Mol Biol Rep DOI 10.1007/s11033-014-3724-2
Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes Dharmendra Kumar Soni • Suresh Kumar Dubey
Received: 9 July 2014 / Accepted: 3 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity. Keywords L. monocytogenes 16S rRNA hlyA Sequencing Phylogenetic analysis
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3724-2) contains supplementary material, which is available to authorized users. D. K. Soni S. K. Dubey (&) Environmental Microbiology Lab, Department of Botany, Banaras Hindu University, Varanasi 221005, India e-mail: [email protected]; [email protected]
Introduction Traditionally, the phenotypic characteristics are used to classify bacteria, involve analysis of different metabolic characteristics. The classification based on the nucleic acid sequence, an outcome of molecular analyses of phylogenetic markers, has become more popular in recent years. Owing to the presence of rRNAs in every living cell, and their highly conserved function, makes such molecules useful in studying the distant relationships. Moreover, rRNAs the mosaic structure composed of a succession of several domains that are more or less conserved as a result of variable evolution rates, makes such studies relatively more useful [1]. Currently, 16S rRNAs (a distinct signature for a bacterial species) are being chosen for identification and differentiation of microorganisms as they form the principal source of phylogenetic information [2]. Klinger et al. [3] previously reported a DNA probe based on a 16S rRNA sequence that detects all the Listeria sp. Studies have also shown a close relationship between the members of Listeria sp. based on the 16S rRNA sequences [4]. While a difference in the 16S rRNA of even closely related Listeria sp. (L. monocytogenes and L. innocua) was reported previously, their study indicated a difference in three distinct single base pair of the V9 region that differentiated L. monocytogenes from L. innocua [5, 6]. Further, Czajka et al. [7] observed, the V9 region to be a clear signature for the discrimination of both the species compared to the V2 region. Moreover, there are several reports that showed the utility of 16S rRNA in the identification, differentiation, genetic relatedness and phylogenetic analysis of L. monocytogenes from different environmental, food and clinical samples [2, 8–16]. Studies on the relationship between different bacterial isolates from the same species are mainly important to
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track source of the food-borne infection and identify the reservoirs of such challenging organisms. A number of different approaches including serotyping, bacteriophage typing, multilocus enzyme electrophoresis (MEE), rRNA ribotyping, and whole-genome restriction digests have been used to distinguish between L. monocytogenes strains [17–20]. Most of such methods with the exception of MEE, are not amenable to phylogenetic analysis, wherein a particular strain can be related to another strain evolutionarily. The present study was focused on the precise identification of L. monocytogenes isolates from different environmental, food and clinical samples through 16S rRNA and hlyA gene sequence similarity analysis.
Materials and methods Study area and sample collection Varanasi is located in the middle Ganges valley of North India in the eastern part of the state of Uttar Pradesh, along the left crescent-shaped bank of the river Ganges, averaging 50 feet (15 m) and 70 feet (21 m) above the river. The ‘‘Varanasi Urban Agglomeration’’—of seven urban subunits, covers 112.26 km2 areas (approx. 43 mi2). The urban agglomeration is stretched between 82° 560 E–83° 030 E and 25° 140 N–25° 23.50 N. Varanasi is often said to be located between two confluences: one of the river Ganges and Varuna, and other of the Ganges and Assi, although the latter is a rivulet. The distance between the two confluences is around 2.5 miles (4.0 km). Among the total of 4,912 samples collected during June 2009–November 2013 and tested, 4,000 were from humans, 412 from milk and milk products, 200 each from vegetable and soil, and 100 from water samples. The human clinical samples (pregnant women with bad obstetric history like repeated abortion, still births and preterm labour) were collected from private and government hospitals of Varanasi city. Vegetable and soil samples were from the agricultural farm of the Indian Institute of Vegetable Research (IIVR), Varanasi, India (25° 080 N latitude; 83° 030 E longitude and 90 m from sea level). Water samples were collected from the river Ganges of Varanasi (25° 200 N and 83°E). Milk and milk products were from the city. All the samples were collected aseptically, quickly transported to the laboratory under chilled condition, and processed within 24 h of collection. Isolation and identification of L. monocytogenes L. monocytogenes were isolated following the standard double enrichment method described by ISO 11290:1 with slight modifications [21]. Briefly, 25 g or mL of water,
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vegetable and soil, 15 mL of milk, 5 g or mL of milk product, placental bit and blood, and cervical or vaginal swab were separately inoculated into 225, 135, 45 or 10 mL of half-Fraser broth (Difco, USA), respectively and incubated (24 h) at 30 °C. Second enrichment was done by adding to 0.1 mL from the overnight grown culture 10 mL of full strength selective agents (Fraser broth, Difco, USA) and incubated (48 h, 37 °C) with the subsequent spreading on PALCAM agar (Difco, USA), and re-incubation (48 h, 37 °C). Grey-greenish colonies with black sunken centre and black halo were picked up and ascertained by Gram staining, biochemical tests such as catalase, methyl redVoges-Proskauer (MR-VP) reaction, nitrate reduction, motility (20–25 °C), acid production from rhamnose, xylose, mannitol, a-methyl-D-mannopyranoside, and CAMP test with Staphylococcus aureus and Rhodococcus equi [22]. L. monocytogenes MTCC1143, S. aureus MTCC1144 and R. equi MTCC1135 served as controls. All the L. monocytogenes isolates and control strains were preserved in tryptic soy agar slants at room temperature for use in the subsequent analysis. DNA isolation Chromosomal DNA was extracted from the isolates grown overnight (37 °C) with shaking (200 rpm) in brain heart infusion broth (BHIB, Difco, USA) using QIAGEN DNeasyÒ Blood and Tissue kit. Harvested cells (maximum 2 9 109 cells) in a microcentrifuge tube (7,500 rpm, 10 min) were re-suspended in 180 lL lysis buffer [20 mM Tris–Cl (pH 8.0), 2 mM sodium EDTA, 1.2 % TritonÒ X-100, 20 mg lysozyme (Sigma) per mL] and incubated (30 min, 37 °C). Proteinase K (25 lL) and 200 lL Buffer AL (without ethanol) were added, mixed by vortexing, and the mixture was incubated (56 °C, 30 min). Thereafter, 4 lL RNase A (100 mg/mL) added and incubated (2 min) at room temperature. Pure ethanol (200 lL, Merck) was added to the sample, and mixed thoroughly by vortexing. The DNA was eluted in Buffer AE and the concentration and purity was ascertained using Nano Drop Spectrophotometer (ND 1000, Nano Drop Technologies, Inc, Wilmington, DE, USA). PCR amplification Total bacterial 16S rRNA gene in the DNA extract of the samples was amplified using bacterial universal primers, 27F (50 -AGAGTTTGATCMTGGCTCAG-30 ) and 1492R (50 -GGYTACCTTGTTACGACTT-30 ) described by Lane [23] using thermal cycler (Bio-Rad, CA, USA) under the following conditions: 94 °C (5 min); 30 cycles of denaturation at 94 °C (1 min), annealing at 60 °C (1 min and 30 s), and extension at 72 °C (1 min) and 72 °C (5 min).
Mol Biol Rep Table 1 Details of source, date of isolation and % of sequence similarity with 16S rRNA and hlyA gene Serial no.
