Comparison of epidemiological markers of Salmonella strains isolated from different sources in Spain

0095-1137/92/123058-07$02.00/0

Copyright X 1992, American Society forMicrobiology

Comparison

of

Epidemiological Markers

of

Salmonella

Strains Isolated

from

Different

Sources in Spain

JUAN J.

BORREGO,1*

DOLORES

CASTRO,1

MANUEL

JIMENEZ-NOTARIO,2

ANTONIO

LUQUE,l

EDUARDO MARTINEZ-MANZANARES,1 CARMEN RODRIGUEZ-AVIAL,3AND JUAN J.PICAZO3

Departamento deMicrobiologia, Universidad de Mdlaga, Campus Universitario Teatinos,

29071-Mdlaga,1

Centro deSalud de Fuengirola, Servicio Andaluz de Salud, Fuengirola

(Mdlaga),

2andServiciode

Microbiologia, Hospital Clinico UniversitarioSanCarlos, Plaza CristoReysin, 28040-Madrid,3Spain

Received 4 April 1992/Accepted 1 September 1992

Acomparative study of thephage

types,

antimicrobial

susceptibility

patterns, and plasmidprofiles of 171 strains ofSalmonelaisolated fromfood,epidemic outbreaks, and water-contaminatedenvironments as well as sporadic human isolates was carried out to determine the most adequate marker in epidemiological investigations.

Typing

based on the plasmid profiles appears to be the most effective method for grouping

strains with the same serotype obtained from a single outbreak and from environmental sources. However, none of the three markers tested allowus total discrimination and identification of related strains from a commonsource forepidemiological tracing. Therefore, the combined use of the three methods is necessary for determining whether common source isolates are related.

Salmonella infectionsappear to be oneof themosttypical

examples of an enteric disease that is transmitted from

animals to humans. Thetransmission occurs both through

food, suchasmeat,

dairy

products, andeggby-products (16,

18), andby contact between animals and humans directly

through the fecal-oral route (26) or indirectly via fecally

contaminatedwaters(22).

More than 2,000 serovars ofSalmonella have been

de-scribed, and all are considered to bepotentially pathogenic

foranimals, includinghumans. Salmonellosis in humans can

producesymptomsranginginseverityfromintestinal

distur-bancestodeath (44).

Infections causedby Salmonella strains, mainlyS.

enter-itidis,haveincreasedoverthe last fewyears (36). Bacterial

epidemic strains havetraditionallybeen defined on the basis

of phenotypic properties of the isolates, such as serotype

and biotype. However, these determinations have not

al-waysbeen usefulinepidemiologicaltracingoftheepidemic

strains(14, 17, 25, 47).

To evaluate the epidemiological

relationship

among

iso-lates from a common source, several different subtyping

methods havebeenapplied,such asserotyping (39),

biotyp-ingandcolicinetyping(4, 19), phage typing(46),

antimicro-bial resistancepatterns (12, 13, 42), and plasmid analysis (8, 31). Several studies have demonstrated that plasmid profile

analysis of the Salmonella isolates could be a reliable

epidemiological

toolfor the differentiation ofepidemic and

nonepidemic strains from outbreaks

(9,

14, 35, 43) or to

elucidate the epidemiology of these food-borne pathogens

(41). However, onlyafew studies of theapplication of this

marker to environmental, food, and animal isolates have

beencarriedout

(7,

29,

34).

Tostudythereliabilityofthesubtypingmethods as useful

epidemiological markers, we compared Salmonella strains

isolated from contaminated food, outbreaks, sporadic hu-mancases, and fecallycontaminated watersbyuseof their

phagetypes(PTs), plasmid profiles (PPs), andantimicrobial

resistancepatterns(APs).

*

Corresponding

author.

MATERIALSANDMETHODS

Bacterial strains. Salmonella strains were selected and

separated into thefollowing fourgroups accordingtotheir

sources and epidemiological significance: (i) isolates from

contaminated food (n = 26); (ii) isolates from different

outbreaks in various locations ofthe provinces of Malaga andMadrid(Spain)andpreviouslydescribedby Castro et al.

(10) (n =

34); (iii)

sporadic human Salmonella isolates(n =

38); and (iv) isolates from contaminated natural fresh and marine waters (Guadalhorce River and Mediterranean Sea,

respectively) (n = 72). All isolates were biochemically

confirmedbyusingthe API20E system(API Systems S.A.,

Montalieu-Vercieu, France) and wereserologically

charac-terizedbytheslide agglutinationtestwith Salmonella poly-valent 0 and group antisera (Difco Laboratories, Detroit,

Mich.) as well as by the tube agglutination test with H

antisera

(Difco).

All strains were sent to theNational

Refer-ence Center ofSalmonella (Majadahonda, Madrid, Spain)

forconfirmationofserotypingresults.

Antimicrobial susceptibility testing. The disk diffusion method (6)onMueller-Hinton agar(Difco)wasused to test

the resistance of the isolates to the following antimicrobial

agents (supplied by Biomerieux, Madrid, Spain): colistin

(Cl),10 ,ug; nalidixic acid(Na), 30 ,ug;gentamicin

(Gm),

10

,ug; streptomycin (Sm), 10 ,ug; kanamycin (Km), 30 p,g;

tetracycline (Tc), 30 ,ug; chloramphenicol

(Cm),

30 p,g;

ampicillin (Ap), 10 ,ug; carbenicillin (Cb), 100 ,Lg;

cephalo-thin(Cf), 30 ,ug; trimethoprim-sulfamethoxazole (SXT), 25

,ug;tobramycin (Tm), 10 ,ug; and

neomycin (Nm),

30 ,ug.

Phagetyping.PTs weredeterminedbytheuseof 25typing

phagesbelongingto a new phagetyping system developed

byCastro et al.(10). PTs wereestablished andidentifiedby

the patterns of reaction to the typing phages described

previously (10).

PP

analysis.

