JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 3058–3061 0095-1137/07/$08.00⫹0 doi:10.1128/JCM.00715-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 9
Stability of Multiple-Locus Variable-Number Tandem Repeats in Salmonella enterica Serovar Typhimurium䌤 K. L. Hopkins,1* C. Maguire,1 E. Best,1 E. Liebana,2 and E. J. Threlfall1 Laboratory of Enteric Pathogens, Health Protection Agency Centre for Infections, London, United Kingdom,1 and Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom2 Received 2 April 2007/Returned for modification 18 June 2007/Accepted 25 June 2007
Variable-number tandem repeats (VNTRs) may evolve so rapidly that multiple profiles emerge during an outbreak. A total of 190 isolates from eight Salmonella enterica serovar Typhimurium outbreaks and 15 isolates from seven patients were analyzed by pulsed-field gel electrophoresis and VNTR typing. Small changes in loci were noted; otherwise, the VNTR profiles were stable during the course of the outbreaks. from seven patients (I to VII), including isolates from different specimen types from the same patient, were analyzed. PFGE. PFGE was performed as previously described (8). Profiles were submitted to the PulseNet Europe database and assigned profile names. VNTR typing. VNTR typing was performed using the method of Lindstedt et al. (5) as described by Best et al. (1). VNTR provided better discrimination than PFGE for some phage types. VNTR typing was able to distinguish between isolates from outbreaks B, C, and G and patient II that were designated definitive phage type (DT) 104 or 104b and indistinguishable by PFGE. All isolates produced PFGE pattern STYMXB.0061, which is the predominant DT104 profile in England and Wales (2). Outbreaks C and G were both caused by STm DT104 resistant to streptomycin, spectinomycin, and sulfonamides, and the receipt of samples at the Laboratory of Enteric Pathogens overlapped by 6 days. VNTR typing was able to distinguish between the two outbreaks based on differences at three loci (see Table 1). This demonstrates how VNTR typing can be used in the definition of outbreak-associated cases of infection during parallel outbreaks of common types with the same phage type or PFGE type. In outbreaks D and F a VNTR type was identified that was stable during the course of each outbreak. Single-locus variants (SLVs) were identified in outbreaks B, C, E, and G and in patient VII. No tendency toward mutation within a particular locus was noted; SLVs were a result of loss or gain of a single 6-bp repeat at locus STTR5 (outbreaks C, E, and G), locus STTR6 (outbreaks C and G), or locus STTR10pl (outbreak B). In patient VII there was a 6-bp difference in locus STTR10pl. These data suggest that small changes in loci are possible within the course of a salmonella outbreak and even during salmonella carriage by the patient. In a similar study, nearly 20% of epidemiologically linked strains of Escherichia coli O157:H7 were SLVs of the outbreak clone (6). Application of a cutoff defined as a difference of two repeats at one or fewer loci allowed correct classification of the isolates. Using this cutoff on our data would allow classification of all the SLVs as part of an outbreak. This is confirmed by PFGE data since each SLV had a PFGE profile identical to the main outbreak profile. Examination of the in vitro mutation rate for each locus in E. coli O157 revealed that the majority of changes
