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Molecular typing of Shigella strains using pulsed field gel electrophoresis and genome hybridization with insertion sequences
(~) INSTITbq"PASTEuR/ELSEVIER Paris 1991
Res. MicrobiaL 199i, 142, 48%498
Molecular typing of Shigella strains using pulsed field gel electrophoresis and genome hybridization with insertion seqaences L. Soldati and J.C. Piffaretti (*) Istituto Cantonale Batteriologico, via Ospedale 6, 6904 Lugano (Switzerland)
SUMMARY
ThG genomes of 18 independent ShigeUaisolates (9 SnJdeltasonnei, 5 Shige/la dysentedae and 4 Shigella fiexner~ as well as of 4 epidemic R, flexneristrains were analysed by pulsed field gel electrophoresis (PFGE}and by the distribution of insertion sequences (iS/, IS2 and IS911). Despite the close relatedness observed among the 9 independent S, sonnel, all of them could be differentiated from each other. The 4 independent $. flexner/isolates ~howcd c~a~!y disting~ishab."e D~A profiles. N~.dy complete genetic identity was detected within the 4 epidemic S. flexneri when analysed by PFGE or for |S 1 and IS2 patterns. However, IS911 was found to be too mobile in these epidemic S, flexneri to be used as a typing probe. The 5 S. dysentetiae isolates could also be distinguished by the techniques used. The diversity found within this species is striking: of the 5 investigated isolates, 3 completely different DNA prof'des were revealed. In conclusion, both PFGE and |S probing demonstrated their potential usefulness in molecular epidemiology and in typing of Shigella strains. The degree of differentiation given by these two methods was generany comparable, although IS probes showed better discrimination of the isolates.
Key-words: Shigella, Genome, MolecuMr typing; Pulsed field gel e~ectrcphoresis, Insertion sequences.
INTRODUCTION Diarrhoeai diseases including shigellosis are among the major causes o f morbidity and mortality in developing countries. Epidemiotogic investigations o f these diseases require typing systems enabling an efficient identification o f the sources o f infection. Serolc:gical analysis o f Shigella based on the specificity o f the O-antigen
is important for species identification, but still u n s a t i s f a c t o r y . C o m m e r c i a l l y available serogrouping and serotyping antisera do not always provide reliable or unambiguous results fEvins et aL, 1989). Furthermore, the presence o f non-typeable :strains zequires the determination o f new provisional serovars, which is timeconsuming and needs exten~ve confirmation to assess their epidemiologic significance (Gross et
Submitted December 3, 1990, accepted December t2, 1990. (*) Correspondingauthor.
490
L. SOLDA TI AND J.C. PIFFARETTI data). IS911 has been recently isolated from S. dysenteriae and is present in varying copy number in all 4 species of Shigetla and in E. coil KI2, but not in E. coil W (Prrre et al., 1990).
aL, 1989). Colicin- and phage-typing were approached by Abbott and Shannon (1958) and Bergan (1979), but these typing systems have not been further improved and at present are not used routinely. Moreover, these techniques are based on variation in phenotypic properties, which in many cases depend on a single or several unidentified genetic loci and therefore do not represent a global and reliable image of the bacterial genome. Plasmid pattern determination, a useful method for typing Sahnonella or other enteric bacteria (Brunner et aL, 1983), showed limitations when used as an epidemiological tool for clinical isolates o f S. sonnei (Prado et aL, 1987L
MATERIALS AND METHODS Bacterial strains and p!asmids
The strains used (listed in table I) were independent clinical isolates comprising 9 S. sonnei, 5 S. aysenteriae and 4 S. flexnerL Four additional S. flexneri strains (3093, 3094, 3 t 02 and 3103) originated from a same epidemic. Ptasmid pKH47 (L. Caro) provided the insertion element fS1, while IS911 was derived from plasmid pOFI39 (Pr~re et al., 1990). The source of the IS2 probe was a pBR322 plasmid derivative (pgalOPIS2; O. Fayet) containing the large Hindlll fragment (800 bp) of tS2 (Saint Giron et at., 1981). E. coli strain 803 was used for the propagation and isolation of these plasmids.
Moiecular methodologies applied to the whole genome are becoming more and more relevant in providing means for accurate bacterial classification and typing systems; however, they have not yet been fully investigated in the genus Shigella. This is the reason why we evaluated two such molecular procedures to identify and compare different Shigella strains, i.e. pulsed fie!d Eel e!ectrophoresis and the use of insertion sequences as genetic markers. On the one hand, we compared the genomic digestion patterns of independent or epidemic Shigella isolates by PFGE after cleavage with the restriction enzyme Notl. This endonuclease recognizes a specific sequence of 8 base pairs and thus produces a limited number of fragments from the genomic DNA. On the other hand, taking advantage of the presence of high copy numbers of some insertion elements in Shigella, we compared the IS patterns of the same strains. We used the insertion sequences IS/, IS2 and IS911. The elements IS/ and IS2 were first isolated from Escheriehia coil, where they are ,gresent at an average number of about 6 to 8 copies (Galas and Chandler, 1988, for review). In Shigella, with the exception of S. boydii, the copy number reaches 40 or more for ISI (Galas and Chandler, t988, for review) and 20 to 30 for IS2 (L. Soldati and J.C. Piffaretti, unpublished
IS = insertionsequence, PFGE = pulsedfield get elc~rophores~s,
Media a~d growth conditions
Shigella and E. coli strains were grown in L broth or on L agar (Maniatis et al., 1982) at 37~C with shaking. When needed, ampicillin (100 rtg/ml) was added. DNA manipulations
Plasmid DNA preparations, DNA digestions with restriction endonucleases and agarose get electrophoresis were as described previously (Fayet et aL, 1982). The method for chromosomal DNA isolation was that of Hartl et al. (1983). Preparation of labelled probes and hybridization experiments
After digestion of the adequate plasmids, DNA framnents containing the IS to be used as a probe were purified from agarose gels using the Genecleankit of Bio-101 (La Jolla, CA). DNA transfer from agarose gels to nylon membranes was done as described by Smith and Summer (1980). Hybridizations and visualization of the results were performed
i i
PMSF = phenyimethylsu]phonytfluoride. TBE = Tris-boricacid-EDTA.
. ~ I O L E C U L A R T Y P I N G O F SHIGELLA S T R A I N S
491
Table i. Bacterial strains. Strain S. sonnei
S. dysenteriae
S. flexneri
E. eoli
Comments 1 3 4 5 9 10 OFB759 199309 298246 1 2 3 6 12 M90T 1 14 1426 3093 3094 3102 3103 803
serotype serotype serotype serotype serotype serotype serotype serotype
Year of isolat ion
Origin or reference
1976 1979 1980 1980 t988 t988
M. Heilz M. Heitz M. Heitz M. Heitz M. Heitz M. Heinz Pr6re et aL (1990) T. Giger T. Giger P. Grimom P. Grimont P. Grimont P. Grimom P. Grimont Sansonetti et al. (t982) T. Giger T. Giger W. Zotliger P. Grimom P. Grimom P. Grimon~ P, Grimont L. Caro
1988 i988 1989 t989 1989 1989 t989
1 2 3 6 12 5 3c 3c
serotype 2a, epidemic strain serotype 2a, epidemic strain serotype 2a, epidemic strain serotype 2a, epidemic strain KI2, supE, supF, hsdM, hsdR
1982 1982 1988 1990 1990 t990 1990
Note: S. fiexneri 3093, 3094. 3102 and 3103 were from a single outbreak.
with the " D N A labeling and dete~ion kit, nonradioactive" (B6hringer Mannheim) using the protocols provided by the manufacturer.
