AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
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 arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doran, G.
Right arrow Articles by Cormican, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doran, G.
Right arrow Articles by Cormican, M.
Agricola
Right arrow Articles by Doran, G.
Right arrow Articles by Cormican, M.
Applied and Environmental Microbiology, December 2005, p. 8236-8240, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8236-8240.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of Salmonella enterica Serovar Typhimurium

Geraldine Doran,1* Dearbhaile Morris,1 Colette O'Hare,1 Niall DeLappe,1 Bernard Bradshaw,3 Geraldine Corbett-Feeney,1,2 and Martin Cormican1,2

National Salmonella Reference Laboratory, University College Hospital, Galway, Ireland,1 Department of Bacteriology, National University of Ireland, Galway, Ireland,2 Bacteriology Division, Central Veterinary Research Laboratory, Dublin, Ireland3

Received 18 April 2005/ Accepted 27 August 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Salmonella enterica serovar Typhimurium is one of the most common serovars isolated from humans, animals, and food in Europe and the United States (3, 4, 5, 13, 16, 22-24). Because the serovar is widely distributed, detection of serovar Typhimurium isolates from two or more sources is not in itself good microbiological evidence of a link between those sources. Routine subtyping of serovar Typhimurium is valuable in tracking the epidemiology of salmonella infection. For many years, phage typing with or without antimicrobial susceptibility testing has been widely used in reference laboratories for this purpose (12, 16, 18, 19). Phage typing is a phenotypic typing method based on the outcome of complex interactions between phage and bacterial cells. Phage typing requires access to the appropriate phage library and training and experience in performance and interpretation of results. It is therefore suitable only for use in reference laboratories; however, it is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. In recent years, standardized pulsed-field gel electrophoresis (PFGE) has increasingly been applied as an additional typing method in salmonella reference laboratories (20). Many laboratories that use phage typing as a primary typing method use PFGE as a supplementary method, but the cost of performing PFGE in addition to phage typing on all isolates is prohibitive.

We have studied the relationship between phage type and PFGE pattern in 503 serovar Typhimurium isolates from humans, animals, and food in Ireland to determine how best to apply PFGE to supplement the existing routine phenotypic subtyping of serovar Typhimurium.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains.
Five hundred three isolates of serovar Typhimurium were analyzed by phage typing, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis. The isolates were from pork (268), human (153) bovine (25), poultry (24), fish (10), and other sources (23). They were collected from more than 20 laboratories throughout Ireland over a 42-month period from 2000 to 2004. The collection included 45 isolates from six small clusters (five outbreaks and an animal feed contamination incident). The bacterial strains were confirmed as Salmonella enterica serovar Typhimurium by API20E (bioMerieux, Marcy l'Etoile, France) and confirmed as serovar Typhimurium according to the Kauffman and White serotyping scheme (8) using slide agglutination with standard antisera (Murex Biotech Ltd., Dartford, England).

Phage typing and antimicrobial susceptibility testing.
Phage typing was performed in accordance with the methods of the Laboratory of Enteric Pathogens, Health Protection Agency, Colindale, London, United Kingdom (2). Antimicrobial susceptibility testing was performed according to the disk diffusion method of the National Committee of Clinical Laboratory Standards (15). The following antimicrobial agents (disk contents indicated in parentheses) were tested: ampicillin (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), kanamycin (30 µg), nalidixic acid (30 µg), sulfonamides (300 µg), streptomycin (10 µg), tetracycline (30 µg) gentamicin (10 µg), minocycline (30 µg), ceftazidime (30 µg), and trimethoprim (5 µg) (OXOID, Hampshire, United Kingdom). Escherichia coli ATCC 25922 was used as the control.

Pulsed-field gel electrophoresis.
PFGE was performed using the CHEF Mapper XA (Bio-Rad, California) system following the standardized protocol of PulseNet (20) using the XbaI restriction enzyme (Roche, Basel, Switzerland). A subset of 16 isolates was analyzed using endonuclease BlnI (Roche, Basel, Switzerland) following the same protocol. These 16 isolates represented different phage types and antibiogram patterns (Fig. 2). Gel images were captured with the Bio-Rad Gel Doc 2000 imaging system. Banding patterns were analyzed with bionumerics software (Applied Maths, Kortrijk, Belgium) using the Dice similarity coefficient, and clustering was created using the unweighted-pair group method using average linkages (UPGMA). The appropriate position tolerance for the final analysis was selected by analyses of multiple runs of the control Salmonella enterica serovar Braenderup, with position tolerance ranging from 1.0% to 2.0%.



