Bacteriocins as alternative agents for control of multiresistant staphylococcal strains

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Bacteriocins as alternative agents for control of multiresistant staphylococcal strains J.S. Nascimento 1, H. Ceotto 1, S.B. Nascimento 1, M. Giambiagi-deMarval 2, K.R.N. Santos 2 and M.C.F. Bastos 1 1 Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Brazil 2 Departamento de Microbiologia Médica, Instituto de Microbiologia Prof. Paulo de Góes, 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 Universitária, , Rio de Janeiro, RJ, Brazil. mcbastos@micro.ufrj.br or mcbastos2@yahoo.com.br 2005/1041: received 13 September 2005, revised and accepted 20 October doi: /j x x Abstract Aims: To investigate the activity of seven staphylococcins, bacteriocins produced by staphylococci, against multiresistant Staphylococcus aureus and coagulase-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 staphylococcins used, Pep5 was able to inhibit 77Æ2% of the CNS strains and 87Æ5% of the Staph. aureus strains tested, including the Brazilian MRSA endemic clone, responsible for a large number of hospital-acquired infections in Brazil. On the other hand, aureocin A53 and epidermin presented a high antagonistic activity only against the Staph. aureus strains, being able to inhibit, respectively, 87Æ5% and 81Æ3% 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. 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 community-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 associated with implanted medical devices (Rupp and Archer 1994). Methicillin-resistant staphylococci (MRS) have become important nosocomial pathogens, requiring effective measures for controlling their spread. They have been isolated 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 Pseudomonas fluorescens that inhibits bacterial growth by binding to isoleucyl trna-synthetase. It is used as a topical agent Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006)

2 Bacteriocins as antimicrobial agents J.S. Nascimento et al. to eradicate methicillin-resistant Staph. aureus (MRSA) nasal carriage (Boyce 1996) and to prevent catheter colonization by staphylococci (Cookson et al. 1990). During the last few years, it has also been proposed as an advisable 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 resistance 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 staphylococci 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 selected 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 characterized species belong to class I or II bacteriocins. Class I bacteriocins, known as lantibiotics, contain post-translationally modified amino acids such as dehydroalanine, dehydrobutyrine, lanthionine and 3-methyllanthionine. Class II bacteriocins are small peptides that do not contain unusual amino acids (Sahl and Bierbaum 1998; Ennahar et al. 2000). Staphylococcins are bacteriocins produced by staphylococci. So far, Staph. epidermidis and Staph. aureus are the most important staphylococcin producers. In Staph. epidermidis, 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 production by staphylococci. Among the Staph. aureus staphylococcins, aureocins A70 and A53, produced by strains isolated from commercial milk, are the best characterized (Netz et al. 2001,2002). Both aureocins are atypical class II bacteriocins. Aureocin A70 is a multipeptide bacteriocin whose inhibitory activity results from four unmodified peptides and aureocin A53 is a highly cationic 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 staphylococcins was evaluated in an attempt to find new alternative agents for control of these important nosocomial pathogens. Materials and Methods Bacterial strains and culture conditions Sixteen Staph. aureus and 57 coagulase-negative Staphylococcus strains (Tables 2 and 3) involved in human infections 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 A70 Aureocin A53 MSWLNFLKYIAKYGKKAVSAAWKYKGKVLEWLNVGPTLEWVWQKLKKIAGL (51) Netz et al. (2002) 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 Tü 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. 216 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006)

3 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 Strains* Source Resistance profile Inhibited by the following 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 electrophoresis (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), erythromycin (Em), gentamicin (Gm), imipenem (Im), kanamycin (Km), mupirocin (Mup), oxacillin (Ox), penicillin (Pc), rifampicin (Rif), sulfamethoxazole/ thrimethoprim (Sft), tetracycline (Tc). Both Mup r 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). Bacteriocinogenic Staph. aureus and Staph. epidermidis strains from previous studies (Table 1) were used as bacteriocin producers 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 (Difco) at 37 C for 18 h. When necessary, the media were supplemented with agar at 1Æ5% (w/v) or 0Æ6% (w/v). Identification of the strains Identification of the staphylococcal strains to the species level was carried out by Gram staining followed by conventional biochemical tests (Bannerman 2003) or, when necessary, using a commercial kit for identification (API Staph, BioMérieux, Marcy-l Etoile, France). Resistance profile of the staphylococcal strains The susceptibility of the nosocomial strains to 18 antimicrobial 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, 10 7 cells of the bacteriocin-producer strains were spotted on mm plates containing 20 ml of BHI agar. After 18 h at 37 C, 10 5 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 Brazilian MRSA clone, proved to be sensitive to aureocin Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006)

