Some coagulase negative Staphylococcus spp. isolated from buffalo can be misidentified as Staphylococcus aureus by phenotypic and Sa442 PCR methods
Almeida et al. BMC Res Notes
Some coagulase negative Staphylococcus spp. isolated from buffalo can be misidentified as Staphylococcus aureus by phenotypic and Sa442 PCR methods
Camila C. de Almeida 0 2
Lucas J. L. Pizauro 0 1
Glenn A. Soltes 0
Durda Slavic 4
Fernando A. de Ávila 1
João M. Pizauro 3
Janet I. MacInnes 0
0 Department of Pathobiology, University of Guelph , 50 Stone Rd. East, Guelph, ON N1G 2W1 , Canada
1 Department of Veterinary Preventive Medicine and Animal Reproduction, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences , Jaboticabal , Brazil
2 Agriculture and Livestock Microbiology Graduation Program, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences , Jaboticabal , Brazil
3 Department of Technology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences , Jaboticabal , Brazil
4 Animal Health Laboratory, University of Guelph , Post Office 3612, Guelph, ON N1H 6R8 , Canada
Objective: Staphylococcus aureus is a commonly reported cause of buffalo mastitis. However, its prevalence may be overestimated. The aim of this study was to compare S. aureus identification by conventional phenotypic and genotypic assays versus Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and novel real-time quantitative PCR tests for the cytochrome oxidase subunit D II (cydB) and staphylocoagulase (coa) genes. Results: From 408 samples obtained from buffalo milk/milking environment, 32 putative S. aureus strains were identified based on characteristic growth on Baird Parker agar, positive catalase reaction, ability to clot rabbit plasma, and positive Sa442 PCR assay. However, in further testing, only 10 of these strains were positive in latex agglutination tests and by MALDI-TOF MS, only eight of the 32 strains were S. aureus while the rest were S. chromogenes (19), S. agnetis (3), S. cohnii (1), or S. xylosus (1). All eight strains identified as S. aureus by MALDI-TOF analysis and confirmed by 16S RNA gene sequencing were positive in a S. aureus-specific cydB PCR test. As well, 7/8 S. aureus strains were PCR positive in a real-time coa PCR test as were 2/69 S. chromogenes and the lone S. xylosus strain tested.
Mastitis; Staphylococcus aureus; Species-specific PCR tests; cydB PCR
Buffalo milk and its derivatives have become
increasingly important worldwide [
] and Staphylococcus aureus
is one of the most significant pathogens responsible for
contagious mastitis in dairy buffaloes [
treatment of S. aureus mastitis is often unsuccessful and
treatment failures can lead to spread of the infection.
As a result, animals with chronic S. aureus infection are
often culled [
The initial identification of S. aureus is based on culture
and phenotype on specific media; other assays commonly
used to identify S. aureus are the Sa442 PCR, nuc gene
PCR, and latex agglutination tests. The Sa442 PCR test,
developed by Martineau et al. [
] targets a chromosomal
DNA fragment thought to be specific for S. aureus, the
nuc gene encodes a species-specific thermonuclease,
while commercially available latex agglutination kits such
as the Staphaurex latex test are based on the interaction
of S. aureus surface-anchored proteins with human IgG
and fibrinogen bound to latex particles [
these tests may not be accurate and can lead to
erroneous identification, and in turn, to unnecessary culling [
]. Here we describe how common testing approaches
can lead to misidentification of “non-S. aureus” strains
as S. aureus and the development of new a cydB
realtime PCR assay that can be used for accurate S. aureus
Milk samples (n = 320) were collected from 80 randomly
selected female buffaloes from a private dairy farm
located in Sao Paulo State, Brazil from November 2013 to
April 2014. After physical examination of the mammary
], teats were cleaned with 70% alcohol and milk
from each quarter was evaluated by strip cup and
California mastitis tests [
]. Hand samples from 16
consenting milkers and 64 samples from liners were collected
using sterile swabs (Pro-Lab Diagnostics) and stored in
peptone water as described previously [
CoPS isolation and identification
Isolation and identification of S. aureus was done
according to compendium of methods for the
microbiological examination of foods [
]. Strains with positive egg
yolk reactions [
] were tested for Gram and catalase
reactions, haemolytic activity, and ability to clot rabbit
plasma using a Coagu-Plasma kit (Laborclin, Pinhais,
Brazil) according to manufacturer’s instructions with S.
