Detection of multidrug-resistant Salmonella typhimurium DT104 by multiplex polymerase chain reaction

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1 FEMS Microbiology Letters 182 (2000) 355^360 Detection of multidrug-resistant Salmonella typhimurium DT104 by multiplex polymerase chain reaction Ashraf A. Khan *, Mohammed S. Nawaz, Saeed A. Khan, Carl E. Cerniglia Division of Microbiology, Food and Drug Administration, National Center for Toxicological Research, Je erson, AR 72079, USA Received 26 August 1999; accepted 18 November 1999 Abstract Salmonella typhimurium definitive type 104 (DT104) is a virulent pathogen for humans and animals with many strains having multiple drug resistance characteristics. The organism typically carries resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant). A multiplex PCR method was developed to simultaneously amplify four genes, florfenicol (flo st ), virulence (spvc), invasion (inva), and integron (int) from S. typhimurium DT104 (ACSSuT-type). Twenty-two ACSSuTresistant DT104 isolates in our collection gave 100% positive reactions to this PCR assay by amplifying 584-, 392-, 321- and 265-bp PCR products, using primers specific to the respective target genes. One Salmonella strain DT23, ACSSuT-resistant, phage type 711 failed to amplify the 584-bp fragment, indicating that this method is specific for DT104-type ACSSuT-resistant S. typhimurium strains. One clinical and one bovine ASSuT-resistant strains that were sensitive to chloramphenicol and florfenicol did not yield a 584-bp fragment, indicating the absence of the flo st gene. This method will be useful for rapid identification of ACSSuT-type DT104 strains from clinical, food and environmental samples. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Multiplex PCR; Salmonella typhimurium DT104; Multi-drug resistant; Detection; ACSSuT-R type 1. Introduction * Corresponding author. Tel.: +1 (870) ; Fax: +1 (870) ; akhan@nctr.fda.gov Salmonella typhimurium de nitive type 104 (S. typhimurium DT104 or DT104) is an increasingly common multiple-antibiotic-resistant strain that has rapidly emerged as a world health problem [1,2]. Most DT104 strains are characterized by chromosomal resistance to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamides (Su), and tetracycline (T) and are referred to as ACS- SuT-type [3,4]. The Centers for Disease Control and Prevention (CDC, Atlanta, GA) have established a Salmonella and Antimicrobial Monitory Resistance Surveillance system [1,4]. In the 1996 National Salmonella Antimicrobial Resistance Monitoring System Study, the CDC serotyped 3903 Salmonella isolates and determined that 976 (25%) were S. typhimurium. Approximately 28% of the S. typhimurium isolates had the R type ACSSuT compared to just 7% in 1990 [5]. Two other antibiotic resistance monitoring programs, Periodic Surveys of Antimicrobial Drug Resistance in Sentinel Counties and the National Antimicrobial Resistance Monitoring System, have reported similar sharp increases in multidrug-resistant Salmonella spp. [1]. The mechanisms for resistance to sulfonamides, streptomycin, ampicillin and orfenicol in DT104 have been described previously [6,7]. Most of the DT104 ACSSuT-type strains contain at least two integrons, one containing the aminoglycoside resistance gene cassette ant (3Q)-la, which encodes resistance to streptomycin, and one containing a L-lactamase resistance gene cassette, pse-1, which encodes resistance to ampicillin [6,7]. A gene encoding sulfonamide resistance (sul-1) was found in the 3P conserved sequences of both integrons [7]. Florfenicol is a uorinated analog of chloramphenicol approved by the FDA in 1996 for the treatment of bovine respiratory pathogens. Previous studies have shown that bacterial isolates which were resistant to chloramphenicol were sensitive to inhibition by uorinated analogs [8,9]. The mechanism of resistance to chloramphenicol and orfenicol in DT104 was recently described [8] and the gene o st confers resistance to both of these antibiotics. Current diagnostic methods used for the identi cation / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (99)

