Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Aureocin A70 production is disseminated amongst

genetically unrelated Staphylococcus aureus involved in
bovine mastitis
H. Ceotto1, R.C. da Silva Dias2, J. dos Santos Nascimento3, M.A.V. de Paiva Brito4, M. Giambiagi-
deMarval1 and M. do Carmo de Freire Bastos1
1 Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
2 Departamento de Microbiologia e Parasitologia, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
3 Instituto Federal de Educacao, Ciencia e Tecnologia do Rio de Janeiro, RJ, Brazil
4 Embrapa Gado do Leite, Juiz de Fora, MG, Brazil

Keywords
bacteriocin, bovine mastitis, staphylococcin,
Staphylococcus aureus.
Correspondence
Maria do Carmo de Freire Bastos, Instituto de
Microbiologia Paulo de Goes UFRJ,
Departamento de Microbiologia Geral, CCS,
Bloco I, Cidade Universitaria, 21941-902 Rio
de Janeiro, RJ, Brazil.
E-mails: mcbastos@micro.ufrj.br or
mcbastos2@yahoo.com.br
2011 1743: received 12 October 2011,
revised 3 February 2012 and accepted 17
February 2012
doi:10.1111/j.1472-765X.2012.03226.x
Abstract
Aims: The main aim of this study was to analyse the genetic relationship
amongst 46 Staphylococcus aureus Bac+ strains isolated in Brazil from 12 geo-
graphically distant dairy herds, including 34 isolates that produce the antimi-
crobial peptide aureocin A70.
Methods and Results: The comparison of 46 Staph. aureus Bac+ strains was
performed by pulsed-field gel electrophoresis (PFGE). Thirteen different pulso-
types were identified, and the subtype A1 was the most prevalent one. Nine
strains belong to pulsotype F, the second most prevalent and mostly confined
to a single herd. The PFGE patterns of the 34 Staph. aureus aureocin A70-pro-
ducers, isolated in Brazil, were also compared with those of strains isolated
from bovine mastitis cases in Argentina and revealed that these strains are not
genetically related.
Conclusions: Although a previous study has suggested that a prevalent pulso-
type of aureocin A70-producer Staph. aureus involved in bovine mastitis is
disseminated in Argentina, this does not occur in Brazil. Additionally, it was
possible to demonstrate that closely related staphylococcal strains can produce
distinct staphylococcins.
Significance and Impact of the Study: This study corroborates the hypothesis
of horizontal gene transfer of aureocin A70 genes amongst distinct staphylococ-
cal strains involved in bovine mastitis, giving them a selective advantage when
colonizing the mammary glands.

Although studies on bacteriocins produced by Staphylo-

Introduction
Bacteriocins (Bac) are antimicrobial peptides or proteins
with bactericidal activity against other bacteria. The bacte-
riocin-producing strains are immune to their own prod-
uct, and this immunity is conferred by an immunity
system, which is generally expressed concomitantly with
the bacteriocin structural genes (Bastos et al. 2009). Bac-
teriocins produced by Staphylococcus spp. are designated
staphylococcins, and most of them are encoded by plas-
mids (Bastos et al. 2009).
coccus aureus are not numerous, some of them have been
identified and characterised to date such as staphylococ-
cin C55 BacR1 (Navaratna et al. 1998), Bsa (bacteriocin
of Staph. aureus) (Daly et al. 2010) and aureocins A53
(Netz et al. 2002), A70 (Netz et al. 2001) and 4185 (Ceot-
to et al. 2010). Both staphylococcin C55 and Bsa are
lantibiotics (small heat-stable peptides containing unusual
amino acids), while aureocins A53 and A70 are class II
bacteriocins, that is, nonmodified small peptides. Aureoc-
ins 4185 have not been fully characterized yet.

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Letters in Applied Microbiology 54, 455461 2012 The Society for Applied Microbiology 455
Spread of Staphylococcus aureus involved in bovine mastitis H. Ceotto et al.

