Draft genome sequence of Lactobacillus plantarum strains E2C2 and E2C5 isolated from human stool culture Academic research paper on "Veterinary science"

Suryavanshi et al. Standards in Genomic Sciences (2017) 12:15 DOI 10.1186/s40793-017-0222-x

Standards in Genomic Sciences

EXTENDED GENOME REPORT Open Access

Draft genome sequence of Lactobacillus plantarum strains E2C2 and E2C5 isolated from human stool culture

Mangesh V. Suryavanshi11, Dhiraj Paul1+, Swapnil P. Doijad2, Shrikant S. Bhute3, Tejashri B. Hingamire4, Rahul P. Gune5 and Yogesh S. Shouche1*

Abstract

Probiotic Lactobacillus species offer various health benefits, thus have been employed in treatment and prevention of various diseases. Due to the differences in the isolation source and the site of action, most of the lactobacilli tested in-vitro for probiotics properties fail to extend similar effects in-vivo. Consequently, the search of autochthonous, efficacious and probably population specific probiotics is a high priority in the probiotics research. In this regards, whole genome sequencing of as many Lactobacillus as possible will help to deepen our understanding of biology and their health effects. Here, we provide the genomic insights of two coherent oxalic acid tolerant Lactobacillus species (E2C2 and E2C5) isolated from two different healthy human gut flora. These two isolates were found to have higher tolerance towards oxalic acid (300 mM sodium oxalate). The draft genome of strain E2C2 consists of 3,603,563 bp with 3289 protein-coding genes, 94 RNA genes, and 43.99% GC content, while E2C5 contained 3,615,168 bp, 3293 coding genes (93.4% of the total genes), 95 RNA genes and 43.97% GC content. Based on 16S rRNA gene sequence analysis followed by in silico DNA-DNA hybridization studies, both the strains were identified as Lactobacillus plantarum belonging to family Lactobacillaceae within the phylum Firmicutes. Both the strains were genomically identical, sharing 99.99% CDS that showed 112 SNPs. Both the strains also exhibited deconjugation activity for the bile salts while genome analysis revealed that the L. plantarum strains E2C2 and E2C5 also have the ability to produce vitamins, biotin, alpha- and beta- glucosidase suggesting potential probiotic activities of the isolates. The description presented here is based on the draft genomes of strains E2C2 and E2C5 which are submitted to GenBank under the accession numbers LSST00000000.1 and LTCD00000000.1, respectively.

Keywords: Human Stool, Bacteria, Firmicutes, Lactobacillus plantarum

Introduction

The genome of lactobacilli is highly diversified which endorses them to occupy wide range of ecological habitats, including carbohydrate-rich environments [1], fermented meats [2], sourdoughs [3], plant-derived substrates [4] and different niches on and in the human body namely respiratory, gastrointestinal and urogenital tract [5, 6]. Owing to the beneficial effects offered by lactobacilli, they have been used as a gold standard in probiotic preparations. Consequently, many strains of lactobacilli

* Correspondence: yogesh@nccs.res.in tEqualcontributors

Microbial Culture Collection, NationalCentre for CellScience, Ganeshkhind, Pune 411007, India

Fulllist of author information is available at the end of the article

(3 BioMed Central

such as Lactobacillus acidophilus, L. amylovorus, L. brevis, L. bulgaricus, L. casei, L. fermentum, L. lactis, L. pentosus, and L. rhamnosus have been well characterized for their ability to produce extracellular proteins, exopo-lysaccharides, and lipoteichoic acids, which influence the health and physiology of the host by interacting with the epithelial cells and enhancing the host immune system [7-12].

From the array of various Lactobacillus species, Lacto-bacillus plantarum, an organism found in a variety of ecological environments, is a well characterized pro-biotic species. Recent genome analysis of Lactobacillus plantarum WCFS1 indicates that this organism is endowed with sets of genes essential for survival in gastrointestinal tract, interactions with other organisms

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in the gut, interactions with the host epithelial barrier and immune system, making it an extremely versatile probiotic bacterium [13] and that the genome of this organism is highly plastic [14]. Despite the extraordinary features possessed by L. plantarum, it suffers from some drawbacks. First, a study involving the pharmacokinetics of L. plantarum has indicated that it is a transient passenger in the gut [15]. Secondly, significant genome editing is required in order to gain the improved probiotic properties [16]. Both of these could be attributed to the incompatibility of the isolation source e.g. human saliva [17] and its implied target (gut). Thus, the search of indigenous L. plantarum strains (e.g. from human gut) is a thrust area in probiotic research and its implications to human health.