aureus ATCC 25293 and S. epidermidis ATCC 12228 as
the positive and negative controls respectively. In
addition, isolates were tested with the Staphyclin latex test
(Laborclin, Pinhais, Brazil) according to the
For DNA extraction, well-isolated colonies were
inoculated into BHI broth, incubated at 37 °C for 18 h and
extracted according to the method of Kuramae-Izioka [
with minor modifications as described previously [
Isolates were characterized using the PCR assay of
Martineau et al. [
] with minor modifications. PCR
primers (Table 1) were used at 10 pmol/µL in 25 µL reaction
mixtures with 50 ng/µL of DNA and 20 µL LightCycler®
480 SYBER Green Master mix (Roche Diagnostics,
Indianapolis, IN). Amplification parameters were: one
cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C
for 10 s, 55 °C for 20 s, and 72 °C for 12 s in a Roche
LightCycler® 480 (LC480) thermocycler. The ramp rates
were 4.4, 2.2, and 4.4 °C/s, respectively. Staphylococcus
chromogenes and Streptococcus suis DNAs and water
were used in negative control reactions and S. aureus
strains COL, NewMan, MW2, Mu50 and ATCC 25923
were used as positive controls. A melting step (62 °C) was
done to confirm single product.
Matrix‑Assisted Laser Desorption IonizationT‑ime of Flight
Mass Spectrometry (MALDI–TOF MS)
Identification of putative Staphylococcus aureus strains
was done using a MALDI Bruker Biotyper system
(Bruker Daltonics Inc., Billerica, MA, USA) at the
Animal Health Laboratory, University of Guelph, Guelph,
Ontario, Canada, as described previously [
16S rRNA gene sequencing
16S rRNA gene sequencing (~ 1000 bp) was also done at
the Animal Health Laboratory and sequences were
compared with the 16S rRNA gene of Staphylococcus aureus
MCRF184 (CP014791.1) and other Staphylococcus spp.
sequences using blastn.
Design of cydB species‑specific primers
PCR primers to the S. aureus cydB gene (cytochrome D
ubiquinol oxidase subunit II; NCBI accession number
NC_007795.1) were designed as described previously
] and produced a 432 bp amplicon. The amplicons
were confirmed by DNA sequencing as described above
(Table 1). As well, an additional 84 putative staphylococci
from buffalo milk/milking environment were evaluated
using the cydB test [
Design of coagulase gene primers
Published coa primers [
] generate products of different
sizes so a new primer pair (coaF and coaR; Table 1) was
designed using PrimerQuest software (Integrated DNA
technologies, Inc. http://www.idtdna.com) to the
coagulase gene of S. aureus JCSC 7638 (AB488509.1). A
conserved region of the gene was identified by aligning 103
strains in GenBank using CLC Sequence View 7 software
(Additional file 1: Fig. S1).
Real-time PCR primers (Table 1) were used at 10 pmol/
µL in 25 µL reaction mixtures containing 50 ng/µL of
DNA and 20 µL Light Cycler 480 (LC480) SYBER Green
Master mix (Roche Diagnostics, Indianapolis, IN). For
cydB and coa gene amplifications the parameters were:
one cycle at 95 °C for 10 min, followed by 40 cycles at
95 °C for 10 s, 55 °C for 20 s, and 72 °C for 25 s in a Roche
Light Cycler 480 (LC480). Ramp rates were 4.4, 2.2, and
4.4 °C/s, respectively; Streptococcus suis DNA and water
were used as negative controls. In addition, a melting
step (62 °C) was done to confirm a single product. S.
aureus strains COL, NewMan, MW2, Mu50 and ATCC
25923 were used as positive controls. All real-time PCR
were performed in triplicate.