Isolates
Source of isolation
Date of isolation
16S rRNA
hlyA
Accession number
% Similarity
Accession number
% Similarity
1
W1
Ravidaas ghat
15.11.2009
KJ765613
99
HQ686043
100
2
W2
Ravidaas ghat
15.12.2009
KJ765614
99
HQ686044
99
3
W3
Ravidaas ghat
15.01.2010
KJ765615
98
HQ686045
96
4
W4
Ravidaas ghat
15.02.2010
KJ765616
97
HQ686046
100
5
W5
Assi ghat
15.12.2009
KJ765617
99
HQ686047
99
6
W6
Assi ghat
15.01.2010
KJ765618
97
HQ686048
100
7 8
W7 W8
Bhadaini ghat Dr. R. P. ghat
15.01.2010 15.01.2010
KJ765619 KJ765620
97 98
HQ686049 HQ686050
92 98
9
Pb1
Human placental bit
03.06.2009
KJ765621
99
KJ504111
100
10
Pb2
Human placental bit
08.08.2009
KJ765622
99
KJ504112
100
11
Pb3
Human placental bit
14.11.2009
KJ765623
99
KJ504113
100
12
Pb4
Human placental bit
17.02.2010
KJ765624
98
KJ504114
100
13
VS1
Human vaginal swab
05.05.2010
KJ765625
97
KJ504115
100
14
M2
Cow milk
05.06.2009
KJ765626
98
KJ504116
99
15
M3
Cow milk
10.07.2009
KJ765627
98
KJ504117
100
16
M14
Cow milk
12.09.2009
KJ765628
97
KJ504118
98
17
M21
Cow milk
17.12.2009
KJ765629
98
KJ504119
97
18
M25
Cow milk
09.02.2010
KJ765630
98
KJ504120
100
19
M28
Cow milk
12.04.2010
KJ765631
97
KJ504121
100
20
M38
Cow milk
15.07.2010
KJ765632
98
KJ504122
100
21
VB1
Vegetable-brinjal
15.10.2011
KJ765633
99
KJ504123
100
22 23
VB2 VB3
Vegetable-brinjal Vegetable-brinjal
15.10.2011 15.10.2011
KJ765634 KJ765635
99 99
KJ504124 KJ504125
99 99
24
VB4
Vegetable-brinjal
15.10.2011
KJ765636
99
KJ504126
99
25
VCF1
Vegetable-cauliflower
15.11.2011
KJ765637
99
KJ504127
99
26
VCF2
Vegetable-cauliflower
15.11.2011
KJ765638
99
KJ504128
99
27
VCF3
Vegetable-cauliflower
15.11.2011
KJ765639
99
KJ504129
99
28
VCF4
Vegetable-cauliflower
15.11.2011
KJ765640
99
KJ504130
99
29
VDB1
Vegetable-dolichos bean
15.12.2011
KJ765641
99
KJ504131
99
30
VDB2
Vegetable-dolichos bean
15.12.2011
KJ765642
99
KJ504132
99
31
VT1
Vegetable-tomato
15.01.2012
KJ765643
99
KJ504133
100
32
VT2
Vegetable-tomato
15.01.2012
KJ765644
99
KJ504134
99
33
VCK1
Vegetable-chappan kaddu
15.01.2012
KJ765645
99
KJ504135
100
34
VCK2
Vegetable-chappan kaddu
15.01.2012
KJ765646
99
KJ504136
100
35
VCK3
Vegetable-chappan kaddu
15.01.2012
KJ765647
99
KJ504137
100
36
VCK4
Vegetable-chappan kaddu
15.01.2012
KJ765648
99
KJ504138
100
37
VC1
Vegetable-chilli
15.02.2012
KJ765649
99
KJ504139
100
38 39
VC2 VC3
Vegetable-chilli Vegetable-chilli
15.02.2012 15.02.2012
KJ765650 KJ765651
99 99
KJ504140 KJ504141
100 99
40
VC4
Vegetable-chilli
15.02.2012
KJ765652
99
KJ504142
99
41
S1
Soil from brinjal field
15.10.2011
KJ765653
99
KJ504143
100
42
S2
Soil from cauliflower field
15.11.2011
KJ765654
99
KJ504144
100
43
S3
Soil from cauliflower field
15.11.2011
KJ765655
99
KJ504145
100
44
S4
Soil from dolichos bean field
15.12.2011
KJ765656
98
KJ504146
99
45
S5
Soil from dolichos bean field
15.12.2011
KJ765657
99
KJ504147
99
46
S6
Soil from tomato field
15.01.2012
KJ765658
99
KJ504148
99
47
S7
Soil from chappan kaddu field
15.01.2012
KJ765659
99
KJ504149
99
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Mol Biol Rep Table 1 continued Serial no.
Isolates
Source of isolation
Date of isolation
16S rRNA Accession number
hlyA % Similarity
Accession number
% Similarity
48
S8
Soil from chappan kaddu field
15.01.2012
KJ765660
99
KJ504150
100
49
S9
Soil from chilli field
15.02.2012
KJ765661
99
KJ504151
100
50
S10
Soil from chilli field
15.02.2012
KJ765662
99
KJ504152
100
51
HPb1
Human placental bit
15.06.2010
KJ765663
99
KJ883238
100
52
HPb2
Human placental bit
02.10.2010
KJ765664
99
KJ883239
99
53
HPb3
Human placental bit
08.01.2011
KJ765665
98
KJ883240
100
54
HPb4
Human placental bit
18.04.2011
KJ765666
99
KJ883241
100
55
HPb5
Human placental bit
03.02.2012
KJ765667
99
KJ883242
100
56
HPb6
Human placental bit
05.08.2012
KJ765668
99
KJ883243
100
57
HPb7
Human placental bit
12.09.2012
KJ765669
98
KJ883244
100
58
HPb8
Human placental bit
16.12.2012
KJ765670
98
KJ883245
100
59
HPb9
Human placental bit
19.04.2013
KJ765671
99
KJ883246
100
60
HPb10
Human placental bit
28.05.2013
KJ765672
99
KJ883247
100
61 62
HPb11 HPb12
Human placental bit Human placental bit
20.07.2013 10.11.2013
KJ765673 KJ765674
99 99
KJ883248 KJ883249
100 100
63
HVS1
Human vaginal swab
13.07.2010
KJ765675
99
KJ883250
100
64
HVS2
Human vaginal swab
06.09.2010
KJ765676
99
KJ883251
100
65
HVS3
Human vaginal swab
09.12.2010
KJ765677
98
KJ883252
100
66
HVS4
Human vaginal swab
12.02.2011
KJ765678
99
KJ883253
100
67
HVS5
Human vaginal swab
18.04.2011
KJ765679
99
KJ883254
100
68
HVS6
Human vaginal swab
24.01.2012
KJ765680
99
KJ883255
100
69
HVS7
Human vaginal swab
03.02.2012
KJ765681
98
KJ883256
100
70
HVS8
Human vaginal swab
07.03.2012
KJ765682
98
KJ883257
100
71
HVS9
Human vaginal swab
11.05.2012
KJ765683
99
KJ883258
100
72
HVS10
Human vaginal swab
22.06.2012
KJ765684
99
KJ883259
100
73
HVS11
Human vaginal swab
12.09.2012
KJ765685
99
KJ883260
100
74
HVS12
Human vaginal swab
16.12.2012
KJ765686
98
KJ883261
100
75
HVS13
Human vaginal swab
25.03.2013
KJ765687
98
KJ883262
100
76 77
HVS14 HVS15
Human vaginal swab Human vaginal swab
28.05.2013 20.07.2013
KJ765688 KJ765689
99 99
KJ883263 KJ883264
100 100
78
HVS16
Human vaginal swab
14.09.2013
KJ765690
99
KJ883265
100
79
HVS17
Human vaginal swab
17.10.2013
KJ765691
98
KJ883266
100
80
HVS18
Human vaginal swab
10.11.2013
KJ765692
98
KJ883267
100
The reaction mixture (25 lL) contained 2.5 lL of 10 9 buffer (Bangalore Genei, India), 0.5 lL (10 mM dNTP each; Bangalore Genei), 1.25 lL each of 10 lM forward and reverse primers (Sigma), 2.5 U Taq DNA polymerase (Bangalore Genei), and 1 lL template. The rest of the volume was maintained by sterilized milliQ water. Further, PCR was performed to detect hlyA gene of L. monocytogenes using primer pairs of 50 -GCAGTTGCAA GCGCTTGGAGTGAA-30 and 50 -GCAACGTATCCTCC AGAGTGATCG-30 as per the assay prescribed by Notermans et al. [24] with suitable modifications. PCR was
123
performed in 25 lL reaction mixture that included 2.5 lL of 10 9 PCR Buffer (Bangalore Genei), 0.5 lL (10 mM dNTP each; Bangalore Genei), 2.5 lL (25 mM MgCl2), 0.25 lL (10 lM) each of forward and reverse primers, 0.5 lL (5 U) Taq DNA polymerase (Bangalore Genei, Bangalore, India), 2.5 lL of DNA template and sterilized milliQ water to make up the reaction volume. The thermal profile for PCR was: initial denaturation at 95 °C (2 min), 35 cycles of denaturation at 95 °C (15 s), annealing at 60 °C (30 s) and extension at 72 °C (1 min 30 s), the final extension at 72 °C (10 min) and then held at 4 °C. Reaction mixture with no DNA template was incorporated as
