Large Conjugative Plasmids from Clinical Strains of Salmonella enterica Serovar Virchow Contain a Class 2 Integron in Addition to Class 1 Integrons and Several Non-Integron-Associated Drug Resistance Determinants

Abstract

Two large conjugative resistance (R) plasmids from clinical strains of Salmonella enterica serovar Virchow carried a class 2 integron with the 5′ conserved sequence (5′CS)-dfrA1-sat1-aadA1-3′CS gene array, which is associated with defective Tn7 transposons. In addition, each contained a different class 1 integron (with 5′CS-aadA1-3′CS or 5′CS-sat-smr-aadA1-3′CS gene arrays) linked to Tn21-Tn9 sequences, and several non-integron-associated R determinants. An intact copy of Tn7 (including the class 2 integron) was present in the chromosome of each strain.

Integrons are gene expression elements that play an important role in the recruitment of antimicrobial drug resistance (R) determinants via site-specific recombination events catalyzed by the integron-encoded integrase (15). Based on the sequence of the integrase genes, several classes of integrons have been described (10, 12).

Integrons of classes 1 and 2 were simultaneously found in six isolates of Salmonella enterica serovar Virchow that were recovered as causal agents of acute gastroenteritis in a northern region of Spain. PCR amplifications (4, 20) and/or sequence analysis revealed 1,000-bp/aadA1 and 2,300-bp/sat-smr-aadA1 variable regions in the class 1 integrons of four and two isolates, respectively. The class 2 integrons from the six isolates were apparently identical (EMBL accession number AM055749) and carried the dfrA1-sat1-aadA1 gene array and the inactive integrase gene characteristic of Tn7 (5, 17, 19). Two serovar Virchow isolates (LSP 231/90 and 205/98), each containing one of the detected integron combinations, were selected for further characterization (Table 1). Both were resistant to six nonrelated antimicrobial drugs but displayed different R patterns. By conjugation experiments using Escherichia coli K-12 J53 as the recipient, transconjugants (Tc) with R phenotypes identical to those of the parental strains were obtained. The extraction of plasmid DNA using the S1-pulsed-field gel electrophoresis (PFGE) method (1) (Fig. 1A) revealed the presence of a large conjugative R plasmid (pUO-SvR1, ca. 275 kb) in LSP231/90 and its transconjugant (Tc-231), while a different conjugative plasmid (pUO-SvR2, of about the same size) was found in LSP205/98 and its transconjugant (Tc-205). PCR amplifications using Tc-231, Tc-205, and the recipient E. coli as the sources of template DNA confirmed the location of the expected R genes and integrons in the two identified plasmids. Of note, as far as we are aware, this is the first report on the presence of class 2 integrons in self-transferable plasmids of Salmonella.

TABLE 1.

Characteristics of serovar Virchow strains and their transconjugants

Bacterial strain	R phenotypea	R genotypeb	VR size/R gene integron(s)		Transposonsd
			Class 1	Class 2	Tn21	Tn9	Tn7
LSP231/90	Amp Chl Gen Sul Str Spt Tmp	blaTEM catA1 aacC2 suI1 strA strB aadA1 dfrA1 sat sat1	2,300/sat-smr-aadA1; qacEΔ1-sul1	2,300/dfrA1-sat1-aadA1	+	+	+
Tc-231	Amp Chl Gen Sul Str Spt Tmp (Rif)c	blaTEM catA1 aacC2 suI1 strA strB aadA1 dfrA1 sat sat	2,300/sat-smr-aadA1; qacEΔ1-sul1	2,300/dfrA1-sat1-aadA1	+	+	Δ
LSP205/98	Chl Kan Sul Str Spt Tet Tmp	catA1 aphA1 suI1 strA strB aadA1 tetA dfrA1 sat sat1	1,000/aadA1; qacEΔ1-suI1e	2,300/dfrA1-sat1-aadA1	+	+	+
Tc-205	Chl Kan Sul Str Spt Tet Tmp (Rif)c	catA1 aphA1 suI1 strA strB aadA1 tetA dfrA1 sat sat	1,000/aadA1; qacEΔ1-sul1e	2,300/dfrA1-sat1-aadA1	+	+	Δ

^aThe strains were tested for susceptibility to antimicrobial drugs by disk diffusion according to the method of the CLSI (formerly NCCLS) (2).

