- Email: [email protected]
Multiple-locus variable-number tandem repeat analysis (MLVA) for genotyping of Salmonella enterica subspecies enterica serotype Infantis isolated from human sources
Accepted Manuscript Multiple-locus variable-number tandem repeat analysis (MLVA) for genotyping of Salmonella enterica subspecies enterica serotype Infantis isolated from human sources Reza Ranjbar, Mitra Ahmadi, Mojtaba Memariani PII:
S0882-4010(16)30380-1
DOI:
10.1016/j.micpath.2016.10.012
Reference:
YMPAT 1974
To appear in:
Microbial Pathogenesis
Received Date: 11 July 2016 Revised Date:
15 September 2016
Accepted Date: 17 October 2016
Please cite this article as: Ranjbar R, Ahmadi M, Memariani M, Multiple-locus variable-number tandem repeat analysis (MLVA) for genotyping of Salmonella enterica subspecies enterica serotype Infantis isolated from human sources, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Multiple-Locus Variable-number Tandem Repeat Analysis (MLVA) for genotyping of Salmonella enterica subspecies enterica serotype Infantis isolated from human sources
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Reza Ranjbar1, Mitra Ahmadi2, Mojtaba Memariani3*
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Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
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Department of Microbiology, Islamic Azad University, Damghan Branch, Damghan, Iran
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Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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*Corresponding Author: Dr. Mojtaba Memariani
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Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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Email: [email protected]
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Tel/Fax: +989124849859
Running title: MLVA analysis for genotyping S. Infantis
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Abstract
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Salmonella is an important cause of food-borne infection worldwide. Detection of outbreaks caused by
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Salmonella spp. relies on suitable and robust methods for genotyping. Little is known about the genetic
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diversity of the Salmonella enterica subspecies enterica serotype Infantis strains isolated from human
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sources in Iran. In this study, 40 isolates of S. Infantis, which were previously recovered from patients
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with gastroenteritis or diarrhea in Tehran between years 2007 and 2009, were subjected to multiple-locus
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variable-number of tandem repeat (VNTR) analysis (MLVA), pulsed-field gel electrophoresis (PFGE),
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and ERIC-PCR. Using MLVA method, 31 types were identified. The MLVA clustering of the isolates by
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the unweighted pair group method with arithmetic mean (UPGMA) revealed the presence of two major
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clusters. The discriminatory power of MLVA was superior to that of PFGE and ERIC-PCR. Overall, our
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data showed that MLVA assay could effectively differentiate closely related strains. It is technically
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simple and inexpensive to perform. Furthermore, MLVA can be used as a helpful method for
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epidemiological investigations.
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Salmonella
enterica
serotype
Infantis,
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Keywords:
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MLVA,
ERIC-PCR,
PFGE,
genotyping
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1. Introduction Salmonellosis is one of the most widespread foodborne zoonotic diseases in developing countries,
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accounts for an estimated 2.8 billion cases of diarrhea annually [1]. However, many cases of
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salmonellosis are significantly underreported; hence, it is very cumbersome to precisely determine the
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actual public health burden of Salmonella globally [2]. Salmonella nomenclature is complex, and
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microbiologists use different systems to refer to and communicate about this genus [3]. To date, over
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2,500 serotypes of Salmonella have been described. In spite of this, only less than 100 serotypes account
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for most human infections. Globally, serotype Infantis was reported among the top 5 common human
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serotypes along with serotypes Enteritidis, Typhimurium, Montevideo, and Typhi [4]. Serotype Infantis
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(S. Infantis) is a host-nonspecific serotype which can infect both humans and animals. Additionally,
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infections caused by this serotype are mainly observed in children, but also adults, sometimes with
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septicaemia and lethal outcome [5]. In Iran, S. Infantis has been increasingly recorded in humans as well
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as foods over the past years [6-10].
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Many epidemiological problems, including detection and interpretation of outbreaks, by tracing
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transmission routes and identifying infection sources, can be addressed by genotyping methods. Several
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molecular-based typing methods such as pulsed-field gel electrophoresis (PFGE) [11-13], plasmid
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profiling [11, 12], ribotyping [14, 15], enterobacterial repetitive intergenic consensus sequence-based
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PCR (ERIC-PCR) [12, 16], and multilocus sequence typing (MLST) [16] have been applied for
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epidemiological investigations of S. Infantis infections worldwide. Of these methods, PFGE is still
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popular and considered as the ‘gold standard’ fingerprinting method for S. Infantis. However, this method
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has certain drawbacks including the need for highly trained staff, expensive equipments, and reduced
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comparability of results between different laboratories [17].
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During the last decade, advances in the Polymerase Chain Reaction (PCR) technology has lead to the
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development of the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA). The
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method targets multiple VNTR loci and relies on the detection of different copy numbers inside each 2
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locus. MLVA genotyping has been successfully employed an effective tool for investigating strains that
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are epidemiologically related or unrelated in specific outbreaks [18, 19]. Whilst S. Infantis is becoming
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increasingly important as an etiologic agent of salmonellosis in Iran [6, 8, 9], there is a paucity of data on
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the genetic diversity of the local strains. In this study, we present an MLVA assay for genotyping of S.
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Infantis strains of human origin. Furthermore, typing results from MLVA are also compared with those of
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PFGE and ERIC-PCR.