Mol Biol Rep b Fig. 1 Phylogenetic analysis of bacterial 16S rRNA gene sequences
and related species by neighbor-joining method obtained from the humans, vegetable, soil, water and cow milk. The scale bars represent 0.005 substitutions per site. GenBank accession numbers are indicated for each sequence clone in parenthesis
the negative control for each run. The resultant PCR products were analysed on 1.5 % agarose gel stained with ethidium bromide (0.5 lg/mL) and visualized by UV transilluminator (AlphaImager EC). The PCR products were pooled and purified using the QIAprep Spin MiniPrep Kit. The primers used for the PCR were from SigmaAldrich, USA. Sequencing and phylogenetic analysis The sequencing of the 16S rRNA and hlyA amplified products as performed on both the strands in ABI PRISM_ 3100 Genetic Analyzer (ABI, Applied Biosystems, Foster City, CA, USA) using the Big Dye Terminator Kit (ver. 3.1; Cycle Sequencing Kit (Applied Biosystems, Rotkreuz, Switzerland)). Electropherograms were edited using the Chromas freeware (ver. 2.01; Chromas lite Technelysium Pvt. Ltd., South Brisbane, Australia). The 16S rRNA and hlyA sequences obtained, were initially recognized and aligned against the known ones in the GenBank database using the BLAST program of the National Centre for Biotechnology Information (NCBI, http://www.ncbi.nlm. nih.gov/). All the sequences were initially aligned using CLUSTAL W [25] available in MEGA4 with opening and at the gap penalty of 10. The phylogenetic relatedness was estimated using the neighbour-joining method [26]. The evolutionary distance was computed using the maximum composite likelihood method [27]. All positions containing gaps and missing data were eliminated from the data set (complete deletion option). One thousand bootstrap replications were performed to place the confidence estimates on the major groups resolved in the tree. The bootstrap consensus tree inferred from 1,000 replicates represents the evolutionary history of the sequences analysed [28]. Branches corresponding to partitions reproduced in \50 % were collapsed. The phylogenetic analysis was carried out using MEGA software ver. 4.0. [28]. Nucleotide accession number The 16S rRNA sequences of present study have been deposited in GenBank under accession numbers KJ765613 to KJ765692 and the hlyA sequences have been deposited under accession numbers HQ686043 to HQ686050, KJ504111 to KJ504152 and KJ883238 to KJ883267.
123
123
W4
W5
W6
W7
W8
Pb1
Pb2
Pb3
Pb4
VS1
M2
M3
M14
M21
M25
M28
M38
VB1
VB2
VB3
VB4
VCF1
VCF2
VCF3
VCF4
VDB1
VDB2
VT1
VT2
VCK1
VCK2
VCK3
VCK4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
W3
3
5
W2
4
W1
2
Vegetable-chappan kaddu
Vegetable-chappan kaddu
Vegetable-chappan kaddu
Vegetable-chappan kaddu
Vegetable-tomato
Vegetable-tomato
Vegetable-dolichos bean
Vegetable-dolichos bean
Vegetable-cauliflower
Vegetable-cauliflower
Vegetable-cauliflower
Vegetable-cauliflower
Vegetable-brinjal
Vegetable-brinjal
Vegetable-brinjal
Vegetable-brinjal
Cow milk
Cow milk
Cow milk
Cow milk
Cow milk
Cow milk
Cow milk
Human vaginal swab
Human placental bit
Human placental bit
Human placental bit
Human placental bit
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges Water
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
98
97
98
98
97
98
98
97
98
99
99
99
98
97
97
99
97
98
99
99
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Source/country
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Source/country
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Source/country
% Similarity
Isolates
Source
16S rRNA similarity with L. monocytogenes in GenBank from different sources
Isolates of present study
1
Sr. no.
Table 2 Similarity between L. monocytogenes isolates with the available ones in GenBank from different habitat and country
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Source/country
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Source/country
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Source/country
Mol Biol Rep
S3
S4
S5
S6
S7
S8
S9
S10
HPb1
HPb2
HPb3
HPb4
HPb5
HPb6
HPb7
HPb8
HPb9
HPb10
HPb11
HPb12
HVS1
HVS2
HVS3
HVS4
HVS5
HVS6
HVS7
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
HVS10
S2
42
HVS9
S1
41
72
VC4
40
71
VC3
39
HVS8
VC2
70
VC1
38
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Soil from chilli field
Soil from chilli field
Soil from chappan kaddu field
Soil from chappan kaddu field
Soil from tomato field
Soil from dolichos bean field
Soil from dolichos bean field
Soil from cauliflower field
Soil from cauliflower field
Soil from brinjal field
Vegetable-chilli
Vegetable-chilli
Vegetable-chilli
Vegetable-chilli
99
99
98
98
99
99
99
98
99
99
99
99
99
99
98
98
99
99
99
98
99
99
99
99
99
99
99
99
98
99
99
99
99
99
99
99
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Source/country
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Source/country
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Source/country
% Similarity
Isolates
Source
16S rRNA similarity with L. monocytogenes in GenBank from different sources
Isolates of present study
37
Sr. no.
Table 2 continued
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Source/country
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Source/country
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Source/country
Mol Biol Rep
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Intestinal/Germany Chicken/Turkey
Chicken/Turkey Sludge and waste water/France
Sludge and waste water/France
Intestinal/Germany
Food/Chile Intestinal/Germany Chicken/Turkey Sludge and waste water/France
Sludge and waste water/France
Intestinal/Germany
Intestinal/Germany Sludge and waste water/France
Chicken/Turkey Sludge and waste water/France
Chicken/Turkey
Chicken/Turkey
Milk product/India
Milk product/India 98
98
HVS18 80
Human vaginal swab
HVS17 79
Human vaginal swab
Milk product/India 99 HVS16 78
Human vaginal swab
Milk product/India 99 HVS15 77
Human vaginal swab
Milk product/India 99 HVS14 76
Human vaginal swab
Milk product/India 98 HVS13 75
Human vaginal swab
Milk product/India
Milk product/India 99
98 Human vaginal swab HVS12 74
Human vaginal swab HVS11 73
Source
Source/country
Source/country
Sludge and waste water/France
Intestinal/Germany
Food/Chile
Results and discussion
Sludge and waste water/France
% Similarity Isolates
Source/country
16S rRNA similarity with L. monocytogenes in GenBank from different sources Isolates of present study Sr. no.
