HAL Id: hal-00532532
https://hal.archives-ouvertes.fr/hal-00532532
Submitted on 4 Nov 2010
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires
Performance of API Staph ID 32, Staph-Zym for identification of coagulase-negative staphylococci
isolated from bovine milk samples
O.C. Sampimon, R.N. Zadoks, S. de Vliegher, K. Supré, F. Haesebrouck, H.W. Barkema, J. Sol, T.J.G.M. Lamf
To cite this version:
O.C. Sampimon, R.N. Zadoks, S. de Vliegher, K. Supré, F. Haesebrouck, et al.. Perfor- mance of API Staph ID 32, Staph-Zym for identification of coagulase-negative staphylococci iso- lated from bovine milk samples. Veterinary Microbiology, Elsevier, 2009, 136 (3-4), pp.300.
�10.1016/j.vetmic.2008.11.004�. �hal-00532532�
Accepted Manuscript
Title: Performance of API Staph ID 32, Staph-Zym for identification of coagulase-negative staphylococci isolated from bovine milk samples
Authors: O.C. Sampimon, R.N. Zadoks, S. De Vliegher, K.
Supr´e, F. Haesebrouck, H.W. Barkema, J. Sol, T.J.G.M. Lamf
PII: S0378-1135(08)00531-2
DOI: doi:10.1016/j.vetmic.2008.11.004
Reference: VETMIC 4268
To appear in: VETMIC Received date: 6-6-2008 Revised date: 3-11-2008 Accepted date: 6-11-2008
Please cite this article as: Sampimon, O.C., Zadoks, R.N., De Vliegher, S., Supr´e, K., Haesebrouck, F., Barkema, H.W., Sol, J., Lamf, T.J.G.M., Performance of API Staph ID 32, Staph-Zym for identification of coagulase-negative staphylococci isolated from bovine milk samples, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2008.11.004
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Accepted Manuscript
Performance of API Staph ID 32 and Staph-Zym for identification of coagulase-
1
negative staphylococci isolated from bovine milk samples
2
3
O.C. Sampimona*, R.N. Zadoksb, S. De Vliegherc, K. Supréc, F. Haesebrouckd, H.W.
4
Barkemae, J. Solaand T.J.G.M. Lama,f
5
aGD Animal Health Service, Arnsbergstraat 7, PO Box 9, 7400 AA Deventer, The
6
Netherlands
7
bQuality Milk Production Services, Cornell University, Ithaca, New York, USA. Current
8
address: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, and
9
Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland, UK
10
cDepartment of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine,
11
Ghent University, Belgium
12
dDepartment of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary
13
Medicine, Ghent University, Belgium
14
eDepartment of Production Animal Health, Faculty of Veterinary Medicine, University of
15
Calgary, Calgary, AB, Canada
16
fDutch Udder Health Centre, Deventer, The Netherlands
17
18
*Corresponding author: o.sampimon@gddeventer.com
19
20 21
Abstract
22
23
In this study, the accuracy of two phenotypic tests, API Staph ID 32 and Staph-Zym, was
24
determined for identification of coagulase-negative staphylococci (CNS) from bovine milk
25
samples in comparison with identification based on DNA-sequencing. A total of 172 CNS
26
isolated from bovine milk were classified into 17 species. The most frequently isolated
27
Manuscript
Accepted Manuscript
epidermidis, followed by Staphylococcus xylosus, and Staphylococcus equorum (37, 13, 9
29
and 6% of isolates, respectively). The API Staph ID 32 correctly identified 41% of the CNS
30
isolates. Best agreement with rpoB sequence based species identification was found for S.
31
epidermidis, Staphylococcus hyicus and S. xylosus (100, 89 and 87%, respectively).The
32
positive predictive value was 89, 100 and 52%, respectively. Poor sensitivity was observed
33
for 3 of the 5 most frequently found species, S. chromogenes(37%), Staphylococcus warneri
34
(15%) and S. equorum (0%) albeit with specificity of 100%. The Staph-Zym needed
35
additional tests for 66% of the isolates and identified 31% of the CNS isolates correctly.
36
Good sensitivity was found for S. epidermidis, S. simulansand S. xyloxus (100, 78 and 73%,
37
respectively). The positive predictive value was 89, 78 and 98%, respectively. Poor
38
sensitivity was observed for S. chromogenes, S. warneri and S. equorum (0, 54 and 0%,
39
respectively) but with a specificity of 100, 99 and 100%, respectively. Both phenotypic tests
40
misidentified a large proportion of CNS isolates and were thus unsuitable for identification of
41
CNS species from bovine milk samples.
