Broth microdilution protocol for minimum inhibitory

doi:10.1111/jfd.12144
Journal of Fish Diseases 2013
Short Communication
Broth microdilution protocol for minimum inhibitory
concentration (MIC) determinations of the intracellular
salmonid pathogen Piscirickettsia salmonis to florfenicol
and oxytetracycline
n
~ ez1,2, K Valenzuela1, C Matzner1, V Olavarrıa1, J Figueroa1,2,
A J Ya
~ o-Herrera2,3 and J G Carcamo1,2
R Avendan
1 Facultad de Ciencias, Instituto de Bioquımica y Microbiologıa, Universidad Austral de Chile, Valdivia, Chile
2 Interdisciplinary Center for Aquaculture Research (INCAR), Concepcion, Chile
3 Laboratorio de Patologıa de Organismos Acuaticos y Biotecnologıa Acuıcola, Facultad de Ciencias Biologicas,
Universidad Andres Bello, Vi~
na del Mar, Chile
Keywords: antimicrobial susceptibility testing, AUSTRAL-SRS broth, florfenicol, minimum inhibitory
concentration,
oxytetracycline,
Piscirickettsia
salmonis.
Piscirickettsia salmonis is a facultative intracellular
Gram-negative bacterium (Mauel, Ware & Smith
2008; Mikalsen et al. 2008) originally isolated
from fish in Chile and constitutes one of the
main problems in farmed salmonids and marine
fish worldwide (see reviews by Almendras &
Fuentealba 1997; Mauel & Miller 2002; Fryer &
Hedrick 2003). This fastidious pathogen was first
observed in southern Chile in 1989 (Bravo &
Campos 1989; Branson & Diaz-Mu~
noz 1991;
Cvitanich, Garate & Smith 1991), then in
western Canada (Brocklebank et al. 1992), Ireland
(Rodger & Drinan 1993), Scotland (Grant et al.
1996), Norway (Olsen et al. 1997), eastern
Canada (Cusack, Groman & Jones 2002) and
southern USA (Arkush et al. 2005). Currently, an
official report from the Chilean authorities
indicates that piscirickettsiosis or salmonid
n
~ ez, R Avendan
~ o-Herrera and
Correspondence A J Ya
J G Carcamo, Interdisciplinary Center for Aquaculture
Research (INCAR), Vıctor Lamas 1290, PO Box 160-C,
Concepcio n, Chile (e-mails: ayanez@uach.cl, gcarcamo@uach.
cl, ravendano@unab.cl)
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1
rickettsial septicaemia (SRS) caused by P. salmonis
has a high prevalence with 68% of fish diagnosed
as positive (www.sernapesca.cl).
Treatments with antimicrobial drugs represent
the only measure to control SRS in farmed fish.
In fact, in 2010, its use in Chilean salmon farms
added up to approximately 103 000 kg (www.
sernapesca.cl). Chemotherapy with a fluorinated
structural synthetic analogue to thiamphenicol and
chloramphenicol, florfenicol (FLO) and oxytetracycline (OTC), two bacteriostatic agents with
broad-spectrum activity, has often been the choice
of drugs for treating the outbreaks. However,
since their first use, the results of antimicrobial
treatments have been inconsistent and have failed
to effectively control SRS (Branson &
Diaz-Mu~
noz 1991; Cvitanich et al. 1991). The
irregular effects of the chemotherapy in controlling P. salmonis in salmonids indicate that the
bacterium has developed drug resistance. The drug
resistance mechanisms for some fish pathogens
have been studied but have not been characterized
for P. salmonis (http://www.who.int/topics/foodborne_diseases/aquaculture_rep_13_16june2006%
20.pdf). Therefore, the appearance of potentially
resistant isolates has raised efforts to provide data
about the antimicrobial susceptibility of P. salmonis isolates to different antibiotics.
