Veterinary Microbiology 154 (2012) 407–412

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Short communication

Genetic analysis and antimicrobial susceptibility of Francisellanoatunensis subsp. orientalis (syn. F. asiatica) isolates from fish

Esteban Soto a,b,*, Matt Griffin c, Judy Wiles a, John P. Hawke a

a Louisiana State University, Department of Pathobiological Sciences, School of Veterinary Medicine, Skip Bertman Dr., Baton Rouge, LA 70803, United Statesb Ross University, Department of Pathobiology, School of Veterinary Medicine, Basseterre, Saint Kitts and Nevisc Thad Cochran National Warmwater Aquaculture Center, Mississippi Agricultural and Forestry Experiment Station and College of Veterinary Medicine, Mississippi

State University, Box 197, Stoneville, MS 38776, United States

A R T I C L E I N F O

Article history:

Received 16 December 2010

Received in revised form 22 July 2011

Accepted 26 July 2011

Keywords:

Francisella noatunensis subsp. orientalis

Francisella asiatica

Antimicrobial

Tilapia

Treatment

A B S T R A C T

Francisella noatunensis subsp. orientalis (syn. F. asiatica) (Fno) is an emergent fish pathogen

that causes acute to chronic disease in a wide variety of freshwater, brackish and marine

fish. Due to the emergent nature of this bacterium, established protocols to measure

antimicrobial susceptibility are lacking. In this project we compare three different

methods to examine the antimicrobial susceptibility (Etest, broth microdilution and disk

diffusion) of 10 different isolates of Fno from two different fish species and four different

geographic outbreaks from 2006 to 2010. PCR mediated genomic fingerprinting (rep-

PCR) performed on the different isolates confirmed genetic homogeneity amongst the

different isolates. The in vitro susceptibility data presented here provides important

baseline data for future research monitoring the development of antibiotic resistance

among Fno isolates as well as provides invaluable data for the development of potential

therapeutics.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

Francisella noatunensis subsp. orientalis (syn. Francisella

asiatica) (Fno) and F. noatunensis subsp. noatunensis (syn. F.

noatunensis) (Fnn) are small pleomorphic, Gram-negativebacteria, belonging to the gamma group of the classProteobacteria that cause piscine francisellosis in a widevariety of fish species (Mikalsen and Colquhoun, 2009;Ottem et al., 2009).

With the increased worldwide production of culturedtilapia, a thorough understanding of pathogens associatedwith its commercial production is of great significanceto fish health professionals and commercial producers.

* Corresponding author at: Ross University, School of Veterinary

Medicine, Island Main Road, West Farm, Basseterre, Saint Kitts and

Nevis. Tel.: +869 465 4161x301; fax: +869 465 6156.

E-mail address: esoto@rossvet.edu.kn (E. Soto).

0378-1135/$ – see front matter � 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2011.07.030

Currently, there are limited published data on the antibioticsusceptibilities of fish pathogens and only three antibioticsare approved by the United States Food and Drug Admin-istration (FDA) for using in United States aquaculture.Additionally, due to the emergent nature of piscinefrancisellosis, there is a dearth of published data regardingantibiotic susceptibility of Francisella spp. that infect fish.

In this study, we first investigated the genetic homo-geneity of Fno isolates from different outbreaks anddifferent geographic locations by PCR mediated genomicfingerprinting (rep-PCR). Secondly we evaluated thebroth micro-dilution, Etest and disk-diffusion (Kirby–Bauer) methods to determine antimicrobial susceptibilityof Fno. Lastly, we were able to determine the in vitro

antibiotic susceptibility of a panel of 10 Fno isolates obtainedfrom different fish species and geographic locations to avariety of antimicrobial agents, any of which might beconsidered as potential therapeutics for the treatment of Fno

infections in aquatic animals.