Aureocin A70 and its variants are staphylococcins with Table 1 Molecular characteristics of the bacteriocin-producer Staphy-
a broad spectrum of activity (Bastos et al. 2009), which lococcus aureus strains isolated in Brazil
are produced by most Bac+ staphylococcal strains (>70%) Staph. aureus Production of
involved in bovine mastitis either in Argentina (Nasci- Bac+ Plasmid profile aureocin
mento et al. 2002) or in Brazil (Ceotto et al. 2009). Pro- Herd strain (size in kb) A70 PFGE type
duction of such bacteriocins by mastitic staphylococci
1 4087 44; 80*; >27 + A1
confers aureocin A70 immunity to the producer strains,
4089 85*; >27 + B
allowing them not only to inhibit other Gram-positive 4091 85*; >27 + G
micro-organisms but also to grow in the presence of 2 4093 24; 38; 135; >27 ) L
either aureocin A70 or its variants, giving them a selective 4100 35 ) E1
advantage when colonizing a given host (Bastos et al. 3 3913 31; 36; 49 ) I
2009). 4 3958C 85*; 95 + A1
Recently, 46 Staph. aureus strains, isolated from 3959 31; 36; 50 ) I
bovine mastitis cases in the southeast region of Brazil, 3962C 85* + A1
3963C 85*; >27 + A1
were shown to produce bacteriocins (Ceotto et al.
5 3705 23; 34; 80* >27 + H
2009). Thirty-four strains produce bacteriocins either
6 4061 43; 80* + A1
identical or similar to aureocin A70, and the remaining 4063 43; 80* + A1
12 Bac+ strains produce antimicrobial peptides that are 4066 43; 80* + A1
distinct from the best characterized staphylococcins 4071 79* + A1
described so far. As all aureocin A70-producer Sta- 4143 45; 80*; >27 + A1
ph. aureus strains involved in bovine mastitis and iso- 4147 43; 81*; >27 + A1
lated from 12 herds located in different provinces of 4148 43; 82*; >27 + A1
Argentina were shown to have the same genetic lineage 4150 30; 42 ) C1
4154 43; 80*; >27 + A1
(Nascimento et al. 2002), the bacteriocin production
4230 30; 55 ) C2
may contribute to this clone spread. This study aimed
7 3633 37; 80* + A1
to confirm this hypothesis amongst strains isolated from 8 4180 34 ) K
different Brazilian herds geographically distant from 4181 84* + M1
each other. Therefore, these 46 Staph. aureus Bac+ 4183 79 ) J
strains isolated from bovine mastitis in Brazil were 4185 115 ) M2
typed by pulsed-field gel electrophoresis (PFGE) and 9 3853 29; 32; 45 ) F2
their pulsotypes were compared with representative puls- 10 4042 81* + B
4044 30; 35; 80* + D
otypes found in Argentina.
4045 ) K
4046 80; >27 ) K
Material and methods 11 2246 81* + A1
12 4314 79* + F1
4315 79* + F1
Bacterial strains and growth conditions
4316 >27 + F1
Fifty-two bacteriocin-producers Staph. aureus strains were 4317 79* + F1
analysed. Most strains (51) were isolated from either clin- 4318 79* + F1
ical (severe) or subclinical (moderate) bovine mastitis 4319 79* + F1
4320 79* + F1
cases. Forty-six strains were isolated, between 1997 and
4322 79* + A1
2001, from different infected animals of 12 different dairy
4323 83*; >27 + E2
herds located in the southeast region of Brazil. Amongst 4324 79* + A1
these 46 strains, 34 carry the genetic determinants for au- 4325 79* + A1
reocin A70 production, previously detected by both PCR 4326 79*; >27 + A1
and DNA DNA hybridization (Ceotto et al. 2009; 4328 79*; >27 + A2
Table 1). Five aureocin A70-producer strains, isolated 4329 79* + F1
between 1989 and 1995, from five different herds located
PFGE, pulsed-field gel electrophoresis; +, aureocin A70 production; ),
in five different provinces of Argentina (Staph. aureus absence of aureocin A70 production.
strains 15, 18, 20, 26 and 32), and Staph. aureus A70, the The plasmid profile and the bacteriocin production by the strains were
first aureocin A70-producer strain described in the litera- retrieved from Ceotto et al. (2009) and confirmed in the present man-
ture (Giambiagi-deMarval et al. 1990), isolated from uscript.
commercial milk in Brazil, were also included in this *Plasmid involved in aureocin A70 biosynthesis.

