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    Phylogenetic analysis of housekeeping genes of Streptococcus agalatiae isolated from bulk tank milk samples in Colombia

    Final assignment for Bioinformatic course winter,  2016*

    University of Prince Edward Island

    Student: 

    Claudia Gisela Cobo-Angel1

    The data for this assignment was collected in collaboration with:

    Ana Sofia Jaramillo1;Sandra Bibiana Aguilar1; Juan Carlos Rodriguez-Lecompte2; Javier Sanchez2;  Ruth Zadoks3; Alejandro Ceballos1

    1Research Group in Biology of Livestock and Animal Science; CLEV group. Universidad de Caldas, Manizales, Caldas, Colombia.

    2Atlantic Veterinary College, University of Prince Edward Island, Canada.

    3Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, United Kingdom

    *This analysis was made by the student as part of the course, the collaborators did not reviewed the manuscript. The information here is only informative for the course, and is not scientific evidence

    Abstract

    S. agalactiae, a Gram positive coccus, aerobic, classified in the group B of Lacefield, is a pathogen of the mammary gland in bovines, causing subclinical mastitis, causing economical loses to the dairy industry. Additionally, it is also a human pathogen; responsible of the disease known as “neonatal sepsis”, in adults, it can cause meningitis, abscess, urinary infections and arthritis.

    The objective of this work is to present a phylogenetic analysis of 11 isolates of S. agalactiae, obtained from bulk tank milk samples from Colombian dairy farms, through the DNA sequences of seven “housekeeping” genes, which are highly conserved and codify for energetic metabolism functions of the pathogen.

    Bacterial DNA extraction and amplification was made for the following genes: Alcohol dehydrogenase (Adhp), Phenylalanine/tRNA ligase (Phes), Serine/Threonine Kinase (Atr), Glutamine synthetase (Glna), Succinate dehydrogenase (Sdha), Glucokinase (glck), and transketolase (Tkt). Sequences of approximately 600 pb were obtained for each gene, by sequencing the amplicons, using sanger technology. 

    The phylogenetic analysis was performed by multiple sequence alignment, and the subsequent phylogenetic tree of each gene using Clustal W®, in order to evaluate possible evolutionary processes among isolates. This comparative phylogenetic analysis showed that the use of only one of the housekeeping genes selected, is not an appropriate phylogenetic marker for S. agalactiae, since the individual analysis of individual genes showed inconsistent relation among isolates. However, the combination of genes in a multilocus analysis such as MLST typing provide a reliable indication of genetic variation and clusters among the species.

    Keywords: Housekeeping, phylogenetic, Streptococcus

     

    Introduction

     Streptococcus agalactiae is a gram positive coccus, aerobic, encapsulated, classified inside the group B by Lancefield (Lancefield, 1934). In bovines is considered a major mastitis pathogen due to the large effect on milk quality and production. It produces mastitis, generally subclinical and chronic, often highly contagious, and often with low probability of self-cure (Keefe, 2012).

    Moreover, S. agalactiae is also a significant human pathogen, and represents the main cause of neonatal septicemia, producing severe infections in newborns, infants and elderly (Bisharat et al., 2004). In women, this pathogen can cause abscess and mastitis during lactation, which is a risk factor for the infant infection (Dinger et al., 2002). In adults, asymptomatic colonization with S. agalactiae is frequent (20-40%) (Manning et al., 2008). However, it may cause meningitis or septicemia, as well as localized infection such as subcutaneous abscesses, urinary tract infections or arthritis (Chaiwarith et al., 2011), reaching mortality rates of approximately 15% in developed countries (Pereira et al., 2010).

    The interspecies transmission is still a major question on the S. agalactiae epidemiology, and to elucidate this, it is necessary to study and compare the phylogenetic relation of the isolates from bovines and humans. Some of the methods include the analysis of highly conserved sequences in a multi locus sequence analysis, or the identification of molecular markers (Zadoks and Schukken, 2006).  This study presents a phylogenetic analysis of S. agalactiae isolated from bovine milk, based on seven housekeeping genes as individual molecular markers and Multi Locus Sequence Type (MLST), analysis to compare both approaches.

    Material and methods

    Bacteria isolation and confirmation

    A total of 11 isolates of S.  agalacatiae were obtained from bulk tank milk in the western of Colombia, identification was made based on culture and CAMP reaction, following the methodology proposed by National Mastitis Council (NMC). Chromosomal DNA was isolated from presumptive positive and negative colonies, using an UltraClean Microbial Isolation Kit (MoBio®). Molecular DNA search was carried out by means the Polymerase Chain Reaction (PCR) to confirm isolates. The species-specific primers used for amplification (V1 and V2): V1 (5’-GCGTGCCTAATACATGCAA-3’) and V2 (5’-TACAACGCAGGTCCATCT-3’), directed to 16S rRNA. Genus—specific primers (C1 and C2) were used C1 (5’-TTTGGTGTTTACACTAGACTG-3’), C2 (5’-TGTGTTAATT ACTCTTATGCG-3’), when the amplification with primers V1 and V2 were negative (Elias et al., 2012). The amplification conditions were: initial denaturation of 94ºC for 4 min, 35 cycles of 94 ºC for 30 s, 45,1º for 30 s, 72º C for 30 s and a final extension of 72 ºC for 5 min.

