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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p. 1673–1680 Vol. 41, No. 40095-1137/03/$08.00�0 DOI: 10.1128/JCM.41.4.1673–1680.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Tandem Tetramer-Based Microsatellite Fingerprinting for Typing ofProteus mirabilis Strains

Tomasz Cieslikowski,1* Dobrosława Gradecka,2 Magdalena Mielczarek,2 and Wiesław Kaca1,2

Centre of Microbiology and Virology, Polish Academy of Sciences,1 and Institute of Microbiology and Immunology,University of Łodz,2 Łodz, Poland

Received 25 July 2002/Returned for modification 15 October 2002/Accepted 7 January 2003

Two microsatellite tandem repeated tetramers, (GACA)4 and (CAAT)4, were used for Proteus mirabilis straindifferentiation. The microsatellite-based PCR tests were applied for the examination of interstrain diversity for87 P. mirabilis strains. Forty-six of the investigated strains were clinical isolates (5 were hospital isolates and39 were outpatient clinic isolates); 42 strains were derived from the Kauffmann-Perch collection of laboratorystrains. Fingerprinting done with the tetramers had a high discrimination ability [0.992 and 0.940 for (GACA)4and (CAAT)4, respectively]. The distributions of clinical isolates among well-defined laboratory strains,determined by numerical analysis (unweighted pair-group method with arithmetic averages; Dice similaritycoefficient), proved their genetic similarity to reference strains in the Kauffmann-Perch collection. Thisanalysis also indicated that it is possible to estimate some phenotypic properties of P. mirabilis clinical isolatessolely on the basis of microsatellite fingerprinting.

Proteus spp. are mobile gram-negative bacteria common inboth the natural environment and animal or human intestinaltracts. Proteus spp. are also known etiologic agents for menin-gitis and numerous bacteremias (8, 20–23, 43). Urinary tractinfections are among the most frequent bacterial infections(19), and Proteus mirabilis strains are one of the most commoncauses of urinary tract infections (7%), third after Escherichiacoli (52%) and Enterococcus spp.(12%) (11). Such infectionsoccur commonly among patients with structural defects of theurinary tract (6, 38, 39). The presence of P. mirabilis rodswithin a urease-induced bladder stone matrix was visualizedrecently (24). Moreover, some results suggest a possible etio-pathogenic role of P. mirabilis in rheumatoid arthritis (9, 31),and some nosocomial transmission events have been reported(31). Because of the increasing spread and clinical significanceof P. mirabilis rods (13, 15, 30, 31, 32), studies of effectivemethods for epidemiological investigations are of great impor-tance.

Out of the numerous types of simple sequence repeats pro-posed as tools for very sensitive bacterial fingerprinting (25, 27,48, 50, 51, 54), many microsatellites have been described asbeing useful for microbial differentiation, especially below thelevel of species (1, 10, 26, 28, 33, 34, 47, 48, 49, 53).

Most of the molecular fingerprinting methods applied forthe differentiation of Proteus (35, 36, 44), however, are notsensitive enough for more detailed interstrain differentiation.In particular, no specific method allowing for P. mirabilis dif-ferentiation, especially below the serotype level, has been de-scribed so far.

In this study, we have focused on microsatellite-based meth-ods supplying patterns specific for particular P. mirabilisstrains. The aim of the study was to verify how effective mic-

rosatellites are for P. mirabilis fingerprinting; in particular, weexamined whether tandem tetramer-based PCR is applicableto Proteus strain differentiation or typing as well as the sensi-tivities of PCR methods based on tandem repeated tetramers.In addition, we compared the efficiencies of these methods andother important Proteus typing methods. Finally, we examinedhow informative these patterns are in relation to other prop-erties of P. mirabilis strains.

Two microsatellite sequences were used for P. mirabilis lab-oratory strain differentiation: (GACA)4 and (CAAT)4. Thestudies were performed with 40 P. mirabilis strains from theserologically defined Kauffmann-Perch (23) collection andwith 42 P. mirabilis clinical isolates.

