Abstract

A highly specific and reproducible approach for the simultaneous detection of enteric pathogenic bacteria was developed using bacterial hsp60 gene and molecular biological tools. A single pair of universal primers was derived from the highly conserved sequence of hsp60 genes encompassing a 600-bp hypervariable region. PCR amplification followed by either dot blot hybridization or restriction enzyme digestion performed on 38 enteric bacteria indicated that this approach could differentiate not only different genera such as Campylobacter, Yersinia and Vibrio, but also species that are closely related genetically, such as between C. jejuni and C. coli, or between Salmonella and Shigella or Escherichia coli.

Introduction

Enteroinvasive Escherichia coli, and members of the genera Campylobacter, Salmonella, Shigella, Yersinia, Aeromonas, Plesiomonas and Vibrio species are the common causes of invasive diarrhea among young children, the elderly, or immunocompromised individuals [1,,,,5]. Infections are often associated with ingestion of contaminated food or water, or contact with farm animals. The traditional and still most commonly used laboratory method for the detection of enteric bacteria associated with invasive diarrhea has been the isolation of these organisms from stool specimens followed by phenotypic identification by biochemical and/or serologic methods [6]. Recent advances in molecular biology have introduced new approaches for the rapid and sensitive diagnosis of these infections by detecting the presence of pathogen-specific DNA sequences in clinical specimens without the need for culturing the bacteria. These genotypic methods offer particular advantages for the detection of fastidious and non-cultivable microorganisms. However, one of the issues that prevent DNA technology from general use in the diagnostic microbiology laboratory is that most of the molecular methods described in the last decade for this purpose involve multiple gene targets and often require the synthesis of different probes and primers for each pathogen. Furthermore, facilities for radioactive material are frequently required. Such an approach is both technically demanding and labor-intensive, and is thus poorly suited for routine use in clinical settings.

We have investigated the use of broad range degenerate primers that amplify the 60-kDa heat shock protein (HSP60) gene that is universally present in all microorganisms [7,8]. Furthermore, we demonstrated previously the presence of species-specific DNA sequences within the 600-bp region of hsp60 among coagulase-negative staphylococci, and that these sequences can be readily amplified by the use of a single pair of universal degenerate primers [7,8]. In this report, we used our universal primers to sequence and compare the 600-bp fragments of the partial hsp60 gene from 18 different ATCC reference strains and clinical isolates of enteric bacteria. An additional 22 strains were used for validation of the approach. Our results suggest that the hsp60 universal primers may provide a fast, sensitive and specific approach that is particularly manageable for clinical diagnostic laboratories for the simultaneous detection and species-specific identification of the majority of enteric bacteria implicated in invasive diarrhea.

Materials and methods

Strains and reagents

The 18 ATCC reference strains and clinical isolates used for sequence comparison of their hsp60 partial sequence are listed in Table 1. These and the additional 22 strains used for validation of the approach include: four strains of Campylobacter jejuni (NCTC 11168, ATCC 33560, ATCC 43433, ATCC 49943), four strains of Campylobacter coli (NCTC 11353, ATCC 33559, ATCC 43488, ATCC 49941), five clinical isolates of Campylobacter fetus subsp. intestinalis, one strain each of Campylobacter fetus subsp. fetus (ATCC 27374) and Campylobacter lari (ATCC 35221), three strains of Vibrio parahaemolyticus (ATCC 17802, and two clinical isolates), two strains of Vibrio vulnificus (ATCC 27562 and a clinical isolate), two strains of Aeromonas hydrophila (ATCC 19570 and a clinical isolate), one strain each of Plesiomonas shigelloides (ATCC 14029), Yersinia enterocolitica (X82212), Yersinia pseudotuberculosis, Shigella boydii, Shigella flexneri, Shigella dysenteriae and Shigella sonnei, three strains of Salmonella typhimurium, one each of Salmonella dublin, Salmonella choleraesuis, and Salmonella typhi (U01039), and five strains of E. coli (ATCC 27165, and laboratory strains AG100, DH5α, EHEC and EPEC).