Staphylococcus spp. isolation and preliminary identification
Thirty-two putative S. aureus strains were selected based
on their characteristic phenotype on Baird Parker agar.
These Gram positive strains were catalase positive and
were positive in the S. aureus species specific Sa442 PCR
assay of Martineau et al. [
]. In further testing, 24
samples were consistently positive in the coagulase test; while
eight gave at least one discordant result. Also, 21 of the
32 putative S. aureus strains were β-haemolytic, two were
α-haemolytic and nine were non-haemolytic.
Latex test, identification by MALDIT‑OF and 16S rRNA sequencing
Only ten of the 32 strains gave a positive latex
agglutination result and MALDI-TOF MS analysis revealed that
only eight of the 32 were S. aureus with the remainder
being S. chromogenes (n = 19), S. agnetis (n = 3), S.
xylosus (n = 1), or S. cohnii (n = 1) (Table 2). All eight strains
identified as S. aureus by MALDI-TOF analysis had
100% identity with the 16S rRNA gene of Staphylococcus
aureus MCRF184 (NZ_CP014791.1). The two latex false
positive strains were S. agnetis and chromogenes by 16S
sequencing and MALDI_TOF.
cydB gene analysis and detection
Alignment of gene sequences (https://www.ncbi.nlm.nih.
gov/genbank/) suggested that the cydB gene is well
conserved in Staphylococcus and thus allowed for the design
of species-specific primers (Additional file 2: Fig. S2). The
eight S. aureus isolates were positive for the S.
aureus-specific cydB primers and resultant amplicons had 99–100%
identity with S. aureus NCTC 8325. Further, the S. aureus
specific cydB primers did not amplify any of the other
CoPS tested in this study nor the 84 strains of other
putative staphylococci evaluated by Pizauro et al. [
coa gene analysis
Alignment of 103 coagulase gene sequences (https://
www.ncbi.nlm.nih.gov/genbank/) revealed that although
coa genes possess many polymorphic areas, a region
between 1300 and 1600 bp has sufficient homology to be
used for detection in S. aureus strains (Table 1, Additional
file 1: Fig. S1). All of the S. aureus positive control strains
tested [COL (NC_002951.2), NewMan (NC_009641.1),
MW2 (NC_003923.1), Mu50 (NC_002745.2) and
Staphylococcus aureus ATCC (25923)] were positive using the
coaF and coaR primer pair.
Coagulase test and coa gene detection
Twenty-four strains clotted rabbit plasma (Table 2). Eight
strains gave discordant results with at least one negative
and one positive. Of these strains, seven of the S. aureus
(n = 8) and two of the S. chromogenes (n = 19) and one S.
xylosus strain were positive for the coa gene while none
of the S. agnetis (n = 3) nor the S. cohnii were positive for
the coa gene. The sequence of the PCR products had 98%
identity with the S. aureus coa gene from strain JCSC
7633 (accession number AB488507.1) and 99%
identity with the coa gene in S. aureus strain MW2 genome
(accession number BA000033.2).
A number of typically coagulase negative Staphylococcus
spp. including more than a quarter of S. chromogenes
isolates (19/69) and at least some S. xylosus (1/1), S. cohnii
(1/1), and S. agnetis (3/17) were coagulase positive. This
finding is consistent with studies of Santos et al. [
which 23/42 CoNS strains clotted rabbit plasma. These
authors suggested that this phenotype was related to
specific PFGE-types, but not with the clumping factor test.
The presence of coagulase is an indicator of pathogenicity
since it enables bacteria to resist phagocytosis and cause
chronic infections [
]. Host specific adaptations can be
acquired though mobile genetic elements (MGEs) [
] from nearby S. aureus  or other CoPS such as S.
