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A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella typhimurium DT104

Marie-Anne Arcangioli, Sabine Leroy-Sétrin, Jean-Louis Martel, Elisabeth Chaslus-Dancla
DOI: http://dx.doi.org/10.1111/j.1574-6968.1999.tb13586.x 327-332 First published online: 1 May 1999

Abstract

A new chloramphenicol resistance gene from Salmonella typhimurium DT104, designated floR, also conferring resistance to florfenicol, was characterized. Sequence analysis of the deduced FloR protein suggested that it belongs to the 12-TMS (transmembrane segments) multidrug efflux pumps family. The floR gene, and the downstream sequenced tetR and tetA tetracycline resistance genes, were surrounded by two class 1 integrons. The first one contained the resistance gene aadA2 and a deleted sulI resistance gene. The second one contained the β-lactamase gene pse1 and a complete sulI gene. Thus, the floR gene is included in a multiresistance locus of at least 12.5 kb. Its particular organization and chromosomal location could be involved in the antibioresistance pattern stability of the DT104 Salmonella typhimurium strains.

Keywords
  • Salmonella typhimurium DT104
  • Chloramphenicol-florfenicol resistance
  • Integron
  • Chromosomal location

1 Introduction

Recently, Salmonella typhimurium DT104 has emerged as an epidemic strain all over the world [1, 2]. These strains were multiresistant to antibiotics, mostly ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfonamides, and tetracyclines. The chromosomal location of the resistance genes was only suggested [3].

Chloramphenicol and florfenicol are two antibiotics of the same family. The first one was prohibited in European veterinary use, in August 1994, and the second one has been agreed since January 1995. Soon after its introduction, strains of S. typhimurium cross-resistant to chloramphenicol and florfenicol were detected by the French National Network of Surveillance of Bovine Antibioresistance (RESABO) [4]. Chloramphenicol acetyl transferases (CAT) are the most frequent enzymes involved in resistance to chloramphenicol, but non-enzymatic mechanisms as efflux, impermeability or ribosomal modification have also been described. Florfenicol was shown to be active against chloramphenicol resistant strains producing CAT enzyme (MIC: 4 µg ml−1) [5]. However, it did not seem to be active against strains harboring ribosomal or porine mutations (MICs: 32 µg ml−1) and, to a lesser extent, against strains harboring genes suspected to determine chloramphenicol efflux such as cml or cmlA from Pseudomonas aeruginosa [6, 7](MIC: 15 µg ml−1). The pp-flo gene from a plasmid of Pasteurella piscicida has also been proposed to confer florfenicol resistance by an efflux mechanism [8].

All these genes have been localized to plasmid or transposon structures. However, cmlA, and more recently cmlA2 from Enterobacter aerogenes [7, 9], were shown to be present on a class 1 integron structure. Class 1 integrons are site-specific integration systems composed of two conserved segments (5′-CS and 3′-CS) flanking a variable region where gene cassettes are inserted [10]. A gene cassette generally includes a single gene and a specific recombination site called the 59-base element (59-BE). An integrase gene (intI1) is located in the 5′-CS and determines the site-specific recombination. There is no insertion in the 3′-CS where the antiseptics and sulfonamides resistance genes, qacEΔ1 and sulI respectively, do not have 59-BE [11, 12]. These 59-BE present common features: they are ranging from 60 to 141 bp, they are located downstream to the cassette's gene, they present a G+C rich sequence, and a 7 bp core site related to the sequence GTTRRRY (or its complement RYYYAAC) at their outer boundaries [13].

We report here a new gene conferring cross-resistance to chloramphenicol and florfenicol, and its link with two integrons within the chromosome of a S. typhimurium DT104 strain.

2 Materials and methods

2.1 Bacterial strains, plasmids and antimicrobial pattern determination

S. typhimurium BN9181, a chloramphenicol-florfenicol DT104 resistant strain, and BN9264, a strain susceptible and not phage typable (Table 1), were clinical isolates of bovine origin, isolated in 1995. For these strains, the absence of CAT production was determined by the enzymatic test of Azemun et al. [14]. Escherichia coli strains harboring the different plasmids were included as controls.

View this table:
Table 1

Bacterial strains and plasmids used in this study

Strain or plasmidRelevant genotype and phenotypeMIC (µg ml−1)Source or reference
CmFfc
S. typhimurium
BN9181DT10425664this study
Ap, Cm, Ffc, Sm, Sp, Sul, Tc
BN9264non-phage typable44this study
susceptible
Plasmids
pSTF1recombinant plasmid containing the 1.985 kb genomic Sau3AI fragment of BN9181 with part of sulI and part of the floR resistance gene44this study
Ap
pSTF2recombinant plasmid containing the 3.983 kb genomic Sau3AI fragment of BN9181 with floR, tetR (G), and part of tetA (G) genes6416this study
Cm, Ffc, Ap
pSTF3recombinant plasmid containing the ca. 12.5 kb genomic XbaI fragment of BN9181 with the multiresistance locus12832this study
Cm, Ffc, Sp, Sm, Tc
pFFrecombinant plasmid containing the 3 kb SacI-BamHI plasmid restriction fragment with the pasppflo sequence10050[8]
Cm, Ffc, Km
pLQ821recombinant plasmid containing the 3.1 kb EcoRI-BglII fragment of pCER100 (Tn1696) with the cmlA gene1288[7]
Cm, Ffc, Ap
  • Resistance abbreviations: ampicillin, Ap; chloramphenicol, Cm; florfenicol, Ffc; kanamycin, Km; streptomycin, Sm; spectinomycin, Sp; sulfonamides, Sul; tetracyclines, Tc.

