Characterization of Salmonella enteritidis Strains

C. Poppe, K.A. McFadden, A.M. Brouwer and W. Demczuk

ABSTRACT

A study was conducted to charac-terize 318 Salmonella enteritidisstrains that were mainly isolatedfrom poultry and their environmentin Canada. Biotype, phagetype (PT),plasmid profile (PP), hybridizationwith a plasmid-derived virulencesequence probe, antibiotic resis-tance, outer membrane proteins(OMPs), and lipopolysaccharide(LPS) profiles were determined.Relationships of these properties toone another, and their diagnosticand pathogenic significance wereassessed. Biotyping indicated thatfailure to ferment rhamnose was

sometimes useful as a marker forepidemiologically related strains.Phagetyping was the most effectivemethod for subdividing S. enteri-tidis; it distinguished 12 PTs. Phage-type 13 was occasionally associatedwith septicemia and mortality inchickens. The strains belonged to15 PPs. A 36 megadalton (MDa)plasmid was found in 97% of thestrains. Only the 36 MDa plasmidhybridized with the probe. Seven-teen percent of the strains weredrug resistant; all strains were sen-sitive to ciprofloxacin. Thirty-five of36 strains possessed the same OMPprofile, and 36 of 41 strains con-tained smooth LPS.

RESUME

Cette etude visait a caracteriser318 souches de Salmonella enteri-tidis isolees au Canada principale-ment a partir de volailles et de leurenvironnement. Le biotype, le typephagique (TP), le profil plas-midique (PP), I'hybridation avecune sonde genomique derivee de lasequence d'un plasmide de viru-lence, la resistance aux antibio-

tiques, les profils de proteines de lamembrane externe (PME) et lelipopolysaccharide (LPS) ont etedetermines. Les relations entre cesdifferentes caracteristiques, ainsique leur importance d'un point devue diagnostique et pathoge'niqueont ete evaluees. Le biotypage ademontre qu'une incapacite a fer-menter le rhamnose etait parfoisutile comme marqueur pour dessouches apparentees epidetmiologi-quement. Le typage phagique etaitla methode la plus efficace poursubdiviser S. enteritidis; 12 TP dif-ferents ont ete identifies. Le TP 13etait parfois associe a une sep-ticemie et de la mortalite chez despoulets. Les souches appartenaienta 15 PP differents. Un plasmide de36 megadalton (MDa) etait presentchez 97 % des souches. Le plasmidede 36 MDa etait le seul a hybrideravec la sonde. Dix-sept pour-centdes souches e'taient resistantes auxantibiotiques; toutes les souchesetaient sensibles au ciprofloxacin.Sur 36 souches, 35 possedaient lememe profil de PME, et 36 sur 41avaient un LPS lisse. (Traduit parDr Serge Messier)

INTRODUCTION

Infections of humans with Salmo-nella enteritidis have increased sig-nificantly during the last ten years,and in many European countries andthe United States S. enteritidis hasreplaced Salmonella typhimurium asthe most commonly isolated serovar(1-3). In the U.K, the U.S., andCanada, reported isolation rates ofS. enteritidis in humans were 53%,20%, and 9%, respectively, of allSalmonella spp. isolated in 1989(2,4,5). In Europe the increase inS. enteritidis infections in humans andanimals has been largely due to

S. enteritidis phagetype (PT) 4 strains(1,6,7) but in the U.S. the increasewas mainly caused by PT 8 and 13a(8-10). In Canada 70% of the S. enter-itidis isolates from humans were PT 8(5). In the U.K. there has been a com-

parable increase in isolations ofS. enteritidis from poultry (4). Similarto the S. enteritidis isolates from peo-ple in the U.K., 71% of the S. enteri-tidis isolates from poultry were PT 4(4). Salmonella enteritidis may infecteggs by transovarian transmission(11), and outbreaks of disease byS. enteritidis in humans have beenassociated with the consumption ofeggs or foods that contain eggs(12,13).A high percentage of S. enteritidis

strains contain a serovar-specific36 MDa plasmid that increases thevirulence of the strains for mice (14).Salmonella choleraesuis, S. dublin,S. gallinarum, S. pullorum, andS. typhimurium also often harbor alarge plasmid that encodes virulencefor mice or chicks (15,16). Virulence-associated plasmids of differentSalmonella serovars possess similargene sequences but the mechanismsby which these genes contribute tovirulence are not fully understood(16-18). Because virulence plasmidshave been detected in nearly 100% ofstrains isolated from animal organs orhuman blood, but only in 48 to 87% instrains of fecal, food or environmentalorigin, it has been suggested thatSalmonella virulence plasmids maybe required for systemic infections inhumans and livestock (18). However,other studies have shown that viru-lence of Salmonella strains encodedby a virulence plasmid is onlyexpressed in Balb/c and other suscep-tible strains of mice (19). Also, thereis a lack of correlation among Salmo-nella strains with respect to virulencefor different species of animals (20).Although typing by plasmid profile is

Can J Vet Res 1993; 57: 176-184

Health of Animals Laboratory, Agriculture Canada, 110 Stone Road West, Guelph, Ontario N 1 G 3W4.

