(~) INSTITUTPASTEUR/ELSEVIER Paris 1992

Res. Mic,'obioL

1992, 143, 703-709

Molecular typing of Salmonella enterica subsp, enterica serovar Wien by rRNA gene restriction patterns S. Pignato (l), G. Giammanco (]), F. Grimont (2) and P.A.D. Grimont (2) a) lstituto di Igiene e Medicina Preventiva, Universitdt di Catania, 1-95 I00 Catania (Italy) an:l (2~ Centre National de Typage Mol#culaire des Ent#robact#ries, Unit# des Ent#robact#ries, U199 INSERM, lnstitut Paste~tr, F-75724 Paris Cedex 15

SUMMARY Analysis of digested DNA from 40 Salmonella enterica subsp, enterlca serovar Wien Isolates revealed five different rRNA gene restriction patterns for Hindlll (H1 to H5) and five for Pstl (P1 to PS). The isolates had been selected on the basis of the year of isolation, the geographic origin and the antibiotic resistance pattern and were distinguished as follows: a) 13 pre-epidemic isolates from different French towns in 1958-69 and from Senegal in 1968; b) 7 epidemic isolates from Algiers in 1969; c) 20 post-epidemic isolates from different French and Italian towns in 1970-90. Three different rRNA patterns (HIP1, H3P3 and H4P4) were observed among the pre-epidemic isolates. Conversely, the epidemic isolates, which were chaJacterized by the previously described large antibiotic resistance patterns and by the presence of 1.3 and 80-109 MDa plasmids, belonged to the same HIP1 ribotype. All but t w o postepidemic isolates were of the HIP1 ribotype. The determination of rRNA gene restriction patterns together with the plasmid content proved to be useful f')r a better characterization of the serovar Wien endemic and epidemic isolates.

Key-words: rRNA, Salmonella enterica, Serovar Wien; Restriction pattern, Molecular typing, Molecular elaidemiology.

INTRODUCTION

cessively, in Italy (Pagano et al., 1979; Scarlata et al., 1980), causing isolatec~ cases and epidemic

Salmonella enterica subsp, enterica serovar Wien, firstly identified in Austria in 1950 (Roschka, 1951), had been rarely isolated from human and animal specimens until 1969, when it caused an epidemic outbreak of gastroenteritis in Algiers (Mered et al., 1970). After 1970 the serovar Wien became the most frequently reported serovar in France (Le Minor, 1972) and, suc-

outbreaks mainly in pediatric wards. The Algerian strains were characterized by an unusual large pattern of antibiotic resistances (Mered et aL, 1970), wifich was also observed in the great majority of the strains isolated in France in 1970-72 (Le Minor, 1972) and in Italy in 1975-76 (Marranzano et al., 1976). Resistance patterns similar to that exhibited by the Algerian strains

Submitted October 10, 1991, accepted June 26, 1992.

