Characterization of Dutch Staphylococcus aureus from bovine mastitis using a Multiple Locus Variable Number Tandem Repeat Analysis

Abstract

Current typing methods for Staphylococcus aureus have important drawbacks. We evaluated a Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) scheme with 6 loci which lacks most drawbacks on 85 bovine mastitis isolates from The Netherlands. For each locus the number of repeat units (RU) was calculated. Each combination of repeat units was assigned a MLVA-type (MT). We compared the MLVA typing result with Multi Locus Sequence Typing (MLST), spa-typing and Pulsed-Field Gel Electrophoresis (PFGE). MLVA typing resulted in 18 MTs, although 3 loci could not always be amplified. Spa-typing distinguished 10 spa-types including 3 dominant and 2 new types. PFGE showed 5 dominant profiles with 15 related profiles and 6 unique profiles. MLST showed 4 dominant STs. Some types appeared to be bovine specific. The Simpson's Indices of diversity for PFGE, MLST, spa-typing and MLVA were 0.887, 0.831, 0.69 and 0.781, respectively, indicating that discriminatory power of MLVA was between MLST and spa-typing, whereas PFGE displayed the highest discriminatory power. However, MLVA is fast and cheap when compared to the other methods. The Adjusted Rand index and Wallace's coefficient indicated that MLVA was highly predictive for spa-type, but not vice versa.Analysis of the region neighboring SIRU05 showed a difference in the genetic element bordering the repeats of SIRU05 that explained the negative SIRU05 PCRs. PFGE, MLST, and MLVA are adequate typing methods for bovine-associated S. aureus.

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Accepted Manuscript
Title: Characterization of Dutch Staphylococcus aureus from
bovine mastitis using a Multiple Locus Variable Number
Tandem Repeat Analysis
Authors: Risma Ikawaty, E.C. Brouwer, M.D. Jansen, E. van
Duijkeren, D. Mevius, J. Verhoef, A.C. Fluit
PII:
DOI:
Reference:
S0378-1135(08)00507-5
doi:10.1016/j.vetmic.2008.10.034
VETMIC 4260
To appear in:
VETMIC
Received date:
Revised date:
Accepted date:
15-7-2008
24-10-2008
29-10-2008
Please cite this article as: Ikawaty, R., Brouwer, E.C., Jansen, M.D., van Duijkeren, E.,
Mevius, D., Verhoef, J., Fluit, A.C., Characterization of Dutch Staphylococcus aureus
frombovinemastitisusingaMultipleLocusVariableNumberTandemRepeatAnalysis,
Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2008.10.034
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Author manuscript, published in "Veterinary Microbiology 136, 3-4 (2009) 277"
DOI : 10.1016/j.vetmic.2008.10.034

