Vol. 29, No. 6ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1986, p. 992-9960066-4804/86/060992-05$02.00/0Copyright © 1986, American Society for Microbiology

Multiple Low-Level Antibiotic Resistance inAeromonas salmonicida

STEPHEN C. WOOD, RUTH N. McCASHION, AND WILLIAM H. LYNCH*Department of Biology, University ofNew Brunswick, Fredericton, New Brunswick E3B 6E1, Canada

Received 26 August 1985/Accepted 11 March 1986

Mutants with multiple low-level antibiotic resistance were isolated from virulent wild-type Aeromonassalmonicida strains exposed to a low concentration of any one of several low-molecular-mass (approximately635 daltons or less) antibiotics. Multiple resistance was toward beta-lactam compounds (penicillin G,ampicillin, cloxacillin), quinolones (flumequine, oxolinic acid, nalidixic acid), tetracyclines, chloramphenicol,and novobiocin. Susceptibilities of the mutants toward several higher-molecular-mass (>700 daltons) hydro-phobic or polycationic antibiotics such as rifampin, erythromycin, polymyxin B, and streptomycin sulfate werenot affected. The mutants-were obtained at frequencies suggesting point mutations. Outer membrane proteinprofiles, examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that all multiplelow-level resistant mutants were deficient in a major protein of approximately 38.5 kilodaltons and containeda major protein of approximately 37 kilodaltons which was not present in significant amounts in the wild-typestrains. In addition, these mutants lacked exoprotease activity. Furthermore, mutants isolated as deficient inexoprotease were found, with the exception of one avirulent strain, to exhibit multiple low-level antibioticresistance and the outer membrane protein changes.

Aeromonas salmonicida is the causative agent offurunculosis in salmonid species of fish. The continued useof antibiotics to treat furunculosis outbreaks in cultured fishhas led to the development of antibiotic resistance in A.salmonicida (4, 18), and the presence of R factors in thismicroorganism has been shown (3, 4). More recently, anti-biotics such as oxolinic acid (5) and flumequine (A. Scallan,Ph.D. thesis, University College, Galway, Republic of Ire-land, 1983) have been promoted for the treatment of thisdisease since plasmid-encoded resistance to these antibioticshas not been reported. However, mutational resistance tosuch antibiotics can occur (30). A. salmonicida strains,resistant to these antibiotics, have been isolated from dis-eased fish (4; this paper).

In addition to mutational resistance to specific antibioticsor groups of related antibiotics, mutations which affect theouter membrane of gram-negative bacteria may alter suscep-tibilities to a much wider range of inhibitory agents (forreview, see reference 22). Although mutations altering outermembrane permeability may provide only low-level multipleresistance (10, 17, 25, 27), this resistance may be additive toother resistance mechanisms residing in the microorganism(17, 26, 27) producing strains with a high level of resistance.The present study was promoted by the isolation of

exoprotease-negative mutants of A. salmonicida which re-tained the virulence of their parent wild-type strain (9) butexhibited a multiple, low-level resistance to a wide range oflow-molecular-mass antibiotics.

MATERIALS AND METHODS

Bacterial strains. Wild-type strains of A. salmonicida wereobtained from various sources (see Table 1), and cultureswere maintained on brain heart infusion (BHI) agar. All BHI

* Corresponding author.

agar plates were incubated at 22°C for 24 h. BHI broths wereincubated at 22°C for 24 h on a shaker (model G-26; NewBrunswick Scientific Co., New Brunswick, N.J.) at 150 rpm.Mutants lacking exoprotease (caseinase) activity (Prt-) wereisolated on skim milk overlay plates with BHI agar as a basalmedium following subculture of lyophilized cultures ortreatment ofthe cultures with ethidium bromide as previouslydescribed (9). Mutants isolated as antibiotic resistant wereselected as colonies growing on BHI agar plates containingampicillin (0.5 ,ug ml-'), chloramphenicol (1.5 jig ml-'),flumequine (0.1 ,ug ml-'), penicillin (4 ,ug ml-'), or rifampin(8 jig ml-'). The plates were surface inoculated from BHIbroth cultures with approximately 2 x 108 cells as estimatedby optical density. The antibiotic-resistant mutants werechecked for exoprotease activity by plating on skim milk agaroverlay plates (9). Mutants lacking the A layer (A-) wereisolated following incubation of cultures at elevatedtemperature, as described by Ishiguro et al. (14), and werechecked for the presence ofA layer by transmission electronmicroscopy (9).

