- Email: [email protected]
Multi-locus variable number tandem repeat analysis for Escherichia coli causing extraintestinal infections
Journal of Microbiological Methods 79 (2009) 211–213
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Multi-locus variable number tandem repeat analysis for Escherichia coli causing extraintestinal infections☆ Amee R. Manges a,b,⁎, Patricia A. Tellis b, Caroline Vincent c, Kimberley Lifeso d, Geneviève Geneau e, Richard J. Reid-Smith f,g, Patrick Boerlin f,h a
Department of Epidemiology, Biostatistics and Occupational Health, McGill University, 1020 Pine Avenue West, 36B, Montréal, Québec, Canada H3A 1A2 McGill University Health Centre, Research Institute, Montréal, Québec, Canada Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada d Faculty of Science, McGill University, Montreal, Quebec, Canada e McGill University and Génome Québec Innovation Centre, Montréal, Québec, Canada f Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario, Canada g Department of Population Medicine, University of Guelph, Guelph, Ontario, Canada h Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada b c
a r t i c l e
i n f o
Article history: Received 2 June 2009 Received in revised form 3 September 2009 Accepted 7 September 2009 Available online 16 September 2009
a b s t r a c t Discriminatory genotyping methods for the analysis of Escherichia coli other than O157:H7 are necessary for public health-related activities. A new multi-locus variable number tandem repeat analysis protocol is presented; this method achieves an index of discrimination of 99.5% and is reproducible and valid when tested on a collection of 836 diverse E. coli. © 2009 Elsevier B.V. All rights reserved.
Keywords: Escherichia coli Molecular epidemiology Multi-locus variable number tandem repeat analysis (MLVA) Variable number tandem repeat (VNTR)
1. Introduction Discriminatory molecular typing methods for non-O157:H7 Escherichia coli are necessary for outbreak investigation, public health surveillance, and molecular epidemiological studies. Pulsed-field gel electrophoresis (PFGE) is the current standard for molecular subtyping, but it is time-consuming, and the results can be difficult to compare across laboratories. Multi-locus variable number tandem repeat analysis (MLVA) has shown promise in differentiating closely related strains of E. coli (Keys et al., 2005; Lindstedt et al., 2004; Noller et al., 2003; Noller et al., 2004). However, these MLVA protocols have been developed specifically for E. coli O157:H7. The objective of this study was to develop a fast and reliable MLVA protocol which could be used alone or in conjunction with other
Abbreviations: ERIC2, enterobacterial repetitive intergenic consensus 2 sequence; PFGE, pulsed-field gel electrophoresis; MLVA, multi-locus variable number tandem repeat analysis; VNTR, variable number tandem repeat. ☆ Ethics committee approval: The study protocol was approved by the McGill University, Institutional Review Board (A01-M04-05A). ⁎ Corresponding author. Department of Epidemiology, Biostatistics and Occupational Health, McGill University, 1020 Pine Avenue West, 36B, Montréal, Québec, Canada H3A 1A2. Tel.: +1 514 398 3267; fax: +1 514 398 4503. E-mail address: [email protected] (A.R. Manges). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.09.006
typing methods such as enterobacterial repetitive intergenic consensus 2 (ERIC2) sequence PCR, to genotype large collections of E. coli. 2. Methods 2.1. E. coli isolates E. coli isolates from three sources were included in the study: 417 isolates were from retail chicken, pork, and beef meat that were systematically selected from the Canadian Integrated Program on Antimicrobial Resistance Surveillance (CIPARS) collection; 74 E. coli isolates were from restaurant and ready-to-eat food items collected by the Division de l'inspection des aliments, Ville de Montréal; and 345 E. coli isolates were from human extraintestinal infections. E. coli was grown in Luria–Bertani broth for 16 h and DNA from 1 mL of overnight culture was extracted and purified using the DNeasy blood and tissue kit (QIAGEN, Inc., Hilden, Germany). 2.2. Multi-locus variable number tandem repeat analysis (MLVA) design and protocol Eight genetic loci containing variable numbers of tandem repeats were identified in silico for this study or from published MLVA protocols (Keys
212