Microbial communities in the human gut are complex and astonishingly diverse in nature [18]. Despite the fact that lactobacilli contribute minutely to these trillions of cells, due to their beneficial roles in gut ecology, they are gaining attention in biomedical research [19]. Consequently, we focused on the isolation of oxalate tolerant Lactobacilli from healthy stool samples. Out of the 16 Lactobacillus isolates grown on MRS media, two isolates E2C2 and E2C5 showed comparatively higher tolerance to oxalic acid and bile salt. Owing to the fact that hyperoxaluria leads to dysbiosis in the human gut [20], these strains of L. plantarum, GRAS category organism, may specifically be useful in ameliorating the hyperoxaluria and associated complications. We, therefore, sequenced the genomes of these isolates using Illumina Miseq platform and compared their metabolic potentials.

Organism information

Classification and features

The two oxalic acid tolerant isolates, E2C2 and E2C5, were isolated from human stool samples by double enrichment method (100 and 200 mM/L sodium oxalate) using MRS (10 g enzymatic digest of animal tissue, 10 g beef extract, 5 g yeast extract, 20 g dextrose, 5 g sodium acetate, 1 g polysorbate 80, 2 g potassium phosphate, 2 g ammonium citrate, 0.1 g magnesium sulfate, 0.05 g manganese sulfate) medium. These bacterial isolates were maintained on MRS agar at the incubation temperature of 30 °C and at pH 6.8.

The strains were tested for phenotypic and biochemical characterization (Table 1). L. plantarum E2C2 and E2C5 isolates are Gram-positive, non-motile, non-spore forming and rod-shape in morphology (Fig. 1 and Table 1). While, in the case of bile salts, both the strains could grow up to 0.40% w/v of Oxgall (Sigma-Aldrich) tested for 24 h incubation at 30 °C. It was observed that these isolates have the ability to deconjugate the glyco-deoxycolate (bile salt) and this activity was confirmed by plate assay and TLC assay methods [21]. Ninhydrin

assay [22] was performed to quantitate the bile salt hydrolase production ability which was found to be maximum at the 72 h, 5.22 U and 5.27 U for glyco-deoxycholic acid as a substrate for E2C2 and E2C5 isolates, respectively (Fig. 2). They were able to utilize a large number of carbon compounds, namely dextrose, fructose, galactose, inulin, L-arabinose, maltose, man-nose, mannitol, melibiose, Na-gluconate, raffinose, salicin, sorbitol, sucrose, trehalose, xylose, etc. during their growth (Table 1).

16S rRNA gene sequencing and isDDH were used for the identification for isolates. 16S rRNA gene sequences were used for phylogenetic analysis using neighbour-joining method, which reveals that the two isolates E2C2 and E2C5 isolates are the members of Lactobacillaceae family, including Lactobacillus plantarum WCFS1, a previously reported probiotic bacterium isolated from human saliva [23] and Lactobacillus plantarum strain 52 [24], earlier isolated and identified from fermented foods (Fig. 3). The isDDH analysis was performed against type strain L. plantarum ATCC 14197T for ANI and GGDC [25, 26]. Both the isolates congruently showed 98.91% ANI and 93.60% GGDC score to the type strain, which are more than recommended thresholds (95% for ANI and 70% for GGDC) for the identification of the species, confirming both isolates as L. plantarum, belonging to the phylum Firmicutes and class Bacilli. Both the strains are deposited in National Collection of Industrial Microorganisms, Pune with accession no. NCIM 5603 (L. plantarum E2C2) and NCIM 5602 (L. plantarum E2C5). The isolates were also deposited in Microbial Culture Collection, Pune with accession no. MCC 3016 (L. plantarum E2C2) and MCC 3190 (L. plantarum E2C5).

Genome sequencing information

Genome project history

The isolates were selected for sequencing as part of an ongoing project investigating the association of gut microbiota with hyperoxaluric condition. Based on metabolic versatility and oxalate tolerance, strains E2C2 and E2C5, were selected and sequenced by Illumina MiSeq platform at Institute of Medical Microbiology, Germany. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession LSST00000000.1 and LTCD00000000.1 (Table 2). The version described in this paper is version LSST00 000000.1 and LTCD00000000.1.