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456 Letters in Applied Microbiology 54, 455461 2012 The Society for Applied Microbiology
H. Ceotto et al.
study. The five strains from Argentina were representative
of the different pulsotypes described by Nascimento et al.
(2002), most of them distinct from the pulsotype of strain
A70.
In some cases, multiple isolates from the same herd
were included in this study, because the plasmids
involved in aureocin A70 production, which generally
have a size ranging from 79 to 85 kb, are mobilizable by
conjugative genetic elements (Coelho et al. 2009). There-
fore, these plasmids might be found in genetically unre-
lated staphylococcal strains.
The staphylococcal strains were cultivated in Tryptic
Soy Broth (TSB; Difco, Detroit, MI, USA) at 37C and
stored in suspensions containing TSB and 40% (v v)
glycerol at )20C. The medium was supplemented with
agar at 15% (w v), for preparation of solid medium,
when required.
Strain typing
All Staph. aureus isolates, including the Argentinean
strains, were typed by PFGE as previously described (Na-
scimento et al. 2002). Chromosomal DNA in the plugs
was digested with SmaI (New England Biolabs, Ipswich,
USA) at 25C according to the manufacturers instruc-
tions. The restriction fragments were separated by PFGE
on 1% (w v) agarose gels, and electrophoresis was carried
out in a CHEF-DR III system (Bio-Rad Laboratories,
Hercules, CA) at 13C and 6 V cm)1 for 21 h with pulse
times ranging from 2 to 35 s. DNA-banding patterns were
interpreted by computer-assisted analysis with GelCom-
par II, ver. 4.01 (Applied Maths, Courtrai, Belgium). A
similarity matrix was obtained by comparison between
pairs of strains using the Dice coefficient of similarity. A
dendrogram was constructed using the unweighted pair
group method with arithmetic mean.
The pulsotypes and subtypes were determined based on
the criteria proposed by van Belkum et al. (2007). The
strains with PFGE banding patterns differing by one to
four bands were assigned to subtypes of the same type.
The strains with banding patterns differing by five or
more bands were classified as distinct types.
DNA isolation and manipulations
The plasmid profiles of 46 strains isolated in the south-
east region of Brazil were confirmed as previously
described. Whole-cell lysates were prepared, and the
plasmid profiles were determined by agarose gel electro-
phoresis [07% (w v)]. Strains MB32 and MB196 were
used as molecular-weight controls for plasmid DNA, as
they contain staphylococcal plasmids of known sizes
(Ceotto et al. 2009).
2012 The Authors
Letters in Applied Microbiology 54, 455461 2012 The Society for Applied Microbiology
Spread of Staphylococcus aureus involved in bovine mastitis
Endonucleases restriction sites present on pRJ6, as well
as the size of the DNA fragments generated upon diges-
tions with these enzymes, were analysed by the program
NEBcutter V2.0 (http://tools.neb.com/NEBcutter2/).
Analyses were made with the 7904-bp sequence of the
plasmid pRJ6 (Coelho et al. 2009), retrieved from Gen-
Bank (accession number: AF241888).
The plasmids pRJ6, from Staph. aureus A70, and
pRJ80, from Staph. aureus 4181, were isolated using
QIAprepSpin Miniprep Kit (Qiagen, Hilden, Germany).
Cultures were harvested after 18 h of growth at 37C.
The cells were lysed by incubation in P1 buffer (provided
by the kit) with lysostaphin (10 U; Sigma, USA), for
30 min at 37C. After this step, the procedure followed
the manufacturers recommendations.
The plasmids were digested with the restriction enzymes
BglII (New England Biolabs), ClaI (New England Biolabs),
EcoRI (New England Biolabs), EcoRV (Promega), HindIII
(New England Biolabs), or PstI (New England Biolabs),
according to the manufacturers recommendations. The
digested DNA was separated by agarose gel electrophoresis
[07% (w v)]. The gels were blotted onto nylon membranes
(Hybond-N; GE Healthcare, Uppsala, Sweden), using stan-
dard methods (Sambrock et al. 1989).
The plasmid pRJ6, purified and labelled with a32P-dCTP,
was used as a probe. The products of digestion of this plas-
mid were used as positive controls. Both the labelling of the
probe and the DNA DNA hybridizations were carried out
using Redprime II Random Prime Labelling System (GE
Healthcare) according to the manufacturers instructions.
The unincorporated nucleotides were removed prior to
hybridization with the ProbeQuantTM G-50 Micro Col-
umns (GE Healthcare). Hybridization was revealed by
exposure to Hyperfilm X-ray film (GE Healthcare), during
1 h, and revealed with Dektol (Kodak, USA).
Results
Cluster analysis of the 46 strains isolated from 12 Brazil-
ian dairy herds
Amongst the 46 Staph. aureus strains isolated from the
dairy herds located in Brazil, 13 different pulsotypes were
identified (A to M) Fig. 1. The pulsotype A was the
most prevalent one, being found in 19 (413%) strains, all
producers of aureocin A70 (Table 1). Two distinct sub-
types (A1 and A2) were identified (Fig. 1). The subtype
A1 was the most prevalent including 18 strains (391%),
described in Table 1. Based on herds location, these
observations suggest that this Staph. aureus clone has a
broad geographical distribution.
Nine strains (196%) belonged to pulsotype F, the sec-
ond most prevalent one. These strains were also assigned
457
Spread of Staphylococcus aureus involved in bovine mastitis H. Ceotto et al.

100
45
50
55
60
65
70
75
80
85
90
95
STRAIN PULSOTYPE
4100 E1
4323 E2
4150 C2
4230 C1
4044 D
4143 A1
4328 A2
4089 B
4315 F1
3853 F2
4093 L Figure 1 Dendrogram generated from
4180 K computer-assisted analysis of the representa-
4181 M1 tive pulsed-field gel electrophoresis banding
4185 M2
patterns observed amongst Staphylococcus
3913 I
4183 J aureus Bac+ strains isolated from bovine
4091 G mastitis cases in the southeast region of
3705 H Brazil.

to two distinct subtypes (F1 and F2). The subtype F1
accounted for eight strains (174%), while only one strain
(3853) belonged to subtype F2 (Table 1).
The pulsotype K included three strains (4045, 4046 and
4180), while the pulsotypes I (strains 3913 and 3959) and
B (strains 4042 and 4089) were found in two strains each,
with identical PFGE patterns. Three pulsotypes, C, E and
M, were also found in two strains each but that exhibited
different PFGE patterns; therefore, they were assigned to
distinct subtypes: C1 (4150) and C2 (4230), E1 (4100) and
E2 (4323), M1 (4181) and M2 (4185). The remaining five
strains (109%) were separated into five different pulso-
types (D, G, H, J and L) Table 1.
Comparative analysis of the aureocin A70 producer
strains
The 34 aureocin A70 producers isolated from Brazilian
herds were grouped into eight pulsotypes: A (subtypes A1
and A2), B, D, E (subtype E2), F (subtype F1), G, H and
M (subtype M1) Table 1. These data indicate that in
Brazil, aureocin A70 production is spread amongst geneti-
cally unrelated bacteria, being specially prevalent in herds
6 and 12, as all strains belonging to pulsotype A and sub-
type F1 produce aureocin A70 (Table 1).
The PFGE patterns of the Brazilian aureocin A70 pro-
ducers were also compared with those of the five aureocin
A70-producer strains isolated from bovine mastitis cases
in Argentina and with that of Staph. aureus A70. These
studies revealed 11 distinct pulsotypes as shown in Fig. 2.
The five representative Staph. aureus strains isolated
from Argentina included in this analysis were assigned to
three different pulsotypes (N, O and P). Staphylococcus
aureus 15 and also Staph. aureus A70 belong to the pulso-
type N, although they were assigned to distinct subtypes:
N1 and N2, respectively. The profile of Staph. aureus 18
was classified as pulsotype O. The strains 20, 26 and 32
were assigned to three distinct subtypes: P1, P2 and P3,
respectively (Fig. 2). These findings agree with the results
previously described by Nascimento et al. (2002).
The 11 distinct pulsotypes observed amongst aureocin
A70 producers could be clustered into two groups. All
strains isolated from Argentina (pulsotypes N, O and P)
were clustered into the same group, II-A, while most
Brazilian strains were clustered into the second group,
II-B (pulsotypes A, B D, E [subtype E2], F and H), by
using a cut-off value of 45% similarity (Fig. 2), con-
firming that these strains are indeed not genetically
related.
Plasmid profiles
The plasmid profiles of the 46 Bac+ strains, isolated in
Brazil, are shown in Table 1.
As previously demonstrated, 45 strains showed at least
one plasmid DNA form and strain 4045 did not present
any plasmid form. Thirty-five strains (761%) exhibited a
plasmid DNA with a size similar to that of pRJ6 (79 kb)
(Ceotto et al. 2009). The single plasmid (84 kb) found in
strain 4181 was named pRJ80.
Comparison between plasmids pRJ6 and pRJ80
As the plasmids with size similar to that of pRJ6 do not
seem to differ from each other more than 500 bp, a preli-
minary physical map was constructed for a representative
one to better estimate its size. Because of the interest in
further studies with the bacteriocin produced by strain
4181, the plasmid pRJ80 was chosen for this analysis.
The sequence analysis of pRJ6 using the program
NEBcutter V2.0 revealed that this plasmid has a single
site for the restriction enzymes BglII and EcoRI, two sites
for the enzymes EcoRV and PstI, and four sites for the
enzymes HindIII and ClaI (Table 2).