    Housekeeping genes amplification and sequencing

    The following housekeeping genes were selected and amplified according to (Jones et al., 2003): Alcohol dehydrogenase (adhp), Phenylalanine/tRNA ligase (phes), Serine/Threonine Kinase (atr), Glutamine synthetase (glna), Succinate dehydrogenase (sdha), Glucokinase (glck), and transketolase (tkt). Primers are shown in Table 1. The amplification conditions were: initial denaturation of 94ºC for 3 min, 30cycles of 94 ºC for 1 min, 55 º for 45 s, 72º C for 1 min and a final extension of 72 ºC for 10 min. Amplicons were sequenced by sanger method using the primers described in the Table 1.

    Table 1. Primers used to amplify and sequencing the housekeeping genes

    Locus

    Forward (5′ to 3′)

    Reverse (5′ to 3′) amplicon size (bp)
    adhP amplification GTTGGTCATGGTGAAGCACT ACTGTACCTCCAGCACGAAC 672
    Sequencing GGTGTGTGCCATACTGATTT ACAGCAGTCACAACCACTCC 498
    pheS amplification GATTAAGGAGTAGTGGCACG TTGAGATCGCCCATTGAAAT 723
    Sequencing ATATCAACTCAAGAAAAGCT TGATGGAATTGATGGCTATG 501
    atr amplification CGATTCTCTCAGCTTTGTTA AAGAAATCTCTTGTGCGGAT 627
    Sequencing ATGGTTGAGCCAATTATTTC CCTTGCTCAACAATAATGCC 501
    glnA amplification CCGGCTACAGATGAACAATT CTGATAATTGCCATTCCACG 589
    Sequencing AATAAAGCAATGTTTGATGG GCATTGTTCCCTTCATTATC 498
    sdhA amplification AGAGCAAGCTAATAGCCAAC ATATCAGCAGCAACAAGTGC 646
    Sequencing AACATAGCAGAGCTCATGAT GGGACTTCAACTAAACCTGC 519
    glcK amplification CTCGGAGGAACGACCATTAA CTTGTAACAGTATCACCGTT 607
    Sequencing GGTATCTTGACGCTTGAGGG ATCGCTGCTTTAATGGCAGA 459
    tkt amplification CCAGGCTTTGATTTAGTTGA AATAGCTTGTTGGCTTGAAA 859
    Sequencing ACACTTCATGGTGATGGTTG TGACCTAGGTCATGAGCTTT 480

    Sequence analysis

    Sequences were examined and assembled using Seqman® software (DNAStar, Madison, WI). Multiple sequence alignment was performed using Clustal-Omega®, with the following parameters: Gap penalty: 15, gap length penalty: 6.66, DNA transition weight: 0.5. Afterwards, the alignment was used to create and bootstrap the phylogenetic tree on DNAStar software, using the method of neighbor joining. Bootstrapping was made with 1000 replicates and a random seed of 111.

    On the other hand, MLST typing was performed, by assigning an allele number for individual genes using alignments of each gene sequence against the database available at http://pubmlst.org/sagalactiae/. Each isolate was therefore designated by a seven-integer number, one per gene, constituting its allelic profile. Isolates with the same allelic profile were assigned to the same sequence type (ST) (Jones et al., 2003).  Concatenated sequences of the seven genes were used to make a multiple sequence alignment and subsequently phylogenetic tree, with the same parameters than the independent sequence for each gene.

    Results and discussion

    Housekeeping genes analyzed independently

    Sequences of housekeeping genes are highly conserved because they codified for metabolic functions, which are essential for living cells. This kind of sequences has been used to identify evolutionary events (Vesth et al., 2010). Suitable molecular markers for identification purposes exhibit the smallest amount of heterogeneity within a species/genomovar and result in maximal separation between the different species/genomovars (Martens et al., 2008).

    In this study, the most conserved sequence among isolates was the atr genes, presenting less than one nucleotides substitutions per 100 residues (Fig 1g), followed by adhp with seven nucleotides substitutions per 100 residues, as maximum (Fig 1b). On the other hand, glck was the most variable gen, with 75 nucleotides substitutions per 100 residues in one of the groups (Fig 1e).

