Folia Microbiol. 48 (2), 162–167 (2003) http://www.biomed.cas.cz/mbu/folia/

Heterogeneity of Escherichia coli Derived from Artiodactyla Animals Analyzed with the Use of rep-PCR Fingerprinting K. BALDY-CHUDZIK, J. NIEDBACH, M. STOSIK Department of Microbiology and Genetics, Institute of Biotechnology and Environmental Protection, University of Zielona Góra, 65-561 Zielona Góra, Poland

Received 15 March 2002 Revised version 3 July 2002

ABSTRACT. Genetic polymorphism of 83 isolates of E. coli, derived from 4 species of artiodactyla ani-mals living in a relatively close contact on the grounds of a theme park ZOO Safarii Świerkocin (Poland) was determined using the rep-PCR fingerprinting method, which utilizes oligonucleotide primers matching interspersed repetitive DNA sequences in PCR reaction to yield DNA fingerprints of individual bacterial isolates based on repetitive extragenic palindrome (REP) primers. The fingerprint patterns demonstrated the essential polymorphism of distribution of REP sequences in genomes of the examined isolates. The arithme-tic averages clustering algorithm (UPGMA) statistical analysis of fingerprints with the use of the Jaccard similarity coefficient differentiated E. coli isolates into three similarity groups containing various numbers of isolates. The groups comprised isolates derived from two, three and four species of the source animals. The isolates derived from each source segregated in the dendrogram in a different way, both within the simi-larity groups and among them, indicating an individual repertoire of E. coli in the examined species of ani-mals. The similarity relations among E. coli derived from the same source, illustrated in a dendrogram with a number of subclusters of a low mutual similarity (≤20 %), indicated an essential interstrain differentiation in terms of the distribution of REP sequences. Our results confirmed the hypothesis of the oligoclonal cha-racters of populations obtained from particular sources. The rep-PCR fingerprinting method with REP pri-mers is simple and highly differentiating and can be recommended for use in explorations of large groups of animals and monitoring the variability of strains.

From the point of view of bacterial ecologists, E. coli is a minority component of the normal biota of the large intestine of vertebrates which occasionally lives in the soil and water (Hultgren et al. 1996). E. coli populations exhibit considerable high dynamics and a clonal character. The observed high genetic variability is affected by the pressure of the environment that finally leads to selection of strains and/or clones which are most successfully adapted both phenotypically and genotypically. The selective influence of the environment of the large intestine results in an individual repertoire of E. coli strains for each verte-brate animal. Understanding this variability is one of the aims of ecology of microorganisms (Schaechter et al. 2001).

rep-PCR fingerprinting is a molecular technique allowing mutual comparison of a large number of closely related isolates (Rademaker et al. 2000). The term “rep-PCR” refers to the general method which utilizes oligonucleotide primers matching interspersed repetitive DNA sequences in PCR reaction to yield DNA fingerprints of individual bacterial isolates (Versalovic et al. 1994). The repetitive extragenic palin-dromic sequences – REP (repetitive extragenic palindrome) belong to the dispersed repetitive DNA sequen-ces. REP sequences are 35–40 bp large. They have been described for numerous enteric (Gilson et al. 1990; Hulton et al. 1991) and phytopathogenic (Fousek et al. 2002) bacteria. The palindromic nature of the REP elements and their ability to form stem-loop structures have led to multiple proposed functions for these highly conserved, dispersed elements (Sharples and Lloyd 1990; van Belkum 1999). Previously, rep-PCR was successfully used for classifying and differentiating among strains of E. coli (Lipman et al. 1995), Rhi-zobium meliloti (de Bruijn 1992), Xanthomonas spp. (Rademaker et al. 2000) and several other bacteria (Versalovic et al. 1994).

Here we describe the use of the rep-PCR (REP) fingerprinting technique for differentiating E. coli isolates derived from 4 species of artiodactyla animals living in a relatively close contact on the grounds of a theme park ZOO Safarii Świerkocin (Poland).

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MATERIALS AND METHODS

Escherichia coli sources and isolation. Samples of feces derived from the following animals were sources of the E. coli isolates: eland (Taurotragus oryx), water buffalo (Bubalus bubalis), waterbuck (Kobus ellipsiprymnus) and aurochs (Bos primigenius). The samples were inoculated on agar m-FC (Merck). After a 1-d incubation at 44 °C, blue colonies (on average 12 colonies from each feces sample) were placed on the MacConkey’s broth. Lactose-positive colonies were tested in the IMViC series. A strain was deter-mined as E. coli when it displayed the ability to produce indole (I+), carried out fermentation Escherichia coli type (M+, V–) and did not grow on Simmons broth (C–) (Dombek et al. 2000). Table I gives the source, the number of isolates derived from each source and the laboratory number (corresponding to the numbering in Figs 1–2).