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458 Letters in Applied Microbiology 54, 455461 2012 The Society for Applied Microbiology
H. Ceotto et al. Spread of Staphylococcus aureus involved in bovine mastitis

100
45
50
55
60
65
70
75
80
85
90
95
STRAIN PULSOTYPE
A70 N1
15 N2
26 P2
32 P3
20 P1
II-A
4091 G
18 O
4181 M1
4143 A1
Figure 2 Dendrogram generated from computer-
4328 A2
assisted analysis of the pulsed-field gel electro- 4315 F1
phoresis pulsotypes observed amongst aureocin 4089 B
A70-producer strains isolated from bovine mastitis 4044 D
cases in Brazil and Argentina. The two main II-B 4323 E2
groups are represented as II-A and II-B. 3705 H

Table 2 Comparison of the restriction sites found in plasmids pRJ80 and pRJ6

Approximate
size (kb) of
Number of the fragments
restriction Expected fragments Number of fragments Number of fragments observed
Restriction sites identified size (kb) after digestion observed after digestion observed after digestion after digestion
Enzyme in pRJ6 of pRJ6 of pRJ6 of pRJ80 of pRJ80

BglII 1 79 1 2 41 and 43
ClaI 4 02, 12, 27 and 38 3* 3 15, 30 and 40
EcoRI 1 79 1 1 80
EcoRV 2 24 and 54 2 3 20, 21 and 43
HindIII 4 04, 11, 13 and 50 4 1 80
PstI 2 17 and 61 2 3 23, 24 and 37

*It was not possible to detect the fourth fragment (230 bp) after agarose gel electrophoresis.

Although pRJ6 presents four fragments generated by

digestion with the enzyme ClaI (230, 1136, 2718 and
3820 bp), only three fragments (1136, 2718 and 3820 bp)
were visualized after agarose gel electrophoresis. The
fourth and smallest fragment could not be visualized. All
other expected fragments were observed (Table 2).
It was possible to observe that, as pRJ6, pRJ80 pre-
sented a single fragment after digestion with EcoRI. A sin-
gle fragment was also seen after digestion with HindIII;
two fragments were visualized after digestion with BglII
and three fragments were visualized after digestion with
EcoRV, ClaI and PstI (Table 2). The digestions of both
plasmids with ClaI generated two fragments of similar
size (ClaI-A, with c. 4 kb, and ClaI-B, with c. 3 kb), and
a third fragment, ClaI-C, of different size in each plasmid:
c. 12 and 15 kb for pRJ6 and pRJ80, respectively.
The products of pRJ80 digestions were transferred to
nylon membranes and used in DNA DNA hybridization
analyses using pRJ6 as a probe. All fragments derived
from pRJ80 hybridized with the a32P-labelled pRJ6 DNA.
Some representative results are shown in Fig. 3.
Discussion
Aureocin A70 is a four-peptide bacteriocin that has been
reisolated many times from Staphylococcus spp. Bac+
strains, including coagulase-negative staphylococci, both
in Brazil and in Argentina (Giambiagi-deMarval et al.
1990; Oliveira et al. 1998b; Gamon et al. 1999; Nascimen-
to et al. 2002, 2005; Ceotto et al. 2009).
The present study investigated the genetic relationship
amongst 46 Staph. aureus Bac+ strains, including 34 au-
reocin A70-producers, isolated from either clinical or sub-
clinical bovine mastitis cases in Brazil. Thirteen different
PFGE pulsotypes (A to M) were identified.
Although the 18 strains that compose the subtype A1,
the most prevalent one, exhibited an identical PFGE
banding pattern, the present study confirmed that these
strains exhibit different plasmid profiles as previously
reported (Ceotto et al. 2009).
The eight strains grouped into the subtype F1 were iso-
lated from the same herd (12). Seven strains (4314, 4315,
4317, 4318, 4319, 4320 and 4329) exhibited the same