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 2. Dendrogram showing the composite comparison of 16 isolates when typed by PFGE using BlnI (patterns A through D) and XbaI (p26). The identification, antibiogram, and phage type of each isolate are also shown. See Table 2, footnote b, for antibiogram designations.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Twenty-nine distinct phage types were identified. In addition, there were six isolates in the category of "react with phage but does not conform to a recognized phage type" (RDNC) and 19 isolates that were nontypeable with phage (NT). For simplicity of presentation, the categories RDNC and NT will be tabulated as phage types (Table 1). The principle phage types are listed in the first column of Table 1. PFGE with XbaI gave 10 to 21 bands per isolate (Fig. 1), and BlnI gave 9 to 13 bands per isolate. A position tolerance of 1% for analysis of multiple runs of serovar Braenderup H9812 showed a similarity of 91.2%. The similarity increased to 98.9% with a position tolerance of 1.5%, and a further increase in tolerance up to 2.0% made no significant difference to the percent similarity. On this basis, a tolerance of 1.5% was accepted for the generation of the dendrogram illustrated in Fig. 1 and 2.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Principle phage types of Salmonella enterica serovar Typhimurium and associated diversity by pulsed-field gel electrophoresis

 


View larger version (61K):
[in this window]
[in a new window]
  		FIG. 1. Dendrogram created by Bionumerics software showing the similarities between the 53 PFGE patterns observed with XbaI digestion. PFGE patterns are designated p1 to p53. The number of isolates (n) in each pattern is indicated. The number of isolates within phage types is included in the right hand column.

 
For direct visual analyses, isolates were regarded as having a distinct PFGE pattern if they differed by one or more bands from other isolates. By these criteria, there were 53 distinguishable XbaI PFGE patterns (labeled p1 through p53) (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Predominant PFGE patterns among 503 Salmonella enterica serovar Typhimurium isolates with associated diversity by phage type and antibiogram

 
The extent of diversity by PFGE among the main phage types is presented in Table 1. Five phage types, U310, DT104b, DT104, DT193, and U302 together, accounted for 343 (68%) of the isolates. For the common phage types U310, DT104, and DT104b, XbaI PFGE provided little additional discrimination, with 86 to 93% of the isolates in each phage type indistinguishable from each other (Table 1). For phage types DT40 and DT132, represented by smaller number of isolates, no PFGE diversity was observed in our series. By contrast, phage types DT193 and U302 are quite heterogeneous by XbaI PGFE and isolates of these phage types are not significantly more closely related to each other than are nontypeable isolates.

Table 2 presents another perspective on the data using the XbaI PFGE pattern as the primary categorization. The data show that isolates that are indistinguishable by XbaI PFGE frequently belong to multiple distinct phage types. For example, the pattern designated p26 includes most isolates of DT104 and DT104b and many of phage type U302. Upon digestion of a subset of 16 p26 isolates with BlnI, all but four remained indistinguishable (Fig. 2). On critical visual review of the XbaI patterns of the isolates with distinct BlnI patterns (Fig. 2), it is apparent that minor differences may also exist in the XbaI patterns for isolates 1099/01, 1014/01, and 756/02 that are very distinct on BlnI. Isolates of phage type DT193, on the other hand, were distributed over 7 of the 10 major PFGE patterns. There is frequently significant antibiogram diversity within a given XbaI PFGE pattern (Table 2); therefore, routine susceptibility testing remains a valuable typing method in addition to its contribution to the surveillance of antimicrobial resistance.

Isolates from six small clusters were included in the study (Table 3). All are from human outbreaks with the exception of the isolates from cluster 5, which relate to animal feed contamination. Five of these clusters (Table 3, clusters 1 to 4 and 6) are referred to briefly elsewhere (16). With the exception of a single isolate, all strains suspected as implicated in a particular outbreak were indistinguishable by both phage type and XbaI PFGE.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Phage type and PFGE applied to isolates from discrete point source outbreaks

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Phage typing has proved effective in the monitoring of epidemic strains of serovar Typhimurium such as DT204, DT204c, and DT104 (7, 17, 21). More recently, during 2001, the emergence of a new strain, since designated U310 (9), was detected by its atypical reaction with the phage panel. In recent years, PFGE has also become an important typing method; however, it is costly, and for most reference laboratories, routine PFGE of all isolates is not affordable. It is appropriate, therefore, to consider how best to apply PFGE in association with older methods for maximum public health impact. One approach is the application of PFGE to groups of isolates that are related to a suspected outbreak (1, 6, 12, 14). When the isolates are of similar phage type and antibiogram, they are most often indistinguishable also by PFGE, and therefore, the PFGE has limited practical impact on outbreak management. Our data suggest that PFGE can be applied in a more systematic way as an adjunct for laboratories performing phage typing.