1

Homology analysis of partial hsp60 and 16S rRNA sequences among enteric bacteriaa

 10 11 12 13 14 15 16 17 18 
E. coli ATCC 27165 (1)  96 96 ND 98 97 98 98 94 92 89 89 89 88 92 73 74 74 
Salmonella typhi U01039 (2) 94  99 ND 94 94 94 91 91 90 86 89 90 85 89 70 69 70 
Salmonella typhimurium (3)b AY044105 93 98  ND 96 96 96 95 94 93 90 89 90 88 92 74 74 74 
Salmonella dublin (4)b AY044102 93 98 98  ND ND ND ND ND ND ND ND ND ND ND ND ND ND 
Shigella boydii (5)b AY044101 98 93 93 93  98 99 97 94 94 89 88 90 87 91 73 73 73 
Shigella dysenteriae (6) ND ND ND ND ND  99 97 94 94 89 88 90 88 91 74 73 74 
Shigella flexneri (7)b AY044103 98 93 93 93 98 ND  97 94 94 89 88 90 87 91 73 73 73 
Shigella sonnei (8)b AY044104 98 93 93 93 98 ND 98  92 92 89 89 88 88 92 73 73 73 
Yersinia enterocolitica X82212 (9) 85 83 84 84 86 ND 86 86  97 89 87 90 89 93 72 71 71 
Yersinia pseudotuberculosis (10)b AY044106 85 83 83 83 85 ND 85 85 92  90 88 90 90 92 72 71 72 
Vibrio cholerae ATCC 14033 (11)b AF230935 80 80 80 80 80 ND 80 80 81 81  92 94 89 90 73 73 74 
Vibrio parahaemolyticus ATCC 17802 (12)b AF230951 76 75 75 75 76 ND 76 76 76 77 80  95 87 89 72 73 73 
Vibrio vulnificus ATCC 27562 (13)b AF230955 75 76 76 76 75 ND 75 75 75 75 80 80  88 90 73 72 73 
Aeromonas hydrophila 7788 (14)b AF230959 81 80 81 80 81 ND 81 81 77 78 78 74 73  89 73 73 74 
Plesiomonas shigelloides ATCC 14029 (15)b AF230960 83 83 83 82 83 ND 83 82 81 82 82 78 76 80  74 73 74 
Campylobacter jejuni NCTC 11168 (16)b AY044099 64 62 63 62 64 ND 65 64 63 62 62 62 62 59 61  96 96 
Campylobacter coli NCTC 11353 (17)b AY044098 63 62 62 61 62 ND 63 63 60 60 59 61 59 58 61 90  97 
Campylobacter lari ATCC 35221 (18)b AY044100 58 57 57 57 61 ND 62 62 61 61 61 59 62 54 60 87 88  
 10 11 12 13 14 15 16 17 18 
E. coli ATCC 27165 (1)  96 96 ND 98 97 98 98 94 92 89 89 89 88 92 73 74 74 
Salmonella typhi U01039 (2) 94  99 ND 94 94 94 91 91 90 86 89 90 85 89 70 69 70 
Salmonella typhimurium (3)b AY044105 93 98  ND 96 96 96 95 94 93 90 89 90 88 92 74 74 74 
Salmonella dublin (4)b AY044102 93 98 98  ND ND ND ND ND ND ND ND ND ND ND ND ND ND 
Shigella boydii (5)b AY044101 98 93 93 93  98 99 97 94 94 89 88 90 87 91 73 73 73 
Shigella dysenteriae (6) ND ND ND ND ND  99 97 94 94 89 88 90 88 91 74 73 74 
Shigella flexneri (7)b AY044103 98 93 93 93 98 ND  97 94 94 89 88 90 87 91 73 73 73 
Shigella sonnei (8)b AY044104 98 93 93 93 98 ND 98  92 92 89 89 88 88 92 73 73 73 
Yersinia enterocolitica X82212 (9) 85 83 84 84 86 ND 86 86  97 89 87 90 89 93 72 71 71 
Yersinia pseudotuberculosis (10)b AY044106 85 83 83 83 85 ND 85 85 92  90 88 90 90 92 72 71 72 
Vibrio cholerae ATCC 14033 (11)b AF230935 80 80 80 80 80 ND 80 80 81 81  92 94 89 90 73 73 74 
Vibrio parahaemolyticus ATCC 17802 (12)b AF230951 76 75 75 75 76 ND 76 76 76 77 80  95 87 89 72 73 73 
Vibrio vulnificus ATCC 27562 (13)b AF230955 75 76 76 76 75 ND 75 75 75 75 80 80  88 90 73 72 73 
Aeromonas hydrophila 7788 (14)b AF230959 81 80 81 80 81 ND 81 81 77 78 78 74 73  89 73 73 74 
Plesiomonas shigelloides ATCC 14029 (15)b AF230960 83 83 83 82 83 ND 83 82 81 82 82 78 76 80  74 73 74 
Campylobacter jejuni NCTC 11168 (16)b AY044099 64 62 63 62 64 ND 65 64 63 62 62 62 62 59 61  96 96 
Campylobacter coli NCTC 11353 (17)b AY044098 63 62 62 61 62 ND 63 63 60 60 59 61 59 58 61 90  97 
Campylobacter lari ATCC 35221 (18)b AY044100 58 57 57 57 61 ND 62 62 61 61 61 59 62 54 60 87 88  