]. Thus, a coa gene in CoNS with the
newly described coa primers may be the result of such
transfer. Coagulase activity in the current study may also
be related to another gene such as the one described in S.
chromogenes that shares 41% identity with the predicted
coagulase gene of the S. pseudintermedius [
In this study, non-S. aureus stains able to clot plasma
(19/19 S. chromogenes, 3/3 S. agnetis, 1/1 S. cohnii and 1/1 S.
xylosus) were also Sa442 positive. This is the first report of
false positive reactions with these species; however, further
stains/herds should be tested to know whether these
findings can be generalized. When the sequences of the Sa442
] were compared with the available genome of S.
chromogenes MU 970 strain (NZ_JMJF00000000.1), no
significant homology was detected; however, this draft whole
shotgun sequence could be missing the region
containing Sa442 sequences. Apart from having been established
as unique for S. aureus, the Sa442 fragment has not been
further characterized [
]. As well, Klaassen et al. [
Heilmann et al. [
] have reported false negative results
with the Sa442 test and the nuc PCR is subject to strain
Latex agglutination tests may also be problematic. In
previous studies, false positive results have been observed
at relatively low frequencies (e.g., 7.9% [
] and 9.3% [
The greater false positive reaction in this study (20%) may
have been related to the population structure and/or the
relatively small sample size.
Given the impact that S. aureus can have on both human
and animal health, its diagnosis is important [
Misidentifying more benign Staphylococcus species as S.
aureus, though arguably less serious, is not without
significant economic consequences. In the current study, the
eight S. aureus strains (as identified by MALDI-TOF/16S
rRNA sequencing) were positive with our novel S.
aureusspecific cydB PCR test while no amplification was observed
with the Sa442 and plasma clotting-positive S.
chromogenes (n = 19), S. agnetis (n = 3), S. xylosus (n = 1) or S. cohnii
(n = 1) strains tested. Also, it might be noted that S. caprae,
S. hyicus, S. hominis, S. epidermidis, S. haemolyticus, S.
warneri, S. equorum, S. sciuri, and S. pasteuri were negative
in the S. aureus-specific cydB real-time PCR test in a
complementary study  (Table 2).
In summary, a significant number of CoNS strains could
clot rabbit plasma and were positive for the Sa442 PCR
test and so, could be misclassified as S. aureus. The bases of
these phenotypes remain to be determined, but they could
be the result of horizontal gene transfer or to the fact that
these species are less homogenous than previously thought.
On the other hand, MALDI-TOF and a species-specific
real-time PCR test for the cydB gene may permit accurate
identification of CoNS.
This study used samples from one buffalo herd which limits
the generalisation of the results. In addition, it was beyond
the scope of this study to determine the basis of the
abnormal coagulase positive phenotype or to determine if there
had been horizontal gene transfer to coagulase negative
Additional file 1: Fig. S1. S. aureus coagulase gene (coa) alignment using
CLC Sequence View 7 software.
Additional file 2: Fig. S2. Alignment using CLC Sequence View 7
software of the cydB gene of 18 closely related Staphylococcus spp. in this
CoNS: coagulase negative Staphylococcus; MALDI-TOF MS: Matrix-Assisted
Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF
MS); qPCR: real-time quantitative PCR; PCR: polymerase chain reaction; CoPS:
coagulase positive Staphylococcus.
CCA, LJLP and GAS developed the proposal, collected the samples in the field,
did the laboratory work and drafted the manuscript. DS, FAA, JMP and JIM
corrected the proposal, supervised the sample collection and laboratory work,
analyzed the data, interpreted the results and provided technical advice and critically
reviewed the manuscript. All authors read and approved the final manuscript.
Our thanks to Dr. David Heinrichs (University of Western Ontario) for the kind
gift of S. aureus strains COL, NewMan, MW2, and Mu50.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets generated during the current study are available from the
corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The study was approved by the Ethics Committee on Animal Use (CEUA) of
the School of Agricultural and Veterinarian Sciences, Jaboticabal—FCAV/
UNESP/Jaboticabal Campus—Brazil (Protocol Number 013737/13). Dairy farm
owner have given verbal consent to use the animals in this work. Farm
workers have also given verbal consent to participate in this work.
This work was conducted during a scholarship supported by the International
Cooperation Program CAPES/COFECUB at the University of Guelph and
financed by CAPES—Brazilian Federal Agency for Support and Evaluation of
Graduate Education within the Ministry of Education of Brazil.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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