E. Soto et al. / Veterinary Microbiology 154 (2012) 407–412408

2. Materials and methods

2.1. Bacterial isolates and growth conditions

Francisella noatunensis subsp. orientalis (Fno) isolateswere obtained from two different fish species from fourdifferent geographic regions between 2006 and 2010(Table 1) (Ostland et al., 2006; Soto et al., 2009, 2011). Fno

isolates, and the quality control Escherichia coli ATCC25922 were grown on Cystine Heart Agar supplementedwith bovine hemoglobin (CHAH) solution (Becton Dick-enson (BD) BBL, Sparks, MD, USA) for 96 h (Fno isolates) or24 h (quality controls) at 28 8C, or in Mueller–Hinton IIcation adjusted broth supplemented with 2% IsoVitaleX(BD BBL, Sparks, MD, USA) and 0.1% glucose (MMH) (Bakeret al., 1985). Broth cultures were grown overnight at 28 8Cin a shaker at 175 rpm and bacteria were frozen at �80 8Cin the broth media containing 20% glycerol for later use.The quality control strain was also grown on Mueller–Hinton agar (Remel, Lenexa, KS) supplemented with 5%sheep blood (MHB) for comparison.

2.2. Genomic fingerprinting

All Fno isolates were cultured as described above andDNA isolation was carried out using the DNEasy Blood andTissue Kit (QIAGEN, Valencia, CA) following the manufac-turer’s suggested protocol for Gram-negative bacteria.Genomic DNA was quantified spectrophotometricallyusing a NanoDrop1 ND-1000 micro-spectrophotometer(NanoDrop Technologies, Wilmington, DE, USA) andanalyzed by PCR mediated genomic fingerprinting (rep-PCR) to determine any genetic variability between isolates.The quality control Escherichia coli ATCC 25922 wasincluded as an outlier/negative control. The rep-PCRanalysis was carried out using the ERIC and BOX primersets following previously published protocols (Versalovicet al., 1991, 1994; Griffin et al., in press). Quantity Onesoftware v. 4.6.5 (Bio-Rad Laboratories Inc., Hercules, CA)was used to calculate the Dice coefficients and generate adendrogram by unweighted pair-group method usingarithmetic averages (UPGAMA).

Table 1

Francisella noatunensis subsp. orientalis isolates used during this study.

Isolate Location Fish

F.#1 CA, USA Hybrid striped bass

(Morone chrysops�M.

saxatilis)

F.#2 CA, USA Hybrid striped bass

(Morone chrysops�M.

saxatilis)

F.#3 CA, USA Hybrid striped bass

(Morone chrysops�M.

saxatilis)

LADL 10-075 #1 Midwest, USA Tilapia (Oreochromis sp.)

LADL 10-075 #5 Midwest, USA Tilapia (Oreochromis sp.)

LADL 10-075 #8 Midwest, USA Tilapia (Oreochromis sp.)

LADL 10-051 Midwest, USA Tilapia (Oreochromis sp.)

LADL 07-285A Alajuela, Costa Rica Tilapia (Oreochromis sp.)

LADL 07-285B Alajuela, Costa Rica Tilapia (Oreochromis sp.)

Victoria Central America Tilapia (Oreochromis sp.)

2.3. Antimicrobial susceptibility

2.3.1. Disk-diffusion (Kirby–Bauer) method

The disk-diffusion procedure was performed accordingto protocols established by the Clinical and LaboratoryStandard Institute (CLSI) (Wayne, PA) with a few mod-ifications (CLSI, 2006a). Mainly, due to the fastidiousnature of Fno and its inability to grow in Mueller–Hintonbased agar, CHAH was utilized for disk-diffusion method.CHAH agar has been previously used in F. tularensis

antimicrobial susceptibility research (Biegeleison andMoody, 1959; Ikaheimo et al., 2000; Tomasoa et al.,2005; Gurcan et al., 2008). Quality control strain (E. coli

ATCC 25922) was tested in both CHAH and MHB forcomparison purposes.