aStrains without ATCC designation are clinical isolates. Numbers shown in boxes are ClustalW alignment scores. The upper triangle indicates the percentage of similarity of the 16S rRNA gene and the lower triangle indicates the percentage of similarity of the hsp60 gene. ND, not determined because the hsp60 partial sequence of S. dysenteriae and the 16S rRNA sequence of S. dublin are not available.

bIndicates that the partial hsp60 sequences of these bacteria were determined in this study and their GenBank accession numbers are indicated. The hsp60 sequences of the remaining bacteria and the 16S rRNA sequences of all the bacteria in this table were obtained from the NCBI databases.

PCR and agarose gel electrophoresis

The PCR mixture contained 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 200 μM of each dNTP, 1 μM each of the HSP60 degenerate primer designated H279 and H280, 2 U Taq polymerase (Gibco BRL, Grand Island, NY, USA), and 20–100 ng of genomic DNA or 5 μl of bacterial cell lysate. Primers H279 and H280, with the nucleotide sequence of 5′-GAATTCGAIIIIGCIGGIGA(TC)GGIACIACIAC-3′ and 5′-CGCGGGATCC(TC)(TG)I(TC)(TG)ITCICC(AG)AAICCIGGIGC(TC)TT-3′, respectively, amplify an expected 600-bp HSP60 DNA as described previously [7]. The H279 primer had an EcoRI restriction enzyme digest site while H280 had a BamHI digest site (both underlined in the above sequences). Genomic DNA from enteric bacteria was prepared using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN, USA) according to the supplier's protocols. Bacterial cell lysates were prepared from a single bacterial colony, resuspended in 50 μl of distilled water, and boiled for 3 min. The tube containing the lysed bacteria was centrifuged at 12 000 rpm for 3 min, and the supernatant was used as input DNA. The thermal cycling conditions were 3 min at 95°C, followed by 30 cycles of 95°C for 30 s, 37°C for 30 s, 72°C for 1 min, and a last step at 75°C for 5 min. After PCR amplification, 10 μl of each reaction mixture was analyzed on a 2.0% Tris-acetate-EDTA (TAE) agarose gel. The DNA fragments were visualized and photographed under UV light after ethidium bromide staining.

Dot blot hybridization

The amplified 600-bp PCR products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, CA, USA). In instances where multiple bands were visualized on the gel, the 600-bp band was cut out and DNA was purified using the QIAquick gel extraction kit. 2 μl (0.05 ng μl−1) of each of the purified, denatured 600-bp PCR products was spotted on a nylon membrane and UV-crosslinked by a Stratalinker (model UV2400, Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Digoxigenin-labeled probes were prepared by PCR from genomic DNA using the universal primers and DIG-containing dNTP mix according to Roche protocol (Roche, Canada). Hybridization was carried out using the Minislot apparatus according to the manufacturer's instructions (Immunetic). Prehybridization was carried out for 1 h at 40°C in hybridization buffer (50% formamide, 5×SSC [1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 2% Boehringer Mannheim blocking reagent, 0.1%N-laurylsarcosine, and 0.02% sodium dodecyl sulfate (SDS) as described by Goh et al. [7]. The heat-denatured digoxigenin-labeled probes were then introduced. After overnight hybridization at 42°C, the probes were flushed from the channels with 100 ml of 0.1×SSC–0.1% SDS. The nylon filter was then removed and washed twice for 5 min each time at room temperature with 0.1×SSC–0.1% SDS followed by two 30-min washes with the same buffer at 68°C. Detection was performed using chemiluminescent substrate according to Roche protocol.