Genomic DNA preparation, restriclion digestions and PFGE The preparation of high molecular weight chromosomal DNA was performed with some modifications from the method of McClenand et aL (1987). Overnight cultures of bacteria were concentrated twice in ET (EDTA t00 raM, Tris 10 mJM ; pH 8) and incubated 10 rain at 37~C. One ml of this suspension was mixed with an equal volume of 1.8 % low gel~ing agarose (freshly prepared and kept at 50'°C), poured immediately between two glass plates (separated by 1.5 ram) and kept ~0 rain at 4°C. Pieces of adequate size were cut and incubated in 8 mt ET buffer with 5 m g / m l lysozyme and 0,05 % sarcosyl for 2 h at 37~C with shaking, The samples were then transferred in 5 ml tysis solution (ET buffer with 0.5 rag/rot proteinase K and 1 % SDS), shaken overnight at 50°C and subsequently dialysed in 50 ml TE (Tris 1 0 m M , EDTA I rmM; pH 8) contaiaing 0.1 mM phenylmethylsulphonyl fluoride (PMSFt for
1 h at room tc-mperature. After dialysis repeated twice without PMSF, the inserts were maintair~ed in TE at 4~C. Fo~"digestion, each sample w ~ incuba[ed in 200 ,ul of' appropriate buffer supplemented with about 15 units of restriction enzyme and incubated h at the appropriate temperature. The inserts were the~ rinsed in one ml TE a~d loaded onto a 0.8 ~ agarose gel. Field inversion gel electrophoresis was performed for 25-29 h and 180-190 volts in TBE (45 mM Tris, 45 rmM boric acid and 0.5 mM EDTA) at 14=C. The temperature was kept conAant by co,ofing the buffer during its recircularization. Field inversion was supplied by a programmable power controller (model PPI-2(~; MJ R~earch~ Londoa).
RESULTS
Comparison of the N o t f genomic restriction patterns of 9 independent S, sonnei s~ra~ns A m o n g ~he 4 species o f the genus Shigetla, S. s o n n e i i s the most homogeneous, tt comprises a single serovar. U s i n g P F G E a f t e r d i g e s t i o n o f
L. SOLDA TI AND J.C. PIFFARETTI
492
the genomes with the endonuclease NotI, we examined 9 independent S. sonnei clinical isoiates (fig. I). Electrophoresis conditions were those allowing the visualization of the highest number o f bands between 40 and 600 Kb. All
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the strains analysed showed similar band patterns; similarity was particularly evident between isolates 1, 3 and 298246, as well as between isolates 4, 5, l0 and OFB759. In order to establish a comprehensive pattern o f all the fragments, we used several other PFGE conditions and confirmed the almost complete identity o f the 4, 10 and OFB759 patterns. These conditions also revealed that the other strains differed by at least three or four bands (results not shown).
Comparison of the 9 S. sonnei strains using IS distribution in their genomes
~d~cd~aid~id ca to c a t ~ to tn to ~ to
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123456789 Fig. l. PFGE analysis of genomic D N A from nine independent S. sonnei isolates. Preparations of high MW D N A were digested with NotI and separated on agarose gel by field inversion dectrophoresis. Numbers at right are the molecu,ar sizes in kilobases obtained using concatemers o f phage tambda D N A .
Figure 2 shows the results o f hybridization with specific IS probes of EcoRl-digested genomic DNA originating from the same S. sonnei strains analysed previously with PFGE. Panel a represents the data obtained with IS/ as a probe. Because o f the high density o f bands at the top of the gel, we did not consider this region in our interpretation. Although the profiles ~ h n ~ a gond erctent o f homogeneity, we could detect significant differences between nearly all strains. A m o n g the three isolates which were indistinguishable from PFGE (4, 10 and OFB759), only the first two were identical, whereas the third showed at least six differences. Strain 199309 exhibited the same I S / p a t t e r n as strains 4 and 10. For the remaining isolates, the n u m ber o f differences between one another varied from one to seven. The patterns obtained with the IS2 probe (panel b, fig. 2) showed a lower n u m b e r o f bands (from 21 to 28) and their interpretation was thus easier. Although less apparent than with IS/, there was still some homogeneity between the profdes. All S. sonneistraJns were here clearly distinguishable from each other by at least six D N A fragments. The number of bands obtained with IS911 as a marker (lanes 4 to 8, panel c, figure 2) was significantly lower t h a n the preceding ones. Nevertheless, all the S. sonnei isolates could be distinguished f r o m each other, with the exception o f strains 4 a n d OFB759, ~ h i c h presented an identical restriction profile.
MOLECULAR TYPING OF SH1GELLA STRAL,%~S
493
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Iliit!lll
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Fig. 2.. IS distribution in the nine S. ~onneiisolates. EcoRI digest.s,of tota~ DNA weresepm-aeued,~a agarose gel, transferred to a n~'ion membranea~d hybfidiz~l w~th~lgoxigenin-~abei~ed iS pT~b~-~-Panc]s a+ b, and c show the patterns obtained w~thprobes ISI. IS2+a~ndISgtL resp~ively.
Comparison of NotI genomie restriction p a t t e r n of S. d t~enteriae, S. sonnei a n d S. flexneri strains We examined 5 S. dysenteriae and 8 S+ flex. neri isolates. All these strains were independent clinical isolates except for S. fleas~eri 3(03,309~, 3t02, and 3103, which originated from a single outbreak. Four S. sonnei strains among those previously analysed were also included here for comparison purposes. Analysis of the five S+ dysenteriae patterns (figure 3) strawed a surprisingly larger number of Notl fragments for strains 3, 6 and t2 than for the other 5higeila isolates. Further etectrophoresis performed under differem conditions confirmed this obsen'ation (data not shown). This could be due to the presence in these peculiar strains of repeated DNA sequences containing the NotI restriction site. Among the S. dysemeriae strains, numbers 3 and 12 shared an identical restriction pattern,
close to number 6 mud t-dgh!y divergem from I and 2, which are themselves dissimilar from one another. The 4 S. ftarneri o u t b r ~ k isolates showed nearly complete identity, Mth the exception of an additional band for 3093 and a1~othev missing for 3 ti)2, which furthermore presented some slight alterations in the size of two restriction fragments. Among the 4 independent S. flexneri isolates, number 1 was the closest to the epidemic strains. Isolates 1426 and Mg0T also showed some similarity to them, but to a Iesser extent. Strain 14 presented the most dissimilar restNction pattern.
Analysis of S. d).enteriae. S+ s onnei and S. flexneri strains using IS distribution in their genome Panel a h'~ figure 4 shows the ~ s t r i b m i o n of I S / i n the strains examined. Two fearares should
494
L. SOLDA TI A N D J.C. P I F F A R E T T I O~
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1
2
3
4
:5 6
7 8
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9 1 0 11 12 t 3 14 15 16 17
Fig. 3. PFGE analysis of genomic DNA from independent clinical isolates comprising five S. dysenteriae (S~ d.), four S. sonnei (S. s.) and four S. flexneri (S. f.), as well as four S. flexneri s t r ~ from an epidemic. Preparations of high MW DNA were digested with NotI and separated on agarose gel by field inversionelectrophoresis. Numbers at right are molecular sizes in kilohases obtained using concatemers of phage lambda DNA,
be pointed out. The first is the absence of I S / in strain S. dysenteriae 2, which is quite surprising for a species k n o w n to carry m a n y o f these transposable elements. The second is the faintness o f the S. f l e x n e r i b a n d s w h e n c o m p a r e d to
the results obtained with other IS elements (fig. 4, panels b and c). This m a y be explained by the I S / p r o b e we used, which is an I S I R deriving f r o m plasmid R I 0 0 and presenting 9.6 % divergence with the isoIS1 (IS/F) usual!y present
MOLECULAR TYPING OF SHIGELLA STRAINS °
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Fig. 4. IS distribution in dependent clinical i~olatescompiling five S. d),gn.'eriaeLS. d.L f~ur S. sonnet iS. s.) and foar S. flexneri (S. f.), as well as foer S. flexneri s~rains from an epidemic. EcoRi digests of total DNA were separated on agarose gel, transferred ~o a nylon me.~br~ne and hybridized with digoxigenindabelled IS probes. Panels a, b and c ~Now~he pa~ems obz~]r*e~ with probes lSl, IS2, and IS91t, respectively.