704

S. PIGNA TO E T AL.

wet~. also shown by the serovar Wien isolates in Great Britain between 1972 and 1977 (McConnell et al., 1979). Several epidemiologic features suggest that a pandemic spread of serovar Wien took place from Algeria to various European countries, including France, Belgium, Italy, Austria, Yugoslavia and Great Britain (Le Minor, 1972; Marranzano et al., 1976; Chasseur-Libotte et al., 1977; WHO, 1975-77). In particular, studies on plasntid conlent strongly suggest a clonai origin for the antibiotic-resistant serovar Wien isolates from different geographic areas in 1970-77 (Avril et al., 1977 ; Maimone et al., 1979; McConnell et al., 1979). More recently serovar Wien infections have afresh become sporadic in Europe, although nosocomial outbreaks have occasionally occurred (Nastasi et al., 1989). The serovar Wien isolates after 1981 in Italy still remain resistant to different antibiotics, but the resistance patterns are different to some degree from the original pattern shown by the great majority of the epidemic isolates until 1980. Therefore, on the basis of antibiotic resistance patterns, it appears that most recent isolates are unrelated to the epidemic clone. In any case, relatedness cannot be excluded, due to the unreliability of the antibiotic resista.".ce pattern as an epidemiologic marker. In recent years, chromosomal D N A fingerprinting techniques have offered a precise means of characterizing bacterial strains (reviewed by Owen, 1989). With this aim, several probes have been used to determine chromosomal D N A restriction patterns. The ribosomal R N A (rRNA) probe from Escherichia ¢oli proposed by Grimont and Grimont (1986) for bacterial strain fingerprinting has been applied to the study of the molecular epidemiology of different organisms because of its broad spectrum of hybridization with bacterial chromosomal D N A (Irino et al., 1988; Stull et al., 1988; Altwegg et al., 1989). In the present paper, we report results of the analysis of forty serovar Wien isolates recovered from widely separated areas in an extended period, from 1958 to 1990. The study was conducted with the aim of assessing the usefulness

of rRNA probe fingerprinting in distinguishing between different epidemic and non-epidemic strains in comparison with other molecular techniques of analysis, such as plasmid analysis.

MATERIALS AND METHODS Bacterial isolates

A total of 40 serovar Wien isolates from human (n. 34 isolates) and swine (n. 2 isolates) faeces, cerebrospinal fluid (n. 2 isolates) and wastewater (n. 2 isolates) were studied. They were selected on the basis of the year of isolation, the geographic origin and the antibiotic resistance pattern and were distinguished as follows: - - pre-epidemic strains: 12 isolates from different French towns from 1958-69 and I !solate from Senegal in 1968; - - epidemic strains: 7 isolates from Algiers, 1969; - - post-epidemic strains: 20 isolates from different French and Italian towns in 1970-90. Gel electrophoresis of endonuclease-cleaved D N A

Bacterial DNA were prepared, cleaved and dectrophoresed as described elsewhere (Grimont and Grimont, 1986). In brief, DNA samples (2 to 3 I~g) were cleaved by restriction enzymes (l unit per p.g of DNA) Hindlll and Pstl following the manufacturer's instructions (Amersham International, Amersham, UK). After 6-h incubation at 37°C, horizontal agarose gel electrophoresis of restricted DNA samples was performed using a 0.8 07o(w/v) agarose gel in tris-acetate buffer (0.04 M tris-acetat¢, 0.002 M EDTA, pH 8.1) for 16 h. Size marker was HindIIIcleaved DNA from Salmonella enterica serovar Typhimurium strain PC. DNA fragments were blotted to a "Hybond-N" nylon membrane (Amershara) using "VacuGene" system (Pharmacia LKB Biotechnology, Uppsala, Sweden). Radioactive labelling of R N A and hybridization

Ribosomal 1 6 + 2 3 S RNA from E. coli (Boehringer, Mannheim, FRG) was end-labelled with -f32P-ATP (Amersham) and a 5' DNA terminus labelling kit with T4-polynucleotide kinase (Bethesda Research Laboratories, Gaithersburg, MD) following the exchange reaction procedure and instructions provided by the manufacturer. Hybridization with -f32P-labelled rRNA probe

M O L E C U L A R T Y P I N G OF SALMONELLA ENTERICA S E R O V A R WIEN

was performed as previously described (Grlmont and Grimont, 1986). Membranes were autoradiographed using "Hyperfilm M P " (Amersham) and an intensifying screen at - 8 0 ° C for 4 days.