Table 2 continued
Source/country
Source/country
Source/country
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A total of 80 isolates of L. monocytogenes were positive for Gram staining, catalase activity, methyl red—Voges Praskauer (MR-VP) reaction, and motility at 20–25 °C but negative for nitrate reduction. Also these were positive for acid production from rhamnose and a-methyl-D-mannopyranoside while contrary to xylose and mannitol. CAMP test was positive with Staphylococcus aureus. These tests indicated the presence of L. monocytogenes in humans, vegetables, soil, water and cow milk based on their morphological and biochemical attributes as earlier. All isolates exhibited amplification of 16S rRNA and hlyA gene up to the expected size 1,500 and 456 bp, respectively (Fig. S1 and S2, Supplementary material). The results from the BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) showed a sequence similarity of 97–99 and 97–100 %, respectively for the 16S rRNA and hlyA gene, except for one isolate (W7) that showed 92 % sequence similarity for hlyA gene. On the basis of sequence similarity, a total of 80 isolates were found to be closely related to L. monocytogenes. Among these 35, 20, 10, 8 and 7 isolates were from humans, vegetable, soil, water and cow milk, respectively. The details of isolation source, date and sequence similarity (%) for 16S rRNA and hlyA gene are shown in Table 1. Listeriosis has emerged as the typical foodborne disease of major public health concern that predominantly affects pregnant women, neonates, elderly, or immunocompromised people. It manifests as abortion, stillbirth, septicemia, meningitis and meningoencephalitis, and is potentially life threatening because of the high mortality rate (20–30 %) and hospitalization (91 %) following infection [29, 30]. The incidence of listeriosis varies between 0.1 and 11.3/1,000,000 in different countries [31]. The epidemiological data on listeriosis in India available to date, are not adequate for assessing the extent of disease. The disease largely remains undiagnosed because of the lack of a suitable and rapid detection test [32]. Polymorphism of rRNA genes is commonly used to characterize bacterial species [10]. The genus Listeria comprises of ten species, among them L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri exhibited extremely high level of sequence relatedness (98.4–99.2 %). L. monocytogenes and L. innocua possessed 99.2 % similarity, corresponding to only 11 nucleotide difference. L. ivanovii, L. seeligeri and L. welshimeri had very high sequence similarity (13–14 base difference), with exhibited slightly greater nucleotide difference (13–23 base) compared to L. monocytogenes and L. innocua [4, 7, 33]. A phylogenetic tree was reconstructed using the 16S rRNA sequence of the Listeria sp. available in GenBank, and the sequence determined for 80 isolates in this study.
Mol Biol Rep Phylogenetic analysis of hlyA gene sequences and related species by neighbor-joining method obtained from the humans, vegetable, soil, water and cow milk. The scale bars represent 0.05 substitutions per site. GenBank accession numbers are indicated for each sequence clone in parenthesis
b Fig. 2
The data revealed that the isolates recovered showed high level of 16S rRNA sequences relatedness (97–99 %) with L. monocytogenes. Nevertheless L. grayi and the newly identified Listeria sp. (L. rocourtiae and L. fleischmannii) representative strain clearly formed the subline distinct from the other high interrelated species (Fig. 1). Due to the close relationship ([99 %) between the members of Listeria species, other Listeria sp. (L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) are also clustered in same L. monocygenes group (Fig. 1). Hence, for Listeria, such a differentiation is difficult because of the highly conserved nature of the 16S rRNA gene among its species. Further, on the basis 16S rRNA gene BLAST similarity, we determined the relationship between L. monocytogenes isolate, and the available sequences in GenBank to track the source and identify the reservoirs (Table 2). All the L. monocytogenes isolates from different environmental, food and clinical samples showed similarity with the isolates from the milk product (KF894986.1), in India and sludge and waste water (AJ535697.1), in France. Additionally, most of the isolates from vegetables also showed similarity with those from food (KF588562.1), in Chile and majority of the isolates from water and human clinical samples showed similarity with the isolates obtained from intestine (HM007564.1) in Germany, and chicken (KF956739.1) in Turkey. Therefore, on the basis of 16S rRNA similarity, it can be suggested that the isolates collected in the present study are similar to the previously reported isolates from different habitats and country. Further, In order to ascertain the presence of pathogens in their respective environment, the detection of one of the major virulence factors is a better option. Among the various virulence factors, LLO (a 58 kDa hemolysin protein encoded by hly gene) is the main virulence factor and pathogenic marker for the detection of Listeria sp. [34]. However, the presence of hemolysin (hlyA) gene in other species (L. ivanovii and L. seeligeri) although, with the difference in sequence similarity [35]. The protein homology between them is 86–91 %, and nucleotide sequence similarity 76–78 % [35, 36]. In the present study, BLAST results of the hlyA gene sequence indicated that all the 80 isolates were closely related (97–100 %), except one isolate (W7) that showed 92 % similarity to L. monocytogenes. Further, a phylogenetic tree was reconstructed using the hlyA gene sequence of Listeria sp. available in GenBank, and the sequence were determined for 80
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isolates examined. The analysis revealed that the isolates could be clustered in same group (Fig. 2). Nevertheless, L. ivanovii and L. seeligeri representative strains clearly formed a subline distinct from the other high interrelated species. Hence, the present study indicates that to ascertain the presence of L. monocytogenes, hlyA gene sequence information is the better option. In conclusion, molecular analyses of phylogenetic markers based on the nucleic acid sequence are useful in classifying Listeria sp. In total 80 isolates from different environmental components (soil, water), food and clinical samples were confirmed to be L. monocytogenes. The 16S rRNA sequence of the Listeria sp. can be used to differentiate L. grayi, and newly identified Listeria sp. (L. rocourtiae and L. fleischmannii strain) from the other highly interrelated species (L. innocua, L. ivanovii, L. seeligeri and L. welshimeri). The 16S rRNA also suggested that these isolates in the present study were similar to the previously reported ones obtained from different habitats and country. The hlyA gene sequence information could be the reliable option to indicate presence of L. monocytogenes. More work, however, is required on L. monocytogenes in order to ascertain its presence in clinical and environmental samples as there is limited information available on such aspects for tropical countries including India. The molecular technologies such as microarray and next generation sequencing are likely to be helpful in rapid acquisition of sequence data to facilitate detection and characterization of other pathogenic strains of L. monocytogenes. Acknowledgments This study was supported by Indian Council of Medical Research (ICMR), Government of India, New Delhi through the research project No. 5/3/3/10/2007-ECD-I.
