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Antimicrobial Agents and Chemotherapy, February 2004, p. 689-690, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.689-690.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

First Canadian Salmonella enterica Serovar Typhi Isolate Harboring an Integron


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Two recent reports in this journal have described multidrug-resistant Salmonella enterica serovar Typhi strains which harbor integrons (7, 9). The emergence of multidrug-resistant S. enterica serovar Typhi poses a serious public health concern, resulting in treatment failures and limiting therapeutic options (11). Multidrug-resistant strains have been isolated in Canada from individuals returning from Asia, primarily from India and Pakistan (2). Recently, an S. enterica serovar Typhi strain was isolated from a 30-year-old male in Ontario, Canada, suffering from bacteremia. There was no recent history of travel; however, the patient's family had emigrated from Sri Lanka approximately 5 years before and had a history of typhoid fever. A strain with an indistinguishable DNA fingerprint generated using pulsed-field gel electrophoresis was also obtained from a stool specimen from an asymptomatic sibling of the patient (13). The clinical isolate, labeled N02-542, was identified using standard biochemical and serological procedures for enteric bacteriology (4). Antimicrobial susceptibility testing was performed initially using agar dilution and was repeated using broth microdilution for additional antimicrobials (4). The strain was found to be resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, streptomycin, tetracycline, and nalidixic acid (5) (Table 1). A second isolate from the same blood culture specimen was also identified as S. enterica serovar Typhi. It was fully susceptible as determined by agar dilution, and the fingerprint was indistinguishable from that of the resistant isolate, suggesting that the strain had lost the multidrug-resistant plasmid (see below). Plasmid DNA was isolated from S. enterica serovar Typhi N02-542 with a commercial plasmid isolation kit (Qiagen) and used to transform electrocompetent Escherichia coli DH10B (Invitrogen). The transformant, E. coli FJ542, was resistant to all the same antimicrobials as the parent strain with the exception of nalidixic acid (Table 1). Plasmid profiling showed that E. coli FJ542 contained the same plasmid as S. enterica serovar Typhi N02-0542, and PCR analysis identified the presence of the tetracycline resistance gene tet(A)B, the chloramphenicol resistance gene catA1, and blaTEM-1. PCR using primers 5'-CS and 3'-CS to detect cassettes of class 1 integrons (3) produced an amplicon of approximately 750 bp in size. Sequence analysis of the amplicon revealed a 617-bp gene cassette (10) which had 100% identity to the cassette containing the dihydrofolate reductase gene dfrA7 from Shigella flexneri (GenBank accession number AF139109) (unpublished data). The dfrA7 gene cassette is also found in integrons in E. coli plasmid pDGO100 (1) and E. coli plasmid R751 (6). The S. enterica serovar Typhi plasmid pHCM1 (218 kb) also carries blaTEM-1, tet(A)B, and catA1; however, it carries a dfrA5 gene and an incomplete class 1 integron (8). The related S. enterica serovar Typhi plasmid R27 (180 kb) contains only the tet(A)B gene and no class 1 integron (12). PCR analysis of the fully susceptible strain of S. enterica serovar Typhi isolated from the patient yielded no amplicons for any of the resistance genes.


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  		TABLE 1. MICs of various antimicrobials for study strains as determined by broth microdilution

 
The plasmid DNA from strain FJ542(pFJ-1) was restricted with HpaI, EcoRI, and BglII, and the sizes of the fragments were used to estimate the size of pFJ-1 to be 180 kb. Southern hybridization analysis with tetA(B), blaTEM, catA1, and dfrA7 probes confirmed the presence of these genes on pFJ-1 (data not shown). Most plasmids associated with S. enterica serovar Typhi have been shown to belong to the H1 incompatibility group, and we have used PCR to confirm that plasmid pFJ-1 also belongs to this group (data not shown) (9).

In addition to the resistance gene content, the plasmid size and the compatibility group of pFJ-1 are identical to those recently described for isolates from India and Vietnam (9). This observation provides evidence to suggest that the integron-harboring plasmid has disseminated to North America. Recently, a 50-kb plasmid harboring a class 1 integron containing six drug resistance genes has been described in an S. enterica serovar Typhi isolate from Korea (7). These two findings stress the need for molecular laboratories to include an integron detection PCR for S. enterica serovar Typhi strains displaying resistance to sulfonamides, as class 1 integrons typically carry this resistance determinant (3).