Plasmid DNA was extracted from lysed

Salmonella isolates by a modification of the

technique

de-scribedbyKado and Liu (23)andperformedbyToranzoet

al.(49).ExtractedplasmidDNAwaselectrophoresedfor 2 h

at150mAon a0.7%horizontal agarosegel

(Sigma

Chemical

Co.,St.Louis,

Mo.)

in Tris-acetate buffer

(40

mMTrisbase,

2 mMdisodiumEDTA;adjustedtopH7.9 withglacialacetic

3058

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TABLE 1. Serotype distribution and numbers of PTs, antimicrobial resistancepatterns, and plasmidprofiles oftheSalmonella isolates according to their origins

No.of No. of:

Source and

serotype

isolates H P P

islts PTs APs PPs

Food

S. entenitidis 16 15 11 14

S. virchow 3 3 3 3

S. typhimuium 2 2 2 2

S. typhi 2 2 1 1

S. anatum 1 1 1 1

S. infantis 1 1 1 1

S. newport 1 1 1 1

Total 26 25 20 23

Outbreakno. 1, S.enteritidis 13 2 4 8

Outbreak no. 2, S. virchow 1 1 1 1

Outbreakno.3, S. enteritidis 9 6 5 2 Outbreak no. 9, S. enteritidis 6 1 6 2 Outbreak no. 11,S. enteritidis 3 3 3 2 Outbreak no. 12, S. enteritidis 2 1 1 1

Outbreak total 34 14 20 16

Sporadicstrains

S. entenitidis 22 8 12 5

S. typhimurium 15 10 8 8

S. virchow 1 1 1 1

S.infantis 1 1 1 1

Total 39 20 22 15

Environmental

S. typhimurium 17 12 12 9

S. enteritidis 13 6 9 3

S. london 9 6 5 3

S. blockley 7 5 4 6

S. ohio 7 6 4 3

S. weltevreden 3 3 3 2

S. infantis 2 2 2 2

S. thompson 2 2 2 1

S. bovismorbificans 1 1 1 1

Self-agglutinable 11 9 8 5

Total 72 52 50 35

acid). Then, the gels were stained with ethidium bromide

solution (0.5 ,ug/ml)and photographed under aUV

transil-luminator(LKB, Pharmacia, Barcelona, Spain) with Plus-X-Pan film anda23 A Wrattenfilter. Theapproximate molec-ular masses of the plasmids (in megadaltons) were determined by comparison with plasmids with known

mo-lecular masses, e.g., R40a (96 MDa) and plasmids from Escherichiacoli V517ranging from 35.8to1.4 MDa.

The nonparametric Friedman test, chi-squaretest, andt

test (one tail) were used for statistical analysis by using StatView 512 Plus software andaMacintosh SE computer.

RESULTS

Table 1 summarizes the serotype distribution and the number ofPTs, APs, andPPs ofthe fourgroupsof Salmo-nella isolatesselected (food, outbreak, sporadic, and envi-ronmental strains). The Salmonella serotypes most fre-quently isolated were S. enteritidis (48.8%) and S. typhimurium (20%), although only S. enteritidiswas

repeat-edlyisolated from all thesamplegroups.

Food isolatespresented awide varietyofPTs, APs, and

PPs, and no epidemiological relationship was observed

among Salmonella isolates (chi-square = 3.71; P > 0.95).

Similar resultswereobtained with theenvironmentalstrains,

which presented a high number of plasmidless strains. On

theotherhand, theisolatesfrom outbreaks possessed com-monserotypes, PTs, and PPs but a great variety of APs.

Numerous PPs were found in the 171 isolates. Many of them were shown toharborlargeplasmids (from82.8 to 110

MDa) that havegenerally beenrelated to virulence

proper-ties(25, 28, 33).Thelargeplasmidswerenotusedforpattern

comparison of isolatesbecauseof theirproveninstabilityon

subculture (24, 38). Plasmids under20 MDa wereused for

comparison ofPPs.AlltheSalmonella isolatesweregrouped

into 29 PPs.

Patterns

containing from one to five plasmids

with lowmolecular masses were found in 72 isolates. The

most frequently detected plasmid groups were D5 (two

plasmids of 2.0 and 2.2 MDa) in 12.5% of strains with

low-molecular-mass plasmidsand 03(one2.0-MDaplasmid)

in 7.6% oftheisolatestested. Ontheotherhand,plasmids of

1.5, 2.0, 2.2, and 3.7 MDa were frequently present in the different

pattern

groups,correspondingto30.6,33.3, 25, and

19.4% of the strains, respectively.

The correlation between serotype and PP is shown in

Table2. Onlyfive serotypes, S. enteritidis,S. typhimurium,

S. virchow, S. blockley, and S. ohio, could be subdivided

into 16, 13, 3, 5, and 2 plasmid groups, respectively. The

otherserotypespresented onlyoneplasmidgroup.S.

typhi,

S. thompson, S. weltevreden, and the nonserological

type-ablestrains couldnotbe includedinanyestablishedplasmid

group.On the otherhand, allplasmidgroups except groups

02,03,

05,

06,07, D2, D3, D5, D7, and D9werelimitedto

a single serotype. The relationship between the serotypes

andthePPsaccordingtotheisolationsourceisalsogiven in

Table 2. Noplasmidgroup was common to the isolates from

all the sources, although the 03, 05, and D5 groups were

detected from strains isolated from three sources. Several

PPs were common to twosources, such as02, 06, andD2

for sporadic and environmental strains; 07 and 08 for

sporadic and food isolates; D9 for strains isolated from

outbreak and sporadic cases; and D3 for outbreak and

environmental strains.