Growth conditions and genomic DNA preparation

The E2C2 and E2C5 bacterial strains of L. plantarum were cultured in MRS agar (MA; Difco) at 30 °C under the aerobic condition for 3 days of incubation. Genomic DNA of the bacterial strains were isolated using a

Table 1 Classification and general features of L. plantarum E2C2 and L. plantarum E2C5

MIGS ID Property

L. plantarum E2C2

L. plantarum E2C5

Evidence codea

Domain

Phylum

Family

Species

Strain

Gram stain

Cell shape

Motility

Sporulation

Temperature range

Optimum temperature

pH range; Optimum

Carbon source

MIGS-6 Habitat

MIGS-6.3 Salinity tolerance

MIGS-22 Oxygen requirement

MIGS-15 Biotic relationship

MIGS-14 Pathogenicity

MIGS-4 Geographic location

MIGS-5 Sample collection

MIGS-4.1 Latitude

MIGS-4.2 Longitude

MIGS-4.4 Altitude

Bacteria

Firmicutes

Bacilli

Lactobacillales

Lactobacillaceae

Lactobacillus

Lactobacillus plantarum

Positive

non-motile spore forming 25 °C -39 °C 30 °C 3.5-6.5; 5

Xylose, Maltose, Fructose, Dextrose, Galactose, Raffinose, Melibiose, Trehalose, Sucrose, L-Arabinose, Mannose, Inulin, Na-gluconate, Salicin, Sorbitol, Mannitol, Cellobiose, Melezitose, ONPG, Esculin, Citrate, Malonate

Human stool

Facultatively anaerobic Free-living non-pathogen India/Asia November 2015 18.5204° N 73.8567° E 562 m a.s.l.

Bacteria

Firmicutes

Bacilli

Lactobacillales

Lactobacillaceae

Lactobacillus

Lactobacillus plantarum

Positive

non-motile spore forming 25 °C -39 °C 30 °C 3.5-6.5; 5

Xylose, Maltose, Fructose, Dextrose, Galactose, Raffinose, Melibiose, Trehalose, Sucrose, L-Arabinose, Mannose, Inulin, Na-gluconate, Salicin, Sorbitol, Mannitol, Cellobiose, Melezitose, ONPG, Esculin, Citrate, Malonate

Human stool

Facultatively anaerobic Free-living non-pathogen India/Asia November 2015 18.5204° N 73.8567° E 562 m a.s.l.

TAS [41] TAS [42, 43] TAS [44] TAS [45] TAS [46] TAS [43, 47-50] TAS [43, 47, 51]

TAS [43] IDA

TAS [43]

TAS [43] TAS [43] IDA

TAS [45] TAS [43] TAS [45] NAS

IDA IDA IDA IDA IDA

aEvidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [52]

Qiagen DNA extraction kit (Hilden, Germany) following manufacturer's instructions. Extracted DNA quality was assessed by 1.0% agarose gel electrophoresis, concentration and purity (A260/A280) were measured using Nano-Drop ND-1000 (NanoDrop technologies, Willingminton, USA). Extracted DNA samples of the strains were preserved at -20 °C until further processing.

assemblers SPAdes 3.5.0 [28] and DNA star assembler v. 11.2.1.25. More than 6 million good quality paired-end reads were obtained from both the strains, which accounted for an approximate 100x sequencing coverage. After assembly, it was found that the draft genomes of L. plantarum E2C2 and L. plantarum E2C5 contained 94 and 99 scaffolds respectively.