   ACKNOWLEDGMENTS
 
We thank Romeo Hizon for his contribution related to antimicrobial susceptibility testing and Shaun Tyler and the staff of the DNA Core Facility at the National Microbiology Laboratory for generating the sequence information and synthesizing oligonucleotides.


   REFERENCES
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  1. Burnside, J. M., and D. J. Groot Obbink. 1996. Plasmid pDGO100 contains a second integron with the trimethoprim resistance gene dfrA7 as the inserted cassette. Plasmid 35:67-70.[CrossRef][Medline]
  2. Harnett, N., S. McLeod, Y. AuYong, J. Wan, S. Alexander, R. Khakhria, and C. Krishnan. 1998. Molecular characterization of multiresistant strains of Salmonella Typhi from South Asia isolated in Ontario, Canada. Can. J. Microbiol. 44:356-363.[CrossRef][Medline]
  3. Levesque, C., L. Piche, C. Larose, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39:185-191.[Abstract]
  4. Murray, P., E. Baron, M. Pfaller, F. Tenover, and R. Yolkin. 1999. Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
  5. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved standard. NCCLS M100-S12, vol. 22 no. 1; M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  6. Ojo, K. K., D. E. Waturangi, H. A. Odelola, A. Suwanto, and S. Schwarz. 2002. Identification of a cassette-borne dfrA7-like gene that shows a 97 bp extension at the 3'-end of the reading frame. J. Antimicrob. Chemother. 49:573-574.[Free Full Text]
  7. Pai, H., J. Byeon, S. Yu, B. K. Lee, and S. Kim. 2003. Salmonella enterica serovar Typhi strains isolated in Korea containing a multidrug resistance class 1 integron. Antimicrob. Agents Chemother. 47:2006-2008.[Abstract/Free Full Text]
  8. Parkhill, J., et al. 2001. Complete genome sequence of a multidrug resistant Salmonella enterica serovar Typhi CT18. Nature 413:848-852.[CrossRef][Medline]
  9. Ploy, M. C., D. Chainier, N. H. Tran Thi, I. Poilane, P. Cruaud, F. Denis, A. Collignon, and T. Lambert. 2003. Integron-associated antibiotic resistance in Salmonella enterica serovar Typhi from Asia. Antimicrob. Agents Chemother. 47:1427-1429.[Abstract/Free Full Text]
  10. Recchia, G. D., and R. M. Hall. 1995. Gene cassettes: a new class of mobile element. Microbiology 141:3015-3027.[Medline]
  11. Rowe, B., L. R. Ward, and E. J. Threlfall. 1997. Multidrug-resistant Salmonella Typhi: a worldwide epidemic. Clin. Infect. Dis. 24:S106-S109.
  12. Sherburne, C. K., T. D. Lawley, M. W. Gilmour, F. R. Blattner, V. Burland, E. Grotbeck, D. J. Rose, and D. E. Taylor. 2000. The complete DNA sequence and analysis of R27, a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer. Nucleic Acids Res. 28:2177-2186.[Abstract/Free Full Text]
  13. Swaminathan, B., T. Barrett, S. B. Hunter, and R. Tauxe. 2001. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Dis. 7:382-389.[Medline]
Michael R. Mulvey*
David Boyd
Lai-King Ng
National Microbiology Laboratory
Health Canada
1015 Arlington St.
Winnipeg, Manitoba R3E 3R2, Canada

Shirley Brown
Marina Lombos
Bruce Ciebin
Aimin Li
Frances Jamieson
Laboratories Branch
Ontario Ministry of Health and Long-Term Care
Toronto, Ontario, Canada

Philip Stuart
Canadian Medical Laboratories
Mississauga, Ontario, Canada

* Phone: (204) 789-2133, Fax: (204) 789-2018, E-mail: michael_mulvey{at}hc-sc.gc.ca

Antimicrobial Agents and Chemotherapy, February 2004, p. 689-690, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.689-690.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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