Submitted December 21, 1992.

176

not as discriminatory as phagetypingfor the primary subdivision ofS. enteritidis strains, different profileshave been found within commonlyoccurring PTs, and it may serve as aneffective adjunct to phagetyping forsubdivision and epidemiological trac-ing of S. enteritidis strains (21).

Salmonella enteritidis strains have ahigh degree of genomic homogeneity(22). When examined by multilocusenzyme electrophoresis, 95% or morebelong to the same genotype (23,24).The use of probes consisting of aninsertion sequence unique to Salmo-nella (25) and of a cloned rRNAoperon derived from Escherichia coli(26) revealed a limited degree of chro-mosomal differences that appear to beless discriminatory than phagetyping(27). Resistance of Salmonella strainsto antimicrobial substances has com-monly been used to subdivide Salmo-nella serovars and for epidemiologicalpurposes. Drug resistance may berelated to plasmid(s) harbored bySalmonella strains (28) and possessionof plasmids encoding drug resistancehas been associated with a change inPT of a S. enteritidis strain (29). Theouter membrane protein (OMP) pro-files of S. enteritidis strains have alsobeen found to vary occasionally inconjunction with possession of plas-mids (15). The length of the lipo-polysaccharide (LPS) side-chain andthe composition of LPS of Salmonellahave been shown to influence viru-lence of the strain. A S. dublin strain,cured of its virulence plasmid, exhib-ited a shorter 0-specific side-chainand a different composition of sugarsin its LPS than its parent, and thechain length was restored by reintro-duction of the virulence plasmid(30,31). Alteration of LPS by curingand reintroduction of the virulenceplasmid was not observed with otherS. dublin strains, or with S. enteritidis,S. choleraesuis, S. typhimurium andtheir derivative strains (31). Further-more, it has been shown that conver-sion of S. enteritidis PT 4 to PT 7involved loss of long-chain LPS, andloss of virulence for intraperitoneallyinoculated Balb/c mice (32).There is a paucity of information

about (i) the reason for the increasedprevalence of S. enteritidis in peopleand animals; (ii) the possibility that

the increased occurrence of S. enteri-tidis infection may be due to a changein characteristics of the pathogen; and(iii) how traits of S. enteritidis relateto one another. The purpose of thisstudy was (i) to characterize S. enteri-tidis strains with respect to PT, bio-type, resistance to antimicrobialagents, PP, hybridization of plasmidswith a probe taken from the virulencesequence of the 60 MDa plasmid ofS. typhimurium, OMP profiles, andLPS profiles, (ii) to determine howthese characteristics relate to oneanother, and (iii) to examine if thesetraits might have additional diagnosticvalue or might explain pathogenic orepidemiological findings.

MATERIALS AND METHODS

BACTERIAL STRAINS

A total of 318 S. enteritidis strainsderived from 77 different sourceswere characterized. Twenty-two sub-missions consisting of 232 S. enteri-tidis strains were isolated in our labo-ratory while conducting Canada-widesurveys for the prevalence of S. enter-itidis in layers and broilers (33,34);the other strains were sent to us forserotyping and/or phagetyping. Ninesubmissions were of foreign origin:two submissions consisted of isolatesfrom fecal samples of visitors toSpain, one submission was a humanisolate from Jamaica, one submissionwas an isolate from a broiler carcassimported from the U.S., one submis-sion consisted of two isolates fromone-day-old poults imported from aprimary breeder in the U.S., threesubmissions were poultry isolatesreceived from Dr. J.F. Timoney,Cornell University, Ithaca, New York,and one submission consisted of fivestrains that were isolated from breederflocks in Venezuela. Another 46 sub-missions were of Canadian origin.Three hundred and eleven of the318 strains were isolated during theyears 1988-1991. The other sevenstrains were isolated in 1985 and1986.

BIOTYPING

All strains were biotyped by deter-mining 30 biochemical tests withGram-Negative Identification (GNI)

cards and the automated diagnosticbacteriology system of Vitek Systems,Hazelwood, Missouri (Table I).

SEROTYPING

The 0 or somatic antigens of Sal-monella isolates were determinedwith slide agglutination tests asdescribed by Ewing (35) whereas theH or flagellar antigens were identifiedby using a microtechnique (36) thatemploys microtiter plates. The anti-genic formulas of Salmonella serovarsas listed by Le Minor and Popoff (37)were used to name the serovars.