^bResistance determinants were identified by PCR amplification using previously described primers and conditions (4, 6). Resistance to streptothricin has not been tested, but the presence of sat genes was established by nucleotide sequencing.

^cResistance from the E. coli recipient strain.

^d+, positive for the indicated transposon; Δ, truncated Tn7.

^eGenes located in the 3′ conserved sequence region of the integron(s).

FIG. 1.

Mapping of integron, transposon, and R genes on the S. enterica serovar Virchow genome. (A) XbaI and S1 profiles of serovar Virchow strains and their transconjugants. (B through H) Hybridizations of panel A with the probes indicated at the left of each panel. Lanes M and L, PFGE marker I (New England BioLabs). Lanes 1 and 2, serovar Virchow LSP231/90 and Tc-231, both carrying pUO-SvR1. Lanes 3 and 4, serovar Virchow LSP205/98 and Tc-205, both carrying pUO-SvR2. Lane 5, serovar Virchow CECT 4154, susceptible to antimicrobial drugs. Lane 6, E. coli K-12 J53, plasmid free. The arrow indicates the ca. 275-kb plasmids.

In order to investigate a possible association between integrons and transposons (7, 17, 19, 22), the presence of Tn21-, Tn9-, and Tn7-related sequences in the two serovar Virchow strains and their transconjugants was investigated by PCR amplifications of individual genes (using already-described primers and primers designed for the present work) (4) (Table 2). The four strains, but not E. coli K-12 J53, yielded right-sized amplicons with primers that were specific for the tnpA, tnpR, and merEDACPTR genes of Tn21. The same result was obtained for the catA1 gene, which was used as an indicator of Tn9. With regard to Tn7, all tested genes (ybfA, ybfB, ybgA, tnsE, tnsD, tnsC, tnsB, and tnsA) could be amplified from LSP231/90 and LSP205/98. However, genes downstream of ybfA and tnsD were not detected in Tc-231 and Tc-205, respectively. Additional amplifications of overlapping fragments (Fig. 2) demonstrated (i) the insertion of the two class 1 integrons within Tn21, which was in turn associated to Tn9, forming complex structures that differed from Tn2670 (7) by the absence of IS1353 (Fig. 2A), and (ii) the existence of an apparently intact copy of Tn7, including the class 2 integron, on the chromosome of each of the serovar Virchow strains and of a second copy of the integron, associated to a truncated Tn7, in each of the conjugative plasmids carried by these strains (Fig. 2B).

TABLE 2.