Application of PFGE either routinely or in outbreak investigation to groups of isolates of DT104, DT104b, or U310 generally yields little additional discrimination. The same may be true for collections of isolates of type DT40 and DT132, although in the case of these phage types, our data are based on smaller numbers of isolates. By contrast, isolates of phage types DT193 and U302 are not significantly more closely related to each other on PFGE than are nontypeable or RDNC isolates. We suggest that routine XbaI PFGE of isolates of DT193, U302, and RDNC and nontypeable isolates should be performed where possible. Among the isolates included in this study were 45 from six point source clusters (Table 3). Phage type proved a generally stable characteristic in the outbreak setting. In five outbreaks (including two small DT193 outbreaks), all isolates suspected on epidemiological grounds as being related were of the same phage type and were indistinguishable by PFGE. Nevertheless, we suggest that, given the overall diversity shown within DT193, caution should be exercised in regarding isolates of phage type DT193 or U302 as likely to be closely related and that PFGE should also be performed.

PFGE with XbaI is a valuable tool for the epidemiological typing of serovar Typhimurium. Knowledge of the diversity within phage types and of the relationships between phage types can facilitate the cost-effective application of this tool to epidemiological investigations. The level of PFGE diversity observed within a given phage type may not be constant in all areas and at all times, and it may be appropriate for reference laboratories to validate our conclusions for their circumstances by a similar analysis of their data.


   ACKNOWLEDGMENTS
 
This investigation was partly funded by the Department of Agriculture and Food of Ireland under the Food Institutional Research Measure. This is project reference 00/R&D/D/32.