Sequencing

For sequencing of the 600-bp regions, PCR was performed with modified primers containing M13 primer binding sites on the ends [9]. DNA sequencing was performed by the Nucleic Acid Protein Service at the University of British Columbia using fluorescent dye-terminator chemistry and an ABI automated sequencer. Sequence alignment was performed using the ClustalW program [10].

Nucleotide sequence accession numbers

The GenBank accession numbers of the partial hsp60 sequence for different enteric bacteria determined in this study are listed in Table 1.

Results and discussions

The universal degenerate primers, H279 and H280 recognize conserved regions of the hsp60 gene and amplify a 600-bp product. The consensus sequence of the partial hsp60 gene from 10 representative Gram-positive and Gram-negative bacteria and the location of primers H279 and H280 are shown in Fig. 1. It is apparent that highly conserved sequences are interspersed within regions of higher variations within the 600-bp fragments in these bacteria amplified by primer H279 and H280 (Fig. 1). Sequence alignment of the 600-bp regions from 18 ATCC reference strains and clinical isolates of enteric bacteria obtained in the current study revealed a similar pattern of nucleic acid sequence similarity and variation within this region (data not shown). As indicated in Table 1, sequence similarity among different genera (E. coli, Yersinia, Vibrio, Aeromonas, Plesiomonas, and Campylobacter) ranged from 63% to 85%, whereas a much higher sequence similarity ranging from 93% to 94% was observed among genetically close-related genera of E. coli, Salmonella and Shigella with about 90–98% sequence similarity between different species within the same genus (Table 1, lower triangle). Of interest, the 16S rRNA sequence of the same panel of bacteria generally showed a higher percentage of sequence similarity compared to their respective hsp60 sequences. For example, 16S rRNA sequence similarity among the genera E. coli, Yersinia, Vibrio, Aeromonas, Plesiomonas, and Campylobacter ranged from 73% to 94%. Similarly, sequence similarity among the close-related genera of E. coli, Salmonella and Shigella ranged from 95% to 99% with about 95–99% sequence similarity between different species within the same genus (Table 1, upper triangle). In particular, the percentage of DNA identity between C. jejuni and C. coli was 90% for their hsp60 partial sequences as compared to 96% for their 16S rRNA sequences. The DNA identity between V. parahaemolyticus and V. vulnificus was 80% for their hsp60 partial sequences as compared to 95% for their 16s rRNA sequences. The hsp60 DNA identity between Yersinia (Y. enterocolitica and Y. pseudotuberculosis) and those of Enterobacteriaceae, Vibrionaceae and Campylobacters were 83–86%, 75–81% and 60–63%, respectively, while their corresponding 16S rRNA sequence similarity with the above three groups were 90–94%, 87–90% and 96–97%, respectively. The lower percent of DNA identity suggests that hsp60 sequences could be more discriminatory for species identification than their 16s rRNA sequences. It is, therefore, possible to rationally use PCR amplicons of the partial hsp60 gene for direct probing and identification of a desired phylogenetic group of enteric bacteria. Accordingly, we further evaluated this approach by dot blot hybridization or restriction fragment length polymorphism (RFLP) analysis of the 600-bp amplicons prepared from the entire panel of 42 different enteric bacteria including both ATCC reference strains and clinical isolates.

1

Sequence alignment of the partial hsp60 gene from 10 different Gram-positive and Gram-negative bacteria. The conserved regions are shown by consensus sequence as indicated by stars. The location of the primers H279 and H280 is indicated by arrows.

1

Sequence alignment of the partial hsp60 gene from 10 different Gram-positive and Gram-negative bacteria. The conserved regions are shown by consensus sequence as indicated by stars. The location of the primers H279 and H280 is indicated by arrows.