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Letters in Applied Microbiology 54, 455461 2012 The Society for Applied Microbiology 459
Spread of Staphylococcus aureus involved in bovine mastitis
Not digested
(a) plasmid EcoRV HindIII PstI
1 2 3 4 5 6 7 8 9 Argentina (Nascimento et al. 2002), this does not occur
80 kb
30 kb
(b)
80 kb
30 kb
Figure 3 Comparison of plasmids pRJ80 and pRJ6. (a) Agarose gel
electrophoresis [07% (w v)] showing the undigested plasmids pRJ6
(lane 1) and pRJ80 (lane 2), and the same plasmids digested with the
enzymes EcoRV, HindIII and PstI. (b) Southern blot hybridization using
the plasmid pRJ6 labelled with a32P- dCTP as probe. Lanes 1, 4, 6 and
8, plasmid pRJ6; Lanes 2, 5, 7 and 9, plasmid pRJ80. Lane 3, 1-kb
DNA ladder. The numbers to the left of the gel represent molecular
size standards.
plasmid profile, suggesting that they represent the same iso-
late, and strain 4316 exhibited a distinct plasmid pattern.
The dissemination of the subtype A1, especially in herd
6, and subtype F1, in herd 12, suggests that the bovine
mastitis cases in these herds were caused by isolates that
represent the same clone. Possibly, the distinct plasmid
profiles observed amongst these clonal isolates resulted
from independent events of horizontal gene transfer,
because the bacterial plasmids are considered mobile
genetic elements commonly found in staphylococci (Ma-
lachowa and DeLeo 2010).
Besides the subtypes A1 and F1, other pulsotypes are
also involved in aureocin A70 production. Such data indi-
cate that aureocin A70 production was not restricted to a
single clone.
When the PFGE patterns of the 34 Staph. aureus aureo-
cin A70-producers isolated in Brazil were compared with
those of strains isolated from bovine mastitis cases in
Argentina and with that of the first aureocin A70 pro-
ducer described in the literature, 11 distinct pulsotypes
460 Letters in Applied Microbiology 54, 455461 2012 The Society for Applied Microbiology
H. Ceotto et al.
were found. Although a previous study has suggested that
a prevalent pulsotype of aureocin A70-producer Sta-
ph. aureus involved in bovine mastitis is disseminated in
in Brazil.
Aureocin A70 is encoded by plasmids either identical or
closely related to pRJ6 (79 kb), which is the first known
bacteriocinogenic mobilizable plasmid found in staphylo-
cocci (Oliveira et al. 1998a; Coelho et al. 2009). Probably,
the prevalence of aureocin A70 amongst Bac+ staphylococci
is related to the ability of pRJ6 and its variants to be mobi-
lized by staphylococcal conjugative plasmids, such as pGO1
(Oliveira et al. 1998a; Gamon et al. 1999; Coelho et al.
2009). This mobilization corroborates the hypothesis of
horizontal transfer of the genes involved in aureocin A70
production amongst bacterial strains of different pulso-
types involved in bovine mastitis in Brazil. Such transfer is
expected to confer a selective advantage to these staphylo-
coccal strains, when colonizing the bovine mammary
glands, because aureocin A70 has a broad spectrum of
activity (Bastos et al. 2009). Moreover, aureocin-A70 pro-
ducers become immune to this bacteriocin, which is the
antimicrobial peptide produced by most Bac+ mastitic
staphylococci (Bastos et al. 2009).
Comparisons between pRJ6 and another plasmid
(pRJ80) related to it revealed that while pRJ6, encoding
aureocin A70, has 79 kb (Coelho et al. 2009), pRJ80,
