Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystrasse 10, 31535 Neustadt-Mariensee, Germany
Received 29 August 2003, returned 6 September 2003; revised 23 September 2003; accepted 3 October 2003
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Abstract |
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Methods: Fourteen porcine E. coli isolates were included in this study and subjected to serotyping, plasmid profiling and macrorestriction analysis. MICs of florfenicol were determined by broth microdilution. The presence of the gene floR was confirmed by hybridization and PCR analysis. Transformation experiments were conducted to isolate florfenicol resistance plasmids. The floR region of a florfenicol resistance plasmid was cloned and sequenced.
Results: All florfenicol-resistant E. coli isolates exhibited MICs of florfenicol >128 mg/L and carried the floR gene. A single isolate had a floR-carrying plasmid of 35 kb, designated pMBSF1. Sequence analysis identified the floR gene flanked by truncated transposase genes. Moreover, a truncated copy of Tn5393 with complete streptomycin resistance genes strA and strB was found upstream of the floR gene of pMBSF1. Chromosomally resistant E. coli isolates, which shared the same BlnI macrorestriction pattern, differed in their floR hybridization patterns.
Conclusion: The plasmid pMBSF1 is the smallest floR-carrying plasmid reported to date. Its floR region differed from those previously found in E. coli isolates from cattle. Variations in the RFLPs of chromosomal EcoRI fragments carrying floR in isolates that had the same macrorestriction pattern might suggest variable chromosomal integration sites.
Keywords: florfenicol resistance, floR gene, transposon Tn5393, transposon Tn1721, recombination
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Introduction |
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While all these data on florfenicol resistance in E. coli were based on strains from cattle or poultry, little is known about the situation in E. coli from pigs. Therefore, we investigated 14 florfenicol-resistant E. coli isolates from pigs for the genetic basis of florfenicol resistance, and compared the data obtained with those from E. coli strains of other animal sources.
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Materials and methods |
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Fourteen florfenicol-resistant porcine E. coli isolates were included in this study. All isolates were obtained from faecal samples of individual animals suffering from diarrhoea. The samples were collected in 2001 and the animals belonged to the same herd. Biochemical confirmation of the isolates as E. coli followed the specifications given by Koneman et al.17 Serotyping was performed at the Federal Institute for Risk Assessment, Dessau, Germany.
In vitro susceptibility testing was performed by agar disc diffusion on MuellerHinton agar (Oxoid, Wesel, Germany) with discs containing ampicillin (10 µg), carbenicillin (100 µg), chloramphenicol (30 µg), florfenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), minocycline (30 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.18 In addition, MICs of florfenicol were determined by the broth microdilution method.18
DNA preparation, PCR analysis, hybridization and PFGE experiments
Plasmid DNA for hybridization studies was prepared according to a previously described modification of the alkaline lysis procedure, which proved to be particularly suitable for the recovery of large enterobacterial plasmids.16 In addition, plasmid DNA for cloning experiments and sequence analysis was prepared by alkaline denaturation and subsequent purification by affinity chromatography on Qiagen columns (Qiagen, Hilden, Germany). The plasmids of E. coli V517 and S. Typhimurium LT2 and the K. pneumoniae plasmid R55 served as size standards. A previously described method for the isolation of whole-cell DNA from salmonellae served for the preparation of whole-cell DNA of the 14 E. coli isolates for Southern blot hybridization experiments.19 Preparation of whole-cell DNA for macrorestriction analysis followed a previously described protocol.20
The gene floR was detected by PCR with primers flo3 and flo4 (flo3, 5'-GCATCCTGAACACGACGCCCGCT-3'; flo4, 5'-GCTGTGGTCGTGACGGTAACGGC-3'). Cycling conditions included an initial denaturation step of 2 min at 94°C followed by 31 cycles, each consisting of 1 min at 94°C, 1 min at 63°C and 1 min at 72°C, followed by a final extension step of 7 min at 72°C. The resulting 1031 bp amplicon comprised almost the entire floR reading frame. The floR gene from a Salmonella enterica serovar Typhimurium DT104 isolate (GenBank accession no. AJ251806) served as a positive control in the PCR experiments. One such amplicon was cloned into pCR Blunt II TOPO (Invitrogen, Groningen, The Netherlands), sequenced and used as a specific gene probe in hybridization experiments. Transfer of uncut plasmid DNA or EcoRI-digested whole-cell DNA from agarose gels to nitrocellulose membranes (Hybond N, Amersham-Buchler, Braunschweig, Germany) was achieved by the capillary blot procedure. The amplicons were labelled either by the non-radioactive enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Freiburg, Germany) or by the DIG-High Prime DNA labelling and detection system (Roche Diagnostics GmbH, Mannheim, Germany). Hybridization and signal detection were carried out strictly according to the manufacturers recommendations. Detection of the streptomycin resistance gene strA by PCR followed a previously described protocol.21
Plasmid or chromosomal location of the floR gene was confirmed as described previously. PFGE was performed using the enzymes BlnI and I-CeuI. The pulse times for BlnI digests were increased from 7 to 12 s for 11 h, and from 20 to 65 s for the next 13 h, whereas those for I-CeuI were increased from 2.2 to 63.8 s for 10.5 h.16 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) with 0.5x TBE as running buffer. The SmaI fragments of Staphylococcus aureus 832522 served as size markers.