Molecular typing is an important tool in detection of outbreaks of Salmonella enterica serovar Typhimurium (STm) when coupled with epidemiologic data. Variable-number tandem repeats (VNTRs) are rapidly evolving genomic elements that demonstrate interstrain variability and therefore have been proposed for molecular typing of STm (5). Pulsed-field gel electrophoresis (PFGE) is currently the “gold standard” for subtyping salmonella strains. VNTR typing is reportedly more discriminatory and reproducible, quicker, and easier to perform, and the data are easier to analyze and share electronically with other laboratories (5). The method has been used in several STm outbreak investigations involving relatively small numbers of isolates (3, 10) and appears to be the only DNAbased typing method with high enough discrimination to differentiate between isolates of DT104 (4). In a recent study, the Statens Serum Institut in Denmark included VNTR typing in routine surveillance over a 2-year period (11). These researchers found VNTR typing to be superior to PFGE for both surveillance and outbreak investigations of STm. Potentially, VNTRs may evolve so rapidly that multiple types could emerge during an outbreak initially caused by a single clone, thereby diminishing its utility for outbreak detection (6). There are few data on the stability of these markers in bacteria and on the correlation between pulsed-field gel electrophoresis (PFGE) and VNTR typing of salmonella strains in the course of outbreak investigations. In this study, we used PFGE and VNTR typing to analyze salmonella isolates belonging to eight outbreaks, as well as multiple isolates from the same patient. These data were compared to the epidemiologic data to determine the concordance between the two methods. Isolates. STm strains (n ⫽ 190) were selected from eight epidemiologically distinct outbreaks (A to H) occurring between 2004 and 2005, as preliminarily identified by local health protection units and confirmed by molecular typing at the Health Protection Agency Laboratory of Enteric Pathogens and by patient questionnaires. In addition, 15 isolates collected * Corresponding author. Mailing address: Salmonella Reference Unit, Laboratory of Enteric Pathogens, 61 Colindale Avenue, London NW9 5EQ, United Kingdom. Phone: 44 (0) 208 327 6107. Fax: 44 (0) 208 905 9929. E-mail:
[email protected]. 䌤 Published ahead of print on 3 July 2007. 3058
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TABLE 1. Isolates characterized in this study Outbreak or patient
Phage type
No. of isolates
Date of receipt
Source (no. of isolates) and geographical locationa
A
U288
7
21 June to 1 July 2005
Human; northeast
B
DT104c
8
12 to 19 October 2005
Human; southeast
C
DT104
71
19 July to 9 August 2004
D
DT208
2
12 November 2004
Human (57), food (14); Northern Ireland (one isolate was from northwestern England) Unknown; northeast
E
DT135
3
1 to 9 September 2005
Human; southeast
F G
DT15 DT104
7 90
17 June to 15 July 2004 3 to 27 August 2004
Human; southeast Human (89), food (1); northeast
H
DT94
2
8 July 2005
Patient I Patient II Patient III
DT104 DT104b DT41
2 2 2
Patient IV Patient V
RDNCd Untypeable
3 2
2 and 17 August 2004 8 and 27 July 2005 9 November and 2 December 2005 13 February 2004 15 February 2005
Human; associated with travel to Tunisia Unknown and feces Feces Unknown and feces
Patient VI Patient VII
Unknown DT8
2 2
20 January 2004 15 March 2006
CSFe, feces, and blood Urine and blood Blood and feces Feces
PFGE pattern(s)/VNTR profile(s)b (no. of isolates)