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2. Material & Methods
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2.1. Bacterial strains and DNA preparation
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A total of 40 S. Infantis isolates (33 from patients less than 12 years of age) were incorporated in the
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study. All of the isolates were previously obtained from patients with gastroenteritis or diarrhea in
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Tehran, Iran between years 2007 and 2009. Identification of S. Infantis [6,7:r:1,5] were conducted
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according to routine biochemical and serological tests [5]. All of the strains were serotyped by
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agglutination with “O” and “H” antigen specific sera (Mast, England). The isolates were revived from
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lyophilized storage (skim milk with 25% glycerol) and grown on blood agar before DNA extraction. A
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single colony was removed from the plate, suspended in 200 µl of sterile water and boiled for 15 min.
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After centrifugation at 4,000 g for 10 min, the supernatant was transferred into a new tube for subsequent
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PCR analysis.
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2.2. MLVA assay
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In order to perform MLVA assay, 8 different VNTR loci were chosen according to previous studies
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[18, 20-22]. Seven of these loci (SE4, SE6, SE7, SE8, SE10, SENTR3, and ENTR6) were previously
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used for typing of S. Enteritidis [18]. The other locus (SENTR2) was first described in genome of S.
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Typhimurium [20]. The locus name, repeat size, primer sequences, and PCR annealing temperatures were
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shown in Table 1. For each locus, PCR was carried out in 25 µl volume including 1X PCR buffer (50
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mmol/L KCL, 10 mmol/L Tris, pH=9), 2.5 mmol/L MgCl2, 0.2 mmol/L of each primer with 1 U of 3
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TaqDNA polymerase (CinnaGen Co., Iran), and 4 µl of the crude DNA extract. The PCR products were
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run on 1.5% (w/v) agarose gels, stained with ethidium bromide (Sigma-Aldrich, Steinheim, Germany),
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and visualized under ultraviolet transillumination. The number of repeats can be easily deduced from the
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PCR product sizes by manual reading. The product sizes were converted into repeat numbers based on
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formula as described previously [23]. A dendrogram of genetic relationships was also generated using the
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unweighted pair group method with arithmetic averages (UPGMA) based on allelic profiles. The
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minimum spanning tree (MST) was also constructed with a categorical coefficient based on allelic
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profiles of the S. Infantis isolates. Furthermore, Hunter-Gaston discriminatory index (HGDI) was
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calculated for each VNTR locus as described previously [23].
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Table 1: Characteristics of VNTR loci used for MLVA genotyping VNTR locus
Coding region
Primer Sequences (5’ to 3’)
Tandem repeat size in base pairs
Optimal annealing temperature (°C)
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58
93
58
175
58
117
58
33
60
61
58
87
58
45
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5’-CAC TGG ACG ATC TGG ATT TCT C SENTR2 (STTR7)
ftsK
Reference
[20]
5’-GTC GCC GTT ACG CAT CAA C
Non identified
ENTR6
5’-CTA AAC AAG CCG CTC ATC CG
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STM1467 homologue
SENTR3
[18]
5’-ACA ACC TGC TGC TGT GCT G 5’TGT GGG GTA AGG ATA CGG GG
[21]
5’GCC AAA GGG AGC AGA CTG TAA AT 5’-ACT TTA GAA AAT GCG TTG AC
valU
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SE-4
[22, 24]
5’-CCC CTA AGC CCG ATA ATG 5’-CCC CTA AGC CCG ATA ATG
SE-6 (STTR3)
bigA
[22]
SE-7
SE-8
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5’-GCC GTT GCT GAA GGT 5’-GAT AAT GCT GCC GTT GGT AA
ygbF
None identified
[22]
5’- ACT GCG TTT GGT TTC TTT TCT 5’-TTG CCG CAT AGC AGA AGT
[22]
5’-GCC TGA ACA CGC TTT TTA ATA GGC T 5’-GCT GAG ATC GCC AAG CAG ATC GTC G
SE-10 (SENTR1, STTR1)
tolA 5’-ACT GGC GCA ACA GCA GCA GCA ACA G
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[20, 22]
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2. 3. PFGE PFGE was performed for 33 S. Infantis isolates according to the criteria suggested by Tenover et al
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[25] using a CHEF Mapper XA apparatus (Bio-Rad Laboratories, Hercules, CA). Restriction analysis of
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chromosomal DNA with XbaI (New England BioLabs, Beverly, MA) was carried out, and separation of
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DNA was performed by using 1% pulsed-field gel agarose (SeaKem Gold agarose; Cambrex Bio Science,
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Rockland, ME). DNA banding patterns were visually compared and interpreted as described elsewhere.
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Isolates having patterns which differed by more than three bands were considered unique and have been
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assigned a different letter. Isolates had a PFGE pattern which includes a letter and number, represent
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highly related strains which differed by less than three bands (subtypes) [8, 12].