A.R. Manges et al. / Journal of Microbiological Methods 79 (2009) 211–213
2.4. Multilocus sequence typing (MLST)
et al., 2005; Lindstedt et al., 2004; Noller et al., 2003; Noller et al., 2004) as follows. Nine fully sequenced and annotated E. coli genomes (CFT073, E24377A, HS, K12, O157, O157 Sakai, UTI89, UTI536, W3110) were queried using the Microorganism Tandem Repeats database (http://minisatellites.u-psud.fr/) to identify candidate loci that exhibited variable copy numbers across 3 or more genomes. A cd-hit-est approach was also used to cluster tandem repeats identified by the Microorganism Tandem Repeats database (Li and Godzik, 2006). Tandem repeats that were greater than 5 bp were considered. Primers to the selected loci were designed using Primer3 (http://frodo.wi.mit. edu/). A search of the literature for published candidate loci from other E. coli studies, particularly studies of E. coli O157:H7 was also performed. Three VNTR loci published earlier by Keys et al (Keys et al., 2005) were selected and primers for two of these loci were modified for this study (see Table 1). A 5′ tail containing GTGTCTT was added to all reverse primers (Brownstein et al., 1996). The primers plus fluorophores FAM (Integrated DNA Technologies, Coralville, Iowa), VIC, NED, and PET (Applied Biosystems, Foster City, California) used for PCR are listed in Table 1. The PCR reaction mixture included magnesium chloride (0.5 mM), PCR Buffer (1x), dNTPs (0.2 mM each), Qiagen HotStart Taq (0.04 U/μl), primers (0.2 μM), and DNA (20 ng). The thermal cycling was as follows: initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s, and a final extension at 72 °C for 7min. Fragments were resolved using capillary electrophoresis (Applied Biosystems Capillary electrophoresis 3730xl DNA analyzer, Foster City, California). Raw fragments lengths for each locus were binned manually using a minimum threshold of ±3-bp to distinguish alleles. E. coli CFT073, K12 and O157:H7 were used as positive controls in every amplification and genotyping run.
MLST on selected E. coli isolates was performed as previously described (Wirth et al., 2006). Gene amplification and sequencing were performed by using the primers specified at the E. coli MLST web site (http://mlst.ucc. ie/mlst/dbs/Ecoli). Allelic profile and sequence type determinations were assigned according to this web site's scheme (Wirth et al., 2006). 2.5. Pulsed field gel electrophoresis (PFGE) The standard protocol for molecular subtyping of E. coli (O157:H7) by PFGE, as established by the Centers for Disease Control and Prevention, was used to genotype selected E. coli isolates from each clonal group (Bender et al., 1997). Isolates exhibiting ≤6 band differences in their PFGE patterns were considered to be related (Tenover et al., 1995). 2.6. DNA sequencing PCR products for all loci for the three positive controls were sent for sequencing at the McGill University and Genome Québec Innovation Centre to confirm the presence and number of predicted tandem repeats. 2.7. Data analyses Indistinguishable MLVA allele profiles for all E. coli isolates were grouped. Nei's estimate of gene diversity (Nei, 1973) was used to estimate the diversity observed for each locus. Simpson's Index of Diversity (Hunter and Gaston, 1988; Hunter, 1990) and 95% confidence intervals were calculated using the Biodiversity Calculator developed by J. Danoff-Burg and C. Xu to estimate an overall discriminatory index (Danoff-Burg and Xu, 2009; Grundmann et al., 2001). The new MLVA method was also evaluated for typeability, reproducibility, and validity. Typeability was measured by the number of isolates that could be resolved by MLVA and the positive call rates for each locus. Reproducibility was judged by evaluating the results of repeated MLVA tests on the positive controls. Validity was assessed by comparing the diversity of MLVA profiles among isolates which were known to be members of different clonal groups and associated with clusters of human extraintestinal infection cases.
2.3. Enterobacterial repetitive intergenic consensus sequence two (ERIC2) PCR All E. coli isolates exhibiting indistinguishable MLVA profiles were screened using ERIC2 PCR fingerprinting to confirm relatedness within each MLVA cluster (Johnson and O'Bryan, 2000; Versalovic et al., 1994; Woods et al., 1993). A clonal group was defined as ≥2 E. coli isolates exhibiting indistinguishable MLVA and ERIC2 PCR patterns.