42 43
Keywords: bovine; mastitis; coagulase-negative staphylococci; genotyping; phenotyping
44
45 46
1. Introduction
47
48
Coagulase-negative staphylococci (CNS) are commonly isolated from cases of
49
subclinical and clinical mastitis, and also from teat canals, teat skin and teat ducts (De
50
Vliegher et al., 2003; Taponen et al., 2006; Sampimon et al., 2007). In several recent
51
prevalence studies, CNS were the most frequently isolated udder pathogens from bovine
52
milk samples with high somatic cell count (SCC) (Pitkälä et al., 2004; Bradley et al., 2007;
53
Piepers et al., 2007). Coagulase-negative staphylococci are a heterogeneous group of
54
organisms. Currently, the genus Staphylococcus consists of more than 40 named species
55
(Bes et al., 2000; Devriese et al., 2002). Coagulase-negative staphylococcal species may
56
Accepted Manuscript
differ in antimicrobial susceptibility, virulence factors, host response to infection and
57
transmissibility. To assess the pathogenic significance of individual CNS species and to
58
develop species-specific management practices, accurate species identification is needed.
59
Several schemes for the identification of CNS based on phenotypic characteristics
60
have been developed (Devriese et al., 1994). These phenotypic schemes require numerous
61
media and are labour intensive. Also, they require extended incubation periods that limit
62
usefulness for routine diagnostics. The ideal test for routine diagnostic laboratory
63
identification of CNS species in milk samples of cows would be reliable, time and cost-
64
effective and easy to use. Commercial test kits, like the API Staph ID 32 (API Test,
65
bioMérieux, France) and the Staph-Zym test (Rosco, Taastrup, Denmark) are used for
66
phenotypic identification of CNS in diagnostic laboratories. However, they may lack accuracy
67
because these tests and their accompanying databases were mainly developed for human
68
isolates (Watts and Washburn, 1991; Thorberg and Brändström, 2000; Taponen et al., 2006;
69
Zadoks and Watts, 2008). Because of the large and increasing diversity of microorganisms
70
and the prevalence of organisms with rare, inconsistent or poorly defined phenotypic
71
characteristics, conventional methods often cannot fully characterize bacterial isolates, and
72
laboratories are increasingly relying on DNA sequencing for microorganism identification
73
(CLSI, 2007; Zadoks and Watts, 2008). For identification of CNS species, sequence data of
74
housekeeping genes such as rpoB, cpn60, dnaJ or tuf can be used (Capurro et al., 2008;
75
Drancourt and Raoult, 2002; Mellmann et al., 2006; Shah et al., 2007; Zadoks and Watts,
76
2008).
77
The aim of the present study was to measure the sensitivity, specificity and positive
78
predictive value of two frequently used phenotypic methods, the API Staph ID 32 and the
79
Staph-Zym test, for identification of CNS isolated from bovine milk samples through
80
comparison with species identification based on DNA-sequence data of housekeeping
81
genes.
82
83
Accepted Manuscript
2. Materials and methods
85
86
2.1. Cultures
87
A total of 175 CNS isolates were selected at random from quarter milk samples of
88
clinical and subclinical mastitis cases submitted for bacteriological examination to the GD
89
Animal Health Service (GD; Deventer, The Netherlands). Only isolates from a pure culture on
90
agar plates were included, with a maximum of one isolate per herd.
91
Bacteriological culturing was carried out according to NMC protocols (Harmon et al.,
92
1990). Briefly, an inoculum of 0.01 ml of milk was spread on a sheep blood agar plate. All
93
plates were incubated at 37ºC and examined after 24 and 48 h. A milk sample was
94
considered CNS-positive when ≥ 500 cfu/ml of CNS were cultured. Coagulase-negative
95
staphylococci were differentiated from coagulase positive staphylococci using Slidex Staph
96
Plus (bioMérieux, France). Isolates with a negative test result on the Slidex Staph Plus were
97
further tested with the tube coagulase test. Coagulase-negative staphylococci isolates were
98
frozen and stored at -70°C.
99 100
2.2. Reference method
101
The identification of the CNS isolates with the API Staph ID 32 and the Staph-Zym
102
test was compared with species identification based on DNA-sequence data. Isolates were
103
grown on Colombia agar with 5% sheep blood (Gibco Technologies, Paisley, Scotland) for
104
24 h at 37°C. To prepare DNA extracts, one calibrated loop (1 µl) of cells was suspended in
105
20 µl lysis buffer (0.25% SDS, 0.05 N NaOH), heated for 5 min at 95°C, and diluted with 180
106
µl distilled water. Cell debris was spun down by centrifugation at 16000g for 5 min and
107
supernatants were used as PCR template. PCR of a 751 bp fragment of the rpoB gene was
108
performed using published primers and run parameters (Drancourt en Raoult, 2002). PCR-
109
products were purified with the PureLink DNA kit (Invitrogen Corporation, Carlsbad, CA).