DNA fragment size determination The algorithm of Schaffer and Sederoff (1981) was used to derive an experimental function relating DNA fragment size to migration distance.

agarose gel in tris-acetate buffer (0.04 M tris-acerate, 0.001 M EDTA, pH 8) for 5 h (5 V/cm). Gels were stained with ethidium bromide and photographed with UV light. The molecular size of plasmtd DNA was estimated by comparison with E. coil plasmid standards: R27 (112 MDa), R40a &96 MDa), 39R861 (08, 42, 23.9, 4.6 MDa), Ri24 (81.7 MDa), R387 (53 MDa), RP4 (34 MDa), Sa (25 MDa), pHHl310a (7.4 MDa), RSFI010 (5.5 MDa), V517 (32, 5.2, 3.5, 3, 2.2, 1.7, 1.5, i.2 MDa). RESULTS

Plasmid analysis Plasmid DNA was extracted from i 8-h broth cultures by the alkaline denaturation method of Birnboim and Doly (1979). Plasmid profile was determined by electrophoresis in 0.7 e/0 horizontal

M3

1 4 "'

1M

Analysis o f dig,~sted D N A f r o m the aO ~erovar Wien isolates revealed five different r R N A patterns for H i n d l l l (HI to H5) (fig. 1 and 2) and five for Pstl (P1 to PS) (fig. 3). ]'he s a m e

Pattern

kbp

Size of rRNA gene restriction fragments Kbp 20 10 5

II I

I, i i ,

HI

IIIII

I-~

IIIII

I

I

I

I

Ill

!11

Ha,

ii II i

!

~:~-22.8 -13.4

I

ill

H3 H5

t

705

IIIII

I

HindlIl rDNA fragments

Fig. 2. Normalized graph showing the different migration patterns of rRNA gene restriction fragments after cleavage by Hindlll and hybridization with labelled E. coil 16+ 23 S rRNA. Dotted lines indicate weaker bands.

- :.'"/m

Pattern

i-6.0

Size of rRNA gene restriction fragments Kbp 20 10 5

II

P1 P2 P3 P5

Hindlll.

i

i

i

lil

I II I IIII IIIII

P4 Fig. 1. Three different patterns of rRNA gene restrietio,~ of serovar Wietl DNA after cleavage with Hindlll and hybridization with labelled E. coil 16+ 23 S rRNA. M=marker, rRNA gene restrictic,n fragments of Salmonella typhimurlum OC DNA after cleavage with

!'II

I

I IIII

Pst l

rDNA

fragments

Fig. 3. Normalized graph showing the different migration patterns ot rRNA gene restriction fragments after cleavage by Pstl and hybridization with labelled 16+ 23 S rRNA.

706

S. P I G N A TO E T AL.

Epidemic Isolates

isolates showed a great variability in the plasmid content and in the antibiotic resistance patterns. S o m e important differences were noted in the pre-epidemic isolates in c o m p a r i s o n with the epidemic and post-epidemic isolates.

All Algerian isolates showed the H 1P 1 r R N A p a t t e r n and were multiresistant, a n d all but one carried a small 1.3-MDa plasmid (table 1I). The largest plasmids (80-109 M D a ) were f o u n d in five out o f the seven e x a m i n e d isolates, while s o m e other.,; o f different molecular weight were variously associated, so that the entire p l a s m i d profiles were rather h e t e r o g e n e o u s .

Pre-epidemic isolates Three different r R N A patterns were observed antong the i3 pre-epidemic isolates (table l). The s a m e H I P I r R N A p a t t e r n was o b t a i n e d f r o m 10 isolates, while H 3 P 3 and H 4 P 4 patterns were observed, respectively, in 1 and 2 other isolates. The 13 isolates were susceptible to the antibiotic tested, with the exception o f three strains resistant to streptomycin. Seven o f the 13 isolates carried plasmids o f different molecular weight; n o presence o f plasmids was shown in the other isolates.