All 42 enteric bacteria (see Section 2.1) produced a 600-bp product after PCR amplification using the primer pair H279 and H280. Fig. 2 shows the 600-bp product from 11 representative bacterial strains. Furthermore, results from dot blot hybridization suggested that the PCR amplicon from Campylobacter, Yersinia, Plesiomonas, and Aeromonas could be used as species-specific DNA probes. For example, the hsp60 amplicon from C. coli, C. fetus and C. jejuni only hybridized to the 600-bp PCR products from their own species (Fig. 3A). Similarly, the PCR amplicon from the Vibrio species, V. vulnificus and V. parahaemolyticus, was also species-specific and did not cross-hybridize with target DNA from the related Aeromonas and Plesiomonas species (Fig. 3B). For Escherichia coli, Salmonella and Shigella species, their hsp60 dot blots cross-hybridized with each other (data not shown) due to the fact that they are more closely related to each other genetically than to others.

2

PCR amplification and agarose gel electrophoresis of the 600-bp fragments from some representative enteric bacteria.

2

PCR amplification and agarose gel electrophoresis of the 600-bp fragments from some representative enteric bacteria.

3

dot blot hybridization of the 600-bp hsp60 amplicons from various enteric bacteria with (A) Campylobacter hsp60 probes, and (B) Vibrio hsp60 probes. Panel A-1: Position of template DNA prepared from C. coli strains NCTC 11353, ATCC 33559, ATCC 43488 and ATCC 49941 (C.c1 to C.c4), C. fetus subsp. intestinalis clinical isolates 7305, 7306, 7307, 7308, 7309 and C. fetus subsp. fetus ATCC 27374 (C.f1 to C.f6); C. jejuni strains NCTC 11168, ATCC 33560, ATCC 43433 and ATCC 49943 (C.j1 to C.j4); and C. lari ATCC 35221 (C.l). Panel A-2: Dot blot hybridization of DNA template with the C. coli hsp60 probe prepared from C.c1. Panel A-3: Dot blot hybridization of DNA template with the C. jejuni hsp60 probe prepared from C.j1. Panel A-4: Dot blot hybridization of DNA template with the C. fetus subsp. intestinalis hsp60 probe prepared from C.f1. Panel B-1: Position of template DNA prepared from V. parahaemolyticus strains ATCC 17802, clinical isolates 97-1605 and 455 (V.p1 to V.p3); V. vulnificus strains ATCC 27562 and clinical isolate 92-1751 (V.v1 to V.v2); A. hydrophila strains ATCC 19570 and clinical isolate 7788 (A.h1 to A.h2); and P. shigelloides (P.s.). Panel B-2: Dot blot hybridization of template DNA with the V. parahaemolyticus hsp60 probe prepared from V.p1. Panel B-3: Dot blot hybridization of template DNA with the V. vulnificus hsp60 probe prepared from V.v1.

3

dot blot hybridization of the 600-bp hsp60 amplicons from various enteric bacteria with (A) Campylobacter hsp60 probes, and (B) Vibrio hsp60 probes. Panel A-1: Position of template DNA prepared from C. coli strains NCTC 11353, ATCC 33559, ATCC 43488 and ATCC 49941 (C.c1 to C.c4), C. fetus subsp. intestinalis clinical isolates 7305, 7306, 7307, 7308, 7309 and C. fetus subsp. fetus ATCC 27374 (C.f1 to C.f6); C. jejuni strains NCTC 11168, ATCC 33560, ATCC 43433 and ATCC 49943 (C.j1 to C.j4); and C. lari ATCC 35221 (C.l). Panel A-2: Dot blot hybridization of DNA template with the C. coli hsp60 probe prepared from C.c1. Panel A-3: Dot blot hybridization of DNA template with the C. jejuni hsp60 probe prepared from C.j1. Panel A-4: Dot blot hybridization of DNA template with the C. fetus subsp. intestinalis hsp60 probe prepared from C.f1. Panel B-1: Position of template DNA prepared from V. parahaemolyticus strains ATCC 17802, clinical isolates 97-1605 and 455 (V.p1 to V.p3); V. vulnificus strains ATCC 27562 and clinical isolate 92-1751 (V.v1 to V.v2); A. hydrophila strains ATCC 19570 and clinical isolate 7788 (A.h1 to A.h2); and P. shigelloides (P.s.). Panel B-2: Dot blot hybridization of template DNA with the V. parahaemolyticus hsp60 probe prepared from V.p1. Panel B-3: Dot blot hybridization of template DNA with the V. vulnificus hsp60 probe prepared from V.v1.