MATERIALS AND METHODS

Bacterial strains. P. mirabilis laboratory strains were from the Czech Collec-tion of Type Cultures, Institute of Microbiology and Epidemiology, Prague,Czech Republic. P. mirabilis strain S1959 was obtained from the Institute ofMicrobiology and Immunology, University of Łodz, Łodz, Poland. Thirty-sixclinical isolates of P. mirabilis from urine were obtained from outpatient clinicsin Łodz and given the prefix “ZOZ”; they were kindly supplied by HalinaSkulimowska (Table 1). An additional six clinical isolates were derived from theMilitary Medical Academy Hospital, Łodz, Poland, and given the prefix “WAM”;they were kindly supplied by Maria Kowalska (Table 1).

Bacterial culture and DNA isolation. Bacteria were cultivated in 3 ml ofLuria-Bertani (LB) medium for 12 h at 37°C. Then, 1 ml of the culture wascentrifuged for 3 min. The pellet was resuspended in 100 �l of Tris-EDTA buffer.After 30 min of incubation with 10 �l of proteinase K solution (20 mg/ml) at37°C, chromosomal DNA was isolated with a genomic DNA isolation kit (A&ABiotechnology, Gdansk, Poland) and then dissolved in 200 �l of Tris (pH 8.2).DNA samples were kept at �20°C until PCR was performed.

The amount of isolated DNA was verified with a UV spectrophotometer(Ultraspec 2000; Pharmacia LKB) at 260 nm and by electrophoresis in 2%agarose (Serva; analytical grade) in 0.04 M Tris-acetate–1 mM EDTA buffer (pH7.8).

Primers. Sequences of oligonucleotides (synthesized by Ransom Hill Bio-science Inc., Ramona Calif.) for genomic DNA analysis were as follows: (GACA)4, 5�-GACAGACAGACAGACA-3� (16 nucleotides) (26), and (CAAT)4, 5�-CAATCAATCAATCAAT-3� (16 nucleotides) (33).

Primer target site computer analysis. The presence of the doubled tetramerrepeats (GACA)4 and (CAAT)4 in the known part of the Proteus genome wasconfirmed. The National Center for Biotechnology Information GenBank data-

* Corresponding author. Mailing address: Centre of Microbiologyand Virology, Polish Academy of Sciences, Łodowa 106, 93-232 Łodz,Poland. Phone: 48 (42) 6771-245. Fax: 48 (42) 6771-230. E-mail:[email protected].

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base was explored for the presence of P. mirabilis DNA sequences. This searchwas followed by a search for tandem repeated tetramers among the 84 P.mirabilis sequences found. For this analysis, two programs, Quicksearch andNesearch, from the PC/Gene packet, were used.

PCR conditions. A master mixture, the same for each reaction type, containedall reagents except for genomic DNA. Each thin-walled vial (MJ Research)contained 25 �l of reaction solution, which consisted of 2.5 mM deoxynucleotidetriphosphate mixture (dATP, dTTP, dCTP, and dGTP; TaKaRa), 2.5 �l of 10�reaction buffer, 100 pM primer, 1 U of thermostable polymerase (Dynazyme;Finnzyme), 18M� ultrapure water (Millipore), and 20 ng of template DNA.Amplification was carried out with a UNO II thermocycler (Biometra). An initial7 min of denaturation at 95°C was followed by 32 cycles of annealing (40°C for1 min), extension (65°C for 1 min), and denaturation (92°C for 30 s). Thereaction was completed by 16 min of extension at 65°C. During gel electrophore-

sis in an MGU 602T unit (CBS Scientific), aliquots of amplification products (5�l) were resolved against molecular weight markers (Ideal, Poland, Gdansk) in2% agarose (Serva; analytical grade). The gels were stained with ethidium bro-mide solution and photographed with the aid of a BioDoc system (Biometra) andthe computer program SM Camera (FAST Multimedia AG 1993, version1.1).

Electrophoretic pattern analysis. For investigation of PCR product diversity,computer-assisted pattern analysis was carried out (GelCompar, version 4.0;Applied Maths, Kortijk, Belgium). The bands chosen for the analysis wereselected manually from the hard-copy photograph and the densitometric curvesof the appropriate electrophoretic paths. For all electropherograms, the samebackground subtraction procedure (the rolling-disk procedure, as suggested bythe program authors) was used. Normalization procedures included the internaland external reference band sets as shown in Fig. 1. Electrophoretic patternswere normalized to common internal sets of amplicons next to the external

TABLE 1. Proteus strains examined in these studies

Strain (serotype) Species Strain (serotype) Species

PrO 10/52 (O3)..................................................................... P. mirabilisPrK 6/57 (O3) ....................................................................... P. mirabilisPrK 7/57 (O3) ....................................................................... P. mirabilisPrK 8/57 (O3) ....................................................................... P. mirabilisPrK 12/57 (O5) ..................................................................... P. mirabilis