PHAGETYPING OF S. ENTERITIDISSTRAINS

Salmonella enteritidis strains werephagetyped with typing phagesobtained from the Division of EntericPathogens at the Central Public HealthLaboratory in London, England (38).

PLASMID DNA PROFILES

Plasmid DNA was prepared andgels were run, stained, and pho-tographed as described previously(33).

HYBRIDIZATION OF PLASMIDS WITHA PROBE OF VIRULENCE GENES

Plasmid DNA of strains with differ-ent PPs, different PTs, or of strainsisolated from different sources withina submission was hybridized with a32P-labelled DNA probe consisting ofa 3750 base pairs (bp) HindIll-Hindlll fragment (17) that wasderived from the virulence region ofthe 60 MDa plasmid of S. typhimu-rium. Preparation of plasmid DNA,digestion of DNA with restrictionendonuclease, nick translation, label-ling with 32P, and hybridization ofplasmid DNA on nitrocellulose mem-branes with the labelled probe werecarried out as described previously(17).

RESISTANCE TO ANTIMICROBIALAGENTS

Antibiotic-susceptibility tests weredetermined with the Repliscan system(39) (Automed, Shoreview, Minne-sota) and the following drugs: ami-kacin (Amk) and kanamycin (Kan)both at 16, 32 and 64 ,ug/mL; ampi-cillin (Amp) at 2, 16 and 64 [ig/mL;carbenicillin (Car) at 32, 128 and256 ,ug/mL; cephalothin (Clt) at 8, 16

177

TABLE I. Biochemical tests performed to determine biotype

BiotypeDescription of test A B C D E F

Glucose fermentation in the presence of2,4,4'-trichloro-2'-hydroxy-diphenyletherAerobic acid production by glucose oxidationGrowth in buffered peptone water containing tryptophanUtilization of acetamideHydrolysis of esculinEnzymatic activity of indoxyl-beta-D-glucosideProduction of urease

Utilization of citrate as sole carbon source

Utilization of malonate as carbon source

Production of tryptophan deaminaseGrowth in the presence of polymyxin BLactose oxidationMaltose oxidationMannitol oxidationXylose oxidationFermentation of raffinoseFermentation of sorbitolFermentation of sucroseFermentation of inositolFermentation of adonitolFermentation of glucose in the presence ofp-coumaric acidProduction of hydrogen sulfide from thiosulfateHydrolysis of o-nitrophenyl-beta-D-galactopyranosideFermentation of rhamnoseFermentation of L-arabinoseFermentation of glucoseProduction of arginine dihydrolaseProduction of lysine decarboxylaseProduction of ornithine decarboxylaseProduction of oxidase

+ + + + +

S. thompson, and of two strains ofeach of S. heidelberg and S. typhi-murium were determined. The LPSprofile of reference strains S. typhimu-rium LB5010 (47), a galE strain,S. typhimurium SA2703 with a TnJOinsertion proximal to the rfa cluster,and S. typhimurium SA3748, an rfaImutant (48) (all obtained fromDr. K.E. Sanderson, Salmonella Gene-tic Stock Centre (SGSC), Calgary,Alberta) were examined as well.

ANALYSIS OF OMP AND LPS BYSDS-PAGE

+ + + + + + The OMP and LPS contents of+ + + + + + Salmonella and reference strains were- - - - - - analysed with a discontinuous SDS-+ + + + + + PAGE system (49). For OMP a 4%

stacking gel and a 10% separating gelcontaining 4 M urea was used,

+ + + - + + whereas a 4% stacking gel and a 14%separating gel containing 4 M urea

+ + + + + + was used for LPS. The molecular

+ + _ + + + weights of the OMPs were estimated+ + + + + + from calibration curves prepared with+ + + + + + molecular weight standards. The

OMP gels were stained with+ + + + + + Coomassie blue (45), whereas the

LPS gels were silver-stained by themethod of Tsai and Frasch (50).

and 256 ,ug/mL; chloramphenicol(Chl) at 8, 16 and 64 ,ug/mL; cipro-floxacin (Cip) at 1 and 2 jig/mL;cotrimoxazole (Cot) at 10, 40 and160 Vig/mL; gentamicin (Gen), andneomycin (Neo) at 4, 8 and16 ,ug/mL; nitrofurantoin (Nit) at64 ,ug/mL; polymyxin B (Pol) at 2, 8and 16 V±g/mL; spectinomycin (Spc)at 16 jiRg/mL; sulfisoxazole (Sul) at256 ,ug/mL; and tetracycline (Tet) at2, 8 and 128 ,ug/mL.