Antibioresistance patterns of strains and recombinant plasmids, as well as MICs were determined as described [15]. Florfenicol discs and drug were purchased from Shering-Plough.

2.2 Molecular biology methods

The PCR reactions were performed on boiled extracts of S. typhimurium strains or on plasmid extract preparations. A consensus sequence from cmlA (transposon Tn1696), cml (plasmid R26), and pp-flo (sequence pasppflo from P. piscicida) permitted the design of two degenerate primers, cml01=TTTGGWCCGCTMTCRGAC and cml15=SGAGAARAAGACGAAGAAG (W=A or T, M=A or C, R=A or G and S=G or C) for detection of the florfenicol resistance gene and preparation of the florfenicol resistance probe. Probe specificity was confirmed by colony hybridization on the control strains harboring pLQ821 or pFF (Table 1) using the labelling Dig High Prime kit (Boehringer-Mannheim).

To clone the resistance gene, partially digested Sau3AI fragments from S. typhimurium BN9181 genomic DNA were ligated into the BamHI site of plasmid vector pUC18. Competent E. coli XL1 blue cells were transformed with the recombinant plasmids. Screening of recombinant plasmids was done by colony hybridization with the florfenicol resistance gene probe. To display the multiresistance locus, the same transformation experiments were done with the recombinant plasmids of XbaI digested BN9181 chromosomal DNA ligated in the XbaI site of pUC18 [1].

DNA sequencing was performed on selected plasmid clones or on PCR products with an Applied Biosystems sequencer (ABI 377, Perkin-Elmer). The nucleotide sequences described in this paper has been deposited in GenBank under accession numbers AF118107 and AF121001.

Pulsed-field gel electrophoresis (PFGE) was performed with strains BN9181 and BN9264, using the I-Ceu I endonuclease (New England Biolabs). DNA fragments were separated in a CHEF-DRII system (BioRad) with pulse times increased from 40 to 65 s the first 3 h and from 60 to 120 s during the next 24 h. The DNA was then transferred by Southern blotting and hybridized to the DNA probes described in Table 2. The probes were labelled by the Dig High Prime (Boehringer-Mannheim) or by the ECL (Amersham) kits.

View this table:
Table 2

DNA probes used in this study

Target geneObtentionReference
floR480 bp internal fragment PCR amplification of BN9181 by cml01-cml15 primersthis study
intI1 (tnpI)692 bp internal fragment PCR amplification of pMON90 by tnp01-tnp15 primers[16]
pseI587 bp internal fragment PCR amplification of BN9181 by pse01-pse15 primers[17]
Ribosomal DNAaction of reverse transcriptase on 16+23S RNA of E. coli[18]

3 Results and discussion

3.1 Characterization of the chloramphenicol and florfenicol resistance gene

The cml01 and cml15 degenerate primers gave rise to an amplification fragment of 498 bp for the S. typhimurium DT104 strain as well as for the plasmids pFF and pLQ821. No amplification was detected for strain BN9264. The PCR product of strain BN9181 was used, after sequencing, as florfenicol gene probe for the screening of the recombinant plasmids. The hybridization, positive for BN9181 and pFF (pp-flo gene in pasppflo sequence) but not pLQ821 (cmlA gene), confirmed the probe's specificity. Thus, after cloning, two plasmid clones, pSTF1 and pSTF2, were obtained carrying the putative florfenicol resistance gene (Table 1) and sequenced. E. coli harboring pSTF1 were susceptible to chloramphenicol and florfenicol (MICs: 4 µg ml−1). Its 1985 bp insert contained an open reading frame (ORF) incomplete in its 3′-end. E. coli harboring pSTF2 were resistant to chloramphenicol (MIC: 128 µg ml−1) and florfenicol (MIC: 16 µg ml−1). Its 3983 bp insert contained the complete above ORF of 1212 bp, with a putative ribosome binding site (RBS), and two putative −35 and −10 promoter sites. The sequence comparison gave 94.8% nucleotide identity over 1917 bp with pasppflo from P. piscicida, so it extended over part of the non-coding sequence and the 1212 bp S. typhimurium ORF was 90 bases longer than the pp-flo gene. Comparison with the cmlA sequence from Tn1696 gave 57.4% nucleotide identity over 1352 bp. Thus the S. typhimurium BN9181 ORF corresponded to a gene conferring cross-resistance to chloramphenicol and florfenicol and was designated floR.