110
DNA-sequencing was carried out in two directions by the BioResource Center (Cornell
111
University, Ithaca, NY) using the PCR primers and Big Dye Terminator chemistry on an ABI
112
Accepted Manuscript
PRISM 3700 DNA analyzer. Sequence data were assembled and proofread using SeqMan
113
(version 5.08, Lasergene, DNASTAR Inc., Madison, WI). Assembled sequence data were
114
compared with sequence data in GenBank using the nucleotide-nucleotide BLAST algorithm
115
of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Although
116
94% DNA sequence homology is considered sufficient for identification of CNS species using
117
rpoB (Mellman et al., 2006), additional methods were used to confirm or determine the
118
identity of isolates with < 98% homology to reference data. Additional identification was
119
performed using the amplification and sequencing of a 600 bp fragment of the housekeeping
120
gene cpn60 (Zadoks et al., 2005) using degenerate primers that amplify this target gene in
121
multiple gram-positive bacterial genera (Goh et al., 1997) or, if species identity was still
122
unresolved, based on sequencing of 16S rDNA (Durak et al., 2005).
123 124
2.3. API Staph ID 32
125
The API Staph ID 32 test strip consists of 32 capsules, 26 of which contain
126
dehydrated biochemical media for colorimetric tests. Tests were performed and interpreted
127
according to the manufacturer’s recommendations (API Test, bioMérieux, Lyon, France).
128
Briefly, the frozen CNS isolates were recultured on agar plates containing 6% sheep blood
129
(Biotrading, Mijdrecht, The Netherlands). After 24 hours of incubation, bacterial suspensions
130
were prepared to a density of 0.5 McFarland in 6 ml of sterile distilled water and distributed
131
into the wells of the API test strip. The strips were incubated at 37ºC and examined visually
132
after 24 h. The Apiweb software was used to calculate the probability of the identification
133
result in a range of 10 to 100% (https://apiweb.biomerieux.com). In this study, only
134
identification with a probability≥ 90% was considered acceptable (Thorberg and Brändström,
135
2000; Taponen et al., 2006). If the probability of a result was < 90%, isolates were
136
considered unidentified.
137 138
2.4. Staph-Zym test
139
Accepted Manuscript
The Staph-Zym test consists of a rigid, transparent plastic strip with 10 upright
140
minitubes divided into four groups of tests. The manufacturer’s instructions (Rosco, Taastrup,
141
Denmark) were followed to conduct the test and interpret the results. Briefly, the frozen CNS
142
isolates were recultured on sheep blood agar plates. Bacterial suspensions were prepared
143
from cultures after 24 hours of incubation and standardized to a 2 McFarland standard in 3
144
ml physiological saline (0.9% NaCl). Approximately 0.25 ml of the bacterial suspension was
145
dispensed into each of the 10 tubes. After an incubation of 24 h at 37ºC, positive reactions
146
were recorded and converted to five-digit numbers. The 5-digit number provided a numerical
147
profile of known CNS species and suggested additional tests, i.e. enzyme reactions,
148
Pyrrolidonyl Aminopeptidase 2h, Vogues Proskauer 4h, anaerobic growth, susceptibility to
149
fosfomycin and nalidixic acid, and acid formation from sucrose, cellobiose, raffinose, and
150
xylose for species delineation. Isolates not recognized by numerical profile and additional
151
tests, were considered unidentified.
152 153
2.5. Data analysis
154
Isolates were considered to be correctly identified by API Staph ID 32 and Staph-Zym if the
155
same species was found as with the reference method. Misidentified was defined as a
156
different species found compared to reference method. For both phenotypic methods,
157
sensitivity, specificity and predictive value positive (PVP) were calculated in comparison with
158
DNA-based species identification. Sensitivity was calculated as the proportion of the true
159
positive isolates that are correctly identified with the phenotypic tests, e.g. the proportion of
160
S. chromogenes isolates based on sequence data that was identified as such by phenotypic
161
testing. Specificity was calculated as the proportion of the true negatives that are correctly
162
identified with the phenotypic tests, e.g. the proportion of isolates other than S. chromogenes
163
based on sequence data that were identified as something other than S. chromogenes by
164
phenotypic testing. Finally, PVP was calculated as the proportion of isolates identified as a
165
specific species based on phenotypic testing that truly represented that particular species,
166
Accepted Manuscript
e.g. the proportion of isolates that were identified as S. chromogenes by phenotypic testing
167
that had been identified as S. chromogenes based on DNA sequence data.