Table 1 MLVA typing method for Escherichia coli of diverse origin. Locus
O157-11
Repeat size 6
O157-33N
17
O157-56N
5
VNTR1
12
VNTR2
95
VNTR5
91
VNTR13
39
VNTR39
91
a b c d e f
Primers + dye
VIC-F-ACCGGCAATCATCGGGCCAACCA R-GATGCTGGAAAAACTGATGCAGACTCGCGT VIC-F-TCCGAAGTTAACCGTCAGGTTATGC R-GTGTCTTCCGCAGTCGGGCAACTAC PET-F-GCTGACGATGCGTGTAATGT R-GTGTCTTCCCCATTCTACGGTCTATGC FAM-F-ATGGTGTGCTGAGAGACAGG R-GTGTCTTGCCATCTCTGCAACGTCTT NED-F-CGTCAGTGTATGTCCGAAGG R-GTGTCTTCTTAGTGTCAGGGGCAGTTTT NED-F-TATTCAACGCCATCGACTTT R-GCGTGGTAATGGGCTATACA FAM-F-TTTACGCCAATTGTTGAACC R-GTGTCTTGGTGTCAGCAAATCCAGAGA PET-F-GAGAAGGTTAACGCTTGGGTA R-GTGTCTTCGAGAAGCTGCTTGAAGAGA
Amplicon sizea 237
Number of repeatsb
Average call ratec
Total allelesd
Nei's indexe
Ref.
6.2
0.99
22
0.91
Keys, 2005 Keys, 2005 Modified from Modified from Modified from Modified from This study This study This study This study This study This study This study This study This study
CFT073
K12
O157:H7
10
3.2
357
2.2
2.2
3.2
0.98
3
0.43
407
8
NULLf
9.2
0.86
5
0.37
230
6.8
2
2.7
0.98
11
0.57
497
4.1
1
1
0.94
8
0.75
444
4.1
1.1
3.1
0.95
10
0.56
131
3.4
5.9
7.1
0.84
10
0.64
392
3.3
1.1
1.1
0.95
11
0.65
Amplicon size is presented for CFT073. Number of repeats was calculated using the bacterial genotyping program at http://minisatellites.u-psud.fr (accessed May 2009). Positive call rate for each locus was the average over five MLVA runs. Total alleles were determined based on MLVA results for the 803 diverse E. coli isolates. Nei's index is a measure of average genetic diversity (Nei, 1973). NULL, null values are defined as either no amplification (missing entire region) or amplification but no tandem repeats present.
Keys, Keys, Keys, Keys,
2005 2005 2005 2005
A.R. Manges et al. / Journal of Microbiological Methods 79 (2009) 211–213
3. Results and conclusions An MLVA method was developed to rapidly and unambiguously group diverse E. coli isolates. A total of 803 diverse E. coli and 33 E. coli isolates belonging to known clonal groups were analyzed with the new MLVA method. All 836 isolates were typeable by MLVA. The average call rates for each locus are presented in Table 1. Sequencing results confirmed the presence of tandem repeats at each locus. Evaluation of the 803 diverse E. coli identified 303 isolates (38%) with unique MLVA profiles and 121 groups containing 2 or more isolates. The Simpson's diversity index for the MLVA method was estimated to be 99.5% (95% CI: 99.4–99.6). The loci exhibited variable discriminatory powers (Table 1). The three positive controls were included in a total of six separate MLVA runs. There was a total of 6 missing PCR products for three of the loci (VNTR13, VNTR39 and O157-56N) and 3 products were of the incorrect size for 2 loci (O157-56 and VNTR2). However, all but one of these errors occurred in the first two trials of the MLVA method, suggesting that reproducibility improved with each successive assay. Initial tests of VNTR13 indicated multiple fragments; primers were re-designed which led to improved reproducibility. The actual fragment sizes (as measured by the standard deviation of the fragment sizes across runs of the positive controls) varied by locus: O157-11 (0.14–0.40); VNTR39 (0.34–2.43); VNTR5; (0.80–1.41); O157-33N (0.31–0.45); O157-56N (0.08–6.1); VNTR1 (0.21–0.39); and VNTR2 (2.0–8.7). The validity of the method was evaluated by comparing the MLVA results among 33 E. coli representing 4 clonal groups (Manges et al., 2001; Manges et al., 2008). E. coli O11/O17/O77/O73:K2:H18–ST69 (n=13 isolates) contained 3 MLVA profiles in the group. E. coli O2:K1:H7–ST95 (n=10 isolates) contained 3 MLVA profiles. E. coli O25:H4–ST131 (n=4 isolates) contained only one MLVA profile. E. coli O2/O6:H1–ST73 (n=6 isolates) contained two MLVA profiles. Within the groups, the only difference in the profiles was attributable to a variable allele for the O15711 locus. ERIC2 PCR was performed on isolates exhibiting indistinguishable MLVA profiles. ERIC2 PCR differentiated additional genotypes within these groups; however, isolates with the same ERIC2 PCR pattern also exhibited different MLVA profiles. This is not surprising as MLVA and ERIC2 PCR measure genetic variability differently. A close inspection of ERIC2 