    Other studies had used some of the genes included in this analysis for bacterial classification, such as glck in Bacillus subtilis (Mesak et al., 2004) and phes in Lactobacillus (Naser et al., 2007). Nonetheless, the phylogenetic analysis of individual housekeeping genes, showed inconsistent relation among isolates (Fig. 1), except with few agreements between phylogenetic trees. For example, tkt and adhp clustered the isolate 1133 away from other isolates (Fig 1a & 1b), but tkt sequences of other isolates were very similar, instead adhp sequences clustered in two more groups.

    On the other hand, glna and adhp had similar distribution of isolates in the phylogenetic tree (Fig 1c & 1d), where the isolate 1038, clustered apart, and the rest of the isolates were close among them.

    The phylogenetic tree of atr was the only one where the isolates were divided in two groups of six and five isolates (Fig 1g), the genes phes and glck neither showed a pattern shared with other gene analysis.

     

    Fig. 1

    Fig 1. Phylogenetic analysis of the seven housekeeping genes, analyzed in this work. Only bootstrap values above 70 are shown.

     

    Some of the phylogenetic trees have low bootstrap values, which reflect the uncertainty of analyzing genes individually. It has been suggested that under favorable conditions, bootstrapping value of more than 70% correspond to a probability of more than 95% that the true phylogeny has been found (Hillis and Bull, 1993), which means that in this study phylogenetic tree of phes gene resulted very unreliable, because the lack of bootstrap values above 70% (Fig 1f).  This fact could indicate that phes gene is not a good molecular marker by itself in this bacteria.

    On the other hand, under certain conditions high bootstrap values can make the wrong phylogeny look good; therefore, the conditions of the analysis must be considered (Hillis and Bull, 1993). For instance, the atr gene did not have high bootstrapping values near to the root, reflecting a lack of consensus at the higher levels, but it had a high value (100%) close to the leafs (Fig 1a). This points to a significant probability of support for incorrect relationships for the isolates included, despite the high bootrapping value (Leekitcharoenphon et al., 2012).

    Even though some researchers has used the housekeeping genes as substitutes for 16S rRNA gen, showing improved efficacy in species identification , it remains unlikely that a single gene can always reflect the subtle differences between genomes of the same species (Leekitcharoenphon et al., 2012). However, these limitations of using a single gene may be improved by the simultaneous analysis of multiple genes, like Multi Locus Sequence Typing (MLST), which has found wide applications, especially in phylogenetic studies.

    MLST analysis

    The MLST typing produced five groups for the 11 isolates (Table 2). The most common group was ST 718 (36.36%), followed by 356 (27.27%) and 248 (18.18%).

    Table 2. Results of MLST typing

    Isolate ST adhp Phes Atr Glna Sdha Glck Tkt
    1106 718 13 1 81 13 1 1 1
    1108 718 13 1 81 13 1 1 1
    1384 718 13 1 81 13 1 1 1
    1134 718 13 1 81 13 1 1 1
    1034 356 13 1 2 41 1 1 5
    1133 356 13 1 2 41 1 1 5
    1125 356 13 1 2 41 1 1 5
    1284 248 16 18 2 2 9 2 2
    1038 248 16 18 2 2 9 2 2
    1393 337 13 1 1 13 1 2 1
    1137 1 1 1 2 1 1 2 2

    The phylogenetic analysis of the concatenated sequences showed the importance of using more than one gene as molecular marker, showing a different distribution compared to the phylogenetic trees of each gene, with bootstrapping values above 70 in all the branches (Fig. 2), indicating that the analysis is accurate and reliable (Hillis and Bull, 1993).  The phylogenetic tree successfully clustered in different groups the different STs, being the ST 248, the most distanced to the other groups (isolates 1125 and 1248). Other authors have reported that the concatenation of a sufficient number of genes overwhelms possible conflicting phylogenetic signals in different genes (Guo et al., 2008).

     

    Fig. 2 Phylogenetic tree of concatenated sequences of the seven housekeeping genes

    Fig. 2 Phylogenetic tree of concatenated sequences of the seven housekeeping genes

     

    Regarding the epidemiological implications of the groups found in this study, it is interesting the fact that one of the isolates were ST 1, which is frequently isolated from colonized pregnant women and infected neonates (Manning et al., 2009), finding this ST in milk could indicate adaptation process of the bacteria to survive in different media. Other authors have found this ST shared in humans and bovines (Manning et al., 2010), which support the hypothesis that interspecies transmission is possible in a farm environment. This needs more research.

    Conclusion

    This comparative phylogenetic analysis showed that the use of only one of the housekeeping genes selected, is not an appropriate phylogenetic marker for S. agalactiae. However, the combination of genes in a multilocus analysis such as MLST typing provide a reliable indication of genetic variation and clusters among the species. Other multilocus schemes should be investigated to find the most suitable marker for this pathogen.

    References

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