168 169 170
3. Results
171
172
3.1. Isolates
173
A total of 175 isolates were analyzed by DNA sequencing. One isolate was identified
174
as Streptococcus uberis and one isolate was identified as Moraxella osloensis based on
175
cpn60 sequencing. PCR with rpoB primers had been negative for both isolates, which is
176
explained by the fact that the primers are specific to the genus Staphylococcus and neither
177
isolate belonged to that genus. Of the remaining 173 CNS isolates, all of which yielded an
178
amplicon with rpoB PCR, 172 were identified at the species level. Based on rpoB, cpn60 or
179
16S sequence data, one isolate could be identified at the genus level but not at the species
180
level, suggesting that it is an unknown species (92% homology to reference sequences for
181
rpoB and cpn60; 99% homology to S. pasteuri as well as S. lugdunensis based on partial
182
16S sequence data). This isolate was not identified to the species level by API Staph ID 32
183
or Staph-Zym typing either. Further analyses were performed on the 172 isolates that were
184
identified to species level. The most frequently isolated species based on gene sequencing
185
were S. chromogenesandS. epidermidis, followed byS. xylosus, S. warneriand S. equorum
186
(Table 1). For 7 isolates with 94% to 97% homology of therpoB sequence to reference data,
187
species identity was also determined based on cpn60 sequence data (4x S. hyicus, and 3x
188
S. warneri). For all 7 isolates, rpoB based species identity was confirmed, with cpn60
189
sequences showing 97% to 100% homology to reference data. In addition, species identity
190
was determined based on cpn60 data for 8 isolate that had yielded noisy or deteriorated
191
rpoB sequence data (3x S. equorum, 2x S. succinus, and 3x S. xylosus). For one isolate,
192
rpoB and cpn60 data were noisy, and isolate identity was determined based on 16S
193
Accepted Manuscript
homology with previously published sequence data were deposited in GenBank under
195
accession number FJ 404467, and FJ 404468 for S. hyicus, and S. warneri, respectively.
196 197
3.2. API Staph ID 32
198
The API Staph ID 32 identified 71 of 172 (41%) isolates correctly (Table 1). The best
199
sensitivity in comparison with rpoB-sequence based identification was found for S.
200
epidermidis, S. hyicus and S. xylosus, (100, 89 and 87%, respectively). The specificity of
201
detection for these species was 98, 100 and 92%, respectively and PVP was 89, 100 and
202
52%, respectively (Table 2). Poor to very poor sensitivity was observed for 3 of the 5 most
203
commonly found species, i.e. for S. chromogenes, S. warneri, and S. equorum(37, 15 and
204
0% sensitivity, respectively) (Table 1). The specificity of detection of these species and PVP
205
were high (100%), with the exception of the PVP for S. equorum (Table 2). Of the 101 (59%)
206
isolates in this set of samples that were not identified correctly, 80 were unidentified and 21
207
were misidentified (Table 1), i.e. the species identification by the Apiweb system (with
208
probability ≥ 90%) was not in agreement with the reference method. Of 9 S. equorum
209
isolates 8 were identified as S. xylosus and one as S. sciuri. Two S. chromogenes isolates
210
were misidentified as S. caprae and S. xylosus, respectively; one S. haemolyticusisolate as
211
S. auricularis; one S. nepalensis isolate as S. xylosus; one S. saprophyticus isolate as S.
212
epidermidis; one S. simulans isolate as S. epidermidis; two S. succinus isolates as S.
213
xylosus;two S. warneri as S. capitisand S. simulans, respectively and finally two S. xylosus
214
isolates as S. epidermidis and as S. simulans, respectively.
215 216
3.3. Staph-Zym test
217
Using the Staph-Zym test, 53 of 172 isolates (31%) were identified correctly, based
218
on a single suggestion for identification. Sensitivity of identification was highest for S.
219
epidermidis (100%), S. simulans (78%) and S. xylosus (73%) (Table 2). The specificity of
220
identification of those species was 98, 99 and 98%, respectively and PVP was 89, 78 and
221
98%, respectively (Table 2). Poor to very poor sensitivity was observed for three of the five
222
Accepted Manuscript
most commonly found species: S. chromogenes, S. warneriand S. equorum (0, 54 and 0%,
223
respectively) (Table 1). The specificity of identification of these species was 100, 99 and
224
100%, respectively. Because sensitivity of detection was 0% for S. chromogenes and S.
225
equorum, PVP could not be calculated. For S. warneri, PVP was 78% (Table 2). No
226
identification was obtained for 101 (59%) isolates. For 86 isolates, the Staph-Zym test
227
yielded more than one species name as a result. For 15 isolates the Staph-Zym test could
228
not suggest any species identification. Misidentification was observed for 18 of 172 (10%)
229
isolates. One S. capitis isolate was misidentified as S. saprophyticus; two S. chromogenes
230
isolates were misidentified as S. haemolyticus; one S. cohniisubsp. cohniias S. xylosus; two
231
S. equorum isolates as S. gallinarum and S. xylosus, respectively; threeS. fleuretti isolates
232
as S. sciuri; three S. haemolyticus isolates as two S. warneri and one S. simulans; one S.
