Abstract
Salmonella enterica serovar Typhimurium phage type 9 isolates resistant to streptomycin and sulfonamide have been recovered from both bovine and human sources in Australia. This study aimed to identify the resistance genes and their location. Polymerase chain reaction was used to screen for resistance genes and sul2 (sulphonamide resistance) and strA and strB (streptomycin resistance) were detected. A small streptomycin and sulfonamide resistance plasmid carrying the three resistance genes was recovered from these isolates by transformation and was shown to be essentially identical to the small IncQ plasmid RSF1010. The sequences of one plasmid, pSRC15, and RSF1010 differed at only a few positions that may be errors in the older sequence. RSF1010 has been recovered from many species and in many countries since its first isolation in the early 1970s. We conclude that this plasmid has persisted unchanged in the environment for over 30 years. The antibiotic resistance gene cluster containing strA, strB, and sul2 genes has clearly arisen from other known entities by a combination of transposition and homologous recombination using a short segment of homology. This resistance gene cluster is now widely distributed in plasmids and genomic islands in a number of contexts.
Introduction
Materials and Methods
Gene identification
Published polymerase chain reaction (PCR) primers were used to detect the sul2, strA, and strB genes; and the mobA region of RSF1010 14 and PCR conditions were essentially as previously described. 12 Briefly, PCR was performed in a final volume of 50 μl containing 10 mM Tris-HCl (pH 8.3), 10 mM KCl, 2 mM MgCl2, 0.2 mM of each deoxyribonucleotide triphosphate, 20 pmol of each primer, and 1 U of Taq DNA polymerase (Roche). Genomic DNA, isolated as previously described, was used as template. 12 Thermal cycling conditions comprised an initial denaturation cycle (95°C, 5 min), followed by 35 cycles of denaturation (95°C, 30 sec), annealing at temperatures ranging from 55°C to 65°C (30–60 sec), and extension (72°C, 90 sec). A final extension cycle (72°C, 5 min) completed the PCR. DNA fragments were resolved by agarose gel electrophoresis using 1%–2% (wt/vol) agarose gels, stained with ethidium bromide (5 μg/ml), visualized with UV light, and imaged using a GelDoc1000 image analysis station (BioRad).
Plasmid isolation and analysis
Plasmid DNA was isolated using an alkaline lysis miniprep procedure and transformed into chemically competent Escherichia coli DH5α (Sigma) using the supplier's instructions. Transformants were recovered by selection for resistance to streptomycin (25 μg/ml). Plasmid DNA was extracted from suitable transformants and digested with restriction enzymes according to the manufacturer's instructions.
The sequence was determined using PCR-amplified fragments of purified plasmid DNA as templates. PCR products were purified for sequencing using the QIAquick PCR Purification Kit (Qiagen) or after separation on an agarose gel using the QIAquick Gel Extraction Kit (Qiagen) following protocols supplied by the manufacturer. Automated sequencing was performed at Supamac (Sydney University Prince Alfred Molecular Analysis Centre) on an Applied Biosystem 3730, 7900 Genetic Analyser and Transgenomic's WAVE-dHPLC (Applied Biosystems) using the Big Dye system. Sequences were assembled using Sequencher version 4.9 (Gene Codes Corporation). To ensure that the Taq polymerase had not introduced errors, all differences from the RSF1010 sequence that were not also present in available sequences of related regions were checked by using more than one PCR product as template.
GenBank accession number
The sequence of pSRC15 has been deposited under accession no. GQ379901. To keep the resistance genes together and thus simplify comparisons of the resistance region with similar regions found elsewhere, for example, in RSF1010, we have deposited the pSRC15 sequence with base 1 corresponding to position 5440 in the RSF1010 sequence in GenBank accession no. M28829.
Results
Several streptomycin and sulphonamide resistant S. enterica serovar Typhimurium isolates of phage type PT9 isolated in 2001 from both human and bovine infections were screened for the presence of a set of relevant antibiotic resistance genes. Only the sul2 gene conferring resistance to sulphonamides and the strA and strB genes conferring resistance to streptomycin were detected. These isolates did not contain plasmids belonging to any of the incompatibility groups detected by the procedure of Carattoli et al. 3 but did contain the mobA gene of the IncQ plasmid RSF1010. 14 Small plasmids were recovered from one human and one bovine isolate by transformation into E. coli with selection for streptomycin resistance. Digestion of the plasmid DNA with restriction enzymes PstI and HinfI gave identical patterns for the human and bovine-derived plasmids, and the fragment sizes were as predicted for RSF1010 from the available sequence (GenBank accession no. M28829).
One plasmid, designated pSRC15, derived from a human isolate was sequenced (GenBank accession no. GQ379901), and the 8.6 kb sequence is nearly identical to the available RSF1010 sequence (GenBank accession no. M28829). RSF1010, one of the earliest plasmids sequenced, 22 is 8.6 kb long and contains three genes required for replication (repA, B′ and C),20,21 three genes required for mobilization (mobA, B, and C),7,15,26, and three antibiotic resistance genes, strA, strB, and sul2, which are clustered together (Fig. 1A). Differences between the RSF1010 and pSRC15 sequences included four bases missing from the M28829 sequence that cause frameshifts in the sul2 gene. These differences clearly represent errors in M28829, as the bases are present in the original sequence of sul2 in RSF1010 17 and in most of the sul2 sequences found in GenBank that are from a wide variety of different plasmids. Close + and − frameshifts in the repC gene of pSRC15 were also found in other repC sequences in GenBank, and two single base differences in strA were present in most reports of this gene. Only a single difference in mobC cannot be confirmed due to a lack of further sequences for this region. We conclude that most of the differences represent errors in the original RSF1010 sequence and, hence, that this small plasmid has persisted in the environment essentially unchanged for at least 30 years.