Primers designed for this work

Region or gene	Name	Sequence (5′ to 3′)	Temp (°C)	Accession no.
IS1 from Tn9	IS1a-F	CTGACGGGGTGGTGCGTAACGGC/	65	NC_002134
	IS1a-R	GTAAAACAGCCAGCGCTGGCGCG
tnpA	tnpA-R100-F	GATCGGCGCGGGGAAGTTCAGGC	65	NC_002134
	tnpA-R100-R	AGCCGGTTGCAGAGGCCGTAGC
tnpR	tnpR-R100-F	GAGCGTCAGCGCGAGGGTATTG	—a	AF_071413
istA	istA-F	GACAGCGTTGGCAAGTTAAGTCC	60	AF_071413
	istA-R	GGCATGCCAGTGGGCATCAAACA
tniA	tniA-F	GATCCTCCGGCGCACGCTGACCC	65	AF_071413
	tniA-R	AATCTGGTCGAACGGTTTGGCGG
urf2	urf2-F	GGAGATCGAACTGACCGAATCGG	65	AF_071413
	urf2-R	CAGGAGCTGGCTGCACAACAGC
merE	merE-F	CGCCCCTGACAAACTGCCGCCC	60	AF_071413
	merE-R	CGAGCGCGGCAACACCCCAATG
merD	merD-F	TACGGCCGGTGGCCTGCACCACG	60	AF_071413
	merD-R	CGCTCGGCTGGCATGGAGGCCAG
merA	merA-F2	AAGGCGCACGTGTCACGCTGATC	70	AF_071413
	merA-R2	CCGCTGCCTTCTTCAACCACCAG
merC	merC-F	AGCGTCGTTTCCGCGATGGGCTG	60	AF_071413
	merC-R	CCGGTCCGCAACGGCGATGCGCC
merP	merP-F	CTCCCTTGCCCTCGCCGCCGCTG	65	AF_071413
	merP-R	GGACGGATAGCCGGCGTCTGCGG
merT	merT-F	GCGCCACGCGAGAAGCGGCCGC	60	AF_071413
	merT-R	GAAATCCAAGCGCGACCAGGAC
merR	merR-F	GGGGTCAACGTGGAGACAATCCG	65	AF_071413
	merR-R	CCTTGAGCTTGTGTTCGGCCAGG
ybfA	ybfA-F	CGCCCAAGTAAGTATCCAGCTGTG/	60	NC_002525
	ybfA-R	CTTGACATCTCATCAATACCACC
ybfB	ybfB-F	CGATGTGGCTACGAGTCTGATC/	55	NC_002525
	ybfB-R	CTTCTCTCTAGATAGCAGGG
ybgA	ybgA-F	CTATGCACTTTGTGACAAGTTGG/	60	NC_002525
	ybgA-R	CTTGAAGTGCAGGCGAGCACG
tnsA	tnsA-F	GCAGCAGCCTTACAAGACGAGCG	60	NC_002525
	tnsA-R	GGCTAATACAGAGATCTGCACAC
tnsB	tnsB-F	TGTAGGTTTGCCAGATGTGTTGC	60	NC_002525
	tnsB-R	TGACCCCGAGTAATACAAACCCC
tnsC	tnsC-F	GGCAATTACAGCTTTTACAACGC	60	NC_002525
	tnsC-R	CTGCGATATCTAGCTGAAGTTGG
tnsD	tnsD-F	ACAGGGATTGGCTAGTTCACTGG/	60	NC_002525
	tnsD-R	CTTTGTGCTTCCTCAGTTATCCG
tnsE	tnsE-F	GGCGGATCGTGTGATTGAGTTTG/	60	NC_002525
	tnsE-R	GCGTACTACCATCACCTGCCTCC

^atnpR-R100-F was used together with tnpA-R100-R at an annealing temperature of 65°C.

FIG. 2.

Integron-transposon associations. (A) Physical linkage between the class 1 integrons, Tn21, and Tn9. The scheme is based on sequencing data from R100 Shigella flexneri plasmid (NC_002134). The class 1 integron, Tn21, and Tn9 sequences are indicated by gray, white, and black arrows, respectively. All of the overlapping fragments that are depicted below the scheme could be amplified from the two serovar Virchow strains and their transconjugants. ORF5, open reading frame 5. (B) Physical linkage between class 2 integrons and Tn7. The schematic representation is based on sequencing data from the R721 plasmid of E. coli (NC_002525). Integron and transposon genes are indicated by gray and white arrows, respectively. All of the overlapping amplicons were generated from the two serovar Virchow strains, except the last three from Tc-205 and the first two from Tc-231. Primers used in PCR amplifications are compiled in Table 2.