233
hominisisolate as S. epidermidis;oneS. simulansisolate asS. epidermidis; one S. succinus
234
as S. xylosus and three S. xylosus isolates as S. epidermidis, S. equorumand S. simulans,
235
respectively. For 113 of 172 (66%) isolates additional tests were needed.
236 237 238
4. Discussion
239
240
Coagulase-negative staphylococci as a group are increasingly seen as important
241
mastitis pathogens (Taponen et al., 2007). The group is comprised of many different
242
staphylococcal species. In a recent study of Taponen et al. (2007), CNS IMI were persistent
243
throughout lactation, significantly increased quarter SCC, and because of their relatively high
244
prevalence contributed to a higher average BMSCC. Because of their impact on milk quality,
245
control measures may need to be developed for CNS. Also for epidemiological investigations
246
and assessment of their pathogenic significance, accurate species identification of CNS is
247
needed.
248
Several commercial test kits are available for CNS identification, all of which are
249
Accepted Manuscript
kits. Genotypic methods have higher typeability and accuracy than phenotypic methods, and
251
identify over 99% of CNS isolates from bovine milk (Zadoks and Watts, 2008). The high
252
proportion of species identification based on DNA sequence data is likely due to the large
253
reference database (GenBank) which is continuously updated with new strains or species
254
including species common in animals but not in humans (Zadoks and Watts, 2008). Routine
255
updates to methodology or databases are not economically feasible for commercial
256
phenotypic test kits. Because of the superior identification obtained with genotypic methods,
257
the implementation of such methods by diagnostic laboratories is encouraged (CLSI, 2007).
258
Among 172 CNS isolates from bovine milk, 17 CNS species were identified with rpoB
259
sequencing, and 11 and 15 species with API Staph ID 32 and Staph-Zym, respectively. The
260
API Staph ID 32 and Staph-Zym identified 41 and 31%, respectively, of the CNS isolates
261
correctly, which was lower than reported in other studies (Watts and Washburn, 1991;
262
Thorberg and Brändström, 2000; Taponen et al., 2006; Carpurro et al., 2008). An explanation
263
could be the different study designs and associated species distribution. Both phenotypic
264
databases were developed to identify CNS isolates from human origin which may explain the
265
relatively high sensitivity for identification of species such as S. epidermidisand S. xylosus,
266
which are commonly found in humans. However, the PVP of the API Staph ID 32 for S.
267
xylosuswas 52% which indicates that only about half of the isolates from bovine milk that are
268
identified asS. xylosusby API Staph ID 32 really belong to this species. With the Staph-Zym,
269
the PVP for S. xylosus was much higher, i.e. 98%, implying that almost all isolates that are
270
identified as S. xylosus by Staph-Zym testing are indeed S. xylosus. However, only about
271
three-quarters of all S. xylosus isolates are recognized as such by Staph-Zym testing. The
272
low sensitivity of the Staph-Zym for most species in our study was mainly caused by the
273
large number of isolates for which identification could not be narrowed down to a single
274
species. Another drawback of the Staph-Zym system was the large number of additional
275
tests needed to obtain final results. Additional tests were needed for 66% of the isolates in
276
our study, compared to 45% of isolates in the study by Thorberg and Brändström (2000) and
277
33% in the study by Carpurro et al. (2008). Additional tests increase cost, labor and time
278
Accepted Manuscript
delay of final outcomes of the test. Despite additional testing, many isolates could not be
279
identified to the species level by the Staph-Zym test, in agreement with results from Capurro
280
et al. (2008).
281
Staphylococcus chromogenes was the most frequently isolated species in our study.
282
Approximately two-thirds of these isolates were misidentified by the API Staph ID 32.
283
However, the specificity and PVP were both 100% which means that the API Staph ID 32
284
had a low number of false-positives in detection of S. chromogenes. The Staph-Zym did not
285
identify a single S. chromogenesisolate correctly, which is a big concern considering that S.
286
chromogenes is among the most common CNS species in bovine milk. Identification of
287
coagulase negative S. aureus as S. chromogenes by the Staph-Zym has been reported
288
(Capurro et al., 2008) but was not observed in our study. Differences between countries
289
seem to exist in predominance of CNS species. In Sweden, USA (Tennessee), and
290
Zimbabwe, S. chromogenes was the predominant species, while in Norway, Finland and
291
Belgium S. simulanswas the most common CNS species (Matthews et al., 1990; Devriese et
292
al., 1994; Waage et al., 1999; Thorberg and Brändström, 2000; Kudinha and Simango, 2002;
293
Taponen et al., 2006; Capurro et al., 2008). These may be true differences, but differences
294
can also be caused by differences in identification methods. Our data suggest that
295
prevalence of S. chromogenes is more likely to be underestimated with the Staph-Zym
296
method than with the API Staph ID 32, whereas the reverse is true for S. simulans.