STYMXB.0213/4-9-5-21-1 (1); STYMXB.0297/4-9-5-21-1 (1); STYMXB.0296/4-9-5-21-1 (1); STYMXB.0258/4-9-5-21-1 (3); STYMXB.0258/4-10-3-21-2 (1)f STYMXB.0061/2-7-12-13-3 (7); STYMXB.0061/2-7-12-14-3 (1) STYMXB.0061/2-11-11-5-3 (66); STYMXB.0061/2-11-12-5-3 (1); STYMXB.0061/2-9-11-5-3 (1); STYMXB.0061/2-7-10-4-3 (1)g; STYMXB.0245/2-11-11-5-3 (2) STYMXB.0249/2-5-6-0-2 (1); STYMXB.0250/2-5-6-0-2 (1) STYMXB.0236/1-8-4-21-3 (2); STYMXB.0236/1-7-4-21-3 (1) STYMXB.0242/3-4-12-2-2 STYMXB.0061/2-7-10-4-3 (82); STYMXB.0061/2-7-9-4-3 (1); STYMXB.0061/2-6-10-4-3 (1); STYMXB.0061/2-12-10-4-3 (1); STYMXB.0199/2-7-10-4-3 (1); STYMXB.0245/2-7-10-4-3 (1); STYMXB.0246/2-7-10-4-3 (1); STYMXB.0294/2-7-10-4-3 (1); STYMXB.0295/2-7-10-4-3 (1) STYMXB.0247/2-4-5-5-3 (1); STYMXB.0248/2-4-5-5-3 (1) STYMXB.0234/2-7-16-0-3 STYMXB.0061/2-7-9-4-3 STYMXB.0060/1-3-13-14-3 STYMXB.0255/2-7-0-0-1 STYMXB.0251/2-13-6-14-1; STYMXB.0254/2-13-6-14-1 STYMXB.0061/3-9-8-27-3 STYMXB.0217/1-1-0-3-3 (1); STYMXB.0217/1-1-0-4-3 (1)
a
In England. The loci of the VNTR profiles are presented in the following order: STTR9-STTR5-STTR6-STTR10pl-STTR3. The number “0” in the VNTR profile represents cases with no amplification of PCR product. PFGE profiles in italics are shown in Fig. 1 and discussed in the main text. c DT, definitive phage type. d RDNC, reacts but does not conform to known patterns. e CSF, cerebrospinal fluid. f Isolate OBS-A. g Isolate OBS-C. b
(85%) involved the addition of one repeat at one particular locus (7) and was consistent with the mutational model proposed by Vogler et al. (12). Analysis of further outbreaks is needed to determine whether this model is applicable to the STm VNTR scheme. Outbreaks A and C each had one isolate, OBS-A and OBS-C, that produced a VNTR profile differing from the prevalent outbreak strain at three loci. Isolate OBS-A had an additional two 6-bp repeats at locus STTR5, four less 6-bp repeats at STTR6, and an additional 33-bp repeat at locus STTR3, whereas isolate OBS-C had four less 6-bp repeats at STTR5 and one less 6-bp repeat at STTR6 and STTR10pl. Noller et al. hypothesized that intralocus differences that occur during outbreaks happen one repeat at a time, whereas unrelated isolates are more likely to differ by more than one repeat (6). Applying these criteria to OBS-A and OBS-C suggests
these isolates were misclassified as part of these outbreaks. Indeed, when we compared the VNTR type of OBS-C to that of the prevalent outbreak strain in outbreak G and took into account the date of receipt for this isolate (9 August 2004), it appeared that OBS-C may be part of outbreak G (for which isolates were received in August 2004) instead of outbreak C (with the exception of isolate OBS-C, isolates were received in July 2004) (Table 1). Both outbreaks were caused by STm strain DT104 with resistance to streptomycin, spectinomycin, and sulfonamides, but the plasmid profile of isolates from outbreak G was different from that of the isolates of outbreak C (13). In addition, the location of the sending laboratories also supports this conclusion: all isolates of outbreak C were received from Northern Ireland with the exception of OBS-C, which was sent from a laboratory in the northeast of England; isolates of outbreak G were all sent from northwestern En-
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FIG. 1. Examples of variation in PFGE profile noted between isolates from the same outbreak or patient. Profiles obtained from U288 isolates were consistently smeary despite repeat subtyping. This has been observed previously in our laboratory (K. L. Hopkins, unpublished data).