Briefly, bacteria were harvested after incubation for96 h (Fno) or 24 h (E. coli ATCC 25922) at 28 8C andsuspended in 1� phosphate buffer saline (PBS) to achieve aturbidity equivalent to that of a 0.5 McFarland standard.The plates were inoculated with 100 ml of a colonysuspension using a Drigalski spatula. Fifteen minutes later(once the plates were dried), the antibiotic discs weredispensed by a disc dispenser, providing sufficientseparation so as to avoid overlapping of inhibition zones.After 30 min, the plates were inverted and incubated at28 8C for 48 h (Fno) or 24 h (E. coli ATCC 25922). Resultswere recorded by measuring the diameter of the inhibitionzones and quality controls were compared with standardsfor antimicrobial disk susceptibility tests supplied by theCLSI (2006a). The antibiotic discs were obtained commer-cially (BD, Sparks, MD) (Table 2).

2.3.2. Broth microdilution method

The minimal inhibitory concentrations (MIC) of 39different antimicrobial agents to Fno isolates and qualitycontrols were tested using the GN2F – sensititre gramnegative plate format and the AVIAN1F – avian one isolateMIC plate (Trek Diagnostic System, West Sussex, UK) usingthe manufacturer’s suggested protocol and previous proto-cols published for Francisella tularensis antimicrobial sus-ceptibility testing (Table 3) (CLSI, 2006b; Baker et al., 1985;Brown et al., 2004; Garcia del Blanco et al., 2004; Urich andPetersen, 2008). Briefly, Fno and E. coli ATCC 25922 qualitycontrol were plated on CHAH agar and incubated from 96 or24 h, respectively at 28 8C. Inocula were prepared bysuspending colonies in 1� PBS to a 0.5 McFarland standard.This suspension was diluted 100-fold (Fno) or 1000-fold (E.

coli ATCC 25922) in MMH, and 50 ml were added to each wellof the sensititre plates containing the different antibiotics.Tested plates were incubated for 48 h (Fno isolates) or24 h (E. coli ATCC 25922 quality control) at 28 8C. Bacterialgrowth was checked visually at 48 h (Fno isolates) or 24 h(E. coli ATCC 25922 quality control) post inoculation.The MIC value was defined as the lowest concentrationexhibiting no visible growth. The MIC assay for the qualitycontrol E. coli ATCC 25922 was determined utilizing MMHat 28 8C following the protocols by CLSI (2006b).

2.3.3. Etest

Minimal inhibitory concentrations (MICs) of 15 anti-microbial agents (Table 4) were determined by Etest on

Table 2

Disk-diffusion (Kirby–Bauer) method was done following the CLSI protocols with modifications (CLSI, 2006a). The mean zone diameters (mm) and standard

deviation is given for F. noatunensis subsp. orientalis (Fno) isolates and quality control when tested in Cysteine Heart Agar supplemented with hemoglobin

(CHAH) or Mueller–Hinton Agar supplemented with blood (MHB) and incubated at 28 8C for 48 h (Fno isolates) or 24 h (E. coli ATTC 25922). CLSIs

recommended ranges for the quality controls are in parenthesis.

Antimicrobial agent tested (concentration) CHAH MHB

F. noatunensis

subsp. orientalis

E.coli ATTC 25922 F. noatunensis

subsp. orientalis

E.coli ATTC 25922

Florfenicol (30) 27.1� 2.55 24.8� 1.8 NA ND 24.4� 1.94 (20–30)

Tetracyclin (30) 33.3� 3.91 24.5� 1.2 NA ND 23.9� 1.16 NA

Nitrofurantoin (300) 40� 3.08 22� 3 NA ND 21.3� 2.3 NA

Enrofloxacin (5) 42� 4.15 30.2� 1.47 NA ND 35� 1.89 NA

Trimethropin/sulfamethoxazole (1.25/23.75) 0� 0 15.2� 1.6 NA ND 25.16� 0.4 (25–32)

Gentamicin (10) 36.9� 2.58 15.7� 1.96 NA ND 23.3� 1.21 (22–29)

Ampicillin (10) 0� 0 22.2� 1.32 NA ND 16.2� 0.75 (14–23)

Erythromycin (15) 28.3� 2.5 0� 0 NA ND 11.5� 1.7 (10–15)

Oxolinic acid (2) 28.2� 3.32 23.3� 1.0 NA ND 26� 1 (25–32)

ND, not determined; NA, not available.