2 356 A.A. Khan et al. / FEMS Microbiology Letters 182 (2000) 355^360 and characterization of ACSSuT-type DT104 strains require several days [10]. Since DT104 ACSSuT-type strains are becoming widespread globally, development of a rapid and sensitive method for the diagnosis of DT104 ACS- SuT-type strain is desirable. PCR technology combines simplicity with a potential for ampli cation of a speci c fragment of nucleic acid and has been used to identify the presence of speci c pathogens directly from clinical specimens, food and water [11,12]. We report a multiplex PCR method with four sets of primers, orfenicol ( o st ), integron (int), invasion (inva), and virulence (spvc) that permitted speci c detection of S. typhimurium DT104 (ACS- SuT-type) strains. In addition, we assessed these four target genes in multidrug-resistant DT104 isolates from di erent sources. 2. Materials and methods 2.1. Bacterial strains The S. typhimurium strains DT104 and other multidrugresistant strains used in this study were obtained from the Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, Washington, DC (Table 1). These isolates were from human, cattle, swine, sewage, clinical and cattle feces samples. All isolates were serotyped at FDA laboratories or by their original sources (Table 1). The bacteria were grown and maintained in Nutrient Broth (Difco Laboratories, Detroit, MI) at 37³C Antibiotic resistance testing The strains were tested for resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and orfenicol, by the disk agar di usion method performed on Mueller-Hinton (Difco Laboratories, Detroit, MI) agar plates. The antibiotic disks used in this study were purchased from Difco Laboratories unless otherwise speci ed. Disks contained the following amounts of antibiotic: ampicillin 10 Wg (A), chloramphenicol 30 Wg (C), tetracycline 30 Wg (T), streptomycin 10 Wg (S), orfenicol 30 Wg (F) (Schering-Plough Animal Health, Kenilworth, NJ), and sul soxazole 250 Wg (Su) (Becton Dickinson and Company, Cockeysville, MD). S. typhimurium ATCC was used as a sensitive control. The sensitivity and resistance were determined as per the criteria of the National Committee for Clinical Laboratory Standards [13] Preparation of the bacterial samples Bacteria were lysed with an equal volume of 0.2% (w/v) Triton X-100 and heated at 100³C for 5 min in a boiling water bath. The samples were allowed to cool immediately in ice for 5 min and used immediately in the PCR. Whenever necessary, chromosomal DNA was puri ed by phenol-chloroform-isoamyl alcohol extraction by the method of Wheatcroft and Watson [14]. Puri ed DNA was precipitated with ethanol, resuspended in sterile bu er (10 mm Tris-HCl, 1 mm EDTA, ph 8.0) at about 1 Wg ml 31, based on the optical density at 260 nm, and stored at 320³C until use Primer design Oligonucleotide sequences of orfenicol ( o st ), integron (int), invasion (inva), and virulence (spvc) genes for the multiplex PCR ampli cation of DT104 are listed in Table 2. The gene primers were designed by using published sequences (GenBank accession numbers AF097407, AF071555, M90846 and M64295, respectively) and DNASTAR primer select software (DNASTAR, Inc., Madison, WI). The o st, int, inva and spvc genes primer sequences were predicted to yield 584, 265, 321 and 392-bp products. The speci city of the primers was con rmed by the GenBank database `Blast' program. The primers used in this study were synthesized by Universal DNA Inc., Tigard, OR PCR ampli cation conditions The ampli cation of the individual target genes for all four pairs of primers from one S. typhimurium (ACSSuTtype DT104) strain was performed by using a DNA thermal cycler (PE Applied Biosystems model 480, Foster City, CA) and the GeneAmp kit with Taq DNA polymerase (PE Applied Biosystems) in 0.5-ml microcentrifuge tubes. The reaction mixture (50 Wl total volume) consisted of Wl of sterile water, 5 Wl of 10UPCR bu er (100 mm Tris-HCl (ph 8.3), 500 mm KCl, 15 mm MgCl 2, 0.1% (w/v) gelatin), 4 Wl of deoxyribonucleoside triphosphates (2.5 mm each datp, dttp, dgtp and dctp), 0.5 Wl of each primer (stock concentration, 100 WM), 