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2. 4. ERIC-PCR
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Clonal relationships of S. Infantis isolates were also determined by ERIC-PCR using primers ERIC1
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(5’-ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC-2 (5’- AAGTAAGTGACTGGGGTGAGCG-3’),
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as described previously [26]. Cycling conditions comprised an initial denaturation (94 °C for 4 minutes),
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followed by 35 cycles of denaturation (94 °C for 1 minute), annealing (52°C for 1 minute), extension
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(65°C for 8 minutes), and a final extension at 65°C for 15 minutes. The PCR products were separated by
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electrophoresis at 50V for 2 hours on 1.5% (w/v) agarose gels, stained with ethidium bromide, and
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visualized using an ultraviolet transilluminator. For convenience, each unique pattern was assigned a
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letter designation.
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3. Results
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MLVA based on 8 VNTR loci was performed to characterize the S. Infantis isolates. Overall, the 40
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isolates were discriminated into 31 distinct MLVA profiles (genotypes). The most common MLVA
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profile was 8-5-2-3-7-8-1-6 accounted for 20% (n=8) of isolates. The genetic diversity based on HGDI
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for 8 VNTR loci ranged from 0.146 to 0.514. VNTR locus SE8 was identified to be the most polymorphic
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loci (HGDI=0.514) while locus SENTR2 had the lowest diversity index (HGDI=0.146). VNTR locus SE6 5
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had the highest number of different repeats (n=5) whereas SE8 and SE10 had the lowest number of
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different repeats (n=3) (Table 2). The UPGMA dendrogram based on VNTR alleles with detailed
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information is shown in Figure 1. The clustering of MLVA profiles revealed the presence of two major
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clusters.
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As for PFGE, a total of 16 distinct patterns were identified among 33 tested isolates (Figure 2). The
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dominant PFGE profile was pattern A, which was observed in 14 isolates (42.4%). Regarding ERIC-PCR,
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among 40 isolates, 8 distinct patterns (A to H) were obtained (Figure 3). Pattern A was the most common,
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comprising 19 isolates (47.5 %), whereas F, G, and H are the least frequently observed types (one isolate
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for each type). MLVA results were also compared to those of PFGE and ERIC-PCR. MLVA Cluster A
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consisted mainly of either PFGE profile A and most of its related subtypes (i.e. A1, A3, A7, A8, A9, and
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A10) or ERIC-PCR type A, whereas MLVA cluster B is more diverse, largely composed of ERIC-PCR
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type B and other PFGE profiles. Distribution of PFGE profiles and ERIC-PCR types among MLVA types
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is also displayed by MST (Figure 4).
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Table 2: Diversity indices, number of alleles, and typeability have shown for each VNTR locus. Hunter-Gatson diversity of index
95% Confidence interval (CI)
No. of alleles
Typeabilitya (%)
0.146
0.000 - 0.293
4
97.5%
0.419
0.248 - 0.590
4
97.5%
0.455
0.285 - 0.626
4
92.5%
0.383
0.212 - 0.555
4
95%
SE-6 (STTR3)
0.395
0.211 - 0.579
5
97.5%
SE-7
0.426
0.248 - 0.603
4
90%
0.514
0.407 - 0.621
3
95%
0.465
0.348 - 0.583
3
97.5%
VNTR locus SENTR2 (STTR7)
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SE-8
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SE-4
SE-10 (SENTR1, STTR1)
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The ability of each VNTR locus to type the isolates was measured as follows: Number of isolates amplified in each VNTR locus/40.
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Figure 1. The UPGMA dendrogram of S. Infantis isolates based on VNTR profile. Isolate code, visual pulsotype, and ERIC-PCR type are also given for all isolates. ND, not determined.
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Figure 2. Example of PFGE gel of XbaI macrorestriction fragments of S. Infantis isolates. Lanes 1 to 11: strains S143 (profile A), S147 (profile B), S112 (profile C1), S104 (profile C), S97 (profile A10), S147 (profile B), S34 (profile A9), S43 (profile A), S131 (profile B2), S64 (profile A8), and S13 (profile A1); MW: PFGE marker.
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Figure 3. Unique patterns of ERIC-PCR types among the S. Infantis isolates.
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Figure 4. Minimum Spanning Tree (MST) of S. Infantis isolates. Each circle represents a unique genotype (MLVA profile). The number of loci which differ between two MLVA profiles is indicated on the lines connecting the MLVA profiles. The letter and color of the circles correspond to PFGE profiles (A) and ERIC-PCR types (B).
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4. Discussion Detection of food-borne outbreaks caused by Salmonella spp. relies on suitable and robust methods for
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typing [22]. Without a discriminatory typing method, it would be difficult to precisely identify the source
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and route of transmission of infection. Therefore, it is nearly impossible to implement prevention
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strategies appropriately. This is particularly important for highly clonal bacterial populations such as S.
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Enteritidis, S. infantis, and S. Typhimurium where limited heterogeneity is observed between isolates
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[18]. Since 1990s, PFGE has been successfully used as a method of choice for identifying and
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investigating Salmonellosis outbreaks, although ribotyping may provide higher discriminatory power than
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dose PFGE with a single enzyme [17, 22]. Recently, MLVA has been introduced as an effective
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molecular typing method for epidemiological investigations. It is based on counting the number of VNTR
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repeats and has advantages such as rapidity, ease, and convenience of interpretation [17, 18].
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In this study, we successfully applied an MLVA method with 8 VNTR loci to analyze S. Infantis
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isolates. Over the past years, several MLVA protocols for genotyping of S. Enteritidis [18, 22, 27], S.