PrK 13/57 (O5) ..................................................................... P. mirabilisPrK 14/57 (O6) ..................................................................... P. mirabilisPrK 15/57 (O7) ..................................................................... P. mirabilisPrK 16/57 (O7) ..................................................................... P. mirabilisPrK 18/57 (O9) ..................................................................... P. mirabilis

PrK 19/57 (O10) ................................................................... P. mirabilisPrK 20/57 (O10) ................................................................... P. mirabilisPrK 21/57 (O10) ................................................................... P. mirabilisPrK 24/57 (O11) ................................................................... P. mirabilisPrK 26/57 (O13) ................................................................... P. mirabilis


PrK 34/57 (O18) ................................................................... P. mirabilisPrK 38/57 (O20) ................................................................... P. mirabilisPrK 41/57 (O23) ................................................................... P. mirabilisPrK 42/57 (O23) ................................................................... P. mirabilisPrK 43/57 (O23) ................................................................... P. vulgaris


PrK 51/57 (O28) ................................................................... P. mirabilisPrK 52/57 (O29) ................................................................... P. mirabilisPrK 53/57 (O30) ................................................................... P. mirabilisPrK 56/57 (O31) ................................................................... P. vulgarisPrK 58/57 (O32) ................................................................... P. mirabilis


PrK 74/57 (O48) ................................................................... P. mirabilisPrK 75/57 (O49) ................................................................... P. mirabilisS1959 (O3) ............................................................................ P. mirabilisZOZ 105................................................................................ P. mirabilis

a The species was not determined.

ZOZ 670 ........................................................................... Proteusa

ZOZ 367 ........................................................................... P. mirabilisZOZ 173 ........................................................................... P. mirabilisZOZ 168 ........................................................................... P. mirabilisZOZ 63A .......................................................................... P. mirabilisZOZ 19 ............................................................................. P. mirabilis

ZOZ 72 ............................................................................. P. mirabilisZOZ 13 ............................................................................. P. mirabilisZOZ 14 ............................................................................. P. mirabilisZOZ 191 ........................................................................... P. mirabilisZOZ 253 ........................................................................... P. mirabilis

ZOZ 87 ............................................................................. P. vulgarisZOZ 302 ........................................................................... P. mirabilisZOZ 303 ........................................................................... P. mirabilisZOZ 304 ........................................................................... P. mirabilisZOZ 352 ........................................................................... P. mirabilis

ZOZ 58 ............................................................................. P. vulgarisZOZ 256 ........................................................................... P. mirabilisZOZ 216 ........................................................................... P. mirabilisZOZ 203 ........................................................................... P. mirabilisZOZ 42 ............................................................................. Proteusa

ZOZ 63B........................................................................... P. mirabilisZOZ 220 ........................................................................... P. mirabilisZOZ 200 ........................................................................... P. mirabilisZOZ 198 ........................................................................... P. mirabilisZOZ 70 ............................................................................. P. mirabilis

ZOZ 99 ............................................................................. P. mirabilisZOZ 186 ........................................................................... P. mirabilisZOZ 383 ........................................................................... P. mirabilisZOZ 349 ........................................................................... P. mirabilisZOZ 376 ........................................................................... P. mirabilis

ZOZ 290 ........................................................................... P. mirabilisZOZ 3 ............................................................................... P. mirabilisZOZ 34 ............................................................................. P. mirabilisZOZ 18 ............................................................................. P. mirabilisZOZ 48 II......................................................................... P. mirabilis

ZOZ 48 I .......................................................................... P. mirabilisZOZ 276 ........................................................................... P. mirabilisZOZ 494 ........................................................................... P. mirabilisWAM 01 ........................................................................... P. mirabilisWAM 02 ........................................................................... P. mirabilis

WAM 03 ........................................................................... P. mirabilisWAM 05 ........................................................................... P. mirabilisWAM 06 ........................................................................... P. mirabilis

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molecular weight markers placed on the peripheral paths of both sides of thegels. The correlations among the investigated species were based on the elec-trophoretic band distribution and the Pearson product-moment correlation co-efficient. For comparison of the electrophoretic patterns and determination oftheir similarities (construction of dendrograms), the UPGMA (unweighted pair-group method with arithmetic averages) clustering algorithm was used (45). Forthe band pattern analysis, the Dice similarity coefficient was used.