1^10 Wl of template, and 0.25 Wl (5UWl 31 )of Taq DNA polymerase. After overlaying with sterile mineral oil, the samples were subjected to PCR ampli cation. Preincubation was at 94³C for 90 s. Thirty PCR cycles were run under the following conditions: denaturation at 94³C for 45 s, primer annealing at 60³C for 45 s, and DNA extension at 72³C for 90 s in each cycle. After the last cycle, the PCR tubes were incubated for 3 min at 72³C and then at 4³C. For multiplex PCR, 100 ng (each) of primers was used. The optimization of multiplex PCR was achieved by individually amplifying all four genes at 1.5, 2.0, 2.5 and 3.0 mm MgCl 2. The annealing temperature was also optimized with all four pairs of primers Detection of ampli ed DNAs Three microliters of the reaction mixture were analyzed by standard submarine gel electrophoresis (1.5% agarose;

3 A.A. Khan et al. / FEMS Microbiology Letters 182 (2000) 355^ Vcm 31 ), and the reaction products were visualized by staining with ethidium bromide (0.5 Wg ml 31 in the running bu er). As negative control, Escherichia coli cells or DNA was used. A reagent blank contained all components of the reaction mixture with the exception of template DNA, for which sterile distilled water was used. This blank was included in every PCR procedure. The thermocycler, tips and pipetters used for preparing the PCR reagents and template DNA were kept in a di erent location from where the gels were loaded, stained and photographed. All reagents used in an experiment were taken from the freezer and discarded at the end of the day Speci city of primers The speci city of multiplex PCR for DT104 ACSSuT strains was evaluated with 33 S. typhimurium strains (Table 1). 3. Results and discussion This paper describes a simple and speci c multiplex PCR method to detect multidrug-resistant (ACSSuTtype) S. typhimurium DT104 strains in a single step. All Salmonella strains listed in Table 1 were tested for drug resistance using a disk agar di usion method. We found that ACSSuT-type S. typhimurium DT104 isolates resistant to chloramphenicol were also resistant to orfenicol, except that one strain (DT23, phage type 771) was sensitive to 30 Wg of orfenicol. This may be due to the absence of the o st gene. Previous studies have shown that most of the bacterial isolates which were resistant to chloramphenicol were sensitive to inhibition by uorinated analogs of chloramphenicol [8,9]. The antibiotic resistance pro les of DT104 strains and other multidrug-resistant S. typhimurium strains are listed in Table 1. Among 32 strains studied, 24 were resistant to A, C, S, Su, T and F, four strains (DT 8, 12, 22 and 25) were resistant to A, S, Su and T. One strain DT23 (phage type 771) was sensitive to F but resistant to A, C, S, Su, and T. Strain DT17 was sensitive to A, C, F and T, and DT16 and 19 were sensitive to A, C, S, F and T. Two strains, DT21 and 24, were U302 phage type and had antibiotic pro les similar to other DT104 (ACSSuT-type) strains and were also resistant to orfenicol. Table 1 Genotypic and phenotypic characteristics of S. typhimurium DT104 isolates Designation Source Serotype Antibiotic resistance S. typhimurium DT1 Cattle DT104 A, C, F, S, Su, T S. typhimurium DT2 Human DT104 A, C, F, S, Su, T S. typhimurium DT3 Bovine DT104 A, C, F, S, Su, T S. typhimurium DT4 Bovine DT104 A, C, F, S, Su, T S. typhimurium DT5 Bovine DT104 A, C, F, S, Su, T S. typhimurium DT6 Bovine DT104 A, C, F, S, Su, T S. typhimurium DT7 Bovine DT104 A, C, F, S, Su, T S. typhimurium DT8 Bovine DT104 A, S, Su, T S. typhimurium DT9 Clinical DT104B A, C, F, S, Su, T S. typhimurium DT10 Bovine DT104B A, C, F, S, Su, T S. typhimurium DT11 Clinical DT104 A, C, F, S, Su, T S. typhimurium DT12 Clinical DT104 A, S, Su, T S. typhimurium DT13 Swine DT104 A, C, F, S, Su, T S. typhimurium DT14 Chicken DT104 A, C, F, S, Su, T S. typhimurium DT15 Human DT104 A, C, F, S, Su, T S. typhimurium DT16 Human DT104 Su S. typhimurium DT17 Sewage DT104 S, Su S. typhimurium DT18 Human DT104 A, C, F, S, Su, T S. typhimurium DT19 Cattle feces DT104 Su S. typhimurium DT20 Human DT104 A, C, F, S, Su, T S. typhimurium DT21 Clinical U302 A, C, F, S, Su, T S. typhimurium DT22 Bovine 208 A, S, Su, T S. typhimurium DT23 Bovine 771 A, C, S, Su, T S. typhimurium DT24 Clinical U302 A, C, F, S, Su, T S. typhimurium DT25 Bovine 771 A, S, Su, T S. typhimurium DT28 Cattle DT104 A, C, F, S, Su, T S. typhimurium DT29 Cattle DT104 A, C, F, S, Su, T S. typhimurium DT30 Cattle DT104 A, C, F, S, Su, T S. typhimurium DT31 Cattle DT104 A, C, F, S, Su, T S. typhimurium DT32 Cattle DT104 A, C, F, S, Su, T S. typhimurium DT33 Porcine DT104 A, C, F, S, Su, T S. typhimurium DT34 NVSL DT104d A, C, F, S, Su, T S. typhimurium ATCC