Cloning and sequence analysis of the floR region of plasmid pMBSF1
Transformation of plasmids into E. coli strain JM109 was achieved using the CaCl2 method and transformants were selected on LuriaBertani agar (Oxoid) supplemented with 20 mg/L florfenicol. E. coli JM109 transformants that grew on the selective medium were analysed for their plasmid content and the presence of the floR gene as described. The floR-carrying plasmid pMBSF1 was subjected to restriction mapping and initially, a 5.4 kb BamHIEcoRI fragment was cloned into pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands). Further on, two SspI fragments of 4.5 kb (one harbouring the EcoRI site, the other harbouring the BamHI site), as well as a 1.1 kb SspI fragment, were cloned into pCR Blunt II TOPO (Invitrogen). To complete the sequence of the streptomycin resistance genes upstream of the floR gene, an RcaI fragment of 1.8 kb was cloned. Sequence analysis was performed using an automated sequencer (ALF DNA Analysis System; Amersham Pharmacia Biotech). For this, the commercially available standard primers M13 universal and M13 reverse (Stratagene) were used first. Twelve further 1620mer oligonucleotide primers, derived from the sequence obtained with the standard primers, were designed to complete the sequence analysis. The sequence of a 10 910 bp segment of plasmid pMBSF1 has been deposited with the GenBank database under the accession number AJ518835. Sequence comparisons were carried out using the BLAST program, available at http://www.ncbi.nlm.nih.gov/BLAST/.
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Results |
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Biochemical analysis confirmed that all 14 isolates belonged to the species E. coli. Serotyping showed that 13 of the 14 isolates were positive for the O antigen 108, but non-typeable by K antigens (O108:K-). The remaining isolate exhibited the serotype O141:K85ac (Table 1). All 14 E. coli isolates showed an extended resistance pattern, including resistances to members of six different classes of antimicrobials (Table 1). MICs of florfenicol were >128 mg/L for all 14 E. coli isolates. Two to five plasmids in the size range between 4 and 95 kb were identified in each of the 14 E. coli isolates (Table 1).
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While all isolates were indistinguishable by their I-CeuI macrorestriction patterns, a total of eight different BlnI macrorestriction patterns were observed (Figure 1). The isolates of serotype O108:K- exhibited seven closely related patterns that differed by one to five bands. Among them, a dominant pattern was detected, which was represented by seven isolates. Each of the other six patterns was represented by a single isolate (Table 1). The remaining BlnI pattern of the O141:K85ac isolate differed distinctly from the other patterns.
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The 35 kb plasmid pMBSF1 mediated resistance to florfenicol and chloramphenicol as well as to streptomycin as confirmed by in vitro susceptibility testing of E. coli JM109:pMBSF1 transformants. PCR assays for the floR gene for combined resistance to florfenicol and chloramphenicol as well as the strA gene for streptomycin resistance showed that both genes were present on pMBSF1. A 10 910 bp segment of pMBSF1, which comprised the entire resistance gene region, was cloned and sequenced. The reading frames detected in this segment are displayed in Figure 3.
