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Genetic Analysis Method for Staphylococcus chromogenes Associated with Goat Mastitis Cover

Genetic Analysis Method for Staphylococcus chromogenes Associated with Goat Mastitis

Polish Journal of Microbiology
Volume 67 (2018): Issue 2 (June 2018)
By: ,  ,  ,  ,   and    
Open Access
|Jun 2018
Figures & tables

Figures & Tables

Table I

Primers used to identify S. chromogenes.

PrimersSequence (5’-3’)ConditionsSize [bp]
16S rRNAF’- CCTATAAGACTGGGATAACTTCGGG
R’- CTTTGAGTTTCAACCTTGCGGTCG		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.791
coaF’- CAAAGCAGATGCGATAG
R’- CCTGTACCAGCATCTCTAT		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.1200
AureolysinF’- GCATTAAACGAAASCTTYTCWG
R’- GTATGCAGCTTTATTTGGWATACC		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.247
HemolysinF’- ATGAATATTGGAATAACTTTAGTCAG
R’- TTAGGAAGCATACAATTGATGT		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.600
Extracelular proteinF’- ACGTGAGCAATATATGAACGC
R’- TTATAAATACCTGTTAATGCGCC		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.133
Zin MetaloproteaseF’- ATTTGCAATCGGGATGG
R’- ATGTTGATCATCTAAAATAATATGCG		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.250
Surface protein SasHF’- GGCWAAAGCRATGAATGC1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m,201
 R’- ACCGATAATKCCRTAACG72°C/1 m/1 cycle 72°C/5 m. 
TRAP (Transduction signal protein)F’-AACTCTATTCACTTATGGAACATATGG
R’- CTGTTCAACATTTTGCTGTTG		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.529
B antigenF’- CAAAAACACTATTAGCGACTGG
R’- CTTTTATGACGATGGAGTTTCC		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.488
Virulence B factorF’- ATGTCTTTTAAAGAAAATGAAATCG
R’- TATGCATGATTTTCACTGTGC		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.905
FemA proteinF’- GTGTGCTTRTACCWYTAGC
R’- CCAGCATAATAAACWASTTC		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 50°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.250
Table II

Primers used to amplify the VNTR.

PrimerSequence (5’-3’)ConditionsSize [bp]
266_07F’- ATTCTGGATTTTGCACAGC
R’- ATCTGCTAAAATGACAGAATTACAAC		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.266
346_06F’- CAAAACAACGATATCTGTATCTGA
R’- TGTCGGTTTATTAGGTTGAGAAG		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.346
360_06F’- CAAGTGCATAACCGTTATTCC
R’- TGTCTGATGTCGGTTTATTAGG		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.360
854_08F’- GAAGCACTTACACTTTCGGAG
R’- GACTTCACTAAGTGAGTCAACAAGTAC		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.854
638_07F’- TGAGGTCTCCGCTGTAGG
R’- GCAGACGTCCCTGTTGAG		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.638
613_12F’- AGTAGCTAACCATTTTGTTAATTGC
R’- GTTAAAGAAAATTCTTCACAGTCG		1 cycle 95°C/5 m, 30 cycles 95°C/1 m, 56°C/1 m, 72°C/1 m/1 cycle 72°C/5 m.613
Table III

Biochemical tests results with Api Staph System and antibiograms.

StrainOriginGoat idFarmProduction system123456789101112131415161718192021222324
  1GuanajuatoA1Extensive++++++−−−++−−−+−+++RSSSS
  2GuanajuatoB1Extensive+++−++−−−++−−−+−+++SSSSS
  3GuanajuatoC1Extensive+++−++−−−++−−−+−+++SSSSS
  4GuanajuatoD1Extensive++++++−−−++−−−+−+++SRSSS
  5GuanajuatoE1Extensive+++−−++−−++−−−+−−++SSSSS
  6GuanajuatoF1Extensive+++−++−−−++−−−+−−++SRSSS
  7QuerétaroG-105R2Intensive++++++−−−++−−−+−+++SRSSS
  8QuerétaroH-105R2Intensive+−++++−−−−+−−−+−+++SRSSS
  9QuerétaroG-60L2Intensive++−−+++−−++−−−+−++−SSSSS
10QuerétaroG-150R2Intensive++−−+++−−++−+−+−++−SSSSS
11QuerétaroI-15R3Intensive++++++−−−−+−−−+−+++RRSSS
12QuerétaroJ-15R3Intensive++++++−−−−+−−−+−+++RRSSS
13QuerétaroK-15L3Intensive++++++−−−−+−−−+−+++RRSSS
14QuerétaroJ-60R3Intensive++++++−−−−+−−−+−+++SRSSS
15QuerétaroK-15L13Intensive++++++−−−++−−−+−+++RRSSS
16QuerétaroK-15L23Intensive++−+++−−−++−−−+−−++RRSSS
17QuerétaroK-15R33Intensive++++++−−−+++−−+−−++SRSSS
18QuerétaroL-15R3Intensive++++++−−−−+−−−+−+++IRSSS
19QuerétaroJ-195R3Intensive++++++−−−−+−−−+−+++IRSSS
20QuerétaroM-15L3Semiintensive++++++−−−++−−−+−−++RRSSS
21QuerétaroN-60R3Semiintensive++++++−−−+++−−+−−++SRSSS
22QuerétaroO-150L3Semiintensive++++++−−−+++−−+−−++IRSSS
23QuerétaroO-150R3Semiintensive++++++−−−++−−−+−−++RRSSS