Journal of Fish Diseases 2013
A J Yan
~ez et al. Broth microdilution for MIC determinations of P. salmonis
Susceptibility presented in the form of minimum inhibitory concentration (MIC) values is an
alternative way to measure antibiotic resistance as
proposed by Isachsen et al. (2012) for Francisella
noatunensis subsp. noatunensis, also an intracellular
pathogen. Smith et al. (1996) described the MIC
values of five antimicrobial agents to four P. salmonis isolates grown in the Chinook salmon embryo
(CHSE-214) cell line. On the other hand,
although the Clinical Laboratory Standard Institute
guideline M49-A (CLSI 2006a) reported the best
framework for developing standard broth dilution
susceptibility test protocols for different bacteria
pathogenic to fish, P. salmonis is not included in
the guidance document. Recently, a marine broth
medium supplemented with L-cysteine, named
AUSTRAL-SRS, which enhances the growth of
P. salmonis isolates, was described (Yan
~ez et al.
2012). The aim of this research was to evaluate the
use of AUSTRAL-SRS broth in the determination
of MIC values and in the interpretation of antimicrobial susceptibility of P. salmonis. Moreover, in
this study, the induction of drug resistance of the
P. salmonis type strain to OTC and FLO under
laboratory conditions was examined.
Two P. salmonis isolates, PPT-04 and PPT-05,
recovered in 2008 from diseased farmed Atlantic
salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), respectively, in
Chile, were used to test the liquid culture media.
The P. salmonis type strain ATCC VR-1361
(equivalent to LF-89) originally isolated from coho
salmon, Oncorhynchus kisutch (Walbaum), was
included for comparative purposes and was
acquired from the American Type Culture Collection (ATCC). All bacteria were confirmed as
P. salmonis using qPCR-based analysis described
by Karatas et al. (2008) and also by an indirect
fluorescent antibody test (IFAT, SRS BiosChile),
according to the manufacturer’s recommendation.
All P. salmonis were maintained frozen at 80 °C
in Criobille tubes (AES Laboratory) and in 1 mL
of L-15 Leibovitz medium containing 20% foetal
bovine serum and 10% dimethyl sulphoxide.
The MIC values were determined using a standardized broth microdilution method recommended by CLSI (2006a), with some
modifications in the medium composition, incubation time and temperature as required for P. salmonis (Yan
~ez et al. 2012). The strains were inoculated
in AUSTRAL-SRS broth medium, which contained twofold dilutions of the antibacterial agents
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2
tested, ranging from 0.016–256 lg mL 1. OTC
and FLO used in the MIC testing were all obtained
from Sigma-Aldrich. Standard stock solutions were
prepared by dissolving 10 mg of each antibacterial
agent with 500 lL of methanol 100% (OTC) and
ethanol 95%, and the final volume was adjusted
with distilled water to 10 mL. All stock solutions
were stored at 4 °C and were used within 24 h.
Each microdilution tray included a growth control
well (without antibiotics) and a negative (uninoculated) well as well as three controls including the
solvents (methanol and ethanol) in amounts corresponding to the highest quantity present in the
assay. Each test was carried out ten times in separate cultures for each strain, and the MIC value
was defined as the lowest concentration exhibiting
no visible bacterial growth at 18 °C for 92–96 h
and/or 116–120 h. In addition, the microtitre
plates were read with a spectrophotometer Synergy™ 2 (Biotex) to compare any possible difference in sensitivity between reading methods. As
recommended in the CLSI M49-A, the Escherichia
coli reference strain ATCC 25922 was included in
this study to check the performance of the MIC
test, as the MIC values of this strain are well
known. It was grown in cation-adjusted Mueller–
Hinton (CAMH) broth and incubated at 18 °C
for 92–96 h and 35 °C for 16–20 h.
The MIC values for the antibacterial agents are
shown in Table 1. No variation between replicates
performed with each P. salmonis isolate was
Table 1 Minimum inhibitory concentration (MIC) of florfenicol and oxytetracycline to P. salmonis isolates and type strain
when tested at 18 1 °C after for 92–96 h and/or
116–120 h
MIC lg mL
Florfenicol
Strain
Origin
PPT-04
Atlantic
salmon
Rainbow
trout
Coho
salmon
PPT-05
ATCC VR-1361
Escherichia
coli ATCC
25922
18 2 °C
after
92–96 h
1
Oxytetracycline
1
0.5
0.5
0.125
0.25
0.5a
0.25
2.5a
Florfenicol
Quality
control
1
Oxytetracycline
35 2 °C
after
16–20 h
4
18 2 °C
after
92–96 h
1
35 2 °C
after
16–20 h
1
a
MIC value obtained by Smith et al. (1996) using chloramphenicol and
oxytetracycline.