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Accepted Manuscript
3584 CX Utrecht, The Netherlands.
1
Characterization of Dutch Staphylococcus aureus from bovine mastitis using a
Multiple Locus Variable Number Tandem Repeat Analysis
Risma Ikawaty1, E.C. Brouwer1, M.D. Jansen1, E. van Duijkeren2, D. Mevius2,3,
J. Verhoef1, A.C. Fluit1
1Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The
Netherlands
2Faculty of Veterinary Medicine Utrecht University, Utrecht, The Netherlands
3Central Veterinary Institute, Lelystad, The Netherlands
Running title: Typing of Dutch Staphylococcus aureus from bovine mastitis
Keywords: Staphylococcus aureus, bovine, mastitis, MLVA, MLST, PFGE, spa-typing
Address for correspondence:
A.C. Fluit
Department of Medical Microbiology,
University Medical Centre Utrecht, Room G04.614,
PO Box 85500
Email: A.C.Fluit@umcutrecht.nl
Manuscript
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aureus
2
Abstract1
Current typing methods for Staphylococcus aureus have important drawbacks. We 2
evaluated a Multiple Locus Variable-number tandem repeat Analysis (MLVA) scheme 3
with 6 loci which lacks most drawbacks on 85 bovine mastitis isolates from The 4
Netherlands. For each locus the number of repeat units (RU) was calculated. Each 5
combination of repeat units was assigned a MLVA-type (MT). We compared the MLVA 6
typing result with Multi Locus Sequence Typing (MLST), spa-typing and Pulsed-Field 7
Gel Electrophoresis (PFGE). MLVA typing resulted in 18 MTs, although 3 loci could not 8
always be amplified. Spa-typing distinguished 10 spa-types including 3 dominant and 2 9
new types. PFGE showed 5 dominant profiles with 15 related profiles and 6 unique 10
profiles. MLST showed 4 dominant STs. Some types appeared to be bovine specific. The 11
Simpson’s Indices of diversity for PFGE, MLST, spa-typing and MLVA were 0.887, 12
0.831, 0.69 and 0.781, respectively, indicating that discriminatory power of MLVA was 13
between MLST and spa-typing, whereas PFGE displayed the highest discriminatory 14
power. However, MLVA is fast and cheap when compared to the other methods. The 15
Adjusted Rand index and Wallace’s coefficient indicated that MLVA was highly 16
predictive for spa-type, but not vice versa. 17
Analysis of the region neighboring SIRU05 showed a difference in the genetic element 18
bordering the repeats of SIRU05 that explained the negative SIRU05 PCRs. PFGE, 19
MLST, and MLVA are adequate typing methods for bovine-associated Staphylococcus 20
21
22
23
24
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discriminatory, reproducible, stable and portable typing method for epidemiological
3
1. Introduction 25
26
27Staphylococcus aureus is a major agent of contagious mastitis in diary cattle. The sources
of bovine mastitis cases mostly are from bovine origin, but S. aureus originating from the 28
farmer are another important source (Zadoks et al., 2002). In 2005 it was reported that29
each year at least 25% of all milking cows in The Netherlands suffer from clinical 30
mastitis which is not only due to S. aureus (32%) as a causative agent (Lam, 2005). Many 31
different typing methods have been used. From a “gold standard” Pulsed-Field Gel 32
Electrophoresis (PFGE) to Multi Locus Sequence Typing (MLST) and S. aureus-specific 33
staphylococcal Protein A typing known as spa-typing (Enright, et al., 2000; Harmsen et 34
al., 2003; Struelens et al.,1992). However, not all methods can be used in all centers 35
because some methods are still limited to well-equipped laboratories. Furthermore, both 36
MLST and spa-typing have insufficient discriminatory power to allow accurate 37
delineation of outbreaks and PFGE is a fingerprinting method which makes 38
interlaboratory comparison difficult (van Belkum et al., 1995). 39
A limited number of molecular studies have been published that explored the population 40
structure and genetic relationships of S. aureus causing bovine mastitis (de Sousa et al., 41
2007; Hata et al., 2006; Jørgensen et al., 2005; Katsuda et al, 2005; Reinoso et al., 2008). 42
The results of our recent study demonstrated that a multiple locus variable number of 43
tandem repeats (VNTR) analysis (MLVA) could be used as a fast, inexpensive, highly 44
45
tracing of human S. aureus. (Ikawaty et al., 2008). Therefore, we aimed to expand the use 46
of this novel MLVA scheme to S. aureus isolated from clinical cases of bovine mastitis47
and to get insight in their genetic relationship with the human S. aureus population.48
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from the manufacturer with the exception that bacterial pellet is resuspended in buffer T1.
4
2. Methods49
50
2.1. Strain collection 51
Eighty five Staphylococcus aureus isolates of clinical or subclinical cases of mastitis 52
were included. Thirty five isolates were obtained by the Faculty of Veterinary Medicine 53
Utrecht University, Utrecht, The Netherlands from at least 26 farms near Utrecht. Isolates 54
from farms sampled twice were taken at least one year apart. Another 50 isolates were 55
collected by the Animal Health Service in Deventer, The Netherlands for the Central 56
Veterinary Institute, Lelystad, The Netherlands from farms throughout The Netherlands. 57
The sources were individual teat milk samples from dairy cattle from all over the country 58
from clinical or subclinical cases of mastitis. Each isolate from CVI represents one farm, 59
the location of the farms is unknown but they are distributed all over the Netherlands. All 60
isolates were methicillin-susceptible Staphylococcus aureus (MSSA) and collected 61
between 1988 and 2005. 62
63
2.2. Genomic DNA preparation64
65
The isolates were grown on blood agar (Trypticase soy agar II containing 5% 66
sheep blood) overnight at 37oC prior to DNA isolation. Preparation of bacterial genomic 67
DNA was performed using the NucleoSpin kit (Macherey-Nagel) following the protocols 68
69
In our method, T1 buffer was replaced by freshly-made lysis buffer that contained 20mM 70
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