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30. Soni DK, Singh RK, Singh DV, Dubey SK (2014) Characterization of Listeria monocytogenes isolated from Ganges water, human clinical and milk samples at Varanasi, India. Infect Genet Evol 14:83–91 31. Anonymous (2004) Microbiological Risk Assessment Series, No. 5, Technical Report. WHO, Rome 32. Barbuddhe SB, Malik SVS, Kumar JA, Kalorey DR, Chakraborty T (2012) Epidemiology and risk management of listeriosis in India. Int J Food Microbiol 154:113–118 33. Sallen B, Rajoharison A, Desvarenne S, Quinn F, Mabilat C (1996) Comparative analysis of 16S and 23S rRNA sequences of Listeria species. Int J Syst Bacteriol 46:669–674 34. Barbuddhe SB, Chaudhari SP, Malik SVS (2002) The occurrence of pathogenic Listeria monocytogenes and antibodies against listeriolysin O in buffaloes. J Vet Med B 49:181–184 35. Rawool DB, Malik SVS, Barbuddhe SB, Shakuntala I, Aurora R (2007) A multiplex PCR for detection of virulence associated genes in Listeria monocytogenes. Int J Food Saf 9:56–62 36. Aurora R, Prakash A, Prakash S, Rawool DB, Barbuddhe SB (2008) Comparison of PI-PLC based assays and PCR along with in vivo pathogenicity tests for rapid detection of pathogenic Listeria monocytogenes. Food Control 19:641–647
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Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes Dharmendra Kumar Soni • Suresh Kumar Dubey
Received: 9 July 2014 / Accepted: 3 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity. Keywords L. monocytogenes 16S rRNA hlyA Sequencing Phylogenetic analysis
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3724-2) contains supplementary material, which is available to authorized users. D. K. Soni S. K. Dubey (&) Environmental Microbiology Lab, Department of Botany, Banaras Hindu University, Varanasi 221005, India e-mail: [email protected]; [email protected]
Introduction Traditionally, the phenotypic characteristics are used to classify bacteria, involve analysis of different metabolic characteristics. The classification based on the nucleic acid sequence, an outcome of molecular analyses of phylogenetic markers, has become more popular in recent years. Owing to the presence of rRNAs in every living cell, and their highly conserved function, makes such molecules useful in studying the distant relationships. Moreover, rRNAs the mosaic structure composed of a succession of several domains that are more or less conserved as a result of variable evolution rates, makes such studies relatively more useful [1]. Currently, 16S rRNAs (a distinct signature for a bacterial species) are being chosen for identification and differentiation of microorganisms as they form the principal source of phylogenetic information [2]. Klinger et al. [3] previously reported a DNA probe based on a 16S rRNA sequence that detects all the Listeria sp. Studies have also shown a close relationship between the members of Listeria sp. based on the 16S rRNA sequences [4]. While a difference in the 16S rRNA of even closely related Listeria sp. (L. monocytogenes and L. innocua) was reported previously, their study indicated a difference in three distinct single base pair of the V9 region that differentiated L. monocytogenes from L. innocua [5, 6]. Further, Czajka et al. [7] observed, the V9 region to be a clear signature for the discrimination of both the species compared to the V2 region. Moreover, there are several reports that showed the utility of 16S rRNA in the identification, differentiation, genetic relatedness and phylogenetic analysis of L. monocytogenes from different environmental, food and clinical samples [2, 8–16]. Studies on the relationship between different bacterial isolates from the same species are mainly important to
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track source of the food-borne infection and identify the reservoirs of such challenging organisms. A number of different approaches including serotyping, bacteriophage typing, multilocus enzyme electrophoresis (MEE), rRNA ribotyping, and whole-genome restriction digests have been used to distinguish between L. monocytogenes strains [17–20]. Most of such methods with the exception of MEE, are not amenable to phylogenetic analysis, wherein a particular strain can be related to another strain evolutionarily. The present study was focused on the precise identification of L. monocytogenes isolates from different environmental, food and clinical samples through 16S rRNA and hlyA gene sequence similarity analysis.
Materials and methods Study area and sample collection Varanasi is located in the middle Ganges valley of North India in the eastern part of the state of Uttar Pradesh, along the left crescent-shaped bank of the river Ganges, averaging 50 feet (15 m) and 70 feet (21 m) above the river. The ‘‘Varanasi Urban Agglomeration’’—of seven urban subunits, covers 112.26 km2 areas (approx. 43 mi2). The urban agglomeration is stretched between 82° 560 E–83° 030 E and 25° 140 N–25° 23.50 N. Varanasi is often said to be located between two confluences: one of the river Ganges and Varuna, and other of the Ganges and Assi, although the latter is a rivulet. The distance between the two confluences is around 2.5 miles (4.0 km). Among the total of 4,912 samples collected during June 2009–November 2013 and tested, 4,000 were from humans, 412 from milk and milk products, 200 each from vegetable and soil, and 100 from water samples. The human clinical samples (pregnant women with bad obstetric history like repeated abortion, still births and preterm labour) were collected from private and government hospitals of Varanasi city. Vegetable and soil samples were from the agricultural farm of the Indian Institute of Vegetable Research (IIVR), Varanasi, India (25° 080 N latitude; 83° 030 E longitude and 90 m from sea level). Water samples were collected from the river Ganges of Varanasi (25° 200 N and 83°E). Milk and milk products were from the city. All the samples were collected aseptically, quickly transported to the laboratory under chilled condition, and processed within 24 h of collection. Isolation and identification of L. monocytogenes L. monocytogenes were isolated following the standard double enrichment method described by ISO 11290:1 with slight modifications [21]. Briefly, 25 g or mL of water,
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vegetable and soil, 15 mL of milk, 5 g or mL of milk product, placental bit and blood, and cervical or vaginal swab were separately inoculated into 225, 135, 45 or 10 mL of half-Fraser broth (Difco, USA), respectively and incubated (24 h) at 30 °C. Second enrichment was done by adding to 0.1 mL from the overnight grown culture 10 mL of full strength selective agents (Fraser broth, Difco, USA) and incubated (48 h, 37 °C) with the subsequent spreading on PALCAM agar (Difco, USA), and re-incubation (48 h, 37 °C). Grey-greenish colonies with black sunken centre and black halo were picked up and ascertained by Gram staining, biochemical tests such as catalase, methyl redVoges-Proskauer (MR-VP) reaction, nitrate reduction, motility (20–25 °C), acid production from rhamnose, xylose, mannitol, a-methyl-D-mannopyranoside, and CAMP test with Staphylococcus aureus and Rhodococcus equi [22]. L. monocytogenes MTCC1143, S. aureus MTCC1144 and R. equi MTCC1135 served as controls. All the L. monocytogenes isolates and control strains were preserved in tryptic soy agar slants at room temperature for use in the subsequent analysis. DNA isolation Chromosomal DNA was extracted from the isolates grown overnight (37 °C) with shaking (200 rpm) in brain heart infusion broth (BHIB, Difco, USA) using QIAGEN DNeasyÒ Blood and Tissue kit. Harvested cells (maximum 2 9 109 cells) in a microcentrifuge tube (7,500 rpm, 10 min) were re-suspended in 180 lL lysis buffer [20 mM Tris–Cl (pH 8.0), 2 mM sodium EDTA, 1.2 % TritonÒ X-100, 20 mg lysozyme (Sigma) per mL] and incubated (30 min, 37 °C). Proteinase K (25 lL) and 200 lL Buffer AL (without ethanol) were added, mixed by vortexing, and the mixture was incubated (56 °C, 30 min). Thereafter, 4 lL RNase A (100 mg/mL) added and incubated (2 min) at room temperature. Pure ethanol (200 lL, Merck) was added to the sample, and mixed thoroughly by vortexing. The DNA was eluted in Buffer AE and the concentration and purity was ascertained using Nano Drop Spectrophotometer (ND 1000, Nano Drop Technologies, Inc, Wilmington, DE, USA). PCR amplification Total bacterial 16S rRNA gene in the DNA extract of the samples was amplified using bacterial universal primers, 27F (50 -AGAGTTTGATCMTGGCTCAG-30 ) and 1492R (50 -GGYTACCTTGTTACGACTT-30 ) described by Lane [23] using thermal cycler (Bio-Rad, CA, USA) under the following conditions: 94 °C (5 min); 30 cycles of denaturation at 94 °C (1 min), annealing at 60 °C (1 min and 30 s), and extension at 72 °C (1 min) and 72 °C (5 min).
