Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Bacteriocins as alternative agents for control

of multiresistant staphylococcal strains
J.S. Nascimento1, H. Ceotto1, S.B. Nascimento1, M. Giambiagi-deMarval2, K.R.N. Santos2
and M.C.F. Bastos1
1 Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro, Brazil
2 Departamento de Microbiologia Medica, Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro, Brazil

Keywords
bacteriocins, coagulase-negative
staphylococci, nosocomial staphylococci,
staphylococcins, Staphylococcus aureus.
Correspondence
Maria do Carmo de Freire Bastos, Instituto de
Microbiologia, UFRJ, Departamento de
Microbiologia Geral, CCS, Bloco I, Cidade
Universitaria, 21941-590, Rio de Janeiro, RJ,
Brazil. E-mail: mcbastos@micro.ufrj.br or
mcbastos2@yahoo.com.br
2005/1041: received 13 September 2005,
revised and accepted 20 October 2005.
doi:10.1111/j.1472-765X.2005.01832.x
Abstract
Aims: To investigate the activity of seven staphylococcins, bacteriocins pro-
duced by staphylococci, against multiresistant Staphylococcus aureus and coagu-
lase-negative staphylococci (CNS) involved in human infections.
Methods and Results: Four bacteriocins produced by Staph. epidermidis (Pep5,
epidermin, epilancin K7 and epicidin 280) and three produced by Staph. aureus
(aureocins A70, A53 and 215FN) were tested. Sixteen Staph. aureus strains,
including a representative strain of the endemic Brazilian methicillin-resistant
clone (MRSA), and 57 CNS strains were used as indicators. Among the sta-
phylococcins used, Pep5 was able to inhibit 772% of the CNS strains and
875% of the Staph. aureus strains tested, including the Brazilian MRSA ende-
mic clone, responsible for a large number of hospital-acquired infections in
Brazil. On the other hand, aureocin A53 and epidermin presented a high ant-
agonistic activity only against the Staph. aureus strains, being able to inhibit,
respectively, 875% and 813% of them, including also the Brazilian MRSA
endemic clone. The remaining bacteriocins inhibited only a low percentage of
the nosocomial staphylococcal strains tested.
Conclusions: Aureocin A53 and epidermin have potential applications against
MRSA, whereas Pep5 seems to be an attractive agent against both MRSA and
CNS, including mupirocin-resistant strains and the Brazilian endemic clone of
MRSA, which is also found disseminated in other countries.
Significance and Impact of the Study: Bacteriocins may represent alternative
agents to control important nosocomial pathogens.

ciated with implanted medical devices (Rupp and Archer

Introduction
The emergence and dissemination of antibiotic resistance
in staphylococci and their association with the use of
antibiotics constitute a serious problem in the hospital
environment. Among the staphylococci, Staphylococcus
aureus is the causal agent of many staphylococcal com-
munity-acquired and nosocomial infections (Aires de
Souza et al. 1998; Bannerman 2003). Coagulase-negative
staphylococci (CNS), such as Staph. epidermidis, Staph.
haemolyticus and Staph. saprophyticus, can also be found
in association with human infections (Rupp and Archer
1994). They are the most frequently reported pathogens
in nosocomial bloodstream infections and are often asso-
1994).
Methicillin-resistant staphylococci (MRS) have become
important nosocomial pathogens, requiring effective
measures for controlling their spread. They have been iso-
lated with increasing frequency worldwide (Schmitz et al.
1998), including in Brazilian hospitals (Santos et al. 1999;
Nunes et al. 2005). Moreover, staphylococcal MRS strains
accumulate additional resistance determinants, making
them difficult to manage therapeutically (Krishnan et al.
2002).
Mupirocin is a natural antibiotic derived from Pseudo-
monas fluorescens that inhibits bacterial growth by binding
to isoleucyl tRNA-synthetase. It is used as a topical agent

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Bacteriocins as antimicrobial agents J.S. Nascimento et al.