PCR patterns for 145 isolates associated with 20 MLVA groups showed that 37% of these isolates exhibited ERIC2 PCR patterns which were indistinguishable for the group and another 37% differed by between 1 and 3 bands. Selected isolates (n=60) were evaluated by MLST and XbaI PFGE. MLST results confirmed the group membership defined by MLVA. A total of 44 specific PFGE patterns were identified (5 isolates were nontypeable), indicating that PFGE is still the more discriminating method. In this very diverse collection we were able to identify three different clonal groups, containing 14 isolates, which were related by PFGE. The new MLVA method is robust and useful for characterizing large collections of diverse E. coli and can aid in public health and epidemiologic investigations. Acknowledgments We wish to thank members of the surveillance team of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (Lucie Dutil and Danielle Daignault); the Division de l'inspection des aliments,
213
Ville de Montréal (Myrto Mantzavrakos and Annie Laviolette) and the Student Health Services Clinical Technician (Christiaine Lacombe). This study was supported by the Public Health Agency of Canada (A.R.M) and by the Canadian Institutes of Health Research, M.Sc. Award (C.V.). References Bender, J.B., Hedberg, C.W., Besser, J.M., Boxrud, D.J., MacDonald, K.L., Osterholm, M.T., 1997. Surveillance for Escherichia coli O157:H7 infections in Minnesota by molecular subtyping. N. Engl. J. Med. 337, 388–394. Brownstein, M.J., Carpten, J.D., Smith, J.R., 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques 20, 1008–1010. Danoff-Burg, J., Xu, C. Biodiversity Calculator. http://www.columbia.edu/itc/cerc/danoff-burg/ MBD_Links.html . 2009. 5-20-2009. Grundmann, H., Hori, S., Tanner, G., 2001. Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J. Clin. Microbiol. 39, 4190–4192. Hunter, P.R., 1990. Reproducibility and indices of discriminatory power of microbial typing methods. J. Clin. Microbiol. 28, 1903–1905. Hunter, P.R., Gaston, M.A., 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's Index of Diversity. J. Clin. Microbiol. 26, 2465–2466. Johnson, J.R., O'Bryan, T.T., 2000. Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli. Clin. Diagn. Lab. Immunol. 7, 265–273. Keys, C., Kemper, S., Keim, P., 2005. Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing. J. Appl. Microbiol. 98, 928–940. Li, W., Godzik, A., 2006. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658–1659. Lindstedt, B.A., Vardund, T., Kapperud, G., 2004. Multiple-locus variable-number tandemrepeats analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis. J. Microbiol. Methods 58, 213–222. Manges, A.R., Johnson, J.R., Foxman, B., O'Bryan, T.T., Fullerton, K.E., Riley, L.W., 2001. Widespread distribution of urinary tract infections caused by a multidrug-resistant Escherichia coli clonal group. N. Engl. J. Med. 345, 1007–1013. Manges, A.R., Tabor, H., Tellis, P., Vincent, C., Tellier, P.P., 2008. Endemic and epidemic lineages of Escherichia coli that cause urinary tract infections. Emerg. Infect. Dis. 14, 1575–1583. Nei, M., 1973. Analysis of gene diversity in subdivided populations. PNAS 70, 3321–3323. Noller, A.C., McEllistrem, M.C., Pacheco, A.G., Boxrud, D.J., Harrison, L.H., 2003. Multilocus variable-number tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates. J. Clin. Microbiol. 41, 5389–5397. Noller, A.C., McEllistrem, M.C., Harrison, L.H., 2004. Genotyping primers for fully automated multilocus variable-number tandem repeat analysis of Escherichia coli O157:H7. J. Clin. Microbiol. 42, 3908. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., et al., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239. Versalovic, J., Schneid, M., de Bruijn, F.L., Lupski, J.R., 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cell. Biol. 5, 25–40. Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L.H., et al., 2006. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60, 1136–1151. Woods, C.R., Versalovic, J., Koeuth, T., Lupski, J.R., 1993. Whole-cell repetitive element sequence-based polymerase chain reaction allows rapid assessment of clonal relationships of bacterial isolates. J. Clin. Microbiol. 31, 1927–1931.