Alltheserotypes couldbe dividedinto severalPTs, e.g.,

S.

enteritidis

into 37 PTs, 29 ofwhich

possessed

1 strain

each;5 PTs with 2strainseach; and 3 PTswithmorethan 5

strainseach(PT84 with 9strains,PT56 with13strains, and

PT 115 with 20 strains). S.

typhimurium

isolates were

divided among 20 PTs, 13 of which possessed one isolate

each, 3 PTs with two isolates each, PTs 114 and 115 with

threeisolateseach,PT 122with fourisolateseach,and PT 61

with fiveisolates. S.

london

could bedividedinto fivePTs,

three of which had one isolate each, and PTs 110 and 135

with three isolates each. The rest of the serotypes were

divided into different PTs comprising one or two isolates

each.

Resistance to one or more of the antimicrobial agents

tested was detected in 140 of the 171

Salmonella

strains

screened(81.9%). According totheirorigins, the antibiotic

resistance

proportions

were 80.8% for foodisolates, 79.5%

forsporadic isolates, 73.5% for outbreak

isolates,

and87.5%

for

environmental

isolates. Withregardto the resistance of the isolates to individual antibiotics, the resistance to Sm was the mostcommonlyfound (overall resistance, 50.9%);

this was followed by resistance to Tc and Na

(33.3

and

31.0%,respectively). On the otherhand,onlyonestrain was

found to beGm resistant.

Itisinterestingthatthe distribution ofantimicrobial

resis-tance of the Salmonella isolates varied according to their

origins.RegardingtheirAPs, theenvironmentalSalmonella

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TABLE 2. Correlation between serotype and PPs of the Salmonellaisolates

No. of

Source andserotype isolates strains

tested stan

Food

S. enteritidis

S. typhimurium

S. virchow

S. infantis

S. typhi

S. anatum S. newport

Outbreak

S. enteritidis

S. virchow

Sporadicstrains

S. enteritidis

S. typhimurium

S. virchow S. infantis

Environmental S. enteritidis

S. typhimunum

S. infantis

S. blockley

S. ohio

S. london

S. weltevreden S. thompson S. bovismorbificans Self-agglutinable

16 1 03, 04, 05, D7

2 D5

10 NP

2 1 08, NP

3 1 07, D5, NP

1 1 NP

2 1 NP

1 1 05

1 1 Dll

33 1 D8, Ti, T5

2 05, D9, D10

3 D6

4 03, D5

5 D3

9 NP

1 1 05

22 1 02, 06, T2, T7

18 NP

15 1 07, D2, D7, T3, T4

2 D1, D9

6 NP

1 1 NP

1 1 D5

13 1 05

12 NP

17 1 06, 09, D4, T6

3 01

10 NP

2 2 NP

7 1 02, 03, Fl, F2, NP

7 1 D3

2 NP

4 02

9 1 D2

8 NP

3 3 NP

2 2 NP

1 1 02

11 11 NP

aNP, Plasmidless strainsorstrains withplasmids whichwerenotincluded intoanestablished PPs. Thefollowing PPswereestablished(molecularmass

expressed inmegadaltons):01(0.7), 02 (1.5), 03 (2.0), 04 (2.2),05(2.4), 06 (2.6),07(3.7), 08(5.2),09(4.1), Dl(1.2,1.5), D2 (1.5,1.8),D3(1.5, 2.2), D4

(1.5,3.7), D5 (2.0,2.2),D6(2.0, 2.6), D7 (2.6, 3.7), D8(1.8,3.7), D9 (1.8,2.4), D10(2.0, 4.4),Dll (4.1,5.2), Ti (1.0,1.5,2.2), T2 (1.5,2.0,2.2), T3 (3.1,3.7, 4.1),T4(2.0, 2.2,3.7),T5(1.8, 3.7,4.8),T6(1.2, 1.5,2.6),T7(1.0, 1.8,2.6),

Fl(1.5, 2.0, 3.1,3.7),and F2(1.0,1.5,3.5, 3.7, 4.4).

strains differedsharplyfrom the strains isolated from other

sources. The strains isolated fromwaterexhibited ahigher

level ofresistance to Sm(62.5%),Tc(45.8%), SXT (5.6%), and Tm(13.9%),butall of the strainsweresusceptibletoCf. Salmonella isolates fromsporadic cases presentedahigher

levelofresistance toCb (25.6%) and Ap(28.2%), and the outbreak isolates showed increased resistance to Km (52.9%)andNm (23.5%).

The most frequent APs displayed by the S. enteritidis

strains was susceptibility to all the antimicrobial agents tested (19 strains); this was followed by resistancetoNa (8

strains). In the case of S. typhimurium, the APs most

frequently obtained was the combination Sm-Na with six

strains.AmongS. ohio and S. london isolates, the

predom-inant AP was resistance to Sm and Tm,respectively. Thedistribution of the multiresistant patterns within the different serotypes is given in Table 3. None of these combinations was common to strains from all sources, although resistancetriplets, such as Tc and Sm-Cm-Na, were detected in strains isolated from food and out-breaks, and the combination Sm-Tc-Na was distributed among isolates from environmental and sporadic human samples. The pattern Sm-Tc-Na was the most common in environmental strains, whereas in sporadic and outbreak strains, the most common patterns were Km-Nm, Sm-Na-Km, andSm-Cm-Tc-Cb-Ap.

Several PPs could beidentified withinonePT, and various PTs possessed the same

PP,

with a total of 24 subtypes among 34 isolates from outbreaks (Table 4). The most common PP of the S. enteritidis isolated from humans

involved in disease outbreaks, PP D3, was included only in PT56.Onthe contrary, the most common PT, PT 56, could be subtyped into six PPs. In Table 5, the relationship

between the APs and the PPs of the outbreak isolates is

given. ThemostfrequentlydetectedAPs among S.

enterit-idis isolates were susceptibility to all the antimicrobial agents(n = 8)andthe resistant pattern Sm-Km-Na(n = 6). Thesusceptible strains could be subtyped into four PPs, with

PP D5 being dominant; the strains with the Sm-Km-Na

resistance pattern couldbe subtyped into four PPs. On the otherhand, D3wasthe mostfrequently observed PP(n=5), and the strainswith this PP were included in threeAPs, with

resistance toNabeingthemostcommon (n = 3).

