Research in Microbiology 158 (2007) 625e630

www.elsevier.com/locate/resmic

Activity of staphylococcal bacteriocins against Staphylococcus aureus

and Streptococcus agalactiae involved in bovine mastitis
Marcus Lvio Varella Coelho a, Janana dos Santos Nascimento a, Patrcia Carlin Fagundes a,
Danielle Jannuzzi Madureira a, Selma Soares de Oliveira a,
Maria Aparecida Vasconcelos de Paiva Brito b, Maria do Carmo de Freire Bastos a,*
a
Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goes, UFRJ, CCS, Bloco I, Cidade Universitaria,
21941-590 Rio de Janeiro, RJ, Brazil
b
EMBRAPA Gado de Leite, Juiz de Fora, MG, Brazil
Received 31 May 2007; accepted 3 July 2007
Available online 14 July 2007

Abstract

The inhibitory activity of seven bacteriocins produced by Staphylococcus aureus (aureocins A70, A53, and 215FN) and Staphylococcus epi-
dermidis (Pep5, epidermin, epilancin K7 and epicidin 280) was tested against strains of both S. aureus (165 strains) and Streptococcus agalactiae
(74 strains) isolated from udders of cows suffering from bovine mastitis. Most strains of the two species were inhibited by epidermin (>85%),
aureocin A53 (>67%) and by a combination of aureocins A70 and A53 (>91%), co-expressed in the genetic background of strain A70, the
native producer of aureocin A70. Synergy between aureocins A70 and A53 was also demonstrated, which broadened the spectrum of strains
inhibited. The remaining staphylococcins inhibited either none of, or a lower percentage (<48%) of, the mastitis-causing pathogens tested.
Our results therefore show that the use of epidermin and/or a combination of aureocins A53 and A70 may represent a new non-antibiotic
alternative for successfully inhibiting both mastitic staphylococci and streptococci.
2007 Elsevier Masson SAS. All rights reserved.

Keywords: Antimicrobial peptides; Aureocins; Bacteriocins; Bovine mastitis; Staphylococcins; Staphylococcus aureus; Streptococcus agalactiae

1. Introduction lactation with or without long-acting formulations during the

Bovine mastitis is a disease caused by infection of cow
udders and is one of the most significant causes of economic
losses to the dairy industry due to rejected milk, degraded
milk quality, early culling of cows, drug costs, veterinary
expenses and increased labor costs for farmers [12,13]. Staph-
ylococcus aureus and Streptococcus spp. are major bacterial
agents involved in this disease [6].
The treatment of intramammary infections (IMIs) makes
use of antimicrobial substances. Therapeutic strategies involve
administration of immediate release formulations during
* Corresponding author. Tel.: 55 21 2562 6742; fax: 55 21 2560 8344.
E-mail addresses: mcbastos@micro.ufrj.br, mcbastos2@yahoo.com.br
(M.doC.de Freire Bastos).
dry (non-lactating) period. Many drugs belonging to various
therapeutic classes have been assessed for dry cow therapy
(DCT) [5,13]. Current treatments have met with limited suc-
cess and cure rates vary considerably and are considered inad-
equate, especially towards S. aureus, which is responsible for
chronic infections and huge economic losses [4,6,13]. More-
over, public health authorities advise prudent use of antibi-
otics, as their use may promote bacterial antibiotic resistance
and leave antibiotic residues in the food chain [5,9,13]. In
addition, the absence of persistence of therapeutic levels of
antibiotics during the dry period and the ineffectiveness of
commercially available DCT products against some mastitis-
causing organisms have prompted recent research efforts
toward finding alternatives to DCT [9,13,28]. One such alter-
natives may lie in the use of bacteriocins.