   FOOTNOTES
 
* Corresponding author. Mailing address: National Salmonella Reference Laboratory, University College Hospital, Galway, Ireland. Phone: 353 91 544628. Fax: 353 91 512514. E-mail: Geraldine.Doran{at}nuigalway.ie. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Agasan, A., J. Kornblum, G. Williams, C. C. Pratt, P. Fleckenstein, M. Wong, and A. Ramon. 2002. Profile of Salmonella enterica subsp. enterica (subspecies I) serotype 4,5,12:i:– strains causing food-borne infections in New York City. J. Clin. Microbiol. 40:1924-1929.[Abstract/Free Full Text]
  2. Anderson, E. S., L. R. Ward, and M. J. de Saxe. 1977. Bacteriophage-typing designations of Salmonella Typhimurium. J. Hyg. 78:297-300.
  3. Botteldoorn, N., L. Herman, N. Rijpens, and M. Heyndrickx. 2004. Phenotypic and molecular typing of Salmonella strains reveals different contamination sources in two commercial pig slaughterhouses. Appl. Environ. Microbiol. 70:5305-5314.[Abstract/Free Full Text]
  4. Centers for Disease Control and Prevention. 2004. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—selected sites, United States, 2003. Morb. Mortal. Wkly. Rep. 53:338-343.[Medline]
  5. Centers for Disease Control and Prevention. 2002. PHLIS surveillance data. Salmonella annual summary 2002. Centers for Disease Control and Prevention, Atlanta, Ga.
  6. Corbett-Feeney, G., and U. N. Riain. 1998. The use of pulsed-field gel electrophoresis for subdivision of Salmonella typhimurium in an outbreak situation. J. Infect. 36:175-177.[CrossRef][Medline]
  7. Cormican, M., C. Butler, D. Morris, G. Corbett-Feeney, and J. Flynn. 1998. Antibiotic resistance amongst Salmonella enterica species isolated in the Republic of Ireland. J. Antimicrob. Chemother. 42:116-118.[Free Full Text]
  8. Difco Laboratories. 1998. Difco manual, 10th ed. Difco Laboratories, Detroit, Mich.
  9. EnterNet. 2004. EnterNet quarterly Salmonella report April to June 2004/2. [Online.] http://www.hpa.org.uk/hpa/inter/enter-net/04q2summ.pdf.
  10. Gudmundsdottir, S., H. Hardardottir, and E. Gunnarsson. 2003. Subtyping of Salmonella enterica serovar Typhimurium outbreak strains isolated from humans and animals in Iceland. J. Clin. Microbiol. 41:4833-4835.[Abstract/Free Full Text]
  11. Health Protection Agency. 2002. Up and coming "new types" of Salmonella in England and Wales. Commun. Dis. Rep. CDR Wkly. 12(28). [Online.] http://www.hpa.org.uk/cdr/archives/2002/cdr2802.pdf.
  12. Lan, R., A. M. Davison, P. R. Reeves, and L. R. Ward. 2003. AFLP analysis of Salmonella enterica serovar Typhimurium isolates of phage types DT 9 and DT 135: diversity within phage types and its epidemiological significance. Microbes Infect. 5:841-850.[Medline]
  13. Martinez-Urtaza, J., E. Liebana, L. Garcia-Migura, P. Perez-Pineiro, and M. Saco. 2004. Characterization of Salmonella enterica serovar Typhimurium from marine environments in coastal waters of Galicia (Spain). Appl. Environ. Microbiol. 70:4030-4034.[Abstract/Free Full Text]
  14. Murase, T., M. Yamada, T. Muto, A. Matsushima, and S. Yamai. 2000. Fecal excretion of Salmonella enterica serovar Typhimurium following a food-borne outbreak. J. Clin. Microbiol. 38:3495-3497.[Abstract/Free Full Text]
  15. NCCLS. 1997. Performance standards for antimicrobial susceptibility tests, 6th edition. Approved standard. NCCLS, Wayne, Pa.
  16. O'Hare, C., G. Doran, N. Delappe, D. Morris, V. Buckley, G. Corbett-Feeney, P. McKeown, W. Anderson, and M. Cormican. 2004. Antimicrobial resistance and phage types of human and non-human Salmonella enterica isolates in Ireland, 1998-2003. Commun. Dis. Public Health 7:193-199.[Medline]
  17. Rabsch, W., H. L. Andrews, R. A. Kingsley, R. Prager, H. Tschape, L. G. Adams, and A. J. Baumler. 2002. Salmonella enterica serotype Typhimurium and its host-adapted variants. Infect. Immun. 70:2249-2255.[Free Full Text]
  18. Refsum, T., E. Heir, G. Kapperud, T. Vardund, and G. Holstad. 2002. Molecular epidemiology of Salmonella enterica serovar Typhimurium isolates determined by pulsed-field gel electrophoresis: comparison of isolates from avian wildlife, domestic animals, and the environment in Norway. Appl. Environ. Microbiol. 68:5600-5606.[Abstract/Free Full Text]
  19. Ribot, E. M., R. K. Wierzba, F. J. Angulo, and T. J. Barrett. 2002. Salmonella enterica serotype Typhimurium DT104 isolated from humans, United States, 1985, 1990, and 1995. Emerg. Infect. Dis. 8:387-391.[Medline]
  20. Swaminathan, B., T. J. Barrett, S. B. Hunter, and R. V. Tauxe. 2001. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Dis. 7:382-389.[Medline]
  21. Threlfall, E. J., L. R. Ward, and B. Rowe. 1978. Epidemic spread of a chloramphenicol-resistant strain of Salmonella typhimurium phage type 204 in bovine animals in Britain. Vet. Rec. 103:438-440.[Medline]
  22. van Duijkeren, E., W. J. Wannet, D. J. Houwers, and W. van Pelt. 2003. Antimicrobial susceptibilities of salmonella strains isolated from humans, cattle, pigs, and chickens in The Netherlands from 1984 to 2001. J. Clin. Microbiol. 41:3574-3578.[Abstract/Free Full Text]
  23. van Duijkeren, E., W. J. B. Wannet, D. J. Houwers, and W. van Pelt. 2002. Serotype and phage type distribution of Salmonella strains isolated from humans, cattle, pigs, and chickens in The Netherlands from 1984 to 2001. J. Clin. Microbiol. 40:3980-3985.[Abstract/Free Full Text]
  24. White, D. G., S. Zhao, R. Sudler, S. Ayers, S. Friedman, S. Chen, P. F. McDermott, S. McDermott, D. D. Wagner, and J. Meng. 2001. The isolation of antibiotic-resistant salmonella from retail ground meats. N. Engl. J. Med. 345:1147-1154.[Abstract/Free Full Text]

Applied and Environmental Microbiology, December 2005, p. 8236-8240, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8236-8240.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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 arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doran, G.
Right arrow Articles by Cormican, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doran, G.
Right arrow Articles by Cormican, M.
Agricola
Right arrow Articles by Doran, G.
Right arrow Articles by Cormican, M.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals