Cultural
Cultural and metagenomic based identifi cation of a microbiome from
and metagenomic based identifi cation of a microbiome from
subclinical mastitis in cows
subclinical mastitis in cows
Bharat B. BhanderiBharat B. Bhanderi11*, Mayurdhvaj K. Jhala*, Mayurdhvaj K. Jhala11, Viral B. Ahir, Viral B. Ahir22, ,
Vaibhav D. Bhatt
Vaibhav D. Bhatt22,, and Chaitanya G. Joshiand Chaitanya G. Joshi22
1
1Department of Veterinary Microbiology, College of Veterinary ScienceDepartment of Veterinary Microbiology, College of Veterinary Science and and Animal Husbandry, Anand Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India
Agricultural University, Anand, Gujarat, India 2
2 Department of Animal Biotechnology, College of Veterinary ScienceDepartment of Animal Biotechnology, College of Veterinary Science and and Animal Husbandry, Anand Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India
Agricultural University, Anand, Gujarat, India
________________________________________________________________________________________ ________________________________________________________________________________________
BHANDERI, B. B., M. K. JHALA, V. B. AHIR, V. D. BHATT, C. G. JOSHI BHANDERI, B. B., M. K. JHALA, V. B. AHIR, V. D. BHATT, C. G. JOSHI: Cultural
Cultural and and metagenomic based identifi cation of a microbiome from subclinical metagenomic based identifi cation of a microbiome from subclinical mastitis in cows
mastitis in cows. Vet. arhiv 84, 215-228, 2014. ABSTRACT
Metagenomic and traditional microbial culture based analyses of milk samples from cows harbouring subclinical mastitis pathogens were carried out to identify the microbial community structure of milk. A total of 77 Triple cross (TP), Kankrej and Gir lactating cows and 301 quarters were screened for subclinical mastitis. A total of 106 isolates belonging to fi ve different microbial genera were recovered from 91 quarters of 41 cows, including 15 quarters having mixed bacterial infections by cultural examination. Pyrosequencing readings obtained from the breed wise pooled DNA of subclinical mastitis milk samples were analyzed using the SEED subsystem database of Meta Genome Rapid Annotation with Subsystem Technology (MG-RAST). Among the fi ve genera, Staphylococcus, Streptococcus, Micrococcus, Bacillus and Escherichia, detected in the subclinical mastitis milk samples by culture based methods, four genera, Staphylococcus, Streptococcus, Bacillus and Escherichia, were identifi ed in the corresponding pyrosequencing data, while Micrococcus was not found. In contrast, the pyrosequencing yielded 28 bacterial species, of which only two species, S. aureus and E. coli, were identifi ed by the cultural method. S. agalactiae, the third species identifi ed by cultural method, was not found in the pyrosequencing data. Metagenomic analysis additionally identifi ed 19 genera and 26 species in comparison with the routine cultural methods. Many of the fastidious / anaerobic bacterial organisms, which are diffi cult to cultivate by routine methods, were identifi ed by metagenomic analyses.
Key words: subclinical mastitis, metagenomic, cows
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*Corresponding author:
Dr. Bharat B. Bhanderi, Assistant Professor, Department of Veterinary Microbiology, College of Veterinary Science and Animal Dr. Bharat B. Bhanderi, Assistant Professor, Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand - 388001, Gujarat, India, Phone: +91 92 2830 9371; E-mail: bbbhanderi@ Husbandry, Anand Agricultural University, Anand - 388001, Gujarat, India, Phone: +91 92 2830 9371; E-mail: bbbhanderi@ gmail.com
Introduction
Subclinical mastitis occurs without visible changes in the appearance of the milk and/or the udder, but milk production decreases by 10 to 20 per cent, with an undesirable e udder, but milk production decreases by 10 to 20 per cent, with an undesirable effect on its constituents and nutritional value, rendering it low quality and unfi t for effect on its constituents and nutritional value, rendering it low quality and unfi t for processing (
processing (HOLDWAY, 1992HOLDWAY, 1992). The annual economic loss due to mastitis in India has ). The annual economic loss due to mastitis in India has been estimated to be 7165 crores (
been estimated to be 7165 crores (BASAL and GUPTA, 2010BASAL and GUPTA, 2010). Mastitis is a multietiological ). Mastitis is a multietiological disease, of which about 95 per cent of the reported cases are caused by
disease, of which about 95 per cent of the reported cases are caused by Streptococcus,Streptococcus, Staphylococcus
Staphylococcus and and E. coli E. coli ((JAIN et al., 2012; KANDEMIR et al., 2013JAIN et al., 2012; KANDEMIR et al., 2013). ).
Metagenomics is presently entering a new phase of development as the Metagenomics is presently entering a new phase of development as the implementation of the massively parallel throughput afforded by second-generation implementation of the massively parallel throughput afforded by second-generation sequencing approaches becomes more widespread and applied to an increasing number sequencing approaches becomes more widespread and applied to an increasing number of environments. Traditional dideoxy termination (Sanger) based DNA sequencing in of environments. Traditional dideoxy termination (Sanger) based DNA sequencing in metagenomics historically relied on large insert libraries, propogated in an
metagenomics historically relied on large insert libraries, propogated in an E. coli E. coli host. host. Genes that may have been otherwise unstable or toxic in the cloning vector host cell can Genes that may have been otherwise unstable or toxic in the cloning vector host cell can be accessed without cloning bias in the sequencing profi le. In addition to circumventing be accessed without cloning bias in the sequencing profi le. In addition to circumventing the need for cloning, the throughput afforded by second-generation sequencing the need for cloning, the throughput afforded by second-generation sequencing technology enables a new approach to comparative metagenomics (
technology enables a new approach to comparative metagenomics (MARGULIES et al., MARGULIES et al., 2005
2005). Sequence representation (abundance) can now be used to contextualize datasets ). Sequence representation (abundance) can now be used to contextualize datasets for driving pattern recognition and uncovering unique properties within natural microbial for driving pattern recognition and uncovering unique properties within natural microbial communities.
communities.
Pyrosequencing is a relatively new molecular technique with an incredible potential Pyrosequencing is a relatively new molecular technique with an incredible potential for metagenomic analysis. It is based upon what is known as a for metagenomic analysis. It is based upon what is known as a “sequencing-by-synthesis” method, utilizing specifi c enzymes to record each nucleotide inserted into a synthesis” method, utilizing specifi c enzymes to record each nucleotide inserted into a complementary DNA strand (
complementary DNA strand (AHMADIAN et al., 2006AHMADIAN et al., 2006). The pyrosequencing technique ). The pyrosequencing technique has been used successfully to evaluate the microbial diversity of soil samples, detect has been used successfully to evaluate the microbial diversity of soil samples, detect medically signifi cant pathogens, and distinguish different species of
medically signifi cant pathogens, and distinguish different species of Mycobacteria Mycobacteria ((JONASSON et al., 2002; ROESCH et al., 2007; TUOHY et al., 2005JONASSON et al., 2002; ROESCH et al., 2007; TUOHY et al., 2005). Recently, work related ). Recently, work related to milk microbiome signatures of subclinical mastitis-affected cattle has been reported to milk microbiome signatures of subclinical mastitis-affected cattle has been reported ((BHATT et al., 2012BHATT et al., 2012), and it highlighted the usefulness of the metagenomic approach ), and it highlighted the usefulness of the metagenomic approach in understanding the possible role of the microbiome pool in subclinical mastitis in a in understanding the possible role of the microbiome pool in subclinical mastitis in a comprehensive way. Although the concept behind pyrosequencing was developed in comprehensive way. Although the concept behind pyrosequencing was developed in the 1980s, the actual procedure was not presented before the mid-1990s by a group of the 1980s, the actual procedure was not presented before the mid-1990s by a group of researchers at the Royal Institute of Technology in Stockholm (
researchers at the Royal Institute of Technology in Stockholm (AHMADIAN et al., 2006; AHMADIAN et al., 2006; RONAGHI et al., 1996
RONAGHI et al., 1996).).
Only 0.001-0.1 per cent of the total microbes in sea water, 0.25 per cent in fresh Only 0.001-0.1 per cent of the total microbes in sea water, 0.25 per cent in fresh water, 0.25 per cent in sediments and only 0.3 per cent soil microorganisms are cultivable water, 0.25 per cent in sediments and only 0.3 per cent soil microorganisms are cultivable in vitro
in vitro ((AMANN et al., 1995AMANN et al., 1995). Further, milk culture may yield no bacteria from truly sub-). Further, milk culture may yield no bacteria from truly sub-clinically infected glands due to the presence of very low numbers of pathogens when clinically infected glands due to the presence of very low numbers of pathogens when samples are collected (
problematic for mastitis laboratories, veterinarians, and dairy producers, and studies have problematic for mastitis laboratories, veterinarians, and dairy producers, and studies have reported failure of growth of bacteria in up to 30 per cent of milk samples from clinical reported failure of growth of bacteria in up to 30 per cent of milk samples from clinical and subclinical bovine mastitis, even after 48 h of conventional culture (
and subclinical bovine mastitis, even after 48 h of conventional culture (SHARMA et al., SHARMA et al., 2009
2009). A number of workers carried out metagenomic studies using different samples ). A number of workers carried out metagenomic studies using different samples ((ANDERSSON et al., 2008; BHAYA et al., 2007; TURNBAUGH et al., 2009ANDERSSON et al., 2008; BHAYA et al., 2007; TURNBAUGH et al., 2009). Looking to the ). Looking to the economic importance of subclinical mastitis, the multiple etiological factors and paucity economic importance of subclinical mastitis, the multiple etiological factors and paucity of documented research on metagenomic analysis of subclinical mastitis samples, the of documented research on metagenomic analysis of subclinical mastitis samples, the present research was carried to determine the complex microbial diversity in mixed present research was carried to determine the complex microbial diversity in mixed populations causing subclinical mastitis in cows.
populations causing subclinical mastitis in cows.
Materials
Materials and and methodsmethods
Samples.
Samples. A total of 77 lactating cows were included, comprising: 31 Triple cross (TP) A total of 77 lactating cows were included, comprising: 31 Triple cross (TP) (Kankrej
(Kankrej Jersey Jersey Holstein Friesian), 29 Kankrej and 17 Gir affi liated with the farms of Holstein Friesian), 29 Kankrej and 17 Gir affi liated with the farms of the College of Veterinary Science and Animal Husbandry, Anand Agricultural University, the College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, India. A total of 301 quarters were screened for subclinical mastitis, following the Anand, India. A total of 301 quarters were screened for subclinical mastitis, following the guidelines of the International Dairy Federation (1987). Seven quarters from fi ve cows guidelines of the International Dairy Federation (1987). Seven quarters from fi ve cows could not be included, because two cows had two nonfunctional quarters each and three could not be included, because two cows had two nonfunctional quarters each and three cows each had one quarter nonfunctional.
cows each had one quarter nonfunctional. Isolation
Isolation and and identifi cation of bacteria from the affected quarters. identifi cation of bacteria from the affected quarters. About 0.01 mL of About 0.01 mL of thoroughly mixed quarter milk samples were inoculated onto 5 per cent sheep blood agar, thoroughly mixed quarter milk samples were inoculated onto 5 per cent sheep blood agar, for primary bacterial isolation. The plates were incubated at 37 °C for 48 h. Following for primary bacterial isolation. The plates were incubated at 37 °C for 48 h. Following the incubation, the plates were examined for bacterial growth and the morphological the incubation, the plates were examined for bacterial growth and the morphological characteristics of the bacterial colonies were recorded. Identifi cation of the bacterial characteristics of the bacterial colonies were recorded. Identifi cation of the bacterial isolates was performed as per the method described by Cowan and Steel (
isolates was performed as per the method described by Cowan and Steel (COWAN and COWAN and STEEL, 1974
STEEL, 1974).).
Isolation of genomic DNA from the subclinical mastitis milk samples.
Isolation of genomic DNA from the subclinical mastitis milk samples. Milk samples of Milk samples of TP, Kankrej and Gir cows, which were found positive for subclinical mastitis, were used for TP, Kankrej and Gir cows, which were found positive for subclinical mastitis, were used for DNA extraction and breed wise pooled DNA was used for metagenomic analysis. Before DNA extraction and breed wise pooled DNA was used for metagenomic analysis. Before being subjected to DNA extraction, milk was fi ltered through 3 μ nitro cellulose fi lters being subjected to DNA extraction, milk was fi ltered through 3 μ nitro cellulose fi lters to remove somatic cells and separate microbial agents, and then the DNA template was to remove somatic cells and separate microbial agents, and then the DNA template was extracted from the subclinical mastitis milk samples according to
extracted from the subclinical mastitis milk samples according to CREMONESI et al. (2006)CREMONESI et al. (2006). . DNA was quantifi ed by ND-1000 spectrophotometer (Nanodrop Technologies, Inc USA). DNA was quantifi ed by ND-1000 spectrophotometer (Nanodrop Technologies, Inc USA).
Pyrosequencing
Pyrosequencing and and Sequence Analysis. Sequence Analysis. The breed wise pooled DNA of all three The breed wise pooled DNA of all three breeds of cows were subjected to a single pyrosequencing run, using a 454 Life Sciences breeds of cows were subjected to a single pyrosequencing run, using a 454 Life Sciences technology based high throughput sequencer (GS FLX 454 Life Sciences). In brief, technology based high throughput sequencer (GS FLX 454 Life Sciences). In brief, the samples were mobilized to generate smaller fragments 600-800 bp in size, and the the samples were mobilized to generate smaller fragments 600-800 bp in size, and the fragments were processed as described by the manufacturer, to apply adaptors at the ends, fragments were processed as described by the manufacturer, to apply adaptors at the ends, emulsion PCR and pyrosequencing. Sequencing was carried out for 200 cycles with the emulsion PCR and pyrosequencing. Sequencing was carried out for 200 cycles with the fl ow of A, T, G and C nucleotides sequentially and image capture. Capture images were fl ow of A, T, G and C nucleotides sequentially and image capture. Capture images were processed by image processing software.
Bioinformatics
Bioinformatics and and statistical analysis. statistical analysis. Data generated after the sequencing were Data generated after the sequencing were transferred to the High throughput cluster work station and then analysed using the inbuilt transferred to the High throughput cluster work station and then analysed using the inbuilt software GS Run Browser provided by Roche, for image processing and signal processing. software GS Run Browser provided by Roche, for image processing and signal processing. The generated data output was in the form of FASTA reads. The reads obtained in FASTA The generated data output was in the form of FASTA reads. The reads obtained in FASTA format of TP, Kankrej and Gir cows were uploaded into MG-RAST server version 2.0 format of TP, Kankrej and Gir cows were uploaded into MG-RAST server version 2.0 (http://metagenomics.nmpdr.org) (
(http://metagenomics.nmpdr.org) (MEYER et al., 2008; OVERBEEK et al., 2005MEYER et al., 2008; OVERBEEK et al., 2005) for further ) for further taxonomic information on the number of signifi cant hits of microorganisms present in taxonomic information on the number of signifi cant hits of microorganisms present in the pooled DNA of subclinical mastitis milk samples of TP, Kankrej and Gir cows, and the pooled DNA of subclinical mastitis milk samples of TP, Kankrej and Gir cows, and to compare our data sets against the database of other microorganisms available in the to compare our data sets against the database of other microorganisms available in the SEED subsystem of MG-RAST, which is referred to as the SEED database (
SEED subsystem of MG-RAST, which is referred to as the SEED database (MEYER et al., MEYER et al., 2008
2008). The IDs given by MG-RAST in the present study were: 4448440.3, 4448441.3 and ). The IDs given by MG-RAST in the present study were: 4448440.3, 4448441.3 and 4448442.3 for TP, Kankrej and Gir cows respectively.
4448442.3 for TP, Kankrej and Gir cows respectively.
Results Results
Isolation
Isolation and and identifi cation of bacteria from the affected quarters. identifi cation of bacteria from the affected quarters. A total of A total of 106 isolates of fi ve different microbial genera viz.:
106 isolates of fi ve different microbial genera viz.: Staphylococcus, Streptococcus, Staphylococcus, Streptococcus, Micrococcus, Escherichia
Micrococcus, Escherichia and and Bacillus, Bacillus, were recovered from 91 quarters of 41 cows, were recovered from 91 quarters of 41 cows, including 15 quarters with mixed bacterial infections on cultural examination. Amongst including 15 quarters with mixed bacterial infections on cultural examination. Amongst the isolates,
the isolates, Staphylococci Staphylococci were the most predominant bacterial species, followed bywere the most predominant bacterial species, followed by Str. Str. agalactiae, Micrococci, E. coli
agalactiae, Micrococci, E. coli and and Bacillus species. Bacillus species.
Metagenomic analysis of subclinical mastitis milk samples.
Metagenomic analysis of subclinical mastitis milk samples. In the present study, the In the present study, the pyrosequencing reads obtained from the breed wise pooled DNA of subclinical mastitis pyrosequencing reads obtained from the breed wise pooled DNA of subclinical mastitis milk samples of TP, Kankrej and Gir cows were analyzed using the SEED subsystem milk samples of TP, Kankrej and Gir cows were analyzed using the SEED subsystem database of MG-RAST. The MG-RAST server was designed for analyzing complete, or database of MG-RAST. The MG-RAST server was designed for analyzing complete, or near-complete, archaeal and bacterial genomes and the set of organisms present in the near-complete, archaeal and bacterial genomes and the set of organisms present in the sample,
sample, and it makes this available via the phylogenetic profi le. In the present study, the via the phylogenetic profi le. In the present study, the Eukaryote group was not further analyzed and was eliminated because of it matches the Eukaryote group was not further analyzed and was eliminated because of it matches the mammalian genome using the SEED subsystem database of the MG-RAST.
mammalian genome using the SEED subsystem database of the MG-RAST. TP cows.
TP cows. The TP MASTITIS READS data set contained 1,960 contigs totaling The TP MASTITIS READS data set contained 1,960 contigs totaling 274,190 basepairs (bp) with an average fragment length of 139.89 bp. The longest 274,190 basepairs (bp) with an average fragment length of 139.89 bp. The longest sequence length was 560 bp and shortest sequence length 40 bp. A total of 54 sequences sequence length was 560 bp and shortest sequence length 40 bp. A total of 54 sequences (2.76%) could be matched to proteins in the SEED subsystem (using an E-value of (2.76%) could be matched to proteins in the SEED subsystem (using an E-value of 111010-5-5). The metagenome received 122 hits against the SEED protein non-redundant ). The metagenome received 122 hits against the SEED protein non-redundant
database (6.22 % of the fragments) and, on the zero hits against the ribosomal RNA database (6.22 % of the fragments) and, on the zero hits against the ribosomal RNA database Greengenes (0.00%) using an E-value of 1
database Greengenes (0.00%) using an E-value of 11010-5-5 and a minimum alignment and a minimum alignment
length of 50 bp. length of 50 bp.
Classifi cation of microbial communities based on SEED subsystem. Only 2.90 per Classifi cation of microbial communities based on SEED subsystem. Only 2.90 per cent (57/1960) of the sequences could be phylogenetically identifi ed to the domain level cent (57/1960) of the sequences could be phylogenetically identifi ed to the domain level in SEED database using an E-value of 1
Bacteria with Eukaryote accounting for 50.88 per cent (29). In the bacterial domain, Bacteria with Eukaryote accounting for 50.88 per cent (29). In the bacterial domain, the different hierarchies of bacterial classifi cation identifi ed were phylum (4), class the different hierarchies of bacterial classifi cation identifi ed were phylum (4), class (6), subclass/order (11), family (15), genus (18) and species (21). The bacterial phylum (6), subclass/order (11), family (15), genus (18) and species (21). The bacterial phylum identifi ed were Chlamydiae/Verrucomicrobia group 3.57 per cent (1), Actinobacteria identifi ed were Chlamydiae/Verrucomicrobia group 3.57 per cent (1), Actinobacteria 10.71 per cent (3), Proteobacteria 64.29 per cent (18) and Firmicutes 21.43 per cent (6). 10.71 per cent (3), Proteobacteria 64.29 per cent (18) and Firmicutes 21.43 per cent (6). Six classes of bacteria identifi ed were Actinobacteria (4), Chlamydiae (1), Bacilli (5), Six classes of bacteria identifi ed were Actinobacteria (4), Chlamydiae (1), Bacilli (5), Alphaproteobacteria (1), Betaproteobacteria (6) and Gammaproteobacteria (11). Eleven Alphaproteobacteria (1), Betaproteobacteria (6) and Gammaproteobacteria (11). Eleven subclasses/order, fi fteen families, eighteen bacteria genus and twenty-one bacteria species subclasses/order, fi fteen families, eighteen bacteria genus and twenty-one bacteria species were identifi ed (Table 1).
were identifi ed (Table 1). Kankrej cows.
Kankrej cows. The KANKREJ MASTITIS data set contained 170 contigs, totaling The KANKREJ MASTITIS data set contained 170 contigs, totaling 17,727 bp, with an average fragment length of 104.28 bp.
17,727 bp, with an average fragment length of 104.28 bp. The longest sequence length The longest sequence length was 327 bp and the shortest sequence length 41 bp. A total of 39 sequences (22.94%) was 327 bp and the shortest sequence length 41 bp. A total of 39 sequences (22.94%) could be matched to proteins in the SEED subsystem (using an E-value of 1
could be matched to proteins in the SEED subsystem (using an E-value of 11010-5-5), based ), based
on 57 hits against the SEED protein non-redundant database (33.53 % of the fragments) on 57 hits against the SEED protein non-redundant database (33.53 % of the fragments) and on 2 hits against the ribosomal RNA database Greengenes (1.18%), using an E-value and on 2 hits against the ribosomal RNA database Greengenes (1.18%), using an E-value of 1
of 11010-5-5 and a minimum alignment length of 50 bp. and a minimum alignment length of 50 bp.
Classifi cation of microbial communities based on the SEED subsystem. Only 7.65 per Classifi cation of microbial communities based on the SEED subsystem. Only 7.65 per cent (13/170) of the sequences could be phylogenetically identifi ed to the domain level in cent (13/170) of the sequences could be phylogenetically identifi ed to the domain level in the SEED database, using an E-value of 1
the SEED database, using an E-value of 11010-5-5 and 100 per cent (13) of these were Bacteria. and 100 per cent (13) of these were Bacteria.
In the bacterial domain, the different hierarchies of bacterial classifi cation identifi ed In the bacterial domain, the different hierarchies of bacterial classifi cation identifi ed were phylum (3), class (5), order (6), family (6), genus (7) and species (7). Amongst the were phylum (3), class (5), order (6), family (6), genus (7) and species (7). Amongst the bacterial phylum identifi ed were Thermotogae 7.69 per cent (1), Proteobacteria 15.38 per bacterial phylum identifi ed were Thermotogae 7.69 per cent (1), Proteobacteria 15.38 per cent (2) and Firmicutes 76.92 per cent (10). Five classes of bacteria identifi ed were Bacilli cent (2) and Firmicutes 76.92 per cent (10). Five classes of bacteria identifi ed were Bacilli (9), Mollicutes (1), Alphaproteobacteria (1), Betaproteobacteria (1) and Thermotogae (1). (9), Mollicutes (1), Alphaproteobacteria (1), Betaproteobacteria (1) and Thermotogae (1). Six orders and families, seven genus and species were identifi ed (Table 2).
Six orders and families, seven genus and species were identifi ed (Table 2). Gir cows
Gir cows. The GIR MASTITIS data set contained 301 contigs totaling 42,548 bp . The GIR MASTITIS data set contained 301 contigs totaling 42,548 bp with an average fragment length of 141.36 bp. The longest sequence length was 454 bp with an average fragment length of 141.36 bp. The longest sequence length was 454 bp and the shortest sequence length 40 bp. A total of 12 sequences (3.99%) could be matched and the shortest sequence length 40 bp. A total of 12 sequences (3.99%) could be matched to proteins in the SEED subsystem (using an E-value of 1
to proteins in the SEED subsystem (using an E-value of 11010-5-5) based on 21 hits against ) based on 21 hits against
the SEED protein non-redundant database (6.98 % of the fragments) and on the 0 hit the SEED protein non-redundant database (6.98 % of the fragments) and on the 0 hit against the ribosomal RNA database Greengenes (0.00%) using an E-value of 1
against the ribosomal RNA database Greengenes (0.00%) using an E-value of 11010-5-5 and and
a minimum alignment length of 50 bp. a minimum alignment length of 50 bp.
Classifi cation of microbial communities based on the SEED subsystem. Only 4.32 Classifi cation of microbial communities based on the SEED subsystem. Only 4.32 per cent (13/301) of the sequences could be phylogenetically identifi ed to the domain per cent (13/301) of the sequences could be phylogenetically identifi ed to the domain level in the SEED database using an E-value of 1
level in the SEED database using an E-value of 11010-5-5. Of these, 84.62 per cent (11) . Of these, 84.62 per cent (11)
were Bacteria, and Eukaryota accounted for 15.38 per cent (2). In the bacterial domain, were Bacteria, and Eukaryota accounted for 15.38 per cent (2). In the bacterial domain, the different hierarchies of bacterial classifi cation identifi ed were phylum (2), class the different hierarchies of bacterial classifi cation identifi ed were phylum (2), class (2), order (4), family (4), genus (5) and species (6). The bacterial phyla identifi ed were (2), order (4), family (4), genus (5) and species (6). The bacterial phyla identifi ed were Proteobacteria 90.91 per cent (10) and Firmicutes 9.09 per cent (1). The two classes Proteobacteria 90.91 per cent (10) and Firmicutes 9.09 per cent (1). The two classes
Table 1. Identifi
cation of bacteria to the species level from subclinical masti
tis milk samples of
TP cows Sr . No. Domain Phylum Class Subclasses/Order Family / Genus Species Hits 1 Bacteria Actinobacteria Actinobacteria Actinobacteridae/ Actinomycetales Microbacteriaceae / Le ifson ia Le ifson ia xyl i subsp. xyl i 1 2 Propionibacteriaceae / Pr op ion ibacter ium Pr op ion ibacter ium acnes 1 3 Streptomycetaceae / Str eptomyces Str eptomyces coel icolor 1 4
Chlamydiae/ Verrucomicrobia group Chlamydiales
Chlamydiae Chlamydiae Chlamydiaceae / Chlamydoph ila Chlamydoph ila abortus 1 5 Firmicutes Bacilli Bacillales Bacillaceae / Bac illus Bac illus sp. 1 6 Staphylococcaceae / Staphylococcus Staphylococcus aur eus subsp. aur eus 1 7 Staphylococcus epiderm id is 2 8 Lactobacillales Lactobacillaceae / Lactobac illus Lactobac illus ac idoph ilus 1 9 Streptococcaceae / Str eptococcus Str eptococcus m it is 1 10 Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae / Caulobacter Caulobacter sp. 1
Table 1. Identifi
cation of bacteria to the species level from subclinical masti
tis milk samples of
TP cows (continued) Sr . No. Domain Phylum Class Subclasses/Order Family / Genus Species Hits 11 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae / Burkholder ia Burkholder ia cenocepac ia 1 12 Burkholder ia cepac ia 1 13 Burkholderiaceae / Ralston ia Ralston ia solanacearum 3 14 Nitrosomonadales Nitrosomonadaceae / Nitr osomonas N itr osomonas eur opaea 1 15 Gammaproteobacteria Alteromonadales Pseudoalteromonadaceae / Pseudoalter omonas Pseudoalter omonas atlant ica 1 16 Enterobacteriales Enterobacteriaceae / Salmonella Salmonella Dublin 1 17 Enterobacteriales Enterobacteriaceae / Serrat ia Serrat ia mar cescens 4 18 Pseudomonadales Pseudomonadaceae / Azotobacter Azotobacter v ineland ii 1 19 Pseudomonadaceae / Pseudomonas Pseudomonas aerug inosa 1 20 Pseudomonas mendoc ina 2 21 Xanthomonadales Xanthomonadaceae / Stenotr ophomonas Stenotr ophomonas maltoph il ia 1
Ta bl e 2. I de nt ifi c at io n of b ac te ri a to th e sp ec ie s le ve l f ro m s ub cl in ic al m as ti ti s m il k sa m pl es o f K an kr ej c ow s us in g Table 2. Identifi
cation of bacteria to the species level from subclinical masti
tis milk samples of Kankrej cows using
py ro se qu en ci ng pyrosequencing Sr . no Domain Phylum Class Subclasses / Order Family / Genus Species Hits 1 Bacteria Firmicutes Bacilli Bacillales Bacillaceae / Bac illus Bacillus subtilis subsp . subtilis 7 2 Bacillaceae / Exiguobacterium Exiguobacterium sp. 1 3 Lactobacillales Lactobacillaceae / Lactobac illus Lactobac illus delbrueck ii subsp . bulgar icus 1 4 Mollicutes Acholeplasmatales Acholeplasmataceae / Phytoplasma Aster yellows witches'-broom phytoplasma
A YWB 1 5 Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae / Parv ibaculum Parv ibaculum lavament ivorans 1 6 Betaproteobacteria Burkholderiales Burkholderiaceae / Ralston ia Ralston ia solanacearum 1 7 Thermotogae Thermotogae Thermotogales Thermotogaceae / Thermococcus Thermos ipho melanes iens is 1 Ta bl e 3. I de nt ifi c at io n of b ac te ri a to th e sp ec ie s le ve l f ro m s ub cl in ic al m as ti ti s m il k sa m pl es o f G ir c ow s us in g py ro se qu en ci ng Table 3. Identifi
cation of bacteria to the species level from subclinical masti
tis milk samples of Gir cows using pyrosequencing
Sr . no Domain Phylum Class Subclasses / Order Family / Genus Species Hits 1 Bacteria Firmicutes Bacilli Bacillales Bacillaceae / Bac illus Bac illus sp. 1 2 Proteobacteria Gamma -proteobacteria Aeromonadales Aeromonadaceae / Aer omonas Aer omonas hydr ophila subsp . hydr ophila 1 3 Enterobacteriales Enterobacteriaceae / Escher ich ia Escher ich ia col i 1 4 Enterobacteriaceae / Sh igella Sh igella boyd ii 1 5 Pseudomonadales Pseudomonadaceae / Pseudomonas Pseudomonas aerug inosa 6 6 7 8 Pseudomonas fl uor escens 1
of bacteria identifi ed were Bacilli (1) and Gammaproteobacteria (10). Four orders and of bacteria identifi ed were Bacilli (1) and Gammaproteobacteria (10). Four orders and families, fi ve genera and six species were identifi ed (Table 3)
families, fi ve genera and six species were identifi ed (Table 3)..
Breed wise comparison between cultural and metagenomic based identifi cation Breed wise comparison between cultural and metagenomic based identifi cation TP cows.
TP cows. In the cultural based methods, fi ve types of the bacteria were identifi ed In the cultural based methods, fi ve types of the bacteria were identifi ed at genus level (
at genus level (Staphylococcus, Streptococcus, Micrococcus, BacillusStaphylococcus, Streptococcus, Micrococcus, Bacillus and and Escherichia)Escherichia) and amongst the genera,
and amongst the genera, S. aureus, Str. agalactiaeS. aureus, Str. agalactiae and and E. coli E. coli were identifi ed at species were identifi ed at species level. In the comparison between cultural and metagenomic based identifi cation, the level. In the comparison between cultural and metagenomic based identifi cation, the genera
genera Staphylococcus, Streptococcus, BacillusStaphylococcus, Streptococcus, Bacillus and and Escherichia, Escherichia, identifi ed by cultural identifi ed by cultural methods, were also identifi ed by pyrosequencing, while
methods, were also identifi ed by pyrosequencing, while Micrococcus, Micrococcus, identifi ed by identifi ed by the cultural method, was not found by pyrosequencing. Fifteen bacterial genera, the cultural method, was not found by pyrosequencing. Fifteen bacterial genera, viz.:
viz.: Leifsonia, Propionibacterium, Streptomyces, Chlamydophila, Lactobacillus, Leifsonia, Propionibacterium, Streptomyces, Chlamydophila, Lactobacillus, Caulobacter, Burkholderia, Ralstonia, Nitrosomonas, Pseudoalteromonas, Salmonella, Caulobacter, Burkholderia, Ralstonia, Nitrosomonas, Pseudoalteromonas, Salmonella, Serratia, Azotobacter, Pseudomonas
Serratia, Azotobacter, Pseudomonas and and Stenotrophomonas, Stenotrophomonas, identifi ed at genus level identifi ed at genus level in pyrosequencing, were not identifi ed by cultural methods. In the cultural methods, in pyrosequencing, were not identifi ed by cultural methods. In the cultural methods, S. aureus,
S. aureus, identifi ed at species level, was also identifi ed in pyrosequencing. However, identifi ed at species level, was also identifi ed in pyrosequencing. However, Str. agalactiae
Str. agalactiae and and E. coli, E. coli, identifi ed by cultural methods, were not identifi ed by identifi ed by cultural methods, were not identifi ed by pyrosequencing. Eighteen species:
pyrosequencing. Eighteen species: Leifsonia xyli, Propionibacterium acnes, Streptomyces Leifsonia xyli, Propionibacterium acnes, Streptomyces coelicolor, Chlamydophila abortus, Staphylococcus epidermidis, Lactobacillus coelicolor, Chlamydophila abortus, Staphylococcus epidermidis, Lactobacillus acidophilus, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, acidophilus, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, Ralstonia solanacearum, Nitrosomonas europaea, Pseudoalteromonas atlantica, Ralstonia solanacearum, Nitrosomonas europaea, Pseudoalteromonas atlantica, Salmonella
Salmonella DublinDublin, Serratia marcescens, Azotobacter vinelandii, Pseudomonas , Serratia marcescens, Azotobacter vinelandii, Pseudomonas aeruginosa, Pseudomonas mendocina
aeruginosa, Pseudomonas mendocina and and Stenotrophomonas maltophilia,Stenotrophomonas maltophilia, identifi ed in identifi ed in pyrosequencing, were not identifi ed by cultural methods.
pyrosequencing, were not identifi ed by cultural methods. Kankrej cows.
Kankrej cows. In the cultural based methods, four genera, In the cultural based methods, four genera, Staphylococcus, Staphylococcus, Streptococcus, Micrococcus
Streptococcus, Micrococcus and and Bacillus, Bacillus, were identifi ed, and amongst these genera,were identifi ed, and amongst these genera, S. S. aureus
aureus and and Str. agalactiae Str. agalactiae were identifi ed at species level. In the comparison between were identifi ed at species level. In the comparison between cultural and metagenomic methods, the genus
cultural and metagenomic methods, the genus Bacillus, Bacillus, identifi ed by cultural methods, was identifi ed by cultural methods, was also identifi ed by pyrosequencing, while
also identifi ed by pyrosequencing, while Staphylococcus, StreptococcusStaphylococcus, Streptococcus and and Micrococcus Micrococcus genera,
genera, identifi ed by cultural methods, were not found by the pyrosequencing method. identifi ed by cultural methods, were not found by the pyrosequencing method. Six bacterial genera:
Six bacterial genera: Exiguobacterium, Lactobacillus, Phytoplasma, Parvibaculum, Exiguobacterium, Lactobacillus, Phytoplasma, Parvibaculum, Ralstonia
Ralstonia and and Thermococcus, Thermococcus, identifi ed at genus level in pyrosequencing, were not found identifi ed at genus level in pyrosequencing, were not found by cultural methods. In the cultural methods,
by cultural methods. In the cultural methods, S. aureus S. aureus and and S. agalactiae, S. agalactiae, identifi ed at identifi ed at species level, were not identifi ed by pyrosequencing. In pyrosequencing, six bacterial species level, were not identifi ed by pyrosequencing. In pyrosequencing, six bacterial species
species Bacillus subtilis, Lactobacillus delbrueckii, Aster yellows witches’-broom Bacillus subtilis, Lactobacillus delbrueckii, Aster yellows witches’-broom phytoplasma, Parvibaculum lavamentivorans, Ralstonia solanacearum
phytoplasma, Parvibaculum lavamentivorans, Ralstonia solanacearum and and Thermosipho Thermosipho melanesiensis
melanesiensis identifi ed, were not found by cultural methodsidentifi ed, were not found by cultural methods. . Gir cows.
Gir cows. In the cultural methods, the genera In the cultural methods, the genera Staphylococcus, Streptococcus, Staphylococcus, Streptococcus, Micrococcus
Micrococcus and and Escherichia Escherichia were identifi ed. In the comparison of cultural methods with were identifi ed. In the comparison of cultural methods with pyrosequencing, the
tthe pyrosequencing method. However, he pyrosequencing method. However, Staphylococcus, StreptococcusStaphylococcus, Streptococcus and and Micrococcus Micrococcus genera were not found in pyrosequencing. The genera
genera were not found in pyrosequencing. The genera Bacillus, Aeromonas, Shigella Bacillus, Aeromonas, Shigella and and Pseudomonas,
Pseudomonas, identifi ed by the pyrosequencing method, were not cultivated by cultural identifi ed by the pyrosequencing method, were not cultivated by cultural methods. In the culture based method, the species
methods. In the culture based method, the species Escherichia coli Escherichia coli identifi ed was also identifi ed was also found in pyrosequencing, but
found in pyrosequencing, but S. aureus S. aureus and and Str. agalactiae, Str. agalactiae, cultivated by cultural methods, cultivated by cultural methods, were not found in the pyrosequencing data. In pyrosequencing, the species
were not found in the pyrosequencing data. In pyrosequencing, the species Aeromonas Aeromonas hydrophila, Shigella boydii, Pseudomonas aeruginosa
hydrophila, Shigella boydii, Pseudomonas aeruginosa and and Pseudomonas fl uorescens Pseudomonas fl uorescens identifi ed were not observed by cultural methods.
identifi ed were not observed by cultural methods.
Discussion Discussion
It is widely accepted that up to 99% of the microbes in the environment cannot be It is widely accepted that up to 99% of the microbes in the environment cannot be readily cultivated (
readily cultivated (KAMAGATA and TAMAKI, 2005; SEKIGUCHI, 2006KAMAGATA and TAMAKI, 2005; SEKIGUCHI, 2006). To overcome ). To overcome these diffi culties and the limitations associated with cultivation techniques, different these diffi culties and the limitations associated with cultivation techniques, different DNA-based molecular methods have been developed for characterizing microbial species DNA-based molecular methods have been developed for characterizing microbial species and assemblages, and these have signifi cantly infl uenced our understanding of microbial and assemblages, and these have signifi cantly infl uenced our understanding of microbial diversity and ecology (
diversity and ecology (DELONG, 2005DELONG, 2005). ).
Overall comparison between cultural and metagenomic based identifi cation. Overall comparison between cultural and metagenomic based identifi cation. An An evaluation of the data obtained by the culture based and metagenomic approaches evaluation of the data obtained by the culture based and metagenomic approaches shows that these two methods display a staggering disparity in the results obtained by shows that these two methods display a staggering disparity in the results obtained by each method. Overall, out of fi ve genera,
each method. Overall, out of fi ve genera, Staphylococcus, Streptococcus, Micrococcus, Staphylococcus, Streptococcus, Micrococcus, Bacillus
Bacillus and and Escherichia, Escherichia, detected in the subclinical mastitis milk samples of TP, Gir and detected in the subclinical mastitis milk samples of TP, Gir and Kankrej breeds by culture based methods, four genera,
Kankrej breeds by culture based methods, four genera, Staphylococcus, Streptococcus, Staphylococcus, Streptococcus, Bacillus
Bacillus and and Escherichia, Escherichia, were identifi ed in the corresponding pyrosequencing data, whilewere identifi ed in the corresponding pyrosequencing data, while Micrococcus,
Micrococcus, identifi ed by culture based methods, was not found in the pyrosequencing identifi ed by culture based methods, was not found in the pyrosequencing data.
data.
In contrast, pyrosequencing yielded 28 bacterial species, of which only two species, In contrast, pyrosequencing yielded 28 bacterial species, of which only two species, S. aureus
S. aureus and and E. coli, E. coli, were identifi ed by the cultural methods. were identifi ed by the cultural methods. Str. agalactiae,Str. agalactiae, the the third species identifi ed by the cultural method, was not found in the pyrosequencing third species identifi ed by the cultural method, was not found in the pyrosequencing data. In pyrosequencing, overall 28 bacterial species were identifi ed from all the three data. In pyrosequencing, overall 28 bacterial species were identifi ed from all the three breeds of cows, viz:
breeds of cows, viz: Leifsonia xyli, Propionibacterium acnes, Streptomyces coelicolor, Leifsonia xyli, Propionibacterium acnes, Streptomyces coelicolor, Chlamydophila abortus, S. aureus, Staphylococcus epidermidis, Lactobacillus Chlamydophila abortus, S. aureus, Staphylococcus epidermidis, Lactobacillus acidophilus, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, acidophilus, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, Ralstonia solanacearum, Nitrosomonas europaea, Pseudoalteromonas atlantica, Ralstonia solanacearum, Nitrosomonas europaea, Pseudoalteromonas atlantica, Salmonella
Salmonella Dublin, Dublin, Serratia marcescens, Azotobacter vinelandii, Pseudomonas Serratia marcescens, Azotobacter vinelandii, Pseudomonas aeruginosa, Pseudomonas mendocina, Stenotrophomonas maltophilia, Bacillus subtilis, aeruginosa, Pseudomonas mendocina, Stenotrophomonas maltophilia, Bacillus subtilis, Lactobacillus delbrueckii,
Lactobacillus delbrueckii, Aster yellows witches’-broom phytoplasma,Aster yellows witches’-broom phytoplasma, Parvibaculum Parvibaculum lavamentivorans, Thermosipho melanesiensis, Aeromonas hydrophila, E. coli, Shigella lavamentivorans, Thermosipho melanesiensis, Aeromonas hydrophila, E. coli, Shigella boydii
boydii and and Pseudomonas fl uorescens. Pseudomonas fl uorescens. Of these, apart from Of these, apart from S. aureusS. aureus and and E. coli, E. coli, 26 26 bacterial species were additionally identifi ed and not by the culture based method. bacterial species were additionally identifi ed and not by the culture based method.
The results revealed that Proteobacteria and Firmicutes were the main phyla in the The results revealed that Proteobacteria and Firmicutes were the main phyla in the milk sampled from three breeds. Proteobacteria is a diverse phylum and includes a wide milk sampled from three breeds. Proteobacteria is a diverse phylum and includes a wide variety of pathogens (
variety of pathogens (MADIGAN and MARTINKO 2005MADIGAN and MARTINKO 2005). Proteobacteria are Gram-negative ). Proteobacteria are Gram-negative and are considered as environmental mastitis pathogens (
and are considered as environmental mastitis pathogens (HOGAN et al., 1999HOGAN et al., 1999), while ), while Gram-positive Firmicutes are generally considered as contagious mastitis pathogens Gram-positive Firmicutes are generally considered as contagious mastitis pathogens ((SMITH and HOGAN, 1995SMITH and HOGAN, 1995).).
Public health importance.
Public health importance. In the present study, out of 28 bacterial species identifi ed by In the present study, out of 28 bacterial species identifi ed by the metagenomic method, seventeen species are known to cause disease or opportunistic the metagenomic method, seventeen species are known to cause disease or opportunistic infections in humans. These are:
infections in humans. These are: Propionibacterium acnes, Chlamydophila abortus, S. Propionibacterium acnes, Chlamydophila abortus, S. aureus, Staphylococcus epidermidis, Streptococcus mitis, Burkholderia cenocepacia, aureus, Staphylococcus epidermidis, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, Salmonella
Burkholderia cepacia, Salmonella DublinDublin, Serratia marcescens, Pseudomonas aeruginosa, , Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas mendocina, Stenotrophomonas maltophilia, Bacillus subtilis, Aeromonas Pseudomonas mendocina, Stenotrophomonas maltophilia, Bacillus subtilis, Aeromonas hydrophila, E. coli, Shigella boydii
hydrophila, E. coli, Shigella boydii and and Pseudomonas fl uorescens.Pseudomonas fl uorescens. Identifi cation of these Identifi cation of these organisms may form a useful database in future to plan a strategy to apply public health organisms may form a useful database in future to plan a strategy to apply public health measures with regards to milk or milk products.
measures with regards to milk or milk products. Unidentifi ed sequences in metagenomic analysis.
Unidentifi ed sequences in metagenomic analysis. The data read were analyzed by The data read were analyzed by metagenome rapid annotation using subsystem technology, Metagenomics RAST server metagenome rapid annotation using subsystem technology, Metagenomics RAST server 2.0 (http://metagenomics.anl.gov) (
2.0 (http://metagenomics.anl.gov) (MEYER et al., 2008MEYER et al., 2008). MG-RAST uses BLAT to fi nd ). MG-RAST uses BLAT to fi nd sequences in the metagenomic dataset which are homologous to sequences in the M5NR sequences in the metagenomic dataset which are homologous to sequences in the M5NR database. The alignments are the comparison of two or more sequences showing the database. The alignments are the comparison of two or more sequences showing the degree of similarity.
degree of similarity.
In the present fi ndings, a major portion of the sequences obtained in the metagenomic In the present fi ndings, a major portion of the sequences obtained in the metagenomic data were not identifi ed in the SEED subsystem of MG-RAST, because of their lower data were not identifi ed in the SEED subsystem of MG-RAST, because of their lower correlation to known organisms. As newly discovered organisms are defi ned and their 16S correlation to known organisms. As newly discovered organisms are defi ned and their 16S sequences added to the MG-RAST databases, these organisms will be positively identifi ed. sequences added to the MG-RAST databases, these organisms will be positively identifi ed. Unifying the fi ndings of metagenomic analysis, it can be inferred that the analysis Unifying the fi ndings of metagenomic analysis, it can be inferred that the analysis yielded an in-depth picture of the possible bacterial organisms involved in the udder yielded an in-depth picture of the possible bacterial organisms involved in the udder environment. The disparity with the cultural method might be due to a focused attempt to environment. The disparity with the cultural method might be due to a focused attempt to apply the routinely used cultural methods, and this might have resulted in missing a few apply the routinely used cultural methods, and this might have resulted in missing a few of the organisms, which could have been obtained by adding more protocols for cultural of the organisms, which could have been obtained by adding more protocols for cultural methods. However, the real scientifi c output, obtained by the metagenomic analysis, may methods. However, the real scientifi c output, obtained by the metagenomic analysis, may be with regards to identifying those organisms, which are diffi cult to cultivate or have be with regards to identifying those organisms, which are diffi cult to cultivate or have never been mentioned as mastitis pathogens / organisms. They also might have been never been mentioned as mastitis pathogens / organisms. They also might have been missed by cultural methods because of their very low concentration in subclinical mastitis missed by cultural methods because of their very low concentration in subclinical mastitis milk, but were promptly detected by the highly sensitive pyrosequencing approach. milk, but were promptly detected by the highly sensitive pyrosequencing approach. The data thus generated may prove to be useful in considering these newly identifi ed The data thus generated may prove to be useful in considering these newly identifi ed organisms in the future course of technical programmes and future approaches to dealing organisms in the future course of technical programmes and future approaches to dealing with the problem of subclinical mastitis.
Conclusions
The present study revealed that Metagenomic analysis of subclinical mastitis samples of TP, Kankrej and Gir cows identifi ed bacterial organisms belonging to phyla (5), class (8), Subclass / order (15), Family (19), Genus (23) and species (28). Metagenomic analysis additionally identifi ed 19 genera and 26 species in comparison with the routine cultural methods. Many of the fastidious / anaerobic bacterial organisms, which are diffi cult to cultivate by routine methods, were identifi ed by metagenomic analyses.
*Part of Ph. D. thesis submitted by fi rst author to AAU, Anand.
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Received: 28 March 2013 Accepted: 19 December 2013 ________________________________________________________________________________________
BHANDERI, B. B., M. K. JHALA, V. B. AHIR, V. D. BHATT, C. G. JOSHI BHANDERI, B. B., M. K. JHALA, V. B. AHIR, V. D. BHATT, C. G. JOSHI: Kulturelna i metagenomska identifikacija mikrobioma kod supkliničkog mastitisa u krava. Vet. arhiv 84, 215-228, 2014.
SAŽETAK
Radi identifi kacije mikrobne zajednice u mlijeku provedena je metagenomska i uobičajena kulturelna pretraga uzoraka mlijeka krava sa supkliničkim mastitisom. Ukupno je 77 trostruko križanih Kankrej i Gir mliječnih krava i 301 četvrt vimena bilo pretraženo na supklinički mastitis. Izdvojeno je bilo 106 izolata svrstanih u pet različitih rodova iz 91 četvrti od 41 krave uključujući i 15 četvrti kod kojih je kulturelnom pretragom bila ustanovljena mješovita bakterijska infekcija. Sljedovi mješavine DNA izdvojeni iz uzoraka mlijeka kod supkliničkog mastitisa očitani pirosekvenciranjem bili su analizirani po podsustavu SEED baze podataka „Meta Genome Rapid Annotation with Subsystem Technology (MG-RAST)“. Iz pretraženih uzoraka mlijeka bilo je izdvojeno pet rodova: Staphylococcus, Streptococcus, Micrococcus, Bacillus i Escherichia. Četiri su bila dokazana postupkom pirosekvenciranja: Staphylococcus, Streptococcus, Bacillus i Escherichia, dok Micrococcus nije bio dokazan. S druge strane, pirosekvenciranjem je bilo dokazano 28 bakterijskih vrsta, od kojih su samo dvije, S. aureus i E. coli, bile dokazane klasičnom kulturelnom pretragom. S. agalactiae, treća vrsta identifi cirana kulturelnom pretragom nije bila dokazana postupkom pirosekvenciranja. Metagenomskom analizom dodatno je bilo dokazano 19 rodova i 26 vrsta u usporedbi s rutinskom kulturelnom pretragom. Mnoge anaerobne bakterije, koje je vrlo teško uzgojiti rutinskim metodama, bile su identifi cirane metagenomskom analizom.
Ključne riječi: supklinički mastitis, metagenomika, krave