0923-2508/$ - see front matter 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2007.07.002
626 M.L. Varella Coelho et al. / Research in Microbiology 158 (2007) 625e630

Bacteriocins are ribosomally synthesized peptides pro- Table 1

duced by bacteria which have inhibitory activities toward Bacteriocin-producing staphylococcal strains used in this study and their
derivatives
other bacteria. In Gram-positive microorganisms, these com-
pounds are generally highly cationic, heat-stable, and primar- Strains Plasmid(s) and relevant Bacteriocin(s) Ref.
features
ily active against bacteria sharing the same ecological niche
[10,19]. Much research has been done to better characterize S. aureus
A70 pRJ6 Aureocin A70 [11]
these compounds because of their potential biotechnological A70 Baca Strain A70 cured of pRJ6 e [11]
use. Up to now, only nisin and pediocin PA1/AcH have found A53 pRJ9 Aureocin A53 [11]
widespread use as food preservatives [7]. Bacteriocins may MB50 pRJ6 and pRJ22 Aureocins A70 and A53 [1]
also be employed in the treatment and prevention of infectious 215FN pRJ35 Aureocin 215FN [22]
diseases in plants [27], animals [20] and humans [29]. 215FN Baca Strain 215FN cured of pRJ35 e [22]
Staphylococcins are bacteriocins produced by bacterial S. epidermidis
strains belonging to the genus Staphylococcus. In S. epidermi- Tu3298 Tu3298 Epidermin [2]
dis, some staphylococcins have been well characterized, such K7 e Epilancin K7 [27]
as the lantibiotics epidermin [2], Pep 5 [16], epilancin K7 [30] BN280 pCR01 Epicidin 280 [14]
and epicidin 280 [14]. Lantibiotics are class I bacteriocins 5 pED503 Pep5 [15]
5 Baca Strain 5 cured of pED503 e [15]
characterized by the presence of modified amino acids such
as lanthionine and b-methyl-lanthionine, among others
a
All strains with the exception of A70 Bac, 215FN Bac and 5 Bac pro-
duce staphylococcins. Bac, bacteriocin.
[19,29].
Our laboratory has been investigating bacteriocin produc-
tion by staphylococcal strains. Among bacteriocins studied A total of 117 strains of S. aureus and all S. agalactiae strains
by our group, aureocins A70 and A53 are the best character- were isolated in the present study from bovine mastitis cases
ized. Both are class II bacteriocins produced by S. aureus. in 56 herds from different states located in the southeast region
Class II bacteriocins are small peptides which do not contain of Brazil. Multiple isolates from the same animal were
modified amino acids [10]. Aureocin A70 is a multipeptide excluded.
bacteriocin composed of four related peptides which are Forty-eight strains of S. aureus were used in a previous
encoded by plasmid pRJ6 [11,23]. Aureocin A53 is a study and represent strains isolated from mastitic cows from
51-amino-acid peptide whose structural gene is located on 48 different herds located in different provinces of Argentina
plasmid pRJ9 [11,24]. [26]. Multiple isolates from the same herd were excluded.
In the present work, the sensitivity of several staphylococ- Staphylococcal and streptococcal strains were grown
cal and streptococcal strains involved in bovine mastitis non-aerobically in BHI medium (Difco), at 37  C for 18e24 h.
toward seven different staphylococcins was evaluated by as- Corynebacterium fimi NCTC 7547, a strain highly sensitive to
says performed in an agar plate system to verify whether these all staphylococcins employed in the present study [22], was
compounds have inhibitory activity toward these pathogens. In used as a positive control for bacteriocin production in all
a previous study, we reported the sensitivity to five aureocins experiments. C. fimi was also grown non-aerobically in BHI
of a smaller number of S. aureus strains (65 strains), most of medium at 37  C for 18 h. Staphylococci and streptococci
them (77%) isolated in Argentina [6]. Therefore, in the present were stored in TSB (Difco) and BHI, respectively, with 40%
study, we decided to test a larger number of strains isolated in glycerol (v/v) at 20  C until use. When necessary, the media
Brazil and also strains of Streptococcus agalactiae, a major were supplemented with agar at 1.5% (w/v) or 0.7% (w/v).
pathogen involved in bovine mastitis [6]. Along with three
aureocins, including both aureocins A70 and A53, four lanti-
2.2. Identification of strains to the species level
biotics produced by S. epidermidis were included in this inves-
tigation, since class I bacteriocins generally exhibit a broad
Staphylococcal strains were identified as S. aureus on the
spectrum of activity [19,29]. Our findings suggested that the
basis of Gram staining and conventional biochemical tests
combination of aureocins A70 and A53 and/or epidermin
described by Bannerman [3]. S. agalactiae strains were iden-
may constitute very promising antimicrobials for successfully
tified on the basis of Gram staining, presence of hemolysis,
inhibiting both staphylococcal and streptococcal pathogens in-
negative results on tests for the presence of catalase and
volved in bovine mastitis.
esculin hydrolysis, and positive results on tests for CAMP
and hydrolysis of sodium hypurate.
2. Materials and methods