A J Yan
~ez et al. Broth microdilution for MIC determinations of P. salmonis
Journal of Fish Diseases 2013
found, regardless of the antibacterial agents tested.
All isolates tested had a high susceptibility and fell
within a narrow range. The isolates showed MIC
values ranging from 0.5 to 1 for FLO and from
0.125 to 0.5 lg mL 1 for OTC. In addition,
P. salmonis ATCC VR-1361 showed an MIC
value of 0.25 lg mL 1 for OTC and FLO. Smith
et al. (1996) classified a type strain and two isolates (EM-90 and SLGO-90) as susceptible to
OTC having MIC values ranging from 0.5 to
3 lg mL 1 (Table 1).
In the case of FLO, the mechanism of action is
similar to that of chloramphenicol; in fact, resistance to FLO is most commonly mediated by
mono- and diacetylation via chloramphenicol acetyltransferase enzymes, which prevents the binding
of FLO to the 50S ribosomal subunit (Shaw
1984). In general, MIC values found by Smith
et al. (1996) for chloramphenicol tested against
the type strain ATCC VR-1361 (equivalent to
LF-89) were similar to that obtained in our study
for FLO. In the case of the isolates PPT-04 and
PPT-05, they exhibited a high susceptibility level,
which was different from the other three P. salmonis isolates studied by Smith et al. (1996).
Interestingly, there are dramatic inconsistencies
with the SRS susceptibility results observed in field
treatments when compared with the biological
results obtained in vitro, probably because during
natural outbreaks of the disease, P. salmonis
invades the fish cells, growing within cytoplasmic
vacuoles of hepatocytes and liver-associated macrophages, as well as in macrophages from the kidney,
spleen and peripheral blood (Branson & DiazMu~
noz 1991; Cvitanich et al. 1991; McCarthy
et al. 2008), thus allowing the antibiotic to reach
the site of action, that is, inside the fish cells. In
addition, it can be speculated that a possible origin
of the variations is also due to the repeated application of FLO and OTC in Chilean farms during
the last years, in doses that are much higher than
that recommended by the pharmaceutical companies (San Martın et al. 2010). In fact, from 2010
to 2011, its use in Chilean salmon farms added up
to 177 597 and 160 266 kg, respectively, for FLO
and OTC (www.sernapesca.cl).
Cohen et al. (1989) have shown that sublethal
antibiotic treatment can result in multidrug resistance induction, which has been associated with
mutations in multidrug efflux pumps (Ma et al.
1993). There is evidence suggesting that antibiotic
resistance mechanisms would be via radicalinduced mutagenesis or induction of the expression of multidrug resistance genes (Kohanski,
DePristo & Collins 2010). Therefore, our further
objective was to study the influence of sublethal
concentrations of OTC and FLO in the susceptibility of P. salmonis ATCC VR-1361 in laboratory conditions.
One hundred microlitres of bacterial cultures
with 0.064 lg mL 1 of OTC and FLO was
transferred into 100-ml flasks containing 50 mL
of AUSTRAL-SRS broth supplemented with each
antibiotic at a concentration of 0.064 lg mL 1.
Each bacterial culture was incubated for 6 days
at 18 °C and then the P. salmonis culture was
washed and adjusted with fresh medium and
used as an inoculum in the MIC assays against
the two antibiotics, ranging from 0.016 to
256 lg mL 1. Each microdilution tray was
determined under the same culture conditions as
described above. The same experiments were
repeated five times.
The results obtained from the MIC analysis for
ATCC VR-1361, which was previously grown in a
sublethal concentration of OTC (0.064 lg mL 1),
showed an increase of twofold dilutions when
compared with the initial MIC value (Fig. 1). For
FLO, natural variation of the broth microdilution
was of onefold dilution, equivalent to
0.50
OTC-0.064 μg mL–1
Figure 1 Minimum inhibitory
concentration using the P. salmonis type
strain previously exposed to
0.064 lg mL 1 of florfenicol and
oxytetracycline under in vitro conditions.