1 The physiological tests used with S. chromogenes were: 1. Glucose; 2. Fructose; 3. Manose; 4. Maltose; 5. Lactose; 6. Trehalose; 7. Manitol; 8. Xilitol; 9. Mellobiose; 10. Nitrates; 11. Alkaline Phosphatase; 12. Voges-Proskauer; 13. Rafifnose; 14. Xylose; 15. Saccharose; 16. Metil-Gluco-Pyranosidase; 17. N-Acetyl-Glucose; 18. Arginin Dihydrolase; 19. Urea; 20. Polymyxin B; 21. Ampicillin; 22. Gentamicine; 23. Tobramycine and 24. Tetracycline.

Table IV

The genes identified in the isolated CNS.

The gen coding forS. chromogenesS. simulansS. xylosusS. sciuriS. warneri
+/total%+/total%+/total%+/total%+/total%
Aureolysin23/231005/18  27.784/9   44.442/21000/10
Hemolysin23/231002/18  11.110/9    00/200/10
Extracellular protein23/2310018/181009/91002/21001/1100
Zin metaloprotease23/2310018/181009/91002/21001/1100
Surface protein SasH23/2310018/181006/9  66.672/21000/10
TRAP23/231003/18  16.673/9  33.332/21000/10
B Antigen23/231002/18  11.110/9    00/200/10
Virulence B factor22/23  95.650/18    03/9  33.332/21001/1100
FemA protein23/2310018/181009/91002/21000/10
Fig. 1.

Cluster analysis according to the phenotypic characteristics and chemotherapeutic susceptibility of 23 S. chromogenes strains according to Ward’s minimum variance method.

Table V

Number of VNTR detected by capillary electrophoresis.

StrainOriginFarmProduction system266_07VNTR346_06VNTR360_06VNTR854_08VNTR
  1Guanajuato1Extensive95527109273118740110111
  2Guanajuato1Extensive955271166781156393013
  3Guanajuato1Extensive96327115377118339110111
  4Guanajuato1Extensive95326116177118139107711
  5Guanajuato1Extensive95226115977118139121013
  6Guanajuato1Extensive93626116478117539120113
  7Querétaro2Intensive453131181791175393293
  8Querétaro2Intensive453131177781177393293
  9Querétaro2Intensive107530117678117639122113
10Querétaro2Intensive108030117879118239122913
11Querétaro3Intensive384111155771176393293
12Querétaro3Intensive383111157771178393293
13Querétaro3Intensive385111156771172393293
14Querétaro3Intensive382111164781181392042
15Querétaro3Intensive382111150771181393293
16Querétaro3Intensive382111155771183393303
17Querétaro3Intensive385111178791181393293
18Querétaro3Intensive989271158771181393293
19Querétaro3Intensive384111156771178393293
20Querétaro3Semi-intensive383111168781186403283
21Querétaro3Semi-intensive386111163781186402052
22Querétaro3Semi-intensive872241192791185403293
23Querétaro3Semi-intensive001187791186403293
Fig. 2.

Cluster analysis according to the VNTR of 23 strains of S. chromogenes using Pearson’s correlation coefficient and the UPGMA algorithm.

Table VI

Diversity Index (Simpson and Hunter-Gaston) and confidence intervals for each VNTR loci for S. chromogenes.

LocusSize [bp]Simpson indexConfidence intervals 95%Hunter-gaston indexConfidence intervals 95%
266_07360.870.803–0.9360.9090.843–0.975
346_06150.9260.896–0.9570.9680.938–0.999
360_06300.7560.650–0.8620.7910.684–0.897
854_08960.6540.449–0.8590.6840.479–0.888
DOI: https://doi.org/10.21307/pjm-2018-019 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331
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Language: English
Page range: 171 - 180
Submitted on: Jul 7, 2017
Accepted on: Oct 4, 2017
Published on: Jun 30, 2018
Published by: Polish Society of Microbiologists
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
genotyping,
mastitis in goats,
MLVA,
Staphylococcus chromogenes,
VNTR
Related subjects:
Life sciences,
Microbiology and virology

© 2018 ROCÍO A. RUIZ-ROMERO, ROBERTO A. CERVANTES-OLIVARES, ANDRÉS E. DUCOING-WATTY, DANIEL MARTÍNEZ-GÓMEZ, EFRÉN DÍAZ-APARICIO, ESTELA T. MÉNDEZ-OLVERA, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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