Species-specific Pcr Aids Bacterial Identification Of Cns

Species-Specific PCR aids Bacterial Identification of Coagulase-Negative Staphylococci

Jessica Cyr

Emporia State University

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Key Words: Coagulase-negative staphylococci, multiplex primers, superoxide dismutase (sodA), species-

specific PCR

Introduction: Microbial forensics faces any classification of crime with microorganisms or biological

influences. Before we can combat biological crimes, bacteria must be reliably identified. Staphylococcus

aureus is a coagulase-positive staphylococci that is viewed as the core for bacterial pathogens leading to

sickness and disease. As more research evolves, common coagulase-negative staphylococci species have

been shown to be the cause of other bacterial diseases. Identifying the origin of these pathogens is

crucial for preventative measures and treatments. Some coagulase-negative staphylococci include S.

epidermidis, S. capitis, S.caprae, and S. warneri. More coagulase-negative staphylococci need to be

identified and more information is needed about their behavior. There is also a wide variety within the

species totaling 74 sequencing types (Otto, 2009). In forensic microbiology, we need to identify these

rarely studied species for scientists to be able to discern between them. In humans, DNA is almost

identical but there can be variability among its microbial flora. There are many approaches to coagulase-

negative staphylococci identification. Some other techniques include: matrix-assisted laser desorption

ionization time-of-flight mass spectrometry (MALDI-TOFMS), API Staph test, and whole-genome DNA-

DNA hybridization analysis (Kim, 2018). Yet, these approaches are either too costly, too labor intensive

or have a lower identification rate. 16S rRNA is a common method for bacterial identification. However,

the 16S rRNA PCR has limitations between differentiating species that are too similar. It also needs

further analyses for clear identification within the Staphylococcus genus (Blaoitta, 2005). The goal of my

research is to reliably identify bacteria, which is incapable using 16S. Due to high similarity in genotypes,

S. pasteuri and S. warneri are two species in a conserved area that are difficult to distinguish. Superoxide

dismutase (sodA) is a gene used in species-specific PCR for genotypic identification. Kim (2018)

experiments further after species-specific PCR including DNA sequencing and homology comparisons. To

be able to differentiate these species, I used multiplex primers and species-specific PCR to confirm the

presence of each strain (Kim, 2018).

Materials and Methods: DNA was isolated using whole-cell polymerase chain reaction (PCR) with

freshly grown cells on agar. Samples were obtained, prior to this study, from random DNA skin cells

swabbed from the inner elbow, behind the ear, or on the forehead and stored at –80⁰ C. Once taken out

of storage, the samples were plated onto liquid broth plates and grown overnight at 37⁰ C. A DNA

colony collected from the agar plate and was mixed into a microfuge tube with 100 μL of TE buffer. Once

mixed 5 μL of sample was put in each GoTaq tube, as well as 5 μL of primer and primer mixes. GoTaq

consists of heat-stable DNA Polymerase, dNTP’s, dye, buffer and MgCl2. Primer sequences were specific

to each species based on the sodA gene, listed below. The stock concentration for the primers was 2.5

mmol. When the primers or primer mixes were added to the reaction, it totaled a concentration of 0.25

mmol. The primers and mixes are listed below show which primers bind to the correlating species. For

initial samples with 16S rRNA PCR, 1% agarose gels were used to observe the DNA samples showing the

presence of DNA with 100-bp DNA markers. DNA sequencing was completed by an outside facility,

Molecular Cloning Laboratories (MCLAB). After reviewing chromatogram peaks and base pairs, the

edited sequences of the samples were put into basic local alignment search tool (BLAST) analysis for

identification. Higher-percent agarose gels are used to observe differences in species that are within 50-

100 bp of each other based on their amplicon size. Amplicon size of tested species was found in Table 2

of Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci (Kim,

2018). 2% agarose gels were used when observing samples that were tested in species-specific PCR with

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multiplex primers. 50–bp DNA markers were used in 2% agarose gels, as well as corresponding positive

controls (pure DNA) for S. epidermidis, S. pasteuri and S. warneri. No positive control was used during

testing for S. capitis and S. caprae due to unavailability. The agarose gels were observed under the

ultraviolet light transilluminator. Samples identified as S. pasteuri and S. warneri by 16S rRNA PCR used

the STAPH-1 PCR conditions below. Samples identified as S. epidermidis, S. capitis, and S. caprae by 16S

rRNA PCR used STAPH-2 PCR conditions below.

Agarose gel recipe

1%: 0.3g of agarose, 30mL of TAE buffer, 2 μL of ethidium bromide

(EtBr) mixed into an Erlenmeyer flask, microwaved until dissolved and

cool.

2%: 0.7g of agarose, 30mL of TAE buffer, 2 μL of ethidium bromide

(EtBr) mixed into an Erlenmeyer flask, microwaved until dissolved and

cool.

Amplicon size (bp)

16S: 1500

S. epidermidis: 194

S. capitis: 103

S. caprae: 252

S. warerni: 110

S. pasteuri: 237

LB agar plate recipe

Add 11 g/mL of agar and 10 g/mL of LB Broth, Lennox to 500 mL of H2O. Autoclave, cool and pour into

plates.

Primer sequences from Integrated DNA Technologies

8F for 16S: 5’AGA GTT TGA CTC AG 3’

1492R (L) for 16S: 5’ GGT TAC CTT GTT ACG ACT T 3’

164 CT103R S. capitis: 5’ CTA CTT CAC CTT TTT CTT CAG A 3’

165 CT103F S. capitis: 5’ TCA GAT ATT CAA ACT GCA GTA G 3’

Figure 1: 1% agarose gel of 16S rRNA showing

initial presence of DNA samples alongside

DNA markers (M).

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166 EpiR S. epidermidis: 5’ TGG CTA ATG GTT TGT CAC CA 3’

167 EpiF S. epidermidis: 5’ GGC AAA TTT GTG GGT CAA GA 3’

172 SW110R S. warneri: 5’ TCT TAC TGC AGT TTG AAT ATC AGA 3’

173 SW110F S. warneri: 5’ GTA ACA AAA TTA AAT GCA GCT G 3’

174 PA237R S. pasteuri: 5’ GCC CGT TAT TTA CTA CTA ACC A 3’

175 PA237F S. pasteuri: 5’ GCT AAT TTA GAC AGT GTA CCT TCT G 3’

PCR conditions for 16S rRNA primer

Cycle 1: 94⁰ C for 1 minute

Cycle 2: 94⁰ C for 1 minute

Cycle 3: 50⁰ C for 1 minute

Cycle 4: 72⁰ C for 1 minute and 30 seconds

Cycle 5: Go back to step 2 and repeat cycles 2-4 29 times until moving to cycle 6

Cycle 6: 72⁰ C for 5 minutes

Cycle 7: 12⁰ C hold indefinitely

PCR conditions for STAPH-1 with 172,173,174,175 primer mix

Cycle 1: 95⁰ C for 5 minutes

Cycle 2: 95⁰ C for 30 seconds

Cycle 3: 55⁰ C for 30 seconds

Cycle 4: 72⁰ C for 30 seconds

Cycle 5: Go back to step 2 and repeat cycles 2-4 29 times until moving to cycle 6

Cycle 6: 72⁰ C for 30 seconds

Cycle 7: 12⁰ C hold indefinitely

PCR conditions for STAPH-2 with 164, 165, 166,167 and 166,167 primer mix

Cycle 1: 95⁰ C for 5 minutes

Cycle 2: 95⁰ C for 30 seconds

Cycle 3: 55⁰ C for 30 seconds

Cycle 4: 72⁰ C for 30 seconds

Cycle 5: Go back to step 2 and repeat cycles 2-4 29 times until moving to cycle 6

Cycle 6: 72⁰ C for 7 minutes

Cycle 7: 12⁰ C hold indefinitely

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Results and Discussion:

Figure 1 above is an example of positive and negative results for presence of bacteria using 16S. The

fluoresced bands show DNA in the agarose gel. Below are the top four results 16S BLAST analysis of

JC362. As you can see the percent identity is 100% for both S. warneri and S. pasteuri.

Staphylococcus warneri strain DK131 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT642942.1

Staphylococcus pasteuri strain ML029 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT636753.1

Staphylococcus pasteuri strain BCVME2 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT598013.1

Staphylococcus warneri strain 513 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT573107.1

There could be presence of both species in the sample’s skin

cells, but we want a reliable confirmation if there is only one

species since they are commonly misidentified as each other.

The advantages of whole-cell PCR include: it is more cost-

efficient than DNA extraction from a kit, it is quick and dirty to

plate on the agar plates, and easily isolated when replated

again. Blaiotta et al. (2004) sought after species-specific PCR

while also confirming identification, but they used singleplex

PCR rather than multiplex PCR. S. epidermidis, S. capitis, and S.

caprae are also misidentified species of coagulase-negative

staphylococci. Ghebremedhin, Layer, Konig, & Konig establish

that the sodA gene found 94% similarity between S. capitis and

S. caprae (2008). Table 1 shows the data regarding the

identified species. The 16S rRNA PCR column shows which

bacterial identification sequenced with BLAST that 16S could

do. Species-specific PCR in the next column shows the

superiority it has over 16S for reliable identification. With the

use of the 172,173,174,175 multiplex primers, S. warneri was

identified in samples JC362, JC126, JC131, JC135, JC137, and

JC140, seen in Figure 2. In Figure 2, lane 1 is the positive control for S. pasteuri, lane 2 is the positive

control for S. warneri and the samples that fluoresced show the identification of S. warneri. Previously

identified S. epidermidis samples from 16S: JC361, JC367, JC373, JC374, JC376, JC377, JC401, JC404,

JC407, JC411, JC412, JC413, JC414, JC417, JC421, JC121, JC122, and JC124 were confirmed as S.

epidermidis with the use of the 166,167-multiplex primers in PCR, shown in Figure 3. The presence of

bands in Figure 3 confirm the bacterial identification as S. epidermidis. Sample JC110 was identified as S.

epidermidis with 164, 165, 166, and 167 multiplex primers. Samples JC111 and JC113 were identified as

S. capitis with 164, 165, 166, and 167 multiplex primers. These positive results can be seen in Figure 4.

Although there were no positive controls for S. capitis or S. caprae, the amplicon size was used for S.

capitis and there were no S. caprae samples that were present at 252 bp. Unconfirmed species using

species-specific PCR were not visually present on agarose gels but were tested twice. There could be a

Figure 2: 2% Agarose gel electrophoresis. M: DNA markers; 1:

S.pasteuri positive control; 2: S. warneri positive control; 3:

JC362 S. warneri; 4: JC365 Unconfirmed; 5: JC126 S. warneri; 6:

JC131 S. warneri; 7: JC135 S. warneri; 8: JC137 S. warneri; 9:

JC140 S. warneri

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variety of reasons why a sample that was tested in PCR does not work. Whole-cell PCR does not have

the purest of DNA being used, experimental errors can occur, and some DNA samples are more difficult

to grow and test. 16S can still provide a baseline of identification that can be further confirmed. The

positive results show that 2% agarose gel electrophoresis with multiplex species-specific PCR can

confirm and differentiate previously identified coagulase-negative staphylococci from 16S rRNA PCR and

my research goal was achieved. Reliable identification of our microbial flora makes it possible to find the

variability from person to person if there is variability between species.

Figure 3: 2% Agarose gel electrophoresis. M: DNA

markers; 1: S. epidermidis positive control; 2: JC

401 S. epidermidis; 3: JC403 unconfirmed; 4: JC404

S. epidermidis; 5: JC405 unconfirmed; 6: JC407 S.

epidermidis; 7: JC411 S. epidermidis; 8: JC412 S.

epidermidis; 9: JC413 S. epidermidis

Figure 4: 2% Agarose gel electrophoresis. M: DNA

markers; 1: S. epidermidis positive control; 2: JC 110

S. epidermidis; 3: JC111 S. capitis; 3: JC113 S. capitis

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Samples

16S RNA PCR

Species-Specific PCR

JC361

S. epidermidis

JC362

S. warneri/pasteuri

S. warneri

JC363

S. epidermidis

Unconfirmed

JC365

S. warneri/pasteuri

Unconfirmed

JC366

S. epidermidis

Unconfirmed

JC367

S. epidermidis

JC373

S. epidermidis

JC374

S. epidermidis

JC376

S. epidermidis

JC377

S. epidermidis

JC401

S. epidermidis

JC403

S. epidermidis

Unconfirmed

JC404

S. epidermidis

JC405

S. epidermidis

Unconfirmed

JC407

S. epidermidis

JC411

S. epidermidis

JC412

S. epidermidis

JC413

S. epidermidis

JC414

S. epidermidis

JC417

S. epidermidis

JC421

S. epidermidis

JC110

S. epidermidis/capitis

S. epidermidis

JC111

S. capitis/caprae/sp.

S. capitis

JC113

S. capitis/sp.

S. capitis

JC121

S. epidermidis

JC122

S. epidermidis

JC124

S. epidermidis

JC126

S. warneri/pasteuri

S. warneri

JC131

S. warneri/pasteuri

S. warneri

JC135

S. warneri/pasteuri

S. warneri

JC137

S. warneri/pasteuri

S. warneri

JC140

S. warneri/pasteuri

S. warneri

Table 1: Comparison of identification with 16S RNA PCR and species-specific PCR

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References:

Blaiotta, G., Casaburi, A., & Villani, F. (2005). Identification and differentiation of Staphylococcus

carnosus and Staphylococcus simulans by species-specific PCR assays of sodA genes. Systematic and

Applied Microbiology, 28(6), 519–526. doi: 10.1016/j.syapm.2005.03.007

Budowle, B. (2003). PUBLIC HEALTH: Building Microbial Forensics as a Response to Bioterrorism. Science,

301(5641), 1852–1853. doi:10.1126/science.1090083

Ghebremedhin, B., Layer, F., Konig, W., & Konig, B. (2008). Genetic Classification and Distinguishing of

Staphylococcus Species Based on Different Partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf Gene

Sequences. Journal of Clinical Microbiology, 46(3), 1019–1025. doi:10.1128/jcm.02058-07

Kim, J., Hong, J., Lim, J.-A., Heu, S., & Roh, E. (2017). Improved multiplex PCR primers for rapid

identification of coagulase-negative staphylococci. Archives of Microbiology, 200(1), 73–

83. doi:10.1007/s00203-017-1415-9

Kiratisin, P., Li, L., Murray, P. R., & Fischer, S. H. (2003). Identification of Bacteria Recovered from Clinical

Specimens by 16SrRNA Gene Sequencing. European Journal of Clinical Microbiology & Infectious

Diseases, -1(1), 1–1. doi:10.1007/s10096-003-1003-6

Mignard, S., & Flandrois, J. P. (2006). 16S rRNA sequencing in routine bacterial identification: A 30-month

experiment. Journal of Microbiological Methods, 67(3), 574–581. doi: 10.1016/j.mimet.2006.05.009

Otto, M. (2009). Staphylococcus epidermidis — the “accidental” pathogen. Nature Reviews Microbiology,

7(8), 555–567. doi:10.1038/nrmicro2182

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Appendix A:

The following results were obtained by 16S rRNA PCR and BLAST analysis:

JC361

Raw Sequence:

NNNNNNNNNNNNNNNGNGNNNNTACNTGCAGTCGAGCGAACAGACGAGGAGCTTGCTCCTCTGACGTTAGCG

GCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCG

GATAATATATTGAACCGCATGGTTCAATAGTGAAAGACGGTTTTGCTGTCACTTATAGATGGATCCGCGCCGCATT

AGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGG

AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGA

GCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAAC

TATGCACGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG

GCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGC

TCAACCGTGGAGGGTCATTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGT

GAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGA

AAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTNGGGGT

TTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGAGTACGACCGCAAGGTTGAAACTCAAAG

GGAATTGACGGGGACCCGCACAAGCGGTGGNGCATGTGGTTTAATTTCGAAGCAACGCGAANAANNTTANCNAA

ATCTTGACATCCTCTGACCCTCTAGAGATAGAGTTTTCCCCTTCGGGGGANNGANTGACNNNNNNGCATGNNNN

NTCGTCAGCTCNNGTCGNGGANATNNNNNNNNNTCNNNNNNCGANNNCNNNNNNNNNCTNNNNNNCNTCN

TTAANNNNNNNNCTNTAANNTNNNCNNNNNNNNNN

Edited Sequence:

GGTTCAATAGTGAAAGACGGTTTTGCTGTCACTTATAGATGGATCCGCGCCGCATTAGCTAGTTGGTAAGGTAACG

GCTTACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGAC

TCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATG

AAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAACTATGCACGTCTTGACGGTACC

TAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTAT

TGGGCGT

Staphylococcus epidermidis strain HP01 16S ribosomal RNA gene, partial sequence

691 691 100% 0.0 100.00% MT586029.1

Staphylococcus epidermidis strain 3039 16S ribosomal RNA gene, partial sequence

691 691 100% 0.0 100.00% MT613456.1

Staphylococcus epidermidis strain ISP111A 16S ribosomal RNA gene, partial sequence

691 691 100% 0.0 100.00% MT605366.1

Staphylococcus epidermidis strain ISP111B 16S ribosomal RNA gene, partial sequence

691 691 100% 0.0 100.00% MT605363.1

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Alignment: Query: None Query ID: lcl|Query_16603 Length: 382

>Staphylococcus epidermidis strain HP01 16S ribosomal RNA gene, partial

sequence

Sequence ID: MT586029.1 Length: 815

Range 1: 113 to 494

Score:690 bits(764), Expect:0.0,

Identities:382/382(100%), Gaps:0/382(0%), Strand: Plus/Plus

Query 1 GGTTCAATAGTGAAAGACGGTTTTGCTGTCACTTATAGATGGATCCGCGCCGCATTAGCT 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 113 GGTTCAATAGTGAAAGACGGTTTTGCTGTCACTTATAGATGGATCCGCGCCGCATTAGCT 172

Query 61 AGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCG 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 173 AGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCG 232

Query 121 GCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 233 GCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTC 292

Query 181 CGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGT 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 293 CGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGT 352

Query 241 AAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAACTATGCACGTCTTGACGGTACC 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 353 AAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAACTATGCACGTCTTGACGGTACC 412

Query 301 TAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGC 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

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Sbjct 413 TAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGC 472

Query 361 GTTATCCGGAATTATTGGGCGT 382

||||||||||||||||||||||

Sbjct 473 GTTATCCGGAATTATTGGGCGT 494

JC362

Raw Sequence:

NNNNNNNNGGNNGCNNNTANTGCAGTCGAGCGACAGATAAGGAGCTTGCTCCTTTGACGTTAGCGGCGGACG

GGTGAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAACAT

ATTGAACCGCATGGTTCAATAGTGAAAGGCGGCTTTGCTGTCACTTATAGATGGATCCGCGCCGTATTAGCTAGTT

GGTAAGGTAACGGCTTACCAAGGCAACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGA

CACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCC

GCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATCAGGGAAGAACAAATGTGTAAGTAACTGTGCACA

TCTTGACGGTACCTGATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGT

TATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGT

GGAGGGTCATTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCG

CAGAGATATGGAGGAACACCAGTGGNGAAGGCGACTTTCTGGNCTGTAACTGACGCTGATGTGCGAAAGCGTGG

GGATCAACAGGGATTAGATACCCTGGNAGTCCACGNCGTAAACGATGANTGCTAGNGTNGGGGGTTTCNGCCCC

TTANNGCTGCAGCTAACGCATTNAGCNNCTCCGNCTGGGNAGTACGACNNNNGGNNGANNTCANNNNNTGAC

GGGGACCNGCNNNGCGGTGNAGCNTNNNNNNNNNNNNAANNNANNNNAANNANNNNNNNATCTTGNNNN

NNNNGACCNNNNNNNNNAANNNNAGNTNNNNNNNNNNGNNNNNNNNNNNGNNNNTGNNNNCNGNNN

Edited Sequence:

GGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGT

CTTCGGATCGTAAAACTCTGTTATCAGGGAAGAACAAATGTGTAAGTAACTGTGCACATCTTGACGGTACCTGATC

AGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGC

GTAAAGCGCGCGTAGGCGG

Staphylococcus warneri strain DK131 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT642942.1

Staphylococcus pasteuri strain ML029 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT636753.1

Staphylococcus pasteuri strain BCVME2 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT598013.1

Staphylococcus warneri strain 513 16S ribosomal RNA gene, partial sequence

439 439 100% 2e-119 100.00% MT573107.1

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Alignment1:

Query: None Query ID: lcl|Query_57565 Length: 243

>Staphylococcus pasteuri strain ML029 16S ribosomal RNA gene, partial

sequence

Sequence ID: MT636753.1 Length: 943

Range 1: 269 to 511

Score:439 bits(486), Expect:2e-119,

Identities:243/243(100%), Gaps:0/243(0%), Strand: Plus/Plus

Query 1 GGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGT 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 269 GGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGT 328