Table I. E. coli isolates used

Source Groupa Number of isolates Numbering of isolatesb

Eland (Taurotragus oryx) A 21 100–109, 111, 112, 114–116, 118, 119–123 Water buffalo (Bubalus bubalis) B 18 172–178, 180–182, 184–187, 190, 191, 193, 195 Waterbuck (Kobus ellipsiprymnus) C 24 148, 149–171 Aurochs (Bos primigenius) D 20 124–131, 135–147

aSee Fig. 2. bCorresponding to numbering in Fig. 1.

Isolation of genomic DNA and the PCR conditions. The input material consisted of 1-d cultures of

E. coli isolates grown in LB (Merck) broth to absorbance A600 = 0.6–0.9. The DNA probes were prepared with a kit (Wizard genomic DNA purification kit; Promega) according to the manufacturer’s instructions. Genomic analysis of the isolates was done by rep-PCR reactions with the single set of primers (Versalovic et al. 1994): REP 1R-I: 5´-III CGI CGI CAT CIG GC-3´ (IDT); REP 2-I: 5´-ICG ICT TAT CIG GCC TAC-3´ (IDT). PCR was carried out according to the Rademarker and de Bruijn (1998) in 25-µL samples: 17 µL reaction buffer with 1.5 mmol/L MgCl2, 10 % Me2SO and bovine serum albumine (20 g/L), 1 µL of a mix-ture of nucleotides (25 mmol/L each of dATP, dTTP, dCTP and dGTP; Sigma), 2 µL each of primers (REP 1R-I, REP 2-I) in a concentration of 30 pmol/L each, 2 µL of DNA polymerase 2.5 U/mL (Finzyme–Poly-gen) and 1 µL of genomic DNA (≈50 ng). Rep-PCR (REP) fingerprinting was carried out in a PTC 200 amplificator (MJ Research Inc., USA) using the following program: initial denaturation (95 °C, 7 min), 30 amplification cycles (92 °C for 30 s, 40 °C for 1 min, 65 °C for 8 min) and a final extension step (65 °C, 8 min) (low temperature in the extension steps – 65 °C – prevents inhibition of the PCR reaction, adapts rep-PCR protocol to the interchangeable usage of the full cell lysate and/or pure DNA as a matrix; Versalovic et al. 1994). Each PCR reaction was done three times. The PCR reaction products were separated on 1 % (W/V) agarose (Serva) gels in TAE buffer (pH 8.0) at a voltage of 2.5 V/cm for 6 h. After staining with ethi-dium bromide, the gels were digitized for computer-aided analysis.

The analysis of electrophoretograms. The electrophoretic paths of rep-PCR fingerprints were analy-zed with the use of the Vilber Lourmat (France) computer software (version 99.03).

The densitometric analysis, supported by normalization including a marker of the molar size in base pairs (MassRuler 1-kb DNA Ladder, Fermentas), was applied to each path. Calculation of the similarity ma-trix was done with the Jaccard algorithm after defining each single band. Hierarchic clustering was achieved by the unweighted pair-group method with arithmetic averages clustering algorithm (UPGMA). The degree of per cent similarity was expressed.

RESULTS AND DISCUSSION

Eighty-three strains of Escherichia coli were identified in the samples derived from 4 species of artiodactyla animals (Table I). All isolates were typeable by rep-PCR fingerprinting with the use of REP pri-mers. The distribution of the amplified fragments of genomic DNA varied both among the isolates derived from the same source and/or species as well as among the isolates derived from various sources (Fig. 1A–D). Fingerprints of isolates derived from eland (group A; Fig. 1A, Table I) represented seven types of band pat-terns. E. coli derived from water buffalo (group B; Fig. 1B, Table I) and waterbuck (group C; Fig. 1C, Table I)

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Fig. 1. rep-PCR fingerprints patterns of eland (A), water buffalo (B), waterbuck (C) and aurochs (D) isolates; at the top of pictures are the numbers of (above) lines of fingerprint and (below) of isolates (x and y, respectively, in Fig. 2); M – the MassRuler 1-kb marker.

revealed 13 types of patterns. Aurochs isolates showed 10 types of patterns (group D; Fig. 1D, Table I). The common feature for all the patterns was the presence of products about 300 bp and smaller in products 1–3. Nearly 46 % of isolates had the 1031-bp product, and about 72 % of them presented 1–7 products exceeding 1031 bp. The complexity of the acquired patterns of REP fingerprints was close to that obtained for laboratory and clinical E. coli strains by Versalovic et al. (1994) but considerably lower than that demon-strated by strains derived from systematically distant animal sources (Dombek et al. 2000).