to eradicate methicillin-resistant Staph. aureus (MRSA)
nasal carriage (Boyce 1996) and to prevent catheter col-
onization by staphylococci (Cookson et al. 1990). During
the last few years, it has also been proposed as an advis-
able antibiotic to be applied when invasive surgeries are
employed, as it could prevent MRSA colonization
(Upton et al. 2003). However, both low- and high-level
resistances have been reported during treatment with
nasal mupirocin and the emergence of a high-level resist-
ance to this drug has threatened its value as a therapeutic
agent (Krishnan et al. 2002). Because of these facts, new
strategies for controlling MRSA and multiresistant staphy-
lococci are required.
Bacteriocins (Bac) are a class of ribosomally synthesized
antimicrobial peptides produced by bacteria (Papagianni
2003). Their inhibitory activity gives them potential use
in either prevention or treatment of bacterial infectious
diseases and, therefore, they can complement or, in selec-
ted cases, substitute for antibiotics (Sahl and Bierbaum
1998; Papagianni 2003).
The bacteriocins produced by Gram-positive bacteria
are divided into three main classes. Most of the character-
ized species belong to class I or II bacteriocins. Class I
bacteriocins, known as lantibiotics, contain post-transla-
tionally modified amino acids such as dehydroalanine,
dehydrobutyrine, lanthionine and 3-methyllanthionine.
Class II bacteriocins are small peptides that do not con-
tain unusual amino acids (Sahl and Bierbaum 1998;
Ennahar et al. 2000).
Staphylococcins are bacteriocins produced by staphylo-
cocci. So far, Staph. epidermidis and Staph. aureus are the
most important staphylococcin producers. In Staph. epi-
dermidis, some bacteriocins have been well characterized,
such as the lantibiotics epidermin, Pep 5, epicidin K7 and
epilancin 280 (Sahl and Bierbaum 1998), whose amino
acid sequences are shown in Table 1.
Our laboratory has been investigating bacteriocin pro-
duction by staphylococci. Among the Staph. aureus sta-
phylococcins, aureocins A70 and A53, produced by
strains isolated from commercial milk, are the best char-
acterized (Netz et al. 2001,2002). Both aureocins are atyp-
ical class II bacteriocins. Aureocin A70 is a multipeptide
bacteriocin whose inhibitory activity results from four
unmodified peptides and aureocin A53 is a highly cati-
onic 51-residue peptide produced and secreted without
any post-translational modification (Table 1). Another
aureocin has also been studied, 215FN, produced by a
strain isolated from healthy cattle (Oliveira et al. 1998).
However, its structure has not been identified yet.
As bacteriocins, in general, inhibit bacteria related to
the bacteriocin-producer strain (Papagianni 2003), in this
work the sensitivity of multiresistant Staph. aureus and
CNS strains involved in human infections to seven sta-
phylococcins was evaluated in an attempt to find new
alternative agents for control of these important nosoco-
mial pathogens.
Materials and Methods
Bacterial strains and culture conditions
Sixteen Staph. aureus and 57 coagulase-negative Staphylo-
coccus strains (Tables 2 and 3) involved in human infec-
tions were isolated from patients in four different
Brazilian hospitals, located in Rio de Janeiro, and used to
determine their sensitivity to bacteriocins. Among the 16

Table 1 Bacteriocinogenic staphylococcal strains previously described and used in this study

Strains Bacteriocin produced Primary structure of the mature peptide (number of amino acid residues) Reference

Staph. aureus
A53 Aureocin A53 MSWLNFLKYIAKYGKKAVSAAWKYKGKVLEWLNVGPTLEWVWQKLKKIAGL (51) Netz et al. (2002)
A70 Aureocin A70*
AurA MGKLAIKAGKIIGGGIASALGWAAGEKAVGK (31)
AurB MGAVAKFLGKAALGGAAGGATYAGLKKIFG (30)
AurC MGALIKTGAKIIGSGAAGGLGTYIGHKILGK (31)
AurD MGAVIKVGAKVIGWGAASGAGLYGLEKILKK (31) Netz et al. (2001)
215FN Aureocin 215FN ND Oliveira et al. (1998)
Staph. epidermidis
Tu 3298 Epidermin IASKFICTPGCAKTGSFNSYCC (22) Sahl and Bierbaum (1998)
5 Pep5 TAGPAIRASVKQCQKTLKATRLFTVSCKGKNGCK (34) Sahl and Bierbaum (1998)
BN280 Epicidin 280 SLGPAIKATRQVCPKATRFVTVSCKKSDCQ (30) Sahl and Bierbaum (1998)
K7 Epilancin K7 SASVLKTSIKVSKKYCKGVTLTCGCNITGGK (31) Sahl and Bierbaum (1998)

