This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lawson, A. J.
Right arrow Articles by Desai, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lawson, A. J.
Right arrow Articles by Desai, M.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 2004, p. 4843-4845, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4843-4845.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Fluorescent Amplified Fragment Length Polymorphism Subtyping of Multiresistant Salmonella enterica Serovar Typhimurium DT104

Andrew J. Lawson,1 John Stanley,2 E. John Threlfall,1 and Meeta Desai2*

Laboratory of Enteric Pathogens,1 Virus Reference Division, Specialist and Reference Microbiology Division, Health Protection Agency, London, United Kingdom2

Received 16 March 2004/ Returned for modification 22 April 2004/ Accepted 8 June 2004


arrow
ABSTRACT
 
Fluorescent amplified fragment length polymorphism (FAFLP) subtyping analysis was used to genotype multiresistant Salmonella enterica serovar Typhimurium definitive phage type 104. Thirteen distinct FAFLP profiles were found among 85 isolates exhibiting identical pulsed-field gel electrophoresis (PFGE) profiles. A single FAFLP profile was shared by 93% of outbreak-associated isolates and 82% of sporadic isolates. This study demonstrates the value of FAFLP as a high-resolution tool for epidemiological investigation of Salmonella.


arrow
TEXT
 
Multiresistant (MR) Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104), with chromosomally encoded resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines (ACSSuT), is an internationally distributed zoonotic pathogen and an important agent of human salmonellosis in England and Wales (9).

While pulsed-field gel electrophoresis (PGFE) has shown some diversity among MR DT104 isolates (6, 7), use of the endonuclease XbaI has shown that the majority (~90%) have a single PFGE profile, xtm 1 (4, 5). Supplementary methods such as resistance typing and plasmid analysis generally provide little additional discrimination (9).

Fluorescent amplified fragment length polymorphism (FAFLP) analysis is a highly discriminatory and reproducible tool for subtyping genetically homogeneous genomes and identifying outbreak genotypes within bacterial genera (1, 2). In this report, the genotypic variation among MR DT104 isolates with the predominant PFGE profile xtm 1 was analyzed by FAFLP.

MR DT104 isolates, all with PFGE profile xtm 1, collected from humans and animals in England and Wales were examined. These comprised randomly selected examples from four distinct outbreaks (n = 45), sporadic isolates (n = 34), and "historic" isolates (n = 6) (Table 1). PFGE, resistance typing, and plasmid analysis were performed as previously described (5).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Isolates of MR S. enterica serovar Typhimurium DT104a used in this study

FAFLP was performed with endonucleases HindIII and HhaI, and amplification was performed with the primer pair HindIII+0 (nonselective) and HhaI+C (one base, C; selective), as described previously (1). FAFLP data consisting of amplified fragments (AFs) of between 50 and 560 bp were analyzed (1, 2). The precision of sizing these AFs was ±0.5 bp. Among the 85 isolates, there were 163 distinct AFs, of which 82 were polymorphic and 81 were common to all profiles. Thirteen distinct FAFLP profiles, each comprising between 118 and 125 AFs, were detected. A dendrogram (Fig. 1) derived by an unweighted pair group method with arithmetic averaging (UPGMA) cluster analysis illustrates the genetic variation within the isolates. Seventy-three isolates (86%), including 10 from animals, shared the FAFLP profile F3, which differed from the other profiles by 1 to 69 AFs. The remaining 12 exhibited unique profiles with AF differences ranging from 1 to 73. FAFLP profile F4 differed from the others by 67 to 73 AFs, constituting 28 to 31% genotypic divergence. The remaining profiles exhibited <5% genotypic divergence, with 1 to 10 AF differences.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 1. Dendrogram showing variability of FAFLP patterns among MR S. enterica serovar Typhimurium DT104 isolates from FAFLP data obtained with the HindIII+0 and HhaI+C primer pair. The dendrogram was derived by UPGMA as previously described (1, 2). Each branch represents a unique FAFLP profile (profile numbers indicate different strains; Table 1). Scale bar, 10% divergence.

Outbreak 1 occurred in an institution. All isolates exhibited profile F3.

Outbreak 2 involved delegates who became ill following a conference. Epidemiologically this appeared to be a point source outbreak. However, FAFLP analysis showed three different profiles among the five isolates. Profiles F7 and F8 differed from each other by four AFs and from profile F3 (shared by three isolates) by two AFs each. This suggests that the cases with unique FAFLP profiles may have acquired their infections elsewhere or that multiple strains were present at the onset of the outbreak.