297
Staphylococcus hyicus was commonly identified in the 1980s (Watts and Owens, 1989), but
298
is not among the most common species in more recent studies. This is probably explained by
299
the fact that S. chromogenes was considered a subspecies of S. hyicus in the 1980s,
300
whereas it is recognized as a separate species nowadays. True misidentification of isolates
301
may also have occurred (Bes et al., 2000; Zadoks and Watts, 2008).
302
In addition to well known species, we found some CNS species that have thus far
303
rarely been associated with bovine mastitis, i.e. S. equorum, S. nepalensisand S. fleuretti.
304
Staphylococcus equorum constituted 6% of our isolates, making it the fifth-most frequently
305
Accepted Manuscript
tests used in our study should have the ability to identify the species (bioMérieux, France;
307
Rosco, Taastrup, Denmark). However, none of the nine S. equorum were identified correctly.
308
In a study in Finland, only one isolate of S. equorum was found (Taponen et al., 2006). We
309
also found S. nepalensis and S. fleuretti in pure culture from bovine samples.
310
Staphylococcus nepalensis was first isolated from the respiratory tract of goats kept in the
311
Himalayan region in 2003 (Spergser et al., 2003). Staphylococcus fleurettiwas isolated from
312
goat’s milk cheeses for the first time in 2000 (Vernozy-Rozand et al., 2000). These examples
313
illustrate that new CNS species continue to be discovered, and explain why phenotypic test
314
methods may not be as up to date as reference databases based on DNA-sequence data. In
315
our study, more CNS species were found with a genotypic method than expected from earlier
316
studies with phenotypic tests. The prevalence and impact of these particular CNS species on
317
udder health is unknown. Further studies would be necessary to determine their impact and
318
relevance.
319
We conclude that, in our study, the API Staph ID 32 and the Staph-Zym had a high
320
number of unidentified or misidentified CNS isolates. Both tests were insufficient to identify
321
CNS species from bovine milk samples as demonstrated by poor sensitivity and positive
322
predictive value for identification of several of the most commonly isolated CNS species.
323
Misidentification or ambiguous test results can lead to erroneous interpretation of
324
data and to false conclusions in epidemiological or monitoring studies. Comparing test
325
results between studies that used different techniques is not meaningful if test results are not
326
accurate. For most routine laboratories it is more appropriate to report “coagulase-negative
327
Staphylococcus species” based on coagulase testing, than reporting of more detailed but
328
potentially inaccurate results. In research and to identify new species, genotypic methods for
329
species identification are to be preferred over phenotypic methods.
330 331 332
Conflict of interest
333
334
Accepted Manuscript
None of the authors (O.C. Sampimon, R.N. Zadoks, S. De Vliegher, K. Supré, F.
335
Haesebrouck, H.W. Barkema, J. Sol and T.J.G.M. Lam) has a financial or personal
336
relationship with other people or organizations that could inappropriately influence or bias the
337
paper entitled “Performance of API Staph ID 32 and Staph-Zym for identification of
338
coagulase-negative staphylococci isolated from bovine milk samples”.
339 340 341
Acknowledgment
342
343
This study is part of the five year mastitis program of the Dutch Udder Health Centre
344
and was financially supported by the Dutch Dairy Board.
345 346 347
References
348
349
Bes, M., Guerin-Faublee, V., Meugnier, H., Etienne, J., Freney, J., 2000. Improvement of the
350
identification of staphylococci isolated from bovine mammary infections using
351
molecular methods. Vet. Microbiol. 71, 287-294.
352
Bradley, A.J., Leach, K.A., Breen, J.E., Green, L.E., Green, M.J., 2007. Survey of the
353
incidence and aetiology of mastitis on dairy farms in England and Wales. Vet. Rec.
354
160, 253-257.
355
Capurro, A., Artursson, K., Waller, K.P., Bengtsson, B., Ericsson-Unnerstad, H., Aspán, A.,
356
2008. Comparison of commercialized phenotyping system, antimicrobial susceptibility
357
testing, and tuf gene sequence-based genotyping for species-level identification of
358
coagulase-negative staphylococci isolated from cases of bovine mastitis. Vet. Mic.
359
doi: 10.1016/j.vetmic.2008.08.028.
360
CLSI (Clinical and Laboratory Standards Institute). 2007. Interpretive criteria for
361
Accepted Manuscript
microorganism identification by DNA target sequencing; proposed guideline. CLSI
362
document MM18-P (ISBN 1-56238-646-8). Clinical and Laboratory Standards
363
Institute, Wayne, PA.
