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Genetic Analysis Method for Staphylococcus chromogenes Associated with Goat Mastitis

ROCÍO A. RUIZ-ROMERO
ROCÍO A. RUIZ-ROMERO
, 
ROBERTO A. CERVANTES-OLIVARES
ROBERTO A. CERVANTES-OLIVARES
, 
ANDRÉS E. DUCOING-WATTY
ANDRÉS E. DUCOING-WATTY
, 
DANIEL MARTÍNEZ-GÓMEZ
DANIEL MARTÍNEZ-GÓMEZ
, 
EFRÉN DÍAZ-APARICIO
EFRÉN DÍAZ-APARICIO
 und 
ESTELA T. MÉNDEZ-OLVERA
ESTELA T. MÉNDEZ-OLVERA
   | 30. Juni 2018
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Article Category: original-paper
Online veröffentlicht: 30. Juni 2018
Seitenbereich: 171 - 180
Eingereicht: 07. Juli 2017
Akzeptiert: 04. Okt. 2017
DOI: https://doi.org/10.21307/pjm-2018-019
Schlüsselwörter
© 2018 ROCÍO A. RUIZ-ROMERO et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

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

Fig. 2.

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

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

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

Primers used to amplify the VNTR.

PrimerSequence (5’-3’)ConditionsSize [bp]
266_07F’- ATTCTGGATTTTGCACAGCR’- ATCTGCTAAAATGACAGAATTACAAC1 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’- CAAAACAACGATATCTGTATCTGAR’- TGTCGGTTTATTAGGTTGAGAAG1 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’- CAAGTGCATAACCGTTATTCCR’- TGTCTGATGTCGGTTTATTAGG1 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’- GAAGCACTTACACTTTCGGAGR’- GACTTCACTAAGTGAGTCAACAAGTAC1 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’- TGAGGTCTCCGCTGTAGGR’- GCAGACGTCCCTGTTGAG1 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’- AGTAGCTAACCATTTTGTTAATTGCR’- GTTAAAGAAAATTCTTCACAGTCG1 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

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

Primers used to identify S. chromogenes.

PrimersSequence (5’-3’)ConditionsSize [bp]
16S rRNAF’- CCTATAAGACTGGGATAACTTCGGGR’- CTTTGAGTTTCAACCTTGCGGTCG1 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’- CAAAGCAGATGCGATAGR’- CCTGTACCAGCATCTCTAT1 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’- GCATTAAACGAAASCTTYTCWGR’- GTATGCAGCTTTATTTGGWATACC1 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’- ATGAATATTGGAATAACTTTAGTCAGR’- TTAGGAAGCATACAATTGATGT1 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’- ACGTGAGCAATATATGAACGCR’- TTATAAATACCTGTTAATGCGCC1 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’- ATTTGCAATCGGGATGGR’- ATGTTGATCATCTAAAATAATATGCG1 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’-AACTCTATTCACTTATGGAACATATGGR’- CTGTTCAACATTTTGCTGTTG1 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’- CAAAAACACTATTAGCGACTGGR’- CTTTTATGACGATGGAGTTTCC1 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’- ATGTCTTTTAAAGAAAATGAAATCGR’- TATGCATGATTTTCACTGTGC1 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’- GTGTGCTTRTACCWYTAGCR’- CCAGCATAATAAACWASTTC1 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

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
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