Outbreak 3 was a major milk-borne outbreak in northwest Lancashire, involving more than 86 cases. The isolates were characterized by additional reduced susceptibility to ciprofloxacin (CpL), caused by a point mutation at codon 87 of gyrA (10). Of 10 isolates analyzed by FAFLP, 3 were from cattle, 1 was from a milk filter, and 6 were from human cases. All shared profile F3, including one cow isolate that possessed an additional 40-MDa plasmid not seen among the other outbreak 3 isolates.

Outbreak 4 involved over 360 laboratory-confirmed cases nationwide (3); the isolates were additionally characterized by the presence of an unusual plasmid "fingerprint" (60 and 2 MDa), usually present in ~5% of MR DT104 isolates (3). For FAFLP analysis 26 human isolates were selected, and all but one shared profile F3. The remaining isolate showed a closely related profile, F13, which differed from profile F3 by one AF.

The 34 contemporaneous isolates with PFGE profile xtm 1 were included for comparison with the outbreak-associated isolates; 28 (82%) of these had the common profile F3. Eight had resistance type ACSSuT, with a 60-MDa plasmid; seven of these had profile F3, and one had profile F5 (differing by six AFs from F3). A further seven isolates had resistance type ACSSuTCpL (also associated with outbreak 3); six of these had profile F3, and one had a closely related profile, F6 (differing from profile F3 by a single AF). The 60- and 2-MDa plasmid fingerprint characteristic of outbreak 4 was present in 20 sporadic isolates; 16 had profile F3, and 4 had distinct profiles: profiles F9 and F10 were from animal isolates, whereas F11 and F12 were from humans.

The six historic isolates were indistinguishable by PFGE, resistance type, and plasmid fingerprint. However, four FAFLP profiles were detected, with only three (50%) isolates sharing the predominant profile F3. The two earliest isolates, from 1984, showed profiles F1 and F2 (differing from F3 and each other by one AF). Two isolates from 1985, one from a human and one from a cow, had F3 and F4 respectively. Profile F4 was the most distinct in this study, differing from the other profiles in approximately two-thirds of the 118 AFs constituting its profile. The remaining isolates, from 1986 and 1988, had profile F3. It is possible that differences observed in FAFLP profiles in the historic isolates occurred due to degradation during storage, but this seems unlikely as serotype, phage type, PFGE profile, plasmids, and resistance type remained unaffected. Reproducibility studies confirm the stability of FAFLP profiles over prolonged storage, subculture, and use of three repeat DNA preparations from the same isolate (results not shown).

In summary, the predominant FAFLP genotype, F3, was present in one-half of the earliest (1980s) MR DT104 isolates from England and Wales. Eighteen years later, the same FAFLP genotype remains prevalent, having caused outbreaks of human illness throughout the 1990s and into the present decade. A small proportion of MR DT104 isolates appear to be genetically heterogeneous, exhibiting unique FAFLP genotypes. Clearly, these FAFLP genotypes possess pathogenic potential and are associated with human enteric disease, but, in this small survey, they were found only as unique types and not in an outbreak context.

In the present study all isolates were identical when hierarchically characterized by serology, phage type, and PFGE. This is in contrast to an earlier study where the PFGE and FAFLP profiles of S. enterica serovar Typhimurium isolates of unknown phage type were compared (8). It is important to note that the predominant PFGE profile, xtm 1, occurs in phage types other than DT104 (4, 5). While the investigation demonstrates the value of subtyping methods, such as resistance typing and plasmid analysis, in the identification of outbreaks caused by common phage types, it also emphasizes the need for high-resolution genotyping methodologies, such as FAFLP, to provide further insight into the microepidemiology of MR DT104 and other salmonellas.


arrow
ACKNOWLEDGMENTS
 
This study was partially funded by a grant (FS3114) from the Food Standards Agency, London, United Kingdom, and was carried out in collaboration with colleagues funded by a grant from the Specialist and Reference Microbiology Division of the Health Protection Agency.