ELIMINATION OF PLASMIDS

Curing of plasmid(s) was by incu-bation of the plasmid-containingstrain at 45°C for two to seven days(40). Alternatively, to eliminate largeplasmids the recipient strain was con-

verted to a nalidixic acid resistant(NalR) strain and a triple-cross matingwas conducted by conjugation of therecipient strain with S. typhimuriumKR1561 (which contains pLL6, a

nonselftransmissible Kan-resistance(KanR) encoding plasmid) andS. typhimurium SA3269 which con-

tains an F'lac plasmid that may trans-fer nontransmissible plasmids (41).

CONJUGATION OF STRAINS

Conjugation of strains to determineif plasmids were selftransmissible andencoded antibiotic resistance was car-

ried out as described by Curtiss (42).The donor S. enteritidis strain was

mated with a recipient NalR E. coliC600 strain (43).

OUTER MEMBRANE PROTEINPREPARATIONS

Outer membrane fractions were

prepared by the methods of Achtmanet al (44) and Davies (45).

LIPOPOLYSACCHARIDE PREPARATIONS

Lipopolysaccharide was preparedby the method of Hitchcock andBrown (46). The method was slightlyaltered in that the lysates were heatedat 100°C for 90 min, and the boiledlysates were incubated with pro-teinase K (Boehringer Mannheim) at60°C for 2 h. To compare the LPSprofiles of the S. enteritidis strainswith the patterns of other strains, theLPS profiles of one strain of each ofS. choleraesuis, S. dublin, S. hadar,S. infantis, S. schwarzengrund, and

RESULTS

BIOTYPING

A total of six biotypes, denotedA-F, were identified. The characteris-tics of these biotypes are shown inTable I. Biotype A was commonestwith 302 isolates, one strain belongedto biotype B, eight strains to bio-type C, four strains to biotype D, twostrains to biotype E, and one strainbelonged to biotype F. The less com-

monly occurring biotypes could occa-

sionally be related to the source of thestrains. Two of four strains from sub-mission 4 were of biotype E. Twostrains from submission 8 were ofbiotype D (one was isolated from theoviduct of a hen and the other fromthe cecal contents of another hen).The eight strains of biotype C (whichdid not ferment rhamnose) were iso-lated from the same production chain:five of them were isolated on differentoccasions in 1989 and 1990 frompoults and the environment of a

turkey breeder, two from one-day-oldpoults of the primary breeder that

178

23456789101112131415161718192021

222324252627282930

I

supplied the turkey breeder, and onestrain from another turkey breederflock supplied by the same primarybreeder. The biotype C strainsbelonged to five different PTs (threestrains of PT 8, one strain of PT 8a,two strains of PT 13, one strain ofPT 23, and one untypable strain) andcontained a 36 MDa plasmid (fivestrains), a 16 MDa plasmid (onestrain) or no plasmids (two strains).The only isolate from the egg contentsof submission 8 was a biotype Fstrain. A small-colony variant of abiotype A isolate from submission 5was a biotype D strain. The biotypescould not be related to any other char-acteristics of the S. enteritidis strains.

PHAGETYPING

The number of strains, the numberof submissions, and the number ofsubmissions of Canadian origin thatbelonged to different PTs or combina-tions of PTs are shown in Table II.Most of the strains belonged to PT 13and PT 8 (Table II). Eleven differentPTs and one untypable group ofstrains were identified. The number ofstrains belonging to PT 13 is highbecause 107 strains, all of which con-sisted of PT 13 strains, were derivedfrom one submission obtained from afarm with layer flocks that wereinfected with S. enteritidis and repeat-edly sampled. The number of submis-sions of strains belonging to differentPTs of S. enteritidis shows that PT 8was more often the cause of contami-nation or disease than PT 13 (44 vs. 17submissions, respectively) (Table II).Exclusion of the nine foreign submis-sions shows that PT 8 was the mostcommon and PT 13 the second mostcommon PT among submissions fromCanadian sources (Table II).Only two PT 4 strains were

obtained from Canadian sources: theywere isolated from scrapings of egg-belts from one layer flock. Twostrains were identified as PT 8a. ThisPT designation was given by theDepartment of Enteric Pathogens atthe Central Public Health Laboratory,Colindale, London, England, tostrains that show a typing pattern asPT 8 strains (38) but that do not reactwith typing phages 4 and 6. The PT 6strains were of Spanish origin,whereas the PT 6a strains were iso-lates from Venezuela.

TABLE II. Number of strains, submissions, and submissions of Canadian origin belonging todifferent PTs, or combinations of PTs

Canadian submissionsPhage types No. of strains No. of submissions No. of submiss. % of submiss.

