Identification and Characterization of Class 1 Integron Resistance Gene Cassettes among Salmonella Strains Isolated from Imported Seafood

A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport strain 62], Typhimurium var. Copenhagen strain 629], and Lansing strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.Salmonella spp. are recognized as major food-borne pathogens of humans worldwide. In the United States, there are an estimated 800,000 to 4 million Salmonella infections annually, and approximately 500 of the cases are fatal (8, 26). A variety of foods have been implicated as vehicles transmitting salmonellosis to humans, including poultry, beef, pork, eggs, milk, cheese, fish, shellfish, fruits, juice, and vegetables (1, 4, 9, 12, 23). Previous studies by field laboratories of the U.S. Food and Drug Administration have shown the prevalences of Salmonella isolates in imported and domestic seafood as 7.2% and 1.3%, respectively (6, 11, 27).Mobile genetic elements, such as plasmids, transposons, and integrons, which disseminate antibiotic resistance genes by horizontal or vertical transfer, as part of either resistance plasmids or conjugative transposons, play an important role in the evolution and dissemination of multidrug resistance (2, 3, 10, 17). Salmonella genomic island 1 (SGI1), the first genomic island reported to contain an antibiotic resistance gene cluster, was identified in the multidrug-resistant Salmonella enterica serovar Typhimurium strain DT 104 (21).Most studies of the prevalence and characterization of antimicrobial resistance genes and integrons in Salmonella spp. have focused on strains from clinical and veterinary sources. However, little is known about the occurrence of SGI1 and its variants in Salmonella spp. isolated from seafood. We have screened a set of drug-resistant S. enterica strains from seafood belonging to 64 different serovars for SGI1 and class 1 integron conserved sequences (CS). We report the presence of a class I variant integron carrying the dfrXII and aadA2 genes on a megaplasmid in serovar Typhimurium var. Copenhagen and on the chromosome in Salmonella enterica serovar Lansing. We also found the variant class 1 integron carrying the dfrA1 and orfC genes in Salmonella enterica serovar Newport strains from seafood.A total of 210 Salmonella enterica strains isolated from seafood imported into the United States between 2000 and 2005 were identified and serotyped by the Pacific Regional Laboratory-Southwest of the FDA, Irvine, CA. The Salmonella strains represented 20 serogroups (Table ​(Table1)1) from various imported seafood items. The Salmonella strains were tested with 16 antibiotics (14) commonly used in either human or veterinary medicine on Mueller-Hinton agar (Difco Laboratories, Detroit, MI), using a disk diffusion method. The sensitivity and resistance were determined by the criteria of the Clinical and Laboratory Standards Institute (1999).

TABLE 1.

Salmonella serotypes isolated from imported foods

No. of strains	S. enterica serovar(s) or Salmonella group(s)
39	Weltevreden
16	Newport
13	Saintpaul
10	Senftenberg
8	Lexington
7	Virchow
6	Enteritidis, Bareily
5	Bovismorbificans, Brunei, Java, Hvittingfoss
4	Paratyphi B var. Java, Thompson
3	Aberdeen, Cubana, Stanley, Derby, Lansing
2	Montevideo, Hadar, Agona, San Diego, Braenderup, Lanka, Salmonella enterica subsp. diarizonae, Oslo, Bareily variant, Salmonella monophasic group C2
1	Ouakam, Cannstatt, Albany, Newport/Bardo, Adelaide, S. enterica subsp. diarizonae, Houten, Giza, Miami, Onderstepoof, Infantis, Salmonella monophasic group D1, Mbandaka, Salmonella monophasic group G2, Ohio, Rutgers, Salmonella monophasic group D2, Amsterdam, Salmonella enterica subsp. IV serotype 43:z4z23, Paratyphi B var. Java, Wentworth, Potsdam, Muenster var. 15+, 34+, Lexington var. 15+, Weltevreden var. 15+, S. enterica subsp. I, Madella, Alachua, London, Singapore, Uphill, Thielallee, Typhimurium var. Copenhagen

Open in a separate windowAll Salmonella strains that were resistant to three or four antibiotics and trimethoprim were screened by PCR for the presence of class 1 integrons, using the CSL1 and CSR1 primers (Table ​(Table2)2) (14). To confirm other antibiotic resistance genes, we used primers and PCR methods described previously (13, 14, 16). To identify SGI1 in multidrug-resistant strains, PCR was performed by using primers U7-L12/LJ-R1 and 104-RJ/104-D (Table ​(Table2),2), corresponding to the left and right junctions of SGI1 in the Salmonella chromosome, respectively (16). For a positive control, serovar Typhimurium DT104 strain DT7 (13) was used. As a negative control, Escherichia coli cells or DNA was used. A reagent blank included in each PCR contained distilled water instead of template DNA. For sequencing, the PCR-amplified integrons were purified and cloned into plasmid vector pCR2.1 (Invitrogen Corp., Carlsbad, CA). The clones were investigated for the presence of inserts by isolating the recombinant plasmid, which was confirmed by digestion with the restriction enzyme EcoRI. Sequencing of both strands was performed. DNA sequences were analyzed with Lasergene (DNASTAR, Inc., Madison, WI) software. Oligonucleotide primers and probes were purchased from MWG Biotech (High Point, NC).

TABLE 2.

Primer pairs for integron PCR and sequencing

Primer	Sequence (5′-3′)	Location	PCR product size (bp)
CSL1	GGC ATC CAA GCA GCA AGC	5′ CS
CSR1	AAG CAG ACT TGA CCT GAT	3′ CS
U7-L12	ACA CCT TGA GCA GGG CAA AG	thdF	500
LJ-R1	AGT TCT AAA GGT TCG TAG TCG
104-RJ	TGA CGA GCT GAA GCG AAT TG	S044
104D	ACC AGG GCA AAA CTA CAC AG	yidY
aadA2F	TGT TGG TTA CTG TGG CCG TA	aadA2	380
aadA2R	GCT GCG AGT TCC ATA GCT TC

Open in a separate windowPlasmid DNA was isolated using an alkaline lysis method with modifications described previously (19). Plasmids were separated by electrophoresis in 1× Tris-acetate-EDTA buffer at 64 V for 2 h on 1.0% agarose gels, stained with 40 μl of ethidium bromide (0.625 mg/ml) for visualization, and then transferred and cross-linked to positively charged nylon membranes (Roche, Indianapolis, IN). The resulting blots were hybridized at 65°C for 18 h with digoxigenin-labeled DNA probes (1.2-kb and 1.9-kb PCR-amplified products), using CSL1 and CSR1 primers specific for class 1 integrons (22).