Genome sequencing and assembly

The bacterial genomes of L. plantarum E2C2 and L. plantarum E2C5 were sequenced by Illumina MiSeq platform using 2x300 paired-end libraries. Sequence quality of both the genomes was analyzed for quality control using FastQC software [27]. After analysis, raw sequences were trimmed and assembled using de novo

Genome annotation

Assembled genomes of both the strains were annotated using RAST version 2.0 [29] and the NCBI Prokaryotic Genome Annotation Pipeline [30]. Protein-encoding genes, tRNA and rRNA genes of the genomes were predicted using Glimmer version 3.02 [31], tRNA_scan-SE [32], and RNAmmer [33], respectively. Protein coding

Lactobacillus plantarum 16 (CP006033.1) Lactobacillus plantarum ZJ316 (CP004082.1) Lactobacillus plantarum subsp.plantarum P-8 (CP005942.2) Lactobacillus plantarum strain JBE245 (CP014780.1) Lactobacillus plantarum E2C2 Lactobacillus plantarum strain Zhang-LL (CP011769.1) Lactobacillus plantarum strain B21 (CP010528.1) Lactobacillus plantarum strain ZS2058 (CP012343.1) Lactobacillus plantarum WCFS1 (AL935263.2) Lactobacillus plantarum strain 5-2 (CP009236.1) Lactobacillus plantarum DOM La (CP004406.1) Lactobacillus plantarum E2C5 Staphylococcus aureus (X70648)

Fig. 1 Neighbour-joining phylogenetic tree is constructed based on 16S rRNA gene sequence. The tree is constructed using Jukes-Cantor distances. Then 1000 bootstraps analyses are conducted. Sequences represented in bold font are derived from isolated strains of this study

genes were analyzed by COG database [34] on WebMGA [35] and Pfam domains were predicted using NCBI Batch CD-Search Tool [36]. Transmembrane helix and signal peptide prediction of the genome was identified by using Phobius [37]. The presence of CRISPR repeats was predicted using the CRISPRFinder tools [38] (Table 4).

Genome properties

The draft genome sequence of L. plantarum strains E2C2 and E2C5 contained 3603,563 bp and 3615,168 bp, with GC content 43.99% and 43.97%, respectively. The reads of L. plantarum strains E2C2 and E2C5 were assembled into 94 and 99 contigs (N50, 235,913 bp, and 256,152 bp, respectively). The genome sequence of L. plantarum strain E2C2 included a total of 3504 genes and 3289 candidate CDS, giving a coding intensity of 94%. The genome was shown to encode at least 94 predicted RNAs, including 15 rRNAs and 75 tRNAs, and also 121 pseudogenes. Whereas, L. plantarum E2C5 genome which contained total 3523 genes and 3293 candidate CDS. L. plantarum E2C5 genome contained 95

predicted RNAs including 16 rRNAs and 75 tRNAs, and also 135 pseudogenes (Table 3). The draft genome size of the strains E2C2 and E2C5 was more than average of L. plantarum genome size that has been reported in public databases. It was found that most of the predicted genes (87.19% and 87.15% of strains E2C2 and E2C5, respectively) code for proteins which involved in major metabolic pathways were assigned to one of the 25 functional COG categories while the remaining genes were assigned as unknown functional proteins (Table 4).

Insights from the genome sequences

Genome sequence analysis of L. plantarum strains E2C2 and E2C5 showed a presence of common subsystem structure, i.e., carbohydrate and protein metabolisms, iron acquisition and metabolism, chemotaxis, stress response, secondary metabolism, nitrogen metabolism, dormancy and sporulation. Genome analysis of both the strains showed that more than 800 genes are present for carbohydrate metabolism indicating a diverse carbohydrate utilization pattern or abilities that include C1- metabolism, organic acids, mono-, di- and polysaccharides

metabolisms. Lactobacillus is well known for its capability to grow in protein-rich environments and contains protein degradation enzymes/machinery, therefore it is well adapted to these conditions. It was observed that both the strains have more than 50 protein degrading

enzymes/transport systems that include metallo-carboxypeptidases, dipeptidase, proteasome and many ATP-dependent uptake systems. A large number of stress response systems that include oxidative stress, heat shock and cold shock are present in both the

Fig. 3 Bile salt hydrolase activity of Lactobacillus plantarum E2C2 and E2C5 isolates (a) Plate assay showing precipitation zones around the line of inoculation in triplicates (b) TLC plate assay showing deconjugation ability and (c & d) Ninhydrin assay indicating quantification of glycine removal by deconjugation ability