*Aureocin A70 consists of four antimicrobial peptides, AurA, AurB, AurC and AurD.
Some amino acids of these bacteriocins of Staph. epidermidis undergo post-translational modifications that are required for antimicrobial activity.
ND, not determined.

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J.S. Nascimento et al. Bacteriocins as antimicrobial agents

Table 2 Nosocomial Staph. aureus strains involved in human infections and used as indicators in the bacteriocin assays

Resistance Inhibited by the following

Strains* Source profile staphylococcins

4 Nasal swab Am, Ap, Cf, Cfo, Cip, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc A53, Pep5, epidermin
130 Nasal swab Am, Ap, Cf, Cfo, Cip, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc A53, Pep5, epidermin
24 Nasal swab Am, Ap, Cf, Cfo, Cip, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc Pep5, epidermin
A/22C Perineal colonization Am, Ap, Cf, Cfo, Cip, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc A53, Pep5, epidermin
C/10C Skin lesion Am, Ap, Cf, Cfo, Cip, Cl, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Sft, Tc A53, Pep5, epidermin
E/7 Blood Am, Ap, Cf, Cfo, Cip, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc A53, epidermin
D/9R Rectal colonization Am, Ap, Cf, Cfo, Cl, Cm, Cro, Em, Gm, Imp, Km, Mup, Ox, Pc, Rif, Sft, Tc A53, Pep5, epidermin
G/4 Blood Am, Ap, Cf, Cfo, Cip, Cl, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc A53, Pep5, epidermin
H/2R Perineal colonization Am, Ap, Cf, Cfo, Cip, Cl, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc Pep5, epidermin
I/26a Perineal colonization Am, Ap, Cf, Cfo, Cip, Cm, Cro, Em, Gm, Imp, Km, Mup, Ox, Pc, Rif, Sft, Tc A53, Pep5
J/18C Perineal colonization Am, Ap, Cf, Cfo, Cip, Cl, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc A53, Pep5
B/14A Lymph node biopsy Am, Ap, Cf, Cfo, Cip, Cl, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Sft, Tc A53, epidermin
F/6 Urine Ap, Cf, Cf, Cfo, Cl, Cm, Cro, Em, Gm, Imp, Km, Ox, Pc, Rif, Sft, Tc A53, Pep5, epidermin
77 Surgical wound Ap, Cm, Em, Km, Pc A53, Pep5, epidermin
177 Surgical wound Ap, Em, Km, Pc, Tc A53, Pep5
195 Surgical wound Ap, Pc A70, A53, 215FN, Pep5, epidermin

*Strains whose designation begins with letters from A to J represent different clones of MRSA strains determined by pulsed-field gel electrophor-
esis (PFGE) of SmaI-digested genomic DNA as previously described (Santos et al. 1999). Strain A/22C represents the Brazilian endemic clone.
Amikacin (Am), ampicillin (Ap), cefalotin (Cf), cefoxitin (Cfo), ciprofloxacin (Cip), clindamycin (Cl), chloramphenicol (Cm), ceftriaxone (Cro), eryth-
romycin (Em), gentamicin (Gm), imipenem (Im), kanamycin (Km), mupirocin (Mup), oxacillin (Ox), penicillin (Pc), rifampicin (Rif), sulfamethoxazole/
thrimethoprim (Sft), tetracycline (Tc). Both Mupr strains exhibited a low-level resistance. In all oxacillin-resistant strains, the presence of the mecA
gene was confirmed by PCR, which resulted in the amplification of a 154-bp internal fragment of the gene (data not shown).