4 358 A.A. Khan et al. / FEMS Microbiology Letters 182 (2000) 355^360 Fig. 1. Agarose gel electrophoresis of S. typhimurium DT18 strain PCR ampli ed products using individual pairs of primers and multiplex PCR. Lanes: 1, 100-bp DNA ladder as a size standard; 2, 392-bp PCR product using VirF and VirR primer pairs; 3, 584-bp PCR product using FloF and FloR primer pairs; 4, 321-bp PCR product using InvF and InvR primer pairs; 5, 265-bp PCR product using IntF and IntR primer pairs; 8, 100-bp DNA ladder as a size standard; 9, multiplex PCR products (584, 392, 321 and 265 bp) using four pairs of primers described above. In preliminary experiments to determine the optimum PCR ampli cation condition for multiplex PCR to simultaneously detect four genes, we utilized puri ed genomic DNA from multidrug-resistant DT104 strain DT18 as a source of template DNA. Individual primer pairs from four di erent genes were selected, targeting the invasion gene, virulence plasmid gene, integrase gene and orfenicol resistance gene. Fig. 1 (lanes 2, 3, 4 and 5) shows the ampli ed product from strain DT18 with four pairs of individual primer sets at 1.5 mm MgCl 2 and an annealing temperature of 60³C. When four pairs of primers were mixed together and subjected to multiplex PCR, using the same annealing temperature and all other conditions, the yields of 548- and 392- bp products were lower than the other two PCR products. Multiplex PCR speci city and sensitivity depends on several parameters like annealing temperature, Mg 2 concentration, and primer concentration. We found that lowering the annealing temperature by 2^5³C yielded several nonspeci c bands. The optimum temperature for these primers was 60³C. The optimum Mg 2 concentration for the multiplex PCR was determined by adding 1.5, 2.0, 2.5 and 3.0 mm MgCl 2 in the PCR reaction. The 2.0 mm MgCl 2 gave speci c and high yields of all four target genes (Fig. 1, lane 9). This protocol was used to screen all Salmonella strains in this study (Fig. 2A,B). Twenty-two ACSSuT-type DT104 strains and two U302 phage type ACSSuT-type strains gave 100% positive (ampli ed all four genes) PCR assays. These strains are all resistant to orfenicol. In this study we included Salmonella (strain #DT23, phage type 771) to verify the speci city of the PCR assay. Although this strain was resistant to chloramphenicol, it did not give any 548-bp PCR product, probably since this strain is sensitive to orfenicol. The multiplex PCR of this strain ampli ed only the inva and int genes, indicating that the o st gene was an important target gene for the detection of ACSSuT-type DT104 strains. A complete nucleotide sequence of the orfenicol resistance gene was determined by Bolton et al. [15] as 1202 bp. They also proposed phenotypic and genotypic methods for the identi cation of multidrug-resistant S. typhimurium DT104 by the use of a probe to determine chloramphenicol and orfenicol resistance. When 44 multidrug-resistant DT104 strains were tested, all of them were found resistant to orfenicol and chloramphenicol [15]. The invasion gene operon, inva, is essential for full virulence in Salmonella and it is thought to trigger the internalization required for invasion of deeper tissues [16]. Our PCR results indicate all were positives for the inva gene (Fig. 2A,B). A similar result was reported in a study where 245 Salmonella isolates from poultry products, wastewater, and human sources contained the inva gene [17]. A plasmid, spvc, that is present in Salmonella spp. interacts with the host immune system