Antibiotic susceptibilities. Inocula were grown in 25-mlErlenmeyer flasks containing 10 ml of BHI broth. Sufficientcells were surface inoculated onto BHI agar plates to pro-duce confluent growth after 24 h. Antibiotic disks wereapplied to the surface of the inoculated plates and zones ofinhibition were recorded. MICs were estimated by usingtwofold or less dilutions of the antibiotics in BHI agar. Theplates were surface inoculated and incubated for 24 h.Readings of less than 10 colonies per approximately 105 cellsinoculated were scored as negative (10). MIC determinationsWere performed at least in triplicate. When variation oc-curred (up to a twofold maximum) in some cases, MICdeterminations were repeated at least eight times and theantibiotic dilution closest to the mean was used.

Isolation of outer membranes. Inocula were from BHIbroth. Skim milk agar overlay plates (150 by 15 mm; threeper strain) were overlaid with cellulose dialysis tubing

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TABLE 1. MICs of antibiotics for wild-typeA. salmonicida strains

MIC (,ug of antibiotic ml-1)bStrainsa Flume- Oxytetra- Streptomycin Sulfa-

quine cycline sulfate diazinec

ATCC 14174, SS70, AsFL, 0.05 0.2 50 50Rest. 80204, AsU,Mac 79-38R

AsB 2 0.2 50 50AsF, AsA, AsD 0.05 50100d 50 50Asl 0.05 25 50 1,000AsL, AsLR, AsLH, AsM, 0.05 25 >1,000 1,000AsH, AsPa ATCC 14174 was from the American Type Culture Collection. SS70,

Rest. 80204, and Asl were from Connaught Research Laboratory. Mac79-38R, AsF, AsD, and AsA were from the Fish Health Unit, Fisheries and.Oceans, Halifax, Nova Scotia. Other strains were from diseased fish obtainedduring furunculosis outbreaks at various facilities and isolated in our labora-tory.

b All wild-type strains showed similar susceptibilities to ampicillin, chlor-amphenicol, cloxacillin, erythromycin, novobiocin, penicillin G, polymyxinB, and rifampin.

c Strains frequently showed very weak growth at sulfadiazine concentra-tions above recorded MICs, especially with incubation time of >24 h.

d MICs of oxytetracycline were 50 F.g ml-' for strains AsD and AsA and100 ,ug ml-' for strain AsF.

(Spectropor 3; Spectrum Medical Industries Ltd., LosAngeles, Calif.) and surface inoculated (1). After 48 h ofincubation, cells were harvested (1) and suspended in 0.033M Tris hydrochloride-20% sucrose-12.5 mM EDTA (29).Cell suspensions were incubated at room temperature withgentle stirring and added lysozyme (final concentration of 1mg ml-' for A' strains incubated for up to 10 h and 0.5 mgml-' for A- strains incubated for up to 4.5 h). Spheroplastformation was followed, and outer membranes were sepa-rated by centrifugation (13), washed, and suspended in 1 mlof Tris hydrochloride containing 2 mM EDTA. The proteincontent of samples was estimated by the Coomassie blueassay (Bulletin 1969, Bio-Rad Laboratories, Richmond,Calif.), using bovine serum albumin as the standard. Outermembrane samples were precipitated and washed with tri-chloroacetic acid (10% [wt/vol] final concentration) at 4°C,followed by three washes in 9 parts acetone-1 part 0.065 MTris hydrochloride, pH 6.8. Supernatants were removed,and samples were air dried and stored at -20°C.SDS-PAGE. The outer membrane sample preparation was