To overcome this difficulty, we explored the possibility of using RFLP analysis of the hsp60 PCR amplicons for species identification of some of the more closely related species. Thus, the 600-bp hsp60 amplicons from these enteric species were digested with the indicated restriction enzymes according to the supplier's instructions. As expected from the restriction map (data not shown), RFLP analysis of the 600-bp amplicons of C. coli, C. jejuni and C. fetus subsp. intestinalis following digestion with the enzyme AluI (Gibco), showed that each species gave a distinct restriction pattern (Fig. 4A). This approach is apparently more advantageous than a similar approach using 16S rRNA as target, since more than one enzyme was required to identify different Campylobacter species, while C. jejuni could not be distinguished from C. coli using 16S rRNA [11,12].

4

Restriction fragment length polymorphism (RFLP) of the 600-bp hsp60 amplicons from: (A) Campylobacter species digested with AluI; (B) Enterobacteriaceae species digested with BstUI. The positions of molecular size standards are as shown.

4

Restriction fragment length polymorphism (RFLP) of the 600-bp hsp60 amplicons from: (A) Campylobacter species digested with AluI; (B) Enterobacteriaceae species digested with BstUI. The positions of molecular size standards are as shown.

RFLP analysis of the 600-bp amplicons from E. coli and Shigella species digested with the enzyme BstUI (New England Biolabs, Beverly, MA, USA) showed that all strains shared a common restriction pattern, while all Salmonella strains shared a common but distinct restriction pattern (Fig. 4B). Therefore, by including a single step of restriction digestion, our hsp60 universal primers could be used to identify and differentiate any given single species. However, as shown in Fig. 4B, we also observed that our hsp60 primers could not accurately differentiate E. coli form Shigella, or different species within the genus Salmonella due to the closeness of these species at the genetic level, a finding which was also reported earlier by other groups using the 16S rRNA target [13].

Conventional methods to discriminate between Campylobacter species by hippurate hydrolysis and antibiotic resistance profiles are not always reliable. In addition, due to the relatedness between C. jejuni and C. coli, antibodies and genetic probes against these two species often cross react with each other [14,,,17]. More recently described molecular methods to differentiate between these two species usually require multiple sets of primers or restriction digests, or primers that are only applicable for a particular Campylobacter species [18,,20]. Likewise, molecular diagnosis of other enteric pathogens is often directed towards a particular organism or requires the use of multiplex PCR and multiple probes. We report here the application of hsp60 gene sequences for the broad range detection and identification of different enteric pathogens that cause invasive diarrhea. The use of the 600-bp hsp60 amplicons as DNA probes in dot blot hybridization allows differentiation of some enteric bacteria at the species level (e.g., C. jejuni from C. coli in the genus Campylobacter). With the help of RFLP analysis, this approach also allows identification to the species level within the genera Vibrio and Yersinia. This method could also identify Salmonella species from E. coli and Shigella. Whereas genotypic methods for laboratory detection and identification of enteric pathogens have limitations and concerns regarding issues of sensitivity, specificity, practicality and cost-effectiveness, as well as inability to perform antibiotic susceptibility testing, we believe that our hsp60 approach may be particularly useful for reference laboratories to assist in accurate speciation and identification of clinical isolates that are taxonomically confusing such as species within Campylobacter and Vibrio. In contrast, closely related organisms such as Salmonella, E. coli and Shigella species can readily be identified by conventional phenotypic tests that may be more discriminative than genotypic methods such as hsp60.

In conclusion, our PCR dot blot or PCR RFLP methods utilize a single set of hsp60 universal primers and accurately identify the majority of enteric pathogens associated with invasive diarrhea. This approach may provide a more practical and less demanding alternative for the detection and identification of these enteric bacteria both in the clinical diagnostic laboratory and the reference laboratory.

Acknowledgements

This project was supported by grants from the Vancouver Foundation (Grant BCM97-0003), and the Canadian Bacterial Diseases Network (Project D8). We thank Drs. B.B. Finlay, L. Mutharia, and W. Johnson for providing some of the bacterial strains.

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Author notes

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