E. Soto et al. / Veterinary Microbiology 154 (2012) 407–412 409

CHAH plates (Fno isolates), and on CHAH and MHB plates(E. coli ATCC 25922) following previously publishedprotocols for F. tularensis (Ikaheimo et al., 2000; Tomasoaet al., 2005; Gurcan et al., 2008). Briefly, bacteria were

Table 3

Minimal inhibitory concentration (MIC) to antimicrobial agents of Francisella noa

quality control after broth micro-dilution testing.

Antimicrobial agent tested (concentration) F. noatunen

Range

Enrofloxacin (2–0.12 mg/ml) <0.12

Gentamycin (8–0.5 mg/ml) <0.5

Ceftiofur (4–0.25 mg/ml) 1 to >4

Neomycin (32–2 mg/ml) <2

Erythromycin (4–0.25 mg/ml) >4

Oxytetracycline (8–0.5 mg/ml) <0.25–0.5

Tetracycline (8–0.25 mg/ml) <0.25–0.5

Amoxicillin (16–0.25 mg/ml) 4 to >16

Spectinomycin (64–8 mg/ml) <8–16

Sulphadimethoxine (256–32 mg/ml) 128 to >25

Trimethoprim/sulfamethoxazole (2/38–0.5/9.5 mg/ml) >2/38

Florfenicol (8–0.25 mg/ml) <1–2

Sulphathiazole (256–32 mg/ml) 128 to >25

Penicillin (8–0.06 mg/ml) 4 to >8

Streptomycin (1024–8 mg/ml) <8

Novobiocin (4–0.5 mg/ml) <0.5–1

Tylosin tartrate (20–2.5 mg/ml) 20 to >20

Clindamycin (4–0.5 mg/ml) >4

Amikacin (64–8 mg/ml) <8

Ampicillin (32–4 mg/ml) 4 to >32

Ampicillin/sulbactam 2:1 ratio (4/2–32/16 mg/ml) <4/2 to >3

Aztreonam (32–8 mg/ml) 16 to >32

Cefazolin (32–4 mg/ml) 16 to >32

Cefepime (32–4 mg/ml) <4 to >32

Cefotetan Na (32–8 mg/ml) >32

Ceftriaxone (64–1 mg/ml) 4 to >64

Ceftazidime (32–1 mg/ml) 4 to >32

Cefoxitin (32–4 mg/ml) 32 to >32

Cefuroxime (32–4 mg/ml) 16 to >32

Ciprofloxacin (4–0.5 mg/ml) <0.5

Imipenem (16–2 mg/ml) <2

Gatifloxacin (8–1 mg/ml) <1

Meropenem (8–1 mg/ml) <1

Piperacillin (128–16 mg/ml) 16 to >128

Nitrofurantoin (128–16 mg/ml) <16

Piperacillin/tazobactam constant 4 (128/4–16/4 mg/ml) <16/4 to >

Ticarcillin/clavulanic acid constant 2 (64/2–16/2 mg/ml) 16/2 to >6

Tobramycin (8–4 mg/ml) <4

Cefpodoxime (16–2 mg/ml) <�2 to 8

harvested after incubation for 96 h (F. noatunensis subsp.orientalis) or 24 h (E. coli ATCC 25922) at 28 8C andsuspended in 1� PBS to achieve a turbidity equivalent of aMcFarland standard 1 (Fno) or 0.5 (E. coli ATCC 25922). The