The levels of effectiveness of the applied fingerprinting methods were com-pared by using the formula described by Hunter (14):

D � 1 � � 1N �N � 1��

j 1

N

aj

where N is the total number of investigated strains, aj is the number of indistin-guishable strains in the experiment, and D is the discrimination power.

RESULTS

Several strains were arbitrarily chosen to examine the repro-ducibility of the typing method and to test the stability of thebacterial strains. The reproducibility of PCR patterns was con-firmed in two series of reactions performed on a DNA matrixisolated from six P. mirabilis laboratory strains, PrK 15/57(O7), PrK 18/57 (O9), PrK 34/57 (O38), PrK 62/57 (O36), PrK66/57 (O40), PrK 75/57 (O49), with the (GACA)4 primer andon a DNA matrix isolated from nine P. mirabilis strains, PrK

15/57 (O7), PrK 18/57 (O9), PrK 34/57 (O38), PrK 38/57(O20), PrK 62/57 (O36), PrK 66/57 (O40), PrK 75/57 (O49),PrO 10/52 (O3), and S1959 (O3), with the (CAAT)4 primer.Each of the strains was isolated three times to check geneticstability. Electrophoresis performed on one gel only with 18probes (six triplets) resulted in 100% reproducibility (i.e., iden-tical strains produced identical outputs) (Fig. 1 and 2). For the(GACA)4 PCR, a lack of full homology was observed withinthe repeats of the same-strain analysis (Fig. 3, series designedA, B, and C). The inclusion of several paths from another gelresulted in a total 6.7% difference in the same-strain analysis(Fig. 3, paths not described). Hence, the cutoff was set at anarbitrary value of 90% interpattern homology. The weakerreproducibility in the last test resulted from the larger numberof similar amplicons in particular paths as well as from thehigher background smear intensity.

For the set of six P. mirabilis laboratory strains, the differ-entiation indices were very high and equal (0.966) with bothprimers. When more electropherograms were investigated si-multaneously (38 and 89), the appropriate differentiation in-dices were 0.992 with the (GACA)4 primer and 0.940 with the(CAAT)4 primer when a cutoff value of 90% was used and0.954 with the (CAAT)4 primer when a cutoff value of 93% wasused, respectively (Table 2; see also Fig. 5). For further anal-ysis of P. mirabilis clinical isolates, only the (GACA)4 primerwas used.

The patterns obtained for the clinical isolates with the(GACA)4 primer were very similar to those obtained for thelaboratory strains. Five bands common to most of the patternswere located at about 2,555, 1,241 (two bands), and 700 bp.Three shorter bands (two pairs separated by a single band)were located at about 500 to 100 bp (Fig. 6). These patterns

FIG. 1. Electrophoretic resolution by (GACA)4 PCR of six repre-sentative P. mirabilis strains derived from the Kauffmann-Perch col-lection. The fingerprinting procedure accuracy was tested three timesfor each strain. Lanes: 1 to 3, PrK 75/57; 4 to 6, PrK 66/57; 7 to 9, PrK62/57; 10 to 12, PrK 34/57; 13 to 15, PrK 18/57; and 16 to 18, PrK 15/57.The repeated procedures included culturing, DNA extraction, andamplification. Experiments were done with 3 ml of inoculum (lanes 3,6, 9, 12, 15, and 18), 1 liter of secondary inoculum (lanes 2, 5, 8, 11, 14,and 17), and 8 liters of culture (lanes 1, 4, 7, 10, 13, and 16). LanesM, molecular weight markers. The white arrows on the left indicatebands applied as internal references for normalization procedure inthe UPGMA analysis.

FIG. 2. Accuracy of (CAAT)4 PCR fingerprinting for six represen-tative P. mirabilis strains derived from the Kauffmann-Perch collection.Lanes: 1 to 3, PrK 66/57; 4 to 6, PrK 62/57; 7 to 9, PrK 38/57; 10 to 12,PrK 34/57; 13 to 15, PrK 18/57; and 16 to 18, PrK 15/57. The testingprocedures included culturing, DNA extraction, and amplification. Seethe legend to Fig. 1 for a further description of lanes. Lanes M,molecular weight markers. The white arrows on the left indicate bandsused as internal reference standards for normalization in the UPGMAanalysis.