2.1. Bacterial strains and culture conditions 2.3. Qualitative assay for bacteriocin sensitivity

Bacteriocinogenic staphylococcal strains used as bacterio- This assay was performed essentially using a previously
cin producers are listed in Table 1. To evaluate bacterial described protocol [22]. Briefly, 107 cells of bacteriocin
sensitivity to these bacteriocins, 165 indicator strains of producer strains were spotted on 18  100 mm Petri dishes
S. aureus and 74 strains of S. agalactiae were employed. containing 25 ml of BHI agar. After 18 h at 37  C, 105 cells
 M.L. Varella Coelho et al. / Research in Microbiology 158 (2007) 625e630
of the indicators in BHI soft agar were sprayed over the plates.
Plates were incubated under the same conditions and inhibition
zones around the producer spots were measured in mm. The
experiments were repeated at least three times. The indicator
strains were considered sensitive to a given staphylococcin
when it exhibited a clear inhibition zone with 14 mm. When
the inhibition zone was either smaller than 14 mm or contained
resistant mutants, the indicator strain was considered resistant to
the corresponding bacteriocin.
2.4. Bacteriocin quantification in culture supernatants
Staphylococcal strains were grown in 5 ml BHI medium at
37  C for 18 h. The culture was diluted into 100 ml BHI broth
to reach an OD600 of 0.04 and incubated at 37  C under shak-
ing (120 rpm) for 8 to 18 h, the time required for maximum
aureocin A70 and aureocin A53 production, respectively
[21]. The cells were removed by centrifugation at 12,000 
g for 10 min at 4  C and supernatant was sterilized by mem-
brane filtration (Millipore; 0.45 mm pores). Antimicrobial
activity in the supernatant was determined by the dilution
method using an agar-well diffusion assay. Serial twofold
dilutions of each crude bacteriocin preparation were made in
100 ml of BHI broth. Wells (with 6 mm diameter) were
made in BHI agar medium seeded with the sensitive strain
(106 C. fimi cells ml1 in 3 ml BHI soft agar poured onto
agar plates) and were filled with 50 ml aliquots of the diluted
supernatant. The plates were incubated for 18 h at 37  C. Bac-
teriocin activity was measured in arbitrary units (AU) per ml,
which represented the reciprocal of the highest supernatant di-
lution showing clear inhibition of bacterial growth multiplied
by 25.
2.5. Synergistic activity between aureocins A70 and A53
To test the combined effect of aureocins A70 and A53,
wells were made in BHI agar medium seeded with the indica-
tor strain (105 cells ml1 in 3 ml BHI soft agar poured onto
agar plates) and were filled with 50 ml aliquots containing
a combination of different amounts of aureocins A70 and
A53 in AU. These amounts depended on the initial AU ml1
found in each bacteriocin crude preparation (1600 AU ml1
for both aureocins) and on the different volume ratios used
to prepare each bacteriocin combination (Table 3). Plates
were incubated for 18 h at 37  C. The inhibition zones ob-
served were measured in mm. In all cases, two independent
experiments were performed.
Fig. 1. Plasmid pRJ9 genetic map showing its open reading frames (orfs) and the transposon Tn551 insertion site to generate plasmid pRJ22. The orfs are
represented as large arrows, which also indicate the direction of transcription. Small dark boxes indicate promoter positions.
627
2.6. Sequencing of insertion of Tn551 into
plasmid pRJ22
The exact position of the insertion of Tn551 into plasmid
pRJ9 to generate plasmid pRJ22 was determined by DNA
sequencing using as a primer an 18 mer (50 -TGTACCACTAA
TAACTCA-30 ), whose 30 end was located 101 bp upstream
of the left end of the transposon. DNA sequencing was per-
formed with an ABI PRISM 377 DNA sequencer (Applied
Biosystems, PerkineElmer).
2.7. Statistical analysis
Statistical analyses were performed using Students t-test
considering p < 0.05. This test was used to perform pairwise
comparison of inhibition zones in mm produced by staphylo-
coccins against either streptococcal or staphylococcal strains.
3. Results and discussion
3.1. Sequencing of insertion of Tn551 into
plasmid pRJ22
The sensitivity of staphylococcal and streptococcal strains
involved in bovine mastitis to seven different staphylococcins
was evaluated by assays done in an agar plate system to verify
whether such compounds might have inhibitory activity
against these pathogens. In these experiments, we also used
strain MB50 that was constructed in a previous study [1].
This strain carries plasmids pRJ6 and pRJ22, and therefore,
should produce both aureocins A70 and A53. pRJ22 is a deriv-
ative of pRJ9 tagged with transposon Tn551. To rule out any
possibility of inactivation of functions involved in aureocin
A53 production upon transposition, the exact position of trans-
poson insertion into pRJ9 was sequenced in the present study,
proving that insertion occurred in a region of pRJ9 that does
not affect aureocin A53 production (Fig. 1). In pRJ22, transpo-
son Tn551 is inserted 11 bp downstream of the putative -10
promoter region of a putative operon, which codes for func-
tions involved in plasmid mobilization [24].
3.2. Inhibitory activity of staphylococcins against
S. agalactiae strains involved in bovine mastitis
Among 74 S. agalactiae strains isolated from cows suffering
from bovine mastitis, 72 (97.3%) were inhibited by epidermin
(Table 2). The two strains that were not inhibited by epidermin
628 M.L. Varella Coelho et al. / Research in Microbiology 158 (2007) 625e630