1 1 1 1 1.54+6a 2+3a 14+8 2+5 1 1 1.56 2 28 104 44 38 55.9

8+8a+23+ 28+1+1+1 1 1 1.5untypable

8+13a 1+2 1 1 1.58a 1 1 1 1.5llb 1 1 1 1.513 148 17 17 25.013a 11 3 3 4.423 2 2 2 2.928 1 1 1 1.5

untypable 1 1 1 1.5Total 318 77 68 100.0

aTwo strains were PT 4 and 3 strains were PT 6a

The PTs were related to the sourceof the strains. Twenty-five of the68 Canadian submissions consisted ofmore than one strain, and the strainsof each of 22 of these 25 submissionsall belonged to the same PT. Theexceptions were three submissions:one of which contained strains ofPT 8, PT 8a, PT 23, and an untypablestrain, another submission which con-tained strains of PT 4 and PT 8, and athird submission which containedstrains of PT 8 and PT 13a (Table II).

In this study, no clear link could beestablished between PT and epidemi-ological evidence of virulence ofstrains. Phagetype 8 and 6a strainswere isolated from people with symp-toms such as diarrhea, abdominalcramps, fever, and malaise. A PT 8strain was associated with persistentinfection in one quarter of the udderand the milk of a cow. In poultry how-ever, PT 8 strains were isolated frombroilers and hens without any signs ofdisease. Phagetype 13 was occasion-ally associated with disease in chick-ens. It was isolated from heart bloodof chickens with septicemia and amortality of 3.7% of the flock at tendays of age, from the yolk sacs ofchickens of a flock with high mortal-ity at two days of age, and from henswith signs of septicemia at 62 weeksof age. However, PT 13 was also iso-lated from the organs or cecal con-tents of layers that did not show anysigns of disease (51).Twelve of 13 PT 13a strains of four

submissions contained a 36 MDa and5.0, 4.0, 3.0 or 2.6 MDa plasmid(s).

However, the possession of a 36 MDaplasmid and a small plasmid (lessthan 20 MDa) was not exclusively acharacteristic of PT 13a strainsbecause 4 of 138 strains of PT 8, 3 of148 strains of PT 13, and strains ofother PTs of S. enteritidis also con-tained such plasmids (Table III).Three of five Venezuelan isolates

contained 60, 36 and 2.4 MDa plas-mids and were PT6a, whereas theother two strains contained 36 and2.4 MDa plasmids and were PT4.Transfer studies demonstrated that the60 MDa plasmid was not related tothe PT of the strains.

PLASMID PROFILES

The 318 strains of the collectioncould be subdivided into 15 differentPPs (Table III). Of the total318 strains 281 (88.4%) contained asingle 36 MDa plasmid, 27 strains(8.5%) contained a 36 MDa and oneor more other plasmids, eight strainscontained no plasmids, one strain con-tained a single 16 MDa plasmid, andone strain harbored a single 2.6 MDaplasmid. Thus, a total of 308 (96.9%)contained a 36 MDa plasmid. Twohundred and seventy-three of the 281strains that harbored a single 36 MDaplasmid belonged to only two PTs,namely PT 8 and PT 13.The PPs of strains could occasion-

ally be related to the source of thestrains, but because of the ubiquitouspresence of a single 36 MDa plasmidthis was not a common occurrence.One hundred and sixteen strains of54 submissions all contained a single

179

TABLE III. Relationship between phagetype and plasmid profile of 318 S. enteritidis strains

Plasmid Molecular mass Number of strains of the following phagetypeprofile in MDa 1 4 6 6a 8 8a 1lb 13 13a 23 28 untypable

1 60365.03.0 - - - 12 60362.4 33 36 30 24 3616 - 25 36 5.0 5 16 36 4.0 37 363.2 18 36 2.6 - 39 362.4 2 110 362.2 - 1 111 36 2.2 1.4 - 1 -12 36 1 2 - 129 1 144 2 213 16 - 1

14 2.6 - 115 none 5 1 1 1

Totals: 1 + 4 + 2 + 3 + 138 + 2 + 1 + 148 + 13 + 3 + 1 + 2 =318

36 MDa plasmid. Examples of strainswhere the PP appeared related to thesource are: three strains of submission2 contained 36 and 2.6 MDa plasmids,whereas the fourth strain containedonly a 2.6 MDa plasmid; five of sixisolates of submission 21 contained36 and 5 MDa plasmids, whereas thesixth strain contained plasmids with amolecular size of 60, 36, 5 and3 MDa; three of five strains of sub-mission 68 contained 60, 36 and2.3 MDa plasmids, whereas the othertwo strains contained 36 and 2.3 MDaplasmids; and two of three isolates ofsubmission 77 harbored plasmids withmolecular sizes of 36 and 4 MDa,whereas the third strain contained asingle 36 MDa plasmid. The absenceof plasmids was also characteristic forS. enteritidis strains belonging to onesubmission; four of five strains ofsubmission 20 did not harbor anyplasmids, while the fifth strain con-tained a single 36 MDa plasmid.