Fromthe statisticalanalysis (Table 6), it canbe deduced

thatonly significant relationshipswere establishedbetween

the markers AP and PT and the markers PP and PT in isolates fromoutbreaks, betweenAPand PP inisolatesfrom

sporadic,andbetween AP and PP and PP and PT inisolates

fromenvironmental sources.

DISCUSSION

For an optimal bacteriological diagnosis and successful

epidemiological tracing, the classification of species into

smaller units is of great importance. Serological and bio-chemicaltypingofSalmonella isolatescanonlyberegarded as a first step inthisrespect(51). However, moderntyping

methods basedon the analysisofgenotypiccharacteristics

mayprovidea useful tool forepidemiological studies.

In the study described here, itwas shown that the

inci-dence of serotypes,PPs, PTs, and APs isextremely highin

Salmonella isolates from all thesourcesexamined(Table 1).

Similar data have beenpublished previously (17, 29, 45).

We would expect that, in outbreaks in which there is a strongepidemiologicalassociation witha commonexposure

(food orwater), the Salmonella isolates wouldprovetobe

thesameregardlessof thetestused.However,the

compar-ison of strains fromfive outbreaks with strains isolated from

sporadic human cases, water, andfood brings to light the

absence of a standard by which a definite classification of relatedorunrelatedcanbe obtained(Table 1). Thus,isolates with the same serotype, PT,

PP,

and AP have different sources.On the otherhand,twostrains which have thesame serotype maydifferbythe lossorgainofaplasmidorbyany

change inphagesusceptibility (7,48).

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TABLE 3. Distribution of the multiresistant patterns (three or more agents) in the differentserotypes ofSalmonella

Food Outbreaksandsporadic humanisolates Environmental

AP Serotype No.of No.

(MMa)

Sero No.of No.(MM) S No.of No.(MM)

isolates ofplasmids e isolates ofplasmids erotye isolates of plasmids

Sm-Cf-Tc Sm-Cm-Tc Sm-Cm-Na Sm-Cm-Na

Sm-Cf-Na

Sm-Tm-Tc Sm-Tc-Na Sm-Tc-Na Sm-Na-Ar Sm-Cm-SX, Sm-Km-Nr' Sm-Km-Ni-Sm-Cb-Ap Sm-Na-Km Sm-Na-Km Sm-Na-Km

Cl-Na-Tc Cl-Na-Ap Nm-Na-Km Nm-Tc-Km Na-Tc-Km Sm-Cm-Na-Tc Sm-Cm-Na-Tc Sm-Cm-Km-Nm Sm-Tc-Km-Na

S.virchow 1 2(2.0, 2.2)

S.enteritidis 1 1(17.7) S.enteritidis

S.enteritidis 1 2(17.7, 36) S. typhimurium

S.typhimurium

S. enteritidis

S.typhimurium

S.typhimurium

S.enteritidis

S. enteritidis

S. enteritidis S. enteritidis S. enteritidis S. enteritidis

S. enteritidis 1 1(36)

S.anatum 1 1(2.6)

S. enteritidis S. enteritidis S. enteritidis S. enteritidis

Sm-Cm-Tc-SXT Sm-Cm-Tc-SXT

Sm-Km-Na-Nm Sm-Km-Na-Tc Sm-Km-Na-Tc

Cf-Tc-Cb-Ap Tm-Na-Cb-Ap Tc-Na-Cb-Ap Sm-Km-Na-Nm-Tc

Sm-Cm-Tc-Cb-Ap Sm-Cm-Tc-Cb-Ap Sm-Cm-Tc-Cb-Ap Sm-Km-Cb-Ap-Tc-Tm-SXT

S. enteritidis S. enteritidis

S.typhimurium

S.enteritidis S. enteritidis S. enteritidis S. enteritidis

S. enteritidis

S. enteritidis

S.enteritidis

2 1(3.7) 1 2(2.6, 3.7)

1 0

1 1(2.6)

1

1 0

1 (3.7)

2 1(32.1) 1 1(36) 1 1(56) 1 2(2.0, 36) 1 3(1.8, 2.4, 16.6) 1 5(2.0, 4.4, 14.8,

30,33.2)

2 0

1 2(2.0, 36) 1 2(2.0, 36) 1 2(2.0, 36)

S. ohio S. typhimurium S. enteritidis

S. weltevreden

S. ohio

1 2(1.5, 2.0)

1 0

2 0

1 1(85)

1 1(1.5)

Self-agglutinable 1 1(39.5)

S. london 1 3(1.5, 1.8,

32.1)

S. typhimurinum 1 2(1.5, 3.7)

S. typhimuinum 1 3(1.2, 1.5,

3.7)

1 0

1 0

1 4(1.8, 2.4, 16.6, 42.7)

1 1(56) 1 2(12, 56)

1 3(19.4, 21, 42)

1 5(2.0, 4.4, 14.8, 30,33.2) 1 2(1.2, 1.5)

1 3(1.2, 1.5, 55)

1 4(1.8, 2.4, 24, 58)

S. typhimurium 1 3(2.6, 40.2,

110)

aMM, molecularmass(inmegadaltons).

Agarosegelelectrophoresis of DNA from 171 Salmonella strainsisolated from different sources revealed a heteroge-neous plasmid population (Table 2). These results are

con-sistent with thoseof other studies ofplasmidsfromstrainsof members of the family Enterobacteriaceae (27) and Salmo-nella isolates (43). Plasmid analysis ofbacteria hasgained acceptanceas atool foridentifyingSalmonella isolates(40).

