a Station de Pathologie Aviaire et Parasitologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; b Institut für Tierzucht und Tierverhalten der Bundesforschungsanstalt für Landwirtschaft (FAL), Dörnbergstraße 2527, 29223 Celle, Germany; c Agence Française de Sécurité Sanitaire des Aliments, 69007 Lyon, France
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Abstract |
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Introduction |
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To date, very little is known about E. coli isolates carrying floR genes in chromosomal DNA.10 We therefore investigated 22 chromosomally florfenicol-resistant E. coli isolates from two countries for their clonal relationships and the genetic environment of the floR genes.
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Material and methods |
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A total of 22 florfenicol-resistant bovine E. coli isolates, 12 from France and 10 from Germany, were included. All were from faecal samples of individual animals of unrelated herds. The French isolates BN10871, BN11282 and BN11285 were obtained from animals suffering from respiratory diseases whereas the remaining 19 isolates were from cases of diseases of the digestive tract. Isolates were collected between 1997 and 2000. Biochemical confirmation of the species assignment followed the specifications given by Koneman et al.11
Resistance patterns were determined by the agar disc diffusion method on diagnostic sensitivity test agar (DST agar; Oxoid, Wesel, Germany), with discs containing ampicillin 10 µg, chloramphenicol 30 µg, florfenicol 30 µg, gentamicin 10 µg, neomycin 30 µg, streptomycin 10 µg, sulfamethoxazole 25 µg, tetracycline 30 µg or trimethoprim 5 µg. The zones of growth inhibition were evaluated according to NCCLS standards.12 In addition, MICs of florfenicol and chloramphenicol were determined by broth micro- and macrodilution methods, respectively, as described previously.9
Serotyping was performed as a slide agglutination test with commercially available monovalent test sera directed against the antigens K99, K88, 987p and F107 (BgVV, Dessau, Germany).
DNA preparation, hybridization experiments and PCR analysis
Plasmid DNA was prepared according to a modification13 of the method of Kado & Liu,14 which is particularly suitable for the recovery of large enterobacterial plasmids. The 150 kb plasmid R55 served as a control to ensure identification of the presence of large plasmids.15 In addition, plasmids were also prepared by alkaline denaturation, and subsequently purified by affinity chromatography on Qiagen columns (Qiagen, Hilden, Germany). The preparation of whole-cell DNA of the 22 E. coli isolates followed a method described previously for the isolation of whole-cell DNA from salmonellae.13 Transformation of plasmids into E. coli strain JM107 or JM109 with subsequent selection on florfenicol-containing (20 mg/L) media were conducted using the CaCl2 method.13 Conjugation experiments with E. coli strain BM14 were performed as described.9,15 PCR-directed detection of the floR gene was achieved with primers flo1 and flo2 (flo1, 5'-GCATCCTGAACACGACGCCCGCT-3'; flo2, 5'-CGACACCAGCACTGCCATTGCCG-3'), which results in the amplification of a 1031 bp amplicon that comprises almost the entire floR reading frame. For Southern blot hybridization the 1031 bp floR segment (GenBank accession no. AJ251806), amplified from the floR gene of S. enterica serovar Typhimurium DT104,3 served as specific probe. Chromosomal location of the floR gene was confirmed by: (i) positive floR-specific PCR results using whole-cell DNA; (ii) positive floR- specific hybridization results using whole-cell DNA; (iii) negative results of repeated plasmid transformation/ conjugation experiments; (iv) negative results of repeated floR-specific hybridization experiments using plasmid profiles as target DNA; and (v) the location of the floR gene on a I-CeuI fragment of chromosomal DNA. For this latter approach, whole-cell DNA was digested with the homing restriction endonuclease I-CeuI, which is known to have its cleavage sites exclusively within the rrn operons of the E. coli genome.16 The fragment patterns were separated by pulsed-field gel electrophoresis. The pulse times were increased from 2.2 to 63.8 s for 10.5 h. The seven fragments were cut from the gel and slices of these gel pieces served as targets for the PCR-directed detection of the floR gene. In this particular case, PCR was performed for 4560 cycles in a total volume of 80 µL.
To compare the chromosomal regions flanking the floR gene in the E. coli isolates, Southern blotting was performed using the 6522 bp segment of the floR-carrying plasmid pEF03 (GenBank accession no. AF231986) as a probe for hybridization with BglI-digested, whole-cell DNA. Labelling of the two probes and hybridization conditions were as reported previously.9,17
Macrorestriction analysis
Macrorestriction analysis using the enzyme BlnI18 was carried out to determine the genomic relationships of the 22 E. coli isolates. This enzyme has proved to be superior in its discriminatory power to other enzymes commonly used for E. coli, such as XbaI or SpeI (S. Schwarz, unpublished data). Preparation of the whole-cell DNA for macrorestriction analysis followed a protocol described previously.18 The pulse times were increased from 7 to 12 s for 11 h and from 20 to 65 s for the next 13 h. Electrophoresis was carried out at 14°C, 5.5 V/cm and 0.15 A in a Bio-Rad CHEF-DRIII system (Bio-Rad, Munich, Germany) using 0.5x TBE as running buffer. The SmaI fragments of Staphylococcus aureus 832519 served as size markers. Cluster analysis using the unweighted pair group average (UPGMA) method was performed with the GelCompar software.