Post-epidemic isolates Characteristics similar to those s h o w n by the Algerian isolates were observed in the isolates f r o m d i f f e r e n t F r e n c h a n d Italian t o w n s (table III). In particular, the H I P I r R N A pattern was o b s e r v e d in all but t w o isolates, which

Table i. Antibiotic rcsi'Aancc pattern, plasmid profile and rRNA gene restriction pattern iu pre-epidemic serovar Wien isolates from 1958 to D69. Strain

Origin

2-58 9-58

SF, Fr ~.n~.e HF, Strasbourg Fr.ance HF, Stra~boutg France HF, Strasbourg France HF, Strasbourg France HF, Strasbourg France HF, Strasbourg France HF, Lyon France HF, Orleans France CSF, Paris France CSF, Senegal WW, Lille France WW, Lille France

1-59 2-59 3-59 4-59 1-62 2-62 1-65 1-66 1-68 3-69 4-69

Resistance pattern

P!asmid profile (MDa)

sens. Sm

rRNA gene restriction pattern with Hindlll

Pstl

none 62; 39

HI HI

PI PI

sens.

58; 35; 2

HI

Pl

Sm

53; 26

HI

PI

sens.

64

H3

P3

sens.

62; 30; 23; 2

HI

PI

sens.

1.8

H4

P4

Sm

none

HI

P1

sens.

42; 2

H4

P4

se,ls,

none

HI

PI

sens. sens.

none none

lq_! HI

Pi PI

sens.

none

Hl

Pl

SF = swinefaeces; HF = human faeces,CSF= cerebrospinalfluid; WW = wastewater; Sm = resistantto s'reptomycin; sens= sensltiv( to ampicillin,tp~racycline,kanamycin, streptomycinand chloramphenicol.

M O L E C U L A R T Y P I N G OF S A L M O N E L , . A ENTERICA S E R O V A R W I E N

707

Table !I. Antibiotic resistance pattern, plasmid profile and rRNA gene restriction pattern in epidemic serovar Wien isolates from h u m a n faeces in Algiers, 1969. Strain

Resistance pattern

Plasmid profile (MDa)

5-69 6-69 9-69 11-69 13-69 14-69 15-69

ApTcKmSm ApTcKmSm ApCmTcKmSm ApTeSm ApCmTcKmSm ApTcSm ApTcSm

36.5; 1.3 109; 48; 24.5 ; 6.4; 2 98; 41; 18; 9.6; 1.3 82; 26; 6.5; i.3 31 ; 1.3 100; 30; 19; 1.3 81.7; 34; 1.3

rRNA gene restriction pattern with HindIII PstI HI H1 HI HI HI H1 HI

PI P1 P! PI PI PI PI

Ap = ampicillin; Cm = chloramphenicol; Tc = tetracycline; Km = kanamycin; Sm = streptomycin.

Table IlL Antibiotic resistance pattern, plasmid profile and rRNA gene restriction pattern in post-epidemic serovar Wien isolates from h u m a n and swine (ME 5-76) faeces in France and in Italy from 1970 to 1990.

Strain

Origin

Resistance pattern

Plasmid profile (MDa)

391-70 863-72 872-72 875-72 164-75 167-75 168-75 2-89 16-89 2-90 CTI-74 PA70-75 MI 2-75 NA 3-75 Me 5-76 Gela 6-80 CT7-82 Gela8-83 CT9-89 CTi0-89

Paris, France Grenoble, France Paris, France Paris, France Paris, France Nevers, France Marseille, France Vichy, France Lille, France Rouen, France Catania, Italy Palermo, l!aly Milan, Italy Naples, Italy Messina, Italy Gela, Italy Catania, Italy Gela, Italy Catania, Italy Catania, Italy

ApCmTcKmSm ApSm ApSm A p C m T c K m S .re. ApCmTcKmSm ApCmTcKmSm ApCmTcKmSm ApCmKmSm ApCmKmSm ApCmKmSm ApCmTcKmSm ApCmTcKm ApCmTcKmSm ApCmTcKmSm ApCmTcKmSm CmTcKm ApTc ApTcSm ApSm Ap