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Multi-locus variable number tandem repeat analysis for Escherichia coli causing extraintestinal infections☆ Amee R. Manges a,b,⁎, Patricia A. Tellis b, Caroline Vincent c, Kimberley Lifeso d, Geneviève Geneau e, Richard J. Reid-Smith f,g, Patrick Boerlin f,h a
Department of Epidemiology, Biostatistics and Occupational Health, McGill University, 1020 Pine Avenue West, 36B, Montréal, Québec, Canada H3A 1A2 McGill University Health Centre, Research Institute, Montréal, Québec, Canada Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada d Faculty of Science, McGill University, Montreal, Quebec, Canada e McGill University and Génome Québec Innovation Centre, Montréal, Québec, Canada f Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario, Canada g Department of Population Medicine, University of Guelph, Guelph, Ontario, Canada h Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada b c
a r t i c l e
i n f o
Article history: Received 2 June 2009 Received in revised form 3 September 2009 Accepted 7 September 2009 Available online 16 September 2009
a b s t r a c t Discriminatory genotyping methods for the analysis of Escherichia coli other than O157:H7 are necessary for public health-related activities. A new multi-locus variable number tandem repeat analysis protocol is presented; this method achieves an index of discrimination of 99.5% and is reproducible and valid when tested on a collection of 836 diverse E. coli. © 2009 Elsevier B.V. All rights reserved.
Keywords: Escherichia coli Molecular epidemiology Multi-locus variable number tandem repeat analysis (MLVA) Variable number tandem repeat (VNTR)
1. Introduction Discriminatory molecular typing methods for non-O157:H7 Escherichia coli are necessary for outbreak investigation, public health surveillance, and molecular epidemiological studies. Pulsed-field gel electrophoresis (PFGE) is the current standard for molecular subtyping, but it is time-consuming, and the results can be difficult to compare across laboratories. Multi-locus variable number tandem repeat analysis (MLVA) has shown promise in differentiating closely related strains of E. coli (Keys et al., 2005; Lindstedt et al., 2004; Noller et al., 2003; Noller et al., 2004). However, these MLVA protocols have been developed specifically for E. coli O157:H7. The objective of this study was to develop a fast and reliable MLVA protocol which could be used alone or in conjunction with other
Abbreviations: ERIC2, enterobacterial repetitive intergenic consensus 2 sequence; PFGE, pulsed-field gel electrophoresis; MLVA, multi-locus variable number tandem repeat analysis; VNTR, variable number tandem repeat. ☆ Ethics committee approval: The study protocol was approved by the McGill University, Institutional Review Board (A01-M04-05A). ⁎ Corresponding author. Department of Epidemiology, Biostatistics and Occupational Health, McGill University, 1020 Pine Avenue West, 36B, Montréal, Québec, Canada H3A 1A2. Tel.: +1 514 398 3267; fax: +1 514 398 4503. E-mail address: [email protected] (A.R. Manges). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.09.006
typing methods such as enterobacterial repetitive intergenic consensus 2 (ERIC2) sequence PCR, to genotype large collections of E. coli. 2. Methods 2.1. E. coli isolates E. coli isolates from three sources were included in the study: 417 isolates were from retail chicken, pork, and beef meat that were systematically selected from the Canadian Integrated Program on Antimicrobial Resistance Surveillance (CIPARS) collection; 74 E. coli isolates were from restaurant and ready-to-eat food items collected by the Division de l'inspection des aliments, Ville de Montréal; and 345 E. coli isolates were from human extraintestinal infections. E. coli was grown in Luria–Bertani broth for 16 h and DNA from 1 mL of overnight culture was extracted and purified using the DNeasy blood and tissue kit (QIAGEN, Inc., Hilden, Germany). 2.2. Multi-locus variable number tandem repeat analysis (MLVA) design and protocol Eight genetic loci containing variable numbers of tandem repeats were identified in silico for this study or from published MLVA protocols (Keys
212
A.R. Manges et al. / Journal of Microbiological Methods 79 (2009) 211–213
2.4. Multilocus sequence typing (MLST)