found in strain 4181, has 84 kb. These two plasmids
exhibited different profiles when digested with the
enzymes BglII, ClaI, EcoRV, HindIII and PstI, thus sug-
gesting that their restriction maps are different. However,
all fragments obtained from digestion of plasmid pRJ80
hybridized with pRJ6 DNA labelled with a32P-dCTP.
Thus, it is possible to conclude that plasmid pRJ80 is
indeed related, but not identical to pRJ6.
The twelve remaining Brazilian Bac+ strains analysed in
the present study do not produce aureocin A70. These
strains produce either aureocins 4185 (Staph. aureus
4185) or antimicrobial peptides (11 strains) that are dis-
tinct from the staphylococcins described so far, but not
characterized yet (Ceotto et al. 2009). Concerning the
PFGE analysis, these 12 Staph. aureus Bac+ strains were
grouped into eight pulsotypes: C (subtypes C1 and C2), E
(subtype E1), F (subtype F2), I, J, K, L and M (subtype
M2), suggesting that these bacteria are not genetically
related.
The strains 4045, 4046 and 4180 (belonging to pulso-
type K), and the strains 3913 and 3959 (belonging to
pulsotype I), isolated from different herds but exhibiting
indistinguishable PFGE profiles, can be considered the
same clone. The distinct plasmid patterns also detected
amongst these strains may result from different events of
horizontal gene transfer, as already mentioned above.
2012 The Authors
H. Ceotto et al.
Based on the results of the present work, it can also be
concluded that strains very closely related, such as 4181
and 4185, can produce distinct staphylococcins, such as
aureocin A70 (Ceotto et al. 2009) and aureocins 4185
(Ceotto et al. 2010), respectively.
In conclusion, this study investigated the genetic rela-
tionship amongst Staph. aureus Bac+ strains isolated from
either clinical or subclinical bovine mastitis cases to test
whether the bacteriocin production would confer a selec-
tive advantage to a single bacterial clone. Although a pre-
valent pulsotype of aureocin A70-producer Staph. aureus
involved in bovine mastitis is disseminated in Argentina,
different pulsotypes are involved in the production of au-
reocin A70 in Brazil. The Brazilian aureocin A70-pro-
ducer strains are not genetically related to the
representative pulsotypes found in Argentina, contradict-
ing the hypothesis that the bacteriocin production may
contribute to the bacterial spread of a single clone
amongst different herds. Additionally, it was demon-
strated that closely related staphylococcal strains can pro-
duce distinct staphylococcins, confirming the hypothesis
of horizontal gene transfer of bacteriocin genes amongst
bacterial strains involved in bovine mastitis.
Acknowledgements
Hilana Ceotto was a recipient of a PhD scholarship from
CNPq Brazil and of a postdoctoral fellowship from
CAPES Brazil. This study was supported by grants from
CAPES, CNPq, PRONEX and FAPERJ to M.C.F.B. We
thank Dr Lucia Martins Teixeira and Dr Katia Regina
Netto dos Santos, for the use of the PFGE apparatus.
References
Bastos, M.C.F., Ceotto, H., Coelho, M.L.V. and Nascimento,
J.S. (2009) Staphylococcal antimicrobial peptides: relevant
properties and potential biotechnological applications.
Curr Pharm Biotechnol 10, 3861.
van Belkum, A., Tassios, P.T., Dijkshoorn, L., Haeggman, S.,
Cookson, B., Fry, N.K., Fussing, V., Green, J. et al. (2007)
Guidelines for the validation and application of typing
methods for use in bacterial epidemiology. Clin Microbiol