in S. flex:wri (Ohtsubo e: --!., !984). There ~s near identity between the IS/ patterns o f S. dysenteriae 3 and 12, and a lower but still s ~ nificant one between these two and S. dysenteriae 6. Despite the faintness o f the S. flexneri bands, we can nevertheless recognize the nearly complete identity o f the I S / p r o f i l e s o f strain 1 and the four epidemic isolates (3093, 3094, 3 t02 and 3103). Interestingly, these five strains all belong to serotype 2a. Between this group and the other three S. flexneri strains, the similarity of the restriction pattern decreased progressively according to the order 1426, MgoT and 14. Similar observations can be made for & flexneri with the IS2 probe (panel b o f figure 4), which furthermore showed sharper a n d less n u m e r o u s bands, thus allowing an easier interpretation. Analysis o f the tS2 patterns obtained wSth the S. dysemeriae strains showed that isolates 1 a n d 2 differ extensively from the others. Strains 3 and 12 are close to each onher but distinguishable and share consistent homology with strain 6. The Observations made for S. dysemeriae
¢~ra~i.s w-ere confirmed using t S g t l as a marker (panel c o f figure 4), Le. a n identical pattern for strains 3 and I2, a good correspondence o f these with strain 6, and extensive divergence o f straff~s wkh 1 and 2, ~hich are, moreover, distant from each other. Surprisingly, ~ e nearly complete identi~" of the fottr epidemic S. flexneri strains outtined by the I S / a n d IS2 profiles was not observed with t S g l l , which reverded few bat significam differences. C o m m o n bands were found ~ a i n between the epidemic isolates and S. fl~r,neri t as well as the e~her strains, but to a lesser extent.
DISCUSSION tn the present work, we examSmed two differem te&miques (PFGE and hybridization with tS elements) in order to assess ~heir usefutness in epidemioio~c tracing of Shigellg ~sola~es. Both methods consider the whole bacterial genome, and require about three days from isolation of' colonies to visualization o f resuks for routine laboratory diagnosis.
496
L. SOLDATt A N D J.C. PIFFARETTI
PFGE can be performed independently of the presence or absence o f insertion elements in the genome and can thus be used whatever the bacterial species. This technique is increasingly employed to map bacterial chromosomes and determine their size (Bancroft et at., 1989; Canard and Cole, 1989; Kauc et al., 1989; Smith el al., 1987; Suwanto and Kaplan, 1989; Tudor et al., 1990). Yet, its use in epidemiologic studies to compare genomic restriction patterns among related bacterial isolates is less frequent. Murray et aL (1990) showed that PFGE is a promising tool to evaluate enteroceccal infections. Using this same technique, Arbeit et al. (1990) could detect different rest;iction fragment prof'des in pathogenic E. eoli strains that were otherwise identical in electrophoretic type o f allozymes, serotype and antibiotic susceptibility. Moreover, Goering and Duensing (1990) used PFGE combined with an rRNA gene probe in the analysis o f staphylococci that were difficult to study epidemiologically by conventional means. However, one disadvantage o f PFGE is the difficulty in monitoring the entire ger,omic restriction pattern within a single PFGE assay. In our hands, application o f this method to the analysis of Shigella strains was successful, since we found sufficient discrimination among independent clinical isolates. Moreover, the epidemic S. flexneri strains presented nearly complete identity, as expected. Marking insertion elements naturally present in bacterial genomes with labelled IS probes is also a promising epidemiologic tool, as recently shown with Mycobacterium tuberculosis isolates (Hermans et al., 1990). Our results with Shigella strains showed that restriction o f the genome followed by electrophoresis and hybridization with tS probes gives good discrimination o f the isolates, provided that a number ~f preliminary conditions are fulfilled: first, the tS used as a probe must be present in all the strains to be tested; second, the copy number of this IS should be sufficiently high to allow a reliable comparison of the patterns obi:aJ~ed, but .~: not too large to enable good resolution o~ ~he fragments. The use o f digoxigenin label instead o f radioactivity allows not only the use o f the same IS probe for several hybridization experi-
ments, but also a better discrimination between fragments o f close molecular weight. With the strains of S. dysenteriae, S. flexneri, and S. sonnei we used in our study, the IS2 element was a satisfactory marker and represented a good compromise between IS al high (IS/) and low (IS911) copy number. Despite their homogeneity, 1S2 enabled a clear differentiation between each single strain o f S. sonnei. This element might therefore be successfully used to investigate outbreaks related to this bacterial species. It is relevant to note that the IS2 marker allowed differentiation among three S. sonnei strains which could not be otherwise distinguished by PFGE. This finding supports the hypothesis of a more rapid evolution o f IS profiles in the genome (due to their transponibility) compared to that o f chromosomal DNA per se. Use of the IS2 elements unequivocally showed the relatedness o f the S. flexneri epidemic strains, but also revealed a nearly identical pattern between them and strain 1, which belongs to the same serotype 2a. In this case, PFGE gave better discrimination between the epidemic isolates and strain 1. This apparent discrepancy, i.e. the similarity o f the IS2 patterns and the difference in the PFGE profiles, may be explained by a common ancestral strain for S. flexneri 1 and the other epidemic isolates. Due to homologous recombination within IS pairs in a cell and its descendant, large inversions in the chromosome may have occurred, gi~Sng rise to S. flexneri t and the epidemic strain lineages. These large inversions would lead to little differences in the IS patterns. Such recombination events have been repeatedly detected in E. coil strains (Birkenbihl and Vielmetter, 1989; Louarn et aL, 1985). A further peculiarity of the epidemic S. flexneri strains we analysed resides in the clear differences in the band proffies given by the IS911 probe and the similarity o f the band patterns obtained with probes I S / a n d IS2. This can be explained by a strong transposition acti~fb' of IS911 in this bacterial host. Preliminary data from our laboratory support this hyr-othesis. Alternatively, I S / a n d IS2 may have !ost the ahili-
MOLECULAR TYPING OF SItlGELLA STRAINS" ty to transpose, either by mutation in their sequence or by the presence of a host-inhibiting factor. Thus, when using IS markers to type strains, it is important to choose insertion elements showing sufficient stability. The presence among the S. dysenteriae strains of an isolate which did not show any I S / e l e ment was quite unexpected, even though N)maan et aL (1983) found considerable variation in IS/ distribution among natural isolates 0*2 E. coil (which is close to Shigella), with copy numbers ranging from 0 to more than 40 copies. In addition, Green etal. (1984) found that natural isolates o f E. coli identical in a l l o z y m e electrophoretic t)q3e may nevertheless differ in the presence, number or chromosomal location of the insertion element IS5. Even though all the S. sonnei strains belong to the same serotype, differences in the DNA profiles were found between the isolates analysed; moreover, in S, dysenteriae, two strains o f different serotype exhibited almost identical DNA patterns, both with PFOE and IS markers. Thus, one should be cautious when correlating serology with genome typing in the genus SMgella, particularly with isolates o f S. dysenteriae. The heterogeneity of this species is outlined by the finding of 3 completely different DNA proFries among the five strains of our collection. Additional investigations at the genetic and molecular level should be performed to better analyse the genetic diversity within this species. In conclusion, both PFGE and genome hybridization with IS elements proved to be potential powerful tools for typing Sf:igella strains. The latter technique, however, appears to distinguish clinical isolates more efficiently, provided a convenient IS probe such as IS2 is chosen.