Table 2 Project information

MIGS ID Property L. plantarum E2C2 L. plantarum E2C25

MIGS 31 Finishing quality High-quality draft High-quality draft

MIGS-28 Libraries used 300 bp 300 bp

MIGS 29 Sequencing platforms Illumina MiSeq Illumina MiSeq

MIGS 31.2 Fold coverage 100 x 100 x

MIGS 30 Assemblers DNASTAR assembler v. 11.2.1.25 DNASTAR assembler v. 11.2.1.25

MIGS 32 Gene calling method RAST RAST

Locus Tag AYO51 AZJ01

Genbank ID LSST00000000.1 LTCD00000000.1

GenBank Date of Release 03/23/2016 03/25/2016

GOLD ID Gs0118511 Gs0120378

BIOPROJECT PRJNA311909 PRJNA313343

MIGS 13 Source Materialldentifier NCIM 5603, MCC 3016 NCIM 5602, MCC 3190

Project relevance Human stool bacteria Human stool bacteria

strains. Stress response genes, namely sodA, sodB, HPI, HPII and CCP for reactive oxygen species; PRP, Rex, OxyR, Fnr, ZUR and FUR for oxidative stress; HrcA, GrpE and fam for heat shock response were identified. In L. plantarum strains E2C2 and E2C5, genes for alpha-glucosidase, choloylglycine hydrolase, alpha-L-rhamnosidase essential for antidiabetic, hydrolysis of bile salt in the small intestine, adaptation to changing nutritional resources are noted. Therefore, the analysis suggests that both the L. plantarum strains (E2C2 and E2C5) can be used in multi-therapeutic aspects. The presence of biotin and other cofactors, vitamins, prosthetic groups and pigment synthesis genes are observed

Table 3 Genome statistics Species Attribute

in the genome of both the strains, suggesting their ability to produce bioactive compounds. Considerable variation was not observed in the remaining subsystems that indicates biochemical homogeneity and similar capabilities of the strains in substrate utilization and processing. In addition, both L. plantarum E2C2 and L. plantarum E2C5 contain sulfur cycling, cobalt, zinc, and cadmium resistance genes.

Extended insights

Comparison of the strains E2C2 and E2C5 genome showed 99.99% shared CDS and only 112 SNPs among the core genome, thus overall demonstrating the high

L. plantarum E2C2

Value % of Total

3,603,563 100.00

2,684,877 74.5

1,585,330 43.9

3504 100

3289 93.8

94 2.6

121 3.4

2416 68.9

2868 81.8

2952 89.7

278 7.9

755 21.5

1 0.028

L. plantarum E2C5

Value % of Total

3,615,168 100.00

2690, 385 74.4

1589, 803 43.9

3523 100

3293 93.4

95 2.6

135 3.8

2426 68.9

2869 81.4

2969 90.1

275 7.8

755 21.4

1 0.028

Genome size (bp)

DNA coding (bp)

DNAG + C (bp)