et al., 2005; Lindstedt et al., 2004; Noller et al., 2003; Noller et al., 2004) as follows. Nine fully sequenced and annotated E. coli genomes (CFT073, E24377A, HS, K12, O157, O157 Sakai, UTI89, UTI536, W3110) were queried using the Microorganism Tandem Repeats database (http://minisatellites.u-psud.fr/) to identify candidate loci that exhibited variable copy numbers across 3 or more genomes. A cd-hit-est approach was also used to cluster tandem repeats identified by the Microorganism Tandem Repeats database (Li and Godzik, 2006). Tandem repeats that were greater than 5 bp were considered. Primers to the selected loci were designed using Primer3 (http://frodo.wi.mit. edu/). A search of the literature for published candidate loci from other E. coli studies, particularly studies of E. coli O157:H7 was also performed. Three VNTR loci published earlier by Keys et al (Keys et al., 2005) were selected and primers for two of these loci were modified for this study (see Table 1). A 5′ tail containing GTGTCTT was added to all reverse primers (Brownstein et al., 1996). The primers plus fluorophores FAM (Integrated DNA Technologies, Coralville, Iowa), VIC, NED, and PET (Applied Biosystems, Foster City, California) used for PCR are listed in Table 1. The PCR reaction mixture included magnesium chloride (0.5 mM), PCR Buffer (1x), dNTPs (0.2 mM each), Qiagen HotStart Taq (0.04 U/μl), primers (0.2 μM), and DNA (20 ng). The thermal cycling was as follows: initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s, and a final extension at 72 °C for 7min. Fragments were resolved using capillary electrophoresis (Applied Biosystems Capillary electrophoresis 3730xl DNA analyzer, Foster City, California). Raw fragments lengths for each locus were binned manually using a minimum threshold of ±3-bp to distinguish alleles. E. coli CFT073, K12 and O157:H7 were used as positive controls in every amplification and genotyping run.
MLST on selected E. coli isolates was performed as previously described (Wirth et al., 2006). Gene amplification and sequencing were performed by using the primers specified at the E. coli MLST web site (http://mlst.ucc. ie/mlst/dbs/Ecoli). Allelic profile and sequence type determinations were assigned according to this web site's scheme (Wirth et al., 2006). 2.5. Pulsed field gel electrophoresis (PFGE) The standard protocol for molecular subtyping of E. coli (O157:H7) by PFGE, as established by the Centers for Disease Control and Prevention, was used to genotype selected E. coli isolates from each clonal group (Bender et al., 1997). Isolates exhibiting ≤6 band differences in their PFGE patterns were considered to be related (Tenover et al., 1995). 2.6. DNA sequencing PCR products for all loci for the three positive controls were sent for sequencing at the McGill University and Genome Québec Innovation Centre to confirm the presence and number of predicted tandem repeats. 2.7. Data analyses Indistinguishable MLVA allele profiles for all E. coli isolates were grouped. Nei's estimate of gene diversity (Nei, 1973) was used to estimate the diversity observed for each locus. Simpson's Index of Diversity (Hunter and Gaston, 1988; Hunter, 1990) and 95% confidence intervals were calculated using the Biodiversity Calculator developed by J. Danoff-Burg and C. Xu to estimate an overall discriminatory index (Danoff-Burg and Xu, 2009; Grundmann et al., 2001). The new MLVA method was also evaluated for typeability, reproducibility, and validity. Typeability was measured by the number of isolates that could be resolved by MLVA and the positive call rates for each locus. Reproducibility was judged by evaluating the results of repeated MLVA tests on the positive controls. Validity was assessed by comparing the diversity of MLVA profiles among isolates which were known to be members of different clonal groups and associated with clusters of human extraintestinal infection cases.
2.3. Enterobacterial repetitive intergenic consensus sequence two (ERIC2) PCR All E. coli isolates exhibiting indistinguishable MLVA profiles were screened using ERIC2 PCR fingerprinting to confirm relatedness within each MLVA cluster (Johnson and O'Bryan, 2000; Versalovic et al., 1994; Woods et al., 1993). A clonal group was defined as ≥2 E. coli isolates exhibiting indistinguishable MLVA and ERIC2 PCR patterns.