,I97
P. Premki (Ge~evao Switzerland) fo~ reading ~he manusvript. This wo~rk~<~.ss~plxa~edb), grgm~31-9396.g£ frum t~e S~,%s National ~ienee Foundation,
Typag¢ mol~culaire de souehes de SMgella par ~lectrophor~e en champ pnis6 et p~.r hy~ridation avec des s~qac;~ccs d'inserfion Le g~nome de 18 spathes de Sh~geIta ~ I f e s ind~pendammem {9 S. sonnet, 5 S. dysemeriae ¢~ 4 S. flexnerO ainsi que celui de 4 souch~ de S. ftex'neri isol&s au coors d'une 6pid~mie, a 6'~,~a~aiys,6 par 61ectrophor~se en .champ put~.6 et par !a ~s~ribreton de s&luences ~qnsertion (ISL tS2, et IsgllL Malgr,5. lem sim;dimdegd~n&iq~e, les 9 soucbes ks& 16esde S. sonnei ont pu &re diffdrenei~es !es runesdm autres. Los 4 isolats ind@endams de S. fl~r.~eri ~at des profits d'ADN clairement diffdreats. Qu~r,t wax 4 souche~de cette memo e~-p&eiso{resau c o ~ 8kme ~pidrmJe, elles se sore rkv~!e2esprm~quernenl iden~iques par ~IectrophrrEse en champ ~ulse ou par ia d~stribution g~omiqne d ~ s~quenee~ d ' i ~ r r o n IS/e-: IS2. Cependant, ~'616mem tSglI s'es~ momrd ~op mobile po~.~r&re m~ise'¢~mme marque~r de ce~ scaches. Les isota~s de S. d),~e~*.eriaeom pu @~reausd distingurs ~ l'aide des deux re&bodes zvd~sges. La diversit~ g~n&ique wase en ~idence c~ez cet*e dero nitre esp~ce s'es~ r&~lre pa~ic~E&~em ~mp~rtame: sur les 5 isotats ar,ab~s~s, ~ro~spro~s d'ADN compl&emen~ diffd-reats om 6,~- mis e~ &~dcace. E n coneh:s~or~, F~lectropho~se en &mm~ p~}s{ ainsi Rue l'analyse de ~a d{~ribu.*io,~ de-~sgqae~.s dqnsertion ont drmon~. Ie~r u~lkd pe~e~tdeI1e po~r le t~age moldvutaire des spathes de Shigeg?a. Lie degrd de diff~rer~datio~ offort par oes deux m&~odes s'e~ rrvrl~ comparable, b~en que h d~-c:qnina.. tion des souches .anatys~esnit e~e md~zu.re pa~ typage par tes ~quevces d'in-:vrdon.
Mo~s.-cI~s: Shigeile, Geaeme~ T?T~a~e mel6aulaire; Elec~rophor/rse e~ ,:hamp puBe, Hyb~dar.:e~ avec des s~quer~cesd'ia~eAoa.
References Ackno~ledgemen~ We ~ank M. C.hamdaer~ToM~ase, France), T. Giger (St. Gallon, S~'iIger~a~:l),P.A.D. Grimoat (P~-~s,France), M. Hd~z(Lansam-~e,Sa'kzerland} and W. Zo]tiger{Aam~a~ Switzerland} for sappbing s~rakn.s.A: Burne=s(Na~o=aJ Center for Foc~dbor~e Diseases, [?,era, Svd~zerla=~l)performed ~rovar det~min'afion for spreeof the srxains.We are also indebted to M. Cham~er, P.A.D. Gr[me=t a~d
R.K. (1'99~LR~o~u~ono-fr~e~a evoI¢:~o~.a,:U ~g,cge~ce am*a~gEscaerie~a co~i¢~<~mre}*;ed ]~:nea~e~:
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Bancroft, I., Walk, C.P. & Oreu, E.V. (1989), Physical and genetic maps of the genome of heterocyst-forming Cyanobacterium Anabaena sp. strain PCC7120. J. Pact., 171, 5940-5948. Bergan, T. (1979), Bacteriophage typing of Shigella. Methods MicrobioL, 13, 17%286. Birkenbihl, R.P. & Viehnelter, V. (1989), Cosm[d derived map of E. coil strain BHB2600 in compatision to the map strain W31 I0. NucL Acids Res,, 17, 5057-5069. Branner, F,, Margadant, A., Peduzzi, R. & Piffaretti, J.C. (I 983L The ptasmid pattern as an epidemiologic tool for Salmonella typhimurium epidemics: comparison with the tysoucpe. J- infect. Dis., 148, 7-It. Canard, B. & Cole, S.T. (1989), Genome organization of the anaerobic pathogen Clost,~dium perfringens. Proc. nat. Acad. SoL (Wash.), 86, 6676-6680. Evins, G.M., Gheesllng, L.L. & Tauxe, R.V. (1989), Quality of commercially produced Sh~gella serogroupiug and serotyping antisera. J. din. MicrobioL, 26, 438-442. Fayet, O., Froment, Y. & Piffaretti, J.C. (1982)o Betatactamase-specifyingplasm.;dsisolated from Neisseria gonorrhoeae have retained an intact right part of a Tn3-1ike transposon. J. Bact., 149, 136-144. Galas, D.J. & Chandler, M. (1988), Bacterial insertion sequences, in "'Mobile DNA" (D.E. Berg & M.M. Howe) (pp. I0%162). Ar~erican Society for Microbiotogy, Washin~on, DC. Goering, R.V. & Duensing, T.D. (1990), Rapid field inversion get electrophotesis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci. J. clin. ]~¢icrobioL, 28, 426-429. Green, L., Miller, R.D., Dykhnizen, D.E. & Hartl, D.L. (1984), Distribution of DNA insertion element IS5 in natural isolates of Escherichia cofi. Proc. nat. Acrid. Sci. (Wash.), 81, 4500-4504. Gross, R.J., Thomas, L.V., Cheasty, T., Rowe, B. & Lindberg, A.A. (1989), Four new provisional serovars of Sh~ella. J. clin. MicrobioL, 27, 829-831. Hartl, D.L., Dykhu~zen, D.E., M~llcr, R.D., Green, L. & De Framond, J. (1983), Transposable dement IS5 improves growth rate orE. colicells without transposition. Cell, 35, 503-5t0. Hermans, P.W.M., Van Soolingen, D., Dale, J.W., Sehnitema, A.R.J., McAdam, R.A., Catty, D. & Embden, J.D.A. (1990), tn~rtion element IS986 from Mycobacterium tuberculosis: a useful toot for diagnosis and epidemio~o_~y of tuberculosis. J. clin. MicrobioL, 28, 205f-2058. Kauc, L., ~,L;tchell,M. & Goodgal, S.H. (t989), Size and physical map of the chromosome of Haemophilus influenzae. J. Baet., 171, 2474-2479. Louarn~ J.M., Bouche, J.P., Legertdre, F., Louarn, 3. &