and is responsible for an increased growth rate in host cells [18]. Bolton et al. [15] found that 98% of all the S. typhimurium tested were positive for inva and 88% percent were positive for spvc gene. Table 2 Sequences of oligonucleotide primers Primer Target gene Sequence PCR product (bp) Accession number FloF o st 5P-ACCCGCCCTCTGGATCAAGTCAAG-3P 584 AF FloR 5P-CAAATCACGGGCCACGCTGTATC-3P VirF spvc 5P-GGGGCGGAAATACCATCTACA-3P 392 M64295 VirR 5P-GCGCCCAGGCTAACACG-3P InvF inva 5P-CGCGGCCCGATTTTCTCTGGA-3P 321 M90846 InvR 5P-AATGCGGGGATCTGGGCGACAAG-3P IntF int 5P-GCCCTCCCGCACGATGAT-3P 265 AF IntR 5P-ATTGGCGGCCTTGCTGTTCTTCTA-3P

5 A.A. Khan et al. / FEMS Microbiology Letters 182 (2000) 355^ genes of DT104 and U302 type ACSSuT-resistant S. typhimurium [21]. Recently, Sandvang et al. [7] have characterized two di erent integrons and found that multiresistant DT104 strains possess these integrons. The rst integron encodes the aminoglycoside resistance cassette ant (3Q)-la, which confers resistance to spectinomycin and streptomycin. The second integron contains the pse-1 L-lactamase gene cassette. Integrons have been documented in other Gram-negative organisms, such as Pseudomonas, E. coli and Shigella spp. [22,23]. Therefore, it is important to have combinations of all these target genes for speci c detection of multiple-drug-resistant Salmonella DT104 strains. These data indicate that multiplex PCR analysis utilizing o st, inva, spvc and int primer sets can speci cally identify multidrug-resistant ACSSuT-type DT104 S. typhimurium strains. This multiplex PCR method, described in this study, will allow the characterization and identi cation of environmental and clinical S. typhimurium DT104 ACSSuT-resistant strains. Acknowledgements Fig. 2. Agarose gel electrophoresis of multiplex PCR products ampli ed from S. typhimurium strains described in Table 1, using four pairs of primers. A: Lanes 1 and 20, standard size marker (100-bp DNA ladder as a size standard); lanes 2^19, DT1^DT18. B: Lanes 1 and 17, standard size marker (100-bp DNA ladder); lanes 2^16, DT18^DT34. Our PCR results also indicated that the spvc gene was present in 97% (31 of 32) of S. typhimurium strains. All multidrug ACSSuT-resistant DT104 strains were positive for spvc. Integrons capture and express mobile genes known as cassettes, which are, in most cases, antibiotic resistance genes [19,20]. The integrase gene is an essential part of all integrons; it encodes a site-speci c recombinase that catalyzes the insertion of the gene cassettes into the integron. The PCR data indicated that 94% (30 of 32) of the strains tested were positive for the int gene. Two DT104 strains (DT16 and 19, sensitive to ACST antibiotics) did not amplify a 265-bp PCR product from the integrase gene. One DT104 strain (DT17, which was resistant to streptomycin and sulfonamide, but sensitive to most of the antibiotics tested) was positive for the int gene. The PCR and antibiotic resistance pro les of ACSSuT-resistant DT104 and U302 phage type were similar; however, the 771 and 208 phage type strains DT22, DT23 and DT25 (Table 1) did not have pro les similar to DT104 strains. These results indicate that these two phage types (DT104 and U302) may be closely related to each other [21]. Similar results were observed by Briggs and Fratamico [21] when they used a long PCR method to amplify the integron sequences between the aada2 and bla CARB 2 The authors thank Dr. Ben D. Tall and Dr. Farukh M. Khambaty, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, DC for providing Salmonella DT104 strains. We thank Dr. John B. Sutherland and Dr. Robert D. Wagner for critical review of the manuscript and Barbara Jacks for illustrations. References [1] Glynn, M.K., Bopp, K.C., DeWitt, W., Dabney, P., Mokhtar, M. and Angulo, F.J. (1998) Emergence of multidrug resistant Salmonella enterica serotype typhimurium DT104 infections in the United States. New Engl. J. Med. 338, 1333^1337. [2] Threlfall, E.J., Frost, J.A., Ward, L.R. and Rowe, B. (1996) Increasing spectrum of resistance in multiresistant Salmonella typhimurium. Lancet 347, 1053^1054. [3] Threlfall, E.J., Frost, J.A., Ward, L.R. and