The UPGMA statistical method illustrated the similarity of E. coli strains placed in a dendrogram (Fig. 2). It comprises three groups with mutual similarity not exceeding 5 %. The analysis of the structures of the particular similarity groups allowed us to demonstrate certain tendency in the manner of distribution of the isolates depending on their origin. The isolates derived from eland (A) were differentiated into four subclusters distributed within two groups (Fig. 2, I.1, II.1, II.3, II.4). Group I is formed mainly by the eland isolates (subcluster I.1). Eland subclusters in group II are localized alternately with water buffalo (B) sub-clusters. Together they form a coherent structure, differentiating them from the other source animal species. Subcluster II.4 was formed by a pair of isolates of a different origin: eland and water buffalo. There were two cases of 100 % similarity among the eland isolates (I.1 and II.3). Strains with identical distribution of REP sequences demonstrated separate distribution of other types of repetitive sequences such as ERIC and BOX (unpublished data). The isolates from water buffalo (B) occurred in seven subclusters (II.2, II.4, II.5, II.6, II.7, III.3, III.7, III.8). Four out of the seven water buffalo subclusters occured in group II and they were in similarity relations with eland isolates. The next three subclusters, localized in group III, revealed low similarity to waterbuck (C) and aurochs (D) isolates. The isolates derived from waterbuck were divided into seven subclusters (I.2, II.8, II.10, II.11, II.12, II.13, III.2) mostly assembled within group II forming a cohe-rent structure together with the isolates from aurochs. Group I comprised only one pair of waterbuck isolates (I.2). One subcluster, i.e. III.2, joined group III. The aurochs (D) isolates formed five subclusters (II. 9, III.1, III.4, III.5, III.6). Only one of them occurred in group II and comprised an isolate from waterbuck. The other four subclusters were localized in group III. Within the dendrogram groups the similarity relations of isolates derived from various sources were low and amounted to 5–20 %; only two cases of higher similarity were found. They concerned subclusters II.1 and II.2 (about 25 %), and subcluster II.9: C, isolate 153, and D, iso-late 136 (both about 40 %).

The rep-PCR (REP) fingerprinting method clearly demonstrates the specific segregation of isolates depending on the source of origin, confirming an individual repertoire of E. coli isolates in each of the source animals. Among the isolates derived from the same source, the number of the found subclusters and the low similarity between them (≤20 %) showed that the individual repertoire of E. coli is of oligoclonal character with a various degree of complexity. As a method of examining the diversity of microorganisms (including E. coli), the rep-PCR fingerprinting has been compared with other molecular biology methods.

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Their discrimination power was discussed in many papers but the similarity relations within the clusters have not been commented (van Belkum 1994; Louws et al. 1994; Versalovic et al. 1998). Dombek et al. (2000) tested the usability of rep-PCR fingerprinting (a tool for rapid determination of fecal pollution) and dif-ferentiated 125 E. coli isolates derived from 6 animal species from healthy animals and humans. The animal isolates included two types of waterfowl (geese and ducks) and common farm animals (cows, pigs, sheep and chickens). Specific segregation of isolates into similarity clusters dependent on the origin was shown. Moreover, analysis of the similarity relations within the clusters revealed that some of the isolates from chickens demonstrated greater similarity to E. coli derived from humans. Our results indicate the usefulness of the method for monitoring the variability and/or propagation of E. coli strains among animals of the same systematical group living in a close contact.

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Fig. 2. Dendrogram of the similarity of repetitive sequence (REP) distribution in the genome of the E. coli isolates derived from four animal sources; A – eland, B – water buffalo, C – waterbuck, D – aurochs; I, II, III – the similarity groups; Lx/y – x is the number of the fingerprint line on gel (see Fig. 1) and y is the number of E. coli isolate; an Arabic numeral followed by a Roman one inside the dendrogram defines a subcluster of a given similarity group.