Patte, J. (1985), Characterization and proI~erfies of very large inversions of the E. coli chromosome ~!e'~ the origin-to-terminus axes. MoL gen. Genet;cs, 201, 467-476. Maniatis, T., Fritsch, E.F. & Sambrook, ~ (~'~.2J, "Molecular cloning. A laboratory manual' . Cold Spring Harbor Laboratory, New York. McClelland, M., Jones, R,, Patel, Y. & Nelson, M° (1987), Restriction endonucleases for puI~,-d field mapping of bacterial genomes. Nucl. Acid~ . ,es., 15 -~%5-6005. Murray, B.E., Singh, K.V., Heath, J D., Sh~tqna, B.R. & Weinstock, G.M. (1990), Compamc ~ ~,? gepomic DNAs of different enterococcal isolates using restriction endonucleases with in~'requent recognition sites. J. clin. MicrobioL, 28, 205%2063. Nyrnan, K., Obtsubo, H., Davidson, D. & Ohtsubo, E. (1983), Distribution of insertion element IS/in natural isolates of EscheHchia coil Mol. gen. Genetics, 189, 516-518. Ohtsubo, E., Ohtsubo, H., Doroskiewicz, W., Nyman, K., Allen, D. & Davidson, D. (1984), An evolutionaD' analysis of Iso-l~l dements from Escherichia coli and Shigella strains. 3". gen. appL MicrobioL, 30, 35%376. Prado, D., Murray, B.E., Clearj,', T.G. & Pickering, L.K. (1987), Lir'~tations of using the plasmid pattern as an epidcmiological tool for clinical isolates of Shigella sonneio d. #~fect. Dis., 155, 31,l-316, Prate, M.F., Chandler, M. & Fayet, O. (1990), Transposition in Shigella dysenteriae: isolation and analysis of IS91i, a new member of the IS3 group of insertion sequences. J. Bact., 172, 4090-4099. Saint Giron, l., Fritz, H.J., Shaw, C., Tillmann, E. & Starlinger, P. (198t), Integration specificity of an artificial kanamycin transposon constructed by the in vitro insertior, of an internal Tn5 fragment into 1S2. MoL gen. Genetics, 183, 45-50. Sansonetti, P.J., Kopecko, D.J. & Formal, S.B. (1982), Involvement of a plasmid in the invasive ability of Shigella flerneri. Inject. Immun., 35, 852-860. Smith, G.E. & Summers, M.D. (1980), The bidirectional transfer of DNA and RNA to nitrocellulose. Analyt. Biochem., 109, 123-129. Smith, C.L., Econome, J.G., Schutt, A., K/co, S. & Cantor~ C.R. (1987), A physical map of the Escho¢ehia coli KI2 genome. Science, 236, 1448-1453. Suwanto, A. & Kaplan, S. (1989), Physica~ and genetic mapping of the Rhodobacter sphaeroides 2.4.1 gehome: genome ~.ze, fragment identification, and gene localization. J. Bact., 171, 5840-5849. ador, J..I., Marri, L., Piggot~ P.J. & Daneo-More, L. (1_990),Size of the Streptococcus mutans GS-5 chromosome as determined by palsed-fidd get electrophoresis, hrfect. Immuno, 58, 838-840.
Res. MicrobiaL 199i, 142, 48%498
Molecular typing of Shigella strains using pulsed field gel electrophoresis and genome hybridization with insertion seqaences L. Soldati and J.C. Piffaretti (*) Istituto Cantonale Batteriologico, via Ospedale 6, 6904 Lugano (Switzerland)
SUMMARY
ThG genomes of 18 independent ShigeUaisolates (9 SnJdeltasonnei, 5 Shige/la dysentedae and 4 Shigella fiexner~ as well as of 4 epidemic R, flexneristrains were analysed by pulsed field gel electrophoresis (PFGE}and by the distribution of insertion sequences (iS/, IS2 and IS911). Despite the close relatedness observed among the 9 independent S, sonnel, all of them could be differentiated from each other. The 4 independent $. flexner/isolates ~howcd c~a~!y disting~ishab."e D~A profiles. N~.dy complete genetic identity was detected within the 4 epidemic S. flexneri when analysed by PFGE or for |S 1 and IS2 patterns. However, IS911 was found to be too mobile in these epidemic S, flexneri to be used as a typing probe. The 5 S. dysentetiae isolates could also be distinguished by the techniques used. The diversity found within this species is striking: of the 5 investigated isolates, 3 completely different DNA prof'des were revealed. In conclusion, both PFGE and |S probing demonstrated their potential usefulness in molecular epidemiology and in typing of Shigella strains. The degree of differentiation given by these two methods was generany comparable, although IS probes showed better discrimination of the isolates.
Key-words: Shigella, Genome, MolecuMr typing; Pulsed field gel e~ectrcphoresis, Insertion sequences.
INTRODUCTION Diarrhoeai diseases including shigellosis are among the major causes o f morbidity and mortality in developing countries. Epidemiotogic investigations o f these diseases require typing systems enabling an efficient identification o f the sources o f infection. Serolc:gical analysis o f Shigella based on the specificity o f the O-antigen
is important for species identification, but still u n s a t i s f a c t o r y . C o m m e r c i a l l y available serogrouping and serotyping antisera do not always provide reliable or unambiguous results fEvins et aL, 1989). Furthermore, the presence o f non-typeable :strains zequires the determination o f new provisional serovars, which is timeconsuming and needs exten~ve confirmation to assess their epidemiologic significance (Gross et
Submitted December 3, 1990, accepted December t2, 1990. (*) Correspondingauthor.
490
L. SOLDA TI AND J.C. PIFFARETTI data). IS911 has been recently isolated from S. dysenteriae and is present in varying copy number in all 4 species of Shigetla and in E. coil KI2, but not in E. coil W (Prrre et al., 1990).
aL, 1989). Colicin- and phage-typing were approached by Abbott and Shannon (1958) and Bergan (1979), but these typing systems have not been further improved and at present are not used routinely. Moreover, these techniques are based on variation in phenotypic properties, which in many cases depend on a single or several unidentified genetic loci and therefore do not represent a global and reliable image of the bacterial genome. Plasmid pattern determination, a useful method for typing Sahnonella or other enteric bacteria (Brunner et aL, 1983), showed limitations when used as an epidemiological tool for clinical isolates o f S. sonnei (Prado et aL, 1987L
MATERIALS AND METHODS Bacterial strains and p!asmids
The strains used (listed in table I) were independent clinical isolates comprising 9 S. sonnei, 5 S. aysenteriae and 4 S. flexnerL Four additional S. flexneri strains (3093, 3094, 3 t 02 and 3103) originated from a same epidemic. Ptasmid pKH47 (L. Caro) provided the insertion element fS1, while IS911 was derived from plasmid pOFI39 (Pr~re et al., 1990). The source of the IS2 probe was a pBR322 plasmid derivative (pgalOPIS2; O. Fayet) containing the large Hindlll fragment (800 bp) of tS2 (Saint Giron et at., 1981). E. coli strain 803 was used for the propagation and isolation of these plasmids.