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Op cal density (625)
0.45
0.40
FLO-0.064 μg mL–1
0.35
OTC
0.30
FLO
0.25
0.20
0.15
0.10
0.05
0.00
0
0.016
0.032
0.064
0.125
0.25
MIC μg mL–1
0.5
1
2
4
Journal of Fish Diseases 2013
A J Yan
~ez et al. Broth microdilution for MIC determinations of P. salmonis
0.5 lg mL 1. However, when we observed the
growth kinetics of the bacterium, some variations
in the distribution of the MIC data were detected,
suggesting that the type strain was able to evolve
and adapt to each drug. Microscopic observations
under phase contrast of P. salmonis cultured into
AUSTRAL-SRS broth did not reveal changes in
the morphology and size of the bacteria, which
were always observed as Gram-negative cocci. In
addition, all samples analysed were positive by the
IFAT test and qPCR analysis, confirming the cultured organism’s identity as P. salmonis. As
expected, liquid culture without the addition of
P. salmonis did not yield any growth.
Although the findings obtained with the type
strain shed light on the increase in resistance, it
needs to be confirmed by testing other isolates
recovered from outbreaks and using molecular
tools. Further studies are being performed to support this hypothesis. Interestingly, the rapid and
combined appearance of resistance caused by the use
of these antibiotics in Chilean fish farms has also been
described for three other fish pathogens: Streptococcus
phocae (Avendaño-Herrera et al. 2012), Flavobacterium psyhrophilum (Henrı́quez-Núñez et al. 2012)
and Vibrio ordalii (Poblete-Morales et al. 2013), but
the mechanism of resistance acquisition by P. salmonis remains unknown.
Our study was performed using AUSTRAL-SRS
broth as the base medium, which showed a good
overall correspondence with the MICs obtained by
Smith et al. (1996). Considering that P. salmonis
replicates by binary fission within membranebound cytoplasmic vacuoles in cells of susceptible
fish hosts or fish cell lines, inducing a characteristic
cytopathic effect (Fryer & Hedrick 2003), the data
obtained in our work indicate that this medium
can be successfully used in susceptibility tests of
P. salmonis isolates and that CLSI should consider
accepting it in the next edition of the guideline.
According to Smith (1998), the composition of
the medium used for the susceptibility tests should
provide enough growth conditions for the strains
to be tested and should not contain materials
interfering with the test itself or react with any
antimicrobial tested. Although CLSI (2006a,b)
suggests that the susceptibility testing (disc diffusion and MIC assays) should be performed using
Mueller–Hinton medium or that it should be
based on some other version, until now P. salmonis has not been included in the guidelines
because it does not grow in this medium.
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4
Despite the fact that the study included a small
number of bacterial strains (n = 3), no differences
were observed among the MIC values obtained
for each strain in the independent experiments
performed (n = 10), which included separate
preparation of the inoculum and/or the test itself.
In the same way, MIC values of E. coli ATCC
25922 grown in CAMH broth showed acceptable
values for all drugs (Table 1), that is, within the
approved quality control ranges. As expected, the
liquid culture without the addition of P. salmonis
did not yield any growth.
In summary, the results suggest that the AUSTRAL-SRS broth is a suitable medium to determine MIC values of P. salmonis strains. Moreover,
it facilitates the in vitro drug susceptibility tests of
this fastidious pathogen and can contribute to an
understanding of the pharmacokinetic data in fish
as well as evaluate the drug resistance of this pathogen and help salmon producers to take treatment
decisions to control the disease.
Acknowledgements
Funding for this study was provided in part by
Grant INNOVA CHILE 07CN13PPT-56, DID
UACh, from the Direccion de Investigacion y
Desarrollo de la Universidad Austral de Chile and
also by Grant FONDECYT 1110219 from the
Comision Nacional de Investigacion Cientıfica y
Tecnologica (CONICYT, Chile). R. A-H, A. JY
and J. GC also acknowledge the CONICYT/
FONDAP/15110027.
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Received: 22 January 2013
Revision received: 7 May 2013
Accepted: 17 May 2013