81.7; 24.5 84: 24.5; 2; 1.3; 1 81.7; 28; 7.6; 1.3; l 96; 34; 7.6; 1.3 86; 3; 1.3 81.7; 2; 1.3 98; 1.3 27; 2 24.5 ; 2; 1.3 84; 12; 1.3 84; 10.6; 2.5; 1.3 96; 53; 7.6; 1.3 98; 7.6; 1.3 98; 1.3 96; 1.3 96; 81.7; 24.5; 2; 1.3 84; 3; 1.3 60; 3; 1.3 42; 5; 1.5 8.7; 5.4; 2; 1.3

rRNA gene restriction pattern with HindIIl Pstl HI HI HI HI Hl HI HI HI H1 HI HI HI H! HI H5 HI HI H2 HI HI

PI Pl Pl P1 Pl Pl Pl Pl "Pl ?l ?1 PI ?1 ,al .~5 PI Pl i'2 Pl PI

Ap = ampicillin; Cm = chlotamphenicol; Tc = tetracycline; Km = kanamycin; Sm = streptomycin.

showed H2P2 and H5P5 patterns. The small 1 . 3 - M D a p l a s m i d was f o u n d in all b u t three isolates, a n d t h e largest p l a s m i d s o f 80-98 M D a in 15 o u t o f 20. Overall p l a s m i d profiles differed s o m e w h a t in t e r m s o f the presence o f v a r i o u s other plasmids.

DISCUSSION P l a s m i d fingerprinting h a s been used in e p i d e m i o l o g i c i n v e s t i g a t i o n s , especially to d e m o n s t r a t e the relatedness o f bacterial isolates f r o m epidemic cases ( M a r k o w i t z et aL, 1982;

708

S. P I G N A T O E T AL.

Schaberg et al., 1981; Shlaas et al., 1986). However, plasmid analysis does not always provide definitive evidence to confirm or exclude the possibility that different bacterial isolates have a c o m m o n origin, since loss of a plasmid can occur, while a given plasmid may disseminate not only in different strains of the same species but among different species (O'Brien et al., 1980). In our serovar Wien epidemic isolates, all plasmid profiles show some degree of heterogeneity, as also observed in recent strain~ f~om a nosocomial epidemic outbreak (Nastasi et al., 1989). Nevertheless, the 80-109-MDa ~,nd the 1.3,1VlDa plasmids, which were absent in the preepidemic isolates, characterize the Algerian and most of the European isolates after 1969. Therefore, in agreement with previous studies on serovar Wien strains isolated in various geographic areas from 1970 to 1976 (Avril et al., 1977; Maimone et al., 1979; McConnell el al., 1979), we suggest that the Algerian and the post-epidemic European isolates have a c o m m o n clo'~al origin. The epidemic clone may have arisen from the pre-epiden.ic H1PI clone by acquisition of resistance ptasmids. Colonna et al. 0 9 8 5 ) Rave found an 84-107-MDa plasmid in 7 of 9 serovar Wien epic'emic isolates. The plasmid belonged to the F l m e group and carried a gene coding a hydroxamate-mediated iron assimilation system (aerobactin-like) flanked by I S / e l e m e n t s . Our study shows that such a plasmid was missing in pre.epidemic isolates. Two Italian isolates differ from the epidemic H1PI type (ribotype) on the basis of the rRNA gent restriction pattern exhibited. The two different isolates belonging to other H2P2 and HSP5 ribotypes are presumably pre-existing endemic strains that acquired the epidemic 1.3 MDa plasmid after the spread of the epidemic bacterial clone. This hypothesis is supported by results of phage typing of serovar Wien strains circulating in Italy after 1975, suggesting thai a minority of cases have been caused by e n d e m i c s t r a i n s d i f f e r e n t f r o m t h e predominant epidemic strain (Scarlata et al., 1982). In spite of the relatively low n u m b e r of iso-