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The pMBSF1 sequences upstream of the Tn5393 element (positions 1320) and downstream of the Tn1721-homologous part (positions 889210 636) did not show significant homology to any sequences deposited in the databases. Moreover, only small reading frames of up to 231 amino acids were detected in these two regions. However, their deduced protein sequences also did not reveal significant homology to proteins with known functions deposited in the databases. Finally, a stretch of 258 bp (positions 10 63710 894 in the pMBSF1 sequence) revealed close homology (95% identity) to the oriT region of the E. coli plasmid R388 (GenBank accession no. X51505).25
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Discussion |
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Two types of mobile elements, both of which are widespread among Enterobacteriaceae, may be considered to carry the floR gene: gene cassettes/integrons or transposons. Although the floR gene is located between two integron structures in the chromosomal multiresistance gene clusters present in several Salmonella serovars,28 analysis of the sequences flanking floR in all floR-associated database entries did not reveal any sequences that suggested direct involvement of gene cassettes/integrons in the spread of floR. Therefore, the location of this gene on a transposable element appeared to be a likely explanation for its various positions on plasmids or in the chromosomal DNA. In this regard, there are parallels between floR and the tetracycline resistance genes tet(A), tet(B) and tet(H), which have been identified as transposon-borne genes. Previous studies on these tet genes in Enterobacteriaceae2628 and Pasteurellaceae29,30 showed that chromosomal integrates of the corresponding transposons, Tn1721 [tet(A)], Tn10 [tet(B)] and Tn5706 [tet(H)], were truncated in the vast majority of the cases. In contrast, analysis of plasmids carrying the genes tet(A) or tet(H) led to the identification of complete copies of the transposons Tn1721 and Tn5706.24,26,27,31 Based on these experiences, our main interest with regard to the identification of a mobile genetic element harbouring floR focused on the floR gene and its flanking regions located on plasmid pMBSF1, to date the smallest known floR-carrying plasmid in Enterobacteriaceae.
The only other detailed report of the floR flanking regions on plasmids in E. coli showed that the floR gene located on the 110 kb plasmid from the bovine E. coli isolate 10 660 was preceded by a truncated copy and followed by a complete copy of a putative transposase gene.14 In the case of plasmid pMBSF1 from porcine E. coli, only the part immediately upstream of floR including the tnp gene was virtually identical to the corresponding region of the floR plasmid from E. coli 10 660.14 However, the complete copy downstream of floR in the plasmid from E. coli 10 660 was replaced by a
tnp gene from Tn1721 in the case of plasmid pMBSF1. Another novel observation was the presence of a Tn5393 relic with complete streptomycin resistance genes strA and strB further upstream of floR in pMBSF1. Thus, the floR gene area of plasmid pMBSF1 consisted of three parts that exhibited homology to Tn5393, the floR plasmid from E. coli 10 660, or Tn1721 (Figure 3). Analysis of these three different parts and their junctions suggested that recombination events obviously played an important role in the development of this region on plasmid pMBSF1.
Although we did not find a novel transposon carrying floR on plasmid pMBSF1, the data presented in this study enlarged the current knowledge of the diversity of floR gene areas in Gram-negative bacteria. Since the sequence of the floR region of plasmid pMBSF1 is only in part similar to the corresponding regions found either on other floR-carrying plasmids from E. coli,14 K. pneumoniae10 or Pasteurella piscicida (now reclassified as Photobacterium damselae subsp. piscicida),32 or on floR-carrying chromosomal multiresistance gene clusters,28,11,12 both interplasmid recombination and recombination between floR-carrying plasmids and the chromosomal DNA must be considered when speculating about the spread of floR. Such recombination events may also play an important role in the development of novel floR-carrying plasmids, which might carry additional resistance genes, such as strA in the present case. Since many of the so far known floR-carrying plasmids from E. coli also mediate resistances to antimicrobials other than florfenicol,14 the spread of these plasmids by co-selection in the presence of the respective antimicrobials might be facilitated. In this regard, detailed information on the floR plasmids and their additional resistance markers are necessary to understand how and under what conditions florfenicol resistance spreads among members of the same and/or different genera of the family Enterobacteriaceae.
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Acknowledgements |
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Footnotes |
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