Table 2 (93.8% and 85.4%, respectively). In contrast, epicidin 280

Activity of staphylococcins against indicator strains tested and epilancin K7 again did not inhibit any strain.
Bacteriocins used Indicator strains tested and percentage of inhibition To rule out the possibility that substances other than the
(alone or combined) S. agalactiae Brazilian Argentinean bacteriocins could be responsible for the inhibition observed,
(74 strains) S. aureus S. aureus the experiments were repeated using as producers strains
(117 strains) (48 strains) A70 Bac, 5 Bac and 215FN Bac which were cured of their
Aureocin A70 1.4 26.5 16.0 Bac plasmids and therefore of bacteriocin production. Ten
Aureocin A53 67.6 74.4 100 staphylococcal strains sensitive to aureocin A70, Pep5 or aur-
Aureocins 91.9 91.5 100
A70 A53a
eocin 215FN were randomly chosen and used as indicators. As
Aureocin 215FN 47.3 38.5 8.0 expected, no inhibition zone was detected in these experiments,
Epicidin280 13.5 0 0 confirming that the inhibition previously observed indeed
EpilancinK7 0 0 0 resulted from bacteriocin activity. This experiment could not
Epidermin 97.3 87.2 85.4 be performed with the remaining staphylococcins (aureocin
Pep5 0 52.1 93.8
A53, epicidin 280, epilancin K7 and epidermin), since bacterio-
The streptococcal and staphylococcal strains were considered sensitive to cin-cured derivatives were not available for them.
a given staphylococcin when it exhibited a clear inhibition zone of
14 mm. When the inhibition zone was smaller than 14 mm or contained
Based on our data, therefore, either epidermin, a lantibiotic,
resistant mutants, the indicator strain was considered resistant to the corre- or a combination of aureocins A53 and A70, both class II bac-
sponding bacteriocin. In all experiments, C. fimi was also used as a sensitive teriocins, showed the most effective antimicrobial activities, in-
indicator and it was inhibited by all staphylococcin-producer strains (data hibiting a remarkable percentage (>85%) of all strains tested,
not shown). which suggested the substantial in vitro effectiveness of these
a
Aureocins A70 A53 refer to both bacteriocins produced by strain MB50.
bacteriocins against staphylococci and streptococci. On the
other hand, epilancin K7, epicidin 280 and aureocin A70
proved to be the most ineffective staphylococcins at inhibiting
were susceptible to a combination of aureocins A53 and A70.
both streptococcal and staphylococcal mastitic strains. Similar
Strain MB50, which produces both aureocins A53 and A70,
results have been found by our group when testing the activity
was able to inhibit 68 strains (91.9%), compared to 50 strains
of these three staphylococcins against S. aureus and coagulase-
(67.6%) inhibited by aureocin A53 alone and only 1 strain
negative staphylococci involved in nosocomial infections [22].
(1.4%) inhibited by aureocin A70 alone. Thus, eighteen strains
Analysis of our data also showed that all 74 strains of
that were not inhibited by either aureocin A70 or aureocin A53
S. agalactiae could be inhibited by a combination of epider-
alone were inhibited by their combination.
min, aureocins A53 and A70. Similarly, among a total of 165
Aureocin 215FN and epicidin 280 were able to inhibit 35
strains of S. aureus, 163 (98.8%) proved to be susceptible to
(47.3%) and 10 (13.5%) of the tested strains, respectively.
a combination of all three of these staphylococcins. Biotechno-
No strain was inhibited by Pep5 and epilancin K7.
logical use of a combination of different bacteriocins has
advantages over the use of single ones [10,18], as discussed
3.3. Inhibitory activity of staphylococcins against below.
Brazilian and Argentinean S. aureus strains involved in None of the staphylococcins inhibited all strains tested,
bovine mastitis whether S. aureus or S. agalactiae. Similar findings have
