Genetic Characterization of Antimicrobial Resistance in Canadian Isolates of Salmonella Serovar Typhimurium DT104

Abstract

PCR was used to identify antibiotic resistance determinants in 31 Canadian Salmonella serovar Typhimurium DT104 isolates. Genes encoding resistance to ampicillin (pse1 or blaP1), chloramphenicol (pasppflo-like), streptomycin-spectinomycin (aadA2), sulfonamide (sulI), and tetracycline [tet(G)] were mapped to a 13-kb region of DNA of one isolate. Two copies of sulI were identified and mapped to the 3′ end of either pse1 or aadA2 integrons. The two integrons were separated by the pasppflo-like gene and the tet(G) gene. The kanamycin resistance determinant (aphA-1) was present on a 2.0-MDa plasmid (five isolates) or on the chromosome (three isolates).

Recently in Canada there has been an increase in occurrence of Salmonella serovar Typhimurium DT104 isolates (17, 18). Many of these isolates are resistant to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamide (Su), and tetracycline (T), like those found in the United Kingdom (23) and the United States (7, 9), and some are also kanamycin resistant (KAN^r). This study describes the identification of the resistant genes involved in 31 Canadian isolates of Salmonella serovar Typhimurium DT104 as well as the location and gene order in a selected isolate (96-5227) with the ACSSuT resistance phenotype.

The antimicrobial susceptibilities (15, 20) of the 31 Salmonella serovar Typhimurium DT104 are shown in Table 1, and the PCR primers designed to identify their resistance genes are shown in Table 2. PCR conditions were optimized based on the melting temperature (T_m) of the primers, and long PCR was performed in accordance with the supplier’s protocol (Roche Diagnostics, Laval, Quebec, Canada). All primers were synthesized with a Beckman Oligo 1000M DNA synthesizer, and DNA sequencing was carried out with an ABI Prism 377 DNA sequencer (Applied Biosystems Division of Perkin-Elmer, Foster City, Calif.). DNA sequences were aligned with sequences in the National Center for Biotechnology Information database by using Blast version 2.0.4 (1).

TABLE 1.

Source, antibiograms, antibiotic resistance genes, and plasmid profiles of Canadian Salmonella serovar Typhimurium DT104 isolates

Source	Total no. of isolates	Antibiograma	Genotype identified	Plasmid size profile (MDa)
Human	6	ACSSuTr	pse1 pasppflo-likecaadA2 sulI tet(G)	60
	3	ACSSuT-KANr	pse1 aadA2 sulI tet(G) aphA-1	60, 2.0
	1	ACSuTr	pse1 sulI tet(G)	60
	1	SSuTr	tet(A)	60
	3	T-KANr	tet(B) aphA-1	60
	6	Susceptibleb		60
	1	Susceptibleb		60, 2.2, 1.4
Nonhuman	2	ACSSuTr	pse1 aadA2 sulI tet(G)	60
	1	ACSSuT-KANr	pse1 aadA2 sulI tet(G) aphA-1	60, 2.0
	5	ACSuTr	pse1 sulI tet(G)	60
	2	Susceptibleb		60
Unknown	14d	ASSu-TMPr	NAf	60, 50, 4.6, 3.7
	16e	ASSu-KANr	sulI aphA-1	60, 3.4, 2.3, 2.0, 1.4

^aTMP^r, trimethoprim resistant. 

^bSusceptible to all antibiotics tested. 

^cPutative chloramphenicol resistance determinant (2, 5), identified in strain 96-5227 by the PCR method. 

^dA phage-type-atypical (AT) isolate containing TMP resistance on a self-transmissible 4.6-MDa plasmid was used as a control for the mating experiment. 

^ePhage type PT120. 

^fNA, unable to be determined in this study. 

TABLE 2.