Moiecular methodologies applied to the whole genome are becoming more and more relevant in providing means for accurate bacterial classification and typing systems; however, they have not yet been fully investigated in the genus Shigella. This is the reason why we evaluated two such molecular procedures to identify and compare different Shigella strains, i.e. pulsed fie!d Eel e!ectrophoresis and the use of insertion sequences as genetic markers. On the one hand, we compared the genomic digestion patterns of independent or epidemic Shigella isolates by PFGE after cleavage with the restriction enzyme Notl. This endonuclease recognizes a specific sequence of 8 base pairs and thus produces a limited number of fragments from the genomic DNA. On the other hand, taking advantage of the presence of high copy numbers of some insertion elements in Shigella, we compared the IS patterns of the same strains. We used the insertion sequences IS/, IS2 and IS911. The elements IS/ and IS2 were first isolated from Escheriehia coil, where they are ,gresent at an average number of about 6 to 8 copies (Galas and Chandler, 1988, for review). In Shigella, with the exception of S. boydii, the copy number reaches 40 or more for ISI (Galas and Chandler, t988, for review) and 20 to 30 for IS2 (L. Soldati and J.C. Piffaretti, unpublished
IS = insertionsequence, PFGE = pulsedfield get elc~rophores~s,
Media a~d growth conditions
Shigella and E. coli strains were grown in L broth or on L agar (Maniatis et al., 1982) at 37~C with shaking. When needed, ampicillin (100 rtg/ml) was added. DNA manipulations
Plasmid DNA preparations, DNA digestions with restriction endonucleases and agarose get electrophoresis were as described previously (Fayet et aL, 1982). The method for chromosomal DNA isolation was that of Hartl et al. (1983). Preparation of labelled probes and hybridization experiments
After digestion of the adequate plasmids, DNA framnents containing the IS to be used as a probe were purified from agarose gels using the Genecleankit of Bio-101 (La Jolla, CA). DNA transfer from agarose gels to nylon membranes was done as described by Smith and Summer (1980). Hybridizations and visualization of the results were performed
i i
PMSF = phenyimethylsu]phonytfluoride. TBE = Tris-boricacid-EDTA.
. ~ I O L E C U L A R T Y P I N G O F SHIGELLA S T R A I N S
491
Table i. Bacterial strains. Strain S. sonnei
S. dysenteriae
S. flexneri
E. eoli
Comments 1 3 4 5 9 10 OFB759 199309 298246 1 2 3 6 12 M90T 1 14 1426 3093 3094 3102 3103 803
serotype serotype serotype serotype serotype serotype serotype serotype
Year of isolat ion
Origin or reference
1976 1979 1980 1980 t988 t988
M. Heilz M. Heitz M. Heitz M. Heitz M. Heitz M. Heinz Pr6re et aL (1990) T. Giger T. Giger P. Grimom P. Grimont P. Grimont P. Grimom P. Grimont Sansonetti et al. (t982) T. Giger T. Giger W. Zotliger P. Grimom P. Grimom P. Grimon~ P, Grimont L. Caro
1988 i988 1989 t989 1989 1989 t989
1 2 3 6 12 5 3c 3c
serotype 2a, epidemic strain serotype 2a, epidemic strain serotype 2a, epidemic strain serotype 2a, epidemic strain KI2, supE, supF, hsdM, hsdR
1982 1982 1988 1990 1990 t990 1990
Note: S. fiexneri 3093, 3094. 3102 and 3103 were from a single outbreak.
with the " D N A labeling and dete~ion kit, nonradioactive" (B6hringer Mannheim) using the protocols provided by the manufacturer.
Genomic DNA preparation, restriclion digestions and PFGE The preparation of high molecular weight chromosomal DNA was performed with some modifications from the method of McClenand et aL (1987). Overnight cultures of bacteria were concentrated twice in ET (EDTA t00 raM, Tris 10 mJM ; pH 8) and incubated 10 rain at 37~C. One ml of this suspension was mixed with an equal volume of 1.8 % low gel~ing agarose (freshly prepared and kept at 50'°C), poured immediately between two glass plates (separated by 1.5 ram) and kept ~0 rain at 4°C. Pieces of adequate size were cut and incubated in 8 mt ET buffer with 5 m g / m l lysozyme and 0,05 % sarcosyl for 2 h at 37~C with shaking, The samples were then transferred in 5 ml tysis solution (ET buffer with 0.5 rag/rot proteinase K and 1 % SDS), shaken overnight at 50°C and subsequently dialysed in 50 ml TE (Tris 1 0 m M , EDTA I rmM; pH 8) contaiaing 0.1 mM phenylmethylsulphonyl fluoride (PMSFt for
1 h at room tc-mperature. After dialysis repeated twice without PMSF, the inserts were maintair~ed in TE at 4~C. Fo~"digestion, each sample w ~ incuba[ed in 200 ,ul of' appropriate buffer supplemented with about 15 units of restriction enzyme and incubated h at the appropriate temperature. The inserts were the~ rinsed in one ml TE a~d loaded onto a 0.8 ~ agarose gel. Field inversion gel electrophoresis was performed for 25-29 h and 180-190 volts in TBE (45 mM Tris, 45 rmM boric acid and 0.5 mM EDTA) at 14=C. The temperature was kept conAant by co,ofing the buffer during its recircularization. Field inversion was supplied by a programmable power controller (model PPI-2(~; MJ R~earch~ Londoa).
RESULTS
Comparison of the N o t f genomic restriction patterns of 9 independent S, sonnei s~ra~ns A m o n g ~he 4 species o f the genus Shigetla, S. s o n n e i i s the most homogeneous, tt comprises a single serovar. U s i n g P F G E a f t e r d i g e s t i o n o f
L. SOLDA TI AND J.C. PIFFARETTI
492
the genomes with the endonuclease NotI, we examined 9 independent S. sonnei clinical isoiates (fig. I). Electrophoresis conditions were those allowing the visualization of the highest number o f bands between 40 and 600 Kb. All
•- ~
O~ O~ ~D U~O~ P~ t~ c~ anO~m 0 U- O~ O~ ~ ' u ~ O~ ~ 0 ~ ~
the strains analysed showed similar band patterns; similarity was particularly evident between isolates 1, 3 and 298246, as well as between isolates 4, 5, l0 and OFB759. In order to establish a comprehensive pattern o f all the fragments, we used several other PFGE conditions and confirmed the almost complete identity o f the 4, 10 and OFB759 patterns. These conditions also revealed that the other strains differed by at least three or four bands (results not shown).
Comparison of the 9 S. sonnei strains using IS distribution in their genomes
~d~cd~aid~id ca to c a t ~ to tn to ~ to
!! ,,..,, 500
4CO
;isn.lii;
300
11;! lt11,: ;
200
;
100
123456789 Fig. l. PFGE analysis of genomic D N A from nine independent S. sonnei isolates. Preparations of high MW D N A were digested with NotI and separated on agarose gel by field inversion dectrophoresis. Numbers at right are the molecu,ar sizes in kilobases obtained using concatemers o f phage tambda D N A .
Figure 2 shows the results o f hybridization with specific IS probes of EcoRl-digested genomic DNA originating from the same S. sonnei strains analysed previously with PFGE. Panel a represents the data obtained with IS/ as a probe. Because o f the high density o f bands at the top of the gel, we did not consider this region in our interpretation. Although the profiles ~ h n ~ a gond erctent o f homogeneity, we could detect significant differences between nearly all strains. A m o n g the three isolates which were indistinguishable from PFGE (4, 10 and OFB759), only the first two were identical, whereas the third showed at least six differences. Strain 199309 exhibited the same I S / p a t t e r n as strains 4 and 10. For the remaining isolates, the n u m ber o f differences between one another varied from one to seven. The patterns obtained with the IS2 probe (panel b, fig. 2) showed a lower n u m b e r o f bands (from 21 to 28) and their interpretation was thus easier. Although less apparent than with IS/, there was still some homogeneity between the profdes. All S. sonneistraJns were here clearly distinguishable from each other by at least six D N A fragments. The number of bands obtained with IS911 as a marker (lanes 4 to 8, panel c, figure 2) was significantly lower t h a n the preceding ones. Nevertheless, all the S. sonnei isolates could be distinguished f r o m each other, with the exception o f strains 4 a n d OFB759, ~ h i c h presented an identical restriction profile.