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Typhimurium [20, 28, 29], S. Typhi [30, 31], S. Paratyphi [32], S. Infantis [33], S. Newport [29, 34], and
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S. Gallinarium [35] have been published. Due to slight differences in genome organization, the
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occurrence and variability of VNTR loci can be divergent between S. enterica serovars. In this regard,
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Ross et al. genotyped 76 epidemiologically unrelated S. Infantis by PFGE, MLVA, and multiple
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amplification of phage loci typing (MAPLT). The VNTR loci used by Ross et al. for S. Infantis were
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quite different from those used in our study. They demonstrated that MLVA with 13 VNTR loci had
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discriminatory power inferior to those of PFGE and MAPLT [33]. Hopkins et al. (2011) proposed an
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MLVA scheme for genotyping of S. Enteritidis based on 5 loci (SENTR7, SENTR5, SENTR6, SENTR4,
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and SE3). They showed that this method was capable of subdividing isolates within a phage type [18].
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Similarly, Malrony et al. (2008) used 9 VNTR loci (SENTR1-7, SE3, and SE-7) to differentiate 240 S.
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Enteritidis isolates belonging to 23 different phage types [27]. In another study conducted by Boxrud et
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al., 38 unique MLVA types were shown among 145 S. Enteritidis isolates from different sources using 9
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VNTR loci somewhat similar to those used in Malrony et al. study. They also found that diversity indices
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for MLVA were higher than those for PFGE and phage typing, revealing that MLVA provided a greater
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number of different subtypes [22]. According to a recent study conducted in our country, a 7-locus
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MLVA typing scheme was compared to MLST to differentiate chicken-derived S. Enteritidis.
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Interestingly, MLST did not represent any nucleotide differences among the isolates whereas 6 genotypes
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were shown by MLVA [36]. In general, most of these studies suggested that combination of MLVA with
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other typing methods such PFGE, phage typing, etc may produce more information about the clonality of
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Salmonella enterica serotypes. For this reason, we therefore used PFGE and ERIC-PCR in addition to
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MLVA. PFGE was not performed on all isolates because seven of them did not survive before PFGE
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analysis. Nevertheless, the discriminatory power of MLVA (HGDI=0.962) was still superior to that of
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PFGE (HGDI=0.818) and ERIC-PCR (HGDI=0.719). In case of PFGE, HGDI was only calculated for 33
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isolates. It has been observed that strains of the same PFGE profile can exhibit different MLVA types.
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For instance, strains with PFGE profile A showed 7 distinct MLVA types. Conversely, some isolates with
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different PFGE profiles had the same MLVA types. For instance, PFGE profiles A6 and A5 showed
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identical MLVA types. Besides PFGE, ERIC-PCR was also performed to evaluate S. Infantis strains.
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Since discovery of ERIC sequences in genome of Enterobacteriaceae, ERIC-PCR has been used for
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genotyping of Salmonella spp. including S. Infantis. Unfortunately, it does not always show complete
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picture of genetic relatedness. Identical bands are based on their size, and not necessarily their genetic
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content [12, 16, 37]. In our study, we also found that discriminatory power of ERIC-PCR was inferior to
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that of PFGE and MLVA.
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For our method, we chose larger VNTRs since the size of alleles can be estimated simply by
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comparing their sizes on a gel electrophoresis. Additionally, most of these MLVA schemes require a high
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precision of DNA length measurement, such as microcapillary electrophoresis or DNA sequencing [18,
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22, 27]. It is also noteworthy that developing countries have limited accessibility to such equipments and
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their expenditures would be a major obstacle for many laboratories [23]. Fortunately, our MLVA protocol 11
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can be performed in a laboratory with simple equipments. According to Centers of Disease Control and
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Prevention (CDC) (http://www.cdc.gov/pulsenet/pathogens/mlva.html), the advantage of MLVA, as a
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complementary technique to PFGE, is permitting an epidemiologist to see more detailed variances
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between the strains which have the same PFGE patterns. However, lack of standardization of the
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methodology and interpretive criteria is problematic and hinders comparison of data between laboratories.
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In conclusion, the present study provided valuable insights into the genetic heterogeneity of S. Infantis
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isolates in Tehran, Iran. MLVA genotypes of the Iranian isolates revealed that our S. Infantis isolates
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were derived from a limited number of clones that undergo minor genetic changes in course of time.
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However, a larger sample size from a variety of regions will be needed to determine which VNTR loci
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provide sufficient resolution for outbreaks and disease surveillance. Furthermore, we found that the
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combination of MLVA with PFGE can lead to an even higher typing discrimination for S. Infantis. In
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addition, due to monomorphic nature of Salmonella enterica serotypes [17], a larger set of VNTR
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markers would be more favorable to obtain a clearer distinction between isolates.
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Acknowledgements
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The authors are much grateful to the laboratory staff of Molecular Biology Research Center,
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Baqiyatallah University of Medical Sciences, Tehran, Iran. Furthermore, the authors are thankful to Dr.
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Hamed Memariani (Pasteur Institue of Iran, Tehran) for his technical assistance.
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Conflict of interest
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The authors confirm that this article content has no conflict of interest.