Rowe, B. (1994) Epidemic in cattle and humans of Salmonella typhimurium DT104 with chromosomally integrated multiple drug resistance. Vet. Rec. 134, 577. [4] Roberts, M.C. (1998) Antibiotic resistance mechanisms in bacteria of oral and upper respiratory origin. Int. J. Antimicrob. Agents 4, 255^ 267. [5] Hosek, G., Leschinsky, S.I. and Safranek, T.J. (1997) Multidrug-resistant Salmonella serotype typhimurium ^ United States, Morbid. Mortal. Weekly Rep. 4, 11^97. [6] Ridley, A. and Threlfall, E.J. (1998) Molecular epidemiology of antibiotic resistance genes in multiresistant epidemic Salmonella typhimurium DT104. Microb. Drug Resist. 4, 113^118. [7] Sandvang, D., Aarestrup, F.M. and Jensen, L.B. (1998) Characteristics of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica typhimurium DT 104. FEMS Microbiol. Lett. 160, 37^41. [8] Cannon, M., Harford, S. and Davies, J.A. (1990) A comparative study on the inhibitory actions of chloramphenicol, thiamphenicol

6 360 A.A. Khan et al. / FEMS Microbiology Letters 182 (2000) 355^360 and some uorinated derivatives. J. Antimicrob. Chemother. 26, 307^ 317. [9] Dorman, C.J. and Foster, T.J. (1982) Nonenzymatic chloramphenicol resistance determinants speci ed by plasmids R26 and R55-1 in Escherichia coli K-12 do not confer high-level resistance to uorinated analogs. Antimicrob. Agents Chemother. 22, 912^914. [10] Low, J.C., Angus, M., Hopkins, G., Munro, D. and Rankin, S.C. (1997) Antimicrobial resistance of Salmonella enterica Typhimurium DT104 isolates and investigation of strains with transferable apramycin resistance. Epidemiol. Infect. 118, 97^103. [11] Khan, A.A. and Cerniglia, C.E. (1997) Rapid and sensitive method for the detection of Aeromonas caviae and Aeromonas trota by polymerase chain reaction. Lett. Appl. Microbiol. 24, 233^239. [12] Way, J.S., Josephson, K.L., Pillai, S.D., Abbaszadegan, M., Gerba, C.B. and Pepper, I.L. (1993) Speci c detection of Salmonella spp. by multiplex polymerase chain reaction. Appl. Environ. Microbiol. 59, 1473^1479. [13] National Committee for Clinical Laboratory Standards (1997) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A4. National Laboratory for Clinical Laboratory Standards, Villanova, PA. [14] Wheatcroft, R. and Watson, R.J. (1988) A positive strain identi cation method for Rhizobium meliloti. Appl. Environ. Microbiol. 54, 574^576. [15] Bolton, L.F., Kelley, L.C., Lee, M.D., Fedorka-Cray, P.J. and Maurer, J.J. (1999) Detection of multidrug-resistant Salmonella enterica serotype typhimurium DT104 based on a gene which confers cross-resistance to orfenicol and chloramphenicol. J. Clin. Microbiol. 37, 1348^1351. [16] Galan, J.E. and Curtiss, R. (1989) Cloning and molecular characterization of gene whose products allow S. typhimurium to penetrate tissue culture cells. Proc. Natl. Acad. Sci. USA 86, 6383^6387. [17] Swamy, S.C., Barnhart, H.M., Lee, M.D. and Dreesen, D.W. (1996) Virulence determinants inva and spvc in salmonellae isolated from poultry products, wastewater, and human sources. Appl. Environ. Microbiol. 62, 3768^3771. [18] Gulig, P.A., Danbara, H., Guiney, D.G., Lax, A.J., Norel, F. and Rhen, M. (1993) Molecular analysis of spv virulence genes of the Salmonella virulence plasmids. Mol. Microbiol. 7, 823^830. [19] Recchia, G.D. and Hall, R.M. (1995) Gene cassettes: a new class of mobile element. Microbiology 141, 3015^3027. [20] Recchia, G.D. and Hall, R.M. (1997) Origins of the mobile gene cassettes found in integrons. Trends Microbiol. 5, 389^394. [21] Briggs, C.E. and Fratamico, P.M. (1999) Molecular characterization of an antibiotic resistance gene cluster of Salmonella typhimurium DT104. Antimicrob. Agents Chemother. 43, 846^849. [22] Levesque, C., Piche, L., Larose, C. and Roy, P.H. (1995) PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39, 185^191. [23] Rajakumar, K., Bulach, D., Davies, J., Ambrose, L., Sasakawa, C. and Adler, B. (1997) Identi cation of a chromosomal Shigella exneri multi-antibiotic resistance locus which shares sequence and organizational similarity with the resistance region of the plasmid NR1. Plasmid 37, 159^168.