tunensis subsp. orientalis isolated from fish and Escherichia coli ATTC 25922

sis subsp. orientalis isolates E.coli ATTC 25922

MIC 50 MIC 90 Range

<0.12 <0.12 <0.12

<0.5 <0.5 1

>4 >4 1

<2 <2 <2

>4 >4 >4

<0.25 <0.25 0.5

<0.25 <0.25 0.5

16 >16 4

<8 16 64

6 256 >256 >256

>2/38 >2/38 <0.5/9.5

<1 2 4

6 256 >256 >256

>8 >8 >8

<8 <8 16

<0.5 1 >4

20 >20 >20

>4 >4 >4

<8 <8 16

>32 >32 <4

2/16 >32/16 >32/16 <4/2

>32 >32 <8

>32 >32 <4

>32 >32 <4

>32 >32 <8

4 >64 <1

4 >32 <1

>32 >32 8

>32 >32 8

<0.5 <0.5 <0.5

<2 <2 <2

<1 <1 <1

<1 <1 <1

>128 >128 <16

<16 <16 <16

128/4 <16/4 >128/4 <16/4

4/2 <16/2 >64/2 32/2

<4 <4 <4

<2 8 <2

Table 4

Minimal inhibitory concentration (MIC) to antimicrobial agents of Francisella noatunensis subsp. orientalis (Fno) isolated from fish and Escherichia coli ATTC

25922 quality control strain after Etest analysis on Cysteine Heart Agar supplemented with hemoglobin (CHAH) (Fno isolates and E.coli ATTC 25922) or

Mueller–Hinton Agar supplemented with blood (MHB) and incubated at 28 8C for 48 h (Fno isolates) or 24 h (E. coli ATTC 25922).

Antimicrobial agent tested (concentration) F. noatunensis subsp. orientalis isolates E.coli ATTC 25922

Range MIC 50 MIC 90 Range in CHAH Range in MHB

Gentamycin 0.19–0.50 0.19 0.5 12 0.75–4

Tetracyclin 0.75–1 1 1 2 1.5–2

Vancomycin >256 >256 >256 >256 >256

Amoxicillin >256 >256 >256 6 6–8

Amykacin 1 1 1 48 2–12

Ceftazidine >256 >256 >256 0.38 0.25–0.38

Levofloxacin 0.016 0.016 0.016 0.125 0.012–0.125

Piperacillin/tazobactam >256 >256 >256 3 3–6

Ampicillin >256 >256 >256 4 4–8

Imipenem 0.38–1 1 1 0.38 0.19–0.38

Trimethoprim/sulfamethoxazole >32 >32 >32 1.5 0.094

Aztreonan >256 >256 >256 0.125 0.047–0.94

Erythromycin 1–4 2 4 256 24–32

Oxacillin >256 >256 >256 >256 >256

Clindamycin 16 to >256 16 >256 >256 >256

E. Soto et al. / Veterinary Microbiology 154 (2012) 407–412410

plates were inoculated with 100 mL of a colony suspensionusing a Drigalski spatula. Fifteen minutes later, two Eteststrips were placed on each plate following the manufac-turer’s suggested protocol (bioMerieux, Durman, USA).MICs were read after 48 h (Fno) or 24 h (E. coli ATCC 25922)incubation at 28 8C.