Schematic maps of antibiotic resistance regions (not to scale). (
Origin of the resistance gene cluster
Since the configuration of sul2 with strAB adjacent to it found in pSRC15 and RSF1010 (Fig. 1A) is seen in many sequenced plasmids, we sought to understand the origins of this resistance gene cluster by examining its structure and relationship to other available sequences. The strA and strB genes encode phosphotransferases that confer resistance to streptomycin but not spectinomycin and are usually found within transposon Tn5393 4 and its insertion sequence (IS)-free parent Tn5393c.11,23 The sul2 gene encodes a sulphonamide-insensitive dihydropteroate synthase. As previously noted, 11 RSF1010 and pSRC15 contain a continuous 1778 bp segment of transposon Tn5393 that includes strAB and the adjacent inverted repeat (IRR). However, the sul2 gene is not in its original context (Fig. 1A). When it occurs alone (e.g., in GenBank accession no. AB277723), sul2 is found adjacent to the readily recognizable end (ori end in Fig. 1B) of the small mobile element CR2. 16 RSF1010 and pSRC15 include only a fragment of CR2 that was originally detected via the homology between the product of a short open reading frame in RSF1010 (designated rcr2Δ in Fig. 2) and the N-terminal part of Rcr1 (formerly Orf513 or Orf 345). 24 Rcr1 and Rcr2 (formerly OrfA) are the enzymes likely to be responsible for initiating movement of CR1 and CR2, respectively, via rolling circle replication. The CR2-derived region, delineated by comparing the RSF1010 sequence with that of other regions containing a complete copy of CR2, covers positions 5058–5580 in pSRC15 (1,809–2,331 in M28829) with the ter end of CR2 at bp 5580 (or 2,331 in M28829) (Fig. 2A).
Boundaries in RSF1010. (
In pSRC15 and RSF1010, the Tn5393 fragment separates the CR2 remnant from the sul2 gene, and 1.6 kb from the center of the CR2-sul2 region is missing (Fig. 1). This configuration suggests that the resistance gene region in RSF1010 arose via transposition of Tn5393 into CR2 in the CR2-sul2 configuration (Fig. 1B) followed by a second event that removed part of Tn5393 and part of CR2 (or via a one-ended transposition event initiated at IRR of Tn5393 and resolved by another event). The second event appears to be a homologous recombination within a short direct duplication (Fig. 2B). A 13-bp segment (with 11/13 matches) that is present in both Tn5393 and downstream of the sul2 gene (indicated by vertical arrows in Fig. 1B) is found at the second junction at bp 3280–3292 in pSRC15 (bp 31–43 in M28829).
Discussion
RSF1010, a small multicopy plasmid that confers resistance to streptomycin and sulfonamides, is mobilizable but not conjugative.19,20 It was first recovered from an Escherichia coli strain isolated from pig feces in Czechoslovakia before 1971.6,10 RSF1010 is one of the prototypes for the IncQ incompatibility group,19,20 the others, believed to be nearly identical, being R1162 13 , which was found in a Pseudomonas aeruginosa in Canada before 1971 2 , and R300B, which was found in a S. enterica serovar Typhimurium in the United Kingdom before 1970. 1 RSF1010 has a very broad host range, because it can replicate in a wide range of organisms, both gram-negative and gram-positive, and has been used as the basis of broad host range cloning vectors that are used in many different organisms. RSF1010 (R1162, R300B, or pSRC15) is also widely disseminated. Identical or near identical plasmids have been detected in many countries over, at least, a 30-year period, being found in many species, in clinical isolates from humans and animals1,2,6,10,19 and in isolates from the environment. 14 Hence, RSF1010 appears to have survived essentially unchanged over that time span.
The sul2 sulphonamide resistance gene 17 can be found alone (e.g., in GenBank accession nos. AB076707 and AB277723 9 ) but is also often found adjacent to strAB in segments that appear to be derived from RSF1010.5,8,18,25 The sul2-strAB cluster of pSRC15 (RSF1010) appears to be very widely disseminated, being found in small plasmids related to RSF1010 25 and also in large resistance plasmids5,18 and chromosomally integrated elements.5,8
Footnotes
Acknowledgments
The project and S. Y. were supported by NHMRC Project Grant 352352, and R.M.H. was supported by NHMRC Grant 358713. X.L. was supported by an ARC Linkage grant (LP0348851). The S.P.D. laboratory also was partly supported by grants from the NSW Department of Primary Industries and the McGarvie Smith Institute. We thank Diane Lightfoot and other staff of the Microbiological Diagnostic Unit (M.D.U.), University of Melbourne, for characterizing and supplying the strains. M.D.U. is supported by The Victorian Department of Human Services.
Disclosure Statement
No competing financial interests exist.