Mol Biol Rep Table 1 Details of source, date of isolation and % of sequence similarity with 16S rRNA and hlyA gene Serial no.
Isolates
Source of isolation
Date of isolation
16S rRNA
hlyA
Accession number
% Similarity
Accession number
% Similarity
1
W1
Ravidaas ghat
15.11.2009
KJ765613
99
HQ686043
100
2
W2
Ravidaas ghat
15.12.2009
KJ765614
99
HQ686044
99
3
W3
Ravidaas ghat
15.01.2010
KJ765615
98
HQ686045
96
4
W4
Ravidaas ghat
15.02.2010
KJ765616
97
HQ686046
100
5
W5
Assi ghat
15.12.2009
KJ765617
99
HQ686047
99
6
W6
Assi ghat
15.01.2010
KJ765618
97
HQ686048
100
7 8
W7 W8
Bhadaini ghat Dr. R. P. ghat
15.01.2010 15.01.2010
KJ765619 KJ765620
97 98
HQ686049 HQ686050
92 98
9
Pb1
Human placental bit
03.06.2009
KJ765621
99
KJ504111
100
10
Pb2
Human placental bit
08.08.2009
KJ765622
99
KJ504112
100
11
Pb3
Human placental bit
14.11.2009
KJ765623
99
KJ504113
100
12
Pb4
Human placental bit
17.02.2010
KJ765624
98
KJ504114
100
13
VS1
Human vaginal swab
05.05.2010
KJ765625
97
KJ504115
100
14
M2
Cow milk
05.06.2009
KJ765626
98
KJ504116
99
15
M3
Cow milk
10.07.2009
KJ765627
98
KJ504117
100
16
M14
Cow milk
12.09.2009
KJ765628
97
KJ504118
98
17
M21
Cow milk
17.12.2009
KJ765629
98
KJ504119
97
18
M25
Cow milk
09.02.2010
KJ765630
98
KJ504120
100
19
M28
Cow milk
12.04.2010
KJ765631
97
KJ504121
100
20
M38
Cow milk
15.07.2010
KJ765632
98
KJ504122
100
21
VB1
Vegetable-brinjal
15.10.2011
KJ765633
99
KJ504123
100
22 23
VB2 VB3
Vegetable-brinjal Vegetable-brinjal
15.10.2011 15.10.2011
KJ765634 KJ765635
99 99
KJ504124 KJ504125
99 99
24
VB4
Vegetable-brinjal
15.10.2011
KJ765636
99
KJ504126
99
25
VCF1
Vegetable-cauliflower
15.11.2011
KJ765637
99
KJ504127
99
26
VCF2
Vegetable-cauliflower
15.11.2011
KJ765638
99
KJ504128
99
27
VCF3
Vegetable-cauliflower
15.11.2011
KJ765639
99
KJ504129
99
28
VCF4
Vegetable-cauliflower
15.11.2011
KJ765640
99
KJ504130
99
29
VDB1
Vegetable-dolichos bean
15.12.2011
KJ765641
99
KJ504131
99
30
VDB2
Vegetable-dolichos bean
15.12.2011
KJ765642
99
KJ504132
99
31
VT1
Vegetable-tomato
15.01.2012
KJ765643
99
KJ504133
100
32
VT2
Vegetable-tomato
15.01.2012
KJ765644
99
KJ504134
99
33
VCK1
Vegetable-chappan kaddu
15.01.2012
KJ765645
99
KJ504135
100
34
VCK2
Vegetable-chappan kaddu
15.01.2012
KJ765646
99
KJ504136
100
35
VCK3
Vegetable-chappan kaddu
15.01.2012
KJ765647
99
KJ504137
100
36
VCK4
Vegetable-chappan kaddu
15.01.2012
KJ765648
99
KJ504138
100
37
VC1
Vegetable-chilli
15.02.2012
KJ765649
99
KJ504139
100
38 39
VC2 VC3
Vegetable-chilli Vegetable-chilli
15.02.2012 15.02.2012
KJ765650 KJ765651
99 99
KJ504140 KJ504141
100 99
40
VC4
Vegetable-chilli
15.02.2012
KJ765652
99
KJ504142
99
41
S1
Soil from brinjal field
15.10.2011
KJ765653
99
KJ504143
100
42
S2
Soil from cauliflower field
15.11.2011
KJ765654
99
KJ504144
100
43
S3
Soil from cauliflower field
15.11.2011
KJ765655
99
KJ504145
100
44
S4
Soil from dolichos bean field
15.12.2011
KJ765656
98
KJ504146
99
45
S5
Soil from dolichos bean field
15.12.2011
KJ765657
99
KJ504147
99
46
S6
Soil from tomato field
15.01.2012
KJ765658
99
KJ504148
99
47
S7
Soil from chappan kaddu field
15.01.2012
KJ765659
99
KJ504149
99
123
Mol Biol Rep Table 1 continued Serial no.
Isolates
Source of isolation
Date of isolation
16S rRNA Accession number
hlyA % Similarity
Accession number
% Similarity
48
S8
Soil from chappan kaddu field
15.01.2012
KJ765660
99
KJ504150
100
49
S9
Soil from chilli field
15.02.2012
KJ765661
99
KJ504151
100
50
S10
Soil from chilli field
15.02.2012
KJ765662
99
KJ504152
100
51
HPb1
Human placental bit
15.06.2010
KJ765663
99
KJ883238
100
52
HPb2
Human placental bit
02.10.2010
KJ765664
99
KJ883239
99
53
HPb3
Human placental bit
08.01.2011
KJ765665
98
KJ883240
100
54
HPb4
Human placental bit
18.04.2011
KJ765666
99
KJ883241
100
55
HPb5
Human placental bit
03.02.2012
KJ765667
99
KJ883242
100
56
HPb6
Human placental bit
05.08.2012
KJ765668
99
KJ883243
100
57
HPb7
Human placental bit
12.09.2012
KJ765669
98
KJ883244
100
58
HPb8
Human placental bit
16.12.2012
KJ765670
98
KJ883245
100
59
HPb9
Human placental bit
19.04.2013
KJ765671
99
KJ883246
100
60
HPb10
Human placental bit
28.05.2013
KJ765672
99
KJ883247
100
61 62
HPb11 HPb12
Human placental bit Human placental bit
20.07.2013 10.11.2013
KJ765673 KJ765674
99 99
KJ883248 KJ883249
100 100
63
HVS1
Human vaginal swab
13.07.2010
KJ765675
99
KJ883250
100
64
HVS2
Human vaginal swab
06.09.2010
KJ765676
99
KJ883251
100
65
HVS3
Human vaginal swab
09.12.2010
KJ765677
98
KJ883252
100
66
HVS4
Human vaginal swab
12.02.2011
KJ765678
99
KJ883253
100
67
HVS5
Human vaginal swab
18.04.2011
KJ765679
99
KJ883254
100
68
HVS6
Human vaginal swab
24.01.2012
KJ765680
99
KJ883255
100
69
HVS7
Human vaginal swab
03.02.2012
KJ765681
98
KJ883256
100
70
HVS8
Human vaginal swab
07.03.2012
KJ765682
98
KJ883257
100
71
HVS9
Human vaginal swab
11.05.2012
KJ765683
99
KJ883258
100
72
HVS10
Human vaginal swab
22.06.2012
KJ765684
99
KJ883259
100
73
HVS11
Human vaginal swab
12.09.2012
KJ765685
99
KJ883260
100
74
HVS12
Human vaginal swab
16.12.2012
KJ765686
98
KJ883261
100
75
HVS13
Human vaginal swab
25.03.2013
KJ765687
98
KJ883262
100
76 77
HVS14 HVS15
Human vaginal swab Human vaginal swab
28.05.2013 20.07.2013
KJ765688 KJ765689
99 99
KJ883263 KJ883264
100 100
78
HVS16
Human vaginal swab
14.09.2013
KJ765690
99
KJ883265
100
79
HVS17
Human vaginal swab
17.10.2013
KJ765691
98
KJ883266
100
80
HVS18
Human vaginal swab
10.11.2013
KJ765692
98
KJ883267
100
The reaction mixture (25 lL) contained 2.5 lL of 10 9 buffer (Bangalore Genei, India), 0.5 lL (10 mM dNTP each; Bangalore Genei), 1.25 lL each of 10 lM forward and reverse primers (Sigma), 2.5 U Taq DNA polymerase (Bangalore Genei), and 1 lL template. The rest of the volume was maintained by sterilized milliQ water. Further, PCR was performed to detect hlyA gene of L. monocytogenes using primer pairs of 50 -GCAGTTGCAA GCGCTTGGAGTGAA-30 and 50 -GCAACGTATCCTCC AGAGTGATCG-30 as per the assay prescribed by Notermans et al. [24] with suitable modifications. PCR was
123