PCR primers used in the identification of integrons and resistance genes

Gene	PCR primer sequences 5′-3′	Expected size (bp)	GenBank accession no.	Reference strain (plasmid)	Source (reference)
Integron	5′-CS, GGC ATC CAA GCA GCA AG; 3′-CS, AAG CAG ACT TGA CCT GA	?a	M73819	NAb	P. Roy (11)
pse1	L, AAT GGC AAT CAG CGC TTC CC; R, GGG GCT TGA TGC TCA CTC CA	586	Z18955	Salmonella serovar Newport E1572	G. Jacoby (10)
aadA2	L, TGT TGG TTA CTG TGG CCG TA; R, GCT GCG AGT TCC ATA GCT TC	381	X68227	Escherichia coli(pSa)	D. Taylor (3)
sulI	qacEΔ1, TAG TTG GCG AAG TAA TCG CA; orf5, AGC TTG TGC AGA TAT GCG G; sulI, TGA AGG TTC GAC AGC AC	1,581	U49101	Proteus mirabilis 88071820	P. Roy (11)
aphA-1	L, TTA TGC CTC TTC CGA CCA TC; R, GAG AAA ACT CAC CGA GGC AG	489	U63147	Escherichia coli JE2571(pHH1457)	D. Taylor (4)
intI1	L, GCC TTG CTG TTC TTC TAC GG; R, GAT GCC TGC TTG TTC TAC GG	558	X12870	NA	P. Roy (11)
pasppflo-like	L, CAC GTT GAG CCT CTA TAT GG; R, ATG CAG AAG TAG AAC GCG AC	868	AFO71555	NA	GenBank (5)
tet(A)	L, GCT ACA TCC TGC TTG CCT TC; R, CAT AGA TCG CCG TGA AGA GG	210	X61367	Escherichia coli D20-15(pSL18)	S. Levy (14)
tet(B)	L, TTG GTT AGG GGC AAG TTT TG; R, GTA ATG GGC CAA TAA CAC CG	659	J01830	Escherichia coli D20-16(pRT11)	S. Levy (13)
tet(C)	L, CTT GAG AGC CTT CAA CCC AG; R, ATG GTC GTC ATC TAC CTG CC	418	J01749	Escherichia coli D20-6(pBR322)	S. Levy (13)
tet(D)	L, AAA CCA TTA CGG CAT TCT GC; R, GAC CGG ATA CAC CAT CCA TC	787	L06798	Escherichia coli D22-2(pSL106)	S. Levy (13)
tet(E)	L, AAA CCA CAT CCT CCA TAC GC; R, AAA TAG GCC ACA ACC GTC AG	278	L06940	Escherichia coli HB101(pSL1504)	S. Levy (12)
tet(G)	L, GCT CGG TGG TAT CTC TGC TC; R, AGC AAC AGA ATC GGG AAC AC	468	S52437	Escherichia coli(pJA8122)	T. Aoki (24)
tet(G)	L, CAG CTT TCG GAT TCT TAC GG; R, GAT TGG TGA GGC TCG TTA GC	844	S52437	Escherichia coli(pJA8122)	T. Aoki (24)
tet(S)	L, CAT AGA CAA GCC GTT GAC C; R, ATG TTT TTG GAA CGC CAG AG	667	X92946	Escherichia coli(pAT451)	M. Roberts (6)

^a?, variable size depending on the inserted gene(s). 

^bNA, not available. 

To obtain a detailed map and elucidate the order of the ACSSuT resistance phenotype genes, Southern blots of isolate 96-5227 DNA digested with BamHI, HindIII, EcoRI, BglI, XbaI, and corresponding double digests were probed sequentially with amplicons of the identified genes.

Class 1 integrons which have conserved regions (5′CS and 3′CS) often contain antimicrobial resistance gene cassettes (8, 19). PCR amplification using primers 3′-CS and 5′-CS on genomic DNA from all of the DT104 ACSSuT-resistant isolates resulted in 1.0- and 1.2-kb amplicons. DNA sequence analyses (1) of the 1.0-kb amplicon from one selected isolate (96-5227) showed a sequence identical to that of a region on InCg, an integron on Corynebacterium glutamicum plasmid pCG4 (emb|;Y14748), containing aadA2, which encodes streptomycin-spectinomycin resistance (3, 16), while the 1.2-kb amplicon contained sequence with 100% identity to pse1 (blaP1) (carb-2, emb|Z18955, coordinates 35 to 1150), which encodes ampicillin resistance (25). In addition, PCR amplification using intragenic primers (Table 2) identified the presence of pse1 in the 1.2-kb amplicons from 18 (8 ACSSuT^r, 4 ACSSuT-KAN^r, and 6 ACSuT^r) isolates and of aadA2 in the 1.0-kb amplicon of 12 (8 ACSSuT^r and 4 ACSSuT-KAN^r) isolates (Table 1). Other studies also found the pse1 and aadA2 genes within 1.0- and 1.2-kb amplicons in DT104 isolates with ACSSuT resistance (5, 20, 21).