already been reported in the literature for other bacteriocins.
The inhibitory activity of staphylococcins was also evalu- Within a given species, some strains may be sensitive and
ated against 117 S. aureus strains isolated in Brazil and others may be resistant to a particular bacteriocin [10,15,22].
48 strains isolated in Argentina, all of them involved in bovine Spontaneous bacteriocin-resistant mutants can also be found
mastitis. in the population of a given bacteriocin-sensitive bacterial
Among Brazilian staphylococcal strains tested (Table 2), strain [15]. Bacteriocin resistance appears to be a complex
107 (91.5%) were inhibited by strain MB50, compared to 87 phenotype involving alterations in the cell wall and/or cyto-
(74.4%) and 31 (26.5%) strains inhibited by aureocins A53 plasmic membrane [8,10,17].
and A70 alone, respectively. Here again, 20 strains that were
not inhibited by either aureocin A70 or aureocin A53 alone 3.4. Synergistic activity in aureocins A70 and A53
were inhibited by their combination.
The bacteriocins epidermin, Pep5 and aureocin 215FN Strain MB50 produces both aureocins A70 (encoded by
were able to inhibit, respectively, 102 (87.2%), 61 (52.1%) plasmid pRJ6) and A53 (encoded by plasmid pRJ22) using
and 45 (38.5%) strains. Eight out of 10 strains that were not the genetic background of the native aureocin A70 producer.
inhibited by a combination of aureocins A53 and A70 were Strain MB50 exhibited the highest percentage of inhibition
susceptible to epidermin. Epicidin 280 and epilancin K7 (91%) of bovine mastitis pathogens when compared to
inhibited none of the Brazilian staphylococcal strains tested. strains A70 (producer of only aureocin A70) and A53 (pro-
All 48 staphylococcal strains isolated in Argentina (Table 2) ducer of only aureocin A53), which suggests the occurrence
were inhibited by strain MB50 and by aureocin A53. Pep5 and of synergy between aureocins A70 and A53. The experiments
epidermin also exhibited a high percentage of inhibition performed also suggest that the major antimicrobial activity of
 M.L. Varella Coelho et al. / Research in Microbiology 158 (2007) 625e630
strain MB50 may rely on the action of aureocin A53, since this
bacteriocin alone inhibited a larger proportion of strains than
did aureocin A70 alone.
When qualitative assays for bacteriocin sensitivity were
performed, it was observed, for streptococcal and staphylococ-
cal strains inhibited by a combination of aureocins A70 and
A53 and by aureocin A53 alone (but not by aureocin A70
alone), that the inhibition zones detected for a combination
of the two aureocins (37  1 mm) were significantly
( p < 0.05) larger than those observed for aureocin A53 alone
(23  2 mm), which also raised the possibility of the occur-
rence of synergism between aureocins A70 and A53.
To confirm synergistic activity between the two aureocins,
three staphylococcal strains (2979, 3852 and 4091) that were
inhibited by neither aureocin A70 nor aureocin A53 alone,
but that were inhibited by strain MB50, were chosen as indi-
cators in an agar well diffusion assay. Combinations of differ-
ent amounts of the two aureocins in AU were employed in
these experiments, summarized in Table 3. As expected, the
two aureocins alone were inactive against all three indicator
strains. However, inhibition of all three indicator strains was
observed when both bacteriocins were combined at an activity
of at least 20 AU. Therefore, our results clearly demonstrated
that synergy between aureocins A70 and A53 indeed seems to
occur and that combining aureocins A70 and A53 can greatly
broaden the target-cell range of these peptides, as already
described for other antimicrobial peptides [18]. Moreover,
the combined action of different bacteriocins might preclude
growth of mutants resistant to bacteriocins appearing during