3.2 FloR belongs to the multidrug resistance (MDR) efflux pump family of proteins

The floR gene product was predicted to be a 404 amino acids protein of ca. 43 kDa. This protein presented 88.8% identity with the Pp-flo protein of P. piscicida, and 48.5% identity with CmlA of P. aeruginosa. Hydropathy analysis of FloR and CmlA revealed 12 hydrophobic putative transmembrane domains. The occurrence of the common conserved motifs with the CmlA family of the 12-TMS (transmembrane segments) MDR efflux systems [19] suggested that the FloR protein can be classified within this family of efflux pumps.

3.3 Chromosomal mapping of floR

Chromosomal DNA cut by I-Ceu-I enzyme yielded six fragments in PFGE with sizes ranging from 40 kb to more than 1000 kb. The florfenicol, carbenicillinase and integrase gene probes labelled an approximately 600 kb fragment as did the ribosomal probe, thus demonstrating the chromosomal location of the antibiotic resistance genes in S. typhimurium DT104 strain BN9181.

PCR mapping was conducted to explore the genetic environment of the floR gene and to identify its location relative to the ampicillin, chloramphenicol, streptomycin, spectinomycin, sulfonamide, and tetracycline resistance genes (Fig. 1). The pSTF3 clone contained almost the entire multiresistance locus, in a ca. 12.5 kb insert (Table 1).

Figure 1

Structure of the multiresistance locus of Salmonella typhimurium DT104, strain BN9181. The genes of the class I integrons are represented by hatched arrows and extra-integron elements by black arrows. Stippled arrows represent the amplified but not sequenced region of the second integron. The direct repeats are indicated by black boxes, their position in sequence AF118107 is indicated. The // type indicates the sequence is discontinuous. The dotted lines represent the three plasmid clones. Position and direction of the primers for PCR mapping and sequencing are shown by short arrows.

We detected two integron structures already described [1, 2] containing either the aminoglycoside resistance gene aadA2 or the β-lactamase pse1 gene. The first integron was deleted of the 311 terminal nucleotides of its 3′-CS sulI gene. The resulting sequence could encode a protein with 147 new amino acids fused in frame with the partial sulfonamide resistance determinant, but did actually not confer resistance to sulfonamides, since the strain harboring pSTF3 was not resistant to sulfonamides. A similar deletion-insertion event has been described for the qacEΔ1 gene, a functional deletion derivative of qacE. However, in this case, the resulting polypeptide (with modification of 21 amino acids) was still active against antiseptics, but not as much as the original QacE protein [12]. To our knowledge this is the first description of class I integrons with partial deletion of the sulI gene.

The tetR and tetA genes of class G tetracycline resistance determinant were located downstream to the floR gene. A part of the tetR sequence was 91% identical to a region located upstream to the floR gene which was also described in pasppflo sequence [8]. The two sequences were actually direct repeat elements and suggested a possible integration by an insertion sequence or a transposition event. As a confirmation we found in pSTF3 a gene encoding a putative resolvase enzyme with more than 50% identity with the Tn3 resolvase family, located upstream of the first integron. Moreover, the features of an integrase site-specific recombination were not found in the S. typhimurium sequence: (i) The floR and tet sequences were located in the 3′-CS of the integron and not in the variable region. Deletion of the 3′-CS has already been described [20], but with insertion of a transposon resolvase. (ii) In pasppflo, we detected a GTT sequence associated with an upstream AAC one, located at the 3′ break-point with floR (Fig. 2). However, this was replaced in the Salmonella sequence by a GTG codon which may be considered as a less efficient recombination-specific site [13], but the distance between AAC and GTG was only 53 bp in size which precluded the presence of a 59-BE.

Figure 2

Sequence alignment in the region of the 3′ break-point between the pasppflo (accession number (AN) D37826), the floR (AN AF118107) and the tetR (AN S52438) sequences. The pasppflo sequence is spanning nucleotide (nt) 2331 to 2394 and the floR sequence nt 2099 to 2167. The tetR sequence is the reverse complement of the GenEMBL sequence and spans nt 890 to 841. The GTT sequence of the putative 59-base element of pasppflo sequence, and the corresponding GTG sequence of floR, as well as the AAC ones, are double underlined. The partial inverted repeat segments are single underlined. The 3′ break-point is represented by the / type.

In conclusion, we identified, in S. typhimurium DT104 strain BN9181, a new chloramphenicol resistance gene, designated floR, also conferring florfenicol resistance. The secondary structure prediction and hydropathy pattern of the resulting protein FloR classified it within the 12 TMS family of MDR efflux transporters. We linked all the resistances expressed in the strain with two integrons in a multiresistance locus and so provided an explanation to the results obtained by Schmieger and Schicklmaier in their transduction experiments [21]. The general organization of these genes in a chromosomal multiresistance locus, also seen in American strains (Briggs and Fratamico, sequence AF071555, just available), could provide an explanation to the large occurrence and apparent stability of the multiresistance pattern of the DT104 S. typhimurium strains. Their corresponding floR gene sequence presented only 2 bp differences with our sequence, involving one amino acid modification.

Acknowledgements

We thank P. Courvalin for providing strains and scientific support, T. Aoki for providing strain, M. Peloille for sequencing, and A. Cloeckaert for helpful discussions.

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