Staph. aureus strains, ten were chosen as representative of
different clones of MRSA, including clone A, the endemic
Brazilian MRSA clone (Santos et al. 1999). Bacteriocino-
genic Staph. aureus and Staph. epidermidis strains from
previous studies (Table 1) were used as bacteriocin pro-
ducers in the inhibition assays. Staphylococcal strains
were stored in TSB (Difco) with 40% glycerol (w/v) at
)20
C until needed. The strains were grown in BHI (Dif-
co) at 37
C for 18 h. When necessary, the media were
supplemented with agar at 15% (w/v) or 06% (w/v).
Identification of the strains
Identification of the staphylococcal strains to the species
level was carried out by Gram staining followed by con-
ventional biochemical tests (Bannerman 2003) or, when
necessary, using a commercial kit for identification (API
Staph, BioMerieux, Marcy-lEtoile, France).
Resistance profile of the staphylococcal strains
The susceptibility of the nosocomial strains to 18 antimi-
crobial agents was performed following the procedures
outlined in the Guidelines of the National Committee for
Clinical Laboratory Standards (NCCLS 2003) except for
oxacillin and mupirocin, which was performed according
to Santos et al. (1999). The MIC was determined for
mupirocin. High-level mupirocin resistance was defined
as an MIC of 512 lg ml)1 and low-level resistance as
MICs of 4 lg ml)1 and 100 lg ml)1 (Santos et al.
1996). The detection of the mecA gene (which confers
resistance to all b-lactamic drugs, including methicillin
and oxacillin) by PCR was performed as described by
Santos et al. (1999).
Assay for antimicrobial activity
This assay was performed as described previously by Netz
et al. (2001), using the nosocomial staphylococcal strains
as indicators. Briefly, 107 cells of the bacteriocin-producer
strains were spotted on 18 100 mm plates containing
20 ml of BHI agar. After 18 h at 37
C, 105 cells of the
indicators in 3 ml of BHI soft agar were sprayed over the
plates. Plates were re-incubated at the same conditions
and the inhibition zones around the producer spots were
observed. The experiments were repeated at least twice.
The nosocomial strains were considered sensitive to a
bacteriocin when it exhibited a clear inhibition zone.
When the inhibition zone was either absent or contained
bacterial colonies, the nosocomial strain was considered
resistant to the corresponding bacteriocin.
Results
Strain A/22C, a strain representative of the endemic Bra-
zilian MRSA clone, proved to be sensitive to aureocin

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Bacteriocins as antimicrobial agents J.S. Nascimento et al.

Table 3 Nosocomial coagulase-negative Staphylococcus strains involved in human infections and used as indicators in the bacteriocin assays