Many investigators have reported the utility of plasmid profile analysis inthestudy of theepidemiologyofinfection by Salmonella (9, 34), as well as in the definition and identification of bacteria originating from the same clone

(32). However, in the present study we were unable to establish strong evidence of theepidemiologyof the strains bymeansof theirplasmid profiles (Table 2). Thesefindings

are in contrast to those of previous reports (31, 35, 43),

whose authorswere able, bymeans ofplasmid profiles, to

group S. typhimurium and S. enteritidis strains from

com-TABLE 4. PPsrelatedtoPTsof outbreak isolates

No.ofisolates with thefollowingPl':

PP

20 21 22 23 35 36 41 48 49 50 54 56 68 79

NP1b 1 1 1 1 1 1 1 0 0 0 1 1 0 0

03 0 0 0 0 0 0 0 3 0 1 0 0 0 0

05 0 0 0 0 0 0 0 0 0 0 0 2 0 0

07 0 0 0 0 0 0 0 0 0 0 0 0 0 2

D3 0 0 0 0 0 0 0 0 0 0 0 5 0 0

D5 0 0 0 0 0 0 0 0 0 0 0 3 1 0

D6 0 0 0 0 0 0 0 0 0 0 0 1 0 0

D8 0 0 0 0 0 0 0 0 0 0 1 0 0 0

D9 0 0 0 0 0 0 1 1 0 0 0 0 0 0

D10 0 0 0 0 0 0 0 1 1 0 0 0 0 0

Ti

0 0 0 0 0 0 0 0 0 0 0 1 0 0

T5 0 0 0 0 0 0 1 0 0 0 0 0 0 0

aAccordingtothemnemonicpatterndescribedbyCastro etal.(10).

bNP,plasmidless strainsorstrainswithplasmids whichwere notincluded

in anestablishedPP.

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TABLE 5. APsrelatedtoPPsof outbreak isolates

No. of isolates with thefollowing PP: AP

NPa 03 05 07 D3 D5 D6 D8 D9 D10 Ti T5

Susceptible 1 0 2 0 0 4 1 0 0 0 0 0

Km 1 0 0 0 0 0 0 0 0 0 0 0

Tc 0 0 0 0 1 0 0 0 0 0 0 0

Na 0 0 0 0 3 0 0 0 0 0 1 0

Km-Na 1 0 0 0 0 0 0 0 0 0 0 1

Sm-Km 2 0 0 0 0 0 0 0 1 0 0 0

Km-Nm 0 0 0 0 0 0 0 1 0 0 0 0

Na-Tc 0 0 0 0 1 0 0 0 0 0 0 0

Sm-Km-Na 3 1 0 0 0 0 0 0 1 1 0 0

Km-Na-Nm 0 1 0 0 0 0 0 0 0 0 0 0

Km-Nm-Tc 0 1 0 0 0 0 0 0 0 0 0 0

Km-Na-Tc 0 1 0 0 0 0 0 0 0 0 0 0

Cm-Sm-Tc 0 0 0 2 0 0 0 0 0 0 0 0

Sm-Km-Na-Nm 1 0 0 0 0 0 0 0 0 0 0 0

Sm-Km-Na-Nm-Tc 0 0 0 0 0 0 0 0 0 1 0 0

aNP,plasmidless strainsorstrains withplasmidswhichwere notincluded in anestablished PP.

mon sources and to characterize thespread of suchstrains.

Olsvik et al. (31) established that the presence of a single

plasmid cannot always be used tocharacterize aclone, and

it may be necessary to carry out restriction endonuclease

digestswhen strainscontainplasmids with similar molecular

weights.

Inthepresentstudy,wecomparedthesmall

plasmids (less

than 20 MDa) present inSalmonella strains because these

plasmidsareprevalentin members ofthefamily

Enterobac-teriaceae (20, 27). Small plasmids

(less

than 5

MDa)

are

foundin both antibiotic-resistant and antibiotic-susceptible

Salmonella strains, but the functions of several of these

smallplasmids are still unknown.Tayloretal. (43) reported

that the strains that weredevoidof smallplasmidscontained

one high-molecular-mass plasmid (greater than 50 MDa for

the drug-resistant strains and greater than 20 MDa for the

drug-susceptible strains). Such plasmids should be capable

of self-transfer, since the transferoperon of the F plasmid

comprises about 15 MDa of DNA

(52).

We have observed

that, whilemostSalmonellastrains

susceptible

toantibiotics

were plasmidless

(42.8%),

a

high

number of these strains

(25%) contained plasmids with both

high

and low molecular

masses. In the case of drug-resistant strains, 52 did not harbor plasmids(37.1%), 32contained onlysmallplasmids

(22.9%), and 23possessed only large

plasmids

(16.4%);

the

remaining strains presented both small and large plasmids

TABLE 6. Statisticalanalysisof theepidemiologic

markers studied

Friedman test

Source (meanrank)

AP/PT PP/PT AP/PP AP PP PT

Food 1.57 1.07 0.64 1.79 1.93 2.29

Outbreak 27.17a 22.0b 15.0 2.25 1.83 1.92 Sporadic 2.40 1.52 9.8c 2.12 1.62 2.25 Environmental 2.64 14.87d 18.63e 2.15 1.40 2.45

ap =0.ooo01. b p =0.0005.

c P=0.002.

dp=0.095. e p= 0.028.

(23.6%).

These strainsmay, in

fact,

harbor

plasmid

aggre-gates

consisting

of a transfer factor and small

plasmids

encoding drug

resistance

(2).

Some small

plasmids

may be

formed

by

dissociation of the

large

plasmids

present inthe

samehostcell

(43).

The

spread

of

drug

resistance inSalmonella isolates is a

relativelyrecent

phenomenon.

Current

investigations

show

a

sharp

increase inantibiotic resistance amongSalmonella

strains,

yielding proportions

greater than 80%

(12, 42).

In this

study,

resistance to one or more antibiotics was

re-corded in 81.9% of the strains

tested,

demonstrating

the

epidemic spread

and

persistence

ofresistant clones of

Sal-monella

in

Spain.