as described by Laemmli (16) except that the samples wereboiled for 1 min and vortexed for 30 s three times. Samplesof 30 ,ul, containing 80 to 140 ,g of protein, were applied tothe gels as described by Ames (2). The sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gelswere prepared as described by Laemmli (16) except that theammonium persulfate concentration was 0.027% (wt/vol) inthe stacking gel and 0.03% (wt/vol) in the separating gel, andthe tetramethyl-ethylenediamine concentration was 0.1%(wt/vol) in the stacking gel and 0.054% (wt/vol) in theseparating gel. The stacking gel was 4.5% acrylamide and 3cm in length, and the separating gel was 12% acrylamide and15 cm long with a thickness of 1.5 mm and a width of 19 cm.The electrode and separating buffer were pH 8.5. Thesamples were stacked at 70-V and separated at 140-V con-stant voltage. The proteins were fixed and stained as de-scribed in LKB application note 321 (LKB-Produckter AB,Bromma, Sweden).Enzyme assays. Cultures (50 ml) were grown in BHI broth

(optical density at 660 nm of 1 to 1.5). The cells were

harvested by centrifugation at 10,000 x g for 10 min at 4°C.Shock fluids (10x concentrated) were obtained by the su-crose-EDTA and MgCl2 procedure of Willis et al. (29).Spheroplasts were prepared from shocked cells as describedabove. The spheroplast pellet was suspended and lysed in 5ml of 0.033 M Tris hydrochloride, pH 7.2, containing a smallamount of DNase, incubated at room temperature untilviscosity was reduced, and centrifuged at 10,000 x g for 10min at 4°C. All fractions were assayed for protease activity,using azocasein, as described by Jensen et al. (15) and forRNase activity as outlined in the Worthington EnzymeManual (Worthington Diagnostics, Freehold, N.J.) exceptthat the reactions were carried out in 0.1 M potassiumphosphate buffer (pH 7.0) for up to 30 min and reactionswere terminated by the addition of trichloroacetic acid (5%[wt/vol] final concentration).

Chemicals. BHI agar, BHI broth, and antibiotic disks werefrom Oxoid Ltd., Basingstoke, England. Antibiotics, EDTA,Tris, and azocasein were obtained from Sigma ChemicalCo., St. Louis, Mo. The SDS-PAGE reagents were fromBio-Rad Laboratories, Richmond, Calif., except for SDSand glycine Analar R, which were purchased from BritishDrug Houses, Poole, England. The low-molecular-weightelectrophoresis calibration kit was from P-L Biochemicals,Inc., Dorval, Canada. DNase I and lysozyme were pur-chased from Boehringer Mannheim Biochemicals, Dorval,Canada.

RESULTSAntibiotic susceptibilities of natural isolates. The wild-type

strains of A. salmonicida displayed similar susceptibilities tomost of the antibiotics tested (Table 1). However, severalnatural isolates showed significantly reduced susceptibilitiesto oxytetracyline, sulfadiazine, and streptomycin sulfate,and one natural isolate demonstrated reduced susceptibilityto the quinolone group (Table 1). Antibiotics such as oxy-tetracycline and, more recently, flumequine and oxolinicacid were used on several occasions to treat furunculosisoutbreaks in fish at the facilities from which we obtainedmost of these latter strains.

Loss of exoprotease activity and acquisition of multiplelow-level antibiotic resistance. Exoprotease-negative (Prt-)mutants were obtained from virulent wild-type strains andfrom avirulent A. salmonicida ATCC 14174. All Prt- mu-tants isolated from virulent wild-type strains were found toexhibit decreased susceptibilities to a wide range of antibi-otics when compared with their parent strains (Table 2). Afour- to eightfold decrease in susceptibility occurred in thesePrt- mutants toward P-lactams (exemplified by ampicillinand cloxacillin), chloramphenicol, flumenquine, nalidixicacid, novobiocin, oxolinic acid, oxytetracycline, and tetra-cycline.