lates that have been examined, sonte conclusions can be drawn. Polymorphism in chromosomal DNA restriction fragments has been shown in serovar Wien. Therefore, this serovar can be divided into differen) types (Nastasi et al., 1990), as observed with other Salmonella serovars using r a n d o m chromosomal D N A fragments as probes (Tompkins et al., 1986). In addition to the isolates presumed to derive from a n epidemic clone which may have maintained the same ribotype and characteristic plasmids over a twenty-year period, it has been possible to distinguish endemic strains that have acquired epidemic ~:lasmids but belong to different ribotypes. These results demonstrate :hat ribotyping ;.~.--. more stable method for the characterization of strains t h a n the analysis of plasmid profiias, particularly when the period of sampling iz long.

Acknowledgements We thank Pr A. Nastasi and Dr C. Mammina for their help in the analysis of plasmid patterns. This work was supported in part by the "Fondo policentrico MURST 40 %, coordinatore Pr P. Crovari".

Typage mol~culaire de Salmonella enterlca subsp, enterica s~rotype Wien par d~termination des profils de restriction des g~nes de I'ARNr Une ~tude r~trospective a dtd effectude sur une collection de 40 isolats de Salmonella enterica s~ro)ype Wien. Les souches ont dtd s~lectionndes sur l'annde d'isolement, l'origine g~ographique, ct la r~sistance aux antibiotiques. La collection dtait constitude de 13 isolats pr~-~pid~miques provenant de diverses villes fran¢aises de 1958 fi 1969 et du S~ndgal en 1968, de 7 isolats dpiddmiques provenant d'Alger en 1969, et de 20 isolats post-dpid~miques provenant de diff~rentes villes fran~:aises et italiennes de 1970 :~ 1990. L'ctude des profils de restriction des g~nes codant pour les ARNr (ribotyp::) a individualis~ 5 profils diffdrents apr~s clivage de I'ADN par Hindill (HI H5) et 5 profils apr~s clivage par Pstl (PI fi P5). Trois ribotypes diffdrents (HIP 1, H3P3, et H4P4) ont ~t~ observes pour les isolats prd~piddmiques. Les souches dpid~miques, caractdrisdes par une multird.sistance aux antibiotiques ant6rieurement d~crite et

MOLECULAR

T Y P I N G O F SALMONELLA ENTERICA S E R O V A R

par la presence de pla~ .mides de ! ,3 et 80/t 109 MDa, avaient le ribotype H1P1. Tous les isolats post~pid6miques, sauf deux, avaient le ribotype H I P I . La d6termination du ribotype combin6e/t celle du contenu plasmidique a contribu6 /l une meillenre caract6risation des isolats end6miques et 6pid6miques de S. enterica s6rotype Wien. Mots-clds: rRNA, Salmonella enterica, S6rotype Wien; Profils de restriction, Typage mol6culaire, Epid~miologie mol6culaire.

References Altwegg, M., Hickmann-Brenner, F.W. & Farmer, J.J. !11 (1989), Ribosomal RNA gene restriction patterns provide increased sensitivity for typing Salmonella typhi strains. J. infect. Dis., 160, 145-149. Avril, J.L., Dabernat, H.J., Gerbaucl, G.R., Horodniceanu, T., Lambert-Zechovsky, N., Le Minor, S., Mendez, B. & Chabbert, Y.A. (1977), Groupes d'incompatibilit~ des plasmides R chez les souches de Salmonella ~pid~mique. Ann. MicrobioL (Inst. Pasteur), 128B, 165-175. Birnboim, H.C. & Doly, J. (1979), A rapid alkaline extraction procedure for screening recombinant plasmid DNA. NucL Ac. Res., 7, 1.513-1523. Chasseur-Libotte, M.L., Ghysels, G., ~uhi, P. & Van Oye, E. (1977), Salmonella wien en Belgique. M~d. Mal. infect., 7, 265-269. Colonna, B., N~coletti, M., Visca, P., Casalino, M., Valenti, P. 8