmonization of
breakpoint interpretation in hospital settings worldwide (http://clsi.org; http://www.eucast.org). Nevertheless, information is scarce in veterinary
medicine, especially regarding fish pathogens: neither epidemiological nor clinical cut-off values for
P. salmonis were available. In recent years, the
European Committee on Antimicrobial Susceptibility Testing (EUCAST) coined the term ECOFF to
describe the upper MIC value of a susceptible peak
in a MIC distribution, which groups micro-organisms with no evidence of acquired or mutational
resistance mechanisms. This concept was applied in
this study, and to avoid misinterpretations, we
based breakpoint setting primarily on MIC distributions modelled by a statistical method (Turnidge
et al. 2006). For quinolones and OTC, proposed
values (>0.125 or >0.250, and >4.0 lg mL 1,
respectively) agreed with the visual inspection of
the corresponding histograms (Fig. 1). The distribution of MICs for quinolones and OTC suggest
that the isolates of NWT subpopulations are likely
bearing a resistant mechanism. In the case of FFC,
the calculated breakpoint (>2 lg mL 1) fell into a
zone of intermediate susceptibility. This finding
may reflect adaptation due to a continuous exposition to enhanced levels of FFC, instead of an acquisition of a resistant mechanism as it has been
observed in environmental bacteria isolated from
Chilean aquaculture settings (Fernandez-Alarcon
et al. 2010).
In regard to the time frame in which resistant
phenotypes have emerged, we can infer from our
data that quinolone resistance appeared before
FFC-intermediate and OTC-resistant phenotypes.
This finding is in accordance with the official
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P Henr quez et al. P. salmonis antibiotic resistance

record for the employment of quinolones, which

were utilized in the period 20052008, while their
use drastically dropped in 2009 (Sernapesca 2011)
because of an agreement signed by the members
of the association of producers to reduce quinolone use. Even though that agreement remains
valid to date and the use of quinolones is practically negligible, our results clearly show that resistance to quinolones persists. Therefore, we may
hypothesize that isolates that exhibit an impaired
susceptibility to FFC may have evolved from those
that were shown to be resistant to quinolones.
Concerning the mechanisms underlying resistance to antibiotics, we previously presented
experimental evidence on P. salmonis quinoloneresistant types and their association with a polymorphism found in gyrA (Henriquez et al. 2015).
Although in this work we did not address the
genetic basis of the resistance to quinolones, our
ongoing research suggests that the reported polymorphism is present in QUI-resistant isolates
analysed to date. In the case of FFC and OTC,
further research is needed in order to elucidate the
molecular mechanisms of the reduced susceptibility found in some isolates.
To the best of our knowledge, this is the first
extensive field study describing the state of the art
on the susceptibility patterns to multiple antibiotics for P. salmonis. Despite the amounts and
prolonged use of FFC and OTC, the analysis of
susceptibility patterns reveals a low frequency of
resistant types within the P. salmonis population,
an unexpected situation considering what has been
described in environmental studies (Miranda et al.
2003; Miranda & Rojas 2007). However, the fact
that resistant types were found points out the
need of developing faster, more reliable and accurate diagnostic systems able to predict phenotypes
with impaired susceptibility in a reasonable time
frame. Thereby, it will be possible to prevent the
emergence and propagation of an even more serious threat: P. salmonis multidrug-resistant strains.
In a situation where the drug choices are limited,
improvements on the surveillance plan and veterinary management are key to relief the pressure
exerted on the pathogen population that prompt
bacterial selection towards antibiotic-resistant
strains. In this line, the dissemination of resistant
isolates open a new perspective on the epidemiology of the disease that should be considered for
limiting SRS outbreaks potentially refractory to
antimicrobial therapies.

Journal of Fish Diseases 2015

Acknowledgements
This research was funded by grants 12BPC213471 and 14IDL2-30005 from the Chilean Economic Development Agency, CORFO.
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Supporting Information
Additional Supporting Information may be found
in the online version of this article:
Table S1. Dataset.
Table S2. Composition of ADL-PSB medium.
Received: 15 July 2015
Revision received: 30 September 2015
Accepted: 30 September 2015