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Results |
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Biochemical analysis confirmed the assignment of all isolates to the species E. coli. Serotyping identified four E. coli isolates (BN10678, BN11157, 9 and 29) as positive for the antigen K99. All E. coli proved to be multiresistant. In addition to florfenicol and chloramphenicol resistance the isolates exhibited in vitro resistance to between four and seven additional antimicrobial agents (Table). The MICs of chloramphenicol varied between 128 and >256 mg/L, and those of florfenicol between 64 and >128 mg/L.
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The floR gene was detected in all isolates using a specific PCR assay in addition to Southern blot hybridization with a specific gene probe. The latter approach identified single copies of the floR gene in 21 of the 22 E. coli, whereas two copies were found in a single French isolate (Table). The assumption that the floR gene is located in the chromosomal DNA of the E. coli isolates was confirmed by: (i) the presence of the 1031 bp amplicon when using wholecell DNA for PCR; (ii) different-sized hybridizing bands using EcoRI-, BamHI- and BglI-digested whole-cell DNA; (iii) repeated negative hybridization results using plasmid DNA of the E. coli isolates; (iv) repeated negative transformation/conjugation results using the plasmids of the E. coli isolates; and (v) the PCR-directed detection of the floR gene on I-CeuI fragments of chromosomal DNA. This latter approach identified the floR gene in most cases on one of the two largest I-CeuI fragments. The French isolate BN10657 harboured its two floR gene copies on two different I-CeuI fragments. To assess the genetic environment of the floR gene in the chromosomal DNA of the E. coli isolates, another set of hybridization experiments was performed using BglI-digested DNA and the 6522 bp EcoRIBamHI insert of plasmid pEF03 as a probe.9 This insert contains in addition to the floR gene approximately 3.3 kb of upstream and 1.8 kb of downstream sequences containing reading frames for putative transposase proteins (orf A' and orf A). The insert exhibited four BglI sites accounting for three BglI fragments in the floR gene area of 1402, 1976 and 906 bp (Figure 2a
). Hybridization of the 22 E. coli isolates revealed a total of nine BglI restriction patterns (Table
and Figure 2b
). The 1976 bp fragment that included the floR gene and the adjacent 906 bp fragment that harboured part of orf A (Figure 2a
) were seen in 17 of the 22 isolates. The 1402 bp fragment representing the floR upstream region including part of orf A' was detected in only two E. coli isolates. Additional hybridization experiments using the floR gene probe and EcoRI- or BamHI-digested whole-cell DNA yielded fragments of 9.3 >23.1 kb or 7.021.8 kb, respectively. These hybridizing fragments were larger than the corresponding fragments expected from plasmid pEF03.
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Discussion |
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The occurrence of the floR gene in the chromosome of E. coli has rarely been observed,10 and compared with the S. enterica serovars Typhimurium46 and Agona,7 very little is known about chromosomal floR genes in E. coli. In this repect two major questions had to be solved: (i) do the chromosomally floR-carrying E. coli isolates represent one clone and (ii) is the genetic environment of the chromosomal floR genes similar or identical to that of plasmid pEF03? To answer the first question, all E. coli isolates were subjected to macrorestriction analysis using BlnI. Two of the German and three of the French isolates proved to be indistinguishable based upon their BlnI fragment patterns, while the majority of the other isolates were distinctly different based on their macrorestriction patterns (Figure 1a). Two clusters that reflected mainly the geographical sources of the isolates were identified (Figure 1b
) and confirmed that floR-carrying bovine E. coli from France and Germany represent independent populations. This was in accordance with the previous observation of strikingly different restriction patterns of floR-carrying plasmids of E. coli from these two countries.9 Based on the macrorestriction patterns and the resulting cluster analysis, it can be concluded that the chromosomally floR-carrying E. coli represented a wide variety of different genotypes.
To analyse the chromosomal regions adjacent to the floR genes, whole-cell DNA was digested with BglI and hybridized with the sequenced 6.522 kb EcoRIBamHI fragment of plasmid pEF03. Of the three conserved BglI fragments, the 2.0 kb fragment carrying the floR gene and the 0.9 kb fragment harbouring part of the downstream-located orf A were seen in all of the German isolates, but only in seven of the 12 French isolates (Table and Figure 2
). The 1.4 kb fragment located upstream of the floR gene in pEF03 was detected in only two isolates, one from France and one from Germany (Table
). Among the remaining five French isolates, two completely different hybridization patterns were observed. In total, nine different BglI hybridization patterns were identified. This confirmed the genetic diversity in the chromosomal regions immediately upstream and downstream of the floR gene. Further hybridization experiments with the floR gene probe identified the floR gene on chromosomal EcoRI or BamHI fragments ranging in size from 7.0 to >23.1 kb.
Assuming that the floR gene is part of a transposable element, different chromosomal integration sites can explain the differences in the hybridization profiles. The detection of two floR gene copies on different I-CeuI fragments of the chromosome of strain BN10657 also supports the hypothesis of different chromosomal integration sites. In addition, there have been a number of structurally different floR-carrying plasmids identified in E. coli. However, the floR-flanking area of only one of them is known. Thus, integration of different floR-carrying plasmidsin part or in totoin the chromosomal DNA of E. coli isolates might represent another possible explanation for the observed structural variations in the chromosomal floR gene area.
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Acknowledgements |
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Notes |
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References |
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Received 4 May 2001; returned 20 July 2001; revised 28 August 2001; accepted 8 September 2001