Strains Source Resistance profile* Inhibited by the following staphylococcins

Staph. epidermidis
118 Surgical wound Am, Ap, Cf, Cfo, Cl, Cm, Cro, Em, Gm, Km, Imp, Mup, Ox, Pc, Sft A53, Pep5, epidermin, epilancin, epicidin
38 Surgical wound Am, Ap, Cf, Cfo, Cl, Cro, Em, Gm, Imp, Mup, Ox, Pc, Sft Pep5, epidermin
16 Surgical wound Am, Ap, Cf, Cip, Cl, Cm, Em, Gm, Imp, Km, Ox, Pc, Sft Pep5
25 Catheter tip Am, Ap, Cip, Cl, Cm, Em, Gm, Imp, Km, Mup, Ox, Pc, Sft Pep5
27 Blood Am, Ap, Cip, Cl, Em, Gm, Imp, Km, Mup, Ox, Pc, Rif, Sft Pep5
29 Blood Am, Ap, Cip, Cl, Cm, Em, Gm, Imp, Km, Mup, Ox, Pc, Sft Pep5, epidermin
86 Blood Ap, Cip, Cl, Cm, Em, Imp, Km, Mup, Ox, Rif, Sft, Pc, Tc Pep5, epidermin
72 Blood Ap, Cip, Cl, Cm, Em, Imp, Km, Mup, Ox, Pc, Rif, Sft Pep5
54 Blood Ap, Cip, Cl, Cm, Em, Km, Mup, Ox, Pc, Rif, Sft Epidermin
148 Blood Ap,Cro, Cfo, Em, Gm, Km, Mup, Ox, Pc, Tc Pep5
81 Blood Am, Ap, Cm, Gm, Imp, Km, Ox, Pc, Rif, Sft Pep5
78 Blood Am, Ap, Cm, Gm, Imp, Km, Ox, Pc, Sft
28 Medullary puncture Ap, Cip, Cl, Em, Gm, Imp, Km, Mup, Ox, Pc, Rif, Sft Pep5
127 Nasal swab Am, Cip, Cl, Em, Gm, Imp, Km, Mup, Ox, Sft Pep5
117 Nasal swab Am, Cip, Cl, Em, Gm, Imp, Km, Ox, Sft A70, Pep5
53 Blood NT
69 Blood NT Pep5
73 Blood NT Pep5
189 Blood NT Pep5
228 Blood NT Pep5
200 Eye secretion NT Pep5
Staph. haemolyticus
87 Blood Am, Ap, Cip, Cl, Cm, Em, Gm, Km, Ox, Pc, Rif, Sft, Tc Pep5, epidermin
74 Blood Am, Ap, Cip, Cl, Cm, Em, Gm, Imp, Km, Ox, Pc, Rif
177 Blood Ap, Cip, Em, Gm, Imp, Km, Pc, Rif, Sft, Ox Pep5
83 Blood Am, Ap, Cip, Em, Gm, Imp, Km, Ox, Pc, Sft, Ox
58 Surgical wound Cf, Cfo, Cm, Cro, Gm, Km, Ox, Pc, Sft, Tc Pep5, epidermin
84 Blood Ap, Co, Gm, Imp, Km, Ox, Pc, Sft Pep5, epidermin
66 Blood Ap, Cm, Gm, Imp, Km, Ox, Pc, Sft Pep5, epidermin
75 Blood Am, Ap, Cip, Gm, Km, Ox, Pc, Sft Pep5, epidermin
100 Blood Am, Ap, Gm, Km, Ox, Pc, Sft
225 Blood Ap, Imp, Ox, Pc, Sft
104 Blood NT
107 Blood NT
109 Blood NT
128 Nasal swab NT Pep5, epidermin, epilancin, epicidin
160 Nasal swab NT Pep5
47 Fistule NT
49 Fistule NT
Staph. hominis
41 Nasal swab NT Pep5, epilancin
45 Fistule NT Pep5, epidermin, epilancin
52 Blood NT Pep5
60 Blood NT A70, A53, epidermin, epilancin
79 Blood NT Pep5, epidermin, epilancin
108 Blood NT Pep5, epidermin
178 Urine NT Pep5, epidermin
226 Blood NT Pep5, epidermin, epilancin
Staph. saprophyticus
46 Fistule NT A53, Pep5, epidermin, epilancin
59 Urine NT A53, Pep5, epidermin, epilancin, epicidin
135 Urine NT Pep5, epidermin, epilancin
139 Urine NT A53, Pep5, epidermin, epilancin, epicidin
Staph. capitis
182 Urine NT A70, A53, Pep5

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Table 3 (contd)

Strains Source Resistance profile* Inhibited by the following staphylococcins

Staph. caprae
95 Blood NT Pep5
Staph. sciuri
175 Femoral secretion NT A53, Pep5, epidermin, epilancin
Staph. simulans
188 Blood NT A53, Pep5, epidermin, epicidin
Staph. warneri
88 Blood NT Pep5
91 Blood NT Pep5
190 Blood NT Pep5

*Only the CNS strains that exhibited resistance to oxacillin were subjected to antibiogram. Amikacin (Am), ampicillin (Ap), cefalotin (Cf), cefoxitin
(Cfo), ciprofloxacin (Cip), clindamycin (Cl), chloramphenicol (Cm), ceftriaxone (Cro), erythromycin (Em), gentamicin (Gm), imipenem (Im), kanamy-
cin (Km), mupirocin (Mup), oxacillin (Ox), penicillin (Pc), rifampicin (Rif), sulfazoprin (Sft), tetracycline (Tc). NT, not tested. The Staph. epidermidis
strains 86, 72, 54 and 148 exhibited high-level mupirocin resistance; the remaining Mupr strains were shown to carry a low-level resistance. In all
oxacillin-resistant strains, the presence of the mecA gene was confirmed by PCR, which resulted in the amplification of a 154-bp internal fragment
of the gene (data not shown).
, no inhibition.