Table 1 MLVA typing method for Escherichia coli of diverse origin. Locus
O157-11
Repeat size 6
O157-33N
17
O157-56N
5
VNTR1
12
VNTR2
95
VNTR5
91
VNTR13
39
VNTR39
91
a b c d e f
Primers + dye
VIC-F-ACCGGCAATCATCGGGCCAACCA R-GATGCTGGAAAAACTGATGCAGACTCGCGT VIC-F-TCCGAAGTTAACCGTCAGGTTATGC R-GTGTCTTCCGCAGTCGGGCAACTAC PET-F-GCTGACGATGCGTGTAATGT R-GTGTCTTCCCCATTCTACGGTCTATGC FAM-F-ATGGTGTGCTGAGAGACAGG R-GTGTCTTGCCATCTCTGCAACGTCTT NED-F-CGTCAGTGTATGTCCGAAGG R-GTGTCTTCTTAGTGTCAGGGGCAGTTTT NED-F-TATTCAACGCCATCGACTTT R-GCGTGGTAATGGGCTATACA FAM-F-TTTACGCCAATTGTTGAACC R-GTGTCTTGGTGTCAGCAAATCCAGAGA PET-F-GAGAAGGTTAACGCTTGGGTA R-GTGTCTTCGAGAAGCTGCTTGAAGAGA
Amplicon sizea 237
Number of repeatsb
Average call ratec
Total allelesd
Nei's indexe
Ref.
6.2
0.99
22
0.91
Keys, 2005 Keys, 2005 Modified from Modified from Modified from Modified from This study This study This study This study This study This study This study This study This study
CFT073
K12
O157:H7
10
3.2
357
2.2
2.2
3.2
0.98
3
0.43
407
8
NULLf
9.2
0.86
5
0.37
230
6.8
2
2.7
0.98
11
0.57
497
4.1
1
1
0.94
8
0.75
444
4.1
1.1
3.1
0.95
10
0.56
131
3.4
5.9
7.1
0.84
10
0.64
392
3.3
1.1
1.1
0.95
11
0.65
Amplicon size is presented for CFT073. Number of repeats was calculated using the bacterial genotyping program at http://minisatellites.u-psud.fr (accessed May 2009). Positive call rate for each locus was the average over five MLVA runs. Total alleles were determined based on MLVA results for the 803 diverse E. coli isolates. Nei's index is a measure of average genetic diversity (Nei, 1973). NULL, null values are defined as either no amplification (missing entire region) or amplification but no tandem repeats present.
Keys, Keys, Keys, Keys,
2005 2005 2005 2005
A.R. Manges et al. / Journal of Microbiological Methods 79 (2009) 211–213
3. Results and conclusions An MLVA method was developed to rapidly and unambiguously group diverse E. coli isolates. A total of 803 diverse E. coli and 33 E. coli isolates belonging to known clonal groups were analyzed with the new MLVA method. All 836 isolates were typeable by MLVA. The average call rates for each locus are presented in Table 1. Sequencing results confirmed the presence of tandem repeats at each locus. Evaluation of the 803 diverse E. coli identified 303 isolates (38%) with unique MLVA profiles and 121 groups containing 2 or more isolates. The Simpson's diversity index for the MLVA method was estimated to be 99.5% (95% CI: 99.4–99.6). The loci exhibited variable discriminatory powers (Table 1). The three positive controls were included in a total of six separate MLVA runs. There was a total of 6 missing PCR products for three of the loci (VNTR13, VNTR39 and O157-56N) and 3 products were of the incorrect size for 2 loci (O157-56 and VNTR2). However, all but one of these errors occurred in the first two trials of the MLVA method, suggesting that reproducibility improved with each successive assay. Initial tests of VNTR13 indicated multiple fragments; primers were re-designed which led to improved reproducibility. The actual fragment sizes (as measured by the standard deviation of the fragment sizes across runs of the positive controls) varied by locus: O157-11 (0.14–0.40); VNTR39 (0.34–2.43); VNTR5; (0.80–1.41); O157-33N (0.31–0.45); O157-56N (0.08–6.1); VNTR1 (0.21–0.39); and VNTR2 (2.0–8.7). The validity of the method was evaluated by comparing the MLVA results among 33 E. coli representing 4 clonal groups (Manges et al., 2001; Manges et al., 2008). E. coli O11/O17/O77/O73:K2:H18–ST69 (n=13 isolates) contained 3 MLVA profiles in the group. E. coli O2:K1:H7–ST95 (n=10 isolates) contained 3 MLVA profiles. E. coli O25:H4–ST131 (n=4 isolates) contained only one MLVA profile. E. coli O2/O6:H1–ST73 (n=6 isolates) contained two MLVA profiles. Within the groups, the only difference in the profiles was attributable to a variable allele for the O15711 locus. ERIC2 PCR was performed on isolates exhibiting indistinguishable MLVA profiles. ERIC2 PCR differentiated additional genotypes within these groups; however, isolates with the same ERIC2 PCR pattern also exhibited different MLVA profiles. This is not surprising as MLVA and ERIC2 PCR measure genetic variability differently. A close inspection of ERIC2 