364
De Vliegher, S., Laevens, H., Devriese, L.A., Opsomer, G., Leroy, J.L., Barkema, H.W., de
365
Kruif, A., 2003. Prepartum teat apex colonization with Staphylococcus chromogenes
366
in dairy heifers is associated with low somatic cell count in early lactation. Vet.
367
Microbiol. 92, 245-252.
368
Devriese, L.A., Laevens, H., Haesebrouck, F., Hommez, J., 1994. A simple identification
369
scheme for coagulase-negative staphylococci from bovine mastitis. Res. Vet. Sci. 57,
370
240-244.
371
Devriese, L.A., Baele, M., Vaneechoutte, M., Martel, A., Haesebrouck, F., 2002. Identification
372
and antimicrobial susceptibility of Staphylococcus chromogenes isolates from
373
intramammary infections of dairy cows. Vet. Microbiol. 87, 175-182.
374
Drancourt, M., Raoult, D, 2002. RpoB-gene sequence-based identification of
375
Staphylococcusspecies. J. Clin. Microbiol. 40, 1333-1338.
376
Durak, M.Z, Fromm, H.I.l., Huck, J.R., Zadoks, R.N., Boor, K.J., 2006. Development of
377
molecular typing methods forBacillusand Paenibacillusspp. isolated from fluid milk
378
products. J. Food Sci. 71, M50-M56.
379
Goh, S. H., Z. Santucci, W. E. Kloos, M. Faltyn, C. G. George, D. Driedger, and S. M.
380
Hemmingsen. 1997. Identification of Staphylococcusspecies and subspecies by the
381
chaperonin 60 gene identification method and reverse checkerboard hybridization. J.
382
Clin. Microbiol. 35:3116–3121.
383
Harmon, R.J., Eberhart, R.J., Jasper, D.E., Langlois, B.E., Wilson, R.A., 1990.
384
Microbiological procedures for the diagnosis of udder infection. National Mastitis
385
Council Inc., Arlington, VA, USA.
386
Kundinha, T., Simango, C., 2002. Prevalence of coagulase-negative staphylococci in
387
bovine mastitis in Zimbabwe. J. S. Afr. Vet. Assoc. 73, 62-65.
388
Matthews, K.R., Oliver, S.P., King, S.H., 1990. Comparison of Vitek Gram-Positive
389
Accepted Manuscript
identification system with API Staph-Trac system for species identification of
390
staphylococci of bovine origin. J. Clin. Microbiol. 28, 1649-1651.
391
Mellman, A., Becker, K., Eiff van C., Keckevoet, U., Schumann, P., Harmsen, D., 2006.
392
Sequencing and staphylococci identification. Emerg. Infect. Dis. 12, 333-336.
393
Piepers, S.L., De Meulemeester, L., De Kruif, A., Opsomer, G., Barkema, H.W., De Vliegher,
394
S., 2007. Prevalence and distribution of mastitis pathogens in subclinically infected
395
dairy cows in Flanders, Belgium. J. Dairy Res. 74, 478-483.
396
Pitkälä, A., Haveri, M., Pyörälä, S., Myllys, V., Honkanen-Buzalski, T., 2004. Bovine mastitis
397
in Finland 2001 prevalence, distribution of bacteria, and antimicrobial resistance. J.
398
Dairy Sci. 87, 2433-2441.
399
Sampimon, O.C., Vernooij, J.C.A., Mevius, D.J., Sol, J., 2007. [Sensitivity for various
400
antibiotics of coagulase-negative staphylococci, isolated from milk samples of Dutch
401
dairy cattle]. Tijdschr. Diergeneeskd. 132, 200-204.
402
Shah, M.M., Iihara, H., Noda, M., Song, S.X., Nhung, P.H., Ohkusu, K., Kawamura, Y.,
403
Ezaki, T. 2007. dnaJ gene sequence-based assay for species identification and
404
phylogenetic grouping in the genus Staphylococcus. Int J Syst Evol Microbiol. 57:25-
405
406
30.Spergser, J., Wieser, M., Taubel, M., Rossello-Mora, R.A., Rosengarten, R., Busse, H.J.,
407
2003. Staphylococcus nepalensis sp. nov., isolated from goats of the Himalayan
408
region. Int. J. Syst. Evol. Microbiol. 53, 2007-2011.
409
Taponen, S., Simojoki, H., Haveri, M., Larsen, H.D., Pyörälä, S., 2006. Clinical
410
characteristics and persistence of bovine mastitis caused by different species of
411
coagulase-negative staphylococci identified with API or AFLP. Vet. Microbiol. 115,
412
199-207.
413
Taponen, S., Koort, J., Björkroth, J., Saloniemi, H., Pyörälä, S., 2007. Bovine intramammary
414
infections caused by coagulase-negative staphylococci may persist throughout
415
lactation according to amplified fragment length polymorphism-based analysis. J.