HYBRIDIZATION STUDIES

Hybridization of plasmid DNA witha probe derived from the virulencesequence of the 60 MDa plasmid ofS. typhimurium was performed onplasmid DNA of isolates that within asubmission differed in PP, in PT, or inorgan or source from which the strainwas isolated, and on plasmid DNA ofall single isolates (one isolate per sub-mission). A total of 194 strains wereexamined: 170 strains that contained asingle 36 MDa, 23 strains harboringother plasmids besides the 36 MDaplasmid, and one strain that contained

a single 16 MDa plasmid. Only the36 MDa plasmids hybridized with theprobe. Although six strains that wereassociated with clinical disease in ani-mals all contained a single 36 MDaplasmid that hybridized with theprobe, such strains were also com-monly isolated from feces, cecal con-tents, internal organs, and the envi-ronment of clinically healthychickens.

ANTIBIOTIC RESISTANCE

Fifty-four of 318 (17%) strains wereresistant to one or more of the antimi-crobial agents employed in the drugtesting panel (Table IV). Resistance tosulfisoxazole was most common(23 strains), followed by resistance tonitrofurantoin (15 strains). Ten of the54 strains showed drug resistance toone or more antibiotics and/or showedintermediate resistance to one or moreother antibiotics. An additional sixstrains showed intermediate resistanceto one (five strains) or three antibi-otics (one strain). All the strains weresensitive to ciprofloxacin, cotrimoxa-zole, and kanamycin at all levelsemployed. Strain SE218 was the onlyisolate of submission 21 that con-tained in addition to 36 and 5 MDaplasmids, a 60 and a 3 MDa plasmid.The strain was AmpR and GenR. Whenthe strain was cured of the 60 MDaplasmid it became Gens but remainedAmpR. When strain SE218 was conju-gated with NalR E. coli C600, theE. coli strain became GenR. The trans-conjugants contained the 60 MDaplasmid, and the 5 and 3 MDa plas-

mid. The latter two plasmids cotrans-ferred with the selftransmissible60 MDa plasmid. No other relation-ship between selftransmissible plas-mids and drug resistance was found.

OUTER MEMBRANE PROTEINS

Salmonella enteritidis strains ofdifferent submissions, strains of dif-ferent PTs within or between submis-sions, strains with different PPs,strains isolated from cecal contents,feces, or from extraintestinal sourcessuch as ovaries, oviduct, and spleen,and strains from the contents of eggsand from environmental samples wereexamined to determine the OMPs theypossessed (Table V). The OMPs ofthe S. enteritidis strains were com-pared with those of other Salmonellaserovars and an E. coli and aCitrobacter freundii strain. Thirty-five of 318 S. enteritidis strains fromour collection, the S. enteritidis PT 4phagetyping reference strain, and aS. choleraesuis strain containedOMP's of 42, 40 and 37 kilodalton(kDa) (Table V). One of the S. enteri-tidis strains of our collection differedfrom the other S. enteritidis strains inthat the OMPs had molecular sizes of42 and 37 kDa (Table V and Fig. 1).This strain was rather unusual in thatit was the only strain that belonged toPT 1 lb, and it did not possess anyplasmids. Two S. typhimurium strainsand two S. heidelberg strains showedthe same OMPs of 40.5, 40 and37 kDa. The OMP patterns obtainedwith other reference strains are shownin Table V.

180

TABLE IV. Resistance of Salmonella enteritidis (SE) strains to antimicrobial agents

AMK64 AMP64AMP64AMP64AMP64AMP64AMP64

Antimicrobial agentsa and concentration in ,ug/mL agar at which growth occurredCAR256CAR256 CHL16bCAR256 CLT16CAR256 CLT256CAR256 CLT16 GEN16CAR256 CLT16

CLT16CLT16CLT16

NIT64

NEO8NEO8NEO16

NIT64NIT64NIT64

SUL256SUL256

POL8

TET8

TET8

SUL256SUL256

SUL256

POL8SUL256

TET8

1 TET8aAMK = Amikacin; AMP = Ampicillin; CAR = Carbenicillin; CHL = Chloramphenicol; CLT = Cephalothin; GEN = Gentamicin; NEO = Neomycin;NIT = Nitrofurantoin; POL = Polymyxin B; SUL = Sulfisoxazole; TET = TetracyclinebLevels of drug concentration in ,ug/mL that were intermediate between resistant and sensitive are underlined