3. Results and discussion

In this study, three different methods were utilized toevaluate the in vitro antimicrobial susceptibility of Fno toseveral antibiotics. The first objective of the study was to

[(Fig._1)TD$FIG]

Fig. 1. Genomic fingerprints of Francisella noatunensis subsp. orientalis isolates fro

(B) primer sets, respectively. Lane 1, molecular weight marker (250 bp ladder); l

lane 6, LADL 10-075 #1; lane 7, LADL 10-075 #5; lane 8, LADL 10-075 #8; lane 9,

051; lane 13, no template control; and lane 14, molecular weight marker (250

evaluate any genetic diversity between isolates recoveredfrom different outbreaks in different fish species anddifferent locations. Polymerase chain reaction mediatedgenomic fingerprinting using the ERIC and BOX primers is awell recognized, reliable method for taxonomic identifica-tion and discrimination of bacteria (Saxena et al., 2002;Tacao et al., 2005; Yuan et al., 2010). For both primer sets,electrophoretic profiles demonstrated high levels ofhomogeneity for the 10 different Fno isolates collectedfrom 4 different geographic outbreaks in different years, allof which were significantly different from the E. coli ATCC25922 outlier (Fig. 1). The similarity matrices and

m fish. Electrophoretic profiles were generated from either BOX (A) or ERIC

ane 2, Escherichia coli ATTC 25922; lane 3, F.#1; lane 4, F. #2, lane 5, F.#3;

LADL 07-285A; lane 10, LADL 07-285B; lane 11, Victoria; lane 12, LADL 10-

bp ladder).

E. Soto et al. / Veterinary Microbiology 154 (2012) 407–412 411

dendograms used a cut-off of 80% for genotypic differ-entiation, which is at or above the level used in similarstudies (Coenye et al., 2002; Woo and Lee, 2006; Maitiet al., 2009; Keymer et al., 2009). Although the profilesgenerated for these isolates were not very complex, at thislevel of discrimination all isolates group together in asingle cluster, regardless of primer set (data not shown). Itshould be noted the BOX primers generated severalamorphous smears at the lower portion of several lanes,which are thought to be primer artifact. However, as theseartifacts are not in every lane, they were included in theanalysis as wide, non-distinct bands. Although inclusion ofthese bands slightly increased the variability betweenisolates, the profiles still demonstrate more than 84%similarity (range 84.9–96.9%), suggesting the same geno-type. Similarly, the ERIC profiles shared more than 83%similarity (range 83.7–98.8%). Given the success theseprimer sets have had in differentiating between closelyrelated isolates of other species of bacteria, the similaritiesdemonstrated by these case isolates make a strongargument these isolates share the same genotype. Furtherresearch involving a larger number of isolates and highercomplexity methodologies may be required to identifygenotypic differences between isolates of Fno fromdifferent outbreaks.

Due to the emergent nature of Fno there are nostandards or established methodologies for testing ofantimicrobial susceptibility. In this study, MICs of morethan 40 different antibiotics were investigated for 10different isolates of Fno. When evaluating the broth mediawith the CLSI quality control (E. coli ATCC 25922) all theMICs for substances which reference values at 28 8C areavailable fell within the range given by the CLSI, indicatingthe broth media was suitable for the evaluation ofantimicrobial susceptibility of fastidious organisms,including Fno (CLSI, 2006b). The fastidious properties ofmembers of the genus Francisella do not allow conven-tional media to be used without the addition of supple-ments (IsoVitalex, Glucose, etc.). In contrast to Baker et al.(1985), the addition of Isovitalex to the broth media in thisstudy did not interfere with the quality controls or the Fno

isolates. The AVIAN and GN2F microplates were chosensince they included two of the antibiotics currentlyapproved by the United States FDA for use in USaquaculture (oxytetracycline and florfenicol) as well as alarge range of antibiotics used in the treatment of diseasescaused by Gram-negative organisms. The microplates andmedia were suitable for the evaluation of antimicrobialsusceptibitly of Fno isolates, since the results wereconsistent and little variability was observed betweenassays (Table 3). There were no differences in antimicrobialMICs for isolates attributable to the fish host or locationfrom which they were isolated.

Recently, several laboratories have used the Etest onCHAH plates for antimicrobial susceptibility research of F.

tularensis, obtaining reproducible and reliable results(Tomasoa et al., 2005; Gurcan et al., 2008; Valade et al.,2008). In this study, we evaluated CHAH as a potentialmedia for use in the Etest and disk diffusion method ofdetermining antimicrobial susceptibility for Fno. The MICvalues obtained with the broth microdilution and Etest

were very similar; and although there are no currentpublished documents or protocols for testing bacteriaisolated from aquatic animals; the Etest appears as asuitable method that needs further exploration (Tables 3and 4).