Antibiotic-resistant mutants were isolated from the wild-type strains and examined for loss of exoprotease activityand acquisition of multiple low-level resistance. Antibiotic-resistant colonies selected on BHI agar plates containingflumequine, chloramphenicol, or ampicillin appeared at fre-quencies of 0.7 x 10-7 to 7 x 1O-7. Greater than 65% of theantibiotic-resistant clones tested were Prt- (e.g., 221 of 308colonies tested from plates containing flumequine). Regard-less of the parent strain or the selective antibiotic, all Prt-antibiotic-resistant mutants exhibited low-level (four- toeightfold) resistance to the same set of drugs. In contrast,antibiotic-resistant Prt+ mutants showed increased resist-ance only to the specific antibiotic (or group of relatedantibiotics) used for their selection.

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TABLE 2. Properties and MICs of antibiotics for A. salmonicida strainsMIC (>g of antibiotic m1l-)

Strain proteasea A layerb Ampi- Chloram- Cloxa- Flume- Nalidixic Novo- Oxolinic Oxytetra- Tetra-cillin phenicol cillin quine acid biocin acid cycline cycline

Rest. 80204C + + 0.2 1 50 0.05 0.2 8 0.05 0.2 0.25Rest. 80204.4" - + 1.0 4 250 0.4 1.0 48 0.25 0.8 1.0Rest. 80204-A1' - + 1.0 4 250 0.4 1.0 48 0.25 0.8 1.0S-Rest. 80204f + - 0.2 1 50 0.05 0.2 8 0.05 0.2 0.25S-Rest. 80204-A1f - - 1.0 4 250 0.4 1.0 48 0.25 0.8 1.0AsFLC + + 0.2 1 50 0.05 0.2 8 0.05 0.2 0.25AsFL A1e _ + 1.0 4 250 0.4 1.0 48 0.25 0.8 1.0AsLC + + 0.2 1 50 0.05 0.2 8 0.05 25 15AsL-P1' - + 1.0 4 250 0.4 1.0 48 0.25 100 60ATCC14174C + - 0.2 1 50 0.05 0.2 8 0.05 0.2 0.25ATCC14174.59 - - 0.2 1 50 0.05 0.2 8 0.05 0.2 0.25

a Determined by plating on skim milk agar overlay plates.b Determined by transmission electron microscopy.c Sources of wild-type strains are given in Table 1.d Exoprotease-negative (Prt-) derivative from subculture of lyophilized culture of Rest. 80204 (9).' Prt- mutants selected in the presence of ampicillin (Rest. 80204-Al, AsFL-A1) or penicillin (AsL-P1).f A-layer-negative strains isolated after incubation at elevated temperature (14).g Spontaneously occurring Prt- derivative of ATCC 14174 (9).

Susceptibility of all Prt- mutants toward rifampin, eryth-romycin, polymyxin B, and streptomycin sulfate was notchanged when compared with the parent strains (data notshown). Antibiotic-resistant colonies (86 tested) isolatedfrom virulent wild-type strains by selection on plates con-taining low inhibitory levels of one of these antibiotics(rifampin) were all Prt+ and showed increased resistanceonly to the antibiotic used for selection.The one exception to the above observations was the

avirulent A. salmonicida ATCC 14174 strain. The Prt-derivatives (five tested) of this strain showed no significantchange in their susceptibilities to any of the test antibiotics(Table 2). Also, no antibiotic-resistant colonies selectedfrom the ATCC 14174 strain were exoprotease negative.A layer and antibiotic susceptibilities. All virulent wild-type