LIPOPOLYSACCHARIDES

The LPS profiles were determinedfor S. enteritidis strains that variedwith respect to presence and size ofplasmids, PT, submission numberand/or source. Thirty-six of 41S. enteritidis strains of our S. enteri-tidis collection contained smoothLPS. The S. enteritidis PT 4 referencestrain from the Central Public HealthLaboratory in Colindale, London,U.K. also possessed smooth LPS.Three S. enteritidis strains of PT 23contained rough LPS, two of thosestrains harbored a 36 MDa plasmid,whereas one contained a 16 MDaplasmid. The other two S. enteritidisstrains with rough LPS were one PT13 strain that harbored a 36 MDaplasmid, and one untypable strain thatdid not contain any plasmids.To compare the LPS profiles of the

S. enteritidis strains with the patternsof other strains, the LPS profiles ofone strain of each of S. choleraesuis,S. dublin, S. hadar, S. infantis,S. schwarzengrund, and S. thompson,and of two strains of each of S. heidel-berg and S. typhimurium were deter-mined. The LPS of all these strainswas smooth. The reference strainsS. typhimurium LB5010, a galEmutant, and S. typhimurium SA2703with a TnJO insertion proximal to therfa cluster, possessed smooth LPS inthe outer membrane, whereas S. typh-imurium SA3748, an rfal mutant, con-

tained rough LPS.

TABLE V. Outer membrane protein (OMP) profiles of S. enteritidis and reference strains

No. ofstrains Genus/Specie

35 S. enteritidis1 S. enteritidisI S. enteritidisI S. choleraesuis1 S. hadar1 S. schwarzengri1 S. thompson1 S. dublinI S. infantis2 S. heidelberg

2 S. typhimurium

1 C. freundii1 E. coli

s

und

Source

H.A.L., GuelphColindale, LondonH.A.L., Guelph

it to

if of

.. It

of ..

It ..

of It

.. to

Strainidentif.a

SE coll.bPT 4SE28588/11SA901912SA901898SA89000288/8SA901901SA901879SE221

SA901942DT433

A.T.C.C., RockvilleSA90 169925922

42424242414141414140.5

OMP's (in kDa)40 3740 37

3740 3740 3740 3740 3738.5 3738 37

5 40 37

40.5 40 37

4039.5 37 36

aStrain identificationbStrains from the S. enteritidis collection

DISCUSSION

Biotyping of the S. enteritidisstrains showed that all eight biotypeC strains that did not ferment rham-nose were isolated from the same

turkey breeder line. The biochemicalcharacteristics of these strains did notcoincide with other characteristics,such as PTs and PPs; the other charac-teristics differed markedly among thebiotype C strains. These findings sug-gest that biotype C strains that do notferment rhamnose possess a stablemarker that is not determined by plas-mids or varies with PT, and that maybe applied for epidemiological pur-poses. The usefulness of biotyping in

studying the epidemiology of Salmo-nella infection has previously beenshown for S. typhimurium, S. para-

typhiB, and other Salmonella serovars

(52).A concomitant rise of a PT or PTs

in poultry and people, which impliesthat poultry and poultry products are

often the source of infection inhumans, has been observed in theU.K. and the U.S. (5,9,53). Some ofthe differences between isolation ratesof PTs may be ,udue to a PT beingidentified in large numbers in an out-break or region as occurred withPT 13 that was identified in a largeoutbreak in people in BritishColumbia, involving cream-filled

181

No. of SEstrains

2131222

115211

23

S......

Fig. 1. Outer membrane protein profiles of S. enteritidis serovars. Molecular weight sl(Biorad) were phosphorylase B (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbikDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa) (Lane1, 3, 4, 5 and 6 are the profiles of S. enteritidis strains SE184, SE285, SE276, SESE057, respectively.

cakes (5) and with PT 14b that wascommonly isolated from poultry andtheir environment in Maine, but notelsewhere in the country (9,54).However, as in the study of Khakhriaet al (5) on the prevalence of the dif-ferent PTs isolated from human andanimal sources in Canada, also in thepresent study of strains mainlyderived from poultry and their envi-ronment in Canada, PT 8 and PT 13rated first and second in percentage ofunrelated submissions (55.9% and25%, respectively) (Table II).