MOLECULAR TYPING OF SH1GELLA STRAL,%~S
493
,..,,,++
,= + ++ d li ,~ ,~ + i l i o+ o+ + ,~ + +,i o'J ¢J +
Iliit!lll
+l.,l +°,:-:-+.'+ "'++, +'"" =
,. :
- +,,,,+++m
+ m m~++'+
+'W+Im~
1 ~
a
b
3
4
5
6
7
~
9
¢
Fig. 2.. IS distribution in the nine S. ~onneiisolates. EcoRI digest.s,of tota~ DNA weresepm-aeued,~a agarose gel, transferred to a n~'ion membranea~d hybfidiz~l w~th~lgoxigenin-~abei~ed iS pT~b~-~-Panc]s a+ b, and c show the patterns obtained w~thprobes ISI. IS2+a~ndISgtL resp~ively.
Comparison of NotI genomie restriction p a t t e r n of S. d t~enteriae, S. sonnei a n d S. flexneri strains We examined 5 S. dysenteriae and 8 S+ flex. neri isolates. All these strains were independent clinical isolates except for S. fleas~eri 3(03,309~, 3t02, and 3103, which originated from a single outbreak. Four S. sonnei strains among those previously analysed were also included here for comparison purposes. Analysis of the five S+ dysenteriae patterns (figure 3) strawed a surprisingly larger number of Notl fragments for strains 3, 6 and t2 than for the other 5higeila isolates. Further etectrophoresis performed under differem conditions confirmed this obsen'ation (data not shown). This could be due to the presence in these peculiar strains of repeated DNA sequences containing the NotI restriction site. Among the S. dysemeriae strains, numbers 3 and 12 shared an identical restriction pattern,
close to number 6 mud t-dgh!y divergem from I and 2, which are themselves dissimilar from one another. The 4 S. ftarneri o u t b r ~ k isolates showed nearly complete identity, Mth the exception of an additional band for 3093 and a1~othev missing for 3 ti)2, which furthermore presented some slight alterations in the size of two restriction fragments. Among the 4 independent S. flexneri isolates, number 1 was the closest to the epidemic strains. Isolates 1426 and Mg0T also showed some similarity to them, but to a Iesser extent. Strain 14 presented the most dissimilar restNction pattern.
Analysis of S. d).enteriae. S+ s onnei and S. flexneri strains using IS distribution in their genome Panel a h'~ figure 4 shows the ~ s t r i b m i o n of I S / i n the strains examined. Two fearares should
494
L. SOLDA TI A N D J.C. P I F F A R E T T I O~
o)
1
2
3
4
:5 6
7 8
~0~0)
O0
9 1 0 11 12 t 3 14 15 16 17
Fig. 3. PFGE analysis of genomic DNA from independent clinical isolates comprising five S. dysenteriae (S~ d.), four S. sonnei (S. s.) and four S. flexneri (S. f.), as well as four S. flexneri s t r ~ from an epidemic. Preparations of high MW DNA were digested with NotI and separated on agarose gel by field inversionelectrophoresis. Numbers at right are molecular sizes in kilohases obtained using concatemers of phage lambda DNA,
be pointed out. The first is the absence of I S / in strain S. dysenteriae 2, which is quite surprising for a species k n o w n to carry m a n y o f these transposable elements. The second is the faintness o f the S. f l e x n e r i b a n d s w h e n c o m p a r e d to
the results obtained with other IS elements (fig. 4, panels b and c). This m a y be explained by the I S / p r o b e we used, which is an I S I R deriving f r o m plasmid R I 0 0 and presenting 9.6 % divergence with the isoIS1 (IS/F) usual!y present
MOLECULAR TYPING OF SHIGELLA STRAINS °
|
°
l!I ,t, r
!lltlm,, ,,!i , !!!lllt "2"
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495
-
-
. C _"_ = ;"= = , -
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" '. ." . ".
.
,-..."':,".'"'---.--. EI
~'~IIl,~-~I~
-
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Fig. 4. IS distribution in dependent clinical i~olatescompiling five S. d),gn.'eriaeLS. d.L f~ur S. sonnet iS. s.) and foar S. flexneri (S. f.), as well as foer S. flexneri s~rains from an epidemic. EcoRi digests of total DNA were separated on agarose gel, transferred ~o a nylon me.~br~ne and hybridized with digoxigenindabelled IS probes. Panels a, b and c ~Now~he pa~ems obz~]r*e~ with probes lSl, IS2, and IS91t, respectively.
in S. flex:wri (Ohtsubo e: --!., !984). There ~s near identity between the IS/ patterns o f S. dysenteriae 3 and 12, and a lower but still s ~ nificant one between these two and S. dysenteriae 6. Despite the faintness o f the S. flexneri bands, we can nevertheless recognize the nearly complete identity o f the I S / p r o f i l e s o f strain 1 and the four epidemic isolates (3093, 3094, 3 t02 and 3103). Interestingly, these five strains all belong to serotype 2a. Between this group and the other three S. flexneri strains, the similarity of the restriction pattern decreased progressively according to the order 1426, MgoT and 14. Similar observations can be made for & flexneri with the IS2 probe (panel b o f figure 4), which furthermore showed sharper a n d less n u m e r o u s bands, thus allowing an easier interpretation. Analysis o f the tS2 patterns obtained wSth the S. dysemeriae strains showed that isolates 1 a n d 2 differ extensively from the others. Strains 3 and 12 are close to each onher but distinguishable and share consistent homology with strain 6. The Observations made for S. dysemeriae
¢~ra~i.s w-ere confirmed using t S g t l as a marker (panel c o f figure 4), Le. a n identical pattern for strains 3 and I2, a good correspondence o f these with strain 6, and extensive divergence o f straff~s wkh 1 and 2, ~hich are, moreover, distant from each other. Surprisingly, ~ e nearly complete identi~" of the fottr epidemic S. flexneri strains outtined by the I S / a n d IS2 profiles was not observed with t S g l l , which reverded few bat significam differences. C o m m o n bands were found ~ a i n between the epidemic isolates and S. fl~r,neri t as well as the e~her strains, but to a lesser extent.
DISCUSSION tn the present work, we examSmed two differem te&miques (PFGE and hybridization with tS elements) in order to assess ~heir usefutness in epidemioio~c tracing of Shigellg ~sola~es. Both methods consider the whole bacterial genome, and require about three days from isolation of' colonies to visualization o f resuks for routine laboratory diagnosis.