4 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 218 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006)

5 J.S. Nascimento et al. Bacteriocins as antimicrobial agents 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), kanamycin (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 Mup r 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 zones of mm. Seventy-two strains were then isolated from clinical specimens in four hospitals, identified to the species level and subjected to antibiogram. The methicillin-resistant phenotype was confirmed by the detection of the meca gene on the bacterial chromosome by PCR (data not shown). Strains that proved to be resistant to mupirocin were subsequently classified as either high- or low-level resistant by MIC determination. Fifteen additional S. aureus strains, including 12 multiresistant strains that were also resistant to mupirocin and/or methicilin (MRSA), were then tested. The results are presented in Table 2 and in Fig. 1. Both aureocin A53 and Pep5 inhibited 87Æ5% of the 16 tested strains, including the Brazilian MRSA endemic clone, A/22C. Epidermin was able to inhibit 13 (81Æ3%) of the Staph. aureus strains, including also the Brazilian endemic clone. The remaining bacteriocins were able to inhibit either one (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 (77Æ2%), 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 colonies and, for this reason, this strain was considered as Pep5 resistant. Epidermin and epilancin K7 inhibited, respectively, 23 (40Æ4%) and 12 (21Æ1%) strains, among Staphylococcins A70 A53 215FN Epilancin K7 Epicidin 280 Epidermin Pep5 them mupirocin and methicillin resistant ones. The remaining bacteriocins, including aureocin A53, inhibited a low percentage of the nosocomial CNS strains (<15%). Discussion Percentage inhibition Figure 1 Inhibition of 57 coagulase-negative staphylococci (CNS) and 16 Staph. aureus strains involved in nosocomial infections by seven staphylococcins produced by either Staph. aureus or Staph. epidermidis. White bars represent Staph. aureus whereas grey bars represent CNS. Aureocin 215N was not able to inhibit any CNS strains tested whereas epilancin K7 and epicidin 280 showed no activity against the Staph. aureus strains tested. The use of broad-spectrum antimicrobial agents associated 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 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006)

6 Bacteriocins as antimicrobial agents J.S. Nascimento et al. (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 staphylococcins. Besides inhibiting this prevalent clone, Pep5, epidermin 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 associated 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 reason for this resistance is presently unknown. Pep5 proved to be the most effective staphylococcin against CNS inhibiting 77Æ2% of the nosocomial strains tested, including methicillin-resistant ones. However, if Staph. haemolyticus is not considered, the percentage of CNS inhibited by Pep5 increases to 90%. Differently from CNS strains involved in bovine mastitis, 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 staphylococcin. These results suggest that staphylococci from different origins show variable sensitivity to the same bacteriocins. Bacteriocin resistance is a complex phenotype 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. However, 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). Interestingly, both mupirocin (Mup R ) and MRSA strains tested were inhibited by Pep5 and aureocin A53, suggesting the potential of both bacteriocins also against Mup R MRSA 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 peptide could also have medical applications of decolonization 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 Bierbaum 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 bacteriocins. 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 prevent undesirable microbial activity (Ennahar et al. 2000). Our results suggest that combination of Pep5 and aureocin 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 important nosocomial pathogens are currently in progress. Acknowledgements This study was supported by grants from CNPq, PRO- NEX and FAPERJ to M.C.F.B. Janaína dos Santos Nascimento and Hilana Ceotto were recipients of scholarships from CNPq for graduate and undergraduate students, respectively. References Aires de Souza, M., Santos Sanches, I., Ferro, M.L., Vaz, M.J., Saraiva, Z., Tendeiro, T., Serra, J. and Lencastre, H. (1998) Intercontinental spread of a multidrug-resistant methicillin-resistant Staphylococcus aureus clone. J Clin Microbiol 39, Bannerman, T.L. (2003) Staphylococcus, Micrococcus and other catalase-positive cocci that grow aerobically. In: Manual of 220 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006)

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