DNA scaffolds

Totalgenes

Protein coding genes

RNA genes

Pseudo genes

Genes in internal clusters

Genes with function prediction

Genes assigned to COGs

Genes with Pfam domains

Genes with signal peptides

Genes with transmembrane helices

CRISPR repeats

Table 4 Number of genes associated with general COG functional categories

Code Lactobacillus plantarum Description

E2C2 E2C5 WCFS1

Value % age Value % age Value

J 213 6.47 213 6.47 197

A 0 0 0 0 0

K 313 9.51 310 9.41 259

L 166 5.04 168 5.10 103

B 0 0 0 0 0

D 49 1.48 48 1.45 33

V 92 2.79 92 2.79 76

T 106 3.22 104 3.15 86

M 186 5.65 184 5.58 158

N 22 0.66 22 0.66 10

U 26 0.79 26 0.78 17

O 99 3.01 100 3.03 83

C 118 3.58 118 3.58 101

G 286 8.69 286 8.68 265

E 218 6.62 219 6.65 183

F 103 3.13 104 3.15 89

H 116 3.52 116 3.52 86

I 97 2.94 96 2.91 80

P 121 3.67 121 3.67 105

Q 19 0.57 20 0.60 23

R 8 5.74 8 5.74 174

S 235 7.14 236 7.16 201

X 94 2.85 97 2.94 55

- 421 12.80 424 12.87 -

The total is based on the total number of protein coding genes in the genome

similarity of the two genomes (Tables 3 and 4). The high similarity of the two isolates, despite the different source of isolation, is an indication of their selective adaptation to the gut environment. But based on COG data analysis it was found that these two strains E2C2 and E2C5 were differed from each other with respect to number of protein coding genes namely signal transduction mechanisms, cell wall/membrane biogenesis, Mobilome: prophages, transposons, etc. Oxalate tolerance ability of the two isolates is an important feature to note. In the hyperoxaluric condition, human gut often acts as a primary excretory organ of oxalate [39] and higher oxalate concentration in the gut has been linked with dysbiosis [20]. In the light of oxalate tolerance ability of the E2C2 and E2C5 isolates, their use as probiotics for hyperoxa-luric patients is anticipated. In addition, genomes of strains E2C2 and E2C5 were compared with the reference strain, Lactobacillus plantarum WCFS1 [17]. The comparison revealed that the three genomes comprised 2639 genes in common at 80% coverage and 90%

Translation, ribosomalstructure and biogenesis RNA processing and modification Transcription

Replication, recombination and repair

Chromatin structure and dynamics

Cellcycle control, Celldivision, chromosome partitioning

Defense mechanisms

Signaltransduction mechanisms

Cellwall/membrane biogenesis

Cellmotility

Intracellular trafficking and secretion

Posttranslationalmodification, protein turnover, chaperones

Energy production and conversion

Carbohydrate transport and metabolism

Amino acid transport and metabolism

Nucleotide transport and metabolism

Coenzyme transport and metabolism

Lipid transport and metabolism

Inorganic ion transport and metabolism

Secondary metabolites biosynthesis, transport and catabolism

Generalfunction prediction only

Function unknown

Mobilome: prophages, transposons

Not in COGs

sequence identity [40]. E2C2 and E2C5 both contained an additional 345 genes while WCFS1 strain contained additional 265 genes. Further, about 344 genes were exclusively found in strains E2C2 and E2C5 as compared to strain WCFS1. When COG categories compared, a significant difference was observed for the functional annotation of the genes. COGs functional categories could be assigned to 2868 and 2869 genes for E2C2 and E2C5 respectively, while in case of WCFS1 only 2384 genes could be categorised by COGs (Table 4).

Conclusions

Considering the high genetic versatility of Lactobacillus plantarum [14], it is important to sequence as many strains as possible to account for the genetic variability and their association with specific probiotic features such as oxalate tolerance. In this study, we provide the in-depth genome analysis of two oxalic acid and bile acid tolerant isolates- L. plantarum E2C2 and L. plantarum E2C5 obtained from healthy human stool samples.

Genomic as well as phenotypic analysis reveals that both 9. the isolates are coherent belonging to a single genetic lineage. The two strains described here can be an intri- 10 guing target to be explored further for their probiotics potentials in managing the specific metabolic disorders such as hyperoxaluria. 11

Abbreviations

ANI: Average nucleotide identity; CDS: Coding DNA sequences or protein coding genes; GGDC: Genome-to-genome distance calculator; GRAS: Generally regarded as safe; isDDH: In silico DNA-DNA hybridisation; MRS medium: De Man, Rogosa and Sharpe medium

Acknowledgements

MS acknowledges Council of Scientific and Industrial Research, New Delhi, for fellowship. Authors are thankful to the Department of Biotechnology, Government of India; Microbial Culture Collection Project (BT/PR10054/NDB/ 52/94/2007) for financial aids. Authors are thankful to Dr. Shashi Ghodake, DDS, Middletown, CT 06457, USA for proofreading of the manuscript.

Authors' contributions

Conceived and designed the experiments: MS, DP, SD, SB, RG, YS. Isolation, biochemical characterization, Genomic DNA, and microscopy: MS, DP, SB, TH. Genome sequencing: SD. Manuscript preparation: DP, MS, SB. Bioinformatics analysis: DP, MS, SD. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests. Author details

1 Microbial Culture Collection, National Centre for Cell Science, Ganeshkhind, Pune 411007, India. institute of Medical Microbiology, Biomedizinisches Forschungszentrum Seltersberg, Schubertstr. 81, Giessen 35392, Germany. 3Department of Zoology, Savitribai Phule Pune University, Ganeshkhind, Pune 411007, India. 4Biochemical Science Division, CSIR-National Chemical Laboratory, Homi Bhabha Road, Pune 411008, India. 5Department of Urology, RCSM Govt. Medical College, CPR Hospital Compound, Bhausingji Rd, Kolhapur 416002, India.

Received: 12 June 2016 Accepted: 7 December 2016 Published online: 30 January 2017

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