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Highlights: •
MLVA genotypes of the Iranian S. Infantis isolates revealed that they were derived from a limited number of clones. Discriminatory power of MLVA was superior to that of PFGE and ERIC-PCR.
•
Combination of MLVA with PFGE can lead to a higher typing discrimination for S. Infantis.
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S0882-4010(16)30380-1
DOI:
10.1016/j.micpath.2016.10.012
Reference:
YMPAT 1974
To appear in:
Microbial Pathogenesis
Received Date: 11 July 2016 Revised Date:
15 September 2016
Accepted Date: 17 October 2016
Please cite this article as: Ranjbar R, Ahmadi M, Memariani M, Multiple-locus variable-number tandem repeat analysis (MLVA) for genotyping of Salmonella enterica subspecies enterica serotype Infantis isolated from human sources, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Multiple-Locus Variable-number Tandem Repeat Analysis (MLVA) for genotyping of Salmonella enterica subspecies enterica serotype Infantis isolated from human sources
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Reza Ranjbar1, Mitra Ahmadi2, Mojtaba Memariani3*
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1
Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
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Department of Microbiology, Islamic Azad University, Damghan Branch, Damghan, Iran
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Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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*Corresponding Author: Dr. Mojtaba Memariani
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Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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Email: [email protected]
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Tel/Fax: +989124849859
Running title: MLVA analysis for genotyping S. Infantis
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Abstract
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Salmonella is an important cause of food-borne infection worldwide. Detection of outbreaks caused by
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Salmonella spp. relies on suitable and robust methods for genotyping. Little is known about the genetic
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diversity of the Salmonella enterica subspecies enterica serotype Infantis strains isolated from human
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sources in Iran. In this study, 40 isolates of S. Infantis, which were previously recovered from patients
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with gastroenteritis or diarrhea in Tehran between years 2007 and 2009, were subjected to multiple-locus
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variable-number of tandem repeat (VNTR) analysis (MLVA), pulsed-field gel electrophoresis (PFGE),
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and ERIC-PCR. Using MLVA method, 31 types were identified. The MLVA clustering of the isolates by
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the unweighted pair group method with arithmetic mean (UPGMA) revealed the presence of two major
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clusters. The discriminatory power of MLVA was superior to that of PFGE and ERIC-PCR. Overall, our
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data showed that MLVA assay could effectively differentiate closely related strains. It is technically
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simple and inexpensive to perform. Furthermore, MLVA can be used as a helpful method for
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epidemiological investigations.
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Salmonella
enterica
serotype
Infantis,
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Keywords:
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MLVA,
ERIC-PCR,
PFGE,
genotyping
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1. Introduction Salmonellosis is one of the most widespread foodborne zoonotic diseases in developing countries,
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accounts for an estimated 2.8 billion cases of diarrhea annually [1]. However, many cases of
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salmonellosis are significantly underreported; hence, it is very cumbersome to precisely determine the
36
actual public health burden of Salmonella globally [2]. Salmonella nomenclature is complex, and
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microbiologists use different systems to refer to and communicate about this genus [3]. To date, over
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2,500 serotypes of Salmonella have been described. In spite of this, only less than 100 serotypes account
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for most human infections. Globally, serotype Infantis was reported among the top 5 common human
40
serotypes along with serotypes Enteritidis, Typhimurium, Montevideo, and Typhi [4]. Serotype Infantis
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(S. Infantis) is a host-nonspecific serotype which can infect both humans and animals. Additionally,
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infections caused by this serotype are mainly observed in children, but also adults, sometimes with
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septicaemia and lethal outcome [5]. In Iran, S. Infantis has been increasingly recorded in humans as well
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as foods over the past years [6-10].
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Many epidemiological problems, including detection and interpretation of outbreaks, by tracing
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transmission routes and identifying infection sources, can be addressed by genotyping methods. Several
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molecular-based typing methods such as pulsed-field gel electrophoresis (PFGE) [11-13], plasmid
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profiling [11, 12], ribotyping [14, 15], enterobacterial repetitive intergenic consensus sequence-based
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PCR (ERIC-PCR) [12, 16], and multilocus sequence typing (MLST) [16] have been applied for
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epidemiological investigations of S. Infantis infections worldwide. Of these methods, PFGE is still
51
popular and considered as the ‘gold standard’ fingerprinting method for S. Infantis. However, this method
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has certain drawbacks including the need for highly trained staff, expensive equipments, and reduced
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comparability of results between different laboratories [17].
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During the last decade, advances in the Polymerase Chain Reaction (PCR) technology has lead to the
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development of the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA). The
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method targets multiple VNTR loci and relies on the detection of different copy numbers inside each 2
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locus. MLVA genotyping has been successfully employed an effective tool for investigating strains that
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are epidemiologically related or unrelated in specific outbreaks [18, 19]. Whilst S. Infantis is becoming
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increasingly important as an etiologic agent of salmonellosis in Iran [6, 8, 9], there is a paucity of data on
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the genetic diversity of the local strains. In this study, we present an MLVA assay for genotyping of S.
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Infantis strains of human origin. Furthermore, typing results from MLVA are also compared with those of
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PFGE and ERIC-PCR.