A53, Pep5 and epidermin (Table 2), exhibiting inhibition A70

zones of 2034 mm. Seventy-two strains were then isola-
A53
Staphylococcins

ted from clinical specimens in four hospitals, identified to

the species level and subjected to antibiogram. The methi- 215FN
cillin-resistant phenotype was confirmed by the detection Epilancin K7
of the mecA gene on the bacterial chromosome by PCR Epicidin 280
(data not shown). Strains that proved to be resistant to
Epidermin
mupirocin were subsequently classified as either high- or
low-level resistant by MIC determination. Pep5
Fifteen additional S. aureus strains, including 12 multi- 0 10 20 30 40 50 60 70 80 90 100
resistant strains that were also resistant to mupirocin Percentage inhibition
and/or methicilin (MRSA), were then tested. The results
Figure 1 Inhibition of 57 coagulase-negative staphylococci (CNS) and
are presented in Table 2 and in Fig. 1. Both aureocin A53 16 Staph. aureus strains involved in nosocomial infections by seven
and Pep5 inhibited 875% of the 16 tested strains, inclu- staphylococcins produced by either Staph. aureus or Staph. epider-
ding the Brazilian MRSA endemic clone, A/22C. Epider- midis. White bars represent Staph. aureus whereas grey bars repre-
min was able to inhibit 13 (813%) of the Staph. aureus sent CNS. Aureocin 215N was not able to inhibit any CNS strains
strains, including also the Brazilian endemic clone. The tested whereas epilancin K7 and epicidin 280 showed no activity
remaining bacteriocins were able to inhibit either one against the Staph. aureus strains tested.

(aureocins A70 and 215FN) or none (epilancin K7 and

epicidin 280) of the strains.
In relation to the nosocomial CNS, the inhibition of 57
strains (among them, at least 24 multiresistant strains) by
the staphylococcins was evaluated and is presented in
Table 3 and in Fig. 1. Pep5 was able to inhibit most of
the strains (772%), including 19 out of 25 strains that
were also resistant to mupirocin and/or methicillin. Three
out of four CNS strains that proved to carry high-level
mupirocin resistance were inhibited by Pep5. The only
exception, strain 54 (Table 3), was also inhibited by Pep5.
However, its inhibition zone presented some resistant col-
onies and, for this reason, this strain was considered as
Pep5 resistant. Epidermin and epilancin K7 inhibited,
respectively, 23 (404%) and 12 (211%) strains, among
them mupirocin and methicillin resistant ones. The
remaining bacteriocins, including aureocin A53, inhibited
a low percentage of the nosocomial CNS strains (<15%).
Discussion
The use of broad-spectrum antimicrobial agents associ-
ated with the use of invasive medical devices has led to
the appearance of multidrug-resistant staphylococci in the
hospital environment (Pannuti and Grinbaum 1995). The
occurrence of nosocomial infections in Brazilian hospitals
because of multiresistant bacteria is widespread (Diekema
et al. 2001) and a large number of hospital-acquired
infections are caused by a unique MRSA endemic clone