gland (Table 1). These data demonstrate how VNTR typing is able to distinguish between concurrent, unrelated strains that may be misclassified as part of an outbreak by initial epidemiologic information or PFGE. Isolates determined to be identical by VNTR typing but with one- to three-band differences in the PFGE profile were occasionally noted in outbreaks A, C, D, G, and H, and in patient V (Fig. 1). This is perhaps not surprising considering the two subtyping methods are determining genomic diversity by very different methods. Tenover’s criteria for interpreting PFGE patterns would imply these isolates had undergone a single genetic event, such as a point mutation, or an insertion or deletion event and therefore should still be considered closely related to the outbreak strain (9). However, DT104 is regarded as a highly clonal phage type, with 36% of DT104 isolates in England and Wales belonging to profile STYMXB.0061 (2). Minor differences in band patterns may therefore be crucial in differentiating between outbreak-related and sporadic cases of infection. These observations highlight the problems of relying on a single subtyping method and highlight the importance of combining laboratory typing data with accurate and meaningful epidemiological information. Our data suggest that VNTR typing is stable enough to be used in outbreak characterization, particularly during parallel outbreaks of common types with the same phage and/or PFGE type. Application of VNTR typing to additional STm outbreaks is needed in order to develop specific guidelines and international consensus for the interpretation of VNTR data, particularly for the consideration of SLVs when analyzing clonal phage types such as DT104 in the context of an outbreak. It may be that stricter criteria need to be applied when analyzing VNTR profiles of clonal phage types than when
analyzing more diverse phage types. Establishment of a VNTR database may facilitate in developing guidelines. This study was funded by the Department for Environment, Food, and Rural Affairs of the United Kingdom (project VM02205). REFERENCES 1. Best, E. L., B. A. Lindstedt, A. Cook, F. A. Clifton-Hadley, E. J. Threlfall, and E. Liebana. 2006. Multiple-locus variable-number tandem repeat analysis of Salmonella enterica subsp. enterica serovar Typhimurium: comparison of isolates from pigs, poultry and cases of human gastroenteritis. J. Appl. Microbiol. doi:10.1111/j.1365-2672.2007.03278.x. 2. Gatto, A. J., T. M. Peters, J. Green, I. S. Fisher, O. N. Gill, S. J. O’brien, C. Maguire, C. Berghold, I. Lederer, P. Gerner-Smidt, M. Torpdahl, A. Siitonen, S. Lukinmaa, H. Tschape, R. Prager, I. Luzzi, A. M. Dionisi, W. K. van der Zwaluw, M. Heck, J. Coia, D. Brown, M. Usera, A. Echeita, and E. J. Threlfall. 2006. Distribution of molecular subtypes within Salmonella enterica serotype Enteritidis phage type 4 and serovar Typhimurium definitive phage type 104 in nine European countries, 2000–2004: results of an international multi-centre study. Epidemiol. Infect. 134:729–736. 3. Isakbaeva, E., B. A. Lindstedt, B. Schimmer, T. Vardund, T. L. Stavnes, K. Hauge, B. Gondrosen, H. Blystad, H. Klovstad, P. Aavitsland, K. Nygard, and G. Kapperud. 2005. Salmonella Typhimurium DT104 outbreak linked to imported minced beef, Norway, October-November 2005. Euro. Surveill. 10:E051110. 4. Lindstedt, B. A., E. Heir, E. Gjernes, and G. Kapperud. 2003. DNA fingerprinting of Salmonella enterica subsp. enterica serovar Typhimurium with emphasis on phage type DT104 based on variable number of tandem repeat loci. J. Clin. Microbiol. 41:1469–1479. 5. Lindstedt, B. A., T. Vardund, L. Aas, and G. Kapperud. 2004. Multiple-locus variable-number tandem repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis. J. Microbiol. Methods 59:163–172. 6. Noller, A. C., M. C. McEllistrem, A. G. Pacheco, D. J. Boxrud, and L. H. Harrison. 2003. Multilocus variable-number tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates. J. Clin. Microbiol. 41:5389–5397. 7. Noller, A. C., M. C. McEllistrem, K. A. Shutt, and L. H. Harrison. 2006. Locus-specific mutational events in a multilocus variable-number tandem repeat analysis of Escherichia coli O157:H7. J. Clin. Microbiol. 44:374–377. 8. Peters, T. M., C. Maguire, E. J. Threlfall, I. S. Fisher, N. Gill, A. J. Gatto,
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