Several

investigators

have used the resistance

typing

of

Salmonella strains for

epidemiological

purposes,

mainly

to

characterize cloneswith a

specific

resistance

(1, 37).

How-ever,wefound that antimicrobial resistance

testing

wasless

specific

than

testing

with other

epidemiological

markers in

the identification of related isolates. These

findings

agree

with those obtained

by

other

investigators

(17,

35).

Resistance to different antimicrobial agents has been

re-ported

to bemediated

by

plasmids. Thus,

Sm and

sulfona-mide resistance in Salmonella isolates is coded for

by

a

plasmid

of

approximately

5.5 MDa

(3, 15).

Tcresistance is

mediatedinS.

typhimurium by

a

plasmid

of24MDa

(31)

or

by

oneof 96 MDa

(29)

and

by

two

plasmids

of 50and 3 MDa in S. dublin

(30).

Sm and Tc resistancesareencoded

by

two

plasmids

of71.1 and 2.5 MDa

(21)

or

by

oneof62 MDa

(29)

or 140 MDa

(17).

In

addition,

multiple drug

resistance

patterns

have been relatedto the presence of

R-type

plas-mids linked to

cryptic plasmids (43).

In the present

study,

resistance to three or more antimicrobial agents was

ob-served in 43

strains,

the most

frequent

resistance pattern

being

the

profiles Sm-Km-Nm, Sm-Na-Km,

and

Sm-Cm-Tc-Cb-Ap

with three isolates each

(Table

3).

The Sm-Km-Nm resistance

pattern

wasobserved in twostrains of S.

enten-tidis with one

plasmid

of 32.1 MDa and in one strain that

harbored a 36-MDa

plasmid.

The Sm-Na-Km resistance

profile

was observed in three strains of S.

entenitidis

with

various

plasmid

contents, one strain with two

plasmids (2

and 36

MDa),

one strain with three

plasmids

(1.8,

2.3 and

16.6

MDa),

andafinalstrain with five

plasmids

(2,

4.4, 14.8,

30,

and 33.2

MDa). Finally,

the

multiple

resistance

pattern

Sm-Cm-Tc-Cb-Ap

wasrecorded in three strainsofS.

typhi-munum with two,

three,

and four

plasmids

each with

mo-lecularmassesof 1.2 and 1.5

MDa;

1.2, 1.5,

and 55

MDa;

and

1.8, 2.3, 24,

and 58

MDa,

respectively.

Plasmids with similar molecularmasses linkedto

antimi-crobial resistancehave been

reported by

other

investigators.

Holmberg

et al.

(17)

described several resistance

profiles

associated with the presence of

plasmids. Thus,

the most

commonpattern

Sm-Tc-Cb-Ap

wasobservedin strains with one

plasmid

(140 MDa),

two

plasmids

(5.7

and73

MDa),

and

three

plasmids (4, 5.5,

and 87

MDa).

In our

study,

this resistance

pattern

seemed to be linked to the presence of

low-molecular-mass

plasmids

(from

1.2to 1.8

MDa).

Naka-mura et al.

(29)

described a

plasmid-linked

resistance to

Sm-Cm-Su-Tc

(the plasmid

was110

MDa).

Asimilar

plasmid

wasobtained inour

study

ina

multiply

resistant strain of S.

typhimurium,

with resistanceto

Sm, Km, Cb, Ap, Tc, Tm,

and

SXT,

although

the strain also harboredtwo additional

plasmids

of 2.6 and 40.2 MDa. It isworthwhileto notethat resistance to SXT was

displayed only

by

environmental strains and was

always

linked to the presence of

large

plasmids.

Similarresultshave beenobtainedin other

enter-obacterial strains

(50).

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Phage typing has been the selected method of differenti-ating among serovars in the reference laboratory. This technique is rapid and provides a reproducible and highly discriminatory method of subdivision (47). Phage typing has been particularly useful in the epidemiological investigation of several salmonellosis outbreaks (5, 11). In the present study, we used a phage typing scheme developed in our laboratory (10) which permitted us to discriminate and group theserotypes ofSalmonella with similar drug resistance and plasmidprofilesinto differentphage types. For isolates from outbreaks, mainly from outbreak 1 (n = 14 isolates), phage typing was the most efficient technique for establishing the fact that the isolates were identical. However, in environ-mental strains, plasmid analysis may be preferable, and antimicrobial resistance could even serve as agoodmarker of relatedstrains in food-borne isolates. Certain PPs and APs were seenrepeatedlyin the various outbreak strainsstudied,

which suggeststhat theyarerelativelystable markers.

ACKNOWLEDGMENTS

Thiswork wassupported by agrant fromUnited Nations

Envi-ronmentProgramme/WorldHealth Organization.

Wethank M.A.Morifiigoforhelpwith thisstudyandreviewing the manuscript and the National Reference Center ofSalmonella (Majadahonda, Madrid, Spain) for the serological confirmation of the isolates.

REFERENCES

1. Anderson, E. S. 1975. The problem and implications of chlor-amphenicol resistance in the typhoid bacillus. J. Hyg. Camb. 74:289-299.

2. Anderson,E.S.,andM.J.Lewis. 1965. Drug resistance and its transfer inSalmonella

typhimurium.

Nature(London) 206:579-583.

3. Araki,Y.,M.Inoue,and H. Hashimoto.1987.Characterization and mobilization of nonconjugative plasmids encoding

resis-tance to streptomycinandsulfanilamide. Microbiol. Immunol.

31:1107-1111.

4. Baker, R. M., and D. C. Old. 1979. Biotyping and colicine typing of Salmonella typhimurium strains ofphage type 141 isolated in Scotland. J. Med. Microbiol. 12:265-276.

5. Baker, R. M., D. C. Old, andJ. C. M. Sharp. 1980. Phage type/biotype groups of Salmonella typhimurium in Scotland

1974-6: variation during spread of epidemic clones. J. Hyg.

Camb. 84:115-125.

6. Barry, A. L., and C. Thornberry. 1991. Susceptibility tests: diffusion testprocedures, p. 1117-1125. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy(ed.), Manual of clinicalmicrobiology,5th ed. Amer-icanSociety forMicrobiology, Washington, D.C.

7. Bezanson,G.,R. Khakhria,and R.Lacroix. 1982. Involvement ofplasmidsondetermining bacteriophage sensitivityin Salmo-nella typhimuium:geneticsandphysical analysisofphagovar

204. Can. J.Microbiol. 28:993-1001.

8. Bezanson, G., R. Khakhria, and D. Pagnutti. 1983. Plasmid profiles of valueindifferentiatingSalmonellamuensterisolates. J.Clin. Microbiol. 17:1159-1160.

9. Brumner, F.,A. Margadant, R. Peduzzi, and J. C. Piffaretti. 1983.Theplasmidpatternas anepidemiological tool for Salmo-nellatyphimurium epidemics. Comparisonwith thelysotype.J.

Infect.Dis. 148:7-11.

10. Castro, D.,M. A.Morifiigo,E. Martinez-Manzanares,R. Cor-nax, M. C. Balebona, A. Luque, and J. J. Borrego. 1992. Developmentand application of a new scheme ofphages for typing and differentiating Salmonella strains from different

sources.J. Clin. Microbiol.30:1418-1423.

11. Chart, H., E. J. Threlfall, and B. Rowe. 1989. Virulence of

Salmonellaenteritidisphage type4isrelatedtopossession ofa

38 MDaplasmid. FEMS Microbiol. Lett. 58:299-304.

12. Chugh, T. D., and A. Suheir. 1983. Drug resistance among

SalmonellaeinKuwait. Trop.Geogr.Med. 35:37-41.

13. Falbo, V.,A.Caprioli,F.MondeUo,M.L.Cacace,S.Luzi,and D. Greco. 1982. Antimicrobial resistance among Salmonella isolates fromhospitalsinRome. J.Hyg. Camb. 88:275-284. 14. Farrar, W. E., Jr. 1983. Molecular analysis of plasmids in

epidemiologicinvestigations.J. Infect. Dis. 148:1-6.

15. Grinter,N.J.,and P. T. Barth.1976.Characterization of SmSu plasmids byrestriction endonucleasecleavageandcompatibility testing.J. Bacteriol. 128:394-400.

16. Gustavsen,S.,and0.Breen. 1984.Investigationsofanoutbreak of Salmonella oranienbwg infections in Norway, caused by contaminated black pepper.Am.J.Epidemiol.119:806-812. 17. Holmberg, S. D., I. K.Wachsmuth, F. W. Hickman-Brenner,

and M. L.Cohen. 1984.Comparisonofplasmidprofile analysis,

phage typing,and antimicrobialsusceptibilitytestingin charac-terizing Salmonella typhimurinum isolates from outbreaks. J. Clin. Microbiol. 19:100-104.

18. Holmberg, S. D., J. G.Wells,and M. L.Cohen. 1984.

Animal-to-man transmission ofantimicrobial-resistant Salmonella: in-vestigationof USoutbreaks, 1971-1983. Science 225:883-885. 19. Ishiguro, N., G.Sato,K.Takeuchi,andA.Nakayama.1979.A

longitudinalepizootiological studyofSalmonella infectionon a

piggery: astudyonthe mode ofcontaminationby biotypingof

Salnonella typhimunum andby the antibiogram. Jpn.J. Vet.

Sci. 41:261-272.

20. Jamieson,A.F.,D. A.Brenner,P. L.Bergquist,and H. E. D. Lane. 1979. Characterization ofplasmidsfrom

antibiotic-resis-tant Shigella isolated by agarose gel electrophoresis. J. Gen. Microbiol. 113:73-81.

21. Jayaratne, A. H. G. P., D. L. Collins-Thompson, and J. T.

Trevors.1987. Plasmid-encoded transferablemultipleantibiotic

resistance inEscherichia coli isolated frommeat. Syst. Appl. Microbiol. 10:78-84.

22. Johnston,W.S.,G.F.Hopkins,G.K.MacLachlan,andJ.C.M. Sharp. 1986. Salmonella in sewage effluent and therelationship

toanimal and human diseaseinthe north of Scotland.Vet.Rec. 119:201-203.

23. Kado, C.I.,andS.T.Lu. 1981.Rapid procedurefor detection and isolation oflargeandsmallplasmids.J.Bacteriol. 145:1365-1373.

24. Litwin,C.M.,A.L.Storm,S.

Chipowsky,

and K.J. Ryan.1991. MolecularepidemiologyofShigellainfections:plasmidprofiles, serotypecorrelation, and restriction endonuclease analysis. J. Clin. Microbiol. 29:104-108.

25. Lujan,R.,A. Echeita,M. A.Usera, J.V.Martinez-Suarez,R.

Alonso, and J. A. Saez-Nieto. 1990. Plasmid profiles as an

epidemiologicalmarkerforSalnonellaenterica serotype

enter-itidisfoodborne outbreaks. Microbiol. SEM 6:45-50.

26. Lyons,R.W., C. L.Samples,N.H.deSilva,K.H.Ross,E. M.

Julian, and P. J. Checko. 1980. An epidemic of resistant

Salnonella

in anursery: animal-to-humanspread.JAMA243:

546-547.

27. Moller, J. K.,H. F.Jorgenson,C.Christiansen,G.Chnstiansen,

A. L.Bak,and A.Senderup.1978.Characterization ofplasmids

fromwild-type Enterobacteriaceae, p. 257-261. InD. Schles-singer (ed.), Microbiology-1978.AmericanSocietyfor Micro-biology, Washington,D.C.

28. Nakamura,M., S.Sato,T.Ohya,S.Suzuki,and S. Ikeda.1985. Possiblerelationshipofa36-megadaltonSalmonellaentenitidis

plasmidtovirulence in mice. Infect. Immun. 47:831-833. 29. Nakamura, M., S.Sato,T.Ohya,S.Suzuki,andS.Ikeda.1986.

Plasmid profile analysis in epidemiological studies of animal

Salnonella typhimurium

infection inJapan.J.Clin. Microbiol. 23:360-365.

30. Nastasi, A.,M. R.Vldlafrate,C.Mammina,M. F.Massenti,D.

Ooliva, and G. Scarlata. 1987. Molecular relationship among Salmonella dublin isolates identifiedatthe Centerof Enterobac-teriaceae of Palermo during the years 1971-85. Epidemiol.

Infect.99:283-290.

31. Olsvik,O.,H.Sorum,K.Birkness,K.Wachsmuth,M.Fjolstad, J. Lassen,K.Fossum,andJ.C.Feeley. 1985. Plasmid

charac-terizationof

SalmoneUla typhimurinum

transmittedfrom animals

tohumans.J.Clin. Microbiol.22:336-338.

on April 12, 2020 by guest

http://jcm.asm.org/

32. Orskov, F., andI. Orskov. 1983. Summary of a workshop on the clone conceptin the epidemiology, taxonomy, and evaluationof theEnterobacteriaceae and other bacteria. J. Infect. Dis. 149: 346-357.

33. Popoff, M., I. Miras, C. Coynault, C.Lasselin, and P. Paradon. 1981. Molecular relationship between virulence plasamids of Salmonella serotypes typhimurium and dublin and large plas-mids of other Salmonella serotypes. Ann. Microbiol. Inst. Pasteur 135A:389-398.

34. Riley,L.W., G. T.DiFerdinando, Jr., T. M. DeMelfi, and M. L.

Cohen. 1983. Evaluation of isolated casesofsalmonellosis by

plasmid profile analysis: introduction and transmission of a bacterial clone byprecookedroastbeef. J.Infect. Dis. 148:12-17.

35. Rodrigue, D. C., D. N. Cameron, N. D.Puhr, F. W. Brenner, M. E. St. Louis, I. K. Wachsmuth, and R. V. Tauxe. 1992. Comparison of plasmidprofiles, phage types, and antimicrobial resistance patterns of Salmonella enteritidis isolates in the United States. J. Clin. Microbiol. 30:854-857.

36. Rodrigue, D. C., R. V. Tauxe, andB.Rowe. 1990.International increase in Salmonellaenteritidis;a newpandemic?. Epidemiol. Infect.105:21-27.

37. Rowe, B., and E. J. Threlfall. 1984. Drug resistance in gram-negative aerobic bacilli. Br. Med. Bull. 40:68-76.

38. Sansonetti, P. J., D. J. Kopecko, and S. B. Formal. 1981. Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence. Infect. Immun. 34:75-83.

39. Saxena, S. N.,M. L.Mago, L.N. RaoBhau,S.Ahuja,andH. Singh. 1983. Salmonella serotypes prevalent in India during 1978-81.IndianJ. Med. Res. 77:10-18.

40. Schaberg,D.R.,L. S.Tompkins, and S.Falkow. 1981. Use of agarosegel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli. J. Clin. Microbiol. 13:1105-1108.

41. Scheinbach, S., and S. I. Hong. 1988. Detection of resident populations ofSalmonella and Escherichia coli in food ingredi-entsby plasmidanalysis. Food Microbiol. 5:235-241.

42. Sojka,W.J.,C.Wray, andI.McLaren.1986.Asurvey of drug

resistance inSalmonellae isolated from animals in England and Wales in 1982 and 1983. Br. Vet.J. 142:371-380.

43. Taylor, D. E., J. G. Levine,and K. L.Kouvelos.1982. Incidence of plasmid DNA in Salmonella strains isolated from clinical sources in Ontario, Canada, during 1979 and 1980. Can. J. Microbiol.28:1150-1157.

44. Taylor, D. N., C. Bopp,K.Birkness, and M. L. Cohen. 1984.An outbreak of salmonellosis associated withfatality in ahealthy child: a large dose and severe illness. Am. J. Epidemiol. 119:907-912.

45. Terakado, N., T. Ohya, H. Ueda, and Y. Isayama. 1980. A surveyondrug resistance and Rplasmids in Salmonella isolated from domestic animals in Japan. Jpn. J. Vet. Sci. 42:543-550. 46. Thiel, W.,and S.Recla.1984.Epidemiologie und lysotypievon

Salmonella typhimunum in Osterreich 1980-1982. Zentralb. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt. 1Orig. ReiheA 258:544-563.

47. Threlfall,E.J.,andJ.A. Frost.1990. Theidentification,typing andfingerprinting ofSalmonella: laboratory aspects and epide-miological applications. J. Appl. Bacteriol. 68:5-16.

48. Threlfall, E. J., L. R. Ward, and B. Rowe. 1978. Epidemic spread ofchloramphenicol-resistant strain ofSalmonella typhi-murium phage type 204 in bovine animals in Britain. Vet. Rec. 103:438-440.

49. Toranzo,A.E., J.L.

Barja,

R. R. Coiwell,and F.M. Hetrick. 1983. Characterization of plasmids in bacterial fish pathogens. Infect. Immun.39:184-192.

50. Tschape, H.,E.Tietze,R. Prager, and H. Heier.1986.

Occur-rence in waterandwaste waterofE.coli and coliform bacteria

carrying R-plasmids. Part 3. Plasmids encoding gentamicin, trimethoprim or streptothricin resistance. Acta Hydrochim. Hydrobiol. 14:167-174.

51. Wachsmuth, K. 1986. Molecular epidemiology of bacterial in-fections; examples of methodology and investigations of out-breaks. Rev.Infect. Dis. 8:682-692.

52. Willetts, N.,and R.A.Skurray. 1980. Theconjugation system of F-likeplasmids.Annu. Rev.Genet.14:41-76.

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http://jcm.asm.org/