When utilizing the Kirby–Bauer method, clear zones ofinhibition could be observed for the quality control in theCHAH for several different antibiotics, but when zonediameters of the quality control was compared with thoseobserved in the MHB plates and the ranges given by theCLSI, only the inhibition zones observed for florfenicol,tetracycline and ampicillin fell within the ranges (Table 2)(CLSI, 2006a). In Mueller–Hinton Media, the levels ofsulfonamide inhibitors, thymidine, thymine, magnesiumand calcium ions are controlled so as not to interfere withsusceptibility testing and to yield good growth. Althoughthe quality controls did not fall within the expected rangesof the CLSI, valuable information can be taken from the diskdiffusion results obtained from the Fno isolates. All the Fno

isolates inhibition zones were consistent and repeatable.Since there is no zone diameters established for Fno for theantimicrobials tested in this study the classifications ofresistant, intermediate, or sensitive cannot be determined.However, due to the large zones of inhibition present whentesting florfenicol, tetracyclin, nitrofurantoin, gentamicin,erythromycin, and oxolinic acid (at 30, 30, 300, 10, 15, or2 mg/ml) coupled with the low MICs observed for thesecompounds, it is reasonable to conclude that Fno isolatesappear intermediately if not highly susceptible to theseantibiotics (Table 2).

In conclusion, according to the combined data obtainedin this project, Fno appears susceptible to enrofloxacin,gentamycin, neomycin, oxytetracycline, tetracycline, flor-fenicol, streptomycin, novobiocin, amikacin, ciprofloxacin,imipenem, gatifloxacin, meropenem, tobramycin, nitro-furantoin, oxolinic acid, and levofloxacin; while resistantto penicillin, amoxicillin, ampicillin, piperacillin, oxacillin,erythromycin, novobiocin, tylosin tartrate, clyndamycin,sulphathiazole, sulphadimethoxine, trimethropin/sulfa-methoxazole, piperacillin/tazobactam constant 4, ticarcil-lin/clavulanic acid constant 2, ampicillin/sulbactam 2:1ratio, aztreonam, ceftiofur, cefazolin, cefepime, cefotetanNa, ceftriaxone, ceftazidime, cefoxitin, cefuroxime andvancomycin. These results are similar to those reported forFno, where in vitro data have shown that different isolateswere resistant to trimethoprim–sulfamethoxazole, peni-cillin, amoxicillin, ampicillin, cefuroxime, and erythromy-cin, yet susceptible to ceftazidime, florfenicol, flumequine,oxolinic acid, oxitetracycline, tetracycline, gentamicin andciproxoxacin (Ottem et al., 2007; Bohle et al., 2009).

Although the risk of Fno developing resistance toantibiotics requires further study, the informationobtained in this study suggests that florfenicol andoxytetracycline, have potential for the treatment of Fno

infections in cultured fish. This is in agreement withprevious studies where both florfenicol and oxytetracy-cline given as medicated feed during early stages ofinfection demonstrated efficacy in treating piscine franci-sellosis (Mauel et al., 2003; Ostland et al., 2006; Soto et al.,2010). Nevertheless, more research and surveillance of Fno

field isolates is necessary to monitor antimicrobial

E. Soto et al. / Veterinary Microbiology 154 (2012) 407–412412

susceptibility for the detection and comparison of resis-tance development and to provide in vitro data for theguidance of therapy and development of potentialtherapeutants.

Acknowledgment

We gratefully thank Dr. John Hansen from the Inter-disciplinary Program in Pathobiology, University ofWashington, Seattle, WA, for sharing some of the Fno

isolates used in the study.

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