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were similar to the patterns obtained with the (CAAT)4

primer, where five bands positioned between 1,827 and 900 bp(Fig. 4) were common to most of the investigated strains. Mostof the strain-specific bands were shorter than 900 bp. Numer-ical analysis of laboratory strains resulted in no evident clus-ters, and the dendrogram had the “stair-shape” structure (Fig.5). The observed interstrain homologies were mostly in therange of about 60 to 95%, similar to those obtained with thedendrogram produced by (GACA)4-based fingerprinting. The

patterns resulting from the (CAAT)4 test were more distinct,although some of the strains were still indistinguishable fromothers, making the interpretation of results difficult (Fig. 5).

DISCUSSION

The studies reported here showed that tandem repeatedtetramers might be used as effective tools for PCR-based P.mirabilis typing. The results of the cluster analysis partially

FIG. 3. Reproducibility of (GACA)4 PCR (left) and (CAAT)4 PCR (right) in UPGMA band pattern analysis (the Dice similarity coefficientwas used). An 0.8% position tolerance value was used. The calculation program GelCompar, version 4.0, was used. Three probes of each strain(A, B, and C) resolved on the same gel and one probe of some of the same strains from another gel (not labeled) are compared. The scalesrepresent the level of homology between the investigated probes.



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corresponded to the distributions of important bacterial sur-face properties.

The levels of effectiveness of the most important Proteustyping methods were compared recently by Senior (42, 43)using phage typing, bacteriocin typing, and Dienes phenome-non-based incompatibility grouping. It was demonstrated that,when a combination of different typing methods is applied,highly sensitive differentiation may be obtained. Pignato et al.(36) found that with two ribotyping methods, 10 examined P.mirabilis strains were clustered together in one ribogroup. Theeffectiveness of ribotyping for P. mirabilis was compared withthose of other genetic differentiation methods by Pfaller et al.(35). The discriminatory indices established were relativelyhigh: 0.92 for ribotyping, 0.979 for PFGE, and 0.980 for theDienes test. The sensitivity of molecular typing methods usedto determine electrophoretic profiles of outer membrane ortotal cell proteins of Proteus strains (17) is much more limited.

PCR-based differentiation methods that have been evalu-ated include randomly amplified polymorphic DNA (RAPD)analysis and repetitive sequence-based PCR. For some Proteusspecies, repetitive sequences can be use as an effective tool forfingerprinting (18, 44). For P. mirabilis, however, the repetitivesequence-based PCR fingerprinting methods described so far(ERIC-PCR, BOXA1R-PCR, BOXA2R-PCR) have a lowersensitivity and only REP-PCR supports greater efficiency (44).Microsatellite-based PCR methods usually have a higher sen-sitivity. Nevertheless, for P. mirabilis, the discrimination abilityof (GTG)5 microsatellite-based PCR analysis is much lower(data not shown).

Both tandem repeated tetramers are short enough for thepresented fingerprints to resemble RAPD types (2, 29). Theresolution of the RAPD method was found to be identical tothat of ribotyping, since both generated the same numbers ofdifferent electrophoretic profiles for the investigated P. mira-bilis strains (36). Therefore, the microsatellite-based finger-printing method described here, with high discriminatory abil-ities [0.992 with the (GACA)4 primer and 0.940 with the(CAAT)4 primer], seems to be promising. Numerical analysisof the total number of investigated strains showed that mostclinical isolates were interspersed among laboratory referencestrains.

For some species, the correspondence between strain phe-notypes and fingerprinting patterns based on sequences notdirectly related to surface antigens has already been reported.The application of ribotyping techniques to Listeria monocyto-genes has demonstrated a significant relationship between se-rotypes and genetic lineages (C. Nadon, D. Woodward, C.Young, F. Rodgers, and M. Wiedmann, Abstr. 100th Gen.Meet. Am. Soc. Microbiol., abstr. P-96, 2000). The efficacy ofphenotypic feature differentiation for some Abiotrophia strainson the basis of several genotyping methods has also beenestablished (16). Also, for uropathogenic Escherichia colistrains, ribotype or RAPD-generated profiles consistent withtheir common clonal origins have been detected. However,interstrain serological similarities may originate from conver-gent evolution or recombination events (37) and therefore donot reflect common clonal origins.