LSP231/90, LSP205/98, and their transconjugants were also analyzed by macrorestriction using XbaI-PFGE (11). The two serovar Virchow strains generated distinctive profiles (Jaccard's coefficient of similarity, 0.79), which included 16 matching and 7 nonmatching fragments (Fig. 1A). Selected probes for classes 1 (qacEΔ1, sul1, and aadA1) and 2 (aadA1, dfrA1) integrons, for Tn21 (merA), Tn9 (catA1), and Tn7 (tnsE and tnsA) transposons, and for R genes that were not associated to integrons or transposons [aacC2, strA, strB, bla_TEM, and tetA(A)] were mapped on the XbaI and S1-PFGE profiles of the four strains (8, 13).

With LSP231/90 and Tc-231, all probes except tnsE mapped on a ca. 75-kb fragment of the XbaI profiles and on the ca. 275-kb band corresponding to pUO-SvR1 (Fig. 1B, C, and E through G). The tnsE probe mapped on a 75-kb fragment from LSP231/90 but failed to hybridize with the XbaI profile of Tc-231 and with pUO-SvR1 (Fig. 1D). These results located the class 1 integron that was linked to Tn21-Tn9 sequences and the class 2 integron inserted into a defective Tn7, as well as all independent R genes within a ca. 75-kb XbaI fragment that was generated from pUO-SvR1. The hybridization of tnsE on a fragment of about the same size suggests the existence of two comigrating bands on the macrorestriction profile of LSP231/90, one from the chromosome and one from pUO-SvR1. In the former, the intact copy of Tn7 (including the class 2 integron) would be located. The high intensity of the relevant fragment on the agarose gel (Fig. 1A) supports this possibility.

In LSP205/98 and its transconjugant, the qacEΔ1-sul1, merA, and catA1 probes (and hence the Tn2670-like transposon with the class 1 integron), as well as the probes for independent R genes, mapped on the ca. 275-kb pUO-SvR2 and on a ca. 65-kb XbaI-fragment that was common to the macrorestriction profiles of the two strains and therefore of plasmid origin (Fig. 1B, F, and H). The dfrA1 probe hybridized with the 275-kb plasmid, a ca. 40-kb XbaI fragment from the transconjugant, and two XbaI fragments (of ca. 75 and 40 kb) from the donor strain (Fig. 1C). These results are consistent with the presence of the class 2 integron in both the plasmid and the chromosome of LSP205/98. This was confirmed by hybridizations with probes for aadA1 (Fig. 1E), a gene cassette shared by the classes 1 and 2 integrons of LSP205/98, and tnsE (Fig. 1D). The former gene mapped on the 275-kb plasmid and on two XbaI fragments (65 and 40 kb) from Tc-205. By comparison with results that were obtained with the qacEΔ1-sul1 and dfrA1 probes, the 65-kb fragment would contain the aadA1 cassette of the class 1 integron and the 40-kb fragment would contain the aadA1 cassette of the class 2 integron. In the XbaI profile of LSP205/98, an additional fragment of ca. 75 kb (where dfrA1 was previously located) hybridized with aadA1 and tnsE, hence verifying the existence of the chromosomal copy of the Tn7 integron (Fig. 1D and E). The association of the extrachromosomal copies of the class 2 integron with truncated Tn7 transposons was finally corroborated by the absence of hybridization of a tnsA probe with the two plasmids and with the XbaI profiles of the transconjugants (not shown).

With regard to public health, serovar Virchow has emerged as the third or fourth most common Salmonella serovar that causes human gastroenteritis in Europe (3, 18) and its incidence is even higher in countries like Australia and Israel, where it has been reported as an important cause of bacteremia in children (9, 14, 16, 21). Taking this into account, the emergence of multiple drug-resistant strains, such as those reported here, is an obvious cause of concern, as it is this wide range of mobile genetic elements that can efficiently contribute to the acquisition, maintenance, and spread of resistance determinants.

Nucleotide sequence accession number.

The sequence for sat-smr-aadA was deposited in the EMBL database under accession number AM055748.