bacteriocin therapy [8,16] and, therefore, might improve their
biotechnological applications [10,18].
Our data also showed that synergistic activity between aur-
eocins A70 and A53 was observed even when activity as low
as 20 AU was used. In fact, most bacterial antimicrobial
peptides are reported to be bactericidal [10,15,19,29] and to
exhibit high potency, with the capacity to kill bacteria even
at nanomolar concentrations [18].
To our knowledge, few bacteriocins have been described as
having an effect against mastitis-causing pathogens. Nisin,
Table 3
Synergistic activity between aureocins A70 and A53
Combination (AU) of aureocin Inhibition of S. aureus strains
A70 aureocin A53
2979 3852 4091
(volume ratio)a
40 40 (1:1) (14 mm) (12 mm) (15 mm)
26.5 53 (1:2) (14 mm) (14 mm) (15 mm)
53 26.5 (2:1) (14 mm) (10 mm) (12 mm)
20 20 (0.50:0.50) (12 mm) (10 mm) (10 mm)
20 10 (0.50:0.25) (11 mm)  (10 mm)
10 20 (0.25:0.50)    epidermidis. Eur. J. Biochem 204, 1149e1154.
Aureocin A70 alone (80 AU)    [3] Bannerman, T.L. (2003). In: P.R. Murray, E.J. Baron, J.H. Jorgensen,
Aureocin A53 alone (80 AU)    M.A. Pfaller, & R.H. Yolken (Eds.), Manual of Clinical Microbiology,
, Inhibition; , no inhibition. The numbers are the diameters of inhibition
zones and represent means of two independent experiments. In both experi-
ments, C. fimi was also used as a sensitive indicator and was inhibited by either
any combination of both aureocins or each aureocin alone (data not shown).
a
The numbers represent total AU present in 50 ml of each bacteriocin prep-
aration volume ratio used in each combination.
629
a lantibiotic produced by Lactococcus lactis subsp. lactis, is
used as an active agent in Wipe-Out, a teat wipe [7]. Lacticin
3147, a lantibiotic also produced by L. lactis subsp. lactis
DPC3147, also proved to be effective in inhibiting those path-
ogens [18,25]. However, a much smaller number of strepto-
coccal and staphylococcal strains (7 and 17, respectively)
were employed in studies performed with lacticin 3147 com-
pared to the number of isolates used in the present work (74
streptococci and 165 staphylococci).
In conclusion, although a considerable number of bacterio-
cins have already been described in the literature, few of them,
including aureocins A53 and A70 and epidermin, have been
shown to have potential veterinary applications. Hence, the
generation of non-antibiotic formulations based on a combina-
tion of aureocins A53 and A70 and/or epidermin for preven-
tion and treatment of bovine mastitis represents an
additional source of antimicrobials for reducing veterinary
dependence upon antibiotics in the control of this persistent
and costly disease. Moreover, unlike traditional antibiotics
which mainly act as enzyme inhibitors, most peptides-
bacteriocins render the membranes of sensitive cells perme-
able, leading to leakage of cellular solutes and eventually
cell death [7,10,17,26]. Hence, complementary use of antibi-
otics with either a combination of aureocins A70 and A53
or epidermin may be another potential approach to preventing
the emergence of resistant microorganisms, since it would be
much more difficult for a bacterium to acquire resistance to
antimicrobials with different and concurrent modes of action
[13]. However, before epidermin and aureocins A53 and
A70 can find clinical applications in vivo, clinical studies
should be undertaken to investigate the potential for treating
infected udder quarters with these bacteriocins. Moreover,
the toxicity of these staphylococcins for normal mammalian
epithelial cells should also be investigated. Such studies are
currently in progress.
Acknowledgements
M.L.V.C., J.S.N., P.C.F. and D.J.M. were recipients of
scholarships from CNPq/Brazil. This study was supported by
grants from CNPq, FAPERJ and PRONEX to M.C.F.B.
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