performed in 25 lL reaction mixture that included 2.5 lL of 10 9 PCR Buffer (Bangalore Genei), 0.5 lL (10 mM dNTP each; Bangalore Genei), 2.5 lL (25 mM MgCl2), 0.25 lL (10 lM) each of forward and reverse primers, 0.5 lL (5 U) Taq DNA polymerase (Bangalore Genei, Bangalore, India), 2.5 lL of DNA template and sterilized milliQ water to make up the reaction volume. The thermal profile for PCR was: initial denaturation at 95 °C (2 min), 35 cycles of denaturation at 95 °C (15 s), annealing at 60 °C (30 s) and extension at 72 °C (1 min 30 s), the final extension at 72 °C (10 min) and then held at 4 °C. Reaction mixture with no DNA template was incorporated as
Mol Biol Rep b Fig. 1 Phylogenetic analysis of bacterial 16S rRNA gene sequences
and related species by neighbor-joining method obtained from the humans, vegetable, soil, water and cow milk. The scale bars represent 0.005 substitutions per site. GenBank accession numbers are indicated for each sequence clone in parenthesis
the negative control for each run. The resultant PCR products were analysed on 1.5 % agarose gel stained with ethidium bromide (0.5 lg/mL) and visualized by UV transilluminator (AlphaImager EC). The PCR products were pooled and purified using the QIAprep Spin MiniPrep Kit. The primers used for the PCR were from SigmaAldrich, USA. Sequencing and phylogenetic analysis The sequencing of the 16S rRNA and hlyA amplified products as performed on both the strands in ABI PRISM_ 3100 Genetic Analyzer (ABI, Applied Biosystems, Foster City, CA, USA) using the Big Dye Terminator Kit (ver. 3.1; Cycle Sequencing Kit (Applied Biosystems, Rotkreuz, Switzerland)). Electropherograms were edited using the Chromas freeware (ver. 2.01; Chromas lite Technelysium Pvt. Ltd., South Brisbane, Australia). The 16S rRNA and hlyA sequences obtained, were initially recognized and aligned against the known ones in the GenBank database using the BLAST program of the National Centre for Biotechnology Information (NCBI, http://www.ncbi.nlm. nih.gov/). All the sequences were initially aligned using CLUSTAL W [25] available in MEGA4 with opening and at the gap penalty of 10. The phylogenetic relatedness was estimated using the neighbour-joining method [26]. The evolutionary distance was computed using the maximum composite likelihood method [27]. All positions containing gaps and missing data were eliminated from the data set (complete deletion option). One thousand bootstrap replications were performed to place the confidence estimates on the major groups resolved in the tree. The bootstrap consensus tree inferred from 1,000 replicates represents the evolutionary history of the sequences analysed [28]. Branches corresponding to partitions reproduced in \50 % were collapsed. The phylogenetic analysis was carried out using MEGA software ver. 4.0. [28]. Nucleotide accession number The 16S rRNA sequences of present study have been deposited in GenBank under accession numbers KJ765613 to KJ765692 and the hlyA sequences have been deposited under accession numbers HQ686043 to HQ686050, KJ504111 to KJ504152 and KJ883238 to KJ883267.
123
123
W4
W5
W6
W7
W8
Pb1
Pb2
Pb3
Pb4
VS1
M2
M3
M14
M21
M25
M28
M38
VB1
VB2
VB3
VB4
VCF1
VCF2
VCF3
VCF4
VDB1
VDB2
VT1
VT2
VCK1
VCK2
VCK3
VCK4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
W3
3
5
W2
4
W1
2
Vegetable-chappan kaddu
Vegetable-chappan kaddu
Vegetable-chappan kaddu
Vegetable-chappan kaddu
Vegetable-tomato
Vegetable-tomato
Vegetable-dolichos bean
Vegetable-dolichos bean
Vegetable-cauliflower
Vegetable-cauliflower
Vegetable-cauliflower
Vegetable-cauliflower
Vegetable-brinjal
Vegetable-brinjal
Vegetable-brinjal
Vegetable-brinjal
Cow milk
Cow milk
Cow milk
Cow milk
Cow milk
Cow milk
Cow milk
Human vaginal swab
Human placental bit
Human placental bit
Human placental bit
Human placental bit
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges water
River Ganges Water
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
98
97
98
98
97
98
98
97
98
99
99
99
98
97
97
99
97
98
99
99
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Source/country
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Source/country
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Source/country
% Similarity
Isolates
Source
16S rRNA similarity with L. monocytogenes in GenBank from different sources
Isolates of present study
1
Sr. no.
Table 2 Similarity between L. monocytogenes isolates with the available ones in GenBank from different habitat and country
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Source/country
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Source/country
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Source/country
Mol Biol Rep
S3
S4
S5
S6
S7
S8
S9
S10
HPb1
HPb2
HPb3
HPb4
HPb5
HPb6
HPb7
HPb8
HPb9
HPb10
HPb11
HPb12
HVS1
HVS2
HVS3
HVS4
HVS5
HVS6
HVS7
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
HVS10
S2
42
HVS9
S1
41
72
VC4
40
71
VC3
39
HVS8
VC2
70
VC1
38
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human vaginal swab
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Human placental bit
Soil from chilli field
Soil from chilli field
Soil from chappan kaddu field
Soil from chappan kaddu field
Soil from tomato field
Soil from dolichos bean field
Soil from dolichos bean field
Soil from cauliflower field
Soil from cauliflower field
Soil from brinjal field
Vegetable-chilli
Vegetable-chilli
Vegetable-chilli
Vegetable-chilli
99
99
98
98
99
99
99
98
99
99
99
99
99
99
98
98
99
99
99
98
99
99
99
99
99
99
99
99
98
99
99
99
99
99
99
99
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Milk product/India
Source/country
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Aquatic foods/China
Source/country
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Sludge and waste water/France
Source/country
% Similarity
Isolates
Source
16S rRNA similarity with L. monocytogenes in GenBank from different sources
Isolates of present study
37
Sr. no.