A. salmonicida isolates from diseased fish and the Prt-,multiple low-level resistance (mir) mutants isolated fromthem possessed an outer protein layer, the A layer (14, 28),as seen by transmission electron microscopy (Table 2).However, the A layer was absent from the avirulent ATCC14174 strain (14) and its Prt- derivatives.To determine whether the A layer was involved in the

differences observed between Prt- mlr mutants derived fromvirulent A' strains and Prt- mutants derived from the A-ATCC 14174 strain, A- mutants were obtained followingincubation at elevated temperature (14). No significant dif-ferences in antibiotic susceptibilities were found betweenvirulent A' Prt+ strains and their A- Prt+ derivatives orbetween A" Prt- mlr mutants and their A- Prt- mlr deriv-atives (Table 2). Furthermore, A- Prt- mutants subse-quently isolated from the A- Prt+ mutants were found toexhibit the multiple low-level antibiotic resistance.Outer membrane proteins of Prt- mlr mutants. Since

susceptibility of the Prt- mlr mutants was decreased towarda number of lower-molecular-mass antibiotics, several ofwhich are reported to have intrinsically lower penetrationrates in porin-deficient mutants of some other genera ofgram-negative bacteria (22), outer membrane proteins of themutants were examined.The outer membrane protein fractions from 6 wild-type

strains and 12 of their Prt- mlr derivatives were examined bySDS-PAGE (Fig. 1). The molecular masses of the major

outer membrane proteins from the wild-type strains werevery similar to those reported for two other strains of A.salmonicida by Nakajima et al. (20). However, a majorchange in the outer membrane protein profiles of all Prt- mlrmutants examined was evident when compared with the

12 4 6 "FIG. 1. SDS-PAGE of the outer membranes of A. salmonicida

wild-type and PrtF mir mutant strains: SS70 (lane 2), SS70-Cl (lane3), AsFL (lane 4), AsFL-Al (lane 5), AsB (lane 6), AsB-F1 (lane 7),AsL (lane 8), and AsL-P1 (lane 9). Lane 1 contained molecular massstandards (in daltons) including phosphorylase b (94,000), bovineserum albumin (67,000), ovalbumin (43,000), carbonic anhydrase(30,000), trypsin inhibitor (20,100), and a-lactalbumin (14,400).Sources of the strains are given in Tables 1 and 2 except forSS70-Cl, selected from SS70 in the presence of chloramphenicol,and AsB-F1, selected from AsB in the presence of flumequine.

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FIG. 2. SDS-PAGE of the outer membranes of A. salmonicidastrains: ATCC 14174 (lane 1), ATCC 14174.4 (lane 2), ATCC 14174.5(lane 3), S-Rest. 80204 (lane 4), S-Rest. 80204-Al (lane 5), and Rest.80204 (lane 6). Sources of the strains are given in Tables 1 and 2except ATCC 14174.4, which was a spontaneously occurring Prt-derivative of ATCC 14174.

profiles obtained for their parent strains. The wild-typestrains possessed a major protein band of approximately 38.5kilodaltons (kDa). In all Prt- mlr mutants, the 38.5-kDa bandwas missing or significantly reduced and a major proteinband of approximately 37 kDa was present which was notobserved in any appreciable quantity in the wild-typestrains. Outer membrane profiles from Prt- mlr mutants alsoshowed reduced amounts of a major protein of approxi-mately 48 kDA (Fig. 1). However, there was no majorprotein band at this location in any of the outer membraneprofiles from A- strains, whether they were Prt+ or Prt-(Fig. 2). The only Prt- mutants in which no significantchanges in the outer membrane protein profiles were ob-served, when compared with their parent strain, were thosemutants isolated from ATCC 14174 (Fig. 2), and they did notdisplay multiple low-level resistance.

DISCUSSIONMutants of A. salmonicida exhibiting a multiple low-level

antibiotic resistance could be isolated from all virulentwild-type strains exposed to low inhibitory concentrations ofany one of several low-molecular-mass antibiotics. Thesemutants were obtained at frequencies suggesting point mu-tations and were found to lack exoprotease activity. Con-versely, selection for loss of exoprotease activity resulted inthe concurrent appearance of multiple low-level resistance.