The small series of P. mirabilis strains were genetically stablein the genome loci including both types of applied sequences:(GACA)4 and (CAAT)4. Moreover, our preliminary resultsindicate that there is some correspondence between microsat-ellite and repetitive sequence genomic distributions and phe-notypic properties of Proteus laboratory strains (4, 5). Theseresults correspond to some other findings indicating that alarge number of P. mirabilis properties are correlated with, e.g.,serological identity and proticine production and sensitivitytype (7). Therefore, as one may expect, a considerable portionof genome sequences should be ordered steadily (i.e., the gen-eral order of genes and/or operons should be to be conserved).If this notion is true, then estimation of one set of bacterialproperties based on a statistical distribution of others is wellgrounded. In particular, microsatellite-based PCR fingerprint-ing may then serve for the estimation of probable serologicalproperties of clinical isolates or the selection of clinical isolateswhich are more strongly associated with uropathogenicity. Ithas been shown that some serological types of P. mirabilis aremore frequent (8, 20–23, 41). The question of whether this

FIG. 4. Electrophoretic resolution by (CAAT)4 PCR of represen-tative P. mirabilis strains derived from the Kauffmann-Perch collection.Lanes: 1, PrK 74/57 (O48); 2, PrK 69/57 (O43); 3, PrK 64/57 (O38); 4,PrK 61/57 (O35); 5, PrK 58/57 (O32); 6, PrK 56/57 (O31) (P. vulgaris);7, PrK 53/57 (O30); 8, PrK 52/57 (O29); 9, PrK 51/57 (O28); 10, PrK50/57 (O27); 11, PrK 49/57 (O26); 12, PrK 47/57 (O24); 13, PrK 46/57(O24); 14, PrK 45/57 (O24); 15, PrK 43/57 (O23); 16, PrK 41/57 (O23);17, PrK 38/57 (O20); and 18, PrK 34/57 (O18). Lanes M, molecularweight markers.

TABLE 2. Differentiation indices (DI) for two investigatedfingerprinting methods based on tandem repeated tetramersa

Cutoff value,% (Fig.)

Value obtained in the following PCR:

(GACA)4 (CAAT)4

N �j1

N

aj DI N �j1

N

ajDI

96 (3) 7 0 1 6 2 0.93390 (3) 7 2 0.952 6 0 193 (4) 89 37 60 0.95590 (4 and 5) 89 66 0.992 37 82 0.938

a For details, see the text.



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FIG. 5. UPGMA (Dice) cluster analysis of 35 P. mirabilis laboratory strains by (CAAT)4 PCR. The calculated values for clustering errors areboxed. The scale at the left represents the homology level. The broken line indicates the accuracy of the method. The arrows indicate the maingroups of indistinguishable strains for the cutoff value established at 90% similarity.

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association really results from general P. mirabilis genome sta-bility or from local linkage disequilibria might be answeredonly by investigations based on a larger number of subtractedsequence types, i.e., additional types.

In conclusion, tetramer-based PCR fingerprinting results areindependent of strain storage and culturing, and the methodused here shows efficient discrimination ability. The presentedfindings suggest that at least some microsatellite-based finger-prints are specific enough for the rapid differentiation of P.mirabilis strains at the level of serogroup and for the effectivedifferentiating of particular clones. Moreover, dendrogramstructures determined independently of the applied calculationalgorithm (40, 42, 52) include additional information whichmay be interpreted in relation to important surface properties.Our results are in agreement with reports of correlations be-tween known surface antigens (of different bacteria) and fin-gerprinting results based on numerous, distinct sequence types(ribotyping, restriction fragment length polymorphism analy-sis, RAPD analysis, and pulsed-field gel electrophoresis) and,along with previous findings, suggest similar correlations withseveral PCR-based fingerprinting methods.

ACKNOWLEDGMENTS

The work was partially supported by grant 4 P05A 092-19 from theState Committee for Scientific Research.

Special appreciation is extended to Maria Olszewska (Institute ofCytology and Plant Physiology, University of Łodz), Leon Sedlaczek(Centre of Microbiology and Virology, Polish Academy of Sciences),and Antoni Rozalski (Institute of Microbiology and Immunology, Uni-versity of Łodz) for encouragement of our research as well as forcomments, advice, and critical discussions. We also thank TomaszSakowicz (Institute of Cytology and Plant Physiology, University ofŁodz) for kindly reading the manuscript.

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