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Journal compilation 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006) 215221 219
Bacteriocins as antimicrobial agents
(Santos et al. 1999). Therefore, in the present study, the
action of seven staphylococcins, bacteriocins produced by
staphylococci, was initially tested against A/22C, an
MRSA strain representative of the Brazilian endemic
clone, which proved to be sensitive to three staphylococc-
ins. Besides inhibiting this prevalent clone, Pep5, epider-
min and aureocin A53 inhibited most of the
multiresistant Staph. aureus strains additionally tested,
suggesting the potential use of these three staphylococcins
as an alternative to treat MRSA and other multiresistant
Staph. aureus strains. Moreover, our results indicated that
a combination of aureocin A53 and Pep5 would result in
inhibition of all nosocomial Staph. aureus strains tested.
Coagulase-negative staphylococci are frequently associ-
ated with nosocomial bloodstream infections and with
implanted medical devices (Rupp and Archer 1994). Their
acquisition of resistance to oxacillin and, recently, to
mupirocin, has drastically increased their importance
(Ferreira et al. 2002). The lower frequency of CNS strains
inhibited by the staphylococcins, when compared to
Staph. aureus strains, resulted from the poor inhibition of
the Staph. haemolyticus strains, as nine out of 17 of them
proved to be resistant to all bacteriocins tested. The rea-
son for this resistance is presently unknown. Pep5 proved
to be the most effective staphylococcin against CNS
inhibiting 772% of the nosocomial strains tested, inclu-
ding methicillin-resistant ones. However, if Staph. haemo-
lyticus is not considered, the percentage of CNS inhibited
by Pep5 increases to 90%.
Differently from CNS strains involved in bovine masti-
tis, which were all inhibited by epidermin (Nascimento
et al. 2005), less than 40% of the CNS strains associated
with nosocomial infections were sensitive to this staphylo-
coccin. These results suggest that staphylococci from dif-
ferent origins show variable sensitivity to the same
bacteriocins. Bacteriocin resistance is a complex pheno-
type involving alterations in cell wall and/or cytoplasmic
membrane (Crandall and Montville 1998; Limonet et al.
2002). Membrane differences are mainly based on the
proportion of unsaturated and saturated fatty acids and
on an altered phospholipid composition. Alternatively,
the presence on the bacterial genome of extra immunity
genes expressed without a cognate bacteriocin has recently
been shown to expand the bacteriocin resistance of the
strain possessing these genes (Fimland et al. 2002).
Mupirocin ointment is still the agent of choice used in
nasal decolonization of MRS, reducing their spread. How-
ever, some studies have shown an increase in the number
of mupirocin-resistant isolates among MRS strains (Boyce
1996; Ferreira et al. 2002; Krishnan et al. 2002). Interest-
ingly, both mupirocin (MupR) and MRSA strains tested
were inhibited by Pep5 and aureocin A53, suggesting the
potential of both bacteriocins also against MupR MRSA
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220 Journal compilation 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006) 215221
J.S. Nascimento et al.
strains, which have been associated with failure to clear
staphylococci in both colonized and infected patients
(Santos et al. 1996). Additionally, most (10 out of 11) of
the CNS that also exhibited resistance to mupirocin were
inhibited by Pep5, reinforcing that this antimicrobial pep-
tide could also have medical applications of decoloniza-
tion against mupirocin-resistant staphylococcal nasal
carriage.
Bacteriocin producers are generally immune to their
own bacteriocin because of a self-protection mechanism
that is specific for the bacteriocin produced (Sahl and Bier-
baum 1998; Ennahar et al. 2000). Therefore, it could be
argued that Pep5, epidermin and aureocin A53 would be
ineffective against all staphylococcal producers of these bac-
teriocins. However, recent results from our group indicate
that this should not be of concern because, among 188
strains of CNS involved in bovine mastitis and 57 strains
associated with human infections, which were used in the
present study, only 16 were shown to produce bacteriocins
and none of them produced Pep5, epidermin and aureocin
A53 (Nascimento et al. 2005; unpublished data). Similarly,
among 619 strains of Staph. aureus tested so far, including
clinical isolates and strains involved in bovine mastitis,
none of them was shown to produce aureocin A53, Pep5 or
epidermin (unpublished data).
None of the staphylococcins inhibited all strains tested.
Interaction of multiple bacteriocins has been proposed to
result in additive or synergistic effects that can better pre-
vent undesirable microbial activity (Ennahar et al. 2000).
Our results suggest that combination of Pep5 and aureo-
cin A53 seems to be an attractive strategy to increase the
spectrum of the staphylococcal strains inhibited. Studies
aiming to analyse the effect of the combined action of
these partially purified bacteriocins against these import-
ant nosocomial pathogens are currently in progress.
Acknowledgements
This study was supported by grants from CNPq, PRO-
NEX and FAPERJ to M.C.F.B. Janana dos Santos Nasci-
mento and Hilana Ceotto were recipients of scholarships
from CNPq for graduate and undergraduate students,
respectively.
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