Table 2 continued
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Chicken/Turkey
Source/country
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Intestinal/Germany
Source/country
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Food/Chile
Source/country
Mol Biol Rep
123
123
Intestinal/Germany Chicken/Turkey
Chicken/Turkey Sludge and waste water/France
Sludge and waste water/France
Intestinal/Germany
Food/Chile Intestinal/Germany Chicken/Turkey Sludge and waste water/France
Sludge and waste water/France
Intestinal/Germany
Intestinal/Germany Sludge and waste water/France
Chicken/Turkey Sludge and waste water/France
Chicken/Turkey
Chicken/Turkey
Milk product/India
Milk product/India 98
98
HVS18 80
Human vaginal swab
HVS17 79
Human vaginal swab
Milk product/India 99 HVS16 78
Human vaginal swab
Milk product/India 99 HVS15 77
Human vaginal swab
Milk product/India 99 HVS14 76
Human vaginal swab
Milk product/India 98 HVS13 75
Human vaginal swab
Milk product/India
Milk product/India 99
98 Human vaginal swab HVS12 74
Human vaginal swab HVS11 73
Source
Source/country
Source/country
Sludge and waste water/France
Intestinal/Germany
Food/Chile
Results and discussion
Sludge and waste water/France
% Similarity Isolates
Source/country
16S rRNA similarity with L. monocytogenes in GenBank from different sources Isolates of present study Sr. no.
Table 2 continued
Source/country
Source/country
Source/country
Mol Biol Rep
A total of 80 isolates of L. monocytogenes were positive for Gram staining, catalase activity, methyl red—Voges Praskauer (MR-VP) reaction, and motility at 20–25 °C but negative for nitrate reduction. Also these were positive for acid production from rhamnose and a-methyl-D-mannopyranoside while contrary to xylose and mannitol. CAMP test was positive with Staphylococcus aureus. These tests indicated the presence of L. monocytogenes in humans, vegetables, soil, water and cow milk based on their morphological and biochemical attributes as earlier. All isolates exhibited amplification of 16S rRNA and hlyA gene up to the expected size 1,500 and 456 bp, respectively (Fig. S1 and S2, Supplementary material). The results from the BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) showed a sequence similarity of 97–99 and 97–100 %, respectively for the 16S rRNA and hlyA gene, except for one isolate (W7) that showed 92 % sequence similarity for hlyA gene. On the basis of sequence similarity, a total of 80 isolates were found to be closely related to L. monocytogenes. Among these 35, 20, 10, 8 and 7 isolates were from humans, vegetable, soil, water and cow milk, respectively. The details of isolation source, date and sequence similarity (%) for 16S rRNA and hlyA gene are shown in Table 1. Listeriosis has emerged as the typical foodborne disease of major public health concern that predominantly affects pregnant women, neonates, elderly, or immunocompromised people. It manifests as abortion, stillbirth, septicemia, meningitis and meningoencephalitis, and is potentially life threatening because of the high mortality rate (20–30 %) and hospitalization (91 %) following infection [29, 30]. The incidence of listeriosis varies between 0.1 and 11.3/1,000,000 in different countries [31]. The epidemiological data on listeriosis in India available to date, are not adequate for assessing the extent of disease. The disease largely remains undiagnosed because of the lack of a suitable and rapid detection test [32]. Polymorphism of rRNA genes is commonly used to characterize bacterial species [10]. The genus Listeria comprises of ten species, among them L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri exhibited extremely high level of sequence relatedness (98.4–99.2 %). L. monocytogenes and L. innocua possessed 99.2 % similarity, corresponding to only 11 nucleotide difference. L. ivanovii, L. seeligeri and L. welshimeri had very high sequence similarity (13–14 base difference), with exhibited slightly greater nucleotide difference (13–23 base) compared to L. monocytogenes and L. innocua [4, 7, 33]. A phylogenetic tree was reconstructed using the 16S rRNA sequence of the Listeria sp. available in GenBank, and the sequence determined for 80 isolates in this study.
Mol Biol Rep Phylogenetic analysis of hlyA gene sequences and related species by neighbor-joining method obtained from the humans, vegetable, soil, water and cow milk. The scale bars represent 0.05 substitutions per site. GenBank accession numbers are indicated for each sequence clone in parenthesis
b Fig. 2
The data revealed that the isolates recovered showed high level of 16S rRNA sequences relatedness (97–99 %) with L. monocytogenes. Nevertheless L. grayi and the newly identified Listeria sp. (L. rocourtiae and L. fleischmannii) representative strain clearly formed the subline distinct from the other high interrelated species (Fig. 1). Due to the close relationship ([99 %) between the members of Listeria species, other Listeria sp. (L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) are also clustered in same L. monocygenes group (Fig. 1). Hence, for Listeria, such a differentiation is difficult because of the highly conserved nature of the 16S rRNA gene among its species. Further, on the basis 16S rRNA gene BLAST similarity, we determined the relationship between L. monocytogenes isolate, and the available sequences in GenBank to track the source and identify the reservoirs (Table 2). All the L. monocytogenes isolates from different environmental, food and clinical samples showed similarity with the isolates from the milk product (KF894986.1), in India and sludge and waste water (AJ535697.1), in France. Additionally, most of the isolates from vegetables also showed similarity with those from food (KF588562.1), in Chile and majority of the isolates from water and human clinical samples showed similarity with the isolates obtained from intestine (HM007564.1) in Germany, and chicken (KF956739.1) in Turkey. Therefore, on the basis of 16S rRNA similarity, it can be suggested that the isolates collected in the present study are similar to the previously reported isolates from different habitats and country. Further, In order to ascertain the presence of pathogens in their respective environment, the detection of one of the major virulence factors is a better option. Among the various virulence factors, LLO (a 58 kDa hemolysin protein encoded by hly gene) is the main virulence factor and pathogenic marker for the detection of Listeria sp. [34]. However, the presence of hemolysin (hlyA) gene in other species (L. ivanovii and L. seeligeri) although, with the difference in sequence similarity [35]. The protein homology between them is 86–91 %, and nucleotide sequence similarity 76–78 % [35, 36]. In the present study, BLAST results of the hlyA gene sequence indicated that all the 80 isolates were closely related (97–100 %), except one isolate (W7) that showed 92 % similarity to L. monocytogenes. Further, a phylogenetic tree was reconstructed using the hlyA gene sequence of Listeria sp. available in GenBank, and the sequence were determined for 80
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isolates examined. The analysis revealed that the isolates could be clustered in same group (Fig. 2). Nevertheless, L. ivanovii and L. seeligeri representative strains clearly formed a subline distinct from the other high interrelated species. Hence, the present study indicates that to ascertain the presence of L. monocytogenes, hlyA gene sequence information is the better option. In conclusion, molecular analyses of phylogenetic markers based on the nucleic acid sequence are useful in classifying Listeria sp. In total 80 isolates from different environmental components (soil, water), food and clinical samples were confirmed to be L. monocytogenes. The 16S rRNA sequence of the Listeria sp. can be used to differentiate L. grayi, and newly identified Listeria sp. (L. rocourtiae and L. fleischmannii strain) from the other highly interrelated species (L. innocua, L. ivanovii, L. seeligeri and L. welshimeri). The 16S rRNA also suggested that these isolates in the present study were similar to the previously reported ones obtained from different habitats and country. The hlyA gene sequence information could be the reliable option to indicate presence of L. monocytogenes. More work, however, is required on L. monocytogenes in order to ascertain its presence in clinical and environmental samples as there is limited information available on such aspects for tropical countries including India. The molecular technologies such as microarray and next generation sequencing are likely to be helpful in rapid acquisition of sequence data to facilitate detection and characterization of other pathogenic strains of L. monocytogenes. Acknowledgments This study was supported by Indian Council of Medical Research (ICMR), Government of India, New Delhi through the research project No. 5/3/3/10/2007-ECD-I.
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