Class 1 integrons may contain the qacEΔ1-sulI (sulfonamide resistance)-orf5 region (8, 19). All 18 isolates that had 5′CS and 3′CS sequence produced a PCR amplicon with qacEΔ1 and orf5 primers (Table 2). Partial DNA sequence analysis and PCR using intragenic sulI primers confirmed the presence of the sulfonamide resistance gene in these isolates. Furthermore, these isolates also contained the integrase (intI1) gene sequence as determined by PCR.

PCR using primers specific for tetracycline resistance genes (Table 2) showed that tet(A) was identified in one isolate with an SSuT resistance profile while tet(B) was found in three isolates with T-KAN^r profiles. In addition, tet(G) was identified in 18 DT104 isolates with ACSSuT resistance, ACSSuT-KAN^r, and ACSuT resistance profiles. DNA sequence analysis of the tet(G) PCR product from isolate 96-5227 (gb|AF11924) showed 100% identity with published sequences from Salmonella serovar Typhimurium DT104 (gb|AF071555) (5) and Pseudomonas plasmid pPSTG2 (gb|AF133140) and 93% identity (CG instead of GC at coordinates 1351 and 1352 of gb|S52437) with that of Vibrio anguillarum (24).

The chloramphenicol resistance determinant in 96-5227 was identified as the previously described pasppflo-like (flor) gene (2, 5) by using PCR primers (Fig. 1) specific for this gene (5) (gb|AF071555).

FIG. 1.

Restriction map of Salmonella serovar Typhimurium DT104 multidrug resistance region from isolate 96-5227. The orientations of the genes are indicated by arrows. The respective positions of the antibiotic resistance genes are shown in boxes. Lines below the map indicate the sizes of products obtained by using the primer sets listed. Restriction enzyme abbreviations: X, XbaI; B, BamHI; Bg, BglI; H, HindII.

All of the identified resistance genes as well as the integrase gene were mapped to an 11.8-kb XbaI fragment, and their order is shown in Fig. 1. Although Ridley and Threlfall (20) have suggested that two integron-associated genes in DT104 isolates map to a single 10-kb XbaI chromosomal fragment, our results showed that the pse1 gene, which has an internal XbaI site, actually extends to an adjacent 4.2-kb XbaI fragment (Fig. 1). This difference is probably due to the elution of the 4.2-kb XbaI fragment from their pulsed-field gel electrophoresis gels (20). The relative positions and orientation of genes on the restriction map were confirmed by using various combinations of PCR primers (Fig. 1). The structure of the integron containing aadA2 is similar to the map published by Briggs and Fratamico (5). However, we did not detect the partial integrase gene (groEL-intI1b) (5) in the 5′ region of the pse1 gene by hybridization with the intI1 probe.

It had been reported that the ACSSuT resistance genes in DT104 were located on the chromosome and were not self-transmissible (22, 23). All ACSSuT-resistant isolates in this study harbored only the 60-MDa virulence plasmid which has not been reported to confer antimicrobial resistance (23). Furthermore, this plasmid was also found in susceptible isolates (Table 1). Therefore, it is likely that the ACSSuT resistance determinants are on the chromosome of isolate 96-5227.

Kanamycin resistance of eight isolates, including four with ACSSuT-KAN^r, was encoded by the aphA-1 gene. The amplicons were verified to contain aphA-1 by AluI and DdeI digestion (gb|U63147). The aphA-1 probe hybridized to a 2-MDa plasmid present in five of the eight isolates (Table 1). This suggests that the other three isolates, containing only the 60-MDa plasmid, have aphA-1 located on the chromosome. Mating experiments showed that the 2-MDa plasmid in a selected Salmonella serovar Typhimurium isolate 97-5025 was not self-transmissible.

In summary, the order, orientation, and location of the ACSSuT resistance genes in the Canadian isolate (96-5227) were similar to those found by others (5, 20, 21). Although the origin of DNA containing the ACSSuT resistance genes remains unknown, our observations together with other published data (5, 20, 21) suggested the possible clonal dissemination of these isolates in the population. The rapid increase of ACSSuT-resistant DT104 isolates in Canada and other countries may suggest that there are other genes in the DNA region which account for the rapid dissemination of this isolate. In an attempt to better understand this unique region of the genome, we have constructed DNA libraries in order to identify other genes and determine the size of the inserted DNA in isolate 96-5227. To this end, sequence analysis and hybridization experiments showed that the insertion site of the unique region is between 24 and 30 kb upstream of the intI1 gene.