PCR patterns for 145 isolates associated with 20 MLVA groups showed that 37% of these isolates exhibited ERIC2 PCR patterns which were indistinguishable for the group and another 37% differed by between 1 and 3 bands. Selected isolates (n=60) were evaluated by MLST and XbaI PFGE. MLST results confirmed the group membership defined by MLVA. A total of 44 specific PFGE patterns were identified (5 isolates were nontypeable), indicating that PFGE is still the more discriminating method. In this very diverse collection we were able to identify three different clonal groups, containing 14 isolates, which were related by PFGE. The new MLVA method is robust and useful for characterizing large collections of diverse E. coli and can aid in public health and epidemiologic investigations. Acknowledgments We wish to thank members of the surveillance team of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (Lucie Dutil and Danielle Daignault); the Division de l'inspection des aliments,
213
Ville de Montréal (Myrto Mantzavrakos and Annie Laviolette) and the Student Health Services Clinical Technician (Christiaine Lacombe). This study was supported by the Public Health Agency of Canada (A.R.M) and by the Canadian Institutes of Health Research, M.Sc. Award (C.V.). References Bender, J.B., Hedberg, C.W., Besser, J.M., Boxrud, D.J., MacDonald, K.L., Osterholm, M.T., 1997. Surveillance for Escherichia coli O157:H7 infections in Minnesota by molecular subtyping. N. Engl. J. Med. 337, 388–394. Brownstein, M.J., Carpten, J.D., Smith, J.R., 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques 20, 1008–1010. Danoff-Burg, J., Xu, C. Biodiversity Calculator. http://www.columbia.edu/itc/cerc/danoff-burg/ MBD_Links.html . 2009. 5-20-2009. Grundmann, H., Hori, S., Tanner, G., 2001. Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J. Clin. Microbiol. 39, 4190–4192. Hunter, P.R., 1990. Reproducibility and indices of discriminatory power of microbial typing methods. J. Clin. Microbiol. 28, 1903–1905. Hunter, P.R., Gaston, M.A., 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's Index of Diversity. J. Clin. Microbiol. 26, 2465–2466. Johnson, J.R., O'Bryan, T.T., 2000. Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli. Clin. Diagn. Lab. Immunol. 7, 265–273. Keys, C., Kemper, S., Keim, P., 2005. Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing. J. Appl. Microbiol. 98, 928–940. Li, W., Godzik, A., 2006. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658–1659. Lindstedt, B.A., Vardund, T., Kapperud, G., 2004. Multiple-locus variable-number tandemrepeats analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis. J. Microbiol. Methods 58, 213–222. Manges, A.R., Johnson, J.R., Foxman, B., O'Bryan, T.T., Fullerton, K.E., Riley, L.W., 2001. Widespread distribution of urinary tract infections caused by a multidrug-resistant Escherichia coli clonal group. N. Engl. J. Med. 345, 1007–1013. Manges, A.R., Tabor, H., Tellis, P., Vincent, C., Tellier, P.P., 2008. Endemic and epidemic lineages of Escherichia coli that cause urinary tract infections. Emerg. Infect. Dis. 14, 1575–1583. Nei, M., 1973. Analysis of gene diversity in subdivided populations. PNAS 70, 3321–3323. Noller, A.C., McEllistrem, M.C., Pacheco, A.G., Boxrud, D.J., Harrison, L.H., 2003. Multilocus variable-number tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates. J. Clin. Microbiol. 41, 5389–5397. Noller, A.C., McEllistrem, M.C., Harrison, L.H., 2004. Genotyping primers for fully automated multilocus variable-number tandem repeat analysis of Escherichia coli O157:H7. J. Clin. Microbiol. 42, 3908. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., et al., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239. Versalovic, J., Schneid, M., de Bruijn, F.L., Lupski, J.R., 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cell. Biol. 5, 25–40. Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L.H., et al., 2006. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60, 1136–1151. Woods, C.R., Versalovic, J., Koeuth, T., Lupski, J.R., 1993. Whole-cell repetitive element sequence-based polymerase chain reaction allows rapid assessment of clonal relationships of bacterial isolates. J. Clin. Microbiol. 31, 1927–1931.