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2. Material & Methods
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2.1. Bacterial strains and DNA preparation
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A total of 40 S. Infantis isolates (33 from patients less than 12 years of age) were incorporated in the
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study. All of the isolates were previously obtained from patients with gastroenteritis or diarrhea in
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Tehran, Iran between years 2007 and 2009. Identification of S. Infantis [6,7:r:1,5] were conducted
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according to routine biochemical and serological tests [5]. All of the strains were serotyped by
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agglutination with “O” and “H” antigen specific sera (Mast, England). The isolates were revived from
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lyophilized storage (skim milk with 25% glycerol) and grown on blood agar before DNA extraction. A
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single colony was removed from the plate, suspended in 200 µl of sterile water and boiled for 15 min.
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After centrifugation at 4,000 g for 10 min, the supernatant was transferred into a new tube for subsequent
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PCR analysis.
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2.2. MLVA assay
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In order to perform MLVA assay, 8 different VNTR loci were chosen according to previous studies
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[18, 20-22]. Seven of these loci (SE4, SE6, SE7, SE8, SE10, SENTR3, and ENTR6) were previously
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used for typing of S. Enteritidis [18]. The other locus (SENTR2) was first described in genome of S.
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Typhimurium [20]. The locus name, repeat size, primer sequences, and PCR annealing temperatures were
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shown in Table 1. For each locus, PCR was carried out in 25 µl volume including 1X PCR buffer (50
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mmol/L KCL, 10 mmol/L Tris, pH=9), 2.5 mmol/L MgCl2, 0.2 mmol/L of each primer with 1 U of 3
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TaqDNA polymerase (CinnaGen Co., Iran), and 4 µl of the crude DNA extract. The PCR products were
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run on 1.5% (w/v) agarose gels, stained with ethidium bromide (Sigma-Aldrich, Steinheim, Germany),
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and visualized under ultraviolet transillumination. The number of repeats can be easily deduced from the
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PCR product sizes by manual reading. The product sizes were converted into repeat numbers based on
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formula as described previously [23]. A dendrogram of genetic relationships was also generated using the
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unweighted pair group method with arithmetic averages (UPGMA) based on allelic profiles. The
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minimum spanning tree (MST) was also constructed with a categorical coefficient based on allelic
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profiles of the S. Infantis isolates. Furthermore, Hunter-Gaston discriminatory index (HGDI) was
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calculated for each VNTR locus as described previously [23].
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Table 1: Characteristics of VNTR loci used for MLVA genotyping VNTR locus
Coding region
Primer Sequences (5’ to 3’)
Tandem repeat size in base pairs
Optimal annealing temperature (°C)
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58
93
58
175
58
117
58
33
60
61
58
87
58
45
60
5’-CAC TGG ACG ATC TGG ATT TCT C SENTR2 (STTR7)
ftsK
Reference
[20]
5’-GTC GCC GTT ACG CAT CAA C
Non identified
ENTR6
5’-CTA AAC AAG CCG CTC ATC CG
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STM1467 homologue
SENTR3
[18]
5’-ACA ACC TGC TGC TGT GCT G 5’TGT GGG GTA AGG ATA CGG GG
[21]
5’GCC AAA GGG AGC AGA CTG TAA AT 5’-ACT TTA GAA AAT GCG TTG AC
valU
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SE-4
[22, 24]
5’-CCC CTA AGC CCG ATA ATG 5’-CCC CTA AGC CCG ATA ATG
SE-6 (STTR3)
bigA
[22]
SE-7
SE-8
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5’-GCC GTT GCT GAA GGT 5’-GAT AAT GCT GCC GTT GGT AA
ygbF
None identified
[22]
5’- ACT GCG TTT GGT TTC TTT TCT 5’-TTG CCG CAT AGC AGA AGT
[22]
5’-GCC TGA ACA CGC TTT TTA ATA GGC T 5’-GCT GAG ATC GCC AAG CAG ATC GTC G
SE-10 (SENTR1, STTR1)
tolA 5’-ACT GGC GCA ACA GCA GCA GCA ACA G
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[20, 22]
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2. 3. PFGE PFGE was performed for 33 S. Infantis isolates according to the criteria suggested by Tenover et al
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[25] using a CHEF Mapper XA apparatus (Bio-Rad Laboratories, Hercules, CA). Restriction analysis of
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chromosomal DNA with XbaI (New England BioLabs, Beverly, MA) was carried out, and separation of
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DNA was performed by using 1% pulsed-field gel agarose (SeaKem Gold agarose; Cambrex Bio Science,
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Rockland, ME). DNA banding patterns were visually compared and interpreted as described elsewhere.
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Isolates having patterns which differed by more than three bands were considered unique and have been
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assigned a different letter. Isolates had a PFGE pattern which includes a letter and number, represent
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highly related strains which differed by less than three bands (subtypes) [8, 12].
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2. 4. ERIC-PCR
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Clonal relationships of S. Infantis isolates were also determined by ERIC-PCR using primers ERIC1
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(5’-ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC-2 (5’- AAGTAAGTGACTGGGGTGAGCG-3’),
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as described previously [26]. Cycling conditions comprised an initial denaturation (94 °C for 4 minutes),
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followed by 35 cycles of denaturation (94 °C for 1 minute), annealing (52°C for 1 minute), extension
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(65°C for 8 minutes), and a final extension at 65°C for 15 minutes. The PCR products were separated by
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electrophoresis at 50V for 2 hours on 1.5% (w/v) agarose gels, stained with ethidium bromide, and
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visualized using an ultraviolet transilluminator. For convenience, each unique pattern was assigned a
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letter designation.