In the members of the Enterobacteriaceae, porin proteinsare known to regulate the outer membrane permeability oflow-molecular-mass solutes, including antibiotics (10, 21, 22,24), with an exclusion limit of approximately 550 to 650 Dafor saccharides (7, 19, 22). It has been reported that A.salmonicida possesses outer membrane proteins in the 35- to45 kDa size range which display charateristics similar toEscherichia coli porins (6). In the Prt- mlr mutants of A.salmonicida, decreased susceptibilities were observed to awide range of low-molecular-mass antibiotics (<635 Da)which have varied chemical structures and sites of action inor on the bacterial cell. The penetration of several of theseantibiotics through the outer membrane has been shown tobe affected in porin-deficient mutants of other gram-negativebacteria (22). Susceptibilities to several high-molecular-mass(>700 Da) hydrophobic or polycationic antibiotics, whichare thought to penetrate the outer membrane by othermechanisms (22), were not significantly affected in the A.salmonicida Prt- mlr mutants. Outer membrane proteinprofiles, as seen by SDS-PAGE, revealed that all Prt- mlrmutants were deficient in a major protein band of approxi-mately 38.5-kDa molecular mass and contained a majorprotein band of approximately 37 kDa which was not presentas a major protein in the wild-type strains. The reducedamounts of a protein band of approximately 48 kDa in A'Prt- mlr mutants would not appear to be involved in alteringantibiotic susceptibilities since no major 48-kDa proteinband was observed in the outer membrane protein profiles ofA- strains, including both A- Prt+ and A- Prt- mlr mutants.Also, antibiotic susceptibilities of A- mutants were notdifferent from their A+ parents. Although there are insuffi-cient data at present to specifically correlate the outermembrane protein changes in Prt- mlr mutants with de-creased susceptibility to antibiotics, it is tempting to specu-late such a relationship. Also, it is interesting to note that theonly exception observed was for the avirulent ATCC 14174strain. The Prt- mutants isolated from this strain did notdemonstrate any apparent change in their outer membraneproteins and did not demonstrate multiple low-level resist-ance.The relationship between the loss of exoprotease activity

and the acquisition of multiple low-level resistance in all Prt-mir mutants isolated from virulent wild-type strains is notapparent. Outer membrane proteins are known to be in-volved in the export of some proteins in gram-negativebacteria (11, 23). Also, exoprotease-deficient mutants of A.hydrophila (12) and Pseudomonas aeruginosa (8) which aredefective in enzyme export across the outer membrane showouter membrane protein changes with similarities to thoseobserved here for A. salmonicida Prt- mlr mutants. With A.hydrophila export mutants, active protease accumulates inthe periplasm (12), whereas with the P. aeruginosa exportmutant protease accumulates in the periplasm but in aninactive form (8). In A. salmonicida wild-type strains prote-ase activity was detected in significant amounts only in theextracellular culture fluid of BHI broth cultures (data notshown). In Prt- mutants, a situation similar to that with theP. aeruginosa export mutant may exist, since no significantprotease activity was found associated with any cell frac-tions. The relationships between outer membrane proteinchanges, decreased susceptibilities to antibiotics, and loss ofexoprotease activity in A. salmonicida Prt- mlr mutants arepresently being investigated.We have not as yet isolated Prt- mlr strains from natural

disease outbreaks, but continued use of antibiotics to treatfurunculosis outbreaks and remove the asymptomatic carrier

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stage of A. salmonicida from fish may in time lead toresistance by this mechanism.

ACKNOWLEDGMENTSThis work was supported by a grant from Supply and Services

Canada (to W. H. L.). R. N. M. was supported by a scholarship fromthe Natural Sciences and Engineering Research Council of Canada.

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