Salmonella enteritidis PT 13 occa-sionally was associated with consider-able mortality in young chicks andsepticemia in hens. Recently, thesame PT was associated with 95 casesof human infection in a hospital out-break in Owen Sound, Ontario (55). Itappears therefore that not only PT 4(6,56), and PT 8 (13), but also PT 13may cause significant clinical diseasein susceptible animal and humanhosts. Twelve of 13 PT 13a strainsharbored the 36 MDa plasmid plussmall plasmids with a size of 2.6 to5.0 MDa. Similar observations weremade by Singer et al (54) whoreported that all 52 S. enteritidisPT 13a strains isolated mainly fromthe environment and organs of poultryin Maine, and also from the samesources outside Maine, contained a40.3 and a 3.0 MDa plasmid, and byRodrique et al (53) who found in 38of 48 PT 13a strains a 36 and a

3.1 MDa plasmid, and in eightten remaining PT 13a str36 MDa and one or two smalmids with molecular weights o10 MDa. Thus, possession of36 MDa and one or two sma]mids appears to be typical forstrains. It is however not uni(PT 13a strains because a few FPT 13 strains of our collectihcontained a 36 MDa and aplasmid.

Possession of a single 36 MEmid was very common amoS. enteritidis strains and occu281 (88.4%) of the 318(Table III). Almost all of these(273) belonged to only two PTpossession of a single 36 MDmid is not a very discriminatoture of S. enteritidis strains andsis of PPs alone would be ofvalue. Phagetyping subdiviclarge group of strains with a36 MDa plasmid into twogroups and is therefore more dinatory in dividing the strainsPP analysis. To maintain the atdistinguish between S. entestrains, both PT and PP shodetermined (21,57).The hybridization studies s

that only the 36 MDa plasiS. enteritidis strains hybridizedprobe that was derived from thlence region of the 60 MDa pof S. typhimurium and confirmvious observations (17). Hyb

tion of genomic DNA with the inser-tion sequence IS200 was not carried

kD out because the method divides5 kD strains into groups of one or more PTs2 (4, 8 and 13a, 11 and 15) (25) and the

273 strains of our collection that0 belonged to PTs 8 and 13 would likely7 not have been further subdivided.

Only 17% of the S. enteritidisstrains were resistant to antimicrobialsubstances. This is similar to the per-centages of antibiotic resistances ofsporadic, outbreak, and animalS. enteritidis strains (23%, 14%, and24%, respectively) isolated in theU.S. (53), but higher than the lowlevel of 4.5% of antibiotic resistant

tandards human S. enteritidis isolates in Spain

2). L(ne (58). All the strains were susceptibleP273 and to the effect of ciprofloxacin,

cotrimoxazole, and kanamycin.Ciprofloxacin is one of the fluoro-

tof the quinolones which have been found to

ains a be more effective in treatment of

11 plas- acute, uncomplicated enterocolitisIf 1.4 to caused by Salmonella infection thana large ampicillin and trimethoprim-11 plas- sulfamethoxazole; the quinolonesPT 13a shorten the duration of clinical symp-que for toms and terminate the excretion ofT 8 and organisms in the feces, and becausean also they eliminate Salmonella fromsmall phagocytes, they are effective in

immunologically incompetent hosts)a plas- (59).ing the The OMP profiles of 35 of 36 strainsrred in of S. enteritidis were the same in thatstrains all the strains contained OMPs of 42,strains 40 and 37 kDa. However, one of the

s. Thus S. enteritidis strains of our collectionla plas- differed from the others in that thery fea- OMPs had molecular sizes of 42 and[analy- 37 kDa. This strain was a ratherlimited unusual strain in that it was the onlyles the strain that belonged to PT 1 lb, and itsingle did not possess any plasmids. Thesemajor findings show similarity to those of

iscrimi- Helmuth et al (15) who reported thatthan is one OMP profile was characteristic of)ility to 30 plasmid-containing and one?ritidis plasmid-free strain, whereas anotherould be pattern was observed for two plasmid-

free strains.,howed Thirty-six of 41 S. enteritidismid of strains contained smooth LPS: on sil-with a ver staining the LPS showed typical

ie viru- ladder-type patterns which extended)lasmid from the bottom to the top part of theLed pre- gel. In five strains only core LPS was)ridiza- present; three of those strains were PT

182

23 strains, one strain was a PT 13strain that harbored a 36 MDa plas-mid, and one strain was an untypablestrain. It is remarkable that all threePT 23 strains were rough.

In summary, the major findings ofthis study were i) phagetypingappeared to be the most discrimina-tory method for subdividing S. enteri-tidis strains, ii) PT 13 strains wereoccasionally associated with sep-ticemia and mortality in chickens,iii) the 36 MDa plasmid was ubiqui-tous among S. enteritidis strains, iv)failure to ferment rhamnose, althoughuncommon, may be an important epi-demiological marker of S. enteritidisstrains, and v) all 318 S. enteritidisstrains were susceptible to the antimi-crobial effects of ciprofloxacin,co-trimoxazole, and kanamycin.

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