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3. Results
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MLVA based on 8 VNTR loci was performed to characterize the S. Infantis isolates. Overall, the 40
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isolates were discriminated into 31 distinct MLVA profiles (genotypes). The most common MLVA
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profile was 8-5-2-3-7-8-1-6 accounted for 20% (n=8) of isolates. The genetic diversity based on HGDI
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for 8 VNTR loci ranged from 0.146 to 0.514. VNTR locus SE8 was identified to be the most polymorphic
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loci (HGDI=0.514) while locus SENTR2 had the lowest diversity index (HGDI=0.146). VNTR locus SE6 5
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had the highest number of different repeats (n=5) whereas SE8 and SE10 had the lowest number of
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different repeats (n=3) (Table 2). The UPGMA dendrogram based on VNTR alleles with detailed
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information is shown in Figure 1. The clustering of MLVA profiles revealed the presence of two major
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clusters.
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As for PFGE, a total of 16 distinct patterns were identified among 33 tested isolates (Figure 2). The
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dominant PFGE profile was pattern A, which was observed in 14 isolates (42.4%). Regarding ERIC-PCR,
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among 40 isolates, 8 distinct patterns (A to H) were obtained (Figure 3). Pattern A was the most common,
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comprising 19 isolates (47.5 %), whereas F, G, and H are the least frequently observed types (one isolate
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for each type). MLVA results were also compared to those of PFGE and ERIC-PCR. MLVA Cluster A
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consisted mainly of either PFGE profile A and most of its related subtypes (i.e. A1, A3, A7, A8, A9, and
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A10) or ERIC-PCR type A, whereas MLVA cluster B is more diverse, largely composed of ERIC-PCR
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type B and other PFGE profiles. Distribution of PFGE profiles and ERIC-PCR types among MLVA types
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is also displayed by MST (Figure 4).
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Table 2: Diversity indices, number of alleles, and typeability have shown for each VNTR locus. Hunter-Gatson diversity of index
95% Confidence interval (CI)
No. of alleles
Typeabilitya (%)
0.146
0.000 - 0.293
4
97.5%
0.419
0.248 - 0.590
4
97.5%
0.455
0.285 - 0.626
4
92.5%
0.383
0.212 - 0.555
4
95%
SE-6 (STTR3)
0.395
0.211 - 0.579
5
97.5%
SE-7
0.426
0.248 - 0.603
4
90%
0.514
0.407 - 0.621
3
95%
0.465
0.348 - 0.583
3
97.5%
VNTR locus SENTR2 (STTR7)
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SE-8
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SE-4
SE-10 (SENTR1, STTR1)
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The ability of each VNTR locus to type the isolates was measured as follows: Number of isolates amplified in each VNTR locus/40.
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Figure 1. The UPGMA dendrogram of S. Infantis isolates based on VNTR profile. Isolate code, visual pulsotype, and ERIC-PCR type are also given for all isolates. ND, not determined.
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Figure 2. Example of PFGE gel of XbaI macrorestriction fragments of S. Infantis isolates. Lanes 1 to 11: strains S143 (profile A), S147 (profile B), S112 (profile C1), S104 (profile C), S97 (profile A10), S147 (profile B), S34 (profile A9), S43 (profile A), S131 (profile B2), S64 (profile A8), and S13 (profile A1); MW: PFGE marker.
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Figure 3. Unique patterns of ERIC-PCR types among the S. Infantis isolates.
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Figure 4. Minimum Spanning Tree (MST) of S. Infantis isolates. Each circle represents a unique genotype (MLVA profile). The number of loci which differ between two MLVA profiles is indicated on the lines connecting the MLVA profiles. The letter and color of the circles correspond to PFGE profiles (A) and ERIC-PCR types (B).
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4. Discussion Detection of food-borne outbreaks caused by Salmonella spp. relies on suitable and robust methods for
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typing [22]. Without a discriminatory typing method, it would be difficult to precisely identify the source
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and route of transmission of infection. Therefore, it is nearly impossible to implement prevention
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strategies appropriately. This is particularly important for highly clonal bacterial populations such as S.
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Enteritidis, S. infantis, and S. Typhimurium where limited heterogeneity is observed between isolates
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[18]. Since 1990s, PFGE has been successfully used as a method of choice for identifying and
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investigating Salmonellosis outbreaks, although ribotyping may provide higher discriminatory power than
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dose PFGE with a single enzyme [17, 22]. Recently, MLVA has been introduced as an effective
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molecular typing method for epidemiological investigations. It is based on counting the number of VNTR
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repeats and has advantages such as rapidity, ease, and convenience of interpretation [17, 18].
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In this study, we successfully applied an MLVA method with 8 VNTR loci to analyze S. Infantis
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isolates. Over the past years, several MLVA protocols for genotyping of S. Enteritidis [18, 22, 27], S.