496
L. SOLDATt A N D J.C. PIFFARETTI
PFGE can be performed independently of the presence or absence o f insertion elements in the genome and can thus be used whatever the bacterial species. This technique is increasingly employed to map bacterial chromosomes and determine their size (Bancroft et at., 1989; Canard and Cole, 1989; Kauc et al., 1989; Smith el al., 1987; Suwanto and Kaplan, 1989; Tudor et al., 1990). Yet, its use in epidemiologic studies to compare genomic restriction patterns among related bacterial isolates is less frequent. Murray et aL (1990) showed that PFGE is a promising tool to evaluate enteroceccal infections. Using this same technique, Arbeit et al. (1990) could detect different rest;iction fragment prof'des in pathogenic E. eoli strains that were otherwise identical in electrophoretic type o f allozymes, serotype and antibiotic susceptibility. Moreover, Goering and Duensing (1990) used PFGE combined with an rRNA gene probe in the analysis o f staphylococci that were difficult to study epidemiologically by conventional means. However, one disadvantage o f PFGE is the difficulty in monitoring the entire ger,omic restriction pattern within a single PFGE assay. In our hands, application o f this method to the analysis of Shigella strains was successful, since we found sufficient discrimination among independent clinical isolates. Moreover, the epidemic S. flexneri strains presented nearly complete identity, as expected. Marking insertion elements naturally present in bacterial genomes with labelled IS probes is also a promising epidemiologic tool, as recently shown with Mycobacterium tuberculosis isolates (Hermans et al., 1990). Our results with Shigella strains showed that restriction o f the genome followed by electrophoresis and hybridization with tS probes gives good discrimination o f the isolates, provided that a number ~f preliminary conditions are fulfilled: first, the tS used as a probe must be present in all the strains to be tested; second, the copy number of this IS should be sufficiently high to allow a reliable comparison of the patterns obi:aJ~ed, but .~: not too large to enable good resolution o~ ~he fragments. The use o f digoxigenin label instead o f radioactivity allows not only the use o f the same IS probe for several hybridization experi-
ments, but also a better discrimination between fragments o f close molecular weight. With the strains of S. dysenteriae, S. flexneri, and S. sonnei we used in our study, the IS2 element was a satisfactory marker and represented a good compromise between IS al high (IS/) and low (IS911) copy number. Despite their homogeneity, 1S2 enabled a clear differentiation between each single strain o f S. sonnei. This element might therefore be successfully used to investigate outbreaks related to this bacterial species. It is relevant to note that the IS2 marker allowed differentiation among three S. sonnei strains which could not be otherwise distinguished by PFGE. This finding supports the hypothesis of a more rapid evolution o f IS profiles in the genome (due to their transponibility) compared to that o f chromosomal DNA per se. Use of the IS2 elements unequivocally showed the relatedness o f the S. flexneri epidemic strains, but also revealed a nearly identical pattern between them and strain 1, which belongs to the same serotype 2a. In this case, PFGE gave better discrimination between the epidemic isolates and strain 1. This apparent discrepancy, i.e. the similarity o f the IS2 patterns and the difference in the PFGE profiles, may be explained by a common ancestral strain for S. flexneri 1 and the other epidemic isolates. Due to homologous recombination within IS pairs in a cell and its descendant, large inversions in the chromosome may have occurred, gi~Sng rise to S. flexneri t and the epidemic strain lineages. These large inversions would lead to little differences in the IS patterns. Such recombination events have been repeatedly detected in E. coil strains (Birkenbihl and Vielmetter, 1989; Louarn et aL, 1985). A further peculiarity of the epidemic S. flexneri strains we analysed resides in the clear differences in the band proffies given by the IS911 probe and the similarity o f the band patterns obtained with probes I S / a n d IS2. This can be explained by a strong transposition acti~fb' of IS911 in this bacterial host. Preliminary data from our laboratory support this hyr-othesis. Alternatively, I S / a n d IS2 may have !ost the ahili-
MOLECULAR TYPING OF SItlGELLA STRAINS" ty to transpose, either by mutation in their sequence or by the presence of a host-inhibiting factor. Thus, when using IS markers to type strains, it is important to choose insertion elements showing sufficient stability. The presence among the S. dysenteriae strains of an isolate which did not show any I S / e l e ment was quite unexpected, even though N)maan et aL (1983) found considerable variation in IS/ distribution among natural isolates 0*2 E. coil (which is close to Shigella), with copy numbers ranging from 0 to more than 40 copies. In addition, Green etal. (1984) found that natural isolates o f E. coli identical in a l l o z y m e electrophoretic t)q3e may nevertheless differ in the presence, number or chromosomal location of the insertion element IS5. Even though all the S. sonnei strains belong to the same serotype, differences in the DNA profiles were found between the isolates analysed; moreover, in S, dysenteriae, two strains o f different serotype exhibited almost identical DNA patterns, both with PFOE and IS markers. Thus, one should be cautious when correlating serology with genome typing in the genus SMgella, particularly with isolates o f S. dysenteriae. The heterogeneity of this species is outlined by the finding of 3 completely different DNA proFries among the five strains of our collection. Additional investigations at the genetic and molecular level should be performed to better analyse the genetic diversity within this species. In conclusion, both PFGE and genome hybridization with IS elements proved to be potential powerful tools for typing Sf:igella strains. The latter technique, however, appears to distinguish clinical isolates more efficiently, provided a convenient IS probe such as IS2 is chosen.
,I97
P. Premki (Ge~evao Switzerland) fo~ reading ~he manusvript. This wo~rk~<~.ss~plxa~edb), grgm~31-9396.g£ frum t~e S~,%s National ~ienee Foundation,
Typag¢ mol~culaire de souehes de SMgella par ~lectrophor~e en champ pnis6 et p~.r hy~ridation avec des s~qac;~ccs d'inserfion Le g~nome de 18 spathes de Sh~geIta ~ I f e s ind~pendammem {9 S. sonnet, 5 S. dysemeriae ¢~ 4 S. flexnerO ainsi que celui de 4 souch~ de S. ftex'neri isol&s au coors d'une 6pid~mie, a 6'~,~a~aiys,6 par 61ectrophor~se en .champ put~.6 et par !a ~s~ribreton de s&luences ~qnsertion (ISL tS2, et IsgllL Malgr,5. lem sim;dimdegd~n&iq~e, les 9 soucbes ks& 16esde S. sonnei ont pu &re diffdrenei~es !es runesdm autres. Los 4 isolats ind@endams de S. fl~r.~eri ~at des profits d'ADN clairement diffdreats. Qu~r,t wax 4 souche~de cette memo e~-p&eiso{resau c o ~ 8kme ~pidrmJe, elles se sore rkv~!e2esprm~quernenl iden~iques par ~IectrophrrEse en champ ~ulse ou par ia d~stribution g~omiqne d ~ s~quenee~ d ' i ~ r r o n IS/e-: IS2. Cependant, ~'616mem tSglI s'es~ momrd ~op mobile po~.~r&re m~ise'¢~mme marque~r de ce~ scaches. Les isota~s de S. d),~e~*.eriaeom pu @~reausd distingurs ~ l'aide des deux re&bodes zvd~sges. La diversit~ g~n&ique wase en ~idence c~ez cet*e dero nitre esp~ce s'es~ r&~lre pa~ic~E&~em ~mp~rtame: sur les 5 isotats ar,ab~s~s, ~ro~spro~s d'ADN compl&emen~ diffd-reats om 6,~- mis e~ &~dcace. E n coneh:s~or~, F~lectropho~se en &mm~ p~}s{ ainsi Rue l'analyse de ~a d{~ribu.*io,~ de-~sgqae~.s dqnsertion ont drmon~. Ie~r u~lkd pe~e~tdeI1e po~r le t~age moldvutaire des spathes de Shigeg?a. Lie degrd de diff~rer~datio~ offort par oes deux m&~odes s'e~ rrvrl~ comparable, b~en que h d~-c:qnina.. tion des souches .anatys~esnit e~e md~zu.re pa~ typage par tes ~quevces d'in-:vrdon.
Mo~s.-cI~s: Shigeile, Geaeme~ T?T~a~e mel6aulaire; Elec~rophor/rse e~ ,:hamp puBe, Hyb~dar.:e~ avec des s~quer~cesd'ia~eAoa.
References Ackno~ledgemen~ We ~ank M. C.hamdaer~ToM~ase, France), T. Giger (St. Gallon, S~'iIger~a~:l),P.A.D. Grimoat (P~-~s,France), M. Hd~z(Lansam-~e,Sa'kzerland} and W. Zo]tiger{Aam~a~ Switzerland} for sappbing s~rakn.s.A: Burne=s(Na~o=aJ Center for Foc~dbor~e Diseases, [?,era, Svd~zerla=~l)performed ~rovar det~min'afion for spreeof the srxains.We are also indebted to M. Cham~er, P.A.D. Gr[me=t a~d
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