Infect 3, 146.
Ceotto, H., Nascimento, J.S., Brito, M.A.V.P. and Bastos,
M.C.F. (2009) Bacteriocin production by Staphylococcus
aureus involved in bovine mastitis in Brazil. Res Microbiol
160, 592599.
Ceotto, H., Brede, D., Salehian, Z., Nascimento, J.S., Fagundes,
P.C., Nes, I.F. and Bastos, M.C.F. (2010) Aureocins 4185,
bacteriocins produced by Staphylococcus aureus 4185 with
potential application in food preservation. Foodborne
Pathog Dis 7, 12551262.
2012 The Authors
Letters in Applied Microbiology 54, 455461 2012 The Society for Applied Microbiology
Spread of Staphylococcus aureus involved in bovine mastitis
Coelho, M.L.V., Ceotto, H., Madureira, D.J., Nes, I.F. and
Bastos, M.C.F. (2009) Mobilization functions of the
bacteriocinogenic plasmid pRJ6 of Staphylococcus aureus.
J Microbiol 47, 327336.
Daly, K.M., Upton, M., Sandiford, S.K., Draper, L.A.,
Wescombe, P.A., Jack, R.W., OConnor, P.M., Rossney, A.
et al. (2010) Production of the Bsa lantibiotic by commu-
nity-acquired Staphylococcus aureus strains. J Bacteriol 192,
11311142.
Gamon, M.R., Moreira, E.C., Oliveira, S.S., Teixeira, L.M. and
Bastos, M.C.F. (1999) Characterization of a novel bacterio-
cin-encoding plasmid found in clinical isolates of Staphylo-
coccus aureus. Antonie Van Leeuwenhoek 75, 233243.
Giambiagi-deMarval, M., Mafra, M.A., Penido, E.G.C. and
Bastos, M.C.F. (1990) Distinct groups of plasmids corre-
lated with bacteriocin production in Staphylococcus aureus.
J Gen Microbiol 136, 15911599.
Malachowa, N. and DeLeo, F.R. (2010) Mobile genetic
elements of Staphylococcus aureus. Cell Mol Life Sci 67,
30573071.
Nascimento, J.S., Santos, K.R.N., Gentilini, E., Sordelli, D. and
Bastos, M.C.F. (2002) Phenotypic and genetic characteriza-
tion of bacteriocin producing strains of Staphylococcus aur-
eus involved in bovine mastitis. Vet Microbiol 85, 133144.
Nascimento, J.S., Fagundes, P.C., Brito, M.A.V.P. and Bastos,
M.C.F. (2005) Production of bacteriocins by coagulase-
negative staphylococci involved in bovine mastitis. Vet
Microbiol 106, 6171.
Navaratna, M.A., Sahl, H.-G. and Tagg, J.R. (1998) Two-com-
ponent anti-Staphylococcus aureus lantibiotic activity pro-
duced by Staphylococcus aureus C55. Appl Environ
Microbiol 64, 48034808.
Netz, D.J.A., Sahl, H.-G., Marcolino, R., Nascimento, J.S.,
Oliveira, S.S., Soares, M.B. and Bastos, M.C.F. (2001)
Molecular characterisation of aureocin A70, a multipeptide
bacteriocin isolated from Staphylococcus aureus. J Mol Biol
311, 939949.
Netz, D.J.A., Pohl, R., Beck-Sinckinger, A.G., Selmer, T.,
Pierik, A.J., Bastos, M.C.F. and Sahl, H.-G. (2002)
Biochemical characterisation and genetic analysis of aureo-
cin A53, a new atypical bacteriocin from Staphylococcus
aureus. J Mol Biol 319, 745756.
Oliveira, S.S., Nascimento, J.S., Povoa, D.C., Araujo, S.A.,
Gamon, M.R. and Bastos, M.C.F. (1998a) Genetic analysis
of the bacteriocin-encoding plasmids pRJ6 and pRJ9 of
Staphylococcus aureus by transposon mutagenesis and clon-
ing of genes involved in bacteriocin production. J Appl
Microbiol 85, 792814.
Oliveira, S.S., Povoa, D.C., Nascimento, J.S., Pereira, M.S.V.,
Junior, J.P.S. and Bastos, M.C.F. (1998b) Antimicrobial
substances produced by Staphylococcus aureus strains iso-
lated from cattle in Brazil. Lett Appl Microbiol 27, 229234.
Sambrock, J., Fritsch, E.F. and Maniaris, T. (1989) Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory.
461