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Typhimurium [20, 28, 29], S. Typhi [30, 31], S. Paratyphi [32], S. Infantis [33], S. Newport [29, 34], and
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S. Gallinarium [35] have been published. Due to slight differences in genome organization, the
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occurrence and variability of VNTR loci can be divergent between S. enterica serovars. In this regard,
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Ross et al. genotyped 76 epidemiologically unrelated S. Infantis by PFGE, MLVA, and multiple
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amplification of phage loci typing (MAPLT). The VNTR loci used by Ross et al. for S. Infantis were
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quite different from those used in our study. They demonstrated that MLVA with 13 VNTR loci had
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discriminatory power inferior to those of PFGE and MAPLT [33]. Hopkins et al. (2011) proposed an
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MLVA scheme for genotyping of S. Enteritidis based on 5 loci (SENTR7, SENTR5, SENTR6, SENTR4,
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and SE3). They showed that this method was capable of subdividing isolates within a phage type [18].
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Similarly, Malrony et al. (2008) used 9 VNTR loci (SENTR1-7, SE3, and SE-7) to differentiate 240 S.
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Enteritidis isolates belonging to 23 different phage types [27]. In another study conducted by Boxrud et
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al., 38 unique MLVA types were shown among 145 S. Enteritidis isolates from different sources using 9
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VNTR loci somewhat similar to those used in Malrony et al. study. They also found that diversity indices
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for MLVA were higher than those for PFGE and phage typing, revealing that MLVA provided a greater
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number of different subtypes [22]. According to a recent study conducted in our country, a 7-locus
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MLVA typing scheme was compared to MLST to differentiate chicken-derived S. Enteritidis.
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Interestingly, MLST did not represent any nucleotide differences among the isolates whereas 6 genotypes
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were shown by MLVA [36]. In general, most of these studies suggested that combination of MLVA with
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other typing methods such PFGE, phage typing, etc may produce more information about the clonality of
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Salmonella enterica serotypes. For this reason, we therefore used PFGE and ERIC-PCR in addition to
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MLVA. PFGE was not performed on all isolates because seven of them did not survive before PFGE
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analysis. Nevertheless, the discriminatory power of MLVA (HGDI=0.962) was still superior to that of
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PFGE (HGDI=0.818) and ERIC-PCR (HGDI=0.719). In case of PFGE, HGDI was only calculated for 33
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isolates. It has been observed that strains of the same PFGE profile can exhibit different MLVA types.
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For instance, strains with PFGE profile A showed 7 distinct MLVA types. Conversely, some isolates with
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different PFGE profiles had the same MLVA types. For instance, PFGE profiles A6 and A5 showed
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identical MLVA types. Besides PFGE, ERIC-PCR was also performed to evaluate S. Infantis strains.
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Since discovery of ERIC sequences in genome of Enterobacteriaceae, ERIC-PCR has been used for
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genotyping of Salmonella spp. including S. Infantis. Unfortunately, it does not always show complete
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picture of genetic relatedness. Identical bands are based on their size, and not necessarily their genetic
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content [12, 16, 37]. In our study, we also found that discriminatory power of ERIC-PCR was inferior to
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that of PFGE and MLVA.
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For our method, we chose larger VNTRs since the size of alleles can be estimated simply by
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comparing their sizes on a gel electrophoresis. Additionally, most of these MLVA schemes require a high
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precision of DNA length measurement, such as microcapillary electrophoresis or DNA sequencing [18,
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22, 27]. It is also noteworthy that developing countries have limited accessibility to such equipments and
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their expenditures would be a major obstacle for many laboratories [23]. Fortunately, our MLVA protocol 11
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can be performed in a laboratory with simple equipments. According to Centers of Disease Control and
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Prevention (CDC) (http://www.cdc.gov/pulsenet/pathogens/mlva.html), the advantage of MLVA, as a
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complementary technique to PFGE, is permitting an epidemiologist to see more detailed variances
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between the strains which have the same PFGE patterns. However, lack of standardization of the
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methodology and interpretive criteria is problematic and hinders comparison of data between laboratories.
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217 5. Conclusion
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In conclusion, the present study provided valuable insights into the genetic heterogeneity of S. Infantis
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isolates in Tehran, Iran. MLVA genotypes of the Iranian isolates revealed that our S. Infantis isolates
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were derived from a limited number of clones that undergo minor genetic changes in course of time.
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However, a larger sample size from a variety of regions will be needed to determine which VNTR loci
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provide sufficient resolution for outbreaks and disease surveillance. Furthermore, we found that the
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combination of MLVA with PFGE can lead to an even higher typing discrimination for S. Infantis. In
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addition, due to monomorphic nature of Salmonella enterica serotypes [17], a larger set of VNTR
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markers would be more favorable to obtain a clearer distinction between isolates.
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Acknowledgements
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The authors are much grateful to the laboratory staff of Molecular Biology Research Center,
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Baqiyatallah University of Medical Sciences, Tehran, Iran. Furthermore, the authors are thankful to Dr.
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Hamed Memariani (Pasteur Institue of Iran, Tehran) for his technical assistance.
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Conflict of interest
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The authors confirm that this article content has no conflict of interest.
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Highlights: •
MLVA genotypes of the Iranian S. Infantis isolates revealed that they were derived from a limited number of clones. Discriminatory power of MLVA was superior to that of PFGE and ERIC-PCR.
•
Combination of MLVA with PFGE can lead to a higher typing discrimination for S. Infantis.
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•