Special thanks to Linda Ward and our colleagues in the Laboratory of Enteric Pathogens for the serotype, phage type, and resistance type data.


arrow
FOOTNOTES
 
* Corresponding author. Present address: Genomics, Proteomics and Bioinformatics Unit, Specialist and Reference Microbiology Division, Health Protection Agency, London NW9 5HT, United Kingdom. Phone: 44 (0)20 83276469. Fax: 44 (0)20 83276768. E-mail: meeta.desai{at}hpa.org.uk. Back


arrow
REFERENCES
 
    1
  1. Desai, M., J. M. Logan, J. A. Frost, and J. Stanley. 2001. Genome sequence-based fluorescent amplified fragment length polymorphism of Campylobacter jejuni, its relationship to serotyping, and its implications for epidemiological analysis. J. Clin. Microbiol. 39:3823-3829.[Abstract/Free Full Text]
    2. 2
  2. Desai, M., E. J. Threlfall, and J. Stanley. 2001. Fluorescent amplified-fragment length polymorphism subtyping of the Salmonella enterica serovar Enteritidis phage type 4 clone complex. J. Clin. Microbiol. 39:201-206.[Abstract/Free Full Text]
    3. 3
  3. Horby, P. W., S. J. O'Brien, G. K. Adak, C. Graham, J. I. Hawker, P. Hunter, C. Lane, A. J. Lawson, M. H. Mitchell, E. J. Threlfall, and L. R. Ward. 2003. A national outbreak of multiresistant Salmonella enterica serovar Typhimurium definitive phage type (DT) 104 associated with consumption of lettuce. Epidemiol. Infect. 130:169-178.[CrossRef][Medline]
    4. 4
  4. Lawson, A. J., H. Chart, M. U. Dassama, and E. J. Threlfall. 2002. Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types. Lett. Appl. Microbiol. 34:428-432.[CrossRef][Medline]
    5. 5
  5. Lawson, A. J., M. U. Dassama, L. R. Ward, and E. J. Threlfall. 2002. Multiply resistant (MR) Salmonella enterica serotype Typhimurium DT 12 and DT 120: a case of MR DT 104 in disguise? Emerg. Infect. Dis. 8:434-436.[Medline]
    6. 6
  6. Liebana, E., L. Garcia-Migura, C. Clouting, F. A. Clifton-Hadley, E. Lindsay, E. J. Threlfall, S. W. McDowell, and R. H. Davies. 2002. Multiple genetic typing of Salmonella enterica serotype Typhimurium isolates of different phage types (DT104, U302, DT204b, and DT49) from animals and humans in England, Wales, and Northern Ireland. J. Clin. Microbiol. 40:4450-4456.[Abstract/Free Full Text]
    7. 7
  7. Markogiannakis, A., P. T. Tassios, M. Lambiri, L. R. Ward, J. Kourea-Kremastinou, N. J. Legakis, the Greek Nontyphoidal Salmonella Study Group, and A. C. Vatopoulos. 2000. Multiple clones within multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104. J. Clin. Microbiol. 38:1269-1271.[Abstract/Free Full Text]
    8. 8
  8. Tamada, Y., Y. Nakaoka, K. Nishimori, A. Doi, T. Kumaki, N. Uemura, K. Tanaka, S. I. Makino, T. Sameshima, M. Akiba, M. Nakazawa, and I. Uchida. 2001. Molecular typing and epidemiological study of Salmonella enterica serotype Typhimurium isolates from cattle by fluorescent amplified-fragment length polymorphism fingerprinting and pulsed-field gel electrophoresis. J. Clin. Microbiol. 39:1057-1066.[Abstract/Free Full Text]
    9. 9
  9. Threlfall, E. J. 2000. Epidemic Salmonella typhimurium DT 104—a truly international multiresistant clone. J. Antimicrob. Chemother. 46:7-10.[Free Full Text]
    10. 10
  10. Walker, R. A., A. J. Lawson, E. A. Lindsay, L. R. Ward, P. A. Wright, F. J. Bolton, D. R. Wareing, J. D. Corkish, R. H. Davies, and E. J. Threlfall. 2000. Decreased susceptibility to ciprofloxacin in outbreak-associated multiresistant Salmonella typhimurium DT104. Vet. Rec. 147:395-396.[Free Full Text]

Journal of Clinical Microbiology, October 2004, p. 4843-4845, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4843-4845.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • de la Torre, E., Tello, M., Mateu, E. M., Torre, E. (2005). Use of Biochemical Kinetic Data To Determine Strain Relatedness among Salmonella enterica subsp. enterica Isolates. J. Clin. Microbiol. 43: 5419-5424 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lawson, A. J.
Right arrow Articles by Desai, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lawson, A. J.
Right arrow Articles by Desai, M.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»