416
Accepted Manuscript
Thorberg, B.M., Brändström, B., 2000. Evaluation of two commercial systems and a new
418
identification scheme based on solid substrates for identifying coagulase-negative
419
staphylococci from bovine mastitis. J. Vet. Med. B Infect. Dis. Vet. Public Health 47,
420
683-691.
421
Vernozy-Rozand, C., Mazuy, C., Meugnier, H., Bes, M., Lasne, Y., Fiedler, F., Etienne, J.,
422
Freney, J., 2000. Staphylococcus fleurettisp. nov., isolated from goat’s milk cheeses.
423
Int. J. Syst. Evol. Microbiol. 50, 1521-1527.
424
Waage, S., Mørk, T., Røros, A., Aasland, D., Hunshamar, A., Odegaard, S.A., 1999. Bacteria
425
associated with clinical mastitis in dairy heifers. J. Dairy Sci. 82, 712-719.
426
Watts, J.L., Owens, W.E., 1989, Prevalence of staphylococcal species in four dairy herds.
427
Res. Vet. Sci. 46, 1-4.
428
Watts, J.L., Washburn, P.J., 1991. Evaluation of the Staph-Zym system with staphylococci
429
isolated from bovine intramammary infections. J. Clin. Microbiol. 29, 59-61.
430
Zadoks, R. N., Schukken, Y. H., Wiedmann, M., 2005. Multilocus sequence typing of
431
Streptococcus uberis provides sensitive and epidemiologically relevant subtype
432
information and reveals positive selection in the virulence gene pauA. J. Clin.
433
Microbiol. 43, 2407-2417.
434
Zadoks, R.N., Watts, J.L. 2008 . Species identification of coagulase-negative
435
staphylococci: genotyping is superior to phenotyping. Vet. Microbiol.,
436
doi:10.1016/j.vetmic.2008.09.012
437
438
Accepted Manuscript
Table 1
439
Identification of coagulase-negative staphylococci with the API Staph ID 32 and the Staph-
440
Zym test, compared with rpoB sequencing as the reference method.
441
rpoB sequencing API Staph ID 32 Staph-Zym
Correct identified
Correct identified
CNS species n n % MI1 UI2 n % MI UI
S. arlettae 1 0 0.0 0 1 0 0.0 0 1
S. capitis 1 0 0.0 0 1 0 0.0 1 0
S. chromogenes 63 23 36.5 2 38 0 0.0 2 61
S. cohnii subsp. cohnii 5 0 0.0 0 5 0 0.0 1 4
S. epidermidis 23 23 100.0 0 0 23 100.0 0 0
S. equorum 10 0 0.0 9 1 0 0.0 2 8
S. fleuretti 5 0 0.0 0 5 0 0.0 3 2
S. haemolyticus 9 0 0.0 1 8 2 22.2 3 4
S. hominis 1 0 0.0 0 1 0 0.0 1 0
S. hyicus 9 8 88.9 0 1 1 11.1 0 8
S. nepalensis 1 0 0.0 1 0 0 0.0 0 1
S. saprophyticus 2 0 0.0 1 1 1 50.0 0 1
S. sciuri subsp. carnaticus 3 0 0.0 0 3 1 33.3 0 2
S. simulans 9 2 22.2 1 6 7 77.8 1 1
S. succinus 2 0 0.0 2 0 0 0.0 1 1
S. warneri 13 2 15.4 2 9 7 53.8 0 6
S. xylosus 15 13 86.7 2 0 11 73.3 3 1
Total 172 71 41.3 21 80 53 30.8 18 101
1MI=misidentified,2UI=unidentified.
442
443
Accepted Manuscript
Table 2
444
Sensitivity, specificity and positive predictive value of the API Staph ID 32 and the Staph-
445
Zym test of most common CNS species in this study, using rpoB sequencing as reference
446
method.
447
CNS species API Staph ID 32 Staph-Zym
Sensitivity Specificity PV+1 Sensitivity Specificity PV+
S. chromogenes 36.5 100.0 100.0 0.0 100.0 n/a
S. epidermidis 100.0 98.0 88.5 100.0 98.0 88.5
S. equorum 0.0 100.0 n/a 2 0.0 100.0 n/a
S. haemolyticus 0.0 100.0 n/a 22.2 98.8 50.0
S. hyicus 88.9 100.0 100.0 11.1 100.0 100.0
S. simulans 22.2 98.8 50.0 77.8 98.8 77.8
S. warneri 15.4 100.0 100.0 53.8 98.8 77.8
S. xylosus 86.7 92.4 52.0 73